Isotope Techniques for Studying Animal Protein Production ...

FOOD

TECHNICAL REPORTS SERIES No. 111

Isotope Techniques for Studying Animal Protein Production

from Non-Protein Nitrogen J O I N T F A O / I A E A D I V I S I O N OF

A T O M I C E N E R G Y IN FOOD A N D A G R I C U L T U R E

I N T E R N A T I O N A L A T O M I C E N E R G Y A G E N C Y , V I E N N A , 1 9 7 0

ISOTOPE TECHNIQUES FOR STUDYING ANIMAL PROTEIN PRODUCTION

FROM NON-PROTEIN NITROGEN

TECHNICAL REPORTS SERIES No. I l l

ISOTOPE TECHNIQUES FOR STUDYING ANIMAL PROTEIN PRODUCTION

FROM NON-PROTEIN NITROGEN

R E P O R T O F A C O N S U L T A N T S ' G R O U P O N T H E A P P L I C A T I O N O F I S O T O P E T E C H N I Q U E S

T O T H E S T U D Y O F A N I M A L P R O T E I N P R O D U C T I O N

F R O M N O N - P R O T E I N N I T R O G E N , O R G A N I Z E D B Y T H E J O I N T F A O / I A E A D I V I S I O N O F

A T O M I C E N E R G Y I N F O O D A N D A G R I C U L T U R E A N D H E L D I N V I E N N A , 25 — 2 8 A U G U S T 1 9 6 9

I N T E R N A T I O N A L A T O M I C E N E R G Y A G E N C Y

V I E N N A , 1 9 7 0

Abstract

ISOTOPE TECHNIQUES FOR STUDYING ANIMAL PROTEIN PRODUCTION FROM NON-PROTEIN NITROGEN. (Technical Reports Series). The Consultants' Group reached the following conclusions. 1. There is a definable role for non-protein nitrogen (NPN) in the feeding of ruminants. NPN can be used as an admixture in concentrate rations for milk and more intensive beef production. 2. The economic feasibility of NPN application must be carefully evaluated, particularly in countries where the relatively ex-pensive quick energy releasing materials would have to be introduced into ruminant feeding. Some developing countries have a relative abundance of by-products rich in protein, and NPN generally could not be recommended under such conditions.

Research methods appropriate for evaluation of NPN sources are outlined. Three isotopes were pointed out as being especially useful and the application of each can be summarized as follows. (1 ) 1 5N for: (a) rate of NH 3 production in rumen, (b ) rate of incorporation of N compounds into microbial protein, (c ) overall conversion of NPN to tissue or milk protein. (2 ) 14С for: (a) rate of hydrolysis of C-containing NPN, (b ) turn-over and entry rate of amino acids, (c) estimates of protein synthesis. (3 ) 30S for: (a) estimates of microbial protein synthesis, ( b ) estimates of microbial contribution to synthesis of milk, wool, muscle.

ISOTOPE TECHNIQUES FOR S T U D Y I N G ANIMAL PROTEIN P R O D U C T I O N FROM NON-PROTEIN N I T R O G E N

IAEA, VIENNA, 1970 S T I / D O C / 1 0 / 1 1 1

Printed by-the IAEA in Austria May 1970

FOREWORD

On average, two-thirds of the protein consumed by human beings throughout the world stems from plants; about one-third is of animal origin. Nevertheless, the populations of some countries in north-west Europe, north America and Australia and also in southern Latin America have about two-thirds of their daily protein intake from animal origin, and trends in develop-ing countries indicate increased demands for animal protein as gross national products rise. As people in the urban areas or industrial centres of developing countries become more prosperous, they tend to consume a greater propor-tion of animal protein. This calls for concerted efforts to bring about efficient systems of low-cost animal production to. satisfy expected increases in demands.

The most efficient use of each potential feed source is a basic require-ment in the planning of future livestock production. Some developing countries have reached, or are near, self-sufficiency in food grains. Surplus coarse or lower-quality feed grains can thus be expected to become available for animal industries in increasing amounts and although such supplies may g o first to the pig and poultry sectors, it is likely that the amounts for ruminant milk and meat production will also increase.

The prospect that cereals and other high-carbohydrate feeds may be-come available to support ruminant production of meat and milk calls for investigation of the possibilities of supplementing these protein-deficient feed stuffs with non-protein nitrogen. Non-protein sources of nitrogen, such as urea, offer an inexpensive supply of the nitrogen essential in improving the nutritive value o f grains for livestock.

The role of isotopes in investigating non-protein nitrogen supplementa-tion of feed stuffs was the subject of a Consultants' Group convened at the Agency headquarters in Vienna to advise the Joint FAO/ IAEA Division of Nuclear Energy in Food and Agriculture. Twenty-five scientists representing 13 countries attended the meeting, which took place from 25 to 28 August 1969. This report gives the results of their deliberations.

CONTENTS

I N T R O D U C T I O N 9

EVALUATION OF NPN SOURCES IN FEED 10

DIRECT EVALUATION — ANIMAL RETENTION STUDIES 11

INDIRECT ASSESSMENT A N D PREDICTIVE METHODS 12 Evaluation of protein synthesis in the rumen 13

Evaluation of protein synthesis beyond the rumen 16

1 5N FOR THE STUDY OF NPN 18

ANALYTICAL TECHNIQUES 19 Mess spectrometry 19

Emission spectrometry 20

CONCLUSIONS A N D RECOMMENDATIONS 20

REFERENCES 25

List of Members of the Consultants' Group 27

INTRODUCTION

The diet of many people in the world is seriously deficient in protein and a number of systems designed to alleviate the problem are being investi-gated. An ancient but still economically attractive method makes use of ruminants, which can convert poor-quality protein and non-protein nitrogen ( N P N ) to high-quality protein for human food. This feature, which is unique to ruminants among the food-producing animals, has prompted much opti-mistic speculation about the addition of NPN to protein-deficient forages and fibrous trash to produce meat and milk. In practice, serious problems have been encountered. Although it has been clearly shown that urea and other NPN sources can be beneficial in ruminant feeding under certain conditions, the restrictions on protein synthesis from urea by micro-organisms in the rumen are severe. The nutritional and biochemical limits must be considered and understood more completely if NPN is to contribute effectively to im-proving animal production. When nutritional limitations are considered, it is possible that in many situations ruminants will be enabled to balance their protein requirements by consumption of urea, or other cheap sources of NPN.

Few areas of ruminant nutrition have received as much attention as the utilization of NPN. Urea and other NPN compounds are of interest because they generally supply nitrogen at less cost than plant proteins. Al-though many factors have been shown to affect the efficiency with which ruminants utilize it, urea has been successfully fed with a wide variety of feeds and under a variety of production circumstances. Management practices which may improve the utilization of dietary urea include full feeding, frequent feeding, feeding urea following consumption of a portion of the diet, and thorough mixing of urea with the daily ration. Dietary factors which influence th^ degree of incorporation of the nitrogen from urea into protein include the digestible carbohydrate content of the diet, percentage of nitrogen in the diet as urea, and the mineral content of the diet. Also, ruminants experience a period of adjustment before urea, or other NPN compounds, are utilized to their maximum extent. ч

The purpose of the Consultants' Meeting was to consider how and in what instances isotopes could be used to simplify and improve the research procedures necessary to investigate the possibilities of use of NPN in the diverse ruminant diets found in developing countries. This report is designed to provide a review of the latest information on approved methods to evaluate the feeding value of NPN with particular emphasis upon those methods requiring isotopes. The nutritional and physiological factors concerned with feeding NPN are only briefly reviewed, since they have been adequately dealt with in recent years in the literature [1-3].

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EVALUATION OF NPN SOURCES IN FEED

Although there are a number of NPN compounds which have been fed to ruminants, urea at present is the only product of commercial significance. The research resulting from feeding trials with NPN compounds has been reviewed in a recent F AO publication [2].

Where low-protein grains, molasses, or other sources of carbohydrate form a substantial part of the ration there is a demonstrated scope for the use of NPN. The optimum level will be influenced by the nitrogen content of the feed and the solubility of NPN and is likely to be lower if the availa-bility of both carbohydrate and nitrogen is rapid.

The potential for using NPN in roughage rations is more difficult to assess and chemical analysis of such rations cannot as yet provide an adequate assessment. If the digestibility is generally low, the nitrogen content per unit of fermentable carbohydrate may well be adequate. In some experiments, however, an increase in digestibility and voluntary intake has resulted from urea administration. Thus, preliminary experiments should be conducted in these circumstances to determine whether an increase in intake occurs as a result of NPN administration. The level of ammonia in the rumen may be used to indicate a deficiency of nitrogen for microbial use.

Feeding trials will always be necessary to evaluate the efficiency of use of NPN additions to diets, and the reproductive performance, growth and milk production are the ultimate measures of the value of the ration. Because feeding trials are considered unsophisticated and require little scientific equip-ment, less attention is often given to their planning than is warranted: feeding experiments must be carefully planned to demonstrate clearly and unequivo-cally any differences due to the addition of NPN to the diet.

Wherever possible, more than one level of comparison should be imposed, including appropriate control diets. The importance of appropriate experimental designs has been discussed in detail by Balch [4], who outlines an experimental approach which makes it possible to interpret results in economic terms.

For effective evaluation, an adequate period must be allowed for ani-mals to adapt to the diet. It is impossible to state arbitrarily the optimjum duration for feeding trials. It should be recognized that in evaluation of production responses it is most desirable to make observations throughout most of the lactation period of dairy cows or throughout the growth period of replacement females or meat-producing animals. The hazards of applying conclusions drawn from short-term trials are well known to animal scien-tists.

Research programs may proceed through a number of investigatory steps, and it is important to remember that pen trials for examining specific aspects of NPN utilization are conducted under conditions of control which almost always differ in a significant way from conditions of application in the field. Not only production response, but also the level of intake and feeding behaviour may be important criteria in defining problems in the utilization of NPN.

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Diets must be systematically sampled at the point of feeding and analysed by standard laboratory techniques. Calculations should be based on such analyses and not on composition predicted from feed tables.

Feed processing can influence NPN utilization to a significant extent. The size of particles of feeds can influence feed intake and its digestion, especially of roughages. Thorough mixing of all dietary components is essential for proper utilization of NPN-containing diets, which can be toxic if not uniformly mixed. Some special treatment.^, which prevent caking of urea during storing will also improve mixing. For the routine assessment of the degree of mixing and homogeneity of mixed feeds, radioactive isotopes such as 24Na and 32P have been used in Yugoslavia.

To decrease the rate of NPN break-down in storage and the release rate of ammonia in the rumen, urea and other NPN compounds have been bound to the gelatinized starch in concentrates. Such treatment might im-prove utilization of NPN by ruminants, enhance the palatability of diet, and decrease the danger of toxicity. Investigation of the utilization of such treated carbohydrate feeds is required and recommended.

Alkaline treatment can in some conditions improve digestibility and utilization of roughages high in crude fibre. Studies involving the combination of NPN and alkali-treated fibrous feed would seem to be desirable.

Radiation treatment of fibrous feeds is a potential method of improving the nutritional value by making the carbohydrate more rapidly available to rumen microbes. However, there seems little likelihood that the high levels of radiation required can ever be furnished at a cost to be economically feasible.

DIRECT EVALUATION — ANIMAL RETENTION STUDIES

Balance studies employed to evaluate NPN sources and to determine the nitrogen requirements of ruminants rely on two alternative methods:

( 1 ) Retention of nitrogen may be derived as the difference between the amounts of nitrogen ingested and excreted.

( 2 ) The amount of nitrogen stored in the body may be determined by direct analysis of the slaughtered animal, or indirectly by predictive methods.

Assuming that all specific vitamins and minerals needed are in the diet, nitrogen storage depends upon both the amount of amino acids and of energy absorbed. For this reason, investigations of efficiency of utilization of NPN probably need to include three or more levels each of nitrogen and energy, chosen within the effective range [4].

Since nitrogen retention is determined as the difference between intake and excretion, it is apparent that any loss of excreta for analysis tends to result in an overestimate of the true retention of nitrogen. Detailed infor-mation on the design and conduct of nitrogen balance trials can be found in most text books on animal nutrition.

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Direct determination of nitrogen retention requires chemical analysis of the whole body of the experimental animal. This technique can also yield energy retention data. However, slaughter and chemical analysis is tedious, expensive and obviously impractical for experiments with lactating and breeding animals.

Much research effort has gone into indirect methods of estimating the body composition of animals and most of the preferred methods involve the use of isotopes [5]. Total body water can be determined readily and quite accurately by either of the hydrogen isotopes, tritium or deuterium. From total body water can be derived an estimate of both total body fat and the residue, frequently called lean body mass. Rumen water can be estimated with the isotopes of soluble but essentially unabsorbed elements (51Cr, 144Ce). Indirect estimates of body protein can be obtained from body potassium estimates derived either from 42K dilution studies or 40K deter-minations in a whole-body counter [5]; however, these techniques are not as well developed as the tritium dilution method.

Total body nitrogen has been determined in living mice with good accuracy by means of neutron activation analysis [6]. It is conceivable that this technique could be applied to farm animals, although a fast neutron source and whole-body counter would be required. A method, however, that has the potential of supplying rapidly a direct measure of total body nitrogen in the live animal is certain to be given serious consideration.

INDIRECT ASSESSMENT AND PREDICTIVE METHODS

Because of the long-term nature of balance and feeding trial studies and the necessity for substantial animal facilities, the need exists for tech-niques to assess the likely efficiency of utilization of NPN added to ruminant diets, without making direct measurements of ultimate production, i.e. growth, lactation. It is advantageous on economic grounds to use relatively small animals. It must be emphasized that such assay procedures are only of bene-fit in screening diets and will only suggest negative or positive responses to NPN supplementation. The ultimate test will still be production by the ruminant animals.

The main means of evaluating the utilization of NPN are as follows:

( 1 ) Digestion in the rumen: (a) Rate of NPN hydrolysis (b ) Ammonia concentration in rumen contents ( c ) Rate of synthesis of microbial protein — the key process.

(2 ) Metabolism in the animal: (a) Ammonia concentration of blood plasma ( b ) Hepatic conversion of ammonia to urea (c) Free amino acids of the blood plasma (d ) Source of tissue proteins (e) Source of milk proteins.

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Evaluation of protein synthesis in the rumen

Isotopes are a unique tool for studying rumen metabolic processes. C-labelled compounds have been used extensively (e.g. Refs[7] and [8]) in

tracing the conversion of 14 С substrates to volatile fatty acids (VFA) by the rumen microbiota and the synthesis and utilization of individual amino acids by pure cultures of rumen bacteria (e.g. Refs [9], [10] and [11]). Many appli-cations for1 4C-, 35S-, 3H- and 15N-labelled compounds in studies o f pathways and in the estimation of pool size and turnover rates of rumen intermediates are cited in Refs [12] and [13].

Of equal importance is the quantitative measurement of microbial pro-tein synthesis in the rumen, or growth of the rumen microbial mass. Although the final efficiency of protein production in the ruminant is measured by the classical methods of growth and lactation experiments the study of rumen microbial protein synthesis per se is of particular interest (a) in understanding its nature as well as the relationship between microbial population and host animal, and ( b ) because methods for measuring this process offer a unique chance of assessing and/or predicting the efficiency of diets in short-term experiments in vivo and in vitro.

The main methodological problems result from the fact that the rumen is a complex open system. Thus ammonia, the principal (but not the sole) nitrogen source for microbial growth is not only produced from dietary pro-tein and NPN compounds but is also rapidly absorbed and recycled. Further-more, the microbial cells are continuously eliminated from the rumen by the passage of digesta and cannot be separated quantitatively from plant debris. Except for protein-free diets, special techniques are therefore needed for determining microbial protein in the presence of dietary protein. Various experimental approaches have been used to overcome these difficulties.

Estimation of microbial protein synthesis from the fermentation process

Various studies have confirmed that microbial growth is limited by the energy available to the microbes. The energy gain of rumen microbes is intimately related to the amount of VFA produced, thus the microbial protein synthesis can be calculated from the rates of VFA production.

In sheep fed on hay about 2 moles of energy-rich phosphate bonds ( A T P ) were estimated for each mole of mixed VFA produced during fermen-tation [14]. The yield of ATP was 10.5 g cell dry weight per mole ATP in pure cultures [15], but may be somewhat higher in the mixed rumen popu-lation [16]. Thus about 21 g cell dry weight may arise from each mole of VFA produced. Since the protein content of bacterial dry cells is given as 6 0 % [17] to 65 % [12], 12.6 - 13.7 g protein per mole VFA may be obtained.

The microbial cell synthesis under optimum conditions is limited by the energy availability. During rumen fermentation this energy is limited by the thermodynamic implications of anaerobiosis. These conditions imply that the micro-organisms can only extract a small amount of energy from carbo-hydrate and limit the amount of cell synthesis per unit of carbohydrate fer-mented.

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From the available literature it would seem that the maximum synthesis of cell nitrogen is in the region of 1.6 g N / 1 0 0 g of carbohydrate fermented. In most rations given to ruminants in temperate countries the amount of nitrogen in the food is in excess of this and therefore no response can be expected.

The rates of rumen VFA production can be measured by isotope di-lution methods if HC-labelled VFA are infused into the rumen and successive samples of the liquid phase of rumen contents are removed. Techniques and results have been reported in Refs [18-22]. The net gain of free energy (ATP) which is available for microbial growth is calculated from the known pathways of VFA formation and the quantitative fermentation data. By additional esti-mates of the yield of ATP (g dry weight of microbial cells per mole ATP) and of the protein content of the microbial cells the amount of microbial protein synthesized per mole of VFA produced is obtained.

Other methods of measuring VFA production have been reviewed in Ref. [23]. The 14C method is the only one which can be applied in vivo and even in animals on pasture. This is a clear advantage over non-isotope tech-niques which are mainly based on the zero time rate method of Hungate. For routine application the yield of ATP under various metabolic rumen con-ditions and the proportion of protein in microbial cells need further study. Certainly, the VFA rate is an indirect means of measuring protein production but it is a useful in-vivo method for field experiments requiring minimal dis-turbance of the animal [22].

Measurement of protein synthesis from 35 S incorporation from sulphate into bacterial protein

As introduced by Henderickx [24, 25], 35SOI* can be used as a measure of microbial protein synthesis from incorporation of the label and establishing the N / S ratio in the microbiota. This method was successfully applied with a rumen fluid system incubated in vitro, to which defined amounts of nutrients were added.

The method involves a multiple-step reaction for sulphate reduction to sulphide (3 5SO!~^— 3 5SOi" —— 35S2"), which is then incorporated into thioamino acids of bacterial cells. The reducing steps may limit 35S2_ availa-bility and make interpretation of the reaction kinetics difficult. The technique may not easily be applied to whole rumen contents because of microbial cells clinging to plant particles which are not included in analysis. Even though quantitative data may not be obtained the method offers the advantage of simplicity for rapid comparative screening in vitro.

Walker and Nader [16] adapted the 33 S method for application to the measurement of microbial protein synthesis in whole rumen contents in-cubated with Na235S in a completely closed in-vitro system. The 35S2~ was used for labelling the sulphide pool and the incorporation of the label into microbial cells was calculated from the size and rate of dilution of the pool together with the radioactivity measured in the microbial cells and the N / S ratio of microbial proteins. Except for a still unexplained non-enzymatic initial binding of label ( 2 0 % during the first 20 min) the incorporation of

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35S2 into microbial protein followed first-order kinetics, Cystine and methio-nine in the protein were found to be heavily labelled and 3oS recovery was 96 — 9 9 % .

Analyses of the N / S ratio in bacteria and protozoa collected by fraction-ated centrifugation and washing procedures of squeezed rumen fluid resulted in almost identical values of 10.7 and 11.0 respectively (which is considerably lower than the ratio of about 25 found in animal proteins). The protein synthesized is calculated from incorporated 35 S by further multiplication by the factor 6.25.

By simultaneous determination of VFA production the efficiency of energy utilization for microbial growth can be estimated. With incubation times of 3, 11 and 20 hours, 8.5 g, 14.4 g and 12.8 g cell dry weight per mole ATP, respectively, were obtained, indicating that the yield of ATP of 10.5 g derived from pure cultures is obviously exceeded in mixed microbial populations. A possible reason for this may be methane synthesis, which is an exergonic process and was not measured in the pure culture studies cited above.

It would appear desirable to adapt this method to incubation times of a few hours since fermentation and hence energy production for microbial growth may change during long-term incubation. Even though this method needs considerable experience it would seem to be the most accurate one for the quantitative measurement of rumen microbial protein synthesis in vitro. The combination of this in-vitro method with the in-vivo technique for VFA production can be used to confirm the factor of protein synthesized per mole VFA produced under various nutritional conditions. Possibly the technique can be adapted to the use of 1 5NH? incorporation as a measure of protein synthesis.

Other application of isotopes to the measurement of protein synthesis in vitro

Phillipson et al. [26] have shown that the label of 15N ammonium chloride incubated with rumen fluid in vitro is concentrated in the trichlor-acetic acid precipitate while the ammonia concentration decreases markedly. Quantitative data on protein synthesis cannot be derived from a rumen fluid system; however, it is worth noting that a substantial increment of 15N in'the protein was obtained in the reasonably short incubation period of 2.5 h. l 0 N may turn out to be a more sensitive indicator for protein synthesis than 33S.

\ ,• •

The suggestion is therefore made to use a closed system in vitro like that of Walker and Nader [16], with whole rumen contents for short in-cubation periods and with 15NHJ for labelling the ammonia pool . According to our present knowledge, short incubation periods of 2 - 3 h ,in a closed system will not markedly affect the fermentation process; long periods may affect it. In rapid fermentations, which may occur with rumen contents of animals on high grain or sugar diets, excess fermentation acids could be eliminated with ion-exchange resin in a dialysing sac included in the closed vessel system.

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Williams [27] has applied 15N in vivo and in vitro in semi-purified diet feeding studies. The in-vitro system was of a continuous flow type which allowed for continuous feeding of nutrients and buffers and for removal of excess population. Within 3 h after introduction of 1 3NHÍ the proportion of label concentrated in the micro-organisms was 6 7 % in vivo but only 24 % in vitro. Glutamic acid, alanine, and aspartic acid were the most highly labelled. Even though the in-vitro system did not respond like the animal, it indicates that short-term incubation may be useful and that 15N is of value in an in-vitro system.

Evaluation of protein synthesis beyond the rumen

The synthesis o f protein from NPN is almost completely restricted to the forestomachs and this protein is digested in the small intestine as in monogastric animals. Several surgical techniques have been developed in recent years which permit entry to the reticulo-rumen and the intestine. Because of continuous flow of materials into and out of the gastrointestinal tract, the concentration of protein or any other metabolic product found in digesta at a given time is the result of synthesis, dilution, absorption and rate of passage. An increase in rate of passage of digesta, for instance, can partially make an increase in microbial protein synthesis.

Omasal and abomasal fistulae have been developed to allow collection or sampling of digesta flowing from the reticulo-rumen for measurement of yield of protein and digestive changes taking place in these organs.

The use of the. re-entrant cannulae in the duodenum is especially valuable since unequivocal records of digesta flow can be obtained over the period of the experiment. When indigestible reference substances are included in the ration or infused into the rumen at constant rates, the observed flow, measured over a relatively short time, can be adjusted to an assumed average flow to provide 100% recovery of the reference substance. The use of refer-ence substances also makes it possible to use animals with single cannulae in various parts of the small intestine instead of re-entrant cannulae which are more difficult to maintain. However, because of the patterns of digesta move-ment in the intestine, reliability of methods used to sample the digesta passing any given point in the small intestine must be closely examined.

Each type of cannula and each point of fistulation have their own limitations which must be critically appraised with respect to the objectives of the experiment.

One most important difficulty encountered in the interpretation of the results is in estimating the proportional contribution of bacterial protein to the total protein in the intestinal contents. Simultaneous analysis of meso-diaminopimelic acid, a characteristic amino acid of almost all the rumen bacteria, is one possible means of overcoming this problem. NPN com-ponents can be labelled with 15N, or 3 5 Scan be used to evaluate the amount of bacterial protein leaving the rumen.

The success of this technique depends on having a satisfactory method for determining microbial protein in the presence of dietary protein and on the proper operation of sample collection. Diaminopimelic acid ( D A P ) has

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been used as a marker for bacterial protein [28, 29] but does not indicate protozoal protein. Furthermore, DAP is restricted to Gram-negative bacteria and its content varies gready between species. Thus, the DAP content may be increased considerably under certain feeding regimes [30] and DAP will only serve as an internal marker under standardized conditions. So 15N or 35S techniques for labelling microbial protein may offer distinct advantages if used with the above collection method.

Current interest in "protected proteins and amino acids" for ruminants means that NPN might be used with such feeding systems if the protein available to rumen microbes is limited.

Conrad et al. [31] have estimated the synthesis of methionine by feeding sodium or barium sulphide— 35S to.lactating cows adjusted to con-trolled levels of constant feed intake by measuring the specific activity of methionine in milk protein. A single methionine pool undergoing simple dilution was indicated and net methionine could be resolved into synthesized and food methionine by regression procedures.

By determining the proportion of methionine in the rumen microbial protein this method could possibly be adapted to estimating microbial protein synthesis with re-entrant cannulae.

Apart from the well known studies of East Germans workers with 15N (see footnote 1 to the next Section), this isotope was also used by Narozny and Sommer [32] in studies on urea-N assimilation in the protein synthesis of fattening bulls. The half-times of 15N incorporation into the rumen microflora in two animals were 1.4 and 3.2 hours, respectively.

The interpretation of 13N data obtained from animal experiments is complicated by N recycling into the rumen. In this study the 15N secreted with the faeces accounted for 11 % of 15N intake and was ascribed to in-digestible N of micro-organisms. This is considerably lower than 15% and 16 % 15N excretion in faeces reported in Ref. [33] for dairy cows.

This clearly indicates that besides the synthesis of rumen microbial protein the digestion of the microbial mass deserves particular attention. Here 15N and 33S labelling is certainly of considerable importance. In this connection, it will also be necessary to consider the secretion of N into t1 e lower gut and its binding by intestinal bacteria which may simulate indi-gestible N of rumen microbes.

Cocimano and Leng [34] used 1JC-labelled urea as a means of as-sessing the nitrogen status of animals. More recently, Leng [22] has des-cribed a similar technique using 15N-labelled urea.

Applications of the isotope dilution technique have been proposed as a tool for evaluating the efficiency of various diets for the synthesis of protein, principally in the form of milk. Black [35] has outlined a scheme involving pulse labelling of plasma with a 14C-labelled indispensable animo acid. Tritiated amino acids are equal in usefulness to "C-labelled acids and much lower priced. Double-labelled compounds may also be used to clarify meta-bolic processes.

An additional contribution that isotope techniques could make in NPN studies is for the evaluation of mineral requirements. In many cases, diets

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containing NPN supplements may require specific investigation of adequacy of minerals.

The importance of sodium and potassium buffers in purified diets has been described by Bunn and Matrone [36]. Radioisotopes, of course, are uniquely suited to almost any study of mineral metabolism.

Finally, there is an important role for radioactive reference substances which are unabsorbed by the animal and permit calculation of flow rates, absorption and dilution changes in the gut. Such measurements have many applications in N P N studies as well as most nutritional experiments; for example, Miller et al. [37] have described the use of 144Ce, and Hogan [38], among others, has used J1Cr complexed with EDTA.

! 5N FOR THE STUDY OF NPN

15N is the only useful stable isotope of nitrogen. Being non-radioactive, — a distinct advantage under certain conditions — it requires different tech-niques for separation, handling and detection, from those using radioisotopes.

It is the most valuable isotope for NPN studies, and to obtain some data (e.g. on ammonia production) it is the only choice. 15N is also neces-sary for studying the nitrogen cycle operating in the ruminant. A disadvantage of 15N is that the insensitivity of the assay techniques using it (about 106

times less sensitive than 14C) makes it almost impossible to use the isotope as a true "tracer". In most instances, an increase in the level of nitrogen compound cannot be avoided, or special procedures must be used to replace part of the N material normally present with 15N material.

Workers at Leipzig have performed very thorough studies of nitrogen metabolism in dairy cows by feeding 15N-labelled ammonium bicarbonate. The series of fourteen papers1 published by this group indicates the variety of questions which can be resolved by using this isotope. The incorporation of 15N added as NPN into microbial protein has been demonstrated by Clark, Ellinger and Phillipson [39], Boggs [40] and Abe and Kandatsu [41] The incorporation of 15N from labelled NPN has been demonstrated in animal tissues by Watson [42], Piva and Silva [43], Schonemann and Kilian [44] and Narozny and Sommer [32].

The major problem in interpreting results of 15N studies arises from the fact that both microbial and animal tissues have the capacity for rapid and almost universal transamination. This includes all dispensable and non-dispensable amino acid with the exception of lysine, which can be deaminated by microbes. Thus, the presence of 13N in any amino acid, other than lysine,

1 ) 4 The papers are written by several authors and appear in a single issue of "Archiv fiir Tierernahrung" Vol. 13, 1963, pp. 263-443 under the general title "Untersuch-ungen zum N-Stofiwechsel beim laktierenden Rind unter Verwendung von oral verabreichtem Harnstoff 15N."

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is not necessarily indicative of de-novo synthesis. This problem is further com-plicated by hepatic synthesis of dispensable amino acids with a consequent dilution of the atom excess per cent of 15N. Virtanen [30] has discussed this relative to his data. 15N present in positions other than the alpha-amino group also increases the problems of interpretation.

ANALYTICAL TECHNIQUES

The IAEA Seibersdorf Laboratory has been involved for a number of years in the application of 15N to agricultural research. General emphasis has been placed on N assimilation in plants, with recent development concerned with protein biosynthesis and the role of various factors which affect plant protein production. In this recent work, emphasis has been placed on the application of 15N in biochemical studies. This work was greatly facilitated by refinements of the emission spectrophotometric techniques which were carried out by IAEA personnel. The technique as now developed permits routine analysis for 15N content of microgram quantities of total N. The method has proven very useful for plant studies and it should find application to many of the animal nutrition problems.

Two methods are available for the determination of 15N abundance in nitrogen gas. Most investigators in the past have used the mass spectrometer which requires at least 0.5 mg of total N2 gas. Emission spectrometry can deal with samples of a few micrograms o f N and even fractions of a microgram of N when special gas preparation techniques are followed [45, 46].

Mass spectrometry

The sample is digested by the Kjeldahl method, NH4 is distilled off into excess HC1. As soon as the titration is completed, the solution is acidi-fied with HC1 and evaporated to a few millilitres volume on a hot plate. An aliquot of the concentrated solution containing approximately 1 mg N is then transferred into one side of a Rittenberg vessel and an alkaline solution of sodium hypobromide is added to the other side. The Rittenberg vessel is connected to a vacuum line with a liquid air trap, to remove the air from the system. After evacuation the sodium hypobromide is poured into the acid extract which reduces the NH4 to N2 gas. The sample is then prepared for introduction into the mass spectrometer.

When the nitrogen content of the sample need not be determined by the Kjeldahl digestion method, the dry sample is mixed with CaO and CuO in a glass tube, evacuated and sealed. The glass ampoules are heated in a muffle furnace to transform the nitrogen in the sample into N2 gas (Dumas principle). The gas samples in Rittenberg flasks are cooled with liquid air and connected to the inlet system of the mass spectrometer. Samples that have been digested according to the Dumas reaction in glass ampoules are

19

inserted into metal cylinders and connected to a rotating cylinder for auto-mated introduction to the mass spectrometer. Holes in the sides of the cylinder permit a metal rod, operated by a switch, to break the ampoule and introduce the N gas into the inlet system.

Emission spectrometry

In principle, the preparation of samples for emission spectrometry is similar to that for mass spectrometry. However, the total quantity of nitrogen involved is much smaller. In particular, when dealing with samples of less than 1 /itg N, there is likely to be contamination with nitrogen from air, filter paper, chemicals and nitrogen adsorbed at glass container walls, if the necessary precautions are not taken. Since the quantity of N2 in which the 15N abundance can be determined is so small, the method can be used for the determination of 15N in fractions separated by paper or thin layer chro-matographic techniques. Blanks and 15N-labelled standards should be used to check for possible contamination of samples.

Specially designed quartz tubes about 20 cm long are heated to about 800 °C to remove adsorbed N. The sample, mixed with the CuO and CaO (often in the form of a pellet), is inserted. The CuO and CaO are heated and the tube is evacuated on a vacuum line. After evacuation, the tubes are sealed and heated in a muffle to liberate N2 by the Dumas reaction. The tubes are now ready for emission spectrometric determination of 15N abundance.

When the samples contain less than 1 /J.g N, the tubes have to be treated with xenon to block the adsorption of N2 gas to the walls. Since xenon prevents the later excitation of the N2 gas by high-frequency discharge, helium is added. Helium lowers the excitation potential in the presence of xenon for reasons not yet fully understood.

The quartz tubes filled with N2 gas are excited, using a 100-megacycle generator (50 watts). By means of a quartz spectrograph the bandheads 14N 14N at 2977 Â and 14N 15N at 2983 A are separated for the determination of the 15N abundance.

The photographic plate used with the spectrograph is replaced in emission spectroscopy by photomultiplier tubes. An amplifier-recorder enables the recording of N28 and N29 peak heights, and the calculation of the per-centage 15N abundance is similar to that used in mass spectrometry.

CONCLUSIONS AND RECOMMENDATIONS

The Consultants' Group considered the effect of environmental factors on the opportunities and problems of NPN application in ruminant industries. It was agreed that the final development of NPN application, and of associ-ated research programs, would have to be based on the prevailing ecosystems and ruminant production patterns. It was recognized that environmental

20

factors could be identified regionally and on the basis of this information it would be possible to decide in what cases NPN application would be advan-tageous. The situation and prospects were seen to be as follows.

1. There is a definable role for NPN in the feeding of ruminants. NPN can be used as an admixture in concentrate rations for milk and more intensive beef production.

2. In most developing countries, use of urea or other NPN sources in combination with agricultural and industrial by-products is, and will continue to be, an important possibility to be considered. For example, NPN in combination with molasses has particular potential.

3. The economic feasibility of NPN application must be carefully evalu-ated, particularly in countries where the relatively expensive high-energy materials would have to be introduced into ruminant feeding.

4. Some developing countries have a relative abundance of by-products rich in protein, and NPN generally could not be recommended under such conditions.

5- Low production levels may be basically caused by disease or poor livestock management, and studies on the application of NPN are not recommended until these factors are under control.

6. In addition to urea, other courses of NPN compounds should be investigated. Biuret has the advantage of being non-toxic; in Poland, ammonium lactate has been produced efficiently from molasses; feed-grade ammonium propionate produced from the by-products of petrol processing is promising. Ammoniated feeds in general have not been important sources of NPN, although many products have been investi-gated. The isotope 15N is suggested for the study of the stability and chemical form of NH3 in ammoniated feeds.

7. It is apparent that in many situations in developing countries where additional dietary nitrogen is in greatest need, experimental results indicate that N P N will be poorly utilized and may be dangerous to feed. However, the potential is so important that additional research can be recommended with the object of improving NPN use with fibrous feeds.

The Consultants' Group outlined research methods which would be appropriate for use in evaluating NPN sources added to any locally formu-lated ration. Emphasis was placed upon the role of isotopic labelling to simplify animal experiments. Some relevant applications of radioisotopes are also presented in the proceedings of two IAEA symposia: 'Radioisotopes in Animal Nutrition and Physiology' (1965) and 'Isotope Studies on the Nitrogen Chain' (1968) .

21

The possible application of isotopes to research on evaluating NPN in ruminant rations is summarized below.

Application

( 1 ) Rate of NH3 production in rumen (2 ) Rate of incorporation of N compounds into

microbial protein ( 3 ) Overall conversion of NPN to tissue or milk

protein

( 1 ) Rate of hydrolysis of C-containing NPN ' ( 2 ) Turnover and entry rate of amino acids ( 3 ) Estimates of protein synthesis

( 1 ) Estimates of microbial protein synthesis ( 2 ) Estimates of microbial contribution to

synthesis of milk, wool , muscle

Body composition

Indicator to study digesta flow rates, dilution rate and extent of absorption of amino acids.

Recommendations to FAO and IAEA

Recognizing the need for high quality protein in human diets and recognizing that NPN can contribute to narrowing the protein gap, the follow-ing recommendations were addressed by the Consultants' Group to the Director General of FAO and to the Director General of IAEA.

The Group:

1. Recommend that FAO assists in the dissemination of the latest research information on NPN to animal nutritionists of member countries.

2. Recommend emphasis upon research designed to make possible the use of N P N to meet protein requirements of ruminants subsisting upon low-protein high-fibre forages.

3. Suggest that the Joint FAO/1AEA Division of Atomic Energy in Food and Agriculture co-ordinate world-wide studies that involve the use of isotopes in NPN research designed to increase animal protein produc-tion.

Isotope

1 5 N

14 С

35 S

3 H , 2 H , 4 0K, 13N

51Cr EDTA and 144Ce

22

4. Suggest further the co-ordination of research programs involving the use of isotopes to study all phases of animal protein synthesis and metabo-lism in domestic animals.

5. Recommend that the Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture make available information on the use of isotopes for N P N Research with particular emphasis upon 15N. Further, it is suggested that facilities be made available preferably at the IAEA Labo-ratory for training in 15N techniques especially those involving the relatively new method of emission spectroscopy.

23

R E F E R E N C E S

BRIGGS, M.H. (Ed.), Urea as a Protein Supplement, Pergamon Press, London (1967). LOOSLI, J.K., McDONALD, I.W., Nonprotein Nitrogen in the Nutrition of Rumi-nants, FA О Agricultural Series No . 75 (1968). COMMONWEALTH BUREAU OF ANIMAL NUTRITION, Use of Urea and Am-monium Compounds for Ruminants, Annotated Bibliography No. 4, Bucksburn, Aberdeen, Scotland (1968). BALCH, C.C., World Rev. Anim. Prod. 3 ( 1967) 84-91. REID, J.T. (Ed.), Body Composition in Animals and Man, Publ. 1598 Nat. Acad. Sci. Wash., D.C., (1968) . NAGAI, T., J. nucl. Med., 10 4 (1969) 192-196. BALDWIN, R.L., W O O D , W.A., EMERY, R.S., J. Bact. 85 (1963) 1346. WALLNOFER, P., BALDWIN, R.L., STAGNO, E., Appl. Microbiol. 14 (1966) 1004. ALLISON, M.J., BRYANT, M.P., DOETSCH, R.N., Arch. Biochem. Biophys. 84 (1959) 246. ALLISON, M.J., BRYANT, M.P., DOETSCH, R.N., J. Bact. 83 (1962) 523. ALLISON, M.J., BRYANT, M.P., Arch. Biochem. Biophys. 101 (1963) 269. HUNGATE, R.E., The Rumen and its Microbes, Academic Press, New York & London (1966). BRUGGEMANN, J., GIESECKE, D „ in Urea as a Protein Supplement, Pergamon Press, London (1967). WALKER, D.J., in Physiology of Digestion in the Ruminant, Butterworths, London (1965). BAUCHOP, T., ELSDEN, S.R., J. Gen Microbiol. 23 ( i 9 6 0 ) 457, 469. WALKER, D.J., NADER, C.J., Appl. Microbiol. 16 (1968) 1124. LURIA, S.E., in The Bacteria, Vol. 1, Academic Press, New York & London ( i 9 6 0 ) . GRAY, F.V., WELLER, R.A., PILGRIM, A.F., JONES, G.B., Austral. J. agrie. Res. U (1966) 69. GRAY, F.V., WELLER, R.A., PILGRIM, A.F., JONES, G.B., Austral. J. agrie. Res. 18 (1967) 625. WELLER, R.A., F.V., PILGRIM, A.F., Brit. J. Nutr. 21 («1969) 97. WELLER, R.A., GRAY, F.V., PILGRIM, A.F., JONES, G.B., Austral. J. agrie. Res. .18 (1967) 107. LENG, R.A. (1969) (unpublished). WARNER, A.C.I., Nutr. Abstr. Rev. 34 ( 1964) 339. HENDERICKX, H.K., Vlaams Diergeneeskundig Tijdschrift 28 ( 1959) 80. HENDERICKX, H.K., Arch. Int. Physiol. Biochim. 69 (1961) 449. PHILLIPSON, A.T., DOBSON, M.J., BLACKBURN, Т.Н., BROWN, M., Brit. J. Nutr. 16 (1961) 151. WILLIAMS, H.H., Proc. Cornell Nutr. Conf. Feed Manufactures (1958) 133. WELLER, R.A., GRAY, F.V., PILGRIM, A.F., Brit. J. Nutr. 12 (1958) 421. EL-SHAZLY, K., HUNGATE, R.E., Appl. Microbiol. 14 (1966) 27. VIRTANEN, A.I., Science 153 (1966) 1603. CONRAD, H.R., MILES, R.C., BUTDORF, J. Nutr. 91 (1967) 337. NAROZNY, J., SOMMER,A., Biologizace a Chemizacei (1968) 456. ULBRICH, M., SCHOLZ, H., Arch. Tierernahr. 5_( 1963) 296; 6 (1963) 366. COCIMANO, M.R., LENG, R.A., Brit. J. Nutr. 21 (1967) 353. BLACK, A.L., in Isotope Studies on the Nitrogen Chain (Proc. Symp. Vienna, 1967), IAEA, Vienna (1968) 287. BUNN, Clara R., MATRONE, G., J. Nutr. 95 (1968) 122. MILLER, J.K., PERRY, S.C., CHANDLER, P.T., CRAGLE, R.G., J. Dairy Sci. 50 ( 1967 )355 . H O G A N , J.P., Austral. J. agrie. Res. 15 (1964) 384. CLARKE, Eileen M.W., ELLINGER, Gabrielle M „ PHILLIPSON, A.T., Proc. Roy. Soc. Series В 166 (1966) 63.

25

[40] BOGGS, D.E., Ph.D. Thesis, Cornell University, Ithaca, N.Y. (1959). [41] ABE, M., KANDATSU, M „ Arch. Tieremahr. 18 (1968) 247. [42] WATSON, C.J., Sci. Agrie. 29 (1949) 185. [43] PIVA, G., SILVA, S., in Isotope Studies on the Nitrogen Chain (Proc. Symp.

i Vienna, 1967) IAEA, Vienna (1968) 239. [44] SCHONEMANN, K., KILIAN, E.F., Arch. Tieremahr. 10 ( i 9 6 0 ) 37. [45] LEICKNAM, J.P., MIDDELBOE, V., PROKSCH, G „ Analyse isotopique de l'anote

par spectrométrie optique pour de faible teneur en 1 5N, Anal. Chim. Acta 40 (1968) 487.

[46] GOLEB, J.A., MIDDELBOE, V., Optical 1 5N analysis of small nitrogen samples with a mixture of helium and xenon to sustain the discharge in an electrodeless tube, Anal. Chim. Acta43 (1968) 229.

26

LIST OF MEMBERS OF THE CONSULTANTS' GROUP

Annison, E. F.

Black, A.L.

Chomyszyn, M.

Egan, A .R .

Giesecke, D .

Halama, A. K.

Hill, K.J.

van 4 Klooster, A :Th .

Kronfeld, D .

Animal Research Division, Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford, United Kingdom

Department of Physiological Sciences, University of California, Davis, California 95616, USA

Institute of Animal Physiology and Nutrition P. A.N., Jablonna (near Warsaw), Poland

Department of Agronomy, Waite Agricultural Research Institute, University of Adelaide, Glen Osmond, South Australia, Australia

Institut für Tierphysiologie der Universitát München, Veterinarstrasse 13, 8 München 22, Federal Republic of Germany

Feed Consultant, Promenade 25, A-2391 Kaltenleutgeben/Wien, Austria

Unilever Research Laboratory, Colworth House, ' Shambrook, Bedford, United Kingdom

Landbouwhogeschool, Laboratorium voor Fysiologie der Dieren, Haarweg 10, Wageningen, Netherlands . .

School of Veterinary Medicine, University of Pennsylvania, Kennett Square, R,D. 1,

.. Pa. .193.48, USA

27

Leng, R. A.

Leskova, R.

McDonald, J. W.

Moustgaard, J.

Oltjen, R. R.

0rskov , E. R.

Reid, J.T.

Rivière, R.

Shaw, J. C.

Shibata, Fumio

Stosid, D .

Department of Biochemical Nutrition, School of Rural Science, University of New England, Armidale, New South Wales 2351, Australia

Institut für Tierernàhrung, Milchhygiene und Lebensmittelkunde,

Tierârztliche Hochschule, Linke Bahngasse 11, A-1030 Vienna, Austria

Division of Animal Physiology, Ian Clunies Ross Animal Research Lab., CSIRO, P.O. Box 144, Parramatta, New South Wales 2150, Australia

Dept. of Physiology, Endocrinology and Bloodgrouping,

Royal Veterinary and Agricultural College, Copenhagen V, Denmark

Nutrition Investigations, Animal Husbandry Research Division, United States Department of Agriculture, Beltsville, Maryland 20705, USA

Rowett Research Institute, Bucksburn, Aberdeen, Scotland, United Kingdom

Department of Animal Science, Cornell University, Ithaca, N.Y. 14850, USA

Institut d'élevage et de médecine vétérinaire des pays tropicaux,

10 rue Pierre-Curie, ; F-94 Maisons-Alfort, France

Merck Sharp and Dohme Nederland N.V. , P.O. Box 581, Haarlem, Netherlands

Laboratory of Animal Nutrition, Nagoya University, Nagoya, Japan

Institute for the Application of Nuclear Energy in Agriculture, Veterinary Medicine and Forestry,

Baranjska 15, Zemun, Yugoslavia

28

Swan, H.

Tsuda, Tsuneyuki

Turek, H.

Verbeke, R.

Walker, D . M .

Yamaguchi, M.

Department of Agriculture, University of Nottingham, Sutton Bonington, Loughborough, United Kingdom

Faculty of Agriculture, Tohoku University, Kita-6-Bancho, Sandai, Japan

Hochschule für Bodenkultur, Gregor Mendelstr. 33, Vienna, Austria

Institute of Physiology, Faculty of Veterinary Sciences, Rijksuniversiteit, Gent, Casinoplein, Belgium

School of Agriculture, Sydney University, Sydney, New South Wales, Australia

Department of Agricultural Chemistry, University of Tokyo, Tokyo,Japan

REPRESENTATIVES OF FAO A N D IAEA

Broeshart, H.

Fried, M.F.

Groenewold, H.

Luse, R.A.

Nagai, T.

Scientific Secretary

Ward, G.M.

Division of Research and Laboratories, IAEA, Vienna

Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture,

IAEA, Vienna

Animal Production Branch, Animal Production and Health Division,

FAO, Rome

Joint FAO/ IAEA Division of Atomic Energy in Food and Agriculture,

IAEA, Vienna

Division of Life Sciences, IAEA, Vienna

Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture,

IAEA, Vienna

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