eChapters

(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).