Study on the Effect of Dietary Supplementation of ...prr.hec.gov.pk/jspui/bitstream/123456789/7705/1/Shakira Ghazanfar Full.pdfShakira Ghazanfar Department of Microbiology Faculty

1

Study on the Effect of Dietary Supplementation of Saccharomyces cerevisiae on Performance of

Dairy Cattle and Heifers

By

Shakira Ghazanfar

Department of Microbiology

Faculty of Biological Sciences

Quaid-i-Azam University

Islamabad

2016

2

Study on the Effect of Dietary Supplementation of Saccharomyces cerevisiae on Performance of

Dairy Cattle and Heifers

A thesis submitted in partial fulfillment of the requirements for the Degree of

Doctor of Philosophy

In

Microbiology

By

Shakira Ghazanfar

Department of Microbiology Faculty of Biological Sciences

Quaid-i-Azam University Islamabad

2016

3

4

Dedication

To

My Loving Parents (Chaudhry Ghazanfar Ali Suleri and Sughra Begum)

Caring Husband (Rana Akmal Hussain)

&

Cute Daughters (Heba and Hamna)

5

Dec larat ion

The material and information contained in this thesis is my original work. I have not

previously presented any part of this work elsewhere for any other degree.

Shakira Ghazanfar

6

LIST OF CONTENTS

Sr. No. Title Page No.

i List of Figures i

ii List of Tables iii

iii List of Abbreviations v

vi Acknowledgements x

v Abstract xi

1 INTRODUCTION 1

1.1 Aims and Objectives 7

2 REVIEW OF LITERATURE 8

3 MATERIAL AND METHODS 57

4 RESULTS 76

5 DISCUSSION 132

6 Conclusion/ Future Prospects 170

7 Recommendations 171

8 References 172

9 Publication 219

7

LIST OF FIGURES

Figure

No. Title Page

No.

2.1 Supply and demand of milk in Pakistan 9

2.2 Nutrients supply from different feed sources for animals in

Pakistan

11

2.3 Modes of action of probiotics 18

2.4 Estimated rumen microbial ecosystem 29

2.5 Representative scheme of the mode of action of S. cerevisiae 30

2.6 Components of fiber and its classification 33

2.7 A model of interaction of yeast cells with rumen microbes 35

3.1 Livestock Research Station, NARC, Islamabad 58

3.2 Animal shed at Livestock Research Station, NARC Islamabad 58

3.3 Commercially available yeast culture (Yac-Sac1026) 59

3.4 Dairy heifers in open paddock at LRS, NARC, Islamabad 59

3.5 Digestibility shed at NARC, Islamabad 60

3.6 Laboratory produced yeast, S. cerevisiae (QAUSC03) 74

3.7 Lactating dairy cattle in shed at open paddock at NARC,

Islamabad

74

4.1 Average monthly dry matter intake pattern of dairy heifers fed on

control feed (control, ♦; no yeast) or commercial probiotic feed

(COM-P, ■; control feed plus commercial yeast)

79

4.2 Average monthly growth pattern (Kg) of dairy heifers fed on

control feed (control, ♦; no yeast) or commercial probiotic feed

(COM-P, ■; control feed plus commercial yeast)

79

4.3 Total aerobic count (CFU/g) in the ruminal gut of dairy heifers fed

on control feed (control, ♦; no yeast) or commercial probiotic feed

(COM-P, ■; control feed plus commercial yeast)

91

4.4 Total Lactobacillus count (CFU/g) in the ruminal gut of dairy

heifers fed on control feed (control, ♦; no yeast) or commercial

probiotic feed (COM-P, ■; control feed plus commercial yeast)

91

4.5 Total coliform count (CFU/g) in the ruminal gut of dairy heifers

fed on control feed (control, ♦; no yeast) or commercial probiotic

feed (COM-P, ■; control feed plus commercial yeast)

92

4.6 Total Lactococcus count (CFU/g) in the ruminal gut of dairy

heifers fed on control feed (control, ♦; no yeast) or commercial

probiotic feed (COM-P, ■; control feed plus commercial yeast)

92

8

4.7 Total Enterococcus count (CFU/g) in the ruminal gut of dairy

heifers fed on control feed (control, ♦; no yeast) or commercial

probiotic feed (COM-P, ■; control feed plus commercial yeast)

93

4.8 Growth pattern of coliforms on machonkey agar (Right) and LAB

on MRS (Left)

94

4.9 Biochemical analysis of different bacterial isolates 94

4.10 Gram staining of Lactobacillus strains on MRS; gram positive rod 95

4.11 Gram staining of Lactococcus and Enterococcus strains on MRS 95

4.12 Gram staining of coliform strains on macconkey agar 95

4.13 Phylogenetic tree of the Lactococcus QAULL04, QAULG03,

QAULG02 species based on 16S rRNA gene sequence.

99

4.14 Phylogenetic tree of the Enterobacter QAUEV13 (KP25621)

based on 16S rRNA gene sequence

100

4.15 Phylogenetic tree of the Enterococcus (KP256016, KP256017,

KP256014, KP256015, KP256018) species based on 16S rRNA

gene sequence

101

4.16 Phylogenetic tree of the Escherichia QAUEV12 (KP256020)

based on 16S rRNA gene sequence

102

,4.17 Simple staining of yeast strains (L) QAUSC05 and (R) QAUSC03 106

4.18 Tolerance rate of isolated yeasts strains in bile salt (% + SEM) 106

4.19 Cholesterol assimilation of isolated yeast strains (%+SEM) 107

4.20 Anti-pathogenic activity of isolated yeast strains QAUSC03

(Strain #3) and QAUSC05 (Strain #5) against ATCC strains with

their zones of inhibition

107

4.21 Monthly variations in total aerobic count of lactating dairy cattle

fed on diet supplemented with a) no yeast (control, ♦), laboratory

yeast (LAB-Y, ■) or commercial yeast (COM-Y, ▲)

124

4.22 Monthly variations in total Lactococcus species count of lactating

dairy cattle fed on diet supplemented with a) no yeast (control, ♦),

laboratory yeast (LAB-Y, ■) or commercial yeast (COM-Y, ▲)

124

4.23 Monthly variations in total Enterococcus species count of lactating

dairy cattle fed on diet supplemented with a) no yeast (control, ♦),

laboratory yeast (LAB-Y, ■) or commercial yeast (COM-Y, ▲)

125

4.24 Monthly variations in total Bacillus species count of lactating

dairy cattle fed on diet supplemented with a) no yeast (control, ♦),

laboratory yeast (LAB-Y, ■) or commercial yeast (COM-Y, ▲)

125

4.25 Phylogenetic tree of the Bacillus (KP25619) based on 16S rRNA

gene sequence

130

9

LIST OF TABLES

Table

No. Title

Page

No.

2.1 Proximate composition of cow and buffaloes milk (per 100 g of

milk)

8

2.2 Milk composition of cow 43

4.1 Ingredient and chemical composition of the control and probiotic

feed fed to dairy heifers

77

4.2 Growth characteristics in dairy heifers fed on control and probiotic

feed

78

4.3 Nutrient digestibility (Means ± SEM) of dairy heifers fed on

control and probiotic feed

80

4.4 Haematological values (Means ± SEM) in dairy heifers fed on

control and probiotic feed

83

4.5 Blood serum metabolites (Means ± SEM) in dairy heifers fed on

control and probiotic feed

84

4.6 Serum macro-minerals (Means ± SEM) in dairy heifers fed on

control and probiotic feed

85

4.7 Total bacteria counts (CFU/g ± SD) in ruminal gut of dairy heifers

fed on control and probiotic feed

90

4.8 Morphological, biochemical identification of bacterial isolates on

MRS and macconkey agar

96

4.9 Identification of isolated strains based on 16S rRNA gene

sequences and their accession numbers published in DNA

database.

97

4.10 Economic efficiency of dairy heifers fed on control versus

probiotic feed

103

4.11 Morphological and biochemical characteristics of isolated strains 105

4.12 The antipathogenic activity of isolated yeast strains against ATCC

strains and their inhibitory zones diameter (mm)

107

4.13 Ingredient and chemical composition of the control, LAB

probiotic and COM probiotic feed

109

4.14 Dry matter intake and milk yield (Means ± SEM) in lactating dairy

cattle fed on control, LAB-probiotic and COM-probiotic feed

110

4.15 Milk composition (Means ± SEM) of lactating dairy cattle fed on

control, LAB-probiotic and COM-probiotic feed

111

4.16 Nutrient digestibility (Means ± SEM) of lactating dairy cattle fed

on control, LAB-probiotic and COM-probiotic feed

112

4.17 Effect of dietary supplementation of yeast on haematological

values (Means ± SEM) in dairy cattle

115

10

4.18 Effect of dietary yeast supplementation on blood parameters

(Means ± SEM) in lactating dairy cattle

117

4.19 Effect of dietary yeast supplementation on blood serum

metabolites (Means ± SEM) in lactating dairy cattle

119

4.20 Total bacteria counts (CFU/g ± SD) in ruminal gut of lactating

dairy cattle fed on control and probiotic feed

122

4.21 Morphological, biochemical identification of selavtive bacterial

isolates on MRS and TSA

127

4.22 Identification of isolated strains based on 16SrRNA gene sequence

and their accession numbers published in DNA database.

128

4.23 Economics of milk production of lactating dairy cattle fed on

probiotic yeast

131

11

LIST OF ABBREVIATIONS

AD Apparent Digestibility

ADF Acid Detergent Fibre

ADFD Acid Detergent Fiber Digestibility

ADG Average Daily Gain

ANOVA Analysis of Variance

AOAC Association of Official Analytic Chemists.

ATCC American Type Culture Collection

B-cells Bone-Marrow Cells

BLAST Basic Local Alignment Search Tool

BSH Bile Salt Hydrolase

Ca Calcium

CF Crude Fiber

CFU Colony Forming Unit

CMC Carboxy Methyl Cellucattlese

COM Commercial

COM-P Commercial Probiotic

CON Control

CO2 Carbon dioxide

Con. Concentration

CP Crude Protein

CPD Crude Protein Digestibility

CPY Commercially Produced Yeast

CDR Complete Randomized Design

12

CTAB Cetyl Trimethyl Ammonium Bromide

CV Crystal Violet

D Day

DC Dendritic Cell

DDBJ DNA Data Bank of Japan

DM Dry Matter

DMI Dry Matter Intake

DMD Dry Matter Digestibility

DNA Deoxyribonucleic Acid

EDTA Ethylene Diaminetetraacetic Acid

EDX Energy Dispersive Scattering

FAO Food and Agriculture Organization

FCR Feed Conversion Ratio

Fig. Figure

g Gram

G Glucose

GC Guanine Cytosine

GIT Gastro-Intestinal Tract

GOS Gcattlecto-Oligoheiferccharide

GRAS Generally Recognised As Safe

Hb Hemoglobin

HMM High Molecular Mass

IBD Inflammatory Bowel Disease

Ig Immunoglobulin

K Potassium

L Liter

IL Interleukin

IR Infrared

LAB Laboratory

LB Luria Broth

13

LAB-P Laboratory Probiotics

LH Luteinizing Hormone

Lit Liter

LMM Low Molecular Mass

LPY Laboratory Produced Yeast

LRS Livestock Research Station

LUB Lactic Acid utilizing Bacteria

m Molar solution

ME Metabolizable Energy

mg Milligram

Min Minute

ml Milliliter

MOS Mannan-Oligosaccharides

MP Microbial Protein

MR Methyl Red

MRL Microbiology Research Laboratory

MRS de Man, Rogosa and Sharpe

MRVP Methyl Red Vogues Proskauer

N Nitrogen

Na Sodium

NaCl Sodium Chloride

NARC National Agriculture Research Council

NCBI National Center for Biotechnology Information

NDF Neutral Detergent Fiber

NDFD Neutral Detergent Fiber Digestibility

NF-β Nuclear Factor Beta

NPN Non-Protein-Nitrogen

NRC National Research Council

NVL National Veterinary Laboratories

nm Nano-Meter

14

OD Optical Density

OGA Oxytetracyclin Glucoheifer Agar

OM Organic Matter

P Phosphorus

PbS Lead sulphide

PC Phyto Chelation

PCV Packed Cell Volume

QAU Quaid-i-Azam University

RDP Rumen Degradable Protein

Rpm Revolution Per Minute

rRNA Ribosomal Nucleic Acid

SC Saccharomyces cerevisiae

SD Standard Deviation

SDS Sodium Dodecyl Sulphate

SIM Sulfide Indole Motility

SME Standard Error of Mean

SNF Solid Not Fat

Sp Species

TGF Transforming Growth Factor

TNTC Too Numerous To Count

TSA Tryptic Soy Agar

TSI Triple Sugar Iron

TSS Tripticaseheiferlt solution

UN United Nation

UVD Ultra Violet Radiation

VFAs Volatile Fatty Acid

Vol Volume

WBC White Blood Cell

WHO World Health Organization

YC Yeast Culture

15

i.e. That is

C Degree Celsius

% Percentage

CO2 Carbon dioxide

O2 Oxygen

NH3 Ammonia

H2 Hydrogen

< Greater than

> Less than

ACKNOWLEDGMENTS

All praises for Allah, The Almighty, Who is the source of knowledge to all mankind.

Please and Blessing be upon on The Holy Prophet Muhammad (S.A.W), Who is

an eternal torch of guidance for humanity as a whole.

I am thankful to my supervisor, Dr. Iftikhar Ahmed, Principle Scientific Officer,

IMCCP, NARC, Islamabad for his guidance, support, and encouragement in the

completion of this thesis.

I owe my gratitude to Dr. Fariha Husan, chairman, Department of Microbiology,

Quaid-i- Azam University, Islamabad for her kind attitude and esteem cooperation

along with valuable guidance.

I wish to extend my appreciation to Dr. Muhammad Imran, Assistant Professor,

Department of Microbiology, Quaid-i-Azam University, Islamabad without whose

innovative idea this uphill task would have been impossible to achieve.

I owe my gratitude to Dr. Muhammad Iqbal, Head, Animal Nutrition NARC,

Islamabad for his great supoport.

With pleasure, I express my gratitude and appreciation to my lab fellow Maria

Qubtia, and Dawood Mustafa Bokhari for their help.

I am very grateful for the prayers of my parents, father in laws and my mother in

laws who have always been a source of inspiration for me. I thankful to my brothers

16

Dr. Mudassar, Alnsar, Eng. Mubasar and Dr. Mustansar, my sister Zumurad and

other family members: Nusrat Thakhar, Dr. Mazia, Rozina, Esba, Dr. Afifa,

Mubeen, Hamza, Zaeem, Sadeem, Hazik, Arham, Usyad, Menal, Abiha, Talha

and Ahfan for their constant support.

Last but not the least I would like to thanks to my caring Husband and cute

daughters for their constant support and unconditional love

Shakira Ghazanfar

17

ABSTRACT

Balance diet is one of the important factors in livestock productivity. Inadequate and unbalanced

diet is considered a major constraint for livestock promotion in Pakistan. The uneven dietary

patterns may result in dysbiosis in rumen. This leads to reduced growth rate, low milk production

and poor reproductive performance. Under such situation, production of livestock can be

increased through treat dysbiosis by supplementation of probiotic-yeast that may stable rumen

pH, increase microbial population, improve gut microbial balance and consequently improve

nutrient utilization and digestion efficiency resulting in enhanced growth rate, feed efficiency

and milk yield. From this line of research, we conduct an experimental study to analyses (i) the

effects of commercially available yeast culture on the growth performance and nutrient

digestibility of dairy heifers (ii) to compare the effects of dietary supplementation of laboratory

produced and commercially available yeast culture on the growth and health status in dairy

heifers (iii) to compare the effects of dietary supplementation of laboratory produced and

commercially available yeast culture on production performance and health status in lactating

dairy cattle and (iv) to study the impact of feed supplements on the changes in fecal microbiota

of dairy cattle and heifers. To achieve these objectives, the experimental work was divided into

three phases. In the first phase of the study, eight dairy heifers (87±5 kg and 6 to 7 months) were

divided into two equal groups of four animals each (control and probiotic) following completely

randomized design. During the trial, heifers in control group were offered control diet (NRC

recommended diet) while in the probiotic group fed with control diet plus commercial available

probiotic yeast (Yea-Sac1026; 5g/animal/corresponding to 2.5×1007 CFU/g S. cerevisiae). The

experimental period was 120 days. Results reveals that dairy heifers fed on probiotic feed gained

significantly (P<0.05) higher average daily weights than dairy heifers fed on control feed. At the

same time, it was observed that probiotic yeast supplemented heifers digested their dietary

nutrient significantly (P<0.05) at higher rate than non-supplemented heifers. Blood urea and

cholesterol levels were significantly (P<0.05) lower in the probiotic yeast fed heifers than those

of control heifers. Feeding probiotic yeast to dairy heifers had no remarkable effect on serum

micro-mineral. The supplementation of the probiotic yeast significantly (P<0.05) decrease the

coliform (CFU/g) species counts and significantly (P<0.05) increases the Lactobacillus (CFU/g)

species counts in the ruminal gut resultantly in improved gut health, increase digestion rate and

better growth performance in dairy heifers. In general, the results of phase I show a clear

18

advantage of probiotic yeast (Yac-Sac1026) regarding improved growth efficiency and health

status of dairy heifers, however, it was not economically efficient. Therefore, to fulfill the need

to isolate an indigenous probiotic yeast strain for our local breeds, in second phase of the study,

two strains of yeast culture; S. cerevisiae (SCQAU03; SCQAU05) were isolated. On basis of

comparatively higher enzymatic potential and as well as probiotic attributes, SCQAU03 strain

was selected for supplementation in animal feed. In the third phase, S. cerevisiae (QAUSC03)

was used in the lactating dairy cattle feed. For this study nine lactating dairy cattle were divided

into three equal groups following completely randomized design. In group I, cows were fed on

control diet (3 kg concentrate, 8 kg maize silage and 20 kg oats fodder). In group II, cows were

fed on control diet plus commercially (COM-Y) available yeast Yac-Sac1026 (10g/day/animal

corresponding to 2.5×1007 CFU/g S. cerevisiae) while in group III, cows were fed on control diet

plus laboratory (LAB-Y) produced yeast (8g/day/animal corresponding to 3.13×1007 CFU/g S.

cerevisiae) for 60 days. Results reveals that LAB-Y group produces significantly (P<0.05) more

milk with high fat content than other groups. However, milk protein, total solid, lactose and

solids not fat remained unchanged. It was found that dry matter, organic matter and protein

digestibility was significantly (P<0.05) better in both probiotic fed groups than the control group

on the other hand, nutrient detergent fibre and acid detergent fibre digestibility were significantly

(P<0.05) better in LAB-Y fed group as compared to other groups. Improved blood hematological

profile and blood chemistry was observed in the probiotic yeast fed groups. Results of the

ruminal gut microflora showed that the average, beneficial Lactococcus species (CFU/g) counts

were increase while pathogenic Enterococcus species (CFU/g) counts were lower in (LAB-Y)

yeast fed groups than other groups which leads to improve GIT microbial balance in this group.

The economic efficiency of LAB-Y fed group was also better than the other groups. It can be

concluded from phase III, that laboratory produced yeast improves the production performance,

gut health and wellbeing of lactating dairy cattle in cost effective manner. Locally isolated yeast

strain may be adopted well in the cattle gut than exotic probiotics.

Finally, we concluded from our experimental study that, dietary supplementations of yeast

culture (Yac-Sac1026) enhance growth efficiency and health status in dairy heifers and locally

isolated yeast economically improves production efficiency and wellbeing in lactating dairy

cattle without any adverse effect.

19

Chapter-1

INTRODUCTION

Livestock plays an essential role in human nutrition by providing essential nutrients in the

form of milk, meat, and egg. Pakistan is an agricultural based country, where livestock

contribution is about 55.9 % of the agriculture value added and 11.8% to national GDP

(Economic Survey of Pakistan, 2013-14). Livestock also contributes towards exports and

8.5-9.0 percent of total exports belong to this sector (Afzal, 2008) Pakistan is top ranked in

livestock population having about 39.7 million buffaloes and 34.6 million cattle (Economic

Survey of Pakistan, 2013-14). Like all other South Asian countries, Pakistan has also been

facing a lot of problems in livestock industry. One of the major problems is the poor growth

and productive performance of the dairy animals. Our livestock sector is mainly based on

traditional lines resulting in low production performance of dairy animals owing to several

factors. These key factors include malnutrition, poor genetic makeup, late age of maturity (Bilal

and Ahmad, 2004; Bilal et al., 2006), traditional line farming poor health condition, insufficient

feed resources and a disorganized marketing and extension services (Ahmad et al., 2012; Arif et

al., 2013).

By several independent analyses, it is conferred that the major constraint in the development

of productive livestock sector is the poor nutritional status of animals. This malnutrition is

due to insufficient feed resources, imbalanced feeding and use of conventionally feeding

scheme (Sarwar et al., 2002; Bilal and Ahmad, 2004; Nkya et al., 2007; Anjum et al., 2012;

Arif et al., 2013), which results in slow growth rate and low productive potential in

ruminants (Jabbar et al., 2006, Bhatti et al., 2007). Malnutrition does not allow full

exploitation of animal’s genetic potential (Raza et al., 2006). Most of the dairy cows enter

into negative energy balance during early lactation leading to poor reproductive efficiency

(Wathes et al., 2007). Moreover, poor nutritional status can affect the activity of certain

enzymes, thereby, impairing the overall immune function of the animal (Spears, 2000). Due

to poor feeing, animals are also at risk to entire bacterial imbalanced and generally suffer

from digestive and respiratory diseases leading to insufficient digestion and consequently

retarded growth and productive performance.

20

It is claimed that Pakistan is at third position in the milk producing countries (FAO, 2010;

IFCN, 2014) and produces 50 million tone milk annually (Economic Survey of Pakistan,

2013-14), but still there is a gap in the demand and supply of milk (Chattha et al., 2013). The

most important reason for this gap is that the human population and consumption per capita

is increasing at the rate of 3% annually, however, the milk production is not increasing at the

same pace. We spend huge amount of foreign reserves to import milk and milk products to

fulfill market needs (Economic Survey of Pakistan, 2008-09; GOP, 2011; Mansoor, 2012). It

is alarming situation that being an agricultural country we have to import milk and milk products

by spending up to 22 million US dollars to fulfill consumer needs (Economic Survey of

Pakistan, 2008-09; Chaudhry et al., 2012). Although the milk production has been increased

overtime; however, at increased animal population (ACO, 2006), average dairy animal yields

of Pakistan is 6-8 times less than that of the advance countries (Hussain et al., 2010).

Therefore, to cope with a huge milk need in future, a holistic approach is required to develop

integrated solution for dairy chain in country in order to meet the requirements. On the other

hand the poor growth rate in growing dairy animals is also major problem in our livestock

industry (Anjum et al., 2012). Several independent studies carried out in Pakistan clearly

indicate that the production performance of the existing animals can be improved by 30 to

35% with proper feeding (Afzal, 2007). Proper feeding management can provide adequate

energy and nutrient during early growth and lactation that leads to a carryover effect

resulting in improved energy and nutrient utilization and ultimately growth, production and

reproductive performance of the ruminants (Mulligan and Doherty, 2008; Gong et al., 2002).

Ruminants can eat different types of feed that are digested by microbial biomass resulting in

better metabolism, which ultimately impacts the dairy animal productivity. The microbial

flora in the gastro intestinal tract (GIT) has a lot of impact on the productive efficiency, health

status and wellbeing of the dairy animals (Guarner and Malagelada, 2003; Eckburg et al., 2005;

Dowd et al., 2008). The diversity and function of ruminal GIT microbes are very important in

feed digestion. The way the nutrients are digested in GIT in ruminants have a crucial impact on

growth, health and productivity (Fuller, 1989). The GIT inhabits multifarious microbial diversity

that helps in generating impassive response regarding nutritious, health, physiology and

productivity of animals (Guarner and Malagelada, 2003). The existing gut microbiota regulates

food safety through shedding of pathogens, interaction with organisms and resource competition

21

in the GIT (Nurmi and Rantala, 1973). Gastrointestinal tract micro-flora aids in stimulation of

the immune system that acts as a barrier against infectious pathogens. It also restrains the

injurious and pathogenic bacteria in gut colonization (Cebra et al., 1999). Different strategies

have been used to enhance the micro-biota of gastrointestinal tract, which ultimately affects

the production potential and growth efficiency of dairy animals. Now a days, the improvement

of microbiota of gastrointestinal tract by using probiotics has become a useful and economical

method to enhance the health and productive performance of animals.

“Probiotics supplements” are natural products containing live microbiota that beneficially affect

the health and production performance of the host by improving gut microbial flora

(Klaenhammer et al., 2012). Early in history (100 years ago), Metschnikoff was the first one who

instigated the interests in probiotics (Metschnikoff, 1907). Lactic acid bacteria are the primary

source of probiotics. It includes Lactobacillus sp., Bifidobacterium sp., Enterococcus sp. and

Pediococcus sp., (Klein et al., 1998) Bacillus sp.,(Senesi et al., 2001) Clostridium butyrium

(Takahashi et al., 2004), yeast “Saccharomycees boulardii” (Elmer et al., 1999),

“Saccharomyces cerevisiae” and Geotrichum candidum (yeast-like fungus) are also appropriate

for probiotics and as animal’s feed sources (Fuller, 1992; Guillot, 1998). Bacterial probiotics

were given better results in young calves, chickens and pigs, whereas yeast/ fungal probiotics

were effective in adult ruminants (Musa et al., 2009). Consequently, probiotic strains carry out

their advantageous phenomena by showing some of its potential activities such as, they should

have their survival rate in upper GIT, they should tolerate bile toxicity and gastric acid

environment (Del Piano et al., 2006) and they should have the ability of mucin-binding and

adherence to intestinal-imitative epithelial linings (Dunne et al., 2001). Furthermore, Antibiotic

sensitivity of probiotic strains must be assessed before its application in food processing (Parvez

et al., 2006). One of the major advantages of the probiotic use in livestock sector is that the

antibiotics uses have been banned in the European Union. Hence, livestock sector demand

from producers for alternative feed additives that can be used to enhance rumen fermentation

and improve productive performance of animals (Ayad et al., 2013). Dietary supplementation

of probiotics is viable and safe option for the farmers to enhance the performance parameters

of the lactating dairy cattle and heifers (Lascano et al., 2012).

22

Recently, consumers s’ demand about safe and healthy food products has been increased

worldwide. Because of that, the advantage of using probiotics is not only to enhance the

productive performance but also to (contribute to) lowing the risk of ruminant GIT carriage

of human pathogen and to reduce excretion of polluting outputs such as nitrogen-based

compounds and methane (Strohlein, 2003). The S. cerevisiae received the Generally

Recognized As Safe (GRAS) status from Food and Drug Administration (FDA) and thus , is

appropriate for use in animal feeds (Auclair, 2001). Some factors i.e. expected response, net

profit, ongoing research, and field responses should be considered to determine when a feed

additive is used for experiment (Hutjens, 1991). Fermented yeast culture has emerged as a

cost-effective product that has many benefits to ruminants (Hutjens, 2003). One of the major

benefits of the probiotic yeast is that yeast has no antibiotic resistance gene (Czerucka et al.,

2007). It has also ability to tolerate to bile salt and gastric acid and to neutralized enterotoxin

and colonizes in the GIT resultantly improve health status and production efficiency of the

dairy animals.

Therefore, presently livestock industry is showing great interest in the use of probiotics for

improving performance of the dairy animal. This ultimately helps in combating the food security

challenge (Puniya et al., 2015). Yeast culture as probiotic may improve growth rate and health

status, increases feed efficiency of poor quality forages and high grain diets (Arambel and Kent,

1990) by increasing the desirable bacterial population and stabilizes the pH inside rumen (Shehu

et al., 2015). Yeast produces many important fermentation metabolites such as protein,

carbohydrates, high potency vitamins and different types of important minerals and enzymes

(Szucs et al., 2013) that makes it useful and highly nutritive feed supplement for ruminants

(Yalcın et al., 2011). These fermentation metabolites may have a number of positive effects on

the rumen including stimulation of the function of desirable bacterial species, increased pH,

increased number of cellulolytic bacteria and they alter the volatile fatty acid concentration

(Dolezal et al., 2012). It supports fermentation by producing many useful digesting enzymes in

the gut and stabilizes the microbial flora within the GIT by supplying the various nutrients which

are essential for their growth purposes.

Earlier scientists reported that yeast culture has significant effects on daily feed intake, feed

conversion ratio, nutrient digestibility and gut health (Lascano et al., 2012; Ayad et al., 2013;

23

Agazzi et al., 2014). Dietary supplementation of yeast culture (S.cerevisiae) improves growth

efficiency in dairy heifers and milk production and its composition in lactating dairy cattle

(Sabbia et al., 2012; Ghazanfar et al., 2015). Hematology and minerals absorption are also

positively affected by yeast culture (YC) resulting in good health and immunity (Dolezal et al.,

2012). The physiological, anatomical and immunological status of the host is strongly

dependent upon micro-biota of gastrointestinal tract which facilitates essential functions to

host. YC has also imparted a positive effect on the balance of fecal microbiota that improves

the gut health of the dairy animals (Agazzi et al., 2014). Another advantage of the use of

yeast culture is that the benefit to cost ratio of YC (S. cerevisiae) supplementation in dairy

cattle is 4:1 (Hutjens, 1991). Many microbial based products are available in the local market

(Szucs et al., 2013). But that may not be effective and economical for our local breed. The

economic advantage of microbial feed additives depends on the price of microbial culture,

microbial strain, concentration of live cell (CFU), age, diet, breed, the lactation stage of the

animal and geographical location of the animal (Yalcın et al., 2011, Vibhute et al., 2011).

In this context, there is adire to propose an empirical study that focuses on probiotic

utilization and its efficiency in local dairy animals. Such empirical study will certainly help

farmers, researchers and government officials to formulate proper guidelines related to the

enhancement of dairy sector in the country. Little work has been conducted in Pakistan

regarding the use of probiotics to enhance the performance parameters of dairy animal. From

this line of research, we conducted an empirical study to check the impact of probiotic yeast

on the performance of dairy cattle and heifers under the control environment.

24

AIMS AND OBJECTIVES

This research work is undertaken with these following objectives.

1. To determine the effect of Saccharomyces cerevisiae (Yea-Sac1026) on growth performance and

wellbeing of dairy heifers.

2. To study the comparative impact of Saccharomyces cerevisiae (Yea-Sac1026) and locally isolated

yeast on nutrient digestibility and blood chemistry of lactating dairy cattle.

3. To study the comparative impact of Saccharomyces cerevisiae (Yea-Sac1026) and locally isolated

yeast on milk yield and milk composition and health status in lactating dairy cattle.

4. To study the impact of feed supplements on the changes in fecal microbiota of dairy cattle and

heifers.

25

Chapter-2

REVIEW OF LITERATURE

2.1 Overview of dairy sector of Pakistan

Being major player in the national economy, livestock sector is considered as an important

economy engine for poverty alleviation in Pakistan. It provides food, income, employment and

foreign exchange. Livestock sector mainly consist of cattle, buffalo, sheep and goat. It is

estimated that Pakistan contains 34.60 million buffaloes, 39.70 million cattle, 29.10 million

sheep and 66.60 million goats (Economic Survey of Pakistan, 2013-14). The main dairy animal

in Pakistan is buffalo, which produced more than 61 % of the total milk produced in the country;

on the other hand 35 % of the total milk produced in the country comes from cattle. Many

important breeds are present in Pakistan that includes Nili Ravi, Kundi and Aza Kheli. Nili Ravi is

considered the best buffalo breed in world and known as Black Gold of Pakistan (Bilal et al.,

2006). Sahiwal, Cholistani and Red Sindhi are the main cattle breeds present in Pakistan (Iqbal et

al., 2015). Sahiwal is an important dairy cattle breed of Pakistan and moreover, due to its heat

and disease resistance qualities, it has gained international recognition (Rehman and Khan,

2012). Within livestock sector, milk is the largest and the single most important commodity and

has long lasting benefits such as bone health, lowing the risk of cardiovascular and blood

pressure diseases, and it is effective against obesity, type 2 diabetics, cancer and dehydration

(Shahid et al., 2012). From nutritious point of view, the milk is a major source of dietary energy,

protein and fat (Table 2.1).

Table 2.1: Proximate composition of cow and buffaloes milk (per 100 g of milk)

Proximate* Energy (KJ) Water

(g)

Total

protein (g)

Total fat

(g)

Lactose

(g)

Ash

(g)

Cow milk 262 87.8 3.3 3.3 4.7 0.7

Buffalo milk 412 83.2 4.0 7.5 4.4 0.8

* Average values After: FAO (2013)

26

Pakistan is 3rd largest milk producing country in the world and produces 50.99 billion liters milk

annually (IFCN, 2014). Besides that huge milk production, Pakistan has been facing a gap

between supply and demand of the milk (Figure 2.1). If we compare the milk yield per animal of

Pakistan with other advanced countries like USA, we observe that Pakistan has 1.6 million more

milking animals than USA but produces 60 billion liter less milk highlighting a significant loss

in potential economic and social value (Shahid et al., 2012; Anonymous, 2009). It is estimated

that per animal milk production is not very high (3.15 liters) in Pakistan as compared to USA

(28.35 liters) (Anonymous, 2006). It is also found that approximately eight milking animals of

Pakistan are equal to one milking animal of the developed world (Shahid et al., 2012;

Anonymous, 2009).

Figure 2.1: Supply and demand of milk in Pakistan. Adopted by (Anonymous, 2011).

2.2 Major constrains in dairy industry of Pakistan

27

There are many factors constraining the improvement in dairy sector of Pakistan. Main factors

represented in the literature includes: poor policy and marketing, weak extension service,

inadequate and inappropriate research, low genetic potential, poor performance of large

population, poor health status, inadequate and poor quality feeds (Nkya et al., 2007; Sarwar et

al., 2010; Ahmad et al., 2012; Anjum et al., 2012; Arif et al., 2013; Iqbal et al., 2015). Feed and

nutrition have been noted as major constructs in animal production performance in south Asia

(ILRI, 1995, Devendara, 2000). So, the significance of improved nutrition in dairy sector is

therefore a major consideration. From nutritional point of view, poor availability of nutrients,

inadequate feed recourses, imbalanced feeding scheme and use of conventional feeding scheme

are major constants that affect the dairy sector of the country (Sarwar et al., 2002; Nkya et al.,

2007; Chattha et al., 2013). Fodder, crops residues, forages, rangelands and agro-industrial by-

products are the main feeding sources for the dairy animals in Pakistan (Hanjra, 1995; Iqbal et

al., 2015). It has been estimated that fodder is the main feeding source for livestock in Pakistan,

but the optimum level of feed and fodder is not available in Pakistan which leading to low

production performance of the dairy animals (Figure 2.2).

2.3 Strategies to enhance performance of in dairy cattle and heifers

The following common strategies can be used to enhance the performance of dairy cattle and

heifers

1. Improve health status of the animals

2. Improve breeding process

3. Improve managemental practices

4. Improve feeding and nutrition

Literature suggests that the milk production depends on the breed, feeding system, and

management, climate of the dairy farm, age and health of the animal at first calving (Rao,

1995; Hussain et al., 2010; Iqbal et al., 2015). Among all these factors, feed and nutrition has a

major effect on the milk production (Hussain et al., 2010).

28

Figure 2.2: Nutrient supply from different feed sources for animals in Pakistan. Adopted by

(Iqbal et al., 2015).

It is estimated that, improvement in the feed quality and quantity could bring improvement in the

livestock production up to 50% from exciting gene pool of the animal (Hasnain, 1983).

2.4 Nutritional strategies to enhance performance of in dairy cattle and heifers

Nutrition plays a crucial role in the performance of dairy animals. Many approaches have been

sought to economically enhance the performance of the dairy animals without affecting the

health status. Some of the approaches represented in the literature includes: use of balance

feeding scheme, use of stair step feeding scheme, use of feed additives, and manipulation of CP:

ME (Lammers and Heinrichs 2000; Ford, 2001; Yalcin et al.,2011; Anjum et al., 2012; Arif et

al., 2013; Ayad et al., 2013).

2.5 Improving diary animal performance by using of feed additives

Feed additive is an ingredient or combination of different feed components that give a useful

response to animals in a non-nutrient role such as, shifting of pH and growth or metabolic

Forages and crop residues,

51%Rangelands,

38%

Post-harvest grazing, 3%

Cereal by-products, 6%

Oil cakes, 2%

29

modifier (Hutjens, 1991). It may also be defined as any chemical incorporated in an animal feed

to improve the weight gain, feed efficiency, production potential or preventing and controlling

disease.

2.5.1 Types of feed additives

Feed additives can be a classified into, nutrient feed additives (which contains; amino acid,

minerals, vitamins) and non-nutrient feed additives (which contains; antibiotics, hormone,

common modulators, coccidiostats, enzymes, antioxidants, mycotoxin binders, anti-caking

agents, feed preservatives, flavoring agents, coloring agents, pellet binders, dietary buffers,

methane inhibitors, propionate promoters and probiotics). Next, we discuss probiotics in detail.

2.5.2 Probiotics

Microbial feed supplementation or ‘Probiotics’ are the living microorganisms specifically,

bacteria and yeast, that grant the beneficial health postures to the host when administered in

adequate amount (Dunne et al., 2001). Generally, probiotics have been employed as a beneficial

source both for humans and animals gut. With reference to UN, FAO, and WHO, probiotics are

defined as: “The livable microorganisms predominantly reside inside the intestinal tract provide

healthy and beneficial impact to host”. As probiotics are considered as the beneficial source of

microorganisms, so they help in stimulating the growth of recommended bacteria and pop out

the detrimental microorganisms, thus, they support the natural defense mechanism of the body

Apart from this, they also induce strong immunological aspects on intestinal immune system,

which causes the displacement of enteric pathogens (Dunne et al., 2001). They also grant

probiotic anti-mutagens and anti-oxidants and some other achievements through cell signaling.

There have been studied various beneficial aspects of probiotics on number of imperative

diseases such as, intestinal infections, some allergy, inflammatory bowel disease (IBD) (Di

Caro et al., 2005) and control of diarrhea (Gao et al., 2010). Probiotics are also behaved as a

potent supplier of vitamin sources (particularly the B group) (Crittenden et al., 2003).

2.5.3 Sources of probiotics

The probiotics are best available in the form of “fermented milk products”, “dairy products”,

“yogurt” and in some “non-dairy food items”. Some of these are describes as following;

2.5.3.1 Food items

30

The food items including mayonnaise, soymilk, fruit drinks, meat products, baby food and

vegetables are rich in probiotic microorganisms. They are also delivered in the form of

supplements (tablets and capsules) (Homayouni et al., 2008).

2.5.3.2 Fermented food items

Dairy and dairy-associated products provide excellent sources of probiotics (Liong, 2011). A

variety of microorganisms, specifically Lactic acid bacteria, Bifidobacterium (from fermented

milk) has been applied as primary source of probiotics since centuries. The conventional milk

fermentation process has intricate lactic acid bacteria composition, thus act as a potent probiotic

source. In a latest designed study, various LAB strains were isolated and identified from different

dairy food stuffs such as from kurut (fermented yak milk), some yeast and Lactobacillus strains

from Koumiss (fermented milk drink), kefir grains and masai milk. Microorganisms screened out

from these sources thus enhance their immunity level (Patrignani et al., 2006; Ya et al., 2008;

Romanin et al., 2010; Audisio and Benitez-Ahrendts, 2011). Naturally, Lactobacillus specie is

evaluated as source of probiotics that is primarily accessible from traditional and natural

fermented products (Lim and Im, 2009, Won et al., 2011) which includes “Weissella” specie

isolated from Nigerian fermented food and is behaved as a potential probiotic (Ayeni et al.,

2011).

2.5.3.3 Non-dairy fermented products

They also set forth the criteria of probiotics strains (Rivera and Gallardo, 2010). A reported

study manipulates the in vitro characteristics of bacterial strains that include; L. sakei, L.

curiatus and Staphylococcus carnosus from meat and L.paracasei and L.plantarum from fruits

exhibit; the same metabolic and functional properties like human intestinal flora (Haller et al.,

2001). Additionally, Lactobacillus strains from brine of naturally fermented Aloren (green table

olive) and L. buchneri P2 from pickled juice have revealed some of the probiotic properties as,

acid and bile tolerance, antimicrobial activity and cholesterol reduction (Zeng et al., 2010).

2.5.3.4 Other sources

Gastrointestinal tract is an effective and primary source of probiotics which inhabit almost more

than 500 bacterial species. L.gasseri and L.reuteri are the most commonly used probiotic species

that reside in the human gastrointestinal tract (Ryan et al., 2008). Similarly, B.longum (Srutkova

et al., 2011) and L.acidophilus (Lin et al., 2009) strains were screened out respectively from

healthy human adult and being marked as probiotics. Besides this, probiotic strains are also

31

profound in animals gastro intestine, commonly in rats, pigs, and poultry sources (Petrof et al.,

2012). Bee gut induces beneficial response to honey bee colonies (Audisio and Benitez-

Ahrendts, 2011). Moreover, GIT of marine and fresh water fish such as, carassiusauratus gibelio

(Chu et al., 2011) and Shrimp (Hill et al., 2009) are also rich sources of probiotics. Furthermore,

in a reported study, human breast milk also provides Lactobacillus strains that act as probiotic

source. This helps in generation of T-cells and natural killer cells and also responsible for

regulatory T cell expansion that simultaneously heighten natural and acquired immunity (Perez-

Cano et al., 2010).

2.5.4 Functional aspects of probiotics

Some clinical trials of probiotics were also implied on animal and human studies (Yan and Polk,

2011). Probiotic effects were analysed and verified by number of trials. They show sudden

responses in repressing diarrhea (Lye et al., 2009), relieving lactose intolerance, anti-colorectal

cancer (Rafter et al., 2007; Liong, 2008) and antimicrobial activities, easiness in post-operative

intricacies (Woodard et al., 2009), reduction of irritable bowel symptoms (Moayyedi et al.,

2010), and hinder inflammatory bowel diseases (Golowczyc et al., 2007). Many experimental

works have manipulated the beneficial effects of non-viable probiotics by means of fermentation

(primarily by LAB), which include the procreation of secondary metabolites e.g. vitamin B,

bioactive peptides, exopolysaccharides, bacteriocin and organic acids. These metabolites are

soluble and can be spray-dried, which are added in the form of dried powder in food milieu.

Non-viable probiotics have certain benefits over viable ones including pro-long shelf life, easier

handling, transportation and storage facilities, and lessen refrigerated storage conditions.

Probiotics have shown direct and indirect effect on functional (fermented) food stuffs. Direct

effect indicates host-organism relationship while in-direct effects demonstrate the biogenic

upshot (due to taking in of microbial metabolites as a result of fermentation). This advances

towards the efficient consequences of probiotics that seems to be applied in non-dairy food items

as products related to chocolate, chewing gum, biscuit, honey, cereals, cakes, dressing, sweetness

and tea (Vinderola, 2008). In general, probiotic bacteria in the food industry provide somehow

difficulty in their multiplication and survival rate because of the distress conditions of

gastrointestinal tract. To ensure shelf-life of probiotics, novel probiotics are being designed

through microencapsulation technology that opposes environmental conditions. Various factors

32

could contribute to the beneficial aspects of probiotics but its proper mechanism of action is still

vague.

2.5.5 Use of probiotics in ruminants

Globally, ruminants are subjected as a momentous quality of tamed animal species, which act as

the suppliers of dairy and meat products. Among them, dairy cattle are demanded at peak and

marked as chief supplier. Ruminants are characterized as herbivores or relating of two suborders

of herbivores that chew the cud and have a complex 3 or 4 chambered stomach. The ruminant’s

stomach is divided into three pre-gastric chambers as rumen, reticulum and omassum. By far,

rumen is the chief component of ruminant’s stomach. Basically, rumen works for the

fermentation and hydrolysation of ingested plant matter and feed materials, while the rest of the

undigested particles and microbial cells surpass into the “abomassum” (the fourth chamber of

ruminant stomach) where gastric digestion takes place. Microbes in rumen provide healthy and

nutritious importance to host and human (Flint et al., 2008). This competency of ruminants has

been proved beneficial for mankind because of the fact that ruminants undergo conversion of

stored energy of plant mass into edible food products. The degree of nutrient consumption by

rumen microbes is based on enzymes that set out a need for the proper utilization of microbial

enzyme system which specifies the purpose and usage of microbial feed additives in an effective

way. Microbial feed cultures when provided orally in the animal body depict tolerance towards

gut environment, though they need to combat and endure the unusual surroundings of gut

(Agarwal et al., 2002). Prior to the establishment of pathogens into the gut, ruminants make use

of probiotic bacteria in such a manner that it supports and maintains the rumen flora and prevents

the animals from diseased and diarrheal conditions. It also reduces the weaning time, sustains the

balanced state of rumen microflora, and enhances the production of enzymes with better

utilization of fibrous foods. Particularly, Ruminobacter and Succinivibrio are supplemented as a

considerable source of probiotics with some unusual features in the rumen. The most commonly

used probiotic microorganisms are the members of lactic acid bacteria such as: Aerococcus,

Bifidobacterium, Brochothrix, Carnobacterium, Enterococcus and Lactobacillus (Chaucheyras-

Durand et al., 2010).

2.5.6 Efficacy of probiotics on farm animals

33

The microbial flora implies a great efficiency on the performance of animal’s gastrointestinal

tract. Microbial feed additives or supplements aids in the development and maintenance of

suitable type of microbiota in the GIT surroundings. Generally, there are two types of microbial

feed additives Lactobacillus and yeast culture. These two types of species play explicit role in

the host’s body i.e: Lactobacillus for the most part helps in the omission of enterotoxigenic

bacteria, while S.cerevisiae primarily aids in proper execution of rumen (Fuller, 1989). There has

been observed an increased mortality rate in calves because of diarrhea. The Lactobacillus

positioned itself in gastrointestinal tract and compete with the pathogens in order to combat

diarrhea (Abu-Tarboush et al., 1996; Fuller, 1989) while yeast culture animate microbiota to

have an effective microbial condition in side rumen (Wallace, 1996; Kumar, 1997; Enjalbert et

al., 1999). Panda et al (1995) reported that S. cerevisiae, as a feed additive, provides positive

impact on nutrients digestion, growth rate, and FCR in calves. Probiotics are used as viable

microbial feed additives in animal feeds that stimulate the growth pattern as well as health

benefits in the host animal (Barrow et al., 1980). Probiotics have imposed valuable consequences

in farm animals, which aids in enhancing digestion process, growth rate, feed conversion and

assimilation of essential nutrients (Fuller, 1989).

2.5.7 Mechanism of action of probiotics

There has been recommended numerous mechanism of action of probiotics which demonstrate

the upshots of probiotics in a positive way. Probiotic strains provide useful aspects with respect

to metabolic activities and survival rate in the gut (Chaucheyras-Duand and Durand 2010;

Chaucheyra-Durand et al., 2010). The probiotics generate its mode of action based on the

specifications of strains (Newbold et al., 1995). In case of monogastric, bacterial probiotics

produce organic, lactic or acetic acid which helps in the reduction of gut pH and prevention of

pathogens from colonisation. Thus, it aids in setting of much approving ecological environment

for the resident microbiota (Servin, 2004). The probiotic strains have the ability to release

“bacteriocins” that are antimicrobial peptides and help in growth inhibition of pathogenic

bacteria. Probiotics have potential to produce enzymes that enables the hydrolysation of bacterial

toxins (Buts, 2004). Several strains of probiotics show elimination of pathogenic bacteria (with

respect to their elevated affinity for nutrients or adhesive sites) (La Ragione and Woodward,

2003). Various probiotics show generation of such growth factors and nutrients that stimulate the

34

favorable microorganisms of gut microbiota. Probiotic also generate host interaction and produce

components that influence the mucosal expansion and metabolism of host’s intestinal cells

(Johnson-Henry et al., 2008). A few probiotics seem to have metabolic and detoxification

phenomena of definite inhibitory compounds for instance, amines, nitrates or hunting for oxygen

(anaerobic system of gut). Thus, the mechanism of probiotics demonstrates beneficial, nutritious

as well as healthful effects both for animal and human gut. Probiotic bacteria incorporate varied

and diverse affects on host. Though their exact mode of action is still unclear but they have

applied its mode of action on the basis of their effect on immune cell (like, epithelial, dendritic,

T and B cells monocytes or macrophages), GIT luminal conditions, function of epithelial barrier,

and the mucosal immunity (Zhang et al., 2007) (Figure 2.3).

.

Figure 2.3: Modes of action of probiotics; Antimicrobial activities are shown in (1)

Bacteriocins/defensins secretion (2) competitive inhibition with pathogens (3) prohibition of

bacteria from translocation or adherence purpose and (4) decreasing luminal pH. Intestinal

barrier function enhancement: (5) increase the production of mucus. Adapted by (Ng et al.,

2009).

2.5.7.1 Enhancement of gut microbial balance

35

Probiotic reduces the luminal pH, inhibits bacterial translocation and adherence properties and

produces antimicrobial substance or defensins. Thus, it aids in the alienation of pathogenic

bacteria from gut. In the gut, the microbial flora inhibits colonisation of pathogenic bacteria by

generating limitations in the physiologically restrictive environment such as pH, redox potential

and hydrogen sulphide production.

2.5.7.2 Enhancement of barrier function

By enrichment the intestinal barrier properties in the course of cytoskeleton modulation and

phosphorylation of tight junction proteins, probiotics promote mucosal (cell-cell) signaling and

provides cellular stability. Numerous organized methods facilitate the maintenance of intestinal

barrier purposes that primarily include chloride, mucus and water secretions and connection of

epithelial cells to their apical junctions through a proteins called tight junction (Watts et al.,

2005).This system aids in health benefit to host and probiotic bacteria restraining it from number

of diseases (Meddings, 2008).

2.5.7.3 Immunomodulation

2.5.7.3.1 Impact of probiotic on epithelial cells

The epithelial cells import significant variation at the point of signal transduction pathway and

production of cytokine which, implicit the differentiation of probiotics and pathogenic bacteria.

The signaling pathway permits different pathways to epithelial cells which enabling the

differentiation between probiotics and pathogenic organisms. In this mechanism, probiotics

hinder the degradation of the counter regulatory factor (IKB), which in turn attenuates the pro

inflammatory responses. On the contrary, pathogenic species stimulate the transcription factor

(NF-B) that provokes pro-inflammatory reaction in the intestinal epithelial cells. Probiotic strains

also facilitate in epithelial recovery or in case hinder apoptosis. Lammers et al. (2002) and Otte

and Podolsky (2004) performed a study on probiotic and found that a probiotic strain

“Lactobacillus rhamnosus GG”, in the epithelial cells of GIT, aids in the prevention of apoptosis

induced by cytokines.

2.5.7.3.2 Impacts of probiotic bacteria on dendritic cells (DCs)

DCs are commonly called as antigen presented cells used for the bacterial identification and

determination of the successive T-cell response. DCs perform specific functions in the gut. They

carry out oral tolerance induction via cytokines (IL-10 and TGF-β) that generate regulation of T

cells and immunoglobulin A producing B cells (Lwasaki and Kelsall, 1999; Akbari et al., 2001;

36

Williamson et al., 2002). GIT DCs assist luminal bacteria directly and indirectly. By direct

means, in the intestine lumen, they surpass their dendrites into the epithelial tight junctions and

indirectly by passing through M cells. Dendritic cells play its role in the junction of innate and

adaptive immunity, which supports reorganization and reaction of bacterial components that

cause initiation of primary immune response and T and B cell responses. There has been reported

a wide impact of probiotic on dendritic cells in various structures (monocyte and bone marrow

derived dendritic cells, dendritic cells of whole blood and dendritic cells of lamina propria) and

also in diverse species of human and mouse.

2.5.7.3.3 Impacts of probiotic bacteria on monocytes and macrophages

Probiotic bacteria induce direct influence on lymphocytes or they depict some nature of

modifications in dendritic cells or on macrophages that alters the stimulation response of

lymphocytes. These impacts have been figured out in B lymphocytes, natural killer cells and T

cells.

2.5.7.4 Effect of pro-prebiotic mixture

Probiotics are non-digestible constituents of food, when added in food or diet confers useful and

healthy affects to host and stimulate the growth of confined quantity of colon bacteria (Quan et

al., 1990). On the whole, probiotics are particular species of micro-organisms that act as a

transient flora and used as a supplements, whereas pre-biotics alternates the gut flora of the host.

The stability of the intestinal flora is frequently confronted by many factors that include:

environmental aspects (age and stress), infectious diseases (gastroenteritis), medications

(antacids and antibiotics) and several other factors. Species, recommended dose of

microorganisms and optimal duration of administration determine the degree of validity

regarding pre and probiotics (Thomas and Greer, 2010).

2.5.8 Pre-requirement for microbiota as feed supplements

Microbiota should exhibit the ability of carbohydrate fermentation and create short chain fatty

acids, which in turn causes reduction in intestinal pH. Short chains fatty acids endorse the

development of intestinal cells and involved in cell differentiation, in that way, promote the

assimilation and absorption process. They also take part in toxin neutralization. They hinder the

growth of various pathogens, act as a barrier via competitive elimination (commensal species

37

compete for the same sources of nutrients as potential pathogens). Intestinal bacteria sponsored

in the maturity of immune system both by structural and functional means, in host gut.

They generate the capability of immunoglobulins production that sustains the proficiency of the

immune system. The probiotic strain are resistant to the enzyme present in oral cavity. It endures

the gastric acid environment, irrespective of the contact with the bile and pancreatic juice (in

upper small intestine). It should not be sensitive against antibiotics. Adherence to the intestinal

cells (epithelial lining) and release of antimicrobial compounds are also important features of

probiotics that behave as a competitor against estrogens (pathogens) which lessen down its

subsistence. Probiotics owned certain characteristics that fulfill the following salient features:

gastric acid and bile salt tolerance (gastro-intestinal conditions), adherence capability to the

mucous lining in GIT tract, non-toxic, non-pathogenic and without any detrimental effects, in-

vivo and in-vitro survival in GIT, and competitive elimination of pathogens from the track

maintenance of viable cell product in sufficient quantity.

2.5.9 Selection criteria for potential probiotics

2.5.9.1 Bile tolerance effect

Bile is a yellowish green aqueous solution mainly consists of cholesterol, phospholipids, biliverd

in pigment and the bile acids (Carey and Duane, 1994, Hofmann, 1994). Bile synthesis occurs in

the pericentral liver cells, stored and accumulated in the gall bladder, and after ingestion,

released into the duodenum. Bile plays its role in solubilisation and emulsifying of lipid contents

and supports fat assimilation. Thus, it can act as a biological detergent that also represents strong

antimicrobial activity by terminating bacterial membranes (Begley et al., 2005). Primarily,

denovo synthesis of cholic, chenodeoxycholic and bile acids takes place in the liver (from

cholesterol). There is an effective preservation of bile salts under usual conditions by means of

“enterohepatic recirculation” process. By means of active transportation, conjugated and

unconjugated bile acids are assimilated in the terminal ileum while in the gut portion by passive

diffusion (Batta et al., 1990). Hepatocytes reabsorbed bile acids in the portal bloodstream, which

is then re-conjugated and re-secreted in the form of bile. Native intestinal flora modifies the

overall bile acid and around 5% of overall bile acid (0.3-0.6g/day) evades epithelial

incorporation (Bortolini et al., 1997). “Deconjugation” is the fundamental step occurs before

modifications. Bile salt hydrolase (BSH) enzyme catalyze the deconjugation process in which

38

amide bond are hydrolyzed and glycine/taurine components are released from the steroid core.

This results in liberation of deconjugated bile acids (Batta et al., 1990).

2.5.9.2 Incidence of BSH activity among bacteria

BSH activity has been reported in many bacterial species including Bacteroides,

Bifidobacterium, Clostridium, Enterococcus, and Lactobacillus. Among them, Bifidobacterium

and Lactobacillus are normally applied as a source of probiotics, whereas, Enterococcus,

Clostridium, and Bacteroides are also remarked under commensal or probiotic category, and act

as normal residents of gastrointestinal tract. Almost all gram positive bacteria of intestinal tract

(except for few bacteroides) possess positive BSH activity, while gram negative bacteria lack

this activity (Elkins and Savage, 1998; Moser and Savage, 2001; Ahn et al., 2003). Listeria

monocytogenes, a notorious pathogen of gastro-intestinal tract and is gram positive. Basically, it

is not believed as a constituent of gastrointestinal flora but it has BSH enzyme. On this basis, its

position is recommended on the edges of commensal and pathogenic species. Besides this,

Enterococcus faecalis act as an opportunistic pathogen also have a BSH homolog (EF0040;

AAM75246) found near pathogenic boundaries, but it is not properly characterized uptil now

(Shankar et al., 2002).

2.5.9.3 Cholesterol assimilation

One of the properties of probiotics is to reduce the effect of cholesterol. The mechanism lies

behind cholesterol assimilation is the deconjugation of bile slats through microbes that show

rapid transit in the small bowel (Gilliland, 1990). Probiotic bacteria boost up or produced a

number of factors by which cholesterol synthesis is restrained in the body (Mann, 1977). In

addition to cholesterol lowering effect, probiotic bacteria also play its vital functions by

mounting phenol tolerance, neutralizing the latent carcinogens, provoking immune response and

metabolic activities, and reducing constipations. Cholesterol-lowering effect by probiotic

bacteria has been suggested in vitro, on the basis of recommended hypothesis that exhibit the

deconjugation of bile acids, binding to cell wall of bacteria, lowering of cholesterol by bacteria,

and the fermentation of short chain fatty acids (particularly propionate) and its end-products by

physiological actions. These purposed mechanisms of cholesterol lowering effect have been

applied on humans and animals studies but the exact mechanism of action on probiotic bacteria

is still ambiguous (Gilliland et al., 1985; Klaver and Vandermees, 1993; Tahri et al., 1996, 1997;

Noh et al., 1997; Usman, 1999; Lin and Chen, 2000).

39

2.5.9.4 Anti-microbial activity

The Lactic acid bacteria (as a potential probiotic) also contribute towards the advancement of

anti-microbial compounds. Among these compounds, bacteriocins are the most noticeable

proteins or peptides, which are synthesised by ribosomes and destroy the pathogenic bacteria

(Corr et al., 2007). Hence, bacteriocins produced by LAB are being used as a putative agent for

probiotics as well as biological control agents. Unusual antimicrobial compounds by LAB are

categorized under high molecular mass and low-molecular mass compounds. High molecular

mass includes bacteriocins like compounds, which can counter act the pathogenic and spoilage

causing bacteria in foods. Low molecular mass include uncharacterized compounds, carbon

dioxide, diacetyl and H2O2 (Jay, 1982; Klaenhammer, 1988. Piard and Desmazeaud, 1991;

1992). Uptil now, a variety of bacteriocins have been ascertained such as: Streptococcus

salivarius has produced a new type of bacteriocin, Enterococcus avium produced avicin A (class

IIa) and another (class IIa) production from Enterococcus faecalis strains. Some sorts of

unknown bacteriocins are also reported in which Lactobacillus gasseri generate two-peptide

gassericin, Lactobacillus fermentum and E. faecalis encodes uncharacterized bacteriocins, two

from L. fermentum and one from E. faecalis.

2.5.10 Classification of probiotics

2.5.10.1 Lactic acid bacteria

Lactic acid bacteria (LAB) act as a chief ingredient in probiotics and are being applied in

production and preservation of fermented probiotic food. LAB assures curative and dietetic

(nutritious) health benefits as: low calorie sugar and vitamins production, lessening the threats of

diarrhea (Briand et al., 2006, Myllyluoma et al., 2007), immunomodulation (Baken et al., 2006),

hindrance of cancer (Chen et al., 2007), anti-mutagenic activities (Hsieh and Chou, 2006), and

diminish serum cholesterol intensity (Xiao et al., 2003, Liong and Shah, 2005). The LAB is

speculated as G+ with low CG content. LAB are generally non-spore producing non-respiring

cocci or rods, acid-tolerant and catalase negative.

Many LAB strains produce “bacteriocins” which are proteinaceous in nature and act as a barrier

against pathogenic microflora. LAB are responsible for decomposing plants and lactic products

resulting in the production of metabolic end-products during carbohydrate fermentation.

Commonly, LAB are more precisely used in the food industry. Industrially, they are recognised

40

under the category of “generally recognised as safe” (GRAS), because of their ubiquitous nature

and their role with healthy microflora present in the mucosal surface of human. Lactic acid

bacteria are comprised of variety of genera which include: Lactobacillus, Leuconostoc,

Pediococcus, Lactococcus, Streptococcus and rest of the genera as Aerococcus, Carnobacterium,

Enterococcus, Oenococcus, Sporolactobacillus, Vagococcus, Tetragenococcus are included in

the order Lactobacillales (Sonomoto, 2011). Some of LAB genera have been described under

following categories.

2.5.10.1.1 Lactobacillus

The genus Lactobacillus is the largest group of Lactobacillaceae family.

Majority of Lactobacillus are rod-shaped and gram-positive based on its morphological

characterization. They are aerotolerant and strictly fermentative; however, they can also be

grown under anaerobic environment (Kandler, 1986). Based on their ability of fermenting

sugars, Lactobacillus are divided into two categories:

1. Homo-fermentative specie (mostly convert sugars into lactic acid)

2. Hetero-fermentative specie (convert sugars into lactic acid, acetic acid, ethanol and CO2)

Lactobacillus species are profound in plants, raw milk, insects, animals, and other ecological

niches. They are being well employed as food preservatives, starter for dairy items, in fermented

vegetables, sausages, fish and in addition to silage inoculants. Based on its prospective

prophylactic and therapeutic properties, Lactobacillus have been nominated as a potent source of

probiotics (Hammes and Vogel, 1995).

2.5.10.1.2 Leuconostoc

Leuconostoc belongs to Leuconostocaceae family. Usually, they exhibit cocci-shaped in the form

of chains, gram-positive, catalase-negative, slime forming and are hetero-fermentative.

Leuconostoc is liable to cabbage (sauerkraut) fermentation along with the combination of

Pediococcus and Leuconostoc, converts sugars into lactic acid that offer sour essence and

provides good keeping qualities. On the fact of their competence of creating “stinking”, they are

also seemed to be competent for human infections for which standard kits are easily available for

their identification (Kulwichit et al., 2007).

2.5.10.1.3 Pediococcus

Pediococcus also belongs to family Lactobacillaceae, a genus of gram positive lactic acid

bacteria. Generally, they suggest themselves in couplet form or in tetrads. Like Leuconostoc, it

41

ids in cabbage fermentation and arise stinky odour. Some isolates of Pediococcus producebutter

scotch aroma in some beer and wine (chardonnay). It is also used in silage inoculants. With

respect to its probiotic efficiency, Pediococci are appended as production of cheese and yogurt

(Haakensen et al., 2009).

2.5.10.1.4 Lactococcus

Previously, Lactococcus were placed in Streptococcus group N1, but now it belongs to genus

LAB. One the basis of its glucose fermentation property, they are recognized as homo-

fermentators, and are gram-positive, catalase negative and non-motile cocci. They are usually

present in singlet, paris and in chain form. It has been further sub-catagorised as: L. lactis, L.

garvieae and L. piscium. These organisms generate wide impact in dairy industry as in

fermentation of dairy products, such as cheeses. They are used in the form of single-chain starter

or in mixed strain culture. The most important function of Lactococcus is being applied in rapid

acidification of milk, which drops the pH and inhibits the growth of spoilage bacteria.

2.5.10.1.5 Streptococcus

The genus streptococcus belongs to the phylum Firmicutes. They usually grow in chains or in

pairs, and are catalase and oxidase negative. Several of them are facultative anaerobes. This

genera approximately include more than 50 species (Facklam, 2002).

2.5.10.1.6 Enterococcus

Enterococcus also shares the same characteristics as of lactic acid bacteria such as, gram positive

cocci and facultative anaerobes (Giraffa, 2003). Enterococci undergo commensalisms and inhibit

the GIT of animal and human. Enterococci seem to be screened out from variety of food sources

(meat, milk, cheese). They have high survival rate in harsh and extreme conditions e.g. they can

resist 65% NaCl, pH as well as high heat. They can also be isolated from variety of soils, raw

plants, surface water and animal products (Giraffa, 2003; Cocolin et al., 2007). Enterococci also

import flavour and contribute in ripening of various types of food.

2.5.10.2 Yeast probiotics

Yeasts are eukaryotic microorganisms and are different from bacteria from structure and

functional point of view (Faria-Oliveira et al., 2013). Yeasts are facultative anaerobes and differ

in terms of their location, shape, reproducing activities and subtracts they utilize and are highly

42

resistant to different antibiotics, like sulfamides and other anti-bacterial substrates (Stone, 1998).

The resistant capability of the yeast cells is natural and genetical. That resistant cannot changed

or transmitted to other microbial species. The size of the yeast cell (5 x 10 μm) is also higher

than bacteria (0.5 x 5 μm). Yeast cells produce many important fermentation metabolites like

protein, carbohydrates, high potency vitamins, and different types of important minerals and

enzymes that make it useful and highly nutritive feed supplement for ruminants (Yalcin et al.,

2011; Sontakke, 2012; Szucs et al., 2013). During the last decades, Saccharomyces cerevisiae

(live yeast) have been used as preventer supplement against diarrhea and other digestive system

problems in livestock (Chaucheyras-Durand and Durand, 2010). Production benefits, together

with reduces digestive problems and better health in case effective manners (Huber, 1997).

Mode of action of YC has been investigated in many studies. The results of these studies

have different outcomes. Speculating regarding the action of YC (S. cerevisiae) has been

brought forward and the proposed mode of actions are discussed here.

2.5.10.3 Mode of action of YC in the rumen

Ruminants stomach consists of reticulum, rumen and omasum and abomasums (Jouany and

Morgavi, 2007). The rumen is an anaerobic chamber, harbors an immense diversity of

microbial community, including, bacteria, archaea, fungi, and single-celled ciliated protozoa

(Carberry et al., 2012) (Figure. 2.4). Bacteria are more numerous microbes in rumen (Fonty and

Chaucheyras-Durand, 2006). Mostly bacteria are associated with feed; some are free living,

attached with mucous membrane and associated with fungi and protozoa. The structure of

rumen microbial community is influenced by many factors, including host species, age,

season, type of feed, geographical location and whether the animal has received any

treatment (Dehority and Orpin, 1997; Weimer et al., 1999; Weinberg, 2003). The balance in

rumen microbial flora plays a crucial role in feed utilization and could result in better animal

productivity (Santra and Karim, 2003). Several hypotheses concerning the mode of action of

probiotic yeast in animal nutrition have been proposed, but most of them emphasize positive

effects by modifying rumen microbial population. The first and most widely supported mode

of action is that the yeast stimulated the growth of bacteria (cellulolytic, amylolytic,

proteolytic) and protozoa (Arakaki et al., 2000; El-Ghani, 2004). The rumen dissolved oxygen

can be measured in situ (Hillman et al., 1985). Loesche (1969) found that majority of rumen

microbial flora are highly sensitive to O2. Probiotic yeasts remove oxygen from rumen and

43

provide a more anaerobic environment for bacterial growth Rose (1987). Sixteen liters of oxygen

can enter inside rumen daily. Mostly that O2 entered during feeding, rumination and salivation

time (Newbold, 1995). Inside rumen, yeast cells use oxygen for their metabolic process. Freshly

ingested feed particles have sugars and small oligosaccharides. Probiotic yeast metabolizes these

small particles and produce peptides, polypeptides and amino acids.

That respiratory activity of probiotic yeast lowers the oxidation reduction potential inside rumen

(Dawson et al., 1990). A negative change in the redox potential (-20mV) has been seen in side

rumen with probiotic yeast addition (Jouany et al., 1999). This change gives more anaerobic

condition inside rumen (Dawson et al., 1990). Above mention environment helps in the

protection of rumen bacteria from damage by oxygen, and stimulation of growth of cellulose

degrading bacteria (Roger et al., 1990). These conditions will also be helpful in the cellulose

degrading process (cellulose digestion). Respiratory-deficient mutants of probiotic yeast cannot

stimulate bacterial growth. As we mention earlier that O2 scavenging property of yeasts is very

important for growth of rumen microbial biomass, so this O2 scavenging property should be kept

in mind when probiotic yeast is selected for ruminants. It has been well studied that yeast culture

can help the establishment of different types of microflora in neonate. Newborn ruminant

digestive system is sterile but with passage of time when he contacts with his mother and other

animals they get microbes from their saliva and feces (Chaucheyras-Durand et al., 2008). The

mother and her young connection is more common in small scale farming systems. On the other

hands in intensive dairy farming systems the neonate is alienated from the mother, and is fed on

solid feeding that provides a negative situation in the development of rumen microflora (Fonty

et al., 1987). This negative situation leads to poor rumen microbial development and making the

neonate to suffer from different diseases. Different diseases of digestive system are most

important factor of low income heifer’s rearing. In has been well studied that yeast culture can

help the establishment of different types of microflora in neonate by removing the oxygen from

rumen.

44

Figure 2.4: Estimated rumen microbial ecosystem

The

rate

of

cellulose degrading microflora population was greater in lambs fed on S. cerevisiae addition

(Chaucheyras-Durand and Fonty, 2008). S. cerevisiae had the ability to provide different types of

organic acids or vitamins, those stimulating ruminal populations of cellulolytic bacteria and LUB

(Chaucheyras-Durand et al., 1995). The cellulose degrading microbial population was also much

stable in the animals fed on yeast addition because protozoa comes in rumen only once the

bacterial species are present rumen. It has been also noted that protozoa appeared earlier in those

animals whose fed on S. cerevisiae addition (Chaucheyras-Durand and Fonty, 2002) (Figure.

2.5). Amylolytic bacterial populations are also affected by yeast in the rumen (Arakaki et al.,

2000). It is because the protozoal concentrations are proliferated and are able to store starch and

postpone bacterial fermentation (Enjalbert et al., 1999).

45

Figure 2.5: Representative scheme of the mode of action of S. cerevisiae. Adapted by

(Wallace, 1994).

Proteolytic bacterial activity was highest in the yeast supplemented animals. Proteins in the feed

are quickly breakdown into peptides, amino acids and NH3 by different protozoa and fungi inside

rumen (Wallace et al., 1997). Some NH3 is converted into microbial protein (MP), and some

ammonia is used by the animal in the form of urea. An important portion of rumen ammonia is

excreted and represents a indicated that nitrogen loss of the dietary nitrogen (N) intake (20 to

25%) (Fonty and Chaucheyras-Durand, 2006). Amino acids and peptides issued from dietary

proteins cannot be directly slipped in the animal intestine, if the diet has highly nutritious value.

46

The same effect on ammonia concentration was seen with daily yeast culture supplementation in

adult ruminants (Kumar et al., 1994). In vitro findings tell that probiotic yeast could alter the

growth and activities of protein degrading bacteria, which ultimately enhanced the protein

digestion inside rumen (Beev et al., 2007). The mode of action of yeast can be explained by a

fight between live S. cerevisiae cells and different bacterial species for energy utilization

(Chaucheyras-Durand et al., 2005). A study on 14 dairy cows field trials addition of yeast strain

in the diet noted that the soluble nitrogen of the diet was a key factor to drive the production

parameters to the probiotics-yeast (Sniffen et al., 2004). However, with other yeast strain no

significant effect was seen on the concentration and fraction of microbial nitrogen in dairy cattle

(Putnam et al., 1997). Further study is needed to explain the effect of probiotic yeast on the

nitrogen microbial metabolism (Chaucheyras-Durand and Durand, 2010). Many study showed

that increased feed intakes are driven by increased flow of absorption nitrogen (Wallace, 1994;

Kamel et al., 2004).

This step stems simultaneously from the proliferation and stimulation of viable cell counts of

anaerobic bacteria population. Higher ammonia nitrogen concentration measured for vessel in

which live yeast was added compared to autoclaved yeast suggest that, the live yeast stimulated

the proteolytic activity of the rumen bacterial species which ultimately influence rumen

fermentation (Oeztuerk, 2009). It was noted that digestibility of crude protein was significantly

higher in animals fed on the mixed fugal (yeast and Aspergillus) supplementation, and

suggesting that fungal supplementation might be promote proteolytic activities by supplying

some types of stimulatory factors (Wiedmeier et al., 1987). Many studies showed that animals

fed on the yeast supplementation has been associated with higher concentration of ammonia

nitrogen, which may suggest that proteolytic bacterial activity has been stimulated by yeast

culture (Kung et al., 1997; Moallem et al., 2009). The second proposed mechanism is that yeast

cell provides the soluble growth factors like, organic acids, branched-chain volatile fatty acids,

vitamins and amino acids, that have a positive effect in stimulating cellulolytic, proteolytic and

lactic acid utilizing bacteria (Wiedmier et al., 1987; Newbold et al., 1996; Callaway and Martin,

1997).

2.5.10.4 Effect on yeast and yeast cultures on rumen fiber digestion

47

Fiber is non-digestible polysaccharides (a complex form of carbohydrate) (Tungland and Meyer,

2002). These polysaccharides give plants their structure-think plant cell wall. In nutrition, the

term fiber define as a components of plant that are not digestible by mammalian enzyme (Moore

and Hatfield, 1994). Cellulose, hemicellulose and lignin are the primary components of

fiber. Cellulose and hemicelluloses constitute 15–70% of most ruminant diet (Hobson and

Stewart, 1997). Cellulose is the most abundant carbohydrate in plant cell wall. Chemically,

cellulose is made up of linear chains of the sugar molecules. In cellulose, glucose molecules are

linked together in a β-1,4 links, and this linkage can only be digested by microbial cellulolytic

enzymes. Cellulose makes up about 40% of plant cell walls. Hemicellulose is also be only

digested by microbial enzymes because it also have β-1,4 linkages. Hemi-cellulose has a strong

negative effect on fiber degradation because of closely association with lignin (Fernando et al.,

2010) (Figure 2.6). The rumen is an important part of the ruminants’ stomach because; cellulose

is broken down into simple sugar that can be used by the animal body inside rumen. The rumen

represents a mobile, self-sustaining fermentation system for plant material (Flint, 1997; Shakira

et al., 2013). It is a complex microbial ecosystem and contain many types of microorganisms like

of bacteria (1010–1011 cells per ml), protozoa (104–106 per ml) and fungi species (103 -105

zoospores per ml) (Kamra, 2005; Fernando et al., 2010).

2.5.10.4.1 Fibrolytic bacteria

Rumen bacteria (1011 viable cells/ml10) dominate the fermentation both in terms of numbers and

metabolic processes. The rumen bacteria are 99.5% obligatory anaerobic. In rumen 200 species

with many subspecies of bacteria are present.

There are different kinds of bacteria in the rumen which aid in fermentation process (Cho et al.,

2006; Chaucheyras-Durand et al., 2008). Fibrobacter and Ruminococcus are the main rumen

fibre

degrading

bacteria in

cattle

48

(Kobayashi et al., 2008; Kim et al., 2011). F. Succinogenes is a gram-negative and rod-shaped

anaerobe first isolated from the cattle (Hungate, 1950). Despite their important role, cellulose

degrading bacteria are thought to only comprise 0.3% of the total bacteria population inside

rumen (Brulc et al., 2011). Rumen bacteria are classified into fibrolytic, amylolytic, pectinolytic,

proteolytic, lipolytica, lactate using bacteria and hydrogen-using bacteria. Amylolytic bacteria

ferment starch while fibrolytic bacteria involve in the fermentation of fiber. Different bacterial

populations dominate the rumen fermentation depending on the type of feed. Cattle fed high

fiber diet will have a ruminal bacterial population that is high in fibrolytic bacteria especially

Ruminococcus ssp. Rumen bacteria mainly involved in the fermentation of fiber, starch and

sugar in the feed.

Figure 2.6: Components of fiber and its classification. (Yarriage, 1981).

2.5.10.4.2 Fibrolytic fungi

Ruminal anaerobic fungi, an emerging group of animal probiotics, account for

only approximately 8% of the total rumen microbial biomass in ruminants (Orpin and Joblin,

1988). Rumen fungi has a crucial role in the degradation of fibre material (Theodorou et al.,

1989; Samanta et al., 2001; Paul et al., 204; Lee et al., 2004; Thareja et al., 2006; Tripoathi et

al., 2007). The fungi have an important role in fiber digestion because of the vegetative thalli

rhizoids (Figure 2.7). The rhizoids have a more penetrating capability to plant cell wall as

compared to bacteria and protozoa. Degradation of lignin of the plant cell wall is an main

characteristic of rumen fungi (Mountfort et al., 1982; Akin and Benner, 1988). Fungi degraded

37–50% of barley straw. The fungi fibrolytic activity is enhanced by hydrogen-utilizing

methanogens which decrease the cruel effect of hydrogen (Orpin and Joblin, 1988; Joblin, 1989).

Fungi play an active and significant role in fibre digestion of low quality roughages by breaking

the beta-1-4 linkages between lignin and hemicelluloses inside the plant cell (Tripathi et al.,

2007). Fungi have a positive role in fiber degradation as evidenced by producing a wide array of

potential hydrolytic enzymes (Williams and Orpin 1978; Samanta et al., 2001; Paul et al., 2003;

Lee et al., 2004).

49

2.5.10.4.3 Protozoa.

In vitro studies have suggested that 19-28% of the total cellulase activity in fiber digestion can

be attributed to protozoa (Gijzen et al., 1988). However, digestion seems to be limited to very

susceptible tissue such as mesophyll cells (Akin, 1989). Studies have demonstrated that

defaunation (removal of protozoa) reduces the rate of fiber/cell wall degradation digestion

(Bonhomme, 1990; Yang and Varga, 1993). However, in the absence of protozoa there is an

increased requirement for non-proteinnitrogen (NPN) because of an increase bacterial

population. A reduction of N may therefore account in the reduction in fiber digestion (Ushida

and Jouany, 1990).

Figure

2.7: A

model of

interactio

n of yeast

cells with

rumen

microbes

(Jouany,

2006).

2.5.10.5 M

ode of action of probiotic yeast in the post-ruminal GIT

The gastrointestinal tract (GIT) inhabits multifarious microbial diversity that helps in generating

impactive response regarding nutritious, health, physiology, and productivity of animals

(Guarner and Malagelada, 2003). The existing gut microbiota regulates food safety through

shedding of pathogens, interaction with organisms, and resource competition in the GIT (Nurmi

and Rantala, 1973). The physiological, anatomical, and immunological status of the host is

50

strongly dependent upon micro-biota of GIT which facilitate essential functions to host. GIT

micro-flora aids in stimulation of the immune system that act as a barrier against infectious

pathogens. It also restrains the injurious and pathogenic bacteria in gut colonization (Cebra et al.,

1999). The micro-flora that resides in GIT mostly belongs to Bacteroides, Bifidobacterium,

Clostridium, Eubacterium, Fusobacterium and Lactobacillus families. Of all intestinal

microbiota, Enterococcus and Escherichia coli represent the least contribution (upto1%),

whereas, anaerobes show dominancy over microaerophiles and facultative anaerobes by 1000:1

(Mestecky and Russell 1998). Lactobacillus and Bifidobacteria are marked as predominant flora

which counts for 90% of the total population in GIT. The fluctuating flora represents their

existence in trace i.e. less than (0.01%) which is usually considered as more diversified and

pathogenic ones (Tournut, 1993). The GIT microbiota protects the host from pathogen that

produced digestive diseases like diarrhea. The performance of the calf is directly related to the

efficient growth together with improve health status (Soberon et al., 2012). Gut microbial flora

plays an important role in the growth and health of the animal. Probiotics put a beneficial effect

on the health of gut by improving its microbial balance. They have antidiarrheal capability and

enhance the growth performance of the animals (Donovan et al., 2002; Soberon et al., 2012).

The intestinal microbiota of cattle performs its vital role in the fermentation process. They help

in methane emission by means of fermentation both from rumen and large intestine (Johnson and

Johnson, 1995).

The microbial diversity in the GIT of the dairy cattle has lot of impact on the productivity and

wellbeing of the cattle (Guarner and Malagelada, 2003; Eckburg et al., 2005; Dowd et al., 2008;

Engelbrektson et al., 2010). There is no direct evidence that yeast or fungal extracts affect

digestion or metabolism in the lower gut. However, the potential for such effects should not be

ignored (Elghandour et al., 2015). Supplementation of yeast can improves the gut microbial

balance of different animals. That improvement can be due to either the effect of mannan-

oligosaccharides (MOS, a component of yeast cell wall) on the immune modulation or direct

effect of yeast on the reduction of pathogenic bacteria and toxic metabolites. According to the

finding of the Heinrichs et al. (2003), MOS has an ability to bind selected pathogen due to

blocking microbial lecithin and preventing pathogens from colonization in host GIT. As noted,

51

previous inquiries regarding feeding DFM to ruminant animals focused on its potential beneficial

effects on the post ruminal GIT. Proposed roles of beneficial yeast are to:

I. Attach to the intestinal mucosa and prevents potential pathogen establishment.

II. Maintain lower pH in the GIT thereby inhibiting growth of pathogens.

III. Produce antibacterial compounds such as bacteriocin and hydrogen peroxide.

IV. Modulate immune cells and stimulate immune function.

V. Modulate microbial balance in the GIT.

VI. Prevent illness caused by intestinal pathogens or stress.

2.5.10.6 Effect of probiotic yeast on the growth performance

Poor growth performance in growing animals is associated with imbalanced nutrition. The use

of probiotic yeast would minimize the expenditure of replacement heifers with optimum growth

rate. Young animals fed on diet supplemented with yeast culture gain more weight than non-

supplemented animals. That improved growth performance in young animals fed on the

probiotic yeast has been reported in many studies (Lascano et al., 2009; Desnoyers et al., 2009).

Increased microbial protein (MP) flow from the rumen for absorption in the intestine and also

the improve supply of amino acid entering the small intestine was the basic reason for improve

growth efficiency in the dairy animals fed on diet supplemented with YC (Devriese et al., 1992;

Panda et al., 1995; Rao et al., 2003; Reddy and Bhima, 2003; Kishan and Ramana, 2008). In an

recent experiment on claves, Terre et al. (2015), noted that daily gain was significantly (P<0.05)

increased (0.68 vs. 0.55 kg/day) in the probiotic yeast supplemented dairy claves than that of

non-supplemented calves during the pre-weaning period. On the other hand, during the post-

weaning period, DMI was significantly (P<0.05) improved (2.34 vs. 2.10 kg) and daily gain was

also greater (P = 0.053) in supplemented claves than non-supplemented calves (0.82 vs. 0.68

kg/d). In the same manners, (Lesmeister and Heinrichs, 2004) reported that probiotic yeast

supplementation (2% per animal) significantly (P<0.05) improve the DMI which leads to

higher ADG (15.6%) in dairy claves when compared with the control. The daily hip width

change was also significantly (P<0.05) improved in the claves compared to the control. Dairy

heifers supplemented with yeast culture (S. cerevisiae) consumed significantly (P<0.05) less

feed to maintained average daily gain as compared to the non-supplemented heifers (Lascano et

al., 2009). Kumar et al. (2011) study the effect of SC (0.25g/day) on the growing claves and

noted significantly (P<0.05) improved (5.49 vs 4.62 kg/day) weight gain and correlated that

52

improved growth efficiency with significantly (P<0.05) improved DMI (5.24 vs 4.60 kg/day). In

contrast, the lack of yeast culture effect on the growth performance has previously seen with

animals. Like, Kellems et al. (1990); Quigley et al. (1992); Kung et al. (1997) and Pinos-

Rodriguez et al. (2008) observed no significant effect of the YC supplementation on the dairy

animals. That inconsistency in YC effects on the ruminant might be due to the different feeding

pattern and different genotype.

2.5.10.7 Effect of YC on the digestive performance

Digestibility measures the amount of a nutrient in a feed available for the process of metabolism

after digestion and absorption (Cheeke, 1991). There are many factors that affect the nutrient

utilization and untimely animal performance. Many researchers suggested that yeast

supplementation has significantly influenced the DM, OM, CP, CF, NDF and ADF digestibility.

2.5.10.7.1 Dry Matter Digestibility (DMD)

DMD has significantly increased in animal fed on the diet supplemented with probiotic yeast

compared to animals fed on non-supplemented diet. Wiedmeier et al. (1987) observed the effect

of dietary supplementation of yeast culture and Aspergillus oryzae in the non-lactating cattle and

found that DMD was increased by the fungal stains supplementation. Similar results were

reported by Lascano et al. (2009), who found significantly (P<0.05) improved DMD (74.98 vs

73.65%) in the dairy heifers fed on the high concentrate diet along with S. cerevisiae at the rate

of 1 g/kg as fed daily. He concluded that improved DMD may contribute to an improved feed

efficiency in the dairy heifers. In the same manners, Mir and Mir (1994) conducted the

experiment on the steers to see the impact of adding S.cerevisiae (5 × 109 live organism) at the

rate of 10 g/day along with diet consists of 96 % corn silage, 75% alfalfa silage, 75 % dry rolled

barley, 25% barley, 25 % alfalfa hay and 4 % soybean meal for 2 years. It was found that DMD

significantly (P<0.05) influenced by the YC supplementation in the second year of the

experiment. In another report, Di Francia et al. (2008) observed the effect of fugal

supplementation in the buffalo claves and noted that significantly (P<0.05) higher (81 vs 71%)

DMD in the animals fed on the diet supplemented with fungus (S. cerevisiae and Aspergillus

oryzae). Miller-Webster et al. (2002) carried out experiment on the lactating cattle to determine

the effect of dietary addition of two different yeast cultures (A-Max and Diamaond; S. cervisiae;

57 g/animal daily) on the nutrient digestibility. It was noted that both yeast cultures products

53

improved DMD in dairy cattle. On the other hand many scientists (Harris et al., 1992; Doreay

and Jouany, 1998; Patrignani et al., 2006; Cooke et al., 2007; Tripathi and Karim, 2010) reported

no significant effect of yeast on the DMD in animals.

2.5.10.7.2 Organic Matter Digestibility (OMD)

In a meta-analysis it is noted that the OMO significantly improved due to yeast supplementation

in the ruminants (Desnoyers et al., 2009). Yoon and Stern (1996) reported that, dietary

supplementation of yeast (S. cerevisiae at the rate of 57 g/ d/animals) along with a basal diet

(32.5% corn silage, 17.5 % alfalfa hay, 35.3% corn grain and 12.7% soybean meal) significantly

(P<0.05) improve OMD in the dairy cattle. Increased OMD effect linearly increased with dose,

however, that improved digestibility was negativity correlated with percentage of concentrate

ration and positivity correlated with dry matter intake (Desnoyers et al., 2009). In another report,

Lehloenya et al. (2008) observed that OMD significantly (P<0.05) increased (75.2 vs 67.6%) by

yeast addition in steers. Marden et al. (2008) carried out research to evaluate the impact of YC

on the OMD in dairy cattle. It was reported that YC significantly (P<0.05) higher (66.6 vs

62.2%) OMD in early lactating Holstein cows. Di Francia et al. (2008) reported that fugal

supplementation was significantly (P<0.05) higher (83 vs 74%) OMD in claves. On the other

hand, El-Ghani (2004) and Tripathi and Karim (2010) reported that yeast supplementation has no

effect on the OMD in animals.

2.5.10.7.3 Crude Protein Digestibility (CPD)

Proteins in the feed are quickly breakdown into peptides, amino acids and NH3 by different

protozoa and fungi (Wallace et al., 1997). Some NH3 is converted into microbial protein (MP),

and some ammonia is used by the animal in the form of urea. An important portion of rumen

ammonia is excreted and represents a indicated that nitrogen loss of the dietary nitrogen (N)

intake (20 to 25%) (Fonty and Chaucheyras-Durand, 2006). Amino acids and peptides issued

from dietary proteins cannot be directly slipped in the animal intestine, if the diet has highly

nutritious value. The same effect on ammonia concentration was seen with daily yeast culture

supplementation in adult ruminants (Kumar et al., 1994). In vitro findings tell that probiotic

yeast could alter the growth and activities of protein degrading bacteria, which ultimately

enhanced the protein digestion inside rumen (Beev et al., 2007). The mode of action of yeast can

be explained by a fight between live S. cerevisiae cells and different bacterial species for energy

utilization (Chaucheyras-Durand et al., 2005).

54

Mir and Mir. (1994) conducted the experiment on the steers to evaluate the effects of adding S.

cerevisiae (10 g/d) on nutrient digestibility. It was noted that CPD significantly (P<0.05)

influenced by the yeast addition in the second year. In another report, Di Francia et al. (2008)

carried out research to evaluate the impact of dietary supplementation of fungal strains in the

claves and noted significantly (P<0.05) higher CPD in the animals fed on the diet supplemented

S. cervisiae and Aspergillus oryzae. In the same manner, (Wohlt et al., 1998) conducted

experiment on early lactating cattle to observe the impact of yeast on nutrient digestibility. Cattle

fed corn silage along with 0, 10 or 20 g of yeast culture per day. He noted significantly (P<0.05)

higher (78.5, 80.8 and 79.5%) CDP with 0, 10 or 20 g yeast per day respectively in yeast fed

groups. A study on 14 dairy cows field trials addition of yeast strain in the diet noted that the

soluble nitrogen of the diet was a key factor to drive the production parameters to the probiotics

yeast (Sniffen et al., 2004). However, with other yeast strain no significant effect was seen on the

concentration and fraction of microbial nitrogen in dairy cattle (Putnam et al., 1997). Some

researchers reported that CPD was no improved by YC supplementation in animals (Arambel

and Kent, 1990; Andrighetto et al., 1993)

2.5.10.7.4 Crude Fibre Digestibility (CFD)

In ruminant, fiber digestion has been regulated by four major factors; 1) structure and

composition of the plant which control bacterial access to nutrients; 2) main fibrolytic

microorganism’s number and their nature; 3) different factors retaled to growth and function of

microfloa that control penetration and degrationa of microbial populations; 4) different factore

related to animals that increase the nutrients utilzation by the process of mastication and

salivation and by digestion kinetics. It has been well studied that dietry supplemenation of live

YC has positive effect on the fibre digestion by stimulated cellulolytic bacteria and increased

protozoal count (Dawson et al., 1990; Martin and Nisbet, 1992). Probiotic yeasts have

established their effectiveness to influence growth and activities of rumen fibrolytic microor-

ganisms (Chaucheyras-Durand and Durand, 2010). YC can improve fungal colonisation of plant

cell wall material (Chaucheyras et al., 1995). On the other hand, thiamin (a vitamin help in the

process of zoosporogenesis) has been seen when S.cerevisiae is added in diet (Chaucheyras-

Durand and Fonty, 2001). Growth of Fibrobacter succinogenes has been stimulated and lags

time for growth of Ruminococcus albus, Ruminococcus flavefaciens and Butyrivibriofibri

55

solvenshas been reduced by addition of yeast culture (Girard and Dawson, 1995). Some studies

noted that the same yeast culture could enhanced the degradation rate of cellulose filter paper by

F. succinogenes and R. flavefaciens (Callaway and Martin, 1997). Using genotoxenic lambs

harbouring only F. succinogenes, R. albus, R. flavefaciens as cellulolytic organisms, it has been

observed that fiber degrading bacteria came earlier in the lambs fed on the active dry yeast

(Chaucheyras-Durand and Fonty, 2001).

Yeast culture also enhanced the polysaccharidase and glycosidehydrolase activities. Better

cellulose degrading activities of the solid- associated bacterial fraction have been seen in sheep

fed a diet supplemented with YC (Michalet-Doreau and Morand, 1997; Jouany, 2006). The

proportions of 16S rRNA of the F. succinogenes, R. albus, R. flavefaciens has been improved

due to yeast culture addition in sheep (Chaucheyras et al., 1996). It should keep in mind that all

such positive effects of yeast could observed in some studies with experimental animals (Plata et

al., 1994; Chaucheyras-Durand and Fonty, 2001) but data on significant benefits of yeasts on

fiber digestion in producing animals are not available yet. The fermented YC provides soluble

growth factors (i.e., organic acids, B vitamins, and amino acids) that stimulate growth of ruminal

bacteria that utilize lactic acid and degrade fibre (Callaway and Martin, 1997). These growth

factors may stimulate synthesis and secretion of IGF I from liver on absorption from

gastrointestinal tract.

The increased digestibility of nutrient may be due to improvement of microbial activities and

increased ruminal anaerobes and cellulolytic bacteria (Jouany, 2001). It wasnoted that SC

improved NDF digestibility in goats fed on the diet containing hay (Fadel, 2007). Therefore, it is

suggested that YC increased the number and functions of total and cellulose degrading bacteria

and improves cellulose degradation in the rumen (Dawson et al., 1990; Newbold et al., 1996;

Miller-Webster et al., 2002). Lascano et al. (2009) demonstrated that SC significantly improved

NDF degradation by ruminal microbiota in the dairy heifers fed on the high energy diet. That

improvement might be due to digestion and utilization of starch and simple sugars and the

removal of fermentable substrates that enhanced the fibre digestion and they suggested that the

YC had a diet-dependent effect. Marden et al. (2008) reported significantly (P<0.05) higher NDF

(41.6 vs 29.6%) and ADF (32.3 vs 18.1 %) digestibility in the early lactating Holstein cows

supplemented with SC at the rate of 5 g/d. He suggested that live yeast prevented accumulation

56

of lactate and allowed better fiber digestion by inducing a lower ruminal Eh and rH (Clark's

Exponent).

In another report, Wiedmeier et al. (1987) noted that supplementation of YC significantly

improved the hemicellulose digestibility and highlighted that improvement might be due to the

increased cellulolytic bacterial population after YC supplementation. Similarly, Wohlt et al.

(1998) reported that dairy cattle fed a corn silage and hay supplemented with 10 g per day fungal

strains early lactating cattle. Yeast supplementation significantly improved digestibility of ADF.

On the other hand, YC supplementation has no effect on NDF (Wiedmeier et al., 1987; Wohlt et

al., 1991; Moallem et al., 2009) ADF (Arambel and Kent, 1990; Cooke et al., 2007; Lehloenya

et al., 2008; Moallem et al., 2009) and hemicellulose, cellulose (Wohlt et al., 1991; Wohlt et al.,

1998; Cooke et al., 2007) digestibility. It has been noted that nutrient digestibility was not

affected when YC in given in high energy and corn gelatinized (Arambel and Kent, 1990; El-

Ghani, 2004; Cooke et al., 2007).

2.5.10.8 Impact of YC on the milk production and composition

Milk is mixture of fat, protein, carbohydrates, water and a number of other constituents present

in very small quantities. All the constituents except fat are collectively named solids not fat

(SNF). Cattle milk contains high amounts of total solids (Table-2.2).

Table 2.2: Milk composition of cow

Species

Fat (%) Protein (%) Lactose (%) Total solids (%)

European cow (Bos Taurus) 3.90 3.47 4.75 12.82

Zebu cow (Bos indicus ) 4.97 3.18 4.59 13.4

After: Bhatt (1999)

2.5.10.8.1 Milk yield

Lactation performance of the dairy animals was improved by probiotic yeast supplementation. It

is found that the increased milk yield might be due to the stimulatory effect of probiotic yeast on

the rumen microbiota, which in turn increase the cellulose digestion and that leads to emptying

rate of the rumen and therefore increase DMI (Wallace, 1994; El-Ghani, 2004). It is well studied

57

that, milk production responses tend to follow dry matter intake and a possible interaction

between the DMI potential and the milk production responses exists. The increased DMI due to

yeast culture supplementation leads to increase production performance of the animals by

providing more nutrients. Move ever, probiotics yeast enhance the absorption of nutrient intake

of vitamin B1 (thiamin), which promotes the colonization of fibre by the rumen, and improve the

diet digestibility (Erasmus et al., 1992; Newbold et al., 1998; Beauchemin et al., 2003). In

addition the improved rumen microbiota could leads to improve the microbial protein (MP) flow

from rumen to the small intestine (Erasmus et al., 1992). Microbial protein has been digested

inside small intestine and there are some indications that yeast supplementation may change the

amino acid proportion entering the small intestine (Erasmus et al., 1992; Newbold et al., 1995;

Beauchemin et al., 2003). Therefore, there is a possibility that increased amino acid supply might

be responsible for the positive impact of YC on the production performance of the ruminant’s

animals. There has been much work on the effect of yeast culture on the milk production in dairy

animals. (Moallem et al., 2009) studied production performance of the dairy cattle fed on the diet

supplemented with 1 g YC (S. cerevisiae) per 4 kg of dry matter intake (DMI). It was reported

that the DMI was 2.5% greater in the yeast supplemented animals compared with the control

animals (24.7 vs 24.1 kg). This increased DMI lead to1.5 kg (4.1%) more milk production for the

yeast supplemented cattle.

Recently, (Salvati et al., 2015) conducted an experiment on the Holstein cows received a diet

(corn silage (37.7%), tifton silage (7.1%), raw soybeans (4.1%), soybean meal (16.5%), finely

ground corn (20.7%), and citrus pulp (11.9%) supplemented with 25 × 1010 CFU of live cells and

5 × 1010 cfu of dead cells. It was noted that SC increased (26.7 vs. 25.4 kg/d) milk production in

dairy cattle. It was concluded that improved production efficiency might be due to the regulation

of body homeothermia in the dairy cattle. In the same manner, Ayad et al. (2013) reported that

supplementation of SC (20 g/day) along with concentrate and forage diet had statistically

significant effect on milk yield (32.7 vs 30.7 kg/d) in the dairy cattle. A meta-analysis of yeast

culture effects reported that DMI increased by 0.44g/kg body weight and milk yield increased by

1.2 g/kg of body weight (Desnoyers et al., 2009). Increasing dose of the yeast has been linearly

related to increasing DMI and milk yield. Concentration levels, NDF, ADF and CP contents of

the feed also related to the increased milk yield. In accordance with this, Chaucheyras-Durand

58

and Fonty (2002) and Stella et al. (2007) reported that yeast supplementation put a positive effect

on the rumen pH which stimulated the cellulolytic bacterial population. These cellulolytic

bacteria degrade the cellulose material inside rumen and enhance the DMI and milk production.

Diet has a crucial role in getting positive effects of the yeast culture. Many studied reported

higher milk production resulted from supplementation of the yeast culture in a diet had 30:70

(Shaver and Garrett, 1997); 50:50 (Piva et al., 1993) and 60:40 (Williams et al., 1991)

concentrated to forage ratio. In the same manners, Shaver and Garrett (1997) and Longuski et al.

(2009) noted increased milk yield with YC supplementation in fermentative carbohydrate diet.

The finding of these experiments shows that the increased milk yield could be due to the

digestive kinetics in the rumen. In another report, it was noted that significantly (p<0.05) more

milk was produce in the heat stress cattle fed on YC.(Bruno et al., 2009). In a yeast culture

review of 22 trials on dairy animals showed that average increase of 7.3% (range 2-30%) in milk

yield and noted that improve milk could be due to the positive effect of yeast on the nutrient

digestibility and DMI (Dawson et al., 1990). Similarly, Yalcın et al. (2011) reported that cattle

fed SC supplementation diet produces significantly (p<0.05) more (24.97 vs. 23.49 kg/d) milk

than in non-supplemented cattle. It was concluded that yeast culture provides soluble growth

factors that stimulate growth of cellulolytic bacteria and cellulose digestion (Callaway and

Martin, 1997). However, some researchers (Soder and Holden, 1999; Schingoethe et al., 2004;

Bagheri et al., 2009) reported no beneficial effects in milk production from feeding yeast to

lactating animals.

2.5.10.8.2 Milk Fat

Fat is the most very important component of the milk. Fat percentage was significantly increased

in animals fed on diet supplemented with yeast. Meller et al. (2014) conducted an experiment to

study the effect of YC on the milk composition of Jersey cows. Cows received 50 g (1.94 × 1010

CFU) and 100 g (4.35 × 1010 Cfu) of SC per animal per day and noted that yeast supplemented

cows consuming (P=0.01) more (0.7 kg/d) DMI, which increased (P<0.05) milk fat by 0.067

kg/d than non-supplemented. In the same manner, Yalcın et al. (2011) noted that dietary

supplementation of YC improves the average fat percentage (31.41 vs 29.63 g/kg) than control

animals. That improve milk fat had also been seen in other studies (Piva et al., 1993; Putnam et

al., 1997; Moallem et al., 2009). All agreed that that improves milk fat might be due to the

59

increased milk yield and increased fibre fermentation in the yeast supplemented animals.

Longuski et al. (2009) reported that milk fat significantly (P<0.05) increased from 1.3 to 1.47 kg

per day the dairy animal fed on high moisture corn grain diet supplemented with YC. He

concluded that milk fat might be improved due to the high fermentative starch diet can be

lessened with yeast culture. Milk fat was significantly lower in yeast supplemented lactating

animals in heat stressed condition (Shaver and Garrett, 1997; Bruno et al., 2009) and in dairy

goats (El-Ghani, 2004; Stella et al., 2007).

That lower milk fat was due to higher milk yield responses obtained for yeast supplementation

which caused a dilation type effect as milk fat yield was not different among the treatments. El-

Din (2015) reported that YC supplementation has no difference in the milk fat parentage possibly

due to forage NDF in the diet which enhance the fermentation process inside rumen and

increased milk fat synthesis. On the other hand, (Bayat et al., 2015) reported that YC had no

effect the fat concentration in the lactating cows fed grass silage diets. A lack of effect of YC

supplementation has been seen for milk fat % (Erasmus et al., 1992; Robinson and Garrett, 1999;

Erasmus et al., 2005; Moallem et al., 2009)and for fat yield (Putnam et al., 1997; Cooke et al.,

2007; Bruno et al., 2009). That non-significant response could be due to the sufficient level NDF

in the diet (Erasmus et al., 2005).

2.5.10.8.3 Milk Protein

Milk protein has been significantly improve in the YC supplemented animals in many studies

(Nocek et al., 2003; White et al., 2008; Bruno et al., 2009; Kalmus et al., 2009). In a study,

Shaver and Garrett (1997) noted that YC significantly (P<0.05) increased (1.17 vs 1.14 kg/day)

milk protein yield in dairy animal fed on basal diet. That increased milk protein in the yeast fed

groups might be the positive impact of YC on the nutrient digestibility and rumen fermentation.

The increased fermentation and digestion rate is due to the increased bacterial population inside

the rumen. Proteins in the feed are quickly breakdown into peptides, amino acids and NH3 by

different protozoa and fungi (Wallace et al., 1997).

Some NH3 is converted into microbial protein (MP), and some ammonia is used by the animal in

the form of urea. The higher MP metabolized in the duodenum can be contributed to higher

protein output from the udder. One of the possible improvement of the milk protein as a result of

YC supplementation lowers the blood urea nitrogen (Bruno et al., 2009). On the other hand, In

vitro findings tell that probiotic yeast could alter the growth and activities of protein degrading

60

bacterial species, by limiting their attack on protein and peptides. YC has positive effect on

microbial growth and negative effect on nitrogen loss (Beev et al., 2007). That process enhances

the ammonia uptake and microbial protein production has been improved and that untimely

increased the milk protein. Meller et al. (2014) found 0.037 kg/d more milk protein in lactating

cattle fed on a high starch diet supplemented with probiotic yeast and noted that increased milk

protein might be due to increased (0.7 kg/d) DMI in yeast fed group. In agreement with this

some researcher (Nocek et al., 2003; Nocek and Kautz, 2006) found that yeast addition has a

positive effect on the milk protein. Some studies showed that yeast culture lower the milk protein

yield (Cooke et al., 2007; Stella et al., 2007; White et al., 2008) and milk protein percentage

(Nocek et al., 2003; Erasmus et al., 2005; Stella et al., 2007; Moallem et al., 2009; Desnoyers et

al., 2009). That negative effect of yeast might be due to the dilution factor of higher milk yield

(Shaver and Garrett, 1997; El-Ghani, 2004).

2.5.10.8.4 Milk Lactose

Moallem et al. (2009) and Bruno et al. (2009) noted that YC addition has a significantly

(P<0.05) effect on the milk lactose percentage and yield respectively in dairy cattle during hot

seasons. They concluded that high milk yield leads to the high milk lactose. On the other hand,

Stella et al. (2007); Bruno et al. (2009) and El-Din (2015) reported that YC supplementation has

no difference in the milk lactose percentage in dairy animals.

2.5.10.8.5 Milk Solid Not Fat (SNF)

Yeast supplementation had a significantly effect on the SNF. Hossain et al. (2014) reported a

significantly (P<0.05) improvement (8.57 vs 8.28 %) in the milk SNF in cross breed cattle fed on

the diet supplemented with 15 g YC. He concluded that improvement can be due to higher milk

yield in the yeast supplemented group than non-supplemented. Similar results were reported by

Bruno et al. (2009) and Vibhute et al. (2011) found that feeding Saccharomyces cerevisiae have

a significant effect on milk SNF.

2.5.10.9 Effect of yeast culture on the blood chemistry

Blood metabolite and minerals levels can be alter by nutritional changes which are directly or

indirectly corrected to regulation of nutrients digestion and subsequently growth and production

61

of the ruminants. Blood plasma metabolites are frequently used to monitor the metabolic health

status of dairy herds (Ametaj, 2009).

2.5.10.9.1 Cholesterol

Probiotic yeast brings changes in the concentration of rumen short chain fatty acids (particularly

propionate, butyrate and valerate) in animals. The increase in these acids is capable of reducing

the synthesis of triglyceride and cholesterol in the liver cells and might be change the lipid

profile in blood (Miller-Webster et al., 2002; Marden et al., 2008).According to Nicolosi et al.

(1999), these polysaccharides reduce the total cholesterol of serum. Fayed (2005) and Kowalik et

al. (2013) reported that YC significantly decreased the cholesterol level in their experiment.

Several other authors found no influence of live YC on triglyceride and total cholesterol

concentration of blood (Galıp, 2006; Masek et al., 2008; Campanile et al., 2008). The increased

triacylglycerol and total cholesterol in cows fed metabolites of YC may explain enhanced

activity of lipolytic enzymes and improved utilization of dietary lipid.

2.5.10.9.2 Glucose

In ruminants, follicles and oocyte maturation have been dependent on the glucose. The glucose

level indicates the physiological condition of animals (Nandi et al., 2008; Hossain et al., 2012).

It is observed that, 40-60 mg/100ml glucose required to maintaining the physiological process of

body. Probiotic yeast has a significant effect on the glucose concentration of dairy animals. In an

study, Hossain et al. (2012) found that, serum glucose was statistically (P<0.05) higher in yeast

supplemented claves as compared to non-supplemented claves. In contracts, Bagheri et al.

(2009), reported that the levels of glucose were not affected by probiotic yeast supplementation

in dairy cow. Similarly, Piva et al. (1993) and Putnam et al. (1997) reported that glucose was

not influenced by YC supplementation. The increased level of glucose is likely due to the

increased nutrient utilization that resulted in an increased DM and OM digestibility (Lascano et

al., 2012). The increased concentration of plasma glucose level could elevate the progesterone

production directly by increasing the LH production. Glucose represents the synthesis of

carbohydrates and is in the form in which carbohydrate is supplied to cell from body fluids. The

age of the animals has exhibited highly significant variation in serum glucose level in the cattle

heifers.

2.5.10.9.3 Blood Urea Nitrogen

62

Yeast culture supplementation has a significant effect on serum urea concentration in dairy

animals. Lower urea level in the blood serum of animal fed on diet supplemented with YC as an

indicator of nitrogen metabolism compared to the animals fed on diet without YC supplemented

suggests a better utilization of protein. Lower blood urea level is often diagnosed in dairy

animals in connection with a higher content of bypass protein in a diet or with a lower value of

diet RDP in the rumen. In a study, Dolezal et al. (2011) reported that probiotic yeast has a

significant effect on urea concentration in dairy animals. Some researchers (Putnam et al., 1997;

Bagheri et al., 2009; Nikkhah et al., 2004) reported that serum urea concentration was not

significantly affected by probiotic yeast.

2.5.10.10 Effect of yeast culture on the serum macro-minerals

Minerals play an important role in health, growth, production (meat and milk) and reproduction

of dairy animals, therefore balance mineral nutrition is crucial to maintain animal health and

productivity. About 17 minerals are identified as essential for dairy animals for maintenance

requirements (include the endogenous fecal losses and insensible urinary losses), growth

(amount of mineral retained /kg body weight gained), pregnancy (amount of mineral retained

within the reproductive tract at each day of gestation) and lactation (the concentration of the

mineral in milk multiplied by the 4 % fat corrected milk yield) requirements. The sum of the

maintenance, growth, pregnancy and lactation requirements is the true requirements of the

tissues for the mineral, and is referred to as the requirement for absorbed mineral. The diet must

supply this amount to the tissues (NRC, 2001). In dairy animals, Ca and P are essentially

important macro nutrients. They are required in larger amounts than other minerals. Over 70

percent of the total minerals in the body are calcium and phosphorus. About 99 percent of the

calcium of the body is present in skeleton where calcium along with phosphate anion, serves to

provide structural strength and support to bone. The probiotic yeast has many positive effects in

the absorption of some minerals and improves the metabolic heath of animals (Cole et al., 1992;

Dolezal et al., 2011).

2.5.10.10.1 Calcium

In adult cattle the normal range of Ca is 9.00 to 10.00 mg/dl (NRC, 2001). Bansal (1978)

suggested that Ca concentration is directly or indirectly related to reproductive performance of

the dairy animals. Ca is also involved in the steroid biosynthesis in ovaries (Shemesh et al.,

1984). Dolezal et al. (2011) reported that calcium levels was significantly (P<0.05) increased by

63

probiotic feed. On the other hand, (Piva et al., 1993), found no significance difference in the Ca

concentration between animals fed on control and probiotic diet.

2.5.10.10.2 Phosphorus

In growing cattle the normal range of P is 6.00 to 8.00 mg/dl and in adult cattle is 4.00 to 6.00

mg/dl (NRC, 2001). Literature suggested that probiotic yeast supplementation lower (1.67 vs

1.74 mmol/L) the P levels in dairy animals (Piva et al., 1993). In the same manner, Dolezal et al.

(2011) suggested that blood P concentration was changed (P<0.05) by addition of probiotic yeast

in Holstein dairy cows.

2.5.10.10.3 Sodium and Potassium

Literature suggested that Na and K were influenced by probiotic yeast supplementation in

animals. In a study, Milewski and Sobiech (2009) found that probiotic yeast supplementation

significantly (P≤0.05) higher the concentrations of Na+ ions in ewes compared with non-

supplemented ewes. On the other hand, Piva et al. (1993) reported that Na and K were

unaffected by probiotic yeast supplementation in dairy cows.

2.5.10.11 Haematological Parameters

Hematological studies present an effective method in monitoring the health and nutritional status

of animals. Study of different hematological constituents of blood is of great importance during

disease conditions, stress, immunity etc. They also have direct clinical application for diagnostic

purposes. These parameters may have direct effect on the reproductive performance of growing

animals. Knowledge on hematological study in growing buffalo heifers is lacking (Jabbar, 2004).

Changes in the hematological profile occur following exposure to stressors in mammals.

2.5.10.11.1 Hemoglobin

Hemoglobin (Hb) is the major substance in red blood cells. It carries oxygen and gives the red

colour to blood cells. The hemoglobin test measures the amount of Hb in blood and is a good

indication of the blood's ability to carry oxygen throughout the body. Some studies reported that

YC has a significant effect on Hb. Milewski and Sobiech (2009) found that ewes fed on the diet

supplemented with yeast had significantly (P≤0.01) higher (101.70 vs 114.00 g/L) Hb

concentration as compared ewes fed on control diet. In the same manner, Dobicki et al. (2005)

reported improve Hb in claves fed on diet supplemented with probiotic yeast (Saccharomyces

cerevisiae).

2.5.10.11.2 Erythrocytic Count

64

Milewski and Sobiech (2009) reported that yeast supplementation significantly (P≤0.01)

increases the RBC count as compared to non-supplemented ewes.

2.5.10.11.3 Leukocytic Count

It was found that ewes when fed a diet supplemented with YC have associated with significantly

(P ≤ 0.05) increased erythrocytes counts as compared to non-supplemented ewes (Milewski and

Sobiech, 2009)

2.5.10.11.4 Haematocrit level

Haematocrit level (also called packed cell volume), measures the amount of space (volume) red

blood cells (RBC) occupy in the blood. The value is given as a percentage (%, vol/vol) of RBC

in a volume of blood. For example, a hematocrit of 38 means that 38 percent of the blood's

volume is composed of red cells. It is the quickest and most accurate measure of the red cell

component of blood. Milewski and Sobiech (2009) reported that yeast supplementation had

associated with significantly (P ≤ 0.05) increased erythrocytes counts as compared to non-

supplemented ewes.

2.5.10.11.5 Lymphocytes

Blood lymphocyte number variation normally indicates the alteration in division rather than

alteration in production (Borghese, 2005). Lymphocytes number also influenced with

environmental atmosphere. (Fagiolo, 2004) observed decreased lymphocytes number during

summer (41%) than winter (77%) in dairy animals. Ciaramella et al. (2005) found significantly

reduced lymphocytes number in buffaloes that are above eight years of age. Khaliq and Rahman

(2010) reported mean lymphocytes 58.23% in lactating Nili-Ravi buffaloes of 7-10 years old fed

mixed ration (sugarcane, berseem fodder and maize oil cake). There was increasing pattern in

lymphocytes number in dairy heifers but were within physiological limit (The Merck Veterinary

Manual).

2.5.10.11.6 Eosinophils

Eosinophils are one of the immune systems components responsible for fighting parasitic

infections in vertebrates. In case of hypersensitivity reactions number of circulating eosinophils

increased. (Fagiolo, 2004) observed 1.64-0.16% eosinophils in early lactating buffaloes in

different seasons. Ciaramella et al. (2005) reported that, buffaloes over ten years of age showed

higher eosinophils levels compared to those in immature females (Canfield, 1984).

65

2.5.10.11.7 Monocytes

Monocytes are important component of immune system. These are produced in the marrow,

circulate briefly in the blood, and migrate into the tissues where they differentiate further to

become macrophages. Monocytes number in the marrow at a given time is very small.

Monocytes in blood are distributed between a marginated and circulating pool. Literature on

monocyte function in dairy animals is scare. Changes in the blood hematological indices suggest

an improvement in body condition and indicative of blood-supply improvement and immunity

enhancement in ruminants. Changes in biochemical indices suggested that the YC had a

stimulating impact on energy metabolism and a protective effect on renal function, and that it

contributed to preventing metabolic acidosis.

2.5.10.12 Effect of yeast on the ruminal gut microbial flora

The gut microflora is known to have a crucial role in shaping key aspects of dairy animal’s life,

such as development of the immune system and influencing the host’s physiology. Different

types of beneficial and harmful microorganisms are present in GIT of dairy animals. Health of

animal may be compromised due to the constant drive for high productive performance, where

eventfully a physiological limit or threshold is reached. Healthy animals have a balanced GIT

microbiota that allows them to performed normal function during their life. The microbial

balance of young animals is different from adult animal. The intestinal micro-biota of young

animals lacks the stability when they confronted the traumatic conditions of severe rearing

system. In that case, there arises the possibility of decreasing population of beneficial bacterial

species (Lactobacilli and Bifidobacteria) followed by increasing number of pathogens (E.coli,

Enterobacter) or other flora. That imbalanced GIT microbiota may leads to poor growth

performance and health status in young animals.

As we know that, DMI of ruminants is the main nutrient source of GIT microbiota; diet plays a

crucial role on their ecosystem and with that on the health of GIT the animals. Increased ruminal

gut fecal Lactobacilli by the supplementation of probiotics have been reported by many

researchers (Ellinger et al., 1980). Coliform includes the gerna Escherichia, Enterobacter and

Citrobacter etc. which are opportunistic pathogens associated with diarrhea. On the other hand

Lactobacillus species have been found in many types of animals, according to literature the

Lactobacillus are predominant in digestive tract and feces of young animals. It adapts and

66

develops beneficial symbiosis with the host claves. (Vlkova et al., 2006). In young ruminants the

rumen (part of GIT) is not yet developed, at that time, probiotics target the GIT, and they

represent an interesting tool to stabilize the small intestinal microbiota and reduced the risk of

pathogens. Literature showed that, when live yeast used to dairy heifers at the beginning life, a

favour microbial colonisation and the set-up of fermentative capacities in the rumen have been

seen (Chaucheyras-Durand and Fonty, 2002).

Yeast supplementation may enhance the health and immune function of animals. The

gastrointestinal tract has acquired a flushing effect, in which any harmful subtracts (toxins and

pathogens) are bound and absorbed to the yeast cell wall fractions in the gut which protect

animals from potential harm (Stone, 1998). According to the findings of numerous authors

(Collins and Gibson, 1999; Heinrich et al., 2003; Li et al., 2005), the immune-stimulating effect

of Saccharomyces cerevisiae can be ascribed to the activity of β-1, 3/1, 6-D-glucan and mannan-

oligosaccharides (MOS) present in yeast cell walls. (Li et al., 2005; Xiao et al., 2004). MOS has

also play an important role in binding of selected pathogenic microbes due to blocking microbial

lectins and preventing pathogens from colonizing in the gut of the host, thus helping in

destruction of bound pathogens by specialized immune cells (Collins and Gibson, 1999; Spring

et al., 2000; Heinrich et al., 2003).

Fecal E.coli was reduced and increased Lactobacillus was seen in the goat fed on YC which

leads to a stable GIT microbial flora. The probiotic effect is contributed by the mannans which

are a chain of mannose sugars consumed by the beneficial bacteria in the gut thereafter

promoting growth of beneficial bacteria which naturally suppresses and inhibits the growth of

the harmful bacteria inside gut, yet as observed in claves receiving CNCM 1-1077

supplementation, the occurrence of diarrhea and pneumonia was narrowed (Pinos-Rodríguez et

al., 2008). Cole et al. (1992) measured no significant effect on the claves performance and

health, through morbid claves required reduced days of antibiotics therapy, and claves

compromised with diseases had maintained higher DMI and body weight. Magalhaes et al.

(2008) demonstrated lower mortality rates, which might have due to improved N, Zn and Fe

metabolism. This is echoed in study by Piva et al. (1993) who had measured improved blood

plasma Zn levels (p=0.14) suggested that yeast may supply the Zn that plays a role in

reproductive performance. Young animals are subjected to a multiple stresses (transport,

nutrition and temperate changes, de-horning), such stressors have been shown to induced

67

imbalance GIT microbiota (Tannock and Savage, 1974). These imbalanced microbiota leads to

increased disease susceptibility in dairy animals (Bayatkouhsar et al., 2013). The use of probiotic

in farm animals restore of beneficially change the microbial flora in young, stressed animal, so

that they can better resist to the infectious diseases (Malago et al., 2014). It is well studied that

supplementation of probiotic as a tool to maintain the microbial balance of intestine, prevents

diarrhea, and improved fecal bacteria flora of ruminants (Abu-Tarboush et al., 1996; Galvao et

al., 2005; Timmerman et al., 2005; Kawakami et al., 2010). It is also effective in improving

resistance to colonization with pathogen and thus it results to improve health of animals

(Jatkauskas and Vrotniakiene, 2010). Since it is difficult to obtain intestinal samples for

microbial analyses, enumeration of fecal microbial flora has been used as an indirect method of

determining bacteria inhabiting in the intestinal tract (Schwab et al., 1980). The fecal flora is

assumed to represent only the luminal flora and not that associated with mucosal epithelial

surfaces and is assumed to vary with different types of diets (Ellinger et al., 1980). Few research

reports are available regarding microbial characterization, diversity and other prospective

applications of cattle fecal material (Yokoyama et al., 2007). The increased growth was higher in

supplemented group than control group suggested that the yeast supplementation has a capability

to improve gut microbial flora and reduce the diarrhea (Kawakami et al., 2010).

Improved growth performance and rumen development in young claves have also been noted by

many scientists (Abu-Tarboush et al., 1996; Galvao et al., 2005; Adams et al., 2008). The GIT

homeostasis is heavily depends on the balance between nutrients and ions absorption and

secretion and the capability of the GIT epithelium to control pathogens and macromolecules. The

interaction between endocrine, neurocrine, stromal and immune cells or the natural intestinal

microbiota controls the epithelial functions (Heyman and Menard, 2002). The biological activity

of probiotic has a direct impact on the metabolic processes, GIT microbiota function and host

resistance (Novik et al., 2006). Probiotics in the intestinal tract prevent colonization of pathogens

and thus lessen down the chances of diarrheal occurrences particularly in young animals (Abe et

al., 1995). Because of the accumulation of pathogenic microorganism in the intestinal tract the

development of young calves is strongly influenced by reduced nutrient assimilation and low

digestion rate. In these scenarios, probiotics act as beneficial source to control diarrhea (Jonsson

and Olsson, 1985). The efficiency of probiotics deviates from one animal to another and of the

same species. That is the reason various authors (Gardiner et al., 2004; Timmerman et al., 2004)

68

have suggested the direction of probiotics (as inoculums) by amalgamation of combining

different strains. The intestinal homeostasis keeps the absorption and secretion stability of

nutrients and ions. The epithelium of intestinal tract also provides resistance against

macromolecules and pathogenic flora (Heyman and Menard, 2002).

2.5.10.13 Effect of probiotic yeast on economic analysis

Improve production efficiency leads to improve profit for dairy farmers; moreover costs

associated with diseases treatment can decrease the profit (Kossaibati and Esslemont, 1997).

Probiotics added to the feed of ruminants to enhanced productive performance, may be cost

effective and safe methods to improve feed utilization in dairy animal. The economic advantage

of microbial feed additives depends on the price of yeast culture, yeast strain, dose, the

lactation stage, age, diet, breed and geographical location of the animal (Yalcın et al., 2011;

Vibhute et al., 2011). S. cerevisiae is cheap probiotic (0.05-0.07 dollar per cow and d) that can

benefit to health and production performance in dairy animal (Eastridge, 2006). Good health of

dairy animal gives a lot of profit to the farmers. Magalhaes et al. (2008) reported that probiotic

yeast improved profit in dairy claves by 48 dollar per calf by decreasing morbidity and mortality

rates. In the same manners, Shaver and Garrett (1997) studied the cost analysis of the

probiotic yeast in dairy animal and observed that YC supplementation has improved milk

yield as 0.23 kg per cow. It is well studied that diseases lower profit of dairy farmers (Kelton

et al., 1998). Clinical mastitis can decrease profit by 735 dollar per lactating cattle (Hultgren

and Svensson, 2009).

Chapter-3

MATERIAL AND METHODS

The present study has been divided into three phases, consisting of different experiments to see

the impact of dietary supplementation of probiotic yeast on the performance of dairy cattle and

69

heifers. All research work was carried out at the National Agricultural Research Centre (NARC),

Islamabad, Pakistan (Lat. 33.7˚N; Long. 73.1˚E; Alt. 508 m) (Figures 3.1, 3.2).

3.1 Phase 1: Determine the impact of Saccharomyces cerevisiae (Yea-Sac1026) on growth

performance and wellbeing of dairy heifers

3.1.1 Probiotic yeast strain:

A commercially available probiotic yeast strain (Yea-Sac1026) (Alltech Inc., Nicholasville, KY)

was used in this phase (Figure 3.3).

3.1.2 Control and probiotic feed for dairy heifers

Control feed was prepared at National Agricultural Research Centre Islamabad, Pakistan to meet

the small dairy-breed heifer’s nutrient requirements (NRC, 2001). For the preparation of

probiotic feed, the control feed was supplemented with commercially available yeast (Yac-

Sac1026, 5g/d/animal; corresponding to 2.5 × 1007 cfu/g).

3.1.3 Animals, treatments and experimental layout

Eight dairy heifers (n=8; average body weight of 87±5 kg) were used in this experiment.

Animals were randomly divided into two equal groups as follow: non-supplemented group

(n=4); which fed on control feed (without any probiotic supplementation) and supplemented

group (n=4); which fed probiotic feed (control feed with yeast supplementation). Feed intake was

recorded on daily basis by measuring the amount of refusal before the morning feeding. Heifers

were allowed access to an exercise lot for 3 hours in the evening time throughout the

experimental

period except

during

digestibility

trial (Figure

3.4). The

study lasted

for four

months.

70

Figure 3.1: Livestock Research Station, NARC, Islamabad.

Figure 3.2: Animal shed at Livestock Research Station, NARC, Islamabad.

71

Figure 3.3: Commercially available yeast (Yac-Sac1026)

Figure 3.4: Dairy heifers in open paddock at NARC, Islamabad

3.1.4 Quantities analysis of performance parameters of dairy heifers

72

The impact of probiotic yeast on dairy heifers was monitored by analysis of following

parameters;

3.1.4.1 Growth performance of dairy heifers

Feed intake was noted daily by subtracting the amount of feed offered from the amount of feed

refusal before the morning feeding. Heifers were weighed at starting and then fortnightly to

calculate the growth performance. Feed efficiency was calculated as feed intake (kg)/live weight

gain (kg).

3.1.4.2 Digestive performance of dairy heifers

During last week of the experiment, a total tract digestibility trial was carried out. Four animals

of almost similar weight from each group were placed in separate digestibility shad (Figure 3.5).

The total collection of feces was collected (5 days of total collection). Feces were weighed mixed

daily, and a random sample (2%) was taken for future analysis. Feed samples were also collected

during the digestibility period. Feed and fecal samples were dried and analyze for proximate

composition (AOAC, 1990), neutral detergent fiber and acid detergent fiber determination (Van

Soest, 1991).

Figure 3.5: Digestibility shed at NARC, Islamabad

3.1.4.3 Microbiological analysis of ruminal-gut microbial flora of dairy heifers

3.1.4.3.1 Sample description and collection

73

Fecal sampling was performed from dairy heifers for ruminal-gut microbial flora study. A total

of 40 fecal samples were collected from dairy heifers at 0, 30, 60, 90 and 120 days of the

experiment directly from rectum in sterile plastics bags. Samples were labeled with respect to

their particular origin and transferred to the laboratory for analysis.

3.1.4.3.2 Sample preparation

One gram of fecal material was mixed in trypticase salt solution (TSS) and vortex for 15-20

minutes and then aseptically shifted in reagent bottle.

3.1.4.3.3 Sample processing

The collected samples were further processed and examined on different types of media such as,

trypticase soy agar (TSA), deMan, Rogosa and Sharpe agar (MRS), M-17 and macconkey. Each

sample was diluted in TSS and 100 μl of the sample was spread on surface of prepared media

plates using spread plate technique. All plates were incubated at 37˚C for 48 hours in the

incubator.

3.1.4.3.4 Microbiological analysis

Different sorts of medias were used for determination of different bacterial count. In this study,

the samples were analysed in the following media:

3.1.4.3.4.1 Trypticase soy agar (TSA)

Total bacterial count (TBC) was performed on TSA medium by using spread plate technique. A

sample was serially diluted (ten folds) up-to specific dilutions and each dilution (100μl) was

spread on TSA medium. This was set to incubate aerobically at 37˚C and 48 hours. After

incubation, the number of colonies was counted down.

3.1.4.3.4.2 Man, Rogosa and Sharpe agar (MRS)

MRS (Oxoid, Basingstoke, UK) media was used for growth of Lactobacillus species. A 100μl of

diluted sample (ten-fold dilutions in normal saline) was spread out in aseptically prepared MRS

agar plates and then placed in incubator aerobically at 37˚C. Results were observed after 48

hours by colony forming unit (CFU) analysis. Colony counts were done manually with numbers

over 300 designated as too numerous to count (TNTC). Lactobacillus screening on MRS is

further confirmed by biochemical tests.

3.1.4.3.4.3 M-17 agar

M-17 growth medium (Oxoid, Basingstoke, UK) was used specifically for Lactococcus and

Enterococcus species. A 100μl of diluted sample (ten-fold dilutions in normal saline) was spread

74

out in aseptically prepared M-17 agar plates and then placed in incubator aerobically at 37˚C for

48 hours. Results were observed by CFU analysis. Colony counts were done manually with

numbers over 300 designated as TNTC. Lactococcus and Enterococcus species screening on M-

17 was further confirmed by different biochemical tests.

3.1.4.3.4.4 Macconkey agar

This media was used for coliform isolation. A 100μl of diluted sample (ten-fold dilutions in

normal saline) was spread out in aseptically prepared macconkey agar plates and then placed in

incubator aerobically at 37˚C. Results were observed after 48 hours by CFU analysis. Colony

counts were done manually with numbers over 300 designated as to TNTC. Coliform species

screening on macconkey media is further confirmed by biochemical tests.

3.1.4.3.5 Purification of Isolates

After the incubation period, the growth appeared on the plates of TSA, MRS, M-17, and

macconkey agar were purified. Single colony from the isolates was streaked out on freshly

prepared plates of respective media with the help of platinum red hot inoculating loop. This step

was further repeated until the culture seemed strictly purified, which was further confirmed by

applying some tests.

3.1.4.3.6 Preservation of pure isolates

The pure isolates were preserved in slants and glycerol stock solution. In its glycerol

preservation, 700μl Luria broth (LB) was inoculated with pure isolates for 24hrs duration. It was

then transferred to 1.5ml eppendorf containing 300μl glycerol that was sterilized in hot oven at

180˚C for 1 hour. This suspension was placed in 4˚C refrigerator for 12 hours which was then

shifted to -20˚C freezer. In its slants preservation, slants of respective media (TSA, MRS, M-17

and macconkey) were prepared in which, after pouring the respective media in test tubes, tubes

were slightly tilted along one side such that there became a butt at the bottom. Pure isolates were

streaked out in those slants and then allowed to incubate at their desired incubation conditions.

After incubation period, these slants were moved in refrigerator (4˚C). These preservation

techniques provide long term storage in which glycerol preservation prevents freeze damaging

and ice-crystal formation and slants preservation provide low contact to air and moisture due to

its narrow size.

3.1.4.3.7 Identification of bacterial isolates

75

The colonies of selected microbes were counted and selected randomly for identification. The

bacterial isolates were identified morphologically and biochemically. The interpretation of

results was done according to “Bergey’s Manual of Determinative Bacteriology”, 8th edition.

3.1.4.3.8 Morphological characteristics:

Gram’s staining is employed for morphological characteristics of pure isolates. In Gram’s

staining procedure, following steps are performed respectively:

Smear fixation

Primary staining (stain with crystal violet)

Mordant fixation (Gram’s iodine)

De-colorization (95% alcohol)

Counter staining (safranin)

Prepared a thin smear by picking up loopfull of bacterial strains in a clean slide and gently mixed

it in a drop of normal saline. It is then air dried and heat fixed over the flame. Stained the smear

with crystal violet (CV) for 1 minute and then rinsed with distilled water. Then put 1 or 2 drops

of Gram’s iodine which act as a mordant for 1 minute. Rinse it too with distilled water properly.

After that, immediately de-colorize it with 95% alcohol. Finally stain it with safranin (counter

stain) for 1 minute. Again rinse with distilled water. Air dried the slide and observed under

microscope using oil-immersion at 100X. In this way, the colony morphology, cell morphology,

and the motility of bacterial isolates from fresh cultures were evaluated.

3.1.4.3.9 Biochemical characterization

A list of biochemical tests were performed for identification purposes. These have been listed

below.

3.1.4.3.9.1 Catalase test

Catalase test suggest, whether the isolates empower such enzyme ‘‘catalase’’ that shows the

capability of neutralizing toxic oxygen and degrading H2O2 into O2 and H2O. In this test, a

smear of 24 hours fresh growth isolates was prepared. Put 2-3 drops of H2O2. Bountiful bubble

formation counts for positive results and no bubble formation meant for negative results.

Bubbles: Catalase positive (+)

No bubbles: Catalase negative (-)

3.1.4.3.9.2 Oxidase test

76

The purpose behind catalase test is to indicate the presence of cytochrome oxidase enzyme in

selected strain of interests. Dimethyl-p-phenylenediamine di hydrochloride is used for this test.

This colourless reagent oxidised and turned into coloured product. In this test, sterilised filter

paper was soaked in oxidase reagent as mentioned as above. A single isolated colony was picked

with red hot sterile loop and rubbed against it. The change in colour indicate positive while no

change support negative results.

Colourless-Dark purple: Positive result

No colour change: Negative result

3.1.4.3.9.3 Citrate utilization test

This citrate utilization test helps to study either the isolated strains utilize sodium citrate as a sole

source of nitrogen (inorganic) and carbon (organic) source or not. To analyse this, loop full

culture of freshly prepared isolated strains were streaked out on Simon’s citrate agar slants.

These slants were then placed in incubator (37˚C±24 hours). If slant’s colour changes from green

to blue, it is an indication of positive result, otherwise it means negative result.

Colour green to blue: Positive result

No colour change: Negative result

3.1.4.3.9.4 Methyl Red (MR) test

This test determines the ability of mixed acid fermentation among isolated microbial strains. For

this test, methyl red voges-proskauer (MRVP) broth is used that contain phosphate buffer,

glucose and peptone. Those organisms that do mixed-acid type of fermentation release acid that

over-whelm the buffering capacity of both resulting in decrease in pH. In this test, autoclaved

MRVP broth was inoculated with freshly pure isolates and incubated at 37˚C for 24 hours. After

incubation, few drops of methyl red used as an indicator was added in the inoculated tubes and

results were examined

Pink – Red: Positive

Pale yellow (no change): Negative

3.1.4.3.9.5 Sulfide Indole Motility (SIM) test

SIM test is used to test motility of organisms, its indole production characteristic and H2S

production ability.SIM test configured three types of things i.e: motility, indole production and

H2S production. Basically, media used for this is semi-solid media. For motility purpose, point

inoculation of desired strains was performed. After 24 hour incubation, haziness pattern from the

77

stab line indicates positive result for motility. In case of indole test, a few drops of kovac’s

reagent were added, where, the appearance of cherry-red ring points out its positive result and no

colour shows negative result. Formation of black residues represents H2S production.

3.1.4.3.9.6 Triple Sugar Iron (TSI) test

This test determines the carbohydrates (sucrose, lactose, and glucose) fermentation ability of

microorganisms. It also facilitates the sulphur reduction (H2S production) phenomena. TSI agar

media was autoclaved and slants were prepared. Pure isolates were then streaked out on these

slants tubes and then allowed to incubate at 37˚C±24 hours. The fermenting conditions were

noted down as:

Slant red / Butt yellow: Glucose fermentation

Butt yellow / Slant red: Lactose and sucrose fermentation

Red only: No fermentation

Slant red / Butt yellow / Black ppt: Glucose fermentation + H2S production

Slant yellow Butt yellow / Black ppt: Sucrose and lactose fermentation + H2S production

3.1.4.3.10 Molecular characterization

Representative isolates were sequenced for 16S rRNA gene to confirm their identification.

Sequencing was done by using commercial service of Macrogen Inc. Korea

(www.dna.macrogen.com). The sequenced data was submitted to NCBI gene data base

(http://www.ncbi.nlm.nih.gov/) and accession numbers were obtained

3.1.4.3.11 Phylogenetic analysis of bacterial isolates

Bio-Edit software (Hall, 1999) was used to assemble the fragment sequences of 16S rRNA gene

sequencing. The gene sequences were submitted to DDBJ (www.ddbj.nig.ac.jp). Using 16S

rRNA gene sequences, the strains were identified by BLAST search on EZTaxon Server. The

sequences of closely related type strains were retrieved for constructing phylogenetic trees.

Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5

(Tamura et al., 2011). A phylogenetic tree was built from unambiguously aligned nucleotides

using the neighbor-joining algorithum (Saitou and Nei, 1987). The stability of the relationship

was assessed by boots trap analysis by performing 1000 resamplings for the tree topology of

neighbor-joining data

3.1.4.4 Blood study of dairy heifers

3.1.4.4.1 Hematological study

78

Blood was collected from the jugular vein of the heifers at 0 and 120 days of the experiment and

put into 5ml heparinised venoject EDTA (K3) tube for future analysis. Erytrocytic counts, total

leukocytic count, packed cell volume, haemoglobin, lymphocytes, monocytes and eosinophils

were determined (Benjamin, 1985) by using Beckman Coulter ®AcT Diff™Haematology

Analyzer.

3.1.4.4.2 Serum biochemical study

Blood was collected from the jugular vein of the heifers at 0 and 120 days of the experiment and

allow to centrifugation at 3000 rpm for 20 minutes. Serum was separated and stored at -20 oC for

future analysis. Serum was used for serum biochemical estimation with commercial kits (Kit

AMP Medizintechnik GmbH, Austria) by using spectrophotometer (UVD-2960, USA). Serum

glucose concentration was measured with kit (BD2901-E Kit AMP Medizintechnik GmbH

Austria). The reaction was employed and absorption was measured at 500 mm for cholesterol

500 mm on spectrophotometer (Barham, 1979). Serum cholesterol concentration was measured

with kit (BD2601-E Kit AMP Medizintechnik GmbH Austria). The reaction was employed and

absorption was measured at 500 mm on spectrophotometer (Roeschlau, 1974). Blood urea

nitrogen concentration was measured with kit (BD4002-E Kit AMP Medizintechnik GmbH

Austria). The reaction was employed and absorption was measured at 340 mm on

spectrophotometer (Berthelot, 1974).

3.1.4.4.3 Serum macro-minerals

Serum was used for calcium determination by using spectrophotometer (UVD-2960, USA) with

commercial kits (Kit AMP Medizintechnik GmbH, Austria, BD7202). The reaction was

employed and absorption was measured at 650 mm on spectrophotometer. Serum P was

estimated by spectrophotometer (UVD-2960, USA) with commercial kits (Kit AMP

Medizintechnik GmbH, Austria, BD3702). The absorbance was measured at a wavelength of 340

nm on spectrophotometer. Na and K concentration in the serum was estimated by using AFP100

Flame photometer (Biotech Engineering Managment Co. Ltd., UK).

3.2 Phase II: Isolation and characterization of locally isolated yeast as a probiotic for

dairy cattle

3.2.1 Isolation of the yeasts strains

79

Two fermented dough samples were collected from local market. These samples were labelled

with respect to their particular origin, transferred and processed in lab. 1gm of sample was mixed

in trypticase salt solution (TSS) and allowed to vortex for 15-20 minutes at 40Hz and then

shifted in reagent bottle in sterile conditions. The purpose of using TSS was to provide better

cellular osmotic and non lytic conditions. The collected samples were further processed and

examined on OGA media. Serial dilution method was applied in which each 1ml of sample was

serially diluted in 9 ml of sterilised normal saline. The first tube dilution was marked as 10-1and

serially diluted up to ten-fold (1/1000000). Thus as a whole 10-1-10-7 dilutions were prepared.

From 10-4, 10-5 and 10-6 dilutions, 0.1ml or 100μl sample was taken and spread on surface of

prepared media plates with the help of sterile glass spreader under proper sterile conditions.

These were then allowed to incubate at its appropriate conditions i.e. 37˚C for 24-28 hours in the

incubator. All the samples were treated with same methodology. Two strains were isolated,

which were selected for further studies.

3.2.2 Identification of yeast

The isolated yeast strains were identified on the basis of morphology, biochemical

characteristics.

3.2.2.1 Morphological identification

The identification of the yeast isolates was carried out according to conventional yeast

identification methods based on the morphological appearance (color, shape, elevation, margins

etc.) and microscopic examination of the colonies after staining them with simple stain

(methylene blue).

3.2.2.2 Biochemical characterization

A list of biochemical tests were performed for identification purposes (Lodder, 1974; Barnett,

1983; Barnett, 1990). These have been listed below.

3.2.2.2.1 Catalase test

Catalase test suggest whether the isolates empower such enzyme ‘‘catalase’’ that show the

capability of neutralizing toxic oxygen and degrading H2O2 into O2 and H2O. In this test, a smear

of 24 hour fresh growth isolates was prepared. After that 2-3 drops of H2O2 were added.

Bountiful bubble formation counts for positive results and no bubble formation meant for

negative results.

Bubbles: Catalase positive (+)

80

No bubbles: Catalase negative (-)

3.2.2.2.2 Urease teat

Urease test involves the streak the surface of a urea agar slant with a portion of well isolated

colony. Alternatively, slant can be incubated with 1-2 drops of overnight brain-heart infusion

broth. Leave the cap on loosely and incubate the test tube at 35oC in ambient air for 48 hours to

75 days. If organism produces urease enzyme, the color of the slant changes from light orange to

magenta. If organism does not produce urease the agar slant and butt remain light orange (No

color change).

Orange to magenta colour: Urease (+)

No bubbles: Urease (-)

3.2.2.2.3 Glucose and sucrose test

The ability of strains to utilize glucose and sucrose as carbon sources was determined in Durham

tubes on YP medium containing the respective sugar plus the pH indicator.

3.2.3 Probiotic characterization of the isolated yeast strains

3.2.3.1 Determination of enzymatic potential

The isolates screened out from fermented food sample on OGA agar were qualitatively analysed

on account of their ability to produce extracellular enzyme. This qualitative assay includes

cellulase, protease, and amylase activity that influence the behavior of organism when performed

qualitatively on its specific media. This assay procedure is described below.

3.2.3.1.1 Detection of amylolytic activity

In order to determine the amylolytic activity of isolates, different ingredients were added to

prepare the media. 1 g nutrient broth, 2 g agar and 1 g starch were dissolved in distilled water

and set on autoclave. Amylase media plates were prepared. These plates were inoculated by

these isolates by means of point inoculation and then allowed to incubate for 48 hours. After

incubation period, iodine crystals were sprinkled over the amylase plates and then let them for

few minutes. Formation of luminous zones around the inoculation point indicates positive result

and no zone is indication for negative result.

81

3.2.3.1.2 Detection of cellulolytic activity

The media prepared for cellulolytic activity by dissolving 0.9 gram nutrient broth, 2 gram agar, 1

gram CMC in 100ml distilled water. The plates were treated same by means of point inoculation.

After incubation period, plates were stained firstly with congo red dye for about 15 minutes and

then stained with NaCl for 15 minutes. The presence of clear zone around the inoculated colony

is indication of positive result and absence of this shows negativity effect.

3.2.3.1.3 Detection of proteolytic activity

1% casein agar media is used for proteolytic activity. Point inoculation was performed on these

plates and set on incubation for 48 hours. After incubation, the plates were immersed in 1%

glacial acetic acid. Bright zone formation brings out positive result and no zone for negative

result.

3.2.3.2 Determination of bile tolerance

Bile tolerance activity was performed among isolates screened out from OGA agar. In this assay,

these 2 selected strains were inoculated in sterilized. Tryptic Soy Broth (TSB) in erlenmeyer

flasks and then kept in shaker incubator at 37˚C, 150 rpm for 24±48 hours. Stock solutions of

bile salts (1.5g/l) and lysozyme (100μg/ml) were prepared. After the incubation period, 750μl

from the stock solution of bile salt and 500μl from the stock solution of lysozyme were added in

the 50ml cultured TSB flask. The pH of the solution was adjusted at 3. TSB media without the

addition of bile salt and lysozyme was set out as a control media. After 30 minute, 60 minute and

90 minute interval, samples were successively taken out and spread out on Tryptic Soy Agar

(TSA) plates via serial dilution method. These plates were incubated at 37˚C for 24-48 hours.

CFU analysis was performed and its CFU was calculated by the formula given below:

CFU/ml = Number of colonies x dilution factor

Tolerance rate = CFU/ml of bile media / CFU/ml of control media

3.2.3.3 Determination of cholesterol lowering effect

Yeast strains were selected and performed for cholesterol assimilation. These selected strains

were inoculated in TSB in Erlenmeyer flasks and set on incubation at their appropriate

conditions. After their incubation period, about 0.1ml of each sample was taken from flasks,

transferred in 10ml FeCl3-acetic acid in the falcon tubes, and then allowed to vortex for 5-10

minutes. Samples were then left for about 15 minute until its complete protein precipitation. For

its comparison, standard was prepared by appending physiological saline (0.1ml) and cholesterol

82

standard solution (10ml). 5ml of FeCl3-acetic acid was taken as a blank and then 3ml of H2SO4

was added in these and mixed well. These were then left for 30 minutes and OD was taken at 560

nm. Percentage of cholesterol assimilation assay was estimated with the help of following

formula (according to Bergey’s Manual of Determinative Bacteriology)

Cholesterol (mg/100ml) = OD of unknown x 100 x 0.2 / OD of known x 0.05

3.2.3.4 Determination of anti-microbial activity

The indicator strains Listeria monocytogenes (ATCC13932), E.coli (ATCC8739),

Staphylococcus aureus (ATCC6538) and Pseudomonas aeruginosa (ATCC9027) (against testing

strains were revived, and 100μl of each indicator strain was suspended in 2.5ml of (0.75% TSA)

soft agar. In order to prepare lawn of indicator strains, soft agar suspension was poured into

freshly prepared TSA plates and allowed it for solidification. Plates were then placed in

incubator at 37°C for about 2-3 hours. Sterile disks were set on the lawn of indicator strains

carefully. After that, 10μl of cell or supernatant was taken from overnight culture of testing

strains and carefully poured on filter paper disks. Then placed were placed in incubator at 37°C

for 24-48 hours. Yeast strains were treated likely, irrespective of its variation in incubation

conditions i.e. 30°C for 48 hours. Results of antimicrobial activity were observed in terms of its

zone diameter (nm). A clear zone formation around the disks, determine the antimicrobial

behavior.

3.2.3.5 Selection and propagation of best performing probiotic strain

After all these tests, we selected the best yeast strain for future study. A stock culture of yeast

stain was inoculated into 5 ml TSA broth and incubated at and aerobically incubated at 37oC for

24 hrs. Samples were taken for viable cell count and analysis of each strain. Pour plate counts in

TSA agar were used to numeration of each culture. 1ml of cultures were transferred into 100 ml

skim milk and allowed to grow for 48 hrs. Every 12 hrs, fresh skim milk was injected into bottles

to assure good growth. The culture was moved into a freezed dried for 72 hrs. The producer of

freeze dried cultures were mixed and added to whey as a carrier. Samples were taken to

determine the total viable count of the finished product. The stability of the product was

determined by the counting of viable and total cell counts. Surviving yeast was numerated by

pour plate counts in TSA agar after incubation at 37oC and counts were expressed as mean log

CFU/g. The product was prepared every 3 weeks and was kept at 4oC.

83

3.3 Phase III: To study the comparative impact of Saccharomyces cerevisiae (Yea-

Sac1026) and locally isolated yeast on productive performance and health status in lactating

dairy cattle

3.3.1 Probiotic yeast strains:

A commercially produced yeast (CPY) (Yea-Sac1026) (Alltech Inc., Nicholasville, KY) and

laboratory produced yeast (LPY) (QAUSC03) were produced in laboratory condition, were used

in this phase (Figure 3.6).

3.3.2 Control and probiotic feed for dairy cattle

The control feed (CON) was consists of 3 kg concentrate feed, 8 kg maize silage and 20 kg oat

fodder without probiotic yeast addition. The concentrate feed was prepared at NARC by using

following formula; 18 % maize grain, 12 % rape seed cake, 18 % rice police, 24 % what bran, 10

% maize gluten 30%, 10 % sugar cane molasses, 5 % sun flower meal, 1.5 % di-calcium

phosphate and 0.5 % each; sodium chloride, mineral premix and limestone power. Concentrate

feed composition was 15% crude protein, 0.88% calcium, 0.81% phosphorus, 22% nutrient

detergent fibre and 14 % acid detergent fibre. For preparation of commercial probiotic feed

(COM-P), the control feed was supplemented with commercially available yeast (Yac-Sac1026;

10g/d/animal; corresponding to 2.5 × 1007 CFU/g) and for preparation of laboratory probiotic

feed (LAB-P) control feed was supplemented with laboratory (LAB) produced yeast

(8g/day/animal, corresponding to 3.13×1007 CFU/g S. cerevisiae).

3.3.3 Animals, treatments and experimental layout

The study was carried out at National Agricultural Research Centre, Islamabad, Pakistan. Nine

lactating dairy cattle (n=9) at first and second lactation and producing 4-5 liters/day were used in

this phase (Figure 3.7). Animals were randomly divided into three equal groups as follow:

control group (CON) (n=3); which fed on control feed; commercially probiotic group (COM-P)

(n=3); which fed commercial probiotic feed and laboratory probiotic group (LAB-P) (n=3);

which fed laboratory probiotic feed. Feed intake was recorded on daily basis by measuring the

amount of refusal before the morning feeding. Animals were allowed access to an exercise lot for

2 hours in the evening time throughout the experimental period except during digestibility trial.

The experiment lasted for 75 days including 15 days adaption period.

3.3.4 Quantities analysis of performance parameters of dairy cattle

84

The impact of probiotic yeast on the dairy cattle was monitored by analysis of following

parameters.

3.3.4.1 Production performance of lactating dairy cattle

Cattles were milked twice a day at 12.00 am and 12.00 pm and milk production was recorded

daily. Milk samples (200 ml) were collected at 0, 15, 30, 45 and 60 days of the experiment.

Samples were analyzed for dry matter and total ash (AOAC, 1990). The milk fat, protein and

lactose were determined by milk analyzer. Solids not fat (SNF) was calculated as total solids

minus fat and lactose was calculated by following equations

% Lactose =100- (% moisture + % fat + % crude protein + % ash)

3.3.4.2 Digestive performance of dairy cattle

Digestive performance was done according to methodology mentioned in section 3.1.4.2 of

methodology of phase I

3.3.4.3 Blood study of dairy cattle

Blood study was done according to methodology mentioned in section 3.1.4.4 of methodology of

phase I

3.3.4.4 Ruminal gut microbial flora study of dairy cattle

Fecal flora study was done according to methodology mentioned in section 3.1.4.3 of

methodology of phase I

85

Figure 3.6: Laboratory produced yeast, S. cerevisiae (QAU03)

Figure 3.7: Lactating dairy cattle in animal shed at NARC, Islamabad

3.3.5 Economic analysis

Economic analysis of data was done according to (Perrin 1979). Data related to feed intake

during whole trial was used to calculate the feed cost of dairy cattle and heifers fed on probiotic

supplemented feed over dairy cattle and heifers fed on control feed.

3.3.6 Statistical analysis:

During phase I, the growth performance and health related collected data were analyzed with a

linear model using student t-test (Steel et al., 1997). Data are given as means plus minus standard

error of the means. During phase III, production and health performance related data collected on

different parameters was subjected to statistical analysis by using Analysis Of Variance

Technique (ANOVA) under completed randomized design (CRD). Means of different

parameters were tested by using least significant difference (Steel, 1984).

86

Chapter 4

RESULTS

4.1 Phase 1: Determine the effect of Saccharomyces cerevisiae (Yea-Sac1026) on growth

performance and wellbeing of dairy heifers

4.1.1 Impact of probiotics on growth performance of dairy heifers

Dietary ingredient and nutritional composition of the control feed is presented in Table 4.1.

Average dry matter intake (DMI) was almost similar (3.72±0.03 vs 3.81±0.06 kg/day) in both

groups (probiotic feed and control feed respectively). During the trail the dairy heifers fed on

probiotic feed gained significantly higher (P=0.037) average daily weights (ADG) (0.60±0.04 vs

0.72±0.02 kg/day) than the dairy heifers fed on control feed. Feed Conversion Ratio (FCR)

reflects the amount of feed (kg) required to gain one kg of body weight. Our results showed that

FCR was improved (P=0.056) in dairy heifers fed on probiotic feed and was reflected in the

increased ADG in this group (Table 4.2). The changes in daily DMI and ADG for dairy heifers

fed on control versus probiotic feed over the entire experimental period are presented in Figures

4.1 and 4.2.

In the present experiment, probiotic feed did not affect skeletal measurements of the dairy

heifers. We noted that the initial body height was almost same (41.84±1.28 vs 42.27 ±1.12 cm)

in both groups. But the final body height was higher (84.13±1.60 vs 78.34±1.14 cm) in dairy

87

heifers fed on the control feed as compared to the dairy heifers fed on probiotic feed. Same is the

case with body length, where the initial body length was similar (39.42±0.92 vs 40.22±1.20 cm)

in both groups, but the final body length was higher (79.36±1.83 vs 78.53±2.12 cm) in the

control diet fed heifers. When we calculated the heart girth of the dairy heifers, we noted that

initial (41.53±1.29 vs 46.23±1.59 cm) and final (86.38±1.56 vs 89.13±0.62 cm) heart girth was

higher in dairy heifers fed on probiotic feed as compared to the dairy heifers fed on control feed.

On the other hand, when we calculated the top line of the dairy heifers, we noted that initial

(54.51±1.31 vs 51.98±0.50 cm) and final (90.56±0.30 vs 89.12±3.50 cm) top line was higher in

dairy heifers fed on control feed as compared to the dairy heifers fed on probiotic feed. All these

differences in the structural measurements of the dairy heifers fed on control and probiotic feed

were non-significant (P>0.05) in our study (Table 4.2).

Table 4.1: Ingredient and chemical composition of the control and probiotic feed fed to

dairy heifers

Items Feeding Scheme

Control1 COM-Probiotic2

Chemical composition (% Dry matter)

Crude protein 15.23 15.23

Neutral detergent fibre 27.88 27.88

Acid detergent fibre 18.04 18.04

Calcium 0.69 0.69

Total phosphorous 0.57 0.57

ME (Mcal/kg)3 2.45 2.45

Feed ingredients (%)

Maize oil cake 17.00 17.00

Cottonseed meal 13.00 13.00

Sunflower meal 1.00 1.00

Canola meal 6.00 6.00

Rice polish 6.00 6.00

Wheat bran 7.00 7.00

Corn gluten feed 4.00 4.00

Corn grains 11.00 11.00

Vegetable oil 2.00 2.00

Wheat straw 24.00 24.00

Cane molasses 6.00 6.00

Urea 0.50 0.50

88

Di-calcium phosphate 1.00 1.00

Limestone power 0.50 0.50

Sodium chloride 0.50 0.50

Minerals premix 0.50 0.50 1Control feed without yeast; 2COM-Probiotic feed compose of control feed supplemented with 2.5×1007 cfu/g

commercially available probiotic yeast (Yac-Sac1026) at the rate of 5 g per animal/day;3 Metabolizable energy

calculated values.

Table 4.2: Growth characteristics (Means ± SEM) in dairy heifers fed on control and

probiotic feed

Items Feeding scheme

p-value Control2 COM-Probiotic3

Average initial body weight (kg)1 88.53±*5.22 87.56±5.52 0.421

Average final body weight (kg) 160.84±2.10 172.38±3.39 0.072

Average daily body weight gain (kg) 0.60±0.04 0.72±0.02 0.037

Average daily DM intake (kg) 3.72±0.03 3.81±0.06 0.416

Feed conversion ratio (kg feed/kg gain) 6.19±0.49 5.41±0.52 0.056

Structural measurements4 (cm)

Body height

Initial 41.84±1.28 42.27±1.12 0.700

Final 84.13±1.60 78.34±1.14 0.107

Body length

Initial 39.42±0.92 40.22±1.20 0.500

Final 79.36±1.83 78.53±2.12 0.750

Heart girth

Initial 41.53±1.29 46.23±1.59 0.106

Final 86.38 ±1.56 89.13±.0.62 0.259

Top line

Initial 54.51±1.31 51.98±0.50 0.345

Final 90.56±0.304 89.12±3.50 0.299

1n=4 per treatment; 2Control feed without yeast; 3COM-Probiotic feed compose of control feed supplemented with

2.5×1007cfu/g commercially probiotic yeast (Yac-Sac1026) at the rate of 5g /day/animal; 4Changes in structural

measurements during the 120 day growth trial; * standard error of the mean

89

4.1.2 Role of probiotics in nutrients digestibility in dairy heifers

Nutrient digestibility of dairy heifers fed on control and probiotic feed is presented in Table 4.3.

The results reveal that probiotic feed significantly (p<0.05) affect DM, OM, CF, CP, NDF and

ADF digestibility in dairy heifers. Apparent DM digestibility of dairy

Fig 4.1: Average monthly dry matter intake pattern (kg) of dairy heifers fed on control feed

(control, ♦; no yeast) or commercial probiotic feed (COM-P, ■; control feed plus

commercial yeast)

2.8

3

3.2

3.4

3.6

3.8

4

7 8 9 10

Dry

Mat

ter

Inta

ke (

kg/a

nim

al)

Age of dairy heifers ( 7-10 months)

Control

COM-P

60

80

100

120

140

160

180

7 8 9 10

Bo

dy

We

igh

ts (

kg/a

nim

al)

Age of dairy heifers (7-10 months)

Control

COM-P

90

Fig 4.2: Average monthly growth pattern (Kg) of dairy heifers fed on control feed (control,

♦; no yeast) or commercial probiotic feed (COM-P, ■; control feed plus commercial yeast)

heifers was higher (60.25 vs 55.52%) in dairy heifers fed probiotic feed than dairy heifers fed

only control feed. This increased DM digestibility was statistically significant (P=0.041) among

the treatments. Same, with the case of OM digestibility, which was also significantly (P=0.047)

higher (64.48 vs 60.31%) in the dairy heifers fed on probiotic feed as compared to dairy heifer

fed on control feed. After analyzing the results of CPD, we noted that, CP digestibility was

significantly (P=0.049) improved (61.02 vs 57.87%) in dairy heifers fed probiotic feed than

control feed. Similarly, apparent total tract digestion of CF was significantly (P=0.043) higher

(57.51 vs 54.28%) in probiotic feed fed group as compared to control feed fed group.

Digestibility coefficient of NDF and ADF were significantly (P<0.05) higher (61.79 vs 58.63%)

and (55.45 vs 53.08%) respectively in dairy heifers fed on probiotic feed than fed on control

feed.

Table 4.3: Nutrient digestibility (Means ± SEM) of dairy heifers fed on control and

probiotic feed

Items Feeding scheme

p-value Control2 COM-Probiotic3

Dry matter (%)1 55.52±*1.10 60.25±0.82 0.041

Organic matter (%) 60.31±1.22 64.48±.36 0.047

Crude protein (%) 57.87±0.71 61.02±0.65 0.049

Crude fibre (%) 54.28±1.40 57.51±1.08 0.043

Neutral detergent fibre (%) 58.63±0.46 61.79±0.59 0.036

Acid detergent fibre (%) 53.08±0.43 55.45±0.48 0.017

1n=4 per treatment; 2Control feed without probiotic yeast; 3COM-Probiotic feed compose of control feed

supplemented with 2.5×1007 cfu/g, commercially available probiotic yeast (Yac-Sac1026) at the rate of 5 g per

animal/day; * Standard error of the mean.

4.1.3 Influence of probiotic on hematological and biochemical parameters in dairy

heifers

4.1.3.1 Hematological parameters

All dairy cattle heifers have shown healthier hematological values in our present study. Before

the treatment (day 0), we observed that RBC counts were non-significantly (P=0.056) higher

91

(10.23±0.90 vs 7.65±0.98x106/µl) in dairy heifers fed on probiotic feed than fed on control fed.

But after the treatment (day 120) we noted significantly (P=0.043) increased (11.54±1.33 vs

8.35±0.67x106/µl mg/dl) RBC in dairy heifers fed on probiotic feed than fed on control fed.

Similarly, when we analysed the results of WBC, we noted that before the treatment (day 0), we

noted that WBC counts were non-significantly (P=0.090) higher (9.99±1.24 vs 8.15±0.43

x103/ml) in dairy heifers fed on probiotic feed than fed on control fed. But at the end of the

treatment (day 120), WBC counts were significantly (P=0.048) increased (10.37±0.85 vs

8.45±0.54x103/ml) in dairy heifers fed on probiotic feed than fed on control feed (Table 4.4). It

was observed that PCV counts were higher at day 0 (26.45±2.43 vs 22.13±2.12%) and at day 120

(30.84±2.55 vs 28.99±1.53%) in dairy heifers fed on probiotic feed than dairy heifers fed on

control fed.

But these difference were non-significantly (P>0.05) among the treatments before (day 0) and

after (day 120) the treatment. Before the treatment (day 0), Hb level was non-significantly

(p=0.076) higher (10.12±0.76 vs 8.45±0.90%) while at the end of treatment (day 120),

significantly (P=0.045) higher (13.26±1.20 vs 9.76±0.55%) in dairy heifers fed on probiotic feed

than dairy heifers fed on control feed. Lymphocytes counts were non-significantly (P>0.05)

higher before treatment (59.71±2.12vs 55.34±1.00 %) and after the treatment (52.81±2.98 vs

49.70±2.09 %) in dairy heifers fed on probiotic feed than fed on control feed. The results of the

monocytes counts showed that a non-significantly (P>0.05) increased monocytes counts were

noted at day 0 (7.34±1.20 vs 6.23±1.30%) and at day 120 (4.70±2.21 vs 4.37 ±0.30%) in dairy

heifers fed on probiotic feed than fed on control feed. Before the treatment (day 0), the

eosinophils levels was non-significant (P=0.796) increased (6.35±1.23 vs 5.34±0.87%) in dairy

heifers fed on probiotic feed than fed on control feed. While after the treatment, significantly

(P=0.017) increased (6.94±0.75vs 4.85±0.46%) eosinophils levels were observed in dairy heifers

fed on probiotic feed than fed on control feed.

4.1.3.2 Blood serum metabolites

The results of some selected serum metabolites like cholesterol, glucose and urea nitrogen in

dairy heifers fed on control and probiotic feed before and at the end of treatment are shown in

92

Table 4.5. All values are in the normal range for dairy cattle, which is a sign of good health. The

40-60 mg/100ml blood glucose level is required to maintain the physiological process of body.

When we analysis the serum glucose concentration in the blood of dairy heifers, we noted that

the serum glucose levels were within the normal range in our study. At start of experiment (day

0), serum glucose level was almost same (60.86±2.80 vs 62.77±4.04 mg/dl) for both groups

(probiotic feed and control feed respectively).But after the treatment (day 120) we noted a

significantly (P=0.034) increased (65.31±2.84 vs 63.31±2.60 mg/dl) in dairy heifers fed on

probiotic feed than dairy heifers fed on control feed.

In our study the serum cholesterol concentration before started of the treatment (day 0) was non-

significantly higher (98.13±1.87 vs 96.50±2.58 mg/dl) in dairy heifers fed on probiotic feed than

dairy heifers fed on control fed. On the other hand after treatment the average cholesterol

concentration was significantly (P=0.012) lower (101.56±1.46 vs 106.45±0.34mg/dl) in dairy

heifers fed on probiotic feed than dairy heifers fed on control feed. Similarly, in our study the

serum urea concentration was non-significantly lower (30.10±0.711 vs 31.14±0.974 mg/dl) in

the dairy heifers fed on control feed as compared to dairy heifers fed probiotic feed at start of the

experiment (day 0). But after the treatment, we noted that the urea concentration was

significantly (P=0.010) lower (29.23±0.494 vs 33.34±0.432 mg/dl) in dairy heifers fed on

probiotic feed than dairy heifers fed on control feed.

Overall results showed that the yeast supplementation significantly (P<0.05) affected the serum

glucose, cholesterol and urea levels in dairy heifers.

Table 4.4: Haematological values (Means ± SEM) in dairy heifers fed on control and

probiotic feed

Items Feeding scheme

p-value Control1 COM-Probiotic2

Erytrocytic count (x10 6/µl) 1

Before treatment4 7.65±*0.98 10.23±0.90 0.056

93

After treatment5 8.35±0.67 11.54±1.33 0.043

Total leukocytic count (x103/ml)

Before treatment 8.15±0.43 9.99±1.24 0.090

After treatment 8.45±0.54 10.37±0.85 0.048

Packed cell volume (%)

Before treatment 22.13±2.12 26.45±2.43 0.980

After treatment 28.99±1.53 30.84±2.55 0.517

Hemoglobin (g/dl )

Before treatment 8.45±0.90 10.12±0.76 0.076

After treatment 9.76±0.55 13.26±1.20 0.045

Lymphocytes (%)

Before treatment 55.34±1.00 59.71±2.12 0.560

After treatment 49.70±2.09 52.81±2.98 0.397

Monocytes (%)

Before treatment 6.23±1.30 7.34±1.20 0.487

After treatment 4.37±0.30 4.70±2.21 0.396

Eosinophils (%)

Before treatment 5.34±0.87 6.35±1.23 0.796

After treatment 4.85±0.46 6.94±0.75 0.017

1n=4 per treatment; 2Control feed without yeast; 3COM-Probiotic feed compose of control feed supplemented with

2.5×10 07 cfu/g commercially available probiotic yeast (Yac-Sac1026) at the rate of 5 g per animal/day; 4before

treatment (day 0);5after treatment (day 120); *standard error of the means.

Table 4.5: Blood serum metabolites (Means ± SEM) in dairy heifers fed on control and

probiotic feed

Items Feeding scheme

p-Value Control2 COM-Probiotic3

Glucose (mg/100ml)1

Before treatment4 *62.67± 4.04 60.86 ± 2.80 0.605

After treatment5 63.31± 2.60 65.47 ± 2.84 0.034

Cholesterol (mg/100ml)

94

Before treatment 96.50 ± 2.58 98.13 ± 1.87 0.587

After treatment 106.45 ± 0.34 101.56± 1.46 0.012

Urea (mg/100ml)

Before treatment 30.10±*0.711 31.14±0.974 0.012

After treatment 33.34±0.432 29.23±0.494 0.01

1n=4 per treatment; 2Control feed without yeast; 3COM-Probiotic feed compose of control feed supplemented with

2.5×10 07 cfu/g commercially available probiotic yeast (Yac-Sac1026) at the rate of 5 g per animal/day; * ± Standard

error of the mean; 4Before treatment (day 0);5after treatment (day 120).

4.1.3.3 Blood serum macro-minerals

The results of the calcium (Ca), phosphorus (P), sodium (Na) and potassium (K) concentration in

blood serum of dairy heifers fed control and probiotic feed before and after the treatment are

given in Table 4.6. We noted that, serum Ca concentration was slightly lower (8.72 ±0.30 vs

8.64±0.18 mg/dl) in dairy heifers fed on the probiotic feed as compared to control feed at the

start of the treatment (day 0). At the end of the treatment (day 120), Ca concentration was again

non-significantly (P=0.286) higher (8.60±0.31 vs 8.36±0.24 mg/dl) in dairy heifers fed on the

probiotic feed as compared to control feed. On the other hand, Phosphorus (P) concentration in

blood serum was non-significantly (P=0.619) higher before the treatment (6.43±0.19 vs

6.33±0.33 mg/dl) in dairy heifers fed on the control feed as compared to dairy heifers fed on

probiotic feed. After the end of the treatment P concentration was again non-significantly

(P=0.869) higher (6.61±0.16 vs 6.57±0.11 mg/dl) in dairy heifers fed on the control feed as

compared to dairy heifers fed on probiotic feed. When we determine the serum K concentration

in blood samples of dairy heifers, we noted slightly higher (P=0.527) K levels at the start of the

experiment (5.43±0.12 vs 5.36±0.11 meq/l) in dairy heifers fed on the control feed as compared

to dairy heifers fed on probiotic feed. But at the end of the experiment the K concentration was

non-significantly (P=0.868) higher (5.49 ±0.13 vs 5.44±0.16 meq/l) in dairy heifers fed on the

control feed as compared to dairy heifers fed on probiotic feed.

Table 4.6: Serum macro-minerals (Means ± SEM) in dairy heifers fed on control and

probiotic feed

Items Feeding regime p-Value

95

Control2 COM-Probiotic3

Calcium (mg/dl)1

Before treatment4 8.64 ±* 0.184 8.72± 0.303 0.866

After treatment5 8.36 ± 0.248 8.60 ± 0.318 0.286

Phosphorus (mg/dl)

Before treatment 6.33 ± 0.33 6.43 ± 0.19 0.619

After treatment 6.61± 0.16 6.57 ± 0.11 0.869

Potassium (meq/l)

Before treatment 5.36±0.11 5.43±0.12 0.527

After treatment 5.49± 0.13 5.44 ± 0.16 0.868

Sodium (meq/l)

Before treatment 119.93 ± 1.04 120.92 ± 0.38 0.387

After treatment 127.67 ± 3.56 129.12 ± 3.54 0.774

1n=4 per treatment; 2Control feed without yeast; 3COM-Probiotic feed compose of control feed supplemented with

2.5×1007 cfu/g commercially available probiotic yeast (Yac-Sac1026) at the rate of 5 g per animal/day; 4before

treatment (day 0);5after treatment (day 120); *±SEM = standard error of the mean

The difference in K concentration values among both groups was found to be statistically non-

significant (P>0.05) in our study. In the same manner, concentration of Nain blood serum of

dairy heifers was non-significantly (P=0.387) lower (119.93±1.04 vs 120.92±0.38 meq/l) before

the treatment in dairy heifers fed on the control feed as compared to dairy heifers fed on

probiotic feed. On the other hand, after the treatment, Na concentration was again non-

significantly (P=0.774) lower (127.67±3.56 vs 129.12±3.54meq/l) in dairy heifers fed on the

control feed as compared to dairy heifers fed on probiotic feed. Overall the results showed that

the P, K and Na concentration were unaffected (P>0.05) by yeast supplementation in the blood

of dairy heifers before and after the end of experiment.

4.1.1 Microbial growth trends in ruminal gut samples of dairy heifers fed on control and

probiotic feed

The average fecal total aerobic bacterial count, Lactobacillus, Enterococcus, Lactococcus and

Coliform species in ruminal gut of dairy heifers fed on control and probiotic feed as determined

at 0, 30, 60, 90 and 120 days of the experiment are given in Table 4.7. The isolated bacteria were

96

identified using morphological and biochemical process and some selected isolated were

identifies using sequencing methods (Tables 4.7; 4.8). When we analyzed that fecal samples at

day 0 of the experiment, we noted that total aerobic bacterial counts were almost similar

(6.16±0.32 vs 6.25±0.17 CFU/g) in both groups (probiotics and control groups respectively).

But, on the other hand, at day 30, the aerobic bacterial population was non-significantly

(P=0.898) higher (6.45±0.57 vs 5.27±0.55 CFU/g) in control group than probiotic group. At day

60, again we noted that the number of total aerobic bacterial count was significantly (P=0.017)

higher (6.81±0.35 vs 6.66±0.19 cfu/g) in dairy heifers fed on the control feed than heifers fed on

probiotic feed. We noted that the total aerobic bacterial counts at day 90, was non-significant

(P=0.131) higher (7.59±0.12 vs 6.76±0.29 CFU/g) counts in the heifers fed on probiotic than

control feed. At day 120, we observed significant (P=0.032) higher (7.97±0.40 vs 6.45±0.32

CFU/g) total aerobic bacterial counts in the heifers fed on probiotic yeast supplemented feed as

compared to control feed with no yeast supplementation. Overall results of the aerobic bacterial

counts showed that, there was no significant (P>0.05) difference was at 0, 30 and 90 days on the

other hand a significantly (P<0.05) difference was seen at 60 and 120 days. The changes in the

number of total aerobic counts as affected by the probiotic yeast are depicted in Figure. 4.3. At

day 0, total Lactobacillus species count was slightly higher (4.61±0.86 vs 4.22±0.94 CFU/g) in

heifers fed on probiotic feed than fed on control feed. At day 30, the numbers of the

Lactobacillus species was non-significantly (P=0.178) highly increased (5.44±0.13 vs

4.63.22±0.38 CFU/g) in heifers fed on the probiotic feed than heifers fed on control feed. On the

other hand, at day 60, Lactobacillus species counts was significant (P=0.039) higher (6.31±0.14

vs 4.86±0.43 CFU/g) in the heifers fed on feed supplemented with probiotic yeast than heifers

fed on feed without probiotic yeast supplementation. Similarly, at day 90, the numbers of the

Lactobacillus species was significantly (P=0.03) higher (6.47±0.10 vs 5.28±0.30 CFU/g) in the

heifers fed on feed supplemented with probiotic yeast than heifers fed on feed without probiotic

yeast supplementation. At day 120, the numbers of the Lactobacillus species was also

significantly (P=0.042) higher (6.98±0.28 vs 5.53±0.49 CFU/g) in the group fed on probiotic

feed as compared to group fed on control feed. The numbers of Lactobacillus were about 6.10 %

higher in probiotic yeast supplemented group as compared to non-supplemented group at the end

of the experiment. The changes in the number of Lactobacillus, species affected by the probiotic

yeast are depicted in Figure. 4.4.

97

After analyzing the results of Coliform species, we noted that at the beginning of experiment

(day 0), the Coliform population almost was similar (3.06±0.32 vs. 3.03±0.27 CFU/g) in both

groups; but after 30 days of experimentation, that number was significantly (P=0.001) higher

(4.11±0.27 vs. 3.17±0.25 CFU/g) in control group than probiotic group and maintained this level

at day 60, where we noted that the number of Coliform species was higher (3.81±0.25 vs

3.14±0.19) in dairy heifers fed on the control feed than heifers fed on probiotic feed. But the

difference was non-significant (P=0.215) between two groups at day 60. We noted that the

coliform species counts at day 90 was almost similar (2.59±0.32 vs 2.56±0.19 CFU/g) between

the heifers fed on probiotic and control feed. At the end of experiment (day 120) we observed

slightly higher (2.79±0.30 vs 3.05±0.52 CFU/g) coliform counts in the heifers fed on probiotic

feed as compared to control feed. Overall results of the coliform counts showed that during 0, 60,

90 and 120 days of the experiment the effect of the YC was statistically non-significant (p>0.05).

Expect at day 30, where we noted a significantly (P=0.001) difference between in control and

probiotic group. The changes in the number of coliform species affected by the probiotic yeast

are depicted in Figure. 4.5. At day 0, total Lactococcus species count was non-significantly

(P=0.789) higher (5.64 ±0.45 vs 5.43±0.85 CFU/g) in heifers fed on probiotic feed than fed on

control feed. At day 30, the numbers of the Lactococcus species were non-significantly

(P=0.231) increased (5.89±0.67 vs 5.66±0.90 CFU/g) in heifers fed on the probiotic feed than

heifers fed on control feed. But, at day 60, Lactococcus species counts was significant (P=0.023)

higher (6.23±0.32 vs 5.90±0.93 CFU/g) in the heifers fed on probiotic feed as compared to

control feed and maintained this level at day 90 and 120. We noted significantly (P=0.039)

higher (6.56±0.70 vs 5.99±0.96 cfu/g) Lactococcus species in the heifers fed on probiotic feed as

compared to control feed at day 90. The numbers of the Lactococcus species was again

significantly (P=0.042) higher (6.54±0.28 vs 6.12±0.49 CFU/g) in the group fed on probiotic

feed as compared to group fed on control feed. The changes in the number of Lactococcus

species, as affected by the probiotic yeast are depicted in Figure. 4.6.

On the other hand, at day 0 we noted that the numbers of Enterococcus species was almost

similar (4.13±0.34 vs. 4.05±0.65 CFU/g) in both groups. At day 30, that number was non-

significantly (P=0.231) lower (4.12±0.29 vs. 4.64±0.44 CFU/g) in probiotic group than control

group. At day 60, again we noted that the number of Enterococcus species was non-significantly

98

(P=0.123) lower (3.01±0.78 vs 4.34±0.44 CFU/g) in probiotic supplemented group than control

group. At day 90, we noted that the Enterococcus species was slightly lower (2.90±0.12 vs

3.12±0.34 CFU/g) in probiotic supplemented group than control group. At the end of experiment

(day 120) we observed significantly (P=0.023) lower (2.79±0.30 vs 4.05±0.52 CFU/g).

Enterococcus species in the heifers fed on probiotic yeast supplemented feed as compared to

control feed with no yeast supplementation. Overall the results of the Enterococcus species

counts showed that at 0, 30, 60 and 90 days of the experiment the effect of YC was non-

significant (P>0.05) expect at day 120 days where we noted a significantly (P˂0.05) difference

between in control and probiotic group. The changes in the number of Enterococcus species, as

affected by the probiotic yeast are depicted in

4.1.2 Isolation, identification and characterization of Lactobacillus, Lactococcus,

Enterococcus and Coliform species from ruminal gut of dairy heifers

MRA agar supported as a specific media for growth of lactic acid bacteria (LAB) and

macconkey agar for growth of Coliform species (Figure 4.8). Their further properties were

examined on the basis of morphology and selective biochemical tests (Table 4.8; Figure 4.9).

4.1.2.1 Colony and cell morphology:

Results of gram stating showed that all isolated strains on MRS were either gram positive rod or

gram positive cocci. This means they exhibited the property of rod and cocci shape and retain its

purple colour (crystal violet) stain, which full fill one of the property of LAB as shown in figure

(Figures 4.10 and 4.11). They appeared as short chains, single rod, cocci and in the form of rod

clusters under microscopy on MRS media. The forms of the colonies of gram positive rod

isolates were mostly round and irregular with smooth, shiny surface. Most of the colonies were

creamish and creamish white colors with entire and convex margin. The results of elevation

characteristics of gram positive rod isolated colonies showed that mostly these are raised and

most of the colonies had opaque in its opacity characteristics point of view.

On the other hand the forms of the colonies of gram positive cocci isolates were mostly circular.

The surface characteristics of the gram positive cocci were mostly found to be smooth, shiny

with creamish white colors with entire margin. The results of elevation characteristics of gram

positive rod isolated colonies showed that mostly these are raised and most of the colonies had

opaque and moist in its opacity characteristics point of view.

99

Table 4.7: Total aerobic bacteria counts (CFU/g ± SD) in ruminal gut of dairy heifers fed

on control and probiotic feed

Days of age Feeding scheme

p-value Control2 COM-Probiotic3

Total aerobic1

0 6.25±*0.17 6.16±0.32 0.817

30 6.45±0.57 5.27±0.55 0.898

60 6.81±0.35 6.66±0.19 0.017

90 6.76±0.29 7.59±0.12 0.131

120 6.45±0.32 7.97±0.40 0.032

Lactobacillus species

0 4.22±0.94 4.61±0.86 0.607

30 4.63±0.38 5.44±0.13 0.178

60 4.86±0.43 6.31±0.14 0.039

90 5.28±0.30 6.47±0.10 0.030

120 5.53±0.49 6.98±0.28 0.042

Coliforms species

0 3.06±0.32 3.03±0.27 0.959

30 4.11±0.27 3.17±0.25 0.001

60 3.81±0.25 3.14±0.19 0.215

90 2.59±0.32 2.56±0.19 0.881

120 2.79±0.30 3.05±0.52 0.521

Lactococcus species

0 5.43±0.85 5.64±0.45 0.789

30 5.66±0.90 5.84±0.67 0.231

60 5.90±0.93 6.23±0.32 0.023

90 5.99±0.96 6.56±0.70 0.039

120 6.12±0.49 6.54±0.28 0.042

Enterococcus species

0 4.05±0.65 4.13±0.34 0.659

30 4.64±0.44 4.12±0.29 0.231

60 4.34±0.49 3.01±0.78 0.123

90 3.12±0.34 2.90±0.12 0.042

120 4.05±0.52 2.79±0.30 0.023 1n=4 per treatment, 2Control feed without yeast; 3COM-Probiotic feed compose of control feed supplemented with

2.5×1007cfu/g commercially probiotic yeast (Yac-Sac1026) at the rate of 5g /day/animal; *±SEM = standard error of

the mean

100

Figure 4.3: Total aerobic count (CFU/g) in the ruminal gut of dairy heifers fed on control

feed (control, ♦; no yeast) or commercial probiotic feed (COM-P, ■; control feed plus

commercial yeast)

Figure 4.4: Total Lactobacillus count (CFU/g) in the ruminal gut of dairy heifers fed on

control feed (control, ♦; no yeast) or commercial probiotic feed (COM-P, ■; control feed

plus commercial yeast)

4.2

4.7

5.2

5.7

6.2

6.7

7.2

7.7

0 30 60 90 120

CFU

/g

Days

Control

COM-P

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

0 30 60 90 120

CFU

/g

Days

Control

COM-P

101

Figure 4.5: Total coliform count (CFU/g) in the ruminal gut of dairy heifers fed on control

feed (control, ♦; no yeast) or commercial probiotic feed (COM-P, ■; control feed plus

commercial yeast)

Figure 4.6: Total Lactococcus count (CFU/g) in the ruminal gut of dairy heifers fed on

control feed (control, ♦; no yeast) or commercial probiotic feed (COM-P, ■; control feed

plus commercial yeast)

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

0 30 60 90 120

CFU

/g

Days

Control

COM-P

5.25

5.45

5.65

5.85

6.05

6.25

6.45

0 30 60 90 120

CFU

/g

Days

Control

COM-P

102

Figure 4.3: Total Enterococcus (CFU/g) in the ruminal gut of dairy heifers fed on control

feed (control, ♦; no yeast) or commercial probiotic feed (COM-P, ■; control feed plus

commercial yeast)

All isolated grown on macconkey were appeared as gram negative macconkey the isolated

strains were appeared as gram negative rod, which full fill one of the property of coliform as

shown in Figure 4.12. They appeared as single rod and in the form of rod clusters under

microscopy on macconkey media. After isolation of pure colonies, we noted that most of the

bacterial isolated on the macconkey agar form the round and smooth colonies. These colonies

were mostly looked as translucent opacity, white yellowish colour, with convex elevation and

entire margin (Figure 4.12).

4.1.2.2 Biochemical characterization

In the biochemical characterization, isolated strains were distinguished according to Bergey‘s

manual of systematic bacteriology. On this basis, the pure isolates of gram positive rod have

respective characteristics i.e. they showed that catalase, indole, citrate, urease, oxidase and MR

tests were negative. These strains were non motile and also there was no gas (H2S) production.

On the basis of data obtained from physical and biochemical test results it was concluded that

these bacterial isolates Lactobacillus species. On the other hand, the pure isolates of gram

positive cocci have particular characteristics i.e. they showed that catalase, indole, urease and

oxidase tests were negative while citrate and MR were positive. These strains were non motile

and also there was no gas (H2S) production. On the basis of data obtained from physical and

biochemical test results we concluded that these bacterial isolates on the MRS agar were

identified as Lactococcus or Enterococcus species. On the basis of a distinguish test (grow at

4.2

4.7

5.2

5.7

6.2

6.7

7.2

7.7

0 30 60 90 120

CFU

/g

Days

Control

COM-P

103

different temperature) between Lactococcus and Enterococcus species grown on the temperature

(15 to 35 oC) and these are belong to Enterococcus species rest of the strains were belong to

Lactococcus species at different time interval. Biochemical test of the bacterial isolates on

macconkey agar showed that these are catalase, oxidase and MR positive while citrate, MR,

indole and citrate negative. These strains were motile and also there were no gas (H2S)

production. According to the experimental data, the isolated bacteria were identified as coliform

(Table 4.8).

Figure 4.8: Growth pattern of coliforms on machonkey agarLAB on MRS (Left)

Figure 4.9: Biochemical analysis of different bacterial isolates: (A) Simmon’s citrate test (B)

Triple sugar iron test (C) Methyl red test (D) Sulfide indole motility test (E) Indole test

104

Figure 4.10: Gram staining of Lactobacillus strains on MRS; gram positive rod

Figure 4.11: Gram staining of Lactococcus and Enterococcus strains on MRS; (A) Strain

QAULG03 and (B) Strain QAULG10. Both are gram + cocci

.Figure 4.12: Gram staining of Coliform strains on macconkey agar; gram + rod.

Table 4.8: Morphological, biochemical identification of bacterial isolates on MRS and mackonkey agar

Characteristics Selective bacterial strains

105

QAULG14 QAUEF07 QAUSG10 QAULG03 QAUEV12 QAUEV13

Morphology characterization

Gram staining +ve +ve +ve +ve -ve +ve

Shape Rod Cocci Cocci Cocci Rod Cocci

Form Round Circular Circular Round Circular Circular

Surface Smooth Smooth/shiny Shiny Smooth/

shiny

Smooth Shiny

Colour Creamish

white

Creamish Creamish white Creamish

white

Pinkish

white

Creamish

white

Margin Entire Convex Entire Entire Entire Entire

Elevation Raised Raised Slightly raised Raised Convex Slightly raised

Opacity Opaque Opaque Moist Opaque Translucent Moist

Biochemical characterization

Catalase -ve -ve -ve -ve +ve -ve

Oxidase -ve -ve -ve -ve +ve -ve

Indole -ve -ve -ve -ve -ve -ve

Citrate -ve +ve +ve +ve -ve +ve

Methyl red -ve +ve +ve +ve +ve +ve

TSI -ve -ve -ve -ve -ve -ve

Urease -ve -ve -ve -ve -ve -ve

Grow at

15°C NT* +ve +ve -ve NT +ve

25°C NT +ve +ve -ve NT +ve

35°C NT +ve +ve -ve NT +ve

Motility teat Non-motile Non-motile Non-Motile Non-motile Motile Non-Motile

H2S No No No No No No

Identified

microorganisms

Lactobacillus Enterococcus Enterococcus Lactococcus Escherichia Enterococcus

*Not tested

4.1.6 Partial 16S rRNA gene sequencing

Four strains were identified on molecular level by blasting the amplified nucleotide sequence

using a BLAST tool at National Centre for Biotechnology Information (NCBI) website. These

106

strains QAUEF07, QAUSG10, QAULG03, QAUEV12 and QAUEV13 were identified as

Enterococcus, Enterococcus, Lactococcus, Escherichia and Enterococcus species on the basis of

genotype. These 16S rRNA sequences were submitted to NCBI GenBank under the accession

numbers KP256014, KP256017, KP256012, KP256020 and KP256021 assigned to QAUEF07,

QAUSG10, QAULG03, QAUEV12 and QAUEV13 respectively (Table 4.9).

Table 4.9: Identification of isolated strains based on 16S rRNA gene sequences andthei1r

accession numbers published in DNA database.

Strain ID Strain name/

genus

Length

of 16Sr

RNA

(ntds)

Accession

number of

16 SrRNA

gene

Closely related

Validly published

species

Similarity %

of 16 S

r RNA gene

sequencing

QAUEF07 Enterococcus 1527 KP256014

Enterococcus

Lactis

ATCCTBT159 T

(GU983697)

99.51

QAUSG10 Enterococcus 902 KP256017

Enterococcus hirae

ATCC

9790 T (CP00304)

100

QAULG03 Lactococcus 959 KP256012

Lactococcus

garvieaeATCC

49156 T

(AP009332)

100

QAUEV12 Escherichia 895 KP256020

Enterobacter

xiangfangensis

ATCC

10-17 T

(HF679035)

99.50

QAUEV13 Enterococcus 817 KP256021

Entrococcus

faecium ATCC T

CGMCC 1.2136

(AJKH01000109)

100

4.1.7 Phylogenetic analyses of bacterial isolates

4.1.7.1 Lactococcus species

The blastn search revealed that Lactococcus QAULG03 (KP256012) had the highest sequence

similarity with the Lactococcus garvieae strain ATCC 49156T (AP009332); The Lactococcus

QAULL04 (KP256013) had the highest sequence similarity with the Lactococcus lactis subsp.

107

tructae L105T (EU770697) and The Bacterium QAULG02 (KP256011) had the highest sequence

similarity with the Lactococcus garvieae ATCC 49156T (AP009332) (Figure 4.13).

4.1.7.2 Enterobacter species

The blastn search revealed that Enterobacter QAUEV13 (KP25621) had the highest sequence

similarity with the Enterococcus faecium ATCCT CGMCC 1.2136 (AJKH01000109)) (Figure

4.14).

4.1.7.3 Enterococcus species

The blastn search revealed that Enterococcus QAUEF07 (KP256014) had the highest sequence

similarity with the Enterococcus Lactis ATCCT BT159T (GU983697). The Enterococcus

QAUSG10 (KP256017) had the highest sequence similarity with the Enterococcus hirae ATCC

9790T (CP00304) and The Enterococcus QAUSG07 (KP256015) had the highest sequence

similarity with the Enterococcus mundtii CECT972T (AJ420806). The Enterococcus QAUSG08

(KP256016) had the highest sequence similarity with the Enterococcus hirae ATCC 9790T

(CP003504). The Enterococcus QAUSKO1 (KP256018) had the highest sequence similarity

with the Enterococcus faecium ATCC CGMCC 1.2136T (AJKH01000109) (Figure 4.14).

108

Figure: 4.13: Phylogenetic tree of the Lactococcus QAULL04, QAULG03, QAULG02

species based on 16S rRNA gene sequence.

Lactococcus lactis subsp. Lactis JCM 5805T (BALX01000047)

Lactococcus lactis subsp. Hordniae NCDO 2181T (AB100804)

Lactococcus lactis subsp. Tructae L105T (EU770697)

Lactococcus QAULL04T (KP256013)

Lactococcus lactis subsp. Cremoris NCDO 607T (AB100802)

Lactococcus taiwanensis0905C15T (AB699722)

Lactococcus chungangensis CAU 28T (EF694028)

Lactococcus raffinolactis DSM 20443T (EF694030)

Lactococcus piscium CCUG 32732T (DQ343754)

Lactococcus plantarum DSM 20686T (EF694029)

Lactococcus formosensis516T (AB775178)

Lactococcus QAULG03T (KP256012)

Bacterium QAULG02T (KP256011)

Streptococcus orisuis NUM 1001T (AB182324)

99

100

92

84

97

67

100

96

0

0.01

109

Figure: 4.14: Phylogenetic tree of the Enterobacter QAUEV13 (KP25621) based on 16S

rRNA gene sequence.

Enterococcus devriesei LMG 14595T (AJ891167)

Enterococcus hermanniensis LMG 12317T (AY396047)

Enterococcus raffinosus NCIMB 12901T (Y18296)

Enterococcus malodoratus ATCC 43197T (ASWA01000002)

Enterococcus gilvus ATCC BAA-350T (AJDQ01000009)

Enterococcus viikkiensisIE3.2T (HQ378515)

Enterococcus pseudoavium NCFB 2138T (Y18356)

Enterococcus xiangfangensis 11097T (HF679036)

Enterococcus mundtii CECT972T (AJ420806)

Enterococcus pallens ATCCBAA-351T (AJAQ01000034)

Enterococcus canintestini LMG 13590T (AJ888906)

Enterococcus thailandicusFP48-3T (EF197994)

Enterococcus sanguinicola SS-1729T (AY321376)

Enterococcus durans CECT411T (AJ420801)

Enterobacter QAUEV13T (KP256021)

Enterococcus villorum ATCC 700913T (AJAN01000023)

Enterococcus ratti ATCC 700914T (AF539705)

Enterococcus hirae ATCC 9790T (CP003504)

Enterococcus lactis BT159T (GU983697)

40

29

49

34

56

63

68 15

21

0

0.0005

110

Figure: 4.15: Phylogenetic tree of the Enterococcus (KP256016, KP256017, KP256014,

KP256015, KP256018) species based on 16S rRNA gene sequence.

Enterococcus avium NCFB 2369T (Y18274)

Enterococcus xiangfangensis 11097 T (HF679036)

Enterococcus devriesei LMG 14595 T (AJ891167)

Enterococcus pseudoavium NCFB 2138 T (Y18356)

Enterococcus raffinosus NCIMB 12901 T (Y18296)

Enterococcus viikkiensis IE3.2 T (HQ378515)

Enterococcus QAUSG08T (KP256016)

Enterococcus durans CECT411 T (AJ420801)

Enterococcus villorum ATCC700913 T (AJAN01000023)

Enterococcus QAUEF07T (KP256014)

Enterococcus QAUSK01T (KP256018)

Entrococcus facium19434T (DQ411813)

Enterococcus lactis BT159T (GU983697)

Enterococcus hirae ATCC 9790T (CP003504)

Enterococcus QAUSG10T (KP256017)

Enterococcus thailandicus FP48-3T (EF197994)

Enterococcus sanguinicola SS-1729T (AY321376)

Enterococcus QAUSG07T (KP256015)

Enterococcus mundtii CECT972T (AJ420806)

Vagococcus penaei CD276T (FJ360897)

96

63

13

57

54

74

50

0

0.002

111

Figure: 4.16: Phylogenetic tree of the Escherichia QAUEV12 (KP25620) based on 16S

rRNA gene sequence.

4.1.8 Economic efficiency of dairy heifers fed on probiotic and non-probiotic feed.

Escherichia QAUEV12T (KP256020)

Enterobacter xiangfangensis 10-17T (HF679035)

Enterobacter asburiae JCM 6051T (AB004744)

Enterobacter cancerogenus LMG 2693T (Z96078)

Klebsiella Michiganensis W14T (JQ070300)

Pantoearodasii LMG 26273T (JF295053)

Pantoea septica LMG 5345T (EU216734)

Klebsiella pneumoniae subsp. pneumoniae DSM 30104T (AJJI01000018)

Klebsiella quasi pneumoniae subsp. quasipneumoniae 01A030T (HG933296)

Erwinia billingiae Eb661T (AM055711)

Kosakonia cowanii CIP 107300T (AJ508303)

Escherichia hermannii GTC 347T (AB273738)

Enterobacter hormaechei ATCC 49162T (AFHR01000079)

Erwinia aphidicola DSM 19347T (AB273744)

Enterobacter aerogenes KCTC 2190T (CP002824)

Cedeceaneteri GTC1717T (AB086230)

Raoultella terrigena ATCC 33257T (Y17658)

Citrobacter youngae CECT 5335T (AJ564736)

Citrobacter braakii CDC 080-58T (AF025368)

Tatumella citrea LMG 22049T (EF688008)

Serratia ureilytica NiVa51T (AJ854062)

Serratia odorifera DSM 4582T (ADBY01000001)

Leminorella grimontii DSM 5078T (AJ233421)

89

77 24

12

96

99 29

39

44

45

67 45

24

17

24

49

38

99

99

0.005

112

The cost of control feed was @ Rs. 11.00/kg used during the experiment. The cost of the

probiotic feed was calculated by sum up the cost of yeast supplementation/day @ Rs. 1.25/g and

the cost of control feed (Rs.11.00). During the experimental period for 4 months, dairy heifers

fed on the control and probiotic feed consumed similar feed (446.40 and 457.20 kg/animal).

Total cost incurred on per animal feed during whole experimental period was Rs. 4464.0 and

5359.50 for control and probiotic groups, respectively. The total feed cost incurred per kg gain of

was similar (62.00 vs. 62.03 Pakistani Rupees) among both groups (probiotic and control group

respectively) (Table 4.10). On the other hand probiotic feed has did not show any detrimental

effect on growth rate, health status and of heifers which is comparable to heifers fed control feed.

Results revealed that heifers reared on probiotic feed are economically not efficient in terms of

saving feed cost as compared to Sahiwal heifers fed control feed. There is a need to isolate an

indigenous probiotic strain for our local breeds which can be economically more efficient than

other available probiotic strains.

Table 4.10: Economic efficiency of dairy heifers fed on probiotic versus non-probiotic feed

Items Feeding scheme

Control1 COM-Probiotic2

Average daily feed intake (kg/animal) 3.72 3.81

Total feed intake (kg) 446.40 457.20

Total feed cost, (Rs3.) (A) 4464.0 4572.0

Daily yeast dietary supplementation (g/animal) 0.00 5.25

Total yeast supplementation (g) 0.00 630.00

Total yeast cost (Rs.) (B) 0.00 787.50

Grand total (A+B) cost (Rs.) 4464.0 5359.50

Average total weight gain (kg) 72.00 86.40

Feed cost per kg gain (Rs2 ) 62.00 62.03

Cost per kg of control feed was Rs. 10.00 and probiotic feed (Rs. 10.00 control feed/kg and Rs. 1.25/g

yeast)1Control feed without yeast; 2Probiotic feed compose of control feed supplemented with 2.5×10 07 cfu/g

commercially available probiotic yeast (Yac-Sac1026) at the rate of 5 g per animal/day; ± Standard error of the

mean.3Rs. Pakistani Rupee

4.2 Phase II: Isolation and characterization of locally isolated yeast as a probiotic for

dairy cattle

4.2.1 Identification of isolated strains

Microbial isolates were identified as S. cerevisiae QAUSC03 and QAUSC05 according to their

morphological and biochemical characteristics (Table 4.11). S. cerevisiae QAUSC03 and

113

QAUSC05 displayed high morphological and biochemical resemblance to the members of S.

cerevisiae (Figures. 4.17, a, b).

4.2.2 Qualitative enzymatic assays of isolated yeast strains

4.2.2.1 Amylolytic activity

Yeast strains (QAUSC03 and QAUSC05) displayed significant amylatic activity. Formation of

clear zone indicated the cellulolytic activity in yeast strains.

4.2.2.2 Cellulolytic activity

Yeast strains (QAUSC03 and QAUSC05) displayed significant cellulolytic activity. Formation

of clear zone indicated the cellulolytic activity in yeast strains.

4.2.2.3 Proteolytic activity

Yeast strains (QAUSC03 and QAUSC05) indicated the proteolytic activity with the formation of

clear zones around the colonies.

4.2.3 Estimation of potential pro-biotic properties of LAB

4.2.3.1 Bile tolerance activity

Yeast strains also showed a high resistance pattern to bile i.e., bile tolerance rate of strain no. 3 at

30, 60, and 90 minutes were 0.848, 1.042, and 1.146 (mean values), respectively. Similarly, the

mean values of strain no.5 at the same time duration were 0.966, 1.07, and 1.176 respectively.

This data thus analyzed the increasing pattern of bile tolerance effect in yeast strains and the

strains were resistant to bile (Figure 4.18).

4.2.3.2 Cholesterol assimilation effect

The cholesterol lowering effect of yeast strains was determined and results showed that the

calculated values of yeast strains S. cerevisiae QAUSC03 was lower than that of S.cerevisiae

QAUSCSC05. The cholesterol level of yeast strains (S. cerevisiae QAUSC03 and S. cerevisiae

QAUSC05) was mentioned in (Figure 4.19).

4.2.3.3 Anti-pathogenic activity

The antimicrobial activity of yeast strains (S. cerevisiae QAUSC03 and S. cerevisiae QAUSC05)

were identified against four ATCC culture strains of Listeria monocytogenes (ATCC13932), E.

coli (ATCC8739), Staphylococcus aureus (ATCC6538) and Pseudomonas aeruginosa

(ATCC9027) Yeast strains (QAUSC03 (Strain #3) and QAUSC05 (Strain #5) showed anti-

pathogenic activity against the three ATCC strains (E. coli, Staphylococcus aureus,

114

Pseudomonas aeruginosa), but it was absent in case of Listeria monocytogenes (Figure 4.20).

Their diameters of zones of inhibition were mentioned in (Table 4.12)

Table 4.11: Morphological and biochemical characteristics of isolated yeast strains

Parameters Yeast strains

QAUSC03 QAUSC05

Morphological characteristics

Cell shape Ellipsoid to elongate Ellipsoid to elongate

Colony morphology Circular Circular

Colony surface Smooth/Slimy Smooth

Colony colour Off-white Pinkish

Colony elevation Pulvinate Umbonate

Colony margin Entire Filamentous

Biochemical characteristics

Gram stain reaction + +

Alcohol production + +

Glucose fermentation + +

Sucrose + +

Urease - -

Enzymatic activities

Amylolytic activity - -

Cellulolytic activity +++ ++

Proteolytic activity ++ +

115

Figure 4.17: Simple staining of isolated yeast strains (L) QAUSC05 and (R) QAUSC03

Figure 4.18: Tolerance rate of isolated yeasts strains in bile salt (% + SEM)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

S. cerevisae QAUSC03 S. cerevisae QAUSC05

30 min 60 min 90 min

Tole

ran

cera

te %

116

Figure

4.19: Cholesterol assimilation of isolated yeast strains (%+SEM)

Figure 4.20: Anti-pathogenic activity of isolated yeast strains QAUSC03 (Strain #3) and

QAUSC05 (Strain #5) against ATCC strains with their zones of inhibition

Table 4.12: The anti-pathogenic activity of isolated yeast strains against ATCC strains and

their inhibitory zones diameter (mm)

Strains

E. coli

(ATCC8739)

Pseudomonas

aeruginosa

(ATCC9027)

Staphylococcus

aureus

(ATCC6538)

Listeria

monocytogenes

(ATCC13932)

QAUSC03 10 18 14 Nil

QAUSC05 10 14 16 Nil

4.3 Phase III: Study of the comparative impact of Saccharomyces cerevisiae (Yea-

Sac1026) and locally isolated yeast on productive performance and health status in

lactating dairy cattle

14

14.5

15

15.5

16

16.5

17

S. cerevisae QAUSC03 S. cerevisae QAUSC05

Ch

ole

ster

ol

ass

imil

ati

on

(%

) C

hole

ster

ol

ass

imil

ati

on

(%

)

117

4.3.1 Effect of probiotic feed on the productive performance of lactating dairy cattle

Ingredients and chemical composition of the control feed is given in Table 4.13. The results of

average dry matter intake (DMI), average daily milk yield, and feed conversion ratio (FCR) of

lactating dairy cattle fed on different treatments are given in Table 4.14. Highest value of

average DMI (10.08 ±0.21kg/d) was observed in lactating dairy cattle fed on LAB probiotic feed

whereas lowest and almost equal values were observed in lactating dairy cattle fed on control

feed (10.01±0.18 kg/d) and dairy cattle fed on COM probiotic feed (10.04±0.19 kg/d). The

difference in the DMI was non-significant (P>0.05) among all the treatments. After measuring

the daily milk yield, we noted that highest average milk yield (6.12±0.70 lit/d) was observed in

lactating dairy cattle fed on LAB probiotic feed followed by lactating dairy cattle fed on COM

probiotic feed (5.80±0.80 lit/d) while lowest (5.40±0.65 lit/d) milk yield was observed in

lactating dairy cattle fed on control feed. There was a significant difference (P<0.05) in milk

production of lactating dairy cattle fed on LAB probiotic feed than fed on control and COM

probiotic feed. Our results showed that FCR was non-significantly improved (P˃0.05) in

lactating dairy cattle fed on LAB probiotic feed and was reflected in the significantly increased

(P˂0.05) milk yield in this group.

4.3.2 Impact of probiotic feed on the milk composition in lactating dairy cattle

The results of the milk protein (MP), fat, total solids (TS), solid not fat (SNT), total ash (TA) and

lactose % of milk of lactating dairy cattle fed on control, LAB probiotic and COM probiotic feed

are given in Table 4.15. After milk analysis, we noted maximum (3.71±0.05%) MP in lactating

dairy cattle fed on LAB probiotic feed and minimum (3.39

Table 4.13: Ingredient and chemical composition of the control, LAB probiotic and COM

probiotic feed

Items Feeding Scheme

Control1 LAB-Probiotic2 COM-Probiotic3

Chemical composition (% DM)

Crude protein 15.23 15.23 15.23

Neutral detergent fibre 27.88 27.88 27.88

Acid detergent fibre 18.04 18.04 18.04

118

Calcium 0.69 0.69 0.69

Total phosphorous 0.57 0.57 0.57

Feed ingredients (%)

Maize oil cake 17.00 17.00 17.00

Cottonseed meal 13.00 13.00 13.00

Sunflower meal 1.00 1.00 1.00

Canola meal 6.00 6.00 6.00

Rice polish 6.00 6.00 6.00

Wheat bran 7.00 7.00 7.00

Corn gluten feed 4.00 4.00 4.00

Corn grains 11.00 11.00 11.00

Vegetable oil 2.00 2.00 2.00

Wheat straw 24.00 24.00 24.00

Cane molasses 6.00 6.00 6.00

Urea 0.50 0.50 0.50

Di-calcium phosphate 1.00 1.00 1.00

Limestone power 0.50 0.50 0.50

Sodium chloride 0.50 0.50 0.50

Minerals premix 0.50 0.50 0.50

1Control feed without yeast;2Probiotic feed compose of control feed supplemented with 2.5×10 07cfu/g

commercially available probiotic yeast (Yac-Sac1026) at the rate of 10g /day/animal; 3Probiotic feed compose of

control feed supplemented with 3.13×1007cfu/g laboratory produced probiotic yeast (QAUSC03) at the rate of 8g

/day/animal. 3 in addition to control feed the silage and fodder were also given to each group. The chemical

composition (% DM basis) of fodder (DM=14.28; CP=12.29; NDF=54.23; ADF=37.39) and maize silage

(DM=34.35; CP=8.04; NDF=50.60; ADF=31.31)

±0.11%) MP in lactating dairy cattle fed on COM probiotic feed respectively as compared to

dairy cattle fed on control feed (3.50±0.12%).Although highest value of MP was present in dairy

cattle fed on LAB probiotic feed but statistically it was non-significant (P>0.05) among all three

treatments in our study.

Table 4.14: Dry matter intake and milk yield (Means ± SEM) in lactating dairy cattle fed

on control, LAB-probiotic and COM-probiotic feed

Parameters Feeding scheme

Control2 LAB-Probiotic3 COM-Probiotic4

119

Av. Dry matter intake (Kg/day)1 10.01± *0.18 10.08 ± 0.21 10.04± 0.19

Milk yield (Lit/day) 05.40b ± 0.65 06.12a ±0.70 05.80b ± 0.83

Feed Conversion Ratio (FCR) 1.87±0.12 1.64±0.11 1.73±0.04

a, b Values on the same row with different superscripts differ significantly (p<0.05); 1n=3 per treatment; 2Control feed

without yeast; 3LAB-Probiotic feed compose of control feed supplemented with 3.13×1007 cfu/g laboratory produces

probiotic yeast (QAUSC03) at the rate of 8g /day/animal 4 COM-Probiotic feed compose of control feed

supplemented with 2.5×1007 cfu/g commercially probiotic yeast (Yac-Sac1026) at the rate of 10g /day/animal;*±SEM

= standard error of the mean.

On the other hand highest (5.46±0.36 %) and significant (P<0.05) value of milk fat has been

recorded in dairy cattle fed on LAB probiotic feed as compared to dairy cattle fed on COM

probiotic and control feed. Dairy cattle fed on COM probiotic feed produced maximum

(15.22±0.23%) TS as compared to dairy cattle fed on LAB probiotic feed (14.51±0.59%) and

control feed (14.40±0.61%). Difference in milk TS values among all treatments was found to be

non-significant (P>0.05) in our study. When we calculated the SNF of the cattle milk, we note

that a non-significant (P>0.05) increased (10.44 ±0.50%) SNF in the dairy cattle fed on COM-

probiotic feed as compared to dairy cattle fed on LAB-probiotic feed (9.84±0.39%) and control

feed (9.05±0.74%). After calculation of milk lactose contents, we found that lactose content of

milk was higher (5.26±0.07 %) in dairy cattle fed on LAB-probiotic feed s as compared to dairy

cattle fed on COM-probiotic feed (5.23±0.08 %) and Control feed (5.09±0.15 %). Overall results

of cattle milk analysis showed that, there was no significant (P>0.05) difference in MP, TS, SNF

and lactose % among the treatments. On the other hand a significant (P<0.05) value of milk fat

has been recorded in dairy cattle fed on LAB probiotic feed as compared to other treatments.

4.3.3 Influences of probiotic on digestive performance in lactating dairy cattle

The results of the nutrient digestibility of dairy cattle fed on control, LAB- probiotic and COM-

probiotic feed are presented in Table 4.16. Results reveals that LAB-probiotic feed significantly

(P<0.05) NAD and ADF digestibility.

Table 4.15: Milk composition (Means ± SEM) of lactating dairy cattle fed on control, LAB-

probiotic and COM-probiotic feed

Parameters Feeding scheme

Control2 LAB-Probiotic3 COM-Probiotic 4

Protein, %1 3.39 ±* 0.11 3.71 ± 0.05 3.50 ± 0.12

120

Fat, % 4.60 ± 0.46b 5.46 ± 0.36a 4.78 ± 0.27b

Total solids, % 14.40 ± 0.61 14.51 ± 0.59 15.22 ± 0.23

Solid not Fat, % 9.84 ± 0.39 9.05 ± 0.74 10.44 ± 0.50

Lactose, % 5.09 ± 0.15 5.26 ± 0.07 5.23 ± 0.08

a, b Values on the same row with different superscripts differ significantly (p<0.05); 1n=3 per treatment; 2Control

feed without yeast; 3LAB-Probiotic feed compose of control feed supplemented with 3.13×1007 cfu/g laboratory

produces probiotic yeast (QAUSC03) at the rate of 8g /day/animal 4 COM-Probiotic feed compose of control feed

supplemented with 2.5×1007 cfu/g commercially probiotic yeast (Yac-Sac1026) at the rate of 10g /day/animal;*

Standard error of the mean.

On the other hand LAB and COM probiotic feeds significantly (P<0.05) dry matter (DM),

organic matter (OM), crude fibre (CF) digestibility as compared to dairy cattle fed on control

feed. In our study, significantly (P<0.05) increased DM digestibility was seen in dairy cattle fed

on LAB-probiotic feed (62.81±0.94 %) and fed on COM-probiotic feed (61.11±1.94 %) as

compared to dairy cattle fed on control feed (56.34±1.13%). Similarly, when we analyzed the

CPD, we noted that a significantly (P<0.05) increased CP digestibility was noted in dairy cattle

fed on LAB-probiotic (63.56±1.12%) and COM-probiotic feed (61.43±1.04%) as compared to

dairy cattle fed on control feed (57.50±1.19 %). On the other hand, we noted a maximum NDF

digestibility (61.55±0.58 %) in dairy cattle fed on LAB probiotic feed and minimum value

(58.33±0.68%) in was seen in the dairy cattle fed on control feed, whereas in dairy cattle fed on

COM probiotic feed intermediate value of NDF digestibility (60.89±0.55%) was recorded. The

difference in the NDF was significantly (P˂0.05) better in dairy cattle fed on LAB-probiotic feed

as compared to dairy cattle fed on control and COM-probiotic feed. In our study, significantly

(P˂0.05) increased value of ADF digestibility (55.27±0.77%) was seen in the LAB probiotic

group as compared to COM-probiotic (54.27±0.56%) and control (54.27±0.56 %) groups. The

overall results reveals that DM and CP digestibility significantly improved (P˂0.05) in LAB and

COM probiotic groups as compared to control group. On the other hand the NDF and ADF

digestibility was significantly (P<0.05) better in the LAB probiotic group as compared to other

groups.

Table 4.16: Nutrient digestibility (Means ± SEM) of lactating dairy cattle fed on control,

LAB-probiotic and COM-probiotic feed

Parameters Feeding scheme

Control2 LAB-Probiotic 3 COM-Probiotic4

121

Nutrient digestibility%1

Dry matter 56.34±*1.13 b 61.11±1.94 a 62.81±0.94 a

Crude protein 57.50±1.19 b 61.43±1.04 a 63.56±1.12 a

Neutral detergent fibre 58.33±0.68 b 61.55±0.58 b 60.89±0.55a

Acid detergent fibre 51.47±0.34 b 55.27±0.77 b 54.27±0.56a

a, b Values on the same row with different superscripts differ significantly (p<0.05); 1n=3 per treatment; 2Control feed

without yeast; 3LAB-Probiotic feed compose of control feed supplemented with 3.13×1007 cfu/g laboratory produces

probiotic yeast (QAUSC03) at the rate of 8g /day/animal 4 COM-Probiotic feed compose of control feed

supplemented with 2.5×1007 cfu/g commercially probiotic yeast (Yac-Sac1026) at the rate of 10g /day/animal;*±SEM

= standard error of the mean.

4.3.4 Influence of probiotics on hematological and biochemical parameters in dairy cattle

4.3.4.1 Hematological parameters:

The results of the red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), packed cell

volume (PCV), lymphocytes, monocytes and eosinophils were estimated before and after the

treatments are presented in the Table 4.17. All cattle have shown healthier hematological values

which is a sigh of good health. When we analysis the blood samples before treatment, we noted

that erytrocytic counts were highest (10.86±0.32 x106/µl) in dairy cattle fed on COM probiotic

feed as compared to dairy cattle fed on LAB probiotic (10.74±0.26 x106/µ) and control feed

(10.83±0.60 x106/µl). After treatment, we found that erytrocytic counts were highest (11.35±0.61

x106/µl) in dairy cattle fed on LAB probiotic feed as compared to dairy cattle fed on control feed

(11.11±0.58 x106/µl) and on LAB probiotic (10.82±0.35 x106/µ). The differences between the

treatments were non-significantly (P>0.05) before and after the treatment. On the other hand, we

noted that total leukocytic count were highest (10.65±0.19 x103/ml) in dairy cattle fed on the

LAB probiotic feed as compared to dairy cattle fed on control (10.30±0.41 x103/ml) and COM

probiotic feed (9.44±0.29 x103/ml) before treatment. After the treatment (day 60), total

leukocytic count were again highest (10.52± 0.20 x13/ml) in dairy cattle fed on LAB probiotic

feed as compared to dairy cattle fed on control and COM probiotic feed having 10.13±0.57

x103/ml and 9.78±0.39 x103/ml RBC counts, respectively. But, these differences in the RBC

counts were non-significantly (P>0.05) among the treatments in our study.

The concentration of PCV were highest (30.12±1.29%) in dairy cattle fed on the control feed as

compared to dairy cattle fed on probiotic feed supplemented with COM yeast (29.57±1.19%) and

supplemented with LAB yeast (29.65±1.66%) before the treatment. After treatment PCV were

122

again highest (32.32±0.47%) in dairy cattle fed on control feed as compared to dairy cattle fed on

LAB probiotic (31.61±1.21%) and COM probiotic feed (29.88±1.33 %). But, that difference was

non-significant (P>0.05) among groups before and after the treatments. On the other hand we

noted that at day 0, (before the treatment) hemoglobin level was non-significantly (P>0.05)

increased (10.64±0.21 %). in dairy cattle fed on control feed as compared to dairy cattle fed on

probiotic feed supplemented with COM yeast (10.55±0.55 %) probiotic feed supplemented with

LAB yeast (10.38±0.28%). On the other hand, at day 60 (after the treatment), haemoglobin level

was non-significantly (P>0.05) increased (10.83±0.24 %) in dairy cattle fed on feed

supplemented with LAB yeast supplemented as compared to dairy cattle fed on COM yeast

supplemented feed (10.60±0.75 %) and control feed with no yeast supplementation (10.41±0.18

%). When we measured the lymphocytes counts of dairy cattle, we noted that lymphocytes

counts were highest (53.15±2.63%) in dairy cattle fed on the control feed without yeast

supplementation as compared to dairy cattle fed on feed supplemented with COM yeast (52.15 ±

1.80%) and supplemented with LAB yeast (52.45± 1.59%) at day 0 (before the treatment). At

day 60 (after the treatment), lymphocytes counts was slightly highest (52.63±1.71%) in dairy

cattle fed on LAB probiotic feed.

Dairy cattle fed on COM yeast supplemented feed and control feed having similar (51.71±2.21

%) and (51.12±2.02%) lymphocytes counts at the end of treatment (day 60). But, that difference

was non-significant (P>0.05) among the treatments at day 0 and 60. At day 0 (before the

treatment), monocytes counts were highest (4.78±0.66%) in dairy cattle fed on the diet with LAB

yeast supplementation as compared to dairy cattle fed on feed supplemented with COM yeast

(4.01±10.79 %) on control feed (3.37±0.58%) supplemented with no yeast. At day 60 (after

treatment), monocytes were again highest (4.48±0.57%) in dairy cattle fed on LAB probiotic

feed as compared to dairy cattle fed on COM probiotic feed (4.34±0.59%) and dairy cattle fed on

control feed (3.67±0.35 %). But, that difference was non-significant (P>0.05) among the

treatments at day 0 and 60. At day 0 (before the treatment), eosinophils counts were highest

(4.41±0.19%) in dairy cattle fed on the diet with LAB yeast supplementation as compared to

dairy cattle fed on control feed (3.52± 0.20%) and dairy cattle fed on feed supplemented with

COM yeast (2.75± 0.32 %). At day 60 (after treatment), monocytes were again highest

(3.86±0.36%) in dairy cattle fed on LAB probiotic feed (4.34±0.59%) as compared to dairy cattle

123

fed on control feed (3.78±0.46 %) and dairy cattle fed on COM probiotic feed (2.64±0.29 %)

But, that difference was non-significant (P>0.05) among the treatments at day 0 and 60.

Table 4.17 Effect of dietary supplementation of yeast on haematological values (Means ±

SEM) in dairy cattle

Parameters Feeding scheme

Control2 LAB-Probiotic 3 COM-Probiotic 4

Erytrocytic count (x10 6/µl) 1

Before treatment5 10.83±*0.60 10.74±0.261 10.86±0.32

After treatment6 11.11±.0.58 11.35±0.617 10.82±0.35

Total leukocyte count (x103/ml)

Before treatment 10.30±0.41 10.65±0.19 9.44±0.29

After treatment 10.13±0.57 10.52±0.20 9.78±0.39

Packed cell volume (%)

Before treatment 30.12±1.29 29.65±1.66 29.57±1.19

After treatment 32.32±0.47 31.61±1.21 29.88±1.33

Haemoglobin (g/dl )

Before treatment 10.64±0.21 10.38±0.28 10.55±0.55

After treatment 10.41±0.18 10.83±0.24 10.60±0.75

Lymphocytes (%)

Before treatment 53.15±2.63 52.45±1.59 52.15±1.80

After treatment 51.12±2.02 52.63±1.71 51.71±2.21

Monocytes (%)

Before treatment 3.37±0.58 4.78±0.66 4.01±0.79

After treatment 3.67±0.35 4.48±0.57 4.34±0.59

Eosinophils (%)

Before treatment 3.52±0.20 4.41±10.19 2.75±0.32

After treatment 3.78±0.46 3.86±0.36 2.64±0.29

a, b Values on the same row with different superscripts differ significantly (p<0.05); 1n=3 per treatment; 2Control

feed without yeast; 3LAB-Probiotic feed compose of control feed supplemented with 3.13×1007 cfu/g laboratory

produces probiotic yeast (QAUSC03) at the rate of 8g /day/animal 4 COM-Probiotic feed compose of control feed

124

supplemented with 2.5×10 07 cfu/g commercially probiotic yeast (Yac-Sac1026) at the rate of 10g /day/animal; 5Before

treatment (day 0);6after treatment (day 120); *±SEM = standard error of the mean

4.3.4.2 Blood serum metabolites

The results of some selected serum metabolites like cholesterol, glucose and urea nitrogen of

dairy cattle fed on different treatments are shown in Table 4.18. All values were in the normal

range for dairy cattle, which is a sign of good health. The 40- 60 mg/100ml blood glucose level

is required to maintain the physiological process of body. When we analyzed the urea levels of

the dairy cattle, we noted that serum urea level was lowest (14.18±0.21 mg/100ml) in dairy cattle

fed on the diet with LAB yeast supplementation as compared to dairy cattle fed on diet

supplemented with COM yeast (15.54±0.32mg/100ml) and dairy cattle fed on control diet

supplemented with no yeast (14.55±0.57 mg/100ml) before the treatment. On the other hand,

after the treatment, urea level was again lower (12.31±0.22 mg/100ml) in dairy cattle fed on diet

supplemented with LAB yeast as compared to dairy cattle fed on diet supplemented with COM

yeast (13.68±0.90 mg/100ml) and dairy cattle fed on control diet (14.18±0.58 mg/100ml). These

differences in serum urea levels were non-significant (P>0.05) among groups before the

treatment. On the other hand, after the treatments, dairy cattle fed on LAB probiotic and COM

probiotic feed differed significantly (P<0.05) from dairy cattle fed on control feed.

The serum cholesterol level was lowest (108.37±1.02 6mg/100ml) in dairy cattle fed on the

control diet without yeast supplementation as compared to dairy cattle fed on feed supplemented

with COM yeast (111.56 ± 4.78 mg/100ml) and dairy cattle fed on feed supplemented with LAB

yeast (110.72± 2.25 mg/100ml) before treatment. After the treatment, cholesterol level was

lower (102.85±1.65 mg/100ml) in dairy cattle fed on feed supplemented with LAB yeast as

compared to dairy cattle fed on feed supplemented with COM yeast (105.69±2.69 mg/100ml)

and fed on control feed (109.71± 1.98 mg/100ml). These differences were non-significant

(P>0.05) among the groups before treatment. On the other hand after the treatments, dairy cattle

fed on LAB probiotic feed differed significantly (P<0.05) from dairy cattle fed on control feed

and COM probiotic feed.

125

Table: 4.18 Effect of dietary yeast supplementation on blood parameters (Means ± SEM) in

lactating dairy cattle

Parameters Feeding scheme

Control2 LAB-Probiotic 3 COM-Probiotic4

Urea (mg/100ml)1

Before treatment5 14.55±*0.57 14.18±0.21 15.54±0.32

After treatment6 14.18b±0.58 12.31a±0.22 13.68a±0.90

Cholesterol (mg/100ml)

Before treatment 108.37 ± 1.02 110.72± 2.25 111.56± 4.78

After treatment 109.71b ± 1.93 102.85a ± 1.65 105.69b±2.69

Glucose (mg/100ml)

Before treatment 75.70 ± 1.24 73.99 ± 2.51 75.08 ± 2.30

After treatment 73.84± 0.71 77.42 ± 1.28 78.97 ± 0.54

a, b Values on the same row with different superscripts differ significantly (p<0.05); 1n=3 per treatment; 2Control

feed without yeast; 3LAB-Probiotic feed compose of control feed supplemented with 3.13×1007 cfu/g laboratory

produces probiotic yeast (QAUSC03) at the rate of 8g /day/animal 4 COM-Probiotic feed compose of control feed

supplemented with 2.5×10 07 cfu/g commercially probiotic yeast (Yac-Sac1026) at the rate of 10g /day/animal; 5Before

treatment (day 0);6after treatment (day 120); *±SEM = standard error of the mean

The serum glucose concentrations before the treatment lower (73.99±2.51 mg/100ml) in dairy

cattle fed on diet supplemented with LAB yeast than dairy cattle fed control diet without any

yeast supplementation (75.70±1.24 mg/100ml) and COM yeast supplementation (75.08±2.30

mg/100ml). After the treatment, glucose concentration was increased (78.97±0.54 mg/100ml) in

the COM probiotic group as compared to LAB probiotic group (77.42±1.28 mg/100ml) and

control group (73.84±0.71 mg/100ml). No significant (P>0.05) effect of the probiotic feed on

glucose concentration was noted before and after the treatment in our study.

4.3.4.3 Blood serum macro-minerals

The results of the calcium (Ca), phosphorus (P), sodium (Na) and potassium (K) concentration in

blood serum of dairy cattle fed different feeding scheme are given in Table 4.19. When we

analyzed the results of Ca concentration of lactating dairy cattle, we noted that before the

treatment, serum Ca concentration was non-significantly (P<0.05) highest (9.38±0.03 mg/100ml)

in LAB probiotic group than COM probiotic (8.71±0.08 mg/100ml) and control (8.68± 0.05

126

mg/100ml) groups. On the other hand, after the treatment, Ca concentration was again non-

significantly (P<0.05) higher (9.28 ±0.03 mg/dl) in LAB probiotic group than COM probiotic

(8.86 ±0.09 mg/100ml) and control (8.65±0.03 mg/100ml) group. After determination of

Phosphorus (P) concentration in blood serum, we found that before treatment, P level was lower

(7.58±0.04 mg/100ml) in the control group as compared to LAB probiotic group (7.89±0.02

mg/100ml) and COM probiotic group (7.85±0.04 mg/100ml) and after the treatment the P

concentration was again lower (7.81±0.04 mg/100ml) in the control group as compared to LAB

probiotic group (8.00±0.02 mg/100ml) and COM probiotic group (7.87±0.02 mg/100ml).

Differences in values among groups were found to be non-significant (P>0.05) before and after

the treatment in our study.

After estimation of serum K concentration, we observed that before the treatment, almost similar

K concentration in control (5.60±0.13 meq/l) LAB probiotic (5.58±0.27 meq/l) and COM

probiotic (5.64±0.15 meq/l) groups. After the treatment, the K concentration was lower

(5.25±0.10 meq/l) in the LAB probiotic group than control group (5.65.±0.10 meq/l) and COM

probiotic group (5.32±0.05 meq/l). Difference in K concentration values among groups were

found to be non-significant (P>0.05) before and after the treatment in present study. When we

determine the Na concentration in the serum of dairy cattle, we noted that, before the treatment,

concentration of Na in blood serum was higher (126.53±2.40 meq/l) in dairy cattle fed on LAB

probiotic feed as compared to dairy cattle fed on COM probiotic feed (125.86±0.19 meq/l) and

control feed (123.86±0.90 meq/l). In the same manners, after the treatment, the Na concentration

was again highest in the dairy cattle fed on LAB probiotic (130.64±1.59 meq/l) than control

group (127.10.±1.03 meq/l) and COM probiotic group (127.17±1.49 meq/l). Difference in Na

concentration values among all treatments was found to be non-significant in present study.

Overall result showed that Ca, P, K and Na concentration were unaffected by probiotic in the

blood of dairy cattle

Table 4.19: Effect of dietary yeast supplementation on blood serum metabolites (Means ±

SEM) in lactating dairy cattle

Parameters Feeding regime

Control2 LAB-Probiotic3 COM-Probiotic 4

Calcium (mg/dl)1

127

Before treatment5 8.68±*0.05 9.38±0.03 8.71±0.08

After treatment6 8.65±0.03 9.28±0.03 8.86±0.09

Phosphorus (mg/dl)

Before treatment 7.58± 0.04 7.89 ± 0.02 7.85± 0.04

After treatment 7.81 ± 0.04 8.00 ± 0.02 7.87 ± 0.02

Potassium (meq/l)

Before treatment 5.60±0.132 5.58±0.27 5.64±0.15

After treatment 5.65± 0.107 5.25 ± 0.10 5.32 ± 0.05

Sodium (meq/l)

Before treatment 123.86 ± 0.90 126.53 ± 2.40 125.86 ± 1.75

After treatment 127.10 ± 1.03 130.64 ± 1.59 127.17 ± 1.49

a, b Values on the same row with different superscripts differ significantly (p<0.05); 1n=3 per treatment; 2Control

feed without yeast; 3LAB-Probiotic feed compose of control feed supplemented with 3.13×1007 cfu/g laboratory

produces probiotic yeast (QAUSC03) at the rate of 8g /day/animal 4 COM-Probiotic feed compose of control feed

supplemented with 2.5×10 07 cfu/g commercially probiotic yeast (Yac-Sac1026) at the rate of 10g /day/animal; 5Before

treatment (day 0);6after treatment (day 120); *±SEM = standard error of the mean

4.3.5 Effect of probiotic on microbial growth trends in ruminal gut samples of dairy cattle

The average total aerobic bacterial count, Enterococcus, Lactococcus, Bacillus and Coliform

species of ruminal gut samples of lactating dairy cattle fed on probiotic feed as determined at 0,

30 and 60 days of the experiment are given in Table 4.20. After analysis of samples we noted

that, at day 0, total aerobic count was higher (7.98±1.23 CFU/g) in lactating dairy cattle fed on

control diet as compared to dairy cattle fed on diet supplemented with LAB yeast

(6.12±1.11CFU/g) and dairy cattle fed on diet supplemented with COM yeast (6.98±1.98

CFU/g). But that difference was non-significantly among the groups at day 0. At day 30 of the

experiment total aerobic counts were non-significantly (P>0.05) increased (7.64±0.87 CFU/g) in

dairy cattle fed on diet supplemented with LAB yeast than dairy heifers fed on control diet

without any yeast supplementation (6.90±0.89 CFU/g) in dairy cattle fed on diet supplemented

with COM yeast (5.44±1.09 CFU/g). At day 60 of the experiment the total aerobic counts were

significantly (P˂0.05) increased (8.12±0.54 CFU/g) in the dairy cattle fed on control diet without

yeast supplementation than the dairy cattle fed on diet supplemented with LAB-yeast

(7.97±0.81CFU/g) and supplemented with COM yeast (7.71±0.38 CFU/g). The changes in the

128

number of total aerobic counts, as affected by the different probiotic yeast are depicted in Fig.

4.21.

At day 0, the numbers of total Lactococcus counts were non- significantly (P>0.05) higher

(4.01±0.34 CFU/g) in lactating dairy cattle fed on control diet as compared to dairy cattle fed on

diet supplemented with LAB yeast (3.54±0.49 CFU/g) and dairy cattle fed on diet supplemented

with COM yeast (3.87±0.98 CFU/g). At day 30 of the experiment total aerobic counts were

slightly increased (4.17±0.39 CFU/g) in dairy cattle fed on diet supplemented with COM yeast

than dairy cattle fed on diet supplemented with LAB yeast (3.98±0.50 CFU/g) and dairy heifers

fed on control diet without any yeast supplementation (4.12±0.58 CFU/g). At day 60 of the

experiment the numbers of total Lactococcus species were significantly (P˂0.05) increased

(4.09±0.71 CFU/g) in the dairy cattle fed LAB probiotic feed than fed on control feed(3.49±0.41

CFU/g)and non-significantly (P>0.05) increased in the dairy cattle supplemented with COM

yeast fed diet (3.90±0.34 CFU/g). The changes in the number of Lactococcus counts, as affected

by the different feed supplements are depicted in Fig. 4.22.

After estimation of Enterococcus counts in ruminal gut samples, we observed that at day 0,

Enterococcus counts in COM-probiotic animals were higher (5.61±0.45 CFU/g) as compared to

LAB probiotic (4.81±0.65 CFU/g) and control (4.19±0.25 CFU/g) groups. On the other hand, at

day 30, the Enterococcus counts were lower (4.06±0.29 CFU/g) in the COM probiotic group

than control group (4.92±0.43 CFU/g) and LAB probiotic group (4.96±0.55 CFU/g). At day 60,

the Enterococcus counts COM-probiotic animals were higher (5.61±0.45 CFU/g) as compared to

LAB probiotic (3.44±0.68 CFU/g) and control (3.94±0.49 CFU/g) groups. Difference in K

concentration values among groups were found to be non-significant (P>0.05) before and after

the treatment in present study. The changes in the number of Enterococcus counts, as affected by

the different probiotic yeast are depicted in Fig. 4.23.

After estimation of Bacillus counts in ruminal gut samples, we observed that at day 0, Bacillus

counts in COM-probiotic animals were higher (5.17±0.78 CFU/g) as compared to LAB probiotic

(5.12±0.34 CFU/g) and control (4.99±0.10 CFU/g) groups. On the other hand, at day 30, the

Bacillus counts were lower (4.34±0.45 CFU/g) in the LAB probiotic group than control group

(4.76±0.98 CFU/g) and COM probiotic group (4.78±0.34 CFU/g). At day 60, the Bacillus counts

129

COM-probiotic animals were higher (5.12±0.12 cfu/g) as compared to LAB probiotic (4.12±0.78

CFU/g) and control (4.23±0.87 CFU/g) groups. Difference in K concentration values among

groups were found to be non-significant (P>0.05) before and after the treatment in present study.

The changes in the number of Enterococcus counts, as affected by the different probiotic yeast

are depicted in Fig. 4.24.

Table 4.20: Total bacteria counts (CFU/g ± SD) in ruminal gut of lactating dairy cattle fed

on control and probiotic feed

Days of age Feeding scheme

Control2 LAB-Probiotic3 COM-Probiotic4

Total aerobic count1

0 7.98±1.23 6.12±1.11 6.98±1.98

30 6.90±0.89 7.64±0.87 5.44±1.09

60 8.12±0.54a 7.97±0.81b 7.71±0.38b

Lactococcus species

0 4.01±0.34 3.54±0.49 3.87±0.98

30 4.12±0.58 3.98±0.50 4.17±0.39

60 3.49±0.41b 4.09±0.71a 3.90±0.34a

Enterococcus species

0 4.19±0.25 4.81±0.65 5.61±0.45

30 4.92±0.43 4.96±0.55 4.06±0.29

60 3.94±0.49 3.44±0.89 5.15±0.67

Bacillus species

0 4.99±0.10 5.12±0.34 5.17±0.78

130

30 4.76±0.98 4.34±0.87 4.78±0.34

60 4.23±0.41 4.12±0.78 5.12±0.12

a, b Values on the same row with different superscripts differ significantly (p<0.05); 1n=3 per treatment; 2Control

feed without yeast; 3LAB-Probiotic feed compose of control feed supplemented with 3.13×1007 CFU/g laboratory

produces probiotic yeast (QAUSC03) at the rate of 8g /day/animal 4 COM-Probiotic feed compose of control feed

supplemented with 2.5×10 07 CFU /g commercially probiotic yeast (Yac-Sac1026) at the rate of 10g

/day/animal;*±SEM = standard error of the mean

4.3.6 Isolation, characterization and identification of Lactococcus, Enterococcus, Bacillus

species from ruminal gut of dairy cattle

MRS agar supported as a specific media for growth of lactic acid bacteria (LAB) and TSA agar

for growth of Bacillus species. Their further properties were examined on the basis of

morphology, gram staining and biochemical tests (Table 4.21).

4.3.6.1 Colony and cell morphology:

Results of gram stating showed that all isolated strains on MRS were gram positive either rod or

cocci. They appeared as long chain, single rod, cocci and in the form of rod clusters under

microscopy on MRS media. The forms of the colonies of gram positive rod isolates were mostly

round and irregular with smooth, rough shiny surface. Most of the colonies were creamish and

creamish white colors with entire and convex margin. The results of elevation characteristics of

gram positive rod isolated colonies showed that mostly these are raised or slightly raised and

most of the colonies had opaque in its opacity characteristics point of view.

On the other hand the forms of the colonies of gram positive cocci isolates were mostly circular.

The surface characteristics of the gram positive cocci were mostly found to be smooth, shiny

with creamish white colors with entire margin. The results of elevation characteristics of gram

positive rod isolated colonies showed that mostly these are raised and most of the colonies had

opaque and moist in its opacity characteristics point of view.

All isolated grown on TSA were appeared as gram negative rod, which full fill one of the

property of Bacillus. They appeared as single rod and in the form of rod clusters under

microscopy on macconkey media (Table 4.21). After isolation of pure colonies, we noted that

most of the bacterial isolated on the TSA agar form the circular with smooth surface colonies.

131

These colonies were mostly looked as translucent opacity, white yellowish colour, with convex

elevation and entire margin

4.3.6.2 Biochemical characterization

The pure isolates of gram positive cocci have particular characteristics i.e. they showed that

catalase, indole, urease, TSI and oxidase tests were negative while citrate and MR were positive.

These strains were non motile and also there was no gas (H2S) production. On the basis of data

obtained from physical and biochemical test results we concluded that these bacterial isolates on

the MRS agar were identified as Lactococcus or Enterococcus species.

Figure 4.21: Monthly variations in total aerobic count of lactating dairy cattle fed on

diet supplemented with a) no yeast (control, ♦), laboratory yeast (LAB-Y, ■) or

commercial yeast (COM-Y, ▲)

5

5.5

6

6.5

7

7.5

8

8.5

9

0 30 60

CFU

/g

Days

Control

LAB-Y

COM-Y

132

Figure 4.22: Monthly variations in total Lactococcus species count of lactating dairy

cattle fed on diet supplemented with a) no yeast (control, ♦), laboratory yeast (LAB-

Y, ■) or commercial yeast (COM-Y, ▲)

Figure 4.23: Monthly variations in total Enterococcus species count of lactating

dairy cattle fed on diet supplemented with a) no yeast (control, ♦), laboratory yeast

(LAB-Y, ■) or commercial yeast (COM-Y, ▲)

3.35

3.45

3.55

3.65

3.75

3.85

3.95

4.05

4.15

4.25

0 30 60

CFU

/g

Days

Control

LAB-Y

COM-Y

3.2

3.7

4.2

4.7

5.2

5.7

6.2

0 30 60

CFU

/g

Days

Control

LAB-Y

COM-Y

133

Figure 4.24: Monthly variations in total Bacillus species count of lactating dairy

cattle fed on diet supplemented with a) no yeast (control, ♦), laboratory yeast (LAB-

Y, ■) or commercial yeast (COM-Y, ▲)

On the basis of a distinguish test (grow at different temperature) between Lactococcus and

Enterococcus species we concluded that most of the gram positive cocci bacterial isolated grown

at MRS were grown on the temperature (15 to 35 oC) and these are belong to Enterococcus

species rest of the strains were belong to Lactococcus species at different time interval. The pure

isolates of gram positive rods have particular characteristics i.e. they showed that catalase, citrate

and oxidase positive and urease, indole, TSI and MR tests were negative. These strains were

motile and there was no gas (H2S) production. On the basis of data obtained from physical and

biochemical test results we concluded that these bacterial isolates on the TSA agar were

identified as Bacillus species. Biochemical test of the gram negative rods bacterial isolates on

macconkey agar showed that these are catalase, oxidase and MR positive while TSI, urease,

indole and citrate negative. These strains were motile and also there was no gas (H2S)

production. According to the experimental data, the isolated bacteria were identified as

Enterobacter species (Table 4.21).

4.3.7 Partial 16S rRNA gene sequencing

Seven strains were identified on molecular level by blasting the amplified nucleotide sequences

using a BLAST tool at National Centre for Biotechnology Information (NCBI) website

http://www.ncbi.nlm.nih.gov/. And, these strains QAUBL11, QAULG02, QAULG04,

4

4.2

4.4

4.6

4.8

5

5.2

0 30 60

CFU

/g

Days

Control

LAB-Y

COM-Y

134

QAUSG07, QAUSG08, and QAUSK01 were identified as Bacillus, Lactococcus, Enterococcus,

Enterococcus, and Enterococcus species on the basis of genotypic analysis. These 16S rRNA

sequences were submitted to NCBI GenBank under the accession numbers KP256019,

KP256011, KP256013, KP256015, KP256016 and KP256018 assigned to strains QAUBL11,

QAULG02, QAULG04, QAUSG07, QAUSG08, and QAUSK01 respectively (Table 4.22).

4.3.8 Phylogenetic analyses of bacterial isolates

The blastn search revealed that Bacillus QAUBL11 (KP25619) had the highest sequence

similarity with the Bacillus Licheniformis ATCC 14580T (AE017333). The blastn search

revealed that Bacterium QAULG02 (KP25611) had the highest sequence similarity Lactococcus

garvieae ATCC 49156T (AP009332).

Table 4.21: Morphological, biochemical identification of selavtive bacterial isolates on MRS and TSA

Characteristics Selected bacterial isolates

QAUBL11 QAULG02 QAULG04 QAUSG07 QAUSG08 QAUSK01

Morphology characterization

Gram staining +ve +ve +ve +ve +ve +ve

Shape Rods Cocci Cocci Cocci Cocci Cocci

Form Circular Circular Circular Round Circular Round

Surface Smooth Smooth Shiny Smooth/shiny Shiny Shiny

Colour Creamish Creamish Creamish

white

Creamish

white

Creamish

white

Creamish white

Margin Undulate Convex Entire Entire Entire Entire

Elevation Raised Raised Slightly raised Raised Slightly raised Raised

Opacity Opaque Opaque Moist Opaque Moist Opaque

Biochemical characterization

Catalase +ve -ve -ve -ve -ve -ve

Oxidase +ve -ve -ve -ve -ve -ve

Indole -ve -ve -ve -ve -ve -ve

Citrate +ve +ve +ve +ve +ve +ve

Methyl red -ve +ve +ve +ve +ve +ve

TSI -ve -ve -ve -ve -ve -ve

Urease -ve -ve -ve -ve -ve -ve

Grow at NT +ve +ve +ve +ve +ve

15°C NT +ve +ve +ve +ve +ve

25°C NT +ve +ve +ve +ve +ve

135

35°C NT +ve +ve +ve +ve +ve

SMT M NM NM NM NM NM

H2S No No No No No No

Identified

Microorganisms

Bacillus Lactococcus Lactococcus Enterococcus Enterococcus Enterococcus

*NT=Not Tested

Table 4.22: Identification of isolated strains based on 16SrRNA gene sequence and their

accession numbers published in DNA database.

Strain ID Strain

name/ genus

Length

of 16S

r RNA

(ntds)

Accession

number of

16S rRNA

gene

Closely related Validly

published species

Similarity

% of 16S r

RNA gene

sequencing

QAUBL11 Bacillus 885 KP256019

Bacillus licheniformis

ATCC 14580T

(AE017333 )

99.66

QAULG02 Bacterium 871 KP256011

Lactococcus garvieae

ATCC 49156T

(AP009332)

99.89

QAULL04 Lactococcus 913 KP256013

Lactococcus lactis subsp.

ATCC Cremoris NCDO

607T (AB100802) 100

QAUSG07 Enterococcus 963 KP256015 Enterococcus mundtii

CECT972T (AJ420806) 99.9

QAUSG08 Enterococcus 925 KP256016 Enterococcus hirae ATCC

9790 T (CP003504) 100

QAUSK01 Enterococcus 903 KP256018

Enterococcus faecium

ATCC CGMCC 1.2136T

(AJKH01000109)

100

The blastn search revealed that Lactococcus QAUBLL04 (KP25613) had the highest sequence

similarity with Lactococcus lactis subsp. ATCC Cremoris NCDO 607T (AB100802). The blastn

search revealed that Enterococcus QAUSG07 (KP25615) had the highest sequence similarity

with Enterococcus mundtii CECT972T (AJ420806). The blastn search revealed that

Enterococcus QAULG08 (KP25016) had the highest sequence similarity Enterococcus hirae

ATCC 9790T (CP003504). The blastn search revealed that Enterococcus QAULSK01

(KP25018) had the highest sequence similarity Enterococcus faecium ATCC CGMCC 1.2136T

(AJKH0100010) (Figure 4.13, 4.15, 4.16).

136

4.3.9 Economic efficiency of dairy cattle fed on probiotic feed

A cost per kg of concentrate was Rs. 11.50; silage was Rs. 8 and fodder was Rs. 6 used during

the experiment. The cost of the commercial available probiotic feed was calculated by sum up

the cost of yeast supplementation/day @ Rs. 1.25/g and the cost of control feed @ 25.50/kg

(concentrate Rs. 11.50; silage Rs. 8 and fodder Rs. 6) and the cost of the laboratory produced

probiotic feed was calculated by sum up the cost of yeast supplementation/day @ Rs. 1.65/g and

the cost of control feed @ 25.50/kg (concentrate Rs. 11.50; silage Rs. 8 and fodder Rs. 6).

During the experimental period for 2 months, dairy cattle fed on the control , COM-P and LAB-P

feed consumed similar feed (29.33; 29.67 and 29.5 kg/animal/d). Total cost incurred on per

animal feed per day during whole experimental period was Rs. 208.48; 223.72 and 222.00 for

control, COM-P and LAB-P group, respectively. The value of milk throughout the experimental

period was 223.52; 265.88 ad 242.00 for control, COM-P and LAB-P group respectively. On the

other hand probiotic feed has did not show any detrimental effect on health status of dairy cattle

which is comparable to dairy cattle fed control feed. Results revealed that cows fed on LAB-P

feed are economically efficient in terms of saving feed cost as compared to fed control and

COM-P feed. The net profit/Lit milk was also better in LAB-P feed fed group compared to

others group (Table 4.23).

137

Figure: 4.16: Phylogenetic tree of the Bacillus (KP25619) based on 16S rRNA gene

sequence.

Table 4.23: Economics of milk production of dairy cattle fed on probiotic yeast

Bacillus tequilensis KCTC 13622T (AYTO01000043)

Bacillus mojavensis RO-H-1T (JH600280)

Bacillus subtilis subsp. inaquosorum KCTC 13429T (AMXN01000021)

Bacillus subtilis subsp. spizizenii NRRL B-23049T (CP002905)

Bacillus subtilis subsp. subtilis NCIB 3610T (ABQL01000001)

Bacillus siamensis KCTC 13613T (AJVF01000043)

Bacillus amyloliquefaciens subsp. amyloliquefaciens DSM 7T (FN597644)

Bacillus amyloliquefaciens subsp. plantarum FZB42T (CP000560)

Bacillus atrophaeus JCM 9070T (AB021181)

Bacillus QAUBL11T (KP256019)

Bacillus sonorensis NBRC 101234T (AYTN01000016)

Bacillus licheniformis ATCC 14580T (AE017333)

Bacillus aerius 24KT (AJ831843)

Bacillus oryzaecorticis R1T (KF548480)

57

45

57

61

88

77

98

54

65

70

0

0.005

138

Parameters Feeding scheme

Cont1 LAB-P2 COM-P3

Feed intake (Kg/d) 3.00 3.00 3.00

Silage intake (Kg/d) 8.00 8.00 8.00

Fooder intake (Kg/d) 18.33 18.67 18.50

Value of feed @4Rs.11.50/ Kg 34.50 34.50 34.50

Value of silage @ Rs.8/ Kg 64.00 64.00 64.00

Value of fodder @ Rs.6/ Kg 109.98 112.02 111.00

Yeast intake (g/d/animal) 0.00 8.00 10.00

Cost of yeast @ Rs.1.25/g COM ; 1.65/g LAB 0.00 13.20 12.50

Total cost (feed+silage+fodder+yeast)/d/animal 208.48 223.72 222.00

Daily milk production (Lit) 05.40 06.12 05.80

Value of milk @ Rs.80/Lit 432.00 489.60 464.00

Profit Rs./d / animal 223.52 265.88 242.00

Economic efficiency(feed cost/kg milk prod ) 38.61 36.56 38.28

Net profit /Lit milk Rs.(net Income/ milk prod/d ) 41.39 43.44 41.72

1Control feed without yeast; 2LAB-Probiotic feed compose of control feed supplemented with 3.13×1007 cfu/g

laboratory produces probiotic yeast (QAUSC03) at the rate of 8g /day/animal 43COM-Probiotic feed compose of

control feed supplemented with 2.5×10 07 cfu/g commercially probiotic yeast (Yac-Sac1026) at the rate of 10g

/day/animal; 4Rs= Pakistani Rupee

Chapter-5

DISCUSSION

139

5.1 Phase 1: Determination of the impact of Saccharomyces cerevisiae (Yea-Sac1026) on

the performance of dairy heifers

5.1.1 Effect of probiotic on the growth performance of dairy heifers

The growth of the animal is usually directly related to the amount of feed intake and nutrients

utilization. Over the entire trial of 4 months (heifer of 6 to 10 months age), dry matter intake

(DMI) did not differ between dairy heifers fed on control and probiotic supplemented feed

whereas, average daily gain (ADG) was 6.2% more in heifers fed on diet supplemented with

probiotic yeast. The improved growth was might be due to increased nutrient digestion and

absorption in our study. YC also has a positive effect on the FCR in current study. The improved

(P˃0.05) FCR reflected in the increased (P˂0.05) average daily gain in dairy heifers fed on

probiotic yeast. Overall results have showed that the probiotic yeast have an ability to improve

growth efficiency of dairy heifers. Probiotic yeast has a single cell protein, which is effective for

improved growth and well-being of ruminants by stimulating rumen acetogens (Halasz and

Lasztity, 1990; Klein et al., 1995; Kurtzman et al., 2011).

The present observations are in agreement with the findings of Kumar et al. (2011), who noted

significantly (P<0.05) higher (549.91 vs 462.13 g/d) ADG and improved FCR in claves fed on

probiotic yeast at the rate of 0.25g/d. That improved growth rate might be due to the higher DMI

(5.24 vs 4.60 kg/d) in claves fed on probiotic supplemented feed than control feed. On the other

hand, Lascano et al. (2009) reported that inclusion of YC tends to require significantly (P<0.05)

less DMI to maintain T-ADG as compared to dairy heifers fed on control diet. Recently, a study

published by Terre et al. (2015) demonstrated similar findings in post weaning calves and noted

(P=0.053) rapid growth (0.82 vs. 0.68 kg/d) fed on diet supplemented with yeast culture. So, we

can inferred here, that improve growth efficiency might be due to the improved DMI (2.34 vs

2.10 kg/d) in yeast fed group.

Lesmeister and Heinrichs (2004) noted significantly (P<0.05) improved (15.6 %) ADG in the

claves fed on starter ration supplemented with 2 % YC than claves fed on diet without YC. The

increased ADG may be an effect of residual gut fill and/or increased bone growth in claves. An

improved ADG in animals is reported due to increased flow of microbial protein leaving the

rumen and an improve amino acid supply in the small intestine (Rao et al., 2003). Our result

showed that skeletal measurements were not changed (P˃0.05) by addition of yeast culture in

140

dairy heifers. Zanton and Heinrichs (2007) and Lascano et al. (2009) found no change in the

structural measurements in the dairy heifers fed diet with YC supplementation. In contrast to our

results, Lesmeister and Heinrichs (2004) reported daily hip width change was also significantly

(P<0.05) improved in the claves fed on 2% YC supplemented diet compared to control diet. In

contrast to this study YC showed no effect on the growth performance in dairy animals (Kung et

al., 1997; Pinos-Rodríguez et al., 2008; Kellems et al., 1990; Quigley et al., 1992). That

inconsistency in YC effects on the animals might be due to the difference in the genotype,

management conditions and the diet.

5.1.2 Effect of probiotic on digestion performance of dairy heifers

5.1.2.1 Dry Matter Digestibility (DMD)

Results of present study indicated that apparent total tract DM digestibility (DMD) during

growing phase was higher (60.25 vs. 55.52 %) in Sahiwal dairy heifers fed on probiotic feed than

control feed. Similar results were reported by Lascano et al. (2009), who observed that YC

supplementation significantly (P<0.05) improved the DMD (74.97 vs. 73.65%) in dairy cattle

heifers fed diets with high concentrate along with Saccharomyces cerevisiae at the rate of 1g/kg

as fed bases cattle heifers. Di Francia et al. (2008) reported significantly (P<0.05) improved

DMD in the claves fed on the diet supplemented with fungus (S. cervisiae and Aspergillus

oryzae) as compared to control group. In another report, Lascano et al. (2012) from USA, found

increased DMD in dairy heifers fed on a diet containing the high starch or low starch, with a

sequence of YC doses (0, 10, 30 and 50 g daily). The lack of impact of YC on the DMD has been

seen in many animals (Harris et al., 1992; Doreau and Jouany, 1998; Cooke et al., 2010; Tripathi

and Karim, 2010). The differences from the present study could be due to the difference in

degree of nutritional scheme.

5.1.2.2 Organic Matter Digestibility (OMD)

In the present study, apparent total tract OM digestibility was significantly (p<0.05) higher

(6.91%) in animals fed on probiotic supplemented feed than control feed. Similarly, Di Francia

et al. (2008) found significantly (P<0.05) higher OMD (83 vs 74%) in yeast fed claves. In

another study by Lascano et al. (2012) from USA, dairy heifers fed on YC showed increased

(P<0.01) OMD than control group. On the other hand, some researchers showed that yeast

addition has no impact on the OMD in ruminants (El-Ghani, 2004; Tripathi and Karim, 2010).

141

5.1.2.3 Crude Protein Digestibility (CPD)

Yeast supplementation has a significant (P<0.05) impact on the CPD in our present study.

Results indicated that CPD was 5.16% higher in the dairy heifers fed on probiotic feed as

compared to the dairy heifers fed on control feed. In another report, Di Francia et al. (2008)

determine the effect of dietary supplementation of fungal strains in the claves and noted that

significantly (P<0.05) higher CPD in the claves fed on the diet supplemented S. cerevisiae and

Aspergillus oryzae. Proteins in the feed are quickly breakdown into peptides, amino acids and

NH3 by rumen protozoa and fungi (Wallace et al., 1997). Some NH3 is converted into microbial

protein (MP), and some ammonia is used by the animal in the form of urea. An important portion

of rumen ammonia is excreted and represents an indication of nitrogen loss of the dietary

nitrogen (N) (20 to 25%). Amino acids and peptides issued from dietary proteins cannot be

directly slip in the animal intestine, if the diet has highly nutritious value (Fonty and

Chaucheyras-Durand, 2006). The same effect on ammonia concentration was seen with daily YC

supplementation in adult ruminants (Kumar et al., 1994). The in vitro findings explain that

probiotic yeast could alter the growth and activities of protein degrading bacteria, which

ultimately enhanced the protein digestion in rumen gut (Beev et al., 2007). The mode of action

of YC can be explained by a competition between live S. cerevisiae cells and different bacterial

species for energy utilization (Chaucheyras-Durand et al., 2005). In a study on fourteen dairy

cows’ field trials an addition of probiotic yeast in the diet noted that the soluble nitrogen of the

diet was a key factor to drive the production parameters to the probiotics-yeast (Sniffen et al.,

2004). However, with other yeast strain no significant effect was observed on the concentration

and fraction of microbial nitrogen in dairy cattle (Putnum et al., 1997; Chaucheyras-Durand et

al., 2010). In contrats, some scientists (Arambel and Kent, 1990; Andrighetto et al., 1993)

observed no effect of yeast culture on CPD with dairy animals.

5.1.2.4 Neutral Detergent Fibre Digestibility (NDFD)

In the current study, apparent total tract NDFD was significantly (P˂0.05) influenced by YC

addition. That improved nutrient digestibility might be due to increased cellulose degrading

microbial biomass population inside rumen. NDFD were also affected (P˂0.05) by YC

supplementation in other studies. The increased digestibility can be due to stable rumen pH and

removal of oxygen from the rumen in the YC supplemented group. That stable rumen pH

provides better environment for growth of rumen microbes, especially cellulose degrading

142

bacteria and fungi. At the same time, the anaerobic condition inside rumen also helped in better

growth of fibrolytic microbial biomass. Consequently, these microbial species helped in better

fibre digestion. The stable pH also enhanced microbial protein synthesis in the rumen. Our data

support the findings Lascano et al. (2012) who reported increased NDFD in the dairy heifers fed

on diet supplemented with YC. Similarly, (Fadel, 2007) noted that YC in the hay diet improved

NDFD in goats. Lascano et al. (2009) and Carro et al. (1992) demonstrated that SC significantly

improved NDF degradation by ruminal microbiota in the dairy heifers fed on the high energy

diet. They suggested that the YC had a diet-dependent effect. Newbold et al. (1990) reported that

Aspergillus oryzae fermentation extract and SC stimulated fiber degradation by their action on

ruminal bacterial population. Wiedmeier et al. (1987) noted that supplementation of YC

significantly improved the hemicellulose digestibility and highlighted that the improvement

might be due to the increased cellulolytic bacterial population after YC supplementation. On the

other hand, Wiedmeier et al. (1987); Wohlt et al. (1991) and Moallem et al. (2009) noted no

significant effect of YC on the NDF digestibility.

5.1.2.5 Acid Detergent Fibre Digestibility (ADFD)

SC has a significant (P˂0.05) effect of the ADFD in current study. Our results indicated that

dairy heifers fed on probiotic feed have 4.27% higher ADFD as compared to the dairy heifers fed

on the control feed. In a report, Lascano et al. (2012), found increased ADFD in the dairy heifers

fed on diet supplemented with increasing YC dose. On the other hand, in contrast of our results,

Lascano et al. (2009) reported that SC has no significant (P>0.05) affect on ADFD in the dairy

heifers fed on the high or low energy diet. It has been noted that nutrient digestibility was not

affected when YC supplemented in high energy and corn gelatinized (Arambel and Kent, 1990;

El-Ghani, 2004; Cooke et al., 2007). The fermented YC provides wide range of enzymes,

vitamins, organic acids and amino acids that stimulate the growth and function of lactic acid

bacteria (Callaway and Martin, 1997). These growth factors may stimulate synthesis and

secretion of IGF I from liver on absorption from gastrointestinal tract. The increased digestibility

of nutrient may be due to improved activities and function of microbial population increased

ruminal anaerobes and cellulolytic bacteria (Jouany, 2001; Chevaux and Mazzia-Fabre, 2007).

5.1.3 Influences of probiotic on the hematological and biochemical parameters of dairy

heifers

143

5.1.3.1 Hematological parameters

In our current study, all hematological values of the dairy heifers before and after the treatment

were within the normal physiological range, which is a sign of good health.

5.1.3.1.1 Erytrocytic count

Supplementation with YC significantly (p<0.05) increased the erytrocytic count end of

treatment. These results are concurrent with results observed in an early study with dairy claves

(Dobicki et al., 2005). He reported that erythrocytes counts increased by 0.80-1.24 thousand, in

calves fed on diet supplemented with YC as compared to the claves fed on control diet. In the

same manner, Milewski and Sobiech (2009) reported significantly (P≤0.01) increased (10.25 vs

8.88 1012/L) erytrocytic count in ewes fed on the diet supplemented with YC.

5.1.3.1.2 Leukocytic Count

Leukocytic counts were significantly (p<0.05) increased in our study. Similarly,Milewski and

Sobiech (2009) noted that YC has associated with significantly (P ≤ 0.05) increased erythrocytes

counts (10.25 vs 8.88 1012/L) during year I of the experiment. Similar results were reported by

Dobicki et al. (2005) who noted that leukocytic counts were increased (1.71-2.54 thousand) in

dairy claves fed on the diet supplemented with YC.

5.1.3.1.3 Packed Cell Volume (PCV)

PCV levels range from 26.45 to 30.84 % in heifers fed on feed supplemented with probiotic

yeast and 22.13 to 28.99 % in heifers fed on control feed without any supplementation during 6

to 10 months of age. PCV levels were not influenced by probiotic yeast in our study. In contrast

to our study, Milewski and Sobiech (2009) noted that YC had associated with significantly (P ≤

0.05) increased erythrocytes counts in ewes during yeast I and II of their experiment.

5.1.3.1.4 Haemoglobin

During the present trial, the haemoglobin (Hb) levels were significantly (p<0.05) higher in dairy

heifers fed on probiotic feed. Agazzi et al. (2014) reported that haemoglobin levels were affected

by probiotic addition in growing claves. In the same manner, Milewski and Sobiech (2009) noted

that YC had significantly (P≤0.01) higher haemoglobin concentration in the supplemented ewes

as compared to non-supplemented ewes (101.70 vs 114.00 g/L). Dobicki et al. (2005) reported

an improved Hb by 0.28-0.78 mmo|/l in claves supplemented with SC as compared to non-

supplemented claves.

5.1.3.2 Lymphocytes

144

Lymphocytes level‘s range was from 52.81 to 59.71% in dairy heifers fed on probiotic feed and

49.70 to 55.34 % in heifers fed on control feed. Lymphocytes levels were not influenced by

probiotic yeast in our study. In contrast to our study, Milewski and Sobiech (2009) reported that

YC supplementation had associated with significantly (P≤0.05) improved erythrocytes counts in

ewes during yeast I and II of the experiment.

5.1.3.2.1 Eosinophils

Eosinophils level‘s range was from 6.35 to 6.94 % in heifers fed on feed supplemented with

probiotic yeast and 4.85 to 5.34 % in heifers fed on control feed without any supplementation

during 6 to 10 months of age. Probiotic yeast has a significant (P<0.05) effect on the eosinophils

levels in our study. In the same manner, Agazzi et al. (2014) reported that probiotic had

associated with significantly (P≤0.05) increased eosinophils levels in claves.

5.1.3.2.2 Monocytes

Monocytes level‘s range was from 4.70 to 7.34% in dairy heifers fed on probiotic feed and 4.37

to 6.23% in heifers fed on control feed. Monocytes levels were not influenced by yeast

supplementation in our study.

5.1.3.3 Blood biochemical parameters

Blood serum constituents reflect the metabolic status of the animal and are frequently used to

assess the reproductive and productive performance of the farm animals. Heifers are the farm

animals, in which period, great care has to be exercised for the earlier reproduction and for better

production thereafter. Under filed conditions, crossbred heifers are usually underfed, which

results in deficiencies of certain nutrients and ultimately reflected in the levels of certain

biochemical constituents.

5.1.3.3.1 Cholesterol

Cholesterol concentration was significantly (P<0.05) influenced by probiotic yeast in our study.

Average cholesterol concentration with references to age of the dairy heifers was significantly

(P<0.05) higher probably to meet growth and other physiological requirement in both groups.

YC supplementation changes the propionate, butyrate and valerate acid in the animals. The

increase in these acids is capable of reducing the synthesis of triglyceride and cholesterol in the

liver cells and may change the lipid profile in blood of the animals (Miller-Webster et al., 2002;

Marden et al., 2008). Nicolosi et al. (1999), studied the effect of YC on the blood parameters of

the ruminants and he noted that the yeast cell is a rich source of β-glucans which reduce the total

145

cholesterol of serum of the animals. Kowalik et al. (2013) reported significantly (P<0.05)

decreased cholesterol in the serum. Contrary to the findings of the present study, some studies

have showed that YC had no influence on triglyceride and total cholesterol concentration of

serum (Galıp, 2006; Masek et al., 2008; Campanile et al., 2008).

5.1.3.3.2 Glucose

Serum glucose was significantly (P<0.05) affected by YC supplementation. Glucose

concentration was 3.29% higher in the dairy heifers fed on probiotic feed. The increased level of

glucose was might be due to increased nutrient utilization that resulted in an increased dry matter

and organic matter digestibility in present study. Hossain et al. (2012) reported that serum

glucose was statistically (P<0.05) higher in YC supplemented claves as compared to non-

supplemented claves. Lascano et al. (2012) studies the effect of YC on the blood metabolites in

dairy heifers. Diet was composed of high and low starch with YC dose (0, 10, 30 and 50 g/day).

He noted that the increasing YC dose tended to increase (P ≤0.10) glucose concentration. He

correlated that increased level of glucose in dairy heifers with increased nutrient utilization that

reflects the diet dependency of the mode of action of yeast culture. SC is a source of glucose

tolerance factor and has stimulated glucose utilization by cell in vitro. The age of the animals has

exhibited highly significant variation in serum glucose level in the cattle heifers.

5.1.3.3.3 Blood Urea Nitrogen

Changes in the serum urea N concentration in cattle heifers fed on probiotic feed and heifers fed

on control feed were significant (P<0.05) at the end of trial. YC supplementation significantly

(P<0.05) lower the serum urea concentration in current study. That lower concentration of urea N

in the dairy heifers fed on probiotic yeast might be due to increased protein digestibility. Because

the intake of N is lower in heifers fed on probiotic feed compared to heifers fed on control feed

that is why concentration of the urea N were lower in the heifer fed probiotic feed compared to

heifers fed on control feed. This observation is in agreement with Dolezal et al. (2011), who

reported that YC significantly lower the urea N in the dairy animals. Contrary to the findings of

our study, some researchers (Putnam et al., 1997; Bagheri et al., 2009; Nikkhah et al., 2004)

reported that urea level was unaffected by YC supplementation.

5.1.3.4 Blood macro-minerals

146

Macro-minerals concentration in the blood serum of dairy heifers was not affected (P>0.05) by

probiotic yeast in our study. Minerals are important to maintain growth requirements of the dairy

animals and play an important role in the action of hormonal and enzymes (NRC, 2001).

5.1.3.4.1 Calcium

Reproductive function are directly or indirectly related to Ca levels (Bansal et al., 1978)and

involved in steroid biosynthesise in ovaries (Shemesh et al., 1984). In adult cattle the normal

range of Ca is 9.00 to 10.00 mg/dl and in dairy heifer is 10.70 mg/dl (NRC, 2001; Jabbar, 2004).

In the current study, serum Ca concentration range from 8.60 to 8.72 mg/dl in heifers fed on

probiotic feed and 8.36 to 8.64 mg/dl in control heifers during 6 to 10 months of age. Piva et al.

(1993) demonstrated that silage, hay and concentrate based diet supplemented with YC had no

effect on the Ca concentration in dairy animals. Contrary to the finding of the present study,

Dolezal et al. (2011) reported that calcium concentration was higher (P<0.05) in dairy animal fed

on probiotic yeast compared to the dairy animal fed on control diet.

5.1.3.4.2 Phosphorus

Serum P is very important macro-mineral in cellular metabolism of all animals. The central

compound in energy metabolism adenosine triphosphtae is phosphorylated compound. In

growing cattle the normal range of Ca is 6.00 to 8.00 mg/dl (NRC, 2001). Serum P concentration

range from 6.43 to 6.57 mg/dl in heifers fed on feed supplemented with probiotic yeast and 6.33

to 86.61 mg/dl in heifers fed on control feed without any supplementation during 6 to 10 months

of age. Similar finding were reported by Bansal et al. (1978) and Dolezal et al. (2011) who

demonstrated that probiotic yeast had no effect on P concentration in blood serum in dairy

animals.

5.1.3.4.3 Potassium

Serum K concentration range was from 5.43 to 5.44 meq/l in dairy heifers fed on probiotic feed

and 5.36 to 5.49 meq/l in heifers fed on control feed during 6 to 10 months of age. Similar results

were reported by Piva et al. (1993) who found that silage, hay and concentrate based diet

supplemented with YC had no effect on the K concentration in dairy animals.

5.1.3.4.4 Sodium

Serum Na concentration range from 120.92 to 129.12 meq/l in dairy heifers fed on probiotic feed

and 119.93 to 127.67 meq/l in heifers fed on control feed during 6 to 10 months of age. Similar

results were reported by Piva et al. (1993) who reported that silage, hay and concentrate based

147

diet supplemented with YC had no effect on the Na concentration in dairy animals. In contrary

Milewski and Sobiech (2009) reported that YC significantly (P≤0.05) higher the concentrations

of Na+ions compared with non- supplemented ewes.

5.1.4 Impact of probiotic on the changes in the ruminal gut microbial flora of dairy

heifers

Different types of microbiota have been studied in the ruminal gut of the growing animals. Such

as, Lactobacillus, Lactococcus, Bacillus, Enterococcus, Clostridia, Coliform, E. coli,

Salmonella, Campylobacter, Bifidobacteria (Kawakami et al., 2010; Ayad et al., 2013;

Bayathouhsar et al., 2013; Agazzi et al., 2014). In current study, coliform and Lactobacillus

species have been affected by yeast supplementation in dairy heifers during 6 to 10 months of

age. There was a variation in levels of coliform and Lactobacillus in ruminal gut at 0 day in both

supplemented and control groups. Because heifers were obtained from different locations,

variation may be due to feeding behavior and different environmental condition. Environmental

circumstances and host factors usually influence the multifarious composition of the gastro-

intestinal micro-flora (Vlkova et al., 2006).Young animals have unbalanced state of intestinal

micro-biota (Lukas et al., 2007) whereas healthy (adult) animals enclose stable state of intestinal

micro-flora that set aside appropriate growth conditions. Less variation was observed for heifers

fed on uniform feeding scheme and gave same management. There were significant (P˂0.05)

effects of yeast supplementation on the numbers of Lactobacillus in heifers’ ruminal gut samples

in 60, 90 and 120 days. Yeast supplementation significance (P˂0.05) increased the numbers of

fecal Lactobacillus with passage of time. Considering the overall growth impact from day 0 up

till day 120, the CFU/g values demonstrated an increased Lactobacillus growth trend in both

groups. The increased growth was higher (P˂0.05) in YC fed group than control group suggested

that the yeast supplementation has a capability to improve gut microbial flora and reduce the

diarrhea. Same trend was reported by Kawakami et al. (2010). The coliform counts (Escherichia,

Enterobacter and Citrobacter) were similar in both groups during 0, 60, 90 and 120 days of the

experiment.

In our study, coliform count was significantly (P=0.001) higher in control group than

supplemented group at day 30. That might be due to the digestive problem in control group

148

during these days. Two heifers of control group got diarrhea which might have led to

significance difference (P=0.001) in both group at 30 days of experiment. With passage of time

the diarrheal condition were eliminated and the number of fecal coliform bacteria were decreased

in supplemented group than control group. Our results indicated that the number of coliform in

supplemented and control group were higher on day 0 compared with day 120. The number of

Lactobacillus species in feces is a widely used index for estimation of balance of intestinal flora ,

however higher numbers of coliform indicates dysbiosis (Fuller, 1989). SC supplementation has

positive effect on Lactobacillus population with respect to age. Our results are in agreement with

finding many researchers who reported that fecal population of Lactobacillus increases with

probiotic supplementation (Agazzi et al., 2014; Bayatkouhsar et al., 2013). Many studies

indicated that coliform range could increase when some disorder occur (Ellinger et al., 1980;

Jenny et al., 1991). However, the result of previous reports are controversial about the effect of

probiotics on the number of fecal coliform species of ruminants. Some researchers reported that

feeding probiotics significantly decreased the counts of fecal coliform species, as reported

byAgarwal et al. (2002) but others for instant Ellinger et al. (1980) reported no effect of

probiotic on coliform counts. During these studies diarrheal condition were not observed

throughout the experimental period and may account for the absence of significant changes in

coliform numbers. The number of coliform is higher than that of Lactobacilli in the animal’s

suffering from diarrhea, but lower in healthy animals, which suggested that coliform numbers L:

C ratio could be used as indicator for estimation of intestinal microbial flora associated with

diarrhea. The L: C ratio hypothesis was also confirmed in these studies. The improved gut health

condition in our study was confirmed by lower incidence of diarrhea in supplemented group as

compared to control group. Similar result were noted by Kawakami et al. (2010), who observed

that feeding yeast and lactic acid bacteria lower the incidence of diarrhea by improving fecal

flora.

In our present study, total aerobic counts were highly affected by probiotic supplementation with

passage of time. The total aerobic counts were similar and no (P˃0.05) difference was seen in

both groups during 0 and 30 days of the experiment. Meanwhile, total counts was significantly

(P˂0.05) higher in control group than supplemented group at day 60, 90 and 120. That might be

due to the increased Lactobacillus species and decreased coliform species during 60, 19 and 120

149

days of the experiment. Lactobacillus species might increase the total aerobic counts in the yeast

fed group. From our present study, microbial growth dynamics were analyzed in dairy heifer’s

fecal samples with respect to age. With respect to age (7, 8, 9 and 10 month), considerably there

was slight differentiation in total bacterial count in heifers fed on probiotic feed as compared to

heifers fed on control feed. This indicates that in dairy animals, age puts significant influence on

total bacterial count. The only factor that might contributes to this variation in bacterial count is

the age of animals, since other factors including diet and health conditions were similar in both

cases. This may be possibly because of the reason that cattle have wide contact with other

animals rather than heifers and heifers have unbalanced state of intestinal flora, too as mentioned

above by (Lukas et al., 2007).

Lactococcus is a genus of lactic acid bacteria and mostly it is non-pathogenic bacteria. In current

study, gram positive and calatase negative cocci were Lactococcus species were non-

significantly (P>0.05) increased in the non-yeast fed group than yeast supplemented group at 0

and 30 days of the experiment. On the other hand these bacterial species were significantly

(P˂0.05) affected by the yeast supplementation at 60 90 and 120 days of the experiment. In

present study, the numbers of Entrococcus species in the dairy heifers were not affected by

probiotic yeast at 0, 30 and 60 days of the experiment. Enterococcus species were significantly

(P˂0.05) increased in the non-yeast fed group than yeast supplemented group a 90 and 120 days.

The pathogenic Enterococcus species decreased in supplemented group than control group

suggested that the yeast supplementation has a capability reduce the infection in the dairy heifers

during their growing phase. Similar finding has been reported by (Jatkauskas and Vrotniakiene,

2010) who reported that probiotic has an ability to reduce the Enterococcus species in GIT. He

also noted that the in the control animals the Enterococcus species numbers were 4.0×103 cfu/g

and in the probiotic treated animals that number was 1.4×108 CFU/g. Likewise, Rada et al.

(2006) reported that probiotic fed group decreased the supplemented group numbers of

Enterococcus species. It was found that in growing animals fed on probiotic supplementation the

numbers of the Enterococcus species were 6.79 CFU/g and in the animals fed only control diet

the Enterococcus species were 7.60 CFU/g. Moreover, the age of cattle also affects the

Enterococcal colonization (Devriese et al., 1992). Results from this study indicate that probiotic

may have an ability to reduce the pathogenic bacteria (i.e. E.coli) and increase the beneficial

150

bacteria (Lactobacillus sp). The probiotic may have beneficial effects on calf gut health by

improving its daily weight gain, feed intake and FCR.

5.1.5 Impact of probiotics on economic efficiency of dairy heifers.

Sahiwal cattle heifers fed on the feed supplemented with probiotic yeast may encourage reducing

the feeding costs. Sahiwal cattle heifers fed on the probiotic feed almost similar (62 vs. 62.03

Pakistan Rupees) feed cost for one kg live weight than heifers fed on control feed over the entire

feeding period. Results showed that dairy cattle heifers fed probiotic feed are economically not

efficient in term of feed cost by consumption of similar DM. Although the weight gain was

better in the probiotic group over control group, but the cost of the probiotic yeast has increased

the total feed cost in that group as compared to the control feed in which no yeast is included.

Feed cost has an important cost factor having direct effect on growth performance of the heifers.

When heifers gained more weight, then relative intake portion used for maintenances should be

decreased, which showed that an improved growth was effective in feed cost saving. In present

study, both groups consumed similar DMI. The probiotic yeast has no potential benefits to lower

the feed cost per unit weight gain in growing heifers in present study. In contracts to our study,

Magalhaes et al. (2008) reported that feeding YC improved profit in dairy claves by 48 dollar

per calf by decreasing morbidity and mortality rates. The difference might be due to the breed

and feeding types. On the other hand the positive effect of the probiotic yeast is that it did not

show any detrimental effect on health status and growth performance of the dairy heifers. The

economic advantage of microbial feed additives depends on the price of yield culture and the

lactation stage of the animal yeast strain age, diet, breed and geographical location of the

animal (Yalçın et al., 2011, Vibhute et al., 2011). In this context, there is an urgent need for

developing clear information on probiotic utilization and its efficiency in local dairy animals.

Such information can definitely help local farmers, scientific, and government official to

formulate suitable plan to enhance the dairy sector in Pakistan. Little work has been

conducted in Pakistan relating to the use of probiotics to enhance the performance

parameters of dairy animal. From this line of motivation, in the second phase of study, we

developed probiotic yeast for our local breed.

151

5.2 Phase II: Isolation and characterization of locally isolated yeast as a probiotic for

dairy cattle

5.2.1 Probiotic characterization of locally isolated yeasts strains

The two fecal isolates were identified as Saccharomyces cerevisiae (QAUSC03 and QAUSC05)

according to their morphological and biochemical characterization. Yeast used as probiotic

addition is commonly delivered in a food system and begin their journey to the lower intestinal

tract via the mouth. Therefore, these useful organisms should be resistant to the lysozyme

enzymes in the oral cavity and α–amylase, lysozyme, and tryps in enzymes in the intestinal tract

(Shukla et al., 2010). In our study, both yeast strains displayed notable cellulolytic and

proteolytic activity but no amylolytic activity. The enzymatic activity of probiotic strains

increases feed utilization efficiency. The yeast isolates exhibit the specific probiotic properties.

Particularly, the strain QAUSC03 appears to be the best probiotic candidate in terms of all three

criteria. Bile tolerance activity, cholesterol assimilation, and production of antimicrobial

compounds are the phenomenal characteristics of yeast strains. Tolerance to bile salt is one of

important characteristics of microbiota for survival and metabolic activity in the gastrointestinal

tract. Probiotic strains must be resistant to bile salts and survive at low pH as stomach maintains

the pH from 2.5-3.3 to restrict bacterial growth (Holzapfel et al., 1998). In this study, yeast

strains (QAUSC03 and QAUSC05) showed better resistance at pH 3 and (1%) bile salt

concentration. Their maximum survival rate was calculated at 90 minutes. Yeast strains showed

a higher resistance to bile, the growth pattern of QAUSC03 strain was not affected in the

presence of bile salt.

Similarly, the growth of QAUSC05 strain was also remained undisturbed in the presence of bile

salt. The number of surviving strains implied that both strains had a relatively high tolerance to

bile salts. These results fully support the bile tolerance activity and show that these tested strains

have the capability of hydrolyzing bile salts by the activity of bile salt hydrolase (BSH) enzyme

(Hofmann and Mysels, 1992). BSH activity has been reported in many species, including

Lactobacillus, Peptostreptococcus, Bifidobacterium, Clostridium and Bacteroide associated with

GIT (Gilliland and Speck, 1977).

152

This enhances the survival rate of probiotics strains and provides resistance against bile salts.

Similar results were reported by (Rajkowska and Kunicka-Styczynska, 2010) in which they

investigated the probiotic ability of yeasts at pH 1.5 and their survival was 85.3 to 92.1%. Chen

et al. (2010) also observed the bile tolerance activity among different yeast strains. He reported

that, yeast strains P. guilliermondii HY18, Trichosporon gracile HJ2, Geotrichum sp. BY2 and

Yarrowiali polytica HY4 showed competent bile tolerance activity at 0.5% bile salt

concentration. The difference in the level of bile tolerance of strains in the study might be due to

differences in their ability to grow and colonize the GIT. (Dunne et al., 2001; Hosono, 1999)

reported the same reasons of the bile tolerance of the yeast strains. Similarly, cholesterol effect

was also observed on the isolated yeast strains (QAUSC03 and QAUSC05). Among these

strains, the yeast strain QAUSC03 showed better cholesterol assimilation than QAUSC05 yeast

strain. This reduction in cholesterol level is probably assumed due to the deconjugation of bile

acids in the liver. (Liong and Shah, 2005) reported that using probiotics strains is one of the most

effective ways to control cholesterol level. Formerly, (Razin et al., 1980) suggested that S.

boulardii, P. kudriavzevii and S. cerevisiae have been estimated as possible probiotics for their

ability to lower the cholesterol from past few years. It is reported that the cholesterol reduction is

a consequence of deconjugation of bile salts (Fukushima and Nakano, 1996). This leads to an

increased excretion of bile acids.

Cholesterol is used as a precursor for the synthesis of new bile acids due to which serum

cholesterol reduces (Tamai et al., 1996; Driessen and de Boer, 1989). An in-vitro study

demonstrated the cholesterol lowering effect by L. fermentum probiotics strain (Pereira et al.,

2003). Klaver and Vandermeer, (1993) in their study also illustrated that some Lactobacillus sp.,

undergo cholesterol assimilation by in-vitro deconjugation of bile salts. Hence, bile tolerance and

cholesterol lowering effect are correlated and is implied as primary factor for the selection of

probiotics strains. The cholesterol lowering capacity of S. cerevisiae strains were determined as

16.1 and 15.5 percent for QAUSC03 and QAUSC05 respectively. The degradation of cholesterol

in rumen leads to lower blood cholesterol in cattle; which ultimately decreases cholesterol

secretion in milk. The antimicrobial activity of yeast strains was estimated against four reference

pathogens strains commonly associated with rumen GIT disorders. It was observed that both

strains showed anti-pathogenic activity, against Escherichia coli (ATCC8739), Staphylococcus

153

aureus (ATCC6538) and Pseudomonas aeruginosa (ATCC9027) but it was absent in case of

Listeria which is probably due to the production of some antimicrobial compounds. In the same

manner Roostita et al. (2011) reported that, fruit yoghurt isolate shown the best antimicrobial

activity. They reported that against Pseudomonas aeruginosa, they showed 35 mm clear zone,

against Staphylococcus aureus they showed 8 mm clear zone and against E. coli they showed 10

mm clear zone. It is observed by many scientists that different organic acid and protein present in

the yeast. These organic acids and proteins were known as antimicrobial compounds that inhibit

the bacteria and mold growth (Roostita, 2004). Yeast has an ability to produced sulphur dioxide

that can inhibit the growth of spoilage lactic acid bacteria (Fleet, 2003).

Phase III: Study the comparative impact of Saccharomyces cerevisiae (Yea-Sac1026) and

locally isolated yeast on productive performance and health status in lactating dairy cattle

5.2.2 Impact of probiotic on productive performance of lactating dairy cattle

Over the entire feeding period, 11.7% and 5.22 % higher milk production was found in cattle fed

on LAB-P feed as compared to dairy cattle fed on control feed and COM-P feed respectively.

The highest milk production in the LAB-P group is might be due to cellulolytic activity of the

locally isolated probiotic yeast (QAUSC03). This activity significantly (P<0.05) improved the

NDF and ADF digestibility and enhanced the overall cellulose digestion rate in our study. That

improved cellulose digestibility enhanced the supply of energy and absorbed nutrients for milk

production. On the other hand, DMI and FCR did not differ between dairy cattle fed on probiotic

and non-probiotic feed in current study. Overall results have showed that the probiotic yeast have

the ability to improve production efficiency of lactating dairy cattle. No previous experiments

(that we know of) have evaluated the effects of laboratory produced YC on milk production of

dairy cattle of Sahiwal and Sahiwal Jersey breed. Higher milk yield without increased DMI due

to commercially available probiotic YC supplementation have previously been noted in dairy

animals.

Our results are in the argument with the recent work by Salvati et al. (2015) who did an

experiment to determine the impact of dietary supplementation of yeast on the lactating dairy

cattle. They used a diet which was composited of silage (44.8%), raw soybean (4.1%), soybean

meal (16.5%), finely ground corn (20.7%) and citrus pulp (11.9%) supplemented with live cells

(25 × 1010 CFU) and dead yeast cells (5 × 1010 CFU). He reported that milk yield was

154

significantly (P<0.05) increased (26.7 vs. 25.4 kg/d) in lactating dairy cattle fed on yeast cells.

They concluded that improved milk yield might be due to the effect of the yeast cell on the

regulation of body homeothermia in the lactating dairy cattle. In the same manner, Hossain et al.

(2012) observed the effect of YC on the productive performance of dairy cattle. The control

animals were fed on control diet consisted of concentrate mixture (3 kg) and roughage (6 kg).

The treatment group fed on control diet along with15 g/cow/day. They concluded that the live

yeast cell consists of enzymes, vitamin, amino acid, oligosaccharides and organic acids which

enhanced the milk yield by 4.1 % in lactating dairy cattle. Similarly, Yalcın et al. (2011) carried

out a study investigate the impact of YC on production performance in dairy cows. Cows were

divided into 2 groups: control and treated group. The control group cow received control diet

which contained; concentrate (10 kg/d), maize silage (26 kg/day), hay (5kg/d) and straw (2 kg/d)

throughout the study period and the test group cow were received control diet plus 50 g YC.

They reported that although both groups consumed similar (20.4 vs. 20.8) DMI however, the

milk yield significantly (P<0.05) increased (24.97 vs. 23.44 kg/d) almost 6.3 % more milk

production in lactating dairy Holstein cattle fed with yeast culture than the control group. They

reported that the YC provides growth metabolites, which stimulates fibrolytic bacteria growth

and enhances the fiber digestion in side rumen. Campanile et al. (2008) performed an experiment

to investigate the impact of YC on the milk yield of buffalo cows. They reported that the DMI

was similar (16.5 vs. 16.5 kg/day) among both groups while the milk yield significantly (P<0.01)

increased (7.9 vs. 7.4 kg/day) in the dairy cow fed on probiotic supplemented feed. They

concluded that improved lactation performance might be due to the increased organic matter

digestibility that provided high energy availability for the milk synthesis.

Kellems et al. (1990) reported that YC had the greatest positive effect on milk yield over control

cows during early lactation stage. They concluded that increased milk flow could be due to

additional minerals provided by the YC as compared to the control groups. Similarly, in another

report it was noted that DMI was not influenced by YC but the production performance showed

that the addition of SC increased the net energy of diet thus leading to increased milk flow in

dairy cow. (Robinson and Garrett, 1999). Similarly, Longuski et al. (2009) did an experiment to

evaluate the impact of YC on the production performance of multiparous Holstein cows. They

offered mix of dry ground corn and soybean meal supplemented with YC to animals. They

155

reported that DMI was not affected by YC but the milk yield was significantly (P<0.05)

increased. In the same manner, Bruno et al. (2009) fed 30 g YC incorporated into the TMR.

They observed that DMI was similar between both groups, but cows fed on diet supplemented

with yeast produce more (1.2 kg/d) milk compared to the cow fed on diet without yeast addition.

Some studied reported that milk flow was increased due to increased DMI. Like, Dawson et al.

(1990) reported that YC had positive impact on the milk yield in the cow when they fed on

60:40% concentrate to forage ratio diet. They noted that milk yield was 1.4 liter/day increased

due to probiotic yeast in dairy cattle. That improvement production performance might be due to

the increased (1.2kg/d) DMI in their study. In an experiment, 2.5% more DMI was consume by

dairy cattle fed on diet supplemented with yeast culture than control group. The daily milk

production of yeast fed group was greater (4.1%) than the milk yield of the dairy cattle fed on the

control diet without any yeast addition. It was concluded that YC improves the rumen

environment, which enhanced the DMI and in consequence improved the productivity and

efficiency of dairy animals (Moallem et al., 2009). Shaver and Garrett (1997) conducted a filed

study with 11 commercial dairy farms and observed that milk production was increased in 8 of

the 11 farm. On the other hand, some researchers showed that YC has no effect on the milk

yield. For instance, (Soder and Holden, 1999; Schingoethe et al., 2004; Bagheri et al., 2009)

reported no effect of yeast supplementation in lactating animals.

In general, improve production performance in response to YC supplementation was

accompanied by greater DMI (El-Ghani, 2004; Wohlt et al., 1998; Stella et al., 2007), whereas

no response in milk yield was accompanied by no effect on DMI (Soder and Holden, 1999;

Schingoethe et al., 2004). In our study DMI and FCR were not affected by yeast addition. Our

results are in agreement with some findings (Arambel and Kent, 1990; Moallem et al., 2009;

Bagheri et al., 2009) who reported that YC did not affect the DMI and FCR. On the other hand,

improved DMI (Stella et al., 2007) and FCR (Erasmus et al., 1992; Schingoethe et al., 2004)

have been observed in dairy animals fed on YC. The variation of the DMI and milk yield

results is might be due to confounding effects of ration formulation, stage of lactation, season

and breed difference.

5.2.3 Effect of probiotic on milk composition of lactating dairy cattle

5.2.3.1 Milk Fat

156

In this study, milk fat was significantly (P<0.05) higher by laboratory produced yeast

supplementation. The average fat percentage was 15.75 % and 12.45% higher in dairy cattle fed

on the LAB-P feed than in the dairy cattle fed on control and COM-P feed respectively. That

increased milk fat in the LAB yeast fed group might be due to the positive effect of the YC on

the milk yield and fiber fermentation in our study. Yeast cells contain the nutrients which

enhance the cellulolytic bacteria population (especially, Ruminococcous albus and

Ruminococccus falivifavis) that ultimately degrades the cellulose and enhances the acetic acid

production inside rumen. In agreement with our results some researcher also reported the

positive effect of probiotic on milk fat. For instance, Meller et al. (2014) reported that Jersey

cows received YC 50 g (1.94 × 1010 CFU/g) and 100 g (4.35 × 1010 CFU/g) and noted that yeast

supplemented cows consuming more (P=0.01) DMI (0.7 kg/d), which increased (P<0.05) milk

fat by 0.067 kg/d than cow fed on no YC. Similar results were reported by Moallem et al. (2009)

who noted that fat yield significantly (P<0.03) improved (7%) by supplementation of YC. They

concluded that the increased fat yield might be attributable to the improved production

performance in cow fed on YC. In the same manner, Ferraretto and Shaver (2012) reported

increased milk fat in the Holstein dairy cows fed on YC. They correlated that improved fat

contents in dairy cow fed on YC with reduced starch content and improved NDF intakes.

Longuski et al. (2009) reported that milk fat significantly (P<0.05) increased from 1.3 to 1.47 kg

per day the dairy animal fed on high moisture corn grain diet. They concluded that milk fat might

improve due to the high fermentative starch diet can be lessened with yeast culture. Similar

finding was observed by Piva et al. (1993) and Wohlt et al. (1998). They reported that positive

response to yeast culture supplementation to milk fat and might be attributable to the increased

milk yield and increased cellulose digestion in the dairy animals fed on YC. Vibhute et al. (2011)

also found significant effects of supplementing yeast in milk fat. In contracts to our finding,

some studies showed lower fat percentage due to YC supplementation. Bruno et al. (2009)

reported that the lower milk fat in dairy cattle fed on diet supplemented with YC resulted in

reduced (P<0.01) concentration of net energy for lactation (NEL) in milk. Arambel and Kent

(1990) found that there was no significant improvement in milk fat percentage after

supplementation with yeast. The variation in the above results may be related to breed and feed

difference.

5.2.3.2 Milk Protein

157

The milk protein was not influenced by inclusion of YC in our study. Milk protein synthesis is

highly affected by energy intake and carbohydrate components in the diet. Similar results were

reported by Jenkins and McGuire (2006) who noted that milk protein was influenced by energy

intake in the diet. A lack of response of YC on the milk protein in present study might be due to

the fact that no energy difference was present among the diet given to the dairy cattle. A similar

response was reported by who observed that YC supplementation has no significant effect on

milk protein. Our results also coincide with the results of Moallem et al. (2009) who observed

that YC has no impact on the milk protein percentage and milk protein yield. Similar finding has

been reported by (Piva et al., 1993; Erasmus et al., 2005). They reported that milk protein was

similar among yeast fed and non-yeast fed groups and that might be due to the similar propionic

concentration in the groups. In contrast to our study, some studies reported increased milk

protein due to YC addition. Gunther (1989) reported 16.5 % more milk protein the dairy for the

cow fed on diet supplemented with YC. He concluded that the increased milk protein might be

due to increased flow of methinonine and lysine in his study. In the same manner, Shaver and

Garrett (1997) noted significantly (P<0.05) increased from milk protein (1.17 vs 1.14 kg/day) in

dairy animal fed on basal diet.

Similarly, milk protein has been significantly improved in dairy animals fed on diet

supplemented with YC (Nocek et al., 2003; White et al., 2008; Kalmus et al., 2009; Bruno et al.,

2009). That increased milk protein in the yeast fed groups might be the positive impact of

probiotic yeast on nutrient digestibility and rumen fermentation. The increased fermentation and

digestion rate are due to the increased bacterial population inside the rumen. Proteins in the feed

are quickly breakdown into peptides, amino acids and NH3 by different protozoa and fungi

(Wallace et al., 1997). Some NH3 is converted into microbial protein (MP), and some ammonia

is used by the animal in the form of urea. The higher MP metabolized in the duodenum can be

contributed to higher protein output from the udder. Second possible improvement of the milk

protein as a result of yeast supplementation lowers the blood urea nitrogen (Bruno et al., 2009).

In vitro findings argue that probiotic yeast could alter the growth and activities of protein

degrading bacteria, by limiting their attack on protein and peptides. Yeast culture has positive

effect on microbial growth and negative effect on nitrogen loss (Beev et al., 2007). That process

enhances the ammonia uptake and microbial protein production has been improved and that

158

untimely increased the milk protein. Likewise, some researcher (Nocek et al., 2003, Nocek and

Kautz, 2006) found that YC has a positive effect on the milk protein. Some studies showed that

YC lower the milk protein yield (Cooke et al., 2007; Stella et al., 2007; White et al., 2008) and

milk protein (Nocek et al., 2003; Erasmus et al., 2005; Stella et al., 2007; Moallem et al., 2009;

Desnoyers et al., 2009). That non-significant effect of yeast might be due to the dilution factor of

higher milk yield (El-Ghani, 2004; Shaver and Garrett, 1997). The variation in the above results

may be related to breed difference.

5.2.3.3 Milk lactose

In the present study, milk lactose was not influenced by YC supplementation. This was

expected, as some researchers showed that milk lactose was not affected by dietary changes or

the change is very small (Sutton, 1989). Jenkins and McGuire (2006) also reported that milk

lactose was not changed by dietary changes but can be changed by serve feeding situation. In the

same manner, some researchers (Bruno et al., 2009; Stella et al., 2007; El-Din, 2015) reported

that yeast supplementation has no difference in the milk lactose percentage. In contracts of our

finding, Moallem et al. (2009) and Bruno et al. (2009) reported that milk lactose was

significantly (P<0.05) effected by YC supplementation in dairy cattle during hot seasons. They

concluded that high milk yield leads to the high milk lactose.

5.2.3.4 Milk Solid Not Fat (SNF)

Yeast supplementation had no significant effect on the SNF in the present study. Similarly, some

studies have shown that YC had no beneficial effect on milk SNF of dairy cows (Arambel and

Kent, 1990; Swartz et al., 1994; Soder and Holden, 1999; Bagheri et al., 2009). In contracts to

our results, Hossain et al. (2012) reported that YC significantly (P<0.05) increased (8.57 vs 8.28

%) the milk SNF. They concluded that improvement can be due to higher milk yield in the yeast

fed group as compared to no yeast fed group.

5.2.3.5 Milk Total Solids (TS)

In the current study, TS contents were not affected by YC supplementation in dairy cattle.

Similarly, some studies have shown that YC had no beneficial effect on milk SNF of dairy cows

(Arambel and Kent, 1990; Swartz et al., 1994; Soder and Holden, 1999; Bagheri et al., 2009).

The reason for the no change in milk TS is not clear.

5.2.4 Effect of probiotic on digestion performance of lactating dairy cattle

5.2.4.1 Dry Matter Digestibility (DMD)

159

Results of the present study indicated that apparent total tract DM digestibility in dairy cattle fed

on LAB-P and COM-P was 11.7% and 5.22% better than dairy cattle fed on control feed. The

higher DMD might be due to higher CP and NDF digestibility in our study. These results are in

accordance with previous study of Miller-Webster et al. (2002), who performed an experiment to

evaluate the impact of YC on the digestibility in lactating dairy cattle and found that YC has a

tendency for increased (P=0.10) DMD as compared. The difference in DMD is due to effect of

buffer salt contamination of the effluent. Our findings also coincide with the results of

(Wiedmeier et al., 1987), who reported that DMD was significantly (P<0.05) influenced by the

yeast supplementation in dairy cattle. Similar results were reported by Mir and Mir (1994) who

conducted the experiment to determine the effects YC in steers. They added SC at the rate of

10g/d per animal in the control diet. They noted that DMD significantly (P<0.05) influenced by

the yeast supplementation. The lack of impact of YC on the DMD has been reported in many

dairy animals (Harris et al., 1992; Doreau and Jouany, 1998; Cooke et al., 2007; Tripathi and

Karim, 2010). The differences from the present study could be due to the difference in the degree

of the nutritional scheme.

5.2.4.2 Crude Protein Digestibility (CPD)

Probiotic has a significant (P<0.05) impact on the CPD in our present study. Results indicated

that CPD was 11.01% and 8.02% higher in the dairy heifers fed on feed supplemented with

LAB-P and COM-P feed respectively as compared to the dairy heifers fed on control. The

higher milk production is might be due to proteolytic activity of the S. cerevisiae QAUSC03.

This activity enhanced the protein digestion rate in our study. In COM yeast fed group, same

reason can be considered that enhanced the CPD in lactating dairy cattle. Our results are

supported by Wohlt et al. (1998) who noted a significantly (P<0.05) higher CPD (78.5, 80.8 and

79.5%) with 0, 10 or 20 g/day probiotic yeast respectively in lactating dairy cattle. They noted

that the improvement in CPD is might be due to increased DMI by cattle fed on diet

supplemented with YC during 5 to 18 weeks of lactation. In the same manner, Mir and Mir

(1994) reported that probiotic has a significant (P<0.05) effect on the CPD steers fed on mixed

the diet (75% alfalfa silage, 25% barley, 96 % corn silage and 4 % soybean meal 75 % dry rolled

barley and 25 % alfalfa hay for 2 years). Wallace et al. (1997) reported that proteins in the feed

are quickly breakdown into smaller units, peptides, amino acids and NH3 by different protozoa

and fungi. Some NH3 is converted into microbial protein (MP), and some ammonia is used by

160

the animal in the form of urea. An important portion of rumen ammonia is excreted and

represents a indicated that nitrogen loss of the dietary nitrogen (N) intake (20 to 25%) (Fonty and

Chaucheyras-Durand, 2006). Amino acids and peptides issued from dietary proteins cannot be

directly slipped in the animal intestine if the diet has highly nutritious value. The same effect on

ammonia concentration was observed with daily yeast culture supplementation in adult

ruminants (Kumar et al., 1994). Beev et al. (2007) reported that in vitro findings tell that

probiotic yeast could alter the growth and activities of protein degrading bacteria, which

ultimately increased CP digestion inside rumen The mode of action of probiotic yeast can be

explained by a fight between live S. cerevisiae cells and different bacterial species for energy

utilization (Chaucheyras-Durand et al., 2005). Sniffen et al. (2004) conducted an experiment on

14 dairy cows fed on dietary supplementation of probiotic yeast and reported that the soluble

nitrogen of the diet was a key factor to drive the production parameters to the probiotics-yeast.

That improved CPD digestibility can be due to stable rumen pH and removal of oxygen from the

rumen. The stable pH enhanced microbial protein synthesis in the rumen. Further study is needed

to explain the effect of dietary supplementation of probiotic yeast on the nitrogen microbial

metabolism (Chaucheyras-Durand et al., 2010). In contrast, some studies reported that CPD was

no influenced by probiotic supplementation (Arambel and Kent, 1990; Andrighetto et al., 1993).

5.2.4.3 Neutral Detergent Fibre Digestibility (NDFD)

Our results showed that apparent total tract NDFD was significantly (P˂0.05) improved by

laboratory produced probiotic yeast. That improvement might be due to the cellulolytic activity

of the S. cerevisiae (QAUSC03), which increased cellulose degradation in our study. Moreover,

yeast also increases the cellulose degrading bacterial population that appears to be the main

mechanism by which yeast improves fibre digestion in many studies. NDFD were also

significantly (P˂0.05) affected by probiotic in others studies highlighting that improved

digestibility can be due to stable rumen pH and removal of oxygen from the rumen. That stable

rumen pH provides better environmental conditions for growth of rumen microbiota. At the same

time the anaerobic condition inside rumen also helped in better growth of fibrolytic microbial

biomass which enhanced the fibre digestion. In the agreement of our findings, Bitencourt et al.

(2011) observed that digestibility of the neutral detergent fiber was significantly (P<0.05) higher

(11.34%) in lactating dairy cows fed a diet based on corn silage and high content of pelleted

citrus pulp supplemented with 10 g yeast culture That improved nutrient digestibility might be

161

due to increased cellulose degrading microbial biomass population inside rumen. In the same

manner, Marden et al. (2008) reported that NDFD was significantly affected by YC and they

suggested that YC enhanced the activity of cellulolytic bacteria in the rumen in early lactating

Holstein cows. Newbold et al. (1990) reported that Aspergillus oryzae fermentation extract and

SC stimulated fiber degradation by their action on fibrolytic bacterial species. Wiedmeier et al.

(1987) noted that supplementation of YC significantly improved the hemicellulose digestibility

and highlighted that improvement might be due to the increased cellulolytic bacterial population

after YC supplementation. On the other hand, Wiedmeier et al. (1987); Wohlt et al. (1991) and

Moallem et al. (2009) noted no significant effect of YC on the NDF digestibility.

5.2.4.4 Acid Detergent Fibre Digestibility (ADFD)

SC has a significant (P˂0.05) effect of the ADFD in the current study. Our results indicated that

dairy cattle fed on LAB produced probiotic feed have 6.9% and 1.8% higher ADFD as

compared to the dairy cattle fed on the control and commercial probiotic feed respectively.

Similar findings were given by Marden et al. (2008) who reported that YC supplementation

significantly (P<0.05) improved (32.3 vs 18.1%) total tract ADF digestibility in the early

lactating Holstein cows. They concluded that YC enhanced the activity of cellulolytic bacteria

in the rumen which untimely improve the ADFD in early lactating Holstein cows. On the other

hand, Wiedmeier et al (1987) noted that ADF apparent digestibility was not influenced by the

supplementation of YC. It has been noted that nutrient digestibility was not affected when YC

in given in high energy and corn gelatinized (Arambel and Kent, 1990; El-Ghani, 2004; Cooke

et al., 2007). The fermented YC provides growth metabolites such as; organic acids, vitamins,

and different amino acids, which stimulate the growth of lactic acid bacteria that utilized lactic

acid and digest cellulolytic material (Callaway and Martin, 1997). These growth factors may

stimulate synthesis and secretion of IGF-I from liver on absorption from the gastrointestinal

tract.

5.2.5 Influences of probiotic on the hematological and biochemical parameters of

lactating dairy cattle

5.2.5.1 Hematological parameters

In our current study, all hematological values of the lactating dairy cattle before and after the

treatment were within the normal physiological range, which is a sign of good health.

162

5.2.5.2 Erythrocytic count (RBC)

YC did not show any effect on the erythrocytic count in lactating dairy cattle. In contracts to our

results, Dobicki et al. (2005) and Milewski and Sobiech (2009) reported that RBC counts

significantly increased in the growing animals. It should be noted that the growing animals have

a more capability to increase their blood profile than older animals.

5.2.5.3 Leukocytic count (WBC)

Leukocytic counts were not influenced by YC in the present study. In the same manner, Dobicki

et al. (2005) and Milewski and Sobiech (2009) reported that RBC counts significantly increased

in the growing animals. It should be noted that the growing animals have a more capability to

increased their blood profile than a the older animals.

5.2.5.4 Packed Cell Volume (PCV)

PCV levels were range from 29.65 to 31.61 % in dairy cattle fed on feed supplemented with

LAB-probiotic yeast, 29.57 to 29.88 % fed feed supplemented with COM-probiotic yeast and

30.12 to 32.32% fed on control feed without any supplementation. PCV levels were not

influenced by probiotic yeast in our study. In contrast, to our study Milewski and Sobiech (2009)

reported that yeast supplementation had associated with significantly (P ≤ 0.05) increased

erythrocytes counts in ewes during year I and II of the experiment. The difference in the PCV

may be due to the different breed, weather and diet.

5.2.5.5 Haemoglobin

During the present trial, the haemoglobin (Hb) levels were not influenced (p<0.05) by YC. These

results are in disagreement with the finding of Agazzi et al. (2014) who reported that

haemoglobin levels were affected by probiotic addition in growing claves. In the same manner,

Milewski and Sobiech (2009) noted that YC had significantly (P≤0.01) higher (101.70 vs 114.00

g/L) haemoglobin concentration in the supplemented ewes as compared to non-supplemented

ewes. In addition, Dobicki et al. (2005) reported improved Hb by 0.28-0.78 mmo|/l in heifer

supplemented with SC as compared to non-supplemented heifers.

5.2.5.6 Lymphocytes

Lymphocytes levels range from 42.45 to 42.63% in dairy cattle fed on LAB-probiotic feed and

42.15 to 51.71 fed on COM probiotic and 43.12 to 41.15 % fed on control feed. Lymphocytes

levels were not influenced by probiotic yeast in our study. In contrast to our study, Milewski and

Sobiech (2009) noted that probiotic yeast supplementation had associated with significantly

163

(P≤0.05) increased erythrocytes counts in ewes during yeast I and II of the experiment. However,

lymphocytes numbers lower with age of dairy animals due to environmental conditions (Fagiolo,

2004). Ciaramella et al. (2005) reported that lymphocytes counts were decreased during summer

(41%) than winter (77%) in early lactating dairy animal. Khaliq and Rehman (2010) reported

that mean lymphocytes were 58.23% in lactating dairy buffaloes of 7-10-year-old.

5.2.5.7 Eosinophils

Eosinophils levels were range from 3.86 to 4.41 % in dairy cattle fed on feed supplemented with

LAB-probiotic yeast, 2.64 to 2.75 % fed feed supplemented with COM-probiotic yeast and 3.52

to 3.78 % fed on control feed. Probiotic yeast has a non-significantly (P>0.05) effect on the

eosinophils levels in our study. We noted that eosinophils levels were higher in dairy cattle as

compared to dairy heifers in our study. Canfiels, (1984) reported that dairy animals over 10 years

of age showed higher levels of eosinophils levels. Probiotics have a significant effect on the

eosinophils levels. Agazzi et al. (2014) reported that probiotic had associated with significantly

(P≤0.05) increased eosinophils levels in claves.

5.2.5.8 Monocytes

Monocytes levels were range from 4.48 to 4.78 % in dairy cattle fed on feed supplemented with

LAB-probiotic yeast, 4.01 to 4.34 % fed feed supplemented with COM-probiotic yeast and 3.37

to 3.67 % fed on control feed. Monocytes levels were not influenced by probiotic yeast in our

study. A slightly lymphopenia associated with monocytosis and neutrophilia was reported by

Agazzi et al. (2014) in their experiment on dairy claves. But that is not related to pathological

condition.

5.2.6 Blood biochemical parameters

5.2.6.1 Cholesterol

Results showed that cholesterol concentration in serum of lactating dairy cattle was significantly

(P<0.05) decreased by probiotic yeast. That lower cholesterol might be due to the cholesterol-

lowing effect of the locally isolated yeast (QAUSC03). A similar effect might be related to the

commercially available yeast culture. In the same manner, Nicolosi et al. (1999) studied the

effect of YC on the blood parameters of the ruminants and he notes that the cell wall of yeast is a

164

rich source of β-glucans which reduce the total cholesterol of serum of the animals. Marden et al.

(2008) and Miller-Webster et al. (2002) reported that YC supplementation increase in

propionate, butyrate and valerate acid levels, which is capable of reducing the synthesis of

triglyceride and cholesterol in the liver cells and may change the lipid profile in blood of the

animals. Similarly, Kowalik et al. (2013) and Fayed (2005) reported significantly (P<0.05)

decreased cholesterol in the serum. Contrary to the findings of the present study, Galıp (2006);

Masek et al. (2008) and Campanile et al. (2008) reported no influence of YC on triglyceride and

total cholesterol concentration of serum in dairy animals.

5.2.6.2 Glucose

In our study, serum glucose was significantly (P<0.05) increased by YC supplementation. The

increased level of glucose in the yeast fed animals was might be due to increased nutrient

utilization that resulted in an increase dry matter and organic matter digestibility. Our results are

in line the recent work by Salvati et al. (2015), who conducted an experiment to determine the

effect of YC on the lactating dairy cattle. The diet was composited of silage (44.8%), raw

soybean (4.1%), soybean meal (16.5%), finely ground corn (20.7%) and citrus pulp (11.9%)

supplemented with live cells (25 × 1010 CFU) and dead yeast cells (5 × 1010 CFU). They reported

significantly (P<0.05) increased plasma glucose with yeast (62.9 vs. 57.3 mg/dl) in lactating

dairy cattle fed on yeast cells. They concluded that improved milk yield might be due to the

effect of the yeast cell on the regulation of body homeothermia in the lactating dairy cattle. A

study from USA by Lascano et al. (2012) reported similar results to the present study. They

reported an increase in YC dose tend to increase (P ≤0.10) glucose levels. They correlated that

increased level of glucose with increased nutrient utilization, which reflects the diet dependency

of the mode of action of yeast culture. Similarly, Hossain et al. (2012) reported that YC

statistically (P<0.05) increased the glucose levels in dairy animal. In another study by (Dolezal

et al., 2011), It was reported that YC significantly YC significantly (P<0.01) increased (2.278 vs.

2.237 mmol.I-1) the glucose concentration in dairy animal. Contrary to the findings of the present

study, Edens et al. (2002) reported that YC is a source of glucose tolerance factor and has

stimulated glucose utilization by cell in vitro. Glucose represents the synthesis of carbohydrates

and is in the form in which carbohydrate is supplied to cell from body fluids. The age of the

animals has exhibited highly significant variation in serum glucose level in the dairy animals.

5.2.6.3 Blood Urea Nitrogen

165

Changes in the serum urea N concentration in lactating dairy cattle fed on probiotic feed and fed

on control feed were significantly (P<0.05) affected at the end of trial. YC supplementation

significantly (P<0.05) lower the serum urea concentration in current study. That lower

concentration of urea N in the dairy cattle fed on probiotic feed might be due to increased protein

digestibility in our study. It is observed that, because the N- intake is lower in cattle fed on

probiotic feed compared to control feed, hence, the concentration of urea-N was lower in the

cattle fed probiotic feed. This observation is in agreement with Dolezal et al. (2011) who

reported that YC significantly YC significantly (P<0.01) decreased (4.807 vs. 4.948 mmol.I-1)

the urea concentration in dairy animal. Contrary to the findings of the present study, Putnam et

al. (1997); Nikkhah et al. (2004) and Bagheri et al. (2009) who reported that serum urea

concentration is not significantly affected by yeast addition. It is possible that the lower ammonia

blood values in YC fed groups were due to the increase of rumen microbial activity as reported

by Williams and Orpin (1987). The concentration of urea in the blood is intimately associated

with the efficiency with which dietary protein is used, and suggests that plasma levels of ureic

ammonia are an indicator of protein degradability in the rumen and protein intake post-rumen.

5.2.7 Blood Serum Macro-Minerals

Minerals are important for normal growth and production performance of the animals. They

serve as the intermediate role in the action of hormones and enzymes at cellular level in an

integrated fashions (NRC, 2001). In our study, macro-minerals concentration in the blood serum

of cattle heifers was not affected (P>0.05) by probiotic yeast.

5.2.7.1 Calcium (Ca)

Reproductive performance of the dairy animals is directly and indirectly related to Ca

concentration (Bansal, 1978) and involved in steroid biosynthesise in ovaries (Shemesh et al.,

1984). In our study, Ca concentration was within the normal serum range of 8 to 10 mg/dl. There

was no response of YC in serum Ca over time in present study. Similar results were reported by

Piva et al. (1993), who demonstrated that silage, hay and concentrate based diet supplemented

with YC had no effect on the Ca concentration in dairy animals. In the same manner, Nursoy and

Baytok (2003), performed an experiment to see the impact of YC on blood chemistry of dairy

cow and found that probiotic yeast has no significant effect on the Ca concentration. On the other

hand, Dolezal et al, (2011) reported that YC significantly (P<0.01) increased (2.328 vs. 2.273

166

mmol.I-1) the Ca concentration in dairy animal. He reported that significant difference in the

blood parameters might be due to the effect of diet and individuality of cow.

5.2.7.2 Phosphorus (P)

P is very important macro-mineral in cellular metabolism of all animals. The central compound

in energy metabolism adenosine triphosphate is phosphorylated compound. Phosphorus content

in blood plasma normally ranges from 4 to 6 mg/dl for adult animals (NRC, 2001). Phosphorus

deficiency is identified as reduced appetite and milk yield; reduce growth rate, lethargy, and

lowered immunity low productive and reproductive performance. Serum P concentration range

from 6.89 to 7.00 mg/dl in dairy cattle fed on feed supplemented with LAB probiotic yeast; 6.33

to 6.61 mg/dl fed on feed supplemented with COM probiotic feed and 6.58 to 6.81 fed in control

feed without any supplementation. All values are in normal range, which is a good sigh of health.

Dietary YC did not affect serum P in our study. The present study complements the study by

Nursoy and Baytok (2003), who found that YC has no significant effect on the P level in dairy

cow. In contract to our study Dolezal et al. (2011) reported that, dietary supplementation of

probiotic yeast significantly (P<0.01) deceased (1.978 vs. 2.062 mmol.I-1) P concentration in

blood serum in dairy animals. P absorption is affected by a number of factors in dairy cattle NRC

1989 (NRC, 2001; Horst, 1986). The primary site of P absorption is the small intestine and it is

influenced by the presence of vitamin D (NRC, 2001; Care, 1994). The total quantity of P

absorbed depends on the Ca to P ratio, type of feed, the age of animal and levels of other

minerals such as Na, K and Ca.

5.2.7.3 Potassium (K)

K concentration was not affected by YC in present study. Similar results were reported by Piva

et al. (1993) calming that silage, hay and concentrate based diet supplemented with YC had no

effect on the K concentration in dairy animals.

5.2.7.4 Sodium (Na)

Serum Na concentration was no affected by YC in our study. Similar results were reported by

Piva et al, (1993), who demonstrated that silage, hay and concentrate based diet supplemented

with YC had no effect on the Na concentration in dairy animals. On the other hand, YC

167

significantly (P≤0.05) increases the concentrations of Na+ ions in ewes compared with control

group (Milewski and Sobiech, 2009).

5.3.6 Impact of probiotic on the changes in the ruminal gut microbial flora in dairy cattle

Literature dealing with the effect of dietary supplementation of probiotic yeast in the gut

microbiota of dairy cattle is scare. This study is unique in its examination of yeast culture on the

gut microbiota in order to account for better feed digestion resulting in better production

performance in lactating dairy cattle. The ruminal gut microbiota of cattle affects animal health

and productivity. It is commonly noted that diet composition has a crucial impact on the changes

in the microbial composition of the GIT and rumen. Although considerable effort has been

expended in isolation and characterization of rumen microbial species, very limited research has

focused on the characterization of the intestinal tract of the dairy animals. Despite the value of

the microbial flora of GIT of the ruminants to host health and productive performance,

knowledge about the GIT microbial diversity and function remains rudimentary. Research has

confirmed that there is a strong relationship between dietary efficiency and GIT microbial

populations and diversity (Ley et al., 2006; Turnbaugh et al., 2009). Probiotics have a curial role

in the improvement of the gut health at the beginning of the lactation period in dairy animals

(Chaucheyras-Durand and Durand, 2010). From our present study, microbial growth dynamics of

total aerobic counts, Enterococcus, Lactococcus and Bacillus counts were analyzed in cattle fecal

samples with respect to a number of days (0, 30 and 60). The Enterococcus species of GIT of

cattle has been studied by many authors (Devriese et al., 1992). Enterococcus is gram positive

cocci and facultative anaerobes and is an important group of intestinal bacteria in ruminant and

performs many important functions including working as an antibiotic resistance reservoir

(Giraffa, 2003). Enterococci undergo commensalisms and inhabit the gastro-intestinal tract of

human and animals. It seemed to be screened out from variety of food sources (meat, milk,

cheese). Enterococci have high survival rate in harsh and extreme conditions, for example, they

can resist 65% NaCl, pH, as well as high heat. They can also be isolated from variety of soils,

raw plants, surface water and animal products (Giraffa, 2003; Cocolin et al., 2007). In the

present study, we found that probiotic has an ability to reduce the Enterococcus species in GIT.

But that reduction was statistically non-significant in dairy cattle fed on probiotic and control

feed. In the same manner, Jatkauskas and Vrotniakiene, (2010) showed a decreased (1.4×108 vs.

168

4.0×103) in numbers of Enterococcus in fecal of dairy claves. Vlkova et al. (2006) determine the

Enterococcus counts in fecal samples of claves at different stages. They found that Enterococcus

species were 7.06 CFU/g at 35 days of age. On the other hand, Rada et al. (2006) found that 8.49

CFU/g Enterococcus species were present in the claves. Previously, Lactococcus were placed in

Streptococcus group N1, but now it belongs to genus Lactic acid bacteria. On the basis of its

glucose fermentation property, they are recognised as homofermentators, and are gram-positive,

catalase negative and non-motile cocci. They are usually present in singlet, pairs and in chain

form. It has been further sub-recognized as L. lactis, L. gravieae, L. piscium. These organisms

generate wide impact in dairy industry as in fermentation of dairy products, such as cheese. They

are used in the form of single-chain starter or in mixed strain culture. The most important

function of Lactococcus is being applied in rapid acidification of milk, which drops the pH and

inhibits the growth of spoilage bacteria (Schleifer et al., 1985). In our present study, we found

that Lactococcus species were not affected by YC supplementation. With respect to days (0, 30

and 60), considerably there was slight differentiation in total aerobic count in cattle fed on LAB-

probiotic feed as compared to cattle fed on the COM-probiotic feed and control feed. This

indicates that in dairy animals, days puts positive influence on total aerobic count. The only

factor that might contribute to this variation in bacterial count is the age of animals, since other

factors including diet and health conditions were similar in both cases. This may be possible

because that cattle have wide contact with other animals rather than heifers and heifers have

unbalanced state of intestinal flora too as mentioned above by (Lukas et al., 2007). According to

day-wise growth dynamics of the Bacillus counts, the microbial growth trends were analyzed in

varying arrangement of days as day 0, 30 and 60. The total Bacillus count (CFU/g) with

reference to these days in dairy cattle has exhibited the wide range and different behavior of

growth patterns using different treatments. Although, the number of the Bacillus counts were

higher in the probiotic feed fed group but that difference was not significant among the

treatments. A similar finding has been reported by Jenny et al. (1991), who reported that

Bacillus were higher in claves fed on probiotic feed compared to control feed. In the same

manner, Kawakami et al. (2010) noted that Bacillus species in GIT of dairy claves were

improved after fed on probiotic feed. As the fecal microbial population in dairy animals are

future quantified, scientists might be in position to correlate microbial populations of kingdoms

or nutrient-utilization guidelines or both with production parameters like feed intake, growth

169

performance, milk production and well beings of the animals. Results from this study indicate

that YC have no effect on the total counts of Enterococcus, Lactococcus, Bacillus and total

aerobic counts in lactating dairy cattle. Diet is the major factor which can change the microbiota

of the GIT and rumen (Bilal, 2004; Russell and Hino, 1985).

5.3.6.1 Modern methods to study the GI microbial flora of ruminants

Gastrointestinal microbiota of ruminants acts a vital role in the production, health status and

well-being of the ruminants (Dowd et al., 2008). The gut microbial populations in cow have been

identified almost 90 % of the total microbial community (Uyeno et al., 2008, 2010). On the other

hand, a certain fraction of the GI tract bacterial community has yet to be identified due to an less

knowledge of the microbial community in gut microbial ecosystem because majority of the 16S

rRNA gene sequences came from feces material are taken from unidentified species (Favier et

al., 2002). Recently, many modern methods of using genomic analysis of communities to

determine changes in microbiota have been used by many scientists. Many studies looking for to

monitor bacterial diversity in the GI tract utilize culture-independent sequencing techniques,

16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing and pyrosequencing and many

more have added a new era to the determine the microbial diversity of GI tract. In a study by

Oikonomou et al, (2013), the changes in microbial population in the GI tract of young calves was

study by using metagemincs and highlights that the average population of Formicates increased

from first week to fourth week of life and then gradually deceased and a reverse pattern was

noted for Bacteroides population. He also noted that the Lactobacillus sp. reached a 14.74%

maximum during the fourth week of calf life and then progressively decreased to reach 2.15%

during the seventh week. Same results were reported in our current study. Escherichia was seen

among treatments throughout the experimental period, and highest at first week of age, averaging

approximately 21 and 20% of all bacteria for calves fed pasteurized and non-pasteurized waste

milk, respectively, and decreasing (2.6 and 1.3%) with passage of time. The microbial profile of

the dairy cattle mastitis was investigated by using the metagenomics (Oikonomou et al., 2012).

Microbial population changes associated with sub-acute ruminal acidosis of dairy cows have

been study using terminal-restriction fragment length polymorphisms of 16S rRNA gens and

PCR. Dowd et al., (2008) use the (bTEFAP), 16S rDNA bacterial tag-encoded FLX amplicon

pyrosequencing to study the fecal flora of cattle and found that the microbial diversity of GI tract

170

of cows are dominated by strict anaerobes such as Bacteroides sp., Clostridium sp.,

and Bifidobacterium sp. Facultative anaerobes; like E.coli, is also seen in his study, but the

population was lower than strict anaerobes. Enterococcus sp. is also present in the fecal samples

of the dairy cattle as a pathogenic microbial species. Uyeno et al. (2010) monitor the faecal

samples of dairy claves of Holstein breed. He analysed the samples by the RNA-based,

sequence-specific rRNA cleavage method and found that the Lactobacillus sp. were deceased

with passage of time (Uyeno et al., 2010). Even accounting for potential bias of latest molecular

methods, it is obvious that these methods are the most dominant tools recently accessible for

monitoring the gut bacterial diversity of dairy animals. Extensive use of molecular

methodologies may show the way in a new era in which such microbial study studies are no

longer limited to a handful of laboratories with abundance of funding and labor.

5.6 Impact of probiotic on economic efficiency of lactating dairy cattle.

Lactating dairy cattle health status is not only considered until there are economic impacts as

dairy sectors often want a low-cost and easy-care way of milk production (Mulligan and

Doherty, 2008). Improved production efficiency of the dairy animals leads to improved profit for

dairy farmers; moreover costs associated with diseases treatment can decrease the profit

(Kossaibati and Esslemont, 1997). Feed additives, like probiotics defined as supplements added

to the feed of ruminants to enhance productive performance, may be cost-effective and safe

methods to improve feed utilization in dairy animal. In our study, animals fed on the feed

supplemented with LAB-probiotic yeast may encourage reducing the feeding costs. Animals fed

on the LAB-probiotic feed has 5.30 and 4.50 % lesser feed cost for one kg milk production than

animals fed on control and COM-probiotic feed respectively. Results revealed that lactating

dairy cattle fed on LAB-probiotic feed are economically more efficient in term of feed cost by

producing more milk. In the present study, similar DMI was consumed by dairy cattle fed on

probiotic and non-probiotic feed. The LAB-probiotic yeast has potential benefits to lower the

feed cost per unit milk production in lactating dairy cattle. Net profit per liter milk production of

LAB yeast fed animals was increased 4.7 % and 3.9 % compared to fed on non-probiotic and

COM yeast probiotic feed. Literature dealing with impact of yeast supplementation the net

profits of dairy cattle is scare. In a study, (Shaver and Garrett, 1997) observed that dietary

supplementation of yeast has improved milk yield as 0.23 kg per cow. It is well studied that

171

diseases can lower the profit of dairy farmers (Kelton et al., 1998). Clinical mastitis can

decrease profit by 735 dollar per lactating cattle (Hultgren and Svensson, 2009). In our study,

probiotic yeast did not show any negative effect on the health status of the lactating dairy cattle.

The economic advantage of probiotic depends on the price of yeast culture, and the lactation

stage, age, diet, breed and geographical location of the animal (Yalcın et al., 2011; Vibhute et

al., 2011; Marrero et al., 2015). Therefore, to achieve an economical effective of probiotic on the

production efficiency of dairy animal of local breeds, it is required to choose an adequate

preparation because not all probiotic yeast strains can stimulate the nutrient digestion in dairy

animal (Pinos-Rodriguez et al., 2008).

172

Conclusions/ Future prospects

In Pakistan, livestock sector is mostly based on traditional lines which lead to unbalance

nutrition resulting in poor growth and productive performance in dairy animals. Now a days,

increasing the performance of dairy animals through the use of probiotic has become a useful

and economical method to overcome the effects of malnutrition. The use of probiotic yeast

enhances the nutrient utilization, which may lead to improved performance and increase

immunity in dairy heifers. Literature reveals that suitability and profitability of the probiotic

yeast depends on many factors including animal breed, age and probiotic strains. From this line

of research we look forward and develop a new probiotic yeast strain for our local breed,

which provide a positive effect on milk yield and fat contents in lactating dairy cattle and

moreover it is cost effective. At the same time, the dietary supplementation of probiotic yeast

could also have an enhancing effect on the microbial balance of the GIT that leads to

improved growth, health and production performance in dairy animal.

In the situation of a high feed cost in Pakistan, probiotic yeast gives a useful nutritional strategy

which allows increasing diet digestibility and consequently enhances the performance parameters

of the dairy animals in cost effective manner. Future research needed to see the impact of the

yeast cells in the GIT of the dairy animals. Future research will also need to address the behavior

of the yeast cells in the digestive environment. We look forward to the development of the new

probiotic strains, which will hopefully mean that the rumen microbiologist in Pakistan

instead of following the nutritious in an exploratory mood as has been the role for so long,

will instead lead advances in ruminant nutrition in year to come.

Recommendations

The recommendations are outline as follows;

173

1. Isolation of new indigenous bacterial and yeast strains.

2. Study the probiotic characterization and genetic potential of the probiotic strains

3. Complete nutritional profile of the probiotic strains for preparation of probiotic feed

4. Application of probiotic strains for more milk and meat production of local breed

animals.

5. Amino acid profile of the milk of dairy animals fed on the probiotic feed.

References

Abe, F., Ishibashi, N. and S. Shimamura. 1995. Effect of administration of bifidobacteria and lactic acid

bacteria to newborn calves and piglets. J. Dairy Sci. 78 (12): 2838-2846.

Abu-Tarboush, H.M., Al-Saiady, M.Y. and A.H. K. El-Din. 1996. Evaluation of diet containing

lactobacilli on performance, fecal coliform, and lactobacilli of young dairy calves. Anim. Feed

Sci. Technol. 57(1): 39-49.

174

ACO. 2006. Agricultural Census Organization. Statistics division, Government of Pakistan.

Adams, M.C., Luo, J. Rayward, D. King, S. Gibson, R. and Moghaddam, G.H. 2008. Selection of a

novel direct-fed microbial to enhance weight gain in intensively reared calves. Anim. Feed Sci.

Technol. 145(1): 41-52.

Afzal, M. 2007. Livestock Development Policy. Ministry of Food, Agriculture and Livestock. Livestock

and Dairy Development Board. Government of Pakistan.

Afzal, M. 2008. Investment Opportunities in Livestock Sector in Pakistan

Published in "The News" Rawalpindi / Islamabad, Lahore and Karachi on September 18, 2006

Agarwal, N., Kamra, D. N. Chaudhary, L. C. Agarwal, I. Sahoo, A. and Pathak, N. N. 2002. Microbial

status and rumen enzyme profile of crossbred calves fed on different microbial feed

additives. Letters Appli. Microbiol. 34(5): 329-336.

Agazzi, A., Tirloni, E. Stella, S. Maroccolo, S. Ripamonti, B. Bersani, C. and Savoini, G. 2014. Effects

of species-specific probiotic addition to milk replacer on calf health and performance during the

first month of life. Annals Anim. Sci. 14(1): 101-115.

Ahmad, S., Hinch, G. Prior, J. Thomas, P. and Burrell, D. 2012. The role of extension in changing the

dairy industry in Pakistan: a review. J. Anim. Plant Sci. 22(2): 113-116.

Ahn, Y. T., Kim, G. B. Lim, K. S. Baek, Y. J. and Kim, H. U. 2003. Deconjugation of bile salts by

Lactobacillus acidophilus isolates. Intern. Dairy J. 13(4): 303-311.

Akbari, O., DeKruyff, R. H. and Umetsu, D. T. 2001. Pulmonary dendritic cells producing IL-10

mediate tolerance induced by respiratory exposure to antigen. Natur. Immunolog. 2(8): 725-731.

Akin, D. E. 1989. Histological and physical factors affecting digestibility of forages. Agronomy J. 81(1):

17-25.

Akin, D. E. and Benner, R. 1988. Degradation of polysaccharides and lignin by ruminal bacteria and

fungi. Appli. Environm. Microbiol. 54(5): 117-1125.

175

Ametaj, B. N., Emmanuel, D. G. V. Zebeli, Q. and Dunn, S. M. 2009. Feeding high proportions of

barley grain in a total mixed ration perturbs diurnal patterns of plasma metabolites in lactating

dairy cows. J. Dairy Sci. 92(3): 1084-1091.

Andrighetto, I., Bailoni, L. Cozzi, G. and Berzaghi, P. 1993. Effects of yeast culture addition on

digestion in sheep fed a high concentrate diet. Small Rumin. Resear. 12(1): 27-34.

Anjum, M. I., Azim, A. Jabbar, M. A. Anwar, M. and Mirza, I. H. 2012. Age and weight at puberty in

Nili-Ravi buffalo heifers reared on three dietary energy restriction periods followed by

compensatory growth. Pak. Vet. J. 32(3): 367-371.

Anonymous. 2006. The white revolution "dhoodh darya". PDDC (2006, June). Retrieved March 4, 2014,

from http://www.pddc.com.pk/DairyPakistan-Publication.pdf.

Anonymous. 2009. Pakistan Dairy Industry – Overview. Retrieved October 9, 2014, from Jassar

Farms.com: http://www.jassarfarms.com/english/?page_id=63.

Anonymous. 2011. Milk Collection and Dairy Development, 2011. Nestal company, Pakistan.

AOAC. 1990. Association of Official Analytical Chemists. (16th edn) , Washington, DC, USA.

Arakaki, L. C., Stahringer, R. C. Garrett, J. E. and Dehority, B. A. 2000. The effects of feeding

monensin and yeast culture, alone or in combination, on the concentration and generic

composition of rumen protozoa in steers fed on low-quality pasture supplemented with

increasing levels of concentrate. Anim. Feed Sci. Technol. 84(1): 121-127.

Arambel, M. J. and Kent, B. A. 1990. Effect of yeast culture on nutrient digestibility and milk yield

response in early-to midlactation dairy cows 1, 2. J. Dairy Sci. 73(6): 1560-1563.

Arif, M., Iqbal, A. Younas, M. Khan, B. B. Sarwarand, M. and Ahmad, S. 2013. Prospects and

limitations of dairying in Gujranwala district (Punjab-Pakistan). J. Anim. Plan Sci. 23: 84-89.

Auclair, E. 2001. Yeast as an example of the mode of action of probiotics in monogastric and ruminant

species. Feed Manufacturing in the Mediterranean Region. Reus, Spain: CIHEAM-IAMZ, 45-53.

176

Audisio, M. and Benitez-Ahrendts, M. 2011. Lactobacillus johnsonii CRL1647, isolated from Apis

mellifera L. bee-gut, exhibited a beneficial effect on honeybee colonies. Beneficial

microbes. 2(1): 29-34.

Ayad, M. A., Benallou, B. Saim, M. S., Smadi, M. A. and Meziane, T. 2013. Impact of feeding yeast

culture on milk yield, milk components, and blood components in Algerian dairy herds. J.

Veterinar. Sci. Technol. 4(135): 2.

Ayeni, F. A., Sanchez, B. Adeniyi, B. A., Clara, G. Margolles, A. and Ruas-Madiedo, P. 2011.

Evaluation of the functional potential of Weissella and Lactobacillus isolates obtained from

Nigerian traditional fermented foods and cow's intestine. Intern. J. food microbiolo. 147(2): 97-

104.

Bagheri, M., Ghorbani, G. R. Rahmani, H. R. Khorvash, M. Nili, N. and Südekum, K. H. 2009. Effect of

live yeast and mannan-oligosaccharides on performance of early-lactation Holstein dairy

cows. Asian-Aust. J. Anim. Sci. 22(6): 812-818.

Baken, K. A., Ezendam, J. Gremmer, E. R. De Klerk, A. Pennings, J. L. Matthee, B. and Van Loveren,

H. 2006. Evaluation of immunomodulation by Lactobacillus caseiShirota: immune function,

autoimmunity and gene expression. Intern. J. Food microbiol. 112(1): 8-18.

Bansal, R., S. Gupta, G. Singh. and F. Chauhan. 1978. Serum levels of phosphorous and calcium in

different phases of reproduction in buffaloes. J. Remount Veterinar. Corps. 17: 95-100.

Barham, D. and Trinder, P. 1979. Methods for determination of blood glucose levels by

spectrophotometer. Analyst. 97: 142.

Barnett, J. A., Payne, R. W. and Yarrow, D. 1983. Yeasts: Characteristics and identification. Cambridge

University Press.

Barnett, J. A., Payne. R. W. and Yarrow, D. 1990. Yeast Characteristic and Identification. 2nd ed.

Cambridge University Press.

177

Barrow, P. A., Brooker, B. E. Fuller, R. and Newport, M. J. 1980. The attachment of bacteria to the

gastric epithelium of the pig and its importance in the microecology of the intestine. J Applied

Bacteriol. 48(1): 147-154.

Batta, A. K., Salen, G. Arora, R. Shefer, S. Batta, M. and Person, A. 1990. Side chain conjugation

prevents bacterial 7-dehydroxylation of bile acids. J. Bio. Chemis. 265(19): 10925-10928.

Bayat, A. R., Kairenius, P. Stefanski, T. Leskinen, H. Comtet-Marre, S. Forano, E. Chaucheyras-

Durand, F. and Shingfield, K. J. 2015. Effect of camelina oil or live yeasts (Saccharomyces

cerevisiae) on ruminal methane production, rumen fermentation, and milk fatty acid composition

in lactating cows fed grass silage diets. J. Dairy Sci. 98(5): 3166-3181.

Bayatkouhsar, J., Tahmasebi, A. M. Naserian, A. A. Mokarram, R. R. and Valizadeh, R. (2013). Effects

of supplementation of lactic acid bacteria on growth performance, blood metabolites and fecal

coliform and lactobacilli of young dairy calves. Anim. Feed Sci. Technol. 186(1): 1-11.

Beauchemin, K. A., Yang, W. Z. Morgavi, D. P. Ghorbani, G. R. Kautz, W. and Leedle, J. A. Z. 2003.

Effects of bacterial direct-fed microbials and yeast on site and extent of digestion, blood

chemistry, and subclinical ruminal acidosis in feedlot cattle. J. Anim. Sci. 81(6): 1628-1640

Bednarek, D. and Kowalski, C. 2007. Effect of prebiotic additives on interleukin 1 activity and

alternations of peripheral blood leukocyte subpopulations in calves. Medycyna

Weterynaryjna, 63(12): 1591-1594.

Beev, G., Todorova P. and Tchobanava, S. 2007. Yeast cultures in ruminant nutrition. Bulgarian J. Agri.

Sci. 13: 357-374.

Begley, M., Gahan, C. G. and Hill, C. 2005. The interaction between bacteria and bile. FEMS Microbio.

Reviews. 29(4): 625-651.

Benjamin, M. M. 1985. Outline of veterinary clinical pathology. 3 ed. The Lowa State Univ. press,

USA.

Berthelot, A. 1974. Clinical Chemistry. 25: 336

178

Bhati, N.P., 1999. Buffaloes. In: Payne, W.J.A., Wilson, T.R. (Eds.), An Introduction to Animal

Husbandry in the Tropics. Blackwell Science Oxford (Oxfordshire), UK, p.815.

Bhatti, S. A., Sarwar, M. Khan, M. S. and Hussain, S. M. I. 2007.Reducing the age at first calving

through nutritional manipulations in dairy buffaloes and cows: a review. Pak. Veterinar. J. 27(1):

42.

Bilal, M. Q. and Ahmad, A. 2004. Dairy hygiene and disease prevention. Faisalabad: Usman and Bilal

printing linkers. Pakistan. Usman and Bilal printing linkers, Faisalabad, Pakistan.

Bilal, M. Q., Suleman, M. and Raziq, A. 2006. Buffalo: black gold of Pakistan. Livestock Res. Rural

Develop. 18(9): 140-151.

Bitencourt, L. L., Silva, J. R. M. Oliveira, B. M. L. D. Dias Junior, G. S. Lopes, F. Siecola Junior, S.

Ozana, D. F. Z. and Pereira, M. N. (2011). Diet digestibility and performance of dairy cows

supplemented with live yeast. Sci. Agricola. 68(3): 301-307.

Bonhomme, A. 1990. Rumen ciliates: their metabolism and relationships with bacteria and their

hosts. Anim. Feed Sci. Technolo. 30(3): 203-266.

Borghese, A. 2005. Buffalo Production and Research. FAO, Rome,Italy.

Bortolini, O., Medici, A. and Poli, S. 1997. Biotransformations on steroid nucleus of bile

acids. Steroids, 62(8): 564-577.

Briand, V., Buffet, P. Genty, S. Lacombe, K. Godineau, N. Salomon, J. and Bouchaud, O. 2006.

Absence of efficacy of nonviable Lactobacillus acidophilus for the prevention of traveler's

diarrhea: a randomized, double-blind, controlled study. Clinical infectious diseases, 43(9): 1170-

1175.

Brulc, J. M., Yeoman, C. J. Wilson, M. K. Miller, M. E. B. Jeraldo, P. Jindou, S. Nigel, G. Harry, J. F.

Raphael, L. Ilya, B. Masa, V. Karen, E. N. Edward, A. B. and White, B. A. 2011. Cellulosomics,

a gene-centric approach to investigating the intraspecific diversity and adaptation of

Ruminococcus flavefaciens within the rumen.PLoS ONE.6(10). e25329.

179

Bruno, R. G. S., Rutigliano, H. M. Cerri, R. L. Robinson, P. H. and Santos, J. E. P. 2009. Effect of

feeding Saccharomyces cerevisiae on performance of dairy cows during summer heat

stress. Anim. Feed Sci. Technol. 150(3): 175-186.

Buts, J.P. 2004. Exemple d’un médicament probiotique: Saccharomyces boulardiilyophilisé. In:

Rambaud, J.C., Buts, J.P., Corthier, G. and Flourié, B. (eds.) Flore microbienne intestinale. John

Libbey Eurotext, Montrouge, France. pp. 221-244.

Callaway, E. S. and Martin, S. A. 1997. Effects of a Saccharomyces cerevisiae culture on ruminal

bacteria that utilize lactate and digest cellulose. J. Dairy Sci. 80(9): 2035-2044.

Campanile, G., Zicarelli, F., Vecchio, D., Pacelli, C., Neglia, G., Balestrieri, A.Rossella D. P.and

Infascelli, F. 2008.Effects of Saccharomyces cerevisiae on in vivo organic matter digestibility

and milk yield in buffalo cows. Livestock Sci. 114(2): 358-361.

Canfield, P. J., Best, F. G. Fairburn, A. J. Purdie, J. and Gilham, M. 1984. Normal haematological and

biochemical values for the swamp buffalo (Bubalusbubalis) in Australia. Aust. Veterinar.

J. 61(3): 89-93.

Carberry, C. A., Kenny, D. A. Han, S. McCabe, M. S. andWaters, S. M. 2012.Effect of phenotypic

residual feed intake and dietary forage content on the rumen microbial community of beef

cattle. Applied Environ. Microbio. 78(14): 4949-4958.

Care, A. D. 1994.The absorption of phosphate from the digestive tract of ruminant animals. British

Veterinar. J. 150(2): 197-205.

Carey, M. C. and W. C. Duane. 1994. The liver: biology and pathobiology. Raven Press, New York,

N.Y.

Carro, M., P. Lebzien, and K. Rohr. 1992. Influence of yeast culture on the in vitro fermentation

(Rusitec) of diets containing variable portions of concentrates. Anim. Feed Sci. Technol. 37:

209-220.

180

Cebra, J.J. Jiang, H.Q. Sterzl, J. and Tlaskalova-Hogenova, H.1999. The role of mucosal microbiota in

the development and maintenance of the mucosal immune system.In. Ogra, R.L. Mestecky, J.

Lamm, ME. Strober, W. Bienenstock, J. Macghee, J.R (eds). Musosal Immunity. 2nd ed. San

Diego: Academic Press, New York. 267-280.

Chattha, M. W. A., A. Fatima, K. Bakhsh, and S. Hassan. 2013. Production and supply response of milk

in Pakistan: Price and non-price determinants. Int. J. Agric. Biol 15: 170-174.

Chaucheyras-Durand, F. and Durand, H. 2010. Probiotics in animal nutrition and health. Benefic.

Microbes. 1: 3-9.

Chaucheyras-Durand, F. and Fonty, G. 2001. Establishment of cellulolytic bacteria and development of

fermentative activities in the rumen of gnotobiotically-reared lambs receiving the microbial

additive Saccharomyces cerevisiae CNCM I-1077. Reprod. Nutr. Dev. 41: 57-68.

Chaucheyras-Durand, F. and Fonty, G. 2002. Yeasts in ruminant nutrition. Experiences with a live yeast

product. Kraftfutter. 85:146-150.

Chaucheyras-Durand, F. Masseglia, S. and Fonty, G. 2005. Effect of the microbial feed additive

Saccharomyces cerevisiae CNCM I-1077 on protein and peptide degrading activities of rumen

bacteria grown in vitro. Curr. Microbiol. 50: 96-101.

Chaucheyras-Durand, F., Walker, N. and Bach, A. 2008. Effects of active dry yeasts on the rumen

microbial ecosystem: Past, present and future. Anim. Feed Sci.Technol. 145: 5-26.

Chaucheyras, F., Fonty, G. Bertin, G. and Gouet, P. 1995. Effects of live Saccharomyces cerevisiae cells

on zoospore germination, growth, and cellulolytic activity of the rumen anaerobic fungus,

Neocallimastix frontalis MCH3. Current Microbiol. 31: 201-205.

Chaucheyras, F., Fonty, G. Bertin, G. Salmon, J. M. and Gouet, P. 1996. Effects of a strain of

Saccharomyces cerevisiae (Levucell SC1), a microbial additive for ruminants, on lactate

metabolism in vitro. Can. J.Microbiol. 42: 927-933.

181

Chaudhry, K., Muhammad, S. Ashraf, I. and Ghafoor, U. 2012. Farmer's perceptions of livestock

production practices introduced by Punjab Rural Support Program (PRSP). Pak J.Agri. Sci. 49:

233-235.

Cheeke, P. R. 1991. Applied animal nutrition: Feeds and feeding, Macmillan PUB Co.866; Third

Advenue NY 10022, USA.

Chen, C. J., Chan, H.M. and Kubow, S. 2007. Kefir extracts suppress in vitro proliferation of estrogen-

dependent human breast cancer cells but not normal mammary epithelial cells. J. Med. Food. 10:

416-422.

Chen, L. S., Ma, Y. Maubois, J. L. He, S. H. Chen, L. J. and Li, H. M. 2010. Screening for the potential

probiotic yeast strains from raw milk to assimilate cholesterol. Dairy Sci. Technol. 90(5): 537-

548.

Chevaux, E. and Mazzia-Fabre, M. 2007. Probiotic yeast in small ruminants. Feed Mix. 15: 28.

Cho, S. J., CHO, K. M. Shin, E. C. Lim, W. J. Hong, S. Y., Byoung, R. C. Jung, M. K. Sun, M. L.

Young, H. K. Kim, H. and Han, D.Y. 2006. 16S rDNA analysis of bacterial diversity in three

fractions of cow rumen. Journal of microbiology and biotechnology 16: 92-101.

Chu, W., Lu, F. Zhu, W. and Kang, C. 2011. Isolation and characterization of new potential probiotic

bacteria based on quorum-sensing system. J. App. Microbiol. 110: 202-208.

Ciaramella, P., Corona, M. Ambrosio, R. Consalvo, F. and Persechino, A. 2005. Haematological profile

on non-lactating Mediterranean buffaloes (Bubalus bubalis) ranging in age from 24 months to 14

years. Resea. Veterinar. Sci. 79: 77-80.

Cocolin, L., Foschino, R. Comi, G. and Fortina, M. G. 2007. Description of the bacteriocins produced

by two strains of Enterococcus faecium isolated from Italian goat milk. Food Microbiol. 24: 752-

758.

Cole, N., Purdy, C. and Hutcheson, D. 1992. Influence of yeast culture on feeder calves and lambs. J.

Anim.. Sci 70: 1682-1690.

182

Collins, M. D. and Gibson. G.R. 1999. Probiotics, prebiotics, and synbiotics: approaches for modulating

the microbial ecology of the gut. Ameri. J. Clin. Nutri. 69: 1052-1057.

Cooke, K., Bernard, J. and West. J. 2007. Performance of lactating dairy cows fed whole cottonseed

coated with gelatinized starch plus urea or yeast culture. J. Dairy Sci. 90: 360-364.

Corr, S. C., Li, Y. Riedel, C.U. Otoole, P.W. Hill, C. and Gahan, C.G. 2007. Bacteriocin production as a

mechanism for the antfinfective activity of Lactobacillus salivarius UCC118. P. Natl. Acad. Sci.

USA. 104(18): 7617-7621.

Crittenden, R. G., Martinez, N. R. and Playne, M. J. 2003. Synthesis and utilisation of folate by yoghurt

starter cultures and probiotic bacteria. Int. J.Food. Microbiol. 80: 217-222.

Czerucka, D., Piche, T. and Rampal. P. 2007. Review article: yeast as probiotics–Saccharomyces

boulardii. Alimentary pharmacology and therapeutics. 26: 767-778.

Dawson, K., K. Newman, and J. Boling. 1990. Effects of microbial supplements containing yeast and

lactobacilli on roughage-fed ruminal microbial activities. J. Anim. Sci. 68: 3392-3398.

Dehority, B. and Orpin, C. 1997. Development of, and natural fluctuations in, rumen microbial

populations The rumen microbial ecosystem. pp.196-245. Springer. Netherlands.

Del Piano, M., Morelli, L. Strozzi, G. P. Allesina, S. Barba, M. Deidda, F. Lorenzini, P. Ballare, M.

Montino.F. Orsello, M. Sartori, M. Garello, E. Carmagnola, S.Pagliarulo, M. and Capurso, L.

2006. Probiotics: from research to consumer. Digestive and Liver Disease., 38: S248-S255.

Desnoyers, M., Giger-Reverdin, S. Bertin, G. Duvaux-Ponter, C. and Sauvant, D. 2009. Meta-analysis

of the influence of Saccharomyces cerevisiae supplementation on ruminal parameters and milk

production of ruminants. J. Dairy Sci., 92: 1620-1632.

Devendara, C. 2000. Strategies for improved feed utilization and ruminant productionsystems in the

Asian regions. . Asian. Aust. J. Anim. Sci., 13: 51.

183

Devriese, L., Colque, J. Herdt, P.D. and Haesebrouck, F. 1992. Identification and composition of the

tonsillar and anal enterococcal and streptococcal flora of dogs and cats. J. Appli. Bacteriolo., 73:

421-425.

Di Caro, S., Tao, H.Grillo, A. Elia, C. Gasbarrini, G. Sepulveda, A. R. and Gasbarrini, A. 2005. Effects

of Lactobacillus GG on genes expression pattern in small bowel mucosa. Digestiv. Liver

Disea., 37(5): 320-329.

Di Francia, A., Masucci, F. De Rosa, G. Varricchio, M. and Proto, V. 2008. Effects of Aspergillus

oryzae extract and a Saccharomyces cerevisiae fermentation product on intake, body weight gain

and digestibility in buffalo calves. Anim. Feed Sci. Technolo., 140: 67-77.

Dobicki, A., Pres, J. Luczak, W. and Szyrner, A. 2005. Influence of dried brewery's yeast on body

weight gains, physiological and biochemical indicators of blood and development of the rumen-

micro-organisms in calves. Medycyna Weterynaryjna., 61: 946-949.

Dolezal, P., Dolezal, J. Szwedziak, K. Dvoracek, J. Zeman, L. Tukiendorf, M. and Havlicek, Z. 2012.

Use of Yeast Culture in the TMR of Dairy Holstein Cows. Iran.J. Applied Anim. Sci., 2(1): 51-

56.

Dolezal, P., Dvoracek, J. Dolezal, J. Cermakovz, J. Zeman, L. and Szwedziak, K. 2011. Effect of

feeding yeast culture on ruminal fermentation and blood indicators of Holstein dairy cows. Acta.

Veterinaria. Brno., 80(2): 139-145.

Donovan, D., Franklin, S. Chase, C. and Hippen, A. 2002. Growth and Health of Holstein Calves Fed

Milk Replacers Supplemented with Antibiotics or Enteroguard 1, 2. J. Dairy Sci., 85: 947-950.

Doreau, M. and Jouany, J. 1998. Effect of a Saccharomyces cerevisiae culture on nutrient digestion in

lactating dairy cows. J. Dairy Sci., 81: 3214-3221.

Dowd, S. E., Callaway, T. R. Wolcott, R. D. Sun, Y. McKeehan, T. Hagevoort, R. G. and Edrington, T.

S. 2008. Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-

encoded FLX amplicon pyrosequencing (bTEFAP). BMC Microbiol., 8(1): 125.

184

Driessen, F. M. and Boer, R.D. 1989. Fermented milks with selected intestinal bacteria: a healthy trend

in new products. Neth. Milk Dairy J., 43: 367–382.

Dunne, C. Liam, M . Lisa, M. Gerardine, T. Darrin, M. Sile, H. Maria, F. Sarah,F. Gerald, F. Charles,

D. Barry, K. Gerald,S. Fergus, S. andKevin, C.2001. In vitro selection criteria for probiotic

bacteria of human origin: correlation with in vivo findings. Amer. J. Clin. Nutri. 73: 386-392.

Eastridge, M. 2006. Major advances in applied dairy cattle nutrition. J Dairy Sci., 89: 1311-1323.

Eckburg, P. B., Bik, E. M. Bernstein, C. N. Purdom, E. Dethlefsen, L.Steven, R. Gill, R.G. Nelson, K.E.

and Relman, D. A. 2005. Diversity of the human intestinal microbial flora. Sci., 308(5728):

1635-1638.

Edens, N. K., Reaves, L. A. Bergana, M. S. Reyzer, I. L. O’Mara, P. Baxter, J. H. and Snowden, M. K.

2002. Yeast extract stimulates glucose metabolism and inhibits lipolysis in rat adipocytes in

vitro. J. Nutri., 132(6), 1141-1148.

El-Din, A. N. 2015. Milk production and some blood metabolites responses to yeast supplemenation in

early lactating Hostein dairy cows. Egyptian J. Anim. Prod., 52: 11-17.

El-Ghani, A. A. 2004. Influence of diet supplementation with yeast culture (Saccharomyces cerevisiae)

on performance of Zaraibi goats. Small Rumin. Resea., 52: 223-229.

Elghandour, M. M. Abdelfattah, Z.M.S. Martinez Castaneda, J.S. Camacho, L.M. Kholif, A.E. and Juan

C.V.C. 2015. Direct-fed microbes: A tool for improving the utilization of low quality roughages

in ruminants. J Integr. Agri, 14: 526-533.

Elkins, C. A. and Savage, D. C. 1998. Identification of genes encoding conjugated bile salt hydrolase

and transport in Lactobacillus johnsonii 100-100. J. Bacteriol., 180: 4344-4349.

Ellinger, D., Muller, L. and Glantz, P. 1980. Influence of feeding fermented colostrum and Lactobacillus

acidophilus on fecal flora of dairy calves. J. Dairy Sci., 63: 478-482.

185

Elmer, G. W., Martin, A. W. Horner, K. L. McFarland, L. V. and Levy, R. H. 1999. Survival of

Saccharomyces.boulardii in the rat gastrointestinal tract and effects of dietary fiber. Microb.

Ecol. Health Dis., 11: 29–34.

Engelbrektson, A., Kunin, V. Wrighton, K. C. Zvenigorodsky, N. Chen, F. Ochman, H. and Hugenholtz,

P. 2010. Experimental factors affecting PCR-based estimates of microbial species richness and

evenness. ISME J., 4(5): 642-647.

Enjalbert, F., Garrett, J. Moncoulon, R. Bayourthe, C. and Chicoteau, P. 1999. Effects of yeast culture

(Saccharomyces cerevisiae) on ruminal digestion in non-lactating dairy cows. Anim. Feed Sci.

Technolo., 76: 195-206.

Erasmus, L., Botha, P. and Kistner, A. 1992. Effect of yeast culture supplement on production, rumen

fermentation, and duodenal nitrogen flow in dairy cows. J. Dairy Sci., 75: 3056-3065.

Erasmus, L., Robinson, P. Ahmadi, A. Hinders, R. and Garrett, J. 2005. Influence of prepartum and

postpartum supplementation of a yeast culture and monensin, or both, on ruminal fermentation

and performance of multiparous dairy cows. Anim. Feed Sci. Technol., 122: 219-239.

Facklam, R. 2002. What happened to the streptococci: Overview of taxonomic and nomenclature

changes. Clin. Microbiol. Rev. 15(4): 613-.630.

Fadel, A. M. A. 2007. Effects of supplemental yeast (Saccharomyces cerevisiae) culture on NDF

digestibility and rumen fermentation of forage sorghum hay in Nubian goat’s kids. J. Agric. Biol.

Sci., 3: 133-137.

Fagiolo, A., O .Lai. Alfieri, A Nardon and R. Cavallina. 2004. Enviromntal factors and different

managemtes that influces metaboltic endocrine and immune responses in water bufflo during

lactating. In: Seventh world buffloes congress., Manila, Philipines: 24-26

FAO. 2010. Food and Agricultural Organization. Agriculture Statistics, Retired from

http://www.fao.org.

186

FAO. 2013. Milk and human nutrition in dairy products. Retired from

www.fao.org/docrep/018/i3396e/i3396e.pdf.

Faria-Oliveira, F., Ferreira, C. and Puga. S. 2013. Yeast: World's Finest Chef. INTECH Open Access

Publisher.

Favier, C. F., Vaughan, E. E., De Vos, W. M., and Akkermans, A. D. 2002. Molecular monitoring of

succession of bacterial communities in human neonates. Applied and environmental

microbiology, 68(1), 219-226.

Fayed, A. M., El-Ashry, M.A. Youssef, K. M. Salem, F. A. and Aziz, H. A. 2005. Effect of feeding

falvomycin or yeast as feed supplement on ruminal fermentation and some blood constituents of

sheep in Sinai. Egyptian J. Nutr. Feeds., 8: 619-634.

Fernando, S. C. Purvis, H. T. Najar, F.Z. Sukharnikov, L.O. Krehbiel, C.R Nagaraja, T. G. Roe, B.A.

and Udaya, D. 2010. Rumen microbial population dynamics during adaptation to a high-grain

diet. Appl. Environ. Microbiol. 76(22): 7482-7490.

Ferraretto, L. and Shaver, R. 2012. Meta-analysis: Effect of corn silage harvest practices on intake,

digestion, and milk production by dairy cows. The Professional Animal Scientist 28: 141-149.

Fleet, G. H. 2003. Yeast interactions and wine flavour. Int. J. Food Microbiol., 86: 11-22.

Flint, H. J. 1997. The rumen microbial ecosystem-some recent developments. Trends in Microbiol 5:

483-488.

Flint, H. J., Bayer, E. A. Rincon, M. T. Lamed, R. and White, B. A. 2008. Polysaccharide utilization by

gut bacteria: potential for new insights from genomic analysis. Nature Reviews Microbiolo., 6:

121-131.

Fonty, G. and Chaucheyras-Durand, F. 2006. Effects and modes of action of live yeasts in the rumen.

Biologia 61: 741-750.

187

Fonty, G., P. Gouet, P. Jouany, J. P. and Senaud, J. 1987. Establishment of the microflora and anaerobic

fungi in the rumen of lambs. J. Genen. Microbiol 133: 1835-1843.

Ford, J. A. and Park,C.S. 2001. Nutrionally direcsted compensatory growth enhances heifers

development and lactation potential. J.Dairy Sci., 84: 1669-1678.

Fukushima, M. and Nakano, M. 1996. Effects of a mixture of organisms, Lactobacillus acidophilus or

Streptococcus faecalis on cholesterol metabolism in rats fed on a fat- and cholesterol-enriched

diet. British J. Nutri.,76: 857-867.

Fuller, R. 1989. Probiotics in man and animals. J. appli. bacteriolo., 66: 365-378.

Fuller, R. 1992. Probiotics.The Scientific Basis. Chapman and Hall, London.

Galıp, N. 2006. Effect of supplemental yeast culture and sodium bicarbonate on ruminal fermentation

and blood variables in rams. J. Animl. Physio. Animal.Nutr. 90: 446-452.

Galvao, K. N. Santos, J. E. Coscioni, A. Villaseñor, M. Sischo, W. M. and Berge, A. C. B. 2005. Effect

of feeding live yeast products to calves with failure of passive transfer on performance and

patterns of antibiotic resistance in fecal Escherichia coli. Repro. Nutr. Devel., 45(4): 427-440.

Gao, X. W., Mubasher, M. Fang, C. Y. Reifer, C. and Miller. L.E. 2010. Dose-Response Efficacy of a

Proprietary Probiotic Formula of Lactobacillus acidophilus CL1285 and Lactobacillus casei

LBC80R for Antibiotic-Associated Diarrhea and Clostridium difficile-Associated Diarrhea

Prophylaxis in Adult Patients. Am. J. Gastroenterol., 105: 1636-1641.

Gardiner, G. E., Casey, P. G. Casey, G. Lynch, P. B. Lawlor, P. G. Hill, C. Fitzgerald, G.F. Catherine, S.

and Ross, R.P. 2004. Relative ability of orally administered Lactobacillus murinus to

predominate and persist in the porcine gastrointestinal tract. Appl. Environ. Microbiol., 70(4):

1895-1906.

Ghazanfar, S., M. Anjum, A. Azim, and I. Ahmed. 2015. Effcets of dietry supplemenation of yeast

(Sacchromyces cerevisiae) culture on growth performance, blood parameters, nutrient

digestibility and fecsl flora od dairy heifers. J Anim. Plant Sci., 25: 53-59.

188

Gijzen, H. J., Lubberding, H. J. Gerhardus, M. J. T. and Vogels, G. D. 1988. Contribution of rumen

protozoa to fibre degradation and cellulase activity in vitro. FEMS Microbiol. Lett., 53: 35-43.

Gilliland, S. E. 1990. Health and Nutritional Benefits from Lactic-Acid Bacteria. Fems Microbiol. Lett.,

87: 175-188.

Gilliland, S. E., Nelson, C. R. and Maxwell. C. 1985. Assimilation of Cholesterol by

LactobacillusAcidophilus. Appl. Environ. Microbiol., 49: 377-381.

Gilliland, S. E. and Speck, M. L. 1977. Deconjugation of bile acids by intestinal lactobacilli. Appl.

Environ. Microbiol. 33: 15–18.

Giraffa, G. 2003. Functionality of enterococci in dairy products. Int. J. Food Microbiol., 88: 215-222.

Girard, I. D. and Dawson, K. A. 1995. Stimulation of ruminal bacteria by different fractions derived

from cultures of Saccharomyces cerevisiae strain 1026. J. Anim. Sci., 73: 264.

Golowczyc, M. A., Mobili, P. Garrote, G. L. Abraham, A. G. and De Antoni, G. L. 2007. Protective

action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar

Enteritidis. Int. J. Food Microbiol., 118: 264-273.

Gong, J., Lee, W. Garnsworthy, P. and Webb, R. 2002. Effect of dietary-induced increases in circulating

insulin concentrations during the early postpartum period on reproductive function in dairy cows.

Repr.,123: 419-427.

GOP. 2011. Government of Pakistan. Economic Survey of Pakistan, 2010-2011. Economic Advisor’s

Wing. Ministry of Finance, Pakistan.

Guarner, F. and Malagelada, J. R. 2003. Gut flora in health and disease. Lancet., 361: 512-519.

Guillot, J. F. 1998. Les probiotiques en alimentation animale. Cahiers Agricultures 7: 49-54.

Gunther, K. D. 1989. Alltech Tech. Publication, Nicholasville, , KY.

189

Haakensen, M., Dobson, C. M. Hill, J. E. and Ziola, B. 2009. Reclassification of Pediococcus

dextrinicus (Coster and White 1964) Back 1978 (Approved Lists 1980) as Lactobacillus

dextrinicus comb. nov., and emended description of the genus Lactobacillus. Int. J. Syst Evol.

Micr., 59: 615-621.

Halasz, A. and Lasztity, R. 1990. Use of yeast biomass in food production. CRC Press.

Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for

Windows 95/98/NT. In: Nucleic acids symposium series. p 95-98.

Haller, D., Colbus, H. Ganzle, M. G. Scherenbacher, P. Bode, C. and Hammes, W. P. 2001. Metabolic

and functional properties of lactic acid bacteria in the gastro-intestinal ecosystem: A comparative

in vitro study between bacteria of intestinal and fermented food origin. Syst. Appl. Microbiol.,

24(2): 218-226.

Hammes, W. P. Vogel, and R. F. 1995. The Genera of Lactic Acid Bacteria. Blackie Academic and

Professional, London.

Hanjra, S. H., David, . J. B. and Akhtar, M.J. 1995. Fodder production. Food and Agricultural

Organization. Small dairy holder dairy development in Punjab,Pakistan. FAO.

Harris, B., Dorminey, D. Smith, W. Van Horn, H. and Wilcox, C. 1992. Effects of feather meal at two

protein concentrations and yeast culture on production parameters in lactating dairy cows. J.

Dairy Sci., 75: 3524-3530.

Hasnain, H. 1983. Feed- The key to more food in Pakistan. In: FAO/PARC workshop on least cost

formulation, Islamabad.

Heinrichs, A., Jones, C. and Heinrichs, B. 2003. Effects of mannan oligosaccharide or antibiotics in

neonatal diets on health and growth of dairy calves. J. Dairy Sci., 86: 4064-4069.

Heyman, M. and Menard, S. 2002. Probiotic microorganisms: how they affect intestinal

pathophysiology. Cell. Mol. Life Sci., 59: 1151-1165.

190

Hill, J. E., Baiano, J. C and Barnes, A.C. 2009. Isolation of a novel strain of Bacillus pumilus from

penaeid shrimp that is inhibitory against marine pathogens. J. Fish Dis. 32: 1007-1016.

Hillman, K., Lloyd, D. and Williams, A. 1985. Use of a portable quadrupole mass spectrometer for the

measurement of dissolved gas concentrations in ovine rumen liquor in situ. Current Microbiol.,

12: 335-339.

Hobson, P. N., and C. S. Stewart. 1997. The rumen microbial ecosystem. Springer Science & Business

Media.

Hofmann, A. F. 1994. The liver: biology and pathobiology. Raven Press, Ltd, New York, N.Y.

Hofmann, A. F. and Mysels, K. J. 1992. Bile-acid solubility and precipitation in vitro and in vivo - the

role of conjugation, pH, and Ca2+ Ions. J. Lipid Res., 33: 617-626.

Holzapfel, W. H., Haberer, P. Snel, J. Schillinger, U. and In't Veld, H.H. 1998. Overview of gut flora

and probiotics. Int. J Food Microbiol., 41(2): 85-101.

Homayouni, A., Azizi, A. Ehsani, M. R. Yarmand, M. S. and Razavi, S. H. 2008. Effect of

microencapsulation and resistant starch on the probiotic survival and sensory properties of

synbiotic ice cream. Food Chem., 111: 50-55.

Horst, R. L. 1986. Regulation of calcium and phosphorus homeostasis in the dairy cow. J. Dairy Sci.,

69: 604-616.

Hosono, A. 1999. Bile tolerance, taurocholate deconjugation, and binding of cholesterol by

Lactobacillus gasseri strains. J. Dairy Sci., 82: 243-248.

Hossain, F., M. Islam, A. Ara, and N. Iliyas. 2014. Supplementing probiotics (Saccharomyces

cerevisiae) in multiparous crossbred cows ration provoke milk yield and composition. Online J.

Anim. Feed Resear., 4: 18-24.

191

Hossain, S. A., Parnerkar, S. Haque, N. Gupta, R. S. Kumar, D. and Tyagi, A. K. 2012. Influence of

dietary supplementation of live yeast (Saccharomyces cerevisiae) on nutrient utilization, ruminal

and biochemical profiles of Kankrej calves. Int. J.Appl.Anim.Sci., 1(1): 30-38.

Hsieh, M. L. and Chou, C. C. 2006. Mutagenicity and antimutagenic effect of soymilk fermented with

lactic acid bacteria and bifidobacteria. Int. J. Food Microbiol., 111: 43-47.

Huber, J. T. 1997. Probiotics in cattle. Probiotics: Applications and Practical Aspects. Chapman and

Hall, London, 162–186 pp.

Hultgren, J. and Svensson, C. 2009. Lifetime risk and cost of clinical mastitis in dairy cows in relation to

heifer rearing conditions in southwest Sweden. J. Dairy Sci., 92: 3274-3280.

Hungate, R. E. 1950. The anaerobic mesophilic cellulolytic bacteria. Bacteriolo. Revie., 14: 1-49.

Hussain, M., Ghafoor, A. and Saboor, A. 2010. Factors affecting milk production in buffaloes: a case

study. Pak.Vertinar. J. 30: 115-117.

Hutjens, M. 2003. Economics of feed additives. In: Proc Penn State Dairy Cattle Nutrition Workshop,

Grantville, PA (www. das. psu. edu/dairynutrition/education/workshop2003/)

Hutjens, M. F. 1991. Feed additives. Vet. Clin. North. Am: Food Animal Practice 7: 525.

IFCN. 2014. Internation Farm Comparison Network, Retrieved March 10, 2014, from

www.ifcndairy.org.

ILRI. 1995. Globalagenda for livestock research. Proc. of Consultation, Int. Livestock Res. Ins.,

Nairobi, Kenya.

Iqbal, M. A., Iqbal, A. Akbar, N. Khan, H. Z. and Abbas, R. N. 2015. A Study on Feed Stuffs Role in

Enhancing the Productivity of Milch Animals in Pakistan-Existing Scenario and Future Prospect.

Global Veterinaria., 14 (1): 23-33.

192

Iwasaki, A. and Kelsall, B. L. 1999. Freshly isolated Peyer's patch, but not spleen, dendritic cells

produce interleukin 10 and induce the differentiation of T helper type 2 cells. J.Exp. Med., 190:

229-239.

Jabbar, L. 2004. Effect of different dietary energy levels on some growth and reproductive aspects and

their relation with age of maturity in growing buffalo heifers, PhD Thesis, University of the

Punjab, Lahore.

Jabbar, M., M. Anjum, S. Rehman, and W. Shahzad. 2006. Comparative efficiency of sunflower meal

and cottonseed cakes in the feed of crossbred calves for meat production. Pakistan Vet. J., 26(3):

126-128.

Jatkauskas, J. and Vrotniakiene, V. 2010. Effects of probiotic dietary supplementation on diarrhoea

patterns, faecal microbiota and performance of early weaned calves. Veterinarni. Medicina., 55:

494-503.

Jay, J. M. 1982. Antimicrobial Properties of Diacetyl. Appl. Enviro. Microbiol., 44: 525-532.

Jenkins, T. and McGuire, M. 2006. Major advances in nutrition: impact on milk composition. J. Dairy

Sci., 89: 1302-1310.

Jenny, B., Vandijk, H. and Collins, J. 1991. Performance and fecal flora of calves fed a Bacillus subtilis

concentrate. J. Dairy Sci., 74: 1968-1973.

Joblin, K. 1989. Physical disruption of plant fibre by rumen fungi of the Sphaeromonas group The Roles

of Protozoa and Fungi in Ruminant Digestion. p 259-260. Penambul Armidale, NSW.

Johnson-Henry, K. C., Donato, K. A. Shen-Tu, G. Gordanpour, A. and Sherman, P.A. 2008.

Lactobacillus rhamnosus strain GG prevents enterohemorrhagic Escherichia coli O157 : H7-

Induced changes in epithelial barrier function. Infec. Immu., 76: 1340-1348.

Johnson, K. A. and Johnson, D. E. 995. Methane emissions from cattle. J. Aanim. Sci., 73: 2483-2492.

193

Jonsson, E. and Olsson, I. 1985. The Effect on Performance, Health and Fecal Microflora of Feeding

Lactobacillus Strains to Neonatal Calves. Swed. J. Agr. Res., 15: 71-76.

Jouany, J. P. and Morgavi, D. 2007. Use of ‘natural’products as alternatives to antibiotic feed additives

in ruminant production.1443-1466.

Jouany, J. P. 2001. Dvacet let výzkumu kvasinkových kultur a jejich masivní nástup v souèasné dobì ve

výzivì pøez- výkavcù. . Evropského Pøednáškového Turné Firmy Alltech, Brno. , Brno.

Jouany, J. P. 2006. Optimizing rumen functions in the close-up transition period and early lactation to

drive dry matter intake and energy balance in cows. Anim. Repro.Sci., 96: 250-264.

Jouany, J. P., Mathieu, F. Senaud, J. Bohatier, J. Bertin, G. and Mercier, M. 1999. Influence of protozoa

and fungal additives on ruminal pH and redox potential. Presented at 9. International Symposium

on Ruminant Physiology, Pretoria, ZAF (1999-10-17 - 1999-10-

22). http://prodinra.inra.fr/record/159212.

Kalmus, P., Orro, T. Waldmann, A. Lindjärv, R. and Kask, K. 2009. Effect of yeast culture on milk

production and metabolic and reproductive performance of early lactation dairy cows. Acta

Veterinaria Scandinavica 51: 32.

Kamel, H., Sekine, J. El-Waziry, A. and Yacout, M. 2004. Effect of Saccharomyces cerevisiae on the

synchronization of organic matter and nitrogen degradation kinetics and microbial nitrogen

synthesis in sheep fed Berseem hay (Trifolium alexandrinum). Small Rumi. Res., 52: 211-216.

Kamra, D. 2005. Rumen microbial ecosystem. Curr. Sci., 89: 124-135.

Kandler, O. 1986. Cell-Wall Chemistry and Early Evolution of Organisms. Origins Life Evol. B., 16:

253-254.

Kawakami, S. I., Yamada, T. Nakanishi, N. and Cai, Y. 2010. Feeding of lactic acid bacteria and yeast

on growth and diarrhea of Holstein calves. J. Anim. Veterinar. Adv., 9: 1112-1114.

194

Kellems, R., Lagerstedt, A. and Wallentine, M. 1990. Effect of feeding Aspergillus oryzae fermentation

extract or Aspergillus oryzae plus yeast culture plus mineral and vitamin supplement on

performance of Holstein cows during a complete lactation. J. Dairy Sci., 73: 2922-2928.

Kelton, D. F., Lissemore, K.D. and Martin, R.E. 1998. Recommendations for recording and calculating

the incidence of selected clinical diseases of dairy cattle. J Dairy Sci., 81: 2502-2509.

Khaliq, T. and Z. Rahman. 2010. Haematological studies of Nili-Ravi buffaloes injected with

recombinant bovine somatotropin. Pak. Vet. J., 30: 53-57.

Kim, M., Morrison, M. and Yu, Z. 2011. Evaluation of different partial 16S rRNA gene sequence

regions for phylogenetic analysis of microbiomes. J. Microbiolo. Meth., 84: 81-87.

Kishan, K. M. and Ramana, D. 2008. Effect of supplementation of yeast culture to calves fed with

complete diet. Indian Veterinar. J., 85: 667-669.

Klaenhammer, T. R. 1988. Bacteriocins of Lactic Acid Bacteria. Biochimie., 70: 337-349.

Klaenhammer, T. R., Kleerebezem, M. Kopp, M. V. and Rescigno, M. 2012. The impact of probiotics

and prebiotics on the immune system. Natu. Revi. Immuno., 12: 728-734.

Klaver, F. A. M. and Vandermeer, R 1993. The assumed assimilation of cholesterol by Lactobacilli and

Bifidobacterium-Bifidum is due to their bile dalt-deconjugating activity. Appl. Enviro.

Microbiol., 59: 1120-1124.

Klein, G., Pack, A. Bonaparte, C. and Reuter, G. 1998. Taxonomy and physiology of probiotic lactic

acid bacteria. Int. J. Food Microbiol., 41: 103-125.

Klein, R. D., Favreau, M. A. Hui, Y. and Khachatourians, G. 1995. The Candida species: biochemistry,

molecular biology, and industrial applications. In Food Biothechnology Microorganisms, eds

Hui, Y. H and. Khachatourians, G. G. VCH publishers. New York: 297-371.

195

Kobayashi, Y., Shinkai, T. and Koike, S. 2008. Ecological and physiological characterization shows that

Fibrobacter succinogenes is important in rumen fiber digestion - review. Folia Microbiol., 53:

195-200.

Kossaibati, M. and Esslemont, R. 1997. The costs of production diseases in dairy herds in England.

Veterinar. J., 154: 41-51.

Kowalik, M. K., Rekawiecki, R. and Kotwica, J. 2013. The putative roles of nuclear and membrane-

bound progesterone receptors in the female reproductive tract. Repro. Bio., 13: 279-289.

Kulwichit, W., Nilgate, S. Chatsuwan, T. Krajiw, S. Unhasuta, C. and Chongthaleong, A. 2007.

Accuracies of Leuconostoc phenotypic identification: A comparison of API systems and

conventional phenotypic assays. BMC. Infect. Dis., 7(69).

Kumar, D. S., Prasad, J. R. and Rao, E. R. 2011. Effect of dietary inclusion of yeast culture

(Saccharomyces cerevisiae) on growth performance of graded murrah buffalo bull calves. Buffa.

Bulletin., 30: 63-66.

Kumar, U., Sareen, V. K. and Singh, S. 1994. Effect of Saccharomyces cerevisiae yeast culture

supplement on ruminal metabolism in buffalo calves given a high concentrate diet. Anim. Sci.,

59: 209-215.

Kumar, U., Sareen, V.K. and Singh, S. 1997. Effect of yeast culture supplement on ruminal microbial

populations and metabolism in buffalo calves fed a high roughage diet. J. Sci. Food Agri., 73:

231–236.

Kung, LKreck, E.M. Tung, R.S. Hessions, A.O. Shaperd, A.C. Cohen, M.A. Swain, H.E. Leeedle,

J.A.Z. 1997. Effects of a live yeast culture and enzymes on in vitro ruminal fermentation and

milk production of dairy cows. J. Dairy Sci., 80: 2045-2051.

Kurtzman, C., Fell, J. and Boekhout, T. 2011. Definition, classification and nomenclature of the yeasts.

The yeasts a taxonomic study: 3-9.

196

La Ragione, R. M. and Woodward, M. J. 2003. Competitive exclusion by Bacillus subtilis spores of

Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens.

Veterinar. Microbiol., 94(3): 245-256.

Lammers, B. and Heinrichs, A. 2000. The response of altering the ratio of dietary protein to energy on

growth, feed efficiency, and mammary developmentin rapidly growing prepubertal heifers. J.

Dairy Sci., 83: 977-983.

Lammers, K. M., Helwig, U. Swennen, E. Rizzello, F. Venturi, A. Caramelli, E. Kamm, M.A. Brigidi,

P. Gionchetti, P. and Campieri. M. 2002. Effect of probiotic strains on interleukin 8 production

by HT29/19A cells. Am. J. gastroenterol., 97(5): 1182-1186.

Lascano, G., Zanton, G. and Heinrichs, A. 2009. Concentrate levels and Saccharomyces cerevisiae

affect rumen fluid-associated bacteria numbers in dairy heifers. Livest Sci., 126: 189-194.

Lascano, G. J., A. Heinrichs, and J. Tricarico. 2012. Substitution of starch by soluble fiber and

Saccharomyces cerevisiae dose response on nutrient digestion and blood metabolites for

precision-fed dairy heifers. J. Dairy Sci., 95: 3298-3309.

Lee, S. S., Choi, C. K. Ahn, B. H. Moon, Y. H. Kim, C. H. and Ha, J. K. 2004. In vitro stimulation of

rumen microbial fermentation by a rumen anaerobic fungal culture. Anim. Feed Sci. Technol.,

115: 215-226.

Lehloenya, K., Krehbiel, C. Mertz, K. Rehberger, T. and Spicer, L. 2008. Effects of propionibacteria

and yeast culture fed to steers on nutrient intake and site and extent of digestion. J. Dairy Sci.,

91: 653-662.

Lesmeister, K. and Heinrichs. A. 2004. Effects of corn processing on growth characteristics, rumen

development, and rumen parameters in neonatal dairy calves. J. Dairy Sci., 87: 3439-3450.

Ley, R. E., Turnbaugh, P. J. Klein, S. and Gordon, J. I. 2006. Microbial ecology: human gut microbes

associated with obesity. Nature., 444: 1022-1023.

197

Li, J., Xing, J., Li, D. Wang, X. Zhao, L. Lv, S. and Huang, D. 2005. Effects of β-glucan extracted from

Saccharomyces cerevisiae on humoral and cellular immunity in weaned piglets. Archives Anim.

Nutr., 59(5): 303-312.

Lim, S. M. and Im. D.S. 2009. Screening and Characterization of Probiotic Lactic Acid Bacteria Isolated

from Korean Fermented Foods. J. Microbiol. Biotechn., 19: 178-186.

Lin, M. Y. and Chen, T. W. 2000. Reduction of cholesterol by Lactobacillus acidophilus in culture

broth. J. Food Drug Anal., 8: 97-102.

Lin, P. P., Hsieh, Y. M. and Tsai, C. C. 2009. Antagonistic activity of Lactobacillus acidophilus RY2

isolated from healthy infancy feces on the growth and adhesion characteristics of

enteroaggregative Escherichia coli. Anaerobe., 15: 122-126.

Liong, M. T. 2008. Safety of probiotics: translocation and infection. Nutr. Rev., 66: 192-202.

Liong, M. T. 2011. Probiotics: biology, genetics and health aspects. Springer-Verlag, Berlin.

Liong, M. T. and Shah, N.P. 2005. Acid and bile tolerance and cholesterol removal ability of

lactobacilli strains. J. Dairy Sci., 88: 55-66.

Lodder, J. 1974. The Yeast, A Taxonomic Study. North Holland Company,

Loesche, W. J. 1969. Oxygen sensitivity of various anaerobic bacteria. Appl. Microbiol., 18: 723-727.

Longuski, R., Ying, Y. and Allen, M. 2009. Yeast culture supplementation prevented milk fat depression

by a short-term dietary challenge with fermentable starch. J. Dairy Sci., 92: 160-167.

Lukas, F., Koppova, I. Kudrna, V. and Kopecny, J. 2007. Postnatal development of bacterial population

in the gastrointestinal tract of calves. Folia. Microbiol., 52: 99-104.

Lye, H. S., Kuan, C.Y. Ewe, J.A. Fung, W.Y. and Liong, M. T. 2009. The Improvement of

Hypertension by Probiotics: Effects on Cholesterol, Diabetes, Renin, and Phytoestrogens. Int. J.

Mol. Sci., 10: 3755-3775.

198

Magalhaes, V. J. A., Susca, F. Lima, F. S. Branco, A. F. Yoon, I. and Santos, J. E. P. 2008. Effect of

feeding yeast culture on performance, health, and immunocompetence of dairy calves. J. Dairy

Sci., 91(4): 1497-1509.

Malago, J. J., Koninkx, J. F. and Marinssek-Logar, R. 2014. Probiotic bacteria and enteric Infections.

Springer. Dordrecht Heidelberg London New York.

Mann, G. V. 1977. Factor in yogurt which lowers cholesteremia in man. Atherosclerosis 26: 335-340.

Mansoor, K. M. C., Muhammad, S. Ashraf, I. and Ghafoor. U. 2012. Farmer’s perceptions of livestock

production practices introduced by Punjab Rural Support Program (PRSP). Pak. J. Agric. Sci. 49:

233-235.

Marden, J. P., Julien, C. Monteils, V. Auclair, E. Moncoulon, R. and Bayourthe, C. 2008. How does live

yeast differ from sodium bicarbonate to stabilize ruminal pH in high-yielding dairy cows? J.

Dairy Sci., 91(9): 3528-3535.

Marrero, Y., Castillo, Y. Ruiz, O. Burrola, E. and Angulo, C. 2015. Feeding of yeast (Candida spp.)

improves in vitro ruminal fermentation of fibrous substrates. J. Integrative Agri., 14: 514-519.

Martin, S. A. and. Nsbet, D. J. 1992. Effect of direct-fed microbials on rumen microbial fermentation. J.

Dairy Sci., 75: 1736-1744.

Masek, T., Zeljko, M. Hrvoje, V. Neven, A. Natasa, M. Zvonko, S. Natalija, F. and Snjezan, P. 2008.

Influence of live yeast culture (Saccharomyces cerevisiae) on milk production and composition,

and blood biochemistry of grazing dairy ewes during the milking period. Acta Veterinaria

Brno 77(4) 547-554.

Meddings, J. 2008. The significance of the gut barrier in disease. Gut., 57: 438-440.

Meller, R., Firkins, F., and Gehman, A. 2014. Efficacy of live yeast in lactating dairy cattle. Professi.

Anim. Scient., 30: 413-417.

199

Mestecky, J. and Russell, M.W. 1998. Passive and active protection against disorders of the gut. Vet.

Quart., 20: 83–87.

Metschnikoff, E. 1907. The prolongation of life. Optimisticstudies. William Heinemann, London, UK.

Michalet-Doreau, B. and Morand, D. 1997. Effect of yeast culture, Saccharomyces cerevisiae CNCM I-

1077, on ruminal fermentation during adaptation to high-concentrate feeding. In: Proceedings

from 4th Renc. Rech. Rum, Volume 4. p 121.

Milewski, S. and Sobiech, P. 2009. Effect of dietary supplementation with Saccharomyces cerevisiae

dried yeast on milk yield, blood biochemical and haematological indices in ewes. Bull. Vet. Inst.

Pulawy., 53: 753-758.

Miller-Webster, T., Hoover, W. Holt, M. and Nocek, J. 2002. Influence of yeast culture on ruminal

microbial metabolism in continuous culture. J. Dairy Sci., 85: 2009-2014.

Mir, Z., and P. Mir. 1994. Effect of the addition of live yeast (Saccharomyces cerevisiae) on growth and

carcass quality of steers fed high-forage or high-grain diets and on feed digestibility and in situ

degradability. J. Anim. Sci., 72: 537-545.

Moallem, U., Lehrer, H. Livshitz, L. Zachut, M. and Yakoby, S. 2009. The effects of live yeast

supplementation to dairy cows during the hot season on production, feed efficiency, and

digestibility. J. Dairy Sci., 92: 343-351.

Moayyedi, P., Ford, A. C. Talley, N. J. Cremonini, F. Foxx-Orenstein, A. E. Brandt, L. J. and Quigley,

E. M. 2010. The efficacy of probiotics in the treatment of irritable bowel syndrome: a systematic

review. Gut., 59(3): 325-332.

Moore, K. J., and R. D. Hatfield. 1994. Carbohydrates and forage quality. In: Forage Quality,

Evaluation, and Utilization. Fahey, G. C. Jr. M. C. Collins, Mertens, D.R. and Moser, L.E. eds.

ASA-CSSA-SSSA, Madison, WI. pp. 229-280.

Moser, S. A. and Savage, D. C. 2001. Bile salt hydrolase activity and resistance to toxicity of conjugated

bile salts are unrelated properties in lactobacilli. Appl. Enviro. Microbiol. 67: 3476-3480.

200

Mountfort, D. O., Asher, R. A. and Bauchop, T. 1982. Fermentation of Cellulose to Methane and

Carbon Dioxide by a Rumen Anaerobic Fungus in a Triculture with Methanobrevibacter sp.

Strain RA1 and Methanosarcina barkeri. Applied and environmental microbiology 44: 128-134.

Mulligan, F. and Doherty, M. 2008. Production diseases of the transition cow. Veterinar. J. 176: 3-9.

Musa, H., Wu, S. Zhu, C. Seri, H. and Zhu, G. 2009. The potential benefits of probiotics in animal

production and health. J. Anim. Vet. Adv., 8: 313-321.

Myllyluoma, E., Kajander, K. Mikkola, H. S. Kyronpalo, S. Rasmussen, M. Kankuri, E. Sipponen, P.

Vapaatalo, H. and Korpela, R. 2007. Probiotic intervention decreases serum gastrin-17 in

Helicobacter pylori infection. Digest. liver dis. 39(4): 516-523.

Nandi, S., Kumar, V. G. Manjunatha, B. Ramesh, H. and Gupta, P. 2008. Follicular fluid concentrations

of glucose, lactate and pyruvate in buffalo and sheep, and their effects on cultured oocytes,

granulosa and cumulus cells. Theriogenology 69: 186-196.

Newbold, C., McIntosh, F. and Wallace. R. 1998. Changes in the microbial population of a rumen-

simulating fermenter in response to yeast culture. Can. J.Anim. Sci., 78: 241-244.

Newbold, C., Wallace, R. Chen, X. and F. McIntosh. F. 1995. Different strains of Saccharomyces

cerevisiae differ in their effects on ruminal bacterial numbers in vitro and in sheep. J. Anim. Sci.,

73: 1811-1818.

Newbold, C., Wallace, R. and McIntosh, F. 1996. Mode of action of the yeast Saccharomyces cerevisiae

as a feed additive for ruminants. Brit. J.Nutr., 76: 249-261.

Newbold, C., Williams, P. McKain, N. Walker, A. and Wallace, R. 1990. The effects of yeast culture on

yeast numbers and fermentation in the rumen of sheep. Proc. Nutr. Soc., 49: 47.

Newbold, C. J., Wallace, R. J. Chen, . X. B. and Mcintosh, F. M. 1995. Different strains of

Saccharomyces cerevisiae differ in their effects on ruminal bacterial numbers in vitro and in

sheep. J. Anim. Sci., 73: 1811-1818.

201

Ng, S. C., Hart, A.L. Kamm, M.A. Stagg, A.J. and Knight, S. C. 2009. Mechanisms of Action of

Probiotics: Recent Advances. Inflamm. Bowel. Dis. 15: 300-310.

Nicolosi, R., Bell, S. J.,Bistrian, B. R. Greenberg, I. Forse, R. A and Blackburn, G. L. 1999. Plasma lipid

changes after supplementation with β-glucan fiber from yeast. Am. J. Clin.nutr., 70(2): 208-212.

Nikkhah, A., Bonadaki, M. D. and Zali, A. 2004. Effects of feeding yeast (Saccharomyces cerevisiae)

on productive performance of lactating Holstein dairy cow. Iranian J. Agric. Sci., 35: 53-60.

Nkya, R., Kessy, B. M. Lyimo, Z. C. Msangi, B. S. J. Turuka, F. and Mtenga, K. 2007. Constraints on

smallholder market oriented dairy systems in the north eastern coastal region of Tanzania. Trop.

Anim.Healh. Prod., 39(8): 627-636.

Nocek, J. and Kautz, W. 2006. Direct-fed microbial supplementation on ruminal digestion, health, and

performance of pre-and postpartum dairy cattle. J. Dairy Sci., 89: 260-266.

Nocek, J., Kautz, W. Leedle, J. and Block, E. 2003. Direct-fed microbial supplementation on the

performance of dairy cattle during the transition period. J. Dairy Sci., 86: 331-335.

Noh, D. O., Kim, S.H. and Gilliland, S.E. 1997. Incorporation of cholesterol into the cellular membrane

of Lactobacillus acidophilus ATCC 43121. J. Dairy Sci., 80: 3107-3113.

Novik, G., Samartsev, A. Astapovich, N. Kavrus, M. and Mikhalyuk, A. 2006. Biological activity of

probiotic microorganisms. Appl. Biochemis. Microbiol., 42: 166-172.

NRC. 2001. Nutrient requirements of dairy cattle. National Reasearch Council. Natioal academy press,

Washeinton, DC, USA.

Nurmi, E. and Rantala, M. 1973. New aspects of Salmonella infection in broiler production. Natur., 241:

210-211.

Nursoy, H. and Baytok, E. 2003. The effects of baker's yeast (Saccharomyces cerevisiae) in dairy cow

diets on milk yield, some rumen fluid parameters and blood metabolites of dairy cow diets.

Turkish J. Veterinar. Anim. Sci., 27: 7-13.

202

Oeztuerk, H. 2009. Effects of live and autoclaved yeast cultures on ruminal fermentation in vitro. J

Anim. Feed Sci., 18: 142-150.

Oikonomou, G., Teixeira, A. G. V., Foditsch, C., Bicalho, M. L., Machado, V. S., & Bicalho, R. C.

(2013). Fecal microbial diversity in pre-weaned dairy calves as described by pyrosequencing of

metagenomic 16S rDNA. Associations of Faecalibacterium species with health and

growth. PLoS One, 8(4), e63157.

Orpin, C. G. and Joblin, K. N. 1988. The rumen anaerobic fungi. In: The rumen microbial ecosystem.

Elsevier. Applied Science Publishers, London, U.K, pp 129–150.

Otte, J. M. and Podolsky, D. K. 2004. Functional modulation of enterocytes by gram-positive and gram-

negative microorganisms. Am. J. Physiol. Gastroint. Liver Physiolo., 286: 613-626.

Economic Survey of Pakistan, 2013-14. Ministry of Finance. Economic Advisors Wing, Islamabad.

Economic Survey of Pakistan, 2008-9. Ministry of Finance. Economic Advisors Wing, Islamabad.

Panda, A., Singh, R. and Pathak, N. 1995. Effect of dietary inclusion of Saccharomyces cerevisiae on

growth performance of crossbred calves. J. Appl. Anim. Res., 7: 195-200.

Parvez, S., Malik, K.A. Kang, S.A. and Kim, H.Y. 2006. Probiotics and their fermented food products

are beneficial for health. J. Appl. Microbiol., 100: 1171-1185.

Patrignani, F., Lanciotti, R. Mathara, J. M. Guerzoni, M. E. and Holzapfel, W. H. 2006. Potential of

functional strains, isolated from traditional Maasai milk, as starters for the production of

fermented milks. Int. J. Food Microbiol., 107: 1-11.

Paul, S. S., Kamra, D. N. Sastry, V. R. Sahu, N. P. and Kumar, A. 2003. Effect of phenolic monomers

on biomass and hydrolytic enzyme activities of an anaerobic fungus isolated from wild nil gai

(Baselophus tragocamelus). Lett. Appl. Microbiol., 36: 377-381.

Paul, S. S., Kamra, D.N. Sastry, V.R.B. Sahu, N.P. and Agarwal, N. 2004. Effect of administration of an

anaerobic gut fungus isolated from wild blue bull (Boselaphus tragocamelus) to buffaloes

203

(Bubalus bubalis) on in vivo ruminal fermentation and digestion of nutrients. Anim. Feed Sci.

Technol., 115: 143-157.

Pereira, D. I. A., McCartney, A. L. and Gibson, G. R. 2003. An in vitro study of the probiotic potential

of a bile-salt-hydrolyzing Lactobacillus fermentum strain, and determination of its cholesterol-

lowering properties. Appl. Enviro. Microbiol. 69: 4743-4752.

Perez-Cano, F. J., Dong, H.L. and Yaqoob, P. 2010. In vitro immunomodulatory activity of

Lactobacillus fermentum CECT5716 and Lactobacillus salivarius CECT5713: two probiotic

strains isolated from human breast milk. Immunobiol. 215: 996-1004.

Perrin PK, D. W., Moscandi, E.R. and Anderson, J.R.1979. From Organic data to farmer

recommendation. Econom. Train. Manu. Info. Bullet., 27, CYMMIT, Mexico.

Petrof, E. O., Dhaliwal, R. Manzanares, W. Johnstone, J. Cook, D. and Heyland, D. K. 2012.

Probiotics in the critically ill: a systematic review of the randomized trial evidence. Crit. Care

Med., 40 (12): 3290-3302.

Piard, J. C. and Desmazeaud, M. 1991. Inhibiting factors produced by lactic-acid bacteria .1. oxygen

metabolites and catabolism end products. Lait., 71: 525-541.

Piard, J. C. and Desmazeaud, M. 1992. Inhibiting factors produced by lactic-acid bacteria .2.

bacteriocins and other antibacterial substances. Lait., 72: 113-142.

Pinos-Rodriguez, J. M., Robinson, P. H. Ortega, M. E. Berry, S. L. Mendoza, G. and Barcena, R. 2008.

Performance and rumen fermentation of dairy calves supplemented with Saccharomyces

cerevisiae 1077 or Saccharomyces boulardii 1079. Anim. Feed Sci.Technol., 140(3): 223-232.

Piva, G., Belladonna, S. Fusconi, G. and Sicbaldi, F. 1993. Effects of yeast on dairy cow performance,

ruminal fermentation, blood components, and milk manufacturing properties. J. Dairy Sci., 76:

2717-2722.

204

Plata, F. P., G. D. M. M, J. R. Barcena-Gama, and S. G. M. 1994. Effect of a yeast culture

(Saccharomyces cerevisiae) on neutral detergent fiber digestion in steers fed oat straw based

diets. Anim. Feed Sci. Technol., 49: 203-210.

Puniya, A. K., Abdelfattah, Z. M. Kumar, S. Sumit, S. D. Gareth, W.G. W. Puniya, M. Ravella, R.

Kumar, N. Dhewa, T. and Kumar, R. 2015. Role of live microbial feed supplements with

reference to anaerobic fungi in ruminant productivity: A review.J. Integrative Agri., 14(3): 550-

560.

Putnam, D. E., Schwab, C. G. Socha, M. T. Whitehouse, N. L. Kierstead, N. A. and Garthwaite, B. D.

1997. Effect of yeast culture in the diets of early lactation dairy cows on ruminal fermentation

and passage of nitrogen fractions and amino acids to the small intestine 1, 2. J. Dairy Sci., 80(2):

374-384.

Quan, R., Barness, L. A. and Uauy, R. 1990. Do infants need nucleotide supplemented formula for

optimal nutrition. J. Pediatr. Gastr. Nutr., 11: 429-434.

Quigley, J., Wallis, L. Dowlen, H. and Heitmann, R. 1992. Sodium bicarbonate and yeast culture

effects on ruminal fermentation, growth, and intake in dairy calves. J. Dairy Sci., 75: 3531-3538.

Rada, V., Vlkova, E. Nevoral, J. and Trojanova, I. 2006. Comparison of bacterial flora and enzymatic

activity in faeces of infants and calves. FEMS microbiol. lett., 258: 25-28.

Rafter, J., Michael, B. Giovanna, C. Yvonne, C. Roisin, H. Pernilla, C. K. Annett,K. Micheal

O’Riordan,Gerald, C. Beatrice, P.Z. Gerhard, R. Monika, R. Ian, R. Maddalena, S. Herbert,

T. Jan, V. Bernhard, W. and John, K.C. 2007. Dietary synbiotics reduce cancer risk factors in

polypectomized and colon cancer patients. Am. J. Clin. Nutr., 85: 488-496.

Rajkowska, K. and Kunicka-Styczynska, A. 2010. Probiotic properties of yeasts isolated from chicken

feces and kefirs. Polskie Towarzystwo Mikrobiologow, Polish Society of Microbiologists., 59:

257-263.

Rao, B. 1995. Factors affecting the milk production: A case study. Indian J. Agri. Econ., 40: 169-170.

205

Rao, T. N., Prasad, J. R. Rao, Z. P. and Prasad, P. 2003. Supplementation of probiotics on growth

performance in sheep. Indian J. Anim. Nutr., 20: 224-226.

Raza, S. H., Riaz, V. and Iqbal, A. 2006. Milk productivity: A changing scenario for future investment. .

In: Productivity and growth in agriculture: Strategies and Interventions, Univ. of Agri.

Faisalabad.

Razin, S., Kutner, S. Efrati, H. and Rottem, S. 1980. Phospholipid and cholesterol uptake by

mycoplasma cells and membranes. Biochim. Biophys. Acta., 598: 628-640.

Reddy, G. and Bhima, B. 2003. Effect of yeast culture based diet on growth and nutrient utilization in

Deoni bull calves. Indian J. Anim. Nutr., 20: 101-104.

Rehman, Z. and Khan, M. 2012. Genetic factors affecting performance traits of Sahiwal cattle in

Pakistan. Pak. Vet. J., 32: 329-333.

Rivera. E. Y. and Gallardo. N. Y. 2010. Non-dairy probiotic products. Food Microbiol., 27: 1-11.

Robinson, P., and J. Garrett. 1999. Effect of yeast culture (Saccharomyces cerevisiae) on adaptation of

cows to postpartum diets and on lactational performance. J. Anim. Sci.,77: 988-999.

Roeschlau, P., Bernt, E. and Gurber, W.J. 1974. Chem.Clin., Biochem., 12: 403.

Roger, V., Fonty, G. Komisarczuk-Bony, S. and Gouet, P.1990. Effects of physicochemical factors on

the adhesion to cellulose avicel of the ruminal bacteria ruminococcus flavefaciens and

fibrobacter succinogenes subsp. succinogenes. Appl. Enviro. Microbiol., 56: 3081-3087.

Romanin, D., Serradell, M. Maciel, D. G. Lausada, N. Garrote, G. L. and Rumbo, M. 2010. Down-

regulation of intestinal epithelial innate response by probiotic yeasts isolated from kefir. Int. J.

Food Microbiolo., 140(2): 102-108.

Roostita, L. 2004. Potensi dan Prospek Yeast (khamir) Dalam Meningkatkan Diversifikasi Pangan di

Indonesia. Pidato Pengukuhan Jabatan Guru Besar Tetap dalam Ilmu Pangan. Fakultas

Peternakan Universitas Padjadjaran. Bandung.

206

Roostita, L., Fleet, G. Wendry, S. Apon, Z. and Gemilang, L. 2011. Determination of yeasts

antimicrobial activity in milk and meat products. Adv. J. Food Sci. Technol. 3 (6): 442-445.

Rose, A. H. 1987. Live yeast, A microorganism for all species: A theoretical look at its mode of action.

Biotechnology in the Feed Industry. Alltech Technical Publications, Nicholasville, Kentucky,

113-118 pp.

Russell, J. B. and Hino, T. 1985. Regulation of lactate production in Streptococcus bovis: a spiraling

effect that contributes to rumen acidosis. J.Dairy Sci., 68: 1712-1721.

Ryan, K. A., Thevaraajan, J. Paul. D.Carlos, C. Susan, C. Fang, F. Quigley, E.M. and Paul, W. O. 2008.

Isolation of lactobacilli with probiotic properties from the human stomach." Lett. Appl.

Microbiol. 47(4): 269-274.

Sabbia, J., Kalscheur, K. Garcia, A. Gehman, A. and Tricarico, J. 2012. Soybean meal substitution with

a yeast-derived microbial protein source in dairy cow diets. J. Dairy Sci., 95: 5888-5900.

Saitou, N. and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing

phylogenetic trees. Mol. Bio. Evo., 4: 406-425.

Salvati, G. G. S., Morais Júnior, N.N. Melo, A.C.S. Vilela, R.R. Cardoso, F.F. Aronovich, M. Pereira,

R.A.N and Pereira. M.N. 2015. Response of lactating cows to live yeast supplementation during

summer. J. Dairy Sci., 98 (6): 4062-4073.

Samanta, A., Walli, T. and Singh, K. 2001. Role of different groups of microbes on fibre utilization.

Indian J.Anim. Sci., 71: 497-498.

Santra, A. and Karim. S. 2003. Rumen manipulation to improve animal productivity. Asian-Austra. J.

Anim. Sci.,16: 748-763.

Sarwar, M., Khan, M.A. and Z. Iqbal, Z. 2002. Feed resources for livestock in Pakistan. Int. J. Agric.

Biol., 4: 186-192.

207

Sarwar, M., Shahzad, M. and Nisa, M. 2010. Small ruminant production in Pakistan. Egyptian J. Sheep

Goat Sci., 5: 17-26.

Schingoethe, D. J., Linke, K. N. Kalscheur, K. F. Hippen, A. R. Rennich, D. R. and Yoon, I. 2004.

Feed efficiency of mid-lactation dairy cows fed yeast culture during summer. J. Dairy

Sci.,87(12): 4178-4181.

Schwab, C., Moore, J. Hoyt, P. and Prentice, J. 1980. Performance and Fecal Flora of Calves Fed a

Nonviable Lactobacillus bulgaricus Fermentation Product 1, 2. J. Dairy Sci., 63: 1412-1423.

Senesi, S., Celandroni, F. Tavanti, A. and Ghelardi, E. 2001. Molecular characterization and

identification of Bacillus clausii Strains marketed for use in oral bacteriotherapy. Appl. Environ.

Microbiol., 67: 834-839.

Servin, A. L. 2004. Antagonistic activities of lactobacilli and bifidobacteria against microbial

pathogens. Fems. Microbiol. Rev., 28: 405-440.

Shahid, H., Shafique, O. and Shokat, A. 2012. Dairy Industry of Pakistan. Euro. J. Busi. Manag., 4: 1-4.

Shakira, G., Mirza, I. and Latif, A. 2013. Scope of common DNA based methods for the study of rumen

bacterial population. Bangl. J. Anim. Sci., 41: 141-146.

Shankar, N., Baghdayan, A.S. and Gilmore, M.S. 2002. Modulation of virulence within a pathogenicity

island in vancomycin-resistant Enterococcus faecalis. Natur., 417: 746-750.

Shaver, R. and J. Garrett, J. 1997. Effect of dietary yeast culture on milk yield, composition, and

component yields at commercial dairies. Professio. Anim. Scient., 13: 204-207.

Shehu, B., Ayanwale, B. Ayo, J. Amodu, J. and A. Ijaiya, A. 2015. Effect of Saccharomyces cerevisiae

supplementation on some serum electrolytes of weaned rabbits. J. Agri. Sci., 7: p206.

Shemesh, M., Hansel, W. and Strauss, J. F.1984. Calcium dependent, cyclic nucleotide-independent

steroidogenesis in the bovine placenta. Proceedings of the National Academy of Sci., 81: 6403-

6407.

208

Shukla, G., Sharma, G. and Goyal, N. 2010. Probiotic characterization of lactobacilli and yeast strains

isolated from whey beverage and therapeutic potential of Lactobacillus yoghurt in murine

giardiasis. Am. J. Biomed. Sci., 2: 248-261.

Sniffen, C., Chaucheyras-Durand, F.Ondarza, M.D. and Donaldson, G. 2004. Predicting the impact of a

live yeast strain on rumen kinetics and ration formulation. 19th Southwest Nutrition and

Management Conference, Phoenix, Arizona, USA. p 53-59.

Soberon, F., Raffrenato, E. Everett, R.and Amburgh, M.V. 2012. Preweaning milk replacer intake and

effects on long-term productivity of dairy calves. J. Dairy Sci., 95: 783-793.

Soder, K. and Holden, L. 1999. Dry matter intake and milk yield and composition of cows fed yeast

prepartum and postpartum. J. Dairy Sci., 82: 605-610.

Sonomoto, K. 2011. Current Progress in Advanced Research. Caister Academic Press.

Sontakke, U. 2012. Benefits of Saccharomyces cerevisiae as a feed additive in ruminants. Nati. Dairy

Res. Inst., Karnal, 1714.

Spears, J. W. 2000. Micronutrients and immune function in cattle. Proceedings of the Nutrition Society

59: 587-594.

Spring, P., Wenk, C. Dawson, K. and Newman, K. 2000. The effects of dietary mannaoligosaccharides

on cecal parameters and the concentrations of enteric bacteria in the ceca of salmonella-

challenged broiler chicks. Poultry Sci., 79: 205-211.

Srutkova, D., Spanova, A. Spano, M. Drab, V. Schwarzer, M. Kozakova, H. and Rittich, B. 2011.

Efficiency of PCR-based methods in discriminating Bifidobacterium longum ssp. longum and

Bifidobacterium longum ssp. infantis strains of human origin. J.Microbiol, Meth., 87(1): 10-16.

Steel, R. G. D. and Torrie, J.H.1984. Principles and Procedures of Statistics; A Biometrical Approach.

3rd ed. McGraw Hill Book Co.Inc, New York, USA.

209

Steel, R. G.D. Torrie, J.H. and Dickey, D.A. 1997. Principles and procedures of statistics. A biochemical

approach. 3rd ed. McGraw Hill Book Co.Inc, New York, USA.

Stella, A. V., Paratte, R. Valnegri, L. Cigalino, G. Soncini, G. Chevaux, E. Dell’Orto, V. and. Savoini,

G. 2007. Effect of administration of live Saccharomyces cerevisiae on milk production, milk

composition, blood metabolites, and faecal flora in early lactating dairy goats. Small

Rumi.Res., 67(1): 7-13.

Stone, C. W. 1998. Yeast products in the feed industry: A practical guide for feed professionals.

Diamond V Mills Inc. Cedar Rapids, IA, USA.

Strohlein, H. 2003. Back to nature.Live yeasts in feed for dairy cows.DMZ, Lebensm. Ind. Milcheirtsh.

124: 68-71.

Sutton, J. 1989. Altering milk composition by feeding. J. Dairy Sci., 72: 2801-2814.

Swartz, D., Muller, L. Rogers, G. and Varga, G.1994. Effect of yeast cultures on performance of

lactating dairy cows: A field study. J. Dairy Sci., 77: 3073-3080.

Szucs, J. P., Suli, A. Halasz, T. Arany, A. and Bodor, Z. 2013. Effect of live yeast culture

Saccharomyces cerevisiae on milk production and some blood parameters. Scient. Pap. Anim.

Sci. Biotechnolo., 46: 40-44.

Tahri, K., Grill, P. J. and Schneider., F. 1996. Bifidobacteria strain behavior toward cholesterol

Coprecipitation with bile salts and assimilation. Curre. Microbiol., 33: 187-193.

Tahri, K., Grill, P.J. and Schneider., F. 1997. Involvement of trihydroxyconjugated bile salts in

cholesterol assimilation by bifidobacteria. Curre. Microbiol., 34: 79-84.

Takahashi, M. et al. 2004. The effect of probiotic treatment with Clostridium butyricum on

enterohemorrhagic Escherichia coli O157:H7 infection in mice. FEMS immunology and medical

microbiology 41: 219-226.

210

Takahashi, M., Taguchi, H. Yamaguchi, H. Osaki, T. Komatsu, A. and Kamiya, S. 2004. The effect of

probiotic treatment with Clostridium butyricum on enterohemorrhagic Escherichia coli O157:

H7 infection in mice. FEMS Immunol. Med. Microbiol. 41(3): 219-226.

Tamai, Y., Yoshimitsu, N. Watanabe, Y. Kuwabara, Y. and Nagai, S. 1996. Effects of milk fermented

by culturing with various lactic acid bacteria and a yeast on serum cholesterol level in rats. J.

Ferment. Bioeng. 81: 181-182.

Tamura, K., Peterson, D. Peterson, N. Stecher, G. Nei, M. and Kumar, S. 2011. MEGA5: molecular

evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum

parsimony methods. Mol. Bio. Evoluti., 28(10): 2731-2739.

Tannock, G. W. and Savage, D.C. 1974. Influences of dietary and environmental stress on microbial

populations in the murine gastrointestinal tract. Infec. Immun. 9: 591-598.

Terre, M., Maynou, G. Bach, A. and Gauthier, M. 2015. Effect of Saccharomyces cerevisiae CNCM I-

1077 supplementation on performance and rumen microbiota of dairy calves. Profess. Anim.

Scienti., 31: 153-158.

Thareja, A., Puniya, A. K. Goel, G. Nagpal, R. Sehgal, J. P. Singh, P. K. and Singh, K. 2006. In vitro

degradation of wheat straw by anaerobic fungi from small ruminants. Arch. Anim. Nutri., 60(5):

412-417.

Theodorou, M. K., Longland, A.C. Dhanoa, M.S. Lowe, S.E. and Trinci, A.P.1989. Growth of

neocallimastix sp. strain R1 on Italian ryegrass hay: removal of neutral sugars from plant cell

walls. Appl. Enviro. Microbiol., 55: 1363-1367.

Thomas, D. W. and Greer, F.R. 2010. Probiotics and prebiotics in pediatrics. Pediatri., 126: 1217-1231.

Timmerman, H. M., Mulder, L. Everts, H. Van Espen, D. C. Van Der Wal, E. Klaassen, G. S. Rouwers,

G. Hartemink, R. Rombouts, F.M. and Beynen. A.C.2005. Health and growth of veal calves fed

milk replacers with or without probiotics. J. Dairy Sci., 88 (6): 2154-2165.

211

Timmerman, H. M., Koning, C.J.M. Mulder, L. Rombouts, F.M. and Beynen, A.C. 2004. Monostrain,

multistrain and multispecies problotics - A comparison of functionality and efficacy. Int. J Food

Microbiol., 96: 219-233.

Tournut, J. 1993. The digestive flora of the pig and its variations. Recl. Med. Vet., 169: 645-652.

Tripathi, M. and Karim, S. 2010. Effect of individual and mixed live yeast culture feeding on growth

performance, nutrient utilization and microbial crude protein synthesis in lambs. Anim. Feed

Sci. echnol., 155: 163-171.

Tripathi, V. K., Sehgal, J.P. Puniya, A.K. and Singh, K. 2007. Effect of administration of anaerobic

fungi isolated from cattle and wild blue bull (Boselaphus tragocamelus) on growth rate and fibre

utilization in buffalo calves. Archi. Anim. Nutri., 61: 416-423.

Tungland, B. and Meyer, D. 2002. Nondigestible oligo‐and polysaccharides (dietary fiber): their

physiology and role in human health and food. Compre. Revi. Food Sci. Food Safy. 1: 90-109.

Turnbaugh, P. J., Micah, H. Tanya, Y. Brandi, L. Cantarel, Duncan, A. Ruth E. Ley, Mitchell L. Sogin,

William J. Jones, Roe, A. Jason P. Affourtit, Egholm, M. Henrissat, B. Heath, A. C. Knight, R.

and Jeffrey, I. G. 2009. A core gut microbiome in obese and lean twins. Natu. 457 (7228): 480-

484.

Ushida, K. and Jouany, J. P. 1990. Effect of defaunation on fibre digestion in sheep given two

isonitrogenous diets. Anim. Feed Sci. Technol., 29: 153-158.

Usman, H. A. 1999. Bile tolerance, taurocholate deconjugation, and binding of cholesterol by

Lactobacillus gasseri strains. J. Dairy Sci., 82: 243–248.

Uyeno, Y., Sekiguchi, Y., & Kamagata, Y. 2008. Impact of consumption of probiotic lactobacilli-

containing yogurt on microbial composition in human feces. International journal of food

microbiology, 122(1), 16-22.

Uyeno, Y., Sekiguchi, Y., & Kamagata, Y. (2010). rRNA‐ based analysis to monitor succession of

faecal bacterial communities in Holstein calves.Letters in applied microbiology, 51(5), 570-577.

212

Van Soest, P. J., Robertson, J.B. and Lewis, B.A. 1991. Methods for dietry fibre, nutral detregent fibre

and non-starch polysacharides in realtion to animal nutrition. J.Dairy Sci., 74: 3583.

Vibhute, V., Shelke, R. Chavan, S. Nage, S. and Vishwavidyalaya, P.D.K. 2011. Effect of probiotics

supplementation on the performance of lactating crossbred cows. Vet. World 4: 557-561.

Vinderola, G. 2008. Dried cell-free fraction of fermented milks: new functional additives for the food

industry. Trends Food Sci. Tech., 19: 40-46.

Vlkova, E., Trojanova, I. and Rada, V. 2006. Distribution of bifidobacteria in the gastrointestinal tract of

calves. Folia Microbiolo., 51: 325-328.

Wallace, R. 1994. Ruminal microbiology, biotechnology, and ruminant nutrition: progress and

problems. J. Dairy Sci., 72: 2992-3003.

Wallace, R. J.1996. The mode of action of yeast culture in modifying rumen fermentation. In

Biotechnol. in the Feed Industry ed. Lyons, T.P. Nicholasville, Kentucky: Alltech Technical

Publications: 217-232.

Wallace, R. J., Onodera, R. and Cotta, M. A. 1997. The Rumen Microbial Ecosystem. Chapman and

Hall, London, pp 283-328 .

Wathes, D. C., Bourne, N. Cheng, Z., Mann, G. E. Taylor, V. J. and Coffey, M. P. 2007. Multiple

correlation analyses of metabolic and endocrine profiles with fertility in primiparous and

multiparous cows. J. Dairy Sci., 90(3): 1310-1325.

Watts, T., Berti, I. Sapone, A. Gerarduzzi, T. Not, T. Zielke, R. and Fasano, A. 2005. Role of the

intestinal tight junction modulator zonulin in the pathogenesis of type I diabetes in BB diabetic-

prone rats. Proceedings of the National Academy of Sciences of the United States of

America, 102(8): 2916-2921.

Weimer, P. J., Waghorn, G. C. Odt, C. L. and Mertens, D. R. 1999. Effect of diet on populations of three

species of ruminal cellulolytic bacteria in lactating dairy cows. J. Dairy Sci., 82(1): 122-134.

213

Weinberg, Z. G. 2003. Effect of Lactic acid bacteria on animal performance. Indian J. Biotechnol., 2:

378-381.

White, R., Harrison, J.Yoon, I. Sanchez, W. and Nicholson, N. 2008. Effect of yeast culture on

efficiency of nutrient utilization for milk production and impact on fiber digestibility and fecal

particle size. Profes. Anim. Scienti., 24: 114-119.

Wiedmeier, R., Arambel, M. and Walters, J. 1987. Effect of yeast culture and Aspergillus oryzae

fermentation extract on ruminal characteristics and nutrient digestibility. J. Dairy Sci.,70: 2063-

2068.

Williams, A. G. and Orpin, C.G. 1987. Polysaccharide-degrading enzymes formed by three species of

anaerobic rumen fungi grown on a range of carbohydrate substrates. Cana. J. Microbiol. 33: 418-

426.

Williams, P., C. Tait, C. Innes, G. and Newbold, C. 1991. Effects of the inclusion of yeast culture

(Saccharomyces cerevisiae plus growth medium) in the diet of dairy cows on milk yield and

forage degradation and fermentation patterns in the rumen of steers. J. Anim. Sci., 69: 3016-

3026.

Williamson, E., Bilsborough, J.M. and Viney, J.L. 2002. Regulation of mucosal dendritic cell function

by receptor activator of NF-kappa B (RANK)/RANK ligand interactions: Impact on tolerance

induction. J. Immunol., 169: 3606-3612.

Wohlt, J., Corcione, T. and Zajac, P. 1998. Effect of yeast on feed intake and performance of cows fed

diets based on corn silage during early lactation.J. Dairy Sci., 81: 1345-1352.

Wohlt, J., Finkelstein, A. and Chung, C. 1991. Yeast culture to improve intake, nutrient digestibility,

and performance by dairy cattle during early lactation. J. Dairy Sci., 74: 1395-1400.

Won, T. J., Kim, B. Lim, Y. T. Song,D. S. Park, S.Y. Park, E. S. Lee, D. I and Hwang, K. W. 2011.

Oral administration of Lactobacillus strains from Kimchi inhibits atopic dermatitis in NC/Nga

mice. J. appl. Microbiol., 110 (5): 1195-1202.

214

Woodard, G. A., Encarnacion, B. John, R. D. Joseph, P. Karen, C. Hernandez, Tina. and Morton, M. J.

2009. Probiotics improve outcomes after Roux-en-Y gastric bypass surgery: a prospective

randomized trial. J. Gastrointesti. Surg. 13 (7): 1198-1204.

Xiao, J. Z., Kondo, S. Takahashi, N. Miyaji, K. Oshida, K. Hiramatsu, A. Iwatsuki, K. Kokubo, S. and

Hosono, A. 2003. Effects of milk products fermented by Bifidobacterium longum on blood lipids

in rats and healthy adult male volunteers. J. Dairy Sci., 86(7): 2452-2461.

Xiao, Z., Trincado, C.A. and Murtaugh, M.P. 2004. β-Glucan enhancement of T cell IFNγ response in

swine. Veterinar. Immunol. Immunopathol., 102: 315-320.

Ya, T., Qijin, Z. Fuliang, C. Justin, M. Menhe, B. Tiansong, S. Ruiting, D. and Heping, Z.

Immunological evaluation of Lactobacillus casei Zhang: a newly isolated strain from koumiss in

Inner Mongolia, China. 2008. BMC Immunol., 9(1): 68.

Yalcin, S., Yalcin, S. Can, P. Gurdal, A. O. Bagci, C. Eltan, O. 2011. The nutritive value of live yeast

culture (Saccharomyces cerevisiae) and its effect on milk yield, milk composition and some

blood parameters of dairy cows. Asi. Aust.J. Anim. Sci., 24(10), 1377-1385.

Yan, F. and Polk, D. B. 2011. Probiotics and immune health. Curr. Opin .Gastroen., 27: 496-501.

Yang, C. M. and Varga, G. A. 1993. The effects of continuous ruminal dosing with dioctyl sodium

sulphosuccinate on ruminal and metabolic characteristics of lactating Holstein cows. Briti. J.

Nutri. 69: 397-408.

Yokoyama, H., Waki, M. Ogino, A. Ohmori, H. Tanaka, Y. 2007. Hydrogen fermentation properties of

undiluted cow dung. J. Biosci. Bioeng., 104: 82-85.

Yoon, I., and Stern, M.1996. Effects of Saccharomyces cerevisiae and Aspergillus oryzae cultures on

ruminal fermentation in dairy cows. J. Dairy Sci., 79: 411-417.

Zanton, G. and Heinrich, A. 2007. The effects of controlled feeding of a high-forage or high-

concentrate ration on heifer growth and first-lactation milk production. J. Dairy Sci., 90: 3388-

3396.

215

Zeng, X. Q., Pan, D.D. and Guo, Y.X. 2010. The probiotic properties of Lactobacillus buchneri P2. J.

appl. Microbiol. 108: 2059-2066.

Zhang, Z., Hinrichs, D. J. Lu, H. Chen, H. Zhong, W. and Kolls, J. K. 2007. After interleukin-12p40,

are interleukin-23 and interleukin-17 the next therapeutic targets for inflammatory bowel

disease? Int. Immunopharmacol., 7(4): 409-416.

216

Publications Related to PhD Thesis

A. Full length paper

Shakira Ghazanfar, Muhammad Iqbal Anjum, Atiya Azim and Iftikhar Ahmed. 2015. Effects of

Dietary Supplementation of Yeast Culture (Saccharomyces cerevisiae) on Growth Performance,

Blood Parameters, Nutrient Digestibility and Fecal Flora of Dairy Heifers” The Journal of

Animal and Plant Science 25(1): 53-59.

B. Presentations in International Conferences (Oral/Posters)

1. Shakira Ghazanfar, M. I. Anjum, F. Hassan, I. Ahmed, M. Qbtiya, M. Afzal and M. Imran.

Effects of dietary supplementation of Saccharomyces cerevisiae on production performance and

health status dairy cattle. 10th Biennial International Conferences of Pakistan Society for

Microbiology Exploring Microbes for Future Endeavors, 25-28, 2015. Lhr. Pakistan

1. Shakira Ghazanfar, M. I. Anjum, F. Hassan, I. Ahmed, M. Qbtiya, M. Afzal and M. Imran.

Dietary Supplementation of Saccharomyces Cerevisiae on Production and Health Status in

Lactating Dairy Cattle. 3rd International workshop on Dairy Science Park. 16-18, 1W-DSP-2015.

University of Agriculture, Peshawar. Pakistan.

2. Shakira Ghazanfar, Qubtia, M. Hassan, F. Muhammad, A. Ahmed, I. Muhammad, I. Isolation and characterization of nutritionally important lactic acid bacteria from cattle gut. 67th Annual Meeting of the European Federation of Animal Science, 29 August-2 September

2016. The Waterfront, Belfast, Northern Ireland (Accepted)

3. Shakira Ghazanfar, Qubtia, M. Hassan, F. Muhammad, A. Ahmed, I. Imran, M. Dietary

supplementation of Saccharomyces cerevisiae on production and health status in dairy cattle.

67th Annual Meeting of the European Federation of Animal Science, 29 August-2 September

2016. The Waterfront, Belfast, Northern Ireland (Accepted)

C. Article Published in Newspaper

http://www.nation.com.pk/E-Paper/islamabad/2013-02-10/page-11