INFLUENCE OF PROBIOTIC SUPPLEMENTATION ON ENDOCRINE AND METABOLIC CHANGES FOR FERTILITY IMPROVEMENT IN REPEAT BREEDING KARAN FRIES (KF) COWS UNDER FARM AND FIELD CONDITIONS THESIS SUBMITTED TO THE ICAR-NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL (DEEMED UNIVERSITY) IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN ANIMAL PHYSIOLOGY BY DANDAGE SHASHIKANT DAMODHAR M.V.Sc. DAIRY CATTLE PHYSIOLOGY DIVISION ICAR-NATIONAL DAIRY RESEARCH INSTITUTE (DEEMED UNIVERCITY) KARNAL-132001 (HARYANA), INDIA 2015 Regn. No. 1090903
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DAIRY CATTLE PHYSIOLOGY DIVISION ICAR …...The study was also conducted at field level reared by farmers of Daradand Indri villages of Karnal. Recently calved cows free from clinical
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INFLUENCE OF PROBIOTIC SUPPLEMENTATION ON ENDOCRINE AND METABOLIC CHANGES FOR FERTILITY IMPROVEMENT
IN REPEAT BREEDING KARAN FRIES (KF) COWS UNDER FARM AND FIELD CONDITIONS
THESIS SUBMITTED TO THE
ICAR-NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL
(DEEMED UNIVERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENT
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY IN
ANIMAL PHYSIOLOGY
BY DANDAGE SHASHIKANT DAMODHAR
M.V.Sc.
DAIRY CATTLE PHYSIOLOGY DIVISION ICAR-NATIONAL DAIRY RESEARCH INSTITUTE
(DEEMED UNIVERCITY) KARNAL-132001 (HARYANA), INDIA
2015
Regn. No. 1090903
Dedicated
to my
Beloved Daughter
“PRANJAL”
ABSTRACT
The present investigation was conducted on Karan-Fries crossbred cattle reared at cattle yard, National Dairy Research Institute (NDRI) Karnal, Haryana, India. The study was also conducted at field level reared by farmers of Darad and Indri villages of Karnal. Recently calved cows free from clinical reproductive tract infection, clinical mastitis and any injury were selected from farm and field conditions. Animals were monitored up to three consecutive services. Pregnancy diagnosis by rectal palpation post 45-60 day after each service was done. Animals conceiving up to three services were considered as regular breeders (RgB) and those that did not conceive up to three services were considered as repeat breeders (RB). There after RB group of animals were divided into two groups (a) supplemented with fermented yeast culture (RB-S) (b) Non supplemented (RB-NS) under both farm and field condition. To RB-S group fermented yeast culture (Saccharomyces cerevisiae; Diamond XP) was supplemented with concentrate @ 12 gm/animal twice daily (5.3 × 105 CFU/g) during experimental period (21st - 40th week).The present study was taken up for finding relationship between plasma IGF-1, Hp, Lactofferin calcium, glucose and urea with respect to repeat breeding problem and availability of energy fuel to lactating dairy cows and also to evaluate the effect of systematic supplementation of fermented yeast culture in alleviating repeat breeding problem. In the present study in vitro effect of IGF-I on expression of Toll-like receptor-4 (TLR-4) and Fas gene in neutrophils was studied in RB group. In the present study concentration of plasma IGF-1, Lactofferin, glucose and calcium were significantly (P<0.001) greater in RgB group when compared with RB group under both farm and field conditions. Concentration of plasma Hp and urea were significantly(P<0.001) lower in RgB group when compared with RB group under both farm and field conditions. DMI, milk yield and body weight were higher in RgB group when compared with RB group under both farm and field conditions, but they were not significantly different. From 22nd week when probiotic (Fermented yeast culture) was supplemented concentration of plasma IGF-1 Lactofferin, glucose and calcium were significantly (P<0.001) greater in RB-S group when compared to RB-NS group under both farm and field conditions. Concentration of plasma Hp and urea were significantly (P<0.001) lower in RB-S group when compared with RB-NS group under both farm and field conditions. DMI, milk yield and body weight were higher in RB-S group, when compared with RB-NS group, but not significantly different under both farm and field conditions. After supplementation, conception rate was significantly (P<0.01) higher in RB-S group when compared to RB-NS group under both farm and field conditions.Relative expression of TLR-4 and Fas gene in blood neutrophils of RgB group was significantly greater (P<0.001) when compared with RB (In vitro IGF-1 supplemented) group. Differential plasma level of IGF-l, Lactoferrin and Haptoglobin were related with Repeat Breeding problem in crossbred Karan Fries cows under both farm and field conditions. Supplementation of fermented yeast culture to repeat breeding crossbred Karan Fries Cows resulted in providing necessary nutrient, improvement in nutrient utilization, rumen functionand production performance which in turn might have reduced repeat breeding problem in crossbred cows under farm and field conditions. Relative expression of TLR-4 and Fas gene in neutrophils may relate with repeat breeding problem in Karan Fries cows.
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CONTENTS
Chapter No.
Title Page No.
1 INTRODUCTION 1-4
2 RIVIEW OF LITERATURE 5-28
2.1 Yeast culture (Saccharomyces cerevisiae) 6
2.1.1 Mode of action of Yeast and its effects on
performance of animals 7
2.1.1.1 Mode of action of yeast in the rumen: 8
2.1.1.2 Effect of Saccharomyces cerevisiae on Rumen
fermentation pattern
10
2.1.1.3 Effect of Saccharomyces cerevisiae on Milk Yield: 11
2.1.1.4 Effect of Saccharomyces cerevisiae on Dry Matter
Intake (DMI)
11
2.1.1.5 Effect of Saccharomyces cerevisiaeon Body Weight 12
2.2 Insulin Like Growth Factors-1 (IGF-1) 12
2.2.1 IGF-l in female reproduction 13
2.3 Haptoglobin (Hp) 15
2.3.1 Biological functions of Haptoglobin (Hp) 16
2.4 Lactoferrin 17
2.5 Blood profile and reproduction 18
2.5.1 Plasma calcium 18
2.5.2 Plasma glucose 19
2.5.3 Plasma urea 20
2.6 Neutrophils 21
2.6.1 Apotosis of neutrophils 22
2.7 Fas gene 24
2.7.1 Mechanisms of neutrophil apoptosis activated by
death receptors (Fas)
24
2.8 Toll-like receptor-4 (TLR-4) 26
2.8.1 Expression of TLR-4 Gene in different cells. 27
2.8.2 Neutrophils and TLR-4 Gene 28
Chapter No.
Title Page No.
3 MATERIALS AND METHODS 29-45
3.1 Location of the study areas and climate 29
3.2 Selection and grouping of experimental animals 29
3.3 Chemicals, glassware and plastic ware 30
3.4 Collection of blood samples 30
3.5 Feed provided under farm and field conditions. 30
3.6 Estimation of feed intake 31
3.6.1 Analysis of feed and fodder samples 31
3.7 Recording of body weight 31
3.8 Recording of milk yield 31
3.9 Estimation of plasma parameters 32
3.9.2 Estimation of blood plasma Lactoferrin 32
3.9.3 Estimation of blood plasma Insulin-like growth factor-
1 (IGF-1)
32
3.9.4 Estimation of blood plasma calcium 32
3.9.5 Estimation of blood urea 33
3.9.6 Estimation of blood glucose 35
3.10 IN VITRO STUDY 36
3.10.1 Chemicals for In Vitro study 36
3.10.2 Chemicals for gene expression studies 36
3.10.3 Separation and enumeration of Neutrophils 37
3.10.4 Determination of number of viable cells 38
3.11 IN VITRO EXPERIMENT 39
3.11.1 Preparation of reagents and glassware for RNA
isolation
40
3.11.2 Quality and quantity of RNA 40
3.11.3 RNA extraction from blood PMN Cells 40
3.11.4 Quality checking of RNA by agarose gel
electrophoresis
41
3.11.5 Quantification of RNA 42
3.11.6 Protocol For first strand cDNA synthesis: 42
Chapter No.
Title Page No.
3.11.7 Primers 43
3.11.8 Reaction mixture for real-time PCR 44
3.11.9 Statistical analysis of relative target gene expression 45
3.12 Statistical analysis 45
4 RESULTS AND DISCUSSION 46-85
4.1 Insulin-Like Growth Factor-1 (IGF-1) 46
4.1.1 Concentration of plasma Insulin-like growth factor-1 (ng/ml) in RgB and RB groups under farm conditions.
46
4.1.2 Concentration of plasma Insulin-like growth factor-1
(ng/ml) in RB-S and RB-NS groups under farm
conditions.
47
4.1.3 Concentration of plasma Insulin-like growth factor-1
(ng/ml) in RgB and RB groups under field conditions.
48
4.1.4 Concentration of plasma Insulin-like growth factor-1
(ng/ml) in RB-S and RB-NS groups under field
conditions.
49
4.2 Haptoglobin (Hp) 51
4.2.1 Concentration of plasma haptoglobin (ng/ml) in RgB
and RB groups under farm Conditions.
51
4.2.2 Concentration of plasma haptoglobin (ng/ml) in RB-S
and RB-NS groups under farm conditions.
52
4.2.3 Concentration of plasma haptoglobin (ng/ml) in RgB
and RB groups under field conditions.
53
4.2.4 Concentration of plasma haptoglobin (ng/ml) in RB-S
and RB-NS groups under field conditions.
54
4.3 Lactoferrin (LF) 56
4.3.1 Concentration of plasma lactoferrin(ng/ml) in RgB
and RB groups under farm conditions.
56
4.3.2 Concentration of plasma lactoferrin(ng/ml) in RB-S and RB-NS groups under farm conditions.
57
4.3.3 Concentration of plasma lactoferrin (ng/ml) in RgB
and RB groups under field conditions.
58
Chapter No.
Title Page No.
4.3.4 Concentration of plasma lactoferrin (ng/ml) in RB-S
and RB-NS groups under field conditions.
59
4.4 Glucose 60
4.4.1 Concentration of plasma glucose (mg/dl) in RgB and
RB groups under farm conditions.
60
4.4.2 Concentration of plasma Glucose (mg/dl) in RB-S
and RB-NS groups under farm conditions.
61
4.4.3 Concentration of Plasma glucose (mg/dl) in RgB and
RB groups under field conditions.
62
4.4.4 Concentration of plasma glucose (mg/dl) in RB-S
and RB-NS groups under field conditions.
63
4.5 Urea 64
4.5.1 Concentration of plasma urea (mg/dl) in RgB and RB
groups under farm conditions.
64
4.5.2 Concentration of plasma urea (mg/dl) in RB-S and
RB-NS groups under farm conditions.
65
4.5.3 Concentration of plasma urea (mg/dl) in RgB and RB
groups under field conditions.
66
4.5.4 Concentration of plasma urea (mg/dl) in RB-S and
RB-NS groups under field conditions.
67
4.6 Calcium 68
4.6.1 Concentration of plasma calcium (mg/dl) in RgB and
RB groups under farm conditions.
68
4.6.2 Concentration of plasma calcium (mg/dl) in RB-S
and RB-NS groups under farm conditions.
69
4.6.3 Concentration of plasma calcium (mg/dl) in RgB and
RB groups under field conditions.
70
4.6.4 Concentration of plasma calcium (mg/dl) in RB-S and RB-NS groups under field conditions.
71
4.7 Dry matter intake (DMI) 72
4.7.1 Dry matter intake (Kg/day) in RgB and RB KF groups
under farm conditions.
72
Chapter No.
Title Page No.
4.7.2 Dry matter intake (Kg/day) in RB-S and RB-NS
groups under farm conditions.
73
4.7.3 Dry matter intake (Kg/day) in RgB and RB groups
under field conditions.
73
4.7.4 Dry matter intake (Kg/day) in RB-S and RB-NS
groups under field conditions.
74
4.8 Milk yield 75
4.8.1 Milk yield (Kg/day) in RgB and RB groups under
farm conditions.
75
4.8.2 Milk yield (Kg/day) in RB-S and RB-NS groups under
farm conditions.
76
4.8.3 Milk yield (Kg/day) in RgB and RB groups under field
conditions.
76
4.8.4 Milk yield (Kg/day) in RB-S and RB-NS groups under
field conditions.
77
4.9 Body weight 78
4.9.1 Body weight (Kg) in RgB and RB groups under farm
conditions.
78
4.9.2 Body weight (Kg) in RB-S and RB-NS groups under
farm conditions.
79
4.9.3 Body weight (Kg) in RgB and RB groups under field
conditions.
79
4.9.4 Body weight (Kg) in RB-S and RB-NS groups under
field conditions.
80
4.10 Conception rate 81
4.10.1 Conception rate in RgBand RB groups under farm
conditions.
81
4.10.2 Conception rate in RB-S and RB-NSgroups under
farm conditions.
81
4.10.3 Conception rate in RgB and RB groups under field
conditions.
82
Chapter No.
Title Page No.
4.10.4 Conception rate in RB-S and RB-NS groups under
field conditions.
82
4.11 In Vitro Study 83
4.11.1 Relative expression of TLR-4 mRNA in neutrophils of
RB, RB (In vitro IGF-1 supplemented) and RgB
group.
83
4.11.2 Relative expression of Fas mRNA in neutrophils of
RB, RB (In vitro IGF-1 supplemented) and RgB
group.
83
5 SUMMARY AND CONCLUSIONS 86-92
6 BIBLIOGRAPHY i-xxvi
LIST OF TABLES
Table No. Titte After
page 3.1 Details of experimental animals under farm conditions. 30 3.2 Details of experimentals animal under field conditions. 30
3.3 Composition of concentrate feed supplemented to animal. 30 3.4 Protocol for the estimation of plasma glucose. 36 3.5 Composition of RPMI -1640 medium. 36 3.6 Details of primers of target and housekeeping genes. 42
3.7 Reaction mixture for Real-Time PCR. 43 3.8 Reaction programme for real time PCR. 43
4.1 Concentration of plasma IGF-1(ng/ml) in RgB and RB groups under
farm conditions. 47
4.2 Concentration of plasma IGF-1(ng/ml) in RB-S and RB-NS groups
under farm conditions. 48
4.3 Concentration of plasma IGF-1(ng/ml) in RgB and RB groups under
field conditions. 48
4.4 Concentration of plasma IGF-1(ng/ml) in RB-S and RB-NS groups
under field conditions. 49
4.5 Concentration of plasma Hp(ng/ml) in RgB and RB groups under
farm conditions. 51
4.6 Concentration of plasma Hp (ng/ml) in RB-S and RB-NS groups
under farm conditions. 52
4.7 Concentration of plasma Hp (ng/ml) in RgB and RB groups under
field conditions. 53
4.8 Concentration of plasma Hp (ng/ml) in RB-S and RB-NS groups
under field conditions. 54
4.9 Concentration of plasma Lactoferrin(ng/ml) in RgB and RB groups
under farm conditions. 56
4.10 Concentration of plasma Lactoferrin (ng/ml) in RB-S and RB-NS
groups under farm conditions. 57
4.11 Concentration of plasma Lactoferrin (ng/ml) in RgB and RB groups
under field conditions.
58
Table No. Titte After
page 4.12 Concentration of plasma Lactoferrin (ng/ml) in RB-S and RB-NS
groups under field conditions. 59
4.13 Concentration of Plasma Glucose (mg/dl) in RgB and RB groups
under farm conditions. 61
4.14 Concentration of Plasma Glucose (mg/dl) in RB-S and RB-NS
groups under farm conditions. 62
4.15 Concentration of Plasma Glucose (mg/dl) in RgB and RB groups
under field conditions. 62
4.16 Concentration of Plasma Glucose (mg/dl) in RB-S and RB-NS
groups under field conditions. 63
4.17 Concentration of Plasma Urea (mg/dl) in RgB and RB groups under
farm conditions. 65
4.18 Concentration of Plasma Urea (mg/dl) in RB-S and RB-NS groups
under farm conditions. 66
4.19 Concentration of Plasma Urea (mg/dl) in RgB and RB groups under
field conditions. 67
4.20 Concentration of Plasma Urea (mg/dl) in RB-S and RB-NS groups
under field conditions. 67
4.21 Concentration of Plasma Calcium in RgB and RB groups under farm
conditions. 69
4.22 Concentration of Plasma Calcium in RB-S and RB-NS groups under
farm conditions. 70
4.23 Concentration of Plasma Calcium (mg/dl) in RgB and RB groups
under field conditions. 70
4.24 Concentration of Plasma Calcium (mg/dl) in RB-S and RB-NS
groups under field conditions. 71
4.25 Dry Matter Intake (Kg/day) RgB and RB groups under farm
conditions. 72
4.26 Dry Matter Intake (Kg/day) in RB-S and RB-NS groups under farm
conditions. 73
4.27 Dry Matter Intake (Kg/day) RgB and RB groups under field
conditions. 74
4.28 Dry Matter Intake (Kg/day) in RB-S and RB-NS groups under field
conditions. 74
Table No. Titte After
page 4.29 Milk Yield (Kg/day) of RgB and RB groups under farm conditions. 75
4.30 Milk Yield (Kg/day) of RB-S and RB-NS groups under farm
conditions. 76
4.31 Milk Yield (Kg/day) of RgB and RB groups under field conditions. 77
4.32 Milk Yield (Kg/day) of RB-S and RB-NS groups under field
conditions. 77
4.33 Body Weight (Kg) of RgB and RB groups under farm conditions. 79 4.34 Body Weight (Kg) of RB-S and RB-NS groups under farm conditions. 79
4.35 Body Weight (Kg) of RgB and RB groups under field conditions. 79 4.36 Body Weight (Kg) of RB-S and RB-S groups under field conditions. 80
4.37 Conception rate of RgB and RB groups under farm conditions 81 4.38 Conception rate of RB-S and RB-NS groups under farm conditions. 81 4.39 Conception rate of RgB and RB groups under field conditions. 82 4.40 Conception rate of RB-S and RB-NS groups under field conditions. 82 4.41 Correlation coefficient of plasma parameters in RgB and RB groups
under farm conditions 82
4.42 Correlation coefficient of plasma parameters in RB-S and RB-NS
groups under farm conditions 82
4.43 Correlation coefficient of plasma parameters in RgB and RB groups
under field conditions 82
4.44 Correlation coefficient of plasma parameters in RB-S and RB-NS
groups under field conditions 82
4.45 Relative expression of TLR-4 and Fas mRNA in neutrophils of RB,
RB (In vitro IGF-1 supplemented) and RgB group. 83
LIST OF FIGURES Figure
No. Title of Figures After
page 2.1 Mode of action of live yeast in ruminants 8
2.2 Mechanism of fermented yeast culture in bacterial growth and
activity
10
2.3 Apoptosis signaling through death receptors 26
3.1 Standard curve for plasma IGF-1 32
3.2 Standard curve for plasma Hp 32
3.3 RT-PCR amplified products of TLR-4, Fas and House keeping
genes on agarose gel 3%
42
3.4 Amplification curve TLR-4 44
3.5 Amplification curve Fas 44
4.1 Concentration of plasma IGF-1 in RgB and during pre and post
supplementation period in RB-S and RB-NS groups under farm
conditions
49
4.2 Concentration of plasma IGF-1 in RgB and during pre and post
supplementation period in RB-S and RB-NS groups under field
conditions
49
4.3 Concentration of plasma Hp in RgB and during pre and post
supplementation period in RB-S and RB-NS groups under farm
conditions
54
4.4 Concentration of plasma Hp in RgB and during pre and post
supplementation period in RB-S and RB-NS groups under field
conditions
54
4.5 Concentration of plasma lactoferrin in RgB and during pre and post
supplementation period in RB-S and RB-NS groups under farm
conditions
59
4.6 Concentration of plasma lactoferrin in RgB and during pre and post
supplementation period in RB-S and RB-NS groups under field
conditions
59
4.7 Concentration of plasma glucose in RgB and during pre and post
supplementation period in RB-S and RB-NS groups under farm
conditions
63
4.8 Concentration of plasma glucose in RgB and during pre and post supplementation period in RB-S and RB-NS groups under field conditions
63
Figure No.
Title of Figures After page
4.9 Concentration of plasma urea in RgB and during pre and post
supplementation period in RB-S and RB-NS groups under farm
conditions
67
4.10 Concentration of plasma urea in RgB and during pre and post
supplementation period in RB-S and RB-NS groups under field
conditions
67
4.11 Concentration of plasma calcium in RgB and during pre and post
supplementation period in RB-S and RB-NS groups under farm
conditions
71
4.12 Concentration of plasma calcium in RgB and during pre and post
supplementation period in RB-S and RB-NS groups under field
conditions
71
4.13 DMI in RgB and during pre and post supplementation period in
RB-S and RB-NS groups under farm conditions
74
4.14 DMI in RgB and during pre and post supplementation period in
RB-S and RB-NS groups under field conditions
74
4.15 Milk yield in RgB and during pre and post supplementation period
in RB-S and RB-NS groups under farm conditions
77
4.16 Milk yield in RgB and during pre and post supplementation period
in RB-S and RB-NS groups under field conditions
77
4.17 Body weight in RgB and RB groups under farm conditions 80
4.18 Body weight in RB-S and RB-NS groups under farm conditions 80
4.19 Body weight in RgB and RB groups under field conditions 80
4.20 Body weight in RB-S and RB-NS groups under field conditions 80
4.21 Relative expression of TLR-4 and Fas mRNA in neutrophils of RB,
RB (In vitro IGF-1 supplemented) and RgB group.
83
LIST OF THE PICTURE
Picture No. Title After
page
3.1 RB-S animal under Darad village conditions 30
3.2 RB-S animal under Darad village conditions 30
5.1 Calf born of RB-S animal under Darad village conditions 82
5.2 Calf born of RB-S animal under Darad village conditions 82
LIST OF ABBREVIATIONS % = Percent
µg = Microgram
µl = Microlitre
µmol = Micromol
@ = at the rate of
°C = Degree centigrade
µg = microgram
BCS = Body condition score
BUN = Blood urea nitrogen
BW = Body weight
Ca = Calcium
DFM = Direct Fed microbials
DM = Dry matter
DMEM = Dulbecco’s modified Eagle’s medium
DMI = Dry matter intake
EDTA = Ethyelene Diamine Tetra-acetic Acid
EtBr = Ethidium Bromide
FCS = Foetal calf serum
g = Gram
h = Hour
KF = Karan Fries
Kg = Kilogram
mg = Milligram
mm = Millimeter
mmol = Milimole
MY = Milk yield
PCR = Polymerase Chain Reaction
RB = Repeat Breeder
RB-NS = Non Supplemented Repeat Breeder
RB-S = Supplemented Repeat Breeder
RgB = Regular Breeder
v/v = Volume by volume
w/v = Weight by volume
SE = Standard error
SEM = Standard error of mean
TLR-4 = Toll Like Receptor-4
FDA = Food and Drug Administration
YC = Yeast Culture
VFA = Volatile fatty acids
IGF-1 = Insulin Like Growth Factors -1
IGFBPs = IGF binding proteins
NSILA = non-suppressible insulin-like activity
Da = Dalton
GH = Growth Hormone
LH = Luteinizing Hormone
APP = Acute phase protein
Hp = Haptoglobin
LF = Lactoferrin
NK = Natural Killer
GnRH = Gonadotropin Releasing Hormone
TNF = Tumor necrosis factor
NGF = Nerve growth factor
FasL = Fas ligand
DD = Death domain
FADD = Fas-associated death domain-containing protein
DED = Death effector domain’
DISC = Death inducing signalling complex’
TRADDs = TNFR-associated death domain-containing proteins
(CD137), and the type I TNF receptor (Simon, 2003).
Accelerated neutrophils death leads to a decrease of neutrophil counts
(neutropenia), augments the chance of contracting bacterial or fungal infections,
and impairs the resolution of such infections. On the other hand, delayed
neutrophil death elevates neutrophils counts (neutrophilia), which is often
associated with bacterial infection, myeloid leukemia, and acute myocardial
infarction (Akgul et al.,2001 and Maianski et al., 2004).
2.6.1 Apotosis of neutrophils
Apotosis of neutrophil is also an essential cellular event for maintaining
neutrophil number during infection and inflammation. Neutrophils are recruited to
the infected tissues to engulf, kill, and digest invading microorganisms. However,
the enzymes and reactive oxygen species (ROS) released by neutrophils can
also damage the surrounding tissues (Murray et al., 1997). To prevent senescent
neutrophils from releasing their toxic contents, these cells become apoptotic and
22
are then recognized, engulfed, and cleared by professional phagocytes such as
tissue macrophages. This safe clearance provides a mechanism of reducing the
number of viable and activated neutrophils without releasing the potentially
harmful enzymes and ROS, thereby facilitating the resolution of inflammatory
response. Delayed death and clearance of neutrophils in tissues causes
unwanted and exaggerated inflammation. Finally, neutrophil apotosis contributes
to neutrophils’ pathogen killing capability. It is an essential step for the
generation and release of neutrophils extracellular traps (NETs), extracellular
structures composed of chromatin, and granule proteins that bind and kill
invading microorganisms. This mechanism allows neutrophils to fulfill their
antimicrobial function even beyond their life span (Simon, 2003). Thus,
regulation of the neutrophil life span by apoptosis is a crucial process in the
resolution of inflammation, and alterations of apoptosis in neutrophils. In these
cells, this mechanism of apoptosis has special features, probably due to
peculiarities of neutrophil mitochondria, which are pivotal for apoptosis (Simon, 2003). Importantly, neutrophil apoptosis is highly regulated. Extrinsic pathway is
mainly stimulated by death-inducing receptors belonging to the tumor necrosis
factor (TNF)/nerve growth factor (NGF) receptor super-family, such as Fas, TNF-
related apoptosis inducing ligand (TNFSF10) receptors, TNFRSF9 (CD137), and
the type I TNF receptor (Simon, 2003).
Various proapoptotic and antiapoptotic signals at the site of inflammation
can interact on neutrophils and regulate their survival (Hughes and Piontkivska,
2008). Neutrophil apoptosis occurs through two main pathways (Fanning et al.,
1999), one that is mediated via exogenous death receptor signaling (Liles et al.,
1996) and the other that occurs spontaneously through mitochondrial membrane
changes under the influence of Bcl-2 family proteins (Lin et al., 1996). Fas (also
called CD95/APO-1) is a type I membrane glycoprotein belonging to the TNF-
receptor superfamily of molecules. Death signals are initiated in neutrophils when
Fas becomes trimerized upon interaction with its ligand, Fas ligand (FasL).
Activation of Fas receptor by FasL recruits numerous death-domain-containing
proteins to the cytoplasmic death domain of Fas, and these act as adaptor
molecules for the ultimate recruitment and activation of caspase 8 to initiate
apoptosis signaling via a variety of mechanisms (Schulze-Osthoff et al.,1998;
23
Sharma et al., 2000; Kuijpers 2002). Ultimately, caspase 8 activation results in
cleavage of proteins involved in cytoskeletal maintenance and DNA repair, leading
to membrane blebbing, nuclear condensation and collapse, and irreversible cell
death (Robertson et al., 2000 and Zimmermann et al., 2001). Fas receptor and
FasL are constitutively coexpressed in human blood neutrophils, rendering the
cells highly sensitive to apoptosis (Liles et al., 1996).
2.7 Fas GENE
Neutrophil apoptosis occurs through two main pathways (Fanning et al.,
1999), one that is mediated via exogenous death receptor signaling (Liles et al.,
1996) and the other that occurs spontaneously through mitochondrial membrane
changes under the influence of Bcl-2 family proteins (Lin et al., 1996). In the
current study, we chose to examine the main exogenous death receptor of
neutrophils, Fas. Fas (also called CD95/APO-1) is a type I membrane glycoprotein
belonging to the TNF-receptor superfamily of molecules. Death signals are
initiated in neutrophils when Fas becomes trimerized upon interaction with its
ligand, Fas ligand (FasL). Activation of Fas receptor by FasL recruits numerous
death-domain-containing proteins to the cytoplasmic death domain of Fas, and
these act as adaptor molecules for the ultimate recruitment and activation of
caspase 8 to initiate apoptosis signaling via a variety of mechanisms (Schulze-
Osthoff et al., 1998; Sharma et al., 2000). Ultimately, caspase 8 activation results
in cleavage of proteins involved in cytoskeletal maintenance and DNA repair,
leading to membrane blebbing, nuclear condensation and collapse, and
irreversible cell death (Robertson et al., 2000 and Zimmermann et al., 2001). Fas
receptor and FasL are constitutively coexpressed in human blood neutrophils,
rendering the cells highly sensitive to apoptosis (Liles et al., 1996).
2.7.1 Mechanisms of neutrophils apoptosis activated by death receptors (Fas)
Death receptors are cell surface receptors that transmit apoptosis signals
initiated by specific death ligands given by Ashkenazi and Dixit, 1998 in Figure
2.3. They include TNFRs, Fas (Apo-1/CD95), DR3 (Apo-3/TRAMP), DR4
(TRAIL-R1), DR5 (TRAIL-R2) and DR6. The two best-characterised death
receptors, TNFR1 and Fas, are both transmembrane protein members of an
24
expanding TNF nerve growth factor (NGF) family that signals for apoptosis in
many cell types (Ashkenazi and Dixit,1998), including neutrophils (Liles et al.,
1996; Murray et al., 1997). Each death receptor contains cysteine-rich
extracellular domains and a motif in the cytoplasmic region termed a ‘death
domain (DD)’ (Ashkenazi and Dixit, 1998). Death domains are mainly involved in
protein-protein interactions and connect the receptors with the components of
the intracellular apoptosis machinery. Associations between death domains
occur upon receptor-ligand binding and these interactions are necessary for
initiation of apoptosis (Ashkenazi and Dixit, 1998; Nagata, 1997). FasL is the
ligand for Fas and is a homotrimeric molecule. Binding of Fas with trimeric FasL
leads to cross-linking of three receptor molecules resulting in clustering of
intracellular death domains. The association of receptor death domains induces
the recruitment of adaptor proteins (Ashkenazi and Dixit, 1998 and Nagata,
1997). The major adaptor protein is the Fas-associated death domain-containing
protein (FADD), and recruited FADD associates with the activated receptor
through its own death domains. FADD also contains a ‘death effector domain’
(DED) that allows its interaction with procaspase- 8 via its respective DEDs.
Fas/FADD/pro-caspase-8 together form a protein complex called the
‘deathinducing signalling complex’ (DISC) (Medema et al., 1997). Pro-caspase-
has been suggested to be activated according to the ‘induced proximity model’ in
which caspase precursor aggregation mediated by FADD induces
autoprocessing and autoactivation through cross-cleavage (Muzio et al., 1998).
Indeed, gene knock-out experiments in mice in which FADD is deleted have
shown that FADD is one of the essential components of the apoptosis machinery
induced by FasL and TNF-a (Ashkenazi and Dixit, 1998), TNF-a, a trimeric
molecule, is the ligand for TNF receptors. Exposure of cells to TNF-a can induce
multiple effects including cell differentiation, proliferation, apoptosis and other
pro-inflammatory effects. TNF binding induces trimerisation of TNFR1 bringing
the death domains of the receptors into close proximity (Ashkenazi and Dixit,
1998; Nagata, 1997). Subsequently, TNFR-associated death domain-containing
proteins (TRADDs) bind to clustered receptors via their respective DDs. TRADD
can also associate with other secondary adaptor molecules including TNFR-
associated factor-2 (TRAF2) and receptor-interacting protein (RIP) leading to the
activation of the transcription factors, NF-kB and AP-1 (Ashkenazi and Dixit
25
1998; Nagata, 1997). However, TRADD can also associate with FADD, thereby
inducing the activation of pro-caspase-8 which leads to apoptosis (Ashkenazi
and Dixit, 1998 and Nagata, 1997). Whilst TNF-a can induce apoptosis through
TNFR1 in some cell types including neutrophils (Murray et al., 1997), triggering
the activation of NF-kB and AP-1 may induce the expression of survival factors,
thereby providing resistance against apoptosis (Ward et al., 1999; Zong et
al.,1999). In this respect, TNF-a is a bifunctional molecule and the response of a
cell to this agent probably depends upon interplay between pro- and anti-
apoptotic signaling effects.
2.8 TOLL LIKE RECEPTOR 4 (TLR-4) The term TOLL originally referred to a cell surface receptor governing
dorsal/ventral orientation in the early Drosophila larvae (Stein et al., 1991). A
variety of proteins homologous with toll-like receptors have been identified in
humans (Rock et al., 1998 and Takeuchi et al., 2000). TLR-4 is a type 1
transmembrane protein with an intracellular domain homologous to that of the
humans IL1 receptor (Medzhitov et al., 1997). Bovine TLR gene was discovered
by White et al. (2003). Bovine TLR-4 shares genomic structure with human and
mouse counterparts. The overall length of bovine TLR-4 is 11kb, which is
comparable to 10 kb for human and 14 kb for mouse. Most of the differences in
length are found in length of the introns. Toll and TLR family proteins are
characterized by the presence of two primary motifs. An extracellular domain
with leucine rich repeats (LRR) and an intracytoplasmic region containing a
All animals were confirmed to be pregnant by 23rd week in RgB group,
where as in RB group none of the animals conceived to 3 services (by AI) till 21st
week under both farm and field conditions.
3.3 Chemicals, glassware and plasticware
Chemicals and utensils required for blood collection and chemical
analysis were purchased from Sisco Research Laboratories., New Mumbai.
3.4 Collection of blood samples
Blood (10 ml) was collected in sterile heparinised vacutainer tubes by
jugular vein puncture, posing minimum disturbance to the animal during
collection. Blood samples were collected at weekly intervals from selected
animals under both farm and field conditions. Blood samples from pregnant
animals were not taken after confirmation of pregnancy by rectal palpation. Immediately after collection, the samples were transported to the laboratory in
ice for further processing.
3.5 Feeding under farm and field conditions.
All cows were fed to meet the nutritional requirements as per NRC (1989)
standards. During postpartum period cows were fed ad lib (at least 15% in
excess of requirement) available green fodders, predominantly, Maize and Jowar
and to some extent Berseem, limited amount of wheat straw as roughage and
concentrates based on body weight and production level of milk. Concentrates
was fed to the individual cow in two major installments, first of which was offered
between 8.00 to 9.00 am. in the morning after weighing of residue left of the
previous day and the second installment was fed between 4.00-5.00 pm. in the
evening before filling their respective feeding manger with another installment of
fodder. Feeding schedule of individual animals was revised at monthly interval
and was reformulated on the basis of body weight of the animal and the milk
yield. The amount of feed and fodder and concentrate offered and residue left by
the individual animals were recorded daily and computed weekly.
30
Table 3.1 Details of experimental animals under farm conditions
Regular Breeder (RgB)
Sr. No. Animal No. Date of Calving Parity Body Weight (Kg)
1 6625 12.03.10 2 395
2 6751 16.04.10 2 415
3 6791 19.03.10 3 380
4 6880 02.05.10 3 400
5 6453 09.04.10 2 412
6 6741 11.04.10 3 392
NonSupplemented Repeat Breeder (RB-NS)
Sr. No. Animal No. Date of Calving Parity Body Weight( Kg)
7 6650 09.04.10 2 395
8 6817 12.03.10 2 401
9 6931 07.05.10 3 380
10 7045 11.03.10 3 400
11 7028 02.04.10 3 408
12 6333 08.04.10 2 392
13 6726 29.03.10 3 385
Supplemented Repeat Breeder (RB-S)
Sr. No. Animal No. Date of Calving Parity Body Weight(KG)
14 6647 21.03.10 2 400
15 6670 19.04.10 3 403
16 6903 25.03.10 2 397
17 6669 16.04.10 3 376
18 5853 02.05.10 2 407
19 6885 03.04.10 3 394
20 6553 30.03.10 3 389
Table 3.2 Details of experimental animals under field conditions
Regular Breeder (RgB)
Sr.
No. Animal No. Name of owner/village Date of Calving Parity
Body
Weight (Kg)
1 RgB 1 Joga Singh/Darar 12.06.10 3 386
2 RgB 2 Gurulal Singh/Darar 23.08.10 2 392
3 RgB 3 Rakesh Kumar/Darar 09.07.10 3 404
4 RgB 4 Deelip Singh/Darar 03.07.10 3 380
5 RgB 5 Amardeep Singh/Darar 09.08.10 2 406
6 RgB 6 Sompal Singh/Darar 11.07.10 3 384
Non Supplemented Repeat Breeder (RB-NS)
7 RB-NS 1 Jasvir Singh/Darar 19.06.10 2 397
8 RB-NS 2 Balvant singh/Darar 07.08.10 2 386
9 RB-NS 3 Amareet Pal/Darar 13.07.10 3 377
10 RB-NS 4 Darampal Singh/Darar 12.07.10 3 388
11 RB-NS 5 Malvinder Singh/Darar 23.08.10 3 376
12 RB-NS 6 Sompal Singh/Indri 24.06.10 2 392
13 RB-NS 7 Pawan Kumar/Indri 08.07.10 3 402
Supplemented Repeat Breeder (RB-S)
14 RB-S 1 Amanjeet Singh/Darar 16.06.10 2 393
15 RB-S 2 Harbhajan singh/darar 29.06.10 3 398
16 RB-S 3 Karnal Singh/Darar 11.07.10 3 409
17 RB-S 4 Raj Singh/Darar 18.08.10 2 394
18 RB-S 5 Pyarelal/Darar 25.07.10 3 391
19 RB-S 6 Rupesh Kumar/Indri 22.07.10 3 389
20 RB-S 7 Rajesh Kumar/Indri 28.08.10 2 381
Picture 3.1. RB-S animal under Darad village conditions
Picture 3.2 RB-S animal under Darad village conditions
Table 3.3 Composition of concentrate feed supplemented to animals
Components of concentrate Per 100 kg
Maize 30 parts
Deoiled mustard cake 25 parts
Soyabean extraction 10 parts
Wheat bran 10 parts
Deoiled rice bran 15 parts
Molasses 7 parts
Mineral mixture 2 parts
Common salt 1 part
3.6 Estimation of feed intake
The daily feed intake was estimated by subtracting the amount of refusals
from the amount of feed offered. During feeding trial, the amount of feed and
fodder and concentrate offered and residue left by the individual animals were
recorded daily and computed on weekly basis.
3.6.1 Analysis of feed and fodder samples
Samples of green fodders and concentrate mixture being fed and residues left
were analyzed for dry matter (DM) every week.
3.7 Recording of body weight
The body weight of the animals were recorded initially at start of experiment and
then at monthly intervals till the end of the experimental period under farm and
field conditions. Body weight of each animal was recorded early in the morning
between 7.30 am to 8.30 am on an electronic weighing scale before providing
feed to the animals.
3.8 Recording of milk yield
Milking of cows was mainly done by machine milking thrice daily at 5:00
am, 12: 00 noon and 7:00 pm, till the end of the experimental period under farm
and field conditions. The individual milk yields (kg) were recorded at each
milking.
31
3.9 Estimation of plasma parameters
3.9.1 Estimation of blood plasma haptoglobin (Hp):
Haptoglobin was estimated as per the instructions provided with ELISA
test kit (CUSABIO BIOTECH. CO. LTD., USA). The range for concentration of
standards varied from 15.6 ng/ml - 200 ng/ml. The minimum detectible
concentration of haptoglobin by this assay was estimated to be 0.1ng/ml.
3.9.2 Estimation of blood plasma lactoferrin
Lactoferrin was estimated as per the instruction provided with the kit
(Lactoferrin ELISA Quantification set, Belthyl Laboratories,Inc., USA). The range
for concentration of standards varied between 7.5 ng/ml to 500 ng/ml. The
minimum detectible concentration of Lactoferrin by this assay was estimated to
be 1.0 ng/ml.
3.9.3 Estimation of blood plasma insulin-like growth factor-1 (IGF-1)
IGF-1 was estimated as per the instructions provided with the kit Bovine
Reaction programme for all the genes was as follows:
Table 3.8 Reaction programme for real time PCR Programme Steps Temperature Time Cycles
Initial denaturation - 95 0C 5 min 1
Extension Denaturation 95 0C 15 sec
45 Annealing 59 0C 15 sec Extension 72 0C 20 sec
Melting curve
Denaturation 95 0C 5 sec
1 Renaturation 65 0C 1 min
Final Denaturation 97 0C Continuous
mode Cooling - 40 0C 1 min 1
Melting peaks for all reactions were analyzed for the presence of primer
dimers or secondary structures (if any) in all samples.
Two housekeeping genes (GAPDH) used as reference gene for
normalization of target gene for relative quantification.
The expression of different genes in different samples was performed as:
ΔCP (experimental samples) = Cp (target gene of sample) – Reference index
ΔCP (calibrator) = Cp (target gene of calibrator) – Reference index
Where: Cp – Crossing point
Reference index = Mean (Cp GAPDH)
Fold expression level of all the genes for all the samples was calculated
as follows:
ΔΔCP (quantity of expression) = ΔCP (experimental samples) - ΔCP(calibrator)
expression= (2)–ΔΔCP
44
Figure 3.4 Amplification curve TLR-4
Figure 3.5 Amplification curve Fas
3.11.9 Statistical analysis of relative target gene expression
Generation of quantitative data by real-time PCR is based on the number
of cycles required for optimal amplification generated fluorescence to reach a
specific threshold of detection (the Quantification cycle) (Bustin et al., 2009). The
relative expression ratio of the target gene was tested for significance as per
method given by Pfaffl, (2001). 3.12 Statistical analysis
All statistical analysis were done using SYSTAT software package. Data
from different experiments are presented as Mean±SE. Significance was tested
within each group by one-way ANOVA with multiple comparisons. The Mean±SE
were analyzed by multiple t test between groups. The significance of Mean±SEM
values was tested by employing unpaired t test (Assuming unequal variance).
The correlation was tested by Spearman Rank Order Correlation.
45
CHAPTER –4
RESULTS AND DISCUSSION
RESULTS AND DISCUSSION
Although comparisons have been made between RgB and RB groups
from 4th week itself, animals were not classified as RB at this stage, they were
classified as RB at the end of 21st week of study tenure. All animals were
confirmed to be pregnant by 23rd week in RgB group, where as in RB group
none of the animal conceived to 3 consecutive services (by AI) post partum till
21st week under both farm and field conditions. In RgB group only two animals
were left by 19th week which were not confirmed pregnant. Supplementation of
fermented yeast culture to RB-S groups was initiated at 22nd week post partum
under both farm and field conditions. For first 21 week post partum RgB group
serve as control for RB group where as from 22nd week till 40th week of
experiment RB-NS serve as control for RB-S under both farm and field
conditions. Results for correlation studies are reported, only if they were
significant at least at P<0.05.
4.1 INSULIN-LIKE GROWTH FACTOR-1 (IGF-1)
4.1.1 Concentration of plasma Insulin-like growth factor-1 (ng/ml) in RgB and RB groups under farm conditions.
The Mean±SE concentration of plasma IGF-1 at weekly interval and
Mean±SEM values for RgB and RB groups under farm conditions is presented in
Table 4.1 and depicted in Figure 4.1.
Under farm conditions, at the beginning of experiment (4th week post
partum), level of plasma IGF-1 in RgB and RB groups was 73.14±2.34 and
69.35±3.81ng/ml respectively, the difference was not significant. The results in
the present study indicate concentration of plasma IGF-1 in RgB exhibited
increasing trend from initial week till 23rd week post partum. Within RgB group it
was observed that concentration of plasma IGF-1 increased significantly
(P<0.05) from initial value at 10th and 16th week post partum. It also increased
significantly (P<0.05) over initial value from 16th week post partum till end of
experiment. The percent increase in concentration of plasma IGF-1 over initial
value at 23rd week post partum in RgB groups was 26.1%. Similar increase was
observed in the concentration of plasma IGF-1 in RB group till 21st week post
46 Results & Discussion
partum but difference was not significant throughout the course of the
experiment. The percent increase in plasma IGF-1concentration over initial value
at 21st week post partum in RB groups was 7.74 %. When Mean±SE values for
concentration of plasma IGF-1 was compared between these two groups,
significant (P<0.05) greater concentration was observed in RgB group at all
week intervals except at 4th, 16th and 18th week post partum. The Mean±SEM
concentration of plasma IGF-1 for RgB and RB groups under farm conditions
was 85.31±2.22 and 72.79±2.46 ng/ml respectively, which was significantly
(P<0.05) greater in RgB when compared with RB group.
Concentration of plasma IGF-1 was significantly positively correlated with
concentration of plasma glucose, LF (P<0.01), calcium (P<0.05) and negatively
correlated (P<0.01) with concentration of plasma urea and Hp for RgB and RB
groups (Table 4.41).
4.1.2 Concentration of plasma Insulin-like growth factor-1 (ng/ml) in RB-S and RB-NS groups under farm conditions.
The Mean±SE concentration of plasma IGF-1 at weekly interval and
Mean±SEM values for RB-S and RB-NS groups under farm conditions is
presented in Table 4.2 and depicted in Figure 4.1.
From 22nd week, post partum when probiotic (Fermented yeast culture)
was supplemented concentration of plasma IGF-1 was observed to be
80.58±3.75 ng/ml and 79.83±2.67 ng/ml in RB-S and RB-NS group respectively.
An increasing trend in concentration of plasma IGF-1 was observed in RB-S
group whereas decreasing trend was observed in RB-NS group till 40th week
post partum. Within both RB-S and RB-NS group difference was not significant
from initial value throughout the course of the experiment. Concentration of
plasma IGF-1 in RB-S group increased to 85.54±0.39 ng/ml and in RB-NS group
deceased to 73.82±4.29 ng/ml over initial concentration at end of 40th week of
study. The percent increase in concentration of plasma IGF-1 over initial value
in RB-S group at end of experiment was 6.15 %. The percent decrease in
concentration of plasma IGF-1 over initial value in RB-NS group at end of
experiment was 7.52 %. When Mean±SE values for concentration of plasma
IGF-1 was compared between groups significantly greater (P<0.05)
47 Results & Discussion
Table 4.1 Concentration of plasma IGF-1(ng/ml) in RgB and RB groups under farm conditions.
Weeks RgB RB
4 73.14A ±2.34 69.35±3.81
5 76.23 a ±1.34 70.13b±2.36
6 81.48 a ±1.18 71.85±1.66
7 78.32 a ±1.91 70.72 b ±2.75
8 82.42 a±1.42a 69.09 b±0.95
9 83.03 a ±3.55 71.02 b ±3.52
10 87.63 Ba ±2.44 70.99 b ±2.75
11 84.34 a ±3.69 69.44 b ±1.06
12 81.84 a ±2.0Aa 71.21 b ±3.39
13 86.34 Ba ±1.02 73.81b±3.71
14 84.52 a±4.00 76.02 b±1.86
15 83.81a±2.27 73.66b±1.11
16 86.59B ±3.87 74.73±3.74
17 90.40 Ba ±2.45 71.06b±2.04
18 88.48 B±1.67 78.60±3.71
19 86.54 Ba ±2.61 77.41b±1.69
20 93.38Ba ±1.60 76.36b±1.66
21 91.67 Ba±2.21 74.72b±2.48
22 93.89B±1.60
23 92.23 B±1.31
Mean±SEM 85.31*±2.22 72.79±2.46
Values with different superscripts (A, B) are significantly different (P<0.05) from initial value within each group. Values with different superscripts (a, b) are significantly different (P<0.05) between RgB and RB groups. * P<0.05
concentration was observed in RB-S group from 33rd to 38th week post partum.
The Mean±SEM concentration of plasma IGF-1 in RB-NS and RB-S groups was
83.20±2.79 and 75.55±3.45 ng/ml respectively, which was significantly (P<0.05)
greater in RB-S when compared with RB-NS group.
Concentration of plasma IGF-1 was significantly positively correlated with
plasma glucose, LF (P<0.01) and calcium (P<0.05) concentration and negatively
correlated (P<0.05) with concentration of plasma urea for both RB-S and RB-NS
groups (Table 4.42).
4.1.3 Concentration of plasma Insulin-like growth factor-1 (ng/ml) in RgB and RB groups under field conditions.
The Mean±SE concentration of plasma IGF-1 at weekly interval and
Mean±SEM values for RgB and RB groups under field conditions is presented in
Table 4.3 and depicted in Figure 4.2.
Under field conditions also, at the begining of experiment (4th week post
partum) level of plasma IGF-1 in RgB and RB groups was 79.91±2.10 and
69.94±0.85 ng/ml respectively. The results obtained in the present study
indicated that concentration of plasma IGF-1 exhibited an increasing trend from
initial concentration till 23rd week post partum in RgB group. The percent
increase in concentration of plasma IGF-1 by 23rd week post partum in RgB
group was 8.68%. Similarly, increasing trend in concentration of plasma IGF-1 in
RB group till 21th week post partum also observed. The difference was not
significant from initial value throughout the course of the experiment within both
RgB and RB groups. Plasma IGF-1 concentration in RB group at start of the
experiment was 69.94±0.85 ng/ml which increased to 73.71±3.09 ng/ml by 21st
week post partum. The percent increase in concentration of plasma IGF-1over
initial value at 21th week post partum in RB groups was 5.4 %. When Mean±SE
values for concentration plasma IGF-1 was compared between groups
significantly (P<0.05) greater concentration was observed in RgB group till end
of the experiment except at 8th and 21st week. The Mean±SEM concentration of
plasma IGF-1 for RgB and RB groups under farm conditions was 82.25±1.73 and
71.62±1.68 ng/ml respectively, which was significantly (P<0.05) greater in RgB
when compared with RB group.
48 Results & Discussion
Concentration of plasma IGF-1 was significantly positively correlated
(P<0.01) with concentration of plasma glucose, LF (P<0.01), calcium (P<0.05)
and negatively correlated (P<0.01) with concentration of plasma urea and Hp for
RgB and RB groups (Table 4.43).
4.1.4 Concentration of plasma Insulin-like growth factor-1 (ng/ml) in RB-S and RB-NS groups under field conditions.
The Mean±SE concentration of plasma IGF-1 at weekly interval and
Mean±SEM values for RB-S and RB-NS groups under field conditions is
presented in Table 4.4 and depicted in Figure 4.2.
At 22nd week post partum when probiotic was supplemented to RB-S
group concentration of plasma IGF-1 was 74.27±1.23 ng/ml and in RB-NS group
was 73.39±1.66 ng/ml. Within RB-S group, concentration of plasma IGF-1
increased from initial value but was significantly (p<0.05) greater only at 30th
week and further from 34th till 37th week. In RB-S group, concentration of plasma
IGF-1 exhibited increasing trend till 40th week. Concentration of plasma IGF-1 in
RB-S group increased to 83.94±1.41 ng/ml from the Mean±SE concentration at
22nd week post partum. The percent increase in plasma IGF-1 in RB-S group at
end of experiment was 13.02 %. In RB-NS group concentration of plasma IGF-1
did not increase significantly from initial value till end of experiment. In RB-NS
group only 2.7% increase was observed in concentration of plasma IGF-1 over
initial value. Concentration of plasma IGF-1 in RB-NS group varied between
72.29 and 77.10 ng/ml during the experimental period. Concentration of plasma
IGF-1 was significantly greater (P<0.05) in RB-S group at 25th, 29th, 33th, 34th,
35th and 40th weeks when compared with RB-NS group. The Mean±SEM
concentration of plasma IGF-1 in RB-NS and RB-S groups was 81.17±1.41 and
74.19±1.75 ng/ml respectively, which was significantly (P<0.05) greater in RB-S
when compared with RB-NS group.
Concentration of plasma IGF-1 was significantly positively correlated
(P<0.01) with plasma glucose, LF and calcium concentration and negatively
correlated (P<0.01) with concentration of plasma urea for RB-S and RB-NS
groups (Table 4.44).
49 Results & Discussion
Table 4.2 Concentration of plasma IGF-1(ng/ml) in RB-S and RB-NS groups under farm conditions.
Weeks RB-S RB-NS
22 80.58±3.75 79.83±2.67
23 80.01±3.12 78.78±3.96
24 78.73±3.52 79.00±4.65
25 80.35±3.71 77.69±2.87
26 82.27±3.06 77.91±3.76
27 80.50±2.71 77.57±2.18
28 80.42±4.79 76.95±3.57
29 80.20±1.27 75.44±2.38
30 86.71±3.57 75.74±4.94
31 83.59 a±1.42 75.32b±2.98
32 84.21±2.79 76.89±4.87
33 85.34 a±2.60 71.9 b±3.33
34 81.21a±2.63 72.93b±4.51
35 86.62a±4.25 73.80b±2.42
36 86.23a±1.86 72.11b±2.33
37 86.94a±4.31 71.95b±4.37
38 86.68a±0.66 73.89b±2.07
39 84.75±3.37 73.93±3.34
40 85.54±0.39 73.82±4.29
Mean±SEM 83.20*±2.79 75.55±3.45
Values with different superscripts (a, b) are significantly different (P<0.05) between RB-S and RB-NS groups. * P<0.05
Table 4.3 Concentration of plasma IGF-1 (ng/ml) in RgB and RB groups under field conditions.
Weeks RgB RB
4 79.91a±2.10 69.94b±0.85
5 75.25a±2.20 68.92b±0.65
6 78.30a±2.23 71.44b±1.71
7 77.09a±3.16 68.71b±1.94
8 77.19±2.48 73.15±0.95
9 78.80a±0.55 70.01b±2.16
10 81.40a±1.12 73.18b±2.27
11 82.11a±1.56 68.43b±1.05
12 81.11a±1.21 69.80b±1.30
13 79.53a±2.71 70.20b±1.37
14 81.61a±4.03 71.76b±2.01
15 83.42a±1.79 72.45b±1.59
16 85.60a±1.17 71.18b±1.37
17 84.41a±0.72 70.89b±2.16
18 86.49a±2.12 74.99b±1.81
19 84.55a±1.51 75.50b±1.59
20 87.59 a±0.72 74.95b±2.27
21 86.29a ±2.12 73.71 b±3.09
22 87.51±0.35
23 86.85±0.71
Mean±SEM 82.25*±1.73 71.62±1.68
Values with different superscripts (a, b) are significantly different (P<0.05) between RgB and RB groups. * P<0.05
Table 4.4 Concentration of plasma IGF-1(ng/ml) in RB-S and RB-NS groups under field conditions.
Weeks RB-S RB-NS
22 74.27A±1.23 73.39±1.66
23 74.96 A ±1.05 75.41±0.99
24 78.14 A ±1.94 73.51±1.00
25 76.16 A a±2.16 74.28 b ±0.90
26 81.84 A ±0.74 74.47±0.98
27 80.31 A ±2.98 72.33±2.54
28 79.61 A ±2.16 74.81±1.09
29 79.43 A a±1.48 75.13b±1.00
30 83.06B±0.85 72.29±1.92
31 82.78 A ±2.08 72.45±1.60
32 79.58 A ±1.37 74.24±1.49
33 82.49 A a±1.10 72.46b±2.30
34 85.48Ba ±0.60 73.28b±1.81
35 83.83Ba±2.01 74.35b±2.08
36 84.42B±0.63 76.66±2.47
37 83.17B±1.78 77.10±2.94
38 85.82 A ±0.57 73.57 b±3.21
39 82.89 A ±0.71 74.57 b±2.17
40 83.94 a±1.41 75.39b±1.18
Mean±SEM 81.17*±1.41 74.19±1.75
Values with different superscripts (A, B) are significantly different (P<0.05) from initial value within each group. Values with different superscripts (a, b) are significantly different (P<0.05) between RB-S and RB-NS groups. * P<0.05
Figure 4.2 Concentration of plasma IGF-1 in RgB and during pre and post supplementation period in RB-S and RB-NS groups under field conditions.
In the present study greater concentration of plasma IGF-1 was observed
in RgB when compared with RB group under both farm and field conditions. In a
variety of species (including farm animals, humans, and laboratory animals)
higher concentration of blood IGF-1 is found in young, well nourished, healthy
individuals (Jones and Clemmons, 1995;Thissen et al.,1994). Animals that are
old, diseased, or mal-nourished exhibited less concentration of plasma IGF-1
that reflect a compromised state of tissue, organ, and cell function (Jones and
Clemmons, 1995 Lucy et al., 1991; Thissen et al., 1994; McNall et al.,1995).
Several studies have established positive relationship between blood IGF-1
concentration and reproductive function of postpartum cattle. Thatcher et al.
(1996), Anandlaxmi et al. (2013) reported that anestrus dairy cows had lower
blood IGF-1 concentration when compared with cows that initiated estrous
cyclicity earlier during the postpartum period. A similar relationship was reported
for beef cattle, the postpartum anestrus cows had lower IGF-1 level when
compared with cyclic cows (Roberts et al., 1997). Blood IGF-1 was correlated
with follicular fluid IGF-1 because the majority of IGF-1 in follicular fluid was
derived from blood (Leeuwenberg et al., 1996). Beam and Butler (1999)
reported positive correlation among serum IGF-1, LH pulsatility and follicular
wave in post partum cows. Wathes (2008) reported that delay or failure of
conception in post partum dairy cows is associated with reduced IGF-1
concentration and also with concentration of urea in plasma.
Low level of IGF-1 and LF reflects lower immune status of repeat
breeding group when compared with regular breeder group. This is un
confirmation with the studies of Nickerson (1989), Oliver and Sordillo (1989) in
different physiological state. Therefore lower concentration of plasma IGF-1 may
be one of the reason for resulting in lower conception rate in RB group under
both farm and field conditions.
The positive correlation between plasma glucose and IGF-1 has also
been reported by Beam and Butler (1999). They observed that plasma level of
IGF-1 is directly related to energy status i.e. glucose and IGF-1 is critical for
ovarian follicular development. In our studies also similar results were observed.
The significant difference in concentration of plasma IGF-1 between RB-S and
RB-NS group as observed under both farm and field conditions, concentration of
50 Results & Discussion
plasma IGF-1 being higher in RB-S and RgB groups favored conception at an
earlier age when compared with their counterparts.
4.2 HAPTOGLOBIN (Hp)
4.2.1 Concentration of plasma Hp (ng/ml) in RgB and RB groups under farm Conditions.
The Mean±SE concentration of plasma Hp at weekly interval and
Mean±SEM values for RgB and RB groups under farm conditions is presented in
Table 4.5 and depicted in Figure. 4.3.
At the begining of experiment (4th week post partum) concentration of
plasma Hp in RgB and RB groups under farm conditions were 499.02±27.67and
763.47±28.86 ng/ml respectively. The results in the present study indicated that
within both RgB and RB groups there was significant (P<0.05) decrease in
Mean±SE values for concentration of plasma Hp from 7th week till end of the
experiment. The percent decrease in concentration of plasma Hp over initial
concentration at 13th week post partum in RgB groups was 52.56 % which was
maximum at 13th week. Similarly, decreasing trend in concentration of plasma
Hp in RB group till 21st week post partum also observed. Concentration of
plasma Hp in RgB group at the beginning of the experiment was 499.02±27.67
ng/ml which derceased to 290.24±13.43 ng/ml at 23rd week post partum. The
percent decrease in concentration of plasma Hp at 23rd week post partum over
initial value in RgB groups was 41.84 %. Concentration of plasma Hp in RB
group at 4th week was 763.47±28.86 ng/ml which decreased to
394.73±21.11ng/ml at 21st week post partum. The percent decrease in
concentration of plasma Hp at 21st week post partum over initial value in RB
groups was 48.29 %. In RgB group Mean±SE values for concentration of plasma
Hp was significantly less (P<0.05) when compared with RB group throughout the
course of the experiment. The Mean±SEM concentration of plasma Hp for RgB
and RB groups was 315.14±22.42 and 546.56±24.41 ng/ml respectively, which
was significantly (P<0.05) greater in RB when compared with RgB group. The
concentration of plasma Hp was within physiological range.
Concentration of plasma Hp was significantly positively correlated
(P<0.01) with concentration of plasma urea and negatively correlated (P<0.01)
51 Results & Discussion
Table 4.5 Concentration of plasma Hp (ng/ml) in RgB and RB groups under farm conditions.
Weeks RgB RB
4 499.02Aa±27.67 763.47Ab±28.68
5 447.02 A a±35.45 727.03 A b±10.86
6 421.86 A a±20.04 678.16 A b±39.92
7 347.86Ba±32.43 585.78Bb±40.50
8 372.86Ba±14.54 608.84Bb ±32.27
9 295.94Ba±12.29 580.43Bb±18.38
10 283.11Ba±23.40 587.26Bb±16.33
11 278.36Ba±14.39 565.69Bb±28.97
12 256.19 Ba ±32.68 527.94 Bb±24.93
13 236.27Ba±21.56 535.03Bb±31.49
14 254.60Ba±37.67 518.92Bb±14.28
15 296.35Ba±14.57 503.82Bb±28.01
16 263.93Ba±12.82 510.03 Bb ±13.51
17 278.35Ba±23.91 470.76Bb±23.06
18 318.85Ba±16.79 454.46Bb±19.57
19 309.85Ba±27.68 418.15Bb±31.11
20 264.08Ba±23.89 407.53Bb ±16.33
21 278.74Ba±19.68 394.73Bb±21.11
22 309.24B±23.46
23 290.24B±13.43
Mean±SEM 315.14±22.42 546.56*±24.41
Values with different superscripts (A, B) are significantly different (P<0.05) from initial value within each group. Values with different superscripts (a, b) are significantly different (P<0.05) between RgB and RB groups. * P<0.05
Table.4.6 Concentration of plasma Hp (ng/ml) in RB-S and RB-NS groups under farm conditions.
Weeks RB-S RB-NS
22 412.14±18.38 422.9±25.54
23 398.93±31.11 411.52±37.73
24 386.52±12.26 411.37±13.91
25 389.16±27.12 410.82±36.88
26 385.25±18.57 424.31±25.07
27 375.36±36.97 444.56±11.11
28 366.35±25.46 425.08±37.98
29 361.35a±18.69 428.64b±22.09
30 350.22±25.57 424.33±43.27
31 350.84a±18.75 445.3 b±11.66
32 348.85±36.76 433.42±28.02
33 312.96a±10.92 420.53 b ±25.36
34 315.16±19.7 403.31±32.87
35 310.43±28.75 392.55±28.09
36 300.26a±12.22 389.37b±35.57
37 309.59±38.29 398.41±27.75
38 299.61a±26.67 390.33 b±17.93
39 305.78±31.49 380.71±34.41
40 295.73±17.82 374.75±27.51
Mean±SEM 346.03±23.97 412.22*±27.50
Values with different superscripts (a, b) are significantly different (P<0.05) between RB-S and RB-NS groups. * P<0.05
Table 4.7 Concentration of plasma Hp (ng/ml) in RgB and RB groups under field conditions.
Weeks RgB RB
4 478.14Aa ±27.27 705.56b±11.89
5 446.14 A a±32.06 708.72b±27.54
6 441.81 A a±43.88 690.25 b ±30.20
7 386.98 A a±27.92 699.87b±.1352
8 371.98 A a±36.82 690.93b±28.47
9 315.73Ba±27.95 630.52b±16.19
10 329.23 A a±40.84 599.3 b±38.38
11 297.48Ba±32.68 577.78Bb±24.57
12 315.31Ba±26.84 590.03b±13.24
13 281.39Ba±13.11 547.12Bb±33.62
14 289.01Ba±19.78 531.01Bb±19.00
15 315.76 A a±27.58 515.91 Bb ±13.71
16 273.34Ba±38.46 472.12Bb±24.99
17 297.76Ba±14.66 482.85 Bb±27.64
18 305.26Ba±23.21 464.55 Bb ±39.60
19 309.26 A a±35.56 430.24Bb±28.69
20 293.09Ba±27.25 417.62Bb±14.16
21 297.54 A a±32.35 446.82Bb±18.68
22 308.04 A ±20.41
23 309.04 A ±31.63
Mean±SEM 333.11±29.01 566.74*±23.56
Values with different superscripts (A, B) are significantly different (P<0.05) from initial value within each group. Values with different superscripts (a, b) are significantly different (P<0.05) between RgB and RB groups. * P<0.05
with concentration of plasma LF, IGF-1(P<0.01), glucose and calcium (P<0.05)
for RgB and RB groups (Table 4.41).
4.2.2 Concentration of plasma Hp (ng/ml) in RB-S and RB-NS groups under farm conditions.
The Mean±SE concentration of plasma Hp at weekly interval and
Mean±SEM values for RB-S and RB-NS under farm conditions is presented in
Table 4.6 and depicted in Figure. 4.3.
At 22nd week post partum when probiotic supplementation was initiated, in
RB-S group concentration of plasma Hp was 412.14±18.38 ng/ml whereas in
RB-NS group was 422.9±25.54 ng/ml. In both the groups, concentration of
plasma Hp decreased at 40th week of experiment. The values were not
significantly different from initial value throughout the course of the experiment
within RB-S and RB-NS groups. Concentration of plasma Hp in RB-S and RB-
NS at 40th week of experimental period was observed to be 295.73±17.82 and
374.75±27.51ng/ml respectively. The decrease observed in Hp concentration in
RB-NS group was less; hence the difference in the Mean±SE concentration of
Hp in this group at the end of 40th week was not significant when compared with
the Mean±SE Hp concentration at 22nd week. The percent decrease in plasma
Hp concentration over initial value in RB-S and RB-NS group at end of
experiment was 28.24 and 11.38 % respectively. When Mean±SE values for
concentration of plasma Hp was compared between groups, concentration was
significantly (P<0.05) less in RB-S group at 29th, 31st, 33rd, 36th and 38th week.
Mean±SEM concentration of plasma Hp in RB-S and RB-NS groups were
346.03±23.97 and 412.22±27.50 ng/ml respectively, which was significantly
(P<0.05) greater in RB-NS when compared with RB-S group.
Concentration of plasma Hp was significantly positively correlated
(P<0.01) with concentration of plasma urea and negatively correlated (P<0.01)
with concentration of plasma LF for both the groups (Table 4.42).
52 Results & Discussion
4.2.3 Concentration of plasma Hp (ng/ml) in RgB and RB groups under field conditions.
The Mean±SE concentration of plasma Hp at weekly interval and
Mean±SEM values for RgB and RB groups under field conditions is presented in
Table 4.7 and depicted in Figure 4.4.
Under field conditions, at the beginning of experiment (4th week post
partum), level of plasma Hp concentration in RgB and RB groups conditions was
478.14±27.27 and 705.56±11.89 ng/ml respectively. The results in the present
study indicate that concentration of plasma Hp when compared within RgB group
it was observed that Mean±SE values for concentration decreased significantly
(P<0.05) at 9th, 11th-14th, 16th-18th and 20th week post partum. The percent
decrease in plasma Hp concentration over initial value at 16th week post partum
in RgB groups was 42.83%, at which Mean± SE concentration of Hp was least.
The percent decrease in plasma Hp concentration over initial value at 23rd week
post partum in RgB groups was 35.4 %. Similar, decreasing trend in
concentration of plasma Hp in RB group from initial value till 21st week post
partum was recorded. Within RB group, concentration of plasma Hp decreased
significantly (P<0.05) at 11th and 13th week and further till end of the experiment.
Mean±SE concentration of plasma Hp in RB group at 1st week of the experiment
was 705.56±11.89 ng/ml which decreased to 446.82±18.68 ng/ml at 21st week
post partum. The percent decrease in plasma Hp concentration over initial value
at 21st week post partum in RB groups was 36.67 %. In RgB group,
concentration of plasma Hp was significantly less (P<0.05) when compared with
RB group throughout the course of the experiment. Mean±SEM concentration of
plasma Hp for RgB and RB groups was 333.11±29.01 and 566.74±23.56 ng/ml
respectively, which was significantly (P<0.01) greater in RB when compared with
RgB group.
Concentration of plasma Hp was significantly positively correlated
(P<0.01) with concentration of plasma urea and negatively correlated with
concentration of plasma LF, IGF-1(P<0.01), glucose and calcium (P<0.05) for
both the groups (Table 4.43).
53 Results & Discussion
4.2.4 Concentration of plasma Hp (ng/ml) in RB-S and RB-NS groups under field conditions.
The Mean±SE plasma Hp concentration at weekly interval and
Mean±SEM values for RB-S and RB-NS groups under field conditions is
presented in Table 4.8 and depicted in Figure 4.4.
Within RB-S and RB-NS groups Mean±SE values for concentration of
plasma Hp exhibited decreasing trend but difference were not significant from
initial value throughout the course of the experiment. In RB-S group, plasma Hp
concentration decreased to 314.54±12.83 ng/ml by 40th week post partum over
the initial value. The percent decrease observed was 25.85 %. Mean±SE in RB-
NS group plasma Hp concentration increased to 424.83±23.54 ng/ml from
416.5±25.39ng/ml. A 2 % percent increase was observed in plasma Hp
concentration over initial value in RB-NS group at end of experiment. When
Mean±SE values for concentration of plasma Hp was compared between
groups, the concentration was significantly less (P<0.05) at 30th, 32nd- 38th and
40th week interval in RB-S group. The Mean±SEM concentration of plasma Hp
RB-NS and RB-S groups was 364.36±23.35 and 430.01±26.82 ng/ml
respectively, which was significantly (P<0.05) greater in RB-NS when compared
with RB-S group.
Concentration of plasma Hp was significantly positively correlated
(P<0.01) with concentration of plasma urea and negatively correlated (P<0.05)
with concentration of plasma lactofferrin for both the groups (Table 4.44).
Results obtained indicated that significant greater (P<0.01) concentration
of plasma Hp was present in RB group when compared with RgB under both
farm and field conditions. Hence, the results indicated that greater concentration
of plasma Hp (P<0.01) prevailed in repeat breeding animals when compared
with their counterpart. To the best of our knowledge no reports are available on
plasma level of Hp in repeat breeding animals under tropical conditions.
Although the Mean±SE concentration of plasma Hp in RB and RB-NS group was
significantly higher than their counterparts, the concentration of plasma Hp under
farm or field conditions was observed to be within physiological range (300-
500ng/ml) (Horadagoda et al., 1999; Anand Laxmi et al.,2013) in cows and does
54 Results & Discussion
not indicate pathological condition. Under pothological conditions it is reported to
increase in microgram range (Horadagoda et al., 1999). It indicates mild stress
or inflammation in the mentioned groups. Higher plasma Hp concentration at an
earlier stage post partum may be due to inflammatory condition persisting at
earlier stage which decreased later on as reflected by Hp concentration. Higher
plasma Hp concentration in cows with post partum reproductive problems has
been reported by Chan et al. (2004) and Petersen et al. (2004). It was concluded
that Hp seems to be a promising marker of health status by reflecting a broad
spectrum of ongoing clinical as well as subclinical diseases in cows. A low
concentration of plasma Hp in RgB group suggests healthy status of group as
supported by Uchida et al. (1993), who suggested that, only a less concentration
of Hp was detectable in normal bovine serum. This may be one of the reasons
due to which RgB group animals might have concieved ≤3 services. Lower
concentration of plasma Hp in RgB group indicates good health which might
have resulted in conception in less than 3 consecutive services under both farm
and field conditions. Number of investigations indicate the ability of Hp as
unspecific markers of clinical and subclinical infections, to discriminate between
acute and chronic disease and for prognostic purposes, since the duration and
magnitude of the response reflect the severity of the disease and the effect of
treatment (Skinner et al., 1991; Horadagoda et al., 1999; Hultén et al., 1999;
Petersen et al., 2002; Hultén and Demmers, 2002; Lauritzen et al., 2003). Higher
concentration of plasma Hp in RB group suggests subclinical infection or
stress due to which it resulted in delay in conception when compared with RgB
and RB-S groups under both farm and field conditions.
Saini and Webert (1991), reported that plasma Hp was considered to be
involved in dynamic process, involving systemic and metabolic changes
providing an early non-specific defence mechanism against infection before
specific immunity is achieved.
To best of our knowledge this is the first investigation which shows the
effect of Probiotic (Saccharomyces cerevisiae) supplementation to repeat
breeding crossbred cows under farm and field conditions. Different studies
revealed beneficial effect of S. cerevisiae on rumen pH, nutrient availability and
health of ruminant (Callaway and Martin, 1997; Kumar et al., 1997; Dann et al.,
55 Results & Discussion
Table 4.8 Concentration of plasma Hp (ng/ml) in RB-S and RB-NS groups under field conditions.
Weeks RB-S RB-NS
22 424.23±26.03 416.50±25.39
23 401.02±17.61 435.12±31.19
24 423.61±24.72 425.64±27.97
25 421.25±11.04 432.32±29.78
26 439.71±28.45 425.81±35.62
27 403.82±25.04 437.73±27.84
28 398.81±12.11 416.58±14.45
29 383.81±28.01 425.14±26.37
30 372.75a±25.89 425.83b±36.10
31 343.37±11.38 446.81±28.73
32 341.38 a±34.88 422.03 b±25.35
33 325.49 a±29.59 410.81b±19.16
34 317.69 a±36.47 444.92b±28.73
35 327.96 a±26.06 431.13b±26.27
36 310.29 a±18.20 425.83b±20.44
37 312.12 a±34.12 446.81b±33.48
38 336.42 a±14.14 444.92 b±11.44
39 324.59±27.07 431.43±37.51
40 314.54a±12.83 424.83b±23.54
Mean±SE 364.36±23.35 430.01*±26.82
Values with different superscripts (a, b) are significantly different (P<0.05) between RB-S and RB-NS groups. * P<0.05
Figure 4.4 Concentration of plasma Hp in RgB and during pre and post supplementation period in RB-S and RB-NS groups under field conditions.
2000). This improvement in health status of animal is reflected parttially due to
the significant lower concentration of plasma Hp in RB-S group when compared
with RB-NS group under farm and field conditions.
4.3 LACTOFERRIN (LF)
4.3.1 Concentration of plasma LF (ng/ml) in RgB and RB groups under farm conditions.
The Mean±SE concentration of plasma LF at weekly interval and
Mean±SEM values for RgB and RB groups under farm conditions is presented
in Table 4.9 and depicted in Figure 4.5.
Under farm conditions, at the beginning of experiment (4th week post
partum) level of plasma LF in RgB and RB groups under farm conditions was
248.33±18.40 and 203.84±25.43 ng/ml respectively. Within RgB group it was
observed that Mean±SE values of plasma LF concentration increased
significantly (P<0.05) only at 17th and 19th week post partum over the initial
value. The results in the present study indicated that concentration of plasma LF
in RgB exhibited an increasing trend from initial value till 23rd week.
Concentration of plasma LF in RgB group at start of the experiment was
248.33±18.40 ng/ml which increased to 376.97±24.54ng/ml at 23rd week post
partum. The percent increase in concentration of plasma LF over initial value at
23rd week post partum in RgB groups was 51.80 %. Similarly, increase in trend
in concentration of plasma LF from initial value till 21st week post partum of RB
group was observed. In RB group, concentration of plasma LF increased but
was not significantly different from initial value till end of experiment.
Concentration of plasma LF in RB group at start of the experiment was
203.84±25.43 ng/ml which increased to 305.06±17.35 ng/ml at 21st week post
partum. The percent increase in concentration of plasma LF over initial value at
21st week post partum in RB groups was 49.65 %. Concentration of plasma LF
was significantly higher (P<0.05) in RgB group at 6th, 10th, 12th, 13th, 15th and 18-
20th weeks when compared with RB group. The Mean±SEM concentration of
plasma LF for RgB and RB groups under farm conditions was 329.68±22.09 and
250.56±22.08 ng/ml respectively, which was significantly (P<0.01) greater in
RgB when compared with RB group.
56 Results & Discussion
Concentration of plasma LF was significantly positively correlated with
concentration of plasma IGF-1, glucose (P<0.01), calcium (P<0.05) and
negatively correlated (P<0.01) with concentration of plasma urea and Hp for RgB
and RB groups (Table 4.41).
4.3.2 Concentration of plasma LF (ng/ml) in RB-S and RB-NS groups under farm conditions.
The Mean±SE concentration of plasma LF at weekly interval and
Mean±SEM values for RB-S and RB-NS groups under farm conditions is
presented in Table 4.10 and depicted in Figure 4.5.
At 22nd week post partum when probiotic was supplemented to RB-S
group concentration of plasma LF was 288.39±24.57 ng/ml and in RB-NS
group was 301.92±17.25 ng/ml. Within both RB-S and RB-NS group difference
in concentration was not significant from the initial value till end of the
experiment. In RB-S group, concentration of plasma LF exhibited increasing
trend till the end of experiment. The percent increase in concentration of plasma
LF over initial value in RB-S group at end of experiment (at 22nd week) was
31.01 %. In RB-NS group change observed at 22nd week for concentration of
plasma LF from initial value was not significant. The range of concentration of
plasma LF in RB-NS group varied between 281.36 and 308.73 25 ng/ml during
the experimental period. Only 2 % increase in concentration of plasma LF over
initial value in RB-NS group at end of experiment was recorded. When Mean±SE
values for concentration of plasma LF was compared between groups, values
were significantly (P<0.05) greater in RB-S group at 29th, 31st- 35th and 37th
week. The Mean±SEM concentration of plasma LF in RB-S and RB-NS groups
was 345.41±20.55 and 296.24±21.32 ng/ml respectively, which was significantly
(P<0.05) greater in RB-S when compared with RB-NS group.
Concentration of plasma LF was significantly (P<0.01) positively
correlated with concentration of plasma IGF-1 and negatively correlated with
concentration of plasma urea (P<0.01) and Hp (P<0.05) for both RB-S and RB-
NS groups (Table 4.42).
57 Results & Discussion
Table 4.9 Concentration of plasma lactoferrin (ng/ml) in RgB and RB groups under farm conditions.
Weeks RgB RB
4 248.33A±18.40 203.84±25.43
5 256.83 A ±27.99 193.96±27.44
6 294.83a±22.63 190.13b±16.18
7 302.80 A ±23.83 234.67±29.18
8 270.17 A ±18.53 210.84±26.11
9 281.97 A ±21.55 229.84±13.99
10 317.47 A a±8.29 209.34 b±24.18
11 293.05 A ±27.99 254.67±14.73
12 339.93 A a±25.57 260.84b±21.90
13 350.02 A a±19.99 259.84b±25.18
14 313.72 A ±12.09 269.14±23.99
15 335.47 A ±18.50 277.70±24.50
16 362.10 A a±29.09 286.90b±16.18
17 376.18B±12.68 287.65±33.99
18 359.68 A a±18.06 278.23b±26.75
19 375.76Ba ±17.83 266.47b±14.85
20 391.85 A a ±17.50 290.99b±15.55
21 375.67 A ±23.95 305.06±17.35
22 370.77 A ±32.89
23 376.97 A ±24.54
Mean±SEM 329.68*±22.09 250.56±22.08
Values with different superscripts (A, B) are significantly different (P<0.05) from initial value within each group. Values with different superscripts (a, b) are significantly different (P<0.05) between RgB and RB groups. * P<0.05
Table 4.10 Concentration of plasma lactoferrin (ng/ml) in RB-S and RB-NS groups under farm conditions.
Weeks RB-S RB-NS
22 288.39±24.57 301.92±17.25
23 293.14±16.18 287.78±22.76
24 310.47±23.29 282.95±25.94
25 332.42±18.23 306.82±15.84
26 326.35±16.15 312.57±27.34
27 351.59±35.22 304.15±16.15
28 322.14±25.76 305.57±25.19
29 326.38 a±27.35 300.66 b ±23.60
30 362.22±26.42 281.36±27.35
31 348.70 a±18.93 272.77b±26.42
32 376.26 a ±15.70 291.97b±28.93
33 373.76 a ±21.93 302.55b±16.70
34 361.50 a±23.76 282.79b±15.18
35 376.05 a ±18.70 290.40b±12.99
36 360.37±17.47 308.73±24.50
37 371.22 a±14.70 300.11b±16.14
38 360.69±13.34 288.90±31.96
39 343.41±15.66 298.58±16.20
40 377.82±17.07 308.02±14.49
Mean±SEM 345.41*±20.55 296.24±21.32
Values with different superscripts (a, b) are significantly different (P<0.05) between RB-S and RB-NS groups. * P<0.05
4.3.3 Concentration of plasma LF (ng/ml) in RgB and RB groups under field conditions.
The Mean±SE concentration of plasma LF at weekly interval and
Mean±SEM values for RgB and RB groups under field conditions is presented in
Table 4.11 and depicted in Figure 4.6.
Under field conditions, at the begining of experiment (4th week post
partum) concentration of plasma LF in RgB and RB groups under field conditions
was 259.11±21.31 and 187.11±24.56 ng/ml respectively. Mean±SE values for
concentration exhibited an increasing trend but difference was not significant
from initial value throughout the course of the experiment in both RgB and RB
groups. The results in the present study indicate that concentration of plasma LF
in RgB showed an increasing trend till 23rd week post partum. The percent
increase in concentration of plasma LF over initial value at 23rd week post
partum in RgB groups was 43.63 %. Similarly, inceasing trend in concentration
of plasma LF in RB group from initial week till 21st week post partum was
recorded. Concentration of plasma LF in RB group at beginning of the
experiment was 187.11±24.56 ng/ml which increased to 302.86±24.93 ng/ml at
21st week post partum. The percent increase in concentration of plasma LF over
initial value at 21st week post partum in RB group was 61.86%. When Mean±SE
values of plasma LF concentration was compared between groups, values were
significantly (P<0.05) greater for RgB group at 7th, 9th, 15th and 20th week. The
Mean±SEM concentration of plasma LF for RgB and RB groups under farm
conditions was 345.41±20.55 and 296.24±21.32 ng/ml respectively, which was
significantly (P<0.01) greater in RgB when compared with RB group.
Concentration of plasma LF was significantly positively correlated with
concentration of plasma IGF-1, glucose (P<0.01), calcium (P<0.05) and
negatively (P<0.01) correlated with concentration of plasma urea and Hp in RgB
and RB groups (Table 4.43).
58 Results & Discussion
4.3.4 Concentration of plasma LF (ng/ml) in RB-S and RB-NS groups under field conditions.
The Mean±SE concentration of plasma LF at weekly interval and
Mean±SEM values for RB-S and RB-NS groups under field conditions is
presented in Table 4.12 and depicted in Figure 4.6.
At 22nd week post partum when probiotic was supplemented to RB-S
group concentration of plasma LF was 317.96±26.12 ng/ml and in RB-NS
group was 322.54±29.18 ng/ml. Within RB-S and RB-NS groups difference was
not significant from the initial value throughout course of the experiment. In RB-S
group, concentration of plasma LF exhibited increasing trend till the end of
experiment. Concentration of plasma LF in RB-S group increased to
392.39±24.07 ng/ml. The percent increase in concentration of plasma LF over
initial value in RB-S group at end of experiment was 23.40 %. In RB-NS group
no significant change was recorded in concentration of plasma LF over initial
value. The range of concentration plasma of LF in RB-NS group varied between
294.81 and 335.96 ng/ml during the experimental period. When Mean±SE
values for concentration of plasma LF was compared between groups,
significantly (P<0.05) greater values were observed in RB-S group at 26th, 30th,
35th- 37th week. The Mean±SEM concentration of plasma LF in RB-S and RB-NS
groups was 368.69±22.27 and 315.43±22.76 ng/ml respectively, which was
significantly (P<0.05) greater in RB-S when compared with RB-NS group.
Concentration of plasma LF was significantly positively correlated
(P<0.01) with concentration of plasma IGF-1 and negatively correlated with
concentration of plasma urea (P<0.01) and Hp (P<0.05) for RB-S and RB-NS
groups (Table 4.44).
Results obtained in present study indicated that significant (P<0.01)
greater concentration of plasma LF in RgB was present when compared with RB
group under both farm and field conditions. Under both farm and field conditions
concentration of plasma LF in RgB and RB groups was within the normal
physiological range. Results of present study were in accordance with those of
Maaks et al. (1989) reported the normal range i.e. as 0.4–2 mg/l under normal
conditions. In present study animals selected were free from clinical infection.
59 Results & Discussion
Table 4.11 Concentration of plasma lactoferrin (ng/ml) in RgB and RB groups under field conditions.
Weeks RgB RB
4 259.11±21.31 187.11±24.56
5 270.41±26.31 188.41±27.57
6 279.61±22.99 217.61±35.18
7 269.92a±15.85 207.92 b ±22.07
8 263.59±27.05 201.59±25.18
9 280.69a±18.10 218.69 b ±14.24
10 269.59±28.71 207.59±19.29
11 290.17±22.00 228.17±27.42
12 277.59±30.95 215.59±18.83
13 319.21a±22.06 217.21b±18.18
14 290.84±33.91 228.84±20.85
15 309.12 a ±17.49 247.12 b ±15.18
16 331.29±26.72 269.29±20.02
17 336.04±35.88 274.04±24.43
18 328.87±25.88 266.87±22.18
19 345.79±27.25 283.79±27.50
20 341.04 a ±19.11 279.04 b±14.43
21 364.86±14.95 302.86±24.93
22 379.96±24.08
23 372.16±12.25
Mean±SEM 308.99*±23.65 235.65±22.34
Values with different superscripts (a, b) are significantly different (P<0.05) between RgB and RB groups. * P<0.05
Table 4.12 Concentration of plasma lactoferrin (ng/ml) in RB-S and RB-NS groups under field conditions.
Weeks RB-S RB-NS
22 317.96±26.12 322.54±29.18
23 310.16±18.26 313.4±27.66
24 333.54±25.29 320.1±26.42
25 325.99±34.27 315.97±22.70
26 346.09 a±15.18 310.22 b±20.44
27 357.33±28.26 304.8±14.13
28 375.54±26.18 298.22±26.90
29 376.79±31.18 294.81±25.35
30 386.29 a±13.80 302.92 b±15.33
31 370.77±24.96 320.01±23.50
32 387.5±32.67 297.54±27.83
33 376.33±18.83 309.21±26.96
34 397.23±19.67 332.38±26.64
35 395.12 a±21.57 321.19 b±20.98
36 401.11 a±15.59 334.99 b±16.99
37 381.95 a ±10.50 316.37b±22.56
38 391.43±15.35 335.96±16.21
39 381.64±21.21 318.5±14.82
40 392.39±24.07 324.01±24.21
Mean±SEM 368.69*±22.27 315.43±22.76
Values with different superscripts (a, b) are significantly different (P<0.05) between RB-S and RB-NS groups. * P<0.05
Figure 4.6 Concentration of plasma LF in RgB and during pre and post supplementation period in RB-S and RB-NS groups under field conditions.
Concentration of plasma LF in RgB and RB remained within normal physiological
range under both farm and field conditions. It is reported that there is 100 fold
increase in concentration of plasma LF during severe inflammation and under
some pathological conditions. It depends on the severity of the condition
(Bennett and Kokocinski, 1978; Maaks et al., 1989).
LF modulates functions of lymphocytes such as cytokine gene activation
(Crouch et al.,1992), cytotoxicity (Shau et al.,1992), B and T cell maturation and
a number of immunopotentiating activities have been ascribed to LF (Zuccotti et
al.,2006). Low level of IGF-1 and LF reflects lower immune status of repeat
breeding groups when compared with regular breeder groups. This is in
confirmation with the reports of Nickerson (1989), Oliver and Sordillo (1989). LF
exhibits diverse biological activities in activation of innate immunity (Miyauchi et
al., 1998). Hence, lower concentration of plasma LF in RB when compared with
RgB group may indicate weaker health and immune status of former group
under both farm and field conditions. This may also be one of reason for reduced
conception rate in RB group under both farm and field conditions.
Chaucheyras et al. (1995), Gattass et al. (2008), Soccol et al. (2010)
reported that S. cerevisiae had the ability to provide growth factors such as
organic acids (methionine and lysine) and vitamins, improving the number of
beneficial rumen microflora, inhibiting growth of pathogens and stimulating
immune function. As probiotic (Saccharomyces cerevisae) shows the positive
effect on immunity of animal which may be one of the reason for significant
greater concentration of plasma LF in RB-S group when compared with RB-NS
group under farm and field conditions. In the present study, supplementation of
fermented yeast culture which is rich source of amino acids and vitamins might
have improved the health status of RB-S group.
4.4 GLUCOSE
4.4.1 Concentration of plasma glucose (mg/dl) in RgB and RB KF cows post partum under farm conditions.
The Mean±SE concentration of plasma glucose at weekly interval and
Mean±SEM values for RgB and RB groups under farm conditions is presented in
Table 4.13 and depicted in Figure 4.7.
60 Results & Discussion
Under farm conditions, at the beginning of experiment (4th week post
partum) level of plasma glucose in RgB and RB groups under farm conditions
was 52.56±0.85 and 50.87±0.21 mg/dl respectively. Within RgB and RB groups,
Mean±SE values for concentration of plasma glucose exhibited increasing trend
but difference was not significant from initial value throughout the course of the
experiment. Concentration of plasma glucose ranged between 52.56 and 54.96
mg/dl in RgB group. Similarly in RB group during the experimental period up to
21st week it ranged between 50.87 and 53.26 mg/dl. When Mean±SE values for
concentration of plasma glucose was compared between groups, concentration
was significantly (P<0.05) greater in RgB group from 7th week except at 9th, 16th,
19th and 20th week. The Mean±SEM concentration of plasma glucose for RgB
and RB groups under farm conditions was 53.98±0.45 and 51.97±0.46 mg/dl
respectively, which was significantly (P<0.05) greater in RgB when compared
with RB group.
Concentration of plasma glucose was significantly positively correlated
(P<0.01) with plasma IGF-1, LF (P<0.01), calcium (P<0.05) and negatively
correlated (P<0.05) with plasma concentration of urea and Hp for both RgB and
RB groups (Table 4.41).
4.4.2 Concentration of plasma glucose (mg/dl) in RB-S and RB-NS groups under farm conditions.
The Mean±SE concentration of plasma glucose at weekly interval and
Mean±SEM values for RB-S and RB-NS groups under farm conditions is
presented in Table 4.14 and depicted in Figure 4.7.
At 22nd week post partum when probiotic was supplemented to RB-S
group concentration of plasma glucose was 52.13±0.36 mg/dl and in RB-NS
group was 52.19±0.016 mg/dl. After supplementation of probiotic to RB-S group,
concentration of plasma glucose increased significantly (P<0.05) from initial
value from 27th week post partum till the end of the experiment. Within RB-NS
group concentration of plasma glucose did not change significantly from the
initial value till end of experiment. Concentration of plasma glucose ranged
between 52.11and 52.80 mg/dl in RB-NS group during the experimental period.
Concentration of plasma glucose was significantly greater (P<0.05) in RB-S
61 Results & Discussion
Table 4.13 Concentration of plasma glucose (mg/dl) in RgB and RB groups under farm conditions.
Weeks RgB RB
4 52.56a±0.85 50.87±0.21
5 52.88 a±0.37 51.16±0.77
6 53.27a±0.71 51.53±0.35
7 52.73 a±0.16 51.09 b±0.31
8 53.40 a±0.50 51.29 b±0.46
9 53.02 a±0.67 51.58±0.16
10 53.32 a±0.37 51.19 b±0.37
11 53.59 a±0.48 51.56 b±0.48
12 54.12 a±0.27 51.95 b±0.76
13 53.89 a±0.15 51.95 b±0.20
14 54.55 a±0.57 52.84 b±0.35
15 54.79 a±0.26 52.79 b±0.52
16 54.60 a±0.78 52.74±0.46
17 54.40 a±0.31 52.80 b±0.55
18 54.66 a±0.55 51.93 b±0.69
19 54.96 a±0.50 53.26±0.71
20 54.92 a±0.24 52.64 b±0.14
21 54.80 a±0.67 52.27±0.75
22 54.49±0.38
23 54.61±0.26
Mean±SEM 53.98*±0.45 51.97±0.46
Values with different superscripts (a, b) are significantly different (P<0.05) between RgB and RB groups. * P<0.05
group from 25th weeks post partum till the end of experiment. The Mean±SEM
concentration of plasma glucose in RB-S and RB-NS groups was 53.76±0.37
and 52.11±0.38 mg/dl respectively, which was significantly (P<0.05) higher in
RB-S when compared with RB-NS group.
Concentration of plasma glucose was significantly positively correlated
(P<0.01) with plasma IGF-1, LF and calcium concentration and negatively
correlated with concentration of plasma urea and Hp for RB-S and RB-NS
groups (Table 4.42).
4.4.3 Concentration of plasma glucose (mg/dl) in RgB and RB groups under field conditions.
The Mean±SE concentration of plasma glucose at weekly interval and
Mean±SEM values for RgB and RB groups under field conditions is presented in
Table 4.15 and depicted in Figure 4.8.
Under field conditions, at the beginning of experiment (4th week post
partum) level of plasma glucose in RgB and RB groups under field conditions
was 52.47±0.47 and 50.38±0.69 mg/dl respectively. Within both RgB and RB
groups, Mean±SE values for concentration of plasma glucose exhibited
increasing trend but difference was not significant from initial value throughout
the course of the experiment. Concentration of plasma glucose ranged between
52.38±0.69 and 54.91±0.62 mg/dl in RgB group during 23 weeks of experimental
period. Similarly concentration of plasma glucose in RB group ranged between
50.38±0.69 and 52.72±0.41 mg/dl. When Mean±SE values for concentration of
plasma glucose was compared between groups, significantly (P<0.05) higher
concentration was observed in RgB group at 4th, 5th, 8th,9th , 11th , 14th , 16-19th
and 21st week post partum. The Mean±SEM concentration of plasma glucose for
RgB and RB groups under farm conditions was 53.68±0.44 and 51.76±0.43
mg/dl respectively, which was significantly (P<0.05) greater in RgB when
compared with RB group.
Concentration of plasma glucose was significantly (P<0.01) positively
correlated with concentration of plasma IGF-1, LF, calcium and negatively
(P<0.01) correlated with concentration of plasma urea and Hp for both RgB and
RB groups (Table 4.43).
62 Results & Discussion
4.4.4 Concentration of plasma glucose (mg/dl) in RB-S and RB-NS groups under field conditions.
The Mean±SE concentration of plasma glucose at weekly interval and
Mean±SEM values for RB-S and RB-NS groups under field conditions is
presented in Table 4.16 and depicted in Figure 4.8.
At 22nd week post partum when probiotic was supplemented to RB-S
group concentration of plasma glucose was 52.16±0.28 mg/dl and in RB-NS
group was 51.98±0.22 mg/dl. Mean±SE values for concentration exhibited
increasing trend but difference was not significant from initial value throughout
the course of the experiment in RB-S and RB-NS groups. After supplementation
of probiotic to RB-S group plasma glucose concentration increased to
54.41±0.18 mg/dl over initial value. Plasma glucose concentration ranged
between 51.63±0.51 and 52.96±0.28 mg/dl in RB-NS group throughout the
experimental period. When Mean±SE values of plasma glucose concentration
was compared between groups, significantly (P<0.05) greater values were
observed in RB-S group at 34th and further from 38-40th week. The Mean±SEM
concentration of plasma glucose in RB-S and RB-NS groups was 53.39±0.41
and 52.48±0.34 mg/dl respectively. Concentration of plasma glucose was
significantly (P<0.05) greater in RB-S when compared with RB-NS group.
Concentration of plasma glucose was significantly positively correlated
(P<0.01) with concentration of plasma IGF-1, LF and calcium and negatively
correlated with concentration of plasma urea and Hp for both RB-S and RB-NS
groups (Table 4.44).
Results of present study indicated significant higher concentration of
plasma glucose in RgB group when compared with RB group under both farm
and field conditions. It is reported by Richards et al. (1989) that higher blood
glucose concentration increases progesterone production by increasing pulse
and mean concentration of LH which would be one of the reasons for RgB and
RB-S groups to conceive earlier. JoeArosh et al. (1998) suggested that
hypoglycemic condition in repeat breeder causes impaired hypothalamic
hypophysial ovarian axis and reduces ovarian activity. Number of studies show
relation between plasma glucose and ovarian function (Highshoe et al., 1991;
63 Results & Discussion
Table 4.14 Concentration of plasma glucose(mg/dl) in RB-S and RB-NS groups under farm conditions.
Weeks RB-S RB-NS
22 52.13 A±0.36 52.19±0.01
23 52.39 A ±0.50 51.86±0.71
24 52.98 A ±0.70 51.95±0.38
25 52.60 A a±0.72 52.25 b±0.11
26 53.32 A a±0.62 52.00b±0.40
27 53.35 Ba±0.38 52.32 b±0.71
28 53.85 Ba±0.06 52.71 b±0.42
29 54.47 Ba±0.22 52.11 b±0.13
30 53.76Ba±0.78 52.90 b±0.52
31 54.30Ba±0.11 51.77 b±0.61
32 53.78Ba±0.35 52.4 b±0.28
33 54.46 Ba±0.24 51.99b±0.33
34 54.07Ba±0.38 51.90b±0.17
35 54.13Ba±0.20 51.87b±0.49
36 54.17Ba±0.28 52.80b±0.28
37 54.46Ba±0.48 51.53b±0.51
38 54.46Ba±0.26 51.92b±0.55
39 54.19Ba±0.17 51.70b±0.47
40 54.61Ba ±0.29 51.97b±0.23
Mean±SEM 53.76*±0.37 52.11±0.38
Values with different superscripts (A, B) are significantly different (P<0.05) from initial value within each group. Values with different superscripts (a, b) are significantly different (P<0.05) between RB-S and RB-NS groups. * P<0.05
Table 4.15 Concentration of plasma glucose (mg/dl) in RgB and RB groups under field conditions.
Weeks RgB RB
4 52.47a±0.47 50.38 b±0.69
5 52.69 a±0.30 50.57 b±0.80
6 52.38±0.69 51.21±0.71
7 52.86±0.47 51.14±0.73
8 52.51±0.47 51.51±0.65
9 53.15a±0.60 51.04b±0.66
10 52.93±0.37 51.80±0.39
11 53.71a±0.46 51.58b±0.23
12 53.23±0.26 52.06±0.47
13 54.10±0.60 52.03±0.14
14 53.70 a±0.47 52.01 b ±0.29
15 53.92±0.68 52.45±0.37
16 54.13 a±0.37 52.12 b±0.30
17 53.85 a±0.50 52.31 b±0.40
18 54.41a±0.28 52.01b±0.23
19 54.73a±0.14 52.19b±0.19
20 54.24±0.60 52.72±0.41
21 54.91a±0.62 52.47b±0.10
22 54.75±0.36
23 54.87±0.22
Mean±SEM 53.68*±0.44 51.76±0.43
Values with different superscripts (a, b) are significantly different (P<0.05) between RgB and RB groups. * P<0.05
Table 4.16 Concentration of plasma glucose (mg/dl) in RB-S and RB-NS groups under field conditions.
Weeks RB-S RB-NS
22 52.16±0.28 51.98±0.22
23 52.72±0.11 51.63±0.51
24 52.13±0.47 52.63±0.44
25 52.51±0.65 52.17±0.33
26 52.34±0.44 52.62±0.56
27 52.73±0.33 51.90±0.38
28 53.09±0.52 52.42±0.21
29 53.36±0.66 52.59±0.18
30 53.24±0.62 52.78±0.58
31 53.46±0.56 52.96±0.28
32 53.64±0.27 52.40±0.47
33 53.74±0.57 52.78±0.38
34 53.78a±0.13 52.89b±0.31
35 54.27±0.38 52.45±0.28
36 54.30±0.55 52.79±0.37
37 54.02±0.42 52.80±0.19
38 54.24a±0.41 52.60b±0.28
39 54.34a±0.48 52.39b±0.34
40 54.41a±0.18 52.36b±0.13
Mean±SEM 53.39*±0.41 52.48±0.34
Values with different superscripts (a, b) are significantly different (P<0.05) between RB-S and RB-NS groups. * P<0.05
Figure 4.8 Concentration of plasma glucose in RgB and during pre and post supplementation period in RB-S and RB-NS groups under field conditions.
Wehrman et al., 1991; Stewart et al., 1995; Rabiee et al., 1997). Miyoshi et al.
(2001) suggested that cows with higher concentration of plasma glucose will
have better ovarian activity. Even though concentration of plasma glucose was
not significantly different but practically, concentration of 50.38±0.69mg/ml in RB
group at 4th week of experiment, had negative effect on reproductive function.
Hence in the present study, RB group of animals had lower glucose
concentration which might have affected ovarian activity and might have
decreased conception rate. This might be one of the reason of infertility in repeat
breeding groups under both farm and field conditions. The finding of this study
are in accordance with the earlier reports by Jani et al. (1995) who observed
high incidence of repeat-breeding and anoestrous problem associated with the
low plasma glucose level. The present findings are also in agreement with El-
Belely (1993) who suggested that lower level of plasma glucose might be the
reason for reduced luteal function in repeat breeding cows. A contradictory
report (Kapadia et al., 2013) is also available stating no significant difference in
concentration of plasma glucose when compared between RgB and RB groups.
Action of Saccharomyces cerevisiae like oxygen uptake, supply of growth
factors and pH stabilization synergistically act for growth of rumen microbes.
Saccharomyces cerevisiae directly stimulated rumen fungi, which may improve
fiber digestion (Chaucheryas et al., 1995). Nisbet and Martin (1991), Gattass et
al. (2008) reported increase in glucogenic VFA production in rumen of cows
supplemented with Saccharomyces cerevisiae in comparision to RB-NS group.
Glucogenic VFA converts into glucose on metabolism. These mechanisms may
explain the significant (P<0.05) greater concentration of plasma glucose in RB-S
group when compared with RB-NS group under farm and field conditions.
4.5 UREA
4.5.1 Concentration of plasma urea (mg/dl) in RgB and RB groups under farm conditions.
The Mean±SE concentration of plasma urea at weekly interval and
Mean±SEM values for RgB and RB groups under farm conditions is presented in
Table 4.17 and depicted in Figure 4.9.
64 Results & Discussion
Under farm conditions, at the beginning of experiment (4th week post
partum) level of plasma urea in RgB and RB groups was 22.29±1.40and
26.03±1.34 mg/dl respectively. Within both RgB and RB groups, Mean±SE
values for concentration exhibited increasing trend but difference was not
significant from initial value till end of the experiment. The results in the present
study indicate that concentration of plasma urea in RgB decreased from initial
value to16.14±1.02 by 23rd week post partum. The percent decrease in
concentration of plasma urea over initial value at 23rd week post partum in RgB
groups was 27.6%. Concentration of plasma urea in RB group at start of the
experiment was 26.03±1.34 mg/dl which decreased to 21.03±0.92 mg/dl at 21st
week post partum. The percent decrease in concentration of plasma urea over
initial value at 21st week post partum in RB groups was 19.2%. When Mean±SE
values for concentration of plasma urea was compared between groups,
significantly (P<0.05) less concentration was observed in RgB group at 5th, 12th,
13th, 17th and 21st week. The Mean±SEM concentration of plasma urea for RgB
and RB groups was 18.6±1.30 and 22.53±1.18 mg/dl. Concentration of plasma
urea was significantly (P<0.01) greater in RB group when compared with RgB
group.
Concentration of plasma urea was significantly positively correlated
(P<0.01) with concentration of plasma Hp and negatively correlated (P<0.01)
with plasma concentration of LF, IGF-1(P<0.01), glucose and calcium (P<0.05)
for both RgB and RB groups (Table 4.41).
4.5.2 Concentration of plasma urea (mg/dl) in RB-S and RB-NS groups under farm conditions.
The Mean±SE concentration of plasma urea at weekly interval and
Mean±SEM values for RB-S and RB-NS groups under farm conditions is
presented in Table 4.18 and depicted in Figure 4.9.
At 22nd week post partum when probiotic was supplemented to RB-S
group concentration of plasma urea was 21.09±1.31 mg/dl whereas in RB-NS
group was 22.55±1.32 mg/dl. Within both RB-S and RB-NS groups,
concentration of plasma urea exhibited decreasing trend but difference was not
significant from initial value till 40th week of experiment. Concentration of plasma
65 Results & Discussion
Table 4.17 Concentration of plasma urea (mg/dl) in RgB and RB groups under farm conditions.
Weeks RgB RB
4 22.29±1.40 26.03±1.34
5 21.75a±0.81 25.31b±1.28
6 22.01±1.47 25.32±1.18
7 20.71±0.89 24.11±1.28
8 21.16±0.94 24.21±1.23
9 21.94±0.79 24.23±0.98
10 20.66±0.64 23.42±2.23
11 18.80±1.47 22.49±0.40
12 17.82a ±0.91 21.62 b ±1.28
13 17.92a ±2.26 20.90 b±1.61
14 16.72±1.57 20.68±1.18
15 17.45±0.90 20.33±1.28
16 17.41±1.92 20.99±1.27
17 16.67a±0.92 21.22 b±0.34
18 17.97±2.27 21.95±0.81
19 17.03±1.54 20.62±1.42
20 16.24±1.27 21.07±1.25
21 15.43a±0.82 21.03 b±0.92
22 15.97±2.15
23 16.14±1.02
Mean±SEM 18.6±1.30 22.53**±1.18
Values with different superscripts (a, b) are significantly different (P<0.05) between RgB and RB groups. ** P<0.01
urea in RB-S group decreased to 15.93±0.36 mg/dl and in RB-NS group
deceased to 21.19±0.94 mg/dl over initial value at 40th week. The percent
decrease in RB-S and RB-NS groups at end of experiment was 24.46 and 6.03
% respectively. When Mean±SE values for concentration of plasma urea was
compared between groups, concentration was significantly (P<0.05) less in RB-
S group from 32nd till end of the experiment except at 38th and 39th week. The
Mean±SEM concentration of plasma urea in RB-S and RB-NS groups was
18.23±1.28 and 22.41±1.13 mg/dl respectively. Concentration of plasma urea
was significantly (P<0.05) greater in RB-NS when compared with RB-S group.
Concentration of plasma urea was significantly positively correlated
(P<0.01) with concentration of plasma Hp and negatively correlated (P<0.01)
with concentration of plasma of IGF-1(P<0.05) and Lactofferrin (P<0.01) for both
RB-S and RB-NS groups (Table 4.42).
4.5.3 Concentration of plasma urea (mg/dl) in RgB and RB groups under field conditions.
The Mean±SE concentration of plasma urea at weekly interval and
Mean±SEM values for RgB and RB groups under field conditions is presented in
Table 4.19 and depicted in Figure 4.10.
Under field conditions, at the beginning of experiment (4th week post
partum) level of plasma urea in RgB and RB groups under field conditions was
23.92±1.23 and 25.55±1.10mg/dl respectively. The results in the present study
indicate that Mean±SE values for concentration decreased significantly (P<0.05)
from initial value from 17th-20th week post partum when compared within RgB
group. The percent decrease in concentration of plasma urea over initial value in
the plasma samples collected at 23th week post partum in RgB groups was
34.33%. Similarly, declining trend in concentration of plasma urea in RB group
from initial value till 21th week post partum was observed. Within in RB group
concentration of plasma urea decreased but difference was not significant from
initial value throughout the course of the experiment. Plasma urea concentration
in RB group at start of the experiment was 25.55±1.10 mg/dl decreased to
20.95±0.93 mg/dl at 21th week post partum. The percent decrease in plasma
urea concentration over initial value at 21th week post partum in RB groups was
66 Results & Discussion
18%. When Mean±SE values of plasma urea concentration was compared
between groups, significantly (P<0.05) less value was observed for RgB group at
8th, 11th, and further from 18th – 21st week. The Mean±SEM concentration of
plasma urea for RgB and RB groups was 18.55±1.21 and 23.03±1.13 mg/dl.
Concentration of plasma urea was significantly (P<0.05) greater in RB when
compared with RgB group.
Concentration of plasma urea was significantly positively correlated
(P<0.01) with concentration of plasma Hp and negatively correlated with
concentration of plasma of IGF-1, LF, glucose (P<0.01) and calcium (P<0.05) for
RgB and RB groups (Table 4.43).
4.5.4 Concentration of plasma urea (mg/dl) in RB-S and RB-NS groups under field conditions.
The Mean±SE concentration of plasma urea collected at weekly interval
and Mean±SEM values for RB-S and RB-NS groups under field conditions is
presented in Table 4.20 and depicted in Figure 4.10.
At 22nd week post partum when probiotic was supplemented to RB-S
group, concentration of plasma urea was 21.07±0.64mg/dl and in RB-NS group
was 21.71±0.95 mg/dl. Within RB-S and RB-NS groups, concentration of plasma
urea exhibited decreasing trend till the end of experiment tenure but the
difference was not significant from the initial value till 40th week. Plasma urea
concentration in RB-S group decreased to 16.10±1.03 mg/dl and in RB-NS
group decreased to 22.08±1.36 mg/dl over initial value. The percent decrease in
concentration of plasma urea from its initial concentration in both RB-S and RB-
NS groups at the end of the experiment were 23.58 and 1.7 % respectively.
When Mean±SE values for concentration of plasma urea was compared
between group, values were significantly (P<0.05) less for RB-S group from 29th
week till end of the experiment. The Mean±SEM concentration of plasma urea in
RB-S and RB-NS groups was 18.02±1.8 and 22.68±1.10 mg/dl respectively.
Concentration of plasma urea was significantly (P<0.05) greater in RB-NS when
compared with RB-S group.
Concentration of plasma urea was significantly positively correlated
(P<0.01) with concentration of plasma Hp and negatively correlated (P<0.01)
67 Results & Discussion
Table 4.18 Concentration of plasma urea (mg/dl) in RB-S and RB-NS groups under farm conditions.
Weeks RB-S RB-NS
22 21.09±1.31 22.55±1.32
23 20.83±1.28 22.58±0.53
24 20.35±1.35 23.28±1.27
25 21.42±2.14 23.07±1.24
26 20.82±1.28 23.75±1.43
27 20.93±1.39 22.42±0.92
28 19.87±1.28 22.88±0.99
29 20.00±1.27 22.80±1.24
30 18.27±1.21 22.08±1.28
31 17.80±1.37 23.20±1.20
32 16.88a±1.37 22.37b±1.55
33 16.06a±2.22 23.37b±1.45
34 16.38a±1.16 22.22b±0.95
35 16.34a±0.94 22.10b±1.15
36 15.73a±1.21 21.81b±1.31
37 16.79a±1.21 20.98b±0.21
38 15.55±0.12 20.98±1.23
39 16.32±1.01 22.24±1.26
40 15.93a±0.36 21.19b±0.94
Mean±SEM 18.23±1.28 22.41*±1.13
Values with different superscripts (a, b) are significantly different (P<0.05) between RB-S and RB-NS groups. * P<0.05
Table 4.19 Concentration of plasma urea (mg/dl) in RgB and RB groups under field conditions.
Weeks RgB RB
4 23.92A±1.23 25.55±1.10
5 22.75 A ±1.97 24.78±1.08
6 21.71 A ±1.31 23.94±1.44
7 22.41 A ±0.98 24.64±1.07
8 21.06 A a±1.27 24.74b±0.71
9 20.39 A ±2.12 24.76±0.52
10 19.32 A ±1.60 23.25±1.36
11 18.70 A a±1.93 23.98b±0.98
12 18.52 A ±0.82 22.05±1.42
13 17.40 A ±2.71 21.43±1.44
14 17.66 A a±1.21 23.13b±0.63
15 17.09 A a±0.96 22.15 b±1.48
16 17.22 A a±1.32 21.44 b±1.10
17 16.53 Ba±1.28 21.80 b±1.33
18 16.60 Ba ±1.28 22.39 b±0.95
19 15.44 Ba ±0.94 20.95 b±1.30
20 16.07 Ba±1.32 22.62 b±2.26
21 16.35 A a±0.95 20.95 b ±0.93
22 15.24 A ±1.30
23 15.71 A ±1.02
Mean±SEM 18.55±1.21 23.03*±1.13
Values with different superscripts (A, B) are significantly different (P<0.05) from initial value within each group. Values with different superscripts (a, b) are significantly different (P<0.05) between RgB and RB groups. * P<0.05
Table 4.20 Concentration of plasma urea (mg/dl) in RB-S and RB-NS groups under field conditions.
Weeks RB-S RB-NS
22 21.07±0.64 21.71±0.95
23 21.36±1.14 22.63±2.30
24 19.82±0.99 22.24±2.20
25 20.92±1.30 22.62±1.23
26 21.35±0.94 22.85±0.96
27 18.81±1.42 22.58±1.51
28 19.59±0.65 21.54±2.45
29 19.47 a±1.30 23.78±1.30
30 17.57a±1.35 22.30 b±0.90
31 17.38a±2.18 22.36b±1.05
32 16.33a±1.37 23.51b±2021
33 17.95 a±0.81 22.57b±1.45
34 15.85a±2.16 23.36b±0.91
35 15.91a±1.24 23.28b±1.21
36 16.71a±0.94 22.89b±2.76
37 17.17a±1.18 24.14b±0.95
38 17.12a±0.94 22.20b±0.49
39 14.93a±1.06 22.28 b±1.17
40 16.10a±1.03 22.08 b±1.36
Mean±SEM 18.02±1.8 22.68*±1.10
Values with different superscripts (a, b) are significantly different (P<0.05) between RB-S and RB-NS groups. * P<0.05
Figure 4.10 Concentration of plasma urea in RgB and during pre and post supplementation period in RB-S and RB-NS groups under field conditions.
with concentration of plasma of IGF-1 (P<0.05) and Lactofferrin (P<0.01) for both
RB-S and RB-NS groups (Table 4.44).
In the present study concentration of plasma urea was greater in RB
when compared with RgB group under both farm and field conditions. The
results are in accordance with (WestWood et al., 1998) who reported negative
relationship between concentration of plasma urea and conception rate in cattle.
Number of studies reported negative relationship between plasma concentration
of urea and pregnancy rate (Elrod and Butler, 1993; Elrod et al., 1993; Ferguson
et al. 1993; Dhali et al., 2006). According to Dhali (2001) greater serum or
plasma urea nitrogen concentration reduces LH binding to ovarian receptors and
reduces or delays ovulation. It has been observed that increased concentration
plasma urea may interfere with the normal inductive actions of progesterone on
the microenvironment of the uterus and, thereby, cause suboptimal conditions
for support of embryo implantation (Hamman et al., 2000; Papadopoulas et al.,
2001). As delayed ovulation and inability of embryo implantation are the reasons
of repeat breeding in cattle. In RB group plasma level of urea was greater than
the physiological level, which might have altered uterine pH level and further
development of embryo. Higher concentration of plasma urea may be one of the
causes for repeat breeding under both farm and field conditions.
In the present study negative correlation was observed between plasma
glucose and urea which is in confirmation with other studies of Westwood et al.
(1998), Shingu et al. (2009) and Shehab-el-deen (2011).
4.6 CALCIUM
4.6.1 Concentration of plasma calcium (mg/dl) in RgB and RB groups under farm conditions.
The Mean±SE concentration of plasma calcium at weekly interval and
Mean±SEM values for RgB and RB groups under farm conditions is presented in
Table 4.21 and depicted in figure 4.11.
Under farm conditions, at the beginning of experiment (4th week post
partum) concentration of plasma calcium in RgB and RB groups was 9.44±0.26
and 9.12±0.30 mg/dl respectively. Within both RgB and RB groups, Mean±SE
values for concentration exhibited increasing trend but difference was not
68 Results & Discussion
significant from initial value throughout the course of the experiment. The
percent increase in concentration of plasma calcium post partum in RgB group
was 15.9 %. Concentration of plasma calcium in RB group at the beginning was
9.12±0.30 mg/dl which increased to 9.64±0.18 mg/dl by 21st week post partum.
Only 0.52 mg/dl increase was observed in concentration of plasma calcium by
21th week post partum in RB group. When Mean±SE values for concentration of
plasma calcium was compared between groups, significantly (P<0.05) greater
concentration was observed for RgB group at 14th, 15th, 18th, 19th and 21st week.
The Mean±SEM concentration of plasma calcium for RgB and RB group under
farm conditions was 10.36±0.28 and 9.52±0.27 mg/dl respectively. Concentration
of plasma calcium concentration was significantly (P<0.05) high in RgB when
compared with RB group.
Concentration of plasma calcium was significantly positively correlated
with concentration of plasma IGF-1, LF (P<0.05), glucose (P<0.01) and
negatively correlated with concentration of plasma urea (P<0.01) and Hp
(P<0.05) for RgB and RB groups (Table 4.41).
4.6.2 Concentration of plasma calcium (mg/dl) in RB-S and RB-NS groups under farm conditions.
The Mean±SE concentration of plasma calcium at weekly interval and
Mean±SEM values for RB-S and RB-NS groups under farm conditions is
presented in Table 4.22 and depicted in figure 4.11.
At 22nd week post partum when probiotic was supplemented to RB-S
group, concentration of plasma calcium was 9.73±0.30 mg/dl and in RB-NS
group was 9.79±0.31mg/dl. Mean±SE values for concentration of plasma
calcium exhibited increasing trend but difference was not significant from initial
value till 40th week in RB-S and RB-NS groups. In RB-S group, concentration of
plasma calcium increased from 22nd to 40th week post partum. Concentration of
plasma calcium in RB-S group increased to 10.50±0.29 mg/dl. The percent
increase in concentration of plasma calcium from 22nd to 40th week post partum
in RB-S and RB-NS group was 7.9 % and 1.73 % respectively. When Mean±SE
values of RB-S and RB-NS group was compared, difference was not significant
throughout the course the experiment. The Mean±SEM concentration of plasma
69 Results & Discussion
Table.4.21 Concentration of plasma calcium (mg/dl) in RgB and RB groups under farm conditions.
Weeks RgB RB
4 9.44±0.26 9.12±0.30
5 9.61±0.36 9.01±0.20
6 9.54±0.30 9.14±0.30
7 9.71±0.21 9.31±0.26
8 9.64±0.44 9.34±0.31
9 10.09±0.40 9.49±0.20
10 10.25±0.28 9.42±0.39
11 10.50±0.43 9.73±0.13
12 10.57±0.33 9.67±0.31
13 10.61±0.18 9.91±0.36
14 10.70a±0.25 9.57 b±0.10
15 10.50a±0.11 9.69 b ±0.17
16 10.52±0.36 9.49±0.40
17 10.72±0.36 9.79±0.24
18 10.94a±0.14 9.76 b±0.20
19 10.50a±0.13 9.57 b±0.34
20 10.81±0.36 9.73±0.40
21 10.82a±0.34 9.64 b±0.18
22 10.76±0.23
23 10.94±0.17
Mean±SEM 10.36*±0.28 9.52±0.27
Values with different superscripts (a, b) are significantly different (P<0.05) between RgB and RB groups. * P<0.05
calcium in RB-S and RB-NS groups was 10.29±0.32 and 9.96±0.25 mg/dl
respectively. The concentration of plasma calcium was not significantly different
between the groups.
Concentration of plasma calcium was significantly positively correlated
(P<0.05) with concentration plasma IGF-1 for RB-S and RB-NS groups (Table
4.42).
4.6.3 Concentration of plasma calcium (mg/dl) in RgB and RB groups under field conditions.
The Mean±SE concentration of plasma calcium at weekly interval and
Mean±SEM values for RgB and RB groups under field conditions is presented in
Table 4.23 and depicted in figure 4.12.
Under field conditions, at the beginning of experiment (4th week post
partum) concentration of plasma calcium in RgB and RB groups under field
conditions was 9.02±0.20 and 8.95±0.28 mg/dl respectively. The result in the
present study indicated that plasma calcium concentration in RgB group
increased from initial value till 23rd week but was significantly (P<0.05) different
from initial value only at 19th and 20th week post partum. The percent increase in
plasma calcium concentration over initial value at 23rd week post partum in RgB
groups was 19.3 %. Within RB group, concentration of plasma calcium did not
increase significantly from initial value till the end of experiment. Concentration of
plasma calcium in RB increased from 8.95±0.28 mg/dl to 9.55±0.13 mg/dl at 21st
week post partum. The percent increase in plasma calcium concentration at 21st
week post partum in RB groups was 6.7 %. When Mean±SE values of
concentration of plasma calcium was compared between groups, significantly
higher (P<0.05) values were observed for RgB group from 11th week till the end
of the experiment. The values were not significant except at 12th, 14th and 16th
weeks. The Mean±SEM concentration of plasma calcium for RgB and RB groups
under field conditions was 9.98±0.27 and 9.28±0.21 mg/dl respectively.
Concentration of plasma calcium was significantly (P<0.05) higher in RgB when
compared with RB group.
Concentration of plasma calcium was significantly (P<0.05) positively
correlated with concentration of plasma IGF-1, LF and glucose and negatively
70 Results & Discussion
correlated (P<0.05) with concentration of plasma urea and Hp for both RgB and
RB groups (Table 4.43).
4.6.4 Concentration of plasma calcium (mg/dl) in RB-S and RB-NS groups under field conditions.
The Mean±SE concentration of plasma calcium at weekly interval and
Mean±SEM values for RB-S and RB-NS groups under field conditions is
presented in Table 4.24 and depicted in figure 4.12.
At 22nd week post partum when probiotic was supplemented to RB-S
group concentration of plasma calcium was 9.44±0.16 mg/dl and in RB-NS
group was 9.59±0.28 mg/dl. Within both RB-S and RB-NS groups, Mean±SE
values for concentration exhibited increasing trend but difference was not
significant from initial value till the end of the experiment. The percent increase in
concentration of plasma calcium over initial value in RB-S group at end of
experiment was 10.27%. In RB-NS group only 6 % increase in concentration of
plasma calcium from initial value was observed. When Mean±SE values for
concentration of plasma calcium was compared between groups, values were
not significantly different till the end of the experiment. The Mean±SEM
concentration of plasma calcium in RB-NS and RB-S groups was 10.19±0.19
and 9.99±0.24 mg/dl respectively. The concentration of plasma calcium was not
significantly different between the groups.
Concentration of plasma calcium was significantly positively correlated
(P<0.05) with concentration of plasma IGF-1 for both RB-S and RB-NS groups
(Table 4.44).
Das et al. (2009) reported significant (p<0.01) decrease in plasma level of
calcium among crossbred cattle with repeat breeding problem. Calcium plays an
important role in gonadotropic regulation of ovarian steroidogenesis (Carnegie
and Tsang, 1984) and ovulation (Peracchia, 1978). Results of present study
indicate significantly greater concentration of plasma calcium in RgB group when
compared with RB group under both farm and field conditions. As disturbed
steriodogenesis and ovulation are causes of infertility, results of present study
may correlate with early conception in RgB group and delay in RB group under
both farm and field conditions.
71 Results & Discussion
Table 4.22 Concentration of plasma calcium (mg/dl) in RB-S and RB-NS groups under farm conditions.
Weeks RB-S RB-NS
22 9.73±0.30 9.79±0.31
23 9.65±0.27 9.71±0.31
24 9.97±0.37 9.93±0.28
25 10.12±0.24 9.98±0.11
26 10.22±0.21 9.83±0.30
27 10.16±0.40 9.87±0.23
28 10.35±0.40 10.10±0.28
29 10.33±0.23 9.96±0.30
30 10.40±0.46 9.93±0.10
31 10.28±0.18 9.98±0.24
32 10.50±0.46 10.06±0.26
33 10.28±0.46 9.94±0.13
34 10.30±0.32 9.86±0.29
35 10.54±0.32 10.04±0.31
36 10.50±0.29 10.06±0.23
37 10.50±0.26 10.14±0.26
38 10.66±0.29 10.02±0.27
39 10.44±0.40 10.10±0.32
40 10.50±0.29 9.96±0.21
Mean±SEM 10.29±0.32 9.96±0.25
Table 4.23 Concentration of plasma calcium (mg/dl) in RgB and RB groups under field conditions.
Weeks RgB RB
4 9.02A±0.20 8.95±0.28
5 9.14 A ±0.32 8.82±0.14
6 9.35 A ±0.15 8.95±0.18
7 9.28 A ±0.22 9.12±0.16
8 9.42 A ±0.32 9.05±0.28
9 9.32 A ±0.28 8.95±0.23
10 9.55 A ±0.36 9.16±0.34
11 9.98 A a±0.25 9.24 b±0.19
12 9.76 A ±0.48 9.08±0.33
13 10.06 A a±0.18 9.32 b±0.22
14 10.02 A ±0.30 9.48±0.26
15 9.92 A a±0.17 9.40 b±0.08
16 10.34 A ±0.30 9.40±0.29
17 10.54 A a±0.26 9.70 b ±0.12
18 10.36 va±0.30 9.67 b ±0.09
19 10.82 Ba±0.30 9.48b±0.25
20 10.63 Ba±0.12 9.74 b±0.26
21 10.84 A a ±0.25 9.55b±0.13
22 10.58 A ±0.28
23 10.76 A ±0.42
Mean±SEM 9.98*±0.27 9.28±0.21
Values with different superscripts (A, B) are significantly different (P<0.05) from initial value within each group. Values with different superscripts (a, b) are significantly different (P<0.05) between RgB and RB groups. * P<0.05
Table 4.24 Concentration of plasma calcium (mg/dl) in RB-S and RB-NS groups under field conditions.
4.16 Milk yield in RgB and during pre and post supplementation period in RB-S and RB-NS groups under field conditions
and for repeat breeding problem. No reports are available with respect to
comparative studies on milk yield in regular and repeat breeders.
Effect of supplementation of Saccharomyces cerevisae on milk yield in RB-
S and RB-NS groups under farm and field conditions are in accordance with study
conducted by Iwanska et al. (1999); Dann et al. (2000); Majdoub-mathiuothi et al.
(2009). They have demonstrated that supplementation of Saccharomyces
cerevisae had no significant effect on milk yield or milk composition in mid or late
lactation period of dairy cows.
Results of present study are contradictory with studies conducted by
others (Arambel, and Kent 1990; Piva et al., 1993; Robinson 1997).They had
demonstrated significant effect on milk yield or milk composition of dairy cows
when Saccharomyces cerevisae was supplemented in early lactation. Phillips
and VonTungelin, (1984) and Barling, (2014) reported increase in feed intake
and nutrient availability on supplementation of Saccharomyces cerevisiae during
stress to cattle to overcome the stress. Therefore yeast culture was thought to
be best utilized by animals under stress. As animals were in early lactation, they
were in stress. This might be one of the reasons for significant effect on milk
yield or milk composition of dairy cows when Saccharomyces cerevisae was
supplemented. Mostly reports on supplementation are available during early
lactation period.
4.9 BODY WEIGHT
4.9.1 Body weight (Kg) of RgB and RB groups under farm conditions.
The Mean±SE body weight recorded at monthly interval and Mean±SEM
values for RgB and RB groups under farm conditions is presented in Table 4.33
and depicted in figure 4.17.
Under farm conditions, at the beginning of experiment (4th week post
partum) body weight of RgB and RB groups under farm conditions was
402.67±9.18 and 379.22±15.6 kg respectively. When Mean±SE values of milk
yield was compared within and between groups, not significance difference was
observed till the end of the experiment. Highest Mean±SE body weight in RgB
group was recorded as. 453.00±16.26 kg at end of the experiment and in RB
group was recorded as 436.25±17.94 kg at 20th week. There after the body
78 Results & Discussion
weight did not change significantly. The Mean±SEM body weight for RgB and
RB groups under farm conditions was 438.30±14.14 and 424.56±17.36 kg
respectively. Body weight recorded for both RgB and RB groups was not
significantly different.
4.9.2 Body weight (Kg) of RB-S and RB-NS groups under farm conditions.
The Mean±SE body weight measured at monthly interval and Mean±SEM
values for RB-S and RB-NS groups under farm conditions is presented in Table
4.34 and depicted in figure 4.18.
At 22nd week post partum when probiotic was supplemented to RB-S
group, Mean±SE body weight was 438.33±13.12 kg and in RB-NS group
Mean±SE body weight was 434.17±17.84 kg. When Mean±SE values of milk
yield was compared between and groups, not significance difference was
observed till the end of the experiment. The percent increase in body weight at
10th month over initial value in RB-S groups was 3 % and in RB-NS group was
1%. The Mean±SEM body weight in RB-S and RB-NS groups under farm
conditions was 442.83±11.91 and 431.77±15.13 kg respectively. The body
weights RB-S and RB-NS groups were not significantly different from each other.
4.9.3 Body weight (Kg) of RgB and RB groups under field conditions.
The Mean±SE body weight recorded at monthly interval and Mean±SEM
values for RgB and RB groups under field conditions is presented in Table 4.35
and depicted in figure 4.19.
Under farm conditions, at the begining of experiment (4th week post
partum) Mean±SE body weight of RgB and RB groups under field conditions was
398.45 ±10.14 and 374.42±15.65 kg respectively. When Mean±SE values of milk
yield was compared between and groups, not significance difference was
observed till the end of the experiment. Highest Mean±SE body weight in RgB
group was recorded as 448.56±17.63 kg and in RB group was recorded as
434.75±18.47 kg at end of the experiment. The Mean±SEM body weight for RgB
and RB groups under field conditions was 429.82±13.00 and 419.03±15.39 kg
respectively. Body weight recorded for both RgB and RB groups was not
significantly different.
79 Results & Discussion
Table 4.33 Body weight (Kg) in RgB and RB groups under farm conditions.
Weeks RgB RB
4 402.67±9.18 379.22±15.6
8 439.33±12.91 428.98±21.20
12 445.00±11.84 438.57±12.16
16 451.50±20.5 439.78±19.84
20 453.00±16.26 436.25±17.94
Mean±SEM 438.30±14.14 424.56±17.36
Table 4.34 Body weight (Kg) in RB-S and RB-NS groups under farm conditions.
Weeks RB-S RB-NS
22 438.33±13.12 434.17±17.84
26 439.8±16.67 426.33±19.54
30 437±12.86 430.60±16.02
34 447.5±9.06 428.00±10.41
38 451.5±7.84 439.75±11.81
Mean±SEM 442.83±11.91 431.77±15.13
4.17 Body weight in RgB and RB groups under farm conditions
4.18 Body weight in RB-S and RB-NS groups under farm conditions
100.00
150.00
200.00
250.00
300.00
350.00
400.00
450.00
500.00
4 8 12 16 20
Body
Wei
ght
(Kg)
Weeks
RgB RB
100
150
200
250
300
350
400
450
500
22 26 30 34 38
Body
Wei
ght
(Kg)
Weeks
RB-S RB-NS
Table 4.35 Body weight (Kg) in RgB and RB groups under field conditions.
Weeks RgB RB
4 398.45±10.14 374.42±15.65
8 435.23±11.10 422.45±16.23
12 432.54±7.84 430.64±12.16
16 434.32±18.30 432.87±14.44
20 448.56±17.63 434.75±18.47
Mean±SEM 429.82±13.00 419.03±15.39
Table 4.36 Body weight (Kg) in RB-S and RB-S groups under field conditions.
Weeks RB-S RB-NS
22 437.23±8.12 432.17±14.04
26 435.9±12.68 428.34±16.50
30 436.12±7.74 429.70±15.02
34 439.7±12.06 430.56±9.42
38 441.3±14.71 431.75±11.05
Mean±SEM 438.05±11.06 430.50±13.21
4.19 Body weight in RgB and RB groups under field conditions
4.20 Body weight in RB-S and RB-NS groups under field conditions
0.00
100.00
200.00
300.00
400.00
500.00
4 8 12 16 20
Body
Wei
ght (
kg)
Weeks
RgB RB
0
100
200
300
400
500
22 26 30 34 38
Body
Wei
ght (
kg)
Month
RB-S RB-NS
4.9.4 Body weight (Kg) of RB-S and RB-NS groups under field conditions.
The Mean±SE body weight measured at monthly interval and
Mean±SEM values for RB-S and RB-NS groups under field conditions is
presented in Table 4.36 and depicted in figure 4.20.
Form 22nd week post partum when probiotic was supplemented Mean±SE
body weight was 437.23±8.12 kg and in RB-NS group Mean±SE body weight
was 432.17±14.04 kg. When Mean±SE values of milk yield was compared
between and groups, not significance difference was observed till the end of the
experiment. The increase in Mean±SE body weight of RB-S group was by 4.07
% and decrease in body weight in RB-NS group by 0.4 % over initial value at
10th month. The Mean±SEM body weight in RB-S and RB-NS groups under
field conditions was 438.05±11.06 and 430.50±13.21 kg respectively. Body
weight recorded for both RB-S and RB-NS groups were not significantly
different.
In the present study, even though the Mean±SE body weight of RgB was
higher than RB group, no significant difference was observed under both farm
and field conditions. Since at the beginning of the study, not significant difference
was observed for the body weight between RgB and RB groups. In adult animal
Saccharomyces cerevisae supplementation might have had the ability to provide
growth factors such as organic acids (methionine and lysine) and vitamins;
stimulating immune function; improving the numbers of beneficial rumen micro
flora and fiber digestion (Chaucheryas et al., 1995; Gattass et al., 2008; Soccol
et al., 2010). Our results suggest Saccharomyces cerevisae supplementation
might have improved health and feed efficiency of the animal which might have
resulted in increase in body weight, although results were not significantly
different. Results are confirmatory with the report of Markusfeld et al. (1997) who
failed to observe any relationship between body weight at calving and first
service conception rate in crossbred cows. Similarly, Wathes et al. (2007) did not
find a relationship between calving BCS, BCS at 60 day post partum, or BCS
change with the conception at first service.
Effect of supplementation of Saccharomyces cerevisae on body weight in
RB-S and RB-NS groups under farm and field conditions are in accordance with
80 Results & Discussion
study conducted by Zhang et al. (2000) who reported that the cows on
supplementation with Saccharomyces cerevisiae had a body weight gain of 1%
greater than that of the control group, but it was not significantly different from
control.
Since at the beginning of the study, the body weight of RB group was not
significantly different from RgB group, Hence body weight might not have been a
critical factor causing the increase or decrease in blood plasma parameters and
for cause of repeat breeding problem.
4.10 CONCEPTION RATE
4.10.1 Conception rate in RgB and RB groups under farm conditions.
Conception rate of RgB and RB groups under farm conditions is
presented in Table 4.37.
Percentage of conception rate of RgB group under farm conditions for first
service was 33.33%. Percentage of conception rate of RgB group under farm
conditions to second service 75%. Percentage of conception rate of RgB group
under farm conditions to third service 100% respectively. In RB group for first 3
services none of the animals conceived. Overall Percentage of conception rate
of RgB and RB groups under farm conditions up to three services was 100 and 0
% respectively.
4.10.2 Conception rate in RB-S and RB-NS groups under farm conditions.
Conception rate of RB-S and RB-NS groups under farm conditions is
presented in Table 4.38.
Percentage of conception rate of RB-S and RB-NS groups under farm
conditions for first service was 28.57 and 14.29 % respectively. Percentage of
conception rate of RB-S and RB-NS groups under farm conditions to second
service was 40.29 and 16.67 % respectively. Percentage of conception rate of
RB-S and RB-NS groups under farm conditions to third service was 33.3 and 20
% respectively. Overall percentage of conception rate of RB-S and RB-NS
groups under farm conditions up to three services was 71.42 and 42.85 %
respectively.
81 Results & Discussion
Table 4.37 Conception rate in RgB and RB groups under farm conditions.
Services RgB RB
1 2/6(33.33%) 0/14(0%)
2 3/4(75%) 0/14(0%)
3 1/1(100%) 0/14(0%)
Overall 6/6(100%) ** 0/14(0%)
Values were presented in percentage. **P<0.01
Table 4.38 Conception rate in RB-S and RB-NS groups under farm conditions.
Services RB-S RB-NS
1 2/7(28.57%) 1/7(14.29%)
2 2/5(40%) 1/6(16.67%)
3 1/3(33.33%) 1/5(20%)
Overall 5/7(71.42%)* 3/7(42.85%)
Values were presented in percentage. * P<0.05
4.10.3 Conception rate in RgB and RB groups under field conditions.
Conception rate of RgB and RB groups under field conditions is presented
in Table 4.39.
Percentage of conception rate of RgB group under farm conditions for first
service was 16.66%. Percentage of conception rate of RgB group under farm
conditions to second service 80%. Percentage of conception rate of RgB group
under farm conditions to third service 100% respectively. In RB group for first 3
services none of the animals conceived. Overall Percentage of Conception rate
of RgB and RB groups under farm conditions up to three services was 100 and 0
% respectively.
4.10.4 Conception rate in RB-S and RB-NS groups under field conditions.
Conception rate of RB-S and RB-NS groups under field conditions is
presented in Table 4.40.
Percentage of conception rate of RB-S and RB-NS groups under field
conditions to first service was 28.57 and 0 % respectively. Percentage of
conception rate of RB-S and RB-NS groups under field conditions to second
service was 40 and 28.56 % respectively. Percentage of conception rate of RB-S
and RB-NS groups under farm conditions to third service was 0 and 20 %
respectively. Overall percentage of conception rate of RB-S and an RB-NS
group under farm conditions up to three consecutive services was 71.42 and
42.85 % respectively.
To the best of our knowledge this is the first investigation which shows the
effect of Probiotic (Saccharomyces cerevisiae) supplementation on conception
rate of repeat breeding crossbred cows under farm and field conditions. As
discused earlier systematic supplementation of probiotic (Saccharomyces
cerevisiae) significantly improved the plasma level of lactofferin, IGF-1, and
glucose in RB-S group. These parameters are positively correlated with health
and reproductive performance of animal. Whereas systematic supplementation
of probiotic (Saccharomyces cerevisiae) significantly decreased plasma level of
urea and Hp in RB-S groups. These parameters were negatively correlated with
health and reproductive performance of animals. These beneficial changes in
plasma level may be the reason for greater conception rate in RB-S when
82 Results & Discussion
compared with RB-NS groups under farm and field conditions. No reports on
supplementation of lactating cows are available for short duration of time up to
120 d post partum in cattle.
4.11 In Vitro Study
4.11.1 Relative expression of TLR-4 mRNA in neutrophils of RB, RB (In vitro IGF-1 supplemented) and RgB groups.
Relative expression of TLR-4 gene in blood neutrophils of RB (In vitro
IGF-1 supplemented) and RgB is presented in Table 4.46 and also depicted in
Figure 4.21. Relative expression of TLR-4 gene in blood neutrophils of RB (In
vitro IGF-1 supplemented) with respect to RB and RgB groups which was
1.13±0.11 and 1.48±0.09 respectively. O’Neill and Bowie (2007) reported
activation of TLR-4 can increase the phagocytic activity of neutrophils and
change their physiology in ways that increase their ability to kill and clear
pathogens. Relative expression of TLR-4 and Fas mRNA in neutrophils may
show immune status of the animal which can be correlate with repeat breeding
problem in Karan Fries cows. Bacterial infection trigger and increase expression
of TLR-4 in neutrophils and lymph node, lower expression of TLR-4 in neutrophil
of RB group does not indicate any bacterial infection but indicating lower immune
status when compared with RgB group which were healthy, did not suffer clinical
infection during study tenure as reported by Wolfram et al., (2008); Goldammer
et al., (2004) increase in TLR-4 expression in neutrophils during bacterial
infection.
4.11.2 Relative expression of Fas mRNA in neutrophils of RB, RB (In vitro IGF-1 supplemented) and RgB groups.
Relative expression of Fas gene in blood neutrophils of RgB and RB (In
vitro IGF-1 supplemented) groups, with respect to RB group is presented in
Table 4.46 and also depicted in Figure 4.21 Relative expression of Fas in blood
neutrophils of RB (In vitro IGF-1 supplemented) and RgB were 1.11±0.10 and
1.41±0.13 which differed significantly (P<0.05).
Regulation of the neutrophil life span by apoptosis is a crucial process in
maintaining number of circulating neutrophils which is regulated by Fas gene
(Simon, 2003). Results of present study suggest that Fas gene has a particular
83 Results & Discussion
Table 4.39 Conception rate in RgB and RB groups under field conditions.
Services RgB RB
1 1/6(16.66%) 0/14(14.29%)
2 4/5(80%) 0/14(16.67%)
3 1/1(100%) 0/14(20%)
Overall 6/6(100%) ** 0/14(0%)
Values were presented in percentage. ** P<0.01
Table 4.40 Conception rate in RB-S and RB-NS groups under field conditions.
Services RB-S RB-NS
1 2/7(28.57%) 0/7(0%)
2 3/5(40%) 2/7(28.56%)
3 0/2(0%) 1/5(20%)
Overall 5/7(71.42%)* 3/7(42.85%)
Values were presented in percentage. * P<0.05
Picture 5.1 Calf born of RB-S animal under Darad village conditions
Picture 5.2 Calf born of RB-S animal under Darad village conditions
Table 4.41 Correlation coefficient of plasma parameters in RgB and RB groups under farm conditions.
Hp IGF-1 Glucose Lactoferrin Urea Calcium
Hp 1.000
IGF-1 -0.679** 1.000
Glucose -0.483* 0.872** 1.000
LF -0.829** 0.634** 0.656** 1.000
Urea 0.797** -0.684** -0.530* -0.848** 1.00
calcium -0.502* 0.374* 0.057** 0.407* -0.5* 1.000
Values with superscripts ** significantly(P<0.01) correlated Values with superscripts * significantly(P<0.05) correlated
Table 4.42 Correlation coefficient of plasma parameters in RB-S and RB-NS groups under farm conditions.
Hp IGF-1 Glucose Lactoferrin Urea Calcium
Hp 1.000
IGF-1 -0.300 1.000
Glucose -0.176 0.820** 1.000
Lactoferrin -0.541* 0.644** 0.310 1.000
Urea 0.635** -0.541* -0.255 -0.770** 1.000
calcium -0.260 0.369* 0.208 0.302 -0.16 1.000
Values with superscripts ** significantly(P<0.01) correlated Values with superscripts * significantly(P<0.05) correlated
Table 4.43 Correlation coefficient of plasma parameters in RgB and RB groups under field conditions.
Hp IGF-1 Glucose Lactoferrin Urea Calcium
Hp 1.000
IGF-1 -0.692** 1.000
Glucose -0.523* 0.892** 1.000
Lactoferrin -0.856** 0.660** 0.670** 1.000
Urea 0.781** -0.684** -0.557** -0.84** 1.00
Calcium -0.532* 0.389* 0.056** 0.41* -0.49* 1.000
Values with superscripts ** significantly(P<0.01) correlated Values with superscripts * significantly(P<0.05) correlated
Table 4.44 Correlation coefficient of plasma parameters in RB-S and RB-NS groups under field conditions.
Hp IGF-1 Glucose Lactoferrin Urea Calcium
Hp 1.000
IGF-1 -0.310 1.000
Glucose -0.197 0.710** 1.000
Lactoferrin -0.561* 0.668** 0.310 1.000
Urea 0.649** -0.566* -0.262 -0.792** 1.000
Calcium -0.274 0.380* 0.220 0.316 -0.174 1.000 Values with superscripts ** significantly(P<0.01) correlated Values with superscripts * significantly(P<0.05) correlated
role to play in the regulation of neutrophil life span which may play role in
immune function of the animal.
Table 4.45 Relative expression of TLR-4 and Fas mRNA in neutrophils of RB, RB (In vitro IGF-1 supplemented) and RgB group.
Gene RB RgB RB+IGF-l
TLR-4 1 a±0.0 1.48 b±0.09 1.13 a±0.11
FAS 1 a±0.0 1.41 b±0.13 1.11 a±0.10
Values with different superscripts are significantly different (P<0.05)
Figure 4.21 Relative expression of TLR-4 and Fas mRNA in neutrophils of
RB, RB (In vitro IGF-1 supplemented) and RgB group.
In the present study it was observed that whether it was under farm or
field Mean±SEM concentration of plasma IGF-1, Hp, LF and Urea exhibited an
important role in influencing conception rate in repeat breeding crossbred Karan
Fries. Greater concentration of IGF-1 and LF had positive effect on reproductive
performance in Regular breeder yielding 100% conception rate. A novel and
economic technique of supplementation of commercial product fermented yeast
culture to repeat breeding cows @12 g/animal/day from 22nd week till 40th week
post partum, could enhance the plasma level of IGF-1 and LF and decrease Hp
and Urea level, which resulted in attaining 71.42% conception rate. Usually
00.20.40.60.8
11.21.41.61.8
RB Regular Breeder RB+IGF-1
Rela
tive
expr
essi
on o
f mRN
A
TLR-4 FAS
84 Results & Discussion
within 23 week, when the repeat breeding animals does not conceive for three
number of services, it is considered as non productive and farmer feels that it’s
an economic loss to him. This simple technology, where repeat breeding
animals, when supplemented with fermented yeast culture, their fertility status
could be upgraded.
In Vitro studies depicted that expression of TLR-4 and Fas genes were
down regulated in neutrophils of repeat breeding cows implicating weak activity
and survival of neutrophils. The dose and time period of incubation for IGF-1 on
neutrophils gene expression parameters were not enough to increase the
expression of genes as observed in control group.
85 Results & Discussion
CHAPTER –5
SUMMARY AND CONCLUSIONS
SUMMARY AND CONCLUSIONS
INSULIN-LIKE GROWTH FACTOR-1 (IGF-1)
Concentration of plasma IGF-1 in both RgB and RB groups increased
from initial value till the end of the experiment period (40th week),underfarm and fieldconditions.Mean±SEM concentration of plasma IGF-1was significantly
higher (P<0.01) in RgB groupwhen comparedwithRB groupunder bothfarm and field conditions.
Systematic supplementation of fermented yeast culture(Saccharomyces