5% IIHHIHNIWJHIWIJHIWWWIWIIHIIHIIHIWHI 57196 NIVERSI ITY LIBRARIES III III III IIIII II ? IIIIII IIIII II III IIIIIII 3 1293 0062 usmv w Midnlgan State University This is to certify that the thesis entitled The Effects of Isoacids on { Performance of Nonruminants presented by Luiz Lehmann Coutinho has been accepted towards fulfillment of the requirements for M. S . degree in Animal Science flaw/a. M Major professor Date June 9, 1987 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove thlo checkout from your record. TO AVOI FINES retun on or before date duo. DATE DUE DATE DUE DATE DUE fiw L_== I—fl MSU Is An Affirmative Action/Equal Opportunity Institution THE EFFECT OF ISOACIDS ON PERFORMANCE OF NONRUMINANTS By Luiz Lehmann Coutinho A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCES Department of Animal Sciences 1987 @OOIIOI ABSTRACT THE EFFECT OF ISOACIDS ON PERFORMANCE OF NONRUMINANTS By Luiz Lehmann Coutinho This study was conducted to evaluate the effects of isoacids (isobutyrate, isovalerate, 2-methy1butyrate, and valerate) and phenylacetate on performance of nonruminants. Six trials were conducted with pigs, rats and chickens. Daily weight gain, feed intake, feed conversion and carcass composition of swine and poultry plus milk production by rats were measured. Isoacids or phenylacetate did not increase milk production in rats or weight gain and feed conversion in swine and poultry (p > 0.10). In swine isoacids decreased back fat thickness by 11% and increased muscle by 5%. However, these differences were not significant at p < 0.10. In poultry, isobutyrate and isobutyrate plus 2-methylbutyrate decreased fat and increased protein (p < 0.05). This study shows that isobutyrate and isovalerate partition nutrients from fat deposition to protein deposition. This may have major application in the livestock industry. To my mother, Julieta, my father Luiz, my brother Felipe, my grandparents, Maria, Lourdes and Otto, and to my best friend Julie, without whom life wouldn’t be as fun. iii ACKNOWLEDGMENTS The author wishes to express gratitude to his major professor Dr. Robert M. Cook for his guidance, attention and friendship during the course of this work. Gratitude is also extended to Dr. E.R. Miller and to the members of the graduate committee Dr. Cal J. Flegal and Dr. J.L. Gill for their attention, guidance and friendship. The author is also grateful to Dr. C.G. Scanes for the growth hormone assay. Special thanks are expressed to all my friends, who have made this program a pleasant and unforgettable experience. Special acknowledgments are expressed to EMBRAPA (Empresa Brasileira de Pesquisa Agropecuaria) for the financial support. iv TABLE OF CONTENTS Page LIST OF TABLES ..................................... vi INTRODUCTION ........................................ 1 LITERATURE REVIEW ................................... 4 Control of growth by exogenous substances ...... 4 Isoacids and animal performance ................ 8 Growth hormone and insulin effects on animal performance .................................. 10 MATERIALS AND METHODS ............................... 15 RESULTS ............................................. 26 DISCUSSION .......................................... 43 REFERENCES .......................................... 50 Table H 10. 11. 12. LIST OF TABLES Page Composition of the diets for swine ............. 22 Composition of the finishing diet for swine....23 Composition of the diet for poultry trial number one ..................................... 24 Composition of the diet for poultry trial number two ..................................... 25 Effects of isoacids on lactation performance of rats ...................................... 30 Effects of isoacids on performance of growing pigs (starter phase) ................. 31 Effects of isoacids on performance of growing pigs (grower phase) ................ ,..32 Effects of isoacids on performance of finishing pigs ............................... 33 Effects of isoacids on carcass composition of pigs ......................................... 34 Effects of isoacids on performance of broilers (twelve days). Trial one ....................... 35 Effects of isoacids on performance of poultry (12 to 26 days). Trial one ..................... 36 Effects of isoacids on carcass composition of broilers. Trial one ....................... 37 13. 14. 15. 16. 17. Effects of isoacids on performance of broilers (day one to fourteen). Trial two ............. 38 Effects of isoacids on performance of broilers (14 to 28 days). Trial two ..................... 39 Effects of isoacids on performance of broilers (28 to 56 days). Trial two ..................... 40 Effects of isoacids on carcass composition of broilers. Trial two ............................ 41 Effects of isoacids on plasma levels of growth hormone of broilers. Trial two ................. 42 vii Introduction The often predicted Malthusian nightmare of population outstriping food production has never materialized. For the past forty years food production has outgrown the increase in population and there is enough food to supply all the population of the world. However, about 730 million people do not have enough food. The world faces a problem of food distribution, because the developing and underdeveloped nations do not have enough purchasing power to buy the needed food (Clausen, 1986). Proper nutrition is one of the most basic needs of mankind, and the whole world community should be involved in solving this problem. The responsibility of the researchers, working in food production, is to develop new technologies that will help to increase the efficiency of production, so a smaller share of a person’s income is used to fulfill the basic need of nutrition. In the last decades the improvement in animal performance has been remarkable. For instance, the poultry industry has reduced the time required to obtain market weight for broilers by half (Chambers et al., 1981 ), and the swine industry has reduced the time required to obtain market weight by 30% (Pond, 1981). Although efficiency of production is still a concern, the public new demands less dietary fat. This means that carcass composition is a l growing problem for the producer. High levels of dietary fat have been correlated with coronary heart disease, cancer, obesity, and reduction of life expectancy in humans (Guyton, 1986; Committee on Diet, Nutrition and Cancer 1982; Creasy, 1985; Caster, 1976; Watkin, 1979; Young, 1978), but there is no conclusive evidence that fat per se is the cause of these problems (Kaunitz, 1975; Hazard, 1976; National Dairy Council Digest, 1974, 1986; Nauss et al. 1983; Kroes et a1., 1986). The increased energy levels of high fat diets seems to be the cause of the negative effects related to dietary fat (Committee on Diet, Nutrition and Cancer 1982; Creasy, 1985; National Dairy Council Digest,1986; Willett, 1984). Fat is a more concentrated source of energy (9 kcal/g) than carbohydrate or protein (4 kcal/g), and is utilized more efficiently (Donato et a1., 1985; Donato, 1986). Partition of nutrients away from adipose tissue should increase animal performance (Etherton and Meserole, 1982 ), because greater energy is required to synthesize 1 kg of adipose tissue than is required to produce 1 kg of protein (van Es,1977). Several exogenous substances have been developed to increase growth rate, feed efficiency, and improve carcass characteristics (Muir, 1985). Recently, a new feed additive (isoacids) was approved by the Federal Food and Drug Administration for use in dairy cows. This product has been shown to increase milk production and feed efficiency of dairy cows (Clark et al., 1986; Rogers et al., 1986 a,b; Papas et al., 1984; Felix et al. 1980 a; Towns and Cook, 1984; Felix, 1976), and performance of heifers and steers (Felix et al., 1980 b; Richardson et al., 1984; Deetz and Richardson, 1984; Richardson et al., 1985). The mode of action of isoacids has not been fully determined. Their action in the rumen has been studied since as early as 1959 (Allison et a1.) and there seems to be no doubt that these compounds enhance rumen fermentation (Machado et al., 1985; Brondani, 1986). Recent studies have postulated that a change in hormone balance is also involved in the mode of action of isoacids . Studies with dairy cows (Towns and Cook, 1984) have reported an increase in growth hormone and insulin, two peptides known to be associated with growth, milk production and carcass composition of livestock. The postabsorptive effect of isoacids opens the possibility that these compounds may improve production and carcass characteristics in nonruminants, and this is the subject of this thesis. Literaturegreview Control ofggrowth by exogenous substances The exogenous, non-nutritive substances that affect animal performance, can be collectively referred to as production improvers. As described by Muir (1985), these substances can be classified in four general classes. Antimicrobial production improvers, rumen additives, anabolic growth promotants, and others. Antimicrobial production improvers, can be further subdivided into growth permittants and growth effectors. Growth permittants (antibiotics, antibacterial, and antifungal), are antimicrobial agents that inhibit non- pathogenic microflora of the intestinal tract. The use of these substances can increase growth rate by approximately 20%. The mode of action of these compounds is not fully understood. The inhibition of the microflora results in reduction of thickness and length of intestinal tract. This suggests a decrease in number and turnover rate of the mucosa, thus decreasing protein and energy requirements (Muir, 1985). It has also been shown that in animals treated with growth permittants, there is an increase in efficiency of nutrient absorption (March and Biely, 1967), inhibition of toxin production (Visek, 1978), nutrient-sparing action (Jukes, 1971), and a decrease in competition for food between the microbes and the host (Muir, 1985). Growth effectors (antibiotics, antibacterial and antifungal) are also antimicrobial agents, but their mode of action is different. These substances restore animal performance by inhibiting microorganisms responsible for a clinical or subclinical infectious disease (Muir, 1985). Rumen additives (antibiotics and isoacids), are compounds that selectively inhibit or induce certain groups of rumen bacteria. The antibiotics used as rumen additives are Rumensin, Lasolacid, Avoparcin, and Teichomycin (Cook, 1985). Rumensin, the most widely used rumen additive, is an ionophore that selects against acetate and hydrogen producing bacteria. The use of rumensin decreases feed consumption, increases weight gain, and improves feed conversion (Rumensin Technical Manual, 1975). The mode of action of rumensin has not been totally elucidated. It causes a shift in the volatile fatty acids production, increasing the percentage of propionate and decreasing acetate. This shift is associated with a decrease of approximately 30% in methane production (Broome, 1980; Chalupa, 1980). These changes in the end products result in an improvement in the efficiency of fermentation, since propionate recovers 109% of the energy of glucose, compared to 62% for acetate (Muir, 1985). It has also been shown that rumensin decreases protein degradation in the rumen, increases efficiency of volatile fatty acid utilization by the animal, and improves nitrogen retention (Broome, 1980). Isoacids are a mixture of three branched-chain fatty acids (isobutyrate, isovalerate, and 2-methylbutyrate) and a straight-chain fatty acid (valerate) (Cook, 1985). This blend of chemicals was the first feed additive of its kind to be developed for dairy cows, and its action on animal performance will be discussed in detail later. Anabolic growth promotants, can be further subdivided in two general classes, steroids and proteins. There are several estrogenic agents on the market (Compudose, Ralgro, Synovex-H, and Synovex-S), that increase gain and feed conversion in ruminants (Muir, 1985), increase carcass protein in lambs (Muir et al., 1983) and steers (Rumsey et al.,1977), increase accumulation of fat in poultry , but have no effect in swine (Trenkle, 1969). The mode of action of estrogenic substances is not fully understood. They seem to increase growth hormone (Struempler and Burroughs, 1957, 1959), and insulin secretion (Trenkle, 1969, 1970). These changes in hormones could explain the increase in carcass protein observed in lambs (Muir et al.,1983) and steers (Rumsey et al.,1977). Progestogenic agents (melengestrol acetate) improves gain and feed conversion up to 10% in cyclic heifers (Davis, 1969). The mode of action of such a compound seems to be through estrus suppression and reduction of estrus related problems, such as low feed intake and hyperactivity(Davis, 1969). Androgenic agents (trenbolone acetate) as reported by Heitzman (1980), improve growth rate, feed conversion and carcass protein percentage of swine and cattle. The mode of action seems to be through its binding to androgen receptors in skeletal muscle. This could directly increase protein deposition (Muir, 1985), or through down regulation of glucocorticoid receptors (Mayer and Rosen, 1975, 1978) decrease protein turnover (Vernon and Buttery, 1976. 1978). Growth hormone, probably the most important hormone required for normal growth, is also capable of stimulating additional growth when its concentration is increased (Scanes, 1985). Growth hormone has also been shown to increase milk production in lactating cows (Peel et al.,1985; Bauman et al.,1985). The mode of action of growth hormone is not fully understood, but it will be discussed further in this dissertation. Other production improvers is a general category which includes substances such as dichlorvos, thyroprotein (Muir, 1985), and clenbuterol. Dichlorvos, an antiparasitic agent has been shown to increase the number of piglets born alive and litter birth weight of sows not infested with parasites (Young et al., 1979). Thyroprotein (iodinated casein), has been shown to increase milk production of dairy cows for a short period of time. The production for the entire lactation period does not seem to be altered (Muir, 1985). This chemical seems to act by increasing the basal rate of the animal (Schmidt et al., 1971). Clenbuterol is a beta-adrenergic agonist that improves feed conversion and carcass protein percentage in lambs (Baker et al., 1984). It increases carcass protein percentages in steers (Ricks et al., 1984), and growth rate, carcass yield, carcass lean tissue in broilers (Dalrymple. 1984). It has also been shown to increase muscle and decrease fat in swine (Ricks, et al., 1984). The mode of action of this compound is not well understood, but it seems to be similar to the hormone adrenalin (Cook, 1985). oa d d ‘ma er a e. Isoacids occur naturally in the rumen as a result of protein degradation by rumen microbes. They stimulate cellulose degradation by rumen microorganisms and can be used for amino acid synthesis by the rumen microflora. Rumen bacteria are unique in that they can carboxylate the carboxyl group of an isoacid to form alpha-ketoacid analogues of essential amino acids (Cook, 1983). Isoacids, either as a complete mixture or as different combinations of the four acids, have been reported to increase nitrogen retention in steers (Oltjen, 1971), feed intake in sheep (Hemsley and Moir, 1963), growth rate in dairy heifers (Lassiter, et al., 1958), growth rate (Deetz, 1984; Richardson et al., 1984 and Owens, 1983), feed efficiency, (Richardson et al., 1984; Owens et al., 1983; Deetz, 1984), and carcass characteristics of beef cattle (Richardson et al., 1984; Owens et al., 1983), milk production and feed efficiency of dairy cows (Felix, 1976; Felix et al., 1980a, Papas et al., 1984; Towns and Cook, 1984; Rogers et al., 1986 a,b; Clark et al., 1986 a,b, Newman et al. 1986). The mode of action of isoacids has not been fully understood. There seem to be two components to its action, one being at the rumen level and another at the postabsorptive level. In the rumen, isoacids increase protein synthesis by the rumen bacteria (Russel, 1983), dry matter digestibility (Gorosito et al., 1985) and nitrogen retention (Oltjen et al., 1971; Felix, 1976). These actions can be explained by the capacity of the rumen bacteria to utilize the carbon skeletons of the isoacids to synthesize amino acids (Allison et al., 1969) and to stimulate rumen cellulolytic activity (Bryant and Doetsch, 1955; Allison, 1970). 10 Postabsorptively, isoacids seem to act through changes in hormone balance. Recent studies have reported that in dairy cows isoacids increase concentrations of growth hormone (Towns and Cook, 1984; Fieo et al., 1984) and insulin (Towns and Cook, 1984; Coutinho et al., 1986, Unpublished). However, there have been contradictory results. Kik and Cook (1986) reported no difference in growth hormone or insulin concentration in dairy cows fed isoacids. In a study with sheep, Brondani (1986) reported no difference in growth hormone or cortisol, but decrease in insulin levels. There is need for more research in this area to clarify the extraruminal effects of isoacids. Growth is a complex phenomenon that involves increase in cell number, increase in cell size and deposition of substances within the cell (Spencer, 1985). Different tissues have different growth rates. The first one to deveIOp in the animal is the nervous system. It is followed by bone, muscle and adipose tissue (Beitz, 1985). The deposition of protein and fat, although similar in early life, differ as the animal develops. In a mature animal protein deposition decreases but fat accretion continues (Bergen, 1974). 11 The endocrine system plays a very important role in controlling growth. Of particular importance for muscle and adipose tissue development are growth hormone and insulin. The importance of insulin in regulating growth can be dramatically noticed in insulin deficient individuals. Induced diabetic pigs have a much lower growth rate (50%) than normal animals (Romsos, 1971 a), and growth can be restored by injection of insulin (Romsos, 1971 b). The fact that insulin is essential for growth, does not mean that high growth rates are associated with high insulin levels. In a study with swine, Steele and Etherton (1983), have demonstrated that injection of insulin (1 U/kg body weight/day) does not alter growth rate, feed efficiency, muscle or adipose tissue mass. There also appears to be little correlation between insulin concentration in the plasma and growth rate (Trenkle and Topel, 1978; Martin et al.,1979; Etherton, 1982). Nevertheless, there seems to be a positive correlation between insulin level and the degree of fatness in nonruminants (Spencer, 1985). The action of insulin on adipose tissue is anabolic. It stimulates glucose uptake and activates lipoprotein lipase in the adipose tissue. It also decreases lipid mobilization by inhibiting the action of the hormone sensitive lipase (Guyton, 1986). 12 In muscle, insulin also has an anabolic effect. It stimulates amino acid transport, protein, RNA, and DNA synthesis (Buttery, 1983). It also has a sparing effect on amino acids, by inhibiting gluconeogenesis (Guyton, 1986). The lack of insulin in diabetic individuals causes an increase in protein catabolism, decrease in protein synthesis, increase in plasma amino acids and an increase in urea excretion. Low insulin levels also increase the use of amino acids for glucose synthesis and as an energy source. Growth hormone is unquestionably required for growth (Muir, 1985). Hypophysectomy decreases growth in pigs, goats, chicken, cattle, rats, and fish (Scanes, 1985). In studies with hypophysectomysed chickens (Scanes, 1985) and pigs (Anderson, 1981), injection of growth hormone partially restored growth rates. It is also reported that administration of growth hormone can improve performance of intact animals. Growth hormone stimulates growth and feed efficiency of pigs (Machline 1972, Chung et al., 1985, Rebhun et al., 1985; Etherton et al., 1986),growth (Wagner and Veenhuzen, 1978), and feed efficiency of sheep (Muir et al., 1983), growth of fish (Higgs et al., 1975) and nitrogen retention in cattle (Mosley et al., 1982). The mode of action of growth hormone has not been fully determined. Several studies have reported that growth hormone increases lipid mobilization. In vitro studies with either rat or chicken adipose tissue, and native or synthetic growth hormone, have demonstrated increased 13 lipolysis (Scanes, 1985). Studies with dairy cows and pigs have been found to increase circulating free fatty acids (Scanes, 1985). There have also been reports that growth hormone decreases lipogenesis. This was observed in rat hepatocytes, chicken hepatocytes and ovine adipose tissue (Scanes, 1985). It should also be noticed that, as reported by Hart (1984), growth hormone seems to have a diabetogenic effect. It increases glucose and insulin in a number of species, including sheep and pig (Scenes, 1985). Growth hormone definitely has an anabolic action on protein metabolism (Buttery, 1983). Growth hormone has been reported positively correlated with growth of lean tissue and negatively correlated with carcass fat in cattle (Trenkle, 1977; Trenkle and Topel, 1978). Recent reports indicate that the growth hormone molecule has one region responsible for its lipolytic activity and another region promoting its growth effects (Hart et al., 1984). The growth promoting activity of growth hormone seems to be modulated by somatomedins, which are produced by the liver (Spencer, 1985). It seems that growth is ultimately regulated by the action of somatomedins. Somatomedins induce hyperplasia and hypertrophy in several tissues. It is important to know that insulin and growth hormone have a major role in determining somatomedin production. Growth hormone receptors in the liver are, at least in part, regulated by insulin. An increase in growth hormone without increase in 14 insulin level may not increase somatomedins if the hormone receptor number in the liver remains the same (Spencer, 1985). It is important to realize that growth is not determined by one growth promoting hormone. It is modulated at least by growth hormone, insulin and somatomedins. Summarizing, there is need to develop new products that will increase the efficiency of production, decrease fat and increase protein in the carcass. Isoacids have proved to increase the efficiency of production in ruminants. Their mode of action involves ruminal and extraruminal effects. The extra ruminal effects appear to be through changes in the hormonal concentration of growth hormone and insulin, hormones known to be involved in growth and carcass characteristics. It is reasonable to expect that nonruminants could also benefit from the extra ruminal effects of isoacids, and this study was developed to investigate the effects of isoacids on growth rate, feed efficiency and carcass composition of nonruminants. t als nd Me ods To evaluate the effects of isoacids on nonruminants three separate studies were conducted, one with rats, one with swine and one with poultry. For the rat study, eighty-eight Sprague-Dawley female rats from the Lab Animal Care Service of Michigan State University were used, and the number of pups per litter was equalized to nine. The animals were housed in an environmentally controlled room. They were individually kept in plastic cages and fed a commercial laboratory rat chow and water ad libitum. The dietary treatments, with at least eight litters per teatment, consisted of one control and six treatments. The treatments were isobutyrate, isovalerate, 2-methylbutyrate, valerate, phenylacetate and an equal weight mixture of the first four acids. The acids were neutralized to pH seven with ammonium hydroxide and mixed with the drinking water at 0.16 % on a weight basis. Water intake was measured during the first week in order to be sure that no palatability problem existed. There was no difference between treatments in water consumption. Milk production was indirectly evaluated by measuring weekly weight gains of the litters for three weeks. A completely randomized design model (I) was used where the random sampling variables Yij may be explained by three additive (independent) effects: u, 15 16 the overall mean; Ai, the fixed (constant) effect of the ith treatment; and the E(i)j, the random experimental error (Gill, 1978 a). (I) Yij = u + Ai + E(i)j where, i (treatment number) = 1, 2,....,7 j (replication number) = unbalanced data Treatment means were compared against the control using the Bonferroni t test (Gill, 1978 a,b). For the swine study, isoacids were fed from weaning to market weight, the study being subdivided into starter, grower and finishing phases. For the starter and the grower phases, ninety six crossbred (York, Landrace and Duroc) pigs from the Michigan State University swine farm were used. Barrows and gilts from different litters were distributed. into twelve groups of eight animals each. All the groups were homogeneous in initial weight with near equalization for sex and litter. For the finishing phase, the animals were regrouped into eight pens with nine animals each, maintaining the same dietary treatments they had before. The new groups were also homogeneous in initial weight with near equalization for sex and litter. This experiment was conducted at the Michigan State University swine farm. The facility is environmentally controlled, and the pens for the starter and grower phases housed eight pigs per unit, while the pens for the finishing phase housed nine animals per 17 unit. The animals were fed ad libitum the starter treatment diet for four weeks, the grower treatment diet from the fifth to the ninth week and the finishing diet from the tenth to the seventeenth week. Water was also available at all times. The composition of the starter and grower diets can be seen in table one, and the finishing diet can be seen in table two. The treatments, with three pens per treatment for the starter and grower phases, and two replications for the finishing phase, consisted of a control diet and three levels of an isoacids mixture at 0.1%, 0.2% and 0.4% of the diet. The mixture contained an equal weight amount of isobutyrate, isovalerate, 2-methylbutyrate and valerate. The liquid solution was neutralized with ammonium hydroxide to pH seven, and completely mixed with the diet. The animals were individually weighed weekly during the starter phase and bi-weekly during the grower and finishing phases. Pen feed intake was also determined at the same intervals. Weight gain, feed intake and feed conversion were calculated for each pen. At the end of the trial, four barrows and four gilts from each treatment were slaughtered and measurements taken to calculate back fat thickness, loin-eye area and muscle percentage in the carcass. The procedure used was described by Kauffman et al. (1981). A completely randomized design model (II) was used where the random sampling variables Yij may be explained by three additive (independent) effects: u, the overall mean; Ai, the fixed (constant) effect of the ith treatment; and E(i)j, the 18 random experimental error (Gill, 1978 a). (II) Yij = u + Ai + E(i)j where, 1 (treatment number) i (replication number) All the treatment means were compared against the control using the Bonferroni t test, with the exception of carcass composition, which was analyzed by multivariate analyses of variance (Gill, 1978 a,b). For the poultry study, two trials were conducted at the Michigan State University Poultry farm. Trial number one was designed to test the effects of five acids, at three different concentrations, in growing chickens. Trial number two was designed to evaluate the synergistic effects of the isoacids from hatching to market weight. In trial number one, six hundred male Hubbard chicks were kept for twenty-six days in heated, wire battery cages with water and feed ad libitum. The batteries had ten birds per unit and were housed in an environmentally controlled building. The composition of the diet used for trial number one is shown in table 3. The treatments, with three replications, consisted of a control diet, three levels of five different acids and three levels of an equal weight mixture of four acids. The acids used as treatments were isobutyrate, isovalerate, 2-methylbutyrate, phenylacetate and valerate. The mixture consisted of a combination of 19 isobutyrate, isovalerate, 2-methylbutyrate and valerate. The acids were neutralized with ammonium hydroxide to pH seven and used at 0.05%, 0.1% and 0.2% of the diet. A completely randomized design model (III) was used where the random sampling variables Yij may be explained by three additive (independent) effects: u, the overall mean; Ai, the fixed (constant) effect of the ith treatment; and the E(i)j, the random experimental error (Gill, 1978 a). (III) Yij = u + Ai + E(i)j where, i (treatment number) = 1, 2,....,19 j (replication number) = 1, 2, 3 All the treatment means were compared against the control using the Bonferroni t test, with the exception of carcass composition which was analyzed by multivariate analyses of variance(Gill, 1978 a,b). In trial number two, two thousand and sixteen male Hubbard chicks were used. The building used was environmentally controlled and the birds were kept in pens with one hundred and twenty-six birds per unit. The pens were equipped with two hanging tube feeders and an automatic waterer. Wood shaving was used as litter over a concrete floor. The animals were fed a starter diet for the first four weeks and a grower diet from the fifth to the seventh week. The composition of the diets is shown in table number 20 four. The treatments, with two replications, consisted of a factorial combination of three acids either present or absent (eight treatment combinations). The acids were A : isobutyrate, B = isovalerate and C = 2-methylbutyrate. The acids were neutralized with ammonium hydroxide to pH seven and used at 0.1% of the diet. A completely randomized design model with three factors (IV) was used where the random sampling variables Yijkl may be explained by nine additive (independent) effects: u, the overall mean; Ai, the average effect of the ith level of factor A; Bj, the average effect of the jth level of factor B; Ck the average effect of the kth level of factor C, (AB)ij, (AC)ik and (BC)ik the effect of the interaction of two factors; (ABC)iJk the effect of the interaction of three factors and the E(iJk)l, the random experimental error (Gill, 1978 a). (IV) Yijkl = u + Ai + BJ + (AB)ij + Ck + (AC)ik + (BC)ik + (ABC)ijk + E(ijk)l where, 1 (level of factor A) H H j (level of factor B) H H 2 2 k (level of factor C) = 1, 2. l (replication number): 1, 2 All the treatment means were compared against the control using the Bonferroni t test, with the exception of carcass composition which was analyzed by multivariate analyses of variance(Gill, 1978 a,b). 21 The animals were individually weighed at the first day, at twelve days and at twenty six days for trial number one. For trial number two, the animals were weighed at fourteen days, twenty-eight days, and fifty-six days. Pen feed intake was measured for each trial following the same schedule used for weight. Weight gain, feed intake and feed conversion were calculated for each pen. For carcass composition, two birds per pen were taken for trial number one and ten birds per pen for trial number two. The feed was withdrawn eighteen hours before the animals were killed. At slaughter the birds were electrocuted, ensanguinated, decapitated, defeathered, feet removed, and frozen. Frozen birds were ground three times in a meat grinder and a representative sample collected and stored frozen. Moisture was determined by freeze drying the samples until constant weight. Fat was determined by ether extraction in a soxhelt apparatus and protein by total nitrogen extraction in a microkjeidahl, with ammonia determination in a Techinicon-autoanalyzer. Three days before the end of trial number two, forty birds per pen were sampled in two consecutive days by heart puncture. Plasma was obtained and samples analyzed for growth hormone (RIA method described by Harvey and Scanes, 1977). 22 TABLE 1: Composition of the diets for swine. Ingredients Starter Grower Corn starch 0.80 0.80 Ground shelled corn 54.50 73.25 Soybean meal (44) 25.50 22.00 Mono-dicalcium phosphate 0.15 0.15 Calcium carbonate 0.10 0.11 Sodium chloride 0.25 0.25 Dried whey 15.00 0.00 MSU vitamin-trace mineral premix (a) 0.75 0.50 Vitamin E-Se premix (b) 0.75 0.50 Antibiotic premix 0.250 0.1d L-lysine HCl (78.4% L-LYS) 0.20 0.00 Total: 100.00 100.00 Calculated analysis Crude protein, % 17.5 16.0 Lysine, % 1.14 0.8 Ca, % 0.87 0.75 P, % 0.72 0.63 (8) Contains per kg: Vitamin A, 660.0 IU; Vitamin D3, 1320,000 IU; Vitamin E, 4,400 IU; Menadione sodium bisulfite, 0.44g; Niacin, 4g; Pantothenic acid, 1.2g; Vitamin B12, 4mg; Choline, 22g; Fe, 13.2g; Zn, 16.5g; Mn, 7.5g; Cu, 2.2g: I, 0.12g. (b) Contains per kg: 3,300 IU of Vitamin E and 20 mg of Selenium. (c) Supplies 110mg of chlortetracycline. 110mg of sulfamethazine and 55mg of penicillin per kg of diet. (d) Supplies 55ppm of chlortetracycline. 23 TABLE 2: Composition of the finishing diet for swine. Ingredients Amount Corn starch 0.80 Ground shelled corn 81.15 Soybean meal (44) 16.00 Mono-dicalcium phosphate 0.80 Calcium carbonate 1.00 Sodium chloride 0.25 MSU vitamin-trace mineral premix (a) 0.50 Vitamin E-Se premix (b) 0.50 Total: 100.00 Calculated analysis Crude protein, % 14.00 Lysine, % 0.65 Ca, % 0.58 P, % 0.47 (a) Contains per kg: Vitamin A, 660.0 IU; Vitamin D3, 1320,000 IU; Vitamin E, 4,400 IU; Menadione sodium bisulfite, 0.44 g; Niacin, 4 g; Pantothenic acid, 1.2 g; Vitamin B12, 4 mg; Choline, 22 g; Fe, 13.2 g; Zn, 16.5 g; Mn, 7.5 g; Cu, 2.2 g: I, 0.12 g. (b) Contains per kg: 3,300 IU of Vitamin E and 20 mg of Selenium. 24 TABLE 3: Composition of the diet for poultry trial number one. Ingredients % Ground shelled corn 48.11 Soybean meal (44) 42.24 Corn oil 5.52 Limestone 1.12 Di. Cal. Phosphate 1.81 Sodium chloride 0.50 D.L. Methionine 0.20 Vitamin trace mineral (a) 0.50 (a) Supplies per kg: Vitamin A, 1,363.6 IU; Vitamin D3, 303 IU; Vitamin E, 2.73 IU; Riboflavin, 1.36 mg; Panthotenic acid, 1.7 mg; Niacin, 9.1 mg; Choline chloride, 113.6 mg; Vitamin B12, 2.7 mg; Folic acid, 0.23 mg; D-biotin, 0.05 mg; Thiamine mononitrate, 1.0 mg; Menodione sodium bisulfite, 0.45 mg; Manganese 60 mg; Zinc, 50 mg; Selenium, 10 mg; Copper, 5 mg and Iodine, 1.05 mg. 25 TABLE 4: Composition of the diet for poultry trial number two. Starter Grower Ingredients % % Ground shelled corn 48.01 56.52 Soybean meal (44) 42.24 33.83 Corn oil 5.52 5.50 Limestone 1.12 1.10 Di. Cal. Phosphate 1.81 1.80 Sodium chloride 0.50 0.50 D.L. Methionine 0.20 0.15 Vitamin trace mineral (a) 0.50 0.50 Coccidiostat 0.10 0.10 (a) Supplies per kg: Vitamin A, 1,363.6 IU; Vitamin D3, 303 IU; Vitamin E, 2.73 IU; Riboflavin, 1.36 mg; Panthotenic acid, 1.7 mg; Niacin, 9.1 mg; Choline chloride, 113.6 mg; Vitamin 812, 2.7 mg; Folic acid, 0.23 mg; D-biotin, 0.05 mg; Thiamine mononitrate, 1.0 mg; Menodione sodium bisulfite, 0.45 mg; Manganese 60 mg; Zinc, 50 mg; Selenium, 10 mg; Copper, 5 mg and Iodine, 1.05 mg. Results In the rat study, isobutyrate when added to the drinking water at 0.16% resulted on 8% increase in milk production, as measured by weight gain of the litter. (table 5). When this acid was combined with isovalerate, 2-methylbutyrate and valerate in an equal weight mixture, it showed no difference in milk production. It is unlikely that the combination with the other acids could antagonize the effect of isobutyrate alone, since none of the other acids present in the mixture reduced milk production when given alone. The lack of a coresponding increase in milk production was probably because when combined with the other acids, the concentration of isobutyrate was reduced to 0.04% of the drinking water, so the level of the treatment could be maintained at 0.16% However, none of the treatments resulted in statistically significant differences (p > 0.10). In the swine study the addition of an equal weight mixture of isobutyrate, isovalerate, 2-methylbutyrate and valerate at 0.1%, 0.2% or 0.4% of the diet did not increase (p > 0.1) average daily gain, average feed consumption, or feed conversion (amount of feed used per unit of gain) in the starter (table 6), grower (table 7) or finisher phase (table 8). However, isoacids had a very interesting result PH 27 the three treatment levels shows that isoacids reduced back fat thickness by 9%, increased loin-eye area by 4% and muscle percentage by 4%. It is important to notice that the improvement in carcass characteristics was obtained even with the smaller dose of the mixture (0.1%). This indicates that in future studies even smaller doses should be tested. The results also indicate that there was no negative effects of the isoacids even at a dose four times higher than the first one used to obtain improvements in carcass composition. This observation indicates that the product is safe even when used at elevated concentrations. However, the results were not significant at p < 0.10, and future studies should be conducted with more experimental units, in order to detect statistical significance. In the first poultry study, during the first twelve days none of the treatments affected weight gain, feed intake or feed conversion (table 10). From the twelfth to the twenty-sixth day all the acids, with the exception of isobutyrate at the 0.05% level, had no effect on average daily gain, feed intake or feed conversion (table 11). Isobutyrate at 0.05% of the diet decreased weight gain by 26% (significant at p < 0.05). However, at 0.1% or at 0.2% of the diet, it did not reduce weight gain. It is interesting that when isobutyrate was combined with isovalerate, 2-methylbutyrate and valerate it did not reduce weight gain, even at the highest concentration of the mixture, when isobutyrate was present at 0.05% of the diet. 28 This observation and the results of the second poultry study, which is shown below, indicate that the negative result obtained with isobutyrate was probably due to an uncontrolled factor and not a result of the treatment. Table number 12 shows the results for carcass composition. There was no significant effect of any of the treatments on moisture, fat or protein percent. However, isobutyrate increased the percentage of protein in the carcass by 8% and 2-methylbutyrate reduced fat in the carcass by 13%, indicating the potential of these two acids to alter carcass composition of broilers. In the second poultry study there was no effect of any of the treatments on weight gain, feed intake and feed conversion during the first fourteen days (table 13), from the fourteenth to the twenty-eighth day (table 14) or from the twenty-eighth to the fifty-sixth day (table 15). However, the results for carcass composition were very interesting and can be seen in table 16. Isobutyrate showed a 12% decrease in fat percent in the carcass (significant at p < 0.1), and the treatment combination of isobutyrate plus 2-methylbutyrate decreased fat and increased protein percent in the carcass (p < 0.05, as showed by multivariate analyses of variance). The plasma growth hormone values (table 17), were not affected by any of the treatment combinations (p > 0.1). The results for this second trial, as mentioned before, indicate that isobutyrate has no negative effect on growth of broilers. They also demonstrate that the potential for the use of 29 isoacids as repartitioning agents suggested in the first trial deserves serious considerations, since the combined effect of isobutyrate and 2-methylbutyrate improves carcass characteristics. 30 TABLE 5: Effects of isoacids on lactation performance of rats. Treatments Initial Weight (a) weight(b) gain(c) Control 65.7 415.2 Isobutyric acid 64.7 449.4 Isovaleric acid 65.6 412.7 2-Methyl butyric acid 64.0 406.7 Valeric acid 65.3 434.3 Phenyl acetic acid 65.4 399.8 Mixture 64.7 416.8 S E M (d) 5 1 36 3 There is no statistical difference for any of the variables (p > 0.1). (a) Treatments were used at 0.16% of the drinking water. (b) Initial litter weight in grams (g). (c) Weight gain (g) from birth to weaning (21 days ). (d) Standard error of the mean. TABLE 6: Effects of isoacids on performance of growing pigs (starter phase). Treatments Init.W. A.D.G. A.D.F.I. Feed (a) (b) (c) (d) conversion Control 8.15 0.36 0 66 1.81 0.1 % 8.06 0.36 0.65 1.81 0.2 % 8.11 0.34 0 64 1.91 0.4 % 8.20 0.36 0 68 1.85 S E M (e) 0 95 0 11 0 24 0 07 There is no statistical difference for any of the variables (p > 0.1). (a) % of the isoacids mixture in the diet. (b) Initial weight (kg). (c) Average daily gain (kg). (d) Average daily feed intake (kg). (e) Standard error of the mean. 32 TABLE 7: Effects of isoacids on performance of growing pigs (grower phase). Treatments A.D.G. A.D.F.I. Feed (a) (b) (0) conversion Control 0 54 1.41 2.60 0.1 % 0.52 1.44 2.76 0.2 % 0.52 1.49 2.87 0.4 % 0 54 1.50 2.77 S E M (d) 0.16 0 73 0 37 There is no statistical difference for any of the variables (p > 0.1). (a) % of the isoacids mixture in the diet. (b) Average daily gain (kg). (0) Average daily feed intake (kg). (d) Standard error of the mean. 33 TABLE 8: Effects of isoacids on performance of finishing pigs. Treatments A.D.G. A.D.F.I. Feed (a) (b) (0) conversion Control 0.78 2.94 3.76 0.1 % 0.78 2.93 3.72 0.2 % 0.75 2.93 3.86 0.4 % 0.80 2 80 3.50 S E M (d) 0.17 0 84 0 33 There is no statistical difference for any of the variables (p > 0.1). (a) % of the isoacids mixture in the diet. (b) Average daily gain (kg). (0) Average daily feed intake (kg). (d) Standard error of the mean. 34 TABLE 9: Effects of isoacids on carcass composition of pigs. Treatments (a) Loin area Back fat Muscle (sq cm) thickness % (cm) Control 31.35 3.22 52.14 0.1 % 33.74 2.93 54.43 0.2 % 32.00 2.97 53.74 0.4 % 32.45 2.87 54.61 S E M (b) 1.90 0 32 2 44 There is no statistical difference for the variables (p > 0.1). (a) % of the isoacids mixture in the diet. (b) Standard error of the mean. TABLE 10: Effects of isoacids on performance of broilers (days one to twelve). Trial one (a). Treatments Initial Weight Feed Feed (b) weight gain intake conversion Control 40.6 268.0 384.8 1.44 Isobutyrate 0.05% 40.8 280.6 414.7 1.48 0.10% 41.4 286.3 409.0 1.43 0.20% 40.9 268.7 379.5 1.41 Isovalerate 0.05% 40.3 272.0 368.5 1.35 0.10% 41.0 266.7 384.0 1.44 0.20% 40.2 275.9 381.5 1.38 2-Methyl butyrate 0.05% 41.2 280.8 406.0 1.45 0.10% 40.3 276.5 424.9 1.54 0.20% 40.0 269.3 382.6 1.42 Phenylacetate 0.05% 40.7 285.0 403.9 1.42 0.10% 40.0 253.3 345.3 1.36 0.20% 40.3 265.1 391.0 1.47 Mixture 0.05% 40.6 283.8 375.5 1.32 0.10% 40.6 267.0 391.7 1.47 0.20% 40.0 283.5 391.7 1.39 Valerate 0.05% 40.3 263.9 395.9 1.51 0.10% 40.0 282.1 418.4 1.48 0.20% 40.2 268.1 390.8 1.46 S E M (c) 1 1 12.4 30 8 10 There is no statistical difference for any of the variables (p > 0.1). (a) All weight units are in grams. (b) Percentage of the acid in the diet. (0) Standard error of the mean. 36 TABLE 11: EffeCts of isoacids on performance of broilers (twelve to twenty-six days). Trial one (a). Treatments Weight Feed Feed (b) gain(a) intake conversion Control 656.8 1537.6 1.66 Isobutyrate 0.05% 487.3 ** 1331.7 1.73 0.10% 639.0 1571.7 1.69 0.20% 594.0 14236. 1.65 Isovalerate 0.05% 635.5 1497.8 1.65 0.10% 554.8 1448.3 1.77 0.20% 683.2 1672.1 1.74 2-Methyl butyrate 0.05% 570.0 1495.4 1.76 0.10% 676.6 1666.8 1.75 0.20% 642.6 1543.0 1.69 Phenylacetate 0.05% 622.6 1614 8 1.77 0.10% 577.5 1357.7 1.63 0.20% 654.9 1569.1 1.71 Mixture 0.05% 607.6 1442.9 .62 0.10% 618.7 15309. 1.74 0.20% 632.0 1457.9 1.59 Valerate 0.05% 691.1 1610.3 1.69 0.10% 632.2 1628.9 1.78 0.20% 640.9 1518.1 1.67 S E M (c) 67.8 142 3 0 09 ** significant effect at p < 0.05. There is no statistical difference for any of the other variables (p > 0.1). (a) All weight units in grams. (b) Percentage of the acid in the diet. (c) Standard error of the mean. 37 TABLE 12: Effects of isoacids on carcass composition of broilers. Trial one. Treatments Moisture Fat Protein (a) % % % Control 68.11 10.92 16.40 Isobutyrate 0.05% 65.97 11.66 16.78 0.10% 66.49 11.3 17.81 0.20% 67.10 11.63 16.72 Isovalerate 0.05% 67.46 10.65 16.45 0.10% 69.53 10.50 15.63 0.20% 67.32 10.01 17.77 2-Methyl butyrate 0.05% 68.53 9.52 16.93 0.10% 67.40 11.03 16.62 0.20% 67.83 10.75 17.20 Phenylacetate 0.05% 69.13 9.72 16.10 0.10% 67.56 10.28 17.60 0.20% 67.27 10.20 17.96 Mixture 0.05% 68.20 10.15 16.29 0.10% 67.74 10.92 17.21 0.20% 68.53 9.86 17.30 Valerate 0.05% 66.90 10.95 17.10 0.10% 67.28 10.76 17.59 0.20% 68.73 10.85 16.53 S.E.M. (b) 1.92 1.53 1.07 There is no statistical difference for any of the variables (p > 0.1). (a) Percentage of the acid in the diet. (b) Standard error of the mean. 38 TABLE 13: Effects of isoacids on performance of broilers (fourteen days). Trial two (a). Treatments Init. Weight Feed Feed (b) weight gain intake conv. Control 42.8 365.8 496.4 1.36 Isobutyrate(IB) ‘ 43.2 365.5 492.1 1.35 Isovalerate(IV) 42.8 365.8 482.8 1.32 2-mehtyl butyrate(2MB) 43.0 358.8 539.6 1 51 IB + IV 42.8 363.9 495.9 1.37 18 + 2MB 42.3 367.3 499.1 1.36 IV + 2MB 43.2 363.6 495.9 1.37 IB + IV + 2MB 42.7 362.3 485.5 1.34 S E M (c) 0.4 5 4 17 3 0 04 There is no statistical difference for any of the variables (p > 0.1). (a) All weight units are grams. (b) Each acid was used at 0.1% of the diet. (0) Standard error of the mean. 39 TABLE 14: Effects of isoacids on performance of broilers (fourteen to twenty-eight days). Trial two (a). Treatments Weight Feed Feed (b) gain intake conv. Control 777.3 1359.1 1.76 Isobutyrate(IB) 726.6 1379.6 1.90 Isovalerate(IV) 722.0 1388.7 1.92 2-mehtyl butyrate(2MB) 732.0 1479.6 2.02 IB + IV 739.7 1411.4 1.91 IB + 2MB 736.3 1434.1 1.95 IV + 2MB 725.6 1422.7 1.96 IB + IV + 2MB 735.9 1422.8 1.93 S E M (c) 9.0 54 7 0 09 There is no statistical difference for any of the variables (p > 0.1). (a) All weight units are grams. (b) Each acid was used at 0.1% of the diet. (c) Standard error of the mean. 40 TABLE 15: Effects of isoacids on performance of broilers (twenty-eight to fifty-six days). Trial two (a). Treatments Weight Feed Feed (b) gain intake conv. Control 1235.4 2781.8 2.25 Isobutyrate(IB) 1191.2 2759.1 2.32 Isovalerate(IV) 1182.3 2940.9 2.49 2-mehtyl butyrate(2MB) 1195.7 2995.5 2.52 IB + IV 1165.8 2743.2 2.36 IB + 2MB 1229.2 2952.3 2.41 IV + 2MB 1162.6 2856.9 2.46 IB + IV + 2MB 1171.1 3104.6 2.65 S E.M. (c) 50.7 167.1 0.12 There is no statistical difference for any of the variables (p > 0.1). (a) All weight units are grams. (b) Each acid was used at 0.1% of the diet. (c) Standard error of the mean. 41 TABLE 16: Effects of isoacids on carcass composition of broilers. Trial two. Treatments Moisture Fat Protein (a) % % % Control 59.39 17.42 18.74 Isobutyrate(IB) 60.08 15.49 * 18.88 Isovalerate(IV) 60.03 16.89 19.13 2-mehtyl butyrate(2MB) 59.33 17.03 18.97 IB + IV 59.30 16.55 19.08 IB + 2MB 60.47 16.22 18.80 IV + 2MB 60.06 16.43 18.65 IB + IV + 2MB 60.74 16.23 18.65 S E M (b) 0.78 0 81 0 35 * significant at p < 0.10 The treatment combination IB + 2MB affects carcass composition (p < 0.05) as tested by multivariate analyses of variance. There is no statistical difference for any of the other variables (p > 0.1). (a) Each acid was used at 0.1% of the diet. (b) Standard error of the mean. 42 TABLE 17: Effects of isoacids on plasma levels of growth hormone of broilers. Trial two. Treatments Growth hormone (a) (ns/ml) Control 294.25 Isobutyrate(IB) 236.85 Isovalerate(IV) 282.75 2-mehtyl butyrate(2MB) 250.20 IB + IV 219.90 IB + 2MB 288.70 IV + 2MB 257.45 IB + IV + 2MB 255.95 S E M (b) 46 36 There is no statistical difference for any of the variables (p > 0.1). (a) Each acid was used at 0.1% of the diet. (b) Standard error of the mean. Discussion Before any discussion of the results obtained in this study it is important to point out the limitation to which this research was subjected. The buildings used for the swine and poultry studies were designed as commercial and not as research facilities, which limits the number of replicates available. The fact that each pen houses several animals contributes to a better estimation of the mean result for each pen, but it does not increase the number of experimental units. This is due to the fact that experimental unit is defined as the unit which receives the treatment. In the case of pen feeding trials each pen receives the treatment and not each individual animal. This resulted in only eight degrees of freedom in the error term for the swine and the poultry study number two, even though 96 and 2016 animals were used for each study respectively. The final consequence of this limitation is the degree of confidence that the hypothesis can be rejected. Because of this problem, some of the results that suggest strong treatment effect were not statistically significant at p < 0.10. Nevertheless, when the results strongly suggested a treatment effect, an appropriate comment was made because it was felt that a larger experiment would have probably statistically confirmed the result. 43 44 This research indicates that isoacids act in nonruminants as a repartitioning agent. Unfortunately there is not enough information to establish the mode of action of the isoacids, but there are several possibilities that deserve consideration. Isoacids could be affecting microbial growth in the digestive tract. As a known requirement for cellulolytic bacteria, isoacids could be enhancing fermentation of fibrous material in the large intestine of the pig, the rat and maybe the chicken. However, this possibility would not explain the results obtained, since the fiber content of the diets offered in this study was low, and any increment in fiber digestion would have minimal effects. There is also the possibility that isoacids would act as an antimicrobial agent, since they are used as a mold inhibitor for hay (Thomas, 1976; Sheaffer and Clarlk, 1975). However, the digestive tract is a different environment, the microorganisms are predominantly anaerobic and the action of isobutyrate as an antimicrobial agent under these conditions is questionable. Isoacids could alternatively be exerting their effects post absorptively. The amphiphatic nature of the membranes constitute a barrier for ionized, highly polar compounds, and this explains why compounds in the nonionized form are more readily absorbed. The state of ionization of a compound depends on its pKa and the pH of the medium. The degree of ionization is given by the Henderson-Hasselbalch equation (Hodgson and Guthrie, 1980). 45 Log [non-ionized form / ionized form] = pKa - pH (for acids) All the acids used in this study are weak acids with their pKa around 4.8 (CRC Handbook of chemistry and Physics,1985-1986). In the stomach, almost all the acid will be in the nonionized form and most of it will be absorbed. If part of the acid is not absorbed, it will reach the intestine, where about 50% will be in the non-ionized from. However, since the absorptive area in the intestine is much greater eventually all the acid will be absorbed. After absorption, the isoacids would enter the circulation through the portal system, because being short chain fatty acids they are not absorbed into the lymphatic system. If isoacids are not metabolized by the liver they could reach the peripheral circulation and increase nitrogen retention. This hypothesis is supported by several studies done with 2-ketoisocaproate. This compound has a very similar structure to isovalerate and has been reported to inhibit protein breakdown and improve animal performance (VandeHaar et. al., 1986, 1987; Flakoll et. al., 1987). Another possibility for the mode of action involves insulin and growth hormone, as suggested by studies with ruminants (Fieo et al., 1984; Towns and Cook, 1984; Coutinho, 1986, unpublished results; Brondani, 1986). A decrease in plasma insulin in sheep, as reported by Brondani (1986), could explain the results obtained in swine and poultry. Insulin has lipogenic and antilipolytic effect. A decrease in insulin level could reduce lipogenesis and release its 46 negative effect on hormone sensitive lipase, and thus allow greater mobilization of fatty acids from the adipose tissue (Guyton, 1986). In ruminants, the decrease in plasma insulin could be explained by the shift in volatile fatty acids produced in the rumen. The presence of isoacids, a requirement for the cellulolytic bacteria, would increase fiber digestion, and thus increase the production of acetate and decrease the acetate-propionate ratio. This reduction in propionate, a stimulant of insulin secretion by the pancreas, would reduce the circulating level of insulin (Brondani, 1986). The fact that this mechanism cannot be applied to nonruminants, and the contradictory reports on the effect of isoacids on insulin secretion (Fieo et al., 1984; Towns and Cook, 1984; Coutinho, 1986 Unpublished results; Brondani, 1986; Kik and Cook, 1986), impose serious questions about the involvement of insulin in the mechanism of action of isoacids in nonruminants. Also, against this idea are the results of a study conducted by Horino et al.(1968). This study was designed to investigate the effects of intravenous infusion of short chain fatty acids on plasma insulin in nonruminants. There was no plasma insulin response in the rat following injection of propionate or butyrate, nor in the rabbit following injection of propionate or valerate, nor in the pig following infusion of propionate. The amounts used were sufficient to provoke 10 to 30 fold plasma insulin increase in sheep. 47 The mode of action of isoacids in nonruminants could be through changes in growth hormone, as reported by Towns and Cook (1984) and Fieo et al.(1984). Growth hormone is know to have a lipolytic effect on adipose tissue (Newsholme and Start, 1981), and an increase in growth hormone could explain the leaner carcass obtained when isoacids were fed. Unfortunately, our attempt to check this hypothesis does not support this theory. Our results indicate no effects of the isoacids on plasma growth hormone in chickens. However, it should be noted that the concentrations were extremely high. Studies have shown that stress can increase growth hormone secretion (Martin et al.,1977) and the heart puncture technique used in the present study to obtain blood samples is known to cause excitement. So, it could be that any difference that might have resulted from feeding isoacids was offset by the excitement during sampling. It should also be mentioned that growth hormone has episodic secretion (Scanes et al. 1984), and one sample may not be very representative of the pattern of growth hormone secretion. Another possibility would be an increase in glucagon secretion. This hypothesis is supported by studies conducted by Phillips et al. (1965). They reported that injection of butyrate induces glycogenolysis. Glucagon is known to be associated with glycogenolysis in the liver and to have lipolytic activity in the adipose tissue (Newsholme and Start, 1981). Also the fact that a greater response was 48 obtained with poultry than with swine supports this theory, since glucagon has a higher lipolytic activity in birds than in other species. Unfortunately no attempt was made to directly check this hypothesis, but the possible effects of isoacids on glucagon should be considered in future studies. It is also important to point out that all the acids used in this trial were neutralized with ammonia hydroxide, in order to prevent a palatability problem due to acidification of the diet. However as reported by Visek (1978), ammonia produced by microbial activity in the intestinal tract causes an increase in wet weight, alteration in nucleic acid synthesis and increase in protein in the intestinal mucosa. These changes in tissue could cause an increase in energy required for maintenance, and thus divert substrate from growth. The normal levels of ammonia in the intestine are in the range of 8 to 10mM, a concentration known to cause in cultured cells the changes mentioned above. For the highest level of acid used in this study, the ammonia concentration in the diet was in the Order of 0.045M. Considering that in the intestine the digesta has approximately 15% dry matter (Kidder and Mannes, 1978), it can be calculated that the diet would be contributing an ammonia concentration of about 9mM to that already in the intestine, a value which could have had some effect on the animals. 49 The results reported in this study show that isoacids reduce fat and increase protein content in the carcass of swine and poultry. The production of leaner animal products could represent a tremendous advance for the animal industry. There is an increasing demand for leaner meat by the public, and isoacids should be considered in future studies as one of the answers for a more acceptable animal product. LI ST OF REFERENCES List of References Allison, M.J., M.P. Bryant and R.N. Doetsch, (1959), Conversion of isovalerate to leucine by Ruminococcus flavefaciens. Arch. Bioch. Biophys. 84:246. Allison, M.J.,(1969), Biosynthesis of aminoacids by ruminal microorganisms. J. of Anim. Sci. 29:797. Allison, M.J.,(1970), Nitrogen metabolism of ruminal microorganism, In: Physiology of digestion and metabolism in the ruminant. A.T.(Ed.) Oriel, New Casttle Upon Tyne, UK. p 456. Anderson, L.L., C.R. Bohnker, R.O. Parker and L.P. Kentiles,(1981), Metabolic action of growth hormone in pigs, J. of Anim. Sci. 53:363. Baker, P.K., R.H. Dalrymple, D.L. Ingle and C.A.Ricks, (1984), Use of Beta-Adrenergic Agonist to Alter Muscle and Fat Deposition in Lambs, J. of Anim. Sci. Vol. 59, No. 5, pp. 1256-1261. Bauman,D.E., Eppard, P.J., DeGeeter, M.J., and Lanza, G.M.,(1985), Response of high producing dairy cows to long term treatment with pituitary- and recombinant-somatotropin, J. of Dairy Sci. 68,1352. Beitz, D.C.,(1985), Physiological and Metabolic Systems Important to Animal Growth: An Overview, J. of Anim. Sci., 61:Suppl. 2, pp. 1-20. ‘ Bergen, W.G.,(1974), Protein synthesis in animal models, J. of Anim. Sci. 38:1979. Brondani, A.V.,(1986), A study on the action of teichomacin a2, avoparcin, and isoacids in sheep, Ph.D. thesis, Michigan State University, East Lansing, MI, p 1-111. Broome, A.W.J.,(1980), Mechanism of action of growth-promoting agents in ruminant animals, In: T.L.J. Lawrence (Ed.) Growth in animals. pp189-205 Butterworths, Boston. Bryant, M.P. and R.N. Doetsch,(1955), Factors necessary for the growth of Bacteroides succinogenes in the volatile acid fraction of the rumen fluid, J.Dairy Sci. 38:340 Buttery, P.J.,(1983), Hormonal control of protein deposition in animals, Proc. Nutr. Soc., 42:137-148. Caster, W.O., (1976), Nutrition,longevity, and aging, M. Rockstein and M.L.Sussman (Eds). New York: Academic Press Inc., pp 29~4h. Eli.) 51 Chalupa, W.,(1980), Chemical control of rumen microbial metabolism, In: Y. Ruckebusch and P. Thivend (Ed.) Digestive physiology and metabolism in ruminants. pp325-347. MTP Press Ltd., Lancaster. Chambers, J.R., J.S. Gavora, and A. Fortin ,(1981), Genetic Changes in Meat-Type Chickens in the LastTwenty Years, Can. J. Anim. Sci. 61:555-563. Chung, C.S., T.D. Etherton, and T.J. Wiggins,(1985), Stimulation of swine growth by porcine growth hormone, J.of Anim. Sci. 60:118. Clark, A.K., A.C. Sperarow, A.A. Jimenez, and D.D. Dildey, (1986), Milk production and carry-over response of cows fed calcium salts of volatile fatty acids at a northern California dairy farm, J. Dairy Sci.(Suppl.1) 69:157. Clausen, A.W.,(1986), An overview of poverty and hunger: Issues and options for food security in developing countries, The World Bank publication summary. Commite on diet, nutrition and cancer, Assembly of life sciences, National research council,(1982), Diet, nutrition and cancer, Washington, D.C.: National Academy Press. Cook, R.M.,(1985), Isoacids, a New Feed Aditive for Lactating Cows, Maryland Nutrition Conference for Feed Manufactures. J.A. Doerr (ed). pp. 41-49. Cook, R.M.,(1983), Isoacids, a new feed additive for lactating dairy cows, Department of Animal Science, Michigan State University, publication with file No. 20.38. CRC Handbook of chemestry and Physics, 60th. Edition 1985-1986, CRC Press, pg d161-162 Creasy, W.A.,(1985), Diet and cancer, Philadelphia, PA: Lea and Febiger. Dalrymple, R.H., P.K. Backer, P.E. Gingher, D.L.Ingle, J.M. Pensack, and C.A. Ricks,(1984), A Repartitioning Agent to Improve Performance and Carcass Composition of Broilers, Poultry Sci. 63:2376-2383. Davis, L.W.,(1969), A new concept in heifer feeding, Proc. 24th. Kansas Formula Feed Conf. pp. 72-82. Kansas State University, Manhattan. 52 Deetz, L.E., and C.R. Richardson,(1984), Performance and characteristics of cattle fed diets containing ammonium salts of branched chain fatty acids and valeric acid, J.of Anim. Sci. 59 (Suppl. 1):438. Donato, K., and D.M. Hegsted,(1985), Efficiency of ultilization of various sources of energy for growth, Proc. Natl. Acad. Sci. USA 82:4866. Donato, K.A.,(1986), Efficiency and ultilization ofvarious energy sources for growth, Symposium on Calories and energy expenditure in carcinogenesis, International life sciences institute-nutrition foundation. Etherton, T.D.,(1982), The role of insulin receptor interactions in regulation of nutrient ultilization by skeletal muscle and adipose tissue: A review, J. of Anim. Sci. 54:58. Etherton, T.D. and Kensinger, R.S.,(1984), Endocrine Regulation of Fetal and Postnatal Meat Animal Growth, J. of Anim. Sci., Vol 59, No. 2 pp. 511-528. Etherton, T.D. and Kensinger, R.S.,(1985), Endocrine Regulation of Fetal and Postnatal Meat Animal Growth, J. of Anim. Sci. 59:No. 2, 511-528. Etherton, T.D. and V.K. Meserole,(1982), New Technology Studied to Improve Animal Growth, Sci. Agr., The Pennsylvania State Univ. 29(4):10. Etherton, T.D., J.P. Wiggins, C.S. Chuang, C.M. Evock, J.F. Rebhun and P.E. Walton,(1986), Stimulation of pig growth performance by porcine growth hormone and growth hormone releasing factor, J. of Anim. Sci. 63:1389. Felix, A.,(1976), Effect of supplementing corn silage with isoacids and urea on performance of high producing cows, Ph.D. thesis, Michigan State University, East Lansing, MI, USA. 1-151. Felix, A., R.M. Cook, and J.T. Huber,(1980 a), Effect of feeding isoacids with urea on growth and nutrient ultilization by lactating dairy cows, J. Dairy Sci. 63:1943. Felix, A., R.M. Cook, and J.T. Huber,(1980 b), Isoacids and urea as a protein supplement for lactating cows fed corn silage, J. Dairy Sci. 63:1103. 53 Fieo, A.G., T.F. Sweeney, R.S. Kensinger, and L.D. Miller, (1984), Metabolic and digestion effects of the addition of the ammonium salts of volatile fatty acids to the diet of cows in early lactation, J. Dairy Sci. 67:117(Suppl.). Flakoll, P., M. VanderHaar and S. Nissen, (1987), Muscle growth and whole-body leucine metabolism of growing lambs fed leucine, 2-ketoisocaproate and isovalerate, Fed. Proc. 46:No.4:1178 (Abstracts). Gill, J.L.,(1978) (a), Design and analysis of experiments in the animal and medical sciences, Vol. 1. The Iowa State University Press, Ames, Iowa. Gill, J.L.,(1978) (b), Design and analysis of experiments in the animal and medical sciences, Vol. 3. The Iowa State University Press, Ames, Iowa. Gorosito, A.R., J.B. Russel, and P.J. Van Soest,(1985), Effect of carbon-4 and carbon-5 volatile fatty acids on digestion of plant cell wall in vitro, J. Dairy Sci. 68:840. Guyton, A.C.,(1986), Textbook of medical physiology, Saunders (Ed.), 7th edition, 826-827. Hart, I.C., P.M.E. Chadwick, T.C. Boone, K.E. Langley, C. Rudman and L.M. Souza,(l984), A comparison of the growth promoting, lipolytic, diabetogenic and immunological properties of pituitary and recombinant DNA derived bovine growth hormone (somatotropin), Biochm. J. 224:93-100. Harvey, S. and 0.6. Scanes,(1977), Purification and radioimmunoassay of chicken growth hormone, J. Endocr. 73:321-329. Harvey, S. and Sanes, C.G.,(1976), Purification and Radioimmunoasssay of Chicken Growth Hormone, J. Endocr., 73, 321-329. Hazard, W.R.,(1976), Nutrition, longevity and aging, M. Rockstein and M.L.Sussman (Eds). New York: Academic Press Inc., pp 143-195. Heitzman, R.J.,(1980), Manipulation of protein metabolism, with special reference to anabolic agent, In: P.J. Buttery and D.B. Linsday (Ed.) Protein deposition in animals. pp 193-203. Butterworths, Boston. 54 Hemsley, J.A., and R.J. Moir,(1963), The influence of higher volatile fatty acids on the intake of urea supplemented low quality cereal hay by sheep, Aust. J. Agric. Res. 14:509. Higgs, D.A., E.M. Donaldson, H.M. Dye and J.R. McBride, (1975), A preliminary investigation of the effect of bovine growth hormone on growth and muscle composition of Coho salmon (Oncorhynchus kisutch), Gen. Comp. Endocrinol. 27:240. Hodgson and Guthrie,(1980),Introduction to biochemical toxicology, chp 2. Horino, M., L.J. Machlin, F. Hertlrndy and D.M. Kipnis, (1968), Effect of short chain fatty acids on plasma insulin in ruminant and nonruminant species, Endocrinology 83:118. Jukes, T.H.,(1971), The present status and background of antibiotics in the feeding of domestic animals, Ann. New York Acad. Sci. 182:362. Kauffman, R.G., R.J. Epley, J.R. Romans and D.G. Topel, (1981), Pork industry handbook, Carcass evaluation, Pork and Pork quality. Extension bulletin E-1222, January 1981, Michigan State University, East Lansing, MI. Kaunitz, H., and R.E. Johnson.,(1975), Proc. IXth Intl. Congr. Nutr. Vol. a, A. Chaves, H. Bourges, and S. Basta(Eds). New York: S. Karger, p. 362. Kidder, D.E. and M.J. Manners, (1978), Digestion in the pig, Scintechnica Bristol, England, chapt. 3. Kik, N. and R.M. Cook,(1986), Effects of bovine somatotropin and IsoPlus on milk production, J. Dairy Sci.69:158 (Suppl.1). Kroes, R., R.B. Beems, M.C. Bolsland, G.S.J. Bunnik, and E.J. Sinkeldman,(1986), Nutritional factors in lung, colon and prostate carcinogenesis in animal models, Fed. Proc. 45:136. Lassiter, C.A., R.S. Emery, and C.W. Duncan,(1958), Effect of alfalfa ash and valeric acid on growth of dairy heifers, J. Dairy Sci. 41:552. Machado, P.E., R.M. Cook, and P. Kone,(1985), Adaptation of a semicontinuous system to test the effects of chemicals on rumen fermentation, In: Reports on XVIII Conference on rumen function, p. 13 (Abs). 55 Machlin, L.J.,(1972), Effect of porcine growth hormone on growth and carcass composition of the pig, J. of Anim. Sci. 35:794. March, R.E., and Biely,(1967), A re-assessment of the mode of action of the growth-stimulating properties of antibiotics, Poul. Sci. 46:831. Martin, J.B., S. Reichlin and G.M. Brown,(1977), Regulation of Growth Hormone Secretion and Its Disorders, Clinical Neuroendocrinilogy, Ch.7. Martin, T.G., T.A. Mollett, T.S. Stewart, R.E. Erb, P.V. Malven and E.L. Veenhuizen,(1979), Comparison of four levels of protein supplementation with and without oral diethylstilbestrol on blood plasma concentration of testosterone, growth hormone and insulin in young bulls, J. of Anim. Sci. 4911489. Mayer, M. and F. Rosen,(1975), Interaction of anabolic steroids with glucocorticoid receptor sites in rat muscle cytosol, Amer. J. Physiol. 229:1381. Mayer, M. and F. Rosen,(1978), Effect of endocrine manipulation on glucocorticoid binding capacity in rat skeletal muscle, Acta Endocrinolo. 88:199. Mosely, W.M., L.F. Krabill and R.E.Olsen,(1982), Effect of bovine growth hormone administered in various patterns on nitrogen metabolism in the holstein steer, J. of Anim. Sci. 55:1062. Muir, L.A.,(1985), Mode of Action of Exogenous Substances on Animal Growth: An Overview, J. of Ani. Sci., 61:Suppl. 2. PP. 154-180. Muir, L.A., S. Wien, P.E. Duquette, E.L. Ricks, and E.H. Cordes,(1983), Effects of exogenous growth hormone and diethylstilbestrol on growth and carcass composition of growing lambs, J. of Anim. Sci. 56:1315. National Dairy Council Digest,(1979), Nutrition, Longevity, and Aging, Dairy Council Digest. National Dairy Council Digest,(1974), Newer Concepts of Coronary Heart Disease, Dairy Council Digest. National Dairy Council Digest,(1986), Diet, Nutrition, and Cancer: New Findings, Dairy Council Digest. Nauss, K.M., M. Locniskar, and P.M. Newberne,(1983), Effect of alterations in the quality and quantity of dietary fat on 1,2-Dimethy1hydrazine induced colon tumorogenesis in rats, Cancer research 43:4083. 56 Newman, D., J.A. Rogers, A.K. Clark, and A.C. Spearow,(1986), Milk production response of dairy cows fed calcium salts of volatile fatty acids on 34 commercial dairy farms, J. Dairy Sci. 69:158 (Suppl.1). Oltjen, R.R., L.L. Slyter, E.E. Williams, Jr., and D.L. Kern, (1971), Influence of branched—chain volatile fatty acids and phenylacetate on ruminal microorganisms and nitrogen ultilization by steers fed urea or isolated soy proteins, J. Nutr. 102:479. Owens, F.N., D.R. Gill, L.E. Deetz and J.J.Martin,(1983), Ammonium salts of volatile fatty acids for feedlot steers, Oklahoma Agric. Exp. Stat., Animal Sci. Res. Report. Papas, A.M., S.R. Ames, R.M. Cook, C.J. Sniffen, C.E. Polan, and L. Chase,(l984), Production responses of dairy cows fed diets supplemented with ammonium salts of isoC-4 and C-5 acids, J. Dairy Sci. 67:276:293. Peel, C.J., Sandles, L.D., Quelch, K.J., and Herington, A.C., (1985), The effects of long term administration of bovine growth hormone on the lactation performance of identical twin dairy cows, Animal Production, 41. Peter,(1983), Hormonal Control of Protein Deposition in Animals, Proc. Nutr. Soc.,42,137. Philips, R.W., A.C. Black and F. Moller, (1965), Butyrate induced glycogenolysis. Life Science 4:521 Pond, W.G.,(1981), Developments in pork production, New protein feeds, vol. 4, Academic Press, Inc. Rebhun, J.F., et al.,(1985), Stimulation of swine growth performance by porcine growth hormone (pGH): Determination of the maximally effective pGH dose, J. of Anim. Sci. 61(Suppl. 1):251. Richardson, C.R., R.L. Preston, R.H. Pritchard and L.E. Deetz ,(1984), Performance, carcass characteristics, and body fat and protein of steers fed diets containing branched chain fatty acids and valerate, J. of Anim. Sci. 59 (Suppl.1);438. Richardson, C.R., L.E. Deetz, R.H. Pritchard and R.L. Preston, (1985), Branched chain fatty acids may improve performance, Feedstuffs 57:12 Number 20. 57 Ricks, C.A., R.H. Dalrymple, P.K. Baker, D.L. Ingle, M.E. Doescher and J. Pankivich (1984), Use of Clenbuterol to alter muscle and fat accretion in swine , Fed. Proc. 43:857. Ricks, C.A., R.H. Dalrymple, P.K. Baker, and D.L. Ingle, (1984), Use of Beta-Agonist to Alter Muscle and Fat Deposition in Steers, J. of Anim. Sci. Vol. 59, No. 5, pp. 1247-1255. Rogers. J.A., and R.M. Cook,(1986 a), Effect of ammonium salts of volatile fatty acids on milk production of dairy cows. 1. Comercial dairy trials - 1982, J.Dairy Sci.(Suppl. 1) 69:157. Rogers. J.A., D. Newman, and R.M. Cook,(1986 b), Effect of ammonium salts of volatile fatty acids on milk production of dairy cows. II. Comercial dairy trials - 1983-1984, J. Dairy Sci.(Suppl. 1) 69:157. Romsos, D.R., G.A. Leveille and G.L. Allee (a),(1971), Alloxan diabetes in the pig (Sus domesticus). Response to glucose, tolbutomide and insulin administration, Comp. Biochm. Physiol. 40A:557. Romsos, D.R., G.A. Leveille and G.L. Allee (b),(1971), In vitro lipogenesis in adipose tissue from alloxan diabetic pigs (Sus domesticus). Comp. Biochm. Physiol. 40A:569. Rumsey, T.S., H.F. Tyrrell, D.A. Dinius, P.W. Moe, and H.R. Cross,(1977), Gain and body composition of beef steers fed DES, J. of Anim. Sci. 45 (Suppl. 1):254. Russel, J.B.,(1983), Effects of C4 and C5 volatile fatty acids on the growth of mixed rumen bacteria in vitro. J. Dairy Sci. 66:(Suppl. 1):152. Scanes, C.G.,(1985), Regulation of Growth in Mammals, Bird and Fish, Regulation of Growth and Lactation in Animals, University of Wisconsin Biotechnology Center Series No. 1, Schuler and First (Eds.). Scanes, C.G., S. Harvey, J.A. Marsh and D.B. King, (1984), Hormones and Growth in Poultry, Poultry Science 63:2062 Schmidt, G.A., R.G. Warner, H.F. Tyrrell and W.Hansel, (1971), Effects of thyroprotein feeding on dairy cows, J. Dairy Sci. 54:481. Sheaffer, C.C. and N.A. Clark, 1975, Effects of organic preservative on the quality of aerobically stored high moisture baled hay, Agronomy J., vol 67:660. 58 Spencer, G.S.G.,(1985), Hormonal Systems Regulating Growth. A Review, Livestock Production Sciences, 12, 31-46. Steele, N.C. and T.D. Etherton,(1983), Nutrient partitioning in the young pig as affected by dietary protein intake and insulin treatment, J. of Anim. Sci. 57(Suppl.1):208. Struempler, A. and W. Burroughs,(1957), The influence of growth hormone and diethylstilbestrol on nitrogen retention im lambs, J. of Anim. Sci. 16:1098. Struempler, A.W. and W. Burroughs,(1959), Stilbestrol feeding and growth hormone stimulation in mature ruminants, J. of Anim. Sci. 18:427. Thomas, J.W., 1976, Recent research on role of heating and preservatives in haylages and silages, DFRC Proceedings, vol 31, p.22. Towns, R., and R.M. Cook,(1984), Isoacids, a new growth hormone releasing factor, AAAS Annual Meeting, New York, NY. (Abs. 347). Trenkle, A.,(1970), Plasma levels of growth hormone, insulin and plasma protein-bound iodine in finishing cattle, J. of Anim. Sci. 31:389. Trenkle, A.,(1977), Relationship of some endocrine measurements to grwoth and carcass composition of cattle, J. of Anim. Sci. 46:1604-1609. Trenkle, A.H.,(1969), The mechanism of action of estrogens in feeds on mammalian and avian growth, In: The use of drugs in animal feeds. pp150-164 National academy of sciences, Washington, DC. Trenkle, A. and D.G. Topel,(1978), Relationship of some endocrine measurements to growth and carcass composittion in cattle, J. of Anim. Sci. 46:1604. VandeHaar, M.J., D.C. Beitz and S. Nissen,(1986), Effect of feeding 2-ketoisocaproate to dairy cows on milk fat composition, Fed. Proc. 45:240. VandeHaar M.J., P.J. Flakoll, D.G. Beitz and S. Nissen,(1987), Effect of leucine metabolites on energy metabolism in growing lamb, Fed. Proc. 46:No.4:1178 (Abstracts) van Es, A.J.H.,(1977), The Energetics of Fat Deposition During Growth, Nutr. Metab. 21:88. 59 Vernon, B.G. and P.J. Buttery,(1976), Protein turnover in rats treated with trienbolone acetate, Brit. J. Nutr. 36:575. Vernon, B.G. and P.J. Buttery,(1978), Protein metabolism of rats treated with trienbolone acetate, Anim. Prod. 26:1. Visek, W.J.,(1978), The mode of growth promotion by antibiotics, J. of Anim. Sci. 46:1447. Wagner, J.F., E.L. Veenhuizen,(1978), Growth performance, carcass deposition and plasma hormone levels in wether lambs when treated with growth hormone and thyroprotein, J. of Anim. Sci. 45 (Suppl. 1):397. Watkin, D.M.,(1979), Nutrition and the world food problem, M. Rechcigl, Jr.(ed). New York: S. Karger, pp 20-62. Weeks, T.E.C.,(1983), The Hormonal Control of Fat Metabolism in Animals, Proc. Nutr. Soc.,42,129. Willet, W.C., and B. MacMahon,(1984), Diet and cancer - An overview, N. Engl. J. Med. 310:697. Young, T., Jr., D.K. Hass and L.J. Brown,(1979), Effect of late gestation feeding of dichlorvos in non-parasitized sows, J. of Anim. Sci. 48:45. Young, V.R.,(1978), Nutrition and aging, Adv. Exptl. Med. Biol. 97:85. "IIIIIIIIIIIIIIIIIII