eChapters

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