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'1 111 111111111 1111111111111 1111 1'111'1'1'1"111111"11'11111'1'1111111'11 .11 """ 11111I1 1111111 11111111111111111'1'1'11;?""11'11'F 1111I 1111111' '11 1I111111'12— 111“? ‘ 4121* ' 1 111111111 1117 «1: 11131;: ';T'_ .1. 1 ,1 , ,,.111111 11 ' ' = - 1111" 1111 '1111,'11111111','111'1 111111 11 111.1 111' 1 111111 1111111111 1111. 11111.1, 1111111111111 : . 1111* , -1' . , 11 I III I! ,_ 11' 3111111111111 1111111111 111 11111111111| 111111 1111,,11I11I11II1I1IIIII1I 1";1u5m1Ig1'-‘ I], '1' 1111111,- I . . , r 1.11. 1111 1 1 . 1: '1 ' ' 11.111 1 1.1,, 1:11:11'1'1111'1111'1'1111.111111111111111“ '11'1111" 11, Of I 1am _ 1'1'1'11111111111 “11114-111 mL'11111m111 5131111111191111151111'1111111111111011111' 1' 11 11:11 ‘ THESIS This is to certify that the thesis entitled PLASMA AMINO ACID RESPONSE TO INTRAPERITONEAL METHIONINE LYSINE AND CYSTEINE INJECTIONS IN HOLSTEIN STEERS presented by ROBE RTO TOWNS has been accepted towards fulfillment of the requirements for M. S. ANIMAL HUSBANDRY Jegree in / Wégaféw/L Major professor é/Jj/MM 0-7639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. PLASMA AMINO ACID RESPONSE TO INTRAPERITONEAL METHIONINE, LYSINE AND CYSTEINE INJECTIONS IN HOLSTEIN STEERS BY Roberto Towns A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry 1979 ABSTRACT PLASMA AMINO ACID RESPONSE TO INTRAPERITONEAL METHIONINE, LYSINE AND CYSTEINE INJECTIONS IN HOLSTEIN STEERS BY Roberto Towns Plasma amino acid (PAA) responses to IP amino acid in- jections in steers were studied in two experiments. In one eXperiment 20 g L—lys or 20 g L-met were injected for 14 days. In the met treatment, plasma met peaked by day eight and tended to accumulate. Plasma lys was not affected by met injections. In the lys treatment, plasma lys peaked at about day 8 and did not accumulate. Plasma met was not affected by lys injections. In the second experiment, sUb and supra Optimal levels of met and met plus cysteine were injected IP to evaluate the met requirement. PAA 2 phase response curves were evaluated using a two slope regression procedure. Two phase breakpoints occurred at an injection level of about 3.7 g met/day. Break- point values plus met in the abomasal flow indicated a met. requirement of 11.41 g/day. A met sparing effect upon administration of cysteine was not observed. ACKNOWLEDGEMENTS I would like to express my gratitude toward the fol- lowing pe0p1e for their help in making this work possible. My academic advisor, Dr. Werner G. Bergen.for his guidance throughout my studies and in the production of this thesis. Drs. John T. Huber and Melvin T. Yokoyama for their advice and constructive criticism as members of my graduate committee and especially for their interest in the improve- ment of this thesis. Dr. Ronald H. Nelson, Head of the Department of Animal Husbandry for making available the resources and facilities utilized in the experimental design. The National Autonomous University of Mexico, Faculty of Veterinary Medicine and Zootechnology (La Universidad Nacional Autonoma de Mexico, Facultad de Medicina Veterinaria y Zootecnia) for making available the funds for my M.S. pro- gram at M.S.U. To you Jessie, for your patient love and loving patience. To my mother for her understanding and financial support. Last but not least, I am deeply indebted to Diane Gartung whose patience and help made it possible to overcome the rigors of winter during the experimental work of this thesis. ii II. III. IV. TABLE OF CONTENTS INTRODUCTIODIO o O O 0 o o O o o o o o o o o O 0 LI TERATURE REVIEX‘J’ . g o g o g o o o o o o o o o A. Protein Metabolism in the Ruminant. . . . . Sulfur: Nitrogen Relationship in the Rumen. Postruminal Fate of Nitrogenous Compounds Rumen Microbial Protein . . . . . . . B. Plasma Amino Acids. . . . . . . . . Plasma Amino Acids in Ruminants . . C. Amino Acid Requirements of Ruminants Qualitative Studies . . . . . . . . Quantitative Studies. . . . . . . . I'MTERIALS AI‘TD I‘IETHODS . O . o o o o o o o o o o A. Experiment One. . . . . General Design. . . . . Sample Processing . . . Chemical Analyses . . . a. Plasma Lysine and methi b. Plasma urea nitrogen. . B. Experiment Two. . . . . General Design. . . . e Sample Processing . . . Chemical Analyses . . . . a. Plasma cysteine, lysin Statistical Analysis. . $0000.30... :3 QOOoooaoooo 05000000000. 50.00.0000. p. 030000000000 3‘ bid-0000000000 (D odoooooooooo O (D RESULTS 0 I O O I O O O O O O 0 Experiment One. . . . . . . . . . . . . . . . Plasma Amino Acid Respons to Long Term Ad- ministration of L-Methionine or L-Lysine. . Experiment Two. . . . . . . . . . . . . . . . . Plasma Amino Acid ReSponse to IP Injections of Amino Acids as Criterion of Amino Acid Requirements. . . . . . . . . . . . . . . . DISCUSSIOrIo o o o o o o o o o o o o o o o o o 0 Experiment One. . . . . . . . . . . . . . . . . Experiment Two. . . . . . . . . . . . . . . . . 24 24 24 27 27 27 28 28 28 31 31 31 31 32 32 32 4O 4O 50 SO 54 VI. VII. A. B. APPENDICES . BIBLIOGRAPHY Plasma Level ReSponse. Quantitation of the Methionine Requirement iv 54 57 64 68 LIST OF TABLES Table Page 1 Ration Used in the Experiments. . . . . . .25 2 Amino Acid Composition of Treatments in Experiment 30 Two 0 O O O O O I O O O O O O O O O I O 0 Mean and Standard Deviation of Plasma Methionine with IP Injection of 20 grams Methionine per day. .34 Mean and Standard Deviation of Plasma Lysine with IP Injection of 20 grams Lysine per day . . . . .35 5 Mean and Standard Deviation of Plasma Lysine with IP Injection of 20 grams Methionine per day . . . .38 6 Mean and Standard Deviation of Plasma Methionine with IP Injection of 20 grams Lysine per day. . . .39 7 PAA Response to IP Injections of Graded Levels ACO Of I‘Iethionine O O O O O O O O O l O O 41 8 PAA Response to IP Injections of Cysteine plus Graded Levels of Methionine . . . .45 9 Nitrogen and Amino Acid Passage in Steers Fed the 9. 5% Crude Protein Ration Utilized in this Experiment. 0 o o o o o o o o o o o o o o 58 10 Quantitation of Methionine and Total Sulfur Amino Acids . . . .60 11 Estimated Daily Essential Amino Acid Requirements of Growing Steers . . . . . . . . . . . . . . . . .63 Appendix Table 1 Plasma Urea Nitrogena Levels With IP Injections of 20 Grams Lysine Pgr day. . . . . . .64 2 Plasma Urea Nitrogen Levels With IP InjectiOns of 20 Grams Methionine Per Day. . . . . . .65 3 Individual Levels of Plasma Amino AcidsaDWith IP Injections of Graded Levels of Methionigg. .66 4 Individual Levels of Plasma Amino Acids With IP Injections of Cysteine Plus Graded Levels of DIethj-onine. O 0 O O O I O I O O O O O O O O O O O .67 LIST OF FIGURES Figure Page Flow Chart of Experiment One. . . . . . . . . . . 25 Flow Chart of Experiment Two. . . . . . . . 29 Plasma Methionine Response to IP Injections of Graded Levels of Methionine. . . . . . . . . . 43 Plasma Lysine and Plasma Cysteine Response . to IP Injections of Graded Levels of Methionine . 44 5 Plasma Methionine Response to IP Injections of Seven g/day Cysteine plus Graded Levels of Iflethionine o o o o o o o o o o o o o o o o o o o o 47 6 Plasma Lysine and Plasma Cysteine Response to IP Injections of Seven g/day Cysteine plus Graded Levels of Methionine . . . . . . . . . . . 48 A (amt-a vi INTRODUCTION The capacity of ruminants to utilize cellulose and non protein nitrogen has given them a unique place as a food producing animal. The action of the rumen ecosystem on dietary protein has became an area of intensive study. It is evident that the extensive degration and resynthesis of protein by the rumen microbiota determine the amount and cOmposition of the amino acids reaching the intestine. Hence, the study of amino acid requirements cannot be carried out by merely changing dietary amino acid levels, but has to be done in a way that avoids the action of the rumen eco- system. The first experiment of the present work was an at- tempt to determine the metabolic response of steers to sustained high levels of amino acid administration. The response was measured through changes in plasma amino acid levels with daily intraperitoneal injections of L-methionine and L-lysine. The secdnd experiment sought to evaluate and quantitate the methionine requirement of growing steers through changes in the plasma amino acid levels in response to intra- peritoneal injections of graded levels of methionine alone and methionine plus cysteine. 1 2 The results of this work should aid in understanding the amino acid metabolism of the ruminant. Nevertheless, it is hOped that the questions this study answers and the interest it might generate will constitute a meaningful c0ntribution in the area of ruminant nutrition. LITERATURE REVIEW Protein Metabolism in the Ruminant Nitrogen Metabolism in the Ruminant The capacity of ruminants to utilize protein and non- protein nitrogen is now a well recognized feature. Loosli gt‘al (1949) showed that rumen microbes are able to synthesize all amino acids. Dietary proteins are broken down into peptides, amino acids and ammonia acCording to their degradability which in turn is largely determined by the rumen solubility of the protein, although the morphological characteristics, rate of passage and interactions among the diverse feedstuffs in the diet also influence the degree to which the protein is degraded. (Bull gt El: 1977; Satter 23 31, 1977). Protein entering the abomasum is thus composed of the undegraded dietary protein that by passed rumen and the microbial protein synthesized by the microbiota (Satter, 1977). The amount of microbial protein produced is regulated by a series of factors; the most important of which is the capacity of rumen microbes to utilize ammonia and carbon skeletons for amino acid synthesis (McDonald, 1948; Loosli gt‘gl, 3 4 1949). There is however, a limit in the capacity of the microbes to utilize the ammonia for protein syn- thesis, Roeffler and Satter (1975) have calculated that ammonia in excess of a concentration of S mg/dl of rumen fluid, is not utilized for ruminal protein synthesis, although this limit may be regulated by factors such as the type and availability of dietary carbohydrates. Excess ammonia is absorbed across the rumen epithelium and is transported to the liver where it is transformed into urea, which is partly recycled to the rumen through salivary secretions (McDonald, 1948). The factors influencing and limiting the growth of rumen microorganisms have been reviewed by Bergen and Yokoyama (1977). The fermentation of carbohydrates into volatile fatty acids in the rumen is the main source of energy. Microbial growth depends on energy metabolism and is related to substrate disappearance, thus it is possible to evaluate the potential for cell production from the amount of ATP generated in the fermentative pathways. The relationship between cell growth and energy production, expressed as growth yield per mole of ATP is known as Y Early work produced ATP' a consensus that the Y was constant for the different ATP rumen microorganisms. A YATP of 10.5 was acCepted (BauchOp and Eldsen, 1960); however, more recent studies, 5 taking into account other aspects of rumen psysiology such as dilution rate, additional pathways for ATP generation (i.e. cytochrome linked electron transfer) and the shifts in microbial pOpulations occupying a specific metabolic niche have calculated Y values ATP ranging from 7 to 25, the main factor influencing the fluctuation in values appears to be the specific growth rates (or dilution rates of the microbes). Sulfer: Nitrogen Relationship in the Rumen The amount of sulfur (S) in the rumen is a limiting factor for protein synthesis (Moir gt 31, 1967; G11 23 a1, 1973; Kennedy 33 a1, 1975). In order to be in- cOrporated into microbial proteins, sulfur must first be reduced to sulfide (Bray and T111, 1975). Rumen microorganisms have a sulfur: nitrogen ratio of ap- proximately 1:15 and optional microbial growth occurs when the dietary prOportion is about 1:10 (Moir 23 a1, 1967). The source of sulfur can influence the growth of rumen microbes. Gil gt a; (1973) working with a culture of rumen microorganisms fermenting glucose and with urea as the sole protein source, found that the addition of methionine hydroxy analog (MHA) elicited an increase of 2.5 times in the logarithmic growth rate of the culture. The addition of other sulfur amino acids also had a stimulant effect but when inorganic sulfur or non sulfur amino acids were added, l i I I J 1 ‘1 ‘l. 6 the growth rate was not stimulated. It was concluded that sulfur amino acids are easily interconverted and that the incorporation of inorganic sulfur into protein is a rate limiting process. High sulfur may also in- fluence microbial protein production through reducing the availability of hydrogen for the growth of methanogens (Bryant £2.§l: 1977). Postruminal Fate of Nitrogenous Compounds Bergen (1978) has reviewed postruminal nitrogen metabolism. Microbial protein, together with dietary protein that escaped ruminal degradation and some free amino acids pass through the omasum and abomasum into the small intestine. The proportion of dietary nitro— gen reaching the intestine varies with nitrogen in the ration. When high levels of soluble protein are fed, nitrogen entering the abomasum might be less than the dietary intake. On the other hand, if the ration is low in protein and high in energy, the amount of nitro- gen reaching the small intestine might be greater than dietary intake (Clarke gt El: 1966). Weller gt‘al (1971) fed sheep a ration containing 8-10% CP and found no net dietary nitrogen loss. These results are in agreement with those of Fenderson and Bergen (1975) who fed a 9.5% CP ration to growing steers and reported a ruminal nitrogen loss of only 2 percent. Post ruminal enzymatic activity resembles that found in non-ruminants although the neutralization of 7 the ingesta in transit is slower in ruminants and the highest proteolytic activity takes place in mid jejunum (Bergen, 1978). The digestibility of nitrogenous compounds ranges from 65 to 85% with nucleic acids being among the more digestible cOmpounds. Hogan (1973) reported a digestibility coefficient of 70% for bulk protein in the small intestine of ruminants. Absorptive processes are also similar to those of non-ruminants, but the highest rate of absorption takes place in the posterior part of the ileum (Ben Ghendalia gt‘al, 1974). According to their absorptive pathways, amino acids can compete for absorption. Hume gt a; (1972) working with sheep, showed an inhibitory effect of leucine on lysine. Johns and Bergen (1973) confirmed this finding lg gitgg working with sheep tissue. Rumen Microbial Protein Depending on the degradability of dietary protein and the limitations in energy, nitrogen and sulfur; micro- bial protein can provide a large prOportion of the protein requirement of the ruminant. Buchholtz and Bergen (1973) measured microbial protein synthesis as a function of microbial phOpholipid synthesis and found that for a 4 liter rumen, the rate of true pro- tein synthesis was 16.1 g Protein/100 g of organic‘ matter digested, enough to meet the needs of growing calves and lambs. 8 Rumen microbial protein has a high biological value (Bergen gt‘al, 1967; Bergen gt 3;, 1968), with protozoa providing the protein of the highest quality (Bergen 35 31, 1968). The rumen ecosystem provides a buffering effect in regard to protein quality and essential amino acid composition. Specially in the microbial protein since the protein quality and bulk essential amino acid make up of rumen bacteria and protozoa tend to remain constant regardless of level or source of dietary nitro- gen (Bergen 33 31, 1968; Fenderson and Bergen, 1972; Williams and Dinusson, 1973). There is however, a dietary effect upon several features of the rumen ecosystem. Protein free diets where all the nitrogen is supplied as nonprotein nitro— gen (NPN), have a depressing effect on the protozoal 'pOpulations. Oltjen and Putnam (1966) studied the effect of NPR or performed protein with purified rations on the rumen microbial population of steers. They found that purified diets containing NPN, reduced the protozoa numbers and nitrogen retention. Plasma levels of branched chain amino acids (val, leu and ile) were also lower in these steers. The rumen c0ntents contained almost no branched chain volatile fatty acids (isobutyrate, isovalerate and valerate) to serve as precursors for branched chain amino acid synthesis. Under such cen- ditions microbial protein synthesis is depressed resulting 9 in lower plasma levels of branched chain amino acids (Bergen gt‘al, 1973). Oltjen 23 a1 (1971) studied the effects of the addition of branched chain volatile fatty acids in steers fed urea or soy protein as protein source and found that steers feed urea had lower plasma levels of valine, isoleucine, leucine and phenylalanime than the steers fed soy protein. The addition of branched chain volatile fatty acids improved nitrogen retention and raised plasma branched chain amino acid levels; however, numbers of rumen protozoa and viable celluloytic bacteria were not altered. Klopfenstein g£_al (1966) assessed the role of faunation on rumen metabolism in sheep and reported that faunation improved dry matter digestion, reduced viable bacteria numbers and increased rumen ammonia but not blood urea. These workers concluded that dietary nitro- gen was digested more readily and utilized more ef— ficiently by faunated sheep. Although the bulk amino acid composition of the microbiota is not influenced by crude protein intake or source, there can be differences in protein quality and amino acid availability within the diverse rumen microbes. Bergen g£.a1 (1967) found significant differences in the protein quality of 22 different strains of rumen bacteria whose amino acid compositions were similar, and suggested 10 that the nitrogen status of the ruminant may be in- fluenced by alterations in the rumen bacterial pOpulation. In a later work with rats, Bergen gt_§1 (1968) showed that histidine was first limiting in protozoal protein and sulfur amino acids were limiting for bacterial pro- tein. These results centrast with those of Klopfenstein gt a; (1966) who, using the "energy induced plasma amino acid technique" in xixg, reported that lysine was first limiting in defaunated sheep, although there was a high proportion of c0rn in the diet, whose lysine content is usually low. The enzymatic release pattern of amino acids from microbial protein in the small intestine may be in- fluenced by the dietary protein source. Burris gt a; (1974) found variations with the source of dietary protein in the enzymatic release pattern of threonine, valine, methionine, phenylalanime and lysine in microbial preparations isolated from the rumen of steers. This may be of physiological importance since the amino acids present at a given absorption site may affect rate of up- take. Competitive interaction during absorption between amino acids of the same transport class, has been studied both in zizg (Hume 25 a1, 1972) and in xitgg (Bergen and Johns, 1973). After assessing the diverse factors af- fecting protein digestion and the length of the intestinal tract, Johns and-Bergen (1973) cOncluded that competitive 11 inhibition of amino acid uptake is not likely to occur lg giyg and suggested an unlimited absorptive capacity throughout the intestinal tract. Plasma Amino Acids Plasma amino acids (PAA) represent a small fraction of the total amino acids of the body. The plasma amino acid pool is a product of the dynamics of amino acid metabolism, affected by amino acids absorbed across the gastrointestinal wall, by those catabolized from tissues and by amino acid synthesis. Plasma amino acids are readily renewed since the daily inflow of dietary amino acids is much larger than the plasma pool. Removal of amino acids from the plasma pool ocCurs as a result of protein synthesis and amino acid catabolism. The re— sponsiveness of the plasma pool to the dynamics of amino acid metabolism, as well as its dependence on dietary amino acid supply, have made it a basic instrument in the study of amino acid nutrition. The evaluation of amino acid nutrition through PAA is a well established technique. Longenecker and Hause (1959) working with dogs, studied the relationship of postprandrial changes in PAA to evaluate the amino acid composition of different protein sources. In a later work, Zimmerman and Sectt (1965) measured the amino acid requirements of chickens according to changes in the PAA levels. The chickens were fed increments of the first l2 limiting amino acids both below and above the supposed requirement. When the dietary amino acid intakes were below the requirement, plasma concentrations of that essential amino acid remained unchanged, but once the requirement was met, the plasma c0ncentration started to rise linearly with each dietary increment of that amino acid. Zimmerman and Scott (1965) indicated that this break point at which the PAA level started to increase, ceincided closely with the dietary level of the essential amino acid in question above which dietary increments would no longer produce improvements in growth rate. The break point method was also implemented by Mitchell 25.31 (1968) who measured the requirements of four essential amino acids in pigs according to the plasma amino acid concentrations and nitrogen retention. Mitchell 2£.él (1968) concluded that changes in plasma amino acid concentration offered a more accurate parameter for the evaluation of amino acid requirements than nitrogen retention. These workers further in- dicated that there must be a period of adaptation to a diet deficient in an essential amino acid (below requirement) before increases of the limiting amino acid can produce a clear break point in the PAA con- centrations. 13 Plasma Amino Acids in Ruminants The rumen ecosystem significantly changes dietary protein and it is necessary to take into account these modifications in order to evaluate amino acid needs in ruminants. Bergen gt a; (1978) have postulated a unifying hypothesis on the effect of dietary nitrogen source and level of PAA in ruminants and suggested that changes in PAA patterns can be best explained by the quantity of protein reaching the intestine of the ruminant. This hypothesis by Bergen gt a; (1973) assumes that amino acid absorption across the rumen epithelium (Leibholz, 1969) is of minor quantitative importance. The behavior of PAA is influenced by a number of factors in addition to protein. Source and level of energy have been shown to induce a decrease in the plasma amino acid concentrations in ruminants (Potter gt a1, 1968; Reilly and Ford, 1971; Fenderson and Bergen, 1972; Eskeland gt.§l, 1974). The effect of several energy sources on the PAA of sheep was studied by Potter 23 a; (1968). Glucose, pro- pionate, acetate and butyrate decreased PAA levels. The sharpest decrease was caused by glucOse, followed in order of efficiency by prOpionate, acetate and butyrate. The above results on energy induced PAA depression were confirmed by Eskeland gt a1 (1974) who also found that glucose was more effective than prOpionate, acetate and butyrate in depressing PAA cOncentrations. 14 Energy induced PAA depressions may be a reflection of the close relationship between protein and energy metabolisms, very probably at the hormonal.level. PAA are known to contribute significantly to gluconeogenesis. Reilly and Ford (1971), using labelled amino acids in sheep, found that 28% of the g1ucOSe was derived from amino acids. The stimulatory effect of amino acids on insulin secretion is well known (McAtee and Trenkle, 1971); Tae gt a1, 1974), as is the capacity of insulin to increase amino acid uptake by muscle cells (Munro, 1964; Wool, 1965; Bergen, 1978). These facts suggest that the energy induced depression on PAA levels arises from increased tissue uptake of amino acids due to stimulation of insulin secretion. The theory is further substantiated by the fact that when fat is used as source of energy, uptake of amino acids by tissue is not af- fected, probably due to lack of stimulus for insulin secretion (Munro, 1964). Starvation and low nitrogen intakes also affect the concentration of PAA in ruminants. Leibholz (1970) studied the effects of these factors in sheep and found that starvation for a period of 12 to 20 days resulted in a decrease in the concentration of serine, glutamine, glycine, alamine, histidine and arginine, while the levels of lysine, 3-methylhistidine and isoleucine in- creased markedly. The ratio of essential to nonessential 15 amino acids increased from .35 to .56 in the starved group. When sheep were fed a low nitrogen diet, the ratio decreased from .40 to .27. Leibholz (1970) sug- gested that starvation elicited the utilization of non- essential amino acids for energy while a low nitrogen intake favored the utilization of essential amino acids for energy. Several relationships have been found among the amino acids in plasma. Zimmerman and Scott (1965) found that lysine tended to accumulate when arginine was deficient in the diet, and that threonine was af- fected by large excesses or deficiencies of lysine, hence it was suggested that a reduction in the plasma levels of threonine does not necessarily mean limiting status. Snyderman and Holt (1967) reported negative relationship between high levels of leucine and the plasma con- centrations of valine, isoleucine, threonine and tyrosine. In a number of experiments Oltjen and Lehmann, 1968; Oltjen 33 a1, 1970; Leibholz, 1970) glycine and serine have been associated with low plasma essential amino acids and poor nitrogen utilization. This tendency sug- gests that glycine and serine have an intrinsic negative effect on nitrogen utilization. The relationship between all sulfur containing amino acids (SAA) is well known. Methionine can be used to synthesize cysteine through the transulfuration pathway 16 (Radcliffe and Egan, 1978) or can also be oxidated for cystine production (Stipanuk and Benevenga, 1977). Al- though methionine can fulfill the SAA dietary require- ment by itself, part of its requirement can be supplied by both cysteine and cystine (Block gt a1, 1969; National Research Council, 1973; Stipanuk and Benevenga, 1977). The National Research Council (1973) summaries suggest that cystine can supply 50 to 70% of the SAA dietary requirement for normal growth in pigs. Although the proportion may vary between species, methionine must be present, Byington and Howe (1972) found in chicks that a methionine: cystine ratio of 70M:30C was superior to 30M:7OC. These results concur with the observations of Featherson and Rogler (1978) who also found an anta— gonistic effect of cystine on methionine in chicks when the dietary level of methionine is suboptimal. Another common feature of SAA is their relatively high toxicity; methionine has been found to be the most toxic nutritionally important amino acid (Sauberlich, 1961, Benevenga, 1974). Methionine can be toxic at levels of only four times its requirements (Benevenga, 1974). Symptoms are varied but are usually reflected in growth depression and tissue damage to organs with high metabolic rates (Benevenga, 1974; Benevenga gtmgl, 1976), the toxicity appears to be caused by aberrations in the metabolism of the methyl group and its conversion to CO 2 (Benevenga, 1974; Benevenga, 1976). 17 Amino Acid Requirements of Ruminants Qualitative Studies Due to the action of the rumen ecosystem, it has not been possible to establish a dietary amino acid re- quirement for ruminants; however, the type and amount of amino acids required at the absorption sites for Optimal nitrogen utilization, have been studied. Techniques such as, plasma amino acid response cUrves (Brookes 25.31, 1973; Reis 32 31, 1973; Tao 33 31, 1974; Broderick and Satter, 1974; Fenderson and Bergen, 1975; Tao 22.2l9 1974; Williams and Smith, 1975; Foldager 22.21: 1977), nitrogen balance studies (Nimrick 33 al, 1970a; Mimrick gt al, 1970b; Fenderson and Bergen, 1975; Hall 23 a1, 1974; Tao 25 a1, 1974; Richardson and Hatfield, 1978), plasma urea nitrogen (PUM) reSponse curves (Tae §§_a1, 1974; Williams and Smith, 1975), urinary nitrogen (Tae gt‘al, 1974; Richardson and Hat- field, 1978) and amino acid oxidation levels (Brookes 35 31, 1973) have produced consistent results in regard to the quality and quantity of amino acid requirements in ruminants. Nimrick 23 a1 (1970) measured changes in nitrogen retention of growing lambs in response to abomasal in- fusion of amino acids, methionine was the only single amino acid to consistently increase nitrogen retention, lysine improved nitrogen retention only when methionine 18- was also supplemented, and threonine increased nitrogen retention only after lysine and methionine were supple- mented; the cdmbination of these three amino acids im- proved nitrogen retention by 60% over the urea infused controls. TryptOphan, histidine and leucine did not improve nitrogen retention when infused with methionine, lysine and threonine. Methionine also appeared as the first limiting amino acid when glutamic acid supplied the non specific nitrogen requirement. Nimrick 22.2l (1970) concluded that the limiting order of essential amino acids was methionine, lysine and threonine in sheep on a NPN diet. The effect of intraperitoneal infusions of amino acids on nitrogen balance and PAA patterns of calves was studied by Hall 23 a; (1974) who found that nitro- gen balance was improved by a mixture of methionine, lysine, tryptophan, histidine and arginine but not by any single amino acid; the PAA patterns after intra- peritoneal infusion of non specific nitrogen suggested that the limiting amino acids were lysine, methionine and histidine thus, they concluded that it was a group of amino acids and not any single one that was limiting. The exclusion of tryptOphan as a limiting amino acid confirms the results of Fenderson and Bergen (1972) who cencluded that tryptOphan was not a limiting amino acid for growing cattle. l9 Nitrogen retention, urinary nitrogen and PAA were measured by Richardson and Hatfield (1978) to determine the limiting amino acids in growing cattle after abo- masal infusions of amino acids. Methionine was the single amino acid to cause the lowest urinary nitrogen value, the infusion of a combination of lysine and methionine improved nitrogen retention over the in- fusion of methionine alone, and methionine, lysine and threonine cembined, improved nitrogen retention over the combination of methionine and lysine. Since the in- fusion of tryptOphan alone or in c0mbination, or his- tidine in combination with methionine and lysine gave lower responses than the other amino acids, the authors concluded that methionine, lysine and threonine, in that order, are the first three limiting amino acids in growing steers. Quantitative Studies Most qualitative studies reviewed heretofore have shown methionine, lysine and threonine to be the first three limiting amino acids in ruminants under most common dietary conditions. As a result, quantitative assess— ments of amino acid requirements in ruminants, have usually been oriented at establishing the requirements of these three amino acids. Mimrick gt al (1970b) infused graded levels of amino acids into the abomasum of growing lambs and evaluated the effect on nitrogen retention to determine 20 the quantitative requirements. They found maximal nitrogen retention at levels equivalent to dietary prOportions of .40% glutamic acid (a non essential ~~ amino acid), .10% methionine, .lO% lysine-HCI and .10% threonine. The reSponse curves confirmed the earlier findings (Nimrick gt_§1, 1970a) that the limiting order of essential amino acids for this species was methionine, lysine and threonine. The lysine requirement of sheep with a weight of 45 kg was evaluated by Brookes gt a; (1973) who measured lysine oxidation after abomasal infusions of graded levels of lysine, the oxidation of lysine was measured as expired radioactivity from the oxidation of radio- active L-lysine hydrochloride. After plotting the oxidation response against the graded levels of in- fusion, a break point was calculated at the infusion level of 2.1 g/d of lysine. Plasma response curves were in close agreement with a break point at 2.4 g/day of lysine. Since the amount of dietary lysine reaching the abomasum was calculated as 4.4 g/day, the lysine requirement for sheep was estimated to be between 6.5 and 6.8 g/day. In another study, Tao 35,31 (1974) compared diverse parameters to evaluate the methionine requirement of sheep, the amounts required were calculated through the 21 responses of urinary nitrogen (UN), plasma urea nitrogen (PUN), urinary urea nitrogen (UUN), nitrogen balance (NB), plasma insulin (PI) to intravenous infusions of methionine. The response curves of nitrogen utilization indicated a methionine requirement of 4.81 to 5.0 g/day; whereas the PAA level curve showed a requirement of 3.63 g/day. The plasma insulin level was influenced by the amounts of methionine infused and was similar to the response in nitrogen utilization. The methionine requirement in preruminant calves was investigated by Williams and Smith (1975), who evaluated the PAA and plasma urea (PU) responses to dietary supplementation of methionine and cysteine. Both PAA and PU were affected within 4 hours after ingestion, the response curves indicated a methionine requirement of 4.5 g/day with the PAA response and 3.9 g/day when the PU response was used as criterion. More recently Foldager gt,al, (1977) supplemented the milk replacer in nursing Holstein calves with graded increments of methionine; the regression analysis of the response in daily gains, nitrogen retention and plasma methionine concentrations indicated a require- ment of sulfur cOntaining amino acids of 3.8 to 4.0 g of SAA/16g of nitrogen. The researchers indicated that only three days on diets were necessary to predict the requirements. 22 The amino acid requirements of growing steers were studied by Fenderson and Bergen (1975); PAA levels and nitrogen balance trials were used to evaluate the re- sponse to abomasal infusions of graded amounts of methionine, lysine, threonine and tryptOphan. The anal- ysis of the response curves indicated a break point at the infusion level of 7 g/day for methionine. No break point was observed in the plasma responses of lysine, threonine and tryptOphan but linear increases in plasma concentrations were recorded with every increment in infusions, suggesting that the requirement for these was met by the digesta entering the abomasum. Con— sidering the amino acid composition of the digesta in the abomasum as well as the methionine required to pro- duce a break point in the response curve, the methionine requirement was set at 14.9 g/day, and the total sulfur amino acid requirement at 18.7 g/day for steers fed a 9.5% protein ration. The investigators suggested that it is possible to extrapolate the requirements of the other amino acids from the methionine requirement, ac; cording to the prOportion of methionine to the require- ments of the other amino acids as reported for swine by the National Research Council (1973). This approach seems to be supported by the fact that the tisSue re- quirements and amino acid cemposition of pigs and cattle are similar (Black g£.al, 1957; Dowes, 1961), and that 23 the prOportion of the requirement of an amino acid per 16 g of nitrogen remains cdnstant regardless of the pro- tein level (Boomgaardt and Baker 1973). MATERIALS AND METHODS Experiment One General Design Eight Holstein steers with an average body weight of 270 kg. were fed a 9.5% C.P. ration (Table 1) once daily at noon, at 2.5% (6.8 kg) of their body weight. The steers were housed individually in 91 x 244 om. metal metabolism stalls with free access to water. Prior to the start of the experimental period, the steers were adapted to the diet for a 21-day period (Figure 1). In the experimental period, 20 g. of amino acid (L-lysine HCI or L-methionine; obtained from Sigma Chemical Co.) were injected intraperitoneally (IP) once daily, at 8:00 a.m. for 14 days. The amino acids were diluted in distilled water before the injections, in prOportions of 1:10 (w/v) for lysine and 1:20 (w/v) for methionine. Blood samples were taken from the jugular vein for amino acid (AA) and blood urea nitrogen (BUM) determinations on days 1, 2, 5, 8, 11 and 14. On days 1, 8 and 14, blood samples were taken at 0, 1, 4 and 8 hr., after the IP injection. On days 2, 5 and 11. blood samples were taken at 0 and 1 hr. after injection. 24 25 TABLE I. RATION USED IN THE EXPERIMENTS Ingredients % Oats, grain (4) 4—03-309 10.00 Wheat, bran (4) 4-05-191 5.00 Corn, dent yellow grain gr 2 US mm wt 54 (4) 4-02-931 51.55 Soybean seeds, solv-ext, grnd mx 7% fiber (5) 5-04-604 3.75 Corn, cobs, grnd (l) 1-02-782 20.00 Sugarcane, molasses, mm 48% invert sugar mm 79.5 degrees br/x (4) 4-04-696 5.00 Wheat, flour by product, fine sifted mx 4% fiber (4) 4—05-203 1.00 Urea (45% N) 0.25 Limestone, grng, mn 33% calcium (6) 6-02-632 be 1.45 Trace mineria salt 2.00 Vitamin A 2,000,000 IV/ton Vitamin De 250,000 IV/ton Vitamin E 55,000 IV/ton Crude Protein (NX6.25) 9.50 aCalcium Carbonate 00., Quincy Illinois bContained in %: Zn, mn 0.35; Mn, mn 0.2; Fe, mn 0.2; Mg, mn 0.15; Cu, mn 0.03; Co,mn 0.05; 12, mn 0.007; NaCl, mx 98.5 International Mineral Co. (DQO Vit A Palmitate (Pfizer Co., Terre Haute, Indiana). Ergocalciferol (Fleichman Irradiated Dried Yeast) Alpha t000pherol acetate (Eastman Kodak, Rochester, N.Y.) H) 26 21 day feed adaptation period 14 day AA infusion period Blood Sample Collection on days (1, 2, 5, 8, 11 and 14) / On days 1, 8 and 14 On days 2, 5 and 11 sampled at hours samples were taken 0, 1, 4 and 8 after at hours 0 and 1 infusion u(///// after infusion Sample Preparation Plasma Obtained by Centrifugation \ 3 ml prepared* ¢//// Rest of sample for for AA analysis . Plasma Urea Nitrogen and froze at -10 C Determination and frozen at -10 C. *AccOrding to the procedure described by Bergen 35 g; (1973) Figure 1. Flow chart of Experiment One. 27 The steers were divided into 2 groups of four steers each; of these, 2 steers were given Lysine in- jections and two were given methionine injections simultaneously. The first group under went the treat- ment in January 1978. The animals were injected with the amino acids and the blood samples obtained by using an outdoor squeeze chute. The sec0nd group was treated in April 1978 and all IP injections and blood samplings were performed in metabolic stalls. During the above experimental period, the steers had an average daily gain of .52 kg/day. Sample Processing Approximately 18 ml. of blood were collected in heparin- ized vacutainer tubes by jugular puncture. Plasma was then obtained by centrifugation and 3 ml were de- proteinized and prepared for AA analysis according to the procedures described in Bergen gt a; (1973).and then frozen at -100 C. until analysis. The remaining plasma was also frozen at ~10° C. for the determination of BUN. Chemical Analyses a. Plasma lysine and methionine Plasma lysine and methionine cdncentrations were determined from the plasma protein free filtrate by means of ion exchange chromatography (with a Technicon TSM Amino Acid Analyser) as described by Bergen gt a; (1973). 28 b. Plasma urea nitrogen (PUN) PUN levels were determined acc0rding to the micro diffusion technique as detailed by Conway (1960). Experiment Two General Design Eight Holstein steers with an average body weight of 325 kg were used for this experiment. The steers were housed as in experiment one and were fed 6.8 kg/ day of the 9.5% CP semipurified ration fed in exper- iment one. The amino acids utilized were L-isomers of methionine and cysteine obtained from Sigma Chemical Company. There was a 21 day period of adaptation to the diet before the start of the experiment (Figure 2). In this study, the steers were injected IP once daily at 8:00 a.m. for 7 days with graded levels of methionine, and cysteine plus methionine; the amounts of amino acids injected during each treatment period are presented in Table 2. Methion- ine was diluted in distilled water in a pr0portion of 1:20 (w/v) and cysteine was diluted in a ratio of 1:10 (w/v). The pH of the solutions c0ntaining cysteine was raised to 6.5 by addition of 6NNa0H to prevent damage to the peritoneum. The steers were divided in 2' groups of 4 steers each and the treatments were organized so that one group was on treatment while the steers of the other group 29 21 day feed adaptatiOn period Steers divided into 2 groups of 4 steers each Group A Group B 7 day IP injection perioda 7 day rest period while while grij B rests group A is treated 7 day rest period to 7 day IP injection eliminate carry over period while group A effects into next rests treatment 7 day IP injection period 7 day rest period to of next treatment eliminate carry over effects into next treatment aBlood samples taken before and one hour after IP injection on days 6 and 7 of injection periods. Blood samples were processed in the same way as in experiment one. Figure 2. Flow Chart of Experiment Two. 30 Table 2. Amino Acid C0mpositiona of Treatments in Experiment Two Treatment Methionine Cysteine 0b 0 0 l 5 0 2 10 0 3 15 0 4 20 0 5 O 7 6 3 7 7 6 7 8 10 7 9 15 7 agrams/day of amino acid bPAA values for this level were obtained from the sample drawn before IP injection of day 1 in the first experiment. 31 were in a 7 day rest period to eliminate carry-over effects into the next treatment. To minimize this risk even further, a 2 week rest period was given between treatments 1-4 (Methionine alone) and 5-9 (Cysteine plus Methionine). Blood samples were taken on days 6 and 7 of the treatment period before and one hour after the in- jection. In this experiment both injections and sam- pling were done in the metabolic stalls. This experi- ment took place between June and September 1978; the steers showed an average daily weight gain of 0.52 kg during that period. Sample Processipg Blood samples were prepared in the same manner de- scribed in experiment I. Chemical Analysis a. Plasmagysteineg_lysine and methionine. All amino acid concentrations were determined from the protein free filtrate by means of Ion Exchange Chromatography (in a Durrum Chromatography Amino- Acid Analyzer Kit) accOrding to the overall pro- cedures outlined by Bergen 23 El (1973) Statistical-Analysis The data obtained were statistically analyzed accOrding to the procedures described by Snedecdr and Cochran (1967). RESULTS Experiment One Plasma Amino Acid Response to Long-Term Administration of L-Methionine or L-Lysine The purpose of this eXperiment was to evaluate a potential adaptive response of plasma amino acids (PAA) in growing steers to IP injections of high (above requirement) quan- tities of methionine and lysine. The response was studied by following changes in the plasma levels of methionine, lysine and plasma urea nitrogen cencentrations over time, after the daily IP injections during a 14 day experimental period. Special attention was also given to the clinical state of the steers during this study due to the potential toxicity of high levels of methionine. Throughout the whole experiment feed intake remained unchanged. Daily feed intakes were 6.8 kg per steer with no significant weigh backs. Usually the feed was con- sumed within the first 4 hours after feeding. The over- all condition of the steers was satisfactory throughout the experiment. Unusual animal behavior due to the potential toxicity of methionine was not Observed during or after the experimental period. 32 33 After the experiment, one of the steers developed an outward convature of the right frong leg but this condition was probably due to lack of exercise and the slotted floor of the metabolic stalls. This steer was subsequently replaced for experiment two. Tables 3 through 6 depict the effect of IP in- jections of lysine or methionine on the cdncentration of PAA. The tables include means and standard de- viations for all four steers as well as the data grouped acc0rding to the type of weather and other environmental effects during the experimental period. Steers A and B were treated in January at temperatures below -100 C as they were led outdoors to a squeeze chute for the IP in- jections and blood samples, whereas steers C and D were treated in April at temperatures above 00 C. The effect of IP methionine injections on plasma methionine appears in Table 3. The daily plasma methionine reSponse at the 0 hr. sample shows an in- crease over the 0 hr. values for the previous days during the first week of the trial. Plasma methionine cen- centrations at 0 hr. peaked at about day eight and then decrease slightly for days 11 and 14. 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