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THESE. ,lv ‘ 7 .i g. ‘ . ~ - _ . a . ,4 1 . ‘jfL‘ ,4 . - ‘3 7' t ‘ I l “. .-~ I ' l “to . 'a v 0 6. 94 ~ .‘ j E" i .av . ‘n :v {... ”.mttg . - win-91:, . «Is. '... I‘ O 3". 32‘ Ma ‘3 '2‘? W WM SVEWE’MWW ' -.o x I“. a. \ v‘ a i This is to certify that the dissertation entitled Spray Dried Fish Solubles, Soy Protein Concentrate and Limestone in Milk Replacers for Young Calves presented by Oriel Fajardo de Campos has been accepted towards fulfillment of the requirements for Ph.D. degree in Animal Science ATW 7 Major orofessor Dategiutisz— MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES » RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. SPRAY DRIED FISH SOLUBLES, SOY PROTEIN CONCENTRATE AND LIMESTONE IN MILK REPLACERS FOR YOUNG CALVES Oriel Fajardo de Campos A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1982 ABSTRACT SPRAY DRIED FISH SOLUBLES, SOY PROTEIN CONCENTRATE AND LIMESTONE IN MILK REPLACERS FOR YOUNG CALVES By Oriel Fajardo de Campos In a first experiment, 168 Holstein calves at Cornell, Kansas State and Michigan State Universities were used to test the partial substitution of spray—dried fish solubles (SDFS) and soy protein concentrate (SPC) for milk protein (MP) in milk replacers. Calves were placed on experiment between 3 and 8 days of age and were fed experi- mental diets as the only source of nutrients for 6 weeks. Replacers were reconstituted with water to 14% solids and fed at 8, 9, 10, 11, 12 and 12% body weight from lst to 6th week. Calves were fed twice daily from open pails. Experimental diets consisted of (1) 13% crude protein (C?) as MP; (2) 19% CP as MP; (3) 19% CP, 13% MP, 6% SDFS; (4) 19% CP, 13% MP, 6% SPC; (5) 19% CP, 13% MP, 3% SDFS, 3% SPC; (6) 23% CP as MP; and, (7) 23% CP, 13% MP, 10% SDFS. Average daily body weight gains were higher for diets containing higher levels of MP (2 and 6), intermediate for 4 containing 6% CP from SPC, and lower for the negative control (diet 1) and those containing SDFS. Fecal socres, treatment for sicknesses, and rectal temperatures were higher for diets containing SDFS. Plasma amino acids sampled Oriel Fajardo de Campos when calves were 3 and 6 weeks old showed higher total essential amino acids for 19 and 23% than 13% MP or milk substitute diets. Xylose absorption test at 6 weeks failed to reveal impaired capacity for gut absorption due to feeding lower quality protein. On diet 7, mortality rate was 30% compared to a mean of 12% for other diets, indicating that 10% protein from SDFS was excessive. The second experiment was a digestibility trial involving diets l to 4 from experiment 1, with four calves per treatment. Calves were placed on experiment between 3 and 8 days of age and were fed and managed similarly to trial 1 for 2 weeks. Lowered digestibilities of dry matter and organic matter, and less retained nitrogen were associated with decreased weight gains in calves fed SDFS or SPC. Necropsy results also raised the possibility of SDFS causing allergy in young calves. A third experiment, involving 16 male calves, was conducted to study the buffering effect of limestone in the small intestine of pre- ruminant calves fed milk replacers containing 50% of the protein from SPC. Experimental diets were: (A) 19% CP as MP; (3) same as A, but containing .8% limestone; (C) 19% CP, 9.5% MP, 9.5% SPC; and, (D) same as C, but containing .8% limestone. Calf management, feeding criteria and dilution rates were the same as in trial 1. It was observed that SPC in milk replacers resulted in 20% lower weight gains, and lower dry matter and protein digestibilities. Apparent crude protein retention was also reduced, but intake of nutrients, feed efficiency, fecal score and rectal temperature were not different between protein sources. Limestone resulted in no significant changes Oriel Fajardo de Campos in the already mentioned parameters, although apparent nutrient digesti— bilities and protein retention were lowered when limestone was fed. Xylose absorption tests performed when calves were 3 and 6 weeks old showed differences due to age, but not treatment. Analysis of digesta obtained from differentsections of the gut of 6 week old calves sacri- ficed 6 hours after feeding revealed that (a) abomasal pH was higher than previously reported for calves fed whole milk; (b) small intestinal pH was above 6 for both protein sources which may explain the ineffec- tiveness of adding limestone to the replacers; (c) the use of SPC resulted in a higher pH in the large intestine and feces. DEDICATION To my wife, Lea, and our children, Oriel Junior, Susana and Adriana. ii ACKNOWLEDGEMENTS I would like to thank Dr. J.T. Huber for assistance and guidance throughout my graduate course. Also, I wish to express my appreciation to Dr. W.G. Bergen, Dr. J.L. Gill and Dr. H. Ritchie for serving on my Graduate Committee. Finally, I wish to thank the Empresa Brasileira de Pesquisa Agropecuaria (EMBRAPA) which provided financial support for my Ph.D. COUI‘SE. iii TABLE OF CONTENTS Page LIST OF TABLES v LIST OF FIGURES viii INTRODUCTION 1 REVIEW OF LITERATURE 3 Protein digestion in the abomasum 3 Protein digestion in the small intestine 9 Soybean products in milk replacers 14 Fish products in milk replacers 22 Processing fish products 22 Nutritional value of fish protein products 23 Other protein sources in milk replacers 30 Addition of enzymes in milk replacers _ 32 Limestone as a buffering agent 33 Summary of the literature review 35 MATERIAL AND METHODS 38 Experiment 1 38 Experiment 2 44 Experiment 3 ' 45 RESULTS AND DISCUSSION 49 Experiment 1 49 Experiment 2 65 Experiment 3 68 CONCLUSIONS 94 APPENDIX A.l Analysis of variance for variables in experiment 1 96 A.2 Analysis of variance for variables in experiment 2 101 A.3 Analysis of variance for variables in experiment 3 102 BIBLIOGRAPHY 1”5 iv Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. Table 8. Table 9. Table 10. Table 11. Table 12. Table 13. LIST OF TABLES Summary of dairy calves and lamb performances on different soy protein sources in milk replacers Summary of dairy calves performance on different fish protein sources in milk replacers Ingredient composition of milk replacer diets (%) Experiments 1 and 2 Chemical composition and pH of protein substitutes and formulated diets (Experiments 1 and 2) Number of calves per treatment in each location (Experiment 1) Ingredient composition of milk replacers (%) (Experiment 3) Chemical composition of diets (Experiment 3) Initial mean body weight, immunoglobulin and protein levels in plasma of calves at Michigan State University (Experiment 1) Least square means of daily weight gains (g/animal) for calves fed milk replacers varying in protein content and source by location, diets, and age (Experiment 1) Effect of diets on plasma free amino acids (pmoles/ 100 ml) for calves fed milk replacers varying in protein level and source (Experiment 1) Effect of the animal age (weeks) on plasma free amino acids in young calves (pmoles/lOO ml) (Experiment 1) Calves omitted from experiment at Michigan State due to unexpected problems or death (Experiment 1) Least square means of dry matter (DM) and crude protein (CP) intakes, and feed efficiency for calves fed milk replacers of varying protein content and source averaged over time and location(Experiment l) V Page 16 25 40 41 42 46 49 51 53 54 56 58 Table Table Table Table Table Table Table Table Table Table 14. 15. 16. 17. 18. 19. 20. 21. Average plasma xylose concentration (mg/100 ml) after an oral dose of .5 g xylose/kg body weight for calves fed milk replacers varying in protein level and source (Experiment 1) Least square means of fecal score and treatments for sickness for calves fed milk replacers varying in protein content and source by location and diet (Experiment 1) Least square means of rectal temperatures for calves fed milk replacers varying in protein content and source by location, diet and week on diet (Experiment 1) Mortality of calves at Michigan State fed milk replacers containing different protein levels and sources (Experiment 1) Influence of protein level and source on daily weight gain, rectal temperature, scour scores, apparent dry matter (DM) and organic matter (0M) digestibilities and apparent nitrogen retention in 2-week-old calves (Experiment 2) Calves omitted from.experiment due to unexpected problems or death (Experiment 2) Mbrtality of calves at Michigan State fed milk replacers containing different levels and sources of protein (Experiment 2) Influence of protein source and limestone on daily weight gain, intake of nutrients, feed efficiency, fecal score and rectal temperature of baby calves (Experiment 3) Influence of protein source and limestone on the apparent digestibilities of dry matter (DM), organic matter (OM), crude protein (CP) on apparent crude protein balance and plasma urea nitrogen on baby calves (Experiment 3) Effect of age on weight gain, intake of nutrients, feed efficiency, fecal score, fecal pH, rectal temperature, apparent nutrient digestibilities, apparent nitrogen retention and plasma urea nitrogen of young calves fed milk replacers as the only source of nutrients (Experiment 3) vi 60 62 63 64 68 69 70 70 71 73 Table 24. Average plasma xylose concentrations (mg/100 ml) 76 after an oral dose of .5 g xylose/kg of body weight, for calves fed milk replacers varying in protein sources (Experiment 3) Table 25. Average plasma xylose concentrations for treatment 77 combination means of protein sources and limestone with calve's age (Experiment 3) Table 26. Weight of contents from different sections of the 85 gastrointestinal tract, size of liver and pancreas and weight at slaughter of 6 week old calves fed experimental milk replacers (Experiment 3) Table 27. pH, dry matter, organic matter, and crude protein 36 in contents from different sections of the gas- trointestinal tract of 6 week old calves fed experi- mental milk replacers'(Experiment 3) Table 28. Effect of protein source (milk or milk + SPC) and 88 limestone incorporation in milk replacers on pH, dry matter, crude protein and organic matter in contents from different seetions of the gastro- intestinal tract of 6 week old calves (Experiment 3) Table 29. Effect of limestone addition on overall gut pH of 91 calves fed milk replacers containing only milk protein or half of dietary protein from SPC (Experiment 3) Appendix Table A.l Analysis of variance for variables in experiment 1 96 Table A.2 Analysis of variance for variables in experiment 2 101 Table A.3 Analysis of variance for variables in experiment 3 102 Figure Figure Figure Figure Figure LIST OF FIGURES Page Mean xylose.concentration in plasma of calves 79 fed milk replacers containing milk (-—-) or soy protein (---). For each hour, points not showing the same letter are different at P<.10 (SEM -':ll.58) Mean xylose concentration in plasma of calves fed 80 milk replacers with (---) or without (——-) lime- stone. For each hour, points not showing the same letter are different at P<.10 (SEM I':ll.58). Mean xylose concentration in plasma of 3 and 6 81 weeks of age calves fed milk replacers contain- ing milk (-—-) or soy (--) protein. For each hour, points in each graph not sharing the same letter are different at P<.Ol (SEM 8.116.37). Mean xylose concentration in plasma of 3 and 6 82 weeks of age calves fed milk replacers with (---) or without (--) limestone.. For each hour, points in each graph not showing the same letter are different at P<.10 (SEM - :d6.37). Mean xylose concentration in plasma of calves 83 with 3 (-—-) or 6 (--) weeks of age. For each hour, points not showing the same letter are different at P<.Ol (SEM 8 jfll.58). viii INTRODUCTION Replacement of milk products in the young dairy calf's diet with plant and/or animal products has been attempted due to the increasing demand for milk in human diets and the consequent high price accompany- ing this demand. Many dairymen utilize colostrum and waste milk as substitutes for whole milk for young females, but young males raised for market beef or veal depend heavily on commercial milk replacers. The proportion of milk consumed by a calf relative to it's mother's production is greater in areas where dairying is less developed and where the milk is less available for human consumption. In such areas, the calf may consume as much as 50% of the dam's production compared to 10% or less for more developed systems of management. Thus, the formu- lation of high quality milk replacers is not only a concern for develOped countries, but also for underdeveloped ones. Schugel (1974) described the history of milk replacers. The first milk replacers were developed in the fifties, and were based on skimmilk, buttermilk, whey and animal fat. 'At that time, problems related to fat incorporation and overheating of the skimmilk during the drying process resulted in a reduced energy content of the replacer, a high incidence of diarrhea and often poor performance of calves. Solving these problems resulted in better formulas and improved performance of calves fed milk replacers. In 1966, the price of skimmilk increased and its inclusion in milk replacers became less economical. Casein and whey were available at moderate cost and the industry shifted to these products with little reduction in quality. Later, as a result of the world's short supply of casein, researchers have investigated non-milk proteins, mainly from the soybean and fish industries; but these protein sources have not proved entirely satisfactory. The objective of this work was to study the partial substitution of milk protein by soybean protein concentrate and/or spray dried fish solubles. Also, the addition of limestone in milk replacers for young calves was evaluated. The parameters measured were growth, feed effi- ciency, scours, digestibility of nutrients, nitrogen retention, xylose absorption in the small intestine, and plasma amino acid concentration. REVIEW OF LITERATURE Efficient digestion and utilization of nutrients require that food pass at a suitable rate through the alimentary tract with appropriate enzymes and absorptive mechanisms that will allow the breakdown of complex molecules and absorption of the products. Young calves are sensitive to protein quality in these liquid rations and only highly digestible proteins with a favorable profile of essential amino acids are desirable in milk replacers. In particular, the first 3 weeks of life are the most critical for high quality protein in young calves (Huber and Slade, 1967; Makdani et al., 1971c; Norman, 1971) since protein digestibility increases with age (Noller et al., 1956; Gorrill et al., 1975; Bell et al., 1979). The sequence of digestive events in the abomasum and small intestine of the young calf fed milk has been determined in systematic studies using fistulated animals (Henschell et al., 1961a; Ash, 1964; Smith, 1964; Mylrea, 1966a). These events will be considered in the development of this review. Protein Digestion in the Abomasum In preruminating calves the stimulus of drinking milk causes the formation of the esophageal groove which directs liquids through the omasum into the abomasum (Titchen and Newhook, 1975). Thus, the first enzymatic action on dietary protein takes place in the abomasum. Hydrochloric acid is produced in the abomasum and its production increases with age Porter (1969). Cholinergic reflexes stimulate acid secretion directly by acting on parietal cells, or indirectly by facilitating gastrin secretion and potentiating its effect on parietal cells (Debas et al., 1974). Before feeding, the abomasal contents consist of a fairly clear, slightly viscous fluid containing small milk clots and has pH of 1-2. During feeding, milk clots rapidly as it enters the abomasum (Hill, 1968), and clotting is complete within a minute or so after finishing the meal. The levalues of abomasal contents increase to around 6 (Mylrea, 1966a) immediately after feeding, but as acid secretion increases pH slowly decreases and reaches pre-feeding values after about 5 hours (Porter, 1969). Only after most of the whey fluids leave the abomasum, doesthe pH of the abomasal fluids become sufficiently low for dissolving of the casein clot by pepsin (Mylrea, 1966a; Ternouth et al., 1974a). There is some evidence that quality of diet affects HCl production in the abomasum. Tagari and Roy (1969) concluded that a diet containing "severely" pre-heated spray dried.skimmilk powder resulted in higher pH values in the pyloric outflow compared to "mildly" pre-heated powder. This difference was probably due to a reduction in acid secretion on the heat damaged diet. Colvin et a1. (1969) showed a higher pH of digesta leaving the abomasum from calves fed soybean flour than in those fed whole milk. At 40% substitution of milk protein, fish protein concentrate appeared a better stimulus than soybean flour for gastric acid secretion (Ternouth, 1971). In the abomasum, milk protein clots through action of rennin and pepsin. The peptic chief cells appear to respond primarily to direct cholinergic neural stimulation, although gastrin is also implicated (Gregory, 1974). Soy, fish and whey proteins may inhibit rennin secre- tion, but pepsin does not seem to be affected by protein substrate (Carnot et al., 1977). Pepsin has only one-third the milk-clotting activity of rennin at near neutral pH but over 20 times the proteolytic activity at a lower pH (Raymond et al., 1973). For clotting by rennin, the optimum pH is 6.5; whereas, the optimum pH for proteolytic activity is 4.0 for rennin and 2.0 for pepsin (Tagari and Roy, 1969). Both enzymes have similar amino acid sequences (Pedersen and Foltmann, 1973), but differ drastically in their activity on ribonuclease as substrate. Pepsin inactivates it and rennin does not (Bang-Jensen et al., 1964). It is uncertain whether efficiency of protein utilization is affected by the nature of the enzymes secreted, though little dif- ference was found between results with pepsin and rennin for the "in vitro" digestion of raw milk (Henschell et al., 1961b). Berridge et a1. (1943) postulated that the changeover from rennin to pepsin secretion is initiated when animals first eat roughage. More recent results indicate that the pattern of protease secretion in the abomasum varied considerably among individual calves, but that, in addition to rennin, some pepsin activity was in most animals sampled during the first 2 weeks of life (Henschell et al., 1961a). Henschell (1973) observed a general increase in total protease activity in the abomasum with age. At weaning, rennin activity in the calf decreases while pepsin remains nearly constant (Thivend et al., 1980). This change is partially reversible. When the animal is given a liquid feed containing casein, pepsin activity is not affected but rennin increases; however, it does not reach preruminant levels (Garnot et al., 1977). Development of a firm curd in the stomach of the preruminant calf has two beneficial functions. It helps digestion by releasing the nutrients slowly into the small intestine where digestive enzymes more effectively hydrolyze nutrients. Secondly, a firm curd prevents excessive quantities of undigested protein from reaching the small intestine where proliferation of pathogenic organisms in the upper gut might occur, leading to illness or even death of the calf. Tagari and Roy (1969) found that replacers containing both low- or high-temperature treated skimmilk powders coagulate; but the former produces a firm, elastic curd, and the latter a floculent, loose curd. A solid clot is retained in the abomasum, whereas a fragile clot breaks down and passes rapidly into the small intestine. Little clotting was obtained by Colvin et al. (1969) and Emmons et a1. (1976) working with soy products. Low digestibilities for non-milk proteins in young calves have been attributed to the lack of coagulation of dietary protein in the abomasum (Gorrill and Nicholson, 1969a; Paruelle et al., 1972). Frantzen et a1. (1971) demonstrated depressed dry matter and protein digestibilities by Z-week-old calves fed milk replacers in which citrate or prior treatment by acid prevented coagulation in the abomasum. On the other hand, Owen and Brown (1958) indicated that citrate addition in whole milk to reduce curd formation did not adversely affect calf growth or health. More recently, Toullec et al. (1971) observed no effects on performance in calves given a non-clotting diet. Several factors may affect curd formation. Emmons and Lister (1976) concluded that curd formation was increased by a lower pH of the skimmilk over a range of 5.6 to 6.6, by higher concentration of rennin, by lower temperatures of heat treatment of skimmilk prior to spray drying, and by higher temperatures of coagulation (37 vs 30°C). Jenkins et a1. (1981) studied several factors affecting "in vitro" clot formation by rennet of milk replacers. They observed that "lower pressure dispersion" of lipid into skimmilk powders produced markedly higher values for curd firmness than did "homogenization" with lipid at all solid and lipid levels tested. Rennet coagulation of skimmilk treated by the "low-pressure dispersion method" promoted firmer curds than that treated by the "homogenization method" when the skimmilk was partially (20-40%) replaced by mixtures of fish protein-whey or soybean protein-whey. Formation of a strong curd may also influence fat digestion since fat globules are enmeshed in the abomasal clot (Hill et al., 1970; Ternough et al., 1980). A longer retention time in the abomasum would allow greater opportunity for fat hydrolysis by pregastric esterase (Radostits and Bell, 1970; Ternough et al., 1980). After coagulation of the casein, the whey in milk moves into the small intestine within 5 minutes after feeding (Radostits and Bell, 1970). Greatest flow of whey from the abomasum occurs shortly after feeding but overall rate is one-half the volume every 2 hours (Porter, 1969). The greater the quantity of milk fed, the longer the time required for all the whey to leave the abomasum (Ternouth et al., 1974a). As digestion in the abomasum proceeds, the clot breaks down 3-4 hours after feeding and chyme passing into the duodenum changes from a whey-like fluid containing predominantly carbohydrate and soluble nitrogenous compounds to a thick, white opaque material containing protein and fat (Mylrea, 1966b; Toullec and Mathieu, 1973). Between 82 and 87% of the digesta fluid passes from abomasum in the first 6 hours after feeding, but less than 50% of the total fat passes (Mathieu et al., 1968; Ternouth et al., 1974a). Feeding causes a considerable increase in the rate of outflow of abomasal contents (Ash, 1964; Mylrea, 1966b), and the rate of flow into the duodenum reaches a peak in the first hour after feeding; whereas, flow is greatest in the jejunum during the first 4 to 5 hours after feeding. There is no marked effect on feeding on the pattern of flow in the ileum (Porter, 1969). Ternouth et al. (1974a) suggested that the major factor regulating abomasal emptying is the tension of the abomasal wall and that the enteric inhibition of abomasal emptying is relatively unimportant for 12 hours postprandially. An increased outflow of undigested protein during the first hour after feeding was reported in calves fed "severely" heat-treated skim- milk powder (Tagari and Roy, 1969) or when milk protein was replaced by soybean flour (Colvin et al., 1969), fish protein concentrate (Ternouth et al., 1975) or even whey powder (Gorrill and Nicholson, 1972a). These results suggest a lower abomasal proteolysis in over- heated milk protein or replacers containing non-milk protein. Guilloteau et al. (1979) concluded that suppressing the transit of digesta into ' the abomasum (by infusion into the duodenum), as well as accelerating the rate of passage, have an adverse effect on nitrogen and lipid digestibilities. In contrast, the slowing down of the rate of passage allows digestibility to increase. On the other hand, Christison and Bell (1976) observed that a more digestible pea protein (60% crude protein digestibility) left the abomasum faster than a poorly digested pea product (41% crude protein digestibility), suggesting that the rate of passage of crude protein through the abomasum was not a major factor in determining the crude protein digestibility. There is also evidence that abomasal secretory activity might be lower when calves are fed diets containing "severely" preheated skimmilk powder, soybean flour or fish protein concentrate as protein sources. Lowered abomasal secretions have been also associated with a high incidence of digestive disturbances in young calves on milk substitute diets (Shillam et al., 1962; Tagari and Roy, 1969;Ternouth and Roy, 1973; Ternouth et al., 1974b; Ternouth et al., 1975; Williams et al., 1976). Protein Digestion in the Small Intestine After pre-digestion in the abomasum, the hydrolysis of dietary protein to small peptides and amino acids continues in the small intes- lO tine by action of trypsin, chymotrypsin and carboxypeptidase present in the pancreatic juice; and by various peptidases secreted into the small intestine or present in intestinal mucosa cells (Porter, 1969). The anatomical relationships of the pancreatic duct, bile duct and duodenum vary between species (Hallenbeck, 1967). In adult cattle, the main pancreatic duct enters the duodenum about 300 mm caudal to the sphincter of Oddi (Sisson and Grossman, 1956). Wass (1965) demonstrated that an accessory pancreatic duct joins the bile duct in most cattle. Pancreatic trypsinogen and chymotrypsinogen are converted to active enzymes a relatively short distance from their entry into the upper small intestine at'a pH lower (5.2 or less) than optimum "in vitro" conditions. There also appears to be rapid digestion of protein at a relatively low pH in the upper part of the small intestine (Gorrill and Nicholson, 1971). Changes in pH in the duodenum reflect those in the abomasum, rising immediately after feeding and then falling. Feeding has little effect on pH values in the distal small intestine, but there is a gradual increase towards the ileum where values range between pH 7 and 8 (Porter, 1969). A more detailed discussion on small intestinal pH, its effects on enzyme activity, and the possible role of buffers will be presented later. Emptying of zymogen granules within the pancreas is mainly under neural rather than entero-hepatic control (Ternouth et al., 1974a), i.e. the secretion of enzymes does not appear related to digestive require- ments of the intestine. There is a large increase of trypsin and chymo- trypsin activities in digesta just after feeding (Gorrill et al., 1967; ll McCormick and Stewart, 1967; Gorrill and Nicholson, 1971; Ternouth et al., 1975). However, response of the pancreas to feeding in calves was much less than for non-ruminants such as dogs (Preshaw et al., 1966). This large output of enzymes from the pancreas during the first 2 hours after feeding does not coincide with the main outflow from abomasum of total nitrogen and lipids occurring 5 to 10 hours after feeding (Ternouth et al., 1975). Ternouth et a1. (1976) observed that the increased secretion of pancreatic fluid but not enzymes 6 to 12 hours after feeding was due to stimulation by secretin. It was classically admitted that regulation of exocrine pancreatic secretion was controlled by neural and hormonal mechanisms (Thomas, 1967; Harper, 1972); however, a feedback mechanism may be involved. Taylor (1962) found that quantity of enzymes secreted by the pancreas decreased when these enzymes were prevented from entering the duodenum. Davicco et a1. (1979) concluded that a pancreatic regulatory mechanism similar to that in rats and pigs exists in young calves. Such regulation acts through a feedback of trypsin which would modulate the release of secretin. Gorrill and Thomas (1965) and Gorrill et al. (1967) reported that adequate amounts of trypsin and chymotrypsin were produced from birth and there was no marked increase in the concentration of these enzymes with age. However, increases in protease activity associated with increases in the flow-rate of pancreatic juice during the first week of calf's life were reported by Huber (1969). Ternouth et a1. (1976) 12 fed Ayrshire calves whole milk and observed in absolute terms and in relation to milk intake or metabolic size, a large increase with age (7 to 63 days of age) in secretion of total protease but no change in trypsin activity. They (Ternouth et al., 1976) also fed Holstein calves milk replacers and concluded that secretion of pancreatic fluid, but not trypsin or total protease increased with age. Reasons for the observed differences were not apparent. Diet quality may affect pancreatic and intestinal enzyme secretions. Gorrill and Nicholson (1972a) observed that proteolytic enzyme secretion from pancreas was about 2.5 times less for calves fed milk replacers containing 50% whey compared to 23% whey. Ternouth et al. (1974a) compared "mildly-" with "severely-" heated skimmilk and concluded that animals receiving "mildly" treated skimmilk secreted more pancreatic fluid, but quantity of trypsin did not differ significantly between diets. Ternouth et a1. (1975) reported that when skimmilk powder was partially replaced in the diets by soybean flour or fish protein concen- trate, neither large reductions in secretion of pancreatic enzymes (Gorrill et al., 1967) nor changes in the pattern of duodenal outflow (Colvin et al., 1969; Smith et al., 1970) were found. Nevertheless, the small decrease in these parameters may have been sufficient to explain the lower growth rate observed for the first 3 weeks of life. Calves fed soybean trypsin inhibitor (SBTI) showed a marked reduc- tion in exocrine pancreatic secretion (Gorrill and Thomas, 1967; Gorrill et al., 1967; Gorrill and Nicholson, 1971). Gorrill and 13 Nicholson (1971) concluded that the reduced pancreatic secretion in calves fed SBTI was not caused by diarrhea because diarrhea induced by neostigmine methyl sulfate or magnesium sulfate did not affect enzyme activities. The diarrhea in calves fed SBTI could be due to protein and peptide accumulation in the large intestine, as postulated by Roy (1969). Indeed, accumulation of undigested protein in the lower intestine of animals fed SBTI was observed in calves (Gorrill and Nicholson, 1971), chicks (Coates et al., 1970) and rats (Carroll et al., 1952). Buraczewski et a1. (1967) found higher concentrations of free amino acids and small peptides in the intestinal contents of rats given severely- heated'cod-fillet protein than in animals given undamaged proteins. Yet, concentrations of free amino acids in portal blood were lower. They postulated that the accumulation of "unavailable peptide" in the intestine, characteristic of rats fed heat-damaged protein, hinders absorption of amino acids by saturating mucosal transport sites. Shorrock and Ford (1978) showed that an extract containing "unavailale" small peptides from severely-heated cod-fillet strongly inhibited leucine uptake in rat intestine, but had no effect on uptake of glucose or its metabolism to lactate. Shorrock and Ford (1978) conjectured that the reduction in amino acid uptake was an allosteric effect resulting from an attachment of the peptides to binding sites adjacent to the sites for amino acid uptake. A further hypothesis was that "unavailable peptides" are trans- ported into the mucosal cells and there interfere with normal cell function. Whatever the true explanation, it remains now to isolate representative "unavailable peptides" and to study their influence on amino acid transport. 14 The utilization of substitute proteins might result in.modification of intestinal nitrogen digestion. .The amount of apparently digestible nitrogen which disappears in the large intestine is only 2 to 4% with milk protein (Weerden et al., 1977) but may reach 8 to 10% with fish concentrate or soybean concentrate when dietary protein is less completely hydrolyzed (Thivend et al., 1980). Thivend et a1. (1980) observed that when ingested proteins came exclusively from milk, the amino acid composition of ileum contents differed greatly from that of milk, feces or fecal bacteria, but was similar to endogenous protein. Thus, when feeding milk protein, true digestibility is nearly complete, so protein appearing in the feces is mainly of endogenous origin. However, when casein is replaced by fish or soybean protein, a greater proportion of protein passing through the lower digestive tract and out the feces will be of dietary origin. Soybean Products in Milk Replacers Acceptable growth has been obtained in calves fed mixtures of plant and milk protein (Murley et al., 1957; Lassiter et al., 1963; Pettyjohn et al., 1963; Stone et al., 1963) but growth from plant protein alone was usually unsatisfactory (Stein et al., 1954; Noller et al., 1956; Fries et al., 1958). Among plant proteins, soybean products have been most successfully incorporated into milk replacers. There are four main soybean products that have been tested. These are: soybean meal, soybean flour, soybean protein concentrate and soy protein isolate. They average 45, 60, 70 and 90% crude protein, and 15 45, 35, 25, and 5% carbohydrate, respectively. Results with preruminant calves vary widely, depending upon the soybean product and concentrations used in milk replacers (Table 1). Increased diarrhea has been reported at high levels of soy protein concentrate (Seegraber and Morrill, 1979; Coblentz et al., 1976). Others have found no diarrhea and some even showed improved fecal consistency (Morrill et al., 1971; Barr et al., 1978; Huber et al., 1978; Gaudreau and Brisson, 1978). Poor performances have been reported for calves fed raw soybeans (Williams and Knodt, 1951; Kakade et al., 1974; Thompson et al., 1974). Reduced digestibilities of nitrogen, fat and ash of milk replacers con- taining soy protein concentrate or soybean meal were reported (Nitsan et al., 1971,1972; Pejic and Kay, 1979). A hypothesis raised to explain these negative results relates to the presence of SBTI. Characteristics were severe decreases in pancrea- tic volume, enzyme concentrations, and specific activities of trypsin and chymotrypsin of 1-week-old calves receiving part of their protein from soybean flour (Gorrill et al., 1967; Gorrill and Thomas, 1967). The reduction in pancreatic secretions in calves directly contrasts to studies with chicks and rats in which extracts from raw soybeans induced pancreatic hypertrophy and hypersecretion of digestive enzymes (Haines and Lyman, 1961; Garlich and Nescheim, 1966; Mickelsen and Yang, 1966). Response of swine pancreas to raw soybeans (Hooks et al., 1965) was similar to that observed with calves. Lepkovisky et a1. (1971) suggested that impaired proteolysis was not the result of decreased activity of proteases in the intestine, l6 TABLE 1. Summary of dairy calves and lamb performances on different soy protein sources in milk replacers. Authors Products Level of Age ADGa DMDb PDc Type of protein animal greplacement 2 wk kg % % Gorrill and sped-502 50 0-6 -.11 Calves Thomas(l967) SPC -7l% 86 0-6 .33 Gorrill and SPC 0(milk) 0-7 .53 90.6 87.3 Calves Nicholson(l969b) 70 0-7 .55 88.8 81.6 Gorrill et a1. SPC 22 0-5 .51 Calves (1971) Gorrill and SPC 0(milk) 2-7 .377 90.0 85.0 Calves Nicholson(l972a) 70 2-7 .348 85.0 77.0 Gorrill and SPC 50 0-5 .197 94.0 92.0 Lambs Nicholson(l972c) Gorrill et a1. Full fat 0(milk) 0-5 90.2 93.6 Lambs (1974) soybean 30 0-5 93.5 95.1 flour 50 0-5 92.2 92.2 Gorrill et a1. Full fat 0(milk) 0-4 .23 Lambs (1975b) soybean 24 0-4 .20 flour 30 0-4 .21 Kakade et a1. Heated 70 2 0 84.0 55.0 Calves (1976) soybean Roy et a1. Soy flour 0(milk) 0-3 95.0 94.0 Calves (1977) 35 0-3 88.0 84.0 65 0-3 79.0 66.0 Barr et a1. Mbdified 0(milk) 0-4 .50 Calves (1978) soy pro- 50 0-4 .46 tein 72 0-4 .45 Bringe and Barr Modified 0(milk) 0-6 .67 Calves (1979) soy pro- 70 0-6 .64 tein Jenkins (1981) SPC 0(milk) 0-4 .443 93.6 90.8 Calves 51 0-4 .198 91.4 85.8 HAverage daily gain. bDry matter digestibility. cProtein digestibility. Soy protein concentrate. 17 but was due to the formation of a trypsin inhibitor-protein complex which resists proteolysis even in the presence of high concentrations of enzymes. Ramsey and Willard (1975) concluded that fully-cooked soy flour contains an inactive form of trypsin inhibitor that is converted to the active form in the pH range of 7 to 9. This inhibitor can be de- stroyed by heating the soy flour in water, but the extent of destruction is partially dependent on concentration of flour in the water. They suggested that the presence of trypsin inhibitor in fully-cooked soy flour might explain why newborn calves performed poorly on milk replacers containing large quantities of the product. Willard and Ramsey (1972) suggested that further removal of small amounts of trypsin inhibitor in heated soybean flour by acid or alkali improved its nutritional value, but this finding was not supported by Sissons and Smith (1976). ‘Gorrill (1970) concluded that "in vitro" pepsin digestion of soybean protein was greater in .05N sodium hydroxide than in water. Opposite effects occurred with milk protein. Alkali treatment on the soybean protein had no effect on proteolysis by trypsin and chymotrypsin after subtracting pepsin digestion from total digestion. More recently, Kakade et a1. (1976) studied the nutritional effects induced in calves by feeding SBTI, and did not find differences due to weight or enzymatic activities of pancreas. They concluded that SBTI plays a minor role, if any, in calf nutrition. Toasting of soybean flour accompanied by acid or alkali treatment (Colvin and Ramsey, 1968, 1969; Colvin et al., 1969) or extraction with 18 alcohol (Nitsan et al., 1962; Smdth and Sissons, 1975) has been claimed to overcome detrimental effects. Thermo-alkali treatment with lower temperatures inactivates SBTI more effectively than thermo-acid treat- ment (Wallace et al., 1971). Colvin et a1. (1969) analyzed digesta collected for 8 hours after feeding soybean flour and concluded that the treatment with acid or alkali had no effect on rate of passage through the abomasum, or on pH changes occurring in the abomasum. Thus, no explanation for the improved nutritional value of acid or alkali treatment of soy flour was found. Kakade et a1. (1971) fed rats lecithinated and non-lecithinated soy flours with and without acid treatment as the only protein and observed that rats receiving treated-lecithinated soy flour grew better than those fed the untreated. No such improvement was observed with treated non- lecithinated soy flour. They suggested that the beneficial effect of acid treatment involves the lecithin component of the soy flour. Diets containing soybean flour reduced gastric acid secretion (Williams et al., 1976) and retention time of protein in the abomasum (Calvin et al., 1969; Smith et al., 1970) with a resultant decrease in proteolysis. Improvement in the nutritive value of soybean products could involve the carbohydrate fraction. The residual carbohydrates in soy concen- trates are pectin-like compounds, such as neutral arabinogalactans, acidic polyssacharides and arabinans (Kellor, 1974). Disappearance in digestibility studies of considerable carbohydrate from soy protein sources (Nitsan et al., 1971,1972; Kakade et al., 1976) is presumably 19 due to intestinal fermentation. However, replacement of 50% of milk protein with soy flour containing 30-40% carbohydrate should not be highly detrimental to calf performance because this fraction would only account for a maximum of 8% of the total solids in the milk replacer. Enzymatic pre-digestion of soy flour did not stimulate growth, even though the carbohydrate fraction underwent extensive degradation (Colvin and Ramsey, 1968). Feeding of high chlorotetracycline to depress intes- tinal femmentation did not improve growth in calves fed untreated soy flour (Colvih and Ramsey, 1969). Moreover, removal of the water-soluble carbohydrates from.soy flour did not improve growth and addition of the water-soluble carbohydrates to,a replacer containing soy flour did not depress growth (Sudweeks and Ramsey, 1972). Crude and refined soybean oils in milk substitutes severely retarded growth and increased diarrhea in young calves (Barker et al., 1952), but level of fat in soy protein concentrate is negligible and therefore presents no practical concern. Methionine is the most limiting amino acid in soybean protein for rats, chicks and pigs (Almiquist et al., 1942; Berry et al., 1962; Hays et al., 1959). Lysine was lower in the alkali-treated than untreated soybean concentrate (Gorrill, 1970), and DeGroot and Slump (1969) reported that lysine, cysteine and serine were reduced when isolated soybean protein was mildly treated with .02N NaOH. Methionine supplementation (0.1% of dry matter) did not increase growth or nitrogen retention in young calves fed milk replacers containing 70% of the dietary protein from soybean protein concentrate (Gorrill and Nicholson, 1969b). On the other hand, 20 Porter and Hill (1964) reported improved calf performance when methionine supplementation of isolated soybean was increased from 1.5 to 2.3 g/100 g of protein. The presence of hemaglutinates in calve's blood has been detected (Smith and Wynn, 1971), especially when milk protein was completely replaced by soybean-flour, and when successive feedings of soybean flour was interpersed with feedings of normal milk diets. Smith and Sissons (1975) and Sissons and Smith (1976) observed digestive disturbances when soy products were fed to calves indicating a gastrointestinal allergic response. Serum antibody responSe was recorded 2 to 3 weeks after feeding milk replacers with soy protein (Kilsham.and Sissons, 1979) and previously sensitized calves responded to reintroduction of soy protein in the replacer with marked increases in antibody level (Barratt et al., 1978). On the other hand, such evidence was not found by Ternouth et a1. (1975) and Roy et a1. (1977). Extraction of soy protein sources with hot aqueous-ethanol rendered it non-immunogenic and seemed to eliminate digestive disturbances nor- mally found with soy protein (Sissons et al., 1979). According to these authors, alcohol treatment denatures the specific antigenic proteins, glycinin and beta—conglycinin. However, other workers reported no benefit from alcohol treatment (Barratt et al., 1978,1979; Barrett and Porter, 1979). Differences in treatment method might be responsible for the conflicting results. Seegraber and Merrill (1980) observed less villi and those villi present were short, blunted and convoluted when calves were fed soy 21 proteins. They concluded that the substitution of one-third of the milk protein with soy protein concentrate resulted in impairment of absorptive ability which was probably associated with morphological changes in intestinal structure. Stobo and Roy (1977) observed a slight thickening of the walls of the small intestine when dried bac- terial cells or yeast supplied 22 or 42% of the dietary protein. As mentioned, the maximum level of milk protein substitution by soy protein varies with the type of soybean product used. Roy et a1. (1977) concluded that 30% replacement of milk protein by soybean flour was possible, but with some detrimental effects on the calf's health and growth. Higher levels of replacement (70%) were worse than lower levels. Satisfactory growth was obtained when soybean protein concentrate supplied more than 50% of the total protein in milk replacers (Gorrill and Thomas, 1967; Gorrill and Nicholson, 1969b,1972a; Polzin, 1978). In contrast, Merrill et a1. (1971) stated that soybean protein concentrate could successfully replace 25%, but not 44% of total protein in milk replacers. More recently, Barr et a1. (1978) and Bringe and Barr (1979) con- cluded that a "modified soybean protein" could replace 72% of the dietary protein without depressing calf growth, feed efficiency or increasing diarrhea. When concentrate and hay were available to calves, in addition to milk replacers, the difference in growth rate of calves fed milk vs. soy proteins diminished (Nitsan et al., 1972). 22 Gorrill et al. (1974,1975a) concluded that specially processed full fat soybean flour could supply 50 to 60% of the total protein in a lamb milk replacer without adversely affecting growth and meat quality. Similar results on growth of lambs have been reported using soybean flour (Jordan et al., 1972) or soy protein concentrate (Gorrill and Nicholson, 1972c). Fish Products in.Milk Replacers One of the major problems in comparing literature results on fish product utilization by calves is the diversity in type and quality of the products tested. Several investigators have observed marked varia- bility in nutritional Quality of products obtained from.different fish Species and processing procedures (Miller, 1956; Morrison and McLaughlan, 1961; Morrison et al., 1962; Makdani et al., 1971a). Processinngish Proteins Fish meal was originally obtained after collection and treatment of the filleting offal by drying and grinding to a meal. Increased demands for such a protein supplement in stock feeds were met by the drying of whole white fish, such as herring. Processing included cooking, followed by pressing to remove most of the oil (Geiger and Borgstrom, 1965). Production of fish flour or fish protein concentrate involves use of the entire fish and yields a dry powder edible by humans. The two processes cleared by the Food and Drug Administration are: a) Isopropanol extraction and b) Ethylene dichloride as the initial solvent, followed 23 by subsequent extraction with isopropanol (Wilcke, 1969). In this paper, products from both processes will be referred to as fish protein concen- trate (FPC). For human consumption (Geiger and Borgstrom, 1965), the finely-ground product is washed repeatedly with alcohol to remove solvent residue and to deodorize. Fish solubles are made of stickwater, an aqueous extract from cooked fish, usually obtained from fish meal plants where fish are cooked and separated by pressure into an aqueous extract and a fish pulp. The pulp is dried and made into fish meal. The stickwater exhibits properties characteristic of both fish muscle extracts and the gelatin- and glue- containing extracts of bone, cartilage, skin and other connective tissues. It is customary to discard this stickwater after removing the oil by "settling tanks," or centrifugation. Stickwater, which contains about 302 of the original fish protein, is then evaporated to yield dried fish solubles (Lassen, 1965). Nutritional Value of Fish Protein Products Morrison and Campbell (1960) confirmed previous results obtained by Sure (1957) in which fish protein concentrate was an excellent source of high quality protein for rats, particularly in diets deficient in lysine. Fish protein concentrate also improved the biological value of cereal based diets for growing infants when used as the main protein source (Yanes et al., 1969). Tavill and Gonik (1969) obtained similar results in Morroco with infants fed diets containing 3% PFC (w/w). Performance of calves on milk replacers containing FPC depends on 24 age, level of FPC, level of crude protein in the replacer, and the use of dry feed in the feeding program (Table 2). Poor performance was obtained when calves were fed high levels of FPC in milk replacers which served as the only source of nutrients (Huber and Slade, 1967; Matre, 1970,197l,l973,1977; Gillespie, 1971; Sleiman and Huber, 1971; Raven, 1972; Ramsey, 1975; Makdani et al., 1971c). Most of the growth depression occurred during the first 3 weeks. Average daily gain and feed efficiency were not significantly depressed when FPC furnished up to 4G to 50% of dietary protein in calves fed milk replacer (Huber and Slade, 1967; Huber and Sleiman, 1971; Sleiman and Huber, 1971; Gorrill et al., 1972; St. Laurent and Brisson, 1972; Ramsey, 1975; Matre, 1977). Good results were also obtained with milk replacers containing higher FPC, but fed with hay and starter (Gelwichs, 1965; Harshberger and Gelwichs, 1965; Williams and Rust, 1968; Danell, 1970; Gorrill, 1970; Sleiman and Huber, 1971; Opstvedt et al.,gl97S). “ix Huber et al. (1978) obtained poorer calf performance when 332'of. dietary protein was replaced by fish hydrolysate compared to all milk. However, relatively good gains were obtained when 33% of milk protein (was replaced by equal parts of fish hydrolysate and soy protein. There are some reports showing that calves fed milk replacers containing 702 of dietary protein from FPC grew as fast as those receiving all milk protein (Van Hellamond, 1967; Sorensen and Lykkeaa, 1968; Danell, 1970). In these experiments, however, milk replacers contained 24 to 26% crude protein and calves were 3 weeks of age or older. The higher total protein and older age of calves may explain why results 25 TABLE 2. Summary of dairy calves performance on different fish protein sources in milk replacers Authors Products Level of Age ADGa DMDb PDc replacement Z wk kg 2 2 Raven (1972) Fish meal 0(milk) 0-8 93.0 86.2 15 0-8 90.7 83.8 Gorrill et al. Herring meal 65 0-3 92.6 81.6 (1975) FPC 62 0-3 92.7 84.8 Huber and Slade FPC 0(milk) 1-6 .397 90.2 90.3 (1976) 33 1-6 .360 88.7 84.3 67 1-6 .273 89.0 85.4 Gorrill et al. FPC 0(milk) 0-3 .365 90.0 79.0 (1972) 25 0-3 .283 89.0 76.0 Roy et al. FPC 35 0-3 .88 87.0 83.0 (1977) 65 0-3 .66 83.0 75.0 Opstvedt et al. FPC 0(milk) 1-9 .514 98.3 96.1 (1978) 36 l-3 .142 94.5 84.4 36 4-5 .337 97.0 93.7 96 1-9 .303 95.3 90.5 Huber et a1. Predigested 0(milk) 0-6 .416 (1978) FPC 33 0—6 .303 snssd 16.5 0-6 .356 3Average daily gain. bDry matter digestibility. cProtein digestibility. dSpray dried fish solubles. were different than obtained in most studies. After weaning, fish protein seems to be good for lambs (Orskov et al., 1971; Fraser and Orskov, 1974; Folman and Eyal, 1978) and calves (Preston et al., 1960,1965; Whitelaw et al., 1961,1963; Kay et al.,1967), probably due to its low degradability in the rumen. 26 The extraction procedure used in FPC production can influence nutritional value of the product. The importance of complete removal of solvent residues was emphasized by Morrison et al. (1962). Isopropanol-extracted fish protein concentrate (IP-FPC) was shown equal or superior to casein as the only protein source for rats and pigs (Power, 1964; Sidwell et al., 1970; Pond et al., 1971). The IP-FPC was also better than dichloroethane-extracted fish protein (DCE-FPC) (Munro and Morrison, 1967b; Ershoff and Rucker, 1969; Makdani, 1969; Makdani et al., l971a,l971c) and hexane-heptane-extracted fish protein concentrate (Makdani et al., 1974). Increased biological value was obtained when DCE-FPC was washed with ethanol, suggesting removal of certain polar-soluble substances that adversely affected protein quality (Makdani et al., l97la). Munro and Morrison (1967a) concluded that extraction with dichloro- ethane resulted in formation of chlorocholine chloride (CCC), a choline derivative toxic to rats. The CCC is a potent inhibitor of acetyl choline esterase (Friess and McCarville, 1954). Later, Munro and Morrison (1967b), suggested that toxicity of DCE-FPC in rats could not be explained by CCC formation alone. Although the toxic factor(s) were extracted by methanol, the resultant material still did not support growth, even when supple- mented with cystine and histidine. The authors suggested that methanol did not remove all of the toxic substances from DCE-FPC. Makdani et al. (l97lb) suggested that FPC extracted with isopropanol was more desirable for humans than DCE-FPC. Surprisingly, calves fed DCE-FPC grew better than those fed IP-FPC (Makdani et al., l971c). Reasons for this discrepancy were not apparent. 27 Protein digestibility and nitrogen retention were depressed when FPC constituted the most of the dietary protein for calves (Huber and Slade, 1967; Matre, 1970; Sleiman and Huber, 1971; Gorrill et al., 1975; Huber, 1975; Roy et al., 1977; Opstvedt et al., 1978). Protein digesti- bilities for FPC have been 10 to 30% lower than milk protein. Ether extract digestibilities were also depressed by inclusion of FPC in milk replacers, but carbohydrate utilization appeared unaltered (Huber and Slade, 1967; Matre, 1970; Roy et al., 1977). Reduced fat digestibility might be explained by the high ash content of FPC which may have increased fecal excretion of calcium.and magnesium soaps (Raven and Robinson, 1959). Negative results on fish protein may also be due to relatively indiges- tible long chain fatty acids in fish oil (Flatlandsmo, 1972). Sleiman and Huber (1967) observed higher digestibilities in young calves when FPC was combined with whey instead of casein. For rats, best nutritive values were obtained when FPC was combined with wheat flour or rice. Gorrill et al. (1972) observed reduced nitrogen retention in the absence of a change in protein digestibility when calves were fed FPC. This finding suggests an amino acid imbalance. Generally, FPC compares favorably with whole egg in terms of amino acid composition, but it is lower in tryptophan and cysteine (Sidwell et al., 1970). Compared to skimmilk, FPC is slightly lower in tyrosine, phenylalanine, leucine, isoleucine and valine, but higher in arginine and lysine (Raven and Robinson, 1959). Under certain conditions, an excess of arginine and lysine were detrimental to chick growth (Huston 28 and Scott, 1966). "In vitro" studies conducted by Ellinger and Boyne (1965) showed that heat treatment (105°C for 36 hr) of cod muscle caused significant losses of serine (8%), tyrosine (6%), lysine (11%), histidine (12%), and cysteine (642). Alanine and methionine appeared the most stable amino acids. Marked differences of FPC sources in availability to rats of certain amino acids such as methionine and histidine, were reported (Morrison, 1963). However, Morrison and Sabry (1963) concluded that unavailability of methionine could not explain entirely the reduced nutritional value of FPC since methionine supplementation did not improve growth. Stillings et a1. (1969) conducted a series of experiments with rats to determine the sequence of limiting amino acids in IP-FPC. bGrouped according to limitation from most to least limiting were: 1) methionine; 2) histidine, tryptophan and threonine; 3) valine, isoleucine and phenylala- nine; 4) leucine, lysine and arginine. Makdani et al. (1971a) also concluded that methionine and histidine were coelimiting in FPC regardless of fish species or extraction processes. Histidine or methionine supple- mentation alone were equal in increasing growth of rats fed FPC diets. Whereas, histidine plus methionine were additive. 0n the other hand, methionine supplementation to a milk replacer containing DCE-FPC did not improve calf growth (Makdani et al., l97lc). More recently, Matre (1977) suggested that isoleucine might be the limiting amino acid for calves fed protein from mackarel flour. Morrison and Munro (1965) demonstrated that 1,2-dichloroethane can react with free sulfhydryl groups of cysteine to yield a thioether, which 29 they hypothesized reduces susceptibility of proteolytic attack (action of pancreatin). Specifically, the dihalide reacts with sulfhydryl groups on adjacent peptide chains and a stable thioether bridge forms which might bind peptide chains more tightly, thus reducing ease of proteolysis. Available histidine was also reduced, but a direct reaction of histidine with ethylene dichloride is unlikely. Rather, the histi- dine may occur adjacent to cysteine and be indirectly affected. Other explanations for the lower performance of calves fed fish protein concentrate than milk protein were contaminants from extraction with DCE causing aplastic anemia (Pritchard et al., 1952); and passage of the diet into the rumen causing bloat (Makdani et al., 1971c). Fish meal has also been tested in milk replacers for calves (Rupel and Wilson, 1962; Wendlandt et al., 1968; Genskow et al., 1968; Gorrill et al., 1975) but performance was generally poorer than for FPC. White muscle disease (WMD) has been associated with calves fed fish products (Genskow, 1969; Michel et al., 1972). Calves are most susceptible to WMD from birth to 6 months of age (Poukka, 1966), probably because of rapid changes in tissue fatty acid composition. According to Horwitt (1956), nutritional WMD in mammals occurs only when certain of the polyunsaturated fatty acids in the muscle lipids reach a high degree of "peroxidizability" in a given time. Therefore, increased linoleic and arachadonic acids in muscle predispose young calves to WMD. The pathogenesis of WMD is not clear and divergent views are found in the literature; but the three factors most often discussed in 30 connection with the disease are vitamin E, selenium and unsaturated fatty acids. Fish fats contain high percentages of unsaturated fatty acids (Medwaddowski et al., 1967; Lovern, 1969) and played an important role in eXperimentally induced WMD (Adams et al., 1954; Blaxter and McGill, 1954). Dam nutrition may also predispose calves to WMD, notably when fed milk high in polyunsaturated fatty acids (Hidiroglou et al., 1968,1977). Vitamin E and selenium function as biological antioxidants, but selenium possesses 50 to 500 times the antioxidant activity of alpha- tocopherol (Hamilton and Tappel, 1963). White muscle disease is cured by vitamin E (Blaxter et al., 1953; Harris and Embree, 1963; Roy, 1964), but amount needed in milk replacers is debatable. Michel et al. (1972) observed muscular degeneration in 8-week-old calves fed fish protein concentrate as the sole protein source despite supplementation with 46 mg vitamin E/kg of dry ration. In a second experiment, 46 mg of vitamin E/kg of dry ration prevented the histopath— ologically-detectable degeneration in FPC-fed calves. The conflicting results may have been due to different batches of FPC in the two experi- ments. Despite the absence of muscle degeneration in calves of the second experiment receiving 46 mg/kg alpha-toeopherol, supplementation of 92 mg E/kg increased growth above that at lower E additions. Other Protein Sources in Milk Replacers Bacterial sludge (containing about 60% crude protein and 30% lactose) in combination with skimmilk and whey did not affect incidence 31 of diarrhea, dry matter and nitrogen digestibilities, and nitrogen retention when substituted for up to 35.6% of the milk protein in milk replacers (Bouchard et al., 1973). Raven (1972) reported decreased nitrogen retention when 13.5% meat meals were substituted for milk protein. Polzin et al. (1976) also reported lower nutrient utilization in calves fed meat meals compared to those fed casein. They observed that meat meals contain mainly collagen, which is approximately 13% hydroxy-proline and is largely unavailable to the calf.- Weight gains and apparent digestibilities of nitrogen, ether extract and energy showed significant linear declines when increasing levels of distillers dried solubles were substituted for dried skimmilk and lactose (Bryant et al., 1967). However, the authors concluded that distillers dried solubles could replace up to 35% of the digestible protein in milk substitute diets for herd replacements without severely impairing growth. Blood meal and blood flour were of comparable value in milk replacers, resulting in slightly lower growth when compared to all-milk protein (Brubaugh and Knodt, 1952). Fababean (up to 25%; Wittenberg and Ingalls, 1979), a rapeseed (70 to 30%; Gorrill et al., 1976), alfalfa (up to 50%; Alpan et al., 1979) and crab meal (up to 20%; Patton et al., 1975) are other protein sources giving acceptable calf performances. 32 Addition of Enzymes to Milk Replacers As mentioned before, one of the hypothesis raised to explain lower digestibilities of non-milk constituents in milk replacers was low concentration of enzymes in the gastrointestinal tract of calves. To be effective, an enzyme fed to young calves must be active in the acid conditions of the abomasum, or must escape destruction in the abomasum to be active in the small intestine. Since calves secrete a limited amount of amylase (Huber et al., 1961a; Morrill et al., 1970a), the inclusion of amylolytic enzymes in milk replacers containing high levels of starch or inclusion of pre- digested starch, has been successful in some studies (Morrill et al., 1970b; Thivend et al., 1979) and unsuccessful in others (Raven and Robinson, 1965; Bell et al., 1974). Chow and Bell (1976) showed that in "in vitro" conditions, the amount of trichloroacetic acid soluble protein could be greatly improved by enzymatic treatment of pea products. Later, Bell et al. (1979) fed young calves milk replacers containing 0 to 75% field pea protein with low or high levels of supplemental proteolytic and amylolytic enzymes. They concluded that gross energy and protein digestibilities were higher for the control replacer than the enzyme-treated rations. Jenkins et al. (1980) tested "in vitro" hydrolysis of milk, soybean and fish proteins by several enzymes. They concluded that all enzymes studied hydrolyzed the milk proteins more extensively than the non—milk proteins, both at their optimum pH and at pH 6.1, which they suggested was comparable to calf abomasal contents immediately after feeding. They 33 also showed that papain and pronase preparations were quite efficient in degrading almost all the milk and non—milk proteins tested. However, enzyme addition or pre-digestion of milk replacers containing soybean protein (Fries et al., 1958; Lassiter et al., 1959; Jenkins, 1981) or fish protein (Toullec et al., 1972; Wilson, 1973; Soliman et al., 1976; Dodsworth et al., 1977; Huber et al., 1978; Petchey et al., 1979) did not improve nutrient digestibilities or calf performance. Limestone As a Buffering Agent Much of the current knowledge about post-abomasal pH has been extrapolated from information available for monogastrics with the assump- tion that digestion in monogastrics and ruminants differs only in the forestomach. However, species differences may extend to the lower tract (Wheeler, 1980a). Gastric contents from the human stomach are rapidly neutralized in the duodenum (Borgstrom et al., 1957), while limited information for ruminants suggests that the acidic digesta entering the small intestine is slowly neutralized. Harrison and Hill (1962) found that pH of duo- denal digesta in sheep increased slowly from 2.7 at the proximal duodenum to around 4.0 beyond the entry of the common bile and pancreatic ducts. Low pH values caudal to these ducts suggest that duodenal secretions of ruminants have a limited neutralizing capacity. This conclusion is supported by Lennox and Garton (1968) and Lennox et a1. (1968), who found that conditions in the upper jejunum of sheep were notably acidic (pH of 2.0 or 3.0) and that pH did not reach values of 6.0 or 7.0 until 34 the lower jejunum. Several other workers (Smith, 1962; Phillipson and Storry, 1965; Topps et al., 1968) indicate a possible limit to the capacity of the small intestine of ruminants to neutralize acidic digesta from the abomasum. The persistent acidity of digesta in the small intestine of ruminants may be attributed to the continuous secretion of hydrochloric acid by the abomasum (Phillipson, 1970) and/or to the weak alkaline nature of secretions entering the small intestine. Reports have indicated that from 300 to 400 ml of pancreatic juice (Magee, 1961; Taylor, 1962) and about 700 ml of bile (Hill, 1970) are secreted daily into the small intestine of sheep. However, these intestinal secretions have little buffering ability compared to duodenal secretions from monogastrics such as dog and man (White et al., 1959). Wheeler (1980a) suggested that reductions in digestibility and physiological abnormalities in ruminants may be associated, at least in part, with changes in the gastrointestinal environment. The use of buffers in diets for dairy cows is extensive, although it is not well understood how and when buffers are beneficial (Muller, 1981). Dietary buffers are often fed to milk cows to counteract acidity and maintain a near normal pH in the digestive tract, especially the rumen. Compounds such as limestone and dolomitic limestone exert a buffering influence in the small intestine of ruminants (Wheeler and Noller, 1976; Wheeler, l979,l980a,l980b). These products add alkaline reserves to the lower digestive tract and help raise the pH in the small intestine. Fecal pH has been considered as a good indicator 35 of intestinal pH (Wheeler and Noller, 1977; Wheeler, 1980a). Limestone instead of sodium or potassium bicarbonate better buffer the intestine, since sodium and potassium are readily absorbed from the forepart of the digestive tract (NRC, 1978). Magnesium oxide has also been shown to increase fecal pH and may function as a buffer in the small intestine (Erdman et al., 1980). The source of particle size of limestone influences its reactivity and effectiveness as a buffer (Wheeler, 1980b). Present recommendation for limestone addition to lactating cow diets is .5 to .8% of the total ration (Muller, 1981). The use of limestone or sodium bicarbonate in pre-ruminant diets has not been extensively studied. Beneficial effects of bicarbonate addition to colostrum were associated with higher immunoglobulin absorp- tion due to pH changes in the small intestine (Foley et al., 1978) and a bacteriostatic action (Harrison and Peat, 1972). Sodium bicarbonate has also been used routinely as fluid therapy for calves with diarrhea (Church, 1971). Summary of the Literature Review From the literature it may be concluded that the substitution for milk protein of soybean or fish products has to be partial, and even in this situation, poorer calf performance may be expected when compared to all-milk protein. Several reasons might explain these negative results of substitute protein sources. First, protein quality of soybean and fish products 36 is generally lower than milk protein. Also, industrial procedures may further alter the quality of these non-milk proteins by denaturation or introduction of toxic compounds during the process. In general, nondmilk proteins have resulted in impaired curd formation in the abomasum, perhaps due to reduced HCl and rennin secre- tions. In some experiments, secretion of pancreatic proteolytic enzymes has been reduced when non-milk proteins were fed resulting in a faster flow of protein through the digestive tract with consequent accumulation of undigested protein in the lower gut. This lowered protein utilization and greater fermentation of undigested nutrients in the lower gut might explain the poor calf performance. With soybean products, the presence of SBTI, problems caused by the polyssacharide and oil fractions, and low levels of certain essential amino acids (methionine and lysine) were frequently claimed as reasons for reduced growth on milk replacers. Industrial processing and amino acid supplementation has improved the quality of soybean products used in milk replacers. More recently, allergic-type reactions in calves have been observed and were associated with changes in the intestinal mucosa structure and reduced nutrient absorption. Quality of the fish products varies widely, depending on the processing method used. Denaturation of protein, presence of toxic compounds, low levels of essential amino acids, high ash content and polyunsaturated fatty acids have all been linked with the negative results observed when these products were fed. The addition of enzymes or hydrolysis of non-milk proteins before 37 incorporation into milk replacers has not, as yet, improved calf performance. For adult ruminants, it has been suggested that reductions in digestibility and physiological abnormalities may be associated, at least in part, with the persistent acidity of digesta in the upper small intestine. Limestone has been shown to raise the pH of the small intestine and improve nutrient utilization. Until now, no information was available on addition of limestone to milk replacer diets for calves. Spray dried fish solubles (SDFS) is a new industrial product, high in soluble protein, desirable features for a milk replacer. Questionable results in a previous experiment at Michigan State (Huber et al., 1978) with SDFS were shown. Experiment 1 was to further test SDFS in a large experiment involving three different locations. Experiment 2 was designed to explain results observed in Experiment 1. Experiment 3 tested limestone as a buffering agent at the small intestine of young calves. Possible beneficial effects on nutrient utilization of milk replacers containing a high level of soybean protein concentrate were investigated. MATERIALS AND METHODS Materials and Methods will be divided into three experiments which had different objectives, different animals and were chronologically separate. Experiment 1 A total of 168 Holstein calves at three locations - Michigan State University (62 males), Kansas State University (37 males and 13 females), and Cornell University (56 males) -- were fed milk replacers containing different protein sources and levels. The objective of this experiment was to study in baby calves the effect of the partial substitution of spray-dried fish solubles and/or soybean protein concentrate for milk protein on growth, health, plasma amino acid concentration and xylose absorptive capability of the small intestine. At Cornell, calves were bought through a calf auction; at Kansas State, five calves were purchased and the remainder were from the Kansas State herd; and at Michigan State all calves were purchased from a large commercial herd and transported to the experimental facilities of Kellogg Farm at Battle Creek, Michigan, when 3 to 8 days old. Preliminary meetings were held among leaders of this project to establish standard procedures and management specifications to be followed 38 39 at the different locations. The day calves started on the experiment they were identified by an ear tag and navels were disinfected with iodine solution. Each calf received an injectable solution containing 500,000 IU of vitamin A, 75,000 IU of vitamin D, and 50 IU of vitamin E, and was randomly assigned to experimental diets. All animals were kept indoors in tie stalls or individual pens bedded with straw. Each calf received the designated milk replacer as the only source of nutrients at 8, 9, 10, ll, 12 and 12% body weight from first to sixth week. Solids content of all milk replacers after mixing with water was 14%. Calves were fed twice daily (12 hr apart) from open pails. Fresh water was available at all times. Experimental diets according to protein percent and sources were: Treatment 1: 13% crude protein (CP); 13% milk protein (MP) (negative control) Treatment 2: 19% CP; 19% MP Treatment 3: 19% CP; 13% MP; 6% spray—dried fish solubles (SDFS) Treatment 4: 19% CP; 13% MP; 6% soy protein concentrate (SPC) Treatment 5: 19% CP; 13% MP; 3% SDFS, 3% SPC Treatment 6: 23% CP; 23% MP Treatment 7: 23% CP; 13% MP; 10% SDFS A.sample was taken from each 22.7 kg sack of milk replacer used and composited for laboratory analyses. Samples of the original ingredients were also obtained. Ingredients and replacers were analyzed for dry matter (forced air oven at 650C), crude protein (macro-Kjeldahl), ether extract and ash as described by A.0.A.C. (1980). Ingredient composition of milk replacer diets is shown in Table 3 and chemical TABLE 3. Ingredient composition of milk replacer diets (%), Experiments 1 and 2 Ingredient Diet Number 1 2 3 4 5 6 7 Protein fat mix (12/50)a 41.00 40.00 40.00 40.00 40.00 40.00 40.00 Dried skimmilk 24.60 42.00 24.00 24.00 24.00 54.00 24.00 Spray dried fish solubles 10.00 ----- 5.00 ----- 17.00 Soy protein concentrate 9.00 4.50 Dextrose 17.40 17.00 17.00 17.00 17.00 5.00 17.00 Lactose 16.40 ----- 8.00 9.00 9.50 ----- 1.00 Vitamin-mineral premixc .63 .63 .63 .63 .63 .63 .63 Chlorotetracycline .01 .01 .01 .01 .01 .01 .01 Lysined .15 .15 .15 ----- .26 Methionined .09 .09 .09 ----- .15 TOTAL 100.04 99.64 99.88 99.88 100.88 99.64 100.05 aSpray-dried mixture prepared to contain 12% milk (45% animal fat and 5% soy licithin). ture in equal amounts of three commercial soy protein concentrates: Procon, Promocalf and Ardex. protein and 50% fat cContaining (per kg), 33,000 IU vitamin A, 6,600 IU vitamin D, 22 mg of vitamin E, Mg, Cu, Co, Zn, Mn and I. dAdded to equal the level of these amino acids in the milk protein at each particular percent of protein studied. composition of ingredients and diets in Table 4. 41 TABLE 4. Chemical composition and pH of protein substitutes and formu- lated dietsa (Experiments 1 and 2) Item 014(2) CP(%) EE(%) Ash(%) pr Whey 92.55 13.80 .72 8.94 5.90 Fish solubles 84.34 61.60 14.44 18.86 4.70 Ardex 90.98 66.25 .99 3.65 6.55 Dextrose 90.76 .06 1.21 .02 6.50 Lactose 96.52 .12 .07 .18 6.30 Promocalf 91.56 67.20 .37 5.95 6.70 Procon 89.39 67.80 .13 5.97 6.70 Diet 1 94.50 13.73 19.91 4.39 6.52 Diet 2 94.43 20.25 21.34 5.75 6.55 Diet 3 94.02 20.30 20.48 6.43 6.05 Diet 4c 94.69 20.14 20.46 4.67 6.60 Diet 5c 94.60 20.08 23.14 5.46 6.27 Diet 6 94.54 23.96 22.86 6.61 6.49 Diet 7 93.24 24.15 24.64 7.12 5.89 aCP - crude protein; EE - ether extract and ash are presented on dry matter basis. . The pH was determined after diluting with water to 14% solids. cEqual portions of Ardex, Promocalf and Procon were mixed in diets 4 and 5. When a calf assigned to the experiment died prior to completion of treatment, the first calf available thereafter was the replacement. All animals which died during treatment at Michigan State were submitted for necropsy at the Veterinary Diagnostic Laboratory. treatment replicates by location is shown in Table 5. Calves were weighed individually for 2 consecutive days at the beginning and at the end of the trial. calves were 3 weeks old. The number of Weights were also taken when 42 TABLE 5. Number of calves per treatment in each location (Experiment 1) Diets Locations Michigan State Kansas State Cornell University 1 9 6 8 2 9 7 8 3 9 8 8 (7)3 4 9 7 8 5 _ 9 7 8 6 9 8 8 7 8 7 8 (7)81 Total 62 50 56 (54) FAt Cornell University one calf from diet 3 and one other from diet 7 were lost when 5 and 3 wk old, respectively. Those calves were not replaced. Daily observations were made on each animal for degree of scouring by rating fecal consistency on an index of 1 to 4 (Larson et al., 1977). Rectal temperatures were taken daily (just before the morning feeding) for each animal during the first 2 weeks of trial. Treatment for illnesses were recorded on weekly basis. When a calf was treated for an illness it was listed as a single treatment eventhough the treatment may have been repeated for more than 1 day in the same week. At Michigan State, blood samples were taken from the jugular vein for immunoglobulin and plasma protein analyses at 24 and 48 hours after arrival of calves. Serum immunoglobulin (zinc sulphate turbidity) and 43 protein (macro-Kjeldahl) were determined, respectively, as described by Pfeiffer et a1. (1977) and A.0.A.C. (1980). At Michigan State, jugular blood samples were taken 8 hours after the morning feed from four randomly assigned calves on each diet when 3 and 6 weeks old. Blood was processed and analyzed for plasma amino acids as described by Foldager et a1. (1977). The xylose absorption test was used at Michigan State State to evaluate intestinal absorptive capacity of calves. Xylose tests were performed for all 62 calves at the termination of the feeding trial. Calves were fasted for 24 hours before administering xylose via nipple pail at .5 g/kg of body weight in a 10% aqueous solution. Jugular blood was sampled just before and .5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 and 5.0 hours after xylose ingestion. Plasma xylose was analyzed by the orcinol/ferric chloride spectrophotometric method as described by Seegraber and Mbrrill (1979). Except for plasma amino acid concentrations, xylose absorption capability, and mortality, all variables were analyzed as completely randomized designs. Plasma amino acid and xylose absorption capability were analyzed as split-plot designs (treatments as plots and time as subplots). Mortality data were analyzed as contingency tables. F-test was used when only two means were compared; tests involving more than two means were as indicated in the table footnotes. Analysis of variance for all variables in this experiment are shown in Table A.l (Appendix) and were processed as described by Gill (1978a, 1978b, 1978c). 44 Experiment 2 This experiment was a digestibility trial conducted at Michigan State University from October 26 to December 13, 1979, involving diets 1 to 4 from experiment 1. Our objective was to determine differences due protein sources in apparent dry matter and organic matter digestibilities and nitrogen retentions. Calves (4 per treatment) were placed on experiment between 3 and 8 days of age and were fed and managed similarly to trial 1. Blood samples were obtained at calve's arrival for plasma protein determinations. All animals were kept in metallic metabolism cages for 2 weeks. Calves were weighed at the beginning and end of the trial, and fecal consistencies were rated from 1 to 4 (Larson et al., 1977). Rectal temperatures were taken daily just before the morning feeding. During the last 5 days of treatment, feces and urine were totally and jointly collected, weighed and homogenized in a blender. Composite samples of the 5-day collections were made by mixing 10% of each day's excreta. Samples were kept at -20°C until analyzed for dry matter (forced- air oven at 65°C), ash (for organic matter), and nitrogen (macro-Kjeldahl) according to A.0.A.C. (1980). When a calf died prior to completion of treatment, the first calf available thereafter was used as a replacement. Mortality data were analyzed as contingency tables and all other variables as completely randomized designs (Table A.2) as described by Gill (l978a,1978b,l978c). Tests chosen for comparison of means are indicated as footnotes in each table. 45 Experiment 3 Thie experiment was conducted at the Dairy Research and Teaching facilities of Michigan State University from November 23, 1981 to January 25, 1982. The main objective was to study limestone as a poten- tial buffer in the small intestine of preruminant calves fed milk replacers in which 50% of the milk protein was replaced by soy protein concentrate. The experimental treatments were as follows: Treatment A: 19% CP; 19% MP Treatment B: As A, but containing .8% limestone Treatment C: 19% CP; 9.5% MP; 9.5% SPC Treatment D: As C, but containing .8% limestone A sample was taken from each 22.7 kg sack of milk replacer used and composited for dry matter (forced—air oven at 65°C), crude protein (macro-Kjeldahl), ether extract and ash determinations as described by A.0.A.C. (1980). Ingredient composition of milk replacers is shown in Table 6, and chemical composition in Table 7. Sixteen male Holstein calves were purchased at 3-4 dayscflfage from a commercial dairy farm and randomly allotted to treatments. Initial care of calves after arriving at Michigan State was the same as Trial 1. For 42 days each calf received its designated milk replacer as the only source of nutrients at 8, 9, 10, 11, 12 and 12% of body weight from weeks 1 to 6, respectively. Total solids of all milk replacers after mixing with water was 14%. Replacers were offered to calves twice daily (12 hr apart) in open pails. Fresh, clean water was available at all times. Animals were kept in individual pens bedded with straw except 46 TABLE 6. Ingredient composition of milk replacers (%) (Experiment 3) Ingredients Diets A B C D Dried whey 19.5 19.34 19.5 19.34 Sodium caseinate 8.2 8.13 ---- ----- Skimmilk 20.3 20.14 14.0 13.89 7/60 High fat mixturea 33.3 33.03 33.3 33.03 Soy protein concentrate —--- ----- 14.2 14.09 Dextrose 17.4 17.26 17.4 17.26 Premix(minerals and vitamins)b 1.37 ' 1.36 1.37 1.36 L-lysine ---- ---- .09 .09 DLdmethionine ---- ---- .15 .15 Limestone ---- .80 ---- .80 100.06 100.01 100.01 TOTAL 100.07 aSpray-dried mixture prepared to contain 7% milk protein and 60% fat (lecithin included). bContaining (per kg) 33,000 IU vitamin A, 6,600 IU vitamin D, and 22 mg vitamin E. Diets l and 3 were calculated to contain 1% calcium, .7% phosphorus and .1 ppm of selenium. TABLE 7. Chemical composition of diets3 (Experiment 3) Items Diets A B C D Dry matter (%) 96.39 96.58 96.43 95.64 Crude protein (%) 18.53 18.15 18.42 18.33 Ether extract (%) 13.24 14.21 14.17 14.25 Ash (%) 6.52 7.06 6.44 7.12 8Crude protein, ether extract and ash are presented on dry matter basis. during collection periods when they were placed in metallic metabolism cages. 47 Animals were weighed individually for 3 consecutive days at the beginning and at the end of the trial and at weekly intervals during treatment. Scour scores and rectal temperatures were recorded as described in trial 1, except that rectal temperatures were taken throughout the entire 6 weeks of the experiment. Calves were moved from individual pens to metabolism cages on days 14 and 35 of experiment. If a particular calf showed diarrhea or was under medication at the beginning of the third week, its transport to the metabolism cage was postponed for 1 week. For this reason, the average age of calves at the beginning of the first collection period was 17, 20, 19 and 20 days for treatments 1, 2, 3 and 4, respectively. Total feces and urine were collected in separate containers for 4 days at 7 AM. At collection, 10% of the total feces and 5% of the total urine were saved, compoSited and kept in the freezer at -20°C until analyses for dry matter (forced-air oven at 65°C), crude protein (macro- Kjeldahl), and ash (for organic matter) as described by A.0.A.C. (1980). Separate samples of fresh feces were taken for pH determination after dilution with distilled water. The xylose absorption test was performed in all calves on days 21 and 40 of treatment, exactly as described for experiment 1. Jugular blood samples were taken from each calf 5 hours after the morning feed on days 23 and 42 for plasma urea nitrogen determinations as described by Fawcet (1960). At the end of the trial, two calves, randomly chosen from each treat- ment, were sacrificed (by electrocution) at the Veterinary Diagnostic 48 laboratory of Michigan State University. After removal of the entire digestive tract, weights were recorded for liver, pancreas, rumen, abomasal, cecal and large intestinal contents. Small intestines were divided in three equal sections (from anterior to posterior) and weights were recorded separately for contents. Samples of all contents were kept in freezer until laboratory analysis for pH, dry matter, crude protein (macro-Kjeldahl), and ash as described by A.0.A.C. (1980). When a calf died prior to completion of treatment, the first calf available thereafter was a replacement. All dead animals were submitted for necropsy at the Veterinary Diagnostic Laboratory at Michigan State. Initial body weights were analyzed as a completely randomized design. All other variables were analyzed as split-plot designs. F-test was used when only two means were compared; tests involving more than two means were as indicated in the table footnotes. Analysis of variance for all variables was as described by Gill (l978a,1978b,l978c), and these analyses are shown on Table A.3 (Appendix). RESULTS AND DISCUSSION Experiment 1 Initial body weights were higher (P<.10) at Kansas (43.6 :16 kg) than at Cornell (42.1 1;.6 kg) or Michigan (41.0 :_.5 kg). This same order for weight continued throughout the experiment. At Michigan State, there was no difference (P>.10) between treatments in initial body weights or plasma protein and immunoglobulin levels (Table 8) of calves at arrival. Initial body weights were also not TABLE 8. Initial mean body weight, immunoglobulin and protein levels in plasma of calves at Michigan State (Experiment 1) Treatment No. of Initial Immunoglo- Plasma calves body bulin protein weight kg mg % % 1. 132 MP 9 41.1a 7.6a 5.03a 2. 191 MP 42.3a 7.93 5.233 3. 132 MP, 6% SDFS 39.7a 10.7a 5.41a 4. 132 MP, 6% SPC 42.6a 9.1a 5.26a 5. 131 MP, 32 SDFS, 3% SPC 40.4a 8.8a 5.333 6. 232 MP 40.1a 9.48 5.198 7. 132 MP, 102 SDFS 40.9a 7.5a 4.92a SEM +1.5 +1.2 10.18 aMeans in columns not sharing the same superscript are different at P<.10 using Tukey's test. different (P>.10) between treatments when data from all three locations 50 were analyzed together, being 41.9:.9, 42.3:.9, 41.3:.8, 43.Q:.9, 41.9:.9, 41.9t.8 and 41.5:.9 kg for treatments 1 through 7, respectively. These results suggest homogenity among treatment groups. Least square means of body weight gains for diets and locations are in Table 9. There was a difference (P<.05) among locations with higher gains at Cornell and Kansas than at Michigan. During the first 3 weeks of trial treatments 2 and 6 produced higher gains (P<.05) than all others. Treatment 7 gave the worst performance although not sig- nificantly different (P>.05) from 1, 3 and 5. During the last 3 weeks, treatments 2 and 6, containing higher amounts of milk protein, were again superior (P<.05) to all others. Treatment 4, containing 6% SPC, was inferior (P<.05) to 2 and 6 but superior (P<.05) to the negative control and to treatments containing SDFS. For the total period (0—6 weeks), treatments were in approximately the same order as for 0 to 3 and 3 to 6 weeks. However, an interaction (P<.01) between diet and location was observed, probably because diet 5 gave poorest gains at Michigan but relatively better gains at Kansas. Detrimental effects of SDFS seemed greater during 0 to 3 weeks when animals were more sensitive to diet quality (Huber, 1975; Roy et al., 1977). Among diets containing SDFS, treatment 5 in which 31% of the replaced protein was shared equally by SPC and SDFS showed the greatest weight gains, although they were not different (P>.05) from the negative control. As the amount of SDFS in the diet was increased, performance was reduced. Huber et a1. (1978) concluded that SDFS successfully replaced SPC at 8 and 16% of the total dietary protein. These results 51 TABLE 9. Least square means of daily weight gains (g/animal) for calves fed milk replacers varying in protein content and source by location, diets, and age (experiment 1) Age Treatments Cornell Kansas Michigan Mean SEM weeks 0-3 1 27 -22 -50 -15bc 26 2 59 160 -10 7o: 25 3 ~67 -19 - 5 -30bc 25 4 54 - 9 -29 5b 26 5 -16 77 -84 8 ° 26 6 65 186 60 104a 25 7 9 -98 -92 -60c 26 Mean 194117 39A+18 -303ii6 4-6 1 389 238 286 304d 28 2 597 605 478 5603d 27 3 361 394 288 345; 27 4 510 447 403 453 27 5 383 435 310 376c 27 6 529 664 482 558ad 26 7 352 296 375 341c 41 Mean 4464118 4398:19 3753:17 0-6e 1 208b° 108° 118° 145Cd 19 2 328a 383a 221ab 315a 19 3 151° 181b° 142b° 160Cd 19 4 282ab 219b 187ab 229b 19 5 184° 256b 104° 184c 19 6 297ab 425a 271a 3313 18 7 181° 99° 142b° 140d 19 Mean 2324112 2404113 1723111 aocuMeans in columns within each age not sharing a common superscript are different at P<.05 using Tukey's test. A3 Means in the same row not sharing a common superscript are different at P<.05 using Tukey's test. e Interaction between diets and locationsums significant (P<.01). were not repeated in the present work, two SDFS products. perhaps due to differences in the replacers containing fish protein were reported by others (Rupel and Negative effects on growth of young calves fed milk Wilson, 1962; Genskow et al., 1968; Gillespie, 1971; Sleiman and Huber, 52 1971; Huber et al., 1978). Digestibility of fish protein incorporatedinto milk replacers was 10 to 30% lower than milk protein (Huber and Slade, 1907; Sleiman and Huber, 1971; Huber, 1975). Ether extract digestibility was also depressed but carbohydrate utilization was unaltered (Huber and Slade, 1967). Roy et a1. (1977) realized that protein digestibility of non-milk sources was low, so diets were formulated with higher protein to compensate for the low digestibility. This rationale was the reason for diet 7, which contained 23% CP, with 10% from SDFS. Because calf growth was inferior and mortality higher on this than on diet 3 (19% CP with 6% from SDFS), we concluded that lack of digested protein was not the primary problem with diets containing high levels of SDFS. Retention of digested nitrogen was lower for calves receiving fish than milk protein, suggesting poor availability of essential amino acids (Sleiman and Huber, 1971). At all locations, lysine and methionine were added to fish and soy protein diets to equal concentrations of these amino acids in the respective milk diets. Diet and age effects on plasma free amino acids are presented separately in Tables 10 and 11 since the interaction between diet and age was not significant for most amino acids (Table A.1 Appendix). An increase in dietary CP from 13 to 19% in diets containing only milk protein resulted in higher (P<.10) plasma total essential amino acids (TEAA). Total nonessential amino acids (TNEAA), sulfur amino acids (SAA) and branched chain amino acids (BCA) and most individual amino acids also increased, but not significantly (P>.10). A further increase 53 TABLE 10. Effect of diets on plasma free amino acids (pmoles/lOO ml) for calves fed milk replacers varying in protein level and source (Experiment 1) Amino Treatments acids (1) (2) (3) (4) (5) (6) (7) 132 MP 192 MP 132 MP+ 132 MP+r 132 MP+ 232 MP 132 MP+ SEM 6% SDFS 6% spc 3z SDFS 102 SDFS +32 SPC No. calves 4 4 4 4 4 4 4 Thr 5.7b 13.8a 7.2ab 7.2ab 9.7ab 8.9ab 9.0ab 2.4 Val 11.2b 18.3ab 12.1b 14.6ab 14.5ab 20.0a 15.8ab 1.8 Met 1.8a 2.8a 2.2a 1.6a 1.7a 2.53 3.0a .4 Ile 4.6b 7.0ab 4.8b 6.4ab 4.9ab 9.2a 6.1ab 1.0 Leu 6.5b 9.8ab 6.6b 8.9ab 8.3ab 13.0a 8.5ab 1.3 Phe 4.8a 6.5a 4.6a 6.3a 6.03 6.6a 6.38 .8 Lys 5.6b 10.2ab 8.3ab 9.2ab 8.7ab 14.2a 12.93 1.7 His 5.0a 7.83 6.7a 5.9a 5.73 7.0a 7.13 .7 Arg 11.0b 15.7ab - 13.7ab 12.9ab 12.7ab 15.6ab 18.7a 1.7 SAAc 2.7a 4.2a 2.83 2.8a 2.88 3.6a 3.6a ,5 BCAAd 22.2b 35.0ab . 23.6b 29.7ab 28.7ab 42.3a 30.4ab 3.9 TEAAe 54.7b 92.4a 66.2ab 72.6ab 71.8ab 97.2a 87.3ab 9.0 TNEAAf 83.7a 119.7a 95.2a 89.9a 82.4a 101.2a 103.3a 9.3 N/Eg 1.5a 1.33 1.4a 1.2a 1.2a 1.0a 1.2a .1 a5Means in each row not Sharing a common superscript are different at P<.10 using Tukey's test. C Sulfur amino acids (sum of Met and Cys). d Branched chain amino acids (sum of Val, Ile and Len). e Total essential amino acids (sum of Thr, Val, Met, Ile, Leu, Phe, Lys, His and Arg). f Total nonessential amino acids (sum of Asp, Ser, Glu, Pro, Gly, Ala, Cys, Nle, and Tyr). 3 Nonessential:essential amino acid ratio. 54 TABLE 11. Effect of the animal age (weeks) on plasma free amino acids in young calves (pmoles/lOO m1) (Experiment 1) Amino acids . Age (week) 3rd 6th SEM (P <) No. calves 28 28 Threonine 8.8 8.8 .8 NS Valine 16.9 13.5 .7 .01 Methionine 2.5 2.0 .2 NS Isoleucine 6.8 5.8 .4 .05 Leucine 9.9 7.7 .7 .05 Phenylalanine 6.6 5.1 .3 .01 Lysine 12.3 7.5 .6 .01 Histidine 7.0 5.9 .3 .05 Arginine 15.8 12.8 .8 .05 SAAa 3.4 3 o 3 NS BCAb 33.6 27.0 1.5 .05 TEAAC 86.5 69.1 3.2 .05 TNEAAd 95.7 97.2 4.9 NS N/se 1.1 1.4 .1 .05 aSulfur amino acids (sum of Met and Cys). bBranched chain amino acids (sum of Val, Ile and Leu). cTotal essential amino acids (sum of Thr, Val, Met, Ile, Leu, Phe, Lys,. His and Arg). Total nonessential amino acids (sum of Asp, Ser, Glu, Pro, Gly, Ala, Cys, Nle and Tyr). eNonessential:essential amino acid ratio. 55 in GP to 23%, with only milk protein (diet 6), did not increase plasma TEAA, TNEAA, BCA, SAA or individual amino acids. Within diets containing 19% CP, higher concentrations for TEAA, TNEAA, BCA, SAA or individual amino acids were observed in calves receiving only milk protein, although differences were not significant (P>.10). The apparent decrease in plasma amino acids when milk was partially replaced by other protein sources could be due to their lower digestibility. On the other hand, calves fed high SDFS (diet 7) showed lower (P<.05) weight gains and higher mortality than those fed milk protein (diet 6), but plasma amino acid concentrations for both diets containing 23% protein were similar. These contradictory results are in agreement with Bergen (1979) who concluded that static measurements of plasma amino acids have limited value as sensitive indicators of protein status because they do not reflect the magnitude of amino acid fluxes in and out of free amino acid pools. The amino acid profiles obtained in the present experiment do suggest, however, that amino acid imbalance in SPC or SDFS diets is unlikely to be the major factor inhibiting growth. The lower concentrations of plasma amino acids at 6 weeks (Table 11) might be explained by a greater utilization, reflected by faster weight gains during this period compared to 3 weeks of age (Munro, 1970). Poor results with fish protein in some experiments were associated with vitamin E deficiency precipitated by residual fish oil, which consists largely of polyunsaturated fats (Makdani et al., l97lc; Michel et al., 1972). Necropsy results at Michigan State of calves failing to complete the experiment (Table 12) showed one case of white muscle 56 .=c«u«v=oo Hmucmawummxm poms: mamom ail.“ snooze Hammaoam we cowumuooas “vocNEuououca Nm Hm N maeoesmcdogocoum mm mm N owcoaaocaonocoum o .NN N Ham: Hmmmaoam mo snowman N NN n pocwsumuocaa NH :m N manganese "Hams HmmmEonm mo 0.2595 2 :m N macoasmca osoawunwm musomnsm N :m N mac axes: N :053 mod coxoum I m N manoaaouaoummm NH ea c mucossocaoaoaoum HN ow m ommomao saunas saws: unmaam oN an m >uauaxou Hoodoo ou=o< «H mm c owcoasmcaonocoun o>wumusaasm ofisouso ouo>om N am N mwcoaoocd evocaunfim ousomnom OH .mq N macoasosm use mouuuouco ousomnaa mam ouso< N we N vmaaahoummna m mm H vocwauouooca NN on H bM%mv .0: Seasons no sumac mo mmamo numon uamo nown AH uaoaauoaxmv snoop no mamunoue condenses: On one oumum somwnofiz um ucmswumdxo Scum vouuwao m0>~mo .NH mam<9 57 disease for treatment 5 (containing SPC and SDFS). Of seven dead animals fed the replacer containing 10% SDFS (diet 7), three had ulceration of abomasal mucosa, two of them showing rupture of the abomasum wall. It is doubtful that a high level of polyunsaturated fatty acids was the main reason for poor growth on SDFS in this experiment because of the inconsistency of the results. For instance, treatment 3 containing 31% of the dietary protein from SDFS resulted in no animal losses, and treatment 5, also containing SDFS, resulted in only one of 11 calves which was diagnosed with white muscle disease. Moreover, there were none on treatment 7 which had highest SDFS. Least square means for dry matter and protein intakes and for feed efficiency are in Table 13. Dry matter intake was higher (P<.05) for diets containing high milk protein and 6% SPC. Intakes were lower for the negative control and diets containing fish solubles. Differences . among treatments in dry matter intake were from the feeding criterion, which adjusted weekly the amount of replacer fed to each animal to body weight of the preceeding week. As expected, crude protein intake was higher (P<.05) for diets con- taining 23% CP, intermediate for 19% CP, and lowest for the negative control. Differences among treatments within each protein percent were mostly due to dry matter intakes. Feed efficiencies, in terms of dry matter or crude protein, were higher (P<.05) for diets containing high milk protein (treatments 2 and 6), intermediate for the SPC diet, and lowest for negative control and diets containing SDFS. This suggests poor utilization for animal growth of nutrients from fish solubles. 58 .umwu m.>oxsa wean: mo.vm um ucouowm«v one uawuuauoasm coEEoo o wawuosm uo: sou oEom ecu cu memoZNovono aa.neo.a eeaaa~.~ ea. em.a ea. mm.a eaaeee.a ea.aoe.~ as“ ma.a axaaaa ao\eaam sense: we on on mo.em~. mo.amm. no.6om. momamm. no.6mm. noaaoa. no. can. daaeea za\aanm named: m name m need m mamas m edema m daze m dmaa m use Aaae\ea\wv menace ao ea amen ma amoe as semen ma mean ma seem as aeoe as soon Aaan\ea\wv eaaeea 2e Na mN em eN em SN MN aaaaaea .62 2mm m 2mm m 2am x 2mm m 2mm m 2mm m zmm m 0mm Nm+ mean Noe . mean am can no mean no +mz «mg m: NMN +mz «ma +m: NMH +m= RNA at RNA at NmH A3 A3 A3 A3 A3 ANV 3V mucoaumoue moanmfium> Aa ueoaauoaamv coauoooa one mafia uo>o voweuo>n season one acoucoo ewououm Newhum> mo enouoaaou xawa com mo>Hmo you aocowowumo room one .moxmucw Amov :fiouona ensue new Azav nouuoa hum mo memos season ammo; .mH mqm¢e 59 Xylose transport in the intestine is similar to glucose (Levitt et al., 1969), but normal levels in blood are much lower (Seegraber and Morrill, 1979). Hence, xylose uptake into blood after feeding a test meal has been used to evaluate malabsorption in the proximal intestine (Levitt et al., 1969; Seegraber and MOrrill, 1979). Plasma xylose concentrations for the different sampling times are in Table 14. They increased to a peak 2 hours after feeding xylose and then decreased after 3 hours. Large variations in xylose patterns for the different diets were observed, but no consistent trend was related to protein source. These results contrast with those of Seegraber and Morrill (1979) who concluded from their xylose uptake tests that absorptive capability was lower in calves fed soy protein concentrate than milk protein. Sissons and Smith (1976) also stated that calves fed heated soybean flour developed a severe disturbance in digestive function probably due to an allergic reaction in the gut. As mentioned, autopsy results from dead calves fed treatment 7 (containing 10% CP from SDFS) Showed necrosis and even rupture in the digestive tract. Hence, it is plausible that high SDFS impaired intestinal absorption, although the xylose test did not indicate a malabsorption syndrome. Plasma xylose concentrations from 2 to 5 hours after ingestion were lowest for diets 2 and 6, which contained only milk protein. The data suggest that animals fed inferior protein Sources or low protein diets have greater difficulty in clearing xylose from their blood which may be due to a slower anabolism of ingested nutrients. It is also possible that less efficient kidney clearance might be part of the broad syndrome (higher rectal temperatures, diarrhea and consequent lower weight gains) 60 .m.e~fl.n memos ucosumouu new mom c.1fl n memos hausc: new nouns unmvcmum .mfimmamco some enemas coaumuucoocoo mmoazx :ofiumuu Imficfiscmnumom scum momentum mums Ac mafia umv memmfim ca ccwumuusoocoo smeazx coaumuumfi=HEomnoum . .umou m.>oxse w wswms o~.vm um ucoummmwb who mafiuomuoasm coesoo o wcwumzm no: :Esfioo mama go 30» same cw mcooZMbomm mN.Nm 0%.2: amm.mNH ncméNH mmeH 0%.moH magma: one.“ macaw: mN.NNH N.N~H m.eNH m.mmH ~.me m.ec~ o.mmH «.mNH N.Hm m mmam NOH .mz Nma .N mm.NoH m.NN w.mod N.oNN a.NNH m.oma m.m- N.mHH H.mm m m3 NMN .o as.m¢a a.aa m.aaa m.wma m.ao~. c.6ma o.mm e.ea a.aa a cam Na .maam Nm .ez NMH .m mm.m- o.Nm o.cH~ H.MMH N.~¢H m.oe~ m.mma m.q- q.m¢ .m cam Re .mz NNH .q m~.mm N.¢m m.mm m.NoH o.No~ m.ma~ o.ao~ m.~oH .N.m¢ m whom No .mz NmH .m mm.mm N.we o.o@ a.ao~ o.w- w.qNH N.mNH o.em m.~q m m: Nma .N mN.cm N.moH o.MHH a.~N~ N.HmH m.oN~ m.HNH m.ow m.mm m m: Nma .H wmcmoz : m a e n m a m.N n N ; m.~ ; a s m. mo>Hmo Amemmam mo Hu\wevmcofiumuucoucco omcdmx «0 .oz mucosummna AN ucoawuoaxmv season was Hm>oH :fiououa :fi wofimum> muoomfieou waE mom mo>~mo now unwfioz anon wx\omo~zx w n. no snow Hmuc cm umumm Aaw\wev cowumuucoocoo omofimx mammaa owmum>< .cm mgm.05) between diets for treatments of sicknesses at Cornell and Kansas. At Michigan,animals receiving diet 7 required more(P<.05) treatment than other diets. The higher (P<.05) fecal scores and treatments for sicknesses during the first 2 or 3 weeks compared to the last 3 or 4 weeks Show the greater sensitivity of the very young calf to illness. Least square means for rectal temperatures during the first 14 days of trial are in Table 16. During the first week, calves fed diet 7 had higher (P<.10) rectal temperatures than those fed diets l and 3, 62 Least square means of fecal score and treatments for sickness TABLE 15. for calves fed milk replacers varying in protein content and source by location and diet8 (Experiment 1) Cornell Kansas Michigan Fecal Score Treatment d d c 1. 13% MP 2.1 f 2.2d 1.5d 2. 192 MP 1.6e 1.9 e 1.2 3. 132 MP, 6% SDFS 2.8; 3.1:e 1.4°g 4. 132 MP, 6% SPC 1.4de 1.9de 1.3: 5. 13% MP, 3% SDFS, 3% SPC 1.8de 2.0e 1.5d 6. 23% MP 1.9b 1.8b 1.1b 7. 13% MP, 10% SDFS 3.7 3.6 1.9 SEM 1.06 1.08 1.08 Week on Diet 1 2.6: 2.3; 1.5: 2 2.7 2.8b 1.6b 3 2.0c 2.5 c 1.5 4 1.8c 2.1c 1.4bc 5 1.9C 2.3c 1.3t 6 1.8C 2.3c 1.3c SEM _-l_-.O6 :.08 :.06 Treatment Tgeatment for gicknessh c 1. 13% MP .27b .22b .51c 2. 19% MP .22b .10b 28c 3. 13% MP, 6% SDFS .31b .14b 39c 4. 13% MP, 6% SPC .25b .26b .31C 5. 13% MP, 3% SDFS, 3% SPC .25b .21b .46c 6. 23% MP .27b .08b .24b 7. 13% MP, 10% SDFS .23 .04 .81 SEM :.04 1.06 _-4_-_.06 3Because of the significant (P<.01) interaction rations x locations, treatment means are compared within each location. deefMeans in columns not sharing the same letter are different at P<.05 8Feca1 consistency was rated from 1 to 4 (1 being normal feces and 4 being using Tukey's test. very fluid feces). hAverage days each calf was treated from an illness. 63 TABLE 16. Least square means of rectal temperatures for calves fed milk replacers varying in protein content and source by location, diet and week on diet (Experiment 1) ‘1' Kansas Week Treatments Cornell Michigan Mean SEM ii i ..aOC_ _ lst 1 38.67 39.01 38.11 38.60a .05 2 38.90 39.08 38.31 38.77 .05 3 38.69 39.02‘ 38.09 38.60 .05 4 38.90 39.13 38.10 38.718 .05 5 38.82 39.04 38.27 38.71a .05 6 38.94 38.97 38.21 38.71: .05 7 38.83 39.03 38.47 38.78 .05 Mean 38.82§1.08 39.048:.09 39.229t.08 2nd 1 38.83 39.52 38.37 38.91a .07 2 38.83 39.28 38.32 38.81: .07 3 38.76 39.13 38.21 38.70a .07 4 38.70 39.62 37.78 38.89a .07 5 38.60 39.32 38.27 38.73 .07 6 38.96 39.32 38.28 38.54: .07 7 38.77 39.27 38.38 38.81 .07 Mean 38.783:.04 39.354:.04 38.119:.04 abcMeans in columns within each week not sharing a common superscript are different at P<.10 using Tukey's test. ABCMeans in the same row not sharing the same superscript are different at P<.10 using Tukey's test.‘ but were similar to those fed diets 2, 4, 5 and 6. During the second week of trial there were no differences (P>.10) between treatments. Among locations, rectal temperatures were highest (P<.10) at Kansas,perhaps because of local factor such as environment or management. Rectal tem- peratures were higher (P<.10) at Cornell than Michigan, but temperatures at all locations were about normal. Mbrtality of calves at Michigan State is shown in Table 17. The hypothesis of independence between diets and death cases may be rejected with 90% confidence. The analysis forpairs of treatments, in 2 x 2 64 contingency tables (by Bonferroni chi-square) indicates that mortality in treatment 7 was higher (P<.10) than in treatments 3, 4 and 6. If replace- ment calves are not considered as "alive calves" on Table 17, the calcu- lated value for "q" changes from 10.8 to 18.0. In this situation, mor— tality in treatment 7 is clearly different, even when compared to treat- ment 2. Across all locations, mortalities for diets 1 through 7 were l4, l4, 8, 17, 11, 4 and 30%. About 90% of the deaths occurred during the first 3 weeks on treatment. These data indicate that 10% SDFS is excessive for inclusion in calf milk replacers. This statement is supported by growth, feed efficiency and fecal score data. In conclusion, spray-dried fish solubles are inferior to milk protein or soy protein concentrate for inclusion in milk replacers for baby calves. The data further suggest that high fish solubles aggravate the health of the young calf. TABLE 17. Mortality of calves at Michigan State fed milk replacers con- taining different protein levels and sources (Experiment 1) Treatments Number of calves Dead Alive Totals 13% MP 2 9 11 19% MP 3 9 12 13% MP, 6% SDFS 0 9 9 13% MP, 6% SPC 1 9 10 13% MP, 3% SDFS, 3% SPC , 2 9 ll 23% MP 1 9 10 13% MP, 10% SDFS 7 8 15 TOTALS 16 62 78 The hypothesis of independence between treatments and death cases is rejected (q I 10.792; x2, 0.1, 6 = 10.64). 65 Experiment 2 Average initial body weights and plasma protein levels when calves arrived at Michigan State were similar (P>.10) between treatments (Table 18). Protein source affected (P<.10) weight gains during the 2 weeks of treatment (Table 18) with animals on diet 3 (6% protein from SDFS) showing lower gains(P<;10) when compared to animals fed diet 2. These data are in general agreement with those reported in Experiment 1 and by other workers (Rupel and Wilson, 1962; Gillespie, 1971: Sleiman and Huber, 1971; Ternouth et al., 1975). There was no difference (P>.lO) between treatments in rectal tem- peratures and all temperatures were within the normal range (Table 18). Scour scores were highest for diets containing SDFS and SPC, although not different (P>.10) from the negative control (Table 18). An impair- ment in milk coagulation'(Tagari and Roy, 1969; Paruelle et al., 1972), followed by faster passage of undigested material through the lower gut (Colvin et al., 1969; Ternouth et al., 1975), might explain the higher incidence of diarrhea in calves fed non-milk protein which was observed in this and other studies (Gorrill and Thomas, 1967; Seegraber and Morrill, 1979). Apparent dry matter and organic matter digestibilities were highest (P<.10) for diet 2 (19% CP as milk protein) although not different from the negative control (Table 18). Similar observations were made for apparent nitrogen retention. These results agree with those reported by other workers (Matre, 1970; Sleiman and Huber, 1971) and partially 66 Imam who aN ucoaumoan .umou m.uuoccsn wean: OH.vm um ucouow Houucoo ecu Lows unapompmeom mama mnu wcwuosm ac: Sou oEmm mnu ca mcoozUpm c.H AH. 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Sansone no snoop no shown some: me0 ucoeumone AN usoannoaxmv sumac no mEoHnona vouoonxmss on one ucoEnnoexo Eonw vaunHEo mo>~mu .ma mqm.10) for any variable listed. Initial body weights were similar (P>.10) for treatment groups. Replacement of 50% of the milk protein with SPC resulted in 20% lower average daily gains. This difference was not significant (P>.10) probably due to the large variation and small number of animals within treatments. Since the initial body weight of calves fed SPC was slightly higher and observed average daily gains were lower, an analysis of covariance using initial weights as the covariant was realized. In this comparison, the F-test did not indicate significant differences among diets. Dry matter and crude protein intakes were not significantly affected (P>.10) by protein sources. Although not significantly different (P>.10), feed efficiency was higher for calves fed only milk protein, probably 70 TABLE 20. Mortality of calves at Michigan State fed milk replacers containing different levels and sources of protein (Experiment 2) Treatments Dead Alive Totals No. of calves 1. 13% MP 0 4 4 2. 192 MP 0 4 4 3. 132 MP, 6% SDFS . ' 4 4 8 4. 132 MP, 6% SPC 0 4 4 Totals 4 16 20 The hypothesis of independence between diets and death cases if reject- ed (q - 7.50;;(,2 .1, 3 - 6.25). because of higher weight gains during the experiment. Likewise, fecal scores and rectal temperatures were not affected (P>.10) by SPC incor- poration into milk replacers. TABLE 21. Influence of protein source and limestone on daily weight gain, intake of nutrients, feed efficiency, fecal score and rectal temperature of baby calves (Experiment 3) Variables ' Protein source Limestone Milk Milk+SPC Present Absent SEM Initial body weight (kg) 38.03 40.78 40.7a 38.0a 1.7 Weight gain (g/an/day) 275 a 223 a 250 a 247 a 41 DM intake (g/an/day) 563 a 558 a 563 a 558 a 33 CP intake (g/an/day) 107 a 107 a 106 a 108 a 6 Weight gain/DM intake .498 .40a .44a .44a .06 Fecal dry matter (7.)C 23.983 23.808 24.70a 23.08b .58 Fecal scores 2.2a 2.0a 2.2a 2.03 .1 Rectal temperature (00) 38.93 38.98 38.98 38.98 .1 abMeans in rows (within protein sources and limestone) not sharing a common superscript are different at P<.10. cAverage dry matter in feces sampled during 4 days when calves were 3 and 6 weeks old. 71 The addition of .8% of limestone to milk replacers did not signifi- cantly change (P>.10) weight gains, intake of nutrients, feed efficiency or rectal temperature (Table 21). However, fecal dry matter of samples obtained when calves were 3 and 6 weeks old were higher (P<.10) in calves receiving limestone. The same tendency was observed in fecal score, although the difference was not significant (P>.10). The effects of protein source and limestone on the apparent digesti- bilities of dry matter, organic matter and crude protein, and on apparent crude protein balance and plasma urea nitrogen are in Table 22. Once again the interaction of protein source x limestone was not significant (P>.10) for all above mentioned variables. TABLE 22. Influence of protein source and limestone on the apparent digestibilities of dry matter (DM), organic matter (OM), crude protein (CP) on apparent crude protein balance and plasma urea nitrogen of baby calves (Experiment 3) Variables ! . Protein source Limestone Milk Milk+SPC Present Absent SEM Apparent DM digestibility (2) 87.648 85.04b 85.20a 87.48b .93 Apparent OM digestibility (2) 87.803 85.953 86.35a 87.403 1.13 Apparent CP digestibility (2) 85.243 76.81b 79.53a 82.518 1.87 Apparent CP balance (g/day) 66.37a 59.68a 59.99a 60.068 6.54 Apparent CP balance (2 of ingested) 50.20a 44.95a 46.54a 48.618 3.42 Plasma urea N (mg/100 ml) 5.63a 6.06a 5.35a 6.33a .71 5bMeans in rows (within protein sources and limestone) not sharing a common superscript are different at P<.10. The substitution of 50% of the dietary protein with SPC resulted in significant (P<.10) reductions in dry matter and crude protein digesti- bilities. Apparent crude protein digestibility was 10% lower when SPC 72 was used. Organic matter digestibility was also reduced, although not significantly (P>.10). Similar results were obtained in experiment 2 and by other workers (Nitsan et al., 1971,1972; Ternouth and Roy, 1973; Pejic and Kay, 1979). Lower nutrient digestibilities may explain in part, the lower weight gains (Table 21) of calves fed SPC. Apparent crude protein balances, expressed either as g/day or as percent of the protein ingested, was 10% lower in calves fed SPC than only milk protein. Although this difference was not significant (P>.10), it is a reflection of the depressed protein digestibility. Plasma urea nitrogen (Table 22) was only slightly higher (P>.10) for calves fed SPC than only milk. Limestone in the replacer slightly reduced nutrient digestibility and nitrogen balance (P>.10). Neither was plasma urea nitrogen signifi- cantly affected (P>.10) by limestone incorporation. The interaction treatments x week was not significant (P>.10) for any variable in Tables 21 and 22, suggesting that there were no specific ages at which effects of SPC or limestone, or both, were greater. Age effects on weight gains, intake of nutrients, feed efficiencies, fecal consistency and pH, rectal temperature, apparent nutrient digesti- bilities, apparent protein retention, and plasma urea nitrogen are in Table 23. As expected most variables were affected (P<.10) by age. Weight losses during the first 2 weeks of treatment emphasizes the importance of good management and nutrition for these young and sensitive animals in order to avoid high mortality. Gains improved (P<.10) with age, but the relatively low gain observed in the sixth week was due to 73 .umou m.%mxsh wane: OH.vm um ncmnmmwnv one unnnomnmosm seem ecu wcwnonm no: mean same man no name: mono mm. acm.m llllllllll mmN.N IIIIIIIIII AHE ooH\wEv 2 men: mammam No. moo.N lllll III-1 amm.c IIIIIIIIII mo Hooom om.H on.qm IIIIIIIIII nmm.oe IIIIIIIIII Aboumowcn mo NV mocmnoa mo uaonmoe< Hm.m oem.Nw III-1 III-I Amm.Ne IIIIIIIIII Ahmm\wv commas: mo unonmaa< mm.H mNo.Nm lllll Ill-I mmo.cm 1111111111 ARV hunannnumownp mo noonmae< Nm. mNm.em IIII- III-l mNm.em 111-- all-I any aunannaumomnv zo neonmem< am. eao.mm --- --- aam.nm ----- --- Ana scannennaawne so neanaaaa n. aa.am so.am ao.an aa.mm an.an aa.wm Aocv ansnanaaEan nausea N. mH.N mm.~ m¢.H mN.N mH.N mm.N monoom Hmoom an. ann.m~ --- --- «No.4N --- d-- any nausea ant Haven en. ems. new. name. ans. ccn.- o-.- dampen zo\anaw pawns: N aamn aamn emnn use can can Aaan\ca\ae banana no an amnn sack amen same names race Ansn\ca\av assess an we ocean «Nae peace ooON vans mama Ahmv\=m\wv snow newnoz 2mm 0 m e m N H axes: moanmwnm> Am unmannoexmv mucownusa mo monsoa Name may as mnoomamon xana vow mo>~mo memo» mo cowonuun mono mammae vcm monocouon cmwonun: uconoaom .monunHunHuoowav unownusc uaonmaam .onsumnanon Nonoon .mn Hoomm .onoom Hmoom .hoaononumo boom .oucownusn mo oxmuafi .cwmm unwwoz so own no nooumm .MN mam.10) by age. Although fecal scores tended to decrease with age, they were higher during the collection periods. Rectal temperatures were not affected (P>.10) by age, all being within the normal range. Apparent dry matter digestibility was lower (P<.10) in older animals (6 vs 3 weeks of age). Instead of decreasing, dry matter digesti- bility was expected to remain the same or increase with age. Apparent crude protein retention was significantly (P<.10) higher for older animals, which supports the higher weight gains in older animals. The higher plasma urea nitrogen (PUN) at 3 than 6 weeks (P<.10) agrees with weight gains and crude protein retention. The higher PUN at 3 weeks might be associated with greater catabolism of muscle protein as reported by Leibholz (1970). As calves became older, protein intake increased and digestibility did not change, but crude protein retention increased,apparently resulting in less gluconeogenesis. Fecal pH was higher (P<.10) in older calves and may be related to less fermentation in the large intestines due to more efficient nutrient absorption. The xylose absorption tests support this hypothesis. 75 Treatment effects on plasma xylose concentrations averaged for age and time of sampling are in Table 24. Analysis of variance (Table A.3) indicates differences (P<.10) for sampling time and age, and for the interaction between treatment and age. Differences due to time of sampling was expected. Xylose concentrations in plasma tend generally to increase up to 1.5-2.5 hr after ingestion and then decrease as observed in Experiment 1. In this experiment, plasma xylose concen- trations (mg %) were: 82.2, 143.7, 162.0, 166.8, 163.5, 153.0, 123.2, and 112.9, respectively for .5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 and 5.0 hr after xylose ingestion. The standard error of these means was 18.2. The interaction between treatment and age may be explained by a differential response to limestone within each protein source for each age (Table 24). When calves were 3 weeks old, average plasma xylose dropped from 134.6 to 96.5 mg 2 when limestone was added to replacers containing only milk protein. At this same age, plasma xylose concen- trations did not change (138.2 vs 144.8 mg %) due to limestone addition to SPC diets. On the other hand, when calves were 6 weeks old, plasma xylose was slightly (151.2 vs 165.8 mg %) increased when limestone was added to only milk protein. Results for limestone incorporation into SPC diets at 6 weeks were similar to those observed for younger calves. Another way of understanding this significant interaction is shown in Table 25 where sampling times are combined and means comparing protein source and limestone addition at the two ages are given. When animals were 3 weeks old, the partial replacement of milk protein by SPC resulted in higher (P<.05) plasma xylose which exactly contradicts the data reported by Seegraber and Morrill (1979). On the other 76 .umou m.%oxse wane: Ho.vm no uaonomwwm one uennomnoeam cosEoo m wcwnmnm nos camaoo oEom ozu cw memozU .umou m.uuoc==a wane: mo.vm no econommnv one nonnomnoosm ooEEoo o wonnonm no: m3on an memo: connonumwonEoolumoa :ooo m< n .onohnoao sump oncNon oofiuonnooocoo omoH%x Eonm pouosvov ono3 Ac oEHu uov mEmoHa an mcowumnucoocoo omoaxx :onumnuwncasmolonmo N.$fl mm.mq~ e.NNH «.mNH «.mqn m.NoH «.mmn N.MNH N.N©H o.w¢ memo: m.nmre.men N.wmflnm.nea m.oHH o.mNH e.mNH m.mo~ w.qu w.omH «.mmn m.NoH m occumoefin+omm .e m.mwom.cmn N.mH.ne.emn e.¢NH a.No~ m.mHN N.mmH «.moa H.oNH m.mm~ m.Na m 0mm .m w.nHAN.Nmn N.mH.nN.moH H.omH o.wmu H.maH o.eNH m.¢o~ N.NNH m.om~ H.om m ocoumosna+xanz .N m.n+nm.NcH N.m+ AN.NmH m.om m.mNH N.o¢~ o.HNH m.em~ m.oma. N.mmH o.ooH m xanz .N memos coo: : o.m : o.¢ a o.m 5 m.N : o.N n m.H a o.H : m. ucoauoonh . xooa @ mo>Hmo Anamoam no as oon\wev oconuonncooaoo omonmx mo .02 mucoEnoona Hawaiian ~53 on: .22 4.4.: 3.: .462 flofi mes asap: New are: o.-n at: 2:2 is: on: teen teen can m 2335358 ... N.wHUnN.mmH m.m- N.Hma H.mNH ¢.HNH N.¢¢H ¢.m¢H m.mNN e.mm m 0mm .m N.mH on.om H.mN m.mw o.QNH N.NHH N.¢NH o.oHH N.ow m.am m ocoumoEaH+anz .N N.w+ono.ema m.m¢. m.oHH m.mna c.~oo Anemone mo HE can\wav moonuonunooaoo omofimx mo .02 mucoEumonH Am ucoaqnooxmv moonooe enouono.cn wcwhno> onoomaaon xfiwe mom oo>~oo now .uzwaoa anon mo mx\omoH>x w m. mo ooou ”one so nouwm ads ocH\wav anonuonucooooo oaouhx mamoao owono>< .QN mqm<9 77 hand, when animals were 6 weeks old, SPC incorporation in the diet tended to reduce plasma xylose concentration, although not significantly (P>.05). TABLE 25. Average plasma xylose concentrations for treatment combination means of protein sources and limestone with calve's age (Experiment 3) Treatments Calve's age 3 weeks 6 weeks Milk protein only ' 115.5b2 158.5Cl c1 c2 Milk protein + SPC 141.5 138.1 No limestone 136.4C1 142.8C1 Limestone 120.7b2 135.8Cl aStandard error for treatment combination means =.:5.8. bcMeans in the same row not sharing a common letter superscript are dif- ferent at P<.Ol. 1’ZMeans in columns (within protein sources and limestone) not sharing the same numerical superscript are different at P<.Ol. More interesting, however, is the effect of age on protein source. As animal becomes older, average plasma xylose concentration increased (P<.05) in calves fed milk protein but not those fed SPC. This observation might lead to the conclusion that absorption in the small intestine does not improve with age in calves fed SPC, but does when only milk protein is fed. However, interpretation of averages of plasma xylose concentrations obtained in a 5 hour period after xylose ingestion are not that simplistic. For instance, higher plasma xylose might result from a greater capacity for absorption or from an impairment in clearing of xylose from blood. One way for interpreting treatment effects on xylose absorption 78 would be through a curve obtained as the function of time after xylose administration. Bolton et al.(1976) and Hill et a1. (1970) suggested that abnormal xylose absorption curves would be characterized by a low peak, a delayed peak, or a flat curve with no distinguishable peak and no marked decline. Even with such criteria, the same restriction listed above persists. Perhaps the best way of interpreting these data is to consider responses during the first 2-3 hours after xylose ingestion as the absorption pattern characteristic, and the behavior during the last 2-3 hours as an indication of xylose clearing from plasma. Figure 1 shows that the asCending portion of the xylose absorption curves for both protein sources were similar. These results agree with those of Experiment 1 when SPC replaced 31% of the milk protein but contradict with those of Seegraber and Morrill (1979), who showed flat curves when SPC was included in the replacer. The addition of limestone to the replacer did not change the xylose absorption pattern (Figure 2). It is interesting to note however, SPC and limestone resulted in higher xylose concentrations 4 to 5 hours after ingestion. Figures 3 and 4 illustrate the already discussed interaction between treatment and age. - When calves were 3 weeks old, SPC presence and limestone absence in the replacer resulted in slightly higher plasma xylose after peak concentrations were reached. When animals are 6 weeks old, the opposite trend was observed. Figure 5 suggests that xylose absorption capability is higher (P<.05) in older calves. A similar conclusion was made by Seegraber and Morrill (1979) and agrees with higher weight gains, feed efficiencies and lower 79 .Amn.~HH.I 2mmv OH.vm no uoonomuno onm nouuoa oEmm och wcw3onm no: nuance .nsc: sumo no; .AI-Iv enouone zoo no Alli sane wancnmucco mnoomnoon anE pow mo>Hmo we oEmmHe on cenumnucoocco omonax coo: Anne sane o6 o2» o.m m.~ arm n; o; p n _ . n L p .H mmDUHm AA IV" om . OHH . CNN r and a CNN r can a o: (z 8m) °ou03 asoIAx emsera 80 .Awm.Hnfl.u :mmv OH.vm no uconouwnm ono nouuoa oEom one wcn3oam no: mucnoo .nscc Loco nom .oCCuuoEnH AIIIV uoozuns nc All-V sun3 mnooofiaon xmne pom mo>~co we mammao on :cnumnucoosco omoa>x coo: .N mmach Anne scan on as 9m m.~ o.~ m4 o4 m. h P b b P p b n A “v . CNN a omn 1 CNN r Goa r oHN 8m) 'ou03 asoIAX emseId % ( (mg Z) Plasma Xylose Conc. Plasma Xylose Conc. (mg %) 200“ 170'1 l40«4 110 - ' AAAJ v 81 fi~ 3 3 weeks 200- 170-4 140‘ llOJ 804 ”l .5 1.0 1.5 2.0 2.5 3.0 4.0 5.0 Time (hr) I ' r t I f T .5 1.0 1.5 2.0 2.5 3.0 4.0 5.0 Time (hr) FIGURE 3. Mean xylose concentration in plasma of 3 and 6 weeks of age calves fed milk replacers con- taining milk (-—-) or soy (-—-) protein. For each hour, points in each graph not sharing the same letter are different at P<.01 (SEM = :16.37). Xylose Conc. (mg %) (mg %) Xylose Conc. 200- 170‘ 140‘ 110: 82 3 weeks V T V I I I r i— .5 1.0 1.5 2.0 2.5 3.0 4.0 5.0 Time (hr) 200‘ If a a I ‘ 6 weeks 170- __3 “~.‘ a a 140 s_--_-_- 1103 I a a a 801 50 I 1 U I I I r l .5 1.0 1.5 2.0 2.5 3.0 4.0 5.0 Time (hr) FIGURE 4. Mean xylose concentration in plasma of 3 and 6 weeks of age calves fed milk replacers with (---) and without (-—-) limestone. For each hour, points in each graph not showing the same letter are different at P<.10 (SEM-= :16.37). 83 .334? u Emmy 89m on economic one nouuon oEcm onu wcw3czm no: menace .nsc; coco new .owm mo mxoo3 e no A-llv m nun3 mo>~mo mo oEmmHe on conuonusoocco omcH>x coo: .m mmson nose sane o.m o.q o.m m.N o.N m.H o.H m. P D P b I D D - AAAA t ‘v‘v om ON co can OmH OmH CNN oma oHN 'ouog asotxx emsetd (z 3m) 84 fecal pH at 6 than 3 weeks of age. Weight of contents from different sections of the digestive tract and size of liver and pancreas of calves selected for slaughter at 6 weeks are in Table 26. Size of pancreas in these calves are similar to those reported by Gorrill and Thomas (1967) except for calf 7550 which had an extremely heavy pancreas. Liver size tended to be heavier in calves fed limestone (23.2 vs. 21.5 g/kg body weight). Reasons for this are not apparent and the difference is not great. Gross examination of rumen mucosa showed little papillary development, typical of calves receiving only liquid diets. Dry matter, organic matter and crude protein contents, and pH values of digesta sampled in different sections of the gut are in Table 27. Data from Tables 26 and 27 were used to construct Table 28, which shows pH values and grams of dry matter, crude protein and organic matter present in different sections of the digestive tract of calves sacrificed 6 hours after a meal. Mylrea (1966a) and Porter (1969) observed thatgfllof abomasal contents 5 hours after a meal reached pre-feeding levels (1.0 to 2.0) in calves fed whole milk. In the present work, samples of abomasal contents obtained 6 hours after feeding showed higher pH values (3.10) as observed by Gorrill and Thomas (1967). Milk replacers containing skimmilk and whey (diets A and B) or SPC (diets C and D) may have resulted in less HCl production than whole milk, as proposed by Tagari and Roy (1969) and Colvin et al. (1969). The higher abomasal pH may in part explain the lower crude protein digestibilities obtained in this experiment (Table 22) than those reported by Noller et a1. (1956) and Jacobson et al. (1965) 85 .AESQoo o£u Cu omoaov .Amsnone ocu on omoaov I "l‘lllil- 01 II I I.“ -- ..r '1' ll- -..-l’. :1'7“-IIIIIQII 0' 0.1.1.. .ocnumouzn owned on» Eonm muSonscoo ocnumoucn HHmEm onu mo pnnnu pnnnu ozu Ecnm mucousco ocnumousw HNoEm osu mo mnnzu mccuom one Ecnm mucouaooo oznumousn Hausa ocu mo pnnsu umnnu ocu Ecnu mucouzcun .nonuowCuNm ozonuoom EsmoEolssasonuonlso52n onu acnw mucousouo o an. on. ms. or. - om. sm.n mo. an.~ so. so. so. no; noon ms\mv oaooocaa m.- o.- o.mm m.n~ o.n~ o.- m.m~ m.on o.n~ m.n~ s.- m.o~ as: soon ma\mv no>no nee can ems man man mnm new omm New osm nan was Ame oocoonco .oon amass son mam con man man nan oaa hem mam man man man Amvoocaoaoo .n.m ohm ppm nae mas mos aom one oom nmm nan ANN com mon own Ameoosoocoo .n.m one saw no as can nan mom or nan nnn man man one awn Amvoocoooco .n.m ohm nan new one son one can omN man one one «no can sn~ Ame scooeoo neonates oo4.~ eno.~ oun.~ mma.n osm.n mmm.~ nmm.~ oom.~ mom.~ oam.n cam.n oma.n Amvoonoocoo .o.a.e on as No as No as no on as mm on so Anne domes: eocm a - soon Name .m-. some omm-. m noon Oman u mean some-..- oaconoan + can can ocooaoeaa + ans: sen: ozosnmonh moanonnw> llll-‘ 1| 1.. III mnoooaoon sane Am usoannoome Housosnnoexo vow mo>Hmo mac xooz c No nounwomam no uzwnos was .omononoe use no>nH no onnm .uoonu Hocnnmoucuonumow one no ocowuoom neonouunv aonm mucouzoo mo.u:wno3 .oN mqmHoo one Ixoosle mo noonu Hocnuooucnonumow onu mo occnuooo economwnv Ecnu announce an enouono omsno moo nouuos oncmwno .nouumE %nu .ze .NN mqm.05). The interaction of protein source times the presence or absence of limestone was significant (P<.05), since limestone addition to replacers containing only milk protein decreased (P<.05) overall gut pH, whereas no changes (P<.05) were observed for calves fed SPC (Table 29). TABLE 29. Effect of limestone addition on overall gut pH of calves fed milk replacers containing only milk protein or half of dietary protein from SPC (Experiment 3) Limestone Protein source Milk SPC Absent 6.28a 5.86a Present 5.98b 5.91at abMeans in the same column with unlike superscript are different at P<.05. Large intestinal and fecal pH was lower (P<.05) in calves fed SPC than milk protein (Table 28). Lower digestibility of nutrients may have resulted in accumulation of more undigested nutrients in the large intestine (Gorrill and Nicholson, 1971), which created conditions for greater fermentation of the organic matter. Gastrointestinal disturbances have been associated with lower nutrient digestibility in calves fed milk replacers containing severely heated skimmilk or non-milk proteins (Shillam et al., 1962; Tagari and Roy, 1969; Williams et al., 1976). Diarrhea in the present experiment however, was not increased by feeding SPC (Table 21). Although data in Table 28 refer only to a particular time (6 hours) after feeding some conclusions can be made. Dry matter and organic 92 matter recovered at each site were not different (P>.05) for only milk protein or milk protein plus SPC. However, the amount of dry matter and organic matter present in the abomasum of animals fed SPC tended to be slightly lower than for calves fed only milk protein. This might be due to a shorter retention time of ingesta in the abomasum of non—milk proteins in preruminant calves, as postulated by Colvin et al. (1969) and Ternouth et al. (1975). However, quantity of protein in the abomasum of calves fed SPC was the same as those fed only milk protein (Table 28). According to Emmons et al. (1976) and Ternouth et a1. (1980), milk protein (and milk fat) should remain in the abomasum longer than other nutrients. The incorporation of .8% limestone in the replacers resulted in more (P<.05) dry matter, organic matter and crude protein in the digestive tract of calves, due to the larger (P<.05) amount present in the rumen- reticulum-omasum section. This observation suggests that calves fed limestone probably had higher intakes of bedding (straw), perhaps because they tried to compensate for the lower nutrient digestibilities and protein retention (Table 22). The interaction of treatment x site in the gut (Table A.3, Appendix) was not significant (P>.10) for any digesta variable measured, suggesting that treatment effects followed the same trends throughout the different sections of the gut. One calf from each treatment died during the experiment and necropsy results did not indicate a diet relationship. In summary the replacement of one-half of dietary protein with SPC in milk replacers resulted in lower weight gains (20%) and decreased dry 93 matter and crude protein digestibilities. Organic matter digestibility and crude protein retention also tended to be reduced, but intake of nutrients, feed efficiencies and fecal scores were not affected. The incorporation of .8% limestone in milk replacers resulted in no signifi- cant changes in the above-mentioned parameters, although apparent nutrient digestibilities and protein retention tended to decrease when limestone was present. The xylose absorption test showed differences in absorption due to calf age, but not between treatments. Similar results were obtained in Experiment 1. Analysis of digesta from different sections of the gastrointestinal tract revealed a higher pH in the abomasum than previously reported for whole milk diets. Abomasal pH was not affected by protein source. Small intestinal pH was always above 6, regardless of the dietary treatment. This observation suggests that digestion in preruminant calves is similar to monogastrics and different from adult ruminants. The high intestinal pH values might explain the ineffectiveness of limestone in improving nutrient digestibilities. The replacement of milk protein by SPC resulted in a lower pH in the large intestine and feces, probably due to fermentation of accumulated organic matter in the large intestine because of a more rapid flow of digesta through the gut. Indeed, animals fed SPC showed lower amounts of dry matter and organic matter in the abomasum than those fed milk protein. CONCLUSIONS Replacing 31% of milk protein in calf milk replacers with soybean protein concentrate (SPC) or spray-dried fish solubles resulted in significantly lower weight gains, dry matter digestibilities and nitrogen retention. Increasing crude protein from 19 to 23% in a replacer containing 10% crude protein from spray-dried fish solubles did not improve the calf's performance. Furthermore, high mortalities indicated that 10% protein from spray-dried fish solubles was excessive. Necropsy results from animals fed large amounts of spray-dried fish solubles suggested that allergy problems may be involved since ulcerations, and in some cases, ruptures of the gastrointestinal wall occurred. Replacing one-half of the milk protein with SPC reduced calf weight gain, dry matter and protein digestibilities, and nitrogen retention and pH in the large intestine and feces. The lowered pH may have been due to fermentation of more undigested material in the large intestine because of a more rapid flow of nutrients through the gut of calves fed SPC. Indeed, animals fed SPC showed lower amounts of digesta in the abomasum 6 hours after a meal than calves receiving only milk protein. Samples from three sections of the small intestine obtained 6 hours after calves were fed, showed pH values above 6 independent of the protein source. These results suggest that unlike adult ruminants, bile and pancreatic juice exert adequate buffering in preruminant calves and 94 probably account for the ineffectiveness of limestone in the replacer fed in this study. The xylose absorption test showed age differences in absorption capabilities of calves but did not show differences between protein sources . APPENDIX 96 TABLE A.1. -- Analysis of variance for variables in experiment 1 Source of d.f. Mean square F Significance variation ratio levela Initial body weight at Michigan State (kg) Treatment 6 10.6661 .51 NS Error 55 20.8753 Immunoglobulin (mg %) Treatment 6 11.0224 .84 NS Error 55 13.1544 Plasma protein (%) Treatment 6 .2494 .91 NS Error 55 .2793 Total weight gains - o to 3 wk (kg)b Location (L) 2 31.3531 4.70 ** Treatment (T) 6 35.1035 5.23 *** L x T 12 10.3144 1.54 NS Error 146 6.7122 Total weight gains - 4‘to 6 wk__(kg)b Location (L) 2 40.2621 5.33 *** Treatment (T) 6 115.7341 15.32 *** L x T 12 11.2236 1.49 NS Error 145 7.5560 Total weight gains - 0 to 6 wk (k‘g)b Location (L) 2 140.1698 9.61 *** Treatment (T) 6 265.9481 18.23 *** L x T _ 12 34.2053 2.34 *** Error 145 14.5875 Dry matter intake - 0 to 6 wk (g)b Location (L) 2 202,208,068.9704 20.42 *** Treatment (T) 6 29,866,844.8616 3.02 *** L x T 12 10,574,208.3345 1.07 NS Error 147 9,900,781.6781 Crude protein intake - 0 to 6 wk (g)b Location (L) 2 8,843,517.9801 19.14 *** Treatment (T) 6 17,858,283.9987 38.65 *** L x T 12 465,579.3709 1.01 NS Error 147 461,939.1200 TABLE A.1. -- Continued 97- Source of d.f. Mean square F Significance variation ratio levela Weight_g§in/dry matter intakeb Location (L) 2 .0712 3.56 ** Treatment (T) 6 .3403 16.36 **n L x T 12 .0432 2.08 ** Error 145 .0208 Weightggain/crude protein intakeb Location (L) 2 .6800 ' 1.11 NS Treatment (T) 6 16.9000 27.70 *** L x T 12 1.0400 1.70 * Error 145 .6100 Rectal temperature - lst week (°F)b Location (L) 2 32.9601 84.49 *** Treatment (T) 6 .3875 .99 NS L x T 12 .2281 .58 NS Error 146 .3901 Rectal temperature - 2nd week (OF)b Location (L) 2 48.2704 69.63 *** Treatment (T) 6 .4698 .68 NS L x T 12 .3188 .46 NS Error 146 .6931 Fecal scoresb Location (L) 2 87.2610 296.47 *** Treatment (T) 6 43.5380 147.92 *** L x T 12 5.6225 19.10 *** Week (W) 5 8.5853 29.17 *** T x W 30 .7940 2.70 88* Error 946 .2943 Treatment of sicknessb Location (L) 2 6.1791 35.22 *** Treatment (T) 6 .4766 2.72 ** L x T 12 .8162 4.65 *** Week (W) 5 3.0410 17.34 *** T x W 30 .1452 .83 NS Error 945 .1754 98 TABLE A.1. -- Continued Source of d.f. Mean Square F Significance variation ratio levela Plasma xylose concentration (mg_3) Treatment (T) 6 6,254.041 .32 NS Animal/T (Error a) 55 19,697.388 Sammle time (S) 7 46,296.715 47.47 *** T x S 42 843.468 .85 NS Error b 385 988.400 Methionine (gmoles/IOO m1) Treatment (T) 6 2.44 2.18 NS Animal/T (Error a) 21 1.12 Week (W) _1 3.92 2.48 NS T x W 6 2.47 1.56 NS Error b 21 ' 1.58 Leucine (pmoles/IOO m1) Treatment (T) 6 38.93 2.72 ** Animal/T (Error a) 21 14.32 Week (W) 1 63.60 5.41 ** T x W 6 6.47 .55 NS Error b 21 11.76 Valine (ymoles/IOO m1) Treatment (T) 6 80.03 3.24 ** Animal/T (Error a) 21 24.73 Week (W) 1 160.01 11.55 *** T x W 6 11.41 .82 NS Error b 21 13.85 Isoleucine (gmolea/loo m1) Treatment (T) 6 19.38 2.53 * Animal/T (Error a) 21 7.65 Week (W) 1 15.06 4.50 ** T x W 6 2.55 .76 NS Error 5 21 3.35 Phenx1a1anine (pmoles/lOO m1) Treatment (T) 6 5.40 1.00 NS Animal/T (Error a) 21 5.39 Week (W) 1 28.54 9.67 *** T x W 6 2.95 1.00 NS Error b 21 2.95 TABLE A.1 -- Continued Source of d.f. Mean square F Significance variation ratio levela Lysine (gmm1es/100 m1) Treatment (T) 6 66.44 2.73 ** Animal/T (Error a) 21 24.31 Week (W) 1 324.00 38.43 *** T x W 6 22.44 2.66 ** Error b 21 8.43 Histidine (Pmoles/IOO m1) Treatment (T) 6 7.66 1.73 NS Animal/T (Error a) 21 4.42 Week (W) 1 15.92 5.34 ** T x W 6 2.19 .73 NS Error b 21 2.98 Arginine_(pmoles/100 m1) Treatment (T) 6 47.68 2.15 * Animal/T (Error a) 21 22.17 Week (W) 1 97.94 5.18 ** T x W 6 50.38 2.66 ** Error b 21 18.93 Threonine (pmoles/lOO m1) 7 Treatment (T) 6 54.33 1.72 NS Animal/T (Error a) 21 31.53 Week (W) 1 0.00 -- NS T x W 6 15.15 .43 NS Error b 21 35.40 Total essential amino acids-TEAA.(pmo1es/100 m1) I Treatment (T) 6 1,890.06 2.90 ** Animal/T (Error a) 21 651.79 Week (W) 1 2,042.29 6.92 ** T x W 6 272.34 .92 NS Error b 21 294.94 Total non-essential amino acids-TNEAA (pmoles/lOO m1) Treatment (T) 6 1,347.70 1.97 NS Animal/T (Error 3) 21 684.90 Week (W) 1 30.01 .05 NS T x W 6 261.06 .39 NS Error b 21 665.46 100 TABLE A.1. -- Continued Source of d.f. Mean square F Significance variation ratio levela Non-essentialzessential amino acids ratio - N/E Treatment (T) 6 .26 2.17 NS Animal/T (Error a) 21 .12 Week (W) 1 1.26 5.48 ** T x W 6 .04 .17 NS Error b 21 .23 ' Branched chain amino acids-BCA (pmoles/lOO m1) Treatment (T) 6 373.84 3.08 ** Animal/T (Error 3) 21 121.30 Week (W) 1 600.50 10.14 *** T x‘W 6 48.84 .82 NS Error b 21 59.25 Sulfur amino acids-SAA gmoles/IOO m1) Treatment (T) 6 2.80 1.28 NS Animal/T (Error a) 21 2.18 Week (W) 1 1.55 .64 NS T x W 6 5.58 2.31 NS Error b 21 2.42 aNS - nonsignificant (P>.10) * - P<.10. ** - P<.05. ***- P<.01. bStatistical analysis realized at Kansas State University. 101 TABLE A.2. -- Analysis of variance for variables in experbment 2 Source of d.f. Mean square F Significance variation ratio levela Initial body weight (kg) Treatment 3 34.68 1.41 NS Error 12 24.53 Plasma protein (2) Treatment 3 .1906 1.44 NS Error 11 .1328 Daily weight gain (kg) Treatment 3 .1906 1.42 NS Error 11 .1328 Rectal temperatures (°C) Treatment 3 .2680 1.38 NS Error 12 .1939 Scour scores Treatment 3 1.09 2.95 * Error 12 .37 Apparent dry matter digestibility (%) Treatment 3 .0215 4.50 ** Error 12 .0047 Apparent ogganic matter digestibility (Z) Treatment 3 .0174 4.34 ** Error 12 .0040 Apparent nitrogen retention (g/day) Treatment 3 43.80 5.78 ** Error 12 7.58 aNS - nonsignificant (P>.10). * - P<.10. ** - P<.05. 102 TABLE A.3. -- Analysis of variance for variables in experiment 3 Source of d.f. Mean square F Significance variation ratio levela Initial body weight (kg) Treatment (T) 3 23.1 1.00 NS Error 11 23.0 Weight gains (g/an/day) Treatment (T) 3 21,132.3666 .27 NS Animal/T (Error a) 11 79,650.3879 Week (W) 5 1,207,505.1794 39.43 *** T x W 15 28,168.6669 .92 NS Error b 59 30,626.0820 Dry matter intake (g/an/day) Treatment (T) 3 1,480.0028 .03 NS Animal/T (Error a) 11 53,704.2871 Week (W) 5 271,263.5080 61.68 *** T x W 15 2,032.8525 .46 ‘ NS Error b 59 4,397.6160 Crude protein intake (g/an/day) Treatment (T) 3 121.3167 .06 NS Animal/T (Error a) 11- 1,957.8288 Week (W) 5 9,883.9058 148.48 *** T x W 15 42.4033 .64 NS Error b 59 66.5687 Weightgain/dry matter intake Treatment ('1‘) 3 . 0967 . 06 NS Animal/T (Error a) 11 .1907 Week (W) 5 3.2827 19.39 *** T x W 15 .1449 .86 NS Error b 59 .1693 Fecal dry matter (2) Treatment (T) 3 13.2848 2.45 NS Animal/T (Error a) 12 5.4319 Week (W) 1 1.0047 .07 NS T x W 3 12.9680 .86 NS Error b 12 15.1504 103 TABLE A.3. -- Continued Source of d.f. Mean square F Significance variation ratio levela Fecal scores Treatment (T) 3 .6020 .66 NS Animal/T (Error a) 11 .9130 Week (W) 5 .5480 1.39 NS T x W 15 .3483 .88 NS Error b 59 .3952 Rectal temperature (°C) Treatment (T) 3 .3767 .70 NS Animal/T (Error a) 11 .5349 Week (W) 5 .1865 .90 NS T x W 15 .1961 .94 NS Error b 59 .2076 Fecalng Treatment (T) (3) (1.9158) (6.27) *** Source (S) 1 5.7207 18.78 *** Limestone (L) 1 .0102 .03 NS S x L 1 .0166 .05 NS Animal/T (Error a) 12 .3056 Week (W) 1 .5486 7.36 ** T x W 3 .0660 .88 NS Error b 12 .0746 Apparent dry matter diggstibility (Z) Treatment (T) (3) (45.2817) (3.25) * Source (S) 1 53.7968 3.86 * Limestone (L) 1 41 .9323 3.01 NS S x L 1 40.1160 2.88 NS Animal/T (Error a) 12 13.9409 Week (W) 1 49.6281 4.43 * T x W 3 14.6532 1.31 NS Error b 12 11.2059 Apparent organic matter digestibility (2) Treatment (T) 3 43.6124 2.12 NS Animal/T (Error a) 12 20.5429 Week (W) 1 .0782 .01 NS T x W 3 13.4667 .99 NS Error b 12 13.5686 104 Error b 120 1,607.9258 Source of d.f. Mean square F Significance variation ratio levela Apparent protein digestibility (2) Treatment (T) (3) (220.7409) (3.96) ** Source (S) 1 570.5574 10.23 *** Limestone (L) 1 72.6897 1.30 NS 8 x L 1 18.9755 .34 NS Animal/T (Error a) 12 55.7702 Week (W) 1 31.8364 .83 NS T x W 3 10.0297 .26 NS Error b 12 38.3330 Apparent protein retention (g/day) Treatment (T) 3 254.2522 .37 NS Animal/T (Error 3) 12 684.4508 Week (W) 1 12,640.5040 51.81 *** T x W 3 113.3984 .46 NS Error b 12 243.9874 . Apparent protein retention (2 of ingested) Treatment (T) 3 109.2886 .58 NS Animal/T (Error a) 12 187.0888 Week (W) 1 1,454.8962 25.18 *** T x W 3 .8671 .01 NS Error b 12 57.7742 Plasma urea nitrogen (mg Z) Treatment (T) 3 3.2069 .39 NS Animal/T (Error 3) 12 8.1549 Week (W) 1 120.9113 10.44 *** T x W 3 16.2117 1.40 NS Error b 12 11.5844 Plasmm~§ylose concentration (@512) Treatment (T) 3 1,640.2619 .09 NS Animal/T (Error a) 8 18,196.1625 Week (W) 1 18,768.0571 11.67 *** Sample (S) 7 21,527.1529 15.25 *** T x W 3 14,092.3394 8.76 *** T x S 21 1,355.9258 .84 NS W x S 7 2,212.3580 1.38 NS T x W x S 21 270.2670 .17 NS 105 TABLE A.3. -- Continued Source of d.f. Mean square F Significance variance ratio levela Gastrointestinalng Treatment (T) (3) (.4169) (10.19) ** Source (8) 1 .6912 16.90 ** Limestone (L) 1 .1850 4.52 NS S x L 1 .3746 9.15 ** Animal/T (Error a) 4 .0409 Cut (G) 5 14.4700 62.77 *** T x G 15 .1450 .52 NS Error b 20 .2783 Gastrointestinal dry matter content (g) Treatment (T) 3 4,050.3542 2.43 NS Animal/T (Error a) 4 1,665.7292 Gut (G) 5 60,199.2375 27.67 *aa T x c 15 1,714.8042 .79 NS Error b 20 2,175.6292 Gastrointestinal organic matter content (Z) Treatment (T) 3 3,221.8333 2.43 NS Animal/T(Error a) 4 1,327.9584 Gut (G) 5 47,869.6333 23.84 *** T x G 15 1,171.2667 .58 NS Error b 20 2,007.6083 Gastrointestinal crude protein content (g) Treatment (T) 3 68.7431 2.05 NS Animal/T (Error a) 2 33.4583 Gut (G) 5 266.3375 11.51 *** T x G 15 22.1931 .96 NS Error b 18 23.1435 aNS - nonsignificant (P>.10). * - P<.10. ** - P<.05. ***- P<.01. BIBLIOGRAPHY BIBLIOGRAPHY Adams, R.S., T.W. Gullickson, H.J. Sautter, and J.E. Gardner. 1954. Effects of tocopherol administration on dairy calves receiving various filled milks. J. Dairy Sci. 37:655 (abstr). Almiquist, H.J., E. Mecchi, H.F. Kratzer, and C.R. Grau. 1942. Soybean protein as a source of amino acids for the chick. J. Nutr. 24:385. Alpan, S.O., J.E. Oldfield, D.W. Claypool, and G.O. Kohler. 1979. Digestibility of alfalfa protein concentrate (Pro-Xan) in milk replacer diets for calves. J. Dairy Sci. 62:86 (Suppl. 1). Ash, R.W. 1964. Abomasal secretion and emptying in suckled calves. J. Physiol. 172:425. Association of Official Agricultural Chemists. 1980. Official Methods of Analysis. 13th edition, A.0.A.C., Washington, D.C. Bang-Jensen, V., B. Foltman, and W. Rombauts. 1964. Studies on rennin. X: On the proteolytic specificity of rennin. C.R.Trav. Lab. Carlsberg 34:326. Barker, H.B., G.H. Wise, and N.L. Jacobson. 1952. Filled milk for dairy calves. III. Comparative value of various soybean oils and butter oil in practical dietary regime. J. Dairy Sci. 35:507. Barr, G.W., S.R. Martin, M.L. Kakade, P.J. Ryan, and F.M. Crane. 1978. Influence of modified soy protein in milk replacers on calf performance and health. J. Dairy Sci. 61:169 (Suppl. 1). Barratt, M.E.J. anle. Porter. 1979. Immunoglobulin classes implicated in intestinal disturbances of calves associated with soya protein antigen. J. Immunol. 123:676. Barratt, M.E.J., P. J. Strachan, and P. Porter. 1978. Antibody mechanisms implicated in digestive disturbances following ingestion of soya protein in calves and piglets. Clin. Exp. Immunol. 31:305. Barratt, M.E.J., P.J. Strachan, and P. Porter. 1979. Immunologically mediated nutritional disturbances associated with soya protein antigens. Proc. Nutr. Soc. 38:143. 106 107 Bell, J.M., B.E. Harvey, and G.I. Christinson. 1979. Effects of the addition of enzymes carboxymethylcellulase to pea flour used for calf milk replacers. Can. J. Anim. Sci. 59:43. Bell, J.M., C.F. Royan, and C.G. Young. 1974. Digestibility of pea protein concentrate and enzyme-treated pea flour in milk replacers for calves. Can. J. Anim. Sci. 54:355. Bergen, W.G. 1979. Free amino acids in blood of ruminants -- Physiological and nutritional regulation. J. Anim. Sci. 49:1577. Berridge, N.J., J.G. Davis, P.M. Ron, and F.R. Spratling. 1943. The production of rennet from living calves. J. Dairy Res. 13:145. Berry, T.H., D.E. Becker, O.G. Rasmussen, A.N. Jensen, and R.W. Norton. 1962. The limiting amino acids in soybean protein. J. Anim. Sci. 21:558. Blaxter, K.L., F. Brown, and A.M. McDonald. 1953. The nutrition of the young Ayrshire. 13. The toxicity of unsaturated acids of cod liver oil. Brit. J. Nutr. 7:287. Blaxter, K.L. and R.F. McGill. 1955. Muscular dystrophy. Vet. Rev. Annot. 10:91. Bolton, J.R., A.M. Merritt, R.F.. Cimprich., C.F. Ramber, and w. Street. 1976. Normal and abnormal xylose absorption in the horse. Cornell Vet. 66:183. Borgstrom, B., A. Dahlquist, G. Lundh, and J. Sjovall. 1957. Studies of intestinal digestion and absorption in the human. J. Clin. Invest. 36:1521. Bouchard, R., G.J. Brisson, and J.P. Julien. 1973. Nutritive value of bacterial sludge and whey powders for protein in calf milk replacers and on chromic oxide as indicator of digestibility. J. Dairy Sci. 56:1445. Bouchard, R., L.F. LaFlame, B. Lachance, and G.L. Roy. 1980. Levels of protein and fat and type of protein in vealer rations. Can. J. Anim. Sci. 60:523. Bringe, A.N. and G.W. Barr. 1979. Effect of protein source of milk replacer on performance of dairy calves in cold housing. J. Dairy Sci. 62:100 (Suppl. 1). Brumbaugh, J.E. and C.B. Knodt. -1952. Milk replacements for dairy calves. J. Dairy Sci. 35:336. Bryant, J.M., C.F. Foreman, N.L. Jacobson, and A.D. McGilliard. 1967. Protein and energy requirements of the young calf. J. Dairy Sci. 50:1645. 108 Burackzewski, S., L. Burackzewska, and J.E. Ford. 1967. The influence of heating of fish proteins on the course of their digestion. Acta Biochim. Pol. 14:121. Carroll, R.W., G.W. Hensley, and W.R. Graham, Jr. 1952. The site of nitrogen absorption in rats fed raw and heat-treated soybean meals. Science 115:36. Chow, C. and J.M. Bell. 1976. Effects of various heat and pH treatments on digestibility of protein in pea protein concentrate (Pisum sativum). Can. J. Anim. Sci. 56:559. Christison, G.I. and J.M. Bell. 1977. The disappearance of vegetable protein from.abomasum of young calves. Can. J. Anim. Sci. 57:882 (abstr). Church, D.C. 1971. Digestive Physiologyand.Nutrition of Ruminants. Vol. 2. . Chapter 28, p. 779. O & B Books, Inc., Corvallis, Oregon. Coates, M.E., D. Hewitt, and P. Golob. 1970. A comparison of the effects of raw and heated soya-bean meal in diets for germ-free and conventional chicks. Brit. J. Nutr. 24:213. Coblentz, E., J.L. Morrill, D.B. Parrish, and A.D. Dayton. 1976. Nutritive value of thermoalkali-processed soy materials for young calves and rats. J. Dairy Sci. 59:481. Colvin, B.M., R.A. Lowe, and H.A. Ramsey. 1969. Passage of digesta from the abomasum of a calf fed soy flour milk replacers and whole milk. J. Dairy Sci. 52:687.. Colvin, B.M. and R.A. Ramsey. 1968. Soy flour in milk replacers for young calves. J. Dairy Sci. 51:898. Colvin, B.M. and R.A. Ramsey. 1969. Growth of young calves and rats fed soy flour treated with acid or alkali. J. Dairy Sci. 52:270. Danell, B. 1970. Officielt forsok med kalvmanna. Astra-Bows A B, Feed protein symposium. 14th September, Stencil 9 pages. Davicco, M.J., L. Lefaivre, P. Thivend, and J.P. Barlet. 1979. Feedback regulation of pancreatic secretion in the young milk-fed calf. Ann. Biol. anim. Bioch. Biophys. 19:1147. Debas, H.T., S.J. Konturek, J.E. Walsh, and M.I. Grossman. 1974. Proof of a pyloric-oxyntic reflex for stimulation of acid secretion. Gastroenterology 66:526. DeGroot, A.P. and P. Slump. 1969. Effects of severe alkali treatment or proteins on amino acid composition and nutritive value. J. Nutr. 98:45. 109' Dodsworth, T.L., J.B. Owen, I.M. Mackie, A. Ritchie, and E.R. Orskov. 1977. Fish-protein hydrolysate as a substitute for milk protein in calf feeding. Anim. Prod. 25:19. Ellinger, C.M. and E.B. Boyne. 1965. Amino acid composition of some fish products and casein. Brit. J. Nutr. 19:587. Emmons, D.B. and E.E. Lister, 1976. Quality of protein in milk replacers for young calves. 1. Factors affecting in vitro curd formation by rennet (chymosin, rennin) from reconstituted skimmilk powder. Can. J. Anim. Sci. 56:317. Emmons, D.E., E.E. Lister, and J.D. Jones. 1976. Quality of protein in milk replacers for young calves. IV. Rennet (chymosin, rennin) coagula- tion of reconStituted skimmilk powder containing added proteins, fat, calcium, phosphate and citrate. Can. J. Anim. Sci. 56:339. Erdman, R.A., R.L. Botts, R.W. Hemken, and L.S. Bull. 1980. Effect of dietary sodium bicarbonate and magnesium oxide on production and physiology in early lactation. J. Dairy Sci. 63:923. Ershoff, B.H. and P.G. Rucker. 1969. Nutritive value of 1,2-dichloro- ethane-extracted fish protein concentrate. J. Food Sci. 34:355. Fawcet, J.K. and J.E. Scott. 1960. A rapid and precise method for the determination of urea. J. Clin. Path. 135156. Flatlandsmo, K. 1972. Marine fat. Digestibility of its fatty acids in young calves. Acta-Vet. Scand. 13:260. Foldager, J., J.T. Huber, and W.G. Bergen. 1977. Methionine and sulfur amino acid requirements in the preruminant calf. J. Dairy Sci. 60:1095. Foley, J.A., A.G. Hunter, and D.E. Otterby. 1978. Absorption of colostral proteins by newborn calves fed unfermented, fermented, or buffered colostrum. J. Dairy Sci. 61:1450. Folman, Y. and E. Eyal. 1978. A note on the performance of Assaf male lambs reared intensively on an all-concentrate diet with herring meal or toasted soya bean as the main protein source. Anim. Prod. 26:331. Frantzen, J.P., R. Toullec, and C.M. Mathieu. 1971. Influence de la coagulation des proteines sur l'utilization digestive d'un lait de replacement par 1e veau preruminant. 10th Congr. Int. Zootech., Versailles. Fraser, C. and E.R. Orskov. 1974. Cereal processing and food utilization by sheep. 1. The effect of processing on utilization of barley by early-weaned lambs. Anim. Prod. 18:75. 110 Fries, C.F., C.A. Lassiter, and C.F. Huffman. 1958. Effect of enzyme supplementation of milk replacers on the growth of calves. J. Dairy Sci. 41:1081. Friess, S.L. and W.J. McCarville. 1954. Nature of the acetyl cholines- terase surface. II. The ring effect in enzymatic inhibitors of the substrate ethylenediamine type. J. Amer. Chem. Soc. 76:2260. Garlich, J.D. and M.C. Nescheim. 1966. Relationship of fractions of soybeans and a crystalline soybean trypsin inhibitor to the effects of feeding unheated soybean meal to chicks. J. Nutr. 88:100. Carnot, P., R. Toullec, J.L. Thapon, P. Martin, M.T. Hoang, C.M. Mathieu, and E.R. Dumas. 1977. Influence of age, dietary protein and weaning on calf abomasal enzymic secretion. J. Dairy Res. 44:9. Gaudreau, J.M. and G.J. Brisson. 1978. Abomasum emptying in young calves fed milk replacers containing animal or vegetable fats. J. Dairy Sci. 61:1435. Geiger, E. and G. Borgstrom. 1965. Fish protein - nutritive “aspects. In: Fish as Food. Edited by George Borgstrom. Vol. II, page 29, Academic Press, New York and London. Gelwichs, T.J. 1965. The effect of fish flour as a protein source in milk replacers on the growth of young dairy calves. M.S. Thesis, University of Illinois, Urbana. Genskow, R.D. 1969. Evaluation of low ash fish protein concentrate for use in calf milk replacer formulas. Ph.D. Thesis, University of Illinois, Urbana. Genskow, R.D., K.E. Harshbarger, and R.M. Wendlandt. 1968. Observations on vitamin deficiencies in calves fed a milk replacer containing lowbash fish meal. J. Dairy Sci. 51:973 (abstr). Gill, J.L. 1978a. Design and Analysis of Experiments in the Animal and Medical Sciences. Vol. 1. The Iowa State University Press, Ames, Iowa. Gill, J.L. 1978b. Design and Analysis of Experiments in the Animal and Medical Sciences. Vol. 2. The Iowa State University Press, Ames, Iowa. Gill, J.L. 1978c. Design and Analysis of Experiments in the Animal and Medical Sciences. Vol. 3 - Appendices. The Iowa State University Press, Ames, Iowa. Gillespi, J.H. 1971. An evaluation of partially deboned fish protein concentrate as an ingredient in calf milk replacers. M.S. Thesis, University of Illinois, Urbana. 111 Gorrill, A.D.L. 1970. Physical and chemical characteristics of soybean and milk proteins before and after treatment with dilute alkali. Can. J. Anim. Sci. 50:745. Gorrill, A.D.L., J.D.Jones, E. Larmond, C.D.T. Cameron, J.E. Cameau, and J.W.G. Nicholson. 1975b. Growth, mortality and meat quality of lambs fed milk replacers containing full-fat soybean flour. Can. J. Anim. Sci. 55:731. Gorrill, A.D.L., J.D. Jones, and J.W.G. Nicholson. 1974. The nutritional value and trypsin inhibitor content of processed soybeans for lambs milk replacers. Can. J. Anim. Sci. 54:337. Gorrill, A.D.L., J.D. Jones, and J.W.G. Nicholson. 1976. Low and high glucosinolate rapeseed flours and rapeseed oil in milk relacers for I calves: their effects on growth, nutrient digestion and nitrogen I retention. Can. J. Anim. Sci. 56:409. Gorrill, A.D.L. and J.W.G. Nicholson. 1969a. Effect of added bulk on growth, nutrient utilization, digestive system and diarrhea in calves fed milk replacers. Can. J. Anim. Sci. 49:305. Gorrill, A.D.L. and J.W.G. Nicholson. 1969b. Growth, digestibility and nitrogen retention by calves fed milk replacers containing milk and soybean proteins, supplemented with methionine. Can. J. Anim. Sci. 49:315. Gorrill, A.D.L. and J.W.G. Nicholson. 1971. Effect of soybean trypsin inhibitor, diarrhea, and diet on flow rate, pH, proteolytic enzymes and nitrogen fractions in calf intestinal digesta. Can. J. Anim. Sci. 51:377. Gorrill, A.D.L. and J.W.G. Nicholson. 19723. Effects of neutralizing acid whey powder in milk replacers containing milk and soybean proteins on performance and abomasal and intestinal digestion in calves. Can. J. Anim. Sci. 52:465. Gorrill, A.D.L. and J.W.G. Nicholson. 1972b. Use of the Williams Polytron to homogenize fat and disperse insoluble ingredients in high-fat liquid milk replacers. Can. J. Anim. Sci. 52:477. Gorrill, A.D.L. and J.W.G. Nicholson. 1972c. Alkali treatment of soybean protein concentrate in milk replacers: its effect on digestion, nitrogen retention and growth of lambs. Can. J. Anim. Sci. 52:665. Gorrill, A.D.L., J.W.G. Nicholson, E. Larmond, and H.E. Power. 1975. Comparison of fish protein sources and milk byproducts in milk replacers for calves. Can. J. Anim. Sci. 55:269. 112 Gorrill, A.D.L., J.W.G. Nicholson, and T.M. Macyntire. 1975a. Effects of formalin added to milk replacers on growth, feed intake, digestion and incidence of abomasal bloat in lambs. Can. J. Anim. Sci. 55:557. Gorrill, A.D.L., J.W.G. Nicholson, and H.E. Power. 1972. Effects of milk, fish and soybean proteins in milk replacers, and feeding fre- quency on performance of dairy calves. Can. J. Anim. Sci. 52:321. Gorrill, A.D.L. and J.W. Thomas. 1965. Proteolytic activity of the bovine pancreas. J. Anim. Sci. 24:882 (abstr). Gorrill, A.D.L. and J.W. Thomas. 1967. Body weight changes, pancreas size and enzyme activity, and proteolytic enzyme activity and protein , digestion in intestinal contents from calves fed soybean and milk protein diets. J. Nutr. 92:215. Gorrill, A.D.L., J.W. Thomas, W.E. Stewart, and J.L. Morrill. 1967. “5 Exocrine pancreatic secretion by calves fed soybean and milk protein 1 diets. J. Nutr. 92:86. Gregory, R.A. 1974. The gastrointestinal hormones. A review of recent advances. J. Physiol. 241:1. Guilloteau, P., R. Toullec, and P. Partureau-Mirand. 1979. Influence de la vitesse d'evacuation gastric des proteines et des lipides sur utilization digestive chez le veau preruminant. Ann. Biol. Anim. Biochem. Biophys. 19:955. Haines, P.C. and R.L. Lyman. 1961. Relationship of pancreatic enzymes secretion to growth inhibition in rats fed soybean trypsin inhibitor. J. Nutr. 74:445. Hamilton, J.W. and A.L. Tappel. 1963. Lipid antioxidant activity in tissues and proteins of selenium-fed animals. J. Nutr. 79:493. Hallenbeck, C.A. 1967. Handbook of Physiology. C.F. Code, editor. Washington, D.C. Harper, A.A. 1972. Progress report. The control of pancreatic secretion. Gut. 13:308. Harris, P.L. and N.D. Embree. 1963. Quantitative considerations of the effect of polyunsaturated fatty acid content of the diet upon the requirements for vitamin E. Amer. J. Clin. Nutr. 13:385. Harrison, F.A. and J.K. Hill. 1962. Digestive secretions and the flow of digesta along the duodenum of the sheep. J. Physiol. 162:225. Harrison, V.C. and G. Peat. 1972. Significance of milk pH in newborn infants. Brit. Med. J. 4:515. 113 Harshbarger, R.F. and T.J. Gelwichs. 1965. Fish flour as a protein source in milk replacers for dairy calves. J. Dairy Sci. 48:788 (abstr). Hays, V.W., V.C. Speer, F.A. Hartman, and D.V. Catron. 1959. The effect of age and supplemental of amino acids on the utilization of milk and soya proteins by the young pig. J. Nutr. 69:179. Henschel, M.J. 1973. Comparison of the development of proteolytic acti- vity in the abomasum of the preruminant calf with that in the stomach of the young rabbit and guinea pig. Brit. J. Nutr. 30:285. Henschel, M.J., W.B. Hill, and J.W.G. Porter. 1961a. The development of proteolytic enzymes in the abomasum of the young calf. Proc. Nutr. Soc. 20 x1. Henschel, M.J., W.B. Hill, and J.W.G. Porter. 1961b. Proteolysis of milk and synthetic milks in the abomasum of the young calf. Proc. Nutr. Soc. 20 xli. Hidiroglou, M., R.B. Carson, and 6.8. Brossard. 1963. Problems associated with selenium deficiency in beef calves. Can. J. Physiol. Pharmacol. 48:854. Hill, F.W.G., D.E. Kidder, and J. Frew. 1970. An xylose absorption test for the dog. Vet. Rec. 87:250. Hill, J.K. 1968. Abomasal function. In: Handbook of Physiology. Section 6. Vol. II - Secretion. C.F. Code,editor. Amer. Physiol. Soc., Washington, D.C. Hill, J.K. 1970. Digestion in the small intestine. In: Duke's Physiology of Domestic Animals. M.J. Swenson,editor. Cornell University Press, Ithaca, New York. Hill, J.K., D.E. Noakes, and R.A. Lower. 1970. Gastric digestive physiology of the calf and piglet. In: Physiology of Digestion and Metabolism in the Ruminant. A.T. Phillipson, editor. Oriel Press, Newcastle Upon Tyne, England. Hooks, R.D., V.W. Hays, V.C. Peer, and J.T. McCall. 1965. Effect of raw soybeans on pancreatic enzyme concentrations and performance of pigs. J. Anim. Sci. 24:894 (abstr). Horwitt, M.K. 1965. Role of vitamin E, selenium, and polyunsaturated fatty acids in clinical and experimental muscle disease. Fed. Proc. 24:68. Huber, J.T. 1958. Relationship of age and diet to digestive enzyme activity in the calf. M.S. Thesis, Iowa State University, Ames, Iowa. 114 Huber, J.T. 1969. Calf nutrition and development of the digestive and metabolic apparatus of the calf. J. Dairy Sci. 52:1303. Huber, J.T. 1975. Fish protein concentrate and fish meal in calf milk replacers. J. Dairy Sci. 58:441. Huber, J.T., N.L. Jacobson, R.S. Allen, and F.A. Hartman. 1961. Digestive enzyme activities in the young calf. J. Dairy Sci. 44:1494. Huber, J.T. and W.E.C. Moore. '1964. Short-chain fatty acid concentra- tions posterior to the stomach of calves fed normal and milk diets. J. Dairy Sci. 47:1421. ‘ Huber, J.T. and L.M. Slade. 1967. Fish flour as a protein source in calf milk replacers. J. Dairy Sci. 50:1296. Huber, J.T. and F.T. Sleiman. 1971. Substitution of isopropanol extracted fish protein concentrate for dried skimmilk in calf milk replacers. MSU Dairy Dept. Report. Huber, J.T., J.W. Thomas, and F.E. Standaert. 1978. Response of calves fed milk replacers containing soybean protein concentrate, an enzymatic hydrolysate of fish or dried fish solubles as partial protein substi- tutes. J. Dairy Sci. 61:176 (Suppl. 1). Huston, R.L. and H.M. Scott. 1966. Concentration of dietary arginine as related to degree of expression of amino acid imbalance. Poultry Sci. 45:1093. Jacobson, N.L., J.M. Bryant, C.F. Foreman, and A.D. McGilliard. 1965. Nutrient source and other factors affecting the utilization of protein and energy by the calf. Proc. Distillers Feed Res. Conf. 20:9. Jarrett, I. Gr., C.B. Jones, and B.J. Potter. 1964. Changes in glucose utilization during development of the lamb. Biochem. J. 90:189. Jenkins, K.J. 1981. Pepsin and pancreatin supplementation of calf milk replacer containing soy protein. Can. J. Anim. Sci. 61:469. Jenkins, K.J., D.B. Emmons, and J.R. Lessard. 1981. Some in vitro observations on factors affecting rennet (chymosin) clotting of calf 'milk replacers. Can. J. Anim. Sci. 61:393. Jenkins, K.J., S. Mahadevan, and D.B. Emmons. 1980. Susceptibility of protein used in calf milk replacers to hydrolysis by various proteolytic enzymes. Can. J. Anim. Sci. 60:907. Jordan, R.M., H.E. Hanke, and J.A. Tichich. 1972. Lamb milk replacer diets containing soybean flour. Can. J. Anim. Sci. 35:1130 (abstr). 115 Kakade, M.L., N.R. Simone, and I.E. Liener. 1971. Nutritive value of acid-treated soy-flour: possible role of lecithin. J. Dairy Sci. 54:1705. Kakade, M.L., R.M. Thompson, W.E. Engelstad, G.C. Behrens, and R.D. Yoder. 1974. Nutritional significance of soybean trypsin inhibitor in calves. J. Dairy Sci. 57:650 (abstr). Kakade, M.L., R.M. Thompson, W.E. Engelstad, G.C. Behrens, R.D. Yoder, and F.M. Crane. 1976. Failure of soybean trypsin.inhibitor to exert deleterious effect in calves. J. Dairy Sci. 59:1484. Kay, M., N.A. MacLeod, G. McKiddie, and E.B. Philip. 1967. The nutrition of the early-weaned calf. X. The effect of replacement of fish meal with either urea or ammonium acetate on growth rate and nitrogen retention in calves fed "ad libitum". Anim. Prod. 9:197. Kellor, R.L. 1974. Defatted soy flour and grits. J. Amer. Oil Chem. Soc. 51:77A. Kilshaw, P.J. and J.W. Sissons. 1979. Gastrointestinal allergy to soybean protein in preruminant calves. Antibody production and digestive disturbances in calves fed heated soybean flour. Res. Vet. Sci. 27:361. Larson, L.L., P.G. Owen, J.L. Albright, R.D. Appleman, R.C. Lamb, and L.D. Muller. 1977. Guidelines toward more uniformity in measuring and reporting calf experimental data. J. Dairy Sci. 60:989. Lassen, S. 1965. Fish solubles. In: Fish as Food. George Borgstrom, editor. Vol. 111, page 281. Academic Press, New York and London. Lassiter, C.A., L.D. Brown, R.M. Grimes, and C.W. Duncan. 1963. Effect of protein level on milk replacers on growth and protein metabolism of dairy calves. J. Dairy Sci. 46:538. Lassiter, C.A., C.F. Fries, C.F. Huffman, and C.W. Duncan. 1959. Effects of pepsin on the growth and health of young dairy calves fed various milk replacer rations. J. Dairy Sci. 42:666. Leibholz, J. 1970. The effect of starvation and low nitrogen intakes on the concentration of free amino acids in the blood plasma and on the nitrogen metabolism of sheep. Australian J. Agric. Res. 23:723. Lennox, A.M. and C.A. Carton. 1968. The absorption of long-chain fatty acids from the small intestine of the sheep. Brit. J. Nutr. 22:247. Lennox, A.M., A.K. Lough, and C.A. Carton. 1968. Observations on the nature and origin of lipids in the small intestine of sheep. Brit. J. Nutr. 22:237. 116 Lepkovisky, S., F. Furuta, and M.K. Dimick. 1971. Trypsin inhibitor and the nutritional value of soya beans. Brit. J. Nutr. 25:235. Levitt, D.C., A.A. Hakim, and N. Lifson. 1969. Evaluation of components of transport of sugars by dog jejunum "in vivo." Amer. J. Physiol. 217:777. Lovern, J.A. 1979. Problems in the development of fish protein concen- trates. Proc. Nutr. Soc. 28:81. ' Magee, D.F. 1961. An investigation into the external secretion of the pancreas in sheep. J. Physiol. 158:132. Makdani, D.D. 1969. Nutritional value of fish protein concentrate. Ph.D. Thesis, Michigan State University, East Lansing, Michigan. Makdani, D.D., W.G. Bergen, O. Mickelsen, and J.T. Huber. l97la. Factors influencing the nutritive value of l,2-dichloroethane-extracted fish protein concentrate in rat diets. Amer. J. Clin. Nutr. 24:1384. Makdani, D.D., J.T. Huber, and W.G. Bergen. 1971b. Effect of histidine and methionine supplementation on the nutritional quality of commer- cially prepared fish protein concentrate in rat diets. J. Nutr. 101:367. Makdani, D.D., J.T. Huber, and R.L. Michel. 1971c. Nutritional value of 1,2-dichloroethane extracted fish protein concentrate for young calves fed milk replacer diets. J. Dairy Sci. 54:886. Makdani, D.D., J.T. Huber, 0. Mickelsen, and W.G. Bergen. 1974. The influence of water fractionation on the nutritional value of fish protein concentrate. Nutr. Rep. Int. 9:309. Mathieu, C.M., P. Thivend, and R. Toullec. 1968. Digestion et utilization du aliments d'allaitement par 1e veau. Aliment Vie. 56:100. Matre, T. 1970. Proteinkvaliteten i keilmjolkerstatningar til kalvar. Meeting on cattle experiments at the Agricultural College of Norway. 3rd-4th December, p. 150. Matre, T. 1971. Proteinkvalitetin i erstatningsmojolk til kavar. Nord. Jordbrukforskning. 53:3368. Matre, T. 1973. Digestibility and feeding value of milk substitutes for calves. Nutr. Abst. Rev. 43:74. Matre, T. 1977. Mackarel flour as protein source in milk replacers for calves. Norges Landbrukshogskole 56. NR 53. McCormick, R.J. and W.E. Stewart. 1967. Pancreatic secretion in the bovine calf. J. Dairy Sci. 50:568. 117 Medwadwski, B.F., J. Vander Veen, and H.S. Olcott. 1967. Nature of residual lipids in fish protein concentrate. J. Food Sci. 32:361. Michel, R.L., D.D. Makdani, J.T. Huber, and A.B. Sculthorpe. 1972. Nutritional myopathy due to vitamin E deficiency in calves fed fish protein concentrate as the sole source of protein. J. Dairy Sci. 55:498. Mickelsen, O. and M.C. Yang. 1966. Naturally occurring toxicants in foods. Fed. Proc. 25:104. Miller, D.S. 1956. The nutritive value of fish proteins. J. Sci. Food Agr. 7:337. Morrill, J.L., R.K. Abe, A.D. Dayton, and C.W. DeYoe. 1970a. Feeding young calves processed starch combined with an amylolytic enzyme. J. Dairy Sci. 53:566. Morrill, J.L., S.L. Melton, A.D. Dayton, B.J. Guy, and M.J. Pallansch. 1971. Evaluation of milk replacers containing a soy protein concen— trate and high whey. J. Dairy Sci. 54:1060. Morrill, J.L., W.E. Stewart, R.J. McCormick, and H.C. Fryer. 1970b. Pancreatic amylase secretion by young calves. J. Dairy Sci. 53:72. Morrison, A.B. 1963. Factors influencing the nutritional value of fish flour. II. Further studies on availability of amino acids. Can. J. Biochem. Physiol. 41:1589. Morrison, A.B. and J.A. Campbell. 1960. Studies on the nutritional value of defatted fish flour. Can. J. Biochem. Physiol. 38:467. Morrison, A.B. and J.M. McLaughlan. 1961. Variability in nutritional value of fish flour. Can. J. Biochem. Physiol. 39:511. Morrison, A.B. and I.C. Munro. 1965. Factors influencing the nutritional value of fish flour. IV. Reaction between 1,2-dichloroethane and protein. Can. J. Biochem. Physiol. 43:33. Morrison, A.B. and 2.1. Sabry. 1963. Factors influencing the nutritional value of fish flour. II. Availability of lysine and sulfur amino acids. Can. J. Biochem. Physiol. 41:649. Morrison, A.B., Z.I. Sabry, and B.J. Middleton. 1962. Factors influencing the nutritional value of fish flour. 1. Effects of extraction with chloroform or ethylene dichloride. J. Nutr. 77:97. Muller, L.D. 1981. Feeding buffers to lactating dairy cows. Proc. Distillers Feed Conf. 36:33. 118 Munro, H.M. 1970. In: Mammalian Protein Metabolism. H.M. Munro, editor. Vol. 4, page 209. Academic Press, New York. Munro, I.C. and A.B. Morrison. 1967a. Factors influencing the nutritional value of fish flour. V. Chlorocholine chloride, a toxic material in samples extracted with 1,2-dichloroethane. Can. J. Biochem. Physiol. 45:1049. Munro, I.C. and A.B. Morrison. 1967b. Toxicity of 1,2-dichloroethane extracted fish protein concentrate. Can. J. Biochem. Physiol. 45:1779. Murley, W.R., T.W. Denton, and R.K. Waugh. 1957. A comparison of systems of feeding milk replacement formulas to dairy calves. J. Dairy Sci. 40:1258. Mylrea, P.J. 1966a. Digestion of milk in young calves. I. Flow and acidity of the contents of the small intestine. Res. Vet. Sci. 7:333. Mylrea, P.J. 1966b. Digestion of milk in young calves. II. The absorp- tion of nutrients from the small intestine. Res. Vet. Sci. 7:394. Nitsan, Z.R., R. Volcani, S. Gordin, and A. Hasdai. 1971. Growth and nutrient utilization by calves fed milk replacers containing milk or soybean protein concentrate toasted to various degrees. J. Dairy Sci. 54:1294. Nitsan, Z.R., R. Volcani, A. Hasdai, and S. Gordin. 1972. Soybean protein substitute for milk protein in milk replacers for suckling calves. J. Dairy Sci. 55:811. Noller, C.H., C.M. Ward, A.D. McGilliard, C.F. Huffman, and C.W. Duncan. 1956. The effect of age of the calf on the availability of nutrients in vegetable milk-replacer rations. J. Dairy Sci. 39:1288. Norman, E. 1971. Fish protein concentrate in milk replacers. Mimeo, Agr. Coll. of Sweden. Res. Infor. Center, Uppsala. NRC. 1978. Nutrient Requirements of Domestic Animals, No. 3. Nutrient Requirements of Dairy Cattle, 5th Revised Edition. National Academy of Sciences. National Research Council. Washington, D.C. Opstvedt, J., G. Sobstad, and P. Hansen. 1978. Functional fish protein concentrate in milk replacers for calves. J. Dairy Sci. 61:72. firskov, E.R., C. Frazer, J.G. Gill, and E.L. Corse. 1971. The effect in an intensive system of type of cereal and time of weaning on the performance of lambs. Anim. Prod. 13:485. Owen, F.G. and C.J. Brown. 1958. Interrelationship of milk temperature, dilution and curd formation in the response of calves to whole milk diets. J. Dairy Sci. 41:1534. 119 Paruelle, J.L., R. Toullec, J.P. Frantzen, and C.M. Mathieu. 1972. Utilisation du proteines per le veau preruminant a 1'engrais. -Annls. Zootech. 21:319. Patton, R.S., F.T. Chandler, and C.G. Gonzalez. 1975. Nutritive value of crab meal for young ruminating calves. J. Dairy Sci. 58:404. Pedersen, V.B. and B. Foltmann. 1973. The amino acid sequence of the first 61 residues of chymosin (Rennin E.C. 3.4.4.3). Fed. Exp. Biol. Soc. Letters 35:250. Pejic, N. and M. Kay. 1979. Soya flour in milk replacers for young calves. Anim. Prod. 28:420. Petchey, A.M., J.B. Owen, T.M. Mackie, A.B. Ritchie, and E.R. orskov. 1979. A comparison of undried and dried fish protein hydrolysate as a protein source for calf milk replacers. Anim. Prod. 28:191. Pettyjohn, J.D., J.P. Everett, and R.D. Mochrie. 1963. Responses of dairy calves to milk replacer fed at various concentrations. J. Dairy Sci. 46:710. Pfeiffer, N.E., T.C. McGuire, R.B. Bendel, and J.M. Weikel. 1977. Quan- tification of bovine immunoglobulins: comparison of single radial immunodiffusion, zinc sulphate turbidity, serum electrophoresis, and refractometer methods. Amer. J. Vet. Res. 38:693. Phillipson, A.T. 1970. Ruminant digestion. In: Duke's Physiology of Domestic Animals. M.J. Swenson, editor. Cornell University Press, Ithaca, New York. Phillipson, A.T. and J.E. Storry. 1965. The absorption of calcium and magnesium from the rumen and small intestine of the sheep. J. Physiol. 181:130. Polzin, H.W. 1978. Soy protein concentrate in milk replacers. In: Proc. 38th Annl. Mtg. Nutr. Council, Amer. Feed Mtg. Assoc., p. 16. Polzin, H.W., D.E. Otterby, J.M. Murphy, and J.B. Williams. 1976. Crude casein and meat solubles in milk replacers. J. Dairy Sci. 59:1842. Pond, W.G., W. Snyder, E.E. Walker, B.R. Stillings, and V. Sidwell. 1971. Comparative utilization of casein, fish protein concentrate and isolated soybean protein in liquid diets for growth of baby pigs. J. Anim. Sci. 33:587. Porter, J.W.G. 1969. Digestion in the preruminant animal. Proc. Nutr. Soc. 28:115. Porter, J.W.G. and W.B. Hill. 1964. Nitrogen balance trials with calves given synthetic milk diets. Nat. Inst. Res. Dairying Ann. Rept., p. 124. Poukka, R. 1966. Tissue lipids in calves suffering from muscular dystrophy. Brit. J. Nutr. 20:245. 120 Power, H.E. 1964. Characteristics and nutritional value of various fish protein concentrates. J. Fish Res. Board Council 21:1489. Preshaw, R.M., A.R. Cooke, and M.I. Grossman. 1966. Quantitative aspects of response pancreas secretion to duodenal acidification. Amer. J. Physiol. 210:629. Preston, T.R., R.D. Ndumbe, F.G. Whitelaw, and E.B. Charlenson. 1960. The effect of partial replacement of groundnut meal by white—fish meal in the diet of early-weaned calves. Anim. Prod. 2:153. Preston, T.R., F.G. Whitelaw, N.A. MacLeod, and E.B. Philip. 1965. The nutrition of the early-weaned calf. VIII. The effect on nitrogen retention of diets containing different levels of fish meal. Anim. Prod. 7:53. ‘ Pritchard, W.R., C.B. Rehfeld, and J.H. Sautter. 1952. Aplastic anemia of cattle associated with ingestion of trichloroethane extracted soybean oil meal. Clinical and laboratory investigation of field cases. J. Amer. Vet. Med. Assoc. 121:1. ' Radostits, C.M. and J.M. Bell. 1970. Nutrition of the pre-ruminant dairy calf with special reference to the digestion and absorption of nutrients: A Review. Can. J. Anim. Sci. 50:405. Ramsey, H.A. 1975. Fish flour as an ingredient of milk replacer. J. Dairy Sci. 58:741 (abstr). Ramsey, H.A. and T.R. Willard. 1975. Soy protein for milk replacers. J. Dairy Sci. 58:436. Raven, A.M. 1972. Nutritional effects of including different levels and sources of protein in milk replacers for calves. J. Sci. Food Agr. 23:517. Raven, A.M. and K.L. Robinson. 1959. Studies of milk substitutes for calves. I. The nutritional value of certain meal mixture as compared with whole milk. Res. & Exp. Record, Ministry of Agr. 8:9. Raven, A-M. and K.L. Robinson. 1965. Studies of the effect of processing and of enzyme supplementation on the utilization of maize in calf milk replacers. Record Agr. Res. 13:117. Raymond, M.N., E. Bricas, R. Salasse, J. Garnier, P. Garnot, and B.R. Dumas. 1973. A proteolytic unit for chymosin (rennin) activity based on a reference synthetic peptide. J. Dairy Sci. 56:419. Roy, J.H.B. 1964. The nutrition of intensively-reared calves. Vet. Res. 76:511. 121 Roy, J.H.B. 1969. Diarrhea of nutritional origin. Proc. Nutr. Soc. 28:160. Roy, J.H.B., I.J.F. Stobo, S.M. Shotton, P. Ganderton, and C.M. Gillies. 1977. The nutritive value of non-milk proteins for the pre-ruminating calf. The effect of replacement of milk protein by soya-bean or fish- protein concentrate. Brit. J. Nutr. 38:167. Rupel, I.W. and K.O. Wilson. 1962. De-fatted fish meal as an ingredient in milk replacers for young calves. Texas Agr. Exp. Sta. Feed Service Rept. No. 24. Schugel, L.M. 1974. Milk replacers for preruminant calves, formulations, PM problems, economics. Proc. 7th Ann. Conf. Amer. Assoc. Bovine Pract. p. 132. Seegrager, F.J. and J.L. Morrill. 1979. Effect of soy protein on intestinal absorptive ability of calves by the xylose absorption test. J. Dairy Sci. 62:972. Seegraber, F.J. and J.L. Morrill. 1980. Effect of soy protein on absorptive ability and on intestinal morphology of calves by scanning electron microscopy. 72nd Ann. Mtg. Amer. Soc. Anim. Sci., p. 394. Cornell University, Ithaca, New York (abstr). Shillam, K.W.G., J.H.B. Roy, and P.L. Ingram. 1962. The effect of heat treatment on the nutritive value of milk for the young calf. Brit. J. Nutr. 16:585. Shorrock, C. and J.E. Ford. 1978. Metabolism of heat-damaged proteins in rat. Brit. J. Nutr. 40:185. Sidwell, V.D., B.R. Stillings, and C.M. Knobl, Jr. 1970. The fish protein concentrate story: 10. U.S. Bureau of Commercial Fisheries. FPC's: Nutritional quality and use in foods. Food Technol. 24:876. Sissons, J.W. and R.H. Smith. 1976. The effect of different diets including those containing soya-bean products, on digesta movement and water and nitrogen absorption in the small intestine of the pre-ruminant calf. Brit. J. Nutr. 36:421. Sissons, J.W., R.H. Smith, and D. Hewitt. 1979. The effect of giving feeds containing soya-bean meal treated or extracted with ethanol on digestive processes in the preruminant calf. Brit. J. Nutr. 42:477. Sissons, S. and J.D. Grossman. 1956. The Anatomy of Domestic Animals. 4th ed. Sanders. Philadelphia, PA. Sleiman, F.T. and J.T. Huber. 1971. Fish protein concentrate and whey protein in milk replacer diets. J. Anim. Sci. 33:1170 (abstr). 122 Smith, R.H. 1962. Net exchange of certain inorganic ions and water in the alimentary tract of the milk fed calf. Biochem. J. 83:151. Smith, R.H. 1964. Passage of digesta through the calf abomasum and small intestine. J. Physiol. 172:305. Smith, R.H., W.B. Hill, and J.W. Sissons. 1970. The effect of diets containing soya products on the passage of digesta through the alimen- tary tract of the preruminant calf. Proc. Nutr. Soc. 29:6A. Smith, R.H. and J.W. Sissons. 1975. The effect of different feeds, including those containing soya-bean products, on the passage of digesta from the abomasum of the preruminant calf. Brit. J. Nutr. 33:329. Smith, R.H. and C.F. Wynn. 1971. Effects of feeding soya products to preruminant calves. Proc. Nutr. Soc. 30:75A. Soliman, H.S., E.R. firskov, T.M. Mackie, and T.L. Dodsworth. 1976. Utilization of fish protein hydrolysate for artificial rearing of lambs. Proc. Nutr. Soc. 35:91A. Sorenson, J. and J. Lykkeaa. 1968. Fish protein som erstatning for maelkeprotein 1 en sodmaelkerstatning (kalvmanna). Landokon. Forsogslab. Autumn Mtg. Yearbook, p. 578. St. Laurent, G.J. and G.J. Brisson. 1972. Nutritive value of FPC for young calves. Can. J. Anim. Sci. 52:585 (abstr). Stein, J.E., C.B. Knodt, and E.B. Ross. 1954. Use of special processed soybean flour and whey solubles in milk replacement formulas for dairy calves. J. Dairy Sci. 37:373. Stillings, B.R., O.A. Hammerle, and D.C. Snyder. 1969. Sequence of limiting amino acids in fish protein concentrate produced by isopropyl alcohol extraction of red hake (Urophycis chuss). J. Nutr. 97:70. Stobo, T.J.F. and J.H.B. Roy. 1977. The use of microbial protein in milk substitute diets for calves. Anim. Prod. 24:143. Stone, J.B., J.C. Rennie, and R.H. Ingram. 1963. A comparison of dif- ferent procedures for the production of veal calves. Can. J. Anim. Sci. 43:320. Sudweeks, F.M. and H.Aa Ramsey. 1972. Growth of calves fed milk replacers prepared from different fractions of fully-cooked soy flour. J. Dairy Sci. 55:705 (abstr). Sure, B. 1957. The addition of small amounts of defatted fish flour to whole yellow corn, whole wheat, whole and milled rye, grain sorghum and millet. I. Influence on growth and protein efficiency. II. Nutritive value of the minerals in fish flour. .J. Nutr. 63:409. 123 Tagari, H. and J.H.B. Roy. 1969. The effect of heat treatment on the nutritive value of milk for the young calf. 8. The effect of the pre-heating treatment of spray-dried skimmilk on the pH and the contents of total protein and nonprotein nitrogen of the pyloric outflow. Brit. J. Nutr. 23:763. Tavill, F. and A. Gonik. 1969. Use of fish protein concentrate in the diets of weanling infants. Amer. J. Clin. Nutr. 22:1571. Taylor, R.B. 1962. Pancreatic secretion in the sheep. Res. Vet. Sci. 3:63. Ternouth, J.H. 1971. Studies of the role of abomasum and pancreas in digestion in the young calf. Ph.D. Thesis, University of Reading, England. Ternouth, J.H. and J.H.B. Roy. 1973. The effect of diet and’feeding technique on digestive function in the calf. Ann. Rech. Vetér. 4:19. Ternouth, J.H., J.H. B. Roy, and S.M. Shotton. 1976. Concurrent studies of the flow of digesta in the duodenum and of exocrine pancreatic secre- tion of calves. 4. The effect of age. Brit. J. Nutr. 36:523. Ternouth, J.H., J.H.B. Roy, and R.C. Siddons. 1974a. Concurrent studies on the flow of digesta in the duodenum and of exocrine pancreatic secretion of calves. 2. The effects of addition of fat to skimmilk and of "severe" pre-heating treatment of spray-dried skimmilk powder. Brit. J. Nutr. 31:13. Ternouth, J.H., J.H.B. Roy, I.F.J. Stobo, P. Gardenton, C.M. Gillies, and S.M. Shotton. 1974b. The effect of experimental variation in the quantity of pancreatic secretion on the digestion and utilization of milk-substitute diets by the calf. Brit. J. Nutr. 32:37. Ternouth, J.H., S.Y. Thompson, and.1.Edwards-Webb. 1980. Concurrent studies of the flow of digesta in the duodenum and of exocrine pan- creatic secretion of calves. 7. Influence of milk substitutes on abomasal lipolysis and salivary secretion. Brit. J. Nutr. 44:141. Ternouth, J.H., J.H.B. Roy, S.Y. Thompson, J. Toothill, C.M. Gillies, and J.D. Edwards-Webb. 1975. Concurrent studies of the flow of digesta in the duodenum and exocrine pancreatic secretion of calves. 3. Further studies on the addition of fat to skimmilk and the use of non-milk proteins in milk substitute diets. Brit. J. Nutr. 33:181. Thivend, P., C.F.S. Clalk, E.R. Orskov, and R.N.B. Kay. 1979. Digestion of partially hydrolyzed starch in milk replacers by the young lamb. Ann. Rech. Vet. 10:422. 124 Thivend, P., R. Toullec, and P. Guilloteau. 1980. Digestive adaptation in the preruminant. In: Digestive Physiology and Metabolism in Ruminants, p. 561. AVI Publishing Company, Inc., Westpoint, Connecticut. Thomas, J.E. 1967. Neural regulation of pancreatic secretion. In: Handbook of Physiology 2:955. Amer. Physiol. Soc., Williams and Wilkins Co. Thompson, R.M., M.L. Kakade, W.E. Engelstad, M.A. Fowler, and F. Friedrich. 1974. Nutritive value of soybeans for calves. J. Dairy Sci. 57:651 (abstr). Titchen, D.A. and J.C. Newhook. 1975. Digestion and metabolism in the ruminant. Univ. New England, Armdale. Topps, J.H., R.N.B. Kay, and E.D. Goodall. 1968. Digestion of concentrate and of hay diets in the stomach and intestines of ruminants. 1. Sheep. Brit. J. Nutr. 22:261. Toullec, R. and C.M. Mathieu. 1973. Influence della composition du lait ingéré'sur 1a vidange stomocale chez le veau preruminant. Annls. Rech. Veter. 4:13. Toullec, R., P. Partureau-Mirand, J.L. Paruelle, and R. Guilhermet. 1972. Utilization of protein by the preruminant fattening calf. Aliment. Vie. 61:57. Toullec, R., P. Thivend, and C.M. Mathieu. 1971. Utilisation des proteines du lactoserum par le veau preruminant a 1' engrais. Annls. Biol. Anim. Bioch. Biophys. 11:435. Van Hellamond, K.K. 1967. A digestibility and N-balance experiment with protaminal in a milk replacer for veal calves. Wageningen Rept. 183. Wallace, C.M., W.R. Bannatyne, and A. Khaleque. 1971. Studies on the processing and properties of soy milk. II. Effect of processing conditions on the trypsin inhibitor activity and the digestibility "in vitro" of proteins in various soy milk preparations. J. Sci. Food Agric. 22:256. - Wass, W.M. 1965. The duct system of the bovine and porcine pancreas. Amer. J. Vet. Res. 26:111. Weerden, van E.J., J. Huisman, and K.K.van.'Hallemond. 1977. Verterings- fysiologisch onderzock enkele ultkomsten. Ten aanzien van het veter- ingsproces in het maagdarmkanaal van het mestkalf. Landbouwk. Tijdschr. 89:217. Wendlandt, R.M., K.E. Harshbarger, and R.D. Genskow. 1968. Growth response of calves fed milk replacers containing fish meal. J. Dairy Sci. 51:972 (abstr). ' 123 Wheeler, W.E. 1979. Starch utilization by ruminants at high feed intakes. Proc. Distillers Fed. Res. Conf. 34:53. Wheeler, W.E. 1980a. Gastrointestinal tract pH environment and the influence of buffering materials on the performance of ruminants. J. Anim. Sci. 51:224. Wheeler, W.E. 1980b. Influence of limestone buffers on nutrient utiliza- tion by ruminants. Proc. 39th Semi. Ann. Amer. Feed. Manuf. Assoc. Meet. 42. Wheeler, W.E. and C.H. Noller. 1976. Limestone buffers in a complete mixed ration for dairy cattle. J. Dairy Sci. 59:1788. Wheeler, W.E. and C.H. Noller. 1977. Gastrointestinal tract pH and starch in feces of ruminants. J. Anim. Sci. 44:131. White, A., P. Handler, E.L. Smith, and D. Stetten. 1959. Principles of Biochemistry. 2nd ed. McGraw-Hill Book Co., New York. Whitelaw, F.G., T.R. Preston, and G.S. Dawson. 1961. The nutrition of the early-weaned calf. II. A comparison of commercial groundnut meal, heat treated groundnut meal and fish meal as the major protein source in the diet. Anim. Prod. 3:127. Whitelaw, F.G., T.R. Preston, and N.A. MacLeod. 1963. The nutrition of the early-weaned calf. V. The effect of protein quality, antibiotics and level of feeding on growth and feed conversion. Anim. Prod. 5:227. Wilcke, H.L. 1969. Potential of animal, fish and certain plant protein sources. J. Dairy Sci. 52:409. Willard, T.R. and H.A. Ramsey. 1972. Effect of treating soy flour with anhydrous hydrogen chloride on its value as a milk replacer ingredient for calves. J. Dairy Sci. 55:704 (abstr). Williams, J.B. and C.B. Knodt. 1951. The supplementation of milk replace- ments with enzymes and other products. J. Anim. Sci. 10:975. Williams, J.B. and J.W. Rust. 1968. Study shows fish flour can be used in milk replacers. Feedstuffs 40:56. Williams, V.J., J.H.B. Roy, and C.M. Gillies. 1976. Milk-substitute diet composition and abomasal secretion in the calf. Brit. J. Nutr. 36:317. Wilson, K.O. 1973. The nutritive value of enzymatic-predigestion of fish protein concentrate for young calves. Dissert. Abstr. 23:5084 B. Wittenberg, K.M. and J.R. Ingalls. 1979. Utilization of fababean protein concentrate in milk substitute diets by preruminant calves. J. Dairy Sci. 62:1626. 126 Yanes, E., D. Ballesper, A. Maccioni, R. Spada, I. Barja, N. Pak, C.O. Chichesper, G. Donoso, and F. Monckeberg. 1969. Fish-protein concen- trate and sunflower prescake meal as protein sources for human con- sumption. Amer. J. Clin. Nutr. 22:878.