eChapters

Introduction

As per statement of Dr. Henneberg of Weende Experiment Station (Göttingen, Germany) the carbohydrates are divided into two groups; crude fiber and nitrogen free extract. The nitrogen free extract, which  comprises the sugars, starch and a large part of the material classed as hemicellulose, is determined by difference. It is represented by the figure obtained when the sum of the water, ash, protein, fat and crude fiber of a feed is subtracted from 100. Since the figure is determined by difference instead of directly, it includes the cumulative errors of the other determinations and thus is not an exact value.

Introduction

Wet ashing is suitable for the determination of Ca, Cu, Fe, Mg, Mn, K, Na, Se and Zn in biological samples and may be applicable to the determination of other elements as well.

The organic matter of the sample is oxidized with concentrated nitric acid and perchloric acids. The acids are partially removed by volatilization and the solube mineral constituents remain dissolved in nitric acid. Any silica present is dehydrated and made insoluble.

Introduction

Wet ashing is suitable for the determination of Ca, Cu, Fe, Mg, Mn, K, Na, Se and Zn in biological samples and may be applicable to the determination of other elements as well.

The organic matter of the sample is oxidized with concentrated nitric acid and perchloric acids. The acids are partially removed by volatilization and the solube mineral constituents remain dissolved in nitric acid. Any silica present is dehydrated and made insoluble.

Definition
The residue of incineration at 550-600ºC is the crude ash.

Reagent
Dilute hydrochloric acid -approximately 5 N prepared from concentrated hydrochloric acid. Dissolve 445 ml conc. hydrochloric acid (about 36 percent HCl) in one litre distilled water. 

Procedure for Total Ash Estimation
Take a vitreosil crucible in oven at 100ºC for six hours, remove and keep it in a desiccator and weigh up to constant weight. About 5-10 g of sample is taken in the weighted vitreosil crucible. The material in the crucible is charred on a low flame and the crucible is then kept in muffle furnace and temperature is allowed to raise to 600ºC and kept constant for two hours. It is removed on cooling and kept in a desiccator  and weight of crucible is taken.

1. Ammonia Nitrogen by Distillation Method
Method I (For Animal Feeds)
Reagents
    1.    Mangnesium oxide (carbonate free)
    2.    Standard sodium hydroxide 0.25 N
    3.    Standard sulphuric acid 0.5 N
    4.    Methyl red indicator

Weigh accurately 2 to 4 g  of the prepared sample. Shake it with water and filter. Wash the residue thoroughly with water. Transfer the filtrate to the distillation flask and dilute to about 200 ml with water. Add about 5 g of the magnesium oxide, Connect the flask to the condenser by means of the connecting bulb tube and distill about 100 ml of liquid into the receiver containing standard sulphuric acid and methyl red indicator solution, Titrate the contents of the receiver with the standard sodium hydroxide solution. Carry out a blank determination using all reagents in the same quantities but without the material to be tested.

True protein nitrogen is also termed a Albuminoid nitrogen in animal feeds and fodders and human foods. If material (Such as seeds, seed residue or oil cake) is rich in alkaline phosphates, 1-2 ml of 10 percent solution of potash alum is added to decompose them. If this is not done Cu3 (PO4) and free alkali may be formed and the protein-copper precipitate may partially dissolve in the alkaline liquid.

Principle

The nitrogen of protein and other compounds is transformed into ammonium sulphate by acid digestion with boiling sulphuric acid. the acid digest is cooled, dilute with water, and made strongly basic with sodium hydroxide. The released ammonia is distilled into a boric acid solution or standard sulphuric acid solution. When boric acid is used to collect ammonia then is the titrated with standard sulphuric acid or standard hydrochloric acid. When standard sulphuric acid is used to collect ammonia then it is titrated with standard sodium hydroxide solution.

Apparatus and Reagents

    1.    Soxhlet extraction asembly. This consists of 3 parts (a) condenser at the top, (b) the soxhlet or extractor in the middle, and (c) the receiver flask at the bottom. The 3 parts are assembled by means of their grounds glass interchangeable joints (Fig. 1).

    2.    Extraction thimble.

    3.    Petroleum ether (B.P. 40ºC to 60ºC)

    4.    Constant temperatue bath or six heater hot plate with temperature controlling device.

Calorimetry

The process of measuring  the amount of heat generated by the combustion of a substance is known as calorimetry. The first successful calorimeter using oxygen under pressure for the combustion of substance in a closed vessel was devised by Berthelot in 1881. Subsequent development by Mahler in 1992, Atwater in 1899, and Parr in 1912, served to improve and to reduce the cost of the original Berthelot apparatus by variations in mechanical details.

The dry matter content of feed samples and other materials is expressed on three aspects as fed, partially dry, and dry.

As fed refers to the feed as it is consumed by the animal, the term as collected is used for materials which are not usually fed to the animal, that is, urine, faeces, etc. If the analysis on a sample is affected by partial drying the analyses are made on the as fed or as collected sample. Similar terms: Air dry, that is hay: as received: fresh; green and wet.

Partially dry, reports to a sample of “as fed” or “as collected” material that has been dried in an oven (usually with forced air) at a temperature usually about 60ºC of freeze-dried and has been equilibrated with the air; the sample after these processes would usually contain more than 88 percent dry matter (12 percent moisture); some materials are prepared in the way so that they may be sampled, chemically analysed and stored. This analysis is referred to as “partial dry matter” percent of “as fed” or “as collected” sample. The partially dry sample must be analysed for dry matter (determined in an oven at 105ºC) to correct subsequent chemical analyses of the samples to a “dry” basis. This analysis is referred to as “dry , percent of partial dry sample similar term - “Air dry” is also used.

Dry matter is generally defined as the constant  weight a sample  attains when heated at 100ºC (occasionally 150ºC). This definition , suitable for inert materials such as sand, has three main lacunae when applied to biological materials.

    1.    In these materials, water is present in various states ranging from extraneous moisture  as rain or dew, through water present in cell sap, water present in various physico-chemical and chemical combinations.

    2.    Biological materials contain active respiratory enzymic systems which continue to function during the early stages of drying; in fact, as the material heats up, activity will be enhanced until it is stopped either  by denaturation of the enzymes or by desiccation. Besides altering the chemical composition of the material, such activity will  lead to loss of dry matter.

    3.    Most biological materials contain organic  compounds which are volatile at 100ºC and which are, therefore, lost on drying. This leads to an under-estimation of dry matter content, for example, in the case of faeces, silage and molasses.

A biological sample received in the laboratory should be processed as below:

Fresh Green Sample: During metabolism trial period it is always advisable to take a sample of fresh green fodder daily so as to  avoid chemical composition variation from place-to-place in a field. Take about two and half kilo sample from the bulk, cut the whole plant including the stem and stalks with a pair of scissors into small pieces and mix thoroughly. Weigh about 100g accurately in the aluminium tray 23156cm approximately for estimation of moisture. It is always recommended to weigh the sample immediately in the laboratory so as to avoid the losses of moisture.

Chemical changes occur during oven-drying because of the direct effect of high temperature, or of enzymic or bacterial changes during the early stages of drying, or of loss of volatile consitituents. Feeds containing molasses should be ground in a mortar.

Dried materials should be ground to pass 1 mm sieve. Samples expected to moisture during the treatments are usually predried. If wet samples are used directly in the assay, sufficient large quantity has to weighted out. In the first stage, roughages about 250-1000g are used, somewhat dependent upon homogeneity and moisture content of the wet material. These samples are weighted into trays and dried in draft oven at 60-70ºC. The dry samples are then again weighed, determined the moisture content. The trays are then left to equilibrate in air at room temperature for two days. before a second reweighing of the trays with samples. Then the sample is milled and filled into airtight containers and labelled as dry samples.

General Views about Weende’s System of Analysis

A team of scientists under the leardship of Henneberg and Stohmann of (University of Göttingen, Germany) developed proximate principles analysis system in the sixties of last century at Weende’s Experimental Station (Germany), located at the University of Göttingen. Main components of different fractions in the proximate analysis of foods are moisture, ash, crude protein, ether extract, crude fiber, and nitrogen-free-extract. These workers emphasized that carbohydrates could be grouped into: (1) the starches and the sugars, and (2) coarse fibrous fractions. Later on an insoluble residue after boiling the fat free food sample first with dilute acid and then with dilute alkali was obtained based on the acid stomach digestion and the subsequent alkaline intestinal digestion of consumed food. At the end of digestion insoluble organic residue was denoted as crude fiber.

The Metric System

At present metric system is followed to calculate final results in analysis work. This system is quite simple in nature as well as in principle. The units of the metric system are: (1) the  metre (linear measure), (2) the litre (measure of capacity), and (3) the gram (measure of mass.)

One litre is the volume of pure water at 4ºC and 760 mm pressure which weigh 1 kilogram (kg). One litre equals 1000.027 cubic centimetres (Cm3) for most work, the units “millilitre”, “cubic centimetre” and used interchangeably.

At present there are three different temperature scales in common use. These are: the Fahrenheit (F), used chiefly in English-speaking countries. the centigrade (C), used universally in scientific work and the Absolute (T) or Kelvin (K) scale, differing from the centigrade only in the position of the zero point, its divisions being of the same size. its zero point is -273ºC. The kelvin scale is used especially in dealing with very high or very low temperature and in computations involving the laws of gases. The following formulae can be used for converting one scale to another:

Normal Solution in Analytical Chemistry

A normal solution is that one that contains one equivalent weight expressed  in grams (one gram equivalent weight) to be dissolved substance per litre of solution, or one gram-milliequivalent weight per millilitre.

In the case of acid a 1N solution contains 1.008 gram of replaceable hydrogen per litre of solution. A 1N solution of a base is one that contains 17.008 of hydroxyl per litre. A 1N solution of a precipitating agent contains a weight of precipitating ion equivalent to 1.008 hydrogen. The quantity of pure reagent necessary per litre of 1N solution of a precipitating solution is calculated by dividing the gram molecular weight by the valency of the precipitating ion. In case of normal solution of oxidising and reducing agents, the amount is calculated by dividing the gram molecular weight by the total valency change in the ion concerned.

Appendix - 1

Differential Diagnosis of Poisons based on Clinical Signs

Chemical warfare involves using the toxic properties of chemical substances to kill, injure or incapacitate an enemy.

Chemical warfare is different from the use of conventional weapons or nuclear weapons because the destructive effects of chemical weapons are not primarily due to any explosive force. The offensive use of living organisms (such as anthrax) is considered to be biological warfare rather than chemical warfare; the use of nonliving toxic products produced by living organisms (e.g., toxins such as botulinum toxin, ricin, or saxitoxin) is considered chemical warfare under the provisions of the Chemical Weapons Convention. Under this Convention, any toxic chemical, regardless of its origin, is considered as a chemical weapon unless it is used for purposes that are not prohibited (an important legal definition, known as the General Purpose Criterion)

Definition 

Radiation poisoning, also called “radiation sickness” or a “creeping dose” is a form of damage to organ tissue due to excessive exposure to  radiation. The term is generally used to refer to acute problems caused by a large dosage of radiation in a short period, though this also has occurred with long term exposure to low level radiation. Many of the symptoms of radiation poisoning occur as ionizing radiation interferes with cell division.

The clinical name for “radiation sickness” is acute radiation syndrome . A short exposure can result in acute radiation syndrome; chronic radiation syndrome requires a prolonged high level of exposure. A chronic radiation syndrome does exist but is very uncommon; 

Pollution is the introduction of contaminants into an environment that causes instability, disorder, harm or discomfort to the physical systems or living organisms they are in.  
Pollution can take the form of chemical substances, or energy, such as noise, heat, or light energy.

Pollutants, the elements of pollution, can be foreign substances or energies, or naturally occurring; when naturally occurring, they are considered contaminants when they exceed natural levels. Sometimes the term pollution is extended to include any substance when it occurs at such unnaturally high concentration within a system that it endangers the stability of that system. For example, water is innocuous and essential for life, and yet at very high concentration, it could be considered a pollutant.

Introduction 

A toxin (Greek: ôïîéêüí, toxikon, lit. (poison) for use on arrows) is a poisonous substance produced by living cells or organisms that is active at very low concentrations. Toxins can be small molecules, peptides, or proteins and are capable of causing disease on contact or absorption with body tissues by interacting with biological macromolecules such as enzymes or cellular receptors. Toxins vary greatly in their severity, ranging from usually minor and acute (as in a bee sting) to almost immediately deadly (as in botulinum toxin)

A toxin is simply a poison that can affect the body by internal or external means. A toxin can be a chemical which occurs naturally or in synthetic form. More than 120,000 human-made chemicals have been introduced into the environment, in one form or another, and this number continues to grow each year at a phenomenal rate. At the same time, microbial toxins, being influenced by the vast numbers of chemicals, are mutating beyond belief.

Introduction

The word mycotoxin is derived from myco, meaning mold and toxin, a poison. A great number of mold metabolites have been identified as mycotoxins. Thus mycotoxins are secondary fungal metabolites (i.e., metabolites not essential to the normal growth and reproduction of the fungus) which cause pathologic, physiologic, and/or biochemical lesions in other species (may include animals, plants, other microbes, etc.).

Mycotoxicosis - Poisoning of a biological system by a mycotoxin.

Mycotoxins  are  defined as toxic metabolites released by moulds under certain conditions conducive for their growth. Acute or chronic toxicoses can result from exposure to feed or bedding contaminated with toxins that may be produced during growth of various saprophytic or phytopathogenic fungi or molds on cereals, hay, straw, pastures, or any other fodder. A few principles characterize mycotoxic diseases:

Introduction 

In the thirst of modernization and industrialization man has contributed pollution to the life and ecology of plants, animals and microbes. Increased demand for food and fiber has lead to the chemicalization of agriculture we have reached to such a stage that modern agriculture is dependent on high yielding varieties, which can only be grown under the influence of fertilizers and pesticides. Pesticides are the man made chemicals which are being used to produce enough cheap food. In India, 90,000 MT of technical grade pesticides are used annually to control pests and plant diseases. The pesticides are classified as insecticides, fungicides, weedicides, herbicides, nematodicides and rodenticides; of which insecticides constitutes 77% of the total pesticides used in different agricultural and animal husbandry practices and in public health operations.

Introduction

Plants are the main source of nutrition to farm animals for their maintenance and production.  But, under certain conditions, plants intoxicate farm animal life due to the presence of some deleterious substances called phytotoxins. Such plants include a variety of fodder plants (Jowar, wheat straw, Lucerne), grasses (Millet grass), ornamental or fencing plants (Crotons, Nerium sps) and weeds.

(Tribulis) in the farm premises.
Generally, farm animals avoid such plants and weeds due to their unpleasant taste and offensive odor but during fodder scarcity caused by drought, floods or overgrazing, animals consume these plants. In addition, animals introduced into new areas, stall fed animals also sometimes lose their sense of discrimination and ingest such plants containing phytotoxins.

Nitrate – Nitrite Poisoning
Sources

Accidental ingestion/grazing of
Plants which are Nitrate accumulators

Arsenic
General Information

    1.    Exists in elemental form (-3), trivalent As+3 and pentavalent As+5 oxidation states
    2.    Arsenic is an essential element for rats, chicks, mini-pigs and goats
    3.    The second most important pollutant for livestock. 

Source

Inorganic form is used in paints.
Pasture near smelters may be contaminated. 
Industrial effluents released into nearby water body.
Insecticides (arsenic trioxide, lead arsenate).
 

Definitions
 
    •    Toxicology: Knowledge (or science) of poisons, including their chemical properties, identification, biological effects, and the treatment of conditions they cause.

    •    Poison: Any solid, liquid, or gas that, when introduced into or applied to the body, can interfere with the life processes of cells of the organism - by its own inherent chemical properties - without acting mechanically and regardless of temperature.

    •    Poison = Toxicant: The term poison is now used less often. Reason: Historical application of the term (malicious) vs. a compound used as a feed additive, agricultural, or other chemical - manufactured to fulfill a perceived human need.

Toxicology is defined as the study of chemical or physical agents which interact with biologic systems to produce a response in the organisms. Toxicity is the relative ability of a substance to cause injury to biologic tissue. Given the broad range of toxicities any substance might eventually invoke in an organism, it is easy to understand the wisdom of Paracelsus (1493-1541) when he said, “All substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy”.

Toxicology is the study of the harmful effects of chemical compounds on biologic systems, including their properties, actions, and effects. The toxic agent is referred to as a toxicant or poison. The term toxin refers to poisons produced by a biologic source (eg, venoms, plant toxins); the redundant term biotoxin is occasionally used. Toxicosis, poisoning, and intoxication are synonymous terms for the disease produced by a toxicant. Toxicity (sometimes incorrectly used instead of poisoning) refers to the amount of a toxicant necessary to produce a detrimental effect. Hazard describes the likelihood of poisoning under conditions of use.

The undesirable constituents includes the toxicants and other adulterants in the feed which are not suppose to be there in the feed and presence of these substances either make the nutrients of feed less utilizable or even the feed become toxic for consumption by animals. Hence it is very essential to find out the presence and also the amount of that substances on which basis decision may be made that the particular feed stuff or ingredient is safe to be consume by the livestock or that has to be rejected. The common undesirable constituents of the feeds are given below in detail:

Complete feed system is a system of ruminants feeding in which roughage and concentrate mixture are mixed homogenously in a predetermined ratio and processed in to pellet, briquette or block form to provide the animals a nutritionally balanced diet. Complete feed system is advantageous over conventional feed system as it reduces the cost of labour, transportation and storage of feed, besides ensuring uniform supply of nutrients and maintaining the optimum rumen environment due to the constant dietary roughage: concentrate ratio, which also avoids the selective feeding.

The requirements for a given nutrient is the minimum quantity of that nutrient that will maintain normal growth and reproduction, when all other nutrients are supplied in adequate quantities and at the same time, prevent the development of symptoms of nutritional deficiency.
 
The accurate way to express the nutrient requirements is in amount per day per animal. Poultry are fed in groups and hence, it is not practicable to express the requirements in amounts per day per animal. So, they are expressed in relation to energy content of the diet as poultry eats within limits to satisfy their energy needs. However, the estimation of energy content of the diet is not possible on routine basis and the calculated values may very much from estimated values. Because of these difficulties, the nutrient requirements of poultry are expressed on unit weight basis. 

Pigs are highly prolific among meat producing livestock and also excellent to all other type of farm animals in the efficiency of converting feed into high quality pork. The Nutrient requirements expressed for the mono-gastric animals like poultry and pigs as percent of the diet or the amount per kg of the diet, since they are generally fed in groups rather than individual feeding practiced in case of cattle and buffaloes (where absolute requirements are given). The latest requirements available are NRC nutrient requirements for swine, 1998 (10th revised edition), BIS, 1986 specifications for pig feeds and ICAR, 1998 for growing and finishing swine. Similarly ARC requirements are also available. NRC requirements are on 90% DM basis and are for breeds like Large White Yorkshire, Landrace, Berkshire etc., while BIS specifications are on 100% DM basis and for indigenous pigs, improved pigs and crossbred pigs.

In the cases of drought and flood when the feed resources made unavailable, creates the problem of animals feeding. In those cases unconventional methods and materials will have to be used to fulfil the hunger and nutrient requirements of animals to sustain them. 

Traditional methods of feeding
    1.    Use of by-products of commercial crops such as oil seed crops, grams etc. as well as drought resistant vegetation in combination with NPN source and molasses (as source of energy) for meeting the immediate nutritional requirements.

    2.     Certain varieties of tree leaves and extractions of inedible oil seeds can be used to meet the essential requirements of intact protein in ruminants. This proved to be successful during draught in many parts of India.
 
    3.    Large scale feeding of bagasse and molasses in combination with urea and mineral supplements may be adopted without any toxic effects. Some of the daily ration schedule that has been successfully tried is given below:

Poor quality roughages includes the wheat straw, commonly fed to ruminants but unable to provide any DCP. Urea ammoniation method, not only improves the nutritive value of straw in terms of DCP and TDN, but it also improves the dry matter intake of animal by improving its palatability. In this method of urea ammoniation, one quintal of wheat straw is treated with 4 kg fertilizer grade urea (4%) by dissolving in 40 litre of water (moisture 40%). The dissolved urea is sprayed over several layers of wheat straw and mixed properly and then kept under airtight condition by covering it with polythene sheet for a period of 4 weeks. With the presence of enzyme urease the urea get broken down in to ammonia and carbon di oxide. This ammonia on one hand improves the nitrogen content of the straw and on another hand alkalinity (by ammonia and by ammonium hydroxide) reacts on the lingo-cellulosic bonds to loosen their strength. So by increasing the nitrogen content of the straw it increase the nutritive value of the straw in terms of increasing the DCP value of the straw and on another hand by reducing the bonds of lingo-cellulose it increase the digestibility of crude fiber and hence it also increase the energy (TDN) content of the straw.

Goats are valued as economical milk producer and main source for meat for a large section of population. The dry matter intake varies from 3% (Meat goat) to 4-6% (Dairy goat) in different breeds of India and it is higher than that of larger farm animals.

1. Nutrient requirements for maintenance

Adult body weight of goats may vary from 15 to 45 kg in different breeds. There are also does and bucks that weight up to 60kg. Nutrient requirements for maintenance of adult goats are, therefore given for body weights ranging from 15 to 60 kg. On an average for maintenance 76 g DM is required for each kg of metabolic body weight and similarly the requirements for DCP and TDN is 3 and 30 g, respectively.

2. Nutrient requirements for pregnant does

Since adult body weight as indicated already varies from 15 to 60 kg, the requirements for pregnant does are also given for body weights ranging from 15 to 60 kg.

Indian breeds of sheep have much lower adult weight (30-40kg) and growth rate in comparison to many foreign sheep breeds (70kg). Hence the requirements are lesser then foreign breeds.

1. Nutrient requirements for maintenance
On the basis of several balance studies and digestibility trials the requirements for Indian sheep breeds has been developed and calculations are made on the basis of 2.73 g of DCP per kg of metabolic body weight and TDN and ME requirements are 27.3 g and 98 Kcal per kg of metabolic body weight respectively. The requirements for different body weight are given in the table below:

The formulation of ration for various categories of livestock depends upon the nutrient requirements for a particular animal or group of animals and the feeds and fodders available for the purpose. Livestock feeds can be grouped into two major categories – Roughages and concentrates, on the basis of bulkiness and chemical composition and combination of both is essential for a balanced ration.

Roughages are the bulky feeds having crude fiber more than 18% and TDN less than 60% while, Concentrates are feed or feed mixture having crude fiber less than 18% and TDN more than 60%. They are rich in either energy or protein and accordingly are called as energy concentrates (when CP is less than 18%) or protein concentrates (when CP value exceeds 18%). Various energy concentrates are grains and seeds (maize, barley, sorghum etc.), milling byproducts (brans and chunis), roots (tapioca tubers, turnip, potatoes) and molasses.

Nutrient requirements of the animals in terms of protein, energy, minerals and vitamins are the basis for scientific feeding. The total requirement of an animal includes two components (i) maintenance (ii) production. While maintenance requirement varies with body size of the animal, the requirement for production depends upon its physiological state and production levels.

Such recommendations are based on the results of a large number of feeding experiments, conducted throughout the world. Feeding standards developed by Nation Research Council (NRC) of USA and Agricultural Research Council (ARC) of UK, for livestock and poultry are considered to be the best general guides for computation of ration. However, limited investigations carried out in this country suggest that adoption of foreign standards always leaves a fair margin of safety so far as the feeding of Indian species are concerned. This owes mainly due to differences in size, breed, nutritional values of feeding stuffs and other practices in the agro-climatic conditions of this tropical country. Some efforts have been made by India scientists in this area.

When we are having two or more than two feed ingredients and by using them we have to prepare a mixture of desired nutrient composition then we will require to use Pearson’s square method. Use of pearson’s square is a simple procedure that allows us to mix two feed stuffs with different nutrients concentrations and come up with a mixture of the desired total concentration. For the pearson’s square to work, the desired diet’ nutrient concentration must be between the nutrient concentrations of the two feed stuffs. Suppose we have a protein source such as ground nut cake (GNC) with 40.9% crude protein (CP) and maize with 8.8% CP, and we need a mixture of the two that will have 18% CP. AS shown in the illustration, we compare the CP percentage of each feed on the left with the desired percentage in the middle of the square. The lesser value is subtracted from the greater value and the answer, in parts of a mixture rather then in percentage, is recorded diagonally, but read horizontally.

It is very essential to know the nutritive value of common feedstuffs used in the diet of ruminants. On the basis of these values the nutrient requirements of ruminants may be fulfilled for different body functions like maintenance, growth, lactation, wool production or others. The nutritive value of various common feedstuffs used in the diet of ruminants is being presented in the table below:

The nutritive value of feedstuffs is generally expressed in terms of protein and energy value. The protein and energy value usually referred in ruminants are DCP (Digestible crude protein) and TDN (Total digestible nutrients), because the feeds used in the diet of ruminants have a vide variability in their digestibility, hence after removal of the variable fraction the requirement of nutrients is expressed. Starch Equivalent (SE) is also the measure of energy but its usefulness is limited to certain extent, due to involvement of tedious and expensive process.

Digestion trial requires feaces collection only but when we have to go for balance studies we have to collect urine also (in addition to the nutrient losses take place through milk in lactating animals) to find out the nutrient losses takes place through urine. As we have discussed in digestion trial also, male animals are usually preferred to perform these types of studies to obtain contamination free urine and feaces to find out clear picture.

Balance studies are usually performed to find out the balance of nitrogen (Nitrogen retention, NR) and minerals (Ca, P etc.) as urine is rich in these nutrients and hence for their balance we have to find out the losses takes place through urine.

Proximate analysis of the feeds and fodders does not give any idea about their nutritive value, because availability of nutrients varies according to the feed stuffs. The major loss of nutrients take place in body after ingestion of feed is through feces. Hence, it is very essential to know the actual amount of feed digested by the animals. Losses which take place after digestion are more or less comparable in different feedstuffs.

Digestibility
Digestibility of a feed can be defined as that proportion of feed which is not being excreted in the faeces and hence assumed to be absorbed by the animal. It is expressed as percentage (% digestibility or digestibility coefficient).

Silage is the green material produced by controlled anaerobic fermentation of the green succulent fodder crop retaining its moisture content. The process of preparing silage is known as Ensiling and the pit in which silage is prepared is called silo.

Crops suitable for silage making
The fundamental behind the conservation of fodder as silage is the acidic condition, which is produced by the anaerobic fermentation of carbohydrates rich fodder. Thus the necessity of the fodder crop being used for silage preparation is the presence of high amount of fermentable carbohydrates and the natural carbohydrate rich crops are the cereal crops like maize, jowar or bajra and grasses or their combination in 3:1 with leguminous crops. Otherwise high moisture leguminous crop after wilting in combination of molasses may be 
used.

To overcome the limitation of proximate analysis P.J. Van Soest in 1963 developed a method which makes use of the concept that the dry matter of plant origin consists of two principal parts:
    1.    Cell contents 
    2.    Cell wall contents

The method is efficient to take care of the defects in the principle of estimating crude fiber and NFE by proximate analysis. Van Soest has partitioned the carbohydrates into various fractions by a system of analysis using detergents. Cell contents are soluble in neutral detergent includes lipids, sugars, organic acids, starch, soluble proteins, NPN compounds and other water soluble materials, where as cell wall (fiber) is insoluble in neutral detergent but it is soluble in acid detergent includes hemicelluloses, cellulose, lignin, lignified nitrogen compounds, heat damages proteins, keratins and silica. This method of partitioning carbohydrate is essential to be expressed in case of ruminants, due to their capacities for making use of different fractions of crude fibre especially the cellulose and hemicellulose, while the simple stomached animals can rarely do so. 

Principle

Phosphorus is the chief acid forming mineral constituent of plant and feeds. Phosphorus present in the ash solution (Mineral extract) is precipitated as ammonium phosphomolybedate. The precipitate is washed with dilute potassium nitrate till free from acid and dissolved in a known excess of standard sodium hydroxide solution. The excess alkali is back titrated against standard acid to arrive at the exact quantity of standard solution required.

Principle

When a solution containing calcium is treated with ammonium oxalate, all the calcium present is precipitated as calcium oxalate. The precipitate when dissolves in sulphuric acid, forming calcium sulphate and liberating free oxalic acid which is quantitatively estimated by titration against standard N/10 KMnO₄ to arrive at the calcium content present in the given solution. Following chemical reactions takes place in the estimation of calcium:

Principle

Carbohydrates are divided into two groups i.e., crude fibre (CF) and nitrogen free extractives (NFE). The NFE comprises the sugars, starch and a large part of the material classed as hemicellulose.

The NFE is not determined by analytical procedures. This is calculated as the figure obtained when the sum of water, ash, protein, fat and crude fibre of a feed is subtracted from 100. Since the figure is determined by difference, it includes the cumulative errors of the other determinations and thus it is not an exact value.

Requirement

Following analysed volumes of a feedstuff are very much essential to derive the nitrogen free extractives of the same feedstuff.

Calculation
The nitrogen free extractives can be derived from the following formula:

Principle

When a sample of moisture and fat free feedstuff is boiled with dilute acid and then with dilute alkali, the soluble carbohydrates and proteins go into the solution and are extracted. The undissolved residue left behind represents cellulose, hemicellulose, lignin and mineral matter. The cellulose, hemicellulose and lignin put together are called crude fibre (CF). When the residue after drying and weighing is ignited, the fibre being organic in nature is burnt whereas the ash or mineral matter is left over. The fibre is obtained by deducting the weight of ash from the weight of the dried residue. In simple way it can also be explained that the crude fibre is determined gravimetrically as part of the substance which is left when protein, soluble carbohydrate, fat, ash and water have been removed.

In feeding of simple stomached animals, the analysis of crude fibre content is very essential because the amount of crude fibre predicts the digestibility of the feedstuff. Higher the fibre content lesser will be the digestibility.

Principle
When a sample of feed, which is free from moisture, is extracted continuously with fat solvent like petroleum ether, the vapour from the ether boiling in the flask passes into the condenser where it condenses and drops back on the sample which dissolves ether soluble materials such as fat, sterols, lecithin, resins and volatile oils in the extraction flask. The extract is termed as crude fat or ether extract (EE) because it also contains other fat like substances in addition to true fat.

Equipment/glass wares
    1.     Analytical balance
    2.     Beaker
    3.     Cotton wool
    4.     Dessicator
    5.     Extraction thimble
    6.     Hot air oven
    7.     Oil flask (150-250 ml)