NUTRITIVE VALUE OF FISH PROTEiN CONCENTRATE Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY D. D. MAKDANI 1969 vv- . . '37’Q' 3.-.... , LIBRARY Michigan State University This is to certify that the thesis entitled Nutritive Value of Fish Protein Concentrate presented by D. D. Makdani has been accepted towards fulfillment of the requirements for ] __Eh..D..__ degree in —Da-:Lr.y—— / (24,44) Major professor Date /":'é—2;/7é ,9 0-169 ABSTRACT NUTRITIVE VALUE OF FISH PROTEIN CONCENTRATE BY D. D. Makdani Experiments were conducted with young Holstein calves and 21-day old weanling rats to evaluate the nutritional quality of commercially prepared fish protein concentrate. Dichloroethane or isopropanol were the extraction solvents. Investigation with young calves revealed that supplementation of methionine to the dichloroethane— extracted fish protein concentrate (DCE-FPC) had no beneficial effect on weight gains. Growth of calves con— suming 20% protein diets containing DCE-FPC or isoprOpanol- extracted fish protein concentrate (IP-FPC) was inferior to that on milk protein rations. Gains on the IP-FPC ration were inferior to those on the DCE-FPC ration. Calves consuming both fish protein sources developed microcytic, normochronic anemia which was typical of that found in protein—deficient animals. Calves fed DCE-FPC rations also showed a higher incidence of muscular dystrophy than those fed milk protein. Addition of chlorocholine D. D. Makdani chloride to the milk ration tended to depress growth but dichloroethane did not effect weight gains. Extraction of DCE-FPC with ethanol improved its nutritive value for calves, but washing with water decreased weight gains. Impaired growth was also observed on IP-FPC diets and was associated with the removal of water-soluble proteins prior to extraction with isopropanol. Improved growth in calves occurred when the DCE—FPC rations were supplemented up to twice the recommended dose of vitamin E. No alteration in weight gains was shown from vitamin E supplementation to milk protein rations. Plasma concentrations of a-toc0pherol seemed related to both level of vitamin E supplemented and source of protein. A greater depression in plasma tocopherol was noted on the DCE-FPC ration compared to the milk protein ration. It is suggested that the daily requirement of vitamin E for young calves on DCE-FPC ration is about 120 mg per 100 kg body weight. Studies with rats receiving diets containing 10% protein from various single sources showed that IP—FPC was superior for growth to casein or DCE-FPC. The nutritive value of DCE-FPC was significantly improved by extracting with methanol, ethanol or washing with water. Addition of methanolic or ethanolic extracts to casein or DCE—FPC diets significantly depressed the weight gains. Addition of chlorocholine chloride or dichloroethane to a casein diet D. D. Makdani did not alter growth responses. In diets containing 20% or 40% protein, weight gains were the same on DCE-FPC and casein suggesting that a toxic factor was not the major reason for depressed growth of rats on DCE-FPC diet. When small amounts of casein were added to DCE-FPC diets, a significant improvement in rat growth was observed. Serum protein, serum albumin and net protein utilization of rats increased in proportion to the superiority of the protein source. Plasma free amino acid levels of rats on all fish protein diets showed lower histidine concentra- tions compared to those of rats fed the casein diet. Addition of 0.075% or 0.15% L-histidine to all fish protein diets increased growth and feed intake of rats compared to unsupplemented diets. No benefit was observed by increasing hestidine level above 0.075%. Addition to fish protein diets of 0.075% L-histidine and 0.20% L— methionine, alone or in combination, resulted in improved growth. An additive effect on growth was observed with a combination of the amino acids. The protein efficiency ratio (PER) of DCE-FPC but not of IP-FPC or ethanol—extracted DCE-FPC was improved by amino acid supplementation. When both histidine and methionine were added to FPC diets, concentrations of total essential amino acids in blood plasma of rats were lowered. When histidine and methionine were supplemented, alone or in combination, their plasma concentrations were raised. Added histidine D. D. Makdani did not influence the methionine level in plasma, however, methionine alone depressed plasma histidine in DCE-FPC and IP-FPC diets but not on ethanol—extracted DCE-FPC. These results suggest that histidine and methionine were limiting rat growth in these fish protein concentrate diets regardless of the method of solvent extraction. NUTRITIVE VALUE OF FISH PROTEIN CONCENTRATE By D. D. Makdani A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1969 <.\ ~.x\ ///y a, / AC KNOWLEDGMENTS The author wishes to express his sincere apprecia- tion to his major professor, Dr. J. T. Huber, for his guidance, encouragement and understanding throughout the course of this work. Appreciation is also extended to Dr. Olaf Mickelson, Dr. R. L. Michel, Dr. E. J. Benne, Dr. H. A. Tucker and Dr. E. D. Convey for serving on his Guidance Committee and valuable advice throughout this study. The author is especially grateful to Dr. R. L. Michel for performing autOpsy on the calves and histo- pathological study. 'Appreciation is extended to the Government of Gujarat (India) and Department of Dairy for financial assistance throughout this study. The author is grateful to Mrs. Elaine Kibbey for typing the rough draft of this manuscript. The sacrifice, patience and encouragement of the author's wife, Sharda, during the course of this study is deeply appreciated. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . LIST OF FIGURES. . . . . . . LIST OF APPENDIX TABLES . . . . INTRODUCTION. . . . . . . . REVIEW OF LITERATURE . . . . . Milk Replacer for Young Calves. Fish Protein in Animal Nutrition Toxicity of Fish Protein Concentrate. Protein Deficiency and Anemia . Vitamin E and Muscular Dystrophy . Protein Quality and Amino Imbalances. . Protein Quality and Plasma Amino Acid Levels Literature Cited . . . . . PART I. FISH PROTEIN CONCENTRATE IN MILK REPLACER FOR YOUNG CALVES Abstract . . . . . . . . . Introduction. . . . . . . . Experimental Procedure . . . . Results and Discussion . . . . Literature Cited . . . . . . PART II. VITAMINE E ADDITION MILK REPLACERS Abstract . . . . . . . . . Introduction. . . . . . . Experimental Procedures . . . . Results and Discussion . . . . Literature Cited . . . . . . iii Page viii ix 10 15 l8 19 22 23 26 34 35 36 42 59 63 64 65 65 71 Page PART III. EVALUATION OF FISH PROTEIN CONCENTRATE AS A SOURCE OF PROTEIN Abstract . . . . . . . . . . . . . . 73 Introduction. . . . . . . . . . . . . 74 Experimental Procedures . . . . . . . . . 75 Results and Discussion . . . . . . . . . 80 Literature Cited . . . . . . . . . . . 95 PART IV. SUPPLEMENTATION OF HISTIDINE AND METHIONINE TO FISH PROTEIN CONCENTRATE DIETS FOR RATS Abstract . . . . . . . . . . . . . . 98 Introduction. . . . . . . . . . . . . 100 Experimental Procedures . . . . . . . . . 101 Results and Discussion . . . . . . . . . 102 Literature Cited . . . . . . . . . . . 109 SUMMARY AND CONCLUSIONS . . . . . . . . . 110 APPENDIX . . . . . . . . . . . . . . 113 iv LIST OF TABLES Table 1. Composition of milk replacer formula . . 2. Effects of extracting fish protein for 16 hours with various solvents on the methionine, histidine and cystine availability and content. . . . . 3. Ingredient composition of rations fed in Trail I O O O O C O O I O O O 4. Ingredient composition of rations used in Trail II . . . . . . . . . . . 5. Chlorocholine chloride and dichloroethane contents of fish protein concentrate used in Trial I and Trial II . . . 6. Growth and hemoglobin levels in calves fed fish protein concentrate supple- mented with methionine and glutamic acid, Trial I . . . . . . . . . 7. Effect of different sources of protein in milk replacers on body weight gains, feed consumption and protein efficiency ratio (PER) in calves, Trial II . . 8. Hematological data on calves fed different sources of protein in the milk replacer, Trial II . . . . . . . . . . 9. Changes in hematological values in calves fed different sources of protein in milk replacers, Trial II . . . . 10. Weekly weight gain changes in calves fed rations with different protein sources (expressed as % of initial weight) . . 11. Effect of chlorocholine chloride (CCC) and dichloroethane (DCE) on growth in calves (Trial III) . . . . . . . . . . Page 14 37 39 41 43 45 48 49 51 55 Table . Page 12. Body weight changes in calves fed FPC extracted with ethanol or washed with water . . . . . . . . . . . . 56 13. Ingredient composition of rations used in Trial v D O O O O O O O O O O 66 14. Growth of calves fed milk or fish protein with or without supplementation vitamin E. . . . . . . . . . . 67 15. Vitamin E concentrations in plasma of claves fed supplemented and unsupple- mented rations with different protein sources (ug/lOO ml) . . . . . . . 68 16. Ingredient composition of diets used in Experiment I. . . . . . . . . . 76 17. Ingredient composition of diets used in Experiment II . . . . . . . . . 78 18. Effect of fish flour on weight grains of rats . . . . . . . . .. . . . 81 19. Effect of fish flour on protein efficiency ratio (PER) of rats . . . . . . . 81 20. Data on growth, PER and weight of organs from rats fed different sources of protein . . . . . . . . . . . 84 21. Effect of different protein sources on weight gains, feed consumption and protein efficiency ratio in rats (10 per treatment) . g. . . . . . . . . 87 22. Essential amino acid composition of milk protein and fish protein concentrate. . 90 23. Plasma free amino acid levels in the plasma of rats fed different protein sources . . . . . . . . . . . 91 24. Net protein utilization (NPU) serum protein and its components in rats fed different sources of protein . . . . 92 vi Table 25. 26. 27. 28. Basic composition of diets used in Experiment I and Experiment II. . . Effect of adding histidine at two different levels on weight gains and protein efficiency ratio. Effect of adding combination of histidine and methionine on weight gains and protein efficiency ratio Amino acid levels in plasma of rats fed different protein sources vii (uM/lOOml). Page 101 103 106 107 LIST OF FIGURES Figure page 1. Changes in body weight of calves fed different protein sources . . . . . 52 2. Changes in hemoglobin values in calves fed different protein sources . . . . 53 3. Relationship between PER and blood protein fractions . . . . . . . . 94 viii Table 10. LIST OF APPENDIX TABLES Summary of description of muscular changes in calves fed various protein sources in milk replacer . . . . . . . Summary of description of muscular changes in calves fed FPC with vitamins E supplementation, Trial V. . . . . . Data on individual calves on Trial I . . Changes in growth and hematological values on individual calves fed milk replacers for eight weeks, Trial II . . . . . Data on individual calves on Trial III. . Data on individual calves on Trial IV . . Data on individual calves fed milk replacers supplemented or unsupple— mented with vitamin E. . . . . . . Brief description of the processes used in preparation of fish protein concentrate. Proximate composition of fish protein concentrate . . . . . . . . . . Mineral content of fish protein concentrate . . . . . . . . . . ix Page 114 115 116 117 118 119 120 121' 123 124 INTRODUCTION In recent years fish protein concentrate (FPC) has attracted attention of nutritionists as a source of protein which can help to alleviate the protein shortage of the world. Its low cost, compared to other animal protein makes it a potential source for use in underdeveloped countries. The nutritive value of FPC has been evaluated by several investigators in various species of animals. Extreme differences in its biological value have been reported in the literature. Unsatisfactory performance of young calves when FPC was used as a sole source of pro- tein was found by Huber and Slade (1967) and Wendlant at 31. (1968). However, the same authors showed that satisfactory growth resulted with FPC as a partial source of protein in the diets of young calves. Morrison and Munro (1965) suggested that the organic solvent used for extracting fish greatly influences the nutritive value of the final product. Munro and Morrison (1967) later isolated chloro- choline chloride from FPC extracted with dichloroethane and proposed that it may be one of several toxic factors present. The fact that the cattle are susceptible to the S-dichlorovinylcysteine produced in trichloroethane extracted soybean oil meal led to the speculation that young calves may be susceptible to the compounds which might result from dichloroethane extraction of FPC. Nutritional studies (Stillings, 1967) with rats have shown that FPC is comparable to casein in biological value. More recent studies by Yanez at 31. (1969) showed normal growth in infants on FPC as the only protein source. The present study was undertaken to evaluate with young calves and rats the nutritional quality of FPC pre- pared by extraction with dichloroethane or is opropyl alcohol and to find out the cause of its inferior biological value. An attempt was also made to test the effect of possible contaminants such as chlorocholine chloride and dichloroethane on performance. REVIEW OF LITERATURE Fish protein concentrate is chiefly used as a rich protein source in human as well as animal nutrition. Con- flicting reports are found in the literature on the nutri- tional value of fish protein concentrate (FPC). When used as a sole source of protein, FPC has generally been unsatisfactory for young calves. Calves on such diets become unthrifty and showed symptoms of vitamin E defi- ciency (Genskow, 1969). Dichloroethane-extracted FPC (DCE-FPC) resulted in inferior growth of rats compared to those fed casein (Morrison, 1963). Certain toxic factors in DCE-FPC have been prOposed (Munro and Morrison, 1967). However, when iSOpropyl alcohol was used as the extraction solvent for FPC fed to rats as the sole source of protein growth was superior to that shown for milk protein (Stellaman, 1969). A discription of the processes used for preparation of fish protein concentrates and further information on chemical composition is given in Appendix Table 8, 9 and 10 respectively.. A brief review of the following tOpics is presented: (1) Protein sources in milk replacers fed to young calves (2) FPC in animal nutrition (3) Toxicity of fish protein concentrates (4) Protein deficiency and anemia (5) Vitamin E and muscular dystrOphy (6) Protein quality and amino acid imbalances (7) Protein quality and plasma amino acid levels Milk Replacer for Young Calves Numerous attempts have been made to raise young dairy calves on milk replacers which would include animal or vegetable products as a source of protein. Though satis- factory gains have been obtained in calves fed a mixture of non-milk protein and milk protein, growth from non-milk protein alone has usually been unsatisfactory. Shoptaw (1936) reported that calves fed a soybean milk replacer did not show a thrifty condition and that there was some difficulty in getting the calves to relish the diet. Williams and Knodt (1950) observed that when raw soybean meal constituted 40 percent of the replacer, all calves died. Studies by Noller gt a1, (1956) showed that the ability of the calves to utilize the soy protein increased after approximately 25 days of age. Lassiter gt 31. (1959) reported that the rates of growth decreased significantly as the amount of dried skim milk in milk replacer decreased and the amount of soybean meal increased. They also observed that adding proteolytic enzymes to the replacers did not improve the utilization of soybean protein. Stein and Knodt (1954a) and Stein gg g1. (1954b) observed that soybean flour could effectively provide a major portion of the protein in a milk replacer containing over 30 percent protein when dried skim milk and whey provided the remainder. When dried skim milk provided less than 43 per cent of the total protein, appetite was depressed and growth was retarded. They also reported that addition of 0.05 and 0.25 percent DL—methionine did not affect growth. Borchers (1961) reported that calves fed soybean meal supplemented with methionine, threonine and valine resulted in higher gains than unsupplemented meal. Colvin and Ramsey (1968) found that predigesting fully-cooked soy flour with various proteolytic enzyme preparations did not improve its nutritive value. However, acid digestion of the soy flour at a pH of 4.0 for five hours at 37°C mark- edly improved gains of calves. Later they (Colvin and Ramsey, 1969) also showed that alkali—treatment of soy flour was equally beneficial for improving calf growth as was acid treatment. Brumbaugh and Knodt (1952) reported that the growth of calves was poor when milk replacer contained less than 35 percent dried skim milk in combination with dried blood meal, dried whey, distillers solubles and soybean meal. Archibald (1928) found that the calves fed a cereal milk replacer were unthrifty and pot-bellied with a higher incident of diarrhea than those fed milk. Williams and Jensen (1955) reported average daily weight gains of only 0.4 lb in calves fed a milk replacer containing 50% dried skim milk, 2% dried rumen contents and 4% blood meal as the protein sources. Slade (1965) obtained extremely poor results in calves fed concentrated rumen fluid as the pro- tein source in milk replacers. Corn distillers dried solubles were substituted for 20, 35 and 55 percent, reSpectively, of dried skim milk and lactose in milk replacers and weight gains of calves decreased with increased levels of the corn solubles (Bryant g5 g1., 1963). Brown and Varnell (1962) tested raw eggs as partial replacement for the nutrients in the whole milk. They con- cluded that the calves could not utilize eggs when fed only with water. Cooking of eggs did not improve the growth. Calves were in negative nitrogen balance when they received only eggs. Weight gains were higher in calves receiving whole milk compared to those on a ration of two eggs plus milk. The poor utilization of eggs by young calves is very interesting in light of the fact that egg protein is con— sidered superior to milk for rats and other simple-stomach animals. The reason for the inferiority of eggs to milk protein in calves is not known but it may be related to the amino acid sequence or spatial configuration of the protein molecules. The inability of the calf to grow well on egg protein draws an interesting parallel to the performance reported on fish protein, which is also considered a high quality protein source for animals other than the young calf. Dichloroethane-extracted fish protein concentrate (DCE-FPC) has been used as an ingredient in milk replacers by several investigators and varying results have been obtained (RUpel and Wilson, 1962; Huber and Slade, 1967; Genshow g5 g1., 1968, Wendlandt g2 g1., 1968). In the studies at Texas A and M, Rupel and Wilson (1962) conducted feeding trials involving 100 Holstein calves. The protein _in the milk replacer formulas was furnished by 0, 12, and 24 percent DCE-FPC and 55, 23 and 0 percent dried skim milk, respectively: Calves on the dried skim milk diet gained 0.63 lb per day while those on 12 percent DCE-FPC and 23 percent dried skim milk gained 0.82 lb per day. Calves receiving DCE-FPC as the only protein source in the milk replacer did not gain during the first 21-day period. There- after, they gained considerably less than the control group. In a series of experiments at Virginia by Huber and Slade (1967), DCE-FPC furnished 0, 20, 33, 40, 60, 66 and 100 percent of the protein in the milk replacers. An ingredient composition of some of the replacers is listed in Table 1. Average daily gains and feed efficiencies were not significantly depressed when FPC furnished up to 40% of the dietary protein. However, when DCE-FPC furnished 60% or more of the protein a significant decrease in TABLE 1.--Composition of milk replacer formula. Ingredients Percent crude protein as FPC 0 20 40 60 100 Dried skim milk 63.9 50.4 37.9 25.2 0 Lactose 26.0 33.6 40.3 47.0 61.0 Fish flour - 5.9 11.7 17.7 28.9 Emulsified lard oil 10.0 10.0 10.0 10.0 10.0 growth rate occurred. In the same study when 100% of the protein came from FPC, the calves became listless, emaciated, refused the ration during the third or fourth week of the treatment and died shortly thereafter. The balance studies showed that digestibilities of dry matter, crude protein, ether extract and ash decreased as a level of DCE—FPC in milk replacers increased. The digestibility of protein in DCE—FPC was reported to be 80% compared to 90% for skim milk protein. van Hellemond (1967) conducted a balance study with isoprOpanol-extracted fish protein concentrate (IP-FPC) and reported a higher apparent digestibility for fish protein (88.5%) than reported by Huber and Slade (1967). However, these studies were conducted on older animals which weighed from 53 to 139 kg. van Hellemond (1967) also observed that the IP-FPC in the reconstituted milk replacer tended to settle at the bottom of the feeding pail and necessitated stirring while being fed to the calves. Wendlandt gg g1. (1968) reported that when DCE-FPC furnished 25% protein (100% of the total protein in the diet) in a milk replacer, the weight gains incalves were significantly lower than those of calves in the milk-fed control group. However, when FPC furnished 12.5% protein (50% of the total protein in the diet), weight gains were comparable to the controls. A higher death loss of calves on milk replacers where DCE-FPC furnished all the protein was also reported. In the same study a digestion trial was carried out using two calves per group at 3 weeks of age. Digestibility of crude protein decreased with increased levels of DCE—FPC in the replacer. Williams and Rust (1968) compared milk replacers containing 10, 5, 10 and 15% DCE-FPC. Dried skim milk was added at 47.0, 64.5, 57.0 and 43.5% to the reSpective rations. Satisfactory gains on all the above replacers for veal calves were reported. Preston gg‘gl. (1960, 1965) did not find a signif- icant difference between milk replacers containing ground- nut meal and groundnut meal plus fish meal. However, calves on milk replacer containing groundnut meal and fish meal gained 6 lb more in an 81 day trial. In another trial comparing groundnut meal and fish meal Whitelaw EE.E£° (1961) observed that nitrogen retention was higher in calves on a fish meal replacer than for those on groundnut meal. 10 Fish Protein in Animal Nutrition Fish protein concentrate has been investigated as a protein source for the monogastric animals including human beings. Bender and Haizelden (1957) examined 27 samples of fish meal and deodorized fish flour for net protein utili- zation (NPU). The NPU's ranged from 18 to 80. The low values were thought to be due to maltreated fish meal, but the defatting and deodorizing process was not associated with poor performance. The Nutrition Division of the Food and Agricultural Organization of the United Nations (1958) reported that the proteins of fish muscle are equivalent in biological value to those of animal muscle and milk. If processed prOperly, fish flour was thought to retain a biological value equal to the original material. Ousterhout gg g1. (1959) examined several samples of fish meal for the availability of amino acids by chick assay and found that arginine, methionine, cystine, histidine and threonine were less available than indicated by their total content in fish meal. Jaffe (1961) obtained a 2.48 protein efficiency ratio (PER) for fish meal fed to rats. The PER of wheat meal was raised from 0.23 to 1.08 by adding 2% fish meal and to 2.90 by adding 7.5% fish meal. In a study with groups of undernourished infants, Graham EE.§1° (1962) obtained comparable weight gains from 11 diets containing fish flour, a wheat—fish flour mixture, a vegetable mixture or modified cow's milk. NitrOgen retention for the vegetable mixture was slightly lower than that of diets with fish or milk protein, probably reflecting poorer digestion and absorption. In 10% pro- tein diets fed to rats of the vegetable mixture Johnson g3 g1. (1962) evaluated a highlyepurified fish flour pre- pared by the VioBin Company in two ways: (1) Protein efficiency ratio and (2) Mitchell biological value. The PER values for fish flour protein was 3.24 and were super- ior to those observed from milk protein (2.85) or beef protein (3.15). Biological value of the fish protein concentrate was 88 percent which was equivalent to that of milk protein. When supplemented at 1 and 3 percent of the diet, fish flour significantly improved the PER of veget- able protein. Deuel g3 g1. (1946) reported considerably higher biological values for fish muscle protein than casein. Fish protein caused a greater recovery in weight gains of rats and a more pronounced stimulation in hemoglobin regeneration. Fish flour was reported by Sure (1957) to be a better protein supplement to corn meal fed as the basal protein source to rats than non-fat milk solids, dried butter milk, defatted soybean flour, brewer's yeast, cultured food yeasts or peanut meal. Addition of 5% fish flour to a corn 12 meal diet produced a lS-fold increase in growth of rats as compared to unsupplemented corn meal. Ferreira (1966) found the limiting amino acids in fish meal were cystine and methionine. Values were 45 to 55 percent, respectively, as high for egg protein as they were in fish meal. Smith g3 g1. (1962) concluded that methionine was the first and arginine the second limiting amino acids in the two fish meal samples studied. In further studies with fish meal, Smith g3 g1. (1965a, 1965b) reported that the addition of 0.25% DL-methionine increased weight gains of rats fed all the fish meal samples used, indicating an overall deficiency of sulfur amino acids in fish proteins. Heat—treated fish meal showed lack of availability of lysine and threonine. Recently, Stillings g3 g1. (1969) carried on a series of experiments with rats to determine the sequence of limit— ing amino acids in iSOpropanol-extracted FPC. The results of his studies have shown methionine to be the most limit— ing amino acid followed by the groups of: (l) histidine, tryptophan and threonine, (2) valine,isoleucine and phenylalanine and (3) leucine, lysine and arginine. In a report on nutritive value of FPC, Stillings (1967) has noted higher value of PER for isopropanol—extracted FPC as compared to casein. Morrison and Munro (1965) reported that the solvent used for extraction of fish greatly influences the nutentive value of fish flour obtained. They found that fish flour extracted with 13 isopropanol was superior to that extracted with ethylene dichloride when tested at a critical level in rats. It was also shown that fish flour extracted with ethylene dichloride contained less available lysine and methionine than that extracted with isopropanol. In further studies with FPC, Morrison (1963) supple- mented DCE-FPC with methionine, histidine, threonine and tryptOphan. When all four amino acids were added to the fish flour diets, weight gains of rats increased signif- icantly. When histidine and methionine were omitted from the diets, weight gains were similar to unsupplemented fish flour and the combination of histidine and methionine was as effective as the combination of the four amino acids. In further studies on the solvent used for extraction of the fish protein, Morrison and Munro. (1965) found that 1,2-dichloroethane reacts with the free sulfhydryl group of cystine to yield S,S'-ethy1enebiscysteine. Munro and Morrison (1967) reported that this reaction product was not toxic to rats. The relationship between the type of solvent used for extraction of fish flour and the amount and availability of the amino acids to enzyme hydrolysis was studied by Morrison and Munro (1965). The results showed that the solvent used markedly influenced the amount and availability of amino acids present in fish flour. (Table 2). Total methionine values were not affected by any of the solvents. However, histidine and cystine reduced when 14 mm.o om.o mH.H mm.m mm.H v.m Hocmcum mo.o vm.o mm.o m>.H mv.o ~.m mpflHoHcoHp mamahcum 04.0 ma.o mH.H mm.~ mm.H m.m Hoemmoum0ma Nv.o No.0 mm.o om.m mn.H H.m wcmxmm mv.o no.0 «N.H nm.~ mm.m w.m sz mumm open open even dump wumm I>HOH©>£ Imaoupwn Iwaoupmc I>HOHU>£ Ihaoupxz I>HOHU>£ mewncm pflom mahncm pflog mfiwncm mmod z m ms\m .mcnumso z m GH\m|.mcaeflumHm z m ma\m .mcflcOAeumz ocm>aom .ucmucoo paw auflaflemafim>w mcaummu can mca mucm>H0m mscflum> rugs mason ma How cflmuoum swam m Home: .mcflcoflcums we» so cfluomuuxm mo muowmmmuu.m mqmda 15 ethylene dichloride was used as extraction solvent. In another experiment it was shown that methionine, histidine and cystine on enzyme hydrolysate prepared from FPC drOpped in proportion to the duration of the time of extraction (Morrison and Munro, 1965). In a study on the effect of method of processing of fish meal on the nutritive value of herring meals, Clandinin (1948) found that meals of equal nutritive value can be produced by either the vacuum or flame methods of drying. Further results showed that meals of equivalent nutritive value were obtained at 10, 14 and 16 inches vacuum but meals dried by flame method at a temperature of 220°F were decidedly inferior to vacuum dried meals or to meals dried by the flame method at a temperature of 185°F. Toxicity of Fish Protein Concentrate Speculation about the toxicity in protein concentrates extracted with organic solvents probably arose from the findings that trichloroethane (TCE) extracted soybean meal was found to be toxic to cattle. This toxicity was due to S-(l,2—dichloroviny1)-L—cysteine (DCVC) which is pro- duced in soybean meal when TCE was used for extraction. Trichloroethane-extracted soybean meal produced aplasticanemia when fed to cattle (Picken gg gl., 1952, Pritchard g5 g1., 1952 and Sautter g3 21:! 1952). The solvent, TCE, by itself was not toxic because cattle are able to convert a large part of an ingested dose to l6 metabolites such as urochloric acid and trichloroacetic acid which are excreted through urine. Schultze g5 g1. (1962) reported that rats are resistant to DCVC. Waibel gg gl. (1967) demonstrated that the turkey, in contrast to the calf, is highly resistant to toxic effects of DCVC. They further reported that the liver and kidney of turkeys have considerably higher activity of DCVC—lyase than the corresponding bovine tissues. In 1962, Morrison g3 g1. observed a toxic effect from a fish flour sample fed to rats to furnish a diet of 20% protein level. The fish flour was prepared by extraction with iSOpropyl alcohol and the bones were partially removed by flolation in chloroform. In a feeding trial using this source of FPC feed intakes at 20% protein were decreased compared to those at 10% protein diet. Weight gains of rats which normally would be superior at 20% protein were not different from those of rats fed 10% fish protein. Renal and hepatic hypertrophy were also observed in rats receiving the 20% diet. In a later study when this fish flour was further extracted with diethyl ether, growth in rats improved. It was concluded that deleterious factor in fish flour was residual chloroform. Recently, Munro and Morrison (1967) showed that the chlorinated solvents react with fish protein to form chlo- rinated compounds which can be toxic to the animals. Two 17 such compounds thought to be produced are S-S'—ethy1enebis- cysteine and chlorocholine chloride (CCC). Very little is known about the toxicity, biochemistry or the presence of S-S'—ethylenebiscysteine in fish flour thus prepared. The other compound (CCC) results from the reaction of dichlo— roethane with trimethylamines (TMA) which are found in raw fish. Fish are known to contain 2 to 5% TMA (Dyre, 1952). Munro and Morrison (1967) reported that the fish flour samples extracted for 24 hrs with dichloroethane contained 400 mg/kg of CCC and when the FPC was fed as 50% of the total diet for rats, mortality was 100%. No mortality resulted when the same FPC was further extracted with methanol. Preliminary studies with rats had showed that the oral LD of CCC was 500 mg/kg body weight. Thus, a 50 100 g rat would need to consume 50 mg CCC daily to attain this level. A diet containing 20% crude protein, all fur— nished from DCE-FPC, would supply only about 1.5 mg CCC daily which is only 3% of the lethal dose. The effect of temperature and duration of extraction on the residual DCE content in fish flour was studied by Ershoff (1967). He observed that when fish flour was extracted for 24rumsat 87°C, the residual DCE was 30,000 ppm. However, when the fish were extracted for 45 minutes at 87°C, only 13 ppm of DCE were present. Also, when fish flour was extracted for 241nm» but at lower temper- ature (40°C), the residual DCE content was only 17 ppm. 18 A significant depression in growth of rats was noted with fish flours extracted at both the higher temperature for the longer period of time. Proggin Deficiency and Anemia Anemia is defined as reduction of hemoglobin in blood. Hemoglobin is a unique protein which makes up about 1% of the body weight. It is estimated that 2.16 x 1011 red blood cells are destroyed and replaced each day in the adult human male. This would mean that 6 g hemo- globin must be produced every day. It is therefore quite logical to conclude that a shortage of protein might cause a subnormal red cell production. Whipple (1942) stated that the hemoglobin formation takes priority over the formation of other proteins and that the body protein store must be greatly reduced before the reduction in hemoglobin formation occurs. However, Platt (1956) thinks that the need for formation of new red cells represents a consider- able proportion of the total body requirement for protein. Robscheit—Robbins and Whipple (1955) have shown that a shortage of protein influences erythropoiesis. Hallgren (1953) investigated a low protein diet compared to one containing 22% egg albumin, and concluded that the animals on low protein diet developed a microcytic, normochromic anemia and that the fall in total hemoglobin on low protein was much more marked. The animals used in these studies were growing rats and competetion for protein by tissue for growth would be at a maximum. 19 Fey and Kondi (1957) suggested that the treatment of some anemias with iron alone is not sufficient to raise the hemoglobin level unless an adequate protein is also provided. Knowles (1957) found marked reduction in hemoglobin con— centration in pigs fed a low protein diet. Anemia in pigs on a low protein diet was alleviated when they were trans- ferred to the diets of higher protein value. Orten and Orten (1946) observed a parallelism between the hemOpoietic value of the protein and its ability to support somatic growth in rats. Various proteins fed at as low level as 2.8% resulted in a mild to moderate anemia in all cases observed. Vitamin_E and Muscular Dystrophy It has long been known that cod liver oil added to calf diets of skim milk, produced a severe muscular disease (Blaxter g£_gl., 1952 and Blaxter EE.El" 1953). Blaxter g5 g1. (1953) also reported that the same disease could be produced in calves by giving them lard oil as a source of fat and it could be prevented by giving them very large amounts of a-tocopherol in addition to the fats. Moore 33,31. (1959) reported that cod liver oil contains 100 to 200 mg/kg of vitamin E. However, this amount is insufficient to protect against the highly unsaturated fatty acids it contains. Adams gg g1. (1959a) and Adams g3 g1. (1959b) have reported that in addition to lard oil and cod liver oil as anti-vitamin E oils, maize oil added to the diet of 20 skim milk produced clinical and histological signs of muscular dystrOphy in calves. In these experiments, the feeding of skim milk alone, hydrOgenation of the maize oil or addition of tocopherol to the maize oil diets prevented muscular dystrophy. Certain synthetic antioxidants structurally unrelated to the tocoperhols were shown to protect vitamin E from oxidative distruction. These were N,N'-dipheny1-p-pheny1enediamine, (Draper g5 g1., 1956) methylene blue (Draper g5 g1., 1958) and ascorbic acid (Blaxter g2 g1., 1953). Cawthorne g_t_ g1. (1968) have suggested that since the toc0pherols are biological antioxidants they also prevent auto-oxidation of vitamin A particularly in the gastro- intestinal tract. Consequently, the calves showing vitamin E deficiency, might also show vitamin A depletion despite normally sufficient dietary levels. Hardin and Hove (1951) have shown that a methionine deficiency is aggravated by rations deficient in vitamin E. Blaxter gE g1. (1953) reported that the muscular dystrOphy induced by diets containing high levels of poly- unsaturated fats can be prevented by giving large amounts of vitamin E, but it is unresponsive to Se. Schwartz and Foltz (1957) observed that Se. is an essential trace element for several species and that it can function as a partial alternative to toc0pherol. Thomas and Okamoto (1955) have indicated that when lard oil was included in milk 21 replacer as a source of fat, plasma tocopherol levels were low compared to those of calves fed whole milk. They also observed that diarrhea in calves causes marked decrease in plasma tocopherol. - Maplesden and Loosli (1960) attempted to produce muscular dystrOphy in calves under laboratory conditions by giving diets of very low vitamin E content and virtually free of fat. From this experiment it was implied that in the absence of unsaturated fat, a very long period on the diet is necessary to produce pathological changes. It has been clearly established that muscular dystrophy symptoms in calves and lambs are seen in severe vitamin E deficiency. The mechanism of action of vitamin E is not understood; however, it has been hypothesized that vitamin E is a physiological antioxidant, preventing the accumulation of undesirable metabolic oxidation products in animal tissues. These products are thought to be lipid peroxides which, in absence of vitamin E or other antioxidants, can break down to give free radicals that react with sensitive cell structures and disrupt the cellular frame (Bunyan g; 31., 1962). Sandlers and Bird (1967) have implicated that there is an indirect relationship between toc0pherol and the sulfhydryl groups on enzymes which are supposedly protected by the antioxidant action of vitamin E. 22 Protein Quality and Amino Imbalances As early as 1914 Osborne and Mendle observed that certain amino acids were essential nutrients and that pro- teins differed in their content of amino acids. Therefore, the nutritive value of protein depends upon its amino acid composition. Later Block and Mitchell (1946) showed that by comparing the amino acid composition of one protein with that of whole egg protein, one can obtain a fairly good estimate of the biological value of that protein. They calculated which essential amino acid was in greatest deficit as compared to whole egg protein and observed that there was a high correlation between the amino acid showing the greatest deficit in that protein and its biological value. Almquist (1954) demonstrated that the efficiency with which a protein can be used for body protein synthesis depends upon the quantity and prOportion of constituent amino acid. Proteins which fall far from the correct pro- portion are termed as unbalanced. Harper and Kumta (1959) have pointed out that the efficiency of utilization of a protein depends upon the actual quantities of essential amino acids a protein contains as well as the proportion in which they occur. The term "amino acid imbalance" according to Harper (1964) can refer to a deficient protein source which is made more deficient by addition of an amino acid(s) other than limiting one, thus resulting in growth retardation 23 much greater than that caused by original deficient protein. The addition of small quantity of limiting amino acid to an imbalanced ration will prevent the growth retardation. Harper (1964) has indicated two types of amino acid imbal— ance. One type results from addition of a protein or a mixture of amino acids lacking in one essential amino acid to a diet containing low or moderate amounts of protein; the other type results from addition of a small amount of amino acid or amino acids to a diet that is low in protein. Such type of addition would cause substantial depression in growth rate. Yoshida EE.El° (1966) have put forward a hypothesis eXplaining the causes for retardation of growth on a diet imbalanced in amino acids. They have suggested that an imbalance leads to more efficient incor- poration of the growth-limiting amino acids into tissues with the result that its concentration in blood plasma decreases within few hours after ingestion of the imbal- anced meal. This phenomenon results in a signal to an appetite regulating centre. Food intake is subsequently depressed and the food intake depression results in retarded growth. Pgotgin Qualigy and Plagma Amino Acid Levels It has long been reCOgnized that the concentration of amino acids in the portal view is greater than those in the jugular vein after ingestion of protein. Denton and Elvehjem (1954) measured free amino acids in plasma of portal 24 and systemic blood of dogs fed zein, casein or beef. Plasma concentration of amino acids increased rapidly when casein or beef were fed and the increase was greater in portal than in systemic blood. Concentration of most of the amino acids decreased when zein was fed. Guggenheim g3 g1. (1960) have also investigated the relationship between plasma amino acid levels and availability of amino acids in protein. They fed rats gluten, zein, soybean protein, casein or lactalbumin. Lysine and methionine were measured in the plasma from the portal vein of the animals. The amounts of these amino acids increased in prOportion to their presence in the protein. McLaughlan (1963) concluded that the concentration of plasma amino acids (PAA) increased after a meal of good quality protein. The extent and duration of the increases were related to both the amount and composition of the protein fed. Zimmerman and Scott (1965) reported that low PAA does not necessarily indicate the deficiency of that amino acid for body functions. They demonstrated that in chicks the plasma amino acid levels did not increase significantly till the dietary intake of amino acid exceeded the amount needed for optimal growth. McLaughlan (1963) demonstrated that PAA levels were not always directly proportional to amino acid levels of the dietary proteins and that PAA levels could not be used directly to predict the nutritional quality of various proteins. 25 Various methods have been proposed to determine the limiting amino acid of the dietary proteins using plasma amino acid levels. Longnacker and Hause (1959) proposed the following formula to calculate the limiting amino acid. B-A R PAA-R x 100 where B - plasma essential amino acid (EAA) concentra- tion after fasting 18 hrs A - plasma BAA concentration in five consecutive blood samples drawn after feeding R - The requirement of the EAA expressed as grams of amino acids per 16 grams of nitrogen, (NRC,1962). For each EAA, a plasma amino acid ratio (PAA-R) is calculated. The lowest ratio indicates the limiting amino acid of the dietary protein. McLaughlan (1964) proposed another method for cal- culating limiting amino acid. In this method, the EAA acid requirement of the experimental animal does not have to be known. PAA level of the protein fed animals PAA's = x 100 PAA level of the fasted animals The lowest PAA score indicates the limiting amino acid. LITERATURE CITED Adams, R. 8., T. W. Gullickson, J. E. Gander and J. H. Sautter. 1959a. Some effects of feeding filled milks to dairy calves. I. Physical condition and weight gains with special reference to low fat ration. J. Dairy Sci. 42:1552. Adams, R. S., J. H. Sautter, T. W. Gullickson and J. E. Gander. 1959b. Some effects of feeding filled milks to dairy calves. IV. Necropsy findings, electro- cardiographic studies and creatinuria ratios. J. Dairy Sci. 42:1580. Almquist, H. J. 1954. Principles of amino acid balance Arch. Biochem. 52:197. - Archibald, J. G. 1928. Calf meal studies. 2. Feeding experiments with cooked and uncooked meal. J. Dairy Sci. 11:119. Bender, A. E. and S. Haizelden. 1957. Biological value of proteins of variety of fish meals. Brit. J. Nutr. 11:42. Blaxter, K. L., W. A. Wood and A. M. MacDonald. 1953. The nutrition of young Ayrshire calf. II. The toxicity of cod liver oil. Brit. J. Nutr. 7:34. Blaxter, K. L., P. S. Watts, and W. A. Woods. 1952. The nutrition of young Ayrshire calf. 8. Muscular dystrophy in growing calf. Brit. J. Nutr. 6:125. Block, R. J. and H. H. Mitchell. 1946. The correlation of the amino acid composition of proteins with their nutritive values. Nutr. Abs. & Rev. 16:249. Borchers, R. 1961. Counteraction of the growth depression of raw soybean oil meal by amino acid supplementation in weanling rats. J. Nutr. 75:330. 26 27 Brown, D. C. and T. R. Varnell. 1962. Value of raw eggs in the ration of young calf. J. Dairy Sci. 45:1394. Brumbaugh, J. H. and C. B. Knodt. 1952. Milk replacement for dairy calves. J. Dairy Sci. 35:336. Bryant, J. M., C. F. Foreman, N. L. Jacobson and N. D. McGilliard. 1963. Utilization of protein and energy in milk replacers by dairy calves. J. Dairy Sci. 46:643. Bunyun, J., A. T. Diplock, E. E. Edwin and J. Green. 1962. Exudative diathesis and lipid peroxidation in the chick. Brit. J. Nutr. 16:519. Cawthorne, M. A., J. Bunyan, A. T. Diplock, E. A. Murrel and J. Green. 1968. On the relationship between vitamin A and vitamin E in the rat. Brit. J. Nutr. 22:133. ‘ Clandinin, D. R. 1948. The effect of method of processing on the nutritive value of herring meals. Poultry Sci. 28:128. Colvin, B. M. and H. A. Ramsey. 1968. Soy flour in milk replacer for young calves. J. Dairy Sci. 51:898. Colvin, B. M. and H. A. Ramsey. 1969. Growth of young calves and rats fed soy flour treated with acid or alkali. J. Dairy Sci. 52:270. Denton, A. E. and C. A. Elvehjem. 1954. Amino acid con- centration in portal vein after ingestion of amino acid. J. Biol. Chem. 206:455. Geiger, E. and G. Schnakenberg. 1946. Studies on nutri— tive value of fish proteins. J. Nutr. 31:175. Draper, H. H., S. Goodyear, K. D. Barbee and B. C. Johnson. 1958. A study of the nutritional role of antioxidants in the diet of rat. Brit. J. Nutr. 12:89. Draper, H. H. and B. C. Johnson. 1956. N,N'-dipheny1-p- phenylenediamine in the prevention of vitamin E deficiency in lambs. J. Animal Sci. 15:1154. Ershoff, B. H. 1967. Animal studies on the nutritional value of fish protein concentrate. Institute of Nutritional Studies, Culver City, Calif. Ferreira, M. F. 1966. Available lysine in fish meal. Nutr. Abs. Rev. 36:46. 28 Foy, H. and A. Kondi. 1957. Anemia of tropics. Relation to iron intake, absorption and losses during growth, pregnancy and lactation. J. TrOp. Med. Hyg. 60:105. Genskow, R. D. ‘1969. Evaluation of a 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. Observation on vitamin deficiencies in the calves fed a milk replacer containing low ash fish protein concentrate. J. Dairy Sci. 51:972. Graham, G. G., J. M. Baertt and A. Cordano. 1962. Evalua— tion of fish flour in the treatment of infantile mal- nutrition. Fish in Nutrition. Edited by Heen, E. and R. Kreuzer. Fishing News Ltd., London. ‘ Guggenheim, K., S. Halevy and N. Friedmann. 1960. Levels of lysine and methionine in portal blood of rats follow— ing protein feeding. Arch. Biochem. BiOphys. 91:6. Hardin, J. O. and E. L. Hove. 1951. Prevention of DL- methionine toxicity in rats by vitamin E, B , folacin, glycine and arginine. Proc. Soc. Expt. Bio . Med. 78:728. Harper, A. E. and U. S. Kumta. 1959. Amino acid balance and protein requirement. Fed. Proc. 18:1136. Huber, J. T. and L. M. Slade. 1967. Fish flour as a pro- tein source in calf milk replacer. J. Dairy Sci. 50:1296. Jaffe, W. G. 1961. Food value of fresh fish, preserved fish and fish meal. Nutr. Abst. and Rev. 31:3613. Johnson, B. C., V. C. Melta and H. E. Schendel. 1962. The nutritive value of fish flour and its use as a protein supplement. Fish in Nutrition. Edited by Heen, E. and R. Kreuzer. Fishing News Ltd., London. Knowles, C. B. 1957. Protein nutrition in pigs. Proc. Nutr. Soc. l6:ix. Lassiter, C. A., G. F. Fries, C. F. Huffman and C. W. Duncan. 1959. Effect of pepsin on the growth and health of young dairy calves fed various milk replacer rations. J. Dairy Sci. 42:666. 29 Maplesden, D. C. and J. K. Loosli. 1960. Nutritional muscular dystrOphy in calves. II. Addition of selenium and toc0pherol to a basal dystrOphogenic diet containing cod liver oil. J. Dairy Sci. 43:645. McLaughlan, J. M. 1963. Relationship between protein quality and plasma amino acid levels. Fed. Proc. 22:1122. Moore, T., I. M. Sharman and R. J. Ward. 1959. Cod liver oil as both source and antagonist of vitamin E. Brit. J. Nutr. 13:100. Morrison, A. B. 1963. Factors influencing the nutritional value of fish flour. III. Further studies on avail— ability of amino acids. Can. J. Biochem. and Physiol. 41:1589. Morrison, A. B. and I. C. Munro. 1965. Factors influenc- ing the nutritional value of fish flour. IV. Reaction between 1,2—dichloroethane and protein. Can. J. Biochem. 43:33. Munro, I. C. and A. B. Morrison. 1967. Factors influenc- ing the nutritional value of fish flour. V. Chloro- choline chloride, a toxic material in samples extracted with 1,2-dichloroethane. Can. J. Biochem. 45: NRC. 1962. Nutrient requirements of laboratory animals. National Academy of Sciences. National Research Council Publication 990. Noller, C. H., C. F. Huffman and C. W. Duncan. 1956. Dried whey and lactose as supplements to a vegetable milk replacer. J. Dairy Sci. 39:992. Nutrition Division, F.A.O. of United Nation. 1958. The use of fish flour as human food. Proc. Nutr. Soc. 17:153. Orten, J. M. and A. U. Orten. 1946. Proteins and hemo- poiesis in rats. J. Nutr. 31:765. Ousterhout, L. E., C. R. Grau and B. D. Lundholm. 1959. Biological availability of amino acid in fish meals and other protein sources. J. Nutr. 69:65. Picken, J. C., H. C. Beister and C. H. Covault. 1959. Trichloroethane extracted soybean oil meal poisoning. Iowa State College Vet. 14:137. 30 Platt, B. S. 1956. Diet and anemia. Nutr. Soc. Proc. 15:103. Preston, T. R., R. D. Ndumlee, F. G. Whitelaw and E. B. Charleson. 1960. The effect of partial replacement of groundnut meal by white—fhish meal in the diet of early weaned calves. Animal Prod. 2:153. Preston, T. R., F. G. Whitelaw, N. A. Macleod and E. B. Philips. 1965. The nutrition of the early weaned calf. VIII. Effect of N-retention of diets containing different levels of fish meal. Animal Prod. 7:53. Pritchard, W. R., C. E. Rehfeld and J. H. Sautter. 1952. Aplastic anemia of cattle associated with ingestion of trichloroethane extracted soybean oil meal. J. Am. Vet. Med. Assoc. 121:1. Robscheit-Robbins, F. S. and G. H. Whipple. 1955. Red cell stroma in dogs. Stroma protein and stroma lipids vary in different types of anemia. J. Exp. Med. 102:705. Rupel, I. W. and K. O. Wilson. 1962. Defatted fhish meal as an ingredient in milk replacers for young calves. Texas Agr. Exp. Sta. Feed Serv. Reprint No. 24. Sandler, N. and J. W. C. Bird. 1967. Relationship between vitamin E and certain enzymes dependent on sulphydryl group. .Nature 216:608. Sautter, J. H., C. E. Rehfeld and W. R. Pritchard. 1952. Aplastic anemia of cattle associated with ingestion of trichloroethane extracted soybean oil meal. J. Am. Vet. Med. Assoc. 121:73. Schultze, M. 0., R. F. Derr, N. S. Mizuno, D. D. Joel and J. H. Sautter. 1962. Effect of phenylalanine on toxicity of S—(dichlorovinyl)-L-cysteine in the rat and the calf. Proc. Soc. Exp. Biol. Med. 111:499. Schwarz, K. and C. M. Foltz. 1957. Selenium as an integral part of factor 3 against dietary neurotic liver degeneration. J. Am. Chem. Soc. 79:3292. Shoptaw, L. 1936. Soybean flour as a substitute for cow milk in feeding dairy calves. J. Dairy Sci. 19:95. Slade, L. M. 1965. Nutritive value of fish flour as a protein source in calf milk replacers. M. S. Thesis. Virginia Polytechnic Institute, Blacksburg. 31 Smith, H. M., W. F. Dean, A. Aguilera and R. E. Smith. 1962. Quality of fish meal in relation to its value as a supplement to corn-soybean meal chick diets. Fish in Nutrition. Fish News Litd., London. Smith, R. E. and H. M. Soctt. 1965a. Biological evaluation of fish meal proteins as sources of amio acids for grow- ing chicks. Poultry Sci. 44:394. Smith, R. E. and H. M. Scott. 1965b. Measurement of amino content of fish meal proteins by chick growth assay. J. Poultry Sci. 44:401. Stein, J. F. and C. B. Knodt. 1954. Further studies on the use of soybean flour and whey solubles in milk replacement formulas for young dairy calves. J. Dairy Sci. 37:655. Stein, J. F., C. B. Knodt and E. E. Ross. 1954. Use of special processed soybean flour and whey solubles in milk replacement formulas for dairy calves. J. Dairy Sci. 37:373. Stellaman J. 1969. Personal Communication. Stillings, B. R. 1967. Nutritional evaluation of fish protein concentrate. Activities Report 19:1:109. Stillings, B. R., O. A. Hammerle and D. G. Snyder. 1969. Sequence of limiting amino acids in fish protein con— centrate produced by isopropyl alcohol extraction of Red Hake (Urophycis Chuss). J. Nutr. 97:70. Sure, B. 1957. The addition of small amounts of defatted fish flour to milled wheat, flour, corn meal and rice. J. Nutr. 61:547. Thomas, J. W. and M. Okamoto. 1955. Plasma tocopherol levels of dairy heifers receiving different diets. J. Dairy Sci. 38:620. van Hellemond, K. K. 1967. A digestibility and N-balance experiment with protonimal in a milk replacer for veal calves. Washington Report 183. Waibel, P. E., J. H. Sautter, V. Perman and M. O. Schultze. 1957. Resistance of turkeys to hematologic effects of S-(l,2-dichloroviny1)-L-cysteine. Poultry Sci. 46:1144. 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. 32 Whipple, G. H. 1942. Hemoglobin and plasma protein. Their production, utilization and interrelation. Am. J. Med. Sci. 203:477. Whitelaw, F. G., T. R. Preston and G. S. Dawson. 1961. 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. Animal Prod. 3:217. Williams, J. B. and C. Jensen. 1955. Dried rumen contents in calf milk replacements. N. Dak. Agr. Exp. Sta. Bi-monthly Bul. 71:91. Williams, J. B. and C. B. Knodt. 1950. The further develop- ment of milk replacer for dairy calves. J. Dairy Sci. 33:809. Williams, J. B. and J. W. Rust. 1968. Fish flour in milk replacer. Diary Herd Management 4:19. Yanez E., D. Ballester, A. Maccioni, R. Spada, I. Barja, N. Pak, C. O. Chichester, G. Donoso and F. Monckeberg. 1969. Fish protein concentrate and sunflour presscake meal as protein sources for human consumption. Yoshida, A., P. M. B. Leung, Q. R. Rogers and A. E. Harper. 1966. Effect of amino acid imbalance on the fate of the limiting amino acid. J. Nutr. 89:80. PART I ~FISH PROTEIN CONCENTRATE IN MILK REPLACER FOR YOUNG CALVES 33 ABSTRACT In four trials with young Holstein calves the nutritional value of fish protein concentrate (FPC) pre- pared by extraction with dichloroethane (DCE—FPC) or isoprOpanol (IP-FPC) was investigated. In trial I the addition of methionine to DCE—FPC showed no beneficial effect on growth in young calves. In trail II calves fed DCE-FPC were inferior in weight gains to those fed a dried skim milk ration. Growth response with IP-FPC was inferior to that on DCE-FPC. Calves on FPC and low protein rations developed microcytic, normochromic anemia. This anemia was associated with jpoor utilitation of FPC protein. A high incidence of Inuscular degeneration was observed in calves on FPC rations. dedition of chlorocholine chloride 400 mg/kg protein in (iiets of calves on trial III tended to depress the growth; ‘flhereas an identical level of dichloroethane did not éadversely affect body weight gains. Trial IV showed that Gixtraction of DCE-FPC with ethanol improved its nutritive \nalue for calves. Washing DCE-FPC with water depressed (growth of calves compared to the original DCE-FPC. The ‘growth depression resulting from washing DCE-FPC with water ‘Nas associated with the removal of water-soluble proteins. 34 Introduction Fish protein concentrate (FPC) is a very rich source of protein. A partial substitute for protein in milk replacers for young calves FPC has resulted in satisfactory growth; however, as a sole protein source poor results have been obtained (Huber and Slade, 1967). In recent studies by Genskow (1969) it was shown that deficiency of vitamin E was the principal cause of death in calves when deboned FPC which had been extracted with dichloro-ethane (DCE) was the sole source of protein in the milk replacers. Plasma amino acid levels of calves on the diet showed histidine, leucine and phenylalanine to be lower than normal. Morrison and Munro (1965) indicated from their studies with rats that the nutritive value of FPC is greatly influe enced by the solvent used for extraction. They suggested that the sulfur amino acids and histidine became less available to the animal because of DCE extraction. Studies have shown that cattle fed trichloroethane-extracted soy- bean meal develops aplastic anemia. (Picken g3 g1., 1952; Pritchard gg gl., 1952; Sautter gg gl., 1952). 35 36 The present study was undertaken to investigate the causes of poor performance of calves fed FPC as the sole source of protein. Blood parameters and the changes in certain tissues were also observed. Because rat data showed a marked improvement in performance from methanol, ethanol or water washing of DCE-extracted FPC, similar treatment was applied to material fed to calves. EXperimental Procedure Four trials were conducted with Holstein bull calves to evaluate the nutritional value of FPC as a sole protein source in milk replacers for young calves. Morrison and Munro, (1965) reported a poor avail- ability of methionine in dichloroethane extractracted FPC (DCE-FPC). Trial I was therefore designed to study the effect of supplementation of methionine to a milk replacer diet in which DCE-FPC furnished all of the protein. The ingredient composition of the milk replacer formulas used in this trial is listed in Table 3. To the control ration 0.40% urea was added in order to make it isonitrogenous with other rations. Rations 2 and 3 were supplemented with 1.24% DL-methionine and 1.24% DL-glutamic acid. Six calves were allotted to three treatments. The calves received colostrum for the first three days after birth and one of the milk replacers as the only source of food from 3 to 56 days. Calves were fed solids at 1.2% of the body weight during the first week of treatment and at 1.3% for the 37 TABLE 3.--Ingredient composition of rations fed in Trial I. Ingredient 7" Rationsa 1 2 3 -------------- (%)-----——----—-— Fish flourb 27.20 27.20 27.20 Lactose 62.15 61.30 61.30 .Emulsified lard oil 10.00 10.00 10.00 Urea 0.40 - - DIrnethionine — 1.24 - (31utamic acid - - 1.24 Iturofac-lo 0.25 0.25 0.25 aVitamin A and D were added to all rations at 3230 IU eand 300 IU, reSpectively. bDichloroethane-extracted fish protein concentrate Cflotained through courtesy of Viobin Corp., Montecello, Ill. Inemainder of the treatment period. The milk replacer for— lTualas were diluted to 15% solids and fed by open pail. CIalf weights were recorded weekly. Blood samples were Cirawn at weekly intervals from the jugular vein and analyzed fior hematocrit by the micro method and for hemoglobin by ‘tdne cyanmethemoglobin method, Wintrobe (1953). At the end C>f the experimental period the calves were sacrificed by eP—lectrocution and autopsies were performed. lAutOpsy of the calves was performed by Dr. R. L. b'Iichel, Department of Pathology, Michigan State University, Elast Lansing, Michigan. 38 Trial II The results of Trial I showed poor growth and a large decrease in hemoglobin irrespective of methionine supplementation. Trial II was therefore designed to further investigate into growth, hematological changes and changes in the various tissues of calves receiving FDC as the only source of dietary protein. Two levels of protein, 20% and 10% from dried skim milk and FPC were .incorporated into the treatment rations. In addition to tflne regular DCE-FPC used in Trial I, a low ash DCE-FPC (fnmtwhich bones were partially removed through mechanical :screening) and also fish extracted with isopropanol (IF-FPC) were tested. Ingredient composition of the six 1:reatment rations is shown in Table 4. Thirty Holstein trull calves were allotted to the six treatments (five per sgroup) and were fed and weighed as described in Trial I. E3100d samples were drawn from jugular vein at weekly j.ntervals and were analyzed for hemoglobin and hematocrit 618 shown previously and for red blood cells and white blood Czedls by using the electronic Coulter Counter. Data were Sitatistically analyzed using analysis of variance and Clifferences between treatments were tested by Duncuns new nwultiple range test. On completion of the eight week treat- fluent period, all calves were sacrificed by electrocution 39 .HHH .mmpcsa ..ocH .meuHmflommm xaazm .ocm>H0m oacmmuo ecu mm Hocwmoum0ma mcwms pmuomuuxm cmwm .cmpm3m ..ocH .muospoum amoausmomsumnm muum< wo xmounsoo :moouzu pmcmacuomu .pm>OEmu xaamAuuma meson I poeumE camouuommo we peuomnuxu .HHH .OHHcoHucoz ..Quoo cHQoH>w .ucm>HOm cacmmuo ecu mm ocmcumouoHnoHp means cornea camouuomum or» >2 pouomuuxw swam macs: .HHH .oflamoaucoz ..QMOO CH20a>p .LHHHO .oomflocchu cam ..ocH .moauaea umcemuom mo xmcLsooo rmscuzu pmzmacusmo _ .sa om .c; was on.o .H .oa.H .oo .wE m.v so .mE 0.0m CN .08 m.mm on cuHE poucoaoagaom osm3 Amx Home mueap Hadn .NHm CHEmuH> mE we can mcflaozo o o.m .paom oflcocuouccm @E o.mH .CHDOHQ mE ma.o .mcflx0papxm OE o.m .cflomac me om .cH>ch02au ms m.m .ocHEmflsu mE o.m .m cflEmufl> as as .o assass> .o.H com .a caemua> .:.H ommm Ame seas encamucoo mamas Adam mm.o mm.o mm.o mm.o mm.o mm.o OHIUMMOHS< I I o.m I o.m I mudcmmozm EDHOHMOHQ 0.0H 0.0H o.oa o.oa 0.0H o.oa maao puma pmeHmHDEu m.mm m.mm n.wm v.om m.wm m.mm omonuoO h.mm o.mm m.mm v.0m o.mm I owmouomq Im.vm o.¢m m.va o.mm I I momuucmocoo Camuoum swam u m U U . I I I I o.mm H.5m xHHE Eflxm pmfluo IIIIIIIIIIIIIIIIIIIIIIIIII vaIIIIIIIIIIIIIIIIIIIIII H> > >H HHH HH H mcoaumm DcmapwumcH hm.HH Hmfiue ca pom: mcofiumu mo cOMLflmOQEoo ucwflwouucHII.w mqm .Amo.ovmv one mH.o oa.v «v.0 I I came pcmfiucmuu mo .m.m HN.HI chmm.nv cam.HI v.Hm m.Hm m womlummImH mav.o cmm.mm nmow.o v.moa o.mm m womlpmcmmuom OmmImOQ am.mI comm.om omo.HI w.om v.voa m onIUmMImUQ oom.o ema.no omm.H m.moa o.mm m womlommImOQ mmm.o mHm.mm QMNH.H N.Hoa v.wm m woaIxHHE wam pmano mm.m I woo.ao pov.v N.@HH o.Hm m womeHHE Eaxm pmfluo 3: ............ 3: ............ .mcoo MMMMMM Hmcam HmauacH poem . . .,. . mmm Hmuoe ucmflm3 moom umHQ ucoEumwuB ll. .HH HMHHB .mm>amo ca Ammmv OHDMH wocwaoflmmm samuoum paw GOADQESmcoo comm .mchm ucmflmz mpOQ co mumomammu xHaE CH campoum mo monDOm DcmHmMMHp mo DomwmmII.n mqm one m¢N.o mno.o cmmE ucmEumme MO .m.m opom0.HI m.am o.Hm nomm.oI m.m o.oa m womIOmmImH chmhv.HI o.mm m.mm noav.0I m.m m.HH m momIpmcmmnom UmmImua UNH5.HI m.om w.mm conv.oI m.m H.NH m wOHIOmMIMOQ ODVMH.HI o.mm «.mm Dmmm.oI m.n m.m m womIOmmImOQ homom.OI m.nm w.vm Doom.OI o.m m.HH m woaIxHaE Eflxm pmwuo ommH.OI o.Hm o.mm emao.ol m.m o.oa m womeaaE Eaxm pmaua IIIIIIIIII AweIuIIIIIIIII IIIIIIIuI-1w\mcIIIuIIIuI mmcmno mmcmco waxed; Hmcflm HeauHcH waxmmz Hmcflm HmHuHcH DAHUODmEmm :flQonOEmm umwp ucwEummuB .HH dawns .Hmomamwu RHHE men CH cflwuonm mo mmousow pcmumwwflp pom mw>Hmo so open HmoamoaoumfimmII.m mange 49 ucmummmap xfiucmoflmflcmam mum DQHHomHDQSm coEEoo mcHumcm uoc mosam>pohwo ovmv nh.o oo.H mv.o some ucmsummuu mo .m.m chom.o+ emo>.mn mom.OI samuoum womIOmmImH hoo0.m+ ohom.wI wom.HI m womIpocmmuom UmmImoa eon.m+ poa.HHI hoo.mI woalommImoo heoo.m+ oom.m- eee.0I d romuodenmoo pooo.HI mom.H+ ooo.o m onIRHHE Eaxm pmano pomN.OI moH.o+ mmH.OI m momeHHE Eme pmflua are Amav Loans Aomozv A>ozv Amuse coHumnucmocoo CanonOEmc DEDHO> cHQonOEmc umasomsmuoo cmwz Hoaoowsmuoo com: Hoasomsmuoo cmmz coHumu ucmfiummue .HH HMMHB .mnmomammu xHaE CH samuonm mo mmousom ucmnmwmap pow mm>Hmo CH mosam> HmonOHoumEmc CH mmmcmcuII.m mqméa 50 is one cause of anemia. Since the milk replacers were adequately supplied with Fe, Cu, Co and vitamin B anemia 12’ observed in these calves is not attributed due to lack of those nutrients. Young animals are usually more aggravated by a low protein ration than older ones since protein is also demanded for body growth. The greater anemia observed in calves on FPC than DSM rations at equal protein intake was attributed primarily to the poor protein quality of the FPC. Hematological indices (Table 9) showed that mean compuscular volumes (MCV)decreased more in the calves on FPC than DSM milk replacers. However, the mean corpuscular hemoglobin concentration(MCHC) increasediJIcmlves fed the DCE-FPC milk replacers, compared to DSM or IP-FPC. It has been reported by Plat (1956), Knowles (1957) and Schalm (1965) that protein deficiency causes normochronic, micro- cytic anemia. The anemia in the present study has been characterized as normochromic, microcytic type. The observation that the reduction in hemoglobin concentration was almost parallel to the depression in growth (Fig. l and 2) suggests that there is competition for protein between demands for body growth and for hemo- globin synthesis. A summary of the autOpsy reports on the calves per- formed at the end of the 8-week experimental period is - " E-L‘Vi' I 51 .. Ice-.4 . P I m.mm m.mm H.Hm spam N.mm m.vm N.mm o.ooa UmmImH mom pmcmmnom m.moa m.voa m.moa o.moH v.00H m.mm m.nm H.wm o.ooa Omelmuo wom o.mm N.mm m.mm m.hm m.mm h.mw N.Ho m.mm o.ooa Ummlmuo woa o.HmH o.maa c.0HH n.moa o.voa ¢.OOH m.wm n.mm o.ooa ummImua mom H.moa v.moa o.moa H.mm 0.5m m.hm «.mm w.mm o.ooa 2mm woa v.mva N.nma w.mmH m.mma o.mHH m.hoa N.NOH o.ooa o.ooa Ema wow m h m m e m N H amAuHcH mxwmz coflumm hsosmeeao rues anchors the .Ausmamz Hmfluacfl mo w mm pmmmmumxwv mmoHDOm cflmuoum mm>amo ca mmmcmco seem ucmHmB maxmszI.oa mange 52 ucwummmflo pom mm>amo mo +e~ It” fioa ommImOQnI I mom ommlmH pmcmmLom Rom ogmlmoa Rea Em: new QLLIQQQ mom Ema mxooz «I— I/ a b q .mmousom :Hmuoum unmamz mpom ca mmmcmnuII.H mnsmam (\l dI-H l ow tom rooa f0.: roma rQMH r03 Tems, (%) nuBIaM Kpos IEIQIUI wOJJ afiueqo 53 woa mom mow 1— .mmonsom samuoum ucmummwflo pom mm>amu CH modam> cHDonOEom ca mmmcmguII.m wusmam mxoms w m o m a m m H oo Ommlmofl oocomsom tom wow QLKIMOD was :3 9-- I .5 Item ULhINOQ QthgH Irom mom 3m; ,rOOH (g) senteA urqotfiomay IBIQIUI won; eBuqu 54 given in Appendix Table I. The main autopsy finding showed that the calves on FPC—milk replacers had a higher incidence of muscular degeneration than those on milk protein, although the recommended levels of vitamin E (Huber and Thomas 1967) snas supplemented to all the rations. Because it appeared that DCE-FPC increased vitamin E requirement of the calves, further study on the relationship of protein source and response to supplemental vitamin E was undertaken and is reported later in this thesis. Trial III As indicated in studies by Munro and Morrison (1967) rand Ershoff (1967), CCC and residual DCE exert depressing (effect on growth in rats. Effect of these two compounds <3n.the growth of calves was studied in Trial III. Results (Df'this trial (Table 11) showed that when CCC was added ‘tx: the diet at the level of 400 mg per kg milk protein, 63 slight but nonsignificant depression in growth was (Distained. The calves consumed 1.53 mg CCC per kg body ‘Véaight. The LD50 of CCC for calves is not known. Munro Eirna Morrison (1967) reported that the LDSO of CCC for rats was 500 mg/kg body weight. Calf numbers in this trial were nOtsufficient to unequivacally conclude a depressing effect of CCC on'calf growth. However, it appears that the calves are more sensitive to this compound than rats who showed no adverse effect on the growth at levels com— patreble to those fed to calves as reported elsewhere in the thesis . 55 TABLE 11.--Effect of chlorocholine chloride (CCC) and dichloroethane (DCE) on growth in calves (Trial III)a. Ration Weekly body weight change (lb) *7 7— Dried skim milk replacer 4.40:0.50a (5)b Dried skim milk replacer + CCC 3.69:0.78 (2) Dried skim milk replacer + DCE 4.56:0.78 (2) as. E. treatment mean. bFigure in parentheses indicate the number of calves used per treatment. Residual DCE in DCE-FPC used in this study was 360 ppm. When 400 mg DCE per kg protein were added to dried skim milk replacer, growth was not altered compared to control diet. In a study by Ershoff (1967) with rats, a diet con- taining FPC extracted with DCE for 24 hrs (which contained 30,000 mg/kg of DCE) exerted marked depression in growth of rats. This level of residual DCE is one hundred-fold higher than found in commercially prepared FPC where the period of extraction is only 1 to 2 hrs. Trial IV Superior weight gains were obtained when DCE—FPC was further extracted with ethanol compared to unextracted DCE—FPC (Table 12). However, weight gains were still lower than those of calves fed dried skim milk replacer. The DCE-FPC used in this study contained 1.43% ether extract. :3 - J—‘T -u 56 TABLE 12.-—Body weight changes in calves fed FPC extracted with ethanol or washed with water. Ration Weekly body weight change —- (1bY * DSM (20% protein) 4.56a 0.61 DCE—FPC (20% Protein) 2.04a 0.70 DCE—FPC extracted with ethanol a (20% protein) 3.35 0.70 DCE-FPC washed with water b (20% Protein) -1.55 0.70 — * — * S. E. of treatment mean. ab . . . . Values not sharing a common superscript are Slgnlf- icantly different (P<0.05). After extraction with ethanol, FPC contained only 0.29% ether extract. Thus, ethanol extraction removed most of the lipids. It is well established that diets high in polyunsaturated fatty acids increase the vitamin E require- ment of calves (Adams gg gl., 1959a). As shown by Genskow (1969) and also later in this study, the addition of vitamin E to a DCE-FPC milk replacers improved weight gains and decreased mortality of calves. The muscular degeneration observed in the calves in Trial III also suggest a vitamin E deficiency on DCE-FPC milk replacers. Studies by Munro and Morrison (1957) showed that further washing with methanol of FPC, originally prepared by extracting 24 hrs in DCE, resulted in a decrease in mortality of rats. 57 In light of the findings of Ershoff (1967), it seems likely that the primary deleterious factor removed by the methanol in Canadian study (Munro and Morrison 1967) was the residual DCE. However, in our studies with rats, as explained in a later section, a marked improvement. in growth also resulted from methanol, ethanol, or water washing of DCE-FPC. This improvement did not appear related to a decrease in residual DCE or CCC, but more specifically to an increase in efficiency of protein utilization. Removal of polyunsaturated fatty acids and of CCC are the probable factors responsible for improved growth of calves observed after ethanol—washing of DCE- FPC . When DCE-FPC was washed with water and included in calf diets as the only protein source, a loss of 1.55 lb body weight per week was observed. This weight loss was comparable to that obtained with the IP—FPC milk replacer in Trial II. During the preparation of IP—FPC, water soluble proteins from fish are removed; but water-soluble constituents of DCE—FPC are conserved during its original preparation. These data suggest that removal of water soluble fractions from FPC milk replacers decreases its nutritive value for calves. These calf data are in general agreement with the findings of Shillam SE.31' (1960) and Shillam and Roy (1963) who observed unsatisfactory growth in calves fed milk replacers in which 72% of the whey pro- tein was denatured by high heat treatment. However, in our .1! . - . Ear. 58 rat studies, removal of the water—soluble constituents markedly improved performance. These data also support the.observation of McCoy g5 31. (1967) who showed an improvement in calf performance when the whey protein non— fat dried milk fed to calves was at least 4.7 mg/g. The reason for need of water—soluble protein in the young calf has not been clarified but one possibility is that the rapid release of certain amino acids are important for efficient protein digestion in the calf and a source com- prised totally of water insoluble protein would not meet this necessity. LITERATURE CITED Ackerman, C. J. and W. D. Salmon. 1960. A simplified and specific method for the estimation of choline. Anal. Biochem. 1:327. Adams, R. S., T. W. Gullickson, J. E. Gander and J. H. Sautter. 1959a. Some effects of feeding filled milks to dairy calves. I. Physical condition and weight gains with special reference to low fat ration. J. Dairy Sci. 42:1552. Ershoff, B. H. 1967. Animal studies on the nutritional value of fish protein concentrate. Institute of Nutritional Studies, Culver City, Calif. Genskow, R. D. 1969. Evaluation of a low ash fish protein concentrate for use in calf milk replacer formulas. Ph.D. Thesis. University of Illinois, Urbana. Huber, J. T. and L. M. Slade. 1967. Fish flour as a protein source in calf milk replacer. J. Dairy Sci. 50:1296. Huber, J. T. and J. W. Thomas. 1967. Vitamin require- ments in young ruminants. Maryland Conference on nutrition of young ruminant. College Park, Maryland. Knowles, C. B. 1957. Protein nutrition in pigs. Proc. Nutr. Soc. l6:ix. McCoy, G., J. Reneau and J. B. Silliams. 1967. Growth and survival of calves fed different sources of nonfat dried milk. J. Dairy Sci. 50:996. Morrison, A. B. and I. C. Munro. 1965. Factors influenc— ing the nutritional value of fish flour. IV. Reac— tion between 1,2-dichloroethane and protein. Can. J. Biochem. 43:33. Morrison, A. B. and Z. I. Sabry. 1963. Factors influenc- Ing the nutritional value of fish protein. II. Avail— ability of lysine and sulfur amino acids. Can. J. Biochem. 41:649. - 59 60 Munro, I. C. and A. B. Morrison. 1967. Toxicity of 1,2- dichloroethane extracted fish protein concentrate. Can. J. Biochem. 45:1779. (Diassterhout, L. E., C. R. Grau and B. D. Lundholm. 1959. Biological availability of amino acid in fish meals and other protein sources. J. Nutr. 69:65. E>ixzken, J. C., H. C. Beister and C. H. Covault. 1952. Trichloroethane extracted soybean oil meal poisoning. Iowa State College Vet. 14:137. ' E>lxatt, B. S. 1956. Diet and anemia. Nutr. Soc. Proc. 15:103. E’rnitchard, W. R., C. E. Rehfeld and J. H. Sautter. 1952. Aplastic anemia of cattle associated with ingestion of trichloroethane extracted soybean oil meal. Clinical and laboratory investigations of field cases. J. Am. Vet. Med. Assoc. 121:1. Srnith, H. M., W. F. Dean, A. Aguilera and R. E. Smith. 1962. Quality of fish meal in relation to its value as a supplement to corn-soybean meal chick diets. Fish in Nutrition. Fishing News, Ltd. London. Sliillam, K. W. G. and J. H. B. Roy. 1963. The effect of heat treatment on the nutritive value of milk for young calf. A comparison of spray-dried skim milks prepared with different preheating treatments and roller- dried skim milk, and the effect of chlortetracycline supplementation of the spray-dried skim milks. Brit. J. Nutr. 17:171. Sflillam, K. W. G., D. A. Dawson, and J. H. B. Roy. 1960. The effect of heat treatment on the nutritive value of milk for young calf. The effect of ultra—high— temperature treatment and of pasteurization. Brit. J. Nutr. 14:403. Stuellaman, J. 1969. Personal communication. Serutter, J. H., C. E. Rehfeld and W. R. Pritchard. 1952. Aplastic anemia of cattle associated with ingestion of trichloroethane extracted soybean oil meal. J. Am. Vet. Med. Assoc. 121:73. StZillings, B. R., O. A. Hammerle and D. G. Snyder. 1969. Sequence of limiting amino acids in fish protein con- centrate produced by isoprOpyl alcohol extraction of Red Hake (Urophycis Chuss). J. Nutr. 97:70. 61 van Hellemond, K. K. 1967. A digestibility and N-balance experiment with protein meal in milk replacer for veal calves. Washington Report 183. VUjRntrobe, M. H. 1953. Clinical hematology. Ed. Lee and Febiger, Philadelphia. PART II VITAMIN E ADDITION TO DCE-FPC MILK REPLACERS 62 ABSTRACT Nineteen Holstein calves were placed on five treat- rneents. Two rations contained dried skim milk with (46 mg/kg (ixfiy matter) or without vitamin E. The other three rations <2<>ntained dichloroethane extracted fish protein concentrate (IDCE—FPC) with 0, 46 and 92 mg/kg vitamin E. Growth rtesponse improved as the level of vitamin E in DCE-FPC rwation increased. However, no alteration in weight gains vnas observed with different levels of vitamin E supple- nmented to milk protein rations. Plasma tocopherol concen- tzrations were related to both level of supplemental vita- rnin E and protein source. More depression in plasma txoc0pherol concentration was noted in calves on the DCE- IE'PC ration compared to the milk protein ration. It is Sliggested that the vitamin E requirement for the young calf tweceiving DCE-FPC as the only protein source is approxi- Huitely 120 mg per 100 kg body weight. 63 Introduction In the preceeding studies with calves fed milk replacers containing dichloroethane extracted fish pro- tein concentrate (DCE-FPC) as a sole source of protein, symptoms of vitamin E deficiency, particularly muscular dystrophy, were observed, although the rations were SUpplemented with recommended levels (Huber and Thomas, 1967) of vitamin E. It has been well documented that muscular dystrophy can be produced in several species of animals by feeding a vitamin E deficient diet (Stafford 31’; 13:” 1954) . Moore g_t_ git. (1961) have reported that COd liver oil contains a considerable amount of vitamin Er the range being 100—200 mg/kg. However, this level of vitamin E is insufficient to protect against the highly Unsaturated fatty acids also present. Thus, animals fed diets containing cod liver oil have an increased require— ment for vitamin E. The purpose of this experiment was to determine the leVel of vitamin E in a DCE-FPC milk replacer ration which will prevent muscular dystrophy and bring about optimum growth in calves. The interaction of protein source and reSponse to vitamin E was also studied. 64 65 Experimgntal Procedures Nineteen Holstein calves (10 bulls and 9 heifers) were placed on five treatments. Ingredient composition of the rations is presented in Table 13. All the rations were supplemented with the vitamins and trace minerals suggested for semi-purified diets for calves (Huber and Thomas, 1967) except vitamin E. The calves were fed and weighed as described in previous experiments. At weekly intervals, blood samples were taken from jugular vein for d-tocopherol determinations. The a-toc0pherol concen- trations were determined according to the method described by Duggan (1959). At the end of the 8-week treatment period, an autopsy and histopathological examination was performed on two bull calves from each diet. Results and Discussion Vitamin E exerted marked effect on weight gains of calves receiving DCE-FPC but did not alter growth on milk protein (Table 14). When 60 mg vitamin E per 100 kg body weight were supplied to calves fed DCE-FPC milk replacer, weight gains improved over the unsupplemented fish protein diet. Growth was further improved by doubling the supple- mental vitamin E. However, none of the DCE-FPC diets were equal to milk protein diet which contained no added vitamin E. 66 TABLE 13.--Ingredient composition of rations used in Trial V.a' Ingredient Rations I II III IV V L— --------------- (%) --------------- Dried skim milk 56.50 56.50 - - - Fish protein concentrate - - 26.50 26.50 26.50 Lactosed 17.00 17.00 31.70 31.70 31.70 Cerelose 16.20 16.20 31.50 31.50 31.50 Emulsified lard oil 10.00 10.00 10.00 10.00 10.00 Aurofac-lo 0.25 0.25 0.25 0.25 0.25 Vitamin BC + - + — ++ aA11 rations contained (per kg) 3230 I.U. vitamin A, 300 I.U. vitamin D, 5.0 mg thiamine, 3.5 mg riboflavin, 20 mg niacin, 5.0 mg pyridoxine, 0.15 mg biotin, 15.0 mg pantothenic acid, 2.0 g choline and 68.0 mg vitamin B12. bAll rations contained (per kg) Fe 22.5 mg, Zn 30.0 mg, Cu 4.5 mg, Co 1.10 mg and I 0.76 mg. CRations I and III contained 46 mg a-tocopherol acetate per kg. Ration V contained 96 mg a-tocopherol acetate per kg. d . . . Furnished through courtesy of Foremost Dairies, Inc., San Francisco, California. Decreases in plasma a-tocopherol were noted for all treatments, but seemed related both to level of vitamin E supplementation and source of protein (Table 15). At the normal level of E supplementation plasma tocopherols were more greatly depressed on fish protein than milk protein. 67 TABLE 14.--Growth of calves fed milk or fish protein with or without supplemented vitamin E. Ration Weight gains per week (1b) . . . d a * I Dried sk1m milk + E 4.56 $0.62 II Dried skim milk — E 4.28aio.62 III Fish protein concentrate + Ed 2.04abt0.71 IV Fish protein concentrate - E 1.06bi0.62 V Fish protein concentrate + 2Ee 3.66ai0.62 * S. E. of treatment mean. abcValues not sharing common superscript are signif- icantly different. (P<0.05) dSupplemented at 46 mg/kg diet with a-tocopherol acetate. eSupplemented at 96 mg/kg diet with d—tocopherol acetate. However, when twice the normal vitamin E dose was added to DCE-FPC replacer, plasma tocopherol concentrations of calves fed this diet were comparable to those found in calves receiv- ing normal dried skim milk ration at recommended level of vitamin E. The very low concentrations of plasma a-tocopherol shown in calves on both protein sources receiving no supple- mented vitamin E raises the question as to the minimum levels of plasma a-tocopherol that can be reached and the calf still live. The concentrations observed in this study were lower than the author could find elsewhere in the 68 .coHumuuceocoo HeHuHcH ecu mo uceouem ecu mucemeumeu mflmeeucewem cw enam>e Aaavom.mm 1a sea.~H Ammcoo.ea Am voo.m Ammvmm.mm xees rum Aaocaa.ama Amavao.ae Ammeoa.os .amcmo.om mamevoo.moa xeez sue ao.ama ea.HmH oe.oaa mm.maa am.~ma HmauacH > >H HHH HH H oceansmm scenes mo mafia .AHE ooa\msv meousom cHeuoum uceuewmap nuaz escape“ peaceseammsmcs pee penceEeHmmom pew me>aeo mo eEmeHm CH mc0aueuuceocoo m caseuw>Il.mH mamfle 69 literature. It is also interesting to note that the unsupplemented calves on dried skim milk grew as well as 'the supplemental group despite the tremendously low plasma cx-tocopherol concentrations. These data strongly suggest ‘that DCE—FPC affords less protection to vitamin E than does dried skim milk. The lipid content of the DCE-FPC used in this study imas 1.43%. A 100 lb calf on DCE-FPC replacer would consume 2.3 g lipid per day. Since fish lipids contain highly ‘unsaturated fatty acids, it is suggested that higher levels (Df vitamin E would be needed in a DCE-FPC than a dried skinIndlk diet to afford adequate protection against uns aturated fatty acids . A higher incidence of diarrhea was observed in calves (n1 the DCE—FPC replacers. It was found that the diarrhea iI1<2a1ves causes marked decrease in plasma toc0pherol (TTquas and Okamoto, 1955). The diarrhea may have further aggravated the vitamin E stress of calves on DCE-FPC diets. Calves on the unsupplemented DCE-FPC replacers were letflmargic, muscularly weak and some were unable to stand ‘Wiiflnout assistance towards the end of the treatment period. Hiestological examination (Appendix Table II) revealed extensive muscular lesions in calves on milk replacers not SuPplemented with vitamin E regardless of protein source; whereas muscular lesions were prevented at all levels of E Slipplementation . 70 Since a-tocopherol is a biological antioxidant, it prevents the auto-oxidation of vitamin A (Cawthorne gg 31°! 1968) . This implies that the calves showing vitamin E deficiency symptoms would need higher levels of vitamin A in the diet. Vitamin A concentrations in the blood of calves were not determined, but no visible symptoms of vitamin A deficiency were evident. These data support those of Genskow (1969) who showed 50 and 100 mg/day of vitamin E supplemented to calves fed milk replacers in which deboned DCE-FPC was the sole source of protein decreased death losses, improved performance and increased plasma a-tocopherol concentrations. No difference was observed between diets furnishing 50 and 100 mg/day of vitamin E. The data also show that amount of vitamin E needed for supplementation to DCE—FPC is over 30 mg/day (the approximate amount the calves on ration IV received). LITERATURE CITED (Cawthorne, M. A., J. Bunyan, A. T. Diplock, E. A. Murrel and J. Green. 1968. On the relationship between vitamin A and vitamin E in diet. Brit. J. Nutr. 22:133. lDuggan, D. E. 1959. Spectroflorometric determination of tocopherols. Arch. Biochem. Biophys. 84:116. (Genskow. R. D. 1969. Evaluation of a low ash fish protein concentrate for use in calf milk replacer formulas. Ph. D. Thesis. University of Illinois, Urbana. thiber, J. T. and J. W. Thomas. 1967. Vitamin requirements in young ruminants. Maryland Conference on Nutrition of the Young Ruminants. College Park, Maryland. bkmore, T. and I. M. Sharman. 1961. Prevention of injurious effects of excessive cod liver oil by its fortification with vitamin E. Brit. J. Nutr. 15:297. Safford, J. W., K. F. Swingle and H. Marsh. 1954. Experi- mental tocopherol deficiencies in young calves. Am. J. Vet. Res. 15:373. Thtnnas, J. W. and M. Okamoto. 1955. Plasma tocopherol levels of dairy heifers receiving different diets. J. Dairy Sci. 38:620. 71 PART III EVALUATION OF FISH PROTEIN CONCENTRATE AS A SOURCE OF PROTEIN 72 ABSTRACT Fish protein concentrates extracted with dichlor- oethane (DCE-FPC) or isopropanol (IP-FPC) were evaluated for their nutritional quality. The IP-FPC pro- duced superior growth response and PER to casein or DCE- FPC. Biological value significantly improved (P<0.05) when DCE—FPC was further extracted with methanol or ethanol or washed with water. Heating of DCE-FPC tended to improve the weight gains in rats. Addition of methanolic and ethanolic extracts of DCE-FPC to casein or DCE-FPC diets depressed rat gains but addition of chlorocholine chloride (CCC) or dichloroethane (DCE) to casein diet did not alter growth response. At 20% and 40% protein, weight gains were same on DCE—FPC and casein suggesting that toxicity Of DCE-FPC was not a major factor depressing growth in rats. When small amounts of casein were added to DCE-FPC diets, a significant improvement (P<0.05) in weight gains Were observed. Serum protein, serum albumin and net pro- tein utilization of rats increased in proportion to the suPeriority of the protein source. Plasma of rats on all FPC diets was lower in histidine than plasma of rats on the casein diet. 73 Introduction The solvent used to remove oil and water from fish to produce fish protein concentrate (FPC) greatly influ- ences the nutritive value of the final product (morrison and Munro, 1965). Chlorocholine chloride (CCC), a reac- tion product of trimethylamine in fish and the dichloro- ethane (DCE), was toxic to rats at high levels (Munro and Morrison, 1967). It was also found that a high residual content of DCE in FPC markedly depressed growth in rats (Ershoff, 1967). Favorable growth responses were reported when dichloroethane extracted FPC (DCE—FPC) was supple- mented with milk protein (Ershoff, 1967) or when FPC was prepared using isoprOpanol as the extraction solvent (Stillings, 1967). The aim of the present study was to determine the importance of certain toxic compounds reportedly present in FPC and whether further extraction or heating of DCE— FPC would alter its nutritive value. An effort was also made to determine the response of rats to varying levels of casein added to DCE-FPC diets. 74 75 Experimental Procedures Experiment I This experiment was designed to gain further infor- mation on the toxicity of DCE-FPC. Three levels of pro- tein were fed and response of rats to methanol extraction of DCE-FPC was also investigated. Fifty, 21-day old weanling male rats of the Sprague- Dawly strain were alloted to the ten diets shown in Table 16. Diets 1, 2 and 3 contained 10, 20, and 40% protein from casein. Diets 4, 5 and 6 contained same levels of protein from DCE-FPC. Identical protein levels from methanol- extracted DCE-FPC were replicated in diets 7, 8 and 9. Diet 10 contained 40% protein from casein plus the methanolic extract from DCE-FPC. The amount of methanolic extract added was equal to that obtained from extraction of DCE— FPC used in diet 9. Rats were housed in individual cages with wire bottom floors. Food and water were given ad libitum. Rats were weighed every week and food intake was measured for each rat. The PER for each rat was calculated from weight gain and food consumption data. Treatment was for 28 days. At the end of treatment rats were sacrificed and bled. Hemoglobin and hematocrit were determined as described previously for calves. HistOpatholOgical exam- ination of tissues were also made as mentioned earlier from selected animals. Because no differences between treat- ments were observed for hemoglobin and hematocrit and all 76 .mm.a .mam "Aoeao he ooaxaev o.m .enmemsoonemn eaaoamo no.a .qor Iecax0pauwm no.a .GH>eHmoQaH m.v .cfloeec “o.m .paoe UHONceQ OCHEMIQ “mm.m .ec0apecee “o.ob .epfluoHno ecflHOLU no.m .Houwmocfl "me epaoe oflouoome no.m .Houecmoooulo “mm.o .1o\oH ooo.ooao o naemoa> 1m.e .1o\sH ooo.oomv a essens> ”Aneao he ooa\ov meanamusoo ensuxHE coflueoameuHOM peep CHEeuH> .pceae>eau ..QHOO maeoHEecoon Hecofluauuszp .smo.o . mz 1m.oH .aomzao.am .aoam x “moo.o .Hx uo.~H .Ho uaooo .omrwm.maaomcaemx umo.o . omcz no.a .aOmoe nea.a .o ma.aonom namo.o .ONrm. omso “a.aa . 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In diet 4 the DCE-FPC was further extracted with ethanol as previously described. Diets 5 and 6 corresponded to diets 3 and 4, respectively, but differed in that ethanolic extracts obtained from extraction of DCE-FPC were added at same level described in Experiment II. The DCE—FPC in diet 7 was moistened with small quantity of ethanol and heated at 65°C for 2 hours. Water—washed DCE-FPC (as described in calf Trial IV) was the protein source in diet 8. Casein was added to DCE-FPC in diets 9, 10 and 11 to furnish 30%, 20% and 10% of the total protein. Diets 12, 13 and 14 contained identical amounts of casein added to ethanol extracted DCE—FPC. Except for protein source, all the diets were of the same basic composition as shown in Table 17. The rats were weighed each week and total feed intake was measured for each rat. At the end of 4 weeks of treatment blood from the abdominal aorta was drawn from five rats each on diets 1, 2, 3, 4 and 8 for determination of plasma free amino acids as well as total serum protein and its components. Samples for plasma free amino acid determination were prepared according to Purser 35.21- (1966) using norleucine as an internal standard. Amino acid analysis was performed on a Technicon TSM Amino Acid Analyzer using a lithium citrate buffer. Total serum protein was determined by modified Lowry method (Miller 1959). Electrophoretic separation WET” .zj. 80 of serum protein components was performed as described by Cowley and Eberhardt (1962) . Net protein utilization (NPU) of rats on treatments 1, 2, 3, 4 and 8 was determined according to method described by Bender (1958) with following modification: An intial group of five rats was sacrificed at the begin- ning of the experiment. Rats on the five treatmentidiets were sacrificed after the four-week treatment period. The contents of the gastrointestinal tract were removed from each rat. The rats were autoclaved and homogenized as described by Mickelsen and Anderson (1959) and the car- cass nitrogen determined by Kjeldahl (AOAC, 1965) . Net protein utilization was calculated by the following formula: Nitrogen reta1ned in carcass x 100 NPU = Nitrogen intake figs ul ts and Discussion Experiment I Growth of rats on the DCE-FPC diet was significantly (P<0. 05)1ower than on the 10% casein diet, (Table 18) . At the 10% protein, methanol extraction of DCE-FPC signif- icant 1y improved growth and PER compared to the original DCE‘FPC, but they still did not equal those observed for casein’ (Table 19) . No significant difference was noted in the 20% or 40% protein levels between casein, DCE-FPC or methanol extracted DCE—FPC. This may have been due to 81 .Amo.ovmv uceuemwflo xauceoflwacmflm ene uQflHomHeQSm COEEOU mcfluecm Doc meSHe> pone mmo.«pom.o IIII IIII Doenpxe oflaocecueE+CHemeO mmo.npom.o mmo.nomn.a mmo.eomm.m Ommlmoo oepoeuueraocecuez mwo.apmm.o mmo.anmo.m mmo.npma.a. OmMImOQ cl 0 0| 0 cl 0 gamma mmo +pmH H mmo +hma m mmo +«ema m . 0 wow mom moa SOHHHGH OPE. CH QHQpOHm IMO HON/m4 eeeo .muen wo Ammmv .Amo.ovmv ucewemmap wapceOAchmflm ewe DQHHomHeQSm coEEoo mcfluecm Doc medae> mOADeH xoceeoawwe caeuowo co Hooflw cmaw mo DoemwmII.mH mqmeq 3.5 moon wo mafiem Leaflez :0 Hoon swam mo o . Umwwmll .mH mqm somepone who.o o~.ww ma.e ceee useEomewu wo .m.m mam.m coho.mwm one.eow cwenonn rammed wow .xm+odeImoo .ew msm.m ha.wmm nma.~ww tampons seemed wow .xm+odsImoo .mw nemm.m mm.cem ea.wmw caenowo caemeo mom .xm+oosImoo .mw momo.m weoona.oom weea.wa awesome seemed wow+odemoo .ww oonmw.m moonw.mom ooo~.ea cwenoso caenmo wo~+odmImoo .ow eoooo.m oonw.mwm moow.ma caenono chewed wom+odmImoo .a mmm.m oonm.ewm one.oow omens; odmImoo .m momo.m meow.mmm owew.ma oeneec omsImoo .s onea~.m woooa.mm~ oeoe.~a nonwoxe+.xm omaImoo .c emm.~ om.eem ow.oe oomnaxe+odsImoo .m onea~.m mocha.~om mooa.ma w.xu omnImoo .e eea.~ wem.oe~ owm.wm odsImoo .m wwa.m me.smm ce.wmw oamIdw .N nmwm.m oonm.cwm oonw.moa caemeo .H Lee Loo cowuoESmcoo mewem med owes prowes wewo .AuceEueeHu wem oao muew c4 owuew NoceHonHe cweuowm one cowumEdmcoo ceew .mCHem ucmwez co meowSOm caeuowm ucewewmwp mo woeMMMII.HN mqmde 88 When the DCE—FPC was washed with water, weight gains were significantly higher (P<0.05) than for the original DCE-FPC. After washing the FPC with water, DCE-FPC was put in the oven at 65°C until completely dry. It is pre- sumed that during washing of FPC, certain water soluble substances which might cause growth depression were removed. It therefore appears that beneficial effect of water wash- ing of DCE-FPC might be due to combined effect of mild heating and the removal of deleterious substances. The ethanolic extract of DCE-FPC resulted in a slight but nonsignificant growth depression when added back to original DCE—FPC or ethanol-extracted DCE-FPC diets. Extraction with ethanol, heating or washing of DCE—FPC with water also improved PERs of the original DCE-FPC. The addition of casein to the original DCE—FPC diets or to the ethanol—extracted DCE-FPC diets at all levels (10, 20 or 30% of the protein) resulted in a beneficial effect on growth of rats, (Table 21). The increased gains were proportional to the amount of casein added. Ethanol- extracted DCE-FPC was superior to the original DCE-FPC at all levels of added casein. MoreoVer, it is interesting to note that when ethanol extracted DCE-FPC furnished 70% and casein 30% of the total dietary protein (diet 12) for rats, growth was superior (P<0.05) to the casein diet. This probably demonstrates the complementary effect of the two protein sources. Er; ”I“ a p I . l .1 89 Amino acid analyses reveal that fish protein is slightly lower than milk protein in tryptophan, isoleucine, leucine, phenylalanine and histidine, (Table 22). It was further shown that availability of histidine, cystine and methionine was reduced when FPC was extracted for a pro— longed period with DCE (Morrison and Munro (1965). If we assume that essential amino acids from casein are fully available to the growing rat, then the addition of casein to the DCE—FPC diet should tend to compensate for the amino acids which may be limiting. Improved growth when casein furnished 10, 20 or 30% of the protein in DCE-FPC diets supports this assumption. The increased growth from addition of casein was associated with higher feed intakes and no change in PER was observed. Previous studies with FPC diets fed to rats established that improvement of the protein source usually resulted in a greater consumption of food (Morrison, 1963). Plasma free amino acid (PFAA) levels in rats on casein, IP-FPC, DCE—FPC and ethanol extracted DCE-FPC are shown in Table 23. Compared to PFAA levels on the casein diet, it was observed that histidine was markedly decreased on all FPC diets. Small differences were noted for some of the other amino acids, with threonine, valine, cystine, leucine, tyrosine, and lysine slightly lower on FPC; whereas, methionine and isoleucine were slightly higher. Because histidine appeared so greatly depressed in the blood of rats on FPC diets, this amino acid was selected for further study. 90 TABLE 22.--Essentia1 amino acid composition of milk protein and fish protein concentrate. f t z. 'I‘ r I Amino acid Milk protein DCE—FPC -------------- g/16 g N————--—-—-— Tryptophan 1.47 1.05 Threonine 4.82 4.54 Fa Isoleucine 6.68 4.63 '3‘ Leucine 10.27 7.90 Lysine . 8.14 8.54 Methionine 2.56 3. 35 p’ Cystine 0.93 0.78 Phenylalanine 5.07 4.30 Valine 5.33. 5.34 Histidine 2.76 2.11 * Alpine Marine Protein Industries, Inc., Bedford, Mass. It has been previously suggested that PFAA levels are somewhat dependent on the amino acid composition of the protein ingested (Richardson g3 g1., 1953; Longenecker and Hause, 1959). Comparison of the relative concentrations of amino acids in casein and FPC with their PFAA concen- trations in rats fed different protein sources tends to support this observation. Total serum protein and serum albumin were closely related to weight gains and PERs, (Table 24). Values for the IP—FPC diet were significantly higher than for all 91 TABLE 23.-—P1asma free amino acid levels in the plasma of rats fed different protein sources. Amino acid Casein IP-FPC DCE-FPC DCE-FPC Ex. ---------------- uM/100 m£----------—----—--— Histidine 5.02 1.43 1.64 0.95 Methionine 2.09 2.95 2.42 2.44 Threonine 26.10 14.09 21.41 24.52 Valine 9.04 9.25 6.77 6.66 Isoleucine 3.74 6.02 4.30 4.45 Cystine 0.47 0.91 0.29 0.37 Leucine 6.38 6.15 5.83 5.60 Tyrosine 5.40 4.62 3.09 3.79 Phenylalanine 2.77 2.80 2.50 2.76 Lysine 26.82 26.82 23.18 23.37 Arginine 4.46 7.05 4.07 4.09 other treatment. These serum components were not significantly different on the casein and DCE-FPC diets. Diets had no sig- nificant incluence on the golbulin levels. It has been generally observed that when animals were kept on a diet low in protein or deficient in a single amino acid, low serum protein and serum albumin levels resulted (Albanese, 1963). The net protein utilization on IP-FPC diet was signif- icantly higher (P<0.05) than on all other treatments. This observation corresponds well with the superior weight gains and PER values on this diet. Values for NPU on DCE-FPC 92 TABLE 24.--Net protein utilization, (NPU), serum protein and its components in rats fed different sources of protein. Diet NPU Serum Serum Serum protein albumin globulin % % % % Casein 77.15a* 5.83b 3.74b 2.09 IP-FPC 81.55b 6.96a 5.06a 1.90 DCE—FPC 72.15d 4.91b 3.24b 1.67 DCE-FPC EX. 75.52ac 5.86b 3.98b 1.88 DCE-FPC washed 74.93C 5.49b 3.76b 1.73 kg S.E. of treatment mean 0.59 0.37 0.26 0.15 adeValues not sharing common superscript are signif— icantly different (P<0.05). diet was significantly lower than for all other diets. Extraction with ethanol or washing with water significantly improved NPU values over the original DCE-FPC. The correla- tiOns between PER and NPU, serum protein and serum albumin were 0.95, 0.95 and 0.96, respectively. In View of the high correlation between these parameters (Fig. 3) the following equation using serum protein or serum albumin as the independent (X) variable can be used for prediction of PER (Y): = 0.446X Y (PER) (Serum Protein)+0'767 ______ ‘—--—(l) = 0.506X +1.371 ---------- (2) Y(PER) (Serum Albumin) 93 The results of this experiment showed that the nutritive value of DCE-FPC can be significantly improved by further extraction with ethanol, or washing with water or by adding a small amount of casein to it. 94 .mcofluomuw aflmuoum pooan pom mmm cmmbmn mflnmcoflumHmmln.m mnsmflm swunbmwllcflobosm m m e w m s m W m campers . cfiESDH< - campers .m> emu caespfia .m> mma oa.o u a Hem.s + Haxmom.o u mans .p a -- no.0 n n Noe.o + axes:.o u mm8> 33 Uquon ‘7 To U m 193 Rouaro. OT LITERATURE CITED Albanese, A. A. 1959. Protein and amino acids in nutrition. Academic Press, New York. Association of Official Agriculture Chemist. 1965. Official methods of analysis. 10th ed. (A.O.A.C.) Washington, D. C. Bender, A. E. 1958. Biological methods of evaluating protein quality. Proc. Nutr. Soc. 17:85. Cowley, L. P. and L. Eberhardt. 1962. Simplified gel electrOphoresis. 1. Rapid technique applicable to the clinical laboratory. Am. J. Clin. Pathol. 38:539. Ershoff, B. H. 1967. Animal studies on the nutritional value of fish protein concentrate. Institute of Nutritional Studies. Culver City, California. Huber, J. T. and L. M. Slade. 1967. Fish flour as a protein source in calf milk replacers. J. Dairy Sci. 50:1296. Longnecker, J. B. and N. L. Hause. 1959. Relationship between plasma amino acids and composition of ingested protein. Arch. Biochem. Biophys. 84:46. Mickelsen, O. and A. A. Anderson. 1959. A method for preparing intact animals for carcass analysis. J. Lab and Clin. Med. 53:282. Miller, G. J. 1959. Protein determination of large number of samples. Anal. Chem. 31:964. Morrison, A. B. 1963. Factors influencing the nutritional value of fish flour. III. Further studies on avail— ability of amino acids. Can. J. Biochem. and Physiol. 41:1589. Ddorrison, 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. 43:33. 95 96 Munro, I. C. and A. B. Morrison. 1967. Toxicity of 1,2- dichloroethane extracted fish protein concentrate. Can. J. Biochem. 45:1779. Purser, D. B., T. J. KlOpfenstein and J. H. Cline. 1966. Dietary and defaunation effects upon plasma amino acid concentration in sheep. J. Nutr. 89:226. Richardson, L. R., L. G. Blaylock and C. M. Lyman. 1953. Influence of dietary amino acids in the blood plasma of chicks. J. Nutr. 51:515. Stellaman,J. 1969. Personal communication. Stillings, B. R. 1967. Nutritional evaluation of fish protein concentrate. Activities Report 19:1:109. PART IV SUPPLEMENTATION OF HISTIDINE AND METHIONINE TO FISH PROTEIN CONCENTRATE DIETS FOR RATS 97 ABSTRACT The nutritive value of fish protein concentrate extracted with isoproanol (IP-FPC), dichloroethane (DCE- FPC) or ethanol extracted DCE—FPC (DCE-FPC-Ex) was studied with male weanling rats. Earlier investigations showed IP-FPC superior for rat growth to casein or DCE- FPC. In Experiment I two levels of L-histidine, (0.075% and 0.15%) were added to FPC diets containing 10% protein. Both histidine levels increased growth and feed intake on all the FPC diets (P<0.05). No benefit was observed by increasing histidine above 0.075%. In Experiment II 0.075% L-histidine and 0.20% L-methionine, alone or in combination were added to FPC diets. Improved growth resulted with added histidine or methionine. Histidine plus methionine produced additive effects on growth. In both trials protein efficiency ratios were improved (P<0.05) by amino acid supplementation to DCE-FPC. Plasma free amino acids in rat blood showed lower levels of total essential amino acids when both histidine and methionine were added to fish protein diets. When histidine and methionine were supplemented, alone or in combination, their plasma concentrations were raised. 98 99 Added histidine did not influence the methionine level in plasma; however, methionine alone depressed plasma histidine in rats on DCE—FPC and IP—FPC but not for those receiving ethanol-extracted DCE-FPC. This study suggests that histidine and methionine were limiting in these FPC diets regardless of method of solvent extraction. Introduction Previous eXperiments with rats on FPC diets showed low biological values of protein in DCE—FPC diets fed to rats compared to casein diets. Improved response obtained by addition of varying amounts of casein to DCE-FPC and the low plasma histidine levels on FPC diets suggested that histidine might be limiting rat growth on FPC diets. Studies with young calves (Genskow, 1969) have indicated low plasma histidine, leucine and phenylalanine on DCE- FPC rations. Rat studies (Morrison and Munro, 1965; Stillings, 1969) proposed that FPC prepared either by extracting with dichloroethane or isopropanol might be limiting in one or more of the following amino acids: methionine, histidine, tryptophan, threonine, valine, isoleucine, phenylalanine, leucine, lysine and arginine. Smith and Scott (1965) compared unheated and heated fish meal by chick biological assay and noted a decrease in availability of lysine and threonine due to heat treatment. In reviewing the amino acid composition of fish protein, it appears that the magnitude of a deficit of amino acids in FPC would not be very great. Since the plasma histidine levels for rats on FPC diets were very low in a previous experiment, this amino acid was selected for 100 101 further study. Addition of methionine alone or in com- bination with histidine was also tested. Experimental Procedures Experiment I Sixty weanling male rats of the Sprague-Dawley strain were alloted to 10 different diets. The basic composition of the diets is shown in Table 25. The three FPC diets were supplemented with 0, 0.075 or 0.15% L- histidine. A11 diets were made isonitrogenous by adding glutamic acid. The rats were fed the diets ad libitum for 14 days and weekly weight gains as well as feed intakes were recorded. Protein efficiency ratios were calculated for each rat. TABLE 25.——Basic composition of diets used in Experiment I and Experiment II. Ingredients Source of protein Casein IP4FPC DCE—FPC DCE-FPC Ex. ___________ ‘:;:;____(%y;_____________-__-____ Casein 11.0 - — - IP-FPC ' - 12.0 - - DCE-FPC - — 13.5 12.8 Corn starch 39.5 39.5 39.5 39.5 Cerelose 39.5 39.7 39.0 39.5 Vegetable oil 5.0 5.0 5.0 5.0 Salt mix 4.0 2.8 2.0 2.0 Vitamin mix 1.0 1.0 1.0 1.0 102 Experiment II In Experiment II L-methionine and L-histidine when added alone or in combination to FPC diets. Casein, IP—FPC, DCE- FPC and ethanol—extracted DCE—FPC were again used as the protein sources. Ninety-six 21—day-old male rats were alloted to 16 treatment diets. The basic composition of all the diets was the same as shown in Table 25. Diets 2, 6, 10 and 14 were supplemented with 0.75% L-histidine; diets 3, 7, 11 and 15 were supplemented with 0.20% L— methionine and diets 4, 8, 12 and 16 were supplemented with 0.075% L—histidine plus 0.20% L-methionine. Diets were again made isonitrogenous by adding glutamic acid. The rats were housed and fed as described in Experiment I. At the end of the 14-day treatment period, blood samples from abdominal aorta from three rats on each treatment diet were collected. Plasma was harvested from each blood sample and samples were pooled from each treatment and prepared for amino acid determination as described earlier. Results and Discussion Experiment I Addition of L-histidine at the rate of 0.075% or 0.15% of diet significantly improved growth rates in all of the FPC diets, (Table 26). Since plasma on all the FPC diets in previous experiment showed very low concentrations of histidine, the improved growth response with histidine 103 TABLE 26.-—Effect of adding histidine at two different levels on weight gains and protein efficiency ratio. ~__ Diet Weight gains PER .1 (g) 1. Casein 64.6f 3.158 2. IP-FPC 74.5e 3.69a 3. IP-FPC+Hg 83.2a 3.72a 4. IP-FPC+2Hh 84.5a 3.67a 5. DCE-FPC 42.2d 2.74f 6. DCE-FPC+H 51.2c 2.95c 7. DCE-FPC+2H 51.0c 2.92C 8. DCE-FPC Ex. 49.3C 3.24b 9. DCE-FPC Ex.+H 58.8b 3.26b 1o. DCE—FPC EX.+2H 57.8b 3.38d S.E. treatment means 1.57 0.038 abcdef icantly different (P<0.05). gRepresents 0.075% supplemental L—histidine. hRepresents 0.15% supplemental L-histidine. supplementation is not too surprising. evidence (Richardson gt 21.,1953) that an increase or Means not sharing common superscript are signif- There is considerable decrease in the dietary level of an essential amino acid results in corresponding change in concentration of that amino acid in plasma. Histidine content of FPC is not markedly low compared to casein but these data suggest that 104 the availability of the histidine in FPC is reduced. Morrison and Munro (1965) found a marked reduction in total histidine released during in vitro digestion of FPC. Results of the present study are substantiated by those of Morrison and Munro (1965) and Stillings (1969) who obtained improved growth of rats by addition of histidine to FPC diets. Addition of 0.15% histidine had no beneficial effect over that observed for 0.075%. Increased PER (P<0.05) was obtained when histidine was supplemented to DCE-FPC, however no change in PER. resulted on other FPC sources due to addition of histidine. The addition of histidine apparently resulted in a more balanced protein and thereby increased feed intake of the rats. Experiment II Growth responses and PER values from Experiment II when histidine or methionine or both were supplemented to the different protein sources are shown in Table 27. Addition of histidine to all FPC diets again resulted in superior growth compared to the unsupplemented FPC. Addition of histidine to casein did not affect growth suggesting that casein is already adequate in its avail- able histidine content. Growth response of the same magnitude as with supplemental histidine was obtained when methionine was added to FPC diets. Addition of methionine to casein produced better growth than unsupplemented casein. 105 Since casein is low in methionine this response was expected. When both histidine and methionine were added to FPC diets, an additive effect of both the amino acids was demonstrated in growth response on all supplemented FPC diets over the unsupplemented. Addition of histidine or methionine or both improved (P<0.05) the PER values on DCE-FPC compared to unsupplemented DCE-FPC, while no improvement was noted for the other FPC diets. Plasma free amino acid (PFAA) levels of rats in Experiment II are shown in Table 28. When both histidine and methionine were added to the diet, a marked lowering in concentrations of total essential amino acid (TEAA) in plasma occurred. It was also on these diets that the best growth was noted. The data suggests that at maximum rates of protein anabolism, a lowering of total PFAA results. These observations are at variance with Hill and Olsen (1963) who correlated low PFAA levels with poor nutrition and poor protein quality. It is also known that feeding a N—free diet adequate in calories results in very low PFAA levels (Bergen and Purser, 1968). Levels of TEAA generally correspond with the growth response obtained with the unsupplemented FPC diets. When histidine and methionine were supplemented alone or in combination, the plasma levels of these amino acids were raised. Histidine supplementation did not influence the methionine levels in plasma; however, methionine 106 TABLE 27.-—Effect of adding combination of histidine and methionine on weight gains and protein efficiency ratio. Diet Weight gains PER _‘(g) 1. Casein 65.8c* 3.09de 2. Casein+Hj 63.0d 3.02e 3. Casein+Mk 69.0b 3.15Cd 4. Casein+H+M 68.0bc 3.15Cd 5. IP-FPC 73.5i 3.73a 6. IP-FPC+H 81.3a 3.74a 7. IP-FPC+M 81.0a 3.82a 8. IP-FPC+H+M 86.0f 3.77a 9. DCE—FPC 42.5h 2.729 10. DCE-FPC+H 51.5fg 2.87f 11. DCE—FPC+M 50.0g 2.88f 12. DCE—FPC+H+M 53.0f 2.91f 13. DCE—FPC Ex. 50.09 3.20de 14. DCE-FPC EX.+H 58.0e 3.21bc 15. DCE-FPC EX.+M 57.0e 3.25b 16. DCE-FPC EX.+H+M 63.0d 3.29b S.E. treatment means 0.88 0.038 adeefgtheans not sharing common superscript are significantly different (P<0.05). JRepresents 0.075% supplemental L-histidine. kRepresents 0.20% supplemental L-methionine 1(37 .maese amass on cases anon measOchme-q aom.o use assessmeslq emeo.oo .w:ec0ecuoElq wom.o nufl3 poucwEwammsm muwx muwflp wmwnh n .mchHumazuq wm5o.o LDHB poucmawammom mHm3 mumep ommzem mm.owa vm.avm HN.5¢N Ho.on vm.mma om.NmH mo.5am mm.vma rc.moa mm.mvm 5N.mmm mm.omm 5o.Hom aa.vHN mH.Nom mo.5om mUHom OCHEG Hmflucmmmm Hmuoe oa.mm v5.mm 5m.mm mm.mm m5.5N mm.mm oo.HN mm.m~ m5.mm 0H.HN H5.oH om.oH mm.5H HH.5N 5m.vN oo.ma maficflmu< om.ma 5q.m mm.va mm.v mm.ma 5¢.m 5m.mm m5.m xm.mH vo.m N5.NH no.5 ov.m~ $5.0H vv.NN 55.MH Ouaflflumam mo.m5 aw.maa mm.moa o~.mm 5o.ab €0.00 mN.o am.mw mm.mx ov.vafl wo.NHH mm.oHH qo.mOH mo.va~ mo.mNH ow.NoH mewmxd 5m.m 5m.m 5N.o 5m.v mo.m 5N.¢ mm.m mw.v at.» 05.5 om.o v0.0 Hw.v oo.m fiv.5 mm.¢ mcflcmamHmcmzm Hm.m 50.0 55.0 om.m om.m Hm.m mm.m mm.v vo.v mH.mN 00.5 mm.c om.oa mm.NH No.0H Po.wa mnemouxfi NH.m mm.w 5m.HH 50.9 ma.v 5m.o 5H.HH om.5 05.5 vo.HH cm.HH mm.oa wm.m 5m.m om.ma oo.HH mcfiwswq om.v om.m 55.m om.m Hm.m mm.m M5.w vo.v ma.m O5.HH vm.5 mm.5 m5.5 5N.o VH.OH mm.m mcflmsmHOmH Hm.v Hm.o 55.m NH.5 55.m wv.o mw.m wv.v mb.¢ no.5 mo.v w5.m mo.© No.0 om.m mo.m mcflcofinumz 5m.m Hm.m ow.MH mo.oa mm.v mv.w mN.NH 5v.m om.5 HH.5H 5m.MH mv.ma mv.HH mm.¢ N5.mH Ho.wa wCflHm> om.H mm.a mo.oo mo.oo H5.oo mo.a 5m.oo mm.oo cm.cc vH.No mm.oo mm.oo wN.H oo.H ma.oo 5H.oo mcflumxu mm.va V5.mv mm.ov m5.mv vo.ma mo.Nm mo.vv av.vm mo.w 5m.mfi oo.ov oo.mm OH.NH mm.m mv.wm vH.mm OGHCOOHLB UZ+I 22 m: I 02+: £2 a: I O2+: 32 m: I 02+: 22 mm ! .XMIUmhImUQ UQLIMUD Umgngw Cflwmmu UHUm OCHEd .AanoH\Zov mDUHZOm aeouona pcmumwwflp pew mums mo mEmde Ce mam>ma pflom OCHE¢II.mN mqmde 108 supplementation alone depressed plasma histidine levels in DCE-FPC and IP—FPC, but not in ethanol extracted DCE- FPC. The general increase in PFAA for all the FPC diets when histidine was supplemented may indicate a catabolic state, despite increased growth response. This suggests that the level of histidine supplementation was in excess of the requirement of the animal. Harper (1964) has indicated that in an imbalance situation only a very small amount of supplemental limiting amino acid is needed to correct the imbalance situation. If histidine and methionine was co-limiting amino acids, plasma methionine level should have decreased when histidine alone was added to the diet and plasma histidine level should have decreased when methionine alone was added. As was mentioned, methionine supplemented to FPC diets did depress plasma histidine but supplemented histidine did not depress plasma methionine, even though growth and PER data strongly suggest that both amino acids were co-limiting. These results indicate that PFAA levels can be used as a guide rather than conclusive evidence for a specific limiting amino acid in a particular protein source . LITERATURE CITED Bergen, W. G. and D. B. Purser. 1968. Effect of feeding different protein sources on plasma and gut amino acids in growing rats. J. Nutr. 95:333. Genskow, R. D. 1969. Evaluation of a low ash fish protein concentrate for use in calf milk replacer formula. Ph. D. Thesis. University of Illinois, Urbana. Harper, H. E. 1964. Amino acid toxicities and imbalances. Mammalian Protein Metabolism. Ed. H. Munro and Allison. Academic Press, New York. Hill, D. C. and E. M. Olsen. 1963. Effect of starvation and a non-protein diet on blood plasma amino acids and observations on the detection of amino acids limiting growth of chick fed purified diets. J. Nutr. 79:303. Morrison, A. B. and I. C. Munro. 1965. Factors influenc- ing the nutritional value of fish flour. IV. Reaction between 1,2-dichloroethane and protein. Can. J. Biochem. 43:33. Richardson, L. R., L. G. Blaylock and C. M. Lyman. 1953. Influence of dietary amino acids in the blood plasma of chicks. J. Nutr. 51:515. Smith, R. E. and H. M. Scott. 1965. Biological evalua- tion of fish meal protein as a source of amino acids for growing chicks. J. Poultry Sci. 44:394. Stillings, B. R., O. A. Hammerle and D. G. Snyder. 1969. Sequence of limiting amino acids in fish protein con— centrate produced by isopropyl alcohol extraction of Red Hake (Urophycis Chuss). J. Nutr. 97:70. 109 SUMMARY AND CONCLUSIONS Experiments were conducted with young calves and weanling rats to evaluate the nutritive value of fish protein concentrate (FPC) as a sole source of protein. The FPC used in the present studies was prepared by extraction with dichloroethane (DCE-FPC) or isopropanol (IP-FPC). Growth response of young calves on FPC milk replacers was significantly inferior to dried skim milk. Growth response on IP-FPC (from which most of the water—soluble substances had been removed before extraction with the solvent) was inferior to DCE-FPC. The data suggest that water-soluble proteins are essential in the diets of young calves but more research on this problem is needed. Calves on FPC milk replacers-developed microcytic, normochronic anemia suggesting a poor utilization of pro— tein from FPC diets. Muscular degeneration occurred in calves on DCE-FPC rations supplemented to provide 60 mg/100 kg body weight of vitamin E. However, raising the level of vitamin E to twice this level prevented muscular degeneration, resulted 110 111 iri normal tocopherol concentrations in blood, and improved sgxnowth compared to that shown for DCE—FPC diets containing lcmner levels of vitamin E. The DCE—FPC contained about 370 mg/kg of chlorocholine czhloride (CCC) and 360 mg/kg of dichloroethane (DCE). Add— :Lng 400 mg CCC per kg protein to a dried skim milk ration 'tended to depress the growth in young calves. Identical .levels of DCE did not exert an adverse effect upon growth. Extraction of DCE—FPC with ethanol produced superior spas emusmEmHaasm oeeumoa “soeumm .coHDMNHHmumcHE Desmouumxp mo mmaocmum mcHw m>mc ou pmummmmm mounflm m>Humumcmmm© ecu mo mEom coHuomm mco CH .Hmaooc oHuocx>m on: can :mHHo3m .pmucmemmum mumB coecz mounem omnmuumom 3mm m mums whose mmv .Hmaooc Hmuucmo omchucoo c0euomm mmouo ce use mounem meow one cofluemuum mmouo mo mmOa mmz muons .Hmaooc Hamo Hmamcocmmwa CH mmmmuocH 080m paw mmubem mo coflumucwammum Oman mmz mumne .oeuocxxm mumz Awaooc News» paw cmaao3m wum3 .cewum :Hmom ecu “Om wueoemmm ommmmuocfl cm pouenflcxm mmunflm Hmconmooo mcoeuomm Hmum>mm CH Ham m CHEMDH> £ua3 poucmswammomco ummnmoo "coaumm HmEuoz owe HmEuoz omm A03 seen as ooa\me owe m seamue> sue: emusmsmeaasm oemumoo "sceumm .HmEuo: maamHucmmmm pmummmdw mHOmSE Hmumamxm mo mcofiuomm umnuo Hmum>mm .Hmaosc oeuocx>m cuHB nephew cmaaozm epmumuumom 3mm m mumB wumcu .coHuowm mmOuo Ce omzwfl> mumz mmuoew may cuecz :H c0euomm umcuocm CH .waamcfloouflmcoH poo mumB mounew ecu :oflc3 CH coHuomm woo CH maamo Hme>cocmmofi an coHumuuHHmcH nufla mounem wo mafiaamzm paw coHumucwEmmum omumuumom won m>emcmuxm aauHmM was mumce mow HmEuoz mmv m CHEmuH> cue3 omucmEmHQQSwas xHHE Eflxm omega ”coHumm HmEuoz mmv HEuoz mom ADB wpon mx ooa\mE omv m CHEmuH> cue3 poucmEmHQQSm xHHEEme pmfluo "coflumm wamo .coHumucmEmHmdsm m CHEmuH> cuflz 0mm pow mm>amo CH mmmcmso HMHSOmSE mo cofluafluommp mo wumEESmII.N mqmde 116 em.e- mm.m- a . o.m- .sao + oamnmoo NH em.e- He.m- a . o.H- .sao + oeaumoa He . em.a- . o.m- .562 + oemnmoa OH . me.a- . o.m- .562 + oem-moo a mo.e- . me.mu o.me+ 0.4+ oamumoo m mm.s- mm.e- o.a I o.e- oemumoa e mama em mama om meme em same om “momammm sees memo mmmcmmw mmmmmeEmm .QH mmmcmcu ucmflmz 0H HMflH“ co “HMO HMSUH>HUGH CO mum0"om mqmde 1.177 TABLE 4.--Changes in growth and hematological values on individual calves fed milk replacers for eight weeks (Trial II) 1 _ m- c- .- -_ :__.:_: :32: - z: .r" ~-~-~1- -—2~r—.n — gglf Milk Replacer Weight Hemoglobin Hematocrit RBC (lb) (0/100 m1) (0) (million/mm3) 709 DSM 20% p 31.0 - 0.4 - 5.0 — 1.0 395 DSM 20% p 23.0 + 1.2 + 4.0 0.8 716C DSM 208 p 42.0 - 0.4 - 3.0 - 9.3 888 DSM 20% p 31.0 - 1.6 - 5.0 - 1.1 380 DSM 20% P 49.0 + 0.5 + 1.0 + 0.3 Avg. 35.2 - 0.14 - 1.60 - 0.26 857 DSM 108 p 11.0 ~ 0.9 - 3.0 - 0.9 970 DSM 10% p 18.0 - 3.1 - 8.0 - 2.4 437 DSM 10% p 9.0 - 2.5 - 7.0 - 1.8 895 DSM 10% p 1.0 — 1.9 - 3.5 - 1.6 436 DSM 108 p 6.0 — 6.0 —l6.0 - 4.7 AVG. 9.0 - 2.88 - 7.50 - 2.28 400 DCE—FPC 20% p 27.0 - 1.2 - 3.0 - 0.3 335 DCE-FPC 208 p 0.0 - 2.8 -10.0 - 1.3 868 DCE-FPC 20% p 9.0 - 2.1 - 9.0 - 2.1 374 DCE-FPC 20% p 21.0 - 1.9 -10.0 - 1.3 376 DCE—FPC 20% p 20.0 - 2.8 -12.0 - 1.0 Avg. 15.4 - 2.16 - 8.80 - 1.20 873 DCE—FPC 10% p 1.0 - 4 3 -14.0 - 0.6 389 DCE-FPC 10% 9 ~20 0 - 3 6 -13.0 - 0.8 969 DCE-FPC 104 p -20 0 — 3.6 -15.0 - 2.5 402 DCE—FPC 108 p — 8.0 - 3 1 -11 0 - 0.4 394:1 DCE—FPC 106 p — 9.0 - 1 3 - 7.0 - 0.9 Avg. ~11.2 - 3 30 -12.00 - 1.04 858 DCE—FPC screened 20: p - 2.0 — 1.5 — 6.0 - 1.0 392 DCE—FPC screened 208 p + 1.0 - 2.6 - 5.0 — 0.7 716D DCE—FPC screened 208 p 4.0 - 4.5 -l6.0 - 2.0 384 DCE-FPC screened 208 18.0 — 3.4 -13.0 ~ 2.1 772 DCE~FPC screened 208 p 11.0 - 4 7 —19 0 - 2.5 Avg 6.4 - 3 34 -11.8 — 1 66 391b IP—FPC 20% p —17 0 - 1.0 - 3.5 + 0.1 401b LP-FPC 20% p -14.0 - 3.6 -11.0 - 2.2 386C IP—FPC 20% p - 9.0 - 1.4 - 7.0 - 1.7 843b IP-FPC 20% p - 4.0 - 0.8 - 3.0 - 0.8 387d IP-FPC 208 p -14.0 - 2.1 - 6.0 - 1.1 Avg. —11.6 - 1.78 - 6.10 - 1.18 4 weeks data 7 weeks data 6 weeks data 5 weeks data 118 TABLE 5.--Data on individual calves on trial III.a Calf . Weight Changes No. Milk Replacer (8 weeks) (lb) 953 DSM + CCC 26.0 438 DSM + CCC 33.0 Avg. 29.5 767 DSM + DCE 41.0 854 DSM + DCE 32.0 Avg. 36.5 aData for calves on DSM ration shown in appendix table 4. 119 TABLE 6.--Data on individual calves on trial IV.a Calf Milk Replacer Weight Changes N (6 weeks) 0. (1b) 446 DCE—FPC EX. 23.0 472 DCE-FPC Ex. 15.0 991 DCE-FPC EX. 22.0 Avg. 20.0 888* DCE-FPC washed -1l.0 468 DCE-FPC washed - 1.0 473 DCE-FPC washed -l4.0 Avg. _ 807 aData on calves on DSM and FPC rations shown 5 weeks data. 120 .m>4 0.00 0.000 0.000 0.00 0.00 0.000 0.000 0.00 H00 + 0001moo 0000 0.00 0.000 0.000 0.00 00 + 0001000 0000 0.00 0.000 0.000 0.00 00 + 0001000 000 0.00 0.000 0.000 0.00 00 + 0001000 000 0.00 0.00 0.000 0.0 .0>4 0.0 0.00 0.000 0.0 m 1 0001mog 0000 0.00 0.00 0.000 0.00 m 1 0001000 0000 0.0 0.00 0.00 0.00 m 1 0001000 000 0.00 0.00 0.00 0.01 m 1 0001000 000 0.00 0.00 0.000 0.00 .0>0 0.00 0.00 0.000 0.00 m + 0001000 0000 0.00 0.00 0.000 0.00 m + 0001000 000 0.00 0.00 0.000 0.00 m + 0001moo 000 0.0 0.00 0.000 0.00 .0>< 0.0 0.00 0.000 0.00 m 1 200 0000 0.0 0.00 0.000 0.00 m 1 :00 0000 0.0 0.000 0.000 0.00 m 1 200 000 0.0 0.00 0.000 0.00 m 1 200 000 0.00 0.000 0.000 0.00 .0>a 0.000 0.000 0.000 0.00 m + 200 0000 0.00 0.000 0.000 0.00 m + 200 000 0.00 0.00 0.000 0.00 m + 200 000 0.00 0.00 0.000 0.00 m + 200 000 1 1 1 1 008 000x050 1 1 1 1 0000 0003 000 0003 000 0600000 000003 00 . IIIIIIIIIIIIIIII.--I!1 umodemm x002 oz . mmcmno unmamz ocoo Houmndooou mEmmHm . mamu .m CHEmuH> c003 omucmEmHQQSmc: uo pmucmEmammom umomHmmH xHHE pom mm>amo Hm5p0>0ch co mum011.5 mqmde 121 .wpoum ucemeum ecu CH pew: HeHHeueE COM peaonEe uce>HOm ecu mm3 HOCeQOHQOmH .eueHmEoo mH mCHuueweU HHqu oeueemeu mH CoHueuemo CoHuomuuxe ecu .pHHom ecu Eoum oe>oseu ceec mec uCe>Hom ecu Heuwd .uoo UeHuueo eH CoHuoeuuxe ecu euec3 xCeu mCHxHE e ouCH peoooouuCH mH uCe>H0m ecB .erHo>e eue eCmcueouoHcoHo exHH uCe>Hom oeueCHuoHcU .muCe>Hom mooHue> cuH3 peuoeuuxe Cecu mH cmHm ecu CH uem eca .gCHuuHum oCe mCHueec uCeumCoo cuH3 oexooo umun mH cmHm ecB .mCeeuom cOCH Nm\H mCHmo HeUCHum m CH oCooum mH CoHuoeuw CHeuoum .CHeuouQ Eoum mCme oCe meCoc eueuemem ou mceeuom cmeEIOm pCe 0H cmsoucu oeCeeuom mH HeHueueE peHuo .muooc em How m wQOH ue ueHuo Ce>o ce CH peHuU mH HeHueueE Ueuoeuuxe ecB .peueuHHw Esooe> UCe meuoCHE mH Com m oomH ou peueec .HOCemoumomH mo ucmHeB muH eoH3u cuH3 oexHE Cecu mH HeHueueE ece .H6eHo mH uCeonme ecu HHqu ueue3 uoc cuH3 oecme3 oCe oeoCMHuuceo mH wuusHm ecB .m oomH um uooc m\H 000 oeuuHum oCe oeumec mH >uusHm oeHMHUHoe 600 606 6066 00060000000000 6003 0.01e.0 ma 06 660006006 .pexHE HHe3 eue HeHueueE pCsoum pCe ueueB mo mucmHe3 Heowm .eNHm cOCH «\H ou uemmoco ueeE m CH pCooum eue cmeHm mCHuecpe oce eCocxoec ecB .oeoueOme oCe Ue>OEeu eue cmHm mo mpeec ece AHV 60000 A00 A>0 A>Hv AHHHV AHHV He>5eCCeoCe> AHV cam CMEUUSO AHV CoHumHuomeo .OHMHUCOOCOU meOOHm CHeuoud cme 00 CoHueuedeum CH pews memmeooud ecu mo CoHumHuomep weHum1|.m mqmce 122 .mCeeE eHceuHCm e xc HmeE ecu Eoum ©e>OEeu mUHQHH uecuo 6C0 HHO wce .UeHuU kHUHmeu mscu mH HmHueueE ece .CoHueumwceU Essoe> ou .CoHumuHme cuHB .Ueuoencsm UCm HHO eHcHUe uecuo no eHceueme> m cuHB UeHuuCHm mH HeHueueE UeuDCHEoo ecB .ECHUeE Hemmceuu umec mo mHec ecu cuH3 U oomlov um Ueueuwxcew Essoe> mH cmHm cmeum .0 com um UeHuc |>muu >HH0CHm 0C0 ueumB cuH3 Uecmez 0©eueuHHm mH HeHueueE ecB .5 Cu Ueumznwe mm ecu UCe ueue3 CH Ueccemmsm HeHueueE Ueuomuuxe ece .Ueueuome>e mH Hocoon Hmscheu .CoHuomuuxe ecu we CoHueHmEou ecu uC .Hocecue mmm UeHwHwHoe cuH3 weuoeuuxe HeHueueE weHHU ecu UCe UeHMCIEqu mH weCHeuco 0m mmmE ece .OH ou mm ecu emHmu ou ewaouvxc ECHGOm msoesve uCeHonwsm 6C0 euHmHCm ECHUOm mo ucmHeB muH mo uCeo Hem H cuH3 uH mCHxHE .cmHm cmeum mCHCCHum mo mumeCOU mmeooum chB .mcsum uCemeum ecu CH Gems HmHueumE 00w veonmEe uCe>H0m ecu mm3 eCecue nouoHcoHo .ueue3 mo e008 0C0 HHO mCHCHmEeu ecu .CemouuHC CHeuoumnCOC 0C0 0000 ecu e>OEeu ou uce>H0m mCHHHoc cuH3 vecmez CHmmm mH uoswoum chB .uem UCe ueumz ecu e>OEeu ou uCe>H0m mCHHHoc mo mecmez e>Hm cuH3 vecm03 mH HmHueume chB .ueUCHum umeE cuH3 UCCoum Ucm ueuez cuH3 wecmez mH cmHm eHocz AHV AHV AHV CoHueuwmceU Essom> muecuoum ue>eq 00000> “my 000 Amv 123 0.0 0.0 m.n ~.0 00000002 0.HH m.mH m.MH m.mH cmd m.o m.H o.m o.m uoenuxe 0ecum m.~m o.m0 m.00 n.00 0000000 00000 00mmlmHV Aommlmoov mecom unocuHB mecom cuHZ muumd eCHde cam muov CHmoH> .eum0uCeoCoo CHeuOHQ cmHm mo COHuHmomEoo 000a0x000nu.m 00000 124 TABLE 10. Mineral content of fish protein concentrate.a Element mg 1100 9. Calcium 3,780 Phosphorus 2,930 Sodium 353 Potassium 593 Chloride 1,000 Bromide 1.5 Iodide 0.1 Flouride 13 Iron 18.9 Selenium 0.2 Copper 0.9 Manganese 0.9 aUnited States Department of the Interior. Fish and Wildlife Service. Bureau of Commercial Fisheries. Fishery leaflet 584. mm 145 0 1293 03