Animal Nutrition and Feeding

Chapter 2

The Feed Elements

Chapter 2

The Feed Elements

Module no 2 entitled Feed Elements talks about the composition of feeds and feedstuffs which
are all essential to animal’s body. It discusses the feed elements as well as its proximate
composition. It also converses the factors that affect the composition of the feed and feedstuff.

Specific Objectives

At the end of the module, the students should be able to:

- Have an overall grasp of the feed elements;
- Discuss and be able to apply in problems sets the theories on the proximate composition
of feedstuffs; and
- Identify the factors that affect the composition of feedstuff.

Duration

Chapter 2: The Feed Elements = 10 hours (4 hours discussion; 6 hours laboratory)

Lesson Proper

Nutrients Required by Plants and Animals

Animals
Depending on animal age and species, animals require a source of nitrogen (N) in the
form of acids essential amino, fat in the form of essential fatty acids, essential mineral
elements, a source of energy that may vary from primarily fat and protein for carnivorous
animals to coarse fibrous plant tissue for some herbivorous species, and some of the fat- and
water-soluble vitamins. The amounts and proportions required are influenced by the type of
gastrointestinal tract, the age of the animal, its level of productivity, what type of productivity
is in question (maintenance of body tissues, work, growth, milk, eggs, conceptus), the dietary
components available, and other factors. Because animals require more than 40 nutrients,
meeting dietary requirements may be difficult, depending on the availability of appropriate
feedstuffs. Humans require the same nutrients as animals, although the amounts of each
nutrient needed for various body functions may differ.

Plants
In contrast to animals, requirements for plants are relatively simple. In general, plants
take up N in the form of nitrate or ammonia and they synthesize their complex proteins by
incorporating these forms of N into amino acids and other intermediate products. Plants require
a large number of inorganic elements. The qualitative requirements for minerals appear to be
essentially the same as for animals. Plants may also require Al, Br, Cs, and Sr. thus, the primary
nutrient needs of plants are the required inorganic elements and N, normally obtained from soil
through the roots. Through the process of photosynthesis, the plant takes in atmospheric CO 2,
releases O2, and synthesizes glucose, the basic biochemical required for plant growth. Using
these basic components, the plant is capable of synthesizing all of the complex biochemical
that is requires for completing its life cycle.
The Feed Elements

The feed an animal consumes may vary from very simple compounds such as salt
(NaCl) or glucose to the extremely complex mixtures provided by some plant and most animal
products. Not all components are usable nutrients. Indeed, some of the material consumed may
be insoluble and/or indigestible, and some may be toxic under certain conditions.

Figure 1. Schematic Chart of Elements and Compounds Present in Food (Pond,

et al 2004)

Water is a major item in most animal diets, although it is not listed in the diagram. The
other ingredients make up the dry matter of the diet, composed of organic compounds (organic
matter) or inorganic elements (mineral matter) in the diagram.

 Water is made up of 2 atoms of Hydrogen (H) and 1 atom of Oxygen (O).

 It is the cheapest and most abundant nutrient.
 Water and dry matter are main components of feeds.
  Water is essential in the transport of metabolic products and wastes and in most
chemical reactions in the body.
 Animals obtain water from drinking water, feed and metabolic water. Metabolic Water
is formed from the oxidation of compounds, such as sugars, as illustrated below:

C6H12O6 + 6 O2 = 6 H2O + Energy

Water has the following functions to the animal:

 As a solvent, it functions in the transport of nutrient to the cell and excretion of
waste products of metabolism;
 It is extremely important in temperature regulation;
 It cushions the nervous system; and
 It lubricates joints and acts as a cerebrospinal fluid.

Lack of water will result in death of an animal faster than a deficiency of any other
nutrients. Limitations in water intake will reduce rate of gain, milk production or egg
production.

Nearly all animal feeds contain proteins, which are complex molecules containing various
amino acids and other non-protein components. Both animal and plant proteins may be very
complex and vary in the content, sequence, and configuration of their constituent amino acids,
resulting in differences in molecular size, solubility, and digestibility of the protein. In addition,
plants have many amino acids not found in proteins, and they may contain other nitrogenous
compounds such as nitrates and nucleic acids.

 Proteins are complex group of compounds, which contain the elements Carbon,
Hydrogen, Oxygen, Nitrogen, Sulfur and Phosphorus
 They are made up of long chains of Amino Acids, which vary in relative amounts and
kind (among different proteins), joined together by a Peptide Bond.
 Amino acids are organic acids, which contain one or more amino group (NH2).
 Proteins are in highest concentration in muscle tissues of animals.
 In ruminants, proteins are first utilized by the microorganisms in the rumen for their
own growth and reproduction. Microbial Fermentation of proteins produces ammonia
and CO2 as main end-products.
 Undigested feed proteins and microorganisms from rumen are passed on the lower
gastrointestinal tract, where these are then enzymatically digested and absorbed as
amino acids. Thus, Microbial Protein becomes an excellent source of amino acids for
ruminants.
 Protein is the most expensive nutrient to furnish in an animal’s diet

AMINO ACIDS ARE CLASSIFIED INTO 2 GROUPS:

A. Dispensable (Non-Essential) – essential to the animal but are normally synthesized by

them or in sufficient amount in the diet.
 B. Non-Dispensable (Essential) – cannot be synthesized by the animal and must always
be present in adequate amounts in the diet to attain optimum performance of an animal

DISPENSABLE(NON-ESSENTIAL) NON-DISPENSABLE (ESSENTIAL)

AMINO ACIDS AMINO ACIDS
Alanine P - Phenylalanine
Asparagine V - Valine
Aspartic Acid T - Threonine
Cysteine M - Methionine
Cystine A - Arginine
Glutamic Acid T - Tryptophan
Glutamine H - Histidine
Glycine I - Isoleucine
Hydroxyproline L -Leucine
Praline L- Lysine
Tyrosine
*Essential Amino Acids - PVT. MAT HILL

PROTEINS ARE IMPORTANT FOR THE FOLLOWING FUNCTIONS

1. It is the basic structural unit of the body
 Collagen (cornea & connective tissues)
 Elastin (tendons, arteries and elastic tissues)
 Keratin (hair, horn, wool)
2. Body metabolism
 Enzymes
 Hormones
 Immune bodies
 Hereditary transmission
3. Excess proteins are de-aminated and used by the animal as source of energy.

Animal diet must satisfy a minimum level of Crude Protein (CP) with adequate and
well-balanced amino acids.
Crude protein is composed of:
 True Protein – made up of amino acids only
 Non-Protein Nitrogen/Amides – contains N that can be converted to protein by
bacterial action

Quality proteins are found in feedstuffs of animal origin. This is because the amino
acid content of these feeds approximates those that are found in animal tissues. These are
usually the best protein source for non-ruminant animals. Ruminant animals have the
capacity to utilize protein sources containing high amounts of NPN, with the help of the
rumen bacteria.

Lipids (FATS AND Ether Extracts) of many different types are found in both plant and
animal tissues. Only two fatty acids, linoleic and linolenic, are thought to be specific dietary
requirements for animals. However, other dietary lipids necessary for animal life include the
fat-soluble vitamins.

 Lipids are made up of chemical elements C, H, and O. oxygen comprises a much

smaller portion of their molecule. For these reason, lipids release more energy upon
oxidation than carbohydrates or proteins.
 Lipids are composed of 1 molecule of Glycerol (a carbohydrate) and 3 molecules of
Fatty Acids. Fatty acids are long chain organic compounds, usually containing an even
number of carbon atoms; they may be saturated (only single bond between carbon
atoms) or unsaturated (double bond between some of the carbon atoms).

FUNCTIONS OF FAT IN THE BODY

 Concentrated source of energy (2.25 times more energy than carbohydrates and
proteins)
 Source of essential fatty acids (vitamin-like role) such as Linoleic, Linolenicand
Arachidonic Acid that are needed in the formation of certain regulatory hormones
 Carrier of fat-soluble vitamins
 Heat, insulation and protection
 Structural component of tissues such as brain and liver

SATURATED FATS Animal Fats (e.g. Solid at room Less

Tallow) temperature digestible
UNSATURATED Vegetable Oil (e.g. Liquid at room More
FATS coconut & soybean oil) temperature digestible

Carbohydrates make up the major fraction of most plant tissues and may be complex
in number and composition. In contrast, carbohydrates make up less than 1% of the tissue in
animals. For the animal, carbohydrates serve as a source of energy and provide sufficient bulk
to keep the digestive tract working smoothly. No specific carbohydrate is required, except for
glucose, which is required by all cells as an energy source. Carbohydrates required by various
organs or tissues can be synthesized in other tissues.

 Carbohydrates are made up of Carbon (C)- 40%, Hydrogen (H)- 7%, and Oxygen
(O)- 53%, with hydrogen and oxygen found in the same ratio as in water.
 In the plant, carbohydrates are formed by photosynthesis, chemically illustrated as
follows:
 6 CO2 + 6 H2O + 673 kcal (sun) – C6H12O6 + 6 O2
 Plants, the chief source of animal feeds, contain about 75% carbohydrates (dry matter).
 The bulk of nutrients found in the animal’s diet are carbohydrates. However, only less
than 1% is found in the animal’s body as the nutrient is continuously metabolized by
the animal.

THE USES OF CARBOHYDRATES ARE AS FOLLOWS:

  Metabolized as a source of energy
 Converted into Glycogen which is sometimes called “animal starch”
 Converted into fat and stored in the body for future use as a source of energy.

The basic unit of carbohydrate structure in livestock and poultry feeds is the Hexose
Unit (6-Carbon Atom Molecule). Smaller amount of pentose (5-carbon atom molecule) are
found in animal feeds; usually, these are less digestible than the hexoses but some animals
(especially ruminants) can utilized these fairly well. Much smaller amounts of diose, triose,
and tetrose (2, 3, and 4 carbon sugars, respectively) are present and are generally
unimportant.

THE CLASSIFICATION OF CARBOHYDRATES IS AS FOLLOWS:

A. MONOSACCHARIDES
Contains one sugar unit; it is usually found as such in feedstuff but serves as a building
unit for more complex carbohydrates.
 Pentoses – Arabinose, Xylose, Ribose
 Hexoses – Glucose, Fructose, Galactose

B. DISACCHARIDES
Formed by two monosaccharides
 Sucrose – Glucose + Fructose (found in cane sugar)
 Maltose – 2 glucose units (obtained from hydrolysis of starch)
 Lactose – Glucose + Galactose (sugar found in milk)

C. POLYSACCHARIDES
Composed of long chains of hexose or pentose units
 Starch – long chains of glucose units joined by alpha linkage between glucose
units; cereal grains and tubers are rich sources of starch.
 Cellulose – composed of long chains of glucose units that are joined together by
beta linkage; found in cell walls of plants, especially in mature grasses.
 Hemicelluloses – it is made up of both 5-carbon and 6-carbon sugar groups; also a
part of plant cell walls but is more digestible than cellulose.

The kind of carbohydrate unit influences the ability of an animal to digest sugars
from a given feedstuff. Generally, starches such as those found in cereals and tubers are
well utilized by animals. Some feedstuff (e.g. barley) containing high amounts of non-
starch polysaccharides (arabinoxylans), however, are poorly digestible and cause viscous
condition in the intestine (most especially non-ruminants). Only ruminant animals could
well utilize feedstuff with high amounts of cellulose and hemicelluloses (e.g. grasses).

Vitamins account for only a small fraction of the weight of almost all feed sources.
Even so, feed sources vary widely in vitamin content, partly because most of the vitamins are
subject to degradation by exposure to heat, light, and environmental variables.
  A vitamin is an organic nutrient required in small quantities necessary for regulating
metabolic processes, but does not become an actual component of body structures.
 Vitamin functions as accessory nutritional factors with no structural or energy yielding
role.
VITAMINS ARE GENERALLY DIVIDED INTO 2 GROUPS.
FAT SOLUBLE VITAMINS WATER SOLUBLE VITAMINS
Vitamin A (Retinol) Vitamin B1 (Thiamine)
Vitamin D (Ergosterol – in plant) Vitamin B2 (Riboflavin)
Vitamin D3-7 (Dehydrocholesterol – in Vitamin B6 (Pyridoxine)
animals)
Vitamin E (Alpha-Tocopherol) Vitamin B12 (Cyanocobalamine)
Vitamin K1 (Phyloquinon) Niacinamide (Nicotinamide)
Vitamin K2 (Menaquinone-7) Panthothenic Acid (Panthotenate)
Vitamin K3 (Menadion) Vitamin H (Biotin)
Folic Acid
Vitamin C (Ascorbic Acid)
Inositol
Choline
Para-Amino Benzoic Acid

The fat soluble vitamins are involved in the regulation of structural portion of the body
e.g.:
 Vitamin D – regulates calcium & phosphorus metabolism
 Vitamin A – maintains the structural integrity of the photoreceptors of the eye

The water soluble vitamins are usually abundant in feeds and these can be readily
utilized by the animal since these are already in active form. They are involved in enzyme
systems which function in the direct metabolism of fats, proteins, carbohydrates as well
as transfer of energy throughout the body (B-Complex), transmission of nerve impulses
(Choline) and Red Blood Cell formation (B12).

Vitamins are involved in the Prevention of Gross Deficiency Symptoms such as:
Thiamine Beriberi
Nicotinamide Pellagra (Corn-Eater Disease)
Folic Acid Sprue (A Tropical Disease)
Cyanocobalamin Anemia (Macrocytic)
Vitamin C Scurvy
Vitamin A Night Blindness
Vitamin D Rickets (Young Animals)
Osteomalacia (Adult)
Vitamin E White Muscle Disease
 Vitamin K Bleeding Disease (White Clover
Poisoning)

Feedstuffs in their fresh form as well as fermentation by-products contain

appreciable quantities of vitamins. However, processing and improper handling may
cause substantial reduction in Biopotency (Vitamin Activity) of these vitamins. Although
some vitamins may be synthesized by some animals (e.g. B-Complex by ruminant
animals and Ascorbic Acid by poultry), amounts are not sufficient to meet their
requirement when these animals are under stress. Hence dietary vitamin supplements are
needed.

General Symptoms Indicative of Marginal or Advanced Vitamin Deficiencies

Poultry
1. Nervous disorder such as convulsion- A, E, B1, B2, B6 and Fol
2. Skin or mouth lesions- A, B2, B6, H, PP and Pantothenic acid
3. Discharge from eyes or swollen pasted eyelids- A and Pantothenic acid
4. Reduced resistance to infectious diseases- A, E, B2, B6, Pantothenic acid and C
5. Poor feathering- A, D, B6, H, Folic acid, PP and Pantothenic acid
6. Bone abnormalities- A, D, H, Folic acid and PP
7. Leg weaknesses or paralysis- A, D, E, B2, B6 and H
8. Egg production reduced full potential- a, D, E, K, B2, B6 and B12
9. Retarded growth- A, E, K, B1, B2, B6, B12, H, Folic acid, PP, Pantothenic acid & C
10. Hatchability reduced/ below full potential- A, D, B2, B6, B12, H, Folic acid and
pantothenic acid

Swine
1. Muscular in coordination or the other nervous signs- A, D, B6, B12 and Pantothenic
acid
2. Reduce feed intake – A, D, B1, B2, B6, B12, H, Folic acid, PP and Pantothenic acid
3. Impaired vision or blindness- A, B2, and B6
4. Scour and / or vomiting- B1, B2, B6, B12 and PP
5. Hair, skin and claw problems- A, B2, B6, B12, H, PP and Pant
6. Anemia- E, K, B6, B12, Folic acid, and pantothenic acid
7. Impaired feed conversion- B1, B6, B12, H, PP and Pantothenic acid
8. Lameness or unsteady gait- A, D, E, B2, B6, and Pantothenic acid
9. Poor reproduction- A, D, E, B1, B6, B12, H, Folic acid
10. Retarded Growth- A, D, E, B1, B6, B12, H and Folic acid

Ruminants

1. Muscular in coordination or other nervous signs- A and B1

 2. Reduced Feed Intake- A, D and PP
3. Impaired vision or Blindness- A
4. Digestive disturbances- A and B1
5. Rough hair coat- A
6. Degeneration of heart and skeletal muscle- E
7. Poor Reproduction- A, D
8. Retarded growth- A, D, and E
9. Bone deformities or swollen joints- A and D

Vitamins Modes of Action Natural occurrences

FAT SOLUBLE In Feeds In Foods
Vitamin A ( Retinol) Promotes the Only as provitamin Liver, egg- yolk,
development of A in green crops; milk, dairy
visual pigments; fish liver oil products
Indispensable for the
formation and
protection of
epithelial tissues;
improved resistance
to infections
Vitamin D Regulates the Sun- dried green Eggs, milk, dairy
(Calciferol) incorporation of Ca forage, fish liver oil products
and P into the bone
matrix and Ca
absorption from the
intestinal lumen
Vitamin E Works as a biological Green crops, cereal Leafy vegetables;
(Tocopherol) antioxidant, as a germs, milling by- some animal
detoxifying agent and products organs, milk butter
participates as a
component of the
respiratory chain;
Functions nucleic
acid metabolism and
in endocrine glans
Vitamin K ( Functions in the Green forage, liver Green vegetables;
Menadione) blood coagulation oils potatoes, fruits;(
system; acts in the tomatoes and
maturation of the strawberries
bone structure
WATER SOLUBLE
Vitamin B1 (Thiamin) Participates in the Cereal germs, Cereals, vegetables,
process of milling by- potatoes, fruits;
carbohydrates products, oil cakes, animal organs, egg-
metabolism yeast yolk, milk
Vitamin B2 Acts in the Some oil seeds, Liver, kidney, eggs,
(Riboflavin) respiratory chain as a yeast, brewer by- milk, dairy
constituent of the products, products
Flavin enzymes vegetables, fish
concerned with meal, meat and
hydrogen transfer bone meals,
skimmed milk
Vitamin B6 Active in amino acid Grains, milling by- Cereals, green
(Pyridoine) metabolism as products, oil cakes, vegetables; red
coenzyme of several yeast meat, liver, egg-
enzyme system yolk, milk
Vitamin B 12 Essential in the Does not occur in Liver, kidney, eggs
(Cyanocobalamin) reduction of one- plants, skimmed yolk
carbon compounds in milk powder, fish
the fat and protein and meat meals
metabolism
Vitamin H (Biotin) Necessary for Occurs in feeds of Vegetables, yeast,
Gluconeogenesis and vegetables and mushrooms, liver,
fatty acid synthesis animal origin, but kidney, meat, egg-
where it acts in only in partly yolk, milk
carboxylation available form
reactions
Folic Acid Acts in the one- Lucerne meal, Dark leafy
carbon metabolism extracted soybean vegetables; liver,
where it is meal; fish meal kidney, muscle,
indispensable in the milk, dairy
formation of amino products
acids and nucleic
acids
Nicotinic acid Acts as an group of Brans, dried green Liver and meat of
(Niacin) different coenzymes crops, yeasts, hoofed animals
which are related to vegetable and
the citric acid cycle animal proteins
Pantothenic acid Part of coenzyme A, Dried green crops, Cereals, legumes,
which occupies a milling by- liver, kidney, egg-
central position in the products, oil cakes, yolk, milk products
intermediary yeast
metabolism by
activating weakly
active acids
Vitamin C (Ascorbic Essential in the Beef, green plants, Potatoes, cabbage,
Acid) formation and skimmed milk lettuce (and other
maintenance of powder vegetables), citrus,
skeletal tissues tomatoes (and other
participates as an fresh fruits)
oxidation- reduction
system in cellular
oxidation processes.
Involved in defensive
mechanisms.
 Inorganic elements found in feed include the macromineral elements, which are the
essential elements required in relatively large amounts, and the micromineral elements or trace
elements, which are required in much smaller amounts. Several mineral elements present in
plants are not believed to have any function in animals, although some may prove to be required
by some animals for specific functions. Minerals, which include some of those that are
essential, may be present in some plant tissues in sufficient amounts to cause toxicity. In the
strict sense, any mineral element (in fact, any nutrient) can become toxic when ingested in
excessive amounts.
 Minerals represent the inorganic nutrients required by animals. A number of mineral
elements are found in the animals’ body. However, their mere presence in the body
does not necessarily indicate that the mineral is essential.
A nutritionally essential mineral has the following features:
 An active part of the structure of the body
 Plays an important role in some enzymes, hormones, or other such compounds
 Its removal from the diet may cause specific deficiency symptom to which could be
reversed by the addition of the mineral in question

2 GROUPS OF MINERALS

1. MACRO MINERALS – these minerals needed in large amount; requirement is

expressed as percentage of the diet.
Calcium (Ca) Required for bone formation; needed for muscle and nerve
function
Phosphorus (P) Required for bone formation and for proper energy utilization
Sodium (Na) Required for the maintenance of osmotic pressure and
Chlorine (Cl) maintenance of acid-base balance (pH)
Potassium (K)
Magnesium Required for bone formation and activation of certain
(Mg) enzymes
Sulfur (S) A component of amino acids Methionine &Cystine

2. MICRO MINERALS – these are minerals needed in small amounts; requirement is

expressed in Parts Per Million (PPM) or Parts Per Billion
Cobalt (Co) Component of the molecule of vitamin B12
Iron (Fe) Component of the hemoglobin molecule which is involved
in oxygen transport in the blood
Copper (Cu) Required in the absorption of iron from the intestine
Iodine (I) Component of Thyroxine which controls metabolic rate
Manganese (Mn) Involved in bone and cartilage formation
Molybdenum Involved in Uric Acid formation in poultry
(Mo)
Selenium (Se) Involved in proper absorption and retention of vitamin E
 Zinc (Zn) Component of Carbonic Anhydrase which is involved in the
transport of carbon dioxide from the cell to the lungs
Fluorine (F) Increases the hardness of bones and teeth
Nickel (Ni) Involved in glucose metabolism
Chromium (Cr)

For some minerals, the borderline between safety and toxicity is very small.
Toxicity dose does not necessarily mean death but can also mean greatly reduced
performance. Copper, Selenium, Fluorine are toxic at relatively low dietary levels.

The Proximate Composition

The 19th century had a significant impact on modern animal nutrition. Developments during
this period include the introduction of fundamental nutrients and the separation of feed into
protein, fat, and carbohydrate components. In this respect, proximate analysis, a combination
of analytical procedures devised more than 100 years ago by German scientists at the Weende
Experiment Station (also known as Weende analysis), paved the way for estimating the nutrient
content of feed samples. Although detailed knowledge of different analytical procedures is not
required, familiarity with different basic feed analyses will enhance learning and understanding
of animal nutrition.

Why Perform Nutrient Analysis of Feedstuffs?

Animal nutrition is the science of feed preparation (formulation) and feeding to meet the
needs of animals at different phases of growth, or life stages. Therefore, nutritionists need to
know the nutrient components of the feed or the raw materials used in ration formulation.
Nutrient analysis serves as a system to analyze the feed and the needs of the animal, enabling
producers to optimize nutrient utilization in feed and helping researchers relate to animal
performance, tackle issues of underperformance, and reduce food production costs.

Reasons for Nutrient Analyses in Feed

 Ration formulation and feeding

 Trouble shooting
 Economics

Sampling Feed for Analyses

Modern chemical methods and equipment need only a small amount of the feed (2 to 10 g)
for analyses. Therefore, sample materials collected and prepared for analyses should
represent the best reasonable estimate of the total feed fed to animals. Sample integrity during
preparation (e.g., grinding, drying), storage (e.g., temperature), and transportation should be
considered. The frequency of feed analysis depends on batches of feed made, variability of
feed sources (e.g., cultivar, location of growth), and cost of analyses. Several core samples
should be taken, combined, ground, and subsampled. Avoid taking a sample directly from
outside of a bale (use common sense)! Weather patterns should also be considered, as they
can affect the moisture content of the sample.
Samples taken for analyses should represent the entire feed, ration, bulk, bale, or load. The
bottom line is that analysis will only be as accurate as the sample collected. If a sample is
inaccurate, analysis is a waste of money.

Analytical Methods

Traditionally, feedstuffs are subjected to different protocols of laboratory analyses (wet

chemistry) for nutrient profiling. These analytical procedures are specific for a given element
(e.g., N), compound, or group of compounds. Chemical methods often employ drastic
degradation of the sample with different acids or other solvents and may not be true estimates
of an animal’s ability to utilize them efficiently. However, considering the time and cost of
other methods using live animals (e.g., explained in chapter 20) that provide more accurate
estimates, laboratory analyses are used widely to get a head start.

Proximate composition is the term usually used in the field of feed/food and means the 6
components of moisture, crude protein, ether extract, crude fiber, crude ash and nitrogen free
extracts, which are expressed as the content (%) in the feed, respectively.

The composition as the feed is classified as shown below:

The measured values of these 6 components in feed are important factors to understand
the nature and the properties of the subject feed.

Table 1. The Six Components in the feed and substances contained in them
Proximate composition Substances in
respective
composition
Moisture Water, volatile
substances
Dry matter Organic matter Crude protein Pure protein, amino
acids, non-protein
compounds
Crude fat (ether Fat, complex lipid,
extract) sterols, fatty acids,
fat-soluble dyes
 Crude fiber Cellulose,
hemicellulose,
lignin
Nitrogen-free Soluble
extracts carbohydrate,
hemicellulose,
lignin, pectin,
organic acids,
tannin, water-
soluble dyes
Inorganic matter Crude ash Pure ash, organic
residue, soil

Analysis methods for proximate composition was examined in the late 1950s mainly
by the Department of Livestock Chemistry, National Institute of Agricultural Sciences
(predecessor of Nutrition Department, National Institute of Animal Industry→ National
Institute of Livestock and Grassland Science → National Institute of Livestock and Grassland
Science, National Agriculture and Food Research Organization) and Analysis Branch, Feed
Department, Livestock Industry Bureau (predecessor of Feed Inspection Station → Fertilizer
and Feed Inspection Services → Food and Agricultural Materials Inspection Center).

At first, the methods were examined referring to Nougei-kagaku Jikkensho (Laboratory

Manual for Agricultural Chemistry) and AOACI methods, etc., and were notified as “Standards
for quantitative analysis testing of feeds” in October 1956, establishing analysis methods for
feed testing. The methods were revised several times to be current analysis methods.

Due to the amendment of the Feed Safety Law in 1976, test methods for assays were
newly defined (currently Note 3, Chapter 1 in “Official Specifications of Feeds” (Public Notice
No. 756 of Ministry of Agriculture and Forestry, 1976), which includes analysis methods for
calcium, phosphorus, water-soluble nitrogen and pepsin digestibility in addition to
conventional proximate composition.

In the early days of nutrition research, it was not uncommon to analyze the whole
animal body. Today, such practices are less feasible because equipment is not adapted to such
methods and the cost would be tremendous. It is seldom feasible today to analyze the whole
bodies even of small animals such as rats or chicks. Modern chemical methods are geared to
procedures that require small amounts of material that must be collected and prepared in a
manner that gives the best reasonable estimate of the total batch. For example, if we are
interested in the protein content of hay produced from a field, where do we begin? We certainly
cannot grind up all of the hay produced; even one bale would tax the facilities of most
laboratories. Consequently, we resort to the use of core samples taken from as many different
bales as is reasonable. Perhaps as many as 25 to 50 core samples may be taken from one stack
of bales that represents the hay from the field in question. This assumption is that each core
will correspond reasonably well to the total composition of the bale from which is came and
that, if we sample enough bales, our composite sample will be representative of the hay crop.
This is an assumption that may not always work out in practice, but it is the appropriate
statistical approach.

The core samples are brought to the laboratory, ground, and mixed well and small
subsamples are taken for analysis. For the common Kjeldahl analysis which is used for crude
protein, a typical sample size is 2g of material. A micro-Kjeldahl procedure now in use allows
the use of a sample that contains about 1mg of N, or a sample of about 100mg of the hay in
question. Thus, we may base our estimate of protein content of the total field on a very small
amount of material. Consequently. The material being analyzed must be representative if
results are to be meaningful.

Similar procedures are used for other commodities. One small sample of grain may be
used to evaluate a carload. Liquids are assumed to be more homogenous than solids, but this is
not always true and errors may creep in if care is not taken in sampling. With respect to the
beef carcass, the 9-10-11 rib cut has been shown to give a relatively accurate estimate of the
total carcass for fat, protein, water, and ash (minerals). As a result, we can obtain this cut from
one side of the carcass, remove the bone, grind it, and analyze it for the constituents of interest.

Moisture

The active ingredients from the view of feed nutrition are present in the part of dry
matter (solid matter); therefore, the level of moisture content is an important factor in both
economy and storage. In summer at high temperature and humidity in Japan, the risk of
putrefaction is predicted due to the proliferation of molds, etc., or self-digestion by enzymes in
the feed when moisture in the feed is not less than about 15 %. For that reason, the moisture
content in formula feed/mixed feed distributed in Japan is usually around 12- 13 %.

As the assay for moisture in the feed measures loss on drying by heating at normal
pressure as moisture, the result includes most of volatile substances other than H2O. Therefore,
it may be more appropriate to be referred to as volatile matter rather than moisture for accuracy.

Organic acids such as acetic acid and butyric acid in silage as well as ammonia and
flavor components in feed materials are also vaporized and thus measured as moisture. Because
the content of these in the feed is extremely low, there has hardly been a need to consider their
influence on the measured value. However, in silage, etc., with high moisture content (low
solid matter content), component contents per solid matter may be slightly affected depending
on the content of volatile acids.

When the feed is spread on sheet and left at rest, moisture absorption or release
proceeds, and under a constant relative humidity comes to equilibrium at the moisture content
corresponding to the constant relative humidity (RH) (equilibrium moisture content), which
indicates comparatively stable water content of the feed in the air-dry state. The equilibrium
moisture content is different by the kind of the feed and is affected by hygroscopic materials
such as salt if they are mixed in the feed. When the annual mean RH in Japan is presumed to
be 65 %, the water content that is at equilibrium with it is in the range of 12- 14 % for most of
feed materials.

Precautions for the procedure of moisture assay are as follows:

1. Check if the temperature display of the dryer indicates the predetermined

temperature (it is needed to check if the thermometer is normal).
2. Errors may be caused such as when the cooling time for the weighing dish is
excessively long.
3. Errors also may be caused by the location to place the weighing dish in the dryer;
therefore, it is needed to check the location in advance before use.
 4. A rough standard for the analysis value can be obtained when the standard sample
(the sample with established analysis values distributed by the Conference for Feed
Quality Improvement, etc.) is analyzed

Methods listed in the Analytical Standard of Feed for Moisture

1. Loss on drying

Weigh accurately 2-5 g of an analysis sample, put it in an aluminum weighing

dish (dried and accurately weighed in advance), dry it at 135±2 °C for 2 hours, let
it stand to cool in a desiccator, and 3 then weigh accurately to calculate the moisture
content in the sample.
However, the drying temperature should be 105±2 °C, and the drying time
should be 3 hours for fish soluble adsorption feed, molasses adsorption feed, gluten
feed and corn distillers dried grains with soluble.
Note that when it is difficult to grind the sample because of the high moisture
content, prepare the analysis sample according to 2 of Chapter 2, and then obtain
the moisture content in the sample after preliminary drying by the assay method
shown above, and calculate the moisture content in the original sample by the
following formula:

Moisture content (%) in the original sample = A + (100 – A) x B/100

where:

A is the moisture content (%) in the original sample after preliminary drying
B is the moisture content (%) in the sample after preliminary drying
This is a method in which an analysis sample is heated with a temperature-
controlled dryer and the loss is quantitated as moisture (loss on drying).

Notes and Precaution in this method

1. The definition of moisture varies depending on the analysis subject or the

analysis purpose; moisture by this analysis method mainly means water of adhesion,
and loss on drying at normal pressure is designated as moisture for the reasons such as
that most of the analysis subjects are organic matter and that it is easy to conduct the
analysis.
The temperature distribution in the temperature-
controlled dryer commonly used varies widely
depending on the location, but the range is
comparatively smaller in the temperature-controlled fan
dryer with forced air-flow. However, care should be
taken because a light sample may be blown off
depending on the location to place the weighing dish.

2. Moisture in grains tends to be changed by grinding.

In order to grind avoiding moisture, change as much as
possible, a device only for rough grinding is preferred,
such as a hand chopper (manual roller mill) shown in Figure 1. Hand chopper
Figure 1.
When a large amount of a sample is ground, the Figure 1 Hand chopper 4 procedure is
accompanied by moisture change regardless of the grinding machine used that is commercially
available. Also, care should be taken for the storage of the
sample after grinding.

A grass weighing dish can also be used, but an

aluminum weighing dish is more advantageous in that it is
less fragile and lighter, shows better thermal conductivity,
better airtightness, and is easier to handle. Put the sample in
the weighing dish, and put it in a dryer with the lid below
or at the side of it. After drying for 2 hours, cover the
container with the lid, and let it stand to cool in a
Figure 2. Aluminum weighing desiccator. (It is recommended to use cotton work gloves,
etc., because the dish is hot.)
dish
A weighing dish of the shape and size as shown in
Figure 2 is generally used. It is convenient to mark the
lid and the dish with a number (the same number).

Additionally, a weighing dish stand as shown in Figure

3 is commercially available, which is convenient
because it can be placed as is in a desiccator (Both
commercially available from Sanshin Industrial.).
Figure 3. Weighing dish stand
1. Collect and spread the sample, and weigh together
with the lid.

2. A desiccator of about 20-22 cm in the diameter of the platform is preferred. Silica gel,
calcium chloride (anhydrous), phosphorus pentoxide, or concentrated sulfuric acid, etc., can
be used as a desiccant; however, use silica gel unless otherwise specified. Silica gel is
supposed to be a common desiccant because it is easy to handle and regenerate; however, it
should be dried again to be used when the blue color of cobalt salt added as the indicator of
moisture absorption fades even if only slightly. Re-drying should be conducted at 130-140
°C for 2-3 hours.

Hygroscopicity is reduced by the adsorption of oil and fat, etc., to silica gel, and thus
care should be taken. To minimize the analytical error due to cooling, it is recommended to
keep the cooling duration in a desiccator to be constant (for 45 minutes for example), and to
always put e.g., 8 weighing dishes in a desiccator. The number of weighing dishes to be
contained in a desiccator is preferably not more than 10 because measurement errors are
likely to occur between the start and the end of weighing when a large number of weighing
dishes are contained in a desiccator.

3. For fish soluble adsorption feed, molasses adsorption feed, gluten feed and corn distillers
dried grains with soluble (DDGS), the method was modified as “drying at 105±2 °C, 3
hours” because there is a risk of vaporization or heat decomposition of volatile substances
other than moisture if the Figure 3.1-2 Aluminum weighing dish Figure 3.1-3 Weighing dish
stand 5 normal methods is applied to them.
 The assay of moisture in muciform feed such as fish soluble and molasses is usually
conducted by the method shown below:
Weigh accurately 2 g of an analysis sample, put it in an aluminum weighing dish (put
10-20 g of sea sand* and a stirrer bar in it, dry, and weigh in advance), mix the sample and
sea sand on a boiling water bath, and then dry for about 15 minutes stirring occasionally.
Then put it in a temperature-controlled dryer, dry at 105±2°C for 3 hours, let it stand to
cool in a desiccator, and then weigh, to calculate the moisture content based on the loss.

* Use sea sand (silica sand) of 350-250 µm (60-80 mesh). Wash sea sand with water,
heat in hydrochloric acid (1+1) for a few hours, wash with water until there is no acid, dry
and store in a desiccator.

Toluene distillation or loss on heating at or 100 °C for 18 hours may be used for a sample
with a high content of volatile components such as silage. For a highly viscous liquid, adsorb
it on filter paper and dry to calculate the moisture content by the loss.

2. Moisture assay methods by international standards

ISO 6496 (1996) Animal feeding stuffs - Determination of moisture and other
volatile matter content

Flow sheet of the analysis method

3. Distillation
 Heat a sample in an organic solvent immiscible with water (such as toluene),
distill water in the sample or the mixed vapor of water and the solvent, cool it and
calculate moisture in the sample based on the volume of water separated from the
solvent. This method is applicable to a thermostable sample that contains volatile
components other than water as well as fat

4. Kahl Fischer method

The method quantitates moisture in a substance utilizing the specific reaction of
Kahl Fischer reagent, which contains iodine, sulfur dioxide and pyridine, with water
under the presence of methanol, and is classified as the volumetric method and
electrometric titration method.
The method is advantageous in that water alone can be quantitated when the
sample contains volatile components other than moisture.

5. Heating furnace control method

A new type of thermogravitic analytical instrument that employs the heating
furnace control method can measure 19 samples simultaneously and can quantitate
ash content in addition to moisture. Full-automatic moisture/ash analyzer TGA701
(manufactured by LECO (US), distributed by LECO Japan)

6. Other methods
When the sample contains thermostable components, dry under constant
temperature and reduced pressure such as “60-70 °C, 26.7-33.3 kPa,” and the loss
is obtained as the amount of moisture.
Also, a moisture meter that employs infrared radiation and can be used for
measurement in the field is also used as a control analysis meter.

Crude Protein

Crude protein is defined as the value obtained by quantitating nitrogen in a sample by

the Kjeldahl method (in which nitrogen compounds in the sample is degraded by sulfuric acid
to become ammonia, sodium hydroxide is added, steam distillation is conducted under the
alkaline conditions, distilled ammonia is absorbed in acid and measured by titration) and
multiplying the result by the factor 6.25 (6.38 for milk products). Therefore, crude protein
includes ammonia, etc., that are not of protein origin.
Generally, the nitrogen content of protein is 16 % on average; thus the inverse number
of this (100/16 = 6.25) is used as the factor. However, as the factor is different between samples
(5.83 for flour; 5.95 for rice), the crude protein of some feeds is different from the pure protein
content; crude protein is measured to be excessively small in materials of milk product origin
such as casein, and excessively large in flour and soybean.

Methods listed in the Analytical Standard of Feed for Crude Protein

1. Kjeldahl method
A. Reagent Preparation

a. 0.1 mol/L sodium hydroxide standard solution

Prepare a saturated solution of sodium hydroxide, close the cap, leave at rest for not
less than 10 days, and to 50 mL of the supernatant, add boiled and cooled water to be 10 L to
prepare the 0.1 mol/L sodium hydroxide standard solution. Moreover, standardize its
concentration by the following procedure:

Weigh accurately 2-2.5 g of amidosulfuric acid (standard reagent) (dried in a desiccator

(vacuum) for 48 hours), put it in a 250-mL volumetric flask, add water to dissolve, and further
add water up to the marked line to prepare the amidosulfuric acid standard solution. Transfer
accurately 25 mL of the amidosulfuric acid standard solution into a 200-mL Erlenmeyer flask,
add a few drops of bromothymol blue test solution, titrate with the 0.1 mol/L sodium hydroxide
standard solution, and calculate the factor (f1) of the 0.1 mol/L sodium hydroxide standard
solution by the following formula:

b. 0.05 mol/L sulfuric acid standard solution

Add 28 mL of sulfuric acid to 1 L of water gradually with stirring, let it

stand to cool, and then add water to be 10 L to prepare the 0.05 mol/L sulfuric
acid standard solution. Moreover, standardize its concentration by the following
procedure:
Transfer accurately 25 mL of the 0.05 mol/L sulfuric acid standard
solution into a 200-mL Erlenmeyer flask, add a few drops of methyl red test
solution, titrate with the 0.1 mol/L sodium hydroxide standard solution,
calculate the factor (f2) of the 0.05 mol/L sulfuric acid standard solution by the
following formula:

Sample solution preparation

Weigh accurately 1-5 g of an analysis sample, put it in a Kjeldahl flask, add 9 g of

potassium sulfate and 1 g of copper sulfate (II) pentahydrate, further add 30-40 mL of sulfuric
acid, and mix by shaking. Heat it gradually, and then strongly after foaming subsides, and heat
for not less than 2 hours after the 9 solution becomes clear, and then let it stand to cool. Transfer
the solution with water into a 250-mL volumetric flask, and add water up to the marked line to
be the sample solution.

Quantification

a. Absorption by the sulfuric acid standard solution

Transfer accurately a certain amount of the sample solution into a Kjeldahl flask, and
add sodium hydroxide solution (50 w/v%) of a volume sufficient to turn the solution strongly
alkaline. Connect the flask to the steam distillation apparatus to which a receiver containing a
certain amount [19] of 0.05 mol/L sulfuric acid standard solution in advance is attached, and
distill until the distillate volume reaches about 120 mL.

Add a few drops of methyl red test solution [10] to the distillate, titrate with the 0.1 mol/L
sodium hydroxide standard solution, and calculate the nitrogen content by the following
formula. Multiply it by 6.25 (6.38 for samples of milk products or milk replacer for calves
which contain milk products not less than 50 %) to calculate the crude protein content in the
sample.

b. Absorption by boric acid solution

Put a certain amount of boric acid solution (4 w/v%) into a receiver instead of the 0.05
mol/L sulfuric acid standard solution, and distill in the same way as a (Absorption by the
sulfuric acid standard solution).
Add a few drops of bromocresol green-methyl red test solution to the distillate, titrate
with the 0.05 mol/L sulfuric acid standard solution, and calculate the nitrogen content by the
following formula. Multiply it by 6.25 (6.38 for samples of milk products or milk replacer for
calves which contain milk products not less than 50 %) to calculate the crude protein content
in the sample.

Generally, a macro-Kjeldahl nitrogen distillation apparatus by the indirect

distillation method is show in Figure 4.
Figure 4. Nitrogen distillation apparatus

Note and Precautions

a. Connect the container containing the sodium hydroxide standard solution prepared
with a soda lime tube or a bottle containing sodium hydroxide solution to avoid the
entrance of carbon dioxide in the air; however, it is desirable to standardize it once
every 2-3 months. Commercially available 0.1 mol/L sodium hydroxide solution
may as well be used.
b. Because sodium hydroxide is highly hygroscopic and is likely affected by carbonic
acid, theoretically it is difficult to obtain a solution of accurate concentration. Use
saturated solution to avoid the effect of carbonic acid (concentrated sodium
hydroxide solution contains little carbon dioxide).
About 80 g of sodium hydroxide is soluble in 74 mL of water at 20 °C, thus it
is recommended to F G E G G G About 3.5 cm 60° About 0.6 cm inner diameter
About 5 cm About 0.7 cm inner diameter About 8 cm About 5 cm B D A About
30 cm About 20 cm About 15 cm About 30 cm C 11 prepare saturated solution by
adding slightly excessive sodium hydroxide and leave at rest to collect clear
supernatant to be used (the concentration is about 20 mol/L at 20 °C).
c. Use purified water that is boiled and then cooled to remove carbon dioxide. See
JIS K 8001 “General rule for test methods of reagents.” Boil water in a flask for
15 minutes, and then shut out carbon dioxide in the air by attaching a gas washing
bottle as shown in Figure 3.2-2 containing potassium hydroxide solution (25
w/v%) or a soda lime tube, and cool. Prepare this water before use.

Figure 5. An example of cooling apparatus for water not containing carbon dioxide
d. Vacuum by suction with a vacuum pump, etc. (not more than 2.0 kPa).
e. The endpoint is where the yellow color disappears and becomes greenish blue and
the tome is maintained for not less than 30 seconds.

During the titration, use the fixed range (such as the graduation range of 10-
20mL) of burette.

f. The number 97.10 means the molecular weight of amidosulfuric acid.

g. Connect the contained containing the sulfuric acid standard solution prepared with a
bottle containing dilute sulfuric acid to avoid the entrance of ammonia gas.
h. Dissolve 0.1 g of methyl red in ethanol to be 100 mL. Filter the solution if needed.
Methyl red-methylene blue mixture test solution (dissolve 0.2 g of methyl red and
0.1 g of methylene blue respectively in ethanol (90 v/v%) to be 100 mL, and mix
them.) may as well be used. When the mixture test solution is used for titration, the
endpoint is where the red-purple color turns to blue and then to green.
i. The number 25 in the calculation formula means the volume of the 0.05 mol/L sulfuric
acid standard solution (25 mL) contained in the Erlenmeyer flask.
j. Potassium sulfate and copper sulfate are used as degradation accelerators.
Degradation accelerators for Kjeldahl degradation includes (a) copper sulfate -
potassium sulfate, (b) copper sulfate - selenium - potassium sulfate, (c) titanium
dioxide - copper sulfate - potassium sulfate, 12 and (d) mercuric - potassium sulfate,
etc. As a measure to prevent environmental pollution and in order to avoid troubles
caused such as by the influence of degradation accelerators when the same sample
solution is used for colorimetric determination of phosphorus, only copper sulfate -
potassium sulfate were employed as degradation accelerators. Copper sulfate is a
catalyst to facilitate degradation, while potassium sulfate elevates the concentration
of sulfuric acid and the boiling point as well as facilitates degradation via the
following reactions:

k. Degradation time may be further reduced by letting it stand to cool after adding
sulfuric acid and heating while gradually adding about 1 mL of hydrogen peroxide
solution (not less than 30 v/v%). Make sure to conduct degradation in a draft
chamber.
l. Care should be taken for samples with high oil content such as plant oil cake because
it may foam violently and spill out of the Kjeldahl flask when heating is strong.
When it foams strongly, stop heating and leave at rest for a while, and then heat again
with lower heat. Additionally, it is recommended to add a small amount of paraffin
to a sample that foams strongly.
m. Because it is not appropriate in some cases to consider that degradation is completed
when the solution becomes clear, it is needed to heat further.
Care should be taken so that the solution is not less than 10 mL because it is
said that there is the loss of ammonia gas when the heating temperature is too high
or the solution volume after degradation is too small.
 Crude protein is degraded by sulfuric acid and turns into the form of
(NH4)2SO4.
n. The two methods, absorption by the sulfuric acid standard solution and absorption
by boric acid solution, are listed in the Analytical Standard of Feed as the
quantification methods for crude protein.
o. This method is frequently used in fertilizer analysis, food analysis and plant analysis,
etc.
p. It is judged by the emergence of the blue color of copper oxide.
q. When 10 mL of the 0.05 mol/L sulfuric acid standard solution is used, distillation
can be conducted according to the rough standards shown below based on the crude
protein content in the sample:

r. At first ammonia is generated in a large amount; care should be taken to avoid the
loss of ammonia by controlling the amount of steam introduced.
s. One milliliter (1 mL) of the 0.1 mol/L sodium hydroxide standard solution
corresponds to 1.40 mg of nitrogen.
t. The value corresponds to the case when the degradation solution of the analysis
sample is accurately 250 mL.
u. This method is widely used in the Japanese Pharmacopoeia, JIS, and food analysis,
etc. The method titrates ammonia with a strong acid when the ammonia (NH3)
generated from an alkalinized sample solution is passed through dilute boric acid
solution and completely is dissociated as shown in the following formula:

The method is advantageous in that it is not needed to strictly define the

concentration of the boric acid solution or the amount to take the boric acid solution,
because the amount of boric acid is not directly involved in titration.
In addition, it is convenient to add methyl red - bromocresol green test solution
in the boric acid solution in advance.
Prepare by dissolving 400 g of boric acid, 100 mL of 0.1 % bromocresol green
solution in ethanol, and 70 mL of 0.1 % methyl red solution in ethanol in 10 L of
water.
Ammonia is absorbed by boric acid, which is a weak acid. Ammonia can be
sufficiently captured at the boric acid concentration of not less than 3 %; however,
the concentration is stipulated as 4 % including the margin of safety.
Additionally, care should be taken for the boric acid solution in the receiver not
to exceed 40 °C. Ammonia absorption decreases at high temperature, leading to loss.
v. Dissolve 0.15 g of bromocresol green and 0.1 g of methyl red in 180 mL of ethanol.
Add water to be 200 mL.
w. The endpoint is when the green color disappears and changes into slightly grayish
blue and then to slightly grayish red purple.
x. One milliliter (1 mL) of the 0.05 mol/L sulfuric acid standard solution corresponds
to 1.40 mg of nitrogen.
y. The value corresponds to the case when the degradation solution of the analysis
sample is accurately 250 mL.
2. Combustion method

Weigh 100-500 mg of an analysis sample, put it in a nitrogen (protein) analyzer,

and run the analyzer to obtain the response peak of nitrogen gas with a detector.
Similarly, weigh the reagent for calibration curve preparation *4 accurately, and
put it in the analyzer to obtain the response peak of nitrogen gas with a detector.
Calculate the area from the response peak obtained to prepare the calibration curve,
calculate the nitrogen [N] amount in the sample, and multiply the nitrogen [N]
amount by 6.25 (6.38 for samples of milk products or milk replacer for calves
which contain milk products not less than 50 %) to be the crude protein content in
the sample.

Requirements for the analysis instrument

1. Capable of thermolysis of a sample in oxygen gas (purity not less than 99.9
%), maintaining the temperature in the reactor at 870 °C at the minimum
2. Capable of separation of free nitrogen gas from the other combustion products
3. Equipped with the system to convert nitrogen oxide (NOx) into nitrogen gas
(N2), or capable of measuring nitrogen as NO2
4. Capable of measuring nitrogen gas with a thermal conductivity detector

1. For samples with high nitrate nitrogen content such as Sudan grass, it is
quantitated as higher crude protein, and thus measure nitrate nitrogen [N]
content separately and subtract the value.
2. All the amount of the analysis sample is screened through a net sieve of 0.5-
mm mesh.
3. Use the instrument according to the combustion method, and measure under
the conditions appropriate for the instrument.
4. Use reagents specified for the nitrogen (protein) analyzer used, such as
disodium dihydrogen ethylenediaminetetraacetate dihydrate, DL-aspartic acid,
etc.

The method is a quantification method using an automatic analyzer applying the

Dumas method, in which a sample is degraded by combustion at high temperature and
released nitrogen gas is quantitated by a thermal conductivity detector (TCD) for crude
protein in feeds. The method is advantageous in that the analysis time is reduced and
that facilities such as a draft are not needed.
The schematic diagram of the analyzer is shown in Figure 6.
 Figure 6. Schematic diagram of the analyzer by the combustion method

Notes and Precautions

1. Particle size is set as 0.5 mm because the measured value may vary in a sample that
passed a net sieve of 1-mm mesh; however, 1 mm will do in some samples.
2. The sampling amount should be adjusted according to the nitrogen content in the
sample and to the specifications of the analyzer used
3. Sampling boats to load a sample are made of quartz, ceramic, etc.
4. Currently available nitrogen (protein) analyzers include SUMIGRAPH NC-220F
(Sumika Chemical Analysis Service), Nitrogen/Protein Analyzer Type TruSpec N
(LECO, distributed by LECO Japan), Dumatherm (Gerhardt, distributed by
Gerhardt Japan), JM3000N (J-Science Lab), vario EL III full automatic element
analyzer (elementar, distributed by DKSH Japan), and FLASH 2000 CHNS-O
(Thermo scientific, distributed by AMCO).

3. Crude protein quantification by international standards

ISO 5983 (1997) Animal feeding stuffs ˗ Determination of nitrogen content and
calculation of crude protein content - Kjeldahl method Flow sheet of the analysis
method
4. Analysis with automated instruments
Recently, automated instruments have been developed to accelerate
quantification procedures applying the Kjeldahl method by degradation with
sulfuric acid.
a. SuperKjel automatic nitrogen/protein analyzer (Actac)
b. Kjeltec auto system (FOSS Tecator, distributed by Foss Japan)
c. Kjeldahl method nitrogen/protein analyzer VAP series (Gerhardt, distributed by
Gerhardt Japan) In the field of crude feed, near-infrared spectrophotometers
have been becoming popular.

Ether extract
This procedure requires that ground up samples be extracted with diethyl ether
for a period of 4 hours or more. Ether-soluble materials include quite a variety of
organic compounds, only a few of which have much nutritional significance. Those of
quantitative importance include the true fats and fatty acid esters, some of the
compound lipids, and fat-soluble vitamins or provitamins such as the carotenoids. The
primary reason for obtaining ether extract data is an attempt to isolate a fraction of
feedstuffs that has a high caloric value. Provided the ether extract is made up primarily
of fats and fatty acid esters, this may be a valid approach. If the extract contains large
percentages pf plant waxes, essential oils, resins, or similar compounds such as these
are of little value to animals.

Ash
Ash is the residue remaining after all the combustible material has been burned
off (oxidized completely) in a furnace heated to 500-600ºC. nutritionally, ash values
have little importance, although excessively high values may indicate contamination
with soil or dilution of feedstuffs with such substances as salt and limestone. In the
proximate analysis, data on ash are required to obtain other values. It should be noted
that some mineral elements, such as iodine and selenium, may be volatile and are lost
in ashing. Normally, these elements represent only very small percentages of the total,
so little error is involved.
Crude Fiber

Crude fiber is determined by boiling an ether-extracted sample in dilute acid,

then boiling it in dilute base, and filtering, drying, and burning it in furnace. The
difference in weight before and after burning is the crude fiber fraction. This is a tedious
laboratory procedure that is not highly repeatable. It is an attempt to simulate digestion
that occurs first in the gastric stomach and then in the small intestine of animals. Crude
fiber is made up primarily of plant structural carbohydrates such as cellulose and
hemicellulose but it also contains some lignin, a highly indigestible material associated
with the fibrous portion of plant tissues. For the nonruminant animal, crude fiber is of
a variable but low value; for ruminants, it is of variable value, but it is much more highly
utilized than it is by nonruminants.

Nitrogen-Free Extract (NFE)

This term is a misnomer in that no extract is involved. It is determined by

difference; that is, BFE is the difference between the original sample weight and the
sum of weights of water, ether extract, crude protein, crude fiber, and ash. It is called
N-free because ordinarily it would contain no N. NFE is made up primarily of readily
available carbohydrates, such as the sugars and starches, but it may also contain some
hemicellulose and lignin, particularly in such feedstuffs as forages. A more appropriate
analysis would be one specifically for readily available carbohydrates-one in which
starches are hydrolyzed to sugars and the mixture analyzed for all sugars present.
Nutritionally, the NFE fraction of grains is utilized to a high degree by nearly all
species, but NFE from forages and other roughages is less well utilized.

Figure 2. Flow Diagram for Proximate Analysis

 Detergent Extraction Methods

Analytical methods primarily intended for forages have been developed by Van Soest
(1982), his co-workers, and other scientists interested in this topic. Micro methods also have
been developed. Use of these methods allows plant components to be divided as follows:

1. Neutral-Detergent Extraction
Samples are boiled for 1 hour in a solution containing primarily sodium laurel
sulfate. This detergent extracts lipids, sugars, organic acids, and other water-soluble
material; pectin (usually classified as a fibrous carbohydrates); nonprotein N
compounds; soluble protein; and some of the silica and tannin. The nonsoluble
material is referred to as neutral detergent residue or more commonly, neutral
detergent fiber (NDF). The NDF contains the major cell wall compartments such as
cellulose, hemicellulose, and lignin. It may also contain minor cell wall
components, including some protein bound N, minerals, and cuticle. The soluble
material, often referred to as cell wall contents (CWC) is highly digestible by all
species, with the possible exception of the pectins and any silica tannin. The NDF
is only partially digestible by any species but can be used to a greater extent by such
animals as ruminants, which depend on microbial digestion for utilization of most
fibrous plant components.

2. Acid-Detergent Extraction
In this technique, samples are boiled for 1 hour in a solution containing cetyl
trimethylammoniium bromide in H2SO4. Components soluble in acid detergent
include primarily hemicelluloses cell wall proteins, and the residue includes
cellulose, lignin and lignified N (indigestible N), cutin, silica, and some pectins. It
is usually referred to as acid-detergent fiber (ADF).
This detergent method is often used alone, but they may be used together or the
ADF method may be substituted for the crude fiber method partly because it is more
repeatable and faster. The ADF fraction can be further extracted with sulfuric acid
to isolate lignin.

3. pH of Feedstuffs
The pH of feedstuffs is rarely used to evaluate materials except for fermented
products such as silage, cannery residues, or other similar mixtures. It should be
pointed out that pH of mineral supplements may be of importance with respect to
palatability or metabolism by the animal. With respect to silage, pH may be
determined by mixing 100g of silage with 100ml of water, expressing the juice, and
measuring with a pH meter. Good quality silages should have a pH between 3.8 and
5.0.

Specialized Analytical Methods

1. Bomb Calorimetry
The oxygen bomb calorimeter is an instrument used to determine energy values
of solids, liquids, or gases. The energy value of a given sample is determined by
burning it in a pressurized oxygen atmosphere. When the sample is burned, the heat
 produced raises
the temperature
of water
surrounding the
container in
which the
sample is
enclosed, and
the temperature
increase
provides the
basis for
calculating the energy value.

Bomb calorimetry finds extensive use for evaluating fuels such as natural gas
and coal. In nutrition, its most useful application is in determining the digestible
energy of feedstuffs or rations. The gross energy value (obtained by burning) of
feedstuffs has little or no direct application, as it is almost impossible to distinguish
between constituents that are well utilized by animals and those that are poorly
utilized.

2. Amino Acid Analysis

Chemical methods for amino acid analysis have been available for a good many
years, but it is only in the last 15 to 20 years that
semi-automated equipment has been available.
This type of equipment is capable of fractioning
protein preparations that have been hydrolyzed
into the constituent amino acids. The
preparations are placed on chromatographic
columns, and various solutions are passed
through the columns, resulting in separation and
measurement of the individual amino acids in a
relatively short time (a few hours). This type of equipment has greatly facilitated
collection of data on amino acid composition of foods and feeds as well as on
metabolism and requirements of amino acids.

3. Atomic Absorption Spectrophotometry

Atomic absorption spectrophotometric instruments have greatly facilitated
analyses for most mineral elements (cations). In the operation of these instruments,
liquid or solid materials are ashed and resuspended in liquid that
may be put
directly into the
[Link]
fluids such as
blood plasma and
urine may be used
directly into the
instrument. Body fluids such as blood plasma and urine may be used directly. The
solution passes through a flame that serves to disperse the molecules into individual
atoms. Radiation from a cathode lamp is passed through the flame, and the atoms
 absorb some of this radiation at specific wavelengths. With instruments such as this,
vast numbers of samples can be analyzed in a short time.

4. Gas-liquid Chromatography
The forerunner of the gas-liquid chromatograph (GLC) was developed to
analyze rumen volatile fatty acids. Since that time (early 50s), tremendous
improvements have occurred in this technique and in the available instrumentation.
Such instruments are capable of handling almost any compound that can be
vaporized or those that are in gas form.

The
sample to be
analyzed is
placed in the
instrument
and is moved
through a
heated chromatographic column by means of gas. This process allows the
quantitative separation of closely related chemical compounds (such as acetic and
propionic acid) quite rapidly. This process allows the quantitative separation of
closely related chemical compounds (such as acetic and propionic acid) quite
rapidly. This process requires only very small samples. In nutrition, GLCs as well
as HPLCs are useful for fatty acid analyses but are capable of handling many other
organic compounds.

5. Automated Analytical Equipment

The gradually increasing cost of labor has stimulated the development of
instrumentation designed to do a number of simultaneous repetitive analyses. Such
equipment has found widespread use in the medical field, particularly, but has
application as well in the nutrition laboratory. For example, it is possible to obtain
simultaneous data on blood serum for glucose, total lipids, cholesterol, Ca, P. Mg,
urea, and total protein as well as other compounds. This is just an example of the
type of information that may be obtained on one tissue.
Increased availability of more complete data on animals would greatly improve
our knowledge of nutrient metabolism of healthy as well as sick animals. The speed
of analysis and the fact that such equipment is highly automated have increased
greatly the volume of information that may be obtained at a given cost, even though
the equipment itself is expensive.
a. Infrared. Use of infrared light rays for feed analyses is of recent origin. Analyses
are obtained by placing a sample in a receptacle and impinging infrared light on
the sample. The reflected light goes back into the instrument and the changes
caused by the sample can be detected and related to composition of the sample
by a built-in computer. Analyses are usually restricted to lipids, protein, fiber
and moisture, although some instruments have been used for Ca, P, salt, and
occasionally other ingredients. These instruments were developed initially for
use with grains but are being used currently with other feedstuffs, including
mixed feeds and ground forages. The major advantage is time since it takes only
about 20 seconds per sample. Obviously, in the grain or feed trade, this speed is
a tremendous asset as compared to most analytical methods, which may have
 turnaround times of one to several days. By that time, the feed may be gone or
have been consumed. However, these instruments have at least two
disadvantages. One is that a range of samples must be available in order to
calibrate the machine. Consequently, samples with at least as much variation as
the test samples must be available for this purpose. In addition, calibration
samples must be available for every type of feed to be used. A second major
disadvantage is the cost. Currently, instruments may cost several thousands of
dollars. A relatively high rate of use would be required to justify such an
expense.
b. Other instrumentation. Other types if instrumentation have been developed in
recent years that may, at times, be used for nutritionally related research. Some
of the instruments or methods available include automated instruments for
measuring blood flow; blood cell counters; high-pressure liquid
chromatography, nuclear magnetic resonance (NIR), DNA synthesizers;
inductively coupled plasma emission spectrophotometers; and flow cytometers.
When available, such instrumentation and methods allow the collection of much
more data than would otherwise be possible or, in some instances, the collection
of data that could not otherwise be obtained.

Factors that Affect Composition of Feedstuff

A. Plant Composition
The chemical composition of whole plants is exceedingly diverse, being
affected greatly by stage of growth and plant species. Generally, plants have the
following relative composition: (1) moderate to low protein; (2) moderate to high
fiber; (3) seeds are high in starch and moderate to low in fiber; (4) moderate in
lipids; (5) low in minerals; (6) moderate to high in vitamins.

Note that water content of pasture grass and the whole corn plant is much
higher than for the other feeds listed.
Also the content of other components is generally lower. However, if we
expressed all components on a water-free basis (dry matter basis), then the protein
content of pasture grass
Water Protein Fat Crude Total Ash Ca P
fiber carbohydrate
Pasture grass 67.8 5.0 1.1 7.9 23.1 2.8 0.12 0.06
(young, leafy)
Corn plant, whole 75.7 2.0 0.6 5.8 20.4 1.3 0.07 0.05
Wheat straw 12.2 3.2 1.4 38.3 76.9 6.3 0.14 0.07
Alfalfa hay 8.6 15.5 1.7 28.0 65.1 9.0 1.29 0.21
Corn grain 14.6 8.9 3.9 2.1 71.3 1.3 0.02 0.27
Soybean meal 10.9 46.7 1.2 5.2 35.3 5.9 0.30 0.65
Meat meal 5.8 54.9 9.4 2.5 5.0 24.9 8.49 4.18
Table 1. Percentage Composition of selected animal feeds

would be about the same as the alfalfa hay (5.0 divided by dry matter content, or 0.321 =
15.57%). This illustrates a common practice that should be used when comparing feedstuffs,
that is, to express nutrient content on a water-free basis. It is much easier to make comparisons
in this manner.

With regard to other comparisons shown in Table 1, note that the protein content of
alfalfa hay and pasture grass is relatively high for plant material. The whole corn plant is lower
and wheat straw is much lower. On the other hand, soybean meal and meat meal are
concentrated sources of protein. Except for meat meal, none of these feeds contains much fat.
With regard to carbohydrate content, meat meal is low, and that present is largely an artifact of
the method of analysis used. Total carbohydrate data are not very meaningful except to show
that plants contain large amounts. Feeding value is generally negatively related to the fiber
content. Mineral content of feed sources is quite variable. Generally, legumes are relatively
high in Ca; soybean meal is moderate and meat meal is quite high. P content is usually high in
feeds high in protein; in this case, soybean meal is moderate and meat meal is high.

B. Composition of Animal body

The composition of the animal body tends to be relatively uniform. Typical body
composition of an adult mammal is about 60% water, 16% protein, 20% fat, and 4% mineral
matter. Carbohydrates (blood and tissue glucose, liver and muscle glycogen) usually are not
listed but amount to less than 15 of body tissue.

Table 2. Composition of the animal body

Species Components
Water Protein Fat Minerals
Calf, newborn 74 19 3 4
Steer, thin 64 19 12 5
Pig, 100 kg 49 12 36 2-3
Hen 57 21 19 3
Horse 60 18 18 4
Rabbit 59 18 8 5
Human 60 18 18 4

Differences among species in body composition are not as large as one may infer from
the values shown in Table 2. In fact, the proportions of water, protein, and ash (inorganic
minerals) in the fat-free bodies of animals are remarkably constant. A literature survey
(Clawson et al, 1991) of nearly 200 published research papers involving several thousand
animals (mammals, bids, and fish) over a wide age range revealed that the water, protein, and
ash content of the ft-free body is in a ratio of about [Link] (74-76% water 20-22% protein, and
3-5% ash) in cattle, goats, mice, rats, sheep, swine, chickens, quail, turkeys, and fish. In the
limited data shown in Table 2, age (and changing fat content) causes more differences than
species. Humans tend to vary more in fat content than most domestic or wild species. Athletes
may have less than 15% body fat, whereas sedate obese adults may have 40% or more fat. Wild
terrestrial species, except for those that hibernate, do not accumulate nearly as much fat as do
domestic species. The aquatic species (such as seals and whales) that accumulate large amounts
of subcutaneous fat do so to improve body insulation as an aid in maintaining their body
temperature above that of the environment.
 Table 3. Composition of steers at increasing body weights
Normal body Components, %*
weight, lb/kg Water Dry matter Protein Fat Ash
100/45 71.8 28.2 19.9 4.0 4.3
200/91 69.5 30.5 19.6 6.3 4.6
300/136 66.3 33.7 19.4 9.8 4.5
400/182 65.8 34.2 19.3 10.6 4.4
500/227 62.9 37.1 19.2 13.7 4.2
600/273 62.2 37.8 19.2 14.0 4.6
700/318 60.8 39.2 18.8 15.9 4.5
800/364 57.9 42.1 18.7 19.2 4.2
900/409 54.1 45.9 17.7 25.5 3.8
1000/454 53.1 46.9 17.6 25.5 3.8
1100/500 48.0 52.0 16.2 31.9 3.9
1200/545 48.6 51.4 15.7 31.1 3.7
*Analyses are shown on the basis of empty body weight (without the contents of
the gastrointestinal tract), and are the result of analyzing the entire bodies of 60
“well-fed animals.”

The changes in body composition with increasing weight, fatness, and age are
illustrated in the table. Although these data are rather old, they are based on the analysis of the
complete bodies of 60 head of cattle, something that is not common in modern laboratories.
Note that the water content of the empty body regularly decreases as the dry matter and fat
content increase. Protein and ash decrease only slightly, whereas fat content increases from 4%
in the very young calf to 31.1% in an animal weighing 545kg. If these data were expressed on
a fat-free basis, changes in composition from 45 to 545 kg would be: ash, 4.44 to 5.33%;
protein, 20.73 to 24.08%; dry matter, 25.17 to 29.415; water, 74.8 to 70.65 – all very normal
changes for cattle increasing 1200% in weight.

A more recent example of changes in empty body composition of cattle is shown in Table
4 for Holstein cattle increasing in weight from 300 to 500kg. when expressed as g/kg of weight
protein content decreased slightly, as did the Ca and P contents. Fat increased 1925 and the
energetic value of the tissues increased by 167%. The changes in protein, fat, water, and ash
content of male sheep from birth to maturity are shown in in Table 5. Note that the edible parts
(offal) contribute a significant part of the total nutrient accretion. The offal is used for animal
feed (dog and cat feed; also meat and bone meal are used for food animal diets) and other
purposes. It is clear from the data presented in Tables 2, 3, and 4 that the two major variables
in animal body composition are the concentrations of water and fat, and that these two
components vary inversely.

Table 4. Change in body composition of cattle with increasing body size

Item* Empty Body Weight, kg
300 400 500
Protein 163 157 152
Fat 299 431 573
Energy 15.6 20.6 26.1
Calcium 14.9 14.2 13.7
Phosphorus 8.1 7.8 7.5
*Expressed as g/kg or, for energy, as Mj/kg
 Table 5. Protein, water, fat and ash (inorganic elements) accretion in edible carcass and
of fat of male sheep from age one day to four years a
Ag Body Pelt- Protein Fat Water Ash
e weight freeb Carcas Offa Carcas Offal, Carcas Offa Carcas Off
, kg empty s,g lc, g s, g g s, g l, g s, g al,
body g
wt, kg
1 5.4 4.2 428 163 100 100 2145 107 136 45
day 9
13 31.9 19.6 2.382 825 2400 800 7984 422 806 21
wks 0 9
6 52.1 37.1 4410 1243 8400 2200 12932 601 1421 34
mo 6 6
1 yr 74.9 56.1 6330 1540 13000 5500 20689 695 1960 46
5 1
2 103.4 64.0 8464 2117 10700 3800 26154 903 2921 71
yrs 4 7
4 110.9 81.8 10606 2224 18000 7400 30899 880 3457 76
yrs 5 3
a
From Jenkins and Leymaster (1993)
bLive body weight minus head, feet, skin, wool, and gastrointestinal tract contents.
cIncludes inedible parts of the body, eg. Lungs, heart, liver, intestinal tract,

reproductive organs.

Mineral composition of the whole body will vary with age, fatness, and to some extent
species. Mineral content of the bones increases as the young animal matures and bone salts
replace much of the cartilage in the skeleton. For cattle, average quoted values for the whole
body are (%): Ca, 1.33; p, 0.74; K, 1.19; Na, 0.16; S, 0.15; Cl. 0.11; and Mg, 0.04. The ratio of
protein to ash (total minerals) in the fat-free dry matter of the body of a wide-array of mammals,
birds, and aquatic animals tends to be similar, but nutritional factors, level of feed intake, and
such factors as age, sex, and genetic background affect the ratio (Clawson et al., 1991). This
variation has important physiological and economic implications in the nutrition of food
animals for efficient production.

Crop Production and Animal Feeding

There have been marked improvements in crop production in the past several decades.
The development of hybrid corn and sorghums has resulted in almost universal use of hybrids
in all areas of intensive production. At the same time, important crops such as corn (maize) and
soy beans have been modified so that they are more adaptable to a wider range of environments.
The so-called “green revolution,” which resulted in the development of high-yielding varieties
of rice and wheat and their greater use on a worldwide basis, has allowed substantial increases
in food grain production. In addition, widespread use of higher level of fertilizer, pesticides,
herbicides, and other chemicals has added to the amount of food and feed that has been
produced. New developments in molecular biology have created avenues for genetic
engineering of plants for disease and pest resistance and for changing their chemical
composition to accommodate desired changes in nutrient content of food for humans and
animals (National Research Council, 1988).
 Currently some counties such as India, Malaysia, and Indonesia, which were net
importers of food grains for some time, are exporting surplus rice. At the same time there has
been an increased consumption of animal products in many of the Asian countries. Although
percentage increases in production of some cereal grains are probably higher than those for
animals, animal productivity has been improved considerably in the past three to four decades.
Production of milk, meat, and eggs is markedly higher on a per animal basis, resulting in more
efficient use of feed, labor, land, and capital. Inputs of energy and protein for production of
milk, beef, swine, and poultry products and the percentage of return for each commodity are
summarized in Table 6. Aquaculture is becoming more important in a number of areas; where
water is available, it is more efficient to produce fish than meats from our typical warm-blooded
animals.

Table 6. Inputs and returns of animal production, b

Product Total energy and protein Human edible energy and protein
Energy Protein Energy Protein
Input, Return, Input, Return, Input, Return, Input, Return,
mcal % kg % mcal % kg %
Milk 19960 23.1 702 28.8 4555 101.1 111.5 181.4
Beef 20560 5.2 823 5.3 1869 57.1 39.9 108.8
Swine 1471 23.2 66 37.8 588 58.0 29.0 86.0
Poultry 23.2 15.0 1.2 30.0 11.2 31.0 0.48 75.0
a Data from Bywater and Baldwin (1980)
b Inputs are calculated as digestible energy and digestible protein and include cost of

maintaining breeding herds and flocks.

Even with the many marked improvements in crop and animal production, there is
much concern that the population growth may outrun the world’s capacity to produce food and
feed because of limited arable land, usable water, and energy. Water for irrigation is in short
supply in many areas. In some instances, where groundwater has been used for irrigation the
water level has been dropping, resulting in greater costs to get it to the surface. Increased energy
costs, unless compensated for by comparable increases in product prices, will make it less
feasible to use groundwater for irrigation. Likewise, increased prices for natural gas and other
petroleum products directly affect fertilizer costs because some manufacturing processes use
natural gas as a primary ingredient.
Many people feed the rising demand for food and feed can be met by continual
technological developments, improvements in marketing, and reduction in wastage. However,
the critical shortage of water predicted by some individuals may, in itself, become a major
constraint on food production.
Animal products seem certain to have a major role in meeting increased demands in
the future, although it is to be expected that animals will be fed less and less of the edible plant
materials that are used in feeds currently, particularly in some of the developed countries,
particularly in some of the developed countries. One reason for predicting less use of animal
products in human diets in the future is that it is less efficient to pass food (edible for humans)
through an animal and then feed the meat, eggs, or milk to a human. Furthermore, grains sold
for human food bring a higher price than animal feeders can usually afford. These statements
apply to items that can be considered edible by humans but not to many of the feed ingredients
consumed by domestic or wild animals. Most of the ingredients in farm animal diets can be
made up of materials that are not edible for humans. In fact, in most areas of the world, the
milk and meat produced by cattle, sheep, buffaloes, and goats is derived directly from grazing
land not in cultivation and from crop residues, milling byproducts, or wastes that normally
never get into the food chain. On the other hand, a considerable amount of feed fed to animals
in some countries is directly competitive for human use. It has been estimated that feed fed to
pets in the United States could feed some 40 million people, although some pet food ingredients
(animal offal of various types such as lungs, condemned livers, etc) generally are not
considered to be edible in the US.
References/Additional Resources/Readings

Chahal U.S., Niranjan P.S., & Kumar, S. (2008) Handbook of General Animal Nutrition;
Department of Animal Nutrition, College of Veterinary Science & Animal Husbandr, Narendra
Deva University of Agriculture & Technology Kumrganj, Faizabad- 224-229 (U.P.), India;
International Book Distributing Co.

McDonald P., R.A. Edwards., J. F. D. Greenhalgh, C. A. Morgan, L. A. Sinclair, R. G.

Wilkinson. Animals Nutrition 7th edition. Pearson, Prentice Hall, Harlow, England. 2010.

Pond, W. G. D. C. Church, K. R. Pond. Basic Animal Nutrition and Feeding 4 th edition.

John Wiley and Sons, Ney York. 2004.
The Philippine Recommends for Livestock Feed Formulation, Philippine Recommends Series
No.64-A, Philippine Council for Agriculture, Aquatic and Natural Resources Research
Development, Department of Science and Technology, Los Baños, Laguna, Philippines

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