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Preface This compendium serves as a ready reference to the scientists engaged in nutritional biochemistry research and teaching. To meet this objective, an attempt has been made to portray latest developments in the analytical and tracer techniques used in nutritional biochemistry research and teaching. The metric system of weights and measures is used throughout this compendium. At present some very good laboratory manuals, written by eminent authors on the theory and practices of chemical biochemistry and nutritional aspects, are available. I however, have not come across, so far, any suitable manual published in India or the Asian subcontinent, etc. to the teacher and students for their practical classes on this important subject, elaborated in a simple way. During my 40 years experience as teacher, I felt that a considerable amount  of time apparently turned to no account, while notes, explanations, directions, etc. are being given to the students. This compendium is written with the object of filling this long felt want. With the help of the material given in this compendium, the students and demonstrators will be able to come prepared to the laboratory with what they have practically to do, and the laboratory assistance would have all the preparation work classes ready at hand all times. Now a days scientists have realized the importance of use of tracer techniques in exploring the changes taking place in every facet of body metabolism and nuclear techniques is the only tool which may provide accurate information about the nutrient metabolism and transportation. Therefore, this compilation aims to provide all the research techniques. Nuclear research laboratories already established in India and Asian subcontinent could use these elaborative techniques specially mentioned in this compendium.

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.

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:

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Limitation of Weende Method The partition of carbohydrate into fiber (CF) and nitrogen free extract (NFE) is presumed to represent a separation of less digestible starch and sugars. However, in 20 to 30 percent feeds listed by Morrison, the NFE is less digestible than the CF. This comes about for several reasons, the most important of which is the CF method (successive boiling with dilute sulphuric acid and sodium hydroxide) does not recover all the fiber and large portions of fibrous constituents  are extracted into the NFE. The most  important of these fractions are lignin and hemicellulose are dissolved by both acid and alkali. The basic error of the NFE concept is the assumption that if constituents are soluble they are digestible. Lignin, the rigid component of wood, not only is indigestible but lowers the digestibility of substances with which it is associated. Another portion of the indigestible part of NFE arises from an artifact in the calculation of faecal NFE, where it is presumed that faecal nitrogen is protein (N x 6.25). Actually, 80-90 percent faecal nitrogen is NPN and is composed of bacterial residue in which the ratio of organic matter to nitrogen is about 1:4. Thus, part of the faecal NFE arises by an underestimation of the organic matter associated with faecal nitrogen.

Principle The residue remaining from the hemicellulose determination is digested with 72 percent sulphuric acid at room temperature. The lignin and acid insoluble  ash are retained in the filter crucible while the digested carbohydrate passes through. The lignin is separated from acid insoluble ash by ashing the residue. Equipment     1.    Acid digestion apparatus. A glass tray about 18 cm x 30 cm, 4 cm deep will hold twelve, 100 ml Griffen beakers. Another tray of the same size can be inverted over the beaker.     2.    Filter crucibles. Same as described for cell wall determination.     3.    Filtering apparatus. Same as described for cell wall determination.

Principle The residue from the cell walls determination is extracted with boiling 8 percent sulphuric acid for one hour to hydrolyse the hemicellulose without dissolving cellulose, lignin or acid insoluble ash. Equipment     1.    Refluxing apparatus.     2.     Filtering apparatus: same as described for cell wall determination     3.    Filtering crucibles: same as described for cell wall determination. Reagents     1.    Sulphuric acid (8%) : Standardize Reagent grade sulphuric acid to specific gravity of 1.052 at 20ºC.     2.    Bromocresol green indicator solution: Same as used for lignin determination.

Estimation of Cellulose in Feeds and Fodders Principle The feed well ground is digested with acetic acid which dissolves all other constituents like protein, disaccharides, fatty acids, etc., and cellulose and mineral contents are unaffected. The residue is made acid and alcohol free by washings. Alcohol removes water, benzene removes pigments and fat and at the end the sample is dried and ashed. The difference between the dried weight before ashing and weight after ashing gives cellulose content of the feed sample.

Principle Dried milled samples are extracted successively with diethyl ether, 0.5 percent ammonium oxalate solution, NH2SO4 and 72 percent sulphuric acid. Soluble carbohydrate in the ammonium oxalate extract determined with anthrone and hydrolysed pentosans and hexosans in the combined N H2SO4. Seventy two percent sulphuric acid extract are determined with aniline acetate and chromotropic acid respectively. The method gives a maximum variation of 4 percent from the mean of duplicates with each carbohydrate component. The residual material is ashed to obtain crude lignin and a nitrogen correction applied. Soluble carbohydrate - this group, which includes sugars, oligosaccharides, and the fructosans of grasses, provides a source of nutrients  readily available to livestock.

Principle Dried milled samples are extracted successively with diethyl ether, 0.5 percent ammonium oxalate solution, NH2SO4 and 72 percent sulphuric acid. Soluble carbohydrate in the ammonium oxalate extract determined with anthrone and hydrolysed pentosans and hexosans in the combined N H2SO4. Seventy two percent sulphuric acid extract are determined with aniline acetate and chromotropic acid respectively. The method gives a maximum variation of 4 percent from the mean of duplicates with each carbohydrate component. The residual material is ashed to obtain crude lignin and a nitrogen correction applied. Soluble carbohydrate - this group, which includes sugars, oligosaccharides, and the fructosans of grasses, provides a source of nutrients  readily available to livestock.

Method I Reagents     1.    20% sodium carbonate.     2.    Conc. hydrochloric acid.     3.    4% sodium hydroxide     4.    10% calcium chloride     5.    Ammonium hydroxide     6.    Potassium permanganate (0.05 N) Procedure Take 2-5 g of powdered sample in a beaker without spout, and add 40 ml of water and boil for half an hour. In case of faeces, add 40 ml of 4 percent NaOH instead of 40 ml of water to form silicates. Now boil, for half an hour with cold water flask over the beaker, then add 10 ml of 20 percent sodium carbonate solution and boil for  another half an hour. During these two cooking processes, the following changes take place.

Introduction Substances found in many plants are generally related to one of the phenols, pyrogallol or catechol. Tannins are solids, usually extracted by hot water insoluble in ether, chloroform, carbon disulphide, benzene; soluble in alcohol ether mixture, alcohol, water and in ethyl acetate, possessing a bitter astringent taste. Tannic acid is Pentadigalloyl glucose, on hydrolysis yields digallic acid and glucose. Tannins are water soluble glucosides of polydepsides, polymeric ester of gallic acid. It is hydrolysed enzymatically and some efforts have, therefore, been made to preserve tannic, if it is to exert its activity in the duodenum. On the basis of physical or chemical principles which they employ, the methods of tannin analysis  can be grouped as follows.

Principle Cyanide is distilled from chloroform and water soultion into KOH solution, forming potassium cyanide. KCN is titrated with silver nitrate; 2 KCN + AgNO3 = KCN. AgCN + KNO3. An excess of AgNO3 produces insoluble AgCN, which is the end point of titration.

Nitrates and nitrites are known to have injurious effects on animals when excess amounts are ingested. Nitrites are much more toxic but nitrates are reduced to nitrites by bacterial action in the digestive tract Nitrites (NO2) are rarely found in plants  in appreciable amounts except under very unusual conditions such as severe disease infections; therefore, the results are usually reported in terms of nitrates (NO3). The usual method is to determine the total nitrate and nitrite in terms of nitrate. However, the method can also be used to determine the nitrite content of a feed separately if a nitrite problem is suspected.

Introduction Aflatoxins are a group of highly subsituted coumarine, of which aflatoxin B1 and G1 are  most  highly hepatotoxic (toxic to liver) to animals. The toxins are produced by some strains of Aspergillus flavus and other species of Aspergillus which develop in many food stuffs, particularly groundnuts. cottonseed, and their cake and flour. Protein concentrates meant for babies as well as adult are now a day  made from groundnut flour and the cakes are used as feed for poultry and livestock. It has been found that even fodgrains such as maize, rice and wheat are attacked by the fungus producing the toxins.

The method described here gives an approximate thioglucoside and isothiocyanates contents in rapeseed meal. Reagents and Apparatus     1.    Barium chloride (5% solution)     2.    Volumetric flask, 600 ml     3.    Steam bath Method To 10 g meal (defatted by soxhlet extraction) add 250 ml distilled water, hydrolyse at 54ºC for one hour and then boil for two hours, keeping volume constant. Filter, retaining filtrate and wash residue three times with 50 ml hot water. Add washings to initial filtrate and make up volume to 600 ml. Precipitate barium sulphate by heating and adding excess barium chloride solution. Leave on a steam bath for a few hours and then filter. Ash in muffle furnace and then weigh precipitate.

Two procedure are described for the determination of free gosssypol . The first for normal meals and the second for meals which have been chemically treated and so contain dianilinogossypol. Reagents       1.    Aqueous acetone:  7 parts acetone, 3 parts distilled water (V/V).     2.    Aqueous acetone-aniline solution: To 700 ml acetone and 300 ml distilled water, add 0.5 ml redistilled aniline. Prepare solution daily.     3.    Aqueous isopropyl alcohol solution: 8 parts isopropyl alcohol. 2 parts distilled water (V/V).     4.    Aniline Distill reagent grade aniline over a small quantity of zinc, dust discarding the first and last 10 percent of the distillate. Store refrigerated in a brown glass stoppered bottle. Solution is stable for several months.

Isolation of RNA from Rat Liver (Method of Schmidt and Thannhauser, 1945) Principle Most of the RNA (ribouncleic acid) of cells is found in the nucleoli and microsomes.These fractions of cells may be isolated by differential centrifugation of cellular homogenates and thus provide an enriched starting material for RNA extraction. Such methods require high speed  centrifugation. An alternate procedure used in the present method is to disrupt the whole cells by homogenization and isolate the nucleic acid of entire homogenate. This procedure consists of the following sequential steps.

Principle (Method of Patterson 1963) The albumin bound long-chain fatty acids of plasma are usually referred to as unesterified or non-esterified fatty acids (NEFA) to distinguish them from the long-chain fatty acids present in the esterified form in neutral fats and phospholipid. In the case of ruminant plasma, it is also important to distinguish these “free fatty acids” from the small molecular weight steam volatile fatty acids of dietary origin which are found in the free state as the carboxylate ion. Basically, the method of Dole (1959a) has been used to estimate plasma NEFA employed by Annison (1960), which is to evaporate the heptane extract of plasma to dryness before titration. This is conveniently done in a stream of nitrogen on a warm hot plate rather than by evaporation in vacua. In place of the 0.05 M- phosphate buffer (pH 6.0) of Annison or the 0.02 N-H2SO4 used by Dole (1956a), in the present method 0.02 N-HCI is used as an extraction medium. This is preferred because owing to its volatility, it is less likely to cause serious accidental contamination of the supernatant heptane layer.

(Method of Joseph Mac Gee, 1959) Principle A simple and rapid enzymatic method for the quantitative estimation of total cis-methylene- interrupted polyenoic acids has been devised. Linoleic, linolenic and arachidonic acids, the more common acids of this group were used to calibrate the method. The potassium salts of the fatty acids are oxidised by atmospheric oxygen in the presence of the enzyme lipo-oxidase, and the absorption of the conjugated diene hydroperoxide is measured at 234 mµ. As little as 5 µg of linoleic acid can be quantitatively measured with good accuracy and precision. The total content of polyunsaturated fatty acids containing the cismethylene-interrupted diene structure of fats, oils, hydrogenated oils, fatty acids, esters blood plasma, microorganisms and plant seeds has been measured directly by this method.

(Method of Bloor, 1928 Modified by Folch et al., 1957) Gravimetric Method — Principle Total lipids in plasma consist of neutral fats, phospholipids, chloesterol esters and free cholesterol. Minor components include non-esterified fatty acids (NEFA), phosphatides and sphingo myelin. Total lipids in serum or plasma are extracted using ethyl alcohol and petroleum  ether, separated and weighed after drying at 105ºC. Procedure Pipette 5 ml serum in a separating funnel, add 5 ml ethyl alcohol and 12.5 ml pertroleum ether. Shake the contents of funnel two times for 3 min. atleast. Keep the contents of funnel as such for one hour. Transfer the contents of funnel in the moisture cup, dry and weigh. Calculate the result by difference method using the following formula.

Introduction (Titrimetric Method of  Cherry and crandall, 1932 Modified by Tietz and Fiereck, 1966). In 1932, Cherry and Crandall employed olive oil as the substrate for pancreatic and serum lipase (LPS. Triacylglycerol lipase) determinations. In subsequent years this method was improved (Tietz et al., 1959; Tietz and Fereck, 1966: Tietz. 1972), reducing the original 24 h, period to 6 h and finally to 3 h. Principle This procedure is based on the hydrolysis of triglycerides in olive oil into fatty acids, diglycerides and to some small extent into monoglycerides and glycerol.

Blood Cholesterol (Colorimetric Method of Lieberman, 1885: Burchard 1889 Modified by Watson, 1960). Introduction According to Liebermann-Burchard reaction, treatment of chloesterol with acetic anhydride and concentrated sulphuric acid in a water-free environment, polymerized unsaturated hydrocarbon with an intense blue-green colour are produced. The values for cholesterol esters may be calculated by deducting free cholesterol from total cholesterol.

(Method of Froesch and Renold, 1956; Froesch et al., 1957) Enzymatic with Glucose-oxidase / Peroxidase Introduction Glucose oxidase enzyme specific for the oxidation of glucose, was discovered in a fungus preparation by Müller in 1928. This enzyme is present in bacteria and in moulds such as (Pencillium notatum), which promotes the oxidation of glucose (b-ring form) to gluconic acid with the production of an equivalent amount of hydrogen peroxide. Normal values for whole blood glucose oxidase are 60 to 90 mg/100 ml. Results by the enzyme methods are lower than by the best “true  sugar” reduction methods largely because of their specificity, but inhibition of glucose oxidase by uric acid may be a contributory factor. In suspected cases of diabetes, values below 120 mg per 100 ml call for further investigation, for example, by glucose tolerance test. Due to the presence of glycolytic enzymes in red cells, blood must be collected into an antiglycolytic/anti-coagulant preservative, if there is to be a delay of more than 1 to 2 h. in processing or estimation. A fluoride/oxalate mixture, containing 2 mg of fluoride per ml of blood, is suitable and the glucose content will then remain unchanged for upto 3 days at 4ºC.

The term “laboratory” refers to the room in the building specifically equipped for the chemical manipulation of radioisotope. The word “shall” is used to indicate procedure which are mandatory.     1.    No unnessary materials are to be brought into the laboratory. Eating drinking, smoking and the using cosmetic in the laboratory are specifically forbidden.     2.    Pipetting or the performance of any similar operation by mouth suction is prohibited.     3.    Laboratory protective clothing (gloves, smocks, etc.) shall be left in the laboratory; gloves shall be removed before using the counting equipment.     4.    Before leaving the laboratory, the hands shall be washed first, then checked with a beta-gamma survey meter. Contamination remaining after thorough washing shall be reported to the instructor.     5.    If, in the course of work, personal contamination is suspected, a survey with a suitable instrument shall be made immediately, to be followed by the required cleansing. Routine precautionary surveys should be made at intervals.     6.    No person shall work with active materials while having abrasions in the skin on the hands without using rubber gloves.

The term “laboratory” refers to the room in the building specifically equipped for the chemical manipulation of radioisotope. The word “shall” is used to indicate procedure which are mandatory.     1.    No unnessary materials are to be brought into the laboratory. Eating drinking, smoking and the using cosmetic in the laboratory are specifically forbidden.     2.    Pipetting or the performance of any similar operation by mouth suction is prohibited.     3.    Laboratory protective clothing (gloves, smocks, etc.) shall be left in the laboratory; gloves shall be removed before using the counting equipment.     4.    Before leaving the laboratory, the hands shall be washed first, then checked with a beta-gamma survey meter. Contamination remaining after thorough washing shall be reported to the instructor.     5.    If, in the course of work, personal contamination is suspected, a survey with a suitable instrument shall be made immediately, to be followed by the required cleansing. Routine precautionary surveys should be made at intervals.     6.    No person shall work with active materials while having abrasions in the skin on the hands without using rubber gloves.

Activation : The process of causing a substance to become artificially radioactive by subjecting it to bombardment by neutrons or other particles. Activation Analysis : Any analytical procedure permitting the detection and measurement of trace quantities of elements following their exposure to a flux of neutrons. Activity :  The strength of a radioactive source. In absolute units, it relates to the number of radioactive atoms decaying per unit of time. It is also a synonym for radioactivity. Absolute activity is usually expressed in curies or millicuries. Alpha Particle : A particle which is identical to the helium nucleus, consisting of two protons and two neutrons. It carries a positive charge of 2. Alpha Rays: A stream of helium nuclei. The helium nucleus has a mass number of 4 and an atomic number of 2. If consists of two protons and two neutrons.

Ionization Chambers for Gases Generally these type of instruments are known as dose-rate meter (e.g., the so called “cutie-pie”). Radiation intensity (i.e., a constant stream of particles or photons) gives rise to a continuous series of pulses, and if these are allowed to merge, they form a weak electric current, which may be amplified and registered by an electronic circuit. The final scale reading will then be a measure of the energy dissipated in the following chamber per unit of time by the ionizing particles or photons. In gas-flow counters, the radioactive samples are placed inside the detector which will be transfused by a gas at atmospheric pressure. This instrument counts particles of low energy, such as the b particles from 14C, effectively (Window-less counting).

We may study the nutrient metabolism in a better way as explained in the following summarized form in Table 1.

For the first time, isotope dilution analysis was introduced by Hevesy and Hafer in 1934, but it was not until 1940, the usefulness of isotope dilution technique was revived by Rittenberg and Foster (1940). There are three general types of isotope dilution methods. These are (a) direct (b) inverse and (c) double isotope dilution. In general isotope dilution methods may be used for the following type of studies.     (a)    In isolation purification and identification of unknown intermediate in a chain of metabolic reactions.     (b)     To obtain evidence of synthesis (incorporation), and precursor-product relationships between known compounds.     (c)    As an analytical tool in the assay of known compounds. In isotope dilution techniques, the labelled intermediate, which is being diluted by unlabelled carrier, is a known compound.

Studies on Volatile Fatty Acids Production and Kinetics of VFA Pool as Determined by Using “Isotope Dilution Technique” (Method of Krishna and Ekern, 1974a,b and c) Details of Experiment Materials     (i)    Animals : Two fistulated sheep of Dala Texel breed weighing about 72 and 76 kg are selected.      (ii)    Experimental Diet :         Basal Diet : Two experimental sheep are maintained on 1.1 kg early cut timothy hay per day. The ration is divided in four equal parts and offered four times a day during the preliminary period of about twenty days. One day before infusion of labelled VFA, the sheep are fed regularly at one hour interval (1.1 kg hay divided in equal parts) to help in increasing the mixing rate of rumen ingesta. The animals are given water adlibitum but they are not allowed to drink water during infusion. Digestible energy and metabolizable energy (ME) of the basal diet is determined by conducting a separate metabolism trial on the same sheep.

(Method of Hyden 1955a,b) I. Use of Polyethylene Glycol (PEG) These compounds are manufactured through the reaction of ethylene oxide with water, ethylene glycol or diethylene glycol to furnish functional  groups (in this case hydroxyl) for the propagation of the reaction. The process results in a mixture of diols of different chain lengths, in which the numbers of molecules of various sizes are presumed to be represented by Poisson’s distribution (Shaffer, Critchfield and Nair, 1950b). Polyethylene glycols 200, 300, 400 and 600 are fluids: compounds 1,000, 1,500, 4000, 6000 and 1,0000 are solids of increasing firmness. PEG compounds of molecular weight above 1000 are not absorbed from the gastrointestinal tract of the rat and man. Polyethylene glycol has been used frequently as a water soluble marker in studies of absorption in man and animals (Sperber et al., 1953; Hyden, 1955a,b; Corbett et al., 1956; Gray et al., 1960; Weller et al., 1962; Ulyatt, 1964a,b; Walker and Hawley, 1965; Tulloh et al., 1965; Sinha et al., 1970; Nagel and Piatkowshi, 1972).

Extent of Passage (Method of Krishna and Ekern, 1974D) Definition of Terms The term  extent of passage is used to describe how far a given ingested material travelled  or was allowed to pass. The point at which  undigested residue of the food comes to or has passed through the alimentary tract may be found by the use of markers without disturbing the subject. In some conditions of digestive system malfunction or disorder , the food residue may be blocked at point along with digestive tract. Transit Time This is the time it takes the digesta of a meal to pass through the alimentary tract or segments of it. This time also represents the  retention time in the tract or the particular segment. A simple way of calculating it is by recording the time of first or last appearance of the marked residue of a meal. Other more useful methods for calculating retention time include “mean retention time” (Castle 1956a), “mean time” (Blaxter et al., 1956) and “turnover time” (Hungate, 1968).

Investigation of the effect of Various Hormones on Fat Synthesis from 14C Labelled Acetate in Chicken Adipose Tissue (Method of OMea and Leveille, 1968, Folch, Less and Solane Stanley, 1957 and Vaughan, 1961) Reagents     1.    Krebs-Ringer calcium-free bicarbonate buffer 7.4         100 parts 0.154M NaCI (0.9001 g in 100ml)         4 parts 0.154M KCI (0.0459 g in 4 ml)         1 part 0.154M  MgSO4 (0.0380 g in 1 ml)         (3 parts 0.110M CaCI2)         21 parts 0.100M NaHCO3 buffered to 7.41 by bubbling through CO2

(Method of Saryre, 1963 and IAEA, 1969) Introduction This technique helps in detection of very small amount of stable elements in fairly large quantitites of extraneous matter. Trace elements like zinc, copper, molybdenum and arsenic which exist in minute amounts in certain organs or tissues. For example, to detect the amount of copper present in one gram of tissue. using 1 mg of thin copper foil as control. Normal copper contains 69 percent 63Cu and 31 percent 65Cu. The (n,r) reactions on these two stable isotope produce 64Cu and 66Cu respectively. The half life of 64Cu is 12.8 h and that of 66Cu is five minutes. Therefore the latter can be ignored, if more than that an hour elapses between the end of the irradiation and the measurement. The specific activity of the unknown separate is compared with that of the known standard separate, and the content of Cu in the original tissue sample is thereby calculated.

(Method of Cocimano and Leng, 1966; Conrad, 1972; Varady & Harmeyer, 1972) Hypothesis The validity of measuring urea kinetics using a single dose of 14C-urea and using urinary excretion as a sampling system is based on specific assumption:     1.    There is a rapid equilibration of 14C-urea with the endogenous urea pool following infusion. This pool is conceived to be a measure of all body compartments permeable to urea and in equilibrium with the plasma urea pool.      2.    Experimental animals are metabolically maintained in a steady state during the test period and metabolic breakdown of urea is random.     3.    Specific radioactivity for urinary urea is proportional to the dilution of replenishment and simultaneous depletion rate of the urea pool.     4.    That the metabolic parameters of urea are correctly measured.

(Method of Visek et al., 1953; Lueker and Lofgreen, 1961) By using isotope dilution technique, It is possible to separate faecal calcium into endogenous and exogenous fractions. The true digestibility can be measured independently by making correction for faecal endogenous loss. The isotope dilution method described by Visek et al., (1953) involves daily intravenous injection of 45Ca for 15 days, followed by a collection of the serum and the faeces for 15 days. However, these numerous veinpunctures result in considerable discomfort to the animal. Lueker and Lofgreen (1961) modified this method in sheep by giving a single subcutaneous injection of 45Ca, followed by a collection of blood and the faeces for 7 days only. The endogenous faecal calcium and true digestibility of calcium is calculated by the formula of Lofgreen and Kleiber (1954) as given below:

Introduction It is a well known fact that the soluble oxalates are broken down by rumen microbes from buffalo and cattle and one of the end product formed is bicarbonates. The rumen microbes are obtained from a fistulated buffalo calf fed on wheat bhusa and sarson cake supplemented with 60 g potassium oxalate daily for about three months. Representative sample of rumen ingesta is drawn before feeding and mixed culture is prepared by the method as described Donefer et al., (1960). Mixed culture and basal nutrient mixture (Quicke et al., 1959) of pH 6.9 mixed in equal quantities. Fifty millilitres of this mixture are added into each incubation flask containing one gram of wheat straw, ground to 40 mesh as substrate. In the incubation flasks are added 35,100 and 550 mg oxalic acid in the form of potassium oxalate and 0.24 µCi sodium oxalate 14C. Blank (containing no substrate, no oxalic acid added) and two controls, one having substrate but no added oxalic acid and the other containing the above plus radioactivity, are also run alongwith. All the incubation flasks are taken in duplicate. The flasks are incubated at 39ºC for 24 h  in an incubator and at the end of this period microbial activity is stopped by the addition of few drops of saturated solution of mercuric chloride.

Principle     Inorganic salts are relativity insoluble in aromatic solvents which has been detrimental to the estimation of 45Calcium by scintillation counting. This technique inolves the counting of 45Calcium as calcium 2-ethyl hexanoate in a solution of toluene phosphor; and the counting as calcium chloride in a tertiary mixture of absolute ethyl alcohol-hydrochloric acid-toluene-phosphor. Reagents     1.    45Calcium. A 45Calcium solution supplied as calcium chloride in hydrochloric acid solution, is suitably diluted.     2.    Calcium chloride, 0.045 N, is prepared by dissolving ACS grade calcium carbonate, dried to constant weight at 110ºC, in 5 N hydrochloric acid.     3.    Calcium acetate, citrate, lactate, acid phosphate, chloride and hydroxide.     4.    Ammonium oxalate solution, 4 percent     5.    2-Ethyl-hexanoic acid.     6.    Toluene-diphenyloxazole solution, 2g of diphenyloxazole are dissolved in 500 ml of toluene.

(Method of Olbrich et al., 1971) Jones and Ekman (1960) showed that 144Ce is absorbed to a very small extent in the gut of the ruminant and suggested its use as a marker in digestibility trials. Huston and Ellis (1968) studied the affinity of radioactive cerium for feedstuffs and digesta particles in vitro experiments and found that cerium is rapidly absorbed onto and remained tenaciously bound to  digesta particles. Ellis and Huston (1968) tested radioactive cerium as a particulate flow marker in sheep with favourable results. Experimental Procedure Three Holstein bulls weighing 315, 239 and 305 kg and one Holstein steer weighing 327 kg are used. Twice daily, at 9.00 am and 9.00 pm, they are fed 3.4 kg of a balanced ration, meeting NRC requirements for normal growth and maintenance.

(Method of Bryant et al., 1959) Strontium and barium are adsorbed on a cation-resin column, and  after a suitable gowth period, yttrium-90, the daughter of strontium-90, is selectively eluted. The yttrium-90 is then adsorbed on and eluted from a second cation-resin column and counted. The radiochemical yield is greater than 97% gravimetric measurement of the recovery of strontium and yttrium carriers is not required. The method, with modifications, is applicable to samples that contain 100 mg of iron or uranium.

General Definitions     1.    Colour : Colour is the name for all sensations arising from the activity of the retina of the eye and its nervous mechanisms, this activity being a specific response to radiant energy of certain wavelength and intensities. Colour is basically psychological and is not synonymous with wavelength.     2.    Lambert’s law : Which states that the proportion of radiant energy absorbed by a substance is independent of the intensity of the incident light.     3.    Beer’s law : Which states that the proportion light absorbed depends only on the total number of absorbing molecules through which it passes independently of their concentration.     4.    Extinction : Extinction is the logarithmic ratio of the intensity of the incident light to that of the emergent light.

Introduction Chromatography was first employed by Ramsey (1905) to separate mixture of gases and vapours. These first experiments used selective adsorption on, or desorption from, solid absorbents such as active charcoals. The  following year Tswett (1906) obtained discrete coloured bands of plant pigments on a chromatographic column. He coined the term “chromatography” (literally “colour writing”) which is obviously a misnomer when used retrospectively. Contribution of Martin and Synge (1941) in a study for which they were awarded the Nobel prize, and later James and Martin introduced gas-liquid chromatography in 1951 and 1952.

Introduction The first application of atomic absorption spectra to chemical analysis was made just over 100 years ago by Kirchoff (1860), who demonstrated the presence of various elements in the solar atmosphere. He and Bunsen, demonstrated shortly afterwards that atomic spectra, whether in emission or absorpiton, could be the basis of a powerful method of chemical analysis. Walsh (1955) recognized the potential advantages of the absorption method over emission method and devised simple and versatile apparatus applicable to the routine analysis of solutions of a wide range of elements. Alkemade and Milatz (1955) published papers describing the application of atomic absorption  techniques to chemical analysis.

Principles of Working (Lundegårdh, 1929-1934 : Mac Intyre, 1961) Flame photometry is the measurement of the concentration of an ionic material in a solution introduced in  to a flame, where the intensity of light emitted by the flame under these conditions is measured. We should give credit to Kirchhoff and Bunsen (1860), who developed the idea of emission spectroscopy into quantitative inorganic analysis, later on Janssen (1970) made lot of developments in this direction. The main parts of flame photometer are: (1) burner, (2) atomizer which disperses the solution as a fine spray into the flame; a means of isolating from the spectrum (i.e., filtering system, diffraction gratting etc.) only that portion of the emitted light which is a specific characteristics of the substance being examined ; a photosensitive detector with or without amplifier; and a method of measuring the desired emission. In flame photometry the diluted biological fluid is atomized directly ino the flame without need of preliminary separation. The resulting emissions are those of all the inorganic ions present. For measurement the emitted light is passed through a monochromater where the wavelength most characteristics of the material analysis and which most closely follows Beer’s law is selected.

Quantitative heat production estimation is necessary to  know the net energy of feeds and fodders. In the case of ruminants approximately 8 per cent of the gross energy consumed is lost as methane, therefore it becomes necessary to know this important loss in terms of methane energy so as to assess the metabolisable energy of any particular feed. In human subjects, heat production studies are conducted to know the effect of planes of nutrition on basal metabolism as affected by different environmental conditions. In the important laboratories of world, heat production and methane production studies were conducted by respiration chamber (open circuit and closed circuit), since in our country respiration chamber has been installed at IVRI, Izatnagar, Bareilly (U.P.) India, therefore, this technique is mentioned in brief in this chapter. Indirect calorimetry involving the use of face mask and Douglas bag is followed at present in our country at NDRI, Karnal (India) so as to study the basal metabolic rate (BMR), heat production in human subjects and animals, Krishna (1973). Some laboratories in the country are interested to study respiratory quotient at different levels of feeding in human subjects and animals. The methods of estimation of RQ (respiratory quotient) is expressed in detail in this chapter.

Assay of Carotene, Tocopherol and Plant Pigments (Method of Astrup et al., 1971). Agricultural University of NORWAY, ÅS-NLH, NORWAY Introduction A method which assays the carotene together with chlorophyll a and b and xanthophyll was given by worker (1957, 1958) and this was a successful approach towards a useful routine procedure. Worker’s way of extraction is based on the use of aqueous acetone a technique which was studied by Davidson (1954). The separation is accomplished on an alumina column. Worker (1958) has used a double successive chromatography, once on magnesia and again on alumina, to obtain its separation from the other lipid components. The method developed by Astrup et al. (1971) permits simultaneous assay of tocopherol, carotene and plant pigments using alumina column.

Riboflavin or Vitamin B2 Introduction Riboflavin (synonymous with lactoflavin, vitamin G and vitamin B2) is a yellow green, fluorescent, water soluble pigment widely distributed in plant and animal cells. The organic formula of riboflavin is (6, 7-dimethyl)-9-(D,1-ribtyl)-iso-alloxazine. The empirical formula is C17H20N4O6. Riboflavin is very sensitive to both visible and ultraviolet light. All manipulations with riboflavin or riboflavin containing materials should be carried out in subdued light or in low actinic glassware. Even subdued daylight may be destructive but artificial light of an intensity of 6 candles per squarefeet or less is permissible. Irradiation of alkaline solutions yields lumiflavin (6, 7, 9-trimethylis-alloxazine) and in acid solutions lumich rome (6.7 dimethyl alloxazine) is formed, which is characterised by a blue fluorescence. One of the distinguishing properties of riboflavin is its yellow-green fluorescence in neutral solutions which reaches a maximum at pH 6.7 to 6.8.

Introduction All the amino acids have in common an amino group and a carboxyl group attached to the -carbon atom; however, they differ in having distinctive side chains, usually called R groups, as is shown in the following  generalised structural formula

A. In vitro Rumen Fermentation Technique for Forage Quality Evaluation The term in vitro means “in a glass” in contrast to in vivo which is defined as “within the living body”. In vitro implies a close relationship to an in vivo measurement an in vitro reaction is used as a substitute for a reaction which normally occurs and is observed within a living body. the terms  “artificial rumen” and “in vitro rumen fermentation” are often used interchangeably. The main advantage of the in vitro techniques rests in the ability to use them to study activity of micro-organisms away form the control and influence imposed by the host animal.

Techniques to Conduct Digestion Trial Under Stall Conditions Digestibilty definition. The digestibility of a food is most accurately defined as that  proportion which is not excreted in the faeces and which is , therefore, assumed to be absorbed by the animal. It is commonly expressed in terms of dry matter and as a percentage, the digestibility coefficient. For example, if a cow ate 11 kg of hay containing 10 kg of dry matter and excreted 4 kg dry matter in its faeces, the digestibility of the hay dry matter would be

The main groups of some diagnostically important enzymes are as below: 1. Oxido Reductases Glucose 6-phosphate dehydrogenase, Isocitrate Dehydrogenase, Lactate dehydrogenase, Malate dehydrogenase, Sorbitol dehydrogenase. 2. Transferases Alanine transaminase (GPT)     Aspartate transaminase (GOT) Creatin binase Hexose-1 phosphate uridyltransferase 3. Hydrolases Acetylcholinesterase phosphatases Amylases Pepsin Trypsin Lipase

Celite Column Chromatography (Method of Wiseman and Irvin, 1957) Principle Celite columns with an internal indicator have been developed for the quantitative separation of silage and rumen volatile fatty acids ranging from butyric to succinic. The aqueous phase employs minimal amounts of sulphuric acid to prevent retention of organic acids, sugar is added to the phase to increase elution resistance. Aqueous samples, 2 ml or less are added directly to a dry column cap. Eluting solvents are mixtures of acetone and petroleum ether. Single zone collections make possible a reduced number of titrations with increased accuracy. The column permits fairly wide separations of lactic acids, often difficult on silicic acid columns. The method employs celite with alphamine red R as an internal indicator. A distinctive feature is the use of concentrated sugar solution as the stationary phase to resist leaching action of solvents.

Principle The use of boric acid as an ammonia absorbent was first introduced by winkle  (1913-15). Conway and O’Malley (1942) used boric acid for ammonia absorption in their original micro diffusion technique. The ammonia liberated in the outer chamber by alkali addition in the usual way is absorbed in boric acid mixture plus indicator in the central chamber (slightly reddish), the pH of this mixture being approximately 5 before absorption. During the ammonia absorption the pH of the mixture rises, and to upwards of 8.0 (the strength of the mixture being such as to prevent it rising further). After the required period, the fluid in the central chamber is titrated to a faint permanent reddish tint with standard acid. A mixture of methyl red and bromocresol green was found satisfactory  to work as an indicator to assist in titration job.

Steps in the Estimation of Trace Elements Estimation of trace elements in feeds and fodders and other biological materials involves the following steps :     1.    Production of an acidic solution of the inorganic elements in the biological samples after removal of the organic matter by dry ashing or wet oxidation.     2.    Removal or masking of interfering substances.     3.    Determination of the selected elements, usually by using colorimetry. Results of trace elements determinations are expressed as parts per million (ppm), mg/kg or µg/g dry matter.

Unit of Length A metre (m) is taken from the international prototype metre at the International Bureau of weights and measures; at 32ºF. (0ºC) = 39.37 inches (in.) = 3.280833 feet (ft.) = centimetres (Cm.). A centimetre (Cm.) = 10 millimetres (mm.) = 10,000 microns (µ) = 0.337 inch (in.) or 10-4cm. A millimetre (mm.) = 1,000 microns (µ) = 0.03937 inch. A micron (mµ) = 1,000 milli-microns (µ) = 3.937x10-5 inch. A milli-micron (mµ) = 0.001 micron or 10-7 cm. A foot (ft.) = 0.3048006 metre (m.) = 30.48006 centimetres (Cm.) = 12 inches (in.)., inch = 2.5 cm. An inch (in.) = 2.539998 centimetres (Cm.) = 25.39998 mm = 25,399.98 microns (µ).

Blood Sampling Blood samples shall be taken in the early morning hours before offering any diet to human subjects or ration to livestock. The syringe and needle should be autoclaved, needle should be free from any blockage etc. In the case of ruminants, blood sample is taken directly from jugular vein, while in swine, blood sample is drawn from ear vein, while in human subjects, blood is taken from prominent vein on the inner surface of the yellow usually the (medium basilic). The container (test tube etc.) should be dried and autoclaved. We should separate plasma (from anticoagulent mixed blood) and serum from ordinary blood by centrifugation immediately. We should remember by heart  that blood samples should not be kept in deep freezer, because there are chances of haemolysis as well as an abnormal distribution of certain ions and enzymes between the cells and the serum. We should never force the blood through the needle as such type of practice may result in extensive hemolysis. We should discard first few drops of blood, while taking sample. We should prepare protein free filtrate immediately. Protein-free filtrate may be kept in a much better way than whole blood or plasma.

Estimation of Calcium in Feeds, Fodders and Faeces (Titrimetric method of AOAC, 1965) Principle When a solution containing calcium is treated with ammonium oxalate, all the calcium present is precipitated as calcium oxalate, The precipitate on treatment with sulphuric acid dissolves, forming calcium sulphate liberating free oxalic acid which is quantitatively estimated by titration against standard N/10 potassium permanganate solution to arrive at the calcium content present in the given solution. The reactions which take place are given below:

Estimation of Phosphorus in Feeds, Fodders and Faeces (Titrimetric method of AOAC, 1965) Principle Metaphosphates and pyrophosphates are converted to the ortho-phosphate, which is treated at 65-70ºC with excess of molybdate in the presence of nitric acid. When acid solutions of phosphates are heated with ammonium molybdate, the phosphoric acid is precipitated as yellow ammonium phosphomolybdate (NH4)3 PO4 (Mo03)12 2HNO3.H2O. Impurities like molybdic oxide, if happened to be formed any, goes into solution. The yellow precipitate formed reacts quantitatively with standard alkali, the excess of which is back titrated with acid.

Estimation of Magnesium in Feeds, Fodders and Faeces (Gravimetric method - AOAC, 1965) Principle Evaporation of calcium free filtrate is carried out to drive off ammonium chloride and nitric acid is added to decompose ammonium salts, which is then subsequently driven off by evaporation. NH4Cl + HNO3 = HCl + NH4NO3 (driven off). Sodium citrate in solution yields citric acid which has got a tremendous effect on aluminium and iron in that the citric acid keeps these materials in solution. At neutrality, first the normal magnesium phosphate is formed, which is then turned to magnesium ammonium phosphate, which on incineration gives rise to magnesium pyrophosphate (Mg2P2O7).

TVFA Estimation in Blood (Method of Friedemann, 1938 modified by Mc Anally, 1944) A method for the determination of volatile fatty acids in various biological materials has been described by Friedemann (1938) which is said to be applicable to blood, then the blood is added to dilute sulphuric acid, then sodium tungstate solution and solid magnesium sulphate are added and the whole is steam distilled. Reagents     1.    Saturated magnesium sulphate solution to which has been added 2.5 per cent by volume of concentrated sulphuric acid.

Separation of Bacteria and Protozoa (Method of Czerkawski, 1975) Rumen contents are obtained just before and two hours after feeding from sheep that had been given an experimental ration. The samples of rumen contents are strained through gauze and fractioned by sedimentation and centrifugation to give preparations of mixed protozoa (PR), large bacteria (LB) and small bacteria (SB). Whole particulate matter is also isolated by centrifugation at 20000g. The detail steps are mentioned in the flow chart given below.

Total Protein in Tissue Extract or Blood, Serum or Plasma (Method of Folin-Ciocalteu, 1927 modified by Lowry et al., 1951) Principle A deep blue dye is formed when protein is treated with the phenol reagent of Folin ciocalteu. The extraordinary sensitivity of the method is due to the fact that two colour reactions are taking place simultaneously.     a.    The biuret reaction of peptide bonds with copper in alkaline solution.     b.    A reduction of phosphomolybdic acid and phosphotungstic acid by the aromatic amino acids, tyrosine and tryptophan, present in the protein.

Pepsin Digestibility of Food Principle Solvent extracted samples are digested for 16 hour with warm acid solution of pepsin under constant agitation. Insoluble residue is centrifuged, dried and weighed and analysed for protein ; or filtered, washed and analysed for protein. Apparatus     1.    Centrifuge : Capable of at least 1750 rpm with conical bottom tubes of 150 ml capacity. If necessary, solution may be centrifuged in 50 ml tubes.     2.    Agitator : or water bath with shaking device and having temperature controlling unit.     3.    Glass fiber filter paper.

Introduction Casein has been widely used as a substrate for measuring the trypsin inhibitor activity of natural trypsin inhibitors such as those which occur in Soybeans and other legume. The most common method employing casein is the one originally described by Kunitz (1947) which involves the spectrophotometric determination of the breakdown products produced by a given concentration of trypsin in the presence and absence of the inhibitor. On the other hand, the tryptic hydrolysis of a synthetic substrate such as benzoyl-DL-arginine-p-nitroanilide (BAPA), first introduced by Erlanger et al. (1961), does follow a zero order reaction, and one obtains, within broad limits, a linear relationship between the quantity of p-nitroaniline released and the concentration of the active enzyme. Although BAPA has been used for assaying the antitryptic activity of soybean fractions (Sambeth et al., 1967).

PEG Estimation Introduction Polyethylene glycol (PEG) is used as a marker for the estimation of rumen water volume and ruminant digestion studies (Hyden and Ekman, 1953). Long back, Hyden (1955) developed a turbidimetric method which is described below : Principle When trichloroacetic acid is added to a solution of PEG, a turbidity is produced, which can be measured photo-electrically. The intensity of the cloud, which consists of finely divided oily droplets, is increased in the presence of barium ions. Protein and sulphate must be completely removed from the sample before the addition of the trichloroacetic acid reagent. Sulphate is precipitated with barium chloride and the proteins are removed essentially according to Samogyi (1945). The method has been developed for determination of PEG in ruminal and abomasal fluids, but it has been tried with other biological materials too.

I. Titrimetric Method of Estimating Salt in Fish Meal (Modified AOAC, 1965 method) Principle The salt content is calculated as sodium chloride from the content of water solubles chlorides. The chlorides are dissolved in water and directly precipitated by titration with a silver nitrate solution using potassium chromate as an indicator.

I. Detection of Castor Husk in Oil Cakes and Oil Seed Meals (Method of ISI, (BIS), IS: 10165-1982) Principle The method is based on the fact that castor husk is not bleached under the conditions which cause the bleaching of almost all other materials of vegetable origin likely to be present in an oilcake. The method consists of the treatment of the material with dilute alkali and acid solutions followed by treatment with bleaching powder solution and the isolation of the unbleached castor husk.

(Method of Schram and Aines, 1959) All the legume seeds and their byproducts are very rich in urease activity which may be responsible for an appreciable loss of urea nitrogen on long duration storage. Therefore it is necessary to estimate urease activity in legume seeds and their byproducts. Reagents     1.    Dimethyleaminobenzaldehyde solution (DMAB). Dissolve 16 g DMAB in 1 litre 95% ethyl alcohol and add 100 ml concentrated hydrochloric acid (Stable one month).     2.    Pyrophosphate buffer. Dissolve 233 g Na4P2O7. 10H2O in approximately 980 ml distilled water. Add 3 ml of conc. Hcl and then dropwise further Hcl until the pH of the buffer is 7.7 to 7.8. Dilute to one litre.     3.    Buffered urea solution. Dissolve 0.4 g urea in one litre pyrophosphate buffer (Stable one week).

(Colorimetric Method of Barker and Summerson, 1941) I. Method for Blood / Plasma Principle In this method, glucose and other interfering material of the protein free blood filtrate is removed by the Van Slyke-Salkowski method of treatment with copper sulphate and calcium hydroxide. A part of the resulting solution is heated with concentrated sulphuric acid to convert lactic acid to acetaldehyde, which is then determined by reaction with p-hydroxydiphenyl in the presence of copper ions.

Preparation of Samples Before withdrawing portions for analytical determinations, bring the sample to a temperature of 26 to 28ºC and mix throughly by pouring gently into a clean dry receptacle and back, until a homogenous mixture is assured. If lumps of cream do not completely disappear, warm the sample to about 40ºC, mix throughly, then cool to 26 to 28ºC. In case a measured volume is required in a determination, bring the temperature of the sample to about 27ºC before pipetting. All samples shall be allowed to stand for three to four minutes after mixing to allow air bubbles to rise ; the sample bottle shall then be inverted three or four times immediately prior to taking the milk for analysis. If a sample is curdled, excessively churned or decomposed, the sample should not be taken up for analysis.

Adulteration in animal and human foods has become common feature in the country, therefore we should be cautious while selecting animal/human feed ingredients. The author of this compendium has developed. “Animal Feed Testing Kit” based on novel cud liquor enzyme complex (innovation certified by ISA, International Search Authority - Patent office, Vienna), registered with WIPO (Geneva). This method is described in detail in this chapter and Patent application is field at Indian Patent Office Delhi. Now-a-days, animal feed is being adulterated with extraneous materials viz, Saw dust, Ric hulls, sand and Silica, Castor husk, oat hulls and urea (higher than 01 percent limit on dry matter basis).

Bibliography : Tracer Techniques     Abou-Akkada, A.R., D.A. Messmex. L.R. Fina, and E.E. Bartley, (1968). J. Dairy Sci,. 51: 78.     Annison, E.F. (1954). Biochem. J., 57: 400.     Ash, R.W. (1961a). J. Physiol., 157: 93.     Ash, R.W. (1961b). J. Physiol., 157: 185.     Balch. C.C. (1961). Movement of digesta through the digestive tract. In D. Lewis’s Digestive Physiology and Nutrition of the Ruminant. London: Butterworth.     Bergrman, E.N., R.S. Reid, M.G. Murry, J.M. Brockway, and F.G Whitelaw, (1965). Biochem. J., 97:53.     Beever, D.E., D.G. Harrison, D.J. Thomson, S.B. Cammell, and D.F. Osbourn, (1974). Brit. J. Nutr., 32:99.     Bernard, G.C., C.V. Boucque, (1968). J. Agric. Food Chem., 16: 105.     Binnerts, W.T., A. T. Klooster, VanT and A.M. Frens, (1958). Vety. Record., 82: 470.     Birks, J.B. (1964). The Theory and Practice of Scintillation Counting. Oxford: Pergamon Press.     Black, A.L. (1968). In isotope studies on the nitrogen chain, pp. 287. (Proc. Symp. Vienna, 1967).     Blackbum, T.H. and P.N. Hobson, (1960). Brit. J. Nutr., 14: 445.