[KELEENCE 483$? 3H3 33:33 0?? TEE UTILIZA’YEGk‘. GF EATS IN TEE CESCKEN .E‘Exeszs §ov the fioqrea 0‘: pi! D. lifiifiiiifififl STATE :E‘IJLRCV Evicdestus X. Same: E972 L I B R A R Y Michigan State University This is to certify that the , thesis entitled INFLUENCE OF BILE SALTS ON THE UTILIZATION OF FATS IN THE CHICKEN presented by Modestus X. Gomez has been accepted towards fulfillment of the requirements for Ph.D. degree in Poultry Science Donald Polin, Ph.D. Associate Professor Avian Nutrition Date November 8, 1972 0-703. ‘ v.-»vv—_~—_‘, __ ._.._.a..__...,__.___ .. ABSTRACT INFLUENCE OF BILE SALTS ON THE UTILIZATION OF FATS IN THE CHICKEN BY Modestus X. Gomez Five experiments were conducted to test the need for supplementary bile salts by the growing chick. In experi- ment I and II (2 x 2 x 4 factorials), the influence of 0.2 percent dietary cholic acid, on the utilization of two high levels each of tallow (TLW), lard (LD), hydrogenated soybean oil (HSBO) and corn oil (CNO) by chicks four weeks and one week of age, respectively, were studied. The effects of substituting fat metabolizable energy (F.M.E.), equicalo- rically for 50 or 100 percent of the glucose from a purified reference diet, were also concomitantly investigated. In the chicks 1 week of age, cholic acid significantly improved fat absorption by 2.7 percent from an overall average of 80.5 percent. At one to four weeks of age cholic acid again improved absorption by 2.1 percent despite a high average absorption of fat of 92.7 percent. The younger chicks absorbed fats more selectively: TLW, 79.3 percent; LD, 69.7 percent; CNO, 92.7 percent; HSBO, 86.4 percent. The response to cholic acid was substantiated by appropriate and significant increases in the M.E. of the diets. According to the weight gain and feed intake data from experiment I, chicks on the 50 percent F.M.E. diets with Modestus X. Gomez supplemental cholic acid were noted to utilize fats effi- ciently. With the 100 percent F.M.E. diets feed intake was generally depressed accompanied by a comparable decline in weight gain. The addition of cholic acid to these 100 percent F.M.E. diets improved weight gain only marginally. Both the younger and older chicks did not tolerate cholic acid when the diets fed had only 2 percent lipid. Depressed feed intake and weight gain were recorded for these chicks. All chicks on diets with cholic acid at 0.2 percent in the diet showed gall bladder distension. The fat M.E. values improved with the age of the chick and with the presence of cholic acid in the diet. In the older chicks LD, HSBO, and CNO gave M.E. values higher than reported, and these values exceeded 9.4 kcal./g. This extracalorific effect appears to be partially derived from the enhanced energy-retaining capacity of the non-lipid components. Not established was the cause for the much higher values in this experiment, as compared to standard values from other experiments. In experiment III,chicks one week of age fed purified diets with 8.2 percent of crude TLW and receiving supplemental bile salts (cholic acid, chenodeoxycholic acid and tauro- cholic acid-—sodium salt) at either 0.025 or 0.05 percent showed an increase in fat absorption from 39.6 to 47.0 per- cent. Chenodeoxycholic acid was most efficient. Both levels of bile salts were equally effective. There were no increases in M.E. to substantiate improvements in fat absorption. Modestus X. Gomez At three weeks of age fat absorption increased from 68.2 percent to 73.6 percent from the addition of bile salts. M.E. of the diets showed an overall increase of 11.4 percent over those values obtained when the birds were younger. Weight gain and feed efficiency data showed no changes from control at both ages. Gall bladders of chicks on chenodeoxy- cholic acid appeared normal indicating that dietary cheno- deoxycholic acid would functionally be the most acceptable of the bile acids tested. In experiment IV cholic acid supplemented at 0.025, 0.05, 0.1 and 0.2 percent had no effect on the utilization of 4 and 8 percent dietary TLW by SCWL (Single Comb White Leghorn) females, 20 weeks of age. In repeating the experi- ment, with HSBO in place of TLW using SCWL females 23 weeks of age, again no effects on fat absorption or M.E. were seen with the supplemental bile salts. TLW and HSBO were absorbed equally well at both levels. Fat absorption im- proved with increasing levels of fat in the diet. Bile salts failed to improve fat absorption in the older birds implying that with age, the bird is able to meet the full demands on bile for fat absorption. Levels of 0.025 and 0.05 percent of cholic acid produced no significant gall bladder distention. INFLUENCE OF BILE SALTS ON THE UTILIZATION OF FATS IN THE CHICKEN BY Modestus XI Gomez A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Poultry Science 1972 ACKNOWLEDGEMENTS The author wishes to express his grateful thanks to Dr. Howard C. Zindel for the encouragement received and for being responsible for his return to Michigan State University for a doctoral program. Thanks are also due to Dr. Zindel for the financial support provided in the form of a Research Assistantship and for full use of the facilities of the Department's laboratories and research farm. The writer also wishes to express his sincere gratitude to Dr. Donald Polin, major advisor, for the excellent guidance, counsel and personal interest throughout the doctoral program. Thanks are specially due to Dr. Polin for the fine critical review of research and research presentation. Appreciation is also extended to Drs. Robert K. Ringer, Olaf Mickelsen, Duane E. Ullrey, Theo H. Coleman and Cal J. Flegal for agreeing to serve on the guidance committee and for their continued interest at all times. The encouragement and guidance of Dr. William J. Thomas is acknowledged in regard to the writer graduating with a joint degree from the Institute of Nutrition. All members of the staff of the Department of Poultry Science, including those at the farm, and fellow graduate students are thanked for the willing and friendly c00peration ii extended during the course of the studies. A special word of thanks to Mrs. Joyce Ingalls and Mr. Sulo Hulkonen for their skilled help in the preparation of this document. The assistance received from Mrs. Joice Adams (Librarian) will be remembered. Above all the author is deeply grateful to his wife Manel and children, Vaughan and Sharon, for their love and enduring patience, and their encouragement towards making this program worthwhile and successful. iii I. II. III. TABLE OF CONTENTS Introduction Review of Literature A. Fats in the diets of growing chicks 1. Factors affecting absorption and utili- zation of fats a. Level of dietary fat and fat toler- ance b. Composition of dietary fat c. Calorie:protein ratio of the diet d. Age of chick Fat energy as a substitute for carbo- hydrate energy Evaluation of fat energy "Extra-calorific" effect of fat Bile salts and fat absorption Physico-chemical role of bile salts in fat absorption Functional bile salts in the chick Effect of supplemental dietary bile salts Experimental Procedure A. B. Introduction The Experiments 1. Experiment I iv 12 l3 l6 17 19 19 23 23 26 26 27 27 Table of Contents (Cont.) IV. VI. VII. VIII. 2. Experiment II 3. Experiment III 4. Experiment IV 5. Experiment V C. Analytical procedures and calculation of data 1. Determination of dry matter content 2. Determination of lipid content 3. Determination of Cr203 content 4. Determination of gross energy content 5. Calculation of fat absorption (%) 6. Calculation of metabolizable energy of the diets 7. Calculation of the metabolizable energy of the fats D. Statistical analysis Results A. Experiment I B. Experiment II C. Experiment III D. Experiment IV E. Experiment V Discussion Conclusion Bibliography Appendix 38 40 44 44 44 45 47 49 50 51 52 52 67 72 85 89 103 114 121 129 Table LIST OF TABLES Composition of purified reference diet (Experiment I) Fatty acid composition of fats used in diets fed to broiler-type chicks (Experiment I) Composition of experimental diets (Experi- ment I) Composition of purified basal diet (Experiment III) Experimental diets showing the different sup- plementary bile salts and the levels added (Experiment III) Composition of reference diet (Experiment IV) Experimental diets showing substitution of ground corn of the reference diet with 4.0 or 8.0% TLW. Five levels of cholic acid were added to each set of TLW diets (Experiment IV). Experimental diets showing substitution of ground corn of the reference diet with 4.0 or 8.0% HSBO. Five levels of cholic acid were added to each set of the HSBO diets (Experiment V). Body weight gain (9.) and feed intake (9.) in broiler-type chicks fed diets in which 50% or 100% of glucose M.E. calories were substituted isocalorically by fat and the diets fed with or without 0.2% cholic acid (Experiment I). vi 31 35 37 39 41 43 53 List of Tables (Cont.) Table 10 Body weight gain (g.) of broiler—type chicks fed TLW, LD, CNO or HSBO substituted for 50% or 100% of the M.E. calories of glu- cose. Diets contained either 0.2% cholic acid or none (Experiment I). ll Feed:gain ratio of broiler-type chicks fed diets in which 50% or 100% glucose M.E. calories were substituted isocalorically by fat and the diets supplemented with 0.2% or without cholic acid (Experiment I). 12 Feed utilization (feed:gain ratio) of broiler- type chicks fed diets with TLW, LD, CNO or HSBO substituted for 50% or 100% of the M.E. calories and the diets supple- mented with 0.2% or without cholic acid (Experiment I). 13 Absorption of fat (%) by broiler-type chicks fed TLW, LD, CNO or HSBO at dietary levels which were isocaloric substitutes for 50% or 100% of the glucose M.E. calories in diets with 0.2% or no cholic acid (Experiment I). 14 Effect of 0.2% cholic acid on M.E. (kcal./g.) of diets in which 50% or 100% of the M.E. calories of glucose were substituted iso- calorically by fat in diets fed to broiler chicks (Experiment I). 15 Effect of cholic acid on M.E. (kca1./g.) values of diets with TLW, LD, CNO or HSBO substituted for 50% or 100% of glucose M.E. calories. Diets contained 0.2% cholic acid or none (Experiment I). 16 M.E. (kcal./g.) value of fats calculated from the 50% or 100% F.M.E. diets, with or without 0.2% cholic acid. The average percentage increases from the standard values are given in parentheses (Experi- ment I). vii List of Tables (Cont.) Table 17 18 19 20 21 22 23 Body weight gains (9.) of broiler-type chicks, at one week of age, when fed diets in which 50% or 100% glucose M.E. calories were substituted by fat M.E. calories, and the diets fed with or without 0.2% cholic acid (Experiment II). Body weight gain (g.) of broiler-type chicks fed TLW, LD, CNO or HSBO substituted for 50% or 100% of the M.E. calories from glucose and fed with or without 0.2% cholic acid (Experiment II). Absorption (%) of fat by broiler-type chicks, one week of age, when fed TLW, LD, CNO or HSBO substituted for 50% or 100% of the M.E. calories from glucose in diets with or without 0.2% cholic acid (Experi- ment II). Effect of 0.2% cholic acid on M.E. (kcal./g.) values of diets, with fat substituted for 50% or 100% of the M.E. calories from glucose. The diets were fed to broiler- type chicks from one to seven days of age (Experiment II). Effect of 0.2% cholic acid on M.E. (kcal./g.) values of diets with TLW, LD, CNO or HSBO substituted for 50% or 100% of the M.E. calories from glucose. The diets were fed to broiler-type chicks from one to seven days of age (Experiment II). M.E. (kcal./g.) values of fats determined in diets with or without cholic acid and fed to broiler-type chicks from one to seven days of age. The values are the means from the 50% and 100% F.M.E. diets. The percentage increases from standard values are given in parentheses (Experiment II). Body weight gains of broiler-type chicks fed diets containing 8.2% of crude TLW with 0.025 or 0.05% cholic acid, chenodeoxycholic acid or taurocholic acid (sodium salt). The data are for the period when chicks were 0 to 7 days of age (Experiment III). viii 69 73 75 77 List of Tables (Cont.) Table Page 24 Feed utilization (feed:gain ratio) by broiler- 78 type chicks during the first week of growth, when fed diets containing 8.2% crude TLW and with 0.025 or 0.05% of cholic acid, chenodeoxycholic acid or taurocholic acid (sodium salt) (Experiment III). 25 Fat absorption by broiler-type chicks one week 79 of age fed diets containing 8.2% crude TLW and with 0.025 or 0.05% of cholic acid, chenodeoxycholic acid or taurocholic acid (sodium salt) (Experiment III). 26 M.E. values (kcal./g.) of diets containing 81 8.2% crude TLW and with 0.025% or 0.05% of cholic acid, chenodeoxycholic acid or taurocholic acid (sodium salt), when fed to broiler-type chicks, during their first week of growth (Experiment III). 27 Body weight gains of broiler-type chicks 82 in the first 18 days when fed diets con- taining 8.2% crude TLW and with 0.025 or 0.05% of cholic acid, chenodeoxycholic acid, or taurocholic acid (sodium salt) (Experiment III). 28 Feed utilization (feed:gain ratios) by broiler- 83 type chicks during the first 18 days, when fed diets containing 8.2% crude TLW and with 0.025 or 0.05% cholic acid, chenodeoxycholic acid or taurocholic acid (sodium salt) (Experiment III). 29 Fat absorption by broiler-type chicks at 84 18 days of age fed diets containing 8.2% crude TLW and with 0.025 or 0.05% of cholic acid, chenodeoxycholic acid or taurocholic acid (sodium salt) (Experiment III). 30 M.E. values of diets containing 8.2% crude 86 TLW and with 0.025 and 0.05% of cholic acid, chenodeoxycholic acid and tauro- cholic acid (sodium salt) when fed to broiler-type chicks, at 18 days of age (Experiment III). ix List of Tables (Cont.) Table 31 32 33 34 35 36 Bile content (g.) of the gall bladders of broiler-type chicks at 18 days of age, when fed diets containing 8.2% crude TLW and with 0.025 or 0.05% cholic acid, chenodeoxycholic acid or taurocholic acid (sodium salt) (Experiment III). Absorption of fat by SCWL females, 20 weeks of age, fed diets with 4 or 8% TLW and graded levels of cholic acid (Experi- ment IV). Effect of graded levels of cholic acid on M.E. (kcal./g.) of diets with 4 or 8% TLW, when fed to SCWL females 20 weeks of age (Experiment IV). Absorption of fat by SCWL females, 23 weeks of age, fed diets with 4 or 8% HSBO and graded levels of cholic acid (Experi- ment V). Effect of graded levels of cholic acid on M.E. (kca1./g.) of diets with 4 or 8% HSBO when fed to SCWL females 23 weeks of age (Experiment V). Effect of graded levels of dietary cholic acid on gall bladder weight (g.) and bile content (9.) of SCWL females, 23 weeks of age, fed diets with 4 or 8% HSBO (Experiment V). 88 9O 93 94 Figure 3a 3b 3c LIST OF FIGURES Trends in M.E. (kcal./gJ increments (M.E.T - M.E.R) of the 50% and 100% F.M.E. diets with or without cholic acid when fed to broiler-type chicks, four weeks of age. (Experiment I) Regression curves showing increases in bile content of gall bladders from adult SCWL females 23 weeks of age. The birds were fed diets with 4 or 8% HSBO and graded levels of 0.025, 0.05, 0.1 and 0.2% cholic acid. (Experiment V) Photographs depicting distention of gall bladders when adult SCWL females 23 weeks of age, were fed diet 71-09-01 (0% HSBO and 0% cholic acid) and diet 71-09-03 (4% HSBO and 0% cholic acid). (Experiment V) Photographs depicting distention of gall bladders when adult SCWL females, 23 weeks of age, were fed diet 71-09-05 (4% HSBO and 0.025% cholic acid) and diet 71—09-06 (4% HSBO and 0.05% cholic acid). (Experiment V) Photographs depicting distention of gall bladders when adult SCWL females, 23 weeks of age were fed diet 71-09-07 (4% HSBO and 0.1% cholic acid) and diet 71-09-08 (4% HSBO and 0.2% cholic acid). (Experiment V) xi 96 98 100 102 I . INTRODUCTION The nutrition of the chicken during its first few weeks of growth is a subject in which fundamental knowledge is still lacking despite the fact that the growing chick has been used in numerous nutritional studies. Many nutri- tional peculiarities of the growing chick are well described in literature. Fats, for instance, though well tolerated, are less well absorbed by the chick during its first few weeks of growth. Fedde etgal, (1960) and Renner and Hill (1960) have shown that saturated fats, such as hog grease and tallow, are poorly absorbed by the chick at two weeks of age, and only recently have efforts been made to adduce reasons for the failure of the young chick to utilize these fats efficiently. Serafin and Nesheim (1970), studying bile acid pools in chicks two weeks of age, demonstrated that these young chicks were unable to replenish bile salts lost by excretion as readily as do the older birds. The poor absorbability of saturated fats by the young chick thus appears to be related to a possible inadequacy of bile secretion. This is a limitation that would apparently need to be overcome if demands are to be made on the chick for efficient utilization of dietary fats at high levels. The field is thus open for investigations and for developing techniques to improve bile salt availability either phys- iologically (endogenously) or by dietary supplementation (exogenously). If the latter approach is pursued, because it seems to be the simpler one, a factor to be taken into account is that bile salts are actively reabsorbed in the lower intestine in an enterohepatic circulation probably controlled by a feedback mechanism. The level and nature of supplementation will, therefore, have to be critical so that the advantages of supplying additional bile salts will not be negated by deleterious or adverse physiological effects that may arise from reabsorption. This study reports on the effect of testing different bile salts, conjugated or unconjugated, in combination with high levels of various saturated and unsaturated fats on growth, feed efficiency and fat absorption and energy utili- zation in broiler-type chicks during the first few weeks of growth. Included in this investigation was a study of the isocaloric substitution of fat for 0, 50, and 100 percent of the M.E. calories from glucose in a purified reference diet. Donaldson 22 31. (1957) and Rand §E_al. (1958) showed that chicks were capable of tolerating fat levels up to 34 percent in the diet. In addition, Rand gt_al. (1958), Renner (1964) and Brambila and Hill (1966) have observed that fat can fully replace carbohydrates in the diets of chicks with little or no adverse effect on growth. Experiments were also conducted to test the effects of dietary bile salts on fat absorption in the adult bird. II. REVIEW OF LITERATURE A. Fats in the Diets of Growing Chicks 1. Factors affecting absorption and utilization of fats a. Level of dietary fat and fat tolerance Supplemental fats in the diets of the growing chick have been variously shown to have deleterious and benefi- cial effects. Studies on the tolerance of fats by chicks were initiated in the early nineteen forties. Most of the early work consisted of varying the levels of edible fats and oils in the diet and observing growth responses. Henderson and Irwin (1940), studying the chicks' tolerance to soybean oil,-showed that chick growth was retarded when soybean oil was added to the diet at levels higher than 10 percent. Fraps (1943) also obtained de- .pressed growth rates in chicks when the level of dietary cottonseed oil was raised from 10 to 30 percent. Kummerow 23 El- (1949), using 25 percent linseed oil in chick diets, reported not only adverse effects on growth but also an increased incidence of perosis. In 1953, Yacowitz fed chicks cottonseed oil at different levels and showed that at the 2.5 to 5.0 percent level there were small but con- sistent growth responses along with improved feed efficiency. Similar results were also recorded with soybean oil and lard at the low levels. When dietary levels of cottonseed oil were increased from 10 to 15 percent, growth was retarded. At about the same time Seidler and Schweigert (1953), tested diets with stabilized choice white grease at 2.0, 4.0 and 8.0 percent and found no effect on body weight gain of chicks to nine weeks of age, but a definite improvement in feed efficiency. In the early fifties, there was a trend to formulate high energy rations for broiler production which led to an increased interest to add fat to broiler diets. Already there was sufficient evidence to support the inclusion of fats at moderate levels to improve feed efficiency. Sunde (1954), Runnels (1955), Biely and March (1954), Seidler gt_§l. (1955) and Scott et 21. (1958) confirmed that the greatest benefit from the addition of fats to diets, at levels up to 7.0 percent, was from improved efficiency of ration utilization. They also showed that the improvements in feed efficiency were seen irrespective of the composition of the fat. Curtin and Raper (1956) demonstrated that even hy- drogenated vegetable fats containing 87 percent or more free fatty acids, at 6 percent in the diet improved feed efficiency. Many workers continued to study the ability of the chick to tolerate high levels of fat and at the same time test the maximum levels the chick could efficiently utilize. Donaldson 22 31. (1957), adding corn oil and stabilized animal grease up to 33.8 percent to chick diets, showed that fats are well tolerated by the growing chick (4 weeks and 7 weeks old). In the experiments he maintained a near equal productive energy calorie to protein ratio (40.0 to 43.0). The high fat diets also having high levels of protein (up to 35.6%) produced not only better feed efficiency but also improved growth responses. In his view the depressed growth seen by previous workers when chicks were fed high fat diets may have been due to the sub—optimal or unadjusted protein levels. In an attempt to evaluate the nutritional role of supplementary fat Baldini and Rosenberg (1957) used 3.6 to 4.0 percent prime tallow in chick diets and contended that the effect of fat was entirely due to the added caloric value of the fat, and that increasing the fat content without increasing the caloric content had no effect on growth, feed efficiency, feed consumption or body composition. Donaldson et_§l. (1957), however, contended that high fat diets contained more nutrient per unit weight and that less diet was required per unit of gain when the fat level was high. With the recognition that consideration has to be given to the interrelationship between fats, protein content of the diet and energy balance, studies on fat supplemented diets began to be more meaningful. Rand §E_al, (1958) substituted graded levels of corn oil from 3.5 to 14.0 percent for glucose on an isocaloric basis and reported a beneficial effect from fat on both weight gain and protein retention even with the highest fat level, which was nearly 57 percent of the total M.E. calories of the diet. They also concluded from their experiments that the chicks' tolerance for fat was unlimited. Vondell and Ringrose (1958), studying the effects of calorie:protein ratio of high fat diets reported significant increases in growth with diets containing up to 15.7 percent tallow. In 1959 Dam gt El: tested corn oil, soybean oil, cottonseed oil, peanut oil, hydrogenated vegetable oil and lard at levels of 5.0, 10.0 and 20.0 percent (at same calorie:protein ratios) with broiler chicks up to 4 weeks of age. They obtained highly significant growth increases when the vegetable oil content of the diet was increased from 5 to 10 percent and suggested that the growth response was possibly from the "associative dynamic action" of fats as described by Forbes and Swift (1944). By 1960 with more reliability being placed on deter- mination of M.E. values of diets and feed ingredients, the benefits of fat supplementation were beginning to be evaluated using M.E. contribution of fat to diets. Renner and Hill (1960) used M.E. values in studying the absorption and utilization of fats. Their data revealed a close cor- relation between M.E. values and absorbability of fats. However, their finding was that a higher dietary level (17.5%) of fat does not have a major effect on the digestion and absorption of other dietary ingredients. Carew gt_al. (1963) examined closely the "caloric density" as a possible cause of the positive growth response of chicks to vegetable fats. They disagreed with the hypothesis of Donaldson gt Ei’ (1957) that diets contain— ing high levels of fats promote growth because of the high energy concentration and showed that enhanced growth rate occurred independent of changes in dietary energy level or caloric density. Accordingly they submitted that the growth stimulating prOperty of vegetable oil is more closely related to its chemical nature than to its energy value. The performance of high fat diets came under further scrutiny with a series of investigations using fat as the sole source of non-protein energy. Renner and Elcombe (1963), Renner and Elcombe (1964a) and Renner (1964), studying the utilization of carbohydrate-free diets by chicks, demon- strated that the chick has the ability to utilize large quantities of fat without depressing growth or nitrogen retention, at levels which completely replace carbohydrate calories. Renner and Elcombe (1964a) and Brambila and Hill (1965), studying the effect of substituting soybean fatty acids for soybean oil in carbohydrate-free diets showed that the separated dietary fatty acids depressed growth although the fatty acids were well absorbed as neutral fat. Brambila and Hill (1965) also noted in another experiment that when the non-protein energy is supplied by synthetic triglycerides not only is there a depression in growth but the birds suffer from a progressive paralysis, the cause of which remained unexplained. b. Composition of dietary fat The fatty acid constituents of triglycerides vary widely in chain length, degree of saturation, location of double bonds and position of the fatty acid in the tri- glyceride molecule. The varying composition gives them different physical and chemical characteristics which in- fluence the absorption and utilization of the fat by the chick. Duokworth et_§l. (1950) demonstrated that linseed oil was more digestible than mutton tallow when fed at 6.0, 9.0 or 12.0 percent to chicks two weeks of age. Carver 22 El; (1955), studying the utilization of fats of different melting points by broiler chicks, showed that hydrogenated fats are poorly utilized when compared to the unhydrogenated fats. They also reported that the feeding of hydrogenated fat as its fatty acids did not improve ab- sorption and, therefore, the inability to hydrolyze fats was not the answer for poor absorption. Sunde (1956) tested a variety of greases, tallows, hydrogenated fats and fatty acids, namely stearic, oleic, linoleic and butyric, at 5.0 percent in chick diets. These fats did not retard growth and, in all cases, excepting hydrogenated vegetable fat and stearic acid, improved feed utilization. He concluded that the chick was unable to utilize effi- ciently saturated fatty acids. March and Biely (1957) made comparative studies using corn oil and a hydrogenated animal fat at different levels. Corn oil at 3.0, 6.0 and 9.0 percent showed improved growth rate and feed efficiency in growing chicks and these improvements were not seen at the 12 percent level. With hydrogenated fat, growth was improved only at the 3 percent level and, thereafter, de- clined progressively at the 6, 9 and 12 percent levels. Hydrogenated animal fat had no effect on feed utilization at all levels. When beef tallow at 12 percent was compared with 12 percent hydrogenated animal fat, the former had no effect on growth and increased feed efficiency, while the hydrogenated fat depressed both growth and feed efficiency. This confirmed the earlier findings that the higher the level of saturation of fatty acids, the poorer the utilization of the fat. More evidence was, however, forthcoming to show the comparatively poor utilizability of saturated fats by the chick. In 1959 Dam gt 31., testing the performance of a number of vegetable oils and a hydrogenated vegetable oil, obtained a highly significant growth response in growing chicks with the unhydrogenated vegetable oil than with its hydrogenated counterpart. The low melting point, soft oils were thus accepted to be better absorbed and utilized by the chick and the level of absorption and utilization was seen to be inversely related to the level of saturation of the fatty acids of the fat or oil. c. Calorie:protein ratio of the diet The ratio or balance between energy and protein in feeds was first demonstrated by Scott gt 21. (1947) who 10 showed that chick growth declined with increasing substi- tution of ground oats for corn. Hill and Dansky (1950 and 1954) obtained normal chick growth with low protein diets, when the energy level of the diet was reduced. However, with the elevation of the energy level in the same low protein diet, growth declined. On this classical work was based a number of subsequent investigations to establish a relationship between energy utilization and the protein level of the diet. Aitken §E_§l, (1954) tested 10 percent beef tallow in broiler rations with 22.0 and 25.0 percent protein. They reported no growth responses and a slight improvement in feed efficiency with the 25.0 percent pro- tein diets. Biely and March (1954) fed chicks diets contain- ing 7 percent tallow with three levels of protein, 19.0, 24.0 and 28.0. They observed that the parameters were depressed if the ration had 7 percent tallow and 19 percent protein. Increasing the protein content in the ration to 24 percent improved feed efficiency but had no effect on growth rate. A further increase in the protein content to 28 percent improved both feed efficiency and growth rate. They concluded from their data that the addition of fat to chick diets was advantageous when high levels of protein were included. Scott‘gt_§l. (1955) studied the effect of stabilized animal fat at 0, 7 and 14 percent with three levels of protein, 20, 25 and 30 percent, ongrowth and feed efficiency. In their fat-free diets there were minor differences in growth for the 11 different protein levels. Their diets with 20 percent pro- tein caused growth to decline with increasing levels of fat substitution. The diets having 25 and 30 percent protein produced growth responses when the fat level was at 7 per- cent and growth retardation for diets with the fat level at 14 percent. Maximum growth was recorded in chicks fed diets having 30 percent protein and 7 percent fat. Sunde (1956) added 5 to 10 percent choice white grease to diets with 20 percent protein and found no effect on growth rate to four weeks. However, when he fed diets with 28 percent protein and 5 percent supplemental fat the chicks grew heavier; raising the fat level to 10 percent resulted in a further improvement in growth. From his studies he concluded that high protein, low energy diets depressed growth. Donaldson gt El' (1956) observed that chicks receiving 7.5 and 15 percent stabilized animal grease tolerated wider calorie: protein ratios before growth was adversely affected, as compared to those on diets with no supplemental fat. There was no adverse effect on growth with the low and high fat diets as long as the calorie:protein ratios were within 44 and 54. They also noted that the feed efficiency improved with increasing dietary energy, that energy had a sparing action on protein, and concluded that at higher calorie: protein ratios, less protein would be required per unit gain. Chicks from their experiments had increased fat and lower water and protein levels in the carcass as calorie: protein ratios were increased. The total fat and water 12 content, which are inversely related, remained constant within a calorie:protein ratio of 50. Finally, they were also able to show a positive correlation between carcass fat and calorie:protein ratio of the diet. Vondell and Ringrose (1958) made further contributions to knowledge of the nutri- tional relationship between fat and protein. They studied the effect of calorie:protein balance on growth and feed conversion in broilers, using tallow at 0 and 15.75 percent in diets containing 16.5, 18.5 or 22.5 percent protein. Feed to gain ratio was found to improve with increasing calorie:protein ratio. Body weights improved with increases in calorie:protein ratio until a ratio of 45 was reached. They concluded from their results that optimum performance is obtained with a specific calorie:protein ratio, irrespec- tive of the level of protein, and that calories from fat do not differ from calories of other nutrients in their effect upon the calorie:protein ratios. Rand gt 21. (1958), using two levels of corn oil (1.0 and 8.0 percent) in 20.0 and 30.0 percent protein diets, substantiated earlier findings that higher protein levels enhance the beneficial effects of fats. Their evidence and conclusions should be more valid as they worked with isocaloric diets. ‘d. Age of chick Duokworth 23.3}: (1950) showed that mutton tallow, fed at levels of 6.0 to 12.0 percent, underwent an improve- ment in "digestibility" as the Chick's age increased from l3 2 to 6 weeks. Fedde gt El: (1960) reported that hog grease used at 20.0 percent in chick diets had an apparent absorp- tion coefficient of 85.4 in chicks at one to two weeks and 93.0 at seven to eight weeks of age. In the same study they found that the absorption coefficient for tallow in- creased from 53.0 at one week of age to 80.0 at twelve weeks of age. Renner and Hill (1960) confirmed the effect of age of chicken on fat digestibility and absorption. They noted that the absorption of tallow was lowest in the chick two weeks of age and reached an absorbability level characteristic of the adult when the chick was eight weeks of age. 2. Fat energy as a substitute for carbohydrate energy Carew and Hill (1958) substituted 10.0 and 20.0 percent corn oil isocalorically for glucose M.E. calories and obtained improved tissue gains. They reported greater tissue fat gains and slightly lower tissue protein gains. Meanwhile Rand 33 El. (1958) showed that the substitu- tion of corn oil (0 to 14.5 percent by weight) for glu- cose isocalorically in diets fed to chicks from one to four weeks of age resulted in improved body weight gains, increased protein and energy utilization and, most im- portant, improved protein retention. The best overall per- formance was obtained when fat contributed 20.0 and 38.0 percent of the total M.E. of the diet. Even at the highest dietary fat level of 14.5 percent (equivalent to 14 57.0 percent of the total M.E. and 97.0 percent of non- protein calories), the effect on body weight gain and pro- tein retention was beneficial. Baldini and Rosenberg (1957) from their experimental data maintained that the nu- tritional value of adding fat containing sufficient essen- tial fatty acids was entirely due to the caloric value of fats. They were also able to show, in contrast to the observations of Rand 93 21. (1958), that increasing the fat content without increasing the caloric content had no effect on growth, feed efficiency, feed consumption and body composition. Renner and Elcombe (1963) completely replaced carbohydrate calories in the diet with those from fat and showed there was no effect on rate of growth. Renner (1964), while investigating the role of protein in the utilization of "carbohydrate-free" diets by the chick,showed that when all the non-protein calories are supplied by fat, neither growth nor nitrogen retention was affected. Renner and Elcombe (1964a) obtained depressed growth in chicks when they substituted soybean fatty acids for soybean oil in "carbohydrate-free" diets. Normal growth was restored by supplemental glycerol or glucose, implying that the abil- ity of the chick to utilize fatty acids in the absence of carbohydrates is limited. Renner and Elcombe (1964b) studied the metabolic effect of feeding "carbohydrate-free" diets in which non-protein calories were supplied by soy- bean oil or soybean fatty acids, and reported that the chicks maintained normal levels of blood glucose, blood 15 lactic acid, muscle glycogen and liver fat but showed a marked depression in liver glycogen. When soybean oil supplied the non-protein calories, blood levels of ketone bodies remained normal and increased when soybean fatty acids replaced the soybean oil. They also showed that chicks fed the "carbohydrate-free" diets with soybean fatty acids utilized nitrogen just as efficiently as those on similar diets having glucose as a source of energy. Renner and Elcombe (1964b) concluded that dietary protein was not diverted from protein to carbohydrate synthesis when soybean fatty acids were the source of non-protein energy, indicating that soybean protein contained glucogenic amino acids in sufficient excess to maintain levels of blood glucose, blood lactic acid and muscle glycogen. The alter- native being that chicks could possibly synthesize carbo- hydrates from fatty acids. Brambila and Hill (1966) fed chicks a synthetic mixed triglyceride, as the sole non-protein energy source and obtained growth retardation, in conjunction with a crippling paralysis. They observed that this effect was probably due to lack of an unidentified essential nutrient found in nat- ural fats. Brambila and Hill (1966) also found that soybean oil fatty acids as the sole supplier of non—protein calories retarded growth. Unlike Renner and Elcombe (1964a) they could not show a restoration to normal growth with glucose or glycerol supplementation. Soybean oil fatty acids were absorbed as well as the neutral fat. In a further set of 16 experiments Brambila and Hill (1966) showed that the food intake by the chick is regulated to avoid excess ingestion of fatty acids. Chicks fed the diet with a higher level of soybean oil (68 percent M.E.) deposited as much fat as the reference group on the fat-free diet. The depot fat of chicks fed the high lipid diets was high in unsaturated fats. Recently, Evans and Scholz (1971) studied the chicks' adaptability to high protein, "carbohydrate-free" diets by assessing the response of several liver and kidney gluco- neogenic enzymes such as g1ucose-6-phosphatase, glycerol kinase, and fructose -l,6-diphosphatase. Chicks on the "carbohydrate-free" diets had increased liver size, kidney hypertrophy, elevated plasma uric acid levels and unchanged plasma glucose levels. The results showed that chicks are able to adapt to "carbohydrate-free" diets and have a marked capacity to switch to g1uconeogenesis from protein. 3. Evaluation of fat energy Fats are perhaps the most difficult of all feeding stuffs to evaluate in terms of metabolizable energy content. Methods for the evaluation of the metabolizable energy of feedstuffs were studied and developed by Hill and Anderson (1958), Anderson'§t_al. (1958) and Potter at al. (1958). They also showed that metabolizable energy is a more valid measure of determining energy content of poultry feeds than is productive energy (Fraps, 1943). Sibbald §t_al. 17 (1960) tested the validity of M.E. values on the basis of its independence of other dietary components and also age of bird. Using only corn as test material, they found significant variations when combined with different basal diets. There was, however, no significant variation with age of bird. Sibbald and Slinger (1963), studying the prob- lems associated with evaluation of M.E. of fats, observed that fats available for animal feeding are extremely variable in composition and M.E. content. There is no apparent relationship between the physical and chemical properties of a fat and its energy content, as demonstrated by Cullen 22 21. (1962) and Sibbald‘gt;al. (1962). Their experimental data showed that with increases in fat levels of the diets, the M.E. values of fats decreased. This was probably from a calorie:protein imbalance. In the light of their findings they concluded that the methods for determining M.E., es- pecially for fats, were not satisfactory and needed further study. 4. "Extra-caloric" effect of fat Sibbald 23 31. (1961) reported that when corn oil re- placed part of the tallow, the M.E. values obtained from the mixed fat diets were higher than the calculated values. From the evidence for increased utilization of the M.E. of tallow, they postulated that corn oil had some factor that increased utilization of energy of tallow. Sibbald gt;§l, (1962) tested various fats at 2.0 and 4.0 percent 18 at similar calorie:protein ratios and obtained a stimulatory effect from the fat-supplemented diets. They attributed the increased response to the "extra-caloric" effect of fats. Cullen 93 21. (1962) fed to growing chicks diets hav- ing 14.0 percent fats and obtained M.E. values exceeding the theoretical maximum for fats. Their values for yellow grease and poultry fat were 9.517 kcal./g. and 10.186 kca1./g. respectively. They concluded that the high values were as a result of fats influencing the energy utilization of other feed ingredients. Touchburn and Naber (1966) clearly dem- onstrated with turkeys that dietary fat improved utiliza- tion of M.E., and also showed that a wider calorie:protein ratio could be tolerated when fat was added. They referred to the increased response as the "extra caloric" effect of fat. Jensen £3 31. (1970), also using turkeys, confirmed that supplementary fat in turkey diets improved utilization of M.E. calories and, therefore, fat had an "extra caloric effect." Their high fat M.E. values (10.165 kcal./g. for yellow grease) were obtained from the recalculation of M.E. values utilizing feed efficiency data and which M.E. value they termed the "adjusted M.E. value." They con- cluded that the "extra caloric effect" of added fat was caused by a lowered heat increment (or specific dynamic effect). 19 B. Bile Salts and Fat Absorption l. Physico-chemical role of bile salts in fat absorption Bile acids are a major constituent of bile. They are synthesized in the liver as important end products of cho- lesterol metabolism and are secreted into the duodenum through bile ducts. After temporary storage and concentration in the gall bladder, bile acids (both conjugated and unconjugated) are secreted into the lumen of the duodenum and subsequently reabsorbed efficiently in the lower intestine and returned to the liver in a controlled enterohepatic cycle (Borgstrom and Danielson, 1958; Borgstrom §E_§l., 1963). The primary bile acids, cholic acid (3,7,12 trihydroxycholanic acid) and chenodeoxycholic acid (3,7,dihydroxycholanic acid) occur in bile, conjugated at C24 with glycine or taurine. The conjugated bile acids together with the cation Na+ form the "bile salts." Cholic acid being metabolically derived from cholesterol has the structural cyclopentanophenanthrene ring and has the following chemical structure: 21 CH OH 19 2 CH-CHz-CHZ-COOH 20 22 23 21+ 20 A number of other bile acids, often isomers, occur in bile. Two well known secondary bile acids, resulting from microbial dehydroxylation are deoxycholic acid (3,12 dihydroxycholanic acid) and lithocholic acid (trans isomer of 3-hydroxycholanic acid). The composition of bile is species specific. In the chick the major bile acid is chenodeoxycholic acid (Wiggins, 1965; Webling and Holdsworth, 1965). The ability of conjugated bile salts to solubilize fatty acids was discovered and studied extensively by Verzar and McDougall (1936). Frazer (1946), in his "parti- tion theory" for absorption of fats, proposed that tri- glycerides were hydrolyzed, and were absorbed in particulate form as a fine emulsion and transported through the lymph system, in contrast to fatty acids which were absorbed as such and transported through the portal system. He also studied the factors related to the formation of stable emulsions of triglycerides and concluded that a combination of an unsaturated fatty acid, saturated monoglyceride and bile salt were requisite for effective emulsion stability, and this system promoted the emulsification of the remain- ing glycerides and other lipids. The function of the bile salt was to provide the charge on the particles. Reiser gg‘gl. (1952) and Mattson and Volpenhein (1962) confirmed Frazer's (1946) restricted lipolysis hypothesis using labeled triglycerides and showed that 25 to 50 percent of dietary triglycerides are absorbed without previous complete 21 hydrolysis. Clark and Hubscher (1961) and Senior and Isselbacher (1962) demonstrated the existence of a re-esteri- fication pathway within the mucosal cell, requiring mono- glyceride as substrate and thus have confirmed the case for direct monoglyceride absorption. Tidwell and Nagler (1952) and Annegers (1952) showed that the action of bile on fats was not due to its emulsifying properties alone, since they found other emulsifiers were ineffective in improving absorption. Hofmann and Borgstrom (1962) and Hofmann (1963), investigating the physico-chemical prerequisites for fat absorption, proposed that an important function of bile salt was in the formation of a highly stable lipid-bile salt micelle. They were convinced that lipids were not absorbed in the form of an emulsion but in a more finely dispersed and a molecularly defined state. They expressed the View that bile salts are atypical detergents and being amphipaths showed a tendency to accumulate at surfaces. When their concentration increased over a critical level (critical micellar concentration), they associated into smaller poly- molecular aggregates termed "micelles" or associated colloids. The lipid-bile salt micelle is more readily formed with polar lipids such as monoglycerides containing unsaturated or short chain fatty acids or oleic acid. Micellar solutions are transparent and have a far smaller particle size (3 to 10 mp) than emulsions (300 to 100 my), form Spon- tanedusly and are totally stable. 22 Norman (1960) showed that micellar solutions of bile salts are able to solubilize non—polar substances. The expanded bile acid-unsaturated monoglyceride micelles can solubilize more non-polar compounds than the bile salt mi- celle alone. Garrett and Young (1964) from their studies on absorption of fatty acids and triglycerides in bile duct cannulated chicks were able to support the proposal that micelle formation is essential for maximum absorption. They were also able to show considerable absorption of fatty acids (36 to 50 percent) in the absence of bile, and con- cluded that the lipid-bile salt micelle is not the only prerequisite for fat absorption. Garrett and Young (1964) also showed that absence of bile from the intestinal lumen markedly decreased absorption of oleic acid from 96 to 52 percent and linoleic acid from 99 to 69 percent, when pre- sented in triglycerides. Similar decreases were also noted with the free oleic and linoleic acids. In the normal chick, absorption of palmitic acid was greatly improved in the pre- sence of high levels of oleic acid and decreased markedly in the absence of bile, confirming the enhanced solubilizing properties of the oleic acid-bile salt micelle. They con- cluded that fatty acids readily forming micelles are affected most in the absence of bile. These workers were also of the view that Verzar's "hydrolytic" theory and Frazer's "parti- culate" theory were no longer valid and that present knowledge showed that fat digestion and absorption was a compromise between the two theories. 23 2. Functional bile salt in the chick Wiggins (1955) and later Webling and Holdsworth (1965) showed that 80 to 85 percent of the total bile acids of chicks was chenodeoxycholic acid while cholic acid com- prised only 13 to 16 percent. Other bile acids appeared in trace amounts. Haslewood and Sjovall (1954) demonstrated that glycine conjugates found in the bile secretions of mammalian bile were not seen in avian bile. Wiggins (1955) followed by showing that the bile acids in the seven-day-old and adult chick occurred as the taurine conjugates. 3. Effect of supplemental dietary bile salts on fat absorption Preliminary investigations on the effect of supplemental bile salts on chick diets were made by Edwards (1962). He fed cholic acid at 0.2 percent in a practical-type ration having 8 percent "blended fat," to broiler chicks, from 0 to 4 weeks and obtained no effect on growth and feed efficiency and only a slight trend towards improvement in fat absorption. Eyssen 2E.2l° (1965) obtained an improved fat absorption with 0.2 percent cholic acid while 0.2 per- cent lithocholic acid caused malabsorption of fats. Leveille 22 21. (1962), Hunt 23 31. (1963), Eyssen and Somer (1963) and Eyssen gt_al. (1965) showed that 0.2 percent cholic acid in chick diets had no effect on liver size, while 0.2 percent lithocholic acid elevated plasma lipids and increased liver size twofold. In the latter case histologi- cal examination of the liver showed a peripheral biliary 24 hyperplasia (also termed the ductular cell reaction) whose etiology was undetermined. Leveille and Fairchild (1965) continued their studies with 0.2 percent chenodeoxycholic acid and found that the bile salt (dietary chenodeoxycholic acid) had no effect on plasma lipids or liver size, but caused liver cholesterol to increase by 12 percent. The observations of Brambila 33 El' (1961) that raw soybeans lowered fat absorption in chicks under four weeks of age led to investigations on the influence of dietary components on bile acid production and eXcretion. Later Nesheim‘eg‘al. (1962) and Sambeth 33 El- (1967) implicated bile deficiency as the cause of poor fat absorption by I chicks on the raw soybean diet. Garlich and Nesheim (1964 and 1965) reported that sodium taurocholate, a bile salt, corrected malabsorption of fat in the chick. In addi- tion, Sambeth 23 a1. (1967) observed empty gall bladders in chicks fed unheated soybeans, and suggested that there may exist a limitation to bile production in these growing chicks. Serafin and Nesheim (1967), using labelled 24 14C cholic acid, administered intraperitoneally, showed that chicks fed diets containing unheated soybean meal excreted 44 percent of the dose in 6 days in comparison to an excre- tion of 23 percent by the chicks fed heated soybean meal. The biological half-life of the cholic acid calculated from the excretion pattern, up to 48 hours following administration of labelled cholic acid was nine days for chicks on the 25 control diet and 4.2 days for chicks on diets with unheated soybean meal. The chicks on diets with unheated soybeans were observed to excrete bile acids twice as rapidly as those on diets with heated soybeans, indicating that chicks on raw soybeans are synthesizing bile acids at a faster rate than the controls, probably to provide more bile acids for normal fat absorption. They concluded that the poor fat absorption seen in young chicks up to two weeks of age (Renner and Hill, 1960) was because of their limited ability to synthesize bile acids. When raw soybeans are fed, this limited ability is overtaxed by heavy excretion which ex- plains the poor absorption of fat in the presence of raw soybeans. Garlich and Nesheim (1965), using diets contain- ing unheated soybeans and supplementary sodium taurocholate at 0.6 percent, showed that young chicks benefit from an exogenous source of bile salts when dietary factors demand more than normal secretion of bile acids. Another side effect of dietary cholic acid as shown by Martin and Patrick (1966) was the increase in volume of bile fluid secreted. These workers also found supplementary dietary taurine to give the same effect. III . EXPERIMENTAL PROCEDURE A. Introduction Five experiments were conducted with growing chicks to examine the effects of dietary bile salts at different levels on the utilization of high levels of fats of varying degrees of saturation. In the first two experiments studies were also made on the isocaloric substitution of fat for 50 (n: 100 percent of the M.E. calories from glucose in a purified reference diet. Experiments I and II were designed to study the effect of 0.2 percent cholic acid on the utilization of tallow (TLW), lard (LD), corn oil (CNO), and hydrogenated soybean oil (HSBO) substituted for 50 or 100 percent of the M.E. calories from glucose in a reference diet. The experiments were conducted with male broiler-type chicks. In experiment I the chicks were on trial from one week of age to four weeks of age; whereas, in experiment II the trial period was from day-old to 7 days of age. Experiment III was conducted to study the effect of 0.025 and 0.05 percent cholic acid, chenodeoxycholic acid or taurocholic acid, sodium salt, on the utilization of crude tallow at 8.2 percent in a purified diet. The diets were 26 27 tested with male broiler-type chicks from one day through 21 days of age. Experiments IV and V involved studies on the effect of four levels of cholic acid (0.025, 0.05, 0.10 and 0.20) on the utilization of tallow and hydrogenated soybean oil at 4.0 and 8.0 percent levels in a practical-type diet. The experiments were conducted with SCWL (Single Comb White Leghorn) females, 20 and 23 weeks of age. B. The Experiments 1. Experiment I Three hundred male broiler-type chicks were fed for one week, ad libitum, on a purified diet (Table 1). At one week of age each chick was weighed and 216 birds were selected for the experiment by omitting birds with extreme upper and lower weights. Groups of 6 of nearly equal average weight were randomly assigned to 36 pens, in three separate multi-deck, battery brooders. The pens were wirefloored, electrically heated and had 24-hour lighting. The room temperature was maintained at 21 1 20 C. The experimental design was a completely randomized 4 x 2 x 2 factorial, involving 4 types of fat, 2 levels of fat, and treatments with and without cholic acid. The ref- erence diet (Table l) was a purified diet with a protein content of 25.1 percent and total calculated M.E. of 3.40 kcal./g. Glucose monohydrate ("Clintose") supplied 67.6 percent (or 2.30 kca1./g.) of the total calculated M.E. 28 Table 1. Composition of purified reference diet (Experiment I) Ingredients % Glucose monohydrate ("Clintose")l 63.2 Soya assay protein (87%)2 28.8 Safflower oil 2.0 Ground limestone 1.0 Dicalcium phosphate 2.15 Salt (iodized) 0.5 Vitamin premix 0.35 Mineral premix 1.02 MHA5 0.72 Tryptophan 0.06 Chromic oxide 0.2 100.00 Calculated analysis Crude protein (%) 25.1 Crude fat (%) 2.09 Metabolizable energy (kca1./g.) 3.40 Protein (%)/M.E. (kca1./g.) ratio 7.38 Calcium:phosphorus ratio 1:0.5 1Clinton Corn Processing Company, Clinton, Iowa. 2Archer Daniels, Midlands, Minnesota. 3Supplied the following per kg. of diet: Vitamin A--ll,000 I.U.; Vitamin D3--l,100 I.C.U.; Vitamin E--ll I.U.; Vitamin K--2.2 mg.; Thiamin--2.2 mg.; Riboflavin--4 mg.; Pantothenic acid--l4.l mg.; Nicotinic acid--31.5 mg.; Pyridoxine--4 mg.; Biotin--0.l mg.; Folic acid--l.3 mg.; Choline--1320 mg.; Vitamin B12--0.01 mg.; and Antioxidant (Santoquin)--12.5 mg. 4Supplied the following per kg. of diet: KZHPO4--8.8 g.; MnSO --l48.6 mg.; MgO--916.6 mg.; FeSO4--273.8 mg.; CuSO4—- 5MHA (Methionine hydroxy analogue,ca1cium--93%), Monsanto Company, St. Louis, Missouri. 29 calories. The 2 percent safflower oil in the diet supplied the minimum requirement of linoleic acid and contributed only 5.0 percent of the total calculated M.E. In the experimental diets were either one of 4 fats, edible beef tallow (TLW), pork lard (LD), corn oil (CNO) and hydrogenated soybean oil (HSBO). They had been selected for the study because they differed in varying degrees of unsaturation (Table 2). Each fat was substituted isocalorically for 50 percent (1.15 kcal./g.) or 100 percent (2.30 kcal./g.) of the glucose M.E. calories of the diet. The fat diets,from hereon, will be referred to as the "50 percent level of fat M.E." (50% FME) or the "100 percent level of fat M.E." (100% FME). To calculate the M.E. of the diets the following M.E. values (kca1./g.) were used: glucose-~3.64; tallow--7.01; lard--8.60; corn oil--8.95; and hydrogenated soybean oil--7.80. The physical and chemi- cal properties and fatty acid composition of the fats used in the experiment are shown in Table 2. The reference and 16 other diets with 4 types of fats, each at 2 levels, with or without 0.2 percent cholic acid, were fed in duplicate lots. The compositions of the 18 experimental diets are outlined in Table 3. All experimental diets were calculated to be isocaloric and isonitrogenous, thus having identical protein:calorie ratios. Cellulose ("Solka floc") and cleaned, washed sand were included in suitable proportions to adjust and balance the diets for equal weight and density. Chromic oxide at 0.2 percent was added as a marker to all diets. 30 Table 2. Fatty acid compositionlof fats used in diets fed to broiler-type chicks (Experiment I) Hydrogenated Edible Pork Soybean Corn Tallow Lard Oil Oil Fatty acids C8:0 0.1 -- —- -- C10:0 -- 0.1 0.1 -- C12:0 0.1 0.1 0.1 -- C14:0 3.5 1.6 0.1 -- C14:1 1'1 " " ” C16:0 25.9 25.8 10.7 12.0 Cl6:l 2.9 3.5 0.1 -- C16:2 0.3 0.2 -- -- C17:0 1.2 0.1 -- -- C18:0 19.2 10.7 6.0 2.7 C18:l 43.0 47.4 72.3 30.1 C18:2 2.3 8.9 5.8 54.7 Cl8:3 0.4 1.2 0.4 1.4 C20:0 -- 0.2 0.1 -- Misc. & unknown -- 0.2 4.3 -- Total saturated 50.0 38.5 21.3 14.7 Total unsaturated 50.0 61.3 78.7 86.2 Ratios C18.1:satd. 0.86 1.23 3.39 2.05 C18.2:satd. 0.05 0.23 0.07 0.63 Unsatd.:satd. 1.00 1.59 3.69 5.86 Iodine value 48 65 81 121 lAnalytical data supplied by Procter & Gamble Company. .mHASmmEmm 3oz .cwaumm .wcmmfiou c3oumv ...m\.Hmox oom.~ ..m.ae umae «0 .m.z mo wm.sm emaaaasmm .wcmmeou cabana w Hopooum mo ammuuooo we omaammsmm .mamoHEmnoon Hmnmcmw Ammmoo “pogo chow oaaoao H N.o mpfixo oweouso oo.o cosmoumwna Nh.c m.o ApoNAUOAV uamm ma.~ mumnamoaa guacamoan o.H mcoumwfiwa ocoouw n o.~ Hao umzoammmm m.m Awamv efimuonm pennaomw somehow w.ma o.va w.ma m.NH «.ma m.ma N.mH v.HH II comm n.ma n.v m.mH ~.v «.mH m.e ~.mH m.m II vA=oon mxHom=V mmoasaamo m.m~ m.mH m.m~ m.vH w.m~ v.ma m.~m «.ma I: new omuopwumnom I: o.Hm II m.Hm II m.Hm In m.Hm m~.mm mmoooao w w w w w w w w w muemaomumcH ooa om OOH om. ooa om ooa om umwo MZm mzm mzm mzm WEE mzm mzm mzm mocmummwm Hao cuoo Hao emmnsom cums xuom soaama manage mouncomoupmm N N .AH ucoEHummxmv Apamm mcwomHmmHUv w~.o um nonsmEmHmmom mm3 meow oaaono .mumwc no new umnuo on» CH .mumflp mo uwm moo show on mmoooam mo moan Hmo IMOm pmumcmmouohn .puma xnom .3oaamu manflom .m.z ecu mo wooa one wom How cmusuwumndm some wuo3 Hwo :Hoo 0cm Hfio coon .mumHU HmucmeHmmxm mo :0wufimomeou .m manna 32 Feed and water were supplied ad libitum during the three weeks of the experiment. Weights of the chicks and feed con- sumption on a pen basis were recorded weekly. At the termi- nation of the experiment all birds were weighed individually. Excreta was collected on aluminum foil under each pen, during the last 4 days of the experiment. Collections were made from the middle of the battery brooder so that contamination from feed spillage was at a minimum. The excreta samples were air dried. Four birds were selected at random for necropsy. Each sample of feed and air-dried excreta were finely ground in a Wiley mill and blended. Assays on these samples were made for moisture, crude fat, Chromic oxide and energy content. Moisture content was determined by heating the sam- ples in a vacuum oven at 600 C. and 38 cm. Hg for 36 hours. Crude fat was assayed using chloroform-methanol 2:1 (v/v) according to the method of Folch et.al. (1957). Chromic oxide was estimated by the method of Czarnocki, Sibbald and Evans (1960). All gross energy determinations were made in a Parr Adiabatic Bomb Calorimeter. M.E. values were calculated by a modification of the formula developed by Hill and Anderson (1958), omitting the correction for retained protein in accor- dance with the concept of Kleiber (1961). The differences among the means were tested using the Duncan's Multiple Range Test according to Garber (1963). 33 2. Experiment II One hundred male broiler-type chicks, one day of age, were individually weighed and divided into groups having a weight spread of 3 g. Seventy-two chicks were selected for the experiment, omitting those from the extreme weight groups. Groups of four of nearly equal average weight were assigned randomly to eighteen pens, in a single multideck battery brooder. The pens were wirefloored, heated electrically and had 24-hour lighting. The room temperature was main- tained at 210 C. The experimental design was identical to that of experiment I. Due to lack of battery space, the experi- ment was phased into an experiment IIA and experiment IIB. Experiment IIA with the full set of 18 treatments was treated as replicate one. Experiment IIB a repetition of experiment IIA was conductted one week after the termi- nation of experiment IIA. The two phases (replicates) of the experiment were conducted under identical environmental and housing conditions. The 18 diets used in experiment IIA and B were identical in composition to those made for experiment I. Feed and water were supplied ad libitum during the one-week duration of the experiment. Feed was supplied in trays covered with wire-mesh, placed inside the pens, on the wire floor. Chick weights and feed intake on a pen basis were recorded at the beginning and at the end of the experimental period. Excreta were collected from the middle 34 dropping pans of the brooder, to minimize contamination from feed spillage. The excreta samples were air-dried. At the termination of the experiment two birds from each pen were selected at random for necropsy. The samples of feed and air-dried excreta were finely ground in a Wiley mill and blended. The samples were assayed for moisture, crude fat, nitrogen, Chromic oxide and gross energy content. The assay techniques were the same as those used on experimental samples from experiment I. 3. Experiment III Two hundred male broiler-type day-old chicks were individually weighed and sorted into groups of uniform weight. One hundred forty-four of these birds each weighing between 35 to 40 g. were randomly selected for the experi- ment. Groups of six of nearly equal average weight were randomly assigned to 24 pens (8 treatments x 3 reps) in 2 separate multideck, battery brooders. The pens were wire floored, electrically heated and had 24-hour lighting. The room temperature was maintained at 21 i 20 C. The experimental design was a completely randomized 3 x 2 factorial, involving three bile salts and two levels of these bile salts. The reference diet was basically the same purified diet used in experiment I (Table 4) except that it contained 8.2 percent crude TLW replacing isocalorically 15.5 percent glucose. In calculating the M.E. of the diets the following M.E. values of the feedstuffs (kcal./g.) were used: glucose--3.64; crude tallow--7.01. The crude 35 Table 4. Composition of purified basal diet (Experiment III) Ingredients % Glucose monohydrate ("Clintose")l 47.40 Soybean isolated protein (87%)2 28.80 Safflower oil 2.00 Crude tallow 8.20 Cellulose 7.60 Limestone 1.00 Dicalcium phosphate 2.15 Salt (iodized)3 0.50 Vitamin premix 0.35 Mineral premix 1.02 MHA 0.72 Tryptophan 0.06 Chromic oxide 0.20 Calculated Analysis Crude protein (%) 25.1 Crude fat (%) 10.2 Metabolizable energy (kcal./g.) 3.40 Protein (%)/M.E. (kca1./g. ratio 7.38 Calcium:phosphorus ratio 1:0.5 1 "Clintose." (Glucose monohydrate), Clinton Corn Processing Company, Clinton, Iowa. 2Soyabean Isolated Protein (87% protein), Archer Daniels, Midlands, Minnesota. 3Supplied the following per kg. of diet: Vitamin A--11,000 I.U.; Vitamin D3--l,100 I.U.; Vitamin E--11 I.U.; Vitamin K--2.2 mg.; Thiamin--2.2 mg.; Riboflavin--4 mg.; Pantothenic acid--14.1 mg.; Nicotinic acid-—3l.5 mg.; Pyridoxine--4 mg.; Biotin--0.l mg.; Folic acid--l.3 mg.; Choline--l320 mg.; Vitamin B12--0.01 mg.; and Antioxidant (Santoquin)--12.5 mg. 4Supplied the following per kg. of diet: KzHPO4--8.8 g.: MnSO4--148.6 mg.; MgO--916.6 mg.; FeSO4--273.8 mg.; CuSO4-- 27.5 mg.; ZnO--55 mg. 5MHA (Methionine hydroxy analogue calcium--93%), Monsanto Company, St. Louis, Missouri. 36 tallow was supplied by the Meats Laboratory at Michigan State University from freshly slaughtered beef cattle. The TLW was from the pelvic and kidney fat depots. The crude TLW was rendered in the laboratory at 600 C. and filtered before being added to the diet. Three bile salts, namely cholic acid, chenodeoxycholic acid or taurocholic acid (sodium salt) at levels of 0.025 percent or 0.05 percent, were added to the basal diet to make six treat- ment diets. The eighth treatment diet was the basal diet having an additional 8.2 percent crude TLW substituted isocalorically for 15.8 percent glucose. The eight treat- ments were replicated three times making a total of 24 ex- perimental units. The composition of the eight experimental diets are outlined in Table 5. All experimental diets were isocaloric and isonitrogenous, thus having identical cal- culated calorie:protein ratios. Chromic oxide was added at 0.2 percent to all diets to serve as a marker. Feed and water were supplied ad libitum during the three-week duration of the experiment. Weights of the chicks and feed consumption on a pen basis were recorded weekly. Excreta collections were made twice during the experiment. The first set of samples were collected from day 4 to 9 and the second set from day 15 to 19. Collec- tions were from the middle of each collecting pan of the battery brooders to minimize contamination from feed spillage. The collecting pans were lined with aluminum foil as an added precaution. The excreta samples were 37 Table 5. Experimental diets showing the different supple- mentary bile salts and the levels added (Experi- ment III). Dietary Supplements Crude TLW Treatment Diet No. % Bile Salt and Level 1 71-12-01 None None 2 71-12-02 8.2 Cholic acid 0.025% 3 71-12-03 8.2 Cholic acid 0.05% 4 71-12-04 8.2 Chenodeoxycholic 0.025% acid 5 71-12-05 8.2 Chenodeoxycholic 0.05% acid 6 71-12-06 8.2 Taurocholic acid 0.025% - (Na--sa1t) 7 71-12-07 8.2 Taurocholic acid 0.05% (Na--sa1t) 8* 71-12-08 16.4 None *Treatment included to calculate M.E. value for crude TLW. 38 air dried. At the termination of the experiment feed intake was determined on a pen basis, and then all birds were weighed individually. Two birds from each pen were selected at random for necropsy. The livers and gall blad- ders were excised and weighed separately. The bile was drained off from the gall bladders and the empty gall bladders weighed. The bile content in the gall bladders was determined from the difference in the weight between the full and empty gall bladder. The samples of feed and air-dried excreta were each finely ground in a Wiley mill and blended. Assays on these samples were made for moisture, crude fat, nitrogen, Chromic oxide and gross energy content using the same techniques employed in the previous experiments. 4. Experiment IV A batch of 48 SCWL females, 20 weeks of age and averaging 1650 grams, were assigned to 12 groups of 4 birds each. The birds for each group were placed in consecutive single cages of a three-tiered laying battery. The groups or treatments were randomly assigned. The room temperature was maintained at 210 C. The experiments were designed to test five levels, 0, 0.025, 0.05, 0.1 and 0.2 percent, of cholic acid in a practical-type diet (Table 6) with two levels, 4.0 and 8.0 percent, of supplemental tallow. The substitutions of tallow for corn of the basal diet were made on a percent basis. The 39 Table 6. Composition of reference diet (Experiment IV) Ingredients % Corn, ground #2 62.8 Soybean meal (49% protein) 12.0 Fishmeal, Menhaden (60% protein) 2.5 Alfalfa meal (17% protein) 6.5 Corn gluten meal (60% protein) 6.0 Limestone, ground 7.5 Dicalcium phosphate (18% protein) 1.8 Salt, iodized 0.25 Choline chloride 0.05 Vitamin and mineral premix1 0.4 Chromic oxide 0.2 TITO—To— Calculated Analysis Crude protein (%) 17.5 Crude fat (%) 3.0 M.E. (kca1./g.) 3.0 Protein (%)/M.E. (kcal./g.) ratio 5.83 Calcium:phosphorus ratio 4.6 1Supplied the following per kg. of diet: Vitamin A--2,200 I.U.; Vitamin D --l,100 I.U.; Vitamin K--2.2 mg.; Riboflavin-- 3.0 mg.; Biotin--25 mg.; Nicotinic acid--4.0 mg.; Vitamin 312"5 mcg.; Ethoxyquin--125 mg.; Manganese--25 mg.; Zinc-- 8 mg.; Ground corn meal to 4 g. 40 treatments and experimental diets showing the substitution level of tallow and the supplemental level of cholic acid are presented in Table 7. For purposes of determining a M.E. value for the tallow, pullets on treatment No. 2 were fed a diet with 16.0 percent glucose substituted for corn of the basal diet. The tallow used in the experimental diets was from the same batch as used in experiments I and II. Chromic oxide at 0.2 percent was added as a marker to all diets. The birds were on experiment for a period of two weeks. Feed and water were supplied ad libitum. Weights of the individual birds were recorded at the beginning and the end of the experimental period. Feed intake was determined on a pen basis. Excreta were collected in aluminum "dropping" pans during the last four days of the experiment. The ex- creta samples were air dried. The feed and air-dried ex- creta samples were finely ground in a Wiley mill and blended. The samples were assayed for dry matter, crude fat, nitrogen, chromic oxide, and gross energy content. The assay techniques were the same as those used for samples from the previous experiments. 5. Experiment V The same batch of 48 SCWL females from experiment III were used in this experiment after a time lapse of 3 weeks. During this intervening period of 3 weeks the birds were fed a practical-type diet with no bile salt supplementation. 41 Table 7. Experimental diets showing substitution of ground corn of the reference diet with 4.0 or 8.0% TLW. Five levels of cholic acid were added to each set of TLW diets (Experiment IV). Supplementary TLW Cholic Acid Treatment Diet No. % % 1 71-08-01 Nil (Basal) Nil 2 71-08-02 Nil (Basal + 16% Nil glucose for corn) 3 71-08-03 4 0 4 71-08-04 8 0 5 71-08-05 4 0.025 6 71-08-06 4 0.05 7 71-08-07 4 0.1 8 71-08-08 4 0.2 9 71-08-09 8 0.025 10 71-08-10 8 0.05 11 71-08-11 8 0.1 12 71-08-12 8 0.2 42 At the commencement of the experiment the 48 birds were randomly divided into 12 groups of 4 birds each. The birds from each group were placed in consecutive single cages, in a three-tiered, laying battery. The groups were randomly assigned to the treatments. The room temperature was main- tained at 210 C. The experimental design was the same as in experiment III. Five levels of cholic acid, 0, 0.025, 0.05, 0.1 or 0.2 percent, were tested with two levels, 4.0 or 8.0 percent, of supplemental HSBO. The basal diet was the same as used in the previous experiment. The fat substitutions were made for corn of the basal diet, on a percent basis. The treat- ments and experimental diets showing substitution level of HSBO, and supplemental level of cholic acid are presented in Table 8. As in the previous experiment, for purposes of determining M.E. of HSBO, birds on treatment no. 2 were fed a diet containing 16.0 percent glucose substituted for corn. The HSBO used in the diets were from the same batch as used in experiments I and II. The birds were on experiment for a period of two weeks. Feed and water were supplied ad libitum. Weights of the in- dividual birds were determined at the beginning and at the end of the experimental period. Feed intake was determined on a pen basis. Excreta were collected on aluminum dropping pans during the last four days of the experiment. The feed and excreta samples were assayed for dry matter, crude fat, chromic oxide and gross energy as in experiment IV. At the 43 Table 8. Experimental diets showing substitution of ground corn of the reference diet with 4.0 or 8.0% HSBO. Five levels of cholic acid were added to each set of the HSBO diets (Experiment V). Supplementary HSBO Cholic Acid Treatment Diet No. % % 1 71-09-01 Nil (Basal) Nil 2 71-09-02 Nil (Basal + 16% Nil glucose for corn) 3 71-09-03 4 0 4 71-09-04 8 0 5 71-09-05 4 0.025 6 71-09-06 4 0.05 7 71-09-07 4 0.10 8 71-09-08 4 0.20 9 71-09-09 8 0.025 10 71-09-10 8 0.05 11 71-09-11 8 0.10 12 71-09—12 8 0.20 44 termination of the experiment two birds from each group were randomly selected for necropsy. The livers and gall bladders were excised and weighed separately. The bile was drained off from each of the gall bladders and the gall bladders weighed when empty. The weight of bile was determined from the difference in weight between the full and empty bladders. C. Analytical Procedures and Calculation of Data 1. Determination of dry matter content The feed and excreta samples were each air-dried, finely ground in a Wiley mill and blended. Two grams of sample were used for each determination. The samples were weighed into tared aluminum drying pans and dried in a convection oven at 1000 C. for 24 hours. Feed samples (containing high levels of glucose), from experiments I, II and III were dried in a vacuum oven at 600 C. and 38 cm. Hg for 24 to 36 hours. After drying in the oven, the samples were cooled in a desiccator and weighed. The dry matter content was calcu- lated and expressed as a percentage. 2. Determination of lipid content The assay technique used was a modification of the method described by Folch et 31. (1957). Finely ground feed and excreta samples were used in the assays. Two grams of sam- ple were weighed into tared 50 ml. centrifuge tubes. Fifteen ml. of the extraction mixture, chloroform:methanol, 2:1 (v/v) were added to the sample and mixed thoroughly with a metal spatula for five minutes. The contents of the tube 45 were centrifuged at 1400 rpm x g. for 5 minutes and the re— sulting supernatant fraction was drawn out carefully using a 15 ml. syringe with a 12 gauge blunt needle. The super- natant fraction was transferred into a second 50 ml. cen- trifuge tube. The extractions were repeated with 15 ml. and 10 ml. of the extraction mixture, and the supernatant frac- tions were pooled with the first collection. Eight ml. of distilled water were added to the chloroform:methanol extract and the mix was centrifuged at 1400 rpm x g. for 5 minutes. The upper distilled water layer was drawn out carefully and discarded. Repeated wash with 8 ml. distilled water, mixed thoroughly, centrifuged and separated off the aqueous layer. The lipid extract remaining in the centrifuge tube was transferred into tared aluminum drying pans and the contents were evaporated to dryness in a vacuum oven at 400 C. and 38 cm. Hg for 12 hours. In the case of the excreta samples the distilled water wash did not completely remove the non-lipid particles at the inter-phase. The lipid extract was therefore filtered through glass wool before final drying. The total crude lipids recovered were weighed and expressed as a percentage of the initial sample. '3. Determination of chromic oxide content The assay technique employed was based on the method described by Czarnocki EE.2£¢ (1961). The sample sizes used in the determinations were 3.0 g. and 1.0 g. for feed and excreta samples, respectively. The finely ground 46 and blended samples were weighed on tared Whatman No. 1 fil- ter paper, then wrapped and transferred into 100 ml. micro Kjeldahl flasks. Ten ml. of concentrated nitric acid were added to each sample and allowed to stand overnight. The flasks and contents were heated for a few hours on a Kjeldahl digesting rack. The final heating to dryness was done using a Bunsen burner flame. The flasks were cooled and 15 ml. of the digestion mixture were added to the contents of each of the flasks. The heating was continued on the Kjeldahl digesting racks until the green color changed to yellow or orange. The flasks were kept on the heating rack for 15 more minutes after the color change, then removed from the rack, and allowed to cool. The contents of the flasks were quantitatively transferred with rinsings of dis- tilled water into graduated 125 ml. Erlenmeyer flasks. The solutions were made up to 100 m1. and the flasks and contents were allowed to stand overnight to precipitate inorganic material. Ten m1. aliquots of the clear solution were pipetted out into 50 m1. volumetric flasks and the solutions were made up to 50 ml. with distilled water. The optical density of the diluted solutions was read at 350 mu. in a Perkin-Elmer spectrophotometer against a reagent blank. The chromic oxide content in the samples were calculated from regression equations derived from standard curves based on the optical density readings of three graded levels of A.R grade chromic oxide. 47 4. Determination of gross energy A "Parr" Adiabatic Oxygen Bomb Calorimeter, Model No. 1241 was used for the determination of gross energy of feed and excreta samples. This model was equipped to handle adjustments in jacket temperature automatically. The finely ground feed and excreta samples were, separately, thoroughly blended and formed into pellets 1.0 cm. in diameter and 0.5 to 1.0 cm. in height, in a pellet press. The weight of the pellets ranged from 1.0 to 1.5 g. for feed samples and 1.0 to 2.0 g. for the excreta samples. The samples were accurately weighed into the Bomb's tared, stainless steel capsules (2.5 cm. diameter and 1.0 cm. deep) and the fuse wires attached. These wires were 10 cm. lengths of Parr 45C10 nickel alloy wire with a heat of combustion of 2.3 cal. per cm. One ml. of distilled water was placed in the bomb before charging with 25 atmospheres oxygen supplied from an industrial oxygen cylinder. The charged bomb was placed in the calorimeter bucket containing 2 litres water. The ini- tial temperature was recorded after the calorimeter was allowed to run for 4 minutes to equilibrate bucket and jacket tem— peratures. The sample was fired using the built-in ignition system and 9 minutes were allowed for the temperatures of the bucket and jacket to equilibrate. At this time a second temperature reading was taken. The bomb was removed from the jacket, the oxygen discharged and the interior surfaces washed down with distilled water. The washings were titrated with a 0.0725 N sodium carbonate solution using methyl orange 48 as indicator. The burette reading in ml. was equivalent to heat of formation of nitric acid in calories (1 calorie per ml.) and served to determine the nitric acid correction. All unburned fuse wire was removed, straightened, and their combined length in cm. measured. From this value the correc- tion for the heat of combustion of the wire was determined by multiplying the length of burned wire in cm. by 2.3 cal. Corrections were made on the thermometer reading from the correction table supplied by the manufacturer. Before each set of samples were assayed the calorimeter was standardized using reagent grade benzoic acid (heat of combustion 6318 cal./g.). The energy equivalent of the calorimeter (W) in calories per degree C. was calculated as follows: o H. m + C1 + c2 W (cal./C ) = t H = 6318 ca1./g. (heat of combustion of benzoic acid) m = weight in g. of benzoic acid (usually 1.0 to 1.2 g.) t = net corrected temperature rise in CO cl = correction for heat of combustion of nitric acid in calories c2 = correction for heat of combustion of fuse wire in calories The gross energy (G.E.) in calories per g. of the feed and excreta samples were calculated as follows: W..t.—.(c m .+ c ) G.E. (cal./g.) = l 2 ’u i. TI. di I’n II 49 t = net corrected temperature rise in C0 W = energy equivalent of calorimeter in calories/ C0 C1 = correction for heat of combustion of nitric acid in calories c2 = correction for heat of combustion of fuse wire in calories m = weight in g. of sample (feed or excreta) 5. Calculation of fat absorption (%) Percent fat absorption (F.A.,%) was calculated as follows: Cr d F (F2 x If?) F.A., 5: = F e x 100 1 All data were expressed on a dry matter (d.m.) basis. Fl = mg. fat per g. diet (obtained from % fat in diets data) F2 = mg. fat per g. excreta (obtained from % fat in excreta data) Cr = mg. Cr203 per g. diet d Cre = mg. Cr203 per g. excreta Crd The factor (F2 x EE—) refers to mg. excreta fat per g. of e diet. NOTE: No corrections were made for endogenous loss as the fat levels under study were high. 50 6. Calculation of metabolizable energy of the diets The procedure by Hill and Anderson (1958) was used for calculating the M.E. values of each of the diets. The corrections for retained nitrogen were omitted from the M.E. calculations in accordance with the concept of Kleiber (1961). The calculations were made from analytical data expressed on a d.m. basis. 7. Calculation of metabolizable energy of the fats The formula by Anderson et 31. (1958) was modified to calculate the M.E. value of each fat because fat substitu- tions were made on an isocaloric and not on a percentage ba- sis. The M.E. values for the fats were calculated accord- ing to the following formula, assuming no change in the M.E. value of the non-lipid portion of the diet: (M.E. - M.E. ) + M.E. r 9 ME = -t 4 ' ‘fat Dec1ma1 proportion of test material in diet (a) (M.E.t - M'E'r) = the determined M.E. of the test diet (M.E.t) compared to the determined M.E. of the reference diet (M.E.r). NOTE: Because test material was substituted on an isocaloric basis, theoretically the net difference between test and reference diet should be zero. (b) M.E.g = the caloric M.E. value of glucose removed per unit weight of the diet. In this case 51 the value was either 1.15 kcal./g. diet (50 percent of glucose M.E. at 31.6 percent of diet = 0.316 x 3.64 kca1./g.), or 2.30 kcal./g. diet (100 percent glucose M.E. at 63.2 percent of diet = 0.632 x 3.64 kca1./g.). The contribution of M.E. by the test material to the test diet is the algebraic sum of (a) and (b). Then to ex- press the M.E. value per unit weight of test material, divide (a) + (b) by the decimal equivalent of the test material in the diet. D. Statistical Analysis All experimental data were subjected to analysis of variance. The data from experiments I and II were statis- tically analyzed using an analysis of variance program at the computer center at Michigan State University. The dif- ferences among the treatment means were tested using the Duncan's Multiple Range Test (1955). IV. RESULTS A. Experiment I 1. Body weight gain Weight gains were examined in relation to net feed in- take per bird during the three-week period of the experi- ment (Table 9). The isocaloric substitution of lipid M.E. calories for 50 percent of the glucose M.E. calories had no effect on body weight gain; feed intake was, however, lower. When fat completely replaced glucose M.E. calories, feed intake was depressed by nearly 18 percent, accompanied by a comparable decrease in weight gain. Cholic acid supplemen- tation in the 50 percent F.M.E. diets tended to promote weight gains with relatively little change in feed intake. At the 100 percent F.M.E. level, cholic acid did not overcome the adverse effect of the fat on weight gain and feed intake. The weight gains of chicks fed diets containing each of the four different types of fats are summarized in Table 10. The analysis of the data revealed that chicks given TLW, CNO and HSBO in place of glucose yielded the most favorable growth responses in relation to feed intake, over those obtained from LD. The addition of cholic acid at 0.2 percent to the diets containing TLW or HSBO consistently promoted weight gains at both levels of substitution. The 52 .7; v... .1 [like .9.»A,. n~na v.—3 3......“ Ix»,\’. 1"... 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Cm. .\ 7:: ,. ... I... '- - ‘11. III! - s 55 level and type of fat influenced weight gains as evidenced by the significant differences (P i .05) of the overall means of the different fat treatments. 2. Feed utilization Feed efficiency was improved by the equicaloric sub- stitution of 50 percent glucose with fat (Table 11). The improvement was marginally significant (P = .07). Cholic acid supplementation seemingly improved the feed:gain ratio for the fat substituted diets, but the effect was not sta— tistically significant; yet, its addition to the reference diet appeared to depress feed efficiency though again not significantly. In examining the feed:gain ratios of each treatment, those within the category of 50 percent F.M.E. treatments showed a trend (P = .07) of being more efficient (Table 12). Feed efficiency values among the treatments were not sig- nificantly different suggesting that there was no specific- ity in the type of fat to obtain an improvement in this statistic. As in the case of body weight gains, the shifts in feed:gain ratios were favorable but not significantly (P i .05) improved by supplementation with 0.2 percent cholic acid into diets containing high levels of fat. 3. Fat absorption The values obtained for fat absorption from all fat treatments were 90 percent or above (Table 13). Absorption of fat, however, varied significantly (P,: .05) according 56 Table 11. Feed:gain ratio of broiler-type chicks fed diets in which 50% or 100% glucose M.E. calories were substituted isocalorically by fat and the diets supplemented with 0.2% or without cholic acid (Experiment I). Feed (g.)/Gain 1g.) Glucose M.E. Cal. Substituted by Fat M.E. Cal. Level of Cholic Acid 0 50% 100% Mean 2 0% (2)ll.66a ( 8)1.ssa ( 8)1.63a (18)1.62 0.2% (2) 1.70a ( 8)1.51a ( 8)1.53a (l8)1.60 Mean (4) 1.68Y3 (16)l.55x (16)1.61xy 1 Number of groups. 2,3 Means not carrying the same superscript are significantly different (P1: .05) in accordance with Duncan's Multiple Range Test. Analysis of variance of feed:gain ratios Source of Variation d.f. Mean Square Cholic acid (A) 1 0.0220 Level of fat M.E. cal. (B) 2 0.0418? Control vs. fat treatments (1) 0.0423 A x B 2 0.0049 Error 30 0.0141 IP = .07. .7 I; :5 ELIH «.4 TIL finite; niuxnultslasfichi QC «OHUEH .euwflhuumusvmvhy :Ofluwmwuwflefluu: ICE}: .n... .i.£tfi. 57 CFO. 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I I I h .l K .. n (J .3 . 4,1... e-JJ.--..~ hnUnd.e2Hd\ It} i 5.0NHN w-crN. 58 .no..w es. seem.n on uouue mmm.~ m o x e x s msm.m m o x m oom.s H o x s eem.m m m x s ssuom.ms H Loy use so ns>sq ssmom.sn m use use eo sees seHmm.mm H A MO TUHSOm AmquEussnu usmv CoHumnosz use w mo soCans> mo mHmMHsCC .umse soCsm sHmHuHCz m.CsoCCo Cqu soCsonooos CH Amo..w e0 qunsHMHo mHquOHMHCmHm sns umHnomnseom sass sCu mCHmnnso uOC mCssz N.H b.mm o.vm h.mm Cssz e.mm m.¢m v.~m m.sm m.mm m.mm m.Hm mmCHs> H.vm m.mm m.mm Nm.nm em.em Qsm.mm sm.Hm ommm «.mm m.vm m.Hm www.mm xo.om Qsm.mm st.mm 020 «.mm e.mm m.vm no.0m Nev.om um.vm nsm.mm CH N.Nm m.mm m.om Nev.vm Nxm.om nsm.mm Hsv.om 3H9 use mo wOOH mom wOOH wom wOOH mom qusussne momma now COHuCuHumnsm .m.z CoHuCuHquCm .m.z use COHuCuHumnCm .m.z use mCssz use now mcssz oHos oHHOCO wm.o oHos UHHOCO so use souuenomns use .AH quEHnsexmv oHos OHHOCo 0C no wm.o Cqu mumHo CH manoHso .m.z smooCHm sCu mo wOOH no mom nom msusuHumnCm OHnOHsUOmH ones C0HC3 me>sH ensusHo us ommm no 020 .QH .3H9 owe meHCo mmhulanHonn >n va use no COHuenomns .mH ansB 59 to the type of fat. TLW substituted at the 50 percent M.E. level for glucose had the lowest absorption value. The fat absorption values for the reference diets not shown in Table 13 averaged 57.6 percent. Absorption of fats from the 100 percent F.M.E. diets was significantly higher (P _<_ .01) than that from the 50 percent F.M.E. diets. Although fat absorption was greater for the 100 percent F.M.E. diets, there was no corresponding significant increase in weight gain (Table 10). In fact, growth tended to lag in these chicks on the 100 percent F.M.E. diets. The addition of 0.2 percent cholic acid to the diets improved fat absorption significantly (P i .01) with the exception of the diets in which 50 percent of glucose M.E. was substituted by corn oil. The increases recorded for the absorption of fat, however, were not appreciable. The absorption of LD and HSBO was significantly higher (P,: .05) than that of TLW and CNO in the 50 percent F.M.E. diets containing cholic acid. A similar pattern was seen for the fats at the 100 percent level. 4. Metabolizable energy M.E. values of the diets were determined to evaluate the effects of cholic acid on the absorption of fats and the utilization of lipid energy. The substitution of glucose isocalorically with 50 percent and 100 percent fat increased the determined M.E. value of those diets significantly (P i .05) (Table 14), demonstrating that the presence of fat 60 Table 14. Effect of 0.2% cholic acid on M.E. (kca1./g.) of diets in which 50% or 100% of the M.E. calories of glucose were substituted isocalorically by fat in diets fed to broiler chicks (Experiment I). M.E. (kca1./g.) Glucose M.E. Cal: Substituted by Fat M.E. Cal. Level of Cholic Acid 0 50% 100% Mean 0 (293.62ab2 ( 8)3.82abC ( 8)3.96bcd (18)3.80 +0.2% (2)3.48a ( 8)4.03bcd ( 8)4.15bcd (18)3.89 Mean (4)3.55x3 (l6)3.93Y (16)4.05Y 1 Number of groups. 2’3Means not carrying the same superscript are significantly different (P i .05) in accordance with Duncan's Multiple Range Test. Analysis of variance of M.E. values Source of Variation d.f. Mean Square Cholic acid (A) 1 242,392* Level of fat M.E. cal. (B) 2 413,960** Control vs. fat treatments (1) 694,923** A x B 2 19,952 Error 30 43,048 **P i .01. *P < .05. 61 enhanced the retention and/or absorption of dietary energy. The addition of cholic acid significantly increased (P :_.05) the M.E. values of the fat diets concomitantly with the observed effect on feed:gain ratio (Table 12) and improved fat absorption (Table 13). On the other hand, cholic acid depressed the M.E. value of the reference diet, producing the same depressed trend as for the values on body weight gain, feed efficiency and fat absorption. In Figure l are shown the kcal./g. increase in M.E. values of the fat diets over the reference diet at the 50 and 100 percent F.M.E. levels. The kca1./g. increases in M.E. of the fat diets obtained with the addition of cholic acid are also shown in Figure 1. At the 50 percent F.M.E. level, TLW, HSBO and CNO showed improved "M.E. increments" as a result of cholic acid supplementations; whereas at the 100 percent F.M.E. level only CNO of the 4 fats tested failed to show an improved "M.E. increment" from cholic acid supplementation. The M.E. values of the fat diets differed according to the type of fat, as one would predict on the basis of their "standard" M.E. values. As indicated in Table 15 the overall differences in M.E. were marginally significant (P = .06 to .08); however, the addition of cholic acid significantly (P i .01) increased the M.E. values of the diets with fat added. .IIL " ‘5 cu. a. n f» h Inn-v. AI Venn...- Tur.~J H as.) A . es\n d: ~35“ch o...~oz Nhnv H... .n. nu sew NV m, Hfiv-en I‘ ‘7‘ .‘ IN! I.u\ I...‘ "I 62 .mo. ou so. u e+ snsavm Csmz .no..w ese smo.o on nouns neo.o m o x m x s eno.o m o x m uoo.o n o x s mso.o m m x s +smn.o n Aug use eo ns>sq +omo.o m “my use eo sees «.tvmm .0 H 33 WHOM OHHOfiU .e.o COHuans> mo monoom mumHo use e0 A.m\.Hsox0 msCHs> .m.z mo moCans> mo memHsCC m.CsoCCo CuHB soCsonooos CH Amo..w e0 qunmeeHo MHquoHeHCmHm mns ueHnomnsmCm sass sCu mCHennso uOC mCsmz .umse smCsm sHmHuHCE N.H mm.m mo.v mm.m Cssz mm.m mo.¢ mm.m mH.v mo.v mm.m mm.m mH.v N~.¢ so.¢ mmm.v axom.v QNH.v nsbm.m ommm mm.m mm.m mm.m xvm.m axvo.v nsvm.m swm.m ozu om.m no.v mm.m meH.¢ xmm.m nsmo.¢ asbm.m CH oo.v vo.¢ mm.m mmm.¢ mmxmo.v nsvb.m Hnsmm.m 3H9 use eo mOOH wom wOOH wom mOOH wom quEussnB momma nOe COHuCuHumndm .m.z COHusuHumnsm .m.z use COHuCuHumnCm .m.z use mCssz use now mCssz oHoC OHHOCO mm.o oHos OHHOCU wo Cubiswua .eé .AH uCoEHnsexmv mCOC no oHos UHHoCo wm.o osCHsuCoo mumHa .manOHso .m.z smooCHm mo «00H no mom noe hHHsOHnOHsOOmH osuCuHumnCm ommm no 020 .04 .349 CuH3 musHo mo moCHs> A.m\.Hsoxv .m.z Co oHos UHHOCo «0 uoseem .mH oHusB 63 5. M.E. value of fats The M.E. values for the fats (Table 16) were calculated assuming no change in the M.E. value of the non-lipid por- tion of the diet. There was no significant difference (P i .05) between the M.E. values obtained on an overall basis at either of the two isocaloric substituted levels, so the values for each fat were pooled and means obtained to show the in- fluence from the presence or absence of cholic acid in the test diets (Table 16). The M.E. values that were calculated exceeded the values in nutrient tables of Scott, Nesheim and Young (1969). The lowest values were from the test diets without cholic acid supplementation; yet, except for the M.E. value of TLW, were still in excess of the theoret- ical maximum energy value of 9.3 kca1./g. for a pure fat. The percentage increase in the M.E. value of a fat, in the absence of cholic acid, in comparison to a standard value was greatest for HSBO followed by LD and TLW, and least for CNO. The fat M.E. values were found to be increased further by cholic acid, with HSBO and TLW values increased more than those of CNO and LD. 6. Gall bladders At the termination of the experiment and on necropsy, all birds on the cholic acid supplemented diets were observed to have gall bladders nearly two to three times larger than normal. The bile content in the gall bladder was a pale 64 .AoHos UHHOCo uCOCqu umHo soCmnsemn e0 .m.20 .m\.Hsox Hw.m u n.m.z mCHmC omusHCOHsO H Am.ovHv om.OH Am.NNHO mm.0H A>.mHHV om.0H Am.¢mH0 m¢.m .m>4 hH.OH bh.m mv.0H om.m .m.z.e wOOH oHos oHHOCo wm.o mh.HH NN.NH mH.0H m0.m .m.z.m wom Am.HmHv hv.m Am.MHHV om.m Ah.mHHv om.0H AH.vHHv oo.m .m>¢ mv.m hH.0H HH.OH ov.h .m.z.e wOOH oHos UHHOCo we mv.m mv.m mv.OH om.w .m.z.e wom HmsCHs> osCHEnsumD AOOHV H 00.5 AOOHV u mm.m AOOHV n 00.0 AOOHV u H0.h mmDHs> Unsvcsum .Uum mo msCHs> .oum mo mmCHs> .oum mo mmCHs> .pum mo mmCHs> w .m.2 w .m.2 w .m.S w .m.z Ommm OZU DH SHE muse mo mmCHs> A.m\.Hsox0 .m.z .AH quEHnsewmv msmsCqunse CH Cs>Hm mns msCHs> onsoCsum onu Bone msmssnoCH omsquonwm smsns>s one .oHos UHHono wm.o uCOCqu no CuH3 .musHo .m.z.e wOOH no mom mCu Bone omusHooHso muse mo oCHs> A.m\.Hson .m.z .mH mHnsB 65 Figure 1. Trends in M.E. (kca1./g.) increments (M.E.T M.E.R) of the 50% and 100% F.M.E. diets with or without cholic acid when fed to broiler-type chicks, four weeks of age. (Experiment I) 0.8 66 FME _—- 0% Cholic acid — 0. 2% Cholic acid 0. 8 0.6 0.4 kcal. lg. 0.2 kcal. lg. 0.4 0.2 0 Figure l FME . 67 yellow compared to the green-blue color of normal chick bile. The livers of the birds appeared normal. The birds on the 100 percent F.M.E. diets appeared to have a greater deposition of abdominal and subcutaneous fat than those on the 50 percent F.M.E. diets. The birds on the reference diets had barely noticeable abdominal and subcutaneous fat. B. Experiment II 1. Body weight gain The isocaloric substitution of lipid M.E. calories for 50 percent of the glucose M.E. calories significantly (P,: .01) improved body weight gain of chicks at one week of age (Table 17). However, with the complete replacement of glucose M.E. calories with fat M.E. calories there was no change in body weight gain. Accurate feed intake data were difficult to obtain on these chicks during the first week of age, due to excessive spillage and contamination of feed, thus the feed intake values were only approximate (see Appendix Table 6). On an overall basis the addition of cholic acid at 0.2 percent to the chick diets depressed body weight gain significantly (P i .05). There were sig- nificant (P i.'01) differences among the weight gains of chicks on the separate fat diets (Table 18). The chicks were unable to tolerate LD particularly at the 100 percent level of substitution; they consumed very little diet and consequently showed no body weight gain. In the statistical analysis of the weight gains a separate set of mean square values were computed excluding the weight gains obtained Table 17. Body weight gains (9.) of broiler-type chicks, at one week of age when fed diets in which 50% or 100% glucose M.E. calories were substituted by fat M.E. calories, and the diets fed with or without 0.2% cholic acid (Experiment II). Weight Gain--(g.) Glucose M.E. Cal. Substituted by Fat M.E. Cal. Level of Cholic Acid 0% 50% 100% Mean 0% (2)1 slab; ( 8) 71b ( 8) 42a (18) 55 (100%) (139%) (82%) +0.2% (2) 37a ( 8) 57ab ( 8) 35a (18) 43 (72%) (112%) (69%) Mean (4) 44x (16) 64y (l6) 39X (36) 49 1 Number of groups. 2Control group = 100%. 3 . . . . . Means not carrying the same superscript are Significantly different (P i .05) in accordance with Duncan's Multiple Range Test. Analysis of variance of b.w. gains for all treatments Source of Variation d.f. MeanSquare Cholic acid (A) 1 1,902* Level of fat M.E. cal. (B) 2 3,129** A x B 2 437 Error 30 296 **P i 0.01. *p :_.05. 69 .mo. W me .Ho. v m«« mmH NH m.mHH oH nonnm mm N m.mm m U x m x < me N H.NNm m U x m mmH H m.o¢H H U x m hH N m.wm m m x « «*HmNN H «em.hNNm H ADV use m0 He>eH emmm N «sm.mmoN m “my use e0 mews nmmOH H so.OHm H “CV oHos UHHOCU ensoom Csez .e ensoom Csez .e .o .unB CHTWCHosHoxm .unB 0H mCHosHo .o CoHuans> eo eonsom CH muCeEusenu use now mCHsm .u3 moon eo eOCans> mo mHmmHsCC .muceEusenu CH mCHoCHoxm H oo mm oo om om mo me oo so we Hmosos Ho mm on ow oo om we ones we om on so so mm so ozo am o as o ow m om on em so no me om we we 3H9 use eo sooH som wooH mom sooH mom ucssusone mseee noe ooHuouHunnom .e.s oouusuuumnom .e.s.e ooHuouHunoom .e.s.e mcmwz “mm HO.“ manme sees oHHono sm.o secs oHHono so Axel oeso usonoz AHH unseHnoexeo sHos oHHoso s~.o usosuus no nuns owe sos smoooHo eone noHnoHso .e.s oau eo sooH no mom noe osuouHumnon omme no ozo .oH .ZHB pee Cens ems mo xee3 eCo us meHCo eeeulneHHonn e0 A.mv CHsm quHeB moom .mH eHnsB 70 from the LD treatments. Significant (P :_.01) decreases in weight gain were recorded for the fats, when they were sub- stituted in diets at the 100 percent level. The addition of cholic acid to the diets depressed body weight gain in all treatments irrespective of level and type of fat. 2. Fat absorption The data regarding the percent fat absorbed (Table 19) from the different fat diets were subjected to arcsin trans- formation before statistical analysis. Overall apparent fat absorption was lower by 11.8 percent from those recorded in the previous experiment with chicks, four weeks of age. As in the case of the data on body weight gain computations of the analysis of variance presented in Table 19 are with or without the values from the LD treatments. Both the level of fat and the composition of fat in the diet influenced significantly (P i.'05) the percent fat absorbed. The sat- urated fats were absorbed less efficiently. The addition of cholic acid at 0.2 percent to the diets significantly (P i .05) improved fat absorption. (This was seen only when the LD treatment values were excluded from the statistical compu- tations.) As in experiment I the fat absorption values were significantly (P':_.05) higher from the 100 percent F.M.E. diets. 3. Metabolizable energy When the M.E. values of diets were studied excluding the M.E. values obtained from the LD treatments, significant um, RH.- 90 r .v u— rebireg \ can a - L4 #1.- L C v. .W .53 FUCC \ Tuvanvflfienv mea>nw IIHHvH. «HAULHAH >3 U new» been a 3\ JIQFN our I‘rihil.(r‘1< .H.o m e+ .mo.o W me .Ho.o v ens 0H NH HH 0H nonne Hm N h m U x m x m Nm N «mm m o x m H H m H U x < mH N 5H m m x s new H N H A00 use e0 He>eH «eemm N «same m Amv use MO emwfi (Hem H mm H Adv oHos OHHOCU ensoom csez .e.o ensoom Csez .H.o COHuans> mo eonoom .una OH mCHoDHoxm .unB OH mCHoCHoCH ACoHusEnoemCsnu CHmons ou oeuoennom ene3 suso easy AmuCeEusenu usev COHuenOmns use m e0 eoCans> Ho mHmeHsC< .muCeEusenu OH mCHoCHoxem I .umee emCse eHeHuHoz s.CsoCoo CuHB eOCsonooos CH Ame. v e0 uCeneeeHo eHquoHeHCmHm ens ueHnomneeCm eEsm eCu mCHennso uOC mCsez 71 N.H m.mm m.em o.¢m m.mm m.mm m.vm m.mm v.mm H.mm m.Nm mCsez v.mm m.mm m.mm um.Hm Nem.mm eoom.mm ovum.me ommm N.Nm m.mm m.om N¢.mm Neo.Hm ee.mm eom.om ozu h.mm m.mm m.mm 3o.mm x3e.Nh nsm.mn sm.mm CH m.me o.me m.me Nexv.mm Nexw.me Qsm.ve Hoonm.m> 3H9 use e0 wOOH mom wooH mom NQOH- wom memes nOe COHuouHquom .e.z COHuouHumnom .e.z.e CoHuouHumuom .e.2.e uCeEusenB mCsez use noe mCsez oHos OHHOCU wN.o oHos UHHOCU we Ase soeuenomns use .AHH uCeEHneexev oHos oHHOCo usonqu no CuHB oeuCeEeHeeom mueHo CH emooon Eone meHnoHso .m.z eCu eo wOOH no son now oeuCuHumnom 0mm: no 020 .CH .BHB pee Cenz .ems e0 xee3 eCo .monCo eeeulneHHonu en use eo «we CoHuenOmu< .mH eHnsB 72 (P 1 .05 and“: .01) improvements were obtained respectively with (a) the substitution of 50 or 100 percent M.E. calories for glucose M.E. calories and (b) the addition of cholic acid to the diet (Table 20). Mean M.E. values of the separate fat diets (Table 21) differed significantly (P i .01) and were in correlation to the data on fat absorption for the different fat diets (Table 19). M.E. of the fat diets and fat absorption percent were greater with diets containing CNO and HSBO. The addition of cholic acid significantly (P 1 .01) improved the M.E. values of the fat diets conforming to the improved effects observed in the fat absorption data. Cholic acid's beneficial effects were more consistent with the 50 percent F.M.E. diets (Table 21). 4. Metabolizable energy of fats The M.E. values for the different fats tested are shown in Table 22. In the absence of cholic acid, the M.E. value for TLW was 9 percent lower and the M.E. values for HSBO and CNO, 18 and 12 percent higher than the standard values reported in nutrient tables (Scott ep,§l., 1969). The M.E. values for the fats from the diets with the added cholic acid were found to be improved in all cases except for LD. The more saturated fats, TLW and HSBO showing the greater increases. C. 'Experiment III ‘ 1. Body weight gain (0 to 7 days of age) Body weight gain of broiler chicks fed diets with 0.025 and 0.05 percent bile salts showed no significant (P :_.05) 73 Table 20. Effect of 0.2% cholic acid on M.E. (kcal./g.) values of diets, with fat substituted for 50% or 100% of the M.E. calories from glucose. The diets were fed to broiler-type chicks from one to seven days of age (Experiment II). M.E. (kcal./g.) Glucose M.E. Cal. Substituted by Fat M.E. Cal. Level of Cholic Acid 0% 50% 100% Mean 0% (2)1 3.34 ( 8) 3.41 ( 8) 3.51 (18) 3.42 +0.2% (2) 3.50 ( 8) 3.87 ( 8) 3.76 (18) 3.71 Means2 (4) 3.42 (16) 3.64 (16) 3.64 (36) 3.57 1 Number of groups. 2Excluding LD treatments. Analysis of variance of M.E. values (kcal./g.) Including LD Trt. Excluding L2 Trt. Source of Variation de. Mean Square d.f. Mean Square Cholic acid (A) 1 0.401 1 0.77** Level of fat M.E. cal. (B) 2 0.075 2 0.025 Control vs fat treatments (1) 0.019 2 0.16* A x B 2 0.133 2 0.14 Error 30 0.142 22 0.035 **P i.'01' *P i .05. 74 .mo. M e« .Ho. v mes mo-o ~H omo.o oH nonne so.o m soo.o m o x m x s eo.o H mmo.o m o x m No.o H «aeH.o H o n s oo.o N smmH.o m m x s Ho.o H meH.o H Loo use eo HsasH emm.o m eseeo.o m Leo use no menu eme.o H «seem.o H Lee oHos oHHoso ensoom Csez .mwm .une oH onusoHooH ensoom Csez we.o .une CH meHosHoxe COHuans> mo eonoom mueHo use mo A.m\.Hsoxv meCHs> .e.z mo eoCans> mo mHmeHsCN .muCeEusenu OH mCHoCHoxe H om.m Nw.m Hm.m wo.m wo.m we.m em.m Hm.m H¢.m HmCsez ve.m me.m vo.m mm.m mm.m em.m om.m ommm me.m me.m mo.m oe.m mo.v om.m Nm.m ozo oo.m om.N eN.m em.N mN.m NH.m NH.m CH Nv.m Nv.m He.m mm.m oo.m mH.m NN.m 3H5 use Ho wOOH wom wOOH mom mooH mom uCeEusene mmeee noe ooHuouHumnom .e.s noHusuHumnom .e.2.e COHuouHumnom .e.z.e mCsez use now mCsez oHod UHHOCU wN.o oHo< UHHOCwIwo A.m\.Hsoxo .e.z .AHH uCeEHneexev ems eo mess Ce>em ou eCo Eone monCo eeeulneHHonn ou see ene3 mueHo eCB .emooCHm Eone meHnOHso .e.z eCu eo moOH no mom n0e oeuCuHumnCm ommm no 020 .CH .ZHB Cqu mueHo eo mesHs> A.m\.Hsoxv .e.z Co oHos oHHono wN.o mo uoeeee .HN eHnsB 75 .AoHos UHHOCU uCOCuHB ueHo eoCeneeen e0 .e.zv .m\.Hsox Hw.m n n.e.z mCHmC oeusHCOHsU H AH.HoHo em.mH ee.emHo mm.NH Am.mo V eH.e Ho.oHHo mm.m sHos oHHoao sm.o+ Im.HHHo oa.m Ie.eHHo mm.oH AH.oo o om.e Ho.Ho o om.o sHos oHHoao so Hm $54.. Ger CGCHEQU OQ IooHo u oo.e HooHo u mo.o LooHo u oo.o LooHo u Ho.e nooHs> snsocsum .oum eo mooHs> .sum eo sooHs> .oum eo sooHs> .sum eo mosHs> s .m.z s .e.z s .e.z s .e.z omme ozo on 3H9 muse e0 meoHs> H.m\.Hsox0 .e.z .HHH uCeEHneexev memeCuCense CH Ce>Hm ens mesHs> onsoCsum Eonm messenOCH emsuCeonee e59 .mueHo .e.z.e wOOH no mom eCu Bone mCseE eCu ens meCHs> ens .ems e0 mess Ce>em ou eCo Eone monCo eeeulneHHonn ou see oCs oHos oHHono uCOCqu no CuH3 mueHo CH oeCHEneueo muse mo meoHs> H.m\.Hsoxv .e.z .NN eHnsB 76 increase in weight gain over chicks receiving no supplemental bile salt (Table 23). There were also no significant dif- ferences in the weight gain of chicks on the diets with the different bile salts, at the two levels fed. 2. Feed utilization (0 to 7 days of age) There was no evidence of any significant differences in the feed efficiencies among the diets containing the different bile salts (Table 24). The level of the bile salt also had no bearing on feed efficiency. 3. Fat absorption (4 to 7 days of age) The overall absorption values for crude TLW were low ranging from 39.6 to 51.2 percent. Significant (P i .01) increases of 13.6 and 25.3 percent in the absorption of crude TLW were calculated when the diets contained either 0.025 or 0.05 percent,respectively,of bile salts (Table 25). Fat absorption was significantly (P i .05) influenced by the type of supplemental bile salt. The chicks on diets with 0.025 or 0.05 percent chenodeoxycho‘ic acid provided the most favorable values (average 51.1 percent) for fat absorption. The two other bile salts, cholic acid (unconjugated) and sodium taurocholic acid (conjugated) appeared to influence fat absorption comparably. 4. Metabolizable energy (4 to 7 days of age) Although the different supplemental bile salts had significantly varying influences on fat absorption, the omo. V m“ Nm.mH HH nonne Nh.mH N H x m mH.o H HHV muHsm eHHn eo He>eH oo.o N Ame muHsm eHHm mm.o H uCeEusenu m> HonuCoo «oH.NHH m msusoHHeme mN.m Amy muCeEusenB ensomm Csez .e.o COHuans> eo eonoom mCHsm quHe3 moon eo eoCans> mo mHmeHsCN .meusoHHeen mo neuECz W H me ono ee_.mo me Loo we use Csez es as 0 me Ame me Ame me Any smo.o mo Am 0 Ne Amy, we Amv we Amy meo.o II I) II we Hamv mo Csez AuHsmIIszv _ oHod UHUN UHHOCU HonuCou uHsm eHHm oHom oHHOCoonosB OHHOCoexerOCeCU eo He>eH muHsm eHHm A.oo aHso usouoz .HHHH uCeEHneexev ems eo mess e ou o ene3 monCo CeCS OOHnee eCu noe ens suso e59 .AuHsm EoHoomv oHos OHHosoonosu no oHos UHHOCoexerOCeCo .oHos UHHOCo mo wmo.o no mNo.o CuHs 3H9 epono wN.m mCHCHsuCoo mueHo see meHCo eeeulneHHonn mo mCHsm quHez eoom .mN eHQsB 78 moo.o NH nonnm mHo.o N H x m omo.o H AHV muHsm eHHn eo He>eH moo.o N Amy muHsm eHHm omo.o H uCeEusenu m> HonuCou oHo.o N meusoHHeee HHo.o on muCeEusenB ensomm Csez .e.o COHuans> e0 eonsom mOHusn CHsmnoeee e0 eoCans> Ho mHmeHsCN m¢.H HNHV m¢.H Amy m¢.H Amy mo.H Amy Csez Hv.H Am 0 mm.H Hmv ow.H Amy h¢.H Amy wmo.o mv.H Am 0 Nm.H Ame o¢.H Amy Hm.H Ame mmNo.o N¢.H Ame so Csez AuHsm szv oHos CHUH UHHOCO HonuCoo uHsm eHHm oHos UHHozoonssB UHHOCoexoeCOCeCO mo He>eH muHsm eHHm 1.ao anso\1.ee ssse .AHHH uses IHneexev AuHsm EdHoOmv oHos UHHonoonCsu no oHos UHHOCoexoeCOCeCo .oHos OHHOCo eo wmo.o no mNo.o CuH3 oCs 3H9 eoono wN.m mCHCHsuCoo mueHo see CeCB .Cu3onm eo xeeB umnHe eCu mCHnCo mHOHCo eeeuineHHonu en AOHusn CHsmuoeeev COHusNHHHus oeee .wN eHQsB 79 .mo. w e4 .Ho. w.ese o.mH NH nonnm m.0N N H x m o.em H HHV uHsm eHHn e0 He>eH «o.mm N Amv muHsm eHHm eno.mmH H uCeEusenu m> HonuCoo m.mN N meusOHHeee neo.Nm Hwy muCeEusenB ensomm Csez .e.o CoHuans> eo eonoom COHuenOmns use no eoCans> e0 mHmeHsCs I .umee emCse eHeHuHoz m.CsoCoQ Cqu eoCsonooos CH Ame. v e0 uCeneeeHo eHquOHeHCmHm ens ueHnomneeom eEsm ecu mCHennso uOC mCsezN .mesonm mo nenfisz H o.h¢ AmHV H.mv A00 H.Hm A00 N.mv A00 Gee: 0.m¢ Am 0 Ov.mv amv 00.Hm Amy Om.mv Amy wmo.o o.mv Am 0 Qsm.Hv Amy ON.Hm Amv UQm.mv Amy meo.o Nsm mmHHmV we ass: qusm szv suos oHos oHHoso Honunoo uHsm oHHm oHos UHHonoonosz OHHOCoexoeCOCeCU eo He>eH muHsm oHem Ase ooHuenomns use .HHHH uCeEHneexev AuHsm ECHUOmV oHos oHHOCoonCsu no oHos UHHOCoexoeCOCeCo .oHos OHHoso eo wmo.o no mNo.o CuH3 oCs 3H9 eoono mN.m mCHCHsuCoo mueHo pee ems eo xeez eCo monCo eeeulneHHonn en COHuenOmns use .mN eHnsB 80 metabolizable energy values of the diets failed to reflect any response to bile salt addition to the diets (Table 26). The determined M.E. values for the control and the treat- ment diets averaged 3.25 kcal./g. 5. Body weight gain (0 to 19 days of age) Chicks at nineteen days of age showed no improvement in body weight gain when fed diets with the supplemental bile salts (Table 27). 6. Feed utilization (0 to 19 days of age) The feed utilization figures (Table 28) indicated that there were no changes in feed utilization with supplemental bile salts. 7. Fat absorption (14 to 19 days of age) At the three-week period, there was an increase in the absorption of crude tallow from an overall average of 45.4 percent to 70.9 percent (Table 29). The chicks on the diets with the supplemental bile salts were able to absorb fats more efficiently (significantly higher (P i .05) than those on the control diet without the bile salts. The average increase in the absorption of tallow with the addition of bile salts was only 8.0 percent as compared to the increase of 18.7 percent when the chicks were a week old. Three bile salts tested did not show significant differences on their individual influence on fat absorption, nor did the levels of bile salts have any significant bearing on fat absorption. 81 .Ho. w e44 Nmoo.o NH nonne «#mmmo.0 N H N m smoo.o H HHV muHsm eHHo eo He>eH omoo.o N Amy muHsm eHHm NHHo.o H uCeEusenu m> HonuCou sememo.o N meusoHHeem mMHo.o Amy muCeEusenB ensomm Csez .e.o COHuans> eo eonoom meCHs> .e.2 eo eOCans> mo mHmeHsCs .mesonm mo neuECz H mN.m AmHv vN.m Lev Hm.m Amv MN.m Amy Csez MN.m Am 0 vN.m Ame. NN.m Amv MN.m Amy wmo.o mN.m Am 0 vN.m Amv ov.m Amy NN.m Amv mmNo.o vN m Heme wo Csez AuHsm szv oHo< oHo¢ oHHOCU HonuCou uHsm eHHm mHos UHHOCoonCsB OHHOCoexoeCOCeCO e0 He>eH muHsm eHHm A.o\.Hsoxo nsoHs> .e.z .HHHH uCeEHneexev Cusonm eo gees umnHe nHeCu mCHnso mHOHCo eeeulneHHonn ou pee CeCB .AuHsm ECHUOmV oHos OHHOCoonosu no oHos UHHOCoexoeCOCeCo .oHos UHHOCU eo wmo.o no mmo.o suns oss 3H9 moons sm.o osHoHsusoo muons eo A.o\.Hsono nsoHs> .e.z .sm oHnse 82 m.emm NH nonne H.00 N H N m m.emm H HHV uHsm eHHn eo He>sH e.mos N Lee suHsn oHHm m.Ho H uCeEusenu m> HonuCoo o.Hom N meusoHHeee m.mMN Amy muCeEusenB ensomm Csez .e.o COHuans> e0 eonoom mCHsm quHeB moon eo eoCans> mo mHmeHst .meoonm Ho neneoz H mNm AmHV 0mm Amy mNm H00 mHm Amy Csez 0Nm Am 0 mNm “NV. CNm.HmV, HHm Amy wmo.o mNm Am 0 Nmm Amy mmm Amy ONm Amv meo.o HNm HAmv we Gsez AuHsm szve UHO¢ UHON UHHOSU HonuCOU uHsm eHHm oHo« omHonoonCsB UHHOCoexoeoOCeCU mo He>eH muHsm eHHm A.m0 mCHsU unmwez moom .HHHH uCeSHneexmv AuHsm ECHCOmv oHos UHHOCoonCsu no oHos OHHOCU lexoeoOCeCo .oHos oHHOCo eo wmo.o no mNo.o CuH3 oCs 3H9 epono wN.m mCHCHsuCoo mueHo see CeCB .meso mH umnHe eCu CH monno eeeulneHHonn Ho mCHsm quHe3 moom .NN eHQsE 83 H nonne. «00.0 N 0H0.0 N H x m 0H0.0 H AHV uHsm eHHQ m0 He>eH mHo.o N Ame muHsm eHHm oHo.o H muCeeusenu m> HonuCoo moo.o N meusoHHeee ooo.o Hwy muCeEusenB enssmm Csez .e.s CoHuans> eo eonCom mOHusn CHsmuoeee eo eOCans> eo mHmeHst .meoonm eo neafisz H mm.H AwHV mN.H A00 mm.H A00 hm.H H00 Csez ¢M.H Am 0 Nm.H Hmv mm.H Amy 0m:H Amy wm0.0 Nm.H Am 0 0N.H Amy Hm.H Amv mm.H Amy me0.0 0N HIHAmv we esoz qusm.szo sues onus oHHono Honunoo uHsm oHHm oHom UHHOCoonosmu UHHOCoexerOCeCo e0 He>eH muHsm sHHm L.oo oHso\L~mo ssoe .AHHH uCeEHneexev AuHsm ECHCOmV oHos OHHOCoonosu no oHos UHHonoexerOCeCo .oHos oHHOCo mmo.o no mNo.o CuH3 oCs 3H9 eosno wN.m mCHCHsuCoo mueHo pee CeC3 meso mH umnHe eCu mCHnCo monCo eeeulneHHonn en AmOHusn CHsmuoeeev COHusNHHHuC oeee .NN eHnsB .mo. v mt «.mH NH nonne m.m N H x m o.om H AHV uHsm eHHn eo He>eH o.mv N Hmv uHsm eHHm so.mm H uCeEusenu .m> Honucoo «o.oe N eusoHHeee e.oe Loo uoosusone ensomm Csez .e.o COHuans> eo eonsom Hwy COHuenomns use Ho eoCans> Ho mHmeHsC< .mesonm eo nenaoz M H m.me AmHv e.HN Amv e.oh Amy m.me Amv Csez m.mh Am 0 m.me Ame m.He Amy m.me Hmv wmo.o N.Nh Am 0 m.He Amv N.oo Amv m.vh Hmv meo.o N.mw any we ass: HuHsm szv sues sues oHHoeo Honueoo uHsm oHHm oHos DHHOCoonCsB OHHOCoexoeoOCesu eo He>eH muHsm eHHm Ase ooHuenosns use .AHHH uCeEHneexev AuHsm ECHoOmV oHos UHHOCoonCsB no oHos UHHOCoexoeCOCeCo .oHos oHHOCo mo mmo.o no meo.o CuH3 oCs 3H9 eosno wN.m mCHCHsuCoo mueHo oem ems mo seso mH us eonCo eeeulneHHonn en CoHuenOmns use .mN eHnsB 85 8. Metabolizable energy (14 to 19 davs of age) The improvements in fat absorption with the addition of bile salts were not reflected in the M.E. values. There was no significant difference between the M.E. values of the control diet and the diets with the bile salts (Table 30). 9. Bile content in gall bladder (14 days of age) The bile content (9.) of the gall bladders of chicks at the termination of the experiment at Day 18 (Table 31) showed that the bile content was higher (of borderline sig- nificance with P = .06) in chicks on diets with cholic acid and sodium taurocholic acid than those on the diets with chenodeoxycholic acid. The bile content of chicks in the latter treatment were comparable to those from the chicks on the control (no bile salt) diet. There was a tendency for increased bile content in the gall bladders of chicks on diets with the higher(0.05 percent)leve1 of dietary bile salt, but this difference was not statistically significant. D. Experiment IV 1. Fat absorption Adult SCWL females 20 weeks of age were fed diets with 8 percent supplemental TLW and were found to be able to absorb a significantly (P i .01) higher percentage (3.5%) of fat than those on the diets with only 4 percent supple- mental TLW (Table 32). Cholic acid at four different levels (0.25, 0.05, 0.1 and 0.2 percent) had no effect on the oeoo.o NH nonnm OHoo.o N H x m mNoo.o H AHV muHsm eHHQ eo He>eH moHo.o N Amv muHsm eHHm mHoo.o H uCeEusenu m> HonuCou mmoo.o N meusOHHeee mvoo.o Rev muCeEusenB ensomm Csez .e.o CoHuans> mo eonoom meCHs> .e.2 Ho eoCans> mo mHmeHsCC .mesonm eo neHECz 6 H 8 N Ho.m AmHo mo.m Loo oe.m Loo me.m Ase ass: so.m la 0 me.m Ame mo.m Ame me.m Ame emo.o mm.m 1a 0 mo.m Ame Hm.m Ame oe.m Ame smmo.o mo m Himo so asoz AuHsm szo sues suns oHHoso Honuooo uHsm sHHm WHUN UHHOCoonCsB OHHOCoexoeoOCeCO mo He>eH mwesm oHHmw H.o\.Hsoxo nooHs> .e.z .AHHH uCeEHneexev ems mo meso mH us mHOHCo eeeulneHHonn ou see CeCS HuHsm ECHCOmV oHos UHHOCoonssu no oHos UHHOCoexoeoOCeCo .oHos OHHOCU mo wmo.o oCs meo.o Cqu oCs 3H9 epono wN.m mCHCHsuCoo mueHo Ho mesHs> .e.z .om eHuse .00. u e+ mH0.0 NH nonnm m00.0 N H x m 0N0.0 H AHV uHsm eHHQ m0 He>eH +mm0.0 N Amy muHsm eHHm oHo.o H uCeEusenu .m> HonuCoo m¢0.0 N meusoHHmee mNo.o Amv muCeEusenB enssmm Csez .m.o COHuans> HO eonuom A.m0 uCeuCoo eHHQ mo eoCans> mo mHmeHsCm .meoonm eo mnenesz H m m0.o AmHv . . me-0 A00 mm.o H00 Hh.o A00 Csez mm.o Am 0 me.o Hmv mm.o Amv H>.o Amv wmo.o mo.o Am 0 no.0 Amv Hm.o Amv He.o Amy mmNo.o H0 0 Hfimv so Csez AuHsm szv UHUN oHod UHHOCU HonuCou uHsm eHHm oHom UHHOCoonssB UHHOCoexoeGOCeCU mo He>eH muHsm eHHm mneoosHm Hst e0 A.m0 uCeuCoo eHHm .AHHH uCeEHneexmv HuHsm ECHCOmv oHos OHHOCoonCsu no oHos UHHosoexoeCOCeCo .oHos oHHOCo wm0.o no mmNo.o Cqu oCs 3H9 eosno wN.m mCHCHsuCoo mueHo oee CeC3 .ems Ho meso mH us monCo eeeulneHHonn mo mneooan HHsm eCu m0 “.m0 uCeuCoo eHHm .Hm eHnsB 88 Table 32. Absorption of fat by SCWL females, 20 weeks of age, fed diets with 4 or 8% TLW and graded levels of cholic acid (Experiment IV). Fat'Absorption Level Cholic Acid 4% TLW 8% TLW Mean 0% 86.15 88.83 87.49 . +0.025% 85.72 89.40 87.56 +0.05% 86.09 89.14 87.62 >87.43 +0.10% 84.75 88.86 86.80 +0.20% 85.73 89.79 87.76 j Mean 85.68 89.21 37.44 Analysis of variance of fat absorption (%) Source of variation d.f. Mean Square Level of TLW 1 22.0** Level of bile acid 4 .05 Control vs treatment (1) .01 Error 4 .01 **P :_.01. 89 absorption of fats from diets supplemented with 4 and 8 percent TLW. 2. Metabolizable energy The determined M.E. values of the diets were improved with the increase in the level of TLW from 4 to 8 percent, substantiating the data obtained from fat absorption. The difference was however only marginally significant (Table 33). Cholic acid supplementation at the four different levels had no effect on the M.E. values of the diets with either 4 or 8 percent TLW. E. Experiment V 1. Fat absorption Fat absorption by the adult SCWL females, 23 weeks of age, increased significantly (P i .05) when the HSBO levels in the diets were raised from 4 to 8 percent (Table 34). The 3.4 percent increase in absorption was almost identical to the increase (3.5 percent) obtained with TLW under simi- lar conditions in experiment IV. The addition of cholic acid at the four levels (0.025, 0.05, 0.1 and 0.2 percent) had no significant effect on the absorption of fat from diets with either 4 or 8 percent HSBO. 2. Metabolizable energy The determined M.E. values of the diets containing 8 percent HSBO were significantly (P :_.01) higher by 8.9 percent over those from diets with the 4 percent HSBO 90 Table 33. Effect of graded levels of cholic acid on M.E. (kcal./g.) of diets with 4 or 8% TLW, when fed to SCWL females, 20 weeks of age (Experiment IV). M.E. Values (kcal,[g.) Level of Cholic Acid 4% TLW 8% TLW Mean 0% 3.27 3.37 3.31 .. +0.025% 3.15 3.46 3.30 ’ +0.05% 3.16 3.39 3.18 )3.24 +0.10% 2.87 3.48 3.18 +0.20% 3.27 3.31 3.29 Mean 3.14 3.40 3.25 Analysis of variance of M.E. values (kcal./g.) Source of Variation d.f. Mean Square Level of TLW l 0.l7+ Level of bile acid 4 0.005 Control vs treatments (1) 0.01 Error 4 0.03 TP = 0.1. 91 Table 34. Absorption of fat by SCWL females, 23 weeks of age, fed diets with 4 or 8% HSBO and graded levels of cholic acid (Experiment V). Fat Absorption (%) Level of Cholic Acid 4% HSBO 8% HSBO Mean 0% 84.5 87.3 85.9 +0.025% 86.3 86.2 86.3 +0.05% 86.5 91.0 88.8 )87.8 +0.10% 87.3 89.9 88.1 +0.20% 84.6 91.5 88.0 j Mean 85.8 89.2 87.4 Analysis of variance of fat absorption % Source of Variation d.f. Mean Square Level of TLW l 23.00* Level of bile acid 4 4.25 Control vs fat (1) 6.00 Error 4 3.25 *P < .05. 92 (Table 35). The inclusion of cholic acid at the four levels (0.025, 0.05, 0.1 and 0.2 percent) had no effect on the M.E. values of the diets containing either 4 or 8 percent HSBO . 3. Bile content of gall bladder At the termination of the experiment and on necropsy the weighed content (9.) of the gall bladders of the SCWL adult females, fed diets with no cholic acid, averaged 0.76 g. This was significantly (P i .01) different from the average bile weight in the gall bladders of the birds on the graded levels of cholic acid (Table 36). The weight of bile in the gall bladders increased as the level of cholic acid in the diets increased. Regression curves were plotted showing the increase in bile content per unit increase of percent dietary cholic acid in diets containing 4 and 8 per- cent HSBO (Fig. 2). The calculated regression equation of Y = 0.79 + 36.5 X represents the average curve depicting these trends up to a level of 0.1 percent cholic acid in the diet. The curve tends to plateau when the dietary level of cholic acid exceeded the 0.1 percent level. Photographs depicting representative distentions of gall bladders are presented in Fig. 3a, Fig. 3b, and Fig. 3c. 93 Table 35. Effect of graded levels of cholic acid on M.E. (kcal./g.) of diets with 4 or 8% HSBO, when fed to SCWL females, 23 weeks of age (Experiment V) M.E. Values (kcaltzg.) Level of Cholic Acid 4% HSBO 8% HSBO Mean 0% 3.24 3.47 3.36 +0.025% 3.28 3.47 3.38 +0.050% 3.28 3.57 3.43 ,3.43 +0.10% 3.34 3.62 3.48 +0.20% 3.22 3.67 3.45 Mean 3.27 3.56 Analysis of variance of M.E. values (kcal./g.) Source of Variation Mean Square Level of HSBO Level of bile acid Error **P < . 94 .m0. H me .Ho. w ess mv.o w nonne ssom.HH AHV uCeEusenu m> HonuCou «smm.m e oHos eHHu e0 He>eH mH.o H ommm Ho He>eH enssmm Csez .e.o COHuans> eo eonsom neoosHo HHsm CH A.m0 eHHn eo eoCans> eo mHmeHsCm eo.e oe.e NH.m NN.m oe.m soN.o no.4 mm.m mo.m om.m mo.e soH.o me.N oe.m mm.m oH.N oe.~ smo.o mm.H oe.H HN.N eo.~ No.N smmo.o oe.o oe.o em.H oe.o oH.H so .A.o0 . H.ov A.oo x.ov mucoucoo + A.oo muosucoo + onus oHHoeo .m.o eH .m.o oH nooosHm HHso .m.o oH noossHm HHso eo HmooH oHHm eo .uz sHHm eo .uz sHHm eo .uz ass: e0 .u: e0 .us mueHQ ommm sm mueHQ ommm so H> unseHnsexev omme ss no a nuns muons owe .oos eo mxoss mm .soHsEoe Hzom eo A.oo unsuooo oHHn oCs A.m0 quHe3 neooan HHsm Co oHos UHHOCU ensueHo Ho mHe>eH oeosnm mo uoeeee .mm eHnsB 95 Figure 2. Regression curves showing increases in bile content of gall bladders from adult SCWL females 23 weeks of age. The birds were fed diets with 4 or 8% HSBO and graded levels of 0.025, 0.05, 0.1 and 0.2% cholic acid. (Experiment V) Bile Content in Gall Bladder (g.) 6.0 5.0 4.0 3.0 2.0 1.0 . 025 96 :+ 4% HSBO in Diet II: + 8% HSBO in Diet ’ y=0. .10 % Cholic Acid in Diet Figure 2 -— Mean 79 + 36.5x (mean curve) .20 97 Figure 3a. Photographs depicting distention of gall bladders when adult SCWL females 23 weeks of age, were fed diet 71-09-01 (0% HSBO and 0% cholic acid) and diet 71-09-03 (4% HSBO and 0% cholic acid). (Experiment V) 98 71-09-01 71‘09-03 Figure 3a 99 Figure 3b. Photographs depicting distention of gall bladders when adult SCWL females, 23 weeks of age, were fed diet 71-09-05 (4% HSBO and 0.025% cholic acid) and diet 71-09-06 (4% HSBO and 0.05% cholic acid). (Experiment V) 100 Figure 3b 101 Figure 3c. Photographs depicting distention of gall bladders when adult SCWL females, 23 weeks of age were fed diet 71—09-07 (4% HSBO and 0.1% cholic acid) and diet 71-09-08 (4% HSBO and 0.2% cholic acid). (Experiment V) 102 7T-Gfi4fl Figure 3c V. DISCUSSION Edwards (1962) and Eysseneg;§l, (1965) have reported earlier that 0.2 percent cholic acid tended to improve fat absorption in chicks. Expanding on these studies and using dietary bile salts at levels of 0.025, 0.05 and 0.2 percent, apparent fat absorption was observed to be enhanced consid- erably during the first few weeks of chick growth, when the chicks were fed diets containing saturated and unsaturated fats ranging from 8.2 to 32.8 percent. These effects indicated a deficiency of bile salt availability, when levels of dietary fat are high. Evidence in literature supports this inference that a deficiency of bile salts exists in the young chick or can be induced. Serafin and Nesheim (1970) demonstrated that unlike older birds, the growing chick is unable to replenish readily bile salts lost by excretion. Goodridge (1968) had shown that the rate of cholesterol synthesis is low at the time of hatching and increases rapidly during the first few weeks of growth. Since bile salts are principally synthesized from cholesterol their deficiency in the young chick is inherent. Raw soybeans are known to induce bile acid deficiency by increasing excretory losses of bile salts and thereby depress fat absorption. Garlich and Nesheim 103 104 (1964) were able to correct the adverse effect of dietary raw soybean meal on fat absorption in the two-week old chick with supplementary bile salts. The evidence from this study has shown that the effect of supplementary bile salts on fat absorption steadily de- clines with the age of the bird. Levels as low as 0.025 percent in the diet appeared to meet the bile salt defi- ciencies in the chick. At this low level the supplementary bile salt promoted fat absorption by nearly 18 percent in the week-old chick. This effective increase in fat utili- zation is meaningful, since the dietary fat tested with the low levels of bile salts was a crude form of tallow, which in the controlled treatments was poorly absorbed. The chick at one week of age did not respond equiv- alently to the presence of different bile salts. It appeared to favor chenodeoxycholic acid over cholic acid and the sodium salt of taurocholic acid for improving fat absorption. Possibly this effect is from the physiological acceptance of chenodeoxycholic acid, due to some physical or chemical attribute of this bile salt. Chenodeoxycholic acid is the predominantly functional bile salt in the chick (Wiggins, 1955; Webling and Holdsworth, 1965). Cholic acid and its conjugated salt, sodium taurocholate, also improved fat absorption equally, though less efficiently than that ob- served with chenodeoxycholic acid. Thus the experimental data reveal that the conjugated form of a supplementary bile salt apparently offers no particular advantage to enhance 105 fat absorption over that observed with its non-conjugated form. Therefore, the deficiency of bile salts in the young chick does not seem to be due to an inability by the chick to conjugate bile acids which according to Webling and Holdsworth (1965) is the predominant form found in bile. By the third week of age the specificity to respond to different bile salts was apparently lost. How much longer the chick can continue to respond in this manner is not known. What is known is that in the adult bird bile salts have no effect on fat absorption. One of the adverse effects observed in the chicks fed diets with 0.2 percent cholic acid was the distended condi- tion of their gall bladders. Whether the observed enlarge- ment of the gall bladders was due to an oversecretion of bile from rapid reabsorption of bile salts via the entero- hepatic route, a stimulation of biosynthesis of bile salts, a functional stasis from high levels of bile salts in the intestinal lumen, or some other undetected factor remains to be settled. Distention of gall bladders was not seen in chicks one week and four weeks of age, when they were fed chenodeoxycholic acid, cholic acid and sodium tauro- cholic acid at 0.025 and 0.05 percent. The chicks on diets with low levels of supplementary chenodeoxycholic acid had the least amount of bile in the gall bladder, whereas those on the low levels of cholic acid and conjugated sodium taurocholic acid had slightly higher bile contents in their gall bladders. Overall low 106 levels of dietary bile salts did not appear to cause any apparent physiological imbalance on the secretion and supply of normal amounts of bile; and at the same time met the requirements for bile required by the young chick for the efficient utilization of fats. Thus it would appear that an effective dietary level of bile salts should not exceed 0.05 percent. When adult birds were tested with graded levels (0.025 to 0.2 percent) of cholic acid, bladder distentions were observed. Gall bladder distentions increased with increasing levels of cholic acid in the diet. The supplementary bile salts had no effect on fat absorption in these adult birds fed diets containing 4.0 or 8.0 percent fat. This indicated that the adult birds were capable of supplying adequate bile salts for absorption. Therefore, the linear relationship between the dietary bile salts and the bile content in the gall bladders appeared to suggest that the supplementary bile salts tended to accumulate in the bile acid pool; and also since they were in excess of the intestinal require- ments of bile for fat absorption may have caused gall bladder stasis. The linear relationship described seemed to be valid at least to the point when the diet contained 0.1 percent of cholic acid. At dietary levels over 0.1 percent where the curve appears to plateau, it is likely that bile acid excretion increases, to deter any harmful or undesirable build-up of systemic bile acids. The condition of the dis- tended gall bladder may appear to be an index of the level 107 of utilization of bile salt by the chicken and also may provide further evidence for the existence of an efficient enterohepatic circulation of bile salts. The graded disten- tions of the gall bladders also indicate that the release of gall bladder contents is particularly influenced by the demand for bile to aid in fat absorption. However whether it is the level of dietary fat and/or the level of bile salts in the intestinal lumen that triggers the cholecystagogic action is an area that needs further investigation. None of the bile salts tested showed any obvious effect on liver size and is in agreement with previous studies that neither cholic acid nor chenodeoxycholic acid at 0.2 percent had any effect on liver size or plasma lipids (Leveille ep‘al., 1962; Hunt ep'al., 1962; Eyssen and Somer, 1963; Eyssen 25,313, 1950; and Leveille and Fairchild, 1965). The studies on the effect of dietary bile salts on the absorption and utilization of fats provided important and interesting data on the status of fat acceptability by the young chick. Duckworth eE.al. (1950), Fedde EE.§$° (1960) and Renner and Hill (1960) have shown that certain fats such as tallow are absorbed to a lesser extent by chicks during their first two weeks of growth than when they are much older. The data from the first three experiments substantiate these observations recorded in the literature that fat absorption improves with age and that the depressed absorption in the young chick is closely associated to the inadequacy of bile salt secretion, as previously discussed. 108 The young chicks though not efficient at absorbing fats were observed to tolerate very high levels of fat which in some diets contributed almost all the non-protein calories. On a percent basis the chicks absorbed more fat with increasing levels of fat, irrespective of the presence of supplementary bile salts, indicating the absence of a well-defined mechanism to regulate the absorption of fat. At one week of age when fat contributed nearly 50 or 100 percent of the non-protein calories, absorption was in the region of 82 percent (average for different fats) and was 92 percent when the birds were four weeks of age. These relatively high percentages for absorption may probably have been due to the refined nature of the fats used in the first two experiments. Absorption of crude tallow, at a level of 8.2 percent in the diet, was 42 percent by chicks at one week of age and 76 percent when the chicks were four weeks old. These absorption levels for young chicks appear to be more valid and realistic as the fat content of the diets were at practical levels. Fat absorption values varied consistently with the composition of the fat. The order of absorbability of the fats were inversely related to the saturated fatty acid: unsaturated fatty acid ratios. Fats with the higher saturated fatty acid:unsaturated fatty acid ratio were the least effi- ciently absorbed. The chick during its first week of growth was observed to absorb fats very selectively. In the adult bird, fatty acid composition and degree of saturation had little effect on the level of fat absorption. However as 109 in the young chick, fat absorption increased when the levels of fat in the diet were raised. The influence of cholic acid on the absorption of fat seemed to be related to the fatty acid composition of the fats. Chicks absorbed larger percentages of HSBO in the presence of supplementary cholic acid. An examination of the fatty acid ratios of the fats revealed that HSBO had the highest oleic acid to saturated fatty acid ratio. Garrett and Young (1964) demonstrated that the absorption of oleic acid was greatly influenced by the presence of sufficient quantities of bile and also that in the presence of oleic acid, absorption of palmitic acid was considerably improved. This offers a probable explanation for hydrogenated soybean oil with its high oleic acid content ‘ to be better absorbed when fed with supplementary cholic acid. The trends in fat absorption with or without cholic acid were in most cases substantiated by data from determinations of M.E. of diets and fats. In other words, in almost all cases the addition of bile salts to the diet improved the M.E. value of the diet and fat. However, many factors as composition, extent of saturation and level of fat in the diet influence the resultant M.E. value. Sibbald and Slinger (1963) from their studies maintained that there seemed to be no correlation between the physical and chemical proper- ties of a fat and its energy content. The M.E. data from this series of experiments indicate that such properties do have an influence in contrast to their View as long as the level of fat in the diet is considered. Generally, and 110 within the limitations of the methods (Hill and Anderson, 1958; Anderson, Hill and Renner, 1958; and Potter ep_al., 1958) currently used for determining M.E. of diets and fats, the M.E. values reflect fat utilization. In reviewing the reason(s) for the unusually high M.E. values, the following should be taken into consideration. The reference diets had determined M.E. values which were within three percent of the calculated M.E. value of 3.40 kcal./g. The analytical procedures for the estimations of chromic oxide, which are the most critical in M.E. evalua- tion (Halloran, 1971), were performed against standard curves with duplicate samples and even run on separate days, with acceptable reproducibility. The Bomb Calorimeter was standardized before and after every run of samples, and the standardizations were within a :0. 2 percent error. The procedure for collecting and drying excreta samples was consistent. The excreta samples collected were reasonably uncontaminated. In retrospect the checks made on the ana- lytical procedures and the precautions taken left no doubts that the methodology was valid. Thus in these circumstances one has to accept the exceptionally high M.E. values determined for the fats, which in most cases exceeded the theoretical gross energy value of 9.4 kcal./g. for a fat. An explanation for the high values should be sought elsewhere as in the validity of the current procedure (Anderson and Hill, 1958) to determine the M.E. values of fats. Cullen'eE_§l, (1962) reported M.E. values for some fats that equalled or exceeded 111 9.4 kcal./g. and attributed the extra-caloric value to the non-lipid sources in the diet. Their high M.E. values for fats were derived from semi-purified or practical-type diets as compared to the purified-type diets used in this study. The methodology to arrive at M.E. values appears to leave no other alternative than to infer that the extra-caloric value is derived from the non-lipid components of the diet. Jensen ep,al. (1970) obtained a quantitative estimation of the extra-caloric effect of dietary fat when they recal- culated M.E. values of diets using feed efficiency values. Their recalculation being based on the principle that in- creasing the actual M.E. value of a diet causes a decrease in feed intake to some extent, and if body weight gain is maintained or is higher then relative efficiency of energy utilization improves. The data obtained at the 50 percent level of substitution of fat for glucose with chicks at four weeks of age, in this study, show that the determined M.E. values for the fats justify such a calculation used by Jensen ep_al. (1970). On a percentage basis M.E. of the diets increased 7.5 percent (3.62 to 3.89 kcal./g.) while feed efficiency improved 5.0 percent (1.61 to 1.54). The magnitude of this change on a determined M.E. value for the fats averaged 17 percent overall (no cholic acid added). The data on the determined M.E. values for fats indicate that the M.E. value of a diet having a substantial fat con- tent cannot be assumed to be the weighted sum of the M.E. values of its individual components. Sibbald ep_al. (1961) 112 obtained higher combined M.E. values for a 50/50 mixture of soybean oil and tallow for the fats fed separately to chicks at dietary levels of 10 and 20 percent, thus demonstrating the non-additive effect of the M.E. values of fats. There- fore, a better perspective is needed of those conditions which influence such a marked improvement in the determined ’M.E. value of a fat. The attempt to measure or relate fat utilization with or without cholic acid to the body weight gain of growing chicks was not conclusive. Although experimental precautions were taken to make the 50 or 100 percent fat M.E. substituted diets theoretically equidense using appropriate levels of sand and cellulose, the feed intake data revealed several discrepancies. Therefore, the weight gain patterns in the chicks had to be necessarily interpreted in relation to feed intake. Chicks fed the purified reference diet in experiment I, having only the 2 percent safflower oil, apparently did not tolerate the bile salt at the level given. Their weight was depressed proportionally to the decline in feed intake. At the other extreme, body weight and feed intake were 20 percent lower when fat replaced glucose completely, and this effect was completely independent of the dietary bile acids. Obviously the consequence of the lower feed intake was an inadequate intake of protein to support growth comparable to the control group, and this occurrence negated growth and feed efficiency values and possibly other parameters for assessing the efficacy of bile salts. Biely and March (1954) 113 and Scott e£.al. (1955) showed that the depressed growth from increasing fat (not substituted isocalorically) in the diet can be overcome by increasing the dietary level of protein. In this experiment the substitution of fat was made on an isocaloric basis for glucose so any reduction of feed intake would have to be a reflection of an enhanced M.E. from fat over the assumed standard value. However, along with this concept one must be willing to accept the possibility that if feed intake is significantly curtailed then the conditions for the assay are no longer valid. The better situation is when protein intakes are adjusted to meet the full demands of growth. The enhanced energy-contributing value of the fats may not be the only factor influencing the sharp drop in intake. One has to consider the physical constitution of the diets. Palatability may be one possible factor, but another worthy of consideration and recently reviewed by Polin and WOlford (1972) would appear to be the "filling effect" of feed based on the concept of "wet density"' (Sibbald SE $1., 1960; Peterson and Baumgardt, 1971) rather than "dry density" value. There was a favorable effect on body weight gain in the older chicks fed diets with 50 per- cent of the M.E. from fat sources. This sparing effect of fat for carbohydrates or protein remains to be explained in terms of changes in carcass composition. VI. CONCLUSIONS In the growing chick supplementary bile salts at 0.025, 0.05 and 0.2 percent improved the absorption of fats at dietary levels ranging from 8.2 to 32.8 percent. Chicks one to four weeks of age were unable to tolerate supplemental bile salts when the diets were totally devoid of fat. Chenodeoxycholic acid, the predominant bile acid in chicks, was the most acceptable and functionally effi- cient of several bile acids tested in diets at 0.025 and 0.05 percent. Cholic acid and taurocholic acid, sodium salt, were also acceptable but less efficient in promoting fat absorption. Dietary cholic acid at 0.2 percent had an adverse effect on chicks, causing gall bladder distentions. The different dietary bile salts tested at 0.025 and 0.05 percent caused no gall bladder distention in chicks up to 3 weeks of age. In the adult female chicken supplemental bile salts did not improve fat absorption. The dietary bile acids apparently accumulated in the systemic bile acid pool, as seen from the graded distentions of the gall bladders from graded levels of dietary bile salts. 114 10. ll. 12. 115 The limitations to the absorption of fats during the first few weeks of age were related to an inadequacy of bile acid biosynthesis. The experimental evidence indicates that bile acid biosynthesis improves with age. Absorption of crude tallow at 8.2 percent in the diet improved from 42 percent in chicks one week of age to 76 percent at four weeks. Absorption of fats were generally observed to increase with increasing levels of dietary fat. Chicks at four weeks of age were able to tolerate very high levels of dietary fats, and at levels when the fats were the only source of non-protein energy. Of the four fats, TLW, LD, HSBO and CNO tested, HSBO showed the greatest improvement in absorption from bile acid supplementation. There is an indication that the oleic acid:saturated fatty acid ratio has a bearing on the promotion and absorption of fats by dietary cholic acid. The metabolizable energy value of fats and diets con- sistently substantiated the data on fat absorption, particularly when dietary fat levels were high. The high determined M.E. values for fats which in most cases exceeded the theoretical maximum gross energy of 9.4 kcal./g. for a fat lend support to the hypothesis of the extra-caloric effect of dietary fats. Based on the current methodology to calculate the M.E. value of of a fat, the extra-caloric value is derived from the 13. 14. 116 capacity of fats to enhance energy retention from the non-lipid components of the diet. When dietary fat levels were high the M.E. value of the diets could not be assumed to be the weighted sum of the M.E. values of the individual components. From a practical standpoint the addition of bile salts to chick starter diets with high levels of fats should prove beneficial, and is an area of study worthy of further investigation. REFLECT IONS In the course of analyzing the data from the experiments just concluded, questions arose for which complete explana- tions or answers were not evident or clearly deducible. The high determined M.E. values for some fats are ESE indicative of the absolute M.E. values for the fats. These high M.E. values for the fats were calculated using the determined M.E. values for the reference and test diets. The accepted procedure of Hill and Anderson (1958) was used in arriving at the determined M.E. values of the reference and test diets. Although on a calculated basis the reference and test diets were isocaloric the M.E. values obtained for the test diets with fat were always higher than that of the reference diet. These increases in M.E. values could be attributed to an increased retention of nutrients as a result of the high levels of fat in the diets or to an improved absorption of fats which would impart the same effect. In addition a depressed feed intake was observed, and this may also have been responsible for the increased retention of nutrients assuming an improvement in the effi- ciency of utilization which occurred. The procedure adopted to calculate the M.E. values of the fats was based on the assumption that there was no change in the M.E. value of the 117 118 non-lipid components of the diet. Therefore any increases in the retention of non-lipid components would be reflected in the M.E. values of the fats. The highest M.E. value of 10.2 kcal./g. fat was ob- tained with CNO at the 100 percent F.M.E. substitution level (treatments without cholic acid). The calculated M.E. value of this test diet was 3.40 kcal./9., while the determined M.E value was 3.94. If the maximum value of 9.4 'diet kcal./g. for CNO was used in calculating the M.E. of the diet, a value of 3.90 kcal./g. diet will be obtained for the maximum M.E. of the diet, only 0.04 units less than that actually determined. However, on the basis of the percent CNO absorbed (93.2%) an estimate of the actual M.E. for CNO would be 8.76 kcal./9.; a value more in line with the 8.95 kcal./g. nutritionists usually use. If one assumes that the increase in dietary M.E. all came from the protein M.E. component of the diet, then the M.E. of the protein would be 4.10 kcal./g. and not 3.57 kcal./g. used in arriving at the calculated value. It is, therefore, feasible to expect that additional calories could come from the 14.8 percent (4.10-3.57/3.57 x 100) increase in the retention of the protein component of the diet. Since the soy assay pro- tein used had an 87 percent protein content, the maximum M.E. of the protein would be (87% of 5.7 kcal./g. = 5.0 kcal./9.). Whether high fat diets actually influence protein absorption could be readily tested from nitrogen retention studies. ”"O -\ :4 n3 - E 5‘» ~\h .1ls «e. .~‘ \‘.I , 119 The CNO used in the experiments had an unsaturated fatty acid content of 86 percent. Dietary cholic acid was hardly able to improve the absorption of this fat. In the case of HSBO, having an equally high unsaturated fatty acid content of 79 percent, cholic acid promoted its absorption consistently. The predominant difference in the composition of the unsaturated fatty acids of CNO and HSBO was that in the latter the oleic acid was nearly 72 percent of the total fatty acid content as compared to 30 percent in CNO. Thus the oleic acid:saturated fatty acid ratio was 5.86 for HSBO and 3.69 for CNO. Bile salts appear to have greater solubilizing affinities to oleic acid than to the other unsaturated or polyunsaturated fatty acids. This has been shown by earlier workers and reviewed under "Discussion." The level of oleic acid in a fat may thus be an important factor if fat ab- sorption is to be promoted by supplemental bile salts. The young chick was able to utilize conjugated and unconjugated bile salts with equal efficiency to promote the absorption of fats. In the chick the bile acid conjugant is taurine, an amino sulfonic acid derived from its precursor cysteine. Whether the equal efficiency of the unconjugated bile salt was achieved at the expense of cysteine needs to be studied. Cysteine it must be noted is derived from the essential amino acid methionine, and therefore continued use of dietary unconjugated bile salts may cause methionine deficiencies. This could be tested by feeding chicks high 120 fat diets marginally deficient in methionine and supplemented with conjugated or unconjugated bile salts. Finally of most concern to nutritional experimentalists would be the observation that certain nutrients because of their physical characteristics altered total feed intake, although their contributions to the diets were equicaloric. The changes in feed intake necessarily altered the intake of other nutrients to the extent of causing dietary imbalances. Thus the use of parameters such as weight gain as indices in testing the efficacy of dietary supplements became invalid. Techniques such as forcefeeding of known quantities of feed may have to be adopted to ensure equal intake of experimental diets and supplemental nutrients. The alternative would be to make up the deficiency of nutrient intake when feed intake is depressed such that the calorie:nutrient ratio is maintained. BIBLIOGRAPHY VII . BIBLIOGRAPHY Aitken, J. R., G. S. Lindblad and W. G. Hunsaker (1954). Beef tallow as a source of energy in broiler rations. Poultry Sci. 33: 1038. Almquist, H. J. (1971). 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Supplementation of corn-soybean oil meal rations with penicillin and various fats. Poultry Sci. 32: 930. APPENDIX 129 Appendix Table 1. Time schedule of experiments Date Date Experiment No. Diet No. Commenced Terminated I 70-ll-(01-l8) 17 Nov. 1970 8 Dec. 1970 II A 71-03-(01-18) 3 Mar. 1971 10 Mar. 1971 II B 71-03-(19-36) 16 Mar. 1971 23 Mar. 1971 III 71-12-(01-08) 7 Dec. 1971 28 Dec. 1971 IV 71-08-(01-12) 17 Aug. 1971 27 Aug. 1971 V 71-09-(01-12) 10 Sept. 1971 30 Sept. 1971 130 H .N cam H mmumowammmll m pom m: mm.a mw.H ems own ome eee ommm mm.mm+ malaanoh me.H me.H ham man mmm mmm ommm ww.ea+ halaanon mm.a hm.a mmm wmh mae HHe ozu wm.mm+ mHIHHIoh oo.H em.H mmm mmh Hmm Ham ozu wm.ma+ maladies mh.H we.H hum mmn mmm mam DH wm.m~+ eHIHH10h mn.H om.H omh mun hme mme oq we.ma+ manaalon me.H me.a mmh th mme mme 3A5 wm.mm+ NHIHHIOh mm.a me.a mmh Hmm mmm mmm BAH we.oa+ HHIHHIOh ow.H mh.a ehm Hen mmm Hue Hmmmm oataauon pflo< owaoso wm.o mm.a mm.H emm mmm Hme omm ommm wm.mm+ moIHHIOB mm.a mm.a mmh mam mom emm ommm wm.ea+ mOIHHIOh mm.a on.H has 5mm mme mee ozu wm.m~+ hOIHHIOh No.a ee.a 5mm mmm mmm com 020 wm.ma+ molaalon mm.H mm.a own emm ome mmm DA wm.mm+ mOIHHIOh H5.H mo.a mmo emm emm HHe an we.ma+ eoIHHIOF em.H Mm.H ewe new eee hwe 399 wm.mm+ monaalon Hm.a me.a mom mam emm omm 349 we.mH+ Nonaalon mm.a mm.H Hum mom mum mne Hmmmm HOIHHIOh UH0< owaoso we mm Hm mm Hm Nm« Hma mocmfloflwwm comm cuwm\fi.mv A.mv mcflmw .uz mwom uwm .Hmmsm .oz umfio mxmuaH cmmm umz .AH ucmfiwummxmv mmm mo mxmmz woman ou xmm3 wco Eoum mxoflno mmmulumafloun 0» 0mm wumfic HousmEHHmmxm .mump mocmHOflmwm comm pom A.mv mxmuca boom .A.mv mcflmm unaflmz mpom .m magma xflpcmmmm 131 .m can H mmumoflammmuumm can Hm: m.ea on.h mh.HH oa.m H.mm d.mm ommm mm.m~+ mHIHHIon h~.ma eH.mH ma.m m.mm m.mm m.mm ommm mm.ea+ nHIHHIOh Hm.m ma.> mo.m m.mm «.mm m.mm ozo wm.mm+ mHIHHIon mm.m om.h eH.m m.em m.mm m.mm ozu wm.ma+ maIHHIOh hm.b mm.h NH.N m.em e.mm m.em on mm.m~+ ealaanon me.oa mm.oa mH.m m.em o.mm «.mm Qq we.ma+ malaalon am.m H>.n mo.m m.mm m.em m.mm ZQB wm.mm+ NHIHHIOS mh.oa mm.ma eH.m m.em d.mm m.mm 3A9 we.ma+ HHIHHIOh mo.ma mo.ma hH.N m.om m.om H.N¢ Hommm OHIHHIOh pflod UHHOQU mm.o mm.h mm.m oa.m e.em m.~m d.mm ommm wm.m~+ monaalo> mm.oa mm.oa ma.m m.~m o.mm m.mm ommm ww.ea+ molaalon em.oa mm.m mo.~ m.mm H.em m.mm ozo wm.mm+ noIHHIOh mm.m mh.oa eH.~ m.em m.em m.mm ozo wm.NH+ molaalon mm.m Hm.h NH.~ H.mm m.mm m.em GA wm.wm+ monaalon oH.HH om.HH ma.~ m.em m.mm ~.mm on we.ma+ eOIHHIOh om.m om.h mo.m H.Hm h.Hm m.mm 3A9 mm.~m+ monaalon om.HH oH.HH eH.N m.Hm h.Hm m.mm 3A9 me.ma+ monaaloh oo.ma OH.MN ha.m w.mm a.Hm d.mm Homom HoIHHIon Uwom UHHono mo mm Hm Hm Nm« Hm Hm« opouoxm pooh muouoxm pooh umm .Hmmsm .oz uowo Arm\.msv momno Awe umuumz sun .AH ucoafluomxmv mamon Houuoe hum o no commonmxo .moamEMm ouonoxo paw coom CH A.m\.mfiv mONHU mo mosam> oocHEnouoU Ugo Amy uouuofi who .m magma xaoqwmmm 132 .N poo H mouooflamomll «m can Hm: m.sm mm.mam mm.amm HN.OH mm.» m.me H.mmm ommm ww.mm+ wataaiom em.mma ma.aha we.HH hm.m o.mm m.mm e.omH ommm ww.eH+ NHIHHIoh ha.omm em.mmm mm.HH mm.m ~.hm m.Hm H.mmm ozo wm.mm+ mHIHHIo> mo.mea oe.meH mh.HH oe.mH m.mm m.Hm m.oma ozo mm.~H+ mHIHHIOh me.amm Ne.em~ mm.HH mm.m m.ae «.mm m.mom DA wm.w~+ eHIHHIoh he.oma mm.mma mm.m he.m e.hm m.mm H.mwa GA weqma+ MHIHHIOh mm.mem Hm.mem mm.m~ mn.ma m.mh m.mm H.mom 3A9 wm.mm+ NHIHHIOh mm.th em.HmH mm.o~ me.na m.~0H m.hoa d.mma RAB we.ma+ HHIHHIOS mm.NH m~.oa HH.0H em.~a o.on m.eoa m.mm Homom oanaanon wwofi UHHO£U wN.o mm.mom mo.mam mh.mm Hm.am H.Nm m.mm m.mmm ommm mm.mm+ monaalon no.mma Hm.mwa mm.ha mm.ma m.mm m.hm m.mha ommm wm.ea+ molaaloh em.eh~ mm.mmm mm.ha mm.Hm e.mm m.am m.amm ozu wm.mm+ nonaaloh me.mea mm.nea em.HH hm.ma m.mm w.mm m.oma ozu mm.~a+ monaaloh em.mm~ mm.am~ mm.ma no.mH e.Hm H.em e.mom an wm.m~+ molaalom om.oma Hm.ema on.m mm.HH H.om m.mm m.mma on we.ma+ eonaalon He.~em mn.mmm mm.~m mm.mm m.hm m.mm e.mmm 3A8 wm.mm+ MOIHHIOh em.~ma hm.mha wm.ma mm.~m m.Hm m.oHH ~.mma 3A9 we.ma+ moIHHIoh mo.HH mH.mH mn.HH No.5 m.hm N.Hm m.~m Homom HoIHHIoh UHO¢ oaaono we mm Hm mm Hm mm« Hm: .III. 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ApoHp .ORmeoxv ououoxm_ pooh pom .Hmmdm .oz uoHO .m.u ououoxm A.M\uHmoxv mmuocm mmouw .AHH uOoEHHoOxmv mHmmn HouuoE mun m O0 commonmxo .moHOEMm mgonoxo cam Uoom OH A.m\.HooxI4m.wv mouoco moonw .m oHQoB xHGGommd 138 .m can m .H mmumoHHmmmnumm new mm .Hm« Hmom meow mHmm momm emHm mmom omo mmo eeo oHN NNN HNN 3H9 mm.m+ mOImHIHh meow emmm hmom mHmm NeHN mmmm moo oon meo omm mmm mHN mo. oHom OHHoco OHDMBIMZ hOINHIHh eoem meow memm oemm mon emHm oeo emo mmo mHN mHm hmm mmo. oHom OHHono ousoelmz ooanIHh mmom meow Hoem meHm mmHm emom emo won mom 0mm 0mm OHN mo. oHoo UHHono nsxowoocmao moanan Ame mmom mmom moom mmHm moom Hmom moo emo mmo on mmm eHN mmo. oHom oHHoco usxomoocmno eoamHuHs Ame mmom ooom oemm mmmm OHHN mmmH who omo eeo mHN omm Hmm mo. oHoo OHHOHU moanIHh mmom meow mmom mmHN eemm eeom mom emo meo omm mHm on mmo. oHom UHHOOU NoumHIHh MHoN ehHm mmom mmHN momH momm mmo eme eoo mHN hHm 5mm Homom HOINHIHh mm mm Hm mm mm Hm mm mm Hm Mme Nm« HMa mHuo mwonmxmucH comm malwmonh.mc .uzwwoom n Noonfiymo .us seem o Hmoalawc .us seem .Hmmsm uHmm mHHm .oz umHo & .AHHH unoEHuomxmv omo mo mxoo3 oousu ou moo oco scum mHOHOo omhuluoHHoun on pom ouo3 muoHo HoucoEHHomxm .mH aoO um muoo oxoucH woom new mH moo can b uoO um H.0v OHom uano3 zoom .OH oHnt XHocomm< 139 .m UOo m .H moumoHHmomll uouuoz >MOH m can mm .HMa cm.> mo.o Hm.o H.mm m.em o.em 3H9 am.m+ moumHan Hm.HH mH.m mo.0H o.mm m.mm H.mm mo.o oHom oHHono onsmaumz noumHan em.m mm.0H HH.m m.mm «.mm m.mm mmo.o oHom oHHoOo ousmeumz ooumHan mm.m on.m eo.0H m.mm m.mm m.mm mo.o oHom oHHoOo umxomoocmno moumHan mH.OH Ho.m mo.m H.mm m.mm m.~m mmo.o oHom oHHono -sxomoocwgo eoanan Ho.HH mm.0H om.m H.em m.mm H.em mo.o oHom UHHoOo moumHan eh.m em.m on.m n.em m.mm m.em mmo.o oHom oHHoso moumHuHs mm.0H mH.m mm.OH o.mm h.mm H.mm Hmmmm Ho-mHan mHumH Nmo mm.m mn.m oo.m o.em m.mm n.mm o.em any w~.m+ moumHan em.> oo.» mm.m o.em m.~m m.mo o.mm mo.o oHom oHHoOo onsmanmz noanuHs eo.n mm.m mo.» o.em o.mm m.em o.mm mmo.o oHum oHHoOo ousmeumz ooumHan No.5 mm.m sm.m n.em o.em m.em o.mm mo.o oHom oHHoso usxomoocmgo moumHuHh oe.m H~.0H Hm.m m.em m.mm m.em o.mm mmo.o oHom oHHono umxomwocmno eoanuHH sh.» me.m Hm.m m.em o.mm m.em o.mm mo.o oHom OHHono moumHuHh om.h em.m Ho.n m.em H.em o.mm o.mm mmo.o oHom oHHoso moanth me.“ eH.m em.n o.em H.mm e.em o.mm Human HoumHqu mm mm Hm mm. mm. 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Hm.e 0H.e 00.e 0m.e 00.0 pHoo oHHOOo umxomoocmso moumHuHs 00.0 00.0 00. oe.e 0H.e 0N.e 0m.e 000.0 UHoo oHHOOo usxomcocmno ecumHuHs 00.0 e0.0 m0. 00.e 00.e 00.0 0m.e 00.0 UHoo UHHOOU m0ImHIHh 00.0 00.0 00. 00.0 o0.e o0.e 0m.e 000.0 oHoo UHHOOU N0ImHIHh H0.0 e0.H 00.0 mm.e oe.e 0H.e 0m.e oOoz Homom H0|~HIH0 mm mm Hm .000 00+ Hm: nuowm.m\.Hoox0 Amuouoxm .m\.Hmox~ .0\.Hoox .HOOOm uHom oHHm .oz uoHO muouoxm .m.0 ououoxm «m.w pooh .m.w 0 Oo oouooHHoo moHOEom ououoxo OOo poom OH A.0\.Hooxl.m.wv mmHoOo mmonw .AHHH HOoEHHomxmv mHmoQ HouuoE 0H6 o O0 commonmxo .0H mom .mH oHOoB xHUOoOOO Appendix Table 16. 144 (Experiment III) Bile content (g.) of gall bladder (g.b.) at Day 19 Wt. of g.b. + bile (g.) % *R1 *R2 Diet No. Bile Salt Suppl. Bdl #1 Bd #2 Bd #1 Bd.#2 .Bd #1 Bd #2 71-12-01 Basal 0.62 0.83 0.62 0.84 0.85 0.73 71-12-02 Cholic acid 0.025 0.83 0.92 1.08 0.94 0.75 0.62 71-12-03 Cholic acid 0.05 0.76 0.85 0.91 0.82 0.75 0.91 71-12-04 Chenodeoxy- cholic acid 0.025 0.57 0.96 0.45 0.82 0.45 0.63 71-12-05 Chenodeoxy- cholic acid 0.05 0.85 1.32 0.45 0.71 0.50 0.64 71-12-06 Na-Tauro cholic acid 0.025 0.45 1.32 0.55 0.91 0.95 0.64 71-12-07 Na-Tauro cholic acid 0.05 0.85 1.25 0.95 1.02 0.83 0.73 71-12-08 +8.2% TLW 1.05 0.65 0.90 0.95 0.63 0.81 Wt. of_g,b. empty (9.) 71-12-01 Basal 0.13 0.13 0.10 0.14 0.13 0.19 71-12-02 Cholic acid 0.025 0.15 0.19 0.15 0.12 0.12 0.14 71-12-03 Cholic acid 0.05 0.14 0.12 0.10 0.13 0.14 0.13 71-12-04 Chenodeoxy- cholic acid 0.025 0.18 0.15 0.10 0.14 0.10 0.14 71-12-05 Chenodeoxy- cholic acid 0.05 0.14 0.19 0.10 0.14 0.18 0.14 71-12-06 Na-Tauro cholic acid 0.025 0.16 0.15 0.12 0.10 0.13 0.16 71-12-07 Na-Tauro ' cholic acid 0.05 0.20 0.16 0.14 0.14 0.13 0.12 71-12-08 +8.2% TLW 0.16 0.18 0.14 0.20 0.18 0.10 Wt. of bile (g.) 71:12-01 Basal 0.49 0.70 0.52 0.70 0.72 0.54 71-12-02 Cholic acid 0.025 0.68 0.73 0.93 0.82 0.62 0.48 71-12-03 Cholic acid 0.05 0.62 0.73 0.81 0.69 0.61 0.78 71-12-04 Chenodeoxy- cholic acid 0.025 0.39 0.81 0.35 0.68 0.35 0.49 71-12-05 Chenodeoxy- cholic acid 0.05 0.71 1.13 0.35 0.57 0.32 0.50 71-12-06 Na-Tauro cholic acid 0.025 0.29 1.17 0.42 0.82 0.82 0.48 71-12-07 Na-Tauro cholic acid 0.05 0.65 1.09 0.81 0.88 0.70 0.61 71-12-08 +8.2% TLW 0.89 0.47 0.76 0.75 0.45 0.71 *R1, R2 and R3--Replicates 1, 2 and 3. 1 Bd = Bird. 145 00.0eH 00.0H 0.0m 0.00H 000.0 00+ 0HI00IH0 00.00H 0e.0H H.00 m.o0H 00H.0 00+ HHI00|H0 00.00H 00.oH 0.0e m.o0H 000.0 00+ 0HI00IH0 0o.0mH o0.oH 0.H0 m.o0H 0000.0 00+ 00l00IH0 00.00 oH.oH H.0e 0.0HH 000.0 0e+ 00l00|H0 00.00 00.0H 0.0m 0.0HH 00H.0 0e+ 00:00IH0 00.00 00.0H 0.00 0.0HH 000.0 0e+ o0|001H0 00.00 0H.oH 0.0e m.mHH 0000.0 0e+ 00:00IH0 00.00 0e.oH 0.H0 m.o0H II 00+ e0|00IH0 00.00 0o.0H 0.00 0.0HH II 0e+ 00:00IH0 em.00 00.HH 0.0m 0.Ho II II 00I00IH0 00.H0 0H.oH 0.00 0.0o II II H0100IH0 A.0\.0EV AaoHO .0\.080 muouoxm ooom oHom UHHOOo 3H9 .oz uoHO oonuomnd pom ouonoxm H.0\.0EV mo Ho>oH mo Ho>oH pom UOoUOOU umm mHmon Hopuoa 0H0 0 O0 powwoumxo .monEMm muouoxo oOo woom OH H>H uOoEHHomxmv uOoUOoo A.0\.0Ev pom .0H oHQMB XHGGond 146 Appendix Table 18. Dry matter (%) and determined values of Cr20 (mg./g.) in feed and excreta samples, expressed on a dry matter basis (Experiment IV). Level of Level of Dry Matter (%) Cr203 (mg./g.) Diet No. TLW Cholic Acid Feed Excreta Feed Excreta 71-08-01 -- -- 89.5 93.5 2.23 4.66 71-08-02 -- -- 89.2 90.1 2.24 7.36 71-08-03 +4% -- 89.6 89.8 2.23 7.44 71-08-04 +8% -- 91.0 90.5 2.19 6.86 71-08-05 +4% 0.025% 87.4 90.9 2.28 6.45 71-08-06 +4% 0.05% 88.6 89.4 2.25 7.24 71-08-07 +4% 0.10% 89.2 91.5 2.24 5.16 71-08-08 +4% 0.20% 88.2 89.9 2.26 6.59 71-08-09 +8% 0.025% 89.9 90.1 2.22 6.96 71-08-10 +8% 0.05% 90.5 90.8 2.21 5.98 71-08-11 +8% 0.10% 89.1 89.7 2.24 7.35 71-08-12 +8% 0.20% 89.1 90.5 2.24 5.45 147 Gross energy (G.E.-kca1./g.) in feed and excreta samples, expressed on a dry matter basis (Experiment Appendix Table 19. Iv) Level of Level of G.E. (kcal./g.) Excreta G.E. Diet No. TLW Cholic Acid Feed Excreta (kcal./g. diet) 71-08-01 -- -- 3.901 2.968 1.420 71-08-02 -- -- 3.801 3.060 .931 71-08-03 +4% -- 4.205 3.128 .937 71-08-04 +8% -- 4.383 3.162 1.009 71-08-05 +4% 0.025% 4.218 3.031 1.071 71-08-06 +4% 0.05% 4.187 3.296 1.024 71-08-07 +4% 0.10% 3.888 2.823 1.226 71-08-08 +4% 0.20% 4.311 3.038 1.042 71-08-09 +8% 0.025% 4.481 3.191 1.018 71-08-10 +8% 0.05% 4.464 2.919 1.079 71-08-11 +8% 0.10% 4.479 3.264 .995 71-08-12 +8% 0.20% 4.530 2.971 1.221 Appendix Table 20. 148 Dry matter (%) and determined values of Cr in feed and excreta samples, expressed on a basis (Experiment V) 2 O 3: 0—7fi Level of Level of ‘ Dry Matter (%) Cr293 (mg./g.) Diet No. HSBO Cholic Acid Feed Excreta .Feed Excreta 71-09-01 -- -- 89.5 90.0 2.23 6.61 71-09-02 -- -- 89.2 89.9 2.24 5.74 71-09-03 +4% -- 89.1 89.8 2.24 7.52 71-09-04 +8% -- 88.9 90.2 2.24 7.41 71-09-05 +4% 0.025% 89.5 90.4 2.23 7.33 71-09-06 +4% 0.05% 89.6 90.7 2.23 7.05 71-09-07 +4% 20.10% 89.1 89.6 2.24 8.04 71-09-08 +4% 0.20% 89.1 90.3 2.24 6.77 71-09-09 +8% 0.025% 88.7 90.8 2.25 6.98 71-09-10 +8% 0.05% 88.1 90.1 2.27 7.99 71-09-11 +8% 0.10% 88.2 90.6 2.27 7.17 71-09-12 +8% 0.20% 88.5 89.5 2.25 8.22 (mgo/9-) y matter 149 Appendix Table 21. Fat (mg./g.) content in feed and excreta samples, expressed on a dry matter basis (Experiment V) Fat Content Fat Level of Level of (mg./g.) Excreta Fat Absorbed Diet No. HSBO Cholic Acid Feed . EXcreta mg./g. Diet (mg./g.) 71-09-01 -- -- 67.03 48.1 16.23 50.80 71-09-02 -- -- 61.66 45.9 17.90 43.76 71-09-03 +4% -- 112.22 55.3 16.46 95.76 71-09-04 +8% -- 157.48 64.0 19.35 138.13 71-09-05 +4% 0.025% 111.98 47.9 14.56 97.42 71-09-06 +4% 0.05% 111.98 45.4 14.36 97.62 71-09-07 +4% 0.10% 111.98 48.4 13.48 98.50 71-09-08 +4% 0.20% 111.98 49.6 16.39 95.59 71-09-09 +8% 0.025% 158.37 65.5 21.10 137.27 71-09-10 +8% 0.05% 158.37 48.1 13.67 144.70 71-09-11 +8% 0.10% 158.37 48.9 15.48 142.89 71-09-12 +8% 0.20% 158.37 47.5 13.01 145.36 150 Gross energy (G.E.-kca1./g.) in feed and excreta samples, expressed on a dry matter basis (Experiment Appendix Table 22. V) Level of Level of G.E. (kcal./g.) Excreta E. Diet No. HSBO Cholic Acid Feed Excreta (kcal./g. Diet) 71-09-01 -- -- 3.923 3.316 1.119 71-09-02 -- -- 3.802 3.119 1.217 71-09-03 +4% -- 4.170 3.117 0.928 71-09-04 +8% -- 4.452 3.263 0.987 71-09-05 +4% 0.025% 4.250 3.174 0.966 71-09-06 +4% 0.05% 4.275 3.156 0.998 71-09-07 +4% 0.10% 4.239 3.246 0.904 71-09-08 +4% 0.20% 4.246 3.106 1.028 71-09-09 +8% 0.025% 4.535 3.298 1.063 71-09-10 +8% 0.05% 4.502 3.287 0.934 71-09-11 +8% 0.10% 4.560 2.952 0.935 71-09-12 +8% 0.20% 4.557 3.257 0.892 151 e0.o 00.0 00.0 00.0 00.0 00.0 000.0 00+ 0H1001H0 00.0 He.0 00.0 eo.0 e0.0 00.o 00H.0 00+ HHI00|H0 00.H H0.0 0o.0 00.0 0e.0 m0.e 000.0 00+ 0HI00IH0 oH.0 00.H 0e.0 He.0 Ho.0 00.H 0000.0 00+ 00100IH0 00.e oo.0 00.0 o0.0 om.0 00.o 000.0 0e+ 00:00IH0 H0.H 00.0 00.0 e0.0 00.0 00.0 00H.0 0e+ 00l00IH0 00.H 0o.0 0o.0 00.0 00.0 00.0 000.0 0e+ o0I00IH0 0e.H H0.0 00.0 00.0 0H.0 H0.0 0000.0 0e+ 00l00|H0 H0.0 00.0 oo.0 0o.0 He.H e0.H II 00+ e0I00IH0 00.H 00.0 He.0 00.0 eo.H 0o.0 II 0e+ 00:00IH0 o0.H 0o.0 00.0 0o.0 0e.0 00.H II II 00l00IH0 00.0 00.H H0.0 0o.0 00.H 00.0 II II H0I00IH0 00 ouHm H0 UHHm 00 onHm H0 qum 0* ouHm H0 ouHm oHod OHHOQU 0000 .02 0oHO .Q.0 OH oHHQ mo .03 .0.0 >0mso 00 .03 oHHn + .H.0 mo .03 Ho>oH mo Ho>oH A> 0OoEHHomxmv .0 OH H.O.0v HooooHO HHmm mo 0Oo0Ooo oHHm .00 oHnme xHoOommd 152 Appendix Table 24. Typical Calculation of the M.E. values of diets and fats Data from chicks fed reference diet and test diets with 16.4% HSBO (Experiment II) M.E. of Diets M.E. per g. diet = G.E. per g. diet - Excreta G.E. per g. diet Reference Diet 3.85 kcal./g. 4.08 kcal./g. . Cr;03 in diet G.E- per g. dlet X Cr203’1n excreta G.E. per g. diet G.E. per g. excreta Excreta energy per g. diet 2.17 = 4.08 x14.98 = 0.59 M.E. (kcal./g.) = 3.85 - 0.59 = 3.26 Test Diet G.E. per g. diet = 4.29 G.E. per g. excreta = 3.51 Cr 03 in diet Cr203 in excreta Excreta energy per g. diet = G.E. per g. diet x = 3.51 X ' = 0.95 M.E. (kcal./g.) = 4.29 - 0.95 = 3.34 M.E. of Fat (M.E.T - M.E.R) Fat =Decimal proportion of test material in diet M.E.R = M.E. of reference diet = 3.26 M.E.T = M.E. of test diet (14.8% HSBO) = 3.34 M.E.G = M.E. of glucose removed/g. diet = 0.316 x 3.64 kcal./g. = 1.15 kcal./g. 0.148 Decimal proportion of HSBO 3.34 - 3.26 + 1.15 0.148 = 8.31 kcal./g. M.E. of HSBO 3 1293 030 'Tflifinmfluflimwuumummlu\wfllfifilu