.0 5‘. 1.4 n a b o. . .u 1 .- .05 n.. g n: .0 I :3“ . $0.; '0‘.“ 0-,... .. Q ‘9. Cy ‘ a as '3: o 1:... - .s u..- t R I 13'... ' ‘ n D c p. Q .l ”on... ...¢ «.3 Q. .0 n O‘— '0. '.' .- n --". ’C . - o h“..v C.U0-‘u 'Q . O its-n? «.3... u!" 3.... c. u ABSTRACT THE REQUIREMENT OF METHIONINE AND TOTAL SULFUR AMINO ACIDS IN THE PRE-RUMINANT CALF BY John Foldager The qualitative and quantitative requirement of amino acids in calves is essentially unknown. The amino acids essential for growth in calves have not been deter- mined but was assumed to be the same as those essential for growth in rats, because they are required at the tissue level in both ovine and bovine animals, and because calves fed gelatin have inferior performance when compared with those fed milk protein. The requirements of methionine and total sulfur amino acids have been studied, but the estimates vary from .23 to more than .58 g per day per kg metabolic weight. The requirement of lysine has been reported to be 1.75 to 1.95% of dry matter. The requirement of methionine and total sulfur amino acids was studied in 20 male Holstein calves employed in a two-period changeover design with five dietary levels of methionine (1.86, 2.48, 3.10, 3.72, and 4.34 g/l6 g N). This was done in ten two by two latin squares where two p. \ I I . Ls“ . .‘ \ ‘r—d' ! ’9 ',: ‘- ~‘ K of» . John Foldager calves represent the rows and two periods (9 to 15 and 21 to 27 days of age) represent the columns. The calves were fed milk replacer containing 25% of the total protein (18.08%) as crystalline L-amino acids as the only feed. Prepared milk (13%) solids were fed at the daily rate of 10% of body weight in two equal meals 12 hours apart. The response criteria were average daily gain, digestibility of dry matter and crude protein, nitrogen balance, and plasma methionine and urea nitrogen levels before and two hours after feeding on the first and the last day of each period. Using these methods plus the difference between fasting and post feeding plasma methionine levels, we estimated methionine requirements ranging from 2.75 to 2.95 g per 16 g N, except when digestibility of dry matter and plasma urea nitrogen were used as response criteria. All diets contained 1.05 g cystine per 16 g N. If the assumption is made that the requirement of sulfur amino acids is that of methionine only or 45% methionine plus 55% cysteine, then the requirement of total sulfur amino acids is 3.80 to 4.00 g per 16 g N, or .25 to .26 g per day per kg metabolic weight. The requirement of the remaining essential amino acids was estimated from the above value and amino acid composition of the 40 week old calf fetus. When poor health due to factors other than treat- ments was encountered, then average daily gain, John Foldager digestibility, nitrogen balance, and plasma urea nitrogen were less sensitive to diet than was plasma methionine. These data suggest that three days on the experimental feed are sufficient to estimate the amino acid requirement in calves by plasma amino acid levels. Poor health due to bacterial infections of the gastrointestinal tract could not be related to dietary methionine but the severity tended to increase at the highest methionine intake. At that dietary level, plasma methionine tended toward a plateau instead of increasing linearly with intake as expected. The cause of the plateau is unknown but it may have been caused by decreased methionine absorption, or increased deamination of amino acids due to gluconeogenesis, as indicated by increased plasma urea nitrogen levels. Whether gluconeogenesis from amino acids is stimulated because energy becomes limiting for vital functions in scouring calves, or is brought about by a direct stimu- lation of glucocorticoid secretion by high level of free methionine in the diet is not known. THE REQUIREMENT OF METHIONINE AND TOTAL SULFUR AMINO ACIDS IN THE PRE-RUMINANT CALF BY John Foldager A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy Science 1976 ACKNOWLEDGMENTS This thesis could not have been completed without generous help and support from numerous people, who I would like to acknowledge. Especially, I want to thank my major professor, Dr. J. T. Huber, for his support and encouragement throughout my graduate program. Thanks are also given to Dr. W. G. Bergen for special interest in the project and for amino acid analysis. I also want to acknowledge the other members of my advisory committee, Drs. J. L. Gill and W. D. Oxender, for their guidance and advise during the graduate study. Gratitude is also extended to professor dr. med. vet. A. Neimann-Sorensen and forsogsleder J. Brolund Larsen, Denmark, for their encouragement and support to take upon the graduate study. Thanks are also given to Dr. R. R. Neitzel, for aid with statistical calculations, Dr. J. W. Peters, for aid with cannulation of calves and Dr. G. Kulasek, for plasma urea nitrogen analysis. Here I would also like to express my appreciation for the financial support from the NATO Science Fellowship Programme, Danmark-Amerika Fondet, and Statens Husdyrbrugsudvalg which brought me to Michigan State University, and the continued support from the ii Department of Dairy Science. Last but not least I wish to extend my deepest and most sincere gratitude to my wife, Karen, for her encouragement, love, understanding and sacrifices during my course of study, research and manuscript preparation. iii TABLE LIST OF TABLES . . . . . . LIST OF FIGURES . . . . . LIST OF ABBREVIATIONS . . INTRODUCTION . . . . . . . REVIEW OF LITERATURE . . . Methods for Assessment Acid Requirements . OF CONTENTS of Protein and Amino Growth and Nitrogen Retention Growth Assay . . . . . . . Nitrogen Retention Methods Plasma Amino Acids . . . . . . Feeding Methods . . . . . Effect of Short Term Fast Breaking Point . . . . . . Carcass Analysis . . . . . . . Urinary Urea . . . . . . . . . Plasma Urea Nitrogen . . . . . Amino Acid Oxidation . . . . . Energy and Protein Requirements of Ruminating Calves . . . . . . . Energy and Milk Requirements of Calves Fed Milk 0 O O O O O O O O 0 Protein Requirement . . . . . Endogenous Losses . . . . Digestibility and Biological Value Milk Proteins . . . . . . . iv Page vi xi xiii ll 13 16 17 18 18 19 20 22 22 24 29 32 Protein Sources in Milk Replacers Amino Acid Requirements Calves . . . Factors Affecting Plasma Amino Levels in Calves Acid Amino Acid Infusion in Ruminating Calves and Lambs Amino Acid Requirements of Other Species . Blood Urea NitrOgen in MATERIALS AND METHODS . Animals . . . . . . Diets . . . . . . . Feeding Schedule . . Body Weight . . . . Nitrogen Balance . . Blood Collection and Experimental Design and Statistics RESULTS 0 O O I O O O O Calves and Lambs Processing. Body Weight, Daily Gain, Digestibility . . . Nitrogen Balance . . Plasma Amino Acids . Plasma Urea Nitrogen Hematocrit . . . . . DISCUSSION . . . . . . . Body Weight, Daily Gain Digestibility. . . . Nitrogen Balance . . Plasma Amino Acids . Plasma Urea Nitrogen Hematocrit . . . . . I Health. Health Sulfur Amino Acid Requirement in CONCLUSIONS . . . . . . APPENDIX . O C C O O O . BIBLIOGRAPHY . . . . . . Baby Calves Page 34 38 38 39 40 41 43 44 44 46 51 53 53 54 56 59 59 66 70 78 90 92 97 97 100 102 104 110 111 112 117 119 139 LIST OF TABLES Table Page 1. Energy requirement of calves fed milk; digestible energy per day . . . . . . . . . . 23 2. Milk requirement of calves fed only milk; liters per day . . . . . . . . . . . . . . . . 24 3. Digestible energy requirements of calves fed milk (kcal/day) . . . . . . . . . . . . . 25 4. Relationships between N retention and the daily supply and requirement of apparently digestible protein in calves . . . 27 5. Relationships between N retention and daily gain in calves . . . . . . . . . . . . . 28 6. Loss of endogenous urinary N (UE) in calves . . . . . . . . . . . . . . . . . . . . 30 7. Loss of metabolic fecal N (MF); g/100 g dry matter ingested . . . . . . . . . . . . . 31 8. Apparent digestibilities of milk dry matter (DM) and milk protein in calves at different ages; % . . . . . . . . . . . . . 33 9. The amino acid requirements of rats, chicks, turkeys, and pigs . . . . . . . . . . 42 10. Calves omitted from the experiment before completion due to poor health or mortality . . . . . . . . . . . . . . . . . . 45 11. Assignment of calves to blocks, date of birth, dam's identification number and treatments . . . . . . . . . . . . . . . . . . 47 12. Composition of the milk replacer . . . . . . . . 48 vi Table Page 13. The amino acid content in whole milk, commercial milk replacer and prepared amino acid mixtures . . . . . . . . . . . . . 49 14. Plan of feeding, weighing, collection, and blood sampling . . . . . . . . . . . . . . 52 15. The effect of dietary methionine on body weight (initial and final), average daily gain, and fecal score of calves fed graded levels of methionine . . . . . . . 60 16. Estimated relationship between dietary methionine levels (X,%) and average daily gain (ADG, g) in calves. Adjusted observations . . . . . . . . . . . . 63 17. The effect of dietary methionine levels on the digestibility of dry matter (DM) and crude protein (CP) and related factors in calves . . . . . . . . . . . . . . . . . . . . 67 18. The effect of dietary methionine (%) on nitrogen balance and related parameters of calves . . . . . . . . . . . . . . . . . . 71 19. The effect of dietary methionine (%) on the daily N-content and N-balance per 100 kg initial body weight (g N/day/100 kg BW) . . . 76 20. Estimated relationships between dietary methionine levels (X, %) and N-balances expressed as total for experimental period (NBl, g), g N daily per 100 kg initial BW (NBZ), and g N daily per kg BW-73 (NB3), respectively. Adjusted observations. . . . . . . . . . . . . . . . . 77 21. The effect of dietary methionine on plasma concentrations of methionine, valine, leucine, and isoleucine . (uM/lOO ml) in calves . . . . . . . . . . . . 80 22. Estimated relationships between dietary methionine levels (X, %) and plasma methionine concentrations (Y, uM/100 m1). Adjusted observations . . . . . . . . . . . . 83 vii Table 23. 24. 25. 26. 27. 28. A1. A2. A3. A4. A5. The effect of dietary methionine levels on plasma urea nitrogen levels in calves on the first and the-last day of the experimental period . . . . . . . . . . . The effect of dietary methionine levels on blood hematocrits in calves . . . . . . . The relationship between average daily gain (ADG, g) and fecal (F, kg), and urinary (U, liters) excretion in calves . Methionine needs of the baby calf deter- mined by various methods . . . . . . . . . The requirement of total sulfur amino acids in the young calf as estimated by several workers . . . . . . . . . . . . . Calculated requirement of essential amino acids in the young calf . . . . . . . . . Analysis of variance for initial and final body weight, average daily gain, and fecal score of calves fed graded levels of methionine . . . . . . . . . . . . . . . . Analysis of variance for the relationship between dietary methionine levels (X, %) and final body weight (BW, kg), average daily gain (ADG, g), and fecal score (FS), respectively. Adjusted observations . . . Analysis of variance for the relationship between fecal excretion (F, kg) and average daily gain (ADG, g), and the estimated regression. Unadjusted observations . . . . . . . . . . . . . . . Analysis of variance for the relationship between fecal excretion (F, kg) and urine excretion (U, liters), and the estimated regression. Unadjusted observations . . . . . . . . . . . . . . . Analysis of variance for the relationship between urine excretion (U, liters) and average daily gain (ADG, g), and the estimated regression. Unadjusted observations . . . . . . . . . . . . . . . viii Page 91 94 99 113 115 116 119 120 121 121 122 Table A6. A7. A8. A9. A10. A11. A12. A13. A14. Page Analysis of variance for dry matter (DM) content (%) in milk replacer, milk weigh back, feces, and digestibilities of DM and crude protein (CP) in calves . . . . 123 Analyses of variation for the relationship between dietary methionine levels and the digestibility of dry matter (DM) and crude protein (CP) in calves. Adjusted observations . . . . . . . . . . . . 124 Analyses of variance for the relationship between fecal excretion (X, kg) and the digestibility of dry matter (DM) and crude protein (CP) in calves . . . . . . . . . 125 Analysis of variance for N-balance data . . . . 126 Analysis of variance for the relationship between urine excretion (U, liters) and urine N-concentration (NU, %), and the estimated regression. Unadjusted observations . . . . . . . . . . . . . . . . . 127 Analysis of variance for the relationship between fecal excretion (F, kg) and the fecal N-concentration (NF, %) for all observations, and for period 1 (NF ) and for period 2 (NFZ), respectively, and the estimated regressions. Unadjusted obser- vations . . . . . . . . . . . . . . . . . . . 128 Analysis of variance for N-balance data expressed as g N/day per 100 kg initial body weight . . . . . . . . . . . . . 129 Analysis of variance for the relationship between dietary methionine levels (X, %) and N-balances expressed as total for the experimental period (NBl, g), g N daily per 100 kg initial BW (NBZ), and g N daily per kg BW-73 (N33), respectively. Adjusted observations . . . . . . . . . . . . 130 Analysis of variance for plasma concentrations of methionine, valine, leucine, and iso- leUCine O O O O O O O O O O O O O O O O O O O 131 ix Table Page A15. Analysis of variance for the relationship between dietary methionine (X, %) and the plasma methionine concentration (M, uM/100 ml). Adjusted observa- tions . . . . . . . . . . . . . . . . . . . . 133 A16. Analysis of variance for the relationship between dietary methionine (X, %) and the plasma methionine concentration (M, uM/lOO m1). Group E deleted. Adjusted observations . . . . . . . . . . . . 135 A17. The relationship between dietary methionine levels (X, %) and plasma urea nitrogen (PUN) in calves on the first (1) and the last (2) day of the experimental period . . . . . . . . . . . 137 A18. The relationship between dietary methionine levels (X, %) and hemato- crits (HEM) in calves on the first (1) and the last (2) day of the experi- mental period . . . . . . . . . . . . . . . . 138 LIST OF FIGURES Figure Page 1. The relationship between graded levels of methionine (X) and average daily gain (ADG, Y) in calves . . . . . . . . . . . 64 2. The relationship between dietary methionine levels and the digest- ibility of dry matter (DM) and crude protein (CP) in milk replacers for calves . . . . . . . . . . . . . . . . . . . . 68 3. The relationship between dietary methionine (X) levels and N-balance (Y) in calves . . . . . . . . . . . . . . . . 79 4. The relationship between dietary methionine levels and plasma methionine concentrations before feeding on the first day of sampling in calves . . . . . . . 86 5. The relationship between dietary methionine (X) levels and plasma methionine (Y) concentrations two hours after feeding on the first day of sampling in calves . . . . . . . . . . . . . . 87 6. The relationship between dietary methionine (X) levels and plasma methionine (Y) concentrations before feeding on the last day of sampling in calves . . . . . . . . . . . . . . . . . . 88 7. The relationship between dietary methionine (X) levels and plasma methionine (Y) concentrations two hours after feeding on the last day of sampling in calves . . . . . . . . . . . . 89 8. The relationship between dietary methionine levels and plasma urea nitrogen (PUN) on the first (1) and the last (2) day of the experimental period . . . . . . . . . . . . . . . . . . . . 93 xi Figure Page 9. The relationship between dietary methionine levels and packed blood cell volume (HEM) in calves on the first (1) and the last (2) day of the experimental period . . . . . . . . . . . . 96 10. The relationship between dietary methionine levels and the difference (DIF) between the linear model for before feeding and the cubic model for two hours after feeding on the first (1) and the last (2) day of sampling in calves . . . . . . . . . . . . . . 109 xii AP BV BW CP DCE DCP DDS DM DSM EAA FS G MF MR LIST OF ABBREVIATIONS average daily gain available protein biological value body weight crude protein 1,2-dichloroethane apparently digestible protein distillers dried solubles dry matter dried skim milk essential amino acid(s) fecal score nitrogen retained in gain metabolic fecal nitrogen milk replacer(s) N-balance = nitrogen balance PAA PUN TP UE plasma amino acid(s) plasma urea nitrogen dermal loss digestible true protein endogenous urinary nitrogen xiii (1' Ci ha (I) 1.1) INTRODUCTION The function of dietary protein is to serve as a source of amino acids for anabolic processes in the body. The most economical protein will be that which provides a mixture of amino acids, both essential and nonessential, in the proportions needed by the body (Munro, 1964b). The importance of this concept was very clearly demon— strated by Dean and Scott (1965) in their development of an amino acid reference diet for early growth in chicks. A final mixture of amino acids containing the equivalent of 17.69% protein gave as good a growth rate as a practical ration of the corn-soybean meal type containing the equivalent of 26.20% protein. With the increasing use of milk replacers in calf nutrition and the desire to replace dried skim milk in the replacers by other protein sources, it becomes increasingly important to obtain knowledge of the quali- tative and quantitative requirement of amino acids in calves to maintain good nutrition. The amino acids essential for growth in calves have not been determined, but may be assumed to be the same as those essential for growth in rats. The amino acids essential for rats are required at the tissue level in both ovine and bovine animals (Downes, 1961; Black 35 31., 1952), and calves fed gelatin (tryptophane deficient) have inferior performance when compared with calves fed milk protein (Blaxter, 1950; Blaxter and Wood, 1952a). Only limited information is available concerning the quantitative requirement of amino acids in non- ruminating calves. At the outset of this study the requirement of methionine had been studied only by Patureau-Mirand EE.El‘ (1973). Since then, an experiment on the requirements of total sulfur amino acids and lysine was reported by Tzeng (1974) and Williams and Smith (1975) investigated the requirement of total sulfur amino acids. REVIEW OF LITERATURE Studies of protein metabolism have traditionally been divided into the intermediary metabolism of protein and amino acids, and protein nutrition (Munro, 1964). Within the studies of protein nutrition two main lines of research have been the evaluation of protein quality and assessment of protein and amino acid requirements (Munro, 1964). The objective of this literature review have been limited to: (a) comparisons of when and how various methods have been used in assessment of dietary need of protein and amino acids and factors influencing interpre- tation of the results, and (b) the energy and protein requirements of the non-ruminating calf. Methods for Assessment of Protein and Amino Acid Requirements Knowledge of the protein and amino acid require- ments of various Species is important in the preparation of highly nutritious feeds which can sustain good health at least cost. The dietary requirementof domestic animals may also be defined as the level which will pro- duce optimal production. However, this can only be done when it is beyond the aspect of "maximum health." The most widely used methods in assessment of dietary needs of protein and amino acids have been the growth assay and the nitrogen retention methods. In the case of amino acids, plasma amino acid (PAA) concentrations, carcass analysis, urine urea, plasma urea nitrogen (PUN) concen- trations, amino acid oxidation, and others also have been suggested. The objective of this section is to compare the various methods with respect to when and how they can be used, and features influencing interpretation of the results. Growth and Nitrogen Retention Balance studies were first conceived by Boussingault (1839; Munro, 1964) in studies with milk cows in which the total intake of C, H, O, and N was compared with the total output of these in urine, feces, and milk. The concept of balance of income and outgo was rapidly adopted and Voit (1831-1908; Munro, 1964) developed the nitrogen balance (N-balance) as a precise tool for the study of protein metabolism. Another important concept in the use of these methods was the principle of diminishing returns. This principle was probably first formulated by Liebig (1855; Brody, 1945) under the name of the "law of the minimum" which may be shown to be a special case of, if not identical with, the principle of diminishing returns (Brody, 1945). This principle has since been widely used in animal experiments by feeding various experimental groups graded levels of the nutrient in question. The rapid response and sensitivity of both growth and N retention methods to changes in the dietary protein and amino acid supply are mainly because: (a) all of the acids needed by higher organisms are obtained together from the proteins of foodstuffs, (b) except for minor quantities required for special purposes, all amino acids are used simultaneously for the synthesis of tissue pro- teins, and (c) there is essentially no storage of free amino acids in the body (Harper, 1964; Munro, 1970). Growth Assay.--The law of diminishing returns applies closely to the amino acid requirement except when physiological limits to response to nutrient variations are attained, i.e., maturity, or at low levels of a specific nutrient intake. Other factors complicating interpretation of growth data are reserve stores, synthesis in the animal, or small residual quantities in the diet may be an important fraction of the supply the animal receives (Almquist, 1947, 1953). Interpretation of the growth assay is made most conveniently by the use of logarithms for the abscissa (Almquist, 1953; Brookes 23 31., 1972). Advantages of this method of interpretation are: (a) bad data are made more conspicuous, (b) requirements can be more readily estimated although no specific data happen to coincide with the full requirements, provided there are sufficient data to establish the limits of requirements, and (c) full use may be made of the submaximal data to establish the response lines (Almquist, 1953). In order to obtain a reasonable degree of con- fidence in the measurements obtained the animals have to be fed the experimental diet for a considerable length of time. With increasing size of experimental animals the daily amount of feed per animal increases. Therefore, the biggest drawback to the applicability of this method in amino acid requirement studies is probably the time factor because of the high cost of mixtures of purified amino acids. Nitrogen Retention Methods.--Nitrogen retention can be measured by either the factorial method or the N- balance method. By the factorial method requirements are estimated by summation of N lost and amounts of protein synthesized, and the assumption that N consumed can be used with 100% efficiency (e.g., Williams et a1., 1974). The formula for the total dietary requirement of digestible true protein (TP) in grams per day is (ARC, 1965): (1) TP = (6.25) (lOO/BV) (UE + S.1 + S2 + MF + G + P + L) 2 :J‘ (D H (D C} (:11 II urinary endogenous loss S1 = loss of N in hair and scurf $2 = N retention in wool by adult sheep MF = metabolic fecal N G = N retention in live-weight gain P = N retention during pregnancy in fetal tissues L = output of N in milk BV = biological value of protein. The corresponding requirement of apparent digestible pro- tein (DCP) is obtained from (1) by subtracting (6.25)(MF) and is therefore: (2) DCP = (6.25)(100/BV)(UE + Sl + $2 + G + P + L) + (6.25)(BV)(100/BV - 1) Both TP and DCP are dependent on MF and hence on the dry matter (DM) intake, which make them inconvenient for pur- poses of tabulation (ARC, 1965). Therefore, the term MF was excluded and the calculated value was termed available protein (AP; ARC, 1965): (3) AP = (6.25)(100/BV)(UE + S1 + $2 + G + P + L) Assessment of the minimum endogenous urinary excretion (UE) is difficult, but a plateau is normally obtained after 5 to 10 days of N depletion (Williams et a1., 1974). In the general case the sum of S1 and $2 is termed the dermal loss (S) and is equal to the total loss of N in sweat, desquamated epithelium, hair and nails, menstrual, seminal, nasal and oral secretions, and excretions from wounds and N retention in hair (Williams et 31., 1974). The coefficient of variation in (UE + S + G) is 15 to 20% (Williams 32 a1., 1974). The metabolic fecal N (MF) often shows variations from the average of more than 80% (Williams et 21., 1974), and the variation is due to: (a) the difficulty in assuring prOper assignment of col- lections to respective metabolic periods and complete fecal collection, (b) fecal N excretion is influenced by the quantity and composition of the food consumed, and (c) values for infants and children are lower in g per day but represent a higher percentage of the total N loss. In case of growing animals, N retained in gain (G) is calculated as the difference of values obtained from animals sacrificed at the beginning and animals sacrificed at the end of the growth period, or by the use of standard values (Williams 33 31., 1974). The latter add error to the method as does the difference method, although to a lesser degree (Williams 33 21., 1974). Furthermore, the sacrifice technique cannot be used for human research, and often not for large domesticated animals (cattle, horses) due to the cost of the experimental unit. The N-balance method is a measurement of the least amount of N that will maintain N equilibrium in adults or satisfactory growth and N retention in the young (Williams 33 31., 1974). The N—balance technique has been discussed by Albanese (1959), Wallace (1959), Allison and Bird (1964), and Williams 33 31. (1974), and the method can be formulated as: (4) NB=I-(U+F+S) where NB = N-balance I = dietary N intake U = N excretion in urine F = N excretion in feces S = dermal N loss Improvement in the N-balance obtained by stepwise additions of the limiting nutrient give a measure of the need in a specifically depleted organism (Albanese, 1959; Allison and Bird, 1964), and may be used for essential amino acid (EAA) requirement determinations only under rigidly con- trolled conditions (Nasset, 1956). The N-balance is the sum of gains and losses of all the tissues of the body and equilibrium may not be identical with good nutrition (Allison and Bird, 1964). The possible errors in conversion of N retention to expected body composition are: (a) the factor used to convert N to proteins, (b) the ash content of the body, (c) the water content of the body, (d) the fat content of the body, and (e) the possibility of cumulative errors (Wallace, 1959). The most pertinent of these factors is 10 the cumulative error (Wallace, 1959), because, even with the most refined and meticulous technique, a finite quan- tity of the measured intake is lost in feeding and, similarly, a finite portion of the excreta is not recovered. When output is subtracted from intake, these losses increase the error. Wallace (1959) concluded that a 2% loss of intake and a similar loss of excreta will result in a 30 to 40% increase in the N-balance when the two, ordinarily very large numbers, are subtracted to obtain what is usually the very small balance value. Further, the common practice of not including the dermal losses may further increase the error by at least 20%. Finally Wallace (1959) pointed out, that the higher the concen- tration of nitrogen in the food, the greater will be the losses and the error in the balance. Changes in body water content in excess of 4 to 5% occur only in abnormal clinical conditions (Wallace, 1959). However, changes in body water have been invoked to explain N retention in excess of correlative body growth. In the 4 month old child 33% of the retained N is being stored without equivalent gain of body weight (BW) . Both the factorial approach and the N-balance method for measuring N retention have disadvantages, but in work with large domesticated animals the N-balance is usually preferable to the sacrifice method because of cost. 11 Plasma Amino Acids Utilization of PAA data in studies of amino acid requirements was suggested by Almquist (1954), and McLaughlan and Morrison (1968), and a direct relationship between plasma and dietary lysine was demonstrated by McLaughlan 33 31. (1961). Since then PAA have been com- pared with growth and N retention methods in growing rats (Morrison 33 31., 1961b; McLaughlan and Illman, 1967; Stockland 33 31., 1970), in growing chicks (Zimmerman and Scott, 1965), in growing pigs (Mitchell 33 31., 1968b), and in young adult men (Young 33 31., 1968, 1971, 1972). Morrison 33 31. (1961b) fed growing rats graded levels of lysine. After an initial lag, plasma lysine rose rapidly in response to added dietary lysine and reached a maximum at a dietary lysine concentration about .2% units greater than that found necessary for maximum growth. When Zimmerman and Scott (1965) fed young growing chicks suboptimal and superoptimal dietary concentrations of lysine, arginine, and valine in a basal diet of crystalline amino acids previously determined to have an Optimal combination of amino acids (Dean and Scott, 1965), the first limiting amino acid remained at a very low level in the blood irrespective of the severity of the amino acid deficiency until the dietary level exceeded that needed to maximize growth. When the dietary amino acid level was in excess of requirement for maximum weight gain, 12 that amino acid accumulated rapidly, and in a linear manner in the plasma, even though the greatest dietary concentration used was more than twice that required. Zimmerman and Scott (1965) did not obtain a maximum plateau in the PAA response curve, and concluded that the shape of the PAA curve (broken-line response curve) can be used to determine the amino acid requirement of the chick. The broken-line response curve and its validity as described by Zimmerman and Scott (1965) has since been confirmed by Mitchell 33 31. (1968b) in studies with grow- ing pigs fed graded levels of lysine, isoleucine, leucine, and histidine when compared with maximum N-balance; and by Stockland 33 31. (1970) in growing rats fed graded levels of lysine when compared with maximum average daily gain and gain per unit feed consumed; and by Young 33 31. (1971, 1972) in young adult men given graded levels of tryptophane, valine, and lysine when compared with N- balances. McLaughlan and Illman (1967) found an almost linear relationship between plasma and dietary concentrations of lysine, isoleucine, leucine, threonine, tryptophane, and histidine. The requirement was considered to be the dietary level at which the plasma level after feeding was equal to the normal fasting level, and was in close agree- ment with average values obtained by other methods. The l3 lack of a broken-line response curve was believed to reflect the duration of the test period. Feeding Methods.--Even though the same typical broken-line response curve has been obtained in most experiments where PAA were used as response criteria the feeding methods used and the length of the fasting period before bleeding have varied. The cost of purified amino acids is a limiting factor in experiments for assessment of the amino acid requirements, and becomes greater with increasing size of the experimental animal. Therefore, if the feeding period necessary in the PAA assay is shorter than that needed for assessment by growth and N retention the PAA method may become increasingly useful for determining amino acid status (Mitchell 33 31., 1968b). Chicks fed suboptimal levels of dietary lysine for up to 33 days showed no accumulation of lysine in the plasma (Zimmerman and Scott, 1965). However, Morrison 33 31. (1961b) did not obtain the typical plasma response curve when rats were fed a test diet for three weeks and blood was collected from non-fasted animals. Test periods of 14 to 17 days in length and blood collection 6 hours after the last food presentation, com- bined with different feeding methods was studied in grow- ing rats by Stockland 33 31. (1970). Feeding for one hour every 12 hours gave the typical response curve. When the l4 diet was fed 33 libitum, plasma response was more variable because the procedure provided no way of controlling the time that the rat last consumed its food. They also com- bined the 33 libitum feeding for 17 days with an additional two day period where feed was offered 33 libitum for one hour every 12 hours before bleeding. In this case the plasma levels started to accumulate prior to dietary levels associated with maximum daily gain and growth per unit feed. It was concluded that continued uptake of the assigned diets combined with slow weight gains during the short period of controlled feeding lead to an accumulation of lysine in the plasma. They suggested a period of "metabolic adaptation" to different feeding methods is needed. Zimmerman and Scott (1965) obtained a typical plasma response curve when chicks were fed a pre-test diet for 7 days and then fed the test diet for 7 days or more and blood was collected after a 24 hours fast. Mitchell 33 31. (1968b) also obtained the typical plasma response curve when pigs were used in a 5 day test experiment (7 days pre-test), where blood was collected 6 hours after feeding on the last day on the test diet. The pigs were offered 3 equal meals at one hour intervals and then the remaining 33_libitum until bleeding. Young 33 31. (1971) found no change in the plasma tryptophane curve in young adult men between days 3 and 6 of feeding the experimental 15 diet. Morrison 33 31. (1961c) fed diets containing 10 or 20% protein to rats and obtained the same response in test periods of 3 and 7 days duration. They also found that rats fasted for 19 hours and then fed diets containing 10% protein of bread or fish flour origin had higher plasma lysine levels when fed fish flour. The response within protein was the same one and 3 days after introduction of the diet. When the diets were fed for two hours only there was no difference between proteins. A broken-line response curve was not obtained and the amino acid requirement could not be determined by the ordinary procedure in the following cases: when McLaughlan and Illman (1967) fed rats pre-test and test diets for 3 days; when McLaughlan 33 31. (1961) fed rats lysine- deficient test diets for one or two days; and when Mitchell 33 31. (1968b) offered pigs the test diet for the first time on the evening before bleeding. Further, Zimmerman and Scott (1967a) did not obtain a broken—line response curve when chicks were fed an isolated soy-protein glucose diet for 4 days, fasted one day, fed a complete amino acid mixture for 3 days, and then fed the test diet 33 libitum for 6 hours, or 1/12 of the feed every 30 minutes for 6 hours. The chicks were bled 4 hours after the 6 hour test period. The evidence presented suggests that the critical length of the feeding period in the PAA assay is 3 to 5 16 days after the diet is introduced or the feeding method has been changed. Zimmerman and Scott (1967a), Mitchell 33 31. (1968b), and Stockland 33 31, (1970) suggested that this length of time is necessary for changes of the meta- bolic system responsible for protein synthesis to utilize all of the first limiting amino acid available in the blood and for depletion of labile endogenous sources of amino acids. Effect of Short Term Fast.--Chicks fasted for 3, 6, 12, or 24 hours showed progressive accumulation of several amino acids in the blood (Zimmerman and Scott, 1967b). When a non-protein diet was fed before fasting, the concentration of EAA was below the levels noted when fasted. The plasma tryptophane concentration was unchanged in young adult men during 12 through 17 hours of an over- night fast (Young 33 31., 1971). After feeding, the tryptophane concentration dropped within 2 hours and was lowest at 3 hours, suggesting accelerated amino acid utilization for hepatic protein synthesis during absorption. Growing rats fed superoptimal levels of lysine for one hour every 12 hours for 14 days and then bled l, 2, 4, and 6 hours post feeding showed linear increases in lysine with increasing dietary lysine (Stockland 33 31., 1970), but plasma lysine levels after a 6 hour fast tended to be less than the responses obtained after 1, 2, and 4 hours of fasting, and the latter three were equal. 17 When animals are "metabolically adapted," uptake and incorporation of amino acids into tissue proteins is very rapid. This was clearly demonstrated by Neale and Waterlow (1974) when they gave 14C-isotopes of lysine and leucine by stomach tube to rats fed a low casein diet. The highest specific radioactivity of CO2 was found one hour after administration. If the fall-off was considered exponential, then the half-life was approximately 1.5 hours. Three hours after leucine isotope administration, protein-bound radioactivity in liver and muscle was 97.7 and 93.7%, respectively. For lysine, the respective values were 92.4 and 75.9%. At all other time intervals (3 hours to 15 days, 15 to 20 days, and 20 to 30 days) all radioactivity was protein-bound and not detectable in the free amino acid fraction. Breaking Point.--Plasma lysine in chicks started to accumulate at a dietary level approximately 10% in excess of that required to maximize weight gain (Zimmer- man and Scott, 1965). However, Mitchell 33 31. (1968b) concluded that deviations in calculated requirements determined by N—balance and PAA response were not larger than what could be explained by the experimental error, and that plasma data seemed to be the more sensitive of the two methods. Part of the discrepancies between the two methods may be related to an underestimation by the 18 N-balance method when dermal losses are not included (Young 33 31., 1971, 1972). Carcass Analysis Williams 33 31. (1954) stated that "an effective method of determining the requirements of a growing animal for these (essential) amino acids may be to determine, first, the requirement in grams per day of one amino acid, such as lysine, and then to estimate the requirements of the others from the proportion existing between the essen- tial amino acids and lysine in the body of the animal, these proportions to be determined by amino acid assays of the entire carcass, or by amino acid assay of a dominant tissue such as muscle." The validity of the method was confirmed by Williams 33 31. (1954). They showed that the amino acid content of the whole carcass of rats, chicks, and pigs had comparable patterns of amino acids within each species at different stages of growth and also a remarkable similarity among species. The similarities in the amino acid pattern is also reflected in estimated requirements. Urinary Urea Brown and Cline (1974) suggested that total urinary urea excretion may indicate protein quality and assess amino acid requirements of swine and other non ruminants. This conclusion was reached because swine fed a corn diet 19 deficient in lysine showed a significant decrease in urinary urea when supplemented with graded levels of lysine. The decrease was linear, but the quadratic effect approached significance on day three. Plasma Urea Nitrogen Lambs fed 12 and 35% protein in a milk replacer showed higher levels of PUN than lambs fed a 24% protein diet (Bergen and Potter, 1975). Increased PUN in lambs fed 12% dietary protein was believed related to the catabolic state of the animals, whereas immediate degra- dation of excessive amino acids appeared to cause elevated PUN levels in lambs fed the 35% protein diet. The low PUN levels in lambs fed 24% dietary protein was explained by better utilization of the amino acids for anabolic purposes. Brown and Cline (1974) and Williams and Smith (1975) showed that PUN levels decreased until the amino acid requirement was met and then remained constant when pigs and calves were fed increasing levels of dietary lysine and methionine, respectively. Williams and Smith (1975) showed good agreement between methionine requirements determined by PUN and PAA, but Brown and Cline (1974) concluded that urinary urea was more precise. 20 Amino Acid Oxidation The validity of amino acid oxidation measurements in assessing amino acid requirements for growth (Brookes 33 31., 1972), and for maintenance (Neale and Waterlow, 1974) have been studied. Brookes 33 31. (1972) injected growing rats with 14 .4 uCi L-lys-U- C—hydrochloride (240 Ci/mole) in .5 ml of .9% saline by heart puncture and collected the expired air with respiration chambers. Release of 14CO2 in the first 6 hours after injection was equal to at least 95% of the total expected for 24 hours. The amount of lysine oxidized remained at a low and relative constant level at suboptimal levels of dietary lysine and accumulated linearly at superoptimal intakes. The response seems analogous to that of PAA, but should be subject to less transient change since amino acid flux is large compared to pool size. The technique appears specific for the amino acid under study. Adaptation of the oxidative mechanisms was complete at day 3 of test diet introduction. The advantage of the oxidation technique over the growth assay is the time factor, although more expensive equipment is required; compared to the PAA method, oxidation is less complex mechanically. In rapidly growing animals, size changes complicate the interpretation of amino acid requirement determined by growth. Of necessity growth data is interpreted more 21 broadly than estimates from amino acid oxidation or PAA (Brookes 33 31., 1972). Requirements determined by amino acid oxidation are usually somewhat lower than by growth assay. Based on the assumption that all carbon chains of amino acids are completely oxidized and not converted to fat, and that the loss of amino acids in urine is negligible, Neale and Waterlow (1974) studied the amino acid requirement for maintenance of rats. They assumed that the maintenance requirement was the net rate of loss from the body which had to be replaced, i.e., the endo- genous loss; and that such a loss could be measured by the output of 14C02. The endogenous rate of amino acid loss as isotope can be measured only if all the amino acids oxidized have the same specific activity. Uniform labelling was reached at day 20 after 14 C-labelled lysine or leucine had been given by stomach tube. Disadvantages to the method are that in order to determine the change in the amount of radioactivity retained in the body, it is necessary to make measurements on different groups of animals at different time intervals. This introduces uncertainties. The loss is the difference between two large values, and therefore cannot be measured very pre- cisely. Due to day to day fluctuations in the endogenous loss of amino acids, caused by variations in physical activity and food intake, it was necessary to calculate 22 the rate of loss from the average excretion of 14CO2 expressed as a fraction of the dose remaining in the body over the dose remaining in the body over the period of observation. The maintenance requirements determined by this method were so different from others that they could not be explained by experimental error (Neale and Water- low, 1974), and prevention of coprophagy was suggested to increase the maintenance requirement. Energy and Protein Requirements of Non Ruminating Calves Nutrient requirements of growing domesticated animals must be considered in terms of age and weight, maintenance, maximum growth, highest retention of ingested nutrients, and the minimum cost per unit of product (Jacobson, 1969). Energy and protein requirements and digestion in the milk-fed calf have been reviewed by Jacobson (1969), Roy (1970), Porter (1969), Huber (1969), ARC (1965), and Radostits and Bell (1970). Energy and Milk Requirements of Calves Fed Milk Requirements of energy for maintenance and growth, and of milk for young calves have been estimated by Roy 33 31. (1958, 1963, 1964), Blaxter and Wood (1951b, 1952a). Brisson 33 31. (1957), Bryant 33 31. (1967), and McGilliard 33 31. (1969; Jacobson, 1969) (Tables 1 and 2). There is 23 Table l.--Energy requirement of calves fed milk; digestible energy per day. Source Maintenance Growth (per kg BW) (per g gain) Blaxter and Wood, 1952a 53.8 —— Blaxter and Wood, 1952a (79.5)b -- Brisson 33 31., 1957 44.7 2.68C McGilliard et a1., 1969 41 3.82 Roy 33 31., 1964 —— 3.02 Bryant 33 31., 1967 48.2d 3.70d Mean 46.9 3 31 aDE = 46.9 BW + 3.31 G; where DE = digestible energy per day (kcal), BW = body weight (kg), G = gain per day (9). bPer m2 body surface at 2.5 times maintenance. CKcal per 9 gain. dBWG .269 DE - 12.96; at 3 to 7 weeks of age BWG = .237 DE - 11.37; at 3 weeks of age = .265 DE - 13.44; at 7 weeks of age where BWG = body weight gain (g/day), DE = digestible energy (kcal) per day. 24 Table 2.--Milk requirement of calves fed only milk; liters per day. BWa Gain; g/day 10% of kg 0 227 454 8wa 27.2 2.15b 2.92 3.68 2.72 36.3 2.75 3.51 4.27 3.63 45.4 3.34 4.11 4.87 4.54 Equation 2.64 3.66 4.68 -- aBW = body weight, kg bRoy 33 31., 1958. CBlaxter and Wood (1952a); G = 222.7 M — 588.75 where G = gain (g/day), M = milk per day (liters). some variation in average energy requirement and the simple mean is slightly below that determined by Roy 33 31. (1958) by covariance analysis of data from 232 shorthorn and 92 ayrshire calves, and since revised by Roy 33 31. (1963) (Table 3). If the simple mean values in Table 1 are used in the formulas estimated by Bryant 33 31. (1967; Table 1), then the values in Table 3 by Roy 33 31. (1963) overestimate the daily gain and the calculated values underestimate it. Protein Requirement The protein requirement of calves has been deter- mined in N-balance and growth response experiments. The 25 .mx .536363 soon zmo .AH magma mmmv o Hm.m + m.mv we a .mema ..mm mm scam immemc mqmea locomv emem imemec «Nee Aomeec Neem OOH Ameemc vemoe Ammmmv mmwm Ameamc Haas lemmmc omev me leemec meem 1mmmmc ammo loeowc mmev nimwmmc cemem em come 0003 com 0 mx mmc\m‘ucwmw 03m .Awmc\amoxv xafle pom mm>amo mo mucmEmuflswmu amumsm manflummmfloan.m magma 26 N-balance studies have shown that the N retention increases with increasing amounts of dietary protein (Brisson 33 31,, 1957); Blaxter and Wood, 1951c, 1952a; Lassiter 33 31., 1963; Bryant 33 31., 1967). Above 24% protein, increases in N retention are not significant. In growth studies, 20% dietary protein has generally been sufficient (Lassiter 33 31., 1963; Brisson 33 31., 1957; Cunningham 33 31., 1958; Huber 33 31., 1964; Bowman 33 31., 1965) (Table 4). However, when fed only 20% protein, calves slaughtered at 91 kg live weight had less edible carcass and more edible fat than those fed 25% protein (Bowman 33 31., 1965). Crane and Hansen (1965) found no effect of 10 or 20% fat in diets containing 24% protein when milk was fed 33 libitum. By the use of the following equation (ARC, 1965): (5) log N = .966 log BW - 1.518; (S.D. i .28) where N nitrogen, kg BW body weight, kg it has been calculated that one kg carcass gain contains 2.5% N. If it is considered that a calf is gaining weight at the rate of .5 kg per day the N gain will be 12.5 g. This is an absolute minimum and slightly lower than estimated by the relationships in Table 5 (13.4 g/day). The positive N retention at zero gain (Table 5) suggests a pronounced ability to conserve protein in the young 27 .mwma .COmQOUMH cHo unmfim3 apon mx\mao am 0 e\ a .CHODOHQ wvH u A .mH u 8 .mm H m “Amm.m x zamv cflmuoum OHQMHHQ>M u m< “Amme\mc e386 n w “mam mxxamoxc smumem maneummwfle n ma “isme\mc seam n mam “z wanfipmmmfio waucmnmmmm n zed «Awmc\mv coflucmuwu z u mz umcowumw>munnamo cfi samuoum manwummmwn maucmummmm mo usmEmuflswmu cam xammsm xaflmo map can coflucmumu z :mm3uwn mmflschHumHmmun.v magma 28 Table 5.--Relationships between N retention and daily gain in calves. Equationa Source NRa'b’c NRC = .0262 so + .703 Blaxter and Wood, 1951c 13.8 NRd = 18.58 Gd + 74.79 Bryant 33 31., 1967 13.0 Mean 13.4 aAbbreviations: NR = N retention; G = body weight gain; BW = body weight. bCalf: BW = 50 kg, G = .5 kg/day. Cg/day- dmg/kg BW. (Bryant 33 31., 1967). Other possible explanations are deposition of lean muscle tissue occurring concomitantly with depletion of lipid and glycogen (or water), as well as biological variation and experimental error (Bryant 33 31., 1967). The water balance becomes a very important factor, especially if diarrhea occurs. The mean retention of 13.4 g N per day in a 50 kg calf gaining weight at the rate of .5 kg per day was applied to the inverse relationships between N retention and the daily requirement of (apparently) digestible protein (Table 4). Results are shown in Table 4 and are in good agreement with recommendations by Roy 33 31. (1970); but slightly below those when protein is supplied 29 through milk (13% DM, 25% of DM as protein) fed at the rate of 10% of BW. If the same feeding procedure were used with milk containing only 20% protein (DM basis) the diet would supply a little less than the calculated digestible N requirement (130 g/day). However, the utilization of calculated available protein is inversely related to the protein content in the diet (Blaxter and Wood, 1951c). Therefore, calculated values are in good agreement with the conclusion that 20% protein is suffi- cient for the milk-fed calf. Endogenous Losses.--In order to estimate the N retention by the factorial method and the biological value (BV) of milk protein, it is necessary to know the loss of endogenous urinary N (UE), the loss of metabolic fecal N (MF), and the loss of N in hair and scurf (S). The loss of UE was determined by Blaxter and Wood (1951a, b, c), Cunningham and Brisson (1957), and Roy 33 31. (1963, 1964); their results are very much alike (Table 6). When Blaxter and Wood (1951c) fed five ayrshire calves semisynthetic milk diets containing 22, 18, and 14% protein on a DM basis, rates of UE losses (slopes) were constant for the three percentages of protein, but the average UE loss increased with amount of protein in the diet. When calves were starved the loss of urinary N was much higher (250 mg/kg BW/day) than in mature .Hmuume 306 :H aHmuoum 33H u H .mH n 2 .mm u m “psmHmz >60n n 3m 16 .z 0HnH lummmflo waucmnmmmm u zed “me .2 wumcfius msocmmoccw n ma "msoflumfl>munndm 30 vomH ..H6 pm 303 «mH m.m6 mm.m + 203 mom. n m: mmmH ..mm mm 30m «mm .. 3mmH .eommHHm 6cm EmrmeHccso 63H m.m6 0HmmH .6003 6am umumem ..u I- mH.m + H203 Hm. u Hm: 0HmmH .6003 6am Hmumem -u- .. He.m + 2204 Hm. u 236 0HmmH .6003 606 Hmumem nu- .. om.m + mzom Hm. u mm: mHmmH .6003 6:6 umumem uuu m.Hm mounom mn.3m 3m coHumsvm mmx\z we .63 6 . .mm>Hmo aw Hmbv z mumcflus msocmmoocw mo mmoqnt.m magma 31 ruminants of the same body size (goat, sheep) or the same species (Blaxter and Wood, 1951b). The excretion of metabolic fecal N (MF) in calves was determined by Harris and Loosli (1944), Lofgreen and Kleiber (1953), Blaxter and Wood (1951a, b, c), Cunningham and Brisson (1957), and Roy 33 31. (1963, 1964) (Table 7). The values obtained by Blaxter and Wood (1951a, c) and Cunningham and Brisson (1957) are as high as those deter- mined for ruminating calves. These studies may have over— estimated MF because N—free diets were fed as milk replacers, whereas Roy 33 31. (1963, 1964) used calves fed whole milk through 10 weeks of age. Lofgreen and Kleiber 32 (1953) used P-labelled casein. Blaxter and Wood (1951a) Table 7.--Loss of metabolic fecal N (MF); g/100 g dry matter ingested. MF Source .27 Lofgreen and Kleiber, 1953 .427 Blaxter and Wood, 1951a N = 1.2 + .3 Pa Blaxter and Wood, 1951c .334 Cunningham and Brisson, 1957 .19 Roy 33 31., 1963, 1964 .372b Harris and Loosli, 1944 aAbbreviations: N = g N in dry feces; P = pct. of total cal. as protein. bRuminating calves. 32 suggested that the quantity of fecal DM rather than DM intake determines the excretion of MP. The loss of N in hair and scurf have been esti— mated to be .02BW-73 (g/day) (ARC, 1965). There is no information on the loss of N in sweat, but it seems unlikely to be of importance (ARC, 1965). Dig3stibility and Bioloq1pal Value of Milk Proteins Digestible protein is more meaningful, if accu- rately determined, than total crude protein in rations for milk fed calves (Jacobson, 1969). Digestibilities of DM and protein in whole milk are high (Table 8), and not affected by age and amount of milk fed (Roy 33 31,, 1964; Blaxter and Wood, 1952a; Bryant 33 31., 1967). The true digestibility of casein is high (Lofgreen and Kleiber, 1953), whereas the apparent digestibility of dried skim milk is lower than whole milk and shows a slight tendency to increase with increasing protein content of the diet (Lassiter 33 31., 1963; Blaxter and Wood, 1951a; Bryant 33 31., 1967; Bowman 33 31., 1965). The estimated biological value of milk proteins varies from 32 to 79 for casein and from 60 to 92 for whole milk and dried skim milk (Blaxter and Wood, 1951c, 1952a, c; Brisson 33 31., 1957; Roy 33 31., 1963, 1964). 33 Table 8.—-Apparent digestibilities of milk dry matter (DM) and milk protein in calves at different ages; %. . Digestibility Protein Source DM Protein Source Whole milk 4 wka 97.5 95.1 Roy e_t_ 31., 1964 7 wk 96.1 93.4 Roy 33_31,, 1964 10 wk 97.2 96.4 Roy 33 31., 1964 Dried skim milk 15.2b 87.0 78.6 Lassiter 33 31., 1963 18.7 88.6 80.7 Lassiter 33 31,, 1963 24.1 88.5 86.2 Lassiter 33_31., 1963 30.9 91.6 90.6 Lassiter 33_31,, 1963 94.0 -- Blaxter and WOod, 1951a 3 wka —— 86.1 Bryant 33_31,, 1967 7 wk -- 90.1 Bryant et a1., 1967 Casein -- 93.5C Lofgreen and Kleiber, 1953 N-free 77.0 -- Blaxter and Wood, 1951a a . Age in weeks. b . . . Percent protein in milk replacer. CTrue digestibility. 34 Protein Sources in Milk Replacers An increasing demand and price for fluid milk for human consumption has caused development of milk replacers (MR). The major protein source in MR is dried skim milk (DSM), but others have been investigated. Mixing one part soy flour with nine parts of warm water was an early attempt to replace whole milk (Shoptow, 1936). When used in an otherwise nutritionally adequate diet replacement of up to 30% of the protein from DSM with soy flour normally resulted in inadequate gains and poor N retention, but results were variable (Noller 33 31., 1956; Porter and Hill, 1963; Neiman-Sorensen 33 31., 1965; Colvin and Ramsey, 1968, 1969; Gorrill and Thomas, 1967; Klausen 33 31., 1969). Soy flour is low in lysine and methionine compared to milk but supplements of these amino acids showed only slight improvement in MR containing 26 to 28% protein (Neiman—Sorensen 33 31., 1965; Klausen 33 31., 1969). Spray drying of 7 parts of soy flour with one part whey solubles was used with moderate success up to a maximum of 43% of the dry non-fat solids in MR, but supplemented methionine (.25%) had no effect on growth of calves (Stein and Knodt, 1954; Stein 33 31., 1954). When soy was subjected to acid (pH 4.0) or alkali for 5 hours at 37 C the nutritional value was markedly improved; but the mechanism of improvement was not identified (Colvin and Ramsey, 1968, 1969). When isolated soy 35 protein (71% protein) supplied 50% of the MR protein without supplemental methionine or 70% with methionine, weight gains were equal to those of calves fed a MR, where DSM or whole milk were the only protein sources (Gorrill and Thomas, 1967; Gorrill and Nicholson, 1969; Schmutz 33 31., 1967). Dry matter and N digestibilities of isolated soy protein were lower than for all the milk protein sources and were not affected by supplemental methionine (Gorrill and Nicholson, 1969). Calves fed isolated soy protein as the only protein source showed N retentions and digestibilities equivalent to casein (Porter and Hill, 1963). The difference between untreated soy flour and isolated soy protein may in part be attrib- uted to a high content of soybean trypsin inhibitor (Gorrill and Thomas, 1967). Soy flour containing 50% protein and supplying 60% of the protein in MR caused weight loss, decreased activities of trypsin and chymo- trypsin in the pancreas and intestinal contents, and decreased 13 31333 protein digestibilities. Isolated soy protein gave results equal to whole milk or replacers containing only milk protein. Weight gains and apparent digestibilities of N, ether extract, and energy showed significant linear declines when increasing levels of distillers dried solubles (DDS) were substituted for DSM and lactose (Bryant 33 31., 1967). However, it was concluded that DDS 36 can replace 35% of the digestible protein in herd- replacement diets without severe impairment of growth. Defatted fish flour substituted for up to 40% of the protein in a MR had no consistent effect on daily gains, incidence of diarrhea, or protein digestion or retention (Slade and Huber, 1965; Harshbarger and Gelwicks, 1965; Huber and Slade, 1967). However, at 60 to 67% replacement, marked decreases in growth and N utilization, and increases in diarrhea were observed; and at 100% death occurred (Huber and Slade, 1967). These data are in contrast to the equal gains in calves fed a commercial MR where defatted fish flour and whey powder were the only protein sources (Sorensen and Lykkeaa, 1968). These dif- ferences may be due to species differences in fishes used, heat damage which led to unavailability of lysine and sulfur amino acids, alkylation of sulfhydryl groups by 1,2-dichloroethane (DCE) to produce thioether linkages with a resultant decrease in cystine, methionine, and histidine, or a marked deficiency of vitamin E, which may be compounded by the presence of about 3% highly unsat- urated fat (Morrison and Sabry, 1963; Morrison and Munro, 1965; Genskow 33 31., 1969; Makdani 33 31., 1971a, b, c). Supplements of histidine (.075%) and methionine (.20%) resulted in additive and parallel increments in growth of rats for all fish flours used, and extraction with isopropanol resulted in a more nutritious fish flour than 37 when DCE was used (Makdani 33 31., 1971a, b). Genskow 33 31. (1969) demonstrated that plasma levels of histidine and tyrosine decline, and that methionine and arginine increase when increasing levels of fish flour (zero to 100% of protein) are added to MR fed to calves. Single cell protein from oil products supported the same rate of gain when substituted for 67% of the DSM protein in a MR containing 20% protein (Lykkeaa 33 31., 1973). When single cell protein replaced more than 67% of the protein, daily gain was significantly lower, probably due to a low methionine content (1.5 versus 2.4% in DSM protein). From the evidence presented it may be concluded that lower digestibility, toxic residues, or inadequacy of essential amino acids or vitamins (due to genetic or preparational factors) make protein sources other than DSM inferior for milk replacers. Supplementation with the limiting amino acids, vitamins, and/or alkali treatment to increase digestibility or removal of toxic factors render alternate sources more suitable. However, most of these sources can be successfully used for partial replacement of milk protein. 38 Amino Acid Requirements Calves.--The young non-ruminating calf is as dependent on its diet for EAA as is man, dog, or rat as clearly demonstrated by Blaxter (1950) and by Blaxter and Wood (1952c) when they fed calves dried skim milk, casein, or gelatin. The requirement of the individual EAA is virtually unknown. Patureau-Mirand 33 31. (1973) deter- mined the methionine requirement of calves by combining results from two experiments where 12 calves were fed 2.6, 3.0 and 3.5, and 3.5 and 4.5 g methionine daily, as a supplement to commercial MR (24.5 and 26.4% protein). The methionine content in the blood (mg/100 9 blood; Y) showed a curvilinear relationship with dietary methionine 73 6.12x. (X, mg met/kg BW- ; Y = .0883e r = .94). From these results they concluded that calves gaining 1.0 kg or more BW per day require .58 g methionine per kg BW-73. Expressed on a MR basis, that equals 9 g methionine per kg MR solids, or 3.5 g methionine per 16 g N when the protein content is 26.4%. They found no effect of graded levels of supplementary cysteine. Moreover, amino acid supplements had no significant effect on growth or feed intake. Tzeng (1974) studied the requirement of methionine and lysine in non-ruminating calves. They fed MR where the protein was replaced by a mixture of crystalline amino acids. The requirement of methionine was determined to be 39 1.65% of DM by weight gain and N retention and 1.15% of DM by PAA. The requirement of lysine was 1.8, 1.75, and 1.95% of DM when determined by weight gain, N-retention, and PAA, respectively. Williams and Smith (1975) estimated the total sulfur amino acid requirement in 2 non-ruminating calves fed successively increasing amounts of methionine (.8 to 4.8 g) as supplement to a diet containing .25 kg whole milk, .53 kg synthetic milk (Smith, 1959). Casein was omitted, and 270 ml aqueous solution of amino acids was fed per kg milk (.05 kg per kg BW). They obtained the typical broken-line response for plasma methionine, and the inverse curve for PUN. From the results obtained the total sulfur amino acid requirement was determined to be 4.2 to 4.8 g per day, or .23 to .26 g per kg BW-73. It was concluded that cow's milk fed to promote .25 kg daily gain provides no surplus of sulfur amino acids (3.2 9 met + 1.0 g cys). The three experiments report widely differing requirements and suggest a need for additional information. Factors Affecting Plasma Amino Acid Levels in Calves.--Leibholz (1966) fed calves a commercial MR from birth to 4 weeks of age and showed significant negative correlations between age and plasma levelsof serine, proline, glutamine, methionine, leucine, lysine, and histidine. Proline declined very rapidly, and the remaining 40 amino acids showed no change with age. Williams and Smith (1973, 1975) found little variation with age. Calves bled before the morning feeding at 10 am and at one hour intervals until 4 pm and thereafter every two hours until 10 am the following morning showed decreased concentrations between 10 am and one pm of total PAA and most individual amino acids (Williams and Smith, 1973, 1975). No marked change in total PAA was observed after the evening feeding at 5 pm. The variation in plasma levels of methionine isoleucine, leucine, phenylalanine, tyrosine, and total PAA is significantly greater between animals than within animals in both non-ruminating and ruminating calves (Williams and Smith, 1973, 1974a, b, c, 1975). Amino Acid Infusion in Ruminating Calves and Lambs.--Amino acids administered orally do not survive rumen degradation in lambs (Papas 33 31., 1974). However, intraperitoneal infusion into calves of a mixture of methionine, lysine, tryptophane, histidine, and arginine improved N-balance compared to a mixture of the remaining EAA or single constituents of the mixture (Hall 33 31., 1974). Williams and Smith (1974a, b, c) infused graded levels of methionine into calves (110 to 160 kg BW) fed 20 g N as decorticated, extracted groundnut meal. Plasma methionine was markedly increased in excess of 4.4 g L-methionine. Estimated flow of methionine and cysteine 41 from the rumen was 9.8 and 4.9 g per day, respectively. This led to an estimated total requirement of sulfur amino acids of 19.1 g per day, and is in good agreement with the estimated requirement of 22.4 g per day for 274 kg steers (Fenderson and Bergen, 1975). Infusion of lysine, threonine, or tryptophane did not show the broken-line response curves, suggesting that they were supplied in adequate amounts from rumen digesta under those circumstances (Williams and Smith, 1974c; Fenderson and Bergen, 1975). Amino Acid Requirements of Other Species.-—Recom- mended dietary levels of the EAA have been given for growth in rats, chicks, turkeys, and pigs (NRC 1971a, 1972a, 1973a), but are essentially unknown for mink, fox, cat, guinea pig, hamster, monkey, mouse, rabbit, horse, sheep, and cattle (NRC, 1968a, b, 1966, 1971b, 1972a, b, 1973b). Variations in requirements were discussed by Hegsted (1963). Table 9 shows recommendations as a per- cent of the diet and as a percent of the dietary protein. The variation in recommended needs of the various species, when expressed as percent of the dry feed, is almost com- pletely eliminated when expressed as a percent of the dietary protein. Boomgaardt and Baker (1973) found that in chicks fed 14 to 23% dietary crude protein, the lysine requirement was constant when expressed as percent of the crude protein. 42 Table 9.--The amino acid requirements of rats, chicks, turkeys, and pigs.a Age, Rat Chick (replacement) Turk3y bPig 35 wk 0-6 6—14 14-20 8—11 11-14 5—10 10-20 Percent of dry diet Arg .67 1.2 .95 .72 1.3 1.1 .28 .23 His .33 .4 .32 .24 .45 .35 .25 .20 Ile .61 .75 .6 .45 .85 .75 .69 .56 Leu .83 1.4 1.1 .84 1.5 1.3 .83 .68 Lys 1. 1.1 .9 .66 1.2 1.0 .96 .79 MetC .67 .75 6 .45 .7 .58 .69 .56 Phed .89 1.3 1.05 .78 1.4 1.20 .69 .56 Thr .56 .7 .55 .42 .8 .70 .62 .51 Trp .17 .2 16 12 2 .17 .18 .15 Val .67 .85 7 .5 .95 .80 .69 56 Percent of dietary protein Arg 5.2 6.0 5.9 6.0 5.9 5.8 1.3 1.3 His 2.5 2.0 2.0 2.0 1.8 1.1 1.1 Ile 4.7 3.8 3.8 3.8 3.9 3.9 3.1 3.1 Leu 6.4 7.0 6.9 7.0 6.8 6.8 3.8 3.8 Lys 7.7 5. 5.6 5. 5.5 5.3 4.4 4.4 Metc 5.2 3.8 .8 3.8 3.2 3.1 3.1 3.1 Phed 6.8 6.5 6.6 6.5 6.4 6.3 3.1 3.1 Thr 4.3 3.5 . 3.5 3.6 3.7 2.8 2.8 Trp 1.3 1.0 . 1.0 .9 .9 .8 .8 Val 5.2 4.2 4.3 4.2 3.1 3.1 aFrom NRC, 1971a, 1972a, 1973a. bWeight, kg. COr45% met and 55% cys. d Or 55% phe and 45% tyr. 43 Blood Urea Nitrogen in Calves and Lambs Increasing PUN with increasing levels of dietary protein (15.2 to 30.9%) have been reported for calves (Lassiter 33 31., 1963), and lambs (Potter and Bergen, 1974). The PUN also increases with increasing age in calves fed whole milk (Roy 33 31., 1964). Milk-fed calves had significantly higher PUN levels than ruminating calves fed zero to 55.6% of the dietary N as urea (Leibholz and Naylor, 1971). In calves fed a commercial MR, Leibholz (1966) found a sharp increase in PUN from birth to one week of age and then an almost linear decline until dry feed was introduced. These results are in contrast to the absence of age effects in grazing lambs, yearling- and adult-sheep (Torell 33 31., 1974), and calves fed whole milk at 35 and 84 days of age (Williams and Smith, 1973, 1975). Torell 33 31. (1974) and Williams and Smith (1973, 1975) found only slight changes with time of sampling after a meal and larger variations between animals than within animals. A semisynthetic diet supplemented with graded levels of methionine led to marked and linear decreases in plasma urea when the supplement was subOptimal and to only low and almost constant responses at superoptimal levels of methionine (Williams and Smith, 1973, 1975). MATERIALS AND METHODS The objective of this study was to determine the effect of feeding graded levels of methionine to the young milk-fed calf in an attempt to learn more concerning its methionine requirement. Weight gain, digestibility, N-balance, plasma methionine, and plasma urea nitrogen were used in assessing methionine effects. Animals.-—Twenty male Holstein calves were employed in a four-week, two-period changeover experiment with five dietary levels of methionine. Thirteen of the calves were born in the M.S.U. dairy herd and 7 were bought from nearby Michigan dairy farms. Due to calf disease problems in the herd, compounded with stress of the metabolism stalls, 12 calves succumbed before completion of the experimental period. Results for these calves were omitted and the first calf born after the death of the original calf was used as a replacement. Scours from bacterial infections unrelated to treatments were the major reason for deaths among the calves (Table 10). At the average age of 3.7 days (range, one to 9 days) the calves were confined to metal metabolism stalls at the M.S.U. Dairy Research Center. The calves were 44 45 .600006H6 H60H0 mH mmHH man .OH .066 mam m .600006H6 H60H0 mH quH «an .em .000 com a .00LHH0ac “60am m vhma .am .mmm vma m .603H36aw “Umao N whaa vbma .Na .adh hma m .600006H6 u60H0 HH HMMH veaH .Hm .000 6mm 6 .mwnuHMaU «00am om mama vhma .ma .Umo mmm m .mwnuumaw “Umao Na mama vhma .6 .UOQ mmm m .mmnuumao “60am ha unmsom vhma .m .>oz mum m .600006H6 H60H0 m HNMH 6emH .oH .000 «mm m mmma ucoum 00ammauu 6mma whma .m .>oz mom 6 .wmnuumav “Umao m Noma whoa .ma .ash mma N .600006H6 160H0 mm HmNH wan .HH .000 mam H mxumemm mmmc .mm« Ema nuuan mo mumo mamo HUOam 30amman . .muaamuuoe Ho Spammn Moon on 056 00au0amEoo 0u0m0n ucmeaummxm map Eoum cwuuan mm>a0011.oa manme 46 blocked in groups of two according to date of birth, and each calf received two dietary levels of methionine; one during period 1 (9 to 15 days of age) and one during period 2 (21 to 27 days of age). The blocks were random— ized as follows: block 3, 7, 8, 4, 10, 2, 6, 5, 1, and 9, before assignment of calves. The assignment of calves to blocks and treatments is given in Table 11 along with the calf's date of birth and the dam's identification number. 21333.--The five dietary levels of methionine representing 75, 100, 125, 150, and 175% of the methionine in milk protein (treatments A, B, C, D, and E, respectively) were obtained by preparing experimental milk replacers containing 20% crude protein from a commercial, non— 1 medicated milk replacer (20% crude protein), crude 2 The commercial lactose,1 and crystalline amino acids. milk replacer was prepared from dried skim milk, dried whole whey, animal fat, casein, premix of vitamins and minerals, and calcium carbonate (Table 12). Its assayed amino acid content showed good agreement with literature values for whole milk (Table 13) when determined by the method previously described by Makdam 33 31. (1971b). 1Supplied by Milk Specialties Co., P. O. Box 278, Dundee, Illinois 60118. 2General Biochemicals, Chagrin Falls, Ohio. 47 Table ll.--Assignment of calves to blocks, date of birth, dam's identification number and treatments. -_—_- .. E-”— ... -mfi— Block Calf Date of Birth Dam §E£i2% Remarks 1 292 Dec. 31, 1974 1196 Aa B 285C 286 Dec. 13, 1974 1260 B A 2 273 Nov. 8, 1974 Bought A C 274 Nov. 9, 1974 Bought C A 126 3 125 Jul. 10, 1974 1227 A D 128 Jul. 16, 1974 1147 D A 4 262 Oct. 29, 1974 1252 A E 270 Nov. 11, 1974 1168 E A 269 5 282 Dec. 1, 1974 1116 B C 287 Dec. 14, 1974 1314 C B 284 6 296 Jan. 22, 1975 1231 B D 275, 283, 288, 294 276 Nov. 10, 1974 Bought D B 7 129 Jul. 20, 1974 1306 B E 130 Jul. 31, 1974 1205 E B 8 259 Oct. 17, 1974 Bought C D 127, 194 260 Oct. 17, 1974 Bought D C 9 289b Dec. 21, 1974 1311 C E 300 Jan. 27, 1975 1328 E C 290, 295 271 Nov. 3, 1974 Bought D E 10 272 Nov. 7, 1974 Bought E D a Treatment. Fed whole milk in period 1 due to lack of feed and amino acids. Data for calf 295 used in period 1. CNumber of calf replaced. 48 Table 12.~-Composition of the milk replacer.a Ingredient % of DM Dried skimmed milkb 32.0 Dried wholg whey 43.8 Animaldfat 20.0 Casein 3.0 Premixe .6 Calcium carbonate .6 Guaranteed analysis: Crude protein, not less than 20.0 Crude fat, not less than 20.0 Crude fiber, not more than .15 Vitamin A, not less than 15,000; Vitamin D3, not less than 3,000 aSupplied by Milk Specialties Co., P. O. Box 278, Dundee, Illinois 60118. b37% protein. CPreserved with BHA. dProcessed into a high fat ingredient before mixing. eSoy lecithin, Polyoxyethylene glycol mono and dioleates, Vitamin A palmitate (stability improved), Vitamin D source: D—activated animal sterol, Vitamin E supplemeng, Vitamin B supplement, Folic acid, Choline chloride, Riboflavin, iacin, Calcium Pantothenate, Thia— mine mononitrate, Calcium carbonate, Copper sulfate, Cobolt sulfate, Zinc sulfate, Ethylene diamine dihydroiodide, Sodium silico aluminate, Manganese sulfate, Magnesium oxide. fUSP units per 1b. 49 5a.6 5a.6 m6.6 mm.m mm.m was 000: 55.6 oa.m ma.m m6.m 30m 0000 m6.m 5o.m mm.m mm.m oum mm.ma no.5a 66.5a mm.ma mm.ma mo.m mm.~ mm.a am.a mam ma.mm mm.mm mm.6m om.mm No.5m 6a.ma mm.ma mm.am 6m.mm sac mo.a mo.a ma.a 5m. 55. 050 000: om.m mm.m 56.6 mm.m 30¢ 000: 6m.m mm.m 6~.m mm.m 0am «dmz mm.m6 06.am 56.66 mm.m6 «6m amuoe a6.m a6.w mm.m ma.m m6.m a6> m~.H m~.H u- m~.H Hm.H 003 nom.6 mm.6 ma.m m5.m mm.m use 56.6 56.6 mm.6 oa.6 m~.6 0:3 mm.m 66.5 mm.6 mm6.m o m6.m m6.~ 6m.m mm.m 002 066.0a ~m.m oa.m 6a.m m~.m 05H «6.x mm.m a~.m 5o.m m6.m :04 am.m am.w 5~.5 mm.m m5.m 0aH am.m am.~ 56.m mo.~ m5.~ mam 66.m 66.m 5m.m mm.m 06.m mufi «mm m o O m a .uuou .uuooao .unou .uuooco pmusuxas oaom ocaEM amucmemammsw H.03000ammuxaaz nxaae 0aon3 paom ocae¢ 6:0 0 umomammu xaaE amaoumfieoo .xaaE 0m .mmusuxae Uaom oca60 nmummmum 0:3 ca ucmucoo naom ocaEM 009:1.ma magma 50 .mua0u0>aca 006nm :6manoa2 .mu606n0sm a6Eas< .muou6uon6q coauauusz uc6cassm .c0mu0m .0 .3 .mo .chau6CaEump06 003:» no 0m630>¢H .00um 0aa< .00um 00a00a£u0zm a .60caEH0u06 uoz .aum .0ca05anq0 m .ma5 .oz 00au60aahsm aaocsou £036000m a600au6z ca 600aauso 0602006 0:» nua3 0036600006 0a 6056006 06ao6 00aE6uq 066mm .Oano .0aa6m caum6zo .0a6an0500am a6u0c006 .maaom 0aocaaaH .006350 ..0um 0aocaaaa 606 H0063 .m5m xom .o .0 ..oo 00aua6ao0mm xaaz an 60aammsmo .H5mH .006H60 N33 .003 N$3 .002 "0000000 .2 0 ma 00¢ 06 oo.ooa am.moa ma.ooa mm.6oa «d a6uoa mm.om a6.mm ~5.mm 5~.6m «dmz a6uoa m o O m d .uuou .HuooaD .uuou .uuooao 6H06 o:HE¢ 60usuxae 6a06 00a56 a6uc0E0ammsm H.03006am0uxaaz nxaaE 0a0£3 . . .60scaucooul.ma 0an6e 51 A mixture containing 15% crude protein was pre- pared from 75% commercial milk replacer and 25% crude lactose. The 5% additional protein was added as a mixture of L-crystalline amino acids. The amino acid mixture con— tained the EAA's, cysteine, and tyrosine in the same ratios as determined for the commercial milk replacer. The remaining non-essential N was formulated from 50% glycine-N and 50% glutamic acid-N (Table 13). In order to make the experimental milk replacers isonitrogenous, the content of glycine and glutamic acid decreased with increasing levels of methionine. Amino acids were first weighed1 and mixed; then they were added to the appropriate amount of crude lactose. This mixture was then blended with the commercial milk replacer in a batch mixer, and stored at room temperature. Feeding Schedule.--The calves were fed colostrum for 3 days and whole milk until placed on experimental diets which were the only feed from 6.5 days (range 4 to 9 days) to 28 days of age. One to two days were used for a gradual change from whole milk to the experimental replacer and from the treatment of period 1 to that of period 2. Each dietary change was followed by a 3-day adjustment period. Periods l and 2 were from 9 to 15 and 21 to 27 days of age, respectively (Table 14). Milk 1Sartorious scale. 52 Table l4.-—Plan of feeding, weighing, collection, and blood sampling. Age Total collection Blood days Treatment Weighing urine + feces samples Remarks Birth 1 Colostrum 2 Colostrum 3 Colostrum + 4 3M + 2Aa’c + Adjustment 5 2M + 3A + Adjustment 6 5A Adjustment 7 A Adjustment 8 A + Adjustment 9 A + + + Period 1 10 A + + b Period 1 11 A + (+) Period 1 12 A + Period 1 13 A + (+) Period 1 14 A + + Period 1 15 A + + + Period 1 16 3A + 23C + Adjustment 1? 2A + 3B Adjustment 18 SB Adjustment 19 B Adjustment 20 B + Adjustment 21 B + + + Period 2 22 B + + Period 2 23 B + (+) Period 2 24 B + Period 2 25 B + (+) Period 2 26 B + + Period 2 27 B + + + Period 2 28 B + aParts of total feed. b . . . Were collected in the first Six calves only. c . . . Abbrev1ations: M = whole milk, A = treatment A, B = treat- ment B. S3 replacer was diluted with water to 13% solids and was fed by nipple pail at 10% of body weight daily in two equal meals (5% each) at 6 am and 6 pm. At each feeding the replacer powder was mixed with warm water (27 C) by use of a hand beater. Initially, the calves had free access to water (calves 125, 128, 129, 130, 259, and 260), but later additional water was restricted to .5 to 1.5 liters after each meal, because some calves became nibblers and would not drink their allotted feed at the following feeding. The first .5 liters was used to wash down milk residue in the nipple pails. Body Weight.--Body weight of the calves was deter- mined on three consecutive days at the beginning and at the end of each experimental period (Table 14). Nitrogen Balance.--The calves were confined to metal metabolism stalls from 4 to 28 days of age during which all urine and feces voided were collected daily (Table 14). Urine was collected in plastic buckets con- taining about 30 ml 33% sulfuric acid. Feces was col- lected in open trays placed underneath the calves. All milk weigh back, urine, and feces were refrigerated until the last sampling in each period, whereupon composited samples were frozen at -20 C until analysis. The nitrogen content in feed, milk weigh back, urine, and feces was determined by the Kjeldahl method. 54 Blood Collection and Processing.--In the first 6 calves blood was collected at 9, ll, 13, and 15 days of age in period 1, and at 21, 23, 25, and 27 days of age in period 2. In the remaining calves blood was collected at 9 and 15 days of age in period 1, and 21 and 27 days of age in period 2 (Table 14). The number of collection days was reduced because initial analyses showed only slight differences between days in plasma methionine levels, the stress introduced by blood sampling would be less, and the high number of analyses decreased. On each day of sampling blood was collected before feeding and l, 2, 4, and 6 hours after feeding. Each sample made up 20 m1. In the first four calves (calf 125, 128, 129, and 130) a permanent jugular catheter was established. Because of difficulty in maintaining the cannula in metabolism stalls the remaining calves were collected by jugular 1 After collection, blood was transferred to 10 puncture. m1 evacuated tubes2 containing 37 mg potassium oxalate as anticoagulant and 37 mg sodium fluoride as glycolysis inhibitor. The tubes were gently inverted and placed in an ice bath until processing. Blood processing was initiated 7 to 10 hours after collection of the first sample. From inverted tubes, whole lSingle Draw Vacutainer Needle (silicone coated, 20 gauge). Becton-Dickinson, Rutherford, N.J. 07070. 2Vacutainer, non-sterile, 10 ml. Becton-Dickinson, Rutherford, N.J. 07070. 55 blood was transferred to hematocrit tubes and the remaining whole blood was centrifuged at 6000 x g for 10 minutes at 0 C.1 Plasma was pipetted into centrifuge tubes, mixed with .1 ml 50% sulfosalicylic acid and .1 m1 Nor-leucine (1 mM) standard per ml plasma, and placed in ice bath from 30 minutes to 2 hours before centrifugation at 35,000 x g for 15 minutes at 0 C. The protein-free supernatant was frozen until analyses. A sample of unprocessed plasma was also frozen for later analysis. Plasma was analyzed for methionine, valine, leucine, and isoleucine by ion-exchange chromatography on an amino acid analyzer2 by Dr. W. G. Bergen.3 Unprocessed plasma was assayed for urea nitrogen by the method described by Fawcett and Scott (1960), and since modified by Kulasek (1972), using the reagents described by Okuda 35 21. (1965). Hematocrit was determined by centrifugation of whole blood in standard hematocrit tubes in an International centrifuge at 65% of maximum speed for 10 minutes by Universal timer. 1Sorvall Superspeed RC2-B Automatic Refrigerated Centrifuge. . ' 2 versity. TSM-l amino acid analyzer,R Michigan State Uni- 3Ruminant Nutrition Laboratory, Animal Husbandry, Michigan State University. 56 Experimental Design and Statistics.--The experi- ment was conducted as a two-period, changeover design with five dietary levels of methionine and 20 calves in 10 two x two latin squares. In each square two calves represent rows and two periods represent columns. All data with equal numbers were analyzed statistically according to the model (6) described by Gill and Magee (1976). A (6) Yijk = u + Di + pj + Tk + E(ijk) A where Yijk = observation in i Eh calf in j Eh period on k Eh treatment, Di = effect of i Eh calf, pj = effect of j Eh period, Tk = effect of k Eh treatment, and E(ijk) = error Treatment means (adjusted for differences in individual calf means) were calculated as described (7) by Gill and Magee (1976): _' = (7) yk y . . . + Tk where yi = adjusted mean for the k Eh treatment, §... = overall mean, a - 2(2'1) 'T = [2(t-l) y.. - Z y...]/t, " 1‘ ind t = number of treatments y..k = observed (unadjusted) mean for the k Eh treatment, and 57 yi.. = observed mean for the i Eh calf among those given the k Eh treatment in either period. Unadjusted treatment means are not reported. Differences between adjusted treatment means were tested by Dunnett's t-test, using treatment B as control (Kirk, 1968). The relationship between dietary methionine levels and assay parameters was analyzed by multiple regression analysis according to a third degree polynomial (8) and its reduced forms: 8A_.'\ /\ A 2 A3 A where Y = predicted assay response, and x relative dietary methionine level (100 = 2.48 g met/l6 g N). The procedure was as follows: The relationship was deter- mined for dietary methionine level and the adjusted mean for the parameter in question if the overall treatment effect was significant (P < .10). If the regression so determined was significant (P < .25), then the regression analysis was repeated on the full data set after obser- vations had been adjusted according to equation (9): = Y (Yobs. trt. - Yadj. trt.) (9) Y adj. obs. obs. where Yadj. obs. = adjusted observation, Y = observed observation, obs. 58 Yobs. trt. observed treatment mean, and §adj. trt. = adjusted treatment mean. Regression coefficients different from zero (P < .05) were then selected and a final prediction equation was calculated. If the relationship was curvilinear, minimum and/or maximums were determined according to (10) for quadratic regressions and approximated according to (11) for regressions involving only linear and cubic terms: (10) xmin/max = - (Bl/282)' and (11) Xmin/max z 1 {817383 (ignoring imaginary part of root, J-I). RESULTS Body Weight, Dailnyain, and Health.--Body weights (BW) at the beginning and at the end of the experimental period, as well as average daily gain (ADG) and fecal score (F8) are given in Table 15, and the corresponding analyses of variance is in Table A1. The average initial BW was 43.2 kg and did not differ between treatment groups. However, the initial BW varied among calves (P < .001), and was lower in period 2 than in period 1 (P < .01). The lower BW at the beginning of period 2 can probably be explained by poor health and weight loss in period 1 as will be discussed later. The average final BW was 42.8 kg and tended to differ between treatment groups (P < .10). The final BW varied among calves (P < .001), but there was no difference between periods. Multiple comparisons among treatment means showed that the calves in group B weighed less than the calves in group A, C, and D (P < .05), but group B did not differ from any other group. The ADC tended to differ between treatment groups (.25 > P > .10), and was less in period 1 than in period 2 (P < .10). There were no significant differences among 59 60 .e \ _mo Ame + v 0 A60 + .z 0 0H\uwz m 06.6 .06000 m 0006 A new .mmomu m 0006 on HOOH :00: :00; .00000 0 coca on Hom sues m .00000 0 000 cu Hmm .0000m 0 0mm N a Ame + o 2N0 + 2003 v 2003 an AHVH 0>66 mo 0>66 mo 0m66 mo 0>66 mo 0x66 mo " QHOUm .OC .OG .OC .OC .OC H60 000:3 0m OOH "0000 002 0>H06H0m0 .Amo. v my 000000006 >H0060fiMflcmflm uoc 0H6 umwuomu0m50 008800 6 mcwu6nm 0:602 6.6 em.H vm.m v~.~ mm.“ amn.~ mmm.H mmm.H mam.~ 6-.~ muoom H6060 H6 06H- 60. am 0 noon- 6~H 600 mm»- «as. m .:060 03066 .m>< m.~v 0.66 0.~v v. H no.av 6H.mv mH.mq n.6m.~v 60.mv Hagen n.~v e.mq «.m6 0. H 66.6w ma.mv 6>.~v mm.~v 6~.mq HmfluficH 0x .060003 0600 m a :60: .m.m m o 0 m 6 6oflu0m HH6H0>O 000506009 .0cfi00flnu0fi mo 0H0>0H 6066mm 60m 00>H60 no 00000 H600m 6:6 .cw6m >HH66 0w6u0>6 .Aa6cflm 6:6 H6wuwcfiv 0:0003 >600 co 0cflcoasu0a6>u6u0w6 mo uo0um0 0£BII.mH 0Hn69 O 61 calves. The weight loss in period 1 explains the lower initial weight in period 2 than in period 1, and the low ADG in period 2 explains why the final BW was the same in period 1 and 2. Multiple comparisons among treatment means for ADG showed that group B gained less (or lost more) than any other group (P < .05), and that differences between groups A, B, C, and D were not significant. A FS was calculated for each calf according to the formula in (12): (12) F8 = [(1)0l + (2)02 + (3)03 + (4)04 + (5)051/7. where FS = fecal score D1 = number of days with < 250 g feces, D2 = number of days with 251 to 500 g feces, D3 = number of days with 501 to 1000 g feces, D4 = number of days with 1001 to 2000 g feces, and D = number of days with > 2000 g feces. S The analysis of variance showed no significant differences between treatment groups or between calves, but the FS was higher in period 1 than in period 2 (P < .001), and may in part explain the low ADG and final BW for period 1. The relationship between dietary methionine and final BW, ADG, and FS, respectively, was determined by the described procedure for multiple regression analyses of the full model and its reduced forms. The determined models for adjusted means and the calculated regressions 62 for adjusted observations are given in Table 16, and the analysis of variance is in Table A2. The calculated regressions showed no significant correlation between dietary methionine levels and final BW or F8. For ADG the analysis showed relatively equal significance levels for the regression coefficients of the full model. The prediction equation for this relationship showed that 26% of the variation in ADG was due to dietary methionine levels. However, only the regression coefficient of the quadratic term was significant, and accordingly, only 10% of the variation in ADG was due to dietary methionine. As may be seen from Table 15 the trends for ADG and FS tend to indicate decreasing ADG with increasing frequency of scouring (increasing FS = increasing fecal excretion). Therefore, the relationship between ADG and fecal excretion was determined for uncorrected observations by multiple regression analyses of a third degree poly- nomial and its reduced forms. The results are given in Table A3, and show that 56% of the variation in ADG was due to variation in fecal excretion (scouring). Further, fecal excretion was found to account for 14% of the vari- ation in urine excretion (Table A4), and urine excretion accounted for 46% of the variation in ADG (Table A5). Therefore, a combined relationship between ADG and fecal and urine excretion is suggested from the multiple regression analyses. A prediction equation was: 63 «x mm + om n » “2H0 H060: Nx mm + x am + om u w "Amt H0602 mx mm + Nx mm + x Hm + om u s "Amt H6602 .mo. v m« .oa. v m0 .A060E 60000n64 I 0608 60>u0mnov I cowu6>u0mno u coflu6>ummno 60005n6mn .z m 6H\u0z m mv.m u ooa ”0:00 002 0>H06H0M6 «mm. In *Amnoavmmom.u II mh.mm mm. v 0 Adv «we. nu «Aauoavvmmh.n oom.na ~.Hmmn mm. v m ANV «Hm. 0Am|oavmm¢~.u ammvm. 0mm.mm| .momm mm. v 0 Amy a m mm mm Hm on .0 mo .cmflm H0602 mucmfloflmm0oo :0H0000mwm 0:608 60000w66 Eoum mcowu6>u0mno 60000n66 800m 0H06OE 60u6H50H6U 60cwEH0006 H060: 0.000wu6>u0mno 60005n64 .00>H60 ca Am .0060 0060 >Hfl66 0m6um>6 606 Am 6.xv 0H0>0H 0:“:ofln008 >u6v0w6 0003000 mwnmaofiu6a0u 6006Ewu0MII.mH 0HQ6B 64 .mo>amo :0 is .0660 :060 saflmo 0m6u0>6 6:6 Axv 00Hnoflnu0e mo 0H0>0H 6066mm c003u0n mfismcoflu6a0u 0sBII.H .mHm B arm—2.20.502. X” 0.. v movur mhw _ .660 x «3.0... 080.00 I 0000 u> Ii. «x $530». ... .80.: 1.001.. > . «x “~60 080.0000; ..I I. 1 com V a ...... 6 i 8. o / .\ .II / / .\ 0m. -\ 0%. s o i on 1 65 f‘ 2 3 ADG = -274.8 - 110.8 F + 16.34 F - .7450 F + 24.76 U - .2753 02; R = .88, where ADG = average daily gain (g), F = fecal excretion in period (kg), and U = urine excretion in period (liters). The equation showed that 77% of the variation in ADG can be explained by variation in fecal and urine excretion. It also shows that the ADG decreases with increasing fecal excretion (scouring), and that a compensatory decrease in urine excretion in milder cases of scouring prevents a dramatic decrease in ADG. It was often observed that scouring calves did not respond to common oral and injected antibiotics (CIBA scour powder, Entefur tablets, Lingomycin,Combiotic, and Penecillin). A fecal swab from one calf showed infection with Kleibsiella bacteria which is resistant to all common antibiotics except Gentamycin and Polymyxin B. Therefore, it may be concluded that weight losses, poor health (scouring), and high death losses in calves before two weeks of age were largely due to bacterial infections of the gastrointestinal tract, which tended to mask dietary methionine effects. When the full model for the relation- ship between dietary methionine levels and ADG was reduced to the quadratic model, dietary methionine levels still accounted for 20% of the variation in ADG (Table 16). This 66 model shows maximum daily gain at the 115% level of methionine; equivalent to 2.85 g per 16 g N. Digestibility.--The average dry matter (DM) content in milk replacer, milk weigh back, and feces are given in Table 17 along with the digestibilities for DM and crude protein (CP). The corresponding analyses of variance are given in Table A6. The DM content in milk replacer and milk weigh back varied among calves (P < .05), and the DM content in milk replacer was higher in period 1 than in period 2 (P < .01). The differences among calves and between periods for milk replacer may be explained by a variable number of feed batches per treatment, whereby a different number of calves per group received the various batches. For DM in milk weigh back, differences among calves may be due to vari- able amounts of milk weigh back (Table A9). The DM content in feces was higher in period 2 than in period 1 (P < .001) and may be explained by more severe scouring in period 1 than in period 2 (Table A1). The DM content in milk replacer, milk weigh back, and feces did not differ among treatment groups. The digestibility of DM did not vary among calves, but tended to be higher in period 2 than in period 1, and to differ among treatments (P < .10). Multiple comparisons among treatment means showed that the digestibility for 67 .260. v av 000006606 5000600600600 006 06000000600 006600 6 6000600 000 006020 .2 6 60 006 6 66.6 n "0000600000000 0000000006 0>00600m6 5.05 6.66 6.66 6.6 0 06.66 66.65 0.60.66 0.66.66 0.66.65 0000006 06000 0.66 5.06 6.66 6.6 0 06.65 0 66.66 66.66 0.66.65 0 66.56 000066 500 m .0000060000000 00.66 66.00 60.50 66.6 0 56.60 06.60 66.50 66.60 60.60 00000 60.66 00.66 60.66 66.600 66.66 06.56 00.56 05.06 66.65 x060 06003 2002 66.56 66.56 65.56 50. 0 66.56 55.56 06.56 65.56 06.56 00060000 0002 .0000000 20 m 0 0602 .0.0 m a U m 6 600000 00600>o 000606009 .00>060 00 0000066 6006000 606 0600 0000006 06000 606 0200 000066 506 00 5000000000606 000 00 6000>00 0000000006 6060006 no 000000 009:).50 0006B 68 90'- o ‘ o BOf- . 39 o t 0 ° 2 -—" ~ 2 7O!— / / O\ \ p. / \ U! /’ ‘\\ “‘ /’ 2 / \ o \ 60L- 0 \\ \ o o 0 DUI 50’ o-—-—0 OF L L 1 1 L 75 100 125 150 175 METHIONINE ; %. Fig. 2.--The relationship between dietary methionine levels and the digestibility of dry matter (DM) and crude protein (CP) in milk replacers for calves. 69 group C was higher than for group E (P < .05) but none of the differences among other groups were significant. The digestibility of CP did not differ among calves but was higher in period 2 than in period 1 (P < .01), and tended to differ among treatments (P < .10). Multiple comparisons among treatments showed that the digestibility was higher for group D than for group E (P < .05). None of the differences between other groups were significant. The relationship between dietary methionine levels and the digestibilities was analyzed by multiple regression, and the results are given in Table A7. For the digest- ibility of DM only the linear and cubic terms are signifi- cant, and only 6% of the variation is due to dietary methionine. The prediction equation showed a maximum dry matter digestibility when methionine was 101% of that in milk. This is equivalent to 2.5 g per 16 g N. For the digestibility of CP dietary methionine levels accounted for 20% of the variation when the full model was used. However, only the cubic term was significant, and if the model was reduced to the linear and the cubic terms, dietary methionine accounted for only 11% of the variation in CF digestibilities. This prediction equation showed a maximum at the 117% level; equivalent to 2.90 g per 16 g N. Since scouring was a problem and digestibilities may be expected to decrease with increased defecation, the 70 relationship was determined by multiple regression of a third degree polynomial and its reduced forms (Table A8). For DM and CP 34 and 56% of the variation in digestibility, respectively, was due to variation in fecal excretion. In healthy calves 89 and 77% of the DM and CP, respectively, were digested. The respective values declined linearly at the rate of 1.3 and 2.7% per kg increase in fecal excretion. Nitrogen Balance.--Data on nitrogen balance (N- balance) and related parameters are given in Table 18, and the corresponding analyses of variance in Table A9. The amount of milk replacer powder varied among calves (P < .001), but did not differ between periods or treat— ments. The variation in feed intake among calves may be explained by the difference in BW of calves, since pre- pared milk replacer containing 13% solids was fed at 10% of BW throughout the trial. The volume of milk weigh back tended to vary among calves (P < .10), but no signifi- cant differences were found between periods or among treatments. The volume of urine varied among calves (P < .01) and was larger in period 2 than in period 1 (P < .05). Correspondingly the amount of feces was greater in period 1 than in period 2 (P < .001). The amount of feces did not vary significantly among calves, and neither the volume of urine nor the amount of feces differed significantly among treatment groups. The higher urine 71 6.6 6.6 6.6 6.6 0 6.5 6.6 0.6 6.6 6. 00060 06003 0002 6.600 5.600 6.600 6.0 0 6.600 6.600 0.600 6.600 6.600 6000606000002 6 .0000000 2 665.0 506. 656.0 666.0 060.0 606.0 606.0 666.0 606.0 00006 666. 066. 666. 060.0 666. 666. 606. 666. 666. 00000 560. 660. 660. 660.0 600. 050. 660. 560. 660. 00060 06003 0002 o o o 0' s o o o o 066 6 606 6 666 6 600 + 0666 6 0 6006 6 .6566 6 6506 6 6606 6 6000606000002 6 .0000600000000 2 6660 6005 6666 66600 6065 6656 6666 6066 6656 6 .00006 I. s 66506 66666 66656 6666+ 066660 0 666606 666606 0 666666 0 665566 05 00000 6660 0660 6660 66000 6606 6660 6600 0650 666 005 .0060 06003 0002 .l 0 5666 6066 0666 66 + 06666 66566 66566 0 66666 0 66666 66 000606000002 05d00> 00006 6 0 0602 .0.0 m 0 U m 0 600006 00600>o 000506009 .00>060 00 0000056066 6006000 606 0006060 00600000 00 060 0000000006 6060006 00 000000 0091:.60 0006B 0 72 .060000 660 00 00000006 000000 .060000 660 00 00000006 000606 6 .z 6 60\002 6 66.6 n 000 “0000 002 0>0060060 .060. v 60 000000006 000 006 06000000600 005500 6 6000600 00602 . 0 6 6.66 6.6 0.00 0.000 66.00- 66.60 0.66.60 6.60.0 0 .6666066 z 6.66 6.06 6.66 6.6 0 66.66 66.00 6.65.06 6.66.66 66660 6.06 6.06 5.06 6.6 0 ~.~0 6.66 0.06 6.56 66006 m 0 666: .6.6 m o 0 6 600006 00600>O 000506008 .6osz0uco0--.60 60660 73 volume in period 2 than in period 1, and the lower amount of feces in period 2 than in period 1, may be explained in part by the apparent inverse relationship between the volume of urine and feces, and a decreased incidence of scouring in period 2 compared to period 1 (Tables 15 and A4). The N concentration in milk replacer varied among calves (P < .01), and tended to be higher in period 1 than in period 2 (P < .10), and to differ among treatment groups (P < .10). Multiple comparisons among treatment means showed that milk replacer E contained less N than milk replacer A and B (P < .05). The differences in the N concentrations in milk replacers may be due to a vari- able number of batches per treatment group (Group A, B, C, D, and E: 3, 3, 2, 3, and 2 batches, respectively), and a different N concentration in commercial milk replacer used for batches one and two, compared to three (20.1 versus 18.1% crude protein in dry matter). The different numbers of batches in different treatment groups was caused by the high loss of calves for certain treatments and the resulting necessity of preparing new feed for the replacement calves. The crude protein con- tent in the milk replacers was equivalent to 18.24, 18.23, 18.04, 18.13, and 17.76% of the dry matter in ration A, B, C, D, and B, respectively. This was lower than the planned 20%. The N concentrations in milk weigh back and urine did not differ between calves, periods, or treat- ments. The fecal N concentration was lower in period 1 74 than in period 2 (P < .001), but no significant differences were found among calves or treatments. The N concentration in urine and feces appears to be inversely related to the excretion of feces and urine (Table 18). The relation- ships were analyzed by a third degree polynomial and its reduced forms. The analyses showed that 72% of the variation in the N concentration in urine was due to variation in urine excretion (Table A10), and that 79% 0f the variation in fecal N concentration was related to variation in the fecal excretion (Table All). The total amount of N in feed varied among calves (P < .001), probably because of BW differences among calves, and different concentrations of N in the feed. No significant differences were found between periods or among treatments for milk replacer, milk weigh back, and urine, nor among calves for the latter two and fecal excretion. The fecal N excretion was greater in period 1 than in period 2, suggesting that the daily N excretion is not constant with a simple inverse relationship between the amount of feces and the fecal N concentration. Nitrogen balances did not differ among calves or between periods, but tended to differ among treatment groups (P < .10). Multiple comparisons among treatment means showed that N retention for group E was less than for group D (P < .05), but was equal for groups A through D. The average N deposition for groups A through D was 75 19.5 g for the total period and equivalent to 17.4 g body protein per day. This is in disagreement with an average weight loss of 13 g per day for the four groups. To eliminate the difference in initial BW, the data for N content and N-balance were recalculated on an equal BW basis (Table 19). The analyses of variance are given in Table A12. The recalculated data show the same differences as the original values, but differences in N consumption among calves were less (P < .05), and N intake even greater for period 2 (P < .05). The differences among calves may be explained by different N concentrations in milk replacers, and the difference between periods by the feeding practice. If a calf lost weight in period 1, the feed was not adjusted downward to correspond with the lower initial BW of period 2. The relationship between dietary methionine levels and the N-balance was also analyzed by multiple regression. The results are given in Tables 20 and A13. When the full model was used differences in dietary methionine levels accounted for 29% of the variation in the N-balance. However, only the cubic term is significant, and accord- ingly, only 5% of the variation in N-balance can be attributed to dietary methionine levels. If the N-balance is recalculated as g N retained per day per kg BW-73, then the linear term approaches significance (Table 20), and the amount of variation explained by dietary methionine 76 .Amo. v 6v 000006006 000 006 06000000600 005500 6 6000600 00602 .2 6 60\062 6 m6.m u 000 "0600 06: 6>0060600 6.6 mm.m 06.0 m0.6 mm.m0 00.6: 660.00 m0.m mm. 60.0 000606nuz 50.6 60.60 66.00 60.00 60.m0 06.0 66.00 mo.m0 00.00 00006 66.06 mm.om m6.om m6.~0 mm.mm m6.00 Nh.o~ m6.m~ 60.60 00000 6m.0 m6. 6N.0 mm. 0 66.m om. 66. n0.m mm. 0060 06003 0002 m6.nm m0.nm 6m.hm 0m. 0 00.nm mm.hm om.hm mo.mm 65.6m 00060600x002 6 .0000000 2 m 0 0602 .0.0 m a U m 0 600006 00600>o 000506009 6x 000 006 000606032 606 0000000Iz 60066 000 00 06V .036 60 oo0\066\z 66 666063 0606 0606060 0 0000000005 6060006 00 000000 00911.60 0006B 77 006 00602 II >" mx mm + om 03 00602 II >-' 0x mm + x 0m + om m0 mm + «x mm + x 00 + 00 u 0 "AMV 00602 .00. v 600 .00. v 60 .00605 60000660 - 0605 60>0000ov I 00006>00000 u 00006>00000 600000600 .2 6 60\z 6 0v.m u 000 "0000 002 0>00600m6 mm. *«Av-o0vmooo.- - - «000. 00. v m 00V 002 cm. ..00-00vmooo.- - 000-0000060. 0600. mm. v m 000 mmz «06m. 0.00-00vmmmo.- AN-O0VOmom. omqvm.- 000.6 mm. v m 000 002 mm. u.0¢-o000m0o.- - - oom.6 00. v m 00V 062 «.mm. 0.0m-o0vmm60.- 00-0000606. 006.6- «.660 mm. v m 000 N02 mm. 0.00-O0Vmovo.- - - mm.- 00. v m 000 002 mm. «.00-0006600.- - o006. 06.00- mm. v m 000 002 ..vm. .«0m-00Vm6mm.- 0000. 60.00- ~.mom mm. v m 000 002 0 mm mm 0m om 0m 00 .:60m 0060: «Ham 000000066000 0000000606 00605 60001666 5006 1006> 000006>00000 60000666 5006 000605 6006000060 6000500006 00602 . 0.000006>00000 60000660 .>00>00006000 .Ammzv .30 6x 006 60066 2 6 606 .000zv 30 0600000 60 000 006 60066 2 6 06 .0026 600006 06mm050006x0 006 06000 06 6000006X0 00006060|z 606 00 6.xv 000>00 0000000005 6060006 0003000 0600000006000 6006500mmll.om 0006B 78 is slightly increased. Inclusion of the linear term in the N-balance for the whole period increases the R2 value slightly, and a maximum is found at 113% methionine; equivalent to 2.80 g per 16 g N (Figure 3). Plasma Amino Acids.--Plasma concentrations of methionine, valine, leucine, and isoleucine before feeding and two hours after feeding are given in Table 21, and the corresponding analyses of variance is in Table A14. Plasma concentrations for the four amino acids did not vary significantly among calves, except for the methionine concentration two hours after feeding on the last day of the experimental period (P < .10). Plasma concentrations of valine, leucine, and isoleucine were higher in period 1 than in period 2 before feeding on the last day of the experimental period (P < .05, P < .10, and P < .10, respectively). These were the only significant differences between periods. However, for all amino acids at all sampling hours, plasma concen- trations tended to be higher in period 1 than in period 2. Excepted from this trend are plasma methionine before feeding on the first day, and two hours after feeding on the last day of the experimental period. Only plasma methionine concentrations were affected by treatments. The plasma concentrations tended to differ due to treatment before feeding on the last day of the experimental period (P < .10), and two hours after 30 :9M 0 N-BALANCE 3 ~10 -20 Fig. 79 _ 1 .\u tr/f L l \f 75 \460 125 150 W5 METHIONINE;% -\ _ — - Y= 22.95 —.4085 (165”3 \. v=(-22.¢3)-.6Ioox-.1592(16‘)x3 \ -—-—- Y= 903.2 - 23.87x +.2041X2—.5578(153)X3 \' 3.--The relationship between dietary methionine (X) levels and N-balance (Y) in calves. 80 am.m ma.m mp.m Ho.HH ov.m Ho.oa H~.m No.5 mm.m mus .mmn umufim nfl.n Hm.m mm.n nv.mfl Hm.m mm.v oH.m mn.oa om.» 0-9 .amo umufim a aH.mH mo.Hm Ha.om mm.afi mm.Hm hm.mH mm.om oo.~m om.om Nue .smo ummq mn.mH oo.ma mh.na mo.NH mm.ma mm.ma om.oa vo.o~ am.na 0-9 .mmo ummq mm.mH em.mfl oq.mH NN.NH mo.mH vo.am m~.ma mn.ofl v~.ma mus .smo umuflm mm.oa am.o~ mm.ma ma.oH Hm.am m~.m mm.nH mo.mm “v.5H one .amn umuam a mm.m mo.m om.m mm. H mo.m np~.m n.mmm.m mmH.m pm.a “mus .mmn puma wo.m mm.m ma.m «a. H mH.¢ n.mwm.m n.mmo.m n.Mmo.m na.~ none .smo ummg mv.m mo.m mm.m om. H .mhv.v o.nao.m o.mon.m amm.H .mmv.~ owns .smc umuflm «H.m mo.m oa.m so. H mo.m Ha.m mm.m om.~ Ho.m No-9 .smo umufim mafiCOwnumz N H cam: .m.m m o o m a poflumm Hamum>o usmfiumwue .m0>ku cw “Ha ooH\z:v mcwosmHOmw new .mcwosma .wcwam> .ocflcoflnuoe mo mcoflumuucmocoo mammam co 0 wcwcofinuos mumumflo mo uoomuw mnanu.am wanna 81 .mcflpmmm uwuwm muse: ozu vmuomaaoo woon .onumm HmucwEHummxm msu 90 wow ummqa .mcfloomm muommn omuomaaoo poon .ooflumm Hmucmsaummxm on» «0 amp among mcflpmmm nonmm mason ozu wouomaaoo poon .GOwnmm Hmucwfifluomxm on» no woo umuwmm .mcflpmwm ouOmwn wouomaaoo coon .ooflumm Hmucmefiummxm 0:» mo amp umufimm .z w oa\umz m mw.m u ooa "ocoo um: m>flumHmmm .Amo. v my useumumav uoc mum umfluomummsm coaeoo m mcwumnm mammz p.0.n.m mv.ma 00.MH 90.0H mm.HH mm.ma 09.09 mm.ma mm.ma «H.0H N-9 .amo ammo m0.0H 0m.ma 00.HH 00.HH mm.mH 00.0 mm.0a HN.MH 00.HH 0-9 .>00 ummq 09.0H 09.ma mm.ma 00.HH m0.ma H0.MH 9H.0H mm.m Av.ma m-9 .900 umufim mm.m 0m.0H HN.0H 9H.H« 09.HH 0m.0H «0.0 00.0 9m.0a 0-9 .900 umuflm wcflosmHOmH 90.0 0~.m 00.0 00. H 00.0 00.9 90.0 no.0 Hm.0 m-9 .900 quq 09.9 mm.m 00.9 m0.HH. 00.0 ma.0 00.0 00.0 «0.9 0-9 .900 ummq m H cum: .m.m m 0 o m m newumm Hamum>o ucmEummua .0mscaucoo-.H~ maQ~9 82 feeding on both the first and the last day of sampling (P < .01). Multiple comparisons among means before feed- ing on the last day of sampling showed that the plasma concentration in group A was lower than the concentration in group B (P < .05). Concentrations for neither group were different from those of groups B, C, and D. The equivalent comparisons two hours after feeding on the first day of sampling showed that plasma methionine concen- trations in A were lower than in D (P < .01), and that B was lower than C (P < .05), D and E (P < .01). Plasma methionine was relatively stable at the lowest level of dietary methionine, but increased with increasing intake. However, at the highest methionine level plasma methionine decreased. The same trend shown two hours after feeding on the first day of sampling was also observed on the last day of sampling with the exception that plasma methionine continued to increase on treatment E even though the rate of increase was less than at lower intakes. The relationship between plasma and dietary methionine concentrations was analyzed by multiple regres- sion, and the results are given in Tables 22 and A15. On the first day of sampling the linear model approached conventional significance for the collection before feeding, whereas both the linear and the cubic regression coefficients were significant for the samples two hours after feeding. On the last day of the experimental 83 «same. A IOHVHHOO.I II A loavmahv. omm.HI mm. v m Amy v H a*«00. Avuoavomma. Am-oavammv. mamm.l om.o~ v m Amv mmm-z ”manmflum> ucoocmmuo «a«~m. nu II AHIOHVNNFH. mumm. 0H. v m AHV *«amm. Avloavaaoo. I: Aauoavawaa. HN¢.H mm. v m Amv «*«mm. Av-oavmmno. Am-oavnamm. mmcm. mm.HHI v m Amv mom-z "wanmflum> unwocmmoo «*«mm. Avaoavnmoo.u I: Aauoavmmom. mmm.al mo. v m Amy *«abw. Av-oavchmm. AH-oavnmma. nmv.an mm.mm v m Amv twat: magmanm> usmoammwo awn. In In AH-oavmmoa. mom.H Ca. V m AHV 5mm. Avaoavovoo. Am-oavmaom. AanoHvom0H.I mmm.m v m Amy OOH-z “manmwum> unoccwmoo mm mm am 00 90 0o Hm>ma .cmam mamvoz m mucmflowmmmoo conmmummm mange Umumswcm Scum mcowum>ummno omumsmpm Eoum pmumHsono proz cmcwEumqu Hmpoz .mcoHum>u0mno owumsn©< .AHE ooa\zn .wv mcowumuucoocoo mascoHSuoE nAw m.xv mam>ma massedsuws humumwu cmw3uwn mmwanOflumHmu vmumeummll.Nm magma mammam new 84 .Hoo. v m««« 0H v ms me + 00 .AHV H000: mxmm + xam + 00 u 9 "Amy H0002 mxmm + Nxmm + me + 00 ”Amy Hmoozm H y II >4 .mcflpoom nouns muson 03p “oofluwm Hmucmeflummxo mnu mo woo umda on» so .ocoo nos mEmMHmm .mcflomwm mnomwn upoflumm Hmucmfiflummxm map m0 wow ummH mnu co .ocoo umE «Encamm .mcapmwm Hound muson 03» “ooflumm Hmucmeflummxm an» MD moo umufim map :0 .0:00 umE mannamo .mcflnmmm mnemmn “poflnmm Hmucmeummxm on» mo mop umuflm mnu co .ocoo umE mammamo .Acmme pwumsmom I come ow>ummnov I coflum>ummno u coaum>ummno Uwumsnpgn .z m mH\umz m mv.m n ooa "coflumuucwocoo um: m>9umHomm «««mo. an nu AH-oavmmav. mom.H- oa. v m AHV H mm mm Hm om .m mo Hm>ma .cmfim oampoz m mucmwoflmumoo cowmmwummm momma cmumsnom Scum m209um>uomno Umumsnom Scum pwumfisoamo H0002 cocfisuwumo Honoz .pmscflucooal.mm wanna 85 period only the linear regression coefficient was signifi— cant for blood collected before feeding and two hours after feeding. For the plasma methionine concentrations two hours after feeding on both the first and the last day of sampling the estimated regressions (where only the significant regression coefficients were included) do not show good agreement with the adjusted means (Figures 5 and 7). Therefore, the estimated full models are also shown in the figures. These estimated full models show an approximately linear increase between 110 and 140% of the methionine in milk. If the point where the linear increase commences is assumed to be the requirement, then the estimated need for methionine in the baby.calf is about 2.73 g per 16 g N. Since the decrease in adjusted means between groups D and E was unexpected, the regression analyses were repeated without group B. The analyses of variance (Table A16) gave the same results as the original analyses except for the sampling two hours after feeding on the first day of the experimental period. For that sampling, the estimated regression was changed to a second degree polynomial with the optimum level at about 93% of that of milk. However, the full model follows the one calculated for all data (including group E) and displays a linear slope between the 110 and 140% level (Figure 5). For the sampling two hours after feeding on the last day of the 86 PLASMA METHIONINE;,-M/1oomg - l 1 ' 1 l I 75 100 125 150 175 METHIONINE; Fig. 4.--The relationship between dietary methionine levels and plasma methionine concentrations before feeding on the first day of sampling in calves. 87 Gr / O / 2 / / \ ' / a / l k J4" /- E / / 2 2 / ' E / / u / z 3 I— / / “ / 5 / ‘< a, \. / / / 2 _ /\.—_. / _ .____. -l -6 3 1 v. (-1.858)+.5063(IO )x— .37no )X y .. 55. 27 — n457x —- .1257(16‘)x2-.3374(1b4) x3 —-— v- 12.02 —,2176x+.1176(152)x2, (group E de'eted) b J . l L L L METHIONINE;% Fig. 5.—-The relationship between dietary methionine (X) levels and plasma methionine (Y) concentrations two hours after feeding on the first day of sampling in calves. 88 4.. E O 2 \ 5 a- J:- E z 9. E 2- I“ 2 < 2 -1 m __ v=.9975+.1722(1o )x 3 1. -2 2 -5 3 n. v.(.11.38)+.3495x—.2817(1o )x +.759(10 )X -1 _.—— v-1.325+.1394(10 )X; (group E deleted) L. l L l l I 75 100 125 150 175 METHIONINE; % Fig. 6.--The relationship between dietary methionine (X) levels and plasma methionine (Y) concentrations before feeding on the last day of sampling in calves. 89 PLASMA METHIONINE;uM/1OO ml 0: r —1 __ v. (—1.396)+.4158 (10) x -2 _ v- 20.35 - .5513x+.4991 (10 )x — .1336 (10‘) x3 —-— v= 20.90—5524 +.4999(102)>?—.1339(10‘)x3,(g-oup E deleted) - I J 1 l 75 100 125 150 175 METHIONINEiyo Fig. 7.--The relationship between dietary methionine (X) levels and plasma methionine (Y) concentrations two hours after feeding on the last day of sampling in calves. 90 experimental period both the second and the third degree polynomials parallel the original full model, and show an approximately linear increase from 110 to 140%. The recalculated regressions did not change the estimated methionine need for the baby calf of 2.73 g per 16 g N. Plasma Urea Nitrogen.——P1asma urea nitrogen (PUN) levels on the first and the last day of the experimental period are given in Table 23. Each value represents the average of 5 sampling hours per calf. Since the data are incomplete (no observations for calves 125, 128, 129, and 130; incomplete for calves 259 and 260), and because of the experimental design used, treatment effects could not be tested by the ordinary analysis of variance. Instead a t-like test with approximate degrees of freedom (Welch, 1938) was used. For the first day of the experimental period PUN was higher in group B than all other groups (P < .05). None of the differences between groups A, B, C, and D were significant. On the last day of the experimental period PUN levels in group A and B, were higher than for groups B, C, and D (P < .05). The difference between periods was not significant. The relationship between dietary methionine levels and PUN was analyzed by a third degree polynomial and its reduced forms. The dependent variable used was not adjusted and was the average of 5 sampling hours for each 91 .mcoflum>ummno mo Hobfiszm .cmmE may 90 90990 photomumm . Emma #CQEuvmmHBm .MHMO .Hmnm mCOHHMCHEHQU®U ®>HM mmmVDHUCH GMQE £06m o .Amo. v mv ucwumMMHp no: mum “manomnwmsm coEEou m mcaumsm mammzo.n.m 00 cm 00 mm em a mu.“ ow.“ mm.“ we.“ 0m.“ .o.m mmN.HH nom.m nma.m nmm.m omH.HH M ummq om om 00 mm om ms 00.9 00.“ mm.“ mm.“ mm.“ m.w.m noo.HH oam.n 0.0mm.m 0mm.m 0.00m.m mm umuflm m o o m m ucwafiumdxm oucwsummua mo woo .oowumm Hmucmefinmmxm 0:» 90 amp umma map can umuwm msu co mm>amo c9 mHm>mH cmmouuwc moms mammaa co mam>ma wcflcowsuma mnmumwv mo pommwm chII.m~ magma 92 calf, because inspection of individual values revealed only small differences. The results of the regression analyses are given in Table A17. On the first day of the experimental period dietary methionine levels accounted for 15% of the variation in PUN and a complicated relation- ship was described by the full model (Figure 8). For the last day of the experimental period only the square term tended to be significant and dietary methionine accounted for only 8% of the variation when the quadratic model was used. The prediction equation shows good agreement with the treatment means (Figure 8) and the minimum PUN at 125% suggest a requirement of 3.1 g methionine per 16 g N. For both days of sampling the low correlation between the independent and the dependent variables probably reflects a larger than normal error because the values were not corrected for incomplete block (calf) differences accord- ing to equation (9). Hematocrit.-—The average effect of dietary methionine on the packed cell volume (hematocrit) is given in Table 24. Since the data are incomplete they were subjected to a t-like test like that used for PUN. On the first day of the experimental period hematocrits were lower for group B and higher for group B than for any of the other groups (P < .05). Differences between group A, C, and D were not significant. On the last day of the experimental period the same trend was observed, but 93 ... N I J é l .5 O I PUN; rug/100ml 0 l _ ‘\\ // 8 \ \ __' / e 7.. e-—~—— PUB“ 6 n o PUN2 L I I I I I 75 100 125 150 175 METHIONINE,- Fig. 8.--The relationship between dietary methionine levels and plasma urea nitrogen (PUN) on the first (1) and the last (2) day of the experi- mental period. 94 .mamo Mom mcoflumcHEuoqu m>Hm mopsaocfl some comm .mcoflum>ummno mo umnEszm .cmmE on» 90 90990 oumocmum m . Emma DC®EHmmHBm o .Amo. v my ucmumMMHU no: mum umfluomummsm :oEEoo m mcwumnm mammzo.n.m om mm mm om on c 90.HH mm.HH H¢.HH mm.“ Hm.“ .m.m 0mm.mq Oom.ov n.0Hh.mm nm>.em 09v.hm m ummq om mm mm on on as om.HH mo.HH mo.HH mm.“ mH.HH m.m.m ohm.vv woo.mm www.0m nnm.vm www.0m mm umuflm m a o m 4 usmEfiummxm cucwaumoue 90 >00 .mm>amo cw muflnooumEms cooHn co mam>ma mcflcoflnume mumuwflw mo pommmm mnBII.vm magma 95 differences between groups B and C, and between D and B were not significant. The relationship between dietary methionine levels and hematocrit was analyzed by a third degree polynomial and its reduced forms. Results are given in Table A18. It was found that dietary methionine levels accounted for 25 and 16% of the variation in hematocrit on the first and the last day of the experimental period when a quadratic prediction equation was used. The equations show good agreement with the treatment means (Figure 9) and suggest the minimum need for methionine is 2.48 to 2.60 per 16 g N. The observed changes in hematocrit are in general agreement with the observed trend for fecal score and fecal excretion data (Tables 15 and 18). 96 “r 39 2 .. I-I-l I 39r- 37f . HEM] 35" O—"_HEM2 8 L I L I 4 l 75 100 125 150 175 METHIONINEf/o Fig. 9.--The relationship between dietary methionine levels and packed blood cell volume (HEM) in calves on the first (1) and the last (2) day of the experimental period. ' DISCUSSION Body Weight, Daily Gain, and Health.--Male Holstein calves fed graded levels of methionine from one to four weeks of age lost weight in the first two weeks after birth. In the following two weeks average daily gain (ADG) was not sufficiently high for full compensation of the weight loss (Table 15). The ADG did not differ significantly among treatment groups fed 75 to 150% of the methionine content in milk protein, but weight loss was greatly increased for the 175% diet (Table 15). The lack of ADG response at the lower levels of dietary methionine is in agreement with observations in calves by Patureau-Mirand 25 31. (1973). However, it is in dis- agreement with an expected response according to the law of diminishing returns (Almquist, 1953). This may be explained by factors such as age, health, and length of the study period. At the age involved in the present study, the ADG in healthy calves will not normally exceed 200 g and may be expected to be even lower in the first two weeks after birth. Any health problems would be superimposed on the age effect and would decrease the ADG. In the present 97 98 study, scouring was a problem in period 1 (9 to 15 days of age) and was caused by bacterial infections of the gastrointestinal tract which seemed unrelated to treatment. It was also found that 56 and 46% of the variation in ADG was due to variation in fecal and urine excretion, respec— tively (Tables A3 and A5). When the two factors were combined they accounted for 77% of the variation in ADG and excluded dietary methionine as a significant predictor in an equation determined by multiple regression. Fecal scores and hematocrit values also suggest an excessive water loss in feces without a sufficient increase in water intake or decrease in urinary excretion to prevent dehydration. The prediction equation indicates that ADG would be maximized in calves when fecal excretion is less than 150 g per day (no scouring) and daily urinary excretion is 5 to 7 liters (Table 25). The optimization effect of urine excretion may be related to a minimum water excretion for removal of excretory metabolites and maintenance of water balance in calves. Because of the age and health factors the length of the study period is critical. The ADG is determined as the difference between two large values (body weight), and a short study period does not allow scouring calves to fully recover and com- pensate for weight losses due to scouring. Even though the ADG response appears insensitive for determination of amino acid requirements in short 99 .Aaoo. v my “mm. “mammnm. I Dmn.vm + mmomwn. I mmm.oa + mm.oaa I m.vn~I u waHmo c9 coflumuoxm Amumuwa .Dv mumcflus can .Amx .oadv :Hmm maflmp mommm>m cmmsumn mflnmcoflumamu mQBII.mm manme 100 term experiments, dietary methionine levels accounted for 26 and 20% of the variation in ADG when fitted to third and second degree polynomials, respectively (Table 16). The third degree polynomial describes an unexpectedly complicated relationship between dietary methionine levels and ADG, but upon reduction to the quadratic form the curve becomes parabolic (Figure 1), and shows maximum gain at the 115% level; suggesting a methionine require- ment of 2.85 g per 16 g N. Digestibility.--The overall digestibility of dry matter (DM) was 83.4% and was only slightly lower than in earlier studies with calves fed similar amounts of protein (Lassiter 2E.2l°r 1963). Digestibilities were lower in period 1 than in period 2 (Table 17) with values for period 2 close to those reported by Lassiter gt 31. (1963). Because scouring was a problem, and the digestibility was negatively correlated with fecal excretion (Table A8), differences between the present study and literature values (Table 8) may be assumed to be caused by age and health factors rather than diet. However, fecal scores showed that increasing levels of dietary methionine caused some increase in scouring (Table 15). Dietary methionine accounted for only 6% of the variation in the digestibility of DM when fitted to a third-degree polynomial without the quadratic term. The prediction equation so determined had a maximum 101 digestibility at 101%; suggesting a methionine require- ment of 2.50 g per 16 g N. The overall digestibility of crude protein (CP) was 65.5% and lower than reported previously for dried skim milk and casein (Table 8; Lassiter gt gt., 1963; Blaxter and Wood, 1951a; Bryant gt gt., 1967; Lofgreen and Kleiber, 1953). The difference may be related to age, protein level, and protein type. Bryant gt gt. (1967) found that digestibility increased with age. Lassiter gt gt. (1963) demonstrated an increased digestibility of CP with increasing amounts in the diet. In the present study the digestibility of CP was 71.7% for period 2; slightly lower than for calves fed 15.2% dietary protein in the study of Lassiter gt gt. (1963). These data suggest that the digestibility of CP decreases when 3 to 5% protein equivalent is replaced by crystalline amino acids; however, the decrease in this study probably was magnified by scouring since the two variables were negatively correlated (Table A8). Dietary methionine levels accounted for 20% of the variation in the digestibility of CP when fitted to a third-degree polynomial (Table A7). However, the quad- ratic term was not significant and consequently only 11% of the variation was accounted for by dietary methionine. When the quadratic term was excluded the polynomial showed 102 a maximum in digestibility at 117%; suggesting a methionine requirement of 2.90 g per 16 g N. The low correlations between dietary methionine and the digestibility of DM or CP, and the high negative correlation between these digestibilities and fecal excretion (scouring), indicate that digestibilities become an insensitive method for determination of the amino acid requirement in calves with diarrhea. Nitrogen Balance.--The overall nitrogen balance (N-balance) for the experimental period was 13.1 9 (Table 18). This is equal to 2.2 g N retained per day per 50 kg body weight (BW), and is much less than in older calves gaining weight at the rate of .5 kg per day (Table 5, Blaxter and Wood, 1951c; Bryant gt_gt., 1967). However, the N-balance was positive even though calves lost weight. Bryant gt gt. (1967) explained such a dis- crepancy in the young calf by biological variation, experimental error, and a pronounced ability to conserve protein by deposition of lean muscle tissue concomitantly with depletion of lipid, glycogen, and water. Experimental error may be the best explanation since a 2% loss of each intake and excreta would result in a 30 to 40% increase in the N-balance (Wallace, 1959). In the present study scouring was a problem. With increasing severity it becomes difficult to secure consumption of allotted feed and total collection of excreta. Error 103 introduced by incomplete collection of excreta is partly reduced because the N concentration in both feces and urine is decreased with increased excretion (Tables A10 and All). A general dehydration in the young calf can be an important factor in the discrepancy between N-balance and ADG. Wallace (1959) found that 33% of retained N is stored without an equivalent gain in BW in the 4 months old child. In the present study a long term general dehydration was probably enhanced by a more rapid dehy- dration due to scouring since increased water excretion often took place without a compensatory increase in intake even if offered. Further, scouring may lead to depletion of glycogen and lipid stores and enhance the inverse relation between ADG and N-balance. This is supported by the observation that digestibilities for both DM and CP decreased linearly with increased fecal excretion (Table A8). The discussed factors, along with an expected low N retention in the first weeks after birth (due to low ADG) make the N-balance method less suitable for determination of amino acid requirements in short term experiments in calves. Even though the N retentions were low, 25% of the variation could be attributed to dietary methionine levels when fitted to a third degree polynomial (Table 20). However, the relationship was unexpectedly complicated 104 (Figure 3). Upon reduction to the linear and cubic terms, it only accounted for 9% of the variation in N retention. The selection of terms to be included was based on the significance of the linear and cubic terms for the relationship between dietary methionine levels 73 (Table A13). The and the daily N retention per kg BW- prediction equation so determined showed a maximum N retention at the 113% level; suggesting a methionine requirement of 2.80 g per 16 g N. Plasma Amino Acids.--The plasma methionine concen- trations reported for the present study (Table 21) are in good agreement with those reported by Patureau-Mirand gt gt. (1973) but a little higher than those determined by Williams and Smith (1973, 1975). Differences among calves only approached signifi- cance for plasma methionine two hours after feeding on the last day Of sampling. This is contrary to the findings by Williams and Smith (1973; 1974a, b, c; 1975) that the variation among calves is greater than the variation within calves. No differences were found between periods (age), except for valine, leucine, and isoleucine before feeding on the last day of sampling. These results are partly in agreement with those by Williams and Smith (1973, 1975), who found little variation with age. However, valine, leucine, and isoleucine concentrations tended to 105 decrease with age at both sampling hours. For methionine, the age effect was variable. These results support those by Leibholz (1966), that plasma methionine and leucine are negatively correlated with age. Plasma methionine was the only amino acid measured that was affected by dietary methionine levels (Table 21). The absence of a response to diet in plasma methionine levels before feeding (12 hours fast; Figure 4 and 6) is in disagreement with findings by Zimmerman and Scott (1965) who found a typical brokeneline response for the amino acid under study when chickens were bled 24 hours after the last feeding, and all other EAA were at require- ment levels. The reason for this difference between species is unknown. At two hours after feeding plasma methionine differed among treatments but the typical broken-line response reported for studies in rats, chicks, pigs, calves, and steers (Stockland gt gt., 1970; Zimmerman and Scott, 1965; Mitchell gt gt., 1968b; Williams and Smith, 1973, 1974a, b, c, 1975; Fenderson and Bergen, 1975) was not observed at the highest dietary concentration (175%; Figures 5 and 7). Morrison gt gt. (1961b) also reported a plate, but did not propose a cause. Since plasma amino acids (PAA) arise from the balance between rates of digestion, absorption, and protein synthesis, as well as tissue breakdown (Albanese, 1959; Gitler, 1964; Harper, 106 1968; Munro, 1970; McLaughlan, 1974), the increased scour- ing at 175% may have impaired absorption of methionine. The above is partly supported by the decreased digestibility for both DM and CP at the highest dietary level. Meth- ionine is a potent stimulator of adrenocortical activity (Munro, 1970). Adrenocortical hormones create a less favorable N-balance (Leathem, 1964), by activation of control and defense mechanisms which increase gluconeo- genesis (Yates gt gt., 1974). Moreover, inappropriately high levels of aldosterone stimulate reabsorption of sodium from luminal fluids which can lead to isotonic expansion of the extracellular fluid volume (Yates gt gt., 1974). Stimulated glucorcorticoid secretion is probably the more dominant effect in calves with diarrhea, because the nutrients are less well absorbed, and gluconeogenesis from amino acids release energy which becomes limiting. The increased PUN levels suggest more gluconeogenic activity at the highest methionine level (Figure 8). Williams and Smith (1973, 1975) found that plasma urea levels in calves stayed low once the methionine require- ment had been met but their studies did not include high enough levels of methionine to create a severe excess (imbalance) as apparently occurred in our study. Dietary methionine is the most toxic amino acid, and dietary excess can cause severe growth depression and histOpathologic changes (Harper gt gt., 1970). In the present study the 107 average free methionine supplemented on treatment E was 2.78 g per day. The young calf may be more susceptible than rats to methionine toxicity, especially if predis— posed by bacterial infections of the gastrointestinal tract. At the four lower dietary levels, plasma methionine increased almost linearly with level fed, and did not show the typical broken-line response. The broken-line response might be shown more clearly if smaller increments of dose of dietary methionine were used. For both the first and the last day of sampling, dietary methionine accounted for 45 to 49% of the variation in plasma levels when fitted to third degree polynomials and showed an approximately linear increase between 110 and 140% methionine. If the 110% level is assumed equal to the breaking point in an ordinary assay it indicates a require- ment of 2.73 g methionine per 16 g N (Figures 5 and 7). McLaughlan and Illman (1967) also found an almost linear relationship between plasma and dietary methionine levels in rats. When they considered the requirement to be at the dietary level at which the post-feeding level was equal to the normal fasting level, the requirement was in close agreement with average values obtained by other methods. In the present study the differences between the linear prediction equation for plasma levels before feeding and the third degree polynomial for plasma levels 108 two hours after feeding were calculated for the first and the last day of sampling and plotted against the dietary methionine level (Figure 10). The curves so obtained cross the zero-difference line at 119 and 111% level for the first and the last day, respectively, and indicate a requirement of 2.95 and 2.75 g per 16 g N. Figure 10 also shows that post-feeding plasma methionine concentrations are lower than the fasting levels at the lowest dietary intake. This may be caused by increased insulin secretion after feeding and increased uptake of methionine by the tissues, i.e., muscle (Leathem, 1964; Munro, 1964c, 1970; Wool and Scharff, 1968). Differences between fasting plasma methionine on the first and the last days of the experimental period, and the comparable differences between the post-feeding levels are small (Table 21). This and the close agreement in methionine requirements determined on the two days of sampling indicate that an experimental period of approxi- mately 3 days is sufficient. This length of time is arrived at becamse blood collected on the first day of the experi- mental period was on the third day that calves received the experimental feed. Originally we planned to analyze samples taken before feeding and 1, 2, 4, and 6 hours after feeding to determine the optimal time of sampling. Only about half of the samples 1, 4, and 6 hours after feeding were 109 L0 DIF; pM/1OO ml J J 150 I75 METHIONINE; DIF --—D|F2 '-L0 “.2 -.4 Fig. 10.--The relationship between dietary methionine levels and the difference (DIF) between the linear model for before feeding and the cubic model for two hours after feeding on the first (1) and the last (2) day of sampling in calves. 110 analyzed because of the large number and the cost of amino acid analyses. Because the plasma levels did not differ between periods, the total data set was pooled and unad- justed observations were analyzed by multiple regression to determine the relationship between plasma and dietary methionine levels at various time intervals after feeding. The prediction equation accounted for 30% of the variation in plasma methionine: A - y = 7.771 - .2096 x1 + .2199 (10 2) xi - .0640 (10-4) Xi + .4697 X2 — .6033 (10-1) X3; R = .55 A where Y = plasma methionine levels (HM/100 ml), X1 = dietary methionine levels (%), and X2 = time of sampling (hours after feeding). A maximum was found at 3.9 hours after feeding. Perhaps better results could have been obtained had the four hour sample been used instead of that taken two hours post- feeding, but little difference between two and four hours was noted. Similar observations were reported in calves (Williams and Smith, 1973, 1975) and rats (Stockland gt gt., 1970). Stockland gt gt. (1970) found that plasma lysine levels in rats were lower 6 hours after feeding with equal concentrations 1, 2, and 4 hours after feeding. Plasma Urea Nitrogen.--On the last day of the experimental period plasma urea nitrogen (PUN) levels were higher for treatments A and E than for others and a 111 minimum was observed at the 125% level; suggesting a methionine requirement of 3.10 g per 16 g N. The increased PUN at the highest methionine level was unexpected because it has been found that PUN remains at a low level after the requirement is reached (Williams and Smith, 1973, 1975). The increased PUN levels may be related to increased gluconeogenesis as a protective mechanism against toxic levels of methionine and/or diarrhea. The higher PUN levels at the lowest dietary methionine may be due to deamination of amino acids in excess of the relative methionine supply, and is in agreement with results in calves by Williams and Smith (1973, 1975) and in pigs by Brown and Cline (1974). The difference between PUN levels on the first and the last day of the experimental period and the increased PUN at the highest methionine level indicate that this measurement cannot be used satisfactorily until the calves have been on full treatment for more than three days. It may also become less useful in scouring calves. Hematocrit.--The hematocrit levels demonstrated a parabolic relationship with dietary methionine levels with a minimum at 100 to 105%; suggesting a methionine requirement of 2.48 to 2.60 g per 16 g N. The cause of the parabolic relationship is unknown, but may be related to a tendency towards more scouring at the highest treat- ment levels. Although the absolute hematocrit tended to 112 be lower in period 2 than 1, the observed trend was the same for both days of sampling, suggesting that hematocrit values give an early sign of dehydration. Sulfur Amino Acid Requirement in Baby Ca1ves.--ln rats, chicks, turkeys, and pigs it is generally accepted that the total requirements for sulfur amino acids can be furnished by methionine alone or by a mixture of as low as 45% methionine and as high as 55% cysteine (NRC, 1971a, 1972a, and 1973a). In the present study, methionine requirement in baby calves (determined by various methods) ranged from 2.50 to 3.10 g per 16 g N (Table 26). Most of the methods had an optimum at 2.75 to 2.95 g per 16 g N, and the average for all methods was 2.76 g. The deter- minations were done in the presence of a constant content of 1.05 g cysteine per 16 g N in all diets. Therefore, the determined requirement of methionine only, or total sulfur amino acids (methionine plus cysteine) is 3.80 to 4.00 g per 16 g N. The validity of the assumption that cysteine can furnish 55% of the total sulfur amino acid requirement in calves needs further investigation since Patureau-Mirand gt gt. (1973) did not find any interaction between graded levels of both methionine and cystine. The total sulfur amino acid requirement of the young calf determined in this experiment, and expressed per kg BW°73 per day, is in good agreement with the requirement determined by Williams and Smith (1975) but 113 Table 26.--Methionine needs of the baby calf determined by various methods. Method Methionine need 9/16 9 N Average daily gain 2.85 N-balance 2.80 Digestibility: Dry matter 2.50 Crude protein 2.90 Plasma methionine: First daya; before feeding -—d First daya; two hours after feeding 2.73 Last dayb; before feeding -—d Last dayb; two hours after feeding 2.73 First daya; fasting levelC 2.95 Last dayb; fasting levelC 2.75 Plasma urea nitrogen: First daya --d Last dayb 3.10 Hematocrit: First daya 2.48 Last dayb 2.60 aFirst day of the experimental period. bLast day of the experimental period. CDifference between prediction equations for levels before and two hours after feeding. dCould not be determined. 114 less than half of that suggested for only methionine by Patureau-Mirand gt gt. (1973) (Table 27). However, Patureau-Mirand gt gt. (1973) also expressed it as 3.50 g methionine per 16 g N when the calves were fed milk with 26.4% protein. This value is in close agreement with that determined in the present study, because cysteine was not included. The amino acid requirement is constant as a percent of crude protein when the protein content of the diet for chicks is increased from 14 to 23% (Boomgaardt and Baker, 1973). If a total sulfur amino acid requirement of .26 g per kg BW-73 per day is accepted, then whole milk would supply a sufficient amount, because an average calf would receive .30 9 when fed whole milk at 10% of BW. Jacobsen (1957) determined the amino acid content in the 40 week old calf fetus, and Williams gt gt. (1954) suggested that the requirement of other amino acids may be calculated by multiplying the relative values for the tissue content by that of the determined requirement of the amino acid under investigation. Requirement values so calculated are given in Table 28. They can only func- tion as a temporary guide until actual determinations have been made for the other amino acids, and may be a slight underestimation since the cysteine content in the calf was not reported. 115 .mcflpmmm um0a 0:0 90990 900: 0co pman 0:0 .mmcflcmmm >HHDO£ x90 CH 0009 0092906 no m\H omm mswowman «0 >00 comv .0950: 00.0H 0:0 oo.oa #0 maw0p 0093p 09:5050 H0sq0 Ummo .mx 0co :900 >HH00 .m>0 .mpofiumm xmszN m>flm «sflmgoum mv.mm ©0cfl0ucoo umflan .uu0m0 0950: NH ma0me H0500 ozu ca @009 >H900 mo N\H 0090 mo.HImH.H II II II ovnma .mcmue II m.va.m mm. I mm. II Umhma .nuHEm 000 mE0HHH93 m. - 0m. 0m.m 00909 ..mm mm_0amuflz-smmusumm on. mm.m 0m. 00.0 0mvsum ucmmmum 20 mo 0 >00\0 0\m9.3m mx\0 2 0 09\0 wousom 00900 0:950 unmasm H0909 .mumxuo3 H0um>0m an 009089000 00 «H00 0:50» 0:» :9 00900 ocHE0 HDMHSm H090» mo usmeuflsvmu 0nBII.0N 0HQ0B 116 Table 28.--Calculated requirement of essential amino acids in the young calf. gggggtgggd AEiEEeiiéd rgéiiiiiéii 9/16 9 N g/kg BW-73/d Arginine 6.75 .75 Histidine 1.47 .16 Isoleucine 2.94 .33 Leucine 6.53 .73 Lysine 7.00 .78 Methionine 1.72 .19c Phenylalanine 3.24 .36 Threonine 3.70 .41 Tryptophane 1.1 .12 Valine 4.20 .47 aFrom Jacobsen (1957); amino acid content in the calf fetus at 40 weeks. The cysteine content was not determined. bDetermined from the content of other amino acids relative to methionine content in the calf and the deter- mined methionine requirement. (Total sulfur amino acids (.26) minus the content of cysteine (.07).) CTotal sulfur amino acid requirement = .26 g. CONCLUSIONS The requirement of total sulfur amino acids in the baby calf was determined to be .25 to .26 g per kg BW-73 per day between 9 and 27 days of age. It was assumed that 55% of the total sulfur amino acid requirement can be furnished by cysteine, but this needs further investi- gation since it has been reported that cysteine does not have a sparing effect on methionine in calves. The methionine requirement was estimated by average daily gain, N-balance, and plasma methionine and urea nitrogen levels. When poor health due to factors other than treatments was encountered, average daily gain, N-balance, and plasma urea nitrogen were less sensitive to diet than plasma methionine. These data suggest that three days on the experimental feed are sufficient to estimate the amino acid requirement in calves by plasma amino acid levels. Because of the high cost of crystalline amino acids, this short period makes the method even more attractive. Plasma methionine was minimized at 3 to 4 hours after feeding but whether this sampling hour might improve the estimate needs further investigation. 117 118 Poor health due to bacterial infections of the gastrointestinal tract was a problem, but the severity of scouring could not be related to dietary methionine in general. However, the severity tended to increase at the highest methionine intake. At this dietary level it was also found that plasma methionine tended to plateau instead of increasing linearly as theoretically expected. The cause is unknown but is suggested to be due to decreased methionine absorption, since the digestibilities of both dry matter and crude protein also decreased linearly with increased defecation; or to increased deamination of amino acids for gluconeogenesis as indicated by the increase in plasma urea nitrogen levels. Whether gluconeogenesis from amino acids is stimulated because energy becomes limiting for vital functions in scouring calves, or is brought about by a direct stimulation of glucocorticoid secretion by high level of free methionine in the diet is not known. The requirements of other amino acids were cal- culated from the knowledge of the amino acid content in the newborn calf and the determined requirement of methionine. The requirement so determined should serve only as guidelines, prior to direct determination of the requirement of these amino acids. APPEND IX .OH. v m "0 uHo. V m u«« uHOO. v m "#00 II 0 mo.H «*«HH.mm mv.H ommm. 0000. om.mH 000m. 00000 H000m 0H.m HHvH.m on. OHo.mv moo.NOH Hum.mm0 «00.0m 0H0m xHH00 .m>¢ 0oo.m H0.H «04H0.mm HHo.H Hmo.m mmv.H >~.mm H0ch NH.H ««mn.oH *«0m0.Hv moo.H 0mm.H mm.HH om.mv H0H0HCH uanws >0om H. H 0H 0H 0 H 0H 2000000 no 0000000 muc0E 000Hu0m 00>H0U Houum muc0a 000Hu0m 00>H0U I000HB I000HB 00Hu0uIm 0090160 c002 .0choHnuma mo 0H0>0H 000000 000 00>H00 mo 09000 H0009 0C0 .cH0m mHH00 0m0u0>0 .usmH03 >003 H0ch 000 H0HuHcH no“ 00C0HH0> mo 0H0>H02¢II.H¢ 0HQ0B XHszmmd 119 120 Table A2.-~Analysis of variance for the relationship between dietary methionine levels (X, %)a and final body weight (BW, kg) average daily gain (ADG, g), and fecal score (FS), respectively. Adjusted observations. Source of Variation d.f. 823::8 F—ratio Siggtitggnce Dgpendent variable: BW B of X 1 7.4689 .26 NS 8 of x2 1 .8377 .03 NS 8 of X3 1 10.6339 .37 NS Error 36 28.5973 ——— —- Dependent variable: ADG B of X 1 202,626 3.98 ** B of x2 1 274,704 5.40 * B of x3 1 170,927 3.36 ** Error 36 50,861 --— -- Dgpendent variable: FS 8 of X l .4986 .42 NS 8 of x2 1 2.1224 1.78 NS 8 of X3 1 1.1793 .99 NS Error 36 1.1935 --- -- aRelative methionine concentration: 100 = 2.48 g Met/16 g N. bAdjusted observation = Observation - (Observed treatment mean - Adjusted treatment mean). C*: P < .05; **: P < .10; NS: non significant. 121 Table A3.--Analysis of variance for the relationship between fecal excretion (F, kg) and average daily gain (ADG, g), and the estimated regression. Unadjusted observations. ' —-.. Source of variation d.f. Mean F—ratio Slgnlflcgnce Square level 3 of F 1 1,288,383 60.57 *4. B of 92 1 51,510 2.42 NS 3 B Of F 1 157,002 7.38 * Error 36 21,272 -— -- Estimated regression: /\ ADG = 120.1 - 40.94 F; R = .75*** a***; p < .001; *: P < .05; NS: non significant. Table A4.--Analysis of variance for the relationship between fecal excretion (F, kg) and urine excretion (U, liters), and the estimated regression. Unadjusted observations. Source of variation d.f. ngzge F-ratio Siggtitgance B of F 1 1,173.45 6.29 * B of F2 1 .06 <.01 NS 8 of F3 l 82.84 .44 NS Error 36 186.50 -- —- Estimated regression: A U = 33.2397 - 1.236 F; R = .38* a . . . *: P < .05; NS: non Significant. 122 Table A5.--Analysis of variance for the relationship between urine excretion (U, liters) and average daily gain (ADG, g), and the estimated regression. Unadjusted observations. Source of variation d.f. Mean F-ratio Significance Square level 8 of U 1 463,601 13.84 *** B of 02 1 577,957 17.26 *** 3 B of U 1 15,639 .47 NS Error 36 33,487 -- —- Estimated regression: A ADG = -791.5 + 43.12 U - .4825 U2; R = .68*** a . . . ***: P < .001; NS: non Significant. 123 .H00. v m .... NH0. v m ... nm0. v m .. u0H. v m 2+ "mumHuomummsmm +H©.N 00m.m ov.H m.mmH «.Hov .mHmH b.0HN :H00oum 00900 +H0.N +mN.0 No.H mv.m© o.m®H h.NmN m0.mw 000005 >00 "a .HHHHHnHummmHn 00. «*5mh.Hm om.H mm.mv mm.mH .thH mh.mm 0000b mm.H Ho.v «0m.m .thH .OONH memo. .HmHm x000 an03 xHHz vm. 0*om.NH *No.m oomo. mmmo. Nvmm. mmho. 0000Hm00 xHHZ ":0H00H0:00:00 Ea 0 H mH mH v H mH 5000000 00 000um0a 00:05 0 oHumm 00>H0O 00000 00:05 0 0H00 00> 0U I00009 0 . I000ue 0 . m H :oH00Hu0> no 000500 00H000Ih 0000300 :00: .00>H00 :H Hmuv :H00oum 00:00 0:0 29 mo 00H0HHHQH000mH0 0:0 .00000 .3000 an03 xHHE .0000Hm0u xHH5 :H Hwy 0:00:00 H200 000005 >00 How 00:0HH0> mo 0H0>H0:¢II.0¢ 0HQ0B 124 Table A7.--Analyses of variation for the relationship between dietary methionine levels and the digestibility of dry matter (DM) and crude protein (CP) in calves. Adjusted observations.b Source of variation d.f. Mean f-ratio Significance square level Dependent variable: DM 8 of X 1 140.2 1.79 *** B of x2 1 36.20 .46 us 8 of X3 1 298.5 3.80 ** Error 36 78.46 -- -- Dgpendent variable: CP B of X 1 367.2 1.61 *** B of X2 1 606.5 2.67 *** B of X3 l 1090. 4.80 * Error 36 227.3 —- -- Estimated regressions: ’~ -1 -4 3 DM = 79.32 + .8835 (10 ) X - .0288 (10 ) X ; R = .24 A -4 3 CP = 35.74 + .4425 x - .1078 (10 ) X ; R = .33** " —l 2 -3 3 CP = 354.3 - 7.965 X + .7010 (10 ) X - .1969 (10 ) X ; R = .45* aRelative methionine level: 100 = 2.48 g per 16 g N. bAdjusted observation = Observation - (Observed mean - Adjusted mean) 0*: p < .05; **: p < .10; ***= p < .25; us: non significant. 125 Table A8.--Analyses of variance for the relationship between fecal excretion (X, kg) and the digestibility of dry matter (DM) and crude protein (CP) in calves. . . Mea . Si ifi a Source of variation d.f. n F-ratio gn C nce square level Dependent variable: DM B of x 1 1221. 21.61 *** B of x2 1 6.936 .12 NS 8 of x3 1 37.91 .67 NS Error 36 56.49 -- -- Dependent variable: CP B of x 1 5061. 39.76 *** B of x2 1 31.15 .24 NS 8 of x3 1 169.4 1.33 NS Error 36 127 3 -— -- Estimated regressions: A DM = 89.03 - 1.260 x; R = .61*** A C? = 76.92 - 2.665 x; R = .73*** a . . . ***: P < .001; NS: non Significant. 126 .OH. v m u+ «mo. v m "0 0 Ho. v m ”00 “H00. vm "00« m +mm.~ ~0.m mm. 00H.m09 H00.H09.H 0mm.Hm~.~ 90H.900 .0000H00-z 0H.N mH.H~ mm.H m09.0MH 000.H0~ mmm.mmm.~ 000.000 00000 00.H H0. 00. 0mm.H00 000.000 mm0.m omm.Hom 00000 mm.H 9H.H m9.H 0mm.0m H90.m0 090.00 000.00 0000 00H03 xHHz H0.H 0m. 0.400.0m 900.0 MH0.0 m~0.N 000.0mm 0000H0000HH2 .m .0:00:00Iz 9m. 4.400.00 H0.H m9mm. «Hmo. 0900.0 0H0m. 00000 00.H 00.H mm. 0000. 0000. 9000. mmmo. 00H00 00.H H0. mm.H mmHo. 0000. «000. 00H0. 0000 00H03 xHHz +00.~ +9m.m 4.00.0 9H00. 0000. 0000. 0000. 0000Hm0uxHHz .:0H0000:00:00Iz 0m.H ...0m.- mm.H 00H.0H0.HH 0H0.000.9H ~m0.090.mm~ 000.000.0H 0 .00009 00.0 .0m.m ..m9.0 000.m~H.90 090.000.00H 000.H0m.0mm 000.009.0Hm He .0000: mm.H 0H. +00.H 0H0.000.m 000.00H.0 9m~.mHH.H mHm.N0m.HH HE .0000 00003 0H0: 00.0 0m. 4..00.H0 000.0 00H.0H Hmo.m m0m.~9m 0 .0000Hm000HH2 0.0-3 0 H 0H 0H 0 H 0H 2000000 00 0000000 00:0500009 000H00m 00>H0O 0000a 00:0500009 000H00m 00>H0o :0H00H00> no 000500 00H000Im 0000000 :00: . .0000 00:0H00Iz 000 00:0H00> mo 0H0>H0:4Il.m« 0Hn09 127 Table AlO.--Analysis of variance for the relationship between urine excretion (U, liters) and urine N-concentration (NU, %), and the estimated regression. Unadjusted observations. Source of variation d.f. sEfide F-ratio Signiiiiance B of U 1 .7613 73.91 *** B of U2 1 .2880 27.96 *** B of U3 1 .0356 3.46 + Error 36 .0103 ——- --- Estimated regressions: A _ _ - NU = 1.314 - .7821 (10 1) u + .1721 (10 2) 02 - .1250 (10 4) U3; R = .86*** A -1 -3 2 NU = .9283 - .3483 (10 ) u + .3406 (10 ) 0 ; R = ,85*** a***: P < .001; +: P < .10. 128 Table All.--Analysis of variance for the relationship between fecal excretion (F, kg) and the fecal N—concentration (NF, %) for all observations, and for period 1 (NF ) and for period 2 (NF ), respectively, and the estimated regressions. Unadjusted observations. Source of variance d.f. Mean F—ratio Significance sQuare level Dependent variable: NF B of F 1 10.35 91.30 *** B of F2 1 4.21 37.17 *** B of F3 1 1.39 12.27 ** Error 36 .ll -- -- Dependent variable: NF1 B of F 1 1.669 44.64 *** B of F2 1 .5898 15.77 *** B of F3 1 .2035 5.44 * Error 16 .0374 -- -— Dependent variable NF2 B of F 1 4.2887 22.12 *** B of F2 1 1.0983 5.66 * B of F3 1 .0964 .50 NS Error 16 .1939 -- -— Estimated regressions: _\ - - NF = 2.716 " .7181 F + .7312 (10 1) F2 - .2304 (10 2) F3; R = .89*** " -1 2 -2 3 NF1 = 2.148 - .4614 F + .4219 (10 ) F — .1243 (10 ) F ; R = .90*** NF2 = 2.840 - .7779 F + .6409 (10 ) F ; R = .79*t* a***: P < .001; **: P < .01; *: P < .05; NS: non significant. 129 .OH. v m 0+ nmo. v m 0* “Ho. v m "0* “H00. v m "000 0 00.0 90.0 90. 00m0.Hm 0900.000 9000.000 mmmo.m9 000000012 00.0 00000.00 +0m.a 9avo.m0 0000.00 0900.000 0000.00 00000 00.0 00. No.0 0000.90 0000.00 0000.0 0090.00 0:00: 00.0 00.0 00.0 HHN9.m mmmm.0 0000.0 0000.0 0000 £0003 0002 90. «000.0 «09.0 0000.0 0009. 0909.0 mvmw.m 00000m00xa02 m0 0 ma m0 0 H ma 8000000 no 0000000 m C0 wwH m OHHQ mw> M HOHH C68 mm 0 Eu 0 0 . 0 0 0 m 00 0 00 0000000 00>000 00000000> 00 000000 00000010 00000wm 0002 .000003 >000 0000000 ox ooH 00m >00\z m 00 000000mx0 0000 000000012 000 0000000> mo 0000000411.N0¢ 00009 130 Table Al3.--Analysis of variance for the relationship between dietary methionine levels (X,a %) and N—balances expressed as total for the experimental period (NB , g), g N daily per 100 kg initial BW (NB ), and g N dailg per kg BW- (N83), respectively. Adjusted observations. Source of variation d.f. Mean square F-ratio Sig:::::ance Dependent variable: NBl B of X 1 1443. 1.83 NS 8 of X2 l 1111. 1.43 NS 8 of x3 1 8752. 11.23 ** Error 36 779.5 --- -- Dependent variable: N82 8 of x 1 151.3 1.73 NS 8 of x2 1 105.4 1.20 NS 8 of X3 1 910.0 10.38 ** Error 36 87.70 --— —- Dependent variable: N83 8 of X 1 .2713 4.10 T B of X2 1 .0189 .29 NS 8 of X3 1 .8574 12.97 ** Error 36 .0661 --- -- aRelative Met conc: 100 = 2.48 g Met/l6 g N. b . . . Adjusted observation = Observation — Adjusted mean). C *t; p < .01; T: P < .10; NS: non significant. (Observed mean - 131 HH.H «H.H mv.H mam.m H¢H.o Hmm.o mnH.m mue .wmc umuflm on. Hm. mm. om.mm om.m~ o~.om m~.~m 0-9 .>mw umuflm a mm.H vm.a mv.H mm.o~ mm.nm Hm.nm vb.m~ mus .mmo ummq mv.H *mm.w mN.H om.am mm.~m mn.oma mv.n~ 0-9 .amo ummq mm. mv. mm.H vn.o~ om.ma vm.~a vo.mm mus .mmo umuflm om. no. vo.H Hm.¢om Hm.mmH vm.nma wo.ma~ oue .amc umuflm Mflmm. «.om.m NV. +mo.m Hono.m ommm.vH Hmmm. vnmm.v um-s .amw ummq +mv.m 0v. mm.H mnmo.a oooo.m Nmmv. Hmmm.a uoue .mmc ummg **vm.o «H. on.a mvvm.a moon.- «bum. moam.m mus .mmn umuwm mu. mo. so. Hmmm.a ommm. «boo. mmmm.a no-9 .mmo umufim mcwcoflnumz v H ma .mH v H ma aonomuu mo momumoa wucmfimwufi OMMMWWMQ m0>HMU HOHHQ mUCUEWMWMMUm GMWMHHOQ m0>HwU COHHMflHMKV NO mOHSOm .ocfiozmHOmw can .mcflosma .mcwam> .mGHCOwnumE mo mcoHumuucwocoo MEmem MOM mommaum> mo mflm>amccnu.va¢ magma 132 .mcflommm umuwm muses o3u omuowaaoo vocam .@Cflwmwm mnemmn omuomaaoo UOOHm .mcflcmmm “mama muso; 03u Umuomaaoo woon .mcflommm muommn @muomaaoo coon .oH. v m “+ umo. v m "« .voHumm Hmucmeflummxm may we xmm ummq .ooflumm Hmucmeflummxm may no mmv umuflm “Ho. v m "««m U .voflumm Hmucmsflummxm 0:» mo wmw ummqo n .woflumm Hmucmefiummxm map mo mac umuflmm mm.H mm.H mm.a mm.oa vn.ma mb.ma mm.ma Nae .>mv pmmq mn.a +mo.v H0.H FH.HH mo.om mo.mv ¢N.HH one .wmv uqu mo.a mo. mv.H Hw.vH Ho.ma 0mm.a mm.Hm NIB .mmv umuflm mm. ms. om. mmm.h oom.m omm.m nhm.m one .umv umuwm mcflosmaomH om.a ov. om.a mvm.q Hmm.m mmn.a hvm.m NIB .mmu ummq vm.a +mn.m ov.H 0mm.m mm.oa mm.om onm.h one .wmo ummq mucmaumoue mwofiumm mw>amo uouum mucmfiummue mnoflumm mm>amu Godumaum> mo wousom moflumuum mmumsvm :mmz . .uoscflucouuu.va¢ magma 133 Table A15.--Ana1ysis of vagiance for the relationship between dietary methionine (x, %) and the plasma methionine concentration (M uM/lOO m1). Adjusted observations.b Source of variation d.f. 523226 F-ratio Signifiiignce Dependent variable: M-lOC B of X 1 5.3572 2.95 + B of x2 1 .0344 .02 NS 8 of X3 1 .0060 <.01 NS Error 36 1.8141 -- -- Dependent variable: M-12d B of x 1 52.8385 17.79 wk 8 of x2 1 .2905 .10 NS 8 of X3 1 32.0197 10.78 ** Error 36 2.9702 -- -— Dependent variable: M-20e B of x 1 14.8264 14.17 *M B of x2 1 .0396 .04 NS 8 of X3 1 1.6217 1.55 NS Error 36 1.0460 -- —- Dependent variable: M-22f B of x 1 86.4282 32.72 *H 8 of X2 l .0179 .01 NS 134 Table AlS.--Continued. Source of variation d.f. Mean F—ratio Significance square level 8 of X3 1 5.0220 1.90 NS Error 36 2.6413 -- —- aRelative methionine concentration: 100 = 2.48 g Met/16 g N. b . . . Adjusted observation = Observation - Adjusted mean). CFirst day of the experimental period. feeding. dFirst day of the experimental period. hours after feeding. eLast day of the experimental period. feeding. fLast day of the experimental period. hours after feeding. (Observed mean - Blood collected before Blood collected two Blood collected before Blood collected two g***: P < .001; **: P < .01; T: P < .10; NS: non significant. 135 Table Al6.--Ana1ysis of vagiance for the relationship between dietary methionine (X, %) and the plasma methionine concentration (M, uM/lOO m1). Group E deleted. Adjusted observations.b —- —.-.. .—._. ——'—.-. .- Source of variation d.f. s::::e F-ratio Signiiiiance Dependent variable: M-lOc B of X 1 2.542 1.47 NS 8 of x2 1 .0673 .04 us 8 of x3 1 .0636 .04 us Error 28 1.734 -- -- Dependent variable: M-12d B of X 1 55.52 19.41 *** B of X2 1 17.30 6.04 * B of X3 1 4.338 1.52 NS Error 28 2.860 -- -- Dependent variable: M-20e B of X 1 4.861 5.74 * B of x2 1 .6172 .73 NS 8 of x3 1 .6548 .77 NS Error 28 .8470 -- -- Dependent variable: M-22f B of X 1 54.49 25.21 *** B of x2 1 2.423 1.12 NS 136 Table Al6.--Continued. *mfi.—.--.— -4 - - M.—..~A-.-—.._ -..._._ ._.- -. .. ---'-—.—.—.-—v-.-v._‘ - .—-—.- "--.- ‘_- -...—. ----- ...- -— Source of variation d.f. Mean F-ratio Slgnlflcance square level 3 0f X3 1 1.541 .71 N5 Error 28 2.1618 -- -— aRelative met conc: 100 = 2.48 g met/l6 g N. b . . . Adjusted observation = Observation — (Observed mean - Adjusted mean). CFirst day of the experimental period. Blood collected before feeding. dFirst day of the experimental period. Blood collected two hours after feeding. eLast day of the experimental period. Blood collected before feeding. fLast day of the experimental period. Blood collected two hours after feeding. g***: p < .001; *: P < .05; NS: non significant. 137 Table Al7.--The relationship between dietary methionine levels (X, %)a and plasma urea nitrogen (PUN) in calves on the first (1) and the last (2) day of the experimental period. Source of variation ngzzzzméf sgszge F-ratio Signisiignce Dependent variable: PUN1 B of x 1 10.95 1.49 # B of x2 1 11.69 1.60 # B of X3 1 14.91 2.03 # Error 29 7.328 -- -- Dependent variable: PUN2 B of X 1 .6251 .06 NS 8 of x2 1 28.24 2.57 # B of x3 1 .4514 .04 us Error 30 11,00 -_ _- Estimated regressions: '\ PUN1 -28.47 + 1.025 X - .9085 (10-2) X l\ — PUN2 22.78 - .2200 X + .8773 (10 3) X2; R = .28# 2 4 3 + .2578 (10’ ) x ; R = .39# aRelative methionine level: 100 = 2.48 g methionine per 16 g N. b#: P < .25; NS: non significant. 138 Table A18.--The relationship between dietary methionine levels (X, %)a and hematocrits (HEM) in calves on the first (1) and the last (2) day of the experimental period. Source of variation Degrees Of Mean F-ratio Significance freedom square level Dependent variable: HEMl B of x 1 192.9 6.22 * B of x2 1 113.4 3.65 1‘ B of X3 1 3.400 .11 NS Error 29 31.03 -- -- Dependent variable: HEM2 B of X 1 144.4 3.77 f B of X2 1 53.12 1.39 # B of x3 1 29.05 .76 NS Error 26 38.27 -- -- Estimated regressions: /\ -2 2 HEM1 = 56.05 - .3852 X + .1827 (10 ) X ; R = .52* " -2 2 HEM2 = 48.57 - .2506 X + .1251 (10 ) X ; R = .40+ aRelative methionine level: 100 = 2.48 g methionine per 16 g N. b*: P < .05; T: P < .10; #: P < .25; NS: non Significant. BIBLIOGRAPHY BIBLIOGRAPHY Albanese, A. A. 1959. Criteria of protein nutrition. in A. A. Albanese, ed., Protein and Amino Acid Nutrition, Academic Press, New York and London, 297-347. Allison, J. B. and J. W. C. Bird. 1964. Elimination of nitrogen from the body. In H. N. Munro and J. B. Allison, eds., Mammalian Piotein Metabolism, Academic Press, New York and London, Vol. 1, 483-512. Almquist, H. J. 1947. 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