ANALYSIS OF THE NUTRITIVE VALUE OF FORAGES LABORATORY PROCEDURES FIRST EDITION 2o14

ANALYSIS OF THE NUTRITIVE VALUE OF FORAGES FEED ANALYTICAL LABORATORY PROCEDURES

VET CARESS| ©http://www.facebook.com/vetcareservices| SPECIAL ISSUE # 1 1

ANALYSIS OF THE NUTRITIVE VALUE OF FORAGES

LABORATORY PROCEDURES FIRST EDITION 2o14

EDITED BY Sa’yem Khan



ANALYSIS OF THE NUTRITIVE VALUE OF FORAGES

LABORATORY PROCEDURES FIRST EDITION 2o14

EDITED BY

Sa’yem Khan



PREFACE

This manualof Analysis Of The Nutritive Value Of Forages, LABORATORY PROCEDUREShas been compiled

and edited by writer desk of|VET CARESS| Veterinary Care Services|Pakistan|.Itwill provide a

comprehensive guide to the nutritive analysis of forages and other feed sample. It is hoped that this

information will contribute to the knowledge of the Students at Feed Analytical Laboratories in National

Institutions. We are well aware that some errors and inaccuracies may have found into the text and hope

that the users of this document will bring these to our attention so that to make corrections in the next

editions.

Editor Sa’yem Khan

__________________________________________________________________________________________________________________________________________________________________________________________________

Copyright: © 2014|VET CARESS| Veterinary Care Services| Pakistan|A Non-Profitable Veterinary Organization. All rights reserved |VET CARESS| Veterinary Care Services| Pakistan|encourages fair use of this material for non-commercial purposes, with proper citation.

Citation: VET CARESS| Veterinary Care Services| Feed Analytical Laboratory Procedures| Special Issue # 1| 2014|. http://www.facebook.com/vetcareservices

It is the policy of the|VET CARESS| Veterinary Care Services| Pakistan| that all persons shall have equal opportunity and access to the Veterinary Sciences Materials and facilities without regard to race, color, sex, religion, national origin, age, marital status, parental status, sexual orientation, or disability. All the presented materials may be available in alternative formats on request from the contributor DrAtiqUllah Khan Sa’yemMarwat (DVM, RVMP, and M.Sc. (H) Animal Nutrition).

http://www.facebook.com/vetcareservices



DEDICATION

I dedicate this effort

To

My beloved Parents for their support throughout my Educational

carrier, my teachers for their encouragement and help in my

studies, my wonderful friends for their cooperation and help, and

to the Veterinarians and Animal Nutritionists for their sense of

dedication and commitment towards the dumb animals.



CONTENTS S.No CHAPTER NAME PAGE NUMBER

01 Laboratory Safety 06

02 Reliability of Laboratory Results 11

03 Introduction 16

04 Factor Affecting Chemical Composition of Forages 22

05 Unit # 1 Sampling and its Preparation 25

06 Unit # 2 Determination of DM, CP, EE, CF, ADF, NDF, ADL 27

07 Unit # 3 Determination of Minerals (Macro and Micro) 46

08 IVD, GAS PRODUCTION TEST AND ISDD 52

09 APPENDICIES 59



VETCARESS (VETERINARY CARE SERVICES)

NUTRITION AND DIETETICS SERVICE


DATED: ______________________

ANALYST: ___________________

Laboratory Safety: Everyone working in the laboratory should be cognizant of the potential hazards they face while working there. Fires with organic solvents, acid and base burns, and toxic fumes and vapors are common hazards in almost any nutrition laboratory. Generally, lab safety is a matter of common sense, but there are several rules that must be followed. Each student is required to read this chapter, which contains a brief synopsis of safety procedures. In addition, students must read the Standard Laboratory Procedures handout that is attached to the end of this chapter. The following material is provided as a brief summary and guide to lab safety. It does not replace assigned reading material, but gives an overview of some important points. Lab Safety from Science Related Materials, Inc. 1980:

1. Laboratory Neatness: Clean and neat work areas avoid risk of damage to clothing and books and injury from spilled chemicals. Neatness also reduces fire hazard.

2. Laboratory Conduct: Fooling around in the laboratory can be hazardous. Keep the lab in its proper place and fun and games in their place.

3. Working with Glassware: Remove frozen glass stoppers with proper equipment. Broken or chipped glassware should be discarded. Properly support glassware with ring-stands and clamps when heating and use cork rings with round-bottom flasks.

4. Working with Glass Tubing: Do not touch heated glass until it has time to cool. Hot glass looks just like cool glass. To remove stoppers from glass tubing or thermometers, grasp tubing close to stopper and push gently with twisting. Use water or glycerin for lubrication.

5. Laboratory Dress: Pull hair back and wear eye protection when required. Sleeves that are too tight prevent freedom of movement, whereas sleeves that are too loose may cause you to overturn apparatus or glassware. Aprons protect clothing from corrosive or staining chemicals. Gloves protect hands from corrosive chemicals. Handle hot objects with insulated gloves. Do not wear open-toe shoes that allow spilled chemicals or broken glass to come in contact with your feet.

6. Working with Test Tubes: Gently heat solids or liquids in a test tube near the liquid or solid surface. Be prepared to remove the tube from heat quickly to prevent eruption. Never point a test tube or reaction vessel at another person. For safety and neatness, place test tubes in a rack.

7. Chemicals in the Eye:Rapid treatment is vital. Run large volumes of water over eyeball until medical help is available. Wash with large volumes of water for at least 15 minutes. Alkaline materials in the eye are extremely hazardous. Know the location of the emergency eyewash station.

8. Safety Shower: Use this for chemical spills or a fire victim. Operate by pulling down on ring and keep the area near the shower clear at all times. Remove clothing from area affected by spills.

LABORATORY SAFETY



9. Fire on Clothing:Do not run or fan flames. Smother fire by wrapping victim in fire blanket or lab coat and use the shower or a carbon dioxide fire extinguisher.

10. Extinguishing a Fire: Using a fire extinguisher:

1. Know its location 2. Remove from mounting 3. Pull pin 4. Squeeze lever 5. Discharge at base of flame 6. Report use and recharge 7. Use dry sand to extinguish burning metals

11. Unauthorized Experiments: Always work under instructor's or lab technician's supervision in the laboratory. 12. Eye Protection: Normal eyeglasses are usually not adequate. Do not wear contact lenses in the lab. Eye protection is especially

important when working with corrosive materials and vacuum and high pressure apparatus. 13. Acid/Alkali Spills: For acid spills, use solid sodium bicarbonate followed by water. For alkali spills, wash with water followed by

dilute acetic acid. 14. Handling Flammable Liquids: Flammable liquids should always be stored in an approved storage cabinet. Extinguish all flames

in the area where flammable solvents are used, as vapors may travel to ignition source and flash back. 15. Types of Fire Extinguishers

i. Rating: A. - For ordinary combustibles; wood, paper, and cloth. B. - For flammable liquids; oil, grease, and gasoline. C. - For use on live electrical equipment.

ii. Number on extinguisher (e.g., 10A:5B) denotes square footage the unit is capable of handling. 16. Handling Mercury: Mercury spills are very hazardous. Droplets should be picked up by suction and a mercury spill kit used to

complete cleanup. Notify lab technician immediately when mercury spills occur. 17. Protection from Toxic Gases: Emergency air masks should be used. However, because our lab is not equipped with such masks,

clear the area where gases are, and notify the lab technician. 18. Waste Disposal: Hot glassware or reactive chemicals should be discarded in a nonmetallic container separate from paper and

other flammable waste. Test-tube quantities of hazardous liquids can be flushed down the sink with plenty of water. Contact lab technician for disposal of large quantities of hazardous materials or anytime you are not sure of how something should be disposed of.

19. Labeling Chemicals: All chemicals should be clearly labeled. Do not use materials from unlabeled containers. Avoid contamination. Never return reagents to their container. Clearly label chemicals as you work.

20. Carrying Chemicals and Equipment: Carry long apparatus such as tubing or burets, in an upright position close to the body. Grasp bottles firmly with both hands and hold them close to the body. Do not carry bottles by the neck. Use a bottle carrier when transporting chemicals any distance.

21. Transferring Liquids: Remember, Acid to Water. Do not pipette by mouth, use a bulb. Use gloves when pouring corrosive liquids. Use a funnel when filling a bottle or flask and prevent an air block by raising the funnel. Pour hazardous liquids over a sink.

22. Fume Hood: Use a fume hood equipped with a safety glass when working with toxic or flammable materials. 23. Gas Cylinders: Protect cylinder valve with cap. Fasten cylinders securely. Transport cylinders on a hand truck, don't roll. Do not

drop cylinders. Mark cylinders when empty. 24. Handling Sodium and Potassium: Fire or explosion may result when metallic Na or K are exposed to water. Store them under

light oil. Metal can be cut safely with a spatula on a paper towel. Destroy residues with alcohol. Cool if necessary.



THINK SAFETY AT ALL TIMES:

1. No smoking 2. No food or beverages 3. No running 4. Know location of exits 5. Keep aisles clear - put books and coats in designated areas 6. Do not leave an experiment unattended 7. Extinguish burners when you leave the work area

ALWAYS BE PREPARED TO HELP FELLOW STUDENTS IN AN EMERGENCY

STANDARD PROCEDURES AND SAFETY RULES: I. Personnel using the facilities of the Laboratory are required to:

A. Read, entirely, all assigned laboratory safety material and sign an affirmation that it has been read. B. Receive, before beginning any activities in the Laboratory, instruction from the

Laboratory staff regarding location and proper use of the following safety equipment: 1. EMERGENCY SHOWER 2. FIRE EXTINGUISHERS 3. ELECTRIC POWER PANEL 4. FIRST AID KIT 5. SAFETY GLASSES, GOGGLES, FACE SHIELDS, PROTECTIVE GLOVES, APRONS, AND LAB COATS 6. HOODS AND VENTS 7. TELEPHONE AND EMERGENCY NUMBERS

C. Read and observe the following rules and procedures. Everyone using the facilities of the Laboratory is required to abide by these procedures.

II. Personnel using the facilities of the Laboratory area are required to demonstrate an understanding, and proficiency in, the use of any equipment and the conduct of any physio-chemical procedures within the premises before use, unless under direct supervision by the Laboratory staff. Ask for proper instruction if in doubt about procedures. III. Personnel using the facilities of the Laboratory are required to be aware of the potential hazard involved in any procedure in which they may be engaged (fire, chemical burn, hot liquids, toxic fumes, poisons, electrical shock, etc.). Personnel who initiate the use of any equipment, facilities, or chemical procedures that involve hazard, or that could become hazardous, are required to remain in that particular area until the procedure is properly terminated. IV. It is not considered good practice to work alone in the Laboratory. Another person should be present or within the range of voice when any potentially hazardous procedure is being conducted. V. Absolutely NO SMOKING in any of the Animal Nutrition Labs or associated rooms.



VI. It is the responsibility of all personnel using flammables to check first to ensure that the area is safe from flames and sparking equipment; it is likewise the responsibility of all personnel using flames or sparking equipment to check first to ensure that the area is free from flammables. VII. Foods and drinks are prohibited in any area of the Laboratory where hazardous chemicals are in use, and eating or drinking are prohibited for all persons during whatever period of time they are engaged in usage or handling of toxic or corrosive chemicals. VIII. Chemicals, equipment, and supplies are to be returned to proper storage immediately on completion of use. Desk tops and work areas are to be kept free of "clutter". IX. Equipment and supplies will not be removed from the premises unless properly checked out. Check out procedure for glassware, equipment, and lab space should be followed. Proper instruction may be obtained from Laboratory personnel. X. All materials, including samples, should be properly labeled. Use proper labeling tape and write legibly. MATERIALS NOT PROPERLY LABELED WILL BE DISCARDED. XI. Work in progress that should not be disturbed must be properly labeled. Every effort must be made to clear ovens, desiccators, and related equipment as soon as possible so that others may use the facilities. XII. Everyone (students, student aides, and graduate students) is responsible for properly washing his or her own glassware and returning it to storage. XIII. A glassware breakage list will be posted in the laboratory area. This list must be signed and any breakage recorded. The purposes of this list are:

A. To keep a record of supplies needed in the Laboratory. B. To instill a greater cautiousness in everyone working in the lab.

XIV. The Laboratory is not an open facility. Permission to use the facility and its equipment must be obtained before use. In the case of proposed extended use of equipment, it is recommended that such use be scheduled, in advance, with the Laboratory supervisor.

(NOTE: The use of facilities by scheduled class groups will take priority over other users).

XV. Before any analytical work on samples is allowed: A. The individual in charge of the samples must make sure each sample has been given a Nutrition Lab code number. Each

sample should then be labeled with such code numbers (includes tissue, blood, rumen, as well as feed, feces, etc.) B. Samples should be adequately described in the code book. Analyses to be performed, project from which samples were

derived and time period samples that are to be saved should be indicated. C. After all analytical work is done; a copy of data resulting from the work should be made available to the Nutrition Lab so it can

be stored for future reference. Samples will be stored for the time period indicated in section (B) above.

XVI. SCAN BULLETIN BOARDS AND CHALKBOARDS IN THE LABS DAILY FOR NOTICES THAT MAY PERTAIN TO THE USE OF EQUIPMENT OR FACILITIES.



NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________






DATED: ______________________

ANALYST: ___________________

Reliability of Laboratory Results: The question of reliability of results is essential to the output of believable, high-quality data from any laboratory. The student who runs a Kjeldahl nitrogen analysis on an alfalfa hay sample should ask the question, "How close is the value I obtained to the true value for this sample?" Moreover, the student should be able to properly evaluate the data to help answer this question. Some definitions will aid our understanding of this concept of reliability of results. Accuracy can be defined as the degree of agreement between a value obtained by a procedure and the actual or true value of the quantity being measured. Because, in most biological settings, the true value is seldom if ever known, the accuracy of any result is seldom known. So, we are still left with the question of how accurate our data really is. Generally, one can increase confidence in the accuracy of a laboratory result by (1) running standards to check for errors in procedures or techniques (e.g., using urea as a standard in the Kjeldahl procedure to check for recovery of nitrogen) and (2) comparing results with others obtained independently. In fact, the use of standards and comparison with other results should be routine procedure in nutrition laboratories. However, these measures still do not ensure accuracy, and for the most part, we must assume the true value is the most probable value from the available data; that is the arithmetic mean of the observations. This is generally a reasonable assumption because the sample mean ( x) is an unbiased estimate of the true mean (population mean, μ). Precision is a term that many students confuse with accuracy, so a clear distinction must be made between the two terms. Precision can be defined as the closeness of a number of similar measurements to a common value. Although precision in laboratory work is very desirable, the attainment of precision does not necessarily imply the measurements are accurate. This concept is illustrated in Figure 2-1. The reason for the divergence of precision and accuracy in Figure 2-1 is a common source of error. In this particular example, we might surmise the rifle is not properly sighted in, causing repeated misses of the bulls-eye and poor accuracy. This same situation can occur in the laboratory, as a constant source of error could cause inaccurate results. Precision is usually evaluated as the deviation of individual measurements from a common value; the common value being x . Precision is the best numerical measure of the repeatability or reliability of a method or instrument, and it is commonly expressed as the standard deviation or coefficient of variation (CV). Although most students are familiar with the method for calculating standard deviation, this information is presented in Table 2-1 for those whom the concept is new.

As one works in the lab, the value of precision in determining how acceptable or reliable one's results are becomes readily evident. Replication of analyses (i.e., duplicate or triplicate observations on the same sample) allows calculation of x ± s and a quick evaluation of a technique or instrument. When should one run triplicate vs. duplicate analyses? This is somewhat a matter of experience, but the acceptable CV for a number of common nutrition lab analyses listed in Table 2-2 should provide some aid in determining whether more replication is needed.

RELIABILITY OF LABORATORY RESULTS



Figure 2-1. Target with x's denoting placement of shots from a rifle. Note that shot placement is very precise (closeness of the x's), but the shooter is not very accurate because of the distance between the x's and the bulls-eye.







NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________






DATED: ______________________

ANALYST: ___________________

INTRODUCTION: Background: Poor nutrition is one of the major constraints to livestock productivity. This is because animals thrive predominantly on high-fiber feeds (straws, stovers and native pasture hay) which are deficient in nutrients (nitrogen, sulphur, minerals, phosphorus etc) essential for microbial fermentation. Consequently, the digestibility and intake of digestible nutrients are unavoidably low. These deficiencies can partly be mitigated by supplementing roughage diets with feeds containing the deficient nutrients. Feeding practices developed in temperate countries are often inappropriate when applied to ruminant production systems in the tropics because temperate animals are fed straw as bulk in high density diets. Forage diets and supplements may differ vastly in quality and therefore in the quantity eaten by the animal. Previously digestibility and chemical composition were used to describe the nutritive value of fibrous feeds. This proved inadequate because these attributes give little indication of the quantity of such feed an animal will eat and the quality of nutrients derived through digestion. An understanding of the factors which affect nutritive value of forage base diet, there rumen microbial degradability and microbial protein production will assist scientists in designing diets that will be utilized more efficiently. In addition to determining responses (performance) from feeds, there is a need to establish causal relationships.

Forage as a Herbivorous Animals Natural Feed: The natural feed of the herbivorous animals is forage and for most of the year this forms all or most of the feed of ruminants. Grasslands/Forage plants may be classified into two main groups as follows. (a) Herbaceous plants (b) Woody plants Among the herbaceous plants, two groups are important as sources of forage. These are:

A. Grasses: They make up the bulk of the plants found in many mixtures of natural vegetation used as forage. They also supply the bulk of the energy content of forages.

B. Legumes:because these have relatively high nitrogen content in the vegetative matter and their ability to fix atmospheric nitrogen. Grasses and legumes are found in the various vegetations that constitutes the grazing resource or sources of feed for ruminant animals. These vegetations are commonly referred to as grasslands which may be divided into two main groups:

INTRODUCTION



1. Natural grasslands which are not cultivated and managed in any form of deliberate human intervention. This is generally referred to as natural pasture. Natural pastures normally include a large number of species of grass, legumes, herbs, shrubs etc.

2. Cultivated grassland which are cultivated and managed generally referred to as cultivated, planted or sown pasture. Cultivated grasslands may consist of single species or mixtures of relatively small numbers of species. . Cultivated grassland may also be sub-divided into permanent and temporary pastures. The latter form part of a rotation of crop whereas permanent pastures is intended to remain as grassland indefinitely.

Growth pattern of Grasses: In tropical climates, soil temperature is high enough to permit the growth of grasses throughout the year, but such growth is commonly restricted by insufficient moisture supply for definite periods of the year. The climate is characterized by clearly defined wet and dry seasons but grass growth is very rapid during the wet season and as the soil dries up towards the dry season, the herbage matures and dies, leaving a feed resource that is some time described as ‘standing hay’. In cold and temperate climates grasses start to grow in the spring when the soil temperature reach 4 to 6oc. There is a rapid production of leaf followed by an increase in the growth of the stem leading to the ultimate emergence of the flowering head and finally to the formation of seed. The rate at which grasses grow is dependent upon climate, available nutrients in the soil and the amount of leaf on the plant which intercepts light. Immediately after harvesting, there is a period of slow re-growth followed by an accelerated rate and finally a period of decreasing growth as the herbage matures. As grass swards increase in leaf area, the photosynthetic capacity of successive newly expanded leaves is progressively reduced because of the increasing shade in which they develop. The rate at which re-growth occurs depends upon the maturity of the crop at the time of harvesting. If the grass is young and leafy it recovers more quickly and starts re-growth earlier than when mature herbage is harvested. Definition of Forage Quality or Feed Value: Animal performance depends on an inter-relationship between a numbers of factors both internal and external to the animal itself. The interrelation is shown in the scheme below. As shown in the scheme below, nutritive value, digestibility and feed intake are the main factors which determine animal performance.

These three factors are, in turn, influenced by a number of other factors related to both the animal (e.g. species, physiological status, age,

grazing experience, management, etc.) and the forage (species, stage of growth/maturity, management, season, location, etc.). Taken

together, these three main factors define what is called FORAGE QUALITY. Chemical composition and digestibility are often linked to the

term nutritive value, which describes the amount and types of nutrients that the animal can derive from the feed.





CHEMICAL COMPOSITION OF FORAGES: Herbages contain a variety of chemical constituents which serve as nutrients for herbivores. Some nutrients are sources while others satisfy specific requirements in the body of the animal. The chemical composition of the dry matter of pasture grass is very variable for instance the crude protein (CP) content may range from as little as 30g/kg (i.e.3%) in very mature herbages to over 300g/kg (or 30%) in young heavily fertilized grass. The fiber content is inversely related to the crude protein content, and the acid detergent fiber may range from 200 to over 450g/kg (20-45%) in very mature tropical grasses. The moisture content is of particular importance when a crop is being harvested for conservation. It is very high in very young material, always in the range of 750 – 780g/kg (75-78%), and falls as the forage matures to about 650g/kg. Weather condition is a major determinant of moisture content. According to Van Soest (1967) the chemical component of forages can be broadly divided into cell wall constituents (CWC) and cell contents (CC) or into digestible and indigestible or poorly digestible fractions. The CC is generally highly digestible and the CWC (commonly referred to as fiber) is either indigestible or poorly digestible. The CC is soluble in neutral detergent while the CWC are only partially soluble in acid detergent. Associations of soluble carbohydrates, starch, organic acids, cellulose and hemicellulose together with lipids (fats) contribute to the energy content of forages. Proteins, vitamins and minerals provide essential components of animal diet and are required in an appropriate balance if animals are to perform optimally. Forage plants may also contain anti-quality factors such as tannins, or even poisonous constituents which may affect animal performance. Proteins: Protein is often the constituent which most limits the performance on animalson pasture. Crude protein (CP) comprises natural proteins (i.e. part of the planttissue constituents) as well as non-protein constituents (NPN). CP is estimatedby multiplying the nitrogen (N) content of the forage by a factor of 6.25.This provides only a gross estimate which does not distinguish between theprotein needs of the micro-flora in the rumen and protein available forabsorption in the lower digestive tract, or the quality or origin of the protein.The protein requirement varies according to species of animals, age, and thephysiological functions of the animal (e.g. lactating, young, pregnant etc.).Generally, the minimum protein requirement of ruminants is between 7 and 8% but high producing animals require levels approaching 13% to 14% andwhere the protein levels is lower than the minimum requirement, proteinneeds to be supplemented. The CP contents vary widely among forage plants but in all species and in allseasons, it declines with increasing age of the forage. N-fertilizer applicationwill normally increase the CP concentration in forages, but much of this may bein the form of NPN which is of little value to the animal and may in fact beharmful and cause nitrate poisoning. Animals should therefore be kept off N-fertilizedareas for about three weeks following top dressing. Minerals: A comprehensive mineral need of livestock is given in tables presented byNational Research Council (1984, 1985). Phosphorus is generally in short supply in most of the tropics for most of theyear. Supplementation with P is therefore often recommended throughout theyear on many types of pasture. When selective grazing is allowed to fullyoperate as in a continuous grazing system, P intake is never constrained asanimals select the young shoots of plants which contain higher proportion of Pthan other plant parts.Other important minerals for good performance of livestock are Na, Ca, K, Mg,S, Zn, Co, Cu, Mn, Mo, I, Se. The concentration of the minerals in forages isdetermined to a large extent by the maturity of the material. Mineralconcentration declines with age and is also influenced by soil type, soil nutrientlevels and seasonal conditions. Structural Constituents (Cell wall or fiber): The structural constituents of plant materials include polysaccharides, ligninand some proteins. The constituents can be divided into matrixpolysaccharides (including hemicellulose and pectin) and fiber polysaccharides(cellulose, lignin, and proteins). All these components have been termed fiber and may be incompletely or variably digested by the animal. The stems of most forage have larger proportion of polysaccharides and ligninthan the leaves. This proportion increases with maturity in both tropical andtemperate forage species. Tropical species appear to have greater cellulosecontent and a higher hemicellulose: cellulose



ratio than temperate species.During digestion, once lignin has been removed, the polysaccharides of the cellwall become more readily digestible.The lignin in plant fiber however resists microbial enzyme attack in the rumenand thus reduces digestibility through its linkage with specific points on thepolysaccharide chains and it prevents physical attachment of rumen bacteriato plant cell walls. Vitamins: These are another group of essential chemical constituents, but are requiredonly in small amounts. The most important of them is vitamin A which isusually well provided for in green forages and well cured leafy hay. Anti-quality and toxic substances: The final group of chemicals that are found in forages are toxic substances.Certain legumes (e.g. Lucerne and clover) contain substances which causebloats. Others contain tannins which reduce the digestibility if forage.



NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________






DATED: ______________________

ANALYST: ___________________

FACTORS AFFECTING CHEMICAL COMPOSITION OF FORAGES: 1. Stage of growth This is the most important factor affecting the composition and nutritivevalue of forages. As plants grow there is need for fibrous tissues andtherefore the main structural carbohydrates (cellulose and hemicellulose)and lignin increase. As the plant ages, the concentration of proteindecreases and the fiber content increases. There is therefore a reciprocalrelationship between the protein and fiber content in given species. 2. Species 3. Soils and fertilizer treatments 4. Grazing system employed 5. Climatic condition and season The nutritive value of a pasture is basically a function of the species in thepasture and the stage of growth, but may be modified by climatic factorsduring growth, soil factors which affect nitrogen and other mineral status,and management factors which affect pasture re-growth rate, swardstructure and botanical composition. Species factor Tropical grasses: The nutritive value of pasture species even at similar stage ofgrowth varies widely both in DMD and Voluntary Intake. Minson and McLeod(1970) showed that tropical grasses were, on the average 13% lower in DMDthan temperate species. Most samples of temperate grasses had digestibilityabove 65% but few tropical grass samples were in that category. Minson andMcleod (1970) suggested that the lower DMD values of tropical grasses may inpart be due to higher growing temperatures but data obtained from studiesthat followed by Reid et al. (1973) showed that selected or improved species oftropical grasses such as Brachiaria, Chloris, Setariaand Panicumhad DMDvalues that are comparable to those of similarly managed temperate species. PLANT FACTORS AFFECTING FORAGE QUALITY The mixture of plants growing in a field or ecosystem determines foragequality depending on growing or harvesting conditions. The plant species,growth stage, and conditions at harvest generally dictate quality of the forage.Each of the plant used as forage has a unique morphology and physiologywhich gives it specific adaptation, growth, and forage quality features. Eventhough some generalization in terms of factors that affect quality can be made,it is important to recognize the differences in quality that exist among groupsof plants, individual species and even cultivars. These differences also interactwith stage of growth and the environment.When reporting animal

FACTORS AFFECTING CHEMICAL COMPOSITION OF FORAGES



results from forage-based experiments, researchersshould describe the forage species, cultivars, stage of maturity andenvironmental conditions during growth and characterize the forage withappropriate quality tests. Some of the plant factors Species: The importance of species is seen in temperate and tropical species. Thetemperate species belong to the C3 category of plants in which the 3-carboncompounds (Phospho-glycerate) is an important intermediate product the inphotosynthetic fixation of carbon dioxide. Most tropical species have C4pathway of photosynthesis in which carbon dioxide is first fixed in a reactioninvolving the four-carbon compound oxalo-acetate. The low protein contentoften found in tropical grasses is an inherent characteristic of C4 plantmetabolism. In temperate grasses fructans are the main storage ofcarbohydrates while in tropical grasses these are replaced by starch. Soil and fertilizer treatment The type of soil may influence the composition of the pasture especially itsmineral content. Plants normally react to mineral deficiency in the soil eitherby limiting their growth or by reducing the concentration of the particularminerals in their tissues, or more usually by both. The acidity of the soil is airimportant factor which can influence the uptake of many trace elements byplants. Liberal dressing of fertilizers can markedly affect the minerals contentof plants and application N-fertilizers is also known to increase leaf area andrate of photosynthesis. As a consequence, the CP content of subsequently theamide and contents are increased. Application of N-fertilizer also depress thewater soluble carbohydrates content of temperate grasses which may have anadverse effect on fermentation if the crop is used preserved as silage.Fertilizers may also affect indirectly the nutritive value of a sward by alteringthe botanical composition. For example, legume do not thrive on a limedeficient soil, while heavy dressing of N encourage growth of grasses anddepress legumes growth. Grazing system In grazing systems that encourage accumulation of mature dry matter due toimproper stocking rate, the nutritive value of the herbage on offer is constantlylow. However, if the available forage is not limiting, animals have theopportunity to graze selectively and they are able to compensate to someextent for the general fall in nutritive value by selecting plants or plant partsthat are higher in nutritive value than the rest .If the grazing pressure is alsoabnormally high, selection by animals is also reduced and the pasture plantsmay also be depleted of foliage and their root reserves are also depleted andthey fail to regrow. Both under-and over grazing of pastures may change theirbotanical composition and therefore the nutritive value of their herbage. Thequality of forage could be better in the rotational system of grazing in whichpastures are grazed for short periods at a high stocking rate and grazingpressure. Under this situation animals harvest most of the herbage on offerand the pasture is then rested for longer periods for recovery. Other factors affecting the nutritive value of forages Factors such as climatic as climate and season may influence the nutritivevalue of pastures .the concentration for example, can be markedly influence bythe amount of sunshine received by the plant .Generally on a dull day thesoluble carbohydrate content of grasses will be lower than on a fine sunny day.Rainfall can affect the minerals composition of pasture herbage. Calcium forexample tends to accumulate in plants during periods of drought but tends tobe present in smaller concentration when the soil moisture is high. On theother hand phosphorus appears to be present in higher concentrations whenthe rainfall is high. Early season forages are also noted to have lower netenergy value than mid-late season forage even when the two are cut at thesame stage of growth and are equal in digestibility



NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________






DATED: ______________________

ANALYST: ___________________

UNIT # 1: Sampling and its preparation 1.1 How to take feed samples: An analysis of feed is only as good as the samples obtained from feed. If the samples are not representative by entire batch area then the evaluation are useless. No matter how extensively the samples are analyzed. Several samples should take from representative area. Thence the sample thoroughly mix together to form a representative sample of entire feed and part of it should take from it so that the final analysis will represent an overall average of entire feed being sampled. All Samples should be kept in plastic bottle or bags. The date place of sampling and an identification number should be placed on each sample. 1.2 How we do sampling from pasture: Pasture: - Forages which are harvesting by grazing animals. When sampling is required from pasture. It is advisable to move in a “Z” or “X” pattern at predetermined intervals e.g after every “5” or “10” steps and is cut fully in “12” by “12” inches. The samples should be cut from a height of mowing height 1-2 feet above the roots or earth level. If there is very little grass at predetermined step then cut what is available and move on. Do not move off the path to cut more densely populated area. Then mix the cut samples thoroughly an ending the walking pattern and bag the samples and send for analysis. 1.3 Preparation in the Laboratory: Sample preparation converts the samples into homogeneous material for the various nutritional analyses. Drying and grinding are the essential operations. Sample preparation is conducted according to sample type and analyses requested. 1.4Drying: The above bag samples are then air dried at 60° C to 65 °C in oven for 48 hrs. To 70 hrs. 1.5 Grinding: Then air dried sample is grind in grinding machine (grinder). Feed samples are ground to 1 mm particle size with a Wiley mill). 1.6 Storage: Samples are stored in airtight containers away from heat and light. For storage transfer to clean, Dry bottle and kept air tight for further chemical analysis.Caution has to be taken to avoid insect damage.

UNIT # 1: Sampling and its preparation



NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________






DATED: ______________________

ANALYST: ___________________

UNIT # 2: DETERMINATION OF DM, EE,CF (NDF+ADF) DM=Dry Matter, EE=Ether Extract, CF=Crude Fiber, NDF=Neutral Detergent Fiber, ADF=Acid Detergent Fiber

2.1 Dry Matter Determination: Samples contain Water and dry mater (Dry Matter is that parts of feed which is not water or total solid it is the sum of crude protein crude fat crude fiber N.E.E and Ash. If we want to calculate dry matter we remove the water or moisture content of sample by drying in oven at 100 – 105 C ever night the left is dry matter. 2.1.2 Equipment:

a. Analytical Balance b. Porcelain crucibles c. Laboratory electric drying oven d. Desiccator filled with silica gel drying agent. e. Fire tongs for holding crucibles f. Marker / Pencil

2.1.3 Procedure: Dry the empty crucible in oven at 100 C for one hr. and cool in a desiccator. Label the crucibles for sample identification and weight it on the balance. Weigh approximately 2g ground sample into the crucible in duplicate or triplicate place the crucibles in oven adjust to 100 C for overnight. Take out the crucibles and transfer to a desiccator. Allow to cool to room temperature. Weigh the crucibles containing the dried sample. Calculate the results as under for dry matter DM% C-A X 100 B-A A = Weight of empty crucible, B= Weight of the crucible + pre dried sample, C= Weight of the crucible + dried sample

UNIT # 2: DETERMINATION OF DM,

EE, CF (NDF+ADF)



NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________






DATED: ______________________

ANALYST: ___________________


2.2 Determination of Ash: 2.2.1 Ash: It is a residue left after in incineration a sample at specified temperature and which represent mineral matter. 2.2.2 Principles: The sample is ignited at 550 °C – 600 °C to burn off all the organic material. The inorganic substance which remains at that temperature is called Ash mineral. 2.2.3 Equipment:

1. Porcelain or aluminum crucibles 2. Electric balance 3. Muffle furnace 4. desiccator Filled with silica get as drying agent 5. fire tong for holding crucibles 6. Glass marking pencil heat resistant

2.2.4 Procedure: We placed clean crucible in muffle furnace ignited at 550 °C – 600 °C for one hrs. Don’t open the door immediately off the furnace and wait until its temperature comes at 200 °C then transfer crucible in desiccator for one hrs to cool down then at room temperature. Weight empty crucible and then add 2-4 gms sample and weight again and then put it into raffle furnace for 4-6 hrs at 550 °C – 600 °C switch off the furnace cool down 100 to 200 °C then transfer the crucibles to desiccator for 15 to 30 minutes to brought its temperature to room temperature. Weight the crucibles for Ash mineral determination. 2.2.5 Formula:

Ash wt X 100 = % Ash Sample wt Before Ashing


EE, CF (NDF+ADF)



NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________






DATED: ______________________

ANALYST: ___________________


2.3 Determination of Crude Fat: 2.3.1 Principle: Diethyl ether or petroleum ether is continuously evaporated then condensed and it passes through sample in thimble placed in extractor. When the extract in the extractor reaches its maximum height it is siphoned into a receiving flash. Then the solvent is again evaporated and passes through the sample for second extraction. This is a continuous cyclic process until all the ether soluble materials have been extracted. 2.3.2 Equipment and Glassware:

1. Soxhlet extraction apparatus 2. Air bath electro thermal 3. Evaporating basin 100 ml 4. Water bath 5. Extraction thimble

2.3.3 Reagents

1. Diethyl ether anhydrous or Petroleum ether 2. extraction thimbles Whatman

2.3.4 Procedure

1. Weigh accurately 1.0 to 2.0 g of a moisture free sample into a clean previously dried extraction thimble. 2. Plug the thimble with absorbent cotton wool. 3. Place the thimble in an extractor and fix under the condenser of the extraction apparatus. 4. Add about 150 ml of the solvent (Petroleum ether) to the receiving flash and connect it to the apparatus. 5. Turn on the Water and the heater. 6. Continue the process 10 hours to recover the solvent for future use.


EE, CF (NDF+ADF)



7. Just before solvent dries in the flask disconnect the extraction and remove the flask. 8. Transfer he extract into a clean dried evaporating basin with ether Washings 9. Evaporate the dryness on water bath. 10. Place the basin in oven at 105 °C for 2 hrs. 11. Cool in desiccator for 30 minutes and weigh.

2.3.5 Calculation:

% Ether extract DM = Weight of Ether Extract X 100 Sample Weight

2.3.6 Converting to as fed basis

% Ether Extract as fed = Weight of ether extract X 100 - % moisture Sample weight



NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________






DATED: ______________________

ANALYST: ___________________


2.4 DEERMINATION OF NITROGEN AND CRUDE PROTEIN

2.4.1 Principle:

The nitrogen of protein and other compounds are transformed into ammonium sulphate by sulphuric acid digestion. The digest is cooled diluted with water and alkalified with sodium hydroxide. The released ammonia is distilled into a boric acid solution or a known volume always in excess of standardized acid. If the distillate is in boric acid then it is titrated with standardized acid to quantify the ammonia evolved. For the latter case a standardized alkali is used to titrate the excess acid so that a quantity of acid neutralized by the ammonia can be found. That is equal to the quantity of ammonia evolved. The following analytical method is combinations of semi-automatic Kjeldahl digestion distillation unite. 2.4.2 Equipment and glassware:

1. Semi-automatic kjeldahl digestion distillation unite 2. Kjeldahl flasks 500 ml or 650 ml 3. Conical flask wide mouth 100ml 4. Volumetric flask 100ml 5. Bulb pipette 5ml 6. Burette 25ml

2.4.3 Reagents:

Concentrated Sulphuric acid technical grade Sodium hydroxide solution 40% W/W Catalyst (Copper Sulphate 7%) Potassium Sulphate 93%), CuSO4 5H2O K2SO4 or Na2SO4 in ratio of 7: 93 and mix up well Methyl red 0.25 percent in 95 percent ethanol Methylene blue 0.2 percent in 95 percent ethanol mix up (a) and (b) in ratio of 3:2 Boric acid solution 2 percent


EE, CF (NDF+ADF)



Standardized sulphuric acid solution 0.02 N 2.4.4 Procedure:

1. Weigh accurately 0.5 - 1.0 g of sample. 2. Transfer the sample to kjeldahl flask. 3. Add 2 to 3g catalyst 5-7 ml concentrate H2SO4. 4. Whirl kheldahl flask to mix its contents thoroughly. 5. Place the flask on the digestion assembly and turn on the heater and exhaust fan. 6. Continue digestion with occasional turning of flask care should be taken for frothing at the early stage of digestion. 7. When the solution in the flask clears and all organic matters have been oxidized continue the digestion for further 30 minutes. 8. Switch off the heater of digestion assembly to cool the flask. 9. Transfer the digest into a 100ml volumetric flask with washing make up the volume to the mark and mix well. 10. Pipette 5 ml from the volumetric flask and introduce it to semi-automatickjeldahl digestion distillation unit. 11. 15ml of NaOH 40 % W/W is added gradually and not remove the stopper otherwise NH3 may escape 12. Plug the funnel firmly add a 10 ml H2O. 13. Distil for 4 minute and collect the distillate into a conical flask containing 5ml of 2 percent Boric acid and 2-3 drops of

methylene red indicator. 14. After 5 minute distillation lower the flask and collect the drippings from the condenser for 1 minute. 15. Wash the tip of condenser into the flask 16. Titrate against standardized H2SO4 run a reagent blank through all the steps of the procedures.

While (N %) = (V1-V2) × 14.01 ×20×.02× 0.5 ×100

(Sample in g) V1= Titration reading of sample V2= Titration reading of blank 14.01= Atomic weight of Nitrogen (N) 20=Dilution factor

2.4.5 Crude protein determination:

Crude protein determined for feed sample, by multiplying the nitrogen content of the sample by 6.25. CP Adjusting to dry matter DM basis

% Crude protein DM = % crude protein as fed x 100 % dry matter of Sample



NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________






DATED: ______________________

ANALYST: ___________________


2.5 Determination of Crude Fiber 2.5.1 Principle: Crude fiber (CF) is defined as an organic matter that remain after being digested first with a weak H2SO4 solution and then with a weak NAOH solution. The residue that collected after digestion is dried and ashed in a furnace. The loss in weight ashing is recorded as CF. 2.5.2 Equipment:

Analytical Balance Heating assembly Glass beaker tall type 400 ml Round bottom flask with inlet and outlet for water circulation used as condenser on the beaker. Filtration apparatus with vacuum pump Sintered Glass filterable crucible porosity-1 Desiccator contain silica get Drying oven Muffle furnace.

2.5.3 Reagents: H2SO4 0.5 M solution 21 ml H2SO4 1.84g /ml is completed to 2.5 liter with distilled water NAOH 1.5 M solution 155 g NAOH per 2.5 liter distilled water HCL acid 0.3 M solution 50 ml HCL 1.14g / ml is completed to 2 liter with distilled water Na2 EDTA disodium ethylene di-aminetetra acetate Dehydrate crystal acetone.


EE, CF (NDF+ADF)



2.5.4 Procedure:

1. Weigh a sample about 2.0 g to the nearest 0.1 mg and transfer into the tall beaker. 2. Add 100 ml H2SO4 solution. 3. Place the condenser on cap of the beaker and the water flow to the condenser. 4. Heat for 5 minutes strongly for quick boiling and then adjust heat to give an even boiling. 5. Continue heating for 30 minutes with cooling on the beakers. 6. Add 50 ml NaOH solution quickly and continue heating for a further 35 minutes. 7. Filter the hot solution through the crucible attached to filtration apparatus. 8. Use hot water for rinsing the beaker. 9. Rinse filtrate in the crucible with 50ml HCL solution 10. Wash with hot water until it is free of acid 11. Wash with 50 ml acetone two time wait a minute before sucking after each addition. 12. Dry the crucible in oven at 100 overnight or at 135 C for two hours. 13. Cool in a desiccator and weight the crucibles. 14. Place crucible containing the dried residue in a muffle furnace and incinerate for two hours at 550c. 15. Cool in a desiccator. 16. Weight the crucible.

2.5.5 Calculation: CF % in sample = wt crucible + cried residise +ushed residue x 10 wt crucible + sample – wt empty crucible CF% in DM= CE% in Sample x 100 DM% in Sample Note

1. If the sample contain 10% fat or is very greasy it must be defatted before CF analysis. 2. CF is very hygroscopic. Therefore do not use 1 desiccator for coolingof more than 4 crucible place in it.



Figure .Schematic representation of the structure of a forage plant cell showing the component layers. The relative amounts of each of

the carbohydrate fractions in the respective layers are depicted by shaded areas, i.e., hemicellulose largely in the secondary wall;

pectin largely in the middle lamella. The figures in parentheses are amounts often found in forage dry matter. (Adapted from Maynard

et al., 1979).



2.5.6 Neutral Detergent Fiber (NDF): Principle: Insoluble fiber in feed is determined as neutral detergent fiber (NDF). Neutral detergent solution recovers its main components cellulose, hemicelluloses and lignin. Neutral detergent solution:

1. 2 l distilled water. 2. 60 g sodium lauryl sulfate (C12H25O4S) 3. 37.22 g disodium dihydrogen EDTA (C10H14N2Na2O8) 4. 13.62 g sodium borate (decahydrate) (Na2B4O7.10H2O) 5. 20 ml 2–ethoxy–ethanol (C4H10O2) 6. Adjust pH 6.9–7.1.

Equipment:

1. Precision balance 2. Refluxing apparatus 3. Vacuum filtering system with trap in line 4. Desiccator 5. Berzelius beakers (600 ml) 6. Sintered glass crucibles (coarse porosity 1)

Procedure:

1. Weigh oven-dry glass crucible (Wt) 2. Add 0.5–1 g sample (Ws) in 600 ml Berzelius beaker 3. Add 100 ml of neutral detergent solution and 0.5 g sodium sulfite (Na2SO3) 4. Boil for one hour in refluxing apparatus 5. Pour through glass crucibles 6. Admit vacuum 7. Rinse crucibles with approximately 50 ml hot water four times until all traces are removed 8. Rinse with acetone repeatedly until drained liquid is cleared. 9. Dry at 105 °C overnight 10. Cool to room temperature in desiccator 11. Weigh sample and crucible (W0) 12. Ash residues for three hours at 550 °C 13. Cool to room temperature in desiccators 14. Weight crucibles and residues (Wa).

Calculations

%NDF = [(W0–Wt)/Ws]×100 Cell soluble material = 100 – %NDF NDF expressed as organic matter: Ash insoluble in neutral detergent %NDFash = [(Ws–Wt )/(W0–Wt)]×100



2.5.7 Acid Detergent Fiber (ADF): Principle: Acid detergent fiber is determinedas the residue remainingafter adding an acidifiedsolution. It is the NDF withoutthe hemicelluloses. Cell soluble,hemicelluloses and soluble mineralsdissolve. Cetyltrimethyl-ammoniumbromide (CTAB) separatesproteins from the remainingcellulose and lignin, and minerals(ash). The acid detergents solutionrecovers cellulose and lignin.ADF determination is a preparationstep for lignin determination. Acid detergent solution:

1. 20 g cetyltrimethylammonium bromide (CTAB) 2. 1l of 1.0 N sulfuric acid (H2SO4).

Equipment:

1. Precision balance (Fig. 5) 2. Refluxing apparatus (Fig. 15) 3. Vacuum filtering system with trap in line (Fig. 16) 4. Desiccator (Fig. 7) 5. Berzelius beakers (600 ml) 6. Sintered glass crucibles (coarse porosity 1) (Fig. 17).

Procedure:

1. Add 0.5–1 g sample (Ws) in600 ml Berzelius beaker 2. Add 100 ml of acid detergentsolution 3. Boil for one hour on refluxingapparatus • Weigh oven–dry glass crucible (Wt) 4. Pour through glass crucibles 5. Admit vacuum 6. Rinse crucibles with approximately 50 ml hot water four times until all traces are removed 7. Rinse with acetone repeatedly until drained liquid is cleared 8. Dry at 105 °C overnight 9. Cool to room temperature in desiccator 10. Weight sample and crucible (W0) 11. Ash residues for three hours at 550 °C 12. Cool to room temperature in desiccator 13. Weight crucibles and residues (Wa).

Calculations:

%ADF = [(W0–Wt)/Ws]×100 ADF expressed as organic matter: %ADFash = [(Wa–Wt)/(W0–Wt)]×100



2.5.8 Acid Detergent lignin:

Principle: Lignin is the indigestible non-carbohydrate component of forages.Residues from ADF determination are treated with sulfuric acid. Lignin represents the indigestible NDF fraction.

Reagents / Solutions: 72% sulfuric acid solution

1. 735 ml concentrated sulfuric acid (98%) 2. Add 265 ml water.

Equipment:

1. Precision balance 2. Muffle furnace (550 °C) 3. Sintered glass crucibles (coarse porosity 1) 4. Desiccator

Procedure:

1. Transfer crucibles with residues from ADF to a flat container or tray 2. Cover the contents of the crucible with cooled 72% sulfuric acid (H2SO4) 3. Stir with glass rod, breaking all lumps 4. Fill crucible about half full with sulfuric acid and stir 5. Refill three times at room temperature 6. Leave for three hours stirring every 15 min with glass rod 7. Filter under vacuum 8. Rinse twice with 400 ml hot water 9. Dry overnight at 105 °C 10. Cool in desiccator and weigh (W0) 11. Ash at 550 °C for three hours 12. Cool in desiccator and weigh (Wa).

Calculations:

%ADL = [(Wa–W0)/Ws]×100



Constituent Characteristic

Lignin Major non-carbohydrate portion of cell wall. Three dimensional polymer of phenylpropanes. Lowers availability of hemicellulose and cellulose it is associated with. Provides structural support for plant.

Cellulose Major skeletal carbohydrate in plants. Polymer of glucose in ± (1→4)

linkages. Cellulase enzyme not secreted by mammals. Digestibility varies with amount of lignin, silica, cutin. Provides structural support for plant.

Hemicellulose Polymer of xylose and other five carbon sugars (arabinose side chains).

Digestibility depends on lignin, etc. Provides structural support for plant.

Pectin Polymer of methyl D-galacturonic acid. Highly digestible, and

availability not greatly influenced by lignin, etc. Cutin Composed of waxes and waxy polymers. May be integrated with lignin

and is measured as lignin in ADL. Lowers availability of cellulose and hemicellulose.

Silica Taken up by grasses more so than by legumes. Content in plants may

vary from l to approximately 22% of dry matter. Plants in sandy soils have higher levels. Has similar effect to lignin on digestibility of cellulose and hemicellulose. May be direct effect on cellulose or hemicellulose or may tie up some trace minerals needed by ruminal microorganisms. Composed of SiO2 polymers.



Figure 5-1.The Van Soest method of

partitioning fiber in feeds. (Source:

H. K. Goering and P. J. Van Soest,

Forage Fiber Analyses.

Agric. Handbook No. 379., ARS,

USDA,1970.). Adapted from

Maynard et al. (1979).



NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________






DATED: ______________________

ANALYST: ___________________

UNIT # 3: Determination of Macro and Micro Minerals 3.1 Principle: Samples are dried and ashed. The ash is dissolved in acid and dilutedwith water. The minerals in the diluted ash are determined. 3.2 Equipment:

1. Vycor® dish 2. Volumetric flask 250 ml 3. Desiccator 4. Air forced oven 5. Muffle furnace

3.3 Procedure: 3.3.1 Sample preparation

1. Mix and homogenize the samples 2. Weigh about 1 g of the sample to nearest mg in crucible (Vycor® dish) 3. Dry at least for 1 h in 105 °C air forced oven 4. Ash in furnace overnight (16 h) at 550 °C 5. Cool in desiccator 6. Add 1 ml HNO3 to dissolve the ash 7. Transfer to 250 ml volumetric flask and make up volume with H2O.

3.4 Equipment/Reagents / Solutions: 3.4.1 Equipment:

1. Shimadzu, Atomic Absorption & Flame Emission Spectrophotometer AA–630–12, Japan (AAS). Determine minerals as shown in table

a. Allow the device to warm up for 10 min with flame and source lamp lit. 7. Spectrophotometer, Hitachi U 2000, Tokyo, Japan. The device is equipped with automatic sampler unit and 1 cm cuvette holder

unit. Determine P at 400 nm. Allow the device to warm up for 10 min with source lit. 8. Flame photometer, Gallenkamp, UK. The device is equipped with filters to determine Na, K and Li.

UNIT # 3: DETERMINATION OF MACRO AND MICRO-MINERALS



3.4.2 Reagents / Solutions: 3.4.2.1Preparation of chemicals for concentrated stock solution

1. Calcium carbonate CaCO3, dry overnight at 200 °C 2. Ammonium dihydrogen phosphate (NH4)H2PO4, dry overnight at 110 °C 3. Sodium chloride NaCl. Dry overnight at 110 °C 4. Potassium chloride KCl. Dry overnight at 120 °C 5. Magnesium stock solution (1000 mg Mg/l)

a. Magnesium metal. Mg 9.95% b. 1000 mg pure Mg metal in 50 ml H2O c. Add slowly 10 ml HCl d. Make up to 1000 ml

6. HCl stock solution(1:3). a. 75 ml distilled water b. Add slowly 25 ml HCl

3.4.2.2 Concentrated standard stock solution

1. 500 mg Ca/l 2. 300 mg P/l 3. 25 mg Mg/l 4. 1000 ml volumetric flask 5. Add 1.249 g CaCO3 6. 1.114 g (NH4)H2PO4 7. 25 ml Mg stock solution 8. Add 30 ml HCl stock solution 9. Mix until dissolved and then make up to volume.

3.4.2.3Diluted standard stock solution (20 mg Ca/l, 12 mg P/l, and 1 mg Mg/l):

1. Add 40 ml concentrated stock solution in 1000 ml volumetric flask 2. Make up to 1000 ml with distilled water.

3.4.2.4Molybdovanadate solution:

While stirring gradually add molybdate solution to vanadate solution. 3.4.2.5Molybdate solution

1. Weigh 1.5 g ammonium metavanadate 2. Add 690 ml hot H2O 3. Add 300 ml HNO3 4. Cool to 20 °C 5. Make up to 1000 ml with distilled water.

3.4.2.6Vanadate solution

1. Weigh 60 g ammonium molybdatetetrahydrate 2. Add 900 ml hot H2O 3. Cool to 20 °C



4. Make up to 1000 ml with distilled water.

Note: Store at room temperature in polyethylene or glass–stopper Pyrex bottle. Reagent is stable in Pyrex bottle. Discard reagent if precipitate forms. 3.4.2.7Lanthanum stock solution (1% La)

1. Weigh 11.73 g La2O3 or 26.74 g LaCl3.7H2O 2. Add slowly 25 ml HNO3 3. Make up solution to 1 l.

Note: Reagents for mineral standards must be ultra–pure (99.95%). Opened reagents should be resealed and stored in desiccator.

3.5Determination of Phosphorus 3.5.1Standards:

1. Pipet 0, 5, 10, 15, 20, 25, 30, and 35ml of diluted stock solution into aseries of 50 ml volumetric flasks. 2. Add 10 ml molybdovanadatereagent and mix 3. Make up volume to 50 ml with H2O

3.5.2Sample:

1. Pipet 10 ml sample solution into 50 ml volumetric flask 2. Add 10 ml molybdovanadate reagent and mix 3. Make up volume to 50 ml with H2O.

3.5.3 Measurements: 1. Set spectrophotometer at wavelength 400 nm. 2. Adjust to 0 absorbance with 0 μg/ml standard, and run testsamples and standards under the same conditions. 3. Determine ppm phosphorus sample solution vs. 4 or 5 standards ofrespective material. 4. Use smaller portion of sample solution if absorbance of samplesolution is beyond range of curve. 5. The correlation of standard linear regression should yield a coefficientof ≥0.999.

3.6Determination of Potassium and Sodium 3.6.1 Standards:

Use 0, 1, 2, 3, 4, 6, 8 and 10 ppm standards.

3.6.2 Sample: 1. Pipet 5 ml sample solution into 50 ml volumetric flask. 2. Make up to volume.

3.6.3Measurements:

1. Adjust flame photometer to 0 absorbance with 0 ppm standard andrun test portions along with standards under the same conditions

2. Determine ppm minerals sample solution vs 4 or 5 standards ofrespective material 3. Use smaller portion of sample solution if absorbance of samplesolution is beyond range of curve



4. The correlation of standard linear regression should yield a coefficientof ≥0.999 5. Linearity is harvested using standards from 0 to 10 ppm; beyond 10ppm the standard curve is not linear.

3.7 Determination of Calcium and Magnesium 3.7.1 Standards:

1. Pipet 0, 2.5, 5, 7.5, 10, 12.5 and 15 ml of diluted stock solution into aseries of 50 ml volumetric flasks 2. Add 5 ml La stock solution, and dilute to volume. You will obtain0–6 μg/ml of Ca and 0–0.3 μg/ml of Mg in 0.1% La.

3.7.2 Samples:

1. Pipet 5 ml sample solution into 50 ml volumetric flask 2. Add 5 ml La stock solution and dilute to volume.

3.7.3Measurements:

1. Set the optimum response of AAS system (Table 1) 2. Adjust to 0 absorbance with 0 μg/ml standard, and run testsamples and standards under the same conditions 3. Determine ppm minerals sample solution vs. 4 or 5 standards ofrespective material 4. Use smaller portion of sample solution if absorbance of samplesolution is beyond range of curve 5. The correlation of standard linear regression should yield a coefficientof ≥0.999.

Note: All glassware should be rinsed in 1:1 HCl and then rinsed withdistilled water. 3.8Calculations:

Plot absorbance vs. μg/ml minerals using Microsoft Excel. Use the Forecastfunction of Microsoft Excel to calculate mineral concentration (C)based on standard data.

Calculate the content of minerals in mg /100 g as follows:

Minerals,mg/100 g = (C×D)/W

Where: C = μg/ml Ca or Mg in the assay solution W = g weight of sample D = dilution factor×factor for transforming to mg/100 g D = [(250×50)/10]×(100/1000) = 125



References: AOAC.2000

Determination of Organic Dry Matter (ODM):

Formula:

%ODM = 100 – %Ash

Determination of NFE: Formula: NFE = 100 – (% Moisture + % Ash + % CF + % EE + % CP)

Determination of TDN:

Formula:

%TDN= % DCP+ %D NFE+% DCF+ (% DEE*2.25)



NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________






DATED: ______________________

ANALYST: ___________________

UNIT # 4: INVITRO DIGESTABILITY, GAS PRODUCTION TEST (HOHENHEIM METHOD), IN SACCO DEGRADABILITY AND DIGESTIBILITY: 4.1Invitro-digestibility: Principle The two steps procedure described is used for in vitro determination of digestibility of forages. In the first step, dried ground forages are incubated in test tubes with rumen fluid for a given period of time. The tubes also contain buffer solution, macro-minerals, trace-minerals, nitrogen sources, and reducing agents to maintain pH and provide nutrients required for growth of rumen bacteria. Because oxygen is toxic to rumen bacteria, solutions are gassed with carbon dioxide to maintain anaerobic conditions, and temperature is held at 39 °C (body temperature) during the incubation. In the second step, after 48 hours of incubation, an enzyme solution is added to simulate digestion in the small intestine. Reagents/ Solutions Rumen liquor Rumen fluid has been taken through a fistula from the fistulated animal.The fluid should be kept in a thermos container to maintain the bodytemperature of the animal (38–39 °C). Liquor filtered through a 2–foldlayer of muslin into a flask while passing CO2 to the flask to displace airfrom above the fluid. Mixed chloride solution:

1. Make up to 2 l with distilled water 2. 47 g sodium chloride (NaCl) 3. 57 g potassium chloride (KCl) 4. 12 g magnesium chloride (MgCl2.6H2O) 5. 4 g calcium chloride (CaCl2.2H2O).

Stock solution:

1. Make up to 2 l with distilled water 2. 37 g di–sodium orthophosphate (Na2HPO4) 3. 98 g sodium hydrogen–carbonate (NaHCO3) 4. 200 ml mixed chloride solution.

Buffer solution:

1. 10.4 ml stock solution

UNIT # 4: IVD, GAS PRODUCTION TEST, IN-SACCO DD



2. 41.6 ml distilled water 3. 1 ml ammonium sulphate (66.07g/l) ((NH4)2SO4).

Pepsin solution:

1. 720 ml distilled water 2. 280 ml hydrochloric acid fuming 3. 36.5% conc. (HCl) 4. 24 g pepsin.

Equipment:

1. Precision balance 2. Centrifuge 3. Air-circulation oven 4. Water bath 5. Vacuum extraction unit 6. Muffle furnace 7. Desiccator 8. Sinta glass gas distribution tubes 9. Nalgene centrifuge tubes 28 mm×160 mm 10. Crucibles

Procedure:

1. Add 0.5 g of ground sample in centrifuge tubes (Ws), include blanks 2. Mix buffer solution and rumen fluid as follows (volumes per one sample):

a. 53 ml of buffer solution and adjust to ph 6.9 gazing with carbon dioxide. b. Add 13 ml of rumen fluid.

3. Add 66 ml of mixture to sample in centrifuge tubes 4. Keep in water bath at 36.5–38 °C for 48 hours (stir 2–3 times per day) 5. Then add 5 ml of pepsin solution carefully in 0.5 ml doses 6. Keep in water bath at 36.5–38 °C for another 48 hours (stir once a day) 7. Centrifuge for 10 min at 4000 rpm 8. Take off excessive liquid under vacuum using sinta glass gas distribution tube 9. Transfer residues to pre-weight oven–dry crucibles, rinsing with small amount of distilled water 10. Dry crucibles overnight at 105 °C and weigh (Wt) 11. Cool to room temperature in desiccators 12. Weigh sample in crucible (W0) 13. Ignite at 550 °C in muffle furnace for four hours 14. Cool to room temperature in desiccators 15. Weigh sample in crucible (Wa).

Calculations:

% DMD = [(Ws×(%DM/100)–(W0–Wt)+blank)×100]/[(Ws×%DM)/100)] % OMD = [(Ws×(%DM)/100)×(%OM/100)–(Wt–Wa)+blank)×100]/ [(Ws×%DM)/100]



4.2 Gas Production Test (Hohenheim Method) Principle: The gas production test is based on the association between rumenfermentation and gas production. The in vitro gas production method can be used to measure the metabolizableenergy of feeds and to quantify utilization of nutrients. Equipment:

1. Precision balance 2. Circulating water bath with holder for syringes 3. Heating plate with stirrer 4. CO2 cylinder 5. Woulff bottle 6. Dispenser 7. 100 ml glass syringes for GPT

Reagents / Solutions: Rumen fluid:

1. Collect rumen fluid before morning feeding from 2 rams 2. Filter through two-layer cheese cloth into thermos container 3. Keep at 39 °C and under carbon dioxide (CO2).

Fermentation buffer solution:

1. 630 ml of bicarbonate buffer 2. 315 ml of macromineral solution 3. 0.16 ml of micromineral solution 4. 1.6 ml of resazurine solution 5. 945 ml distilled water 6. 60 ml of fresh prepared reducing solution 7. 660 ml rumen fluid.

Bicarbonate buffer:

1. 35 g sodium bicarbonate (NaHCO3) 2. 4 g ammonium carbonate 3. Dissolve in 500 ml distilled water and then make up to 1litre.

Macromineral solution:

1. 6.2 g potassium dihydrogen phosphate (KH2PO4) 2. 5.7 g disodium hydrogen phosphate (Na2HPO4) 3. 0.6 g magnesium sulphate (MgSO4.7H2O) 4. Dissolve in 500 ml distilled water and then make up to 1 l.



Micromineral solution:

1. 10 g manganese chloride (MnCl2.4H2O) 2. 13.2 g calcium chloride (CaCl2.2H2O) 3. 1 g cobalt chloride (CoCl2.6H2O) 4. Dissolve in 50 ml distilled water and then make up to 100 ml.

Resazurine:

1. 0.1 g resazurine in 100 ml distilled water. Reducing solution:

1. 996 mg sodium sulphide (Na2S.9H2O) 2. Dissolve in 94 ml distilled water 3. 6 ml 1 N sodium hydroxide solution (NaOH) 4. 1 N NaOH = 4 g NaOH in 100 ml distilled water.

Procedure:

1. Weigh 200 mg of feed sample (1 mm ground) and insert carefully in 100 ml calibrated glass syringe 2. Include blank syringes without feed 3. Include syringes with standard feed (concentrate or/and hay) 4. Keep syringes overnight in circulating water bath at 39 °C 5. Prepare fermentation buffer solution except rumen fluid and reducing solution in 2 l Woulff bottle 6. Keep bottle in water bath at 39 °C with stirrer overnight.

Next morning:

1. Keep under CO2 2. Add reducing solution and wait 20 minutes until color changes from blue to purple to colorless 3. Add rumen fluid 4. Keep stirring under CO2 for 10 minutes 5. Fill 30 ml into glass syringes 6. Incubate syringes in 39 °C 7. Shake syringes every hour during first four hours, then twice every hour 8. Record gas production at 8 hours and push back piston to 30 ml if gas production exceeds 70 ml 9. Record gas production at 24 hours (V24) and terminate experiment.

Calculations: Calculation for gas production G24 = [(V24–V0–G0)×FSt×200]/Ws If pushed back after 8 hours: G24 = [(V24–V0+V8–G0)×FSt×200]/Ws G24 = gas production value (ml/200 mg) at 24 hours G0 = gas production of blank syringes (ml) V0 = volume in ml at begin V8 = volume in ml at 8 hours



V24 = volume in ml at 24 hours Ws = weight of dried sample in mg FSt = GSt/GSt measured

Reference value (FSt) is gas production GSt of the standard sample (e.g. hayand/or concentrate) as per supplier (e.g. Landesarbeitskreis. Fütterung,Baden Württemberg e.V. (LAF), Hohenheim, 70578 Stuttgart, PF 7200220)compared to the measured gas production in the test (GStmeasured). Calculation for organic matter digestibility (OMD) %OMD = 14.88+0.889 G24+0.45 CP CP = Crude Protein Calculation for metabolizable energy (ME) ME(MJ/kg DM) = 2.2+0.136 G24+0.057 CP Calculation for net lactation energy (NEL) NEL(MJ/kg Ws) = 0.0663 G24+0.095 CP+0.228 CF+0.079 Nfree– 3.49 Nfree = N-free extract in % dry weight CF = Crude Fat

4.3 In saccoDegradability and Digestibility Principle: The incubation of feeds in nylon bags inside the rumen is used to measurethe degradation of the feedstuff. Degradability is expressed as lossof weight during incubation. Digestibility can be measured by analyzingthe residues for NDF. Equipment:

1. Precision balance 2. Air circulating oven 3. Muffle furnace 4. Nylon bags of 9x16 cm made from indigestible material; 40–60 micron mesh size 5. Plastic tubes with attachments for nylon bags

Procedure for degradability:

1. Weigh 3 g of dried and ground feed (3 mm mesh size) in each nylon bag (Ws) 2. Attach 3–5 bags to each plastic tube and insert in rumen 3. Up to 15 bags can be inserted in rumen 4. Use at least three animals 5. Diet of animals same as feed in nylon bags 6. Incubation time: 4, 8, 16, 24, 36, 48, 72 and 96 hours 7. Insert bags at different time intervals to take them out at the same time 8. Take out bags and put immediately in cold water to stop fermentation 9. Wash in washing machine for 20 minutes at 22–25 °C (record revolutions per minutes, rpm) 10. Dry bags at 65 °C for 30 hours 11. Weigh residues (Wr).



Calculations for dry matter degradability (DMdeg): Calculate dry matter as under for feed sample (DMs) and residue (DMr) DMdeg = [(DMs–DMr)/DMs ]×100 Procedure for digestibility: • Transfer contents of nylon bag to Berzelius beaker • Proceed with NDF determination for residue (NDFr). Calculation of digestibility (D): D = (NDFr/DMr )×100



NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________



Appendix # 1. Preparing Standard Solutions A. Molar (M) solutions 1 Molar solution (1M) contains 1 mole of solute dissolved in a solutiontotaling 1 l. Molecular weight is MW. 1 M = 1 g MW of solute per liter of solution

Example: NaCl • MW of sodium chloride (NaCl) is:Na (23)+Cl (35.5) = 58.5 grams/mole • 1 M aqueous solution of NaCl, dissolve 58.5 grams of NaCl in 1 l. ofdistilled water. • 0.1 M solution of sodium chloride contains 5.85 grams per 1 l. Example: H2SO4 • Molar (1 M) aqueous solution of H2SO4 • Check on bottle label for Molar concentration (e.g. 18.0)x ml = 1 ml×1 mole/ 0.018 molesx ml = 55.6 ml of H2SO4 Note: Add H2SO4 slowly to 500 ml of distilled water and then completeto 1 l. Never add water to sulfuric acid.

B. Normal (N) solutions Equivalent weight (EW) = molecular weight g/n mEW = millieauivqlent weight n = valence of solute N = Weight of solute/ mEW of solute×Volume (ml) of dilution

Example: Normal solution with salt 2.9216 grams of NaCl in 500 ml distilled water MW of NaCl = 58.44 EW = 58.44/1 mEW = 0.05844 N = 2.9216 g/ (0.05844×500 ml) N = 0.099

Example: Normal solution with acids EW = MW/n n = number of replaceable hydrogen atoms in the reaction HCl n = 1 HNO3 n = 1 H2SO4 n = 2 HF n = 1 For HCl MW = 36.46, EW = 36.46 1 N = 36.46 g/l For H2SO4 MW = 98.08, EW = 98.08/ 2 = 49.04 g/l N = 49.04



Example: Normal solution with alkalis EW = MW/n n = number of hydrogen ions that are required to neutralize the base NaOH n = 1 Ca(OH)2 n = 2 For NaOH MW = 40, EW = 40 1 N = 40 g/l For Ca(OH)2 MW = 74, EW = 74/2 = 37 1 N = 37 g/l

C. Preparation of normal solutions from concentrated reagents Example: H2SO4 Prepare 2 l. of 0.2N 96% H2SO4 Density = 1.84 g/ml MW = 98.08 EW = MW/2 = 49.04 g/l V (ml) = Volume of H2SO4 needed Therefore, slowly add 11.105 ml of 96% H2SO4 to 1 l of distilled water andthen add up to 2 l to get a 0.2 N solution. Example: HCl Prepare 2 l. of a 0.2 N 38% HCl Density = 1.188 g/ml MW = 36.461 EW = MW/1 = 36.461 V (ml) = Volume of HCl needed

V = N needed × Volume of solution × EW/density x concentration reagent = 0.2 × 2 × 36.461/(1×1.188 × 0.38) = 32.604 ml

Therefore, slowly add 32.604 ml of 38% HCl to 500 ml of distilled water and then add up to 2 l to get a 0.2 N solution.



NOTES

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________

_____________________________________________________________________________________________________________



Appendix # 2. Abbreviations

°C Degree Celsius ADF Acid Detergent Fiber ADIN Acid Detergent Insoluble Nitrogen ADL Acid Detergent Lignin

AOAC Association of Official Analytical Chemists cm centimeter CMT California Mastitis Test CP Crude Protein CT Condensed Tannins CTAB Cetyl Trimethyl Ammonium Bromide DM Dry Matter FID Flame Ionization Detector g gram GC Gas Chromatograph GPT Gas Production Test L liter m meter

M Molarity ME Metabolizable energy mg milligram ml milliliter mm millimeter

μg microgram

μl microliter

μm micrometer

N Nitrogen

N Normality NDF Neutral Detergent Fiber

ODM Organic Dry Matter



Appendix # 3. LABORATORY TEST RESULT TABELS

FORAGE AND FEED TEST RESULTS

Determinations of % Dry Matter, % Moisture, % Protein, % Fat, % Fiber (ADF,NDF), % Calcium, % Phosphorus, %

Magnesium, % Potassium,TDN, NEl (MCal/lb), NEm (MCal/lb),NEg (MCal/lb) in Feed and Forages on

Dry Matter and As Fed Basis

KEY: ADF=Acid Detergent Fiber, NDF=Neutral Detergent Fiber, NEl=Net Energy for Lactation, NEm=Net Energy for

Maintenance, NEg=Net Energy for Growth, TDN=Total Digestible Nutrients

Sample #

EXAMPLE

1

Lab Number 80426

Sample Type Hay-Orchardgrass-Trimothy

Sample Weight (gm.) X

Moisture (%) 8.46

Dry Matter (%) 91.54

DM BASIS AS FED BASIS




Protein (%) 10.39 9.51

Fat (%) 2.48 2.27

Fiber-ADF (%) 37.14 34.00 Fiber-NDF (%) 62.58 57.29

Calcium (%) 0.46 0.42

Phosphorus (%) 0.25 0.23

Magnesium (%) 0.12 0.11

Potassium (%) 2.67 2.44 TDN 61 55

NEl (MCal/lb) 0.62 0.57

NEm (MCal/lb) 0.61 0.56

NEg (MCal/lb) 0.35 0.32

RFV 89



FORAGE AND FEED TEST RESULTS

Determinations of % Dry Matter, % Moisture, % Ash and % Organic Matter in Feed and Forages

KEY: Cru = Crucible, S = Sample, DM = Dry Matter, OM = Organic Matter, Wt = Weight

A B C D E F G H I J K L M N O

Name of Sample

Number of Cru

Wt of Cru

Wt of S + Cru

S + Cru Wt after drying

S + Cru Wt

after Ashing

Wt of Sample

DM in sample

Ash in Sample

Moisture in Sample

OM in Sample

% DM

% Moistu

re

% Ash

% Organi

c Matter

EXAMPLE

1

50g

51g

50.60g

50.07g

D-C

E-C

F-C

G-H

H-I

H*100 G

J*100 G

I*100 G

K*100 G

Mott Grass

1.0g

0.6g

0.07g

0.4g

0.53g

60%

40%

7%

53%



Appendix # 4. LABORATORY EQUIPMENTS

LABORATORY EQUIPMENTS















REFERENCES AND SUPPLEMENTAL READING:

1. Animal Nutrition and Product Quality Laboratory Manual by Monika Zaklouta, Muhi El-DineHilali, Ali Nefzaoui and Mohammad Haylani. Edited by Monika Zaklouta

Citation: Zaklouta M., Hilali M., Nefzaoui A. and Haylani M. 2011.Animal nutrition and product quality laboratory manual.ICARDA, Aleppo, Syria.viii + 92 pp.

2. AOAC. 1995.AOAC. 2000.

3. Food for Thought: Equine Nutrition and Feeding Management Bridgett J. McIntosh, PhDDepartment of Animal

ScienceUniversity of Tennessee (Dr. Bridgett McIntosh, UT Department of Animal Science, [email protected],931‐486‐2777)

4. FORAGE QUALITY AND CONSERVATION Course Lectures: Dr. J.A. Olanite; Dr. O.M. Arigbede; Mrs. V.O.A. Ojo, DEPARTMENT OF PASTURE AND RANGE MANAGEMENT,COLLEGE OF ANIMAL SCIENCE AND LIVESTOCKPRODUCTION, UNIVERSITY OF AGRICULTURE, ABEOKUTA.

5. Goering HK and Van-Soest PJ. 1970. 6. LABORATORY PROCEDURES IN ANIMAL NUTRITION RESEARCH by M. L. Galyean, Department of Animal and Food

Sciences, Texas Tech University, Lubbock, Originally prepared August, 1980.Revised, July 1982, June 1983, June 1984, May 1985, May 1986, May l987, June 1988, May 1989, May 1990, August 1991, August 1992, May, 1997, and May 2010.

7. Makkar HPS. 2002. 8. Menke KH, Raab L, Salewski A, Steingass H, Fritz D and Schneider W. 1979. 9. Methodenbuch Bd.1988.

10. Osuji P O, Nsahlai I V and Khalili H. 1993. Feed evaluation. ILCA Manual 5. ILCA (International Livestock Centre for Africa),

Addis Ababa, Ethiopia. 40 pp. ISBN 92–9053–278–5 11. Ørskov ER, Hovell FD DeB and Mould F. 1980.

12. Tilley JMA and Terry RA. 1963.

13. Undersander D, Mertens DR and Thiex N. 1993.

14. Van Soest PJ, Robertson JB and Lewis BA.1991.

mailto:[email protected],931?486?2777



THIS PAGE INTENTIONALY LEFT BLANK!



EDITOR: Pen Name: Sa’yem Khan DrAtiqUllah Khan Sa’yemMarwat

DVM (GCVS). GOMAL UNIVERSITY. DERA ISMAIL KHAN RVMP (PVMC).ISLAMABAD. M.Sc. (H) ANIMAL NUTRITION (In Progress). UNIVERSITY OF AGRICULTURE PESHAWAR.

.

|VET CARESS| Veterinary Care Services| Pakistan|



ANALYSIS OF THE NUTRITIVE VALUE OF FORAGES LABORATORY PROCEDURES FIRST EDITION 2o14

Documents