ABSTRACT FATTY LIVER STUDIES IN LAYING HENS by Thomas Lionel Barton A series of seven experiments was conducted relative to the production and/or prevention of fatty livers. A specific high-energy (3,000 Metabolizable Energy Cal/Kg), commercially available diet was used to induce fatty livers in a commercial strain of Single Comb White Leghorn hens. Experiment I was conducted in floor pens and the re- mainder of the tests were conducted in individual-bird cages. A scoring system from one to four was devised to evaluate the livers when birds were sacrificed. A score of one was a mahogany— colored "normal" liver; two was a mild degree of fat infiltration pro- ducing a slightly yellow color; three was a very fatty appearing liver, very yellow in color, but with no hemorrhages. If the livers had hemorrhages and/or scar tissue present, a score of four was given. Birds were sacrificed at different intervals in the various experiments, but all birds were evaluated in each experiment. Fatty liver incidence was much higher in the caged birds than in floor birds fed the same ration and under similar environmental condi- tions. Methionine supplementation of the basal high-energy diet was not effective in reducing the incidence of fatty livers in caged birds. A specific low—energy, high-fiber diet was very effective in preventing Thomas Lionel Barton fatty livers from developing. Most non—laying birds had normal appearing livers, indicating that hormonal balance is involved in this condition. Varying protein and energy levels were fed in one experiment and higher liver scores were obtained as the energy content of the diet was increased, Higher protein (18?) had no effect on the inci- dence of fatty livers. In an experiment concerning methods of correcting fatty livers once they had been induced, the low-energy, high-fiber diet was again very effective in correcting fatty livers. Fifteen percent of wheat bran and three percent of fish meal were also effective in this respect, but required a longer period of time than the low—energy diet. Choline, vitamin B and vitamin E showed very erratic results and a combination 12 of aureomycin and furazolidone was ineffective in correcting fatty livers. MOst of the mortality from fatty liver in these experiments occurred during periods of hot weather. One experiment in which birds were artificially heated to 32° C for 12 weeks showed that mortality was higher in a high temperature environment than in a cool environment (10 - 15.50 C). However, fatty liver incidence of living birds was just as high in the cool environment when fed a fatty liver producing diet. In a preliminary fractionation experiment, a water extract of wheat bran and its residue corrected the fatty livers to some extent. NUmbers of birds were not large in this experiment, but the results sug- gest possible factor(s) in wheat bran that correct fatty livers. Thomas Lionel Barton That the fatty liver syndrome is a nutritional problem is sug- gested by the fact that fatty livers were induced and/or corrected by diet manipulation. Hormonal balance is also a factor in this condition since most non-layers did not have fatty livers. FATTY LIVER STUDIES IN LAYING HENS BY Thomas Lionel Barton A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Poultry Science 1967 ACKNOWLEDGEMENTS The author wishes to express his appreciation to Dr. Philip J. Schaible, Professor of Poultry Science, for his encouragement, interest and guidance during this period of study and research and for his help- ful suggestions in the preparation of this manuscript. The author also appreciates the assistance of Drs. R. K. Ringer and H. C. Zindel of the Department of Poultry Science, Dr. Dorothy Arata of the Department of Foods and Nutrition, and Dr. D. A. Reinke of the Department of Pharmacology in planning the course of study and for their careful review of this manuscript. I would also like to thank Mr. Sulo Hulkonen for photography work, Mrs. Maryann Duke for liver fat analyses and other members of the Department of Poultry Science for their assistance. Finally, the author is indebted to his wife, Martha, for her encouragement, sacrifice and understanding, and to his two sons, James and Steven, for their added stimulus during this period of study and research. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . ii TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . v LIST OF APPENDIX TABLES . . . . . . . . . . . . . . . . . . . . vii INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . . . . 3 EXPERIMENT I Procedure . . . . . . . . . . . 17 Results and Discussion . . . . . . . . . . . . . . . . . . 23 EXPERIMENT II Procedure . . . . . . . . . . . . . . . . . . . . . . . . . 26 Results and Discussion . . . . . . . . . . . . . . . . . . 29 EXPERIMENT III Procedure . . . . . . 35 Results and Discussion . . . . . . . . . . . . . . . . . . 37 EXPERIMENT IV Procedure . . . . . . . . . . . . . . . . . . . . . . . . . 41 Results and Discussion . . . . . . . . . . . . . . . . . . 43 EXPERIMENT V Procedure . . . . . . 49 Results and Discussion . . . . . . . . . . . . . . . . . . 55 EXPERIMENT VI Procedure . . . . . . 57 Results and Discussion . . . . . . . . . . . . . . . . . . 60 iii Page EXPERIMENT VII Procedure . . . . . . . . . . . . . . . . . . . . . . . . . 68 Results and Discussion . . . . . . . . . . . . . . . . . . 70 GENERAL DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 71 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . 77 APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 iv Table 10 ll 12 13 14 15 l6 17 18 LIST OF TABLES Composition (Z) of experimental diets used in Experiment I Summary of data collected in Experiment I . Fat content of livers from birds sacrificed after 5 and 7 28-day periods Liver score values obtained in Experiment I . Average body weights by liver scores Experimental outline for Experiment II Composition of diet 7 used in Experiment II . Average egg production, feed consumption and final body weight obtained in Experiment II . Fat content of livers from birds sacrificed after 3 and 5 periods in Experiment II . . Liver scores obtained in Experiment II Average body weight by liver scores . Composition (Z) of low—energy diet used in Experiment III Summary of results for Experiment III . Liver scores, Experiment III Composition (Z) of experimental diets used in Experiment IV . Egg production (Z hen-day) obtained in Experiment IV . . . . . . . . Average feed intake (gms/bird/day) for Experiment IV. . . . . . . . . . Average body weight (gms) obtained in Experiment IV. . . . . . Page 18 20 21 22 25 27 28 30 31 32 34 36 38 39 42 44 45 46 LIST OF TABLES Cont'd. Table 19 20 21 22 23 24 25 26 27 28 29 30 31 Page Liver scores, Experiment IV . . . . . . . . . . . . . . . 47 Experimental outline for Experiment V . . . . . . . . . . 50 Percent hen—day egg production in Experiment V . . . . . 51 Feed intake (gms/bird/day) in Experiment V . . . . . . . 52 Body weight change (gms) in Experiment V . . . . . . . . 53 Average liver scores obtained in Experiment V . . . . . . 54 Experimental outline for Experiment VI . . . . . . . . . 58 Composition (Z) of diet 5 used in Experiment VI . . . . . 59 Egg production (percent hen—day) for Experiment VI . . . 61 Feed intake (gms/bird/day) for Experiment VI . . . . . . 62 Body weight changes (gms) in Experiment VI . . . . . . . 63 Average liver scores for Experiment VI . . . . . . . . . 64 Data collected in Experiment VII . . . . . . . . . . . . 69 vi LIST OF APPENDICES Table Page 1 Appendix Table I. Summary of data collected in Experiment I by 28—day periods . . . . . . . . . . . . . 81 2 Appendix Table 2. Summary of data collected in Experiment II by 28-day periods . . . . . . . . . . . . . 82 3 Appendix Table 3. Summary of data collected during the first 12 weeks in Experiment IV by 2-week periods . . . . . . . . . . . . . . . . . . . . 83 vii INTRODUCTION Many changes have occurred in the laying hen industry during the past 15 years. Feed efficiency has been improved by the addition of fat to the feed and substitution of corn for the more fibrous ingre- dients. Due to the competitive nature of the poultry industry, in- creasing the bird density has been one method of lowering production costs. A.much higher percentage of laying hens are now kept in cages because of economics. One problem that has developed in the midst of this progress is a condition called "fatty liver syndrome". In September, 1964, a conference concerning this problem was held at Michigan State University with various feed industry represen- tatives and members of the Department of Poultry Science participating. It was the opinion of this group that the fatty liver syndrome is a complex problem. It is quite general throughout the united States and Canada and may cause two percent mortality a month. Some of the in- dustry representatives had tried to produce the syndrome experimentally by using different levels of energy or different types of fat. These attempts were unsuccessful. It was the opinion of this group that fatty livers had to be produced experimentally so that a fundamental approach could be made. This study was initiated as a result of that conference. The objectives of this study were: (1) to try to create the fatty liver syndrome as it occurs under field conditions; (2) to determine whether 1 2 the condition is more prevalent in cage or floor pen operations and; (3) to study ways of correcting the fatty liver condition once it had occurred. REVIEW OF LITERATURE The pathogenesis of fatty liver is complex. Popper and Shaffner (1957) stated that excess fat deposition in the liver results from a disturbance of one or more pathways of the movement of fat to and from the liver. These are: (1) deposition of dietary fat; (2) mobilization of fat from fat depots; (3) transformation of dietary carbohydrate and protein into fat; (4) lipogenesis from the metabolic pool; (5) oxidation of fat in the liver; and (6) release of fat to the depots. Hershey (1930) discovered that egg-yolk lecithin could replace raw pancreases in the diet of the depancreatized dog and restore liver fat levels to normal. Best_g£_§l: (1932) reported that varying amounts of crude or purified lecithin prepared from egg yolk or beef liver also prevented an increase in liver fat of normal rats fed saturated fat in the ration. The active ingredient in lecithin was reported to be choline and this was further substantiated by the same group later that year (Best and Huntsman, 1932a). In a report by Gavin and MbHenry (1941), fatty liver produced in rats by feeding beef liver fraction was only slightly affected by giving choline, but was completely prevented by the simultaneous administration of lipocaic. Inositol prevented the development of the fatty liver and the accumulation of cholesterol in the liver. They suggested that the action of inositol resembles that of lipocaic. Similarly, Abels_g£nal, (1943) reported reduced liver fat of human patients with carcinoma of the gastro-intestinal tract who each 3 4 received eight grams of lipocaic during the night prior to laparotomy. The lipotropic properties of the lipocaic could be accounted for by its content of inositol alone. Amdur_g£ugl. (1946), in studies on the effect of manganese and choline on bone formation in the rat, found that manganese as well as choline prevented the deposition of excess fat in the liver. At a given level of choline, more fat was observed to be present in the livers of manganese-deficient rats than in the livers of rats receiving adequate manganese. The lipotropic action of manganese was much greater when the choline content of the diet was low, thus indicating an inter- action between manganese and choline. Singal_g£ El. (1949), while studying the nicotinic acid-tryptophan relationship in rats fed a nine percent casein diet containing five per- cent fat and 0.2 percent cystine and choline, observed yellowish livers at autopsy. Upon analysis, the liver lipids were 16 percent. Supple- mentary nicotinic acid, tryptophan, valine, histidine, phenylalanine or serine were without effect. The addition of threonine reduced the liver lipids to 5.1 percent. Harper 35 a1. (1954) failed to obtain greater deposition of fat in the liver of rats by increasing the amount of fat in the diet. This indicated that dietary fat Ee£_§g_did not accumulate to any great extent in the livers of the animals used in this study. Since the amount of fat which was deposited in the liver was decreased slightly as the amount of dietary fat was increased, it appeared that the liver fat arose from the conversion of carbohydrate or protein to fat. Addi- tionally, evidence supporting this conclusion was obtained from the 5 observation that high fat deposition occurred in the livers of rats receiving fat-free diets. Yoshida and Harper (1960), using C14 labeled acetate and palmitate, reported that stimulation of fat synthesis accompanied the fat accumula— tion in the livers and carcasses of rats fed a low protein diet deficient in threonine. The effects of choline deficiency were less clear-cut. They included increased fat synthesis in the liver, but not in the car— cass, and some evidence of impaired transport of fat from the liver. In a later report, Yoshida_e£flal. (1961) found that a moderate restric- tion of caloric intake (70Z of controls) prevented the development of fatty livers in rats fed a threonine-deficient diet, but an equivalent restriction of caloric intake did not prevent the accumulation of liver fat in rats fed a choline—deficient diet. Similarly, Donovan and Balloun (1955) reported that chicks con- suming a high-fat (lOZ) diet accumulated no more fat in their livers than those consuming a low-fat (lZ) diet. They concluded that dietary fat £e£_se_was not deposited in the chick liver to any great extent. Shils and Stewart (1954a) observed that rats subsisting on a diet containing 76 percent corn and three percent or less casein devel— oped a fatty liver Characterized by initial and preponderant accumula- tion of lipid in the portal areas. Rats of the same strain on a diet in which the only protein was casein developed a centrolobular type of fatty liver. Methionine, choline and vitamin B were able to decrease 12 the liver lipid. However, methionine was rarely capable of preventing the deposition of some excess fat, while under certain conditions, the lipotropic action of vitamin B was unpredictable. Later, Shils gt 31. 12 (1954) reported that a portal type of fatty liver was rapidly and 6 consistently produced in stock weanling rats by feeding diets in which the protein was exclusively of plant origin (corn, rice, wheat or cassava). Within one week on a corn meal diet excess fat appeared in the portal region of the liver lobule and increased in amount over the 12-week period of study. Shils and Stewart (1954b) found that DL—tryptophan (0.5Z) and L-lysine (lZ), alone and together, greatly reduced or prevented the accumulation of hepatic fat usually seen on the corn meal diet. Threo— nine had little or no effect. Additionally, Vennart 35 a1. (1958) re- ported that the type of fatty liver produced by corn diets was reversed by the addition of lysine and tryptophan. In no instance was there any demonstrable permanent damage. They suggested a possible relationship between protein and lipid metabolism that has not yet been elucidated. Gyorgy_gt El: (1951) reported that a delay in the production of hepatic necrosis in rats fed a low choline diet was obtained from aureomycin and, to a lesser extent, from terramycin and streptomycin. Chloromycetin, polymixin and penicillin were not protective. The beneficial effect of aureomycin was not limited to the delay of hepatic necrosis but manifested itself also in the prevention of hepatic cirrhosis in rats fed a low-protein (casein)—high-fat diet. They sug— gested an antimicrobial effect on the intestinal flora. Baxter and Campbell (1952) found that the renal lesions and mortality of rats caused by a purified diet deficient in choline were largely prevented by supplementing the diet with rather high levels of crystalline aureomycin. Fatty changes in the liver also appeared to be reduced to some extent. The levels of choline in liver and kidney 7 tissue and in feces of deficient animals appeared to be increased slightly as a result of aureomycin administration. Di Luzio and Zilversmit (1956) reported that the triglyceride concentration in livers of dogs maintained for three weeks on a high— fat, low-protein, choline-deficient diet was lowered by daily injections of heparin and by supplementation of the diet with choline. Oral ad- ministration of aureomycin (0.5 grams twice daily) increased liver triglyceride concentrations. One gram of aureomycin per day fed to dogs on normal rations was without any visible or chemical effect on liver lipids. Rutenburg_g£”gl. (1957) also studied the role of intestinal bacteria in the development of dietary cirrhosis in rats. They found that absorbable broad spectrum antibiotics added to the daily diet did not prevent the development of fatty infiltration, but they delayed the development of cirrhosis for about 100 days more. Non-absorbable antibiotics added to the daily diet prevented the development of cirrhosis in most rats for as long as 750 days but did not prevent fatty infiltration. Since non-absorbable antibiotics were superior to absorbable antibiotics, the authors excluded systemic activity of the antibiotic as having anything to do with the protective effect. They asserted that cirrhosis in rats on a choline—deficient diet is caused by intestinal bacteria and not by the choline deficiency. Leevy (1962) reported on observations in 270 human patients with fatty liver demonstrated by percutaneous liver biopsy in a large munici- pal hospital. Seventy-seven percent of the patients were alcoholic. Similar degrees of hepatic steatosis were encountered in non-alcoholic patients with a variety of diseases, diabetes and heart failure being 8 most frequent in this series. Dietary abnormalities were present in each of the patients with fatty liver and appeared to play a central role in its evolution. All dietary regimes tested including a general hospital diet, a high—fat diet, or a low-protein, low-choline diet were associated with elimination of all liver fat in the malnourished alcoholic in 4 to 6 weeks. Couch (1956) first reported the fatty liver syndrome in laying hens. He stated that the syndrome was first called to his attention by veterinarians at the Poultry Diagnostic Laboratory of the School of Veterinary Medicine, Texas A & M University who stated that the syndrome had first been observed in late 1954. The symptoms were described as follows; "The hens usually in— crease in weight from 25 to 30 percent, and the rate of egg production decreases approximately one-third. The birds appear to be healthy and in good condition. A post-mortem examination reveals an excess of abdominal fat, fatty livers, capillary hemorrhages in the liver and hematoma." The author stated that the syndrome may occur as a result of increased strain brought on by such developments as improved breeding and higher egg production, increased energy content of feeds and the caged layer system of management. For prevention, it was recommended that laying mash formulas of the high—energy type have a minimum of 500 grams of added choline, 12 milligrams of vitamin B12 and 5,000 to 10,000 units of added vitamin E per ton and that the ration contain at least 17 percent protein. Shortly thereafter, Fisher and Weiss (1956) in a preliminary re- port, stated that a similar, if not identical, condition to the fatty liver disease was first induced in their laboratory during the fall of 9 1955. Animal fat was the variable in their studies with the diets well fortified with choline, vitamin B 2, methionine and higher than normal 1 protein levels. Young pullets fed a ten percent animal fat diet for four months after the start of egg production showed a significant in— crease in the lipid and cholesterol content of the blood with fatty encapsulation and infiltration of the kidneys. Fatty livers, while observed to a larger extent in fat—fed birds, also occurred with remarkable frequency in well-laying pullets on fat- free diets. No heavy fat deposits were noticeable in these young birds. 0n the other hand, quite heavy visceral fat deposits were observed after only eight weeks on a five percent animal fat ration when the birds used were over one year old and thus at the end of their first laying season. In view of the high levels of choline, protein and vitamin B12 in the animal-fat containing diets, they concluded that these nutrients were not helpful in alleviating or preventing this condition. A few months later in a more extensive report, Weiss and Fisher (1957) reported that, depending on the age of birds and the duration of feeding trials, 5 to 10 percent of added fat to laying rations resulted in the development of an apparent derangement in lipid metabolism characterized by one or more of the following symptoms: elevated plasma cholesterol and total plasma lipid; excess deposits of body fat; friable and fatty livers; fatty deposits in and around the kidneys; and greater severity of aortic atherosclerosis. The incidence of abnormal appearing livers and kidneys, as deter- mined by gross observation at autopsy, was approximately twice as high in the fat supplemented birds as in control birds. Abdominal fat 10 scores indicated a significant deposition of excess body fat in the fat supplemented birds. Histological examination of livers and kidneys from some of the treated and control birds revealed evidence of fatty metamorphosis in both organs. However, the incidence and severity seemed about the same in the liver of the two groups and only slightly greater in the kidneys of the treated birds. Sutton 35 31. (1957) found that mature White Leghorn hens housed in individual cages and fed normal low-fat diets exhibited a tendency to increase liver fat deposition. The addition of one percent cholesterol to this diet increased their blood cholesterol level materially and also the total liver fat. Choline chloride (lZ) fed with cholesterol exerted a lipotropic action and was effective in reducing both the blood choles- terol level and the total liver fat. Methionine (lZ) reduced blood cholesterol levels but did not reduce total liver fat. Donaldson and Millar (1958) reported on the use of 3 percent added animal fat in a laying ration containing 19 percent crude protein. Fat addition over a 350-day test period resulted in significantly poorer hen-housed egg production and feed conversion but greater energy con- sumption, body weight gains and mortality during the laying year. The entire increase in mortality was associated with obesity and/or fatty degeneration and hemorrhage in the liver and kidneys. Turk 25 a1. (1958) fed diets ranging from 690 to 1210 Calories of productive energy per pound during the growing and laying periods. Energy level did not affect egg production. Hens fed more energy deposited large amounts of fat in the viscera and liver but this could be removed by a cool environment and a low-energy ration. ll MtDaniel gt 31, (1957) studied the effect of dietary caloric density in relation to protein level on the plasma lipids and liver fat of caged layers. Liver fat values, expressed on a dry weight basis, were found to be higher in caged birds as compared to birds in floor pens. On a given diet, non-laying birds showed more liver fat accumu- lation than did birds in production. Among birds maintained on litter, this was not the case. The non—layers on litter exhibited the same or lower liver fat when compared to the layers. Liver fat was increased slightly with an increase in energy content of the diet and decreased slightly with an increase in dietary protein at a given calorie level. Later, MbDaniel e£_§l, (1959) reported on the effect of different dietary protein and calorie levels in the presence of dietary fat on performance and on the occurrence of the fatty liver syndrome, obesity and atherosclerotic conditions in caged layers. Caloric consumption per hen per day differed appreciably between the low and higher energy levels. Liver fat values were found to be similar except in a 15 per— cent protein, 935 Calorie productive energy/lb ration that contained no added fat. Liver fat values were about double in this treatment. All diets tested, except a 15 percent pretein - 750 Calorie diet, were scored as producing a high incidence of fatty livers. The presence of added fat in the diet, either from an animal or vegetable source, did not appear to influence the fatty liver condition. Excessive deposition of abdominal fat was observed in approxi- mately the same degree and incidence as the fatty liver condition. Abdominal fat appeared to be closely related to the increase in body weight. Fatty encapsulation of the kidneys was observed in all hens with normal to excessive amounts of abdominal fat. This condition was 12 considered to be normal in that the kidney itself showed no fat infil— tration, as evidenced by ether extraction and histological examination. A highly significant correlation was obtained between egg production and percent liver fat. Treat_et El. (1960) also reported that liver fat and liver weight of caged layers were higher in treatments with no added fat than in any of the groups receiving added fat. Donaldson and Gordon (1960) added three percent animal fat to laying hen diets in a floor pen experiment and reported that all fat- fed groups gained more body weight than the controls, but the gains were appreciably greater in heavy as compared to light breeds. Added fat resulted in higher mortality with heavy breeds but not with light breeds. March and Biely (1962) studied mortality in White Leghorn pullets subjected to three dietary regimens under which the birds received 2.5, 7.5 or 12.5 percent of fat from the time of hatching until they were three years old. Mortality from liver derangement was approximately doubled when either 7.5 or 12.5 percent fat was fed but no other unto- ward effects on mortality rate were noted. In a later study, March and Biely (1963) introduced supplementary fat into the diet of mature birds which had been fed a ration without added fat. There was no effect on mortality attributable to the level of fat. ' Ringer and Sheppard (1963) reported the occurrence of fatty liver syndrome in a Michigan caged layer operation. Postmortem examination revealed excessive fat lining all the visceral organs, an enlarged and very friable liver of a light yellowish-brown color indicative of fat infiltration, a massive hemorrhage from the liver and multiple antemortem 13 hematomas of the liver. Egg production, based on a percentage of birds surviving did not drop below that for birds of an equal age. The addi— tion of choline chloride to the diet did not alleviate the condition. Couch (1964) reported on attempts by veterinarians to transmit the fatty liver condition to normal laying hens through the feeding of fecal material, transplantation of respiratory discharge and through the injection of blood from affected birds into normal ones. All such attempts were without effect. It was recommended at this time that treatment of fatty liver include 12 milligrams of vitamin B 1,000 12’ grams of choline chloride and 10,000 units of vitamin E per ton added on top of the current micronutrient fortification for four weeks along with furazolidone at 200 grams per ton for two weeks and at 50 grams per ton for two weeks. Summers_etual. (1966) recommended that if a flock becomes affected with fatty liver syndrome, 2 ounces of copper sulfate, 500 grams of choline, 3 milligrams of vitamin B and 5,000 I.U. of vitamin E should 12 be added per ton of laying diet. Increasing the level of dietary pro- tein by one to two percent was also reported to help alleviate the condition. Quisenberry (1966) added iodinated casein (Protamone) at 100 and 200 grams per ton to diets containing either 937 or 1,051 Calories of Productive Energy per Pound. One hundred grams of Protamone decidedly reduced the visual fat scores of the livers taken from birds on autopsy at the end of the experimental period of eleven months. In spite of the lower liver fat score the mortality of the Protamone—fed birds was con- siderably increased. Liver scores from birds fed 200 grams Protamone per ton were higher than those of birds fed 100 grams per ton but less 14 than livers of birds from the unsupplemented diets. The reason for this was not clear. It was stated that solution of the fatty liver problem has been delayed because of the inability to create the condi- tion at will. Sunde (1966) fed pullets in floor pens on eight different energy levels ranging from 848 to 1190 Productive Energy Calories per pound and, after five months of production, five birds from each pen were killed. The livers were scored for fatty livers by visual means and later analyzed for fat content. As the energy level increased, the liver fat increased although a plateau seemed to occur during the middle range of energy levels. Liver scores determined visually also seemed to increase. Visual scores were not as dependable as would be desired due to the range in fat content for each visual score. By determining moisture content of the livers, percent solids was a better guide to the liver fat content than a visual score. However, the visual fat score was obtained much faster. The hens that remained in each experimental group after the first phase of the experiment was concluded were placed on a low-energy diet in a cool environment. Several weeks later additional hens were killed. At this time, all average liver weights had decreased and liver fat had also dropped to much lower levels. Data were presented which indicated that laying hens usually have higher fat in their livers than non- layers. The author reported inability to reproduce the fatty liver con- dition in the laboratory as it occurs in field cases. Thornton and MtPherron (1962), in an effort to alleviate exces- sive body and liver fat often observed in chickens maintained for long periods of time in cages, added twenty percent brewers dried grains to 15 a normal ration at the expense of corn, milo and soybean meal. The gain in body weight and liver fat accumulation during the last fifty weeks was less in the experimental birds to a highly significant degree. The experimental birds also grew faster, matured earlier and produced a greater number of eggs. The results suggested that brewers grains may either contain an unknown factor or that the biological availability of recognized nutrients is greater. Heald (1963) reported that the metabolism of carbohydrate by slices of liver from the domestic fowl is quantitatively different from that of mammalian liver in several ways. Slices of liver from the domestic fowl metabolized fructose with an increased oxygen uptake, in contrast with mammalian liver where fructose metabolism does not in— crease the oxygen uptake. Determination of the respiratory quotient, R.Q., showed that slices of fowl liver from fed birds metabolized fructose with an R.Q. of 1.10, and occurred in both laying and non— laying pullets. The increased R.Q. was not found with slices of liver from birds starved for 18 - 24 hours. Heald and Rookledge (1964) found that estrogen treatment in— creased the levels of plasma-free fatty acids simultaneously with those of the total lipids and lipophosphoprotein in the immature fowl. Tes- tosterone and thyroxine decreased the levels of plasma lipids and of lipophosphoprotein in the laying fowl, but had no effect upon the levels of free fatty acids in either the immature or laying fowl. Gonadotrophins were without effect upon the plasma free fatty acids in the immature pullet but increased the levels in laying or molting birds. They considered the increases in plasma free fatty acids found when 16 the bird comes into lay to be an indirect result of ovarian stimulation by pituitary hormones. Hawkins and Heald (1966) reported that slices of liver from the domestic fowl, when incubated in media containing Cl4 palmitic acid, incorporated the isotope into the neutral fat fraction, principally triglycerides. Both on a dry weight basis and a DNA basis, slices of liver from the laying hen incorporated more palmitate into the neutral lipid fraction than did slices from immature birds. Treatment of immature birds with estrogen enhanced the ability of the liver to in— corporate palmitate into the neutral lipid fraction. Based on their data, it was calculated that the liver of the laying hen synthesizes 15 to 25 times as much lipid per unit time as does that of immature birds when compared on a cellular basis. Interestingly, in a report by Leach_e£H31, (1966) on the choline requirements of chicks and hens, it would appear that the hen is able to synthesize substantial amounts of choline providing there are suf- ficient methyl groups present in the diet. Most responses to dietary choline were achieved with marginal levels of methionine. The limiting factor in the hen's ability to synthesize choline appears to be in the formation of methyl groups. Thus, the hen differs from the young chick, which cannot synthesize choline even in the presence of adequate methyl groups. Therefore, the objectives of this study were: (1) to try to create the fatty liver syndrome as it occurs under field conditions; (2) to determine whether the condition is more prevalent in cage or floor pen operations; and (3) to study ways of correcting the fatty liver condition once it had occurred. EXPERIMENT I Procedure: This experiment was conducted with birds in floor pens. All subsequent experiments were conducted in cages. The birds used, as in all experiments in this thesis, were a commercial strain of Single Comb White Leghorns. In this experiment the birds were raised to 21 weeks of age using standard procedures of the Michigan State University Poultry Science Research and Teaching Center. At this time, the birds were transferred to the laying house and randomly distributed into the pens, eliminating the large and small extremes in size and any obvious culls. Wing badges were applied for identification purposes and "Specs" to prevent cannibalism. Twenty—four pens of 25 birds each were fed the experimental diets shown in Table 1. Each diet was fed to six pens. Diet 1 is a commercially available, all-mash laying formula that has experienced some problem with the fatty liver syndrome under field conditions. All birds were fed this diet from 21 to 26 weeks of age when the experiment started. This formula is available in a concentrate and can be mixed by using 75 percent ground corn, 20 percent concentrate and 5 percent ground limestone. This diet will be referred to in this and following experiments as the basal diet. When the experiment started, six pens of birds were changed to diet 4 in which 12 milligrams of vitamin B 5,000 I.U. of vitamin E 12’ 17 18 Table 1. Composition (Z) of experimental diets used in Experiment I Ingredient Diet 1 Diet 2 Diet 3 Diet 4 Ground yellow corn 75.00 71.00 67.50 75.00 Soybean meal (50Z) 11.90 15.90 16.36 11.90 Alfalfa meal (17Z) 0,90 0.90 1.24 0.90 Meat and bone meal (SOZ) 2.20 2.20 3.02 2.20 Fish meal (60Z) 0.40 0.40 0.55 0.40 Dried fish solubles 0.10 0.10 0.14 0.10 Feather meal 0.20 0.20 0.27 0.20 Blood meal 1.00 1.00 1.38 1.00 Dicalcium phosphate 1.40 1.40 1.92 1.40 Ground limestone 5.85 5.85 6.17 5.85 Salt 0.35 0.35 0.48 0.35 Fat (animal and vegetable) 0.50 0.50 0.69 0.50 Vitamin-trace mineral premix 0.20* 0.20* 0.28 0.20* Methionine hydroxy analogue, Ca —- —- 0.05 -- Vitamin B12 -- -- -- 600 mcg. Vitamin E -- -- —- 250 I.U. Choline __ -- _ -- -- 50 gms. Total 100.00 100.00 100.00 100.00 Z Protein (calc.) 15.20 16.84 17.70 15.20 Metab. energy (Cal/kg) 3004 2986 2905 3004 * Supplied the following per kg. of diet: Vitamin A, 4400 IU; Vitamin B12, 3.52 mcg; Vitamin D3, 1198.5 ICU; Riboflavin, 2.64 mg; Niacin, 17.0 mg; Calcium pantothenate, 4.8 mg; Choline, 172 mg; BHT, 11.35 mg; Methionine, 74.9 mg; Vitamin K, .84 mg; Manganese, 80 mg; Iodine, 0.8 mg; Iron, 16 mg; Copper, 4 mg; Zinc, 40 mg; Cobalt, 4 mg. 19 and 1,000 grams of choline chloride were added per ton of basal diet. Eighteen pens of birds remained on the basal diet to allow further changes as the experiment progressed. After 12 weeks, six pens of birds were changed from diet 1 to diet 2. In diet 2, 4 percent soybean meal (50Z protein) was substituted for corn. After 20 weeks, six pens of birds were changed from diet 1 to diet 3. Diet 3 was formulated by increasing the percentage of concentrate to 27.5 percent of the ration and adding .05 percent methionine hydroxy analogue. Data were recorded for each pen by 28-day periods and included hen-day production, feed consumption, and body weight. These data are shown in Table 2 by periods at which ration changes were made. Data for each 28-day period are shown in Appendix Table 1. One bird was sacrificed from each pen after 20 and 28 weeks on test for liver fat determination and observation. These data are shown in Table 3. Due to the large numbers of birds involved in this and subsequent experiments and the time required to perform liver fat analyses, a scoring system was devised to evaluate the livers when birds were sacrificed. The scores were from one to four with a score of one being a mahogany-colored "normal" liver. A score of two was indicative of a mild degree of fat infiltration being slightly yellow in color. A score of three was an indication of a very fatty appearing liver, very yellow in color but with no hemorrhages. If the livers had hemorrhages and/or scar tissue present, then a score of 4 was used. After ten 28- day periods, all remaining birds were sacrificed and the livers visually scored for incidence and degree of fatty livers. Liver score values are presented in Table 4. 20 Table 2. Summary of data collected in Experiment I* T 4-week periods Treatment* 0 - 3 4 - 5 6 - 10 Overall' Average egg production (Percent hen-day) 73.4 72.5 67.6 70.5 74.0 72.0 68.7 71.2 71.1 69.4 73.1 71.6 74.0 70.9 68.2 70.7 Average feed consumption (gms/bird/day) 1 105 113 103 105 2 108 114 105 108 3 105 113 107 105 4 106 111 103 106 Average body weight (grams) Initial 1730 1675 1734 1816 1703 1643 1730 1798 1707 1639 1716 1857 1712 1634 1716 1789 * Each diet is an average of 6 pens of 25 birds each. 21 Table 3. Fat content of livers from birds sacrificed after 5 and 7 28—day periods Percent fat (wet weight) after Treatment 5 28-day periods 7 28-day periods 1 15.5 1'. 2.8* 19.2 i 5.0 2 11.6 i;l.8 11.8;: 1.7 3 11.1 i 3.0 11.4 i 1.7 4 10.9 i 1.2 14.4 i 2.8 * Mean i standard error. Each mean is an average of 6 birds. 22 Table 4. Liver score values obtained in Experiment I Liver score expressed as Z of treatment Average No. liver Treatment birds No. 1 No. 2 No. 3 No. 4 score 1 115 50 35 15 0 1.66 2 106 73 24 3 0 1.31 3 104 83 9 8 l 1.27 4 114 79 17 3 1 1.28 23 Pen average hen-day egg production, feed consumption and body weights were subjected to analysis of variance (Snedecor, 1956) and Duncan's multiple range test (Duncan, 1955) was used where applicable. Results and Discussion: The basal diet used in this experiment was a high energy diet with most of the energy supplied from carbohydrate sources. With this energy level, the 15.2 percent protein content was considered marginal. At the end of four weeks, birds on all treatments had lost weight (Appendix Table l). The weight loss continued through the third 4—week period of production. During the second 4-week period, a drop in egg production was noted. During the early stages of lay, body weight usually increases rapidly for 6 to 8 weeks and then increases gradually until mature body weight is reached. Likewise, egg production should rapidly increase until peak production is attained at approximately 32 — 34 weeks of age. The weight loss and subsequent drop in egg production observed in the early weeks of this experiment could have been due to inadequate protein intake. Therefore, the protein content was increased in diet 2 after 12 weeks and in diet 3 after 20 weeks of the experiment. There were no significant differences between treatments for egg production, feed consumption or body weights. The response in egg pro- duction when birds were switched to diet 3 after 20 weeks on test (Table 2) approached but was not significant at the five percent level. Two birds on each of diets 1, 2 and 4 were diagnosed as having died from a fatty liver during the eighth and ninth periods. This was during July and August, 1965. Couch (1956) reported increased mortality 24 in warmer weather and the mortality due to fatty liver in this experi— ment would tend to confirm this. However, when all remaining birds were sacrificed after forty weeks on test, there was not much evidence of fatty liver occurrence. As shown in Table 4, only two birds had hemorrhages present in their livers. Most of the livers were considered normal. Since Couch (1956) reported that birds with fatty liver are usually 25 to 30 percent heavier than normal birds, final body weights obtained in this experiment were plotted by liver scores. These data are presented in Table 5. As body weight increased, the birds tended to have higher liver scores. However, the birds in this experiment were smaller, on average, than would normally be expected of birds of this strain. Likewise, the birds from diet 3 were larger than the other treatments and yet had a lower average liver score. 25 Table 5. Average body weights by liver scores No. Treatment birds No. 1 No. 2 No. 3 No. 4 l 115 1739 1866 1948 -- 2 106 1775 1884 2102 -- 3 104 1798 1784 2229 2225 4 114 1757 1898 2102 2088 EXPERIMENT II Procedure: The purpose in starting this experiment nine weeks later than Experiment I was to take advantage of any early observations that developed as Experiment I progressed. Since the birds used in Experi— ment I experienced a weight loss and drop in egg production, it seemed desirable to add methionine to one diet (Diet 6). Supplemental vitamin E was added since Couch (1956) reported that it was beneficial in pre- venting fatty livers. Ten percent oats were substituted for corn as a method of lowering the energy content of the ration. Experiment II was conducted in cages with birds of the same population as those in Experiment I. The birds were placed in in- dividual 8 x 16-inch cages at 21 weeks of age and fed the basal diet designated as diet 1 in Experiment I until the experiment started when the birds were 35 weeks of age. Egg production (hen-day), feed con- sumption and body weight were measured by 28-day periods during the 28- week experiment. Four groups of 8 birds were fed each experimental diet and the experimental outline is shown in Table 6. The composition of diet 7 is shown in Table 7. One bird from each treatment was sacrificed after 3 and 5 28—day periods on test for liver fat analysis and observation for fatty livers. This corresponds to the fifth and seventh period in Experiment I. At the end of the test all remaining birds were sacrificed and livers were scored for fatty liver incidence according to the system described 26 27 Table 6. Experimental outline for Experiment II Diet 7 Supplement 5 Basal diet as in Experiment I 6 Diet 5 + 0.1Z methionine hydroxy analogue 7 See Table 7 8 Diet 7 + 10,000 I.U. vitamin E/ton 9 Diet 7 with 10Z oats replacing corn 28 Table 7. Composition of diet 7 used in Experiment II Ingredient Percent of ration Ground yellow corn 66.85 Soybean meal (50Z) 17.50 Fish meal (57Z) 3.00 Alfalfa meal (17Z) 2.00 Animal fat 1.50 Ground limestone 7.00 Dicalcium phosphate 1.50 Salt 0.35 Methionine hydroxy analogue, Ga 0.05 Vitamin-trace mineral premix 0.25* Total 100.00 Percent protein (calc.) 16.8 Metabolizable energy (Cal/kg) 2944 * Supplied the following per kg of feed; Vitamin A, 6,600 I.U.; Vitamin D3, 1,650 ICU; Vitamin E, 8.25 10; Riboflavin, 4.4 mg; Niacin, 22 mg; d-Pantothenic acid, 7.04 mg; Choline chloride, 220 mg; Vitamin 312, 11 mcg; Folic acid, 0.275 mg; Menadione sodium bisulfite complex, 2.2 mg; BHT, 124.7 mg; Manganese, 60 mg; Zinc, 27.5 mg; Iodine, 1.2 mg; Iron, 20 mg; Copper, 2 mg; Cobalt, 0.2 mg. 29 in Experiment 1. Average egg production, feed consumption and final body weight data are shown in Table 8. Data for these measurements are presented by 28-day periods in Appendix Table 2. Liver fat analyses for birds sacrificed after 3 and 5 28-day periods are shown in Table 9. Fat content of livers from birds fed a low—energy, high—fiber diet from an unrelated experiment are also presented in this table. Average liver scores and the percentage of each treatment having the various scores are presented in Table 10. Results and Discussion: The birds used in this experiment became considerably larger than those in Experiment I. These birds apparently did not experience a weight loss during the early stages of production and thus were consid- erably larger at 35 weeks of age than the birds from Experiment I (See Appendix Table 2). There were no statistical differences between treatments for egg production, feed consumption or final body weight when the entire experimental period was considered. Egg production was higher at the start but lower at the end of this experiment than in Experiment I. Egg production was lower for the entire experiment than in Experiment I, but it should be pointed out that peak production had occurred before this experiment started and thus is not included. Birds sacrificed at the end of the third and fifth periods (equivalent to the 5th and 7th periods in Experiment I) had higher liver fat content than did the birds in Experiment I (See Table 9). Liver fat content was higher after five periods than at the earlier interval. Four birds that died during the fifth period were diagnosed as having 30 Table 8. Average egg production, feed consumption and final body weight obtained in Experiment II* Egg Feed production consumption Final body Diet (Z Hen-day) (gms/bird/day) weight (gms) 5 65.8 109 2007 6 66.6 113 2106 7 69.9 109 2116 8 68.9 108 2070 9 69.6 109 2052 * Each diet is an average of four groups of eight birds. 31 Table 9. Fat content of livers from birds sacrificed after 3 and 5 periods in Experiment II Percent fat (wet weight) Diet 3 periods 5 periods 5 21.9 22.6 6 26.8 42.1 7 13.3 19.4 8 30.7 39.1 9 24.8 30.1 Low-energy, high-fiber 5.8 9.8 II II II II 7. 2 6 9 H H II N 17. S 32 Table 10. Liver scores obtained in Experiment II Liver score expressed as percent of treatment Average Treatment liver score No. 1 No. 2 No. 3 No. 4 5 2.79 13 25 33 29 6 2.46 21 29 32 18 7 1.88 40 40 12 8 8 2.10 34 35 l7 l4 9 2.04 35 35 23 7 33 died from a fatty liver (2 birds were from diet 5 and one each from diets 8 and 9). This was during June and the early part of July which was slightly earlier than the fatty liver mortality experienced in Experiment I. The mortality during this period and the fat contents of sacrificed birds indicate that fatty liver was present during this time and of greater severity than in Experiment I. Liver fat levels of some birds fed a low—energy, high-fiber diet in an unrelated experiment were much lower than those of birds sacrificed in this experiment. This observation was the basis of Experiment III 'and will be discussed later. When all remaining birds were sacrificed at the end of the experi- ment, liver fat scores were much higher than those obtained in Experiment I (See Table 10). Twenty-nine percent of the birds fed the basal diet had hemorrhages present. Supplementation of the basal diet with methio- nine had little effect, but the average liver score of birds fed a higher quality, higher protein diet (Diet 7) were considerably less than those from the basal diet. There was no effect from supplemental vitamin E or the substitution of ten percent oats for corn. Average body weights were again plotted for each treatment by liver scores and are presented in Table 11. As body weight increased, the average liver score also increased. However, this was only true within treatments since the birds fed diet 7 were larger at the end of the test but had lower liver scores. The results of this experiment indicate that fatty liver is more prevalent in cages. Consequently, all subsequent experiments were con- ducted in cages. 34 Table 11. Average body weight by liver scores Treatment No. 1 No. 2 No. 3 No. 4 5 1740 2012 1901 2140 6 1869 1969 2260 2360 7 1875 2293 2194 2315 8 1870 2179 2252 2247 9 1851 2017 2285 2360 EXPERIMENT III Procedure: As mentioned previously, it was observed that birds from an un- related experiment fed a low—energy, high-fiber ration did not develop fatty livers. Experiment III was conducted, therefore, to compare this low-energy, high—fiber ration with the basal diet used in Experiments I and II. Twenty-four groups of four birds each that had been fed the low-energy ration for approximately six months of production were ran- domly distributed into 12 x l8-inch cages. TWelve groups of four birds each were fed the basal diet and a like number of birds remained on the low-energy, high-fiber diet. In this experiment, the basal diet was designated as diet 1 and the low-energy, high-fiber diet was designated as diet 2. Diet 2 was quite different in composition from the high~ energy basal but for purposes of discussion in this study, will be referred to as the low-energy, high-fiber diet. The objective of this experiment was to determine if fatty livers could be created in a relatively short time during the summer months. The experiment started June 30, 1965 and was conducted for a period of 14 weeks. Feed consumption, body weight and hen-day egg production were measured by two-week periods. At the end of 14 weeks all birds were sacrificed and livers were scored for fatty livers as described in Experiment I. The composition of the low-energy, high-fiber diet is presented in Table 12. Egg production, feed consumption and body 35 36 Table 12. Composition (Z) of low-energy diet used in Experiment III Ingredient Percent Ground yellow corn 34.5 Ground oats 20.0 Wheat bran 15.0 Wheat middlings 10.0 Alfalfa meal (17Z) 3.0 Dried skim milk 2.0 Fish meal (60Z) 2.5 Meat and bone scraps 3.0 Soybean meal (44Z) 2.5 Oyster shell flour 5.0 Steamed bone meal 1.5 Salt 0.6 Fish oil (2000 A; 400 D) 0.4 Total 100.0 Percent protein (calc.) 14.8 Metabolizable energy (Cal/kg) 2359 37 weight data are presented in Table 13 and liver score results are presented in Table 14. Results and Discussion: The low—energy diet used in this experiment was formulated in the mid 1930's and the only supplemental vitamins added were vitamins A and D from the fish oil. It also contained relatively large amounts of oats, wheat bran and wheat middlings, which are of lower energy content than corn. When birds were switched from this diet to the high-energy basal diet used in Experiment I, they did not readily adjust to the higher energy content by lowering feed intake (Table 13). During the third two—week period, birds fed the high-energy basal diet consumed less feed than birds remaining on the low-energy diet and this trend was maintained for the remainder of the experiment. Feed intake differences over the entire experimental period approached but were not significantly different (P < 0.05). Thus, birds fed the high-energy basal diet con- sumed approximately 55 M.E. Calories more per day than birds fed the low-energy diet. Body weight was a reflection of energy intake and thus birds fed the basal diet were heavier at the end of the test than birds receiving the low—energy diet, but this difference was not statistically significant (P < 0.05). No significant differences were observed between treatments for egg production, although birds switched to the high—energy basal diet laid at a lower rate. When all birds were sacrificed at the end of the test, there was a wide difference in the average liver scores of the two treatments. Six of the livers from the birds fed the high-energy basal diet had 38 moan meN moNa amNN mmNH HQNN Essa mass N soma Nash waN Nmma News owma amma «men a Housmawuomxoloum Amawv uszoS mmom o.maa was NNH mNH NNH and NEH NNH Aumsam-nwas .awumam-3oav N N.oNN as «as «as mod ooh «NH mNN Aammms .Nwsssm-ewaev a Ammv\nufin\mawv oxmuufi moom m.HN e.No m.NN N.mo q.NN m.NN m.NN m.oN Aumsam-ewas .smnmamasoav N o.No H.Nm N.ss a.wo a.No N.oo H.0N w.sN Aammas .smumam-ewasv a Ahmmlconv cofluosmoua wwm ucooumm Namumso N e n e m N H “man wthnom 3663:038 HHH unmawumaxm How mufismou mo humaasm .mH manna 39 Table 14. Liver scores, Experiment III Liver score expressed as percent of treatment Average Diet liver score No. 1 No. 2 No. 3 No. 4 1 (Basal, High-energy) 2.44 11 47 29 13 2 (Low-energy, high-fiber) 1.77 85 13 2 0 40 hemorrhages and/or scar tissue present and only five of the birds on this treatment had mahogany-colored livers. On the other hand, 85 per- cent of the birds fed the low-energy diet had livers that were scored one and only one bird was scored a number three liver. The average liver score of birds fed the basal diet was lower in this experiment than in Experiment II, but probably was a result of this shorter test period. Although the two diets fed in this experiment were quite dif- ferent in composition, one of the major differences was energy content and resulting energy intake. The results of this experiment indicated that fatty liver could be developed in as short as 14 weeks during the summer months. EXPERIMENT IV Procedure: Since the birds in Experiment III did not readily adjust feed intake downward when switched to a high-energy diet, Experiment IV was designed to test the effect of varying energy and protein levels on the incidence of fatty livers. Sixteen birds of the same strain used in previous experiments were fed individually in 8 x l6-inch cages each of the six experimental diets shown in Table 15. Wheat bran and wheat middlings were used to achieve the low-energy levels and these in~ gredients were removed and animal fat was added in the higher energy diets. Corn and soybean meal were altered to keep the protein constant at either 15 or 18 percent. Egg production, feed consumption and body weight were determined individually by two-week periods during the course of the 24—week experiment. The birds in this experiment were purchased at 20 weeks of age as started pullets and were fed a standard laying ration until they were 34 weeks of age when the experiment started. After 12 weeks on the test diets, four birds were sacrificed from each treatment for liver scoring. However, if a bird had died from a given treatment, only three birds were sacrificed so as to leave 12 birds remaining on each treatment. A similar number of birds were sacrificed from each treatment after 16, 20 and 24 weeks of the test. The results for egg production, feed consumption and body weight 41 42 Table 15. Composition (Z) of experimental diets used in Experiment IV Diets Ingredient l 2 3 4 5 6 Ground yellow corn 50.58 62.36 72.92 58.41 63.16 57.68 Soybean meal (50Z) 7.82 10.04 12.48 17.99 20.24 21.22 Alfalfa meal (ZOZ) 2.00 2.00 2.00 2.00 2.00 2.00 Wheat bran 14.00 7.00 —- 5.00 -— -- Wheat middlings 14.00 7.00 -— 5.00 —- ~- Fish meal (60Z) 3.00 3.00 3.00 3.00 3.00 3.00 Ground limestone 6.50 6.50 6.50 6.50 6.50 6.50 Dicalcium phosphate 1.50 1.50 1.50 1.50 1.50 1.50 Salt 0.35 0.35 0.35 0.35 0.35 0.35 Animal fat -- -- 1.00 -- 3.00 7.50 Vitamin premix* 0.25 0.25 0.25 0.25 0.25 0.25 Total 100.00 100.00 100.00 100.00 100.00 100.00 Z protein (calc.) 15.00 15.00 15.00 18.00 18 00 18.00 M.E. Cal/kg 2472 2716 2991 2715 2984 3127 * Supplied the following per kilogram of feed: Vitamin A, 6600 I.U.; Vitamin D3, 1650 ICU; Vitamin E, 8.25 IU; Riboflavin, 4.4 mg; Niacin, 22 mg; d—Pantothenic acid, 7.04 mg; Choline chloride, 220 mg; Vitamin B 2, 11 mcg; Folic acid, 0.275 mg; Menadione sodium bisulfite complex, 2.2 mg; BHT, 124.7 mg; Manganese, 60 mg; Zinc, 27.5 mg; Iodine, 1.2 mg; Iron, 20 mg; Copper, 2 mg; Cobalt, 0.2 mg. 43 corresponding to the intervals at which birds were sacrificed are shown in Tables 16, 17 and 18, respectively. The results for these measurements during the first 12 weeks of the experiment are presented by two—week periods in Appendix Table 3. Average liver scores for the various intervals are presented in Table 19. Results and Discussion: Feed intake was essentially the same for all treatments during the first two weeks of the experiment (See Appendix Table 3). Thus, the birds fed the lower energy diets lost weight during this period while birds fed the higher energy diets gained weight. During the second two-week period, birds on all treatments adjusted their intake to the energy content of the diet and thus birds on all treatments gained weight. However, complete adjustment to the energy level of the diet did not occur, since birds fed higher energy diets consumed more calories (See Appendix Table 3). A portion of this difference in energy intake could be due to the fact that birds fed higher energy diets did not lose weight during the initial two-week period. Thus, birds fed higher energy diets were larger and would require more calories for maintenance. Protein intake was decreased as the energy level of the diet increased since energy level was the primary factor governing feed in- take. Possibly the increased intake on the low-protein diets was an attempt by the birds to consume adequate protein. Feed intake was significantly different (P < 0.01) between treatments as shown in Table 17. There was a step-wise decrease in feed intake as the energy content of the diet was increased. Feed 44 Table 16. Egg production (Z hen-day) obtained in Experiment IV 2—week periods Diet 0 - 6 7 - 8 9 - 10 11 - 12 1 80.4 75.6 73.7 72.3 2 81.0 80.6 78.1 65.2 3 83.8 76.2 65.6 76.8 4 77.6 72.0 59.4 73.2 5 80.1 77.6 70.1 77.4 6 79.2 74.0 73.2 71.4 Table 17. Average feed intake (gms/bird/day) for Experiment IV* 45 2—week periods Diet 0 - 6 7 - 8 9 - 10 ll - 12 l 111 Aa 115 Aa 110 Aa 103 ABab 2 107 ABab 107 ABb 97 ABbc 99 ABCabc 3 100 BCde 102Bb 94 Bbc 90 BCcd 4 105 ABabc 107 ABb 103 ABab 108 Aa 5 100 BCcd 102 Bb 98 ABbc 95 ABCde 6 95 Cd 93 CC 90 Bc 88 Cd _._‘ __.._. * For any given time interval, means having different letters are significantly different according to Duncan's Multiple Range Test. Capital letters denote P < 0.01; small letters denote P < 0.05. 46 Table 18. Average body weight (gms) obtained in Experiment IV* 2-week periods Diet 0 - 6 7 ~ 8 9 - 10 ll - 12 l 1719 Aa 1778 a 1756 ab 1806 a 2 1758 Aab 1782 ab 1706 b 1715 a 3 1782 ABabc 1816 abc 1800 ab 1686 a 4 A 1755 ABab 1825 abc 1841 ab 1855 a 5 1825 ABbc 1885 bc 1906 a 1867 a 6 1862 Bc 1906 c 1895 a 1845 a * For any given time interval, means having different letters are significantly different. Capital letters denote P < 0.01; small letters denote P < 0.05. 47 Table 19. Liver scores, Experiment IV Average liver scores - Overall Diet lZ-week 16—week 20-week 24-week average 1 2.25 2.38 2.00 2.87 2.38 2 2.75 2.63 2.25 2.25 2.47 3 2.83 3.00 2.75 3.00 2.90 4 2.33 2,50 2.67 2.12 2.41 5 3.00 3.00 2,83 2.67 2.88 6 2.90 3.00 2.50 2.38 2.70 48 intake was more variable between treatments during the latter part of the test. This was because fewer birds were in each treatment during this time and thus the values were less repeatable. Body weights shown in Table 18 were significantly different (P < 0.01) after 12 weeks on test. These weights increased as the energy content of the diet was increased. After 16 and 20 weeks on test, body weights between treatments were not as great, but were significantly different (P < 0.05). Body weights at the end of the test were not significantly different since only four birds remained on each treatment. Liver scores presented in Table 19 indicate that there was a trend toward higher liver scores as the energy content of the diet was increased. However, there was a considerable degree of fatty livers in all treatments, indicating that other factors are involved besides energy. None of the birds that died during this test were diagnosed as having died from a fatty liver. Four birds (one each from diets l and 3; two from diet 6) were scored as having hemorrhages and/or scar tissue present. The results of this experiment indicate that energy level of the diet is a factor in the incidence of fatty livers, but other factors are also involved since some fatty livers occurred in all treatments. EXPERIMENT V Procedure: At this point in the study, it seemed desirable to test various possibilities of correcting fatty livers once they had developed. Approximately 180 birds from the same population as those used in Experiment IV were placed on the basal diet described in Experiment I at 38 weeks of age. Forty similar birds were fed the low-energy, high- fiber diet used in Experiment III. The birds were fed these diets until they were 57 weeks of age. During the week prior to the start of the experiment all non-layers and extremes in size were eliminated. Eight birds fed the basal diet and six birds fed the low—energy, high- fiber diet were sacrificed to determine the incidence of fatty livers. The experiment started June 30, 1966 and was conducted for a period of nine weeks. Six groups of four birds each were fed diets 1 - 6 and three groups of four birds each were fed diets 7 and 8. The experimental outline of these diets is shown in Table 20. Egg produc- tion, feed consumption and body weight changes were measured by groups of four birds and are presented in Tables 21, 22 and 23, respectively. Two groups of four birds were sacrificed from each of the first six treatments after 3, 5 and 9 weeks on test and scored for fatty livers as described previously. One group of four birds was sacrificed from treatments 7 and 8 at these intervals. The average liver scores are presented in Table 24. 49 50 Table 20. Experimental outline for Experiment V Previous Treatment treatment Treatment during Experimental period 1 Basal diet, Exp. 1. No change 2 ” " " " Switch to low-energy, high-fiber diet used in Experiment III 3 ” ” " " Basal diet with 15% wheat bran and 3% fish meal replacing corn 4 " ” " " Basal diet plus 1,000 gms choline chloride, 12 mg Vitamin B12 and 5,000 I.U. vitamin E/ton 5 " " " " Diet 4 + 100 gms aureomycin and 150 gms furazolidone/ton 6 ” ” ” " Basal diet + the following per Kg of feed; 4.4 mg riboflavin, 8.8 mg pantothenic acid, 22 mg niacin, 0.55 mg folic acid and 3.3 mg pyridoxine 7 Low-energy, high—fiber diet, Experiment III No change 8 " " ” Switch to basal diet 51 Table 21. Percent hen-day egg production in Experiment V Weeks Treatment Pre-exp.* 0 - 4 - 5 6 — 9— 1 67.7 63. 57.6 54.67- 2 69.5 61. 57.6 61.3 3 69.8 62.. 56.7 48.1 4 66.7 59.. 60.5 57.1 5 71.6 66.. 65.7 60.0 6 70.6 65.. 61.6 68.8 7 64.6 65. 60.7 44.2 8 60.4 60. 50.9 40.0 * Two weeks. 52 Table 22. Feed intake (gms/bird/day) in Experiment V Weeks Treatment 0 - 3 4 : 5 6 - . 1 91 7 91.7 98 2 83.7 94.9 112. 3 89.4 90.8 97. 4 88.3 96.9 101. 5 94.9 96.9 96. 6 90.5 93.2 103. 7 93.3 100.5 90. 8 91.7 88.9 101. 53 Table 23. Body weight change (gms) in Experiment V* Weeks Initial Treatment weight 0 - 3 4 - 5 6 - 9 1 1789 — 26 - 6 a - l8 2 1774 — 82 + 11 a + 40 3 1800 - 18 - 31 a + 10 4 1791 0 - 11 a + 10 5 1800 — 2 — 5 a - 6 6 1783 - 33 + 59 b - 32 7 1630 - 36 - 30 a + 21 8 1654 — 2 - 4 a + 29 * Means having different letters are significantly different (P < 0.05). 54 Table 24. Average liver scores obtained in Experiment V Treatment Pre—exp. 3 weeks 5 weeks 9 weeks 1 3.13 3.13 3.14 2.94 2 1.94 1.31 1.37 3 3.00 1.71 1.64 4 2.63 2.50 2.06 5 2.88 2.29 3.00 6 3.06 2.94 3.19 7 1.67 1.50 1.25 1.13 8 2.37 2.50 2.88 55 Results and Discussion: Two birds from the basal diet were diagnosed as having died from fatty livers during the month preceding the start of the experiment. Three birds died as a result of fatty livers during the first two weeks of the experiment (one each from diets 1, 4 and 5). No other birds died as a result of fatty liver during the nine-week experimental period. Body weight change was used in this experiment to reflect the status of remaining birds, since one-third of the birds fed each diet were sacrificed at each time interval. During the first three weeks of the experiment, birds on all treatments except treatment 4 lost weight (See Table 23). Birds switched to the low-energy, high-fiber diet lost the most weight and this was probably a result of failing to adjust to the energy content of the diet during this period. The weather was quite hot (over 909 on several days) during this period and feed intake of birds on all treatments was lowered, which accounts for the weight loss during this period. During the fourth and fifth weeks of the test birds fed treatment 6 gained significantly more weight (P < 0.05) than birds fed any other diet. The large fluctuation in body weight of this group is not readily explainable. No other significant differences between treatments for body weight change were noted in this experiment. The average liver scores presented in Table 24 indicate wide differences between treatments. Birds sacrificed before the experiment started indicated that the basal diet once again caused fatty livers while other birds did not develop fatty livers when fed the low-energy, 56 high-fiber diet. Birds switched from the basal diet to the low-energy diet (treatment 2) showed complete correction of fatty livers after five weeks on test and these were largely corrected after three weeks. Wheat bran and fish meal supplementation of the basal diet (Treatment 3) had no effect after three weeks on test, but had corrected the fatty livers after 5 and 9 weeks. Supplemental choline, vitamin B12 and vitamin E (treatment 4) showed very erratic results. In some birds, there were no fatty livers while other birds had very fatty livers with hemorrhages present. The same was true for treatment 5 in which aureomycin and furazolidone were added along with these vitamins. It is interesting to note that Quisenberry gt 31' (1967) also obtained no benefit from either low or high levels of supplemental choline, 312 or E as far as mortality due to fatty liver was concerned. Birds fed the low-energy, high-fiber diet during both the pre— experimental periods and the experimental period had the lowest liver scores. The average liver score for treatment 8, in which birds were switched from the low-energy diet to the basal diet, increased at each time interval. The results of this experiment indicate that fatty livers can be created in laying hens by switching them to the basal diet described previously. Fatty livers can also be corrected by placing the birds on the low—energy, highefiber diet described in Experiment III. Wheat bran and fish meal substitution for corn will also correct fatty liver, but require longer than the loweenergy diet to do so. Multiple B—vitamin supplementation (Treatment 6) had no effect and the recommendations of Couch (1956) were erratic if not ineffective. EXPERIMENT VI Procedure: In previous experiments, mortality from fatty liver had occurred during periods of hot weather. This experiment was designed to test the effect of high temperatures on the incidence and severity of fatty livers. The birds used in this experiment were of the same population as those used in Experiments IV and V and had previously been used in an unrelated floor pen experiment. At the end of July, 1966, approxi- mately 400 of these birds were force-molted by withdrawal of feed and water in total darkness for 48 hours. Then, only water and eight hours of light were provided for three additional days. After the fifth day, feed and water were supplied §g_libitum but light was restricted to eight hours per day for seven additional weeks at which time the birds were placed in individual 8 x 16-inch cages. The feed used after the molt was initiated was the basal diet described in Experiment I. These force-molted birds were used since field cases of fatty liver syndrome occurs more frequently during the latter stages of lay. The forced-molt was accomplished to eliminate any previous dietary effects and to increase the level of production of this group of birds. The fact that birds used in this experiment were force-molted was not considered relevant to the experimental results obtained. When the experiment started, three groups of eight birds were fed each of the diets described in Table 25 in an environment of 10 to 57 58 Table 25. Experimental outline for Experiment VI Diet Supplement 1 Basal diet, Experiment I 2 Diet 1 with 15% wheat bran substituted for corn 3 Diet 1 with 1.5% dicalcium phosphate substituted for 1% ground limestone and 0.5% corn 4 Low-energy diet, Experiment III 5 Diet 4 + 12% animal fat (See Table 26) 59 Table 26. Composition (%) of diet 5 used in Experiment VI Ingredient Percent Ground yellow corn 19.5 Ground oats 20.0 Wheat bran 15.0 Wheat middlings 10.0 Alfalfa meal (17%) 3.0 Dried skim milk 2.0 Fish meal (60%) 2.5 Meat and bone scraps 3.0 Soybean meal (44%) 5.5 Oyster shell flour 5.0 Steamed bone meal 1.5 Salt 0.6 Fish oil (2000 A; 400 D) 0.4 Animal fat 12.0 Total 100.00 Percent protein (calc.) 14.8 Metabolizable energy (Cal/kg) 2789 60 15.50 C (50 to 60° F). A like number of birds were fed the same diets in an environment artificially heated to 32° C (90° F). Both of the rooms were equipped with thermostatically controlled exhaust fans. Egg production, feed consumption and body weight change were measured at weekly intervals for one month, two-week intervals for the second month, and for the final month during the 12-week study. Sufficient extra birds were fed the basal diet to sacrifice six birds in each environ- ment after 1, 2 and 3 weeks on test. Eight birds were sacrificed from each of the treatments in both environments after 4, 8 and 12 weeks on test. The data for egg production, feed consumption and body weight change are presented in Tables 27, 28 and 29, respectively. The average liver scores are presented in Table 30. This experiment started December 6, 1966, and ended February 28, 1967. Results and Discussion: Two birds fed diet 1 died of a fatty liver in the unheated en— vironment and eight birds died of a fatty liver in the heated environ— ment (one each from diets 2 and 3; two from diet 5; four from diet 1) during the experimental period. This indicates that laying hens are less able to withstand fatty livers during periods of high environmental temperatures. The effect of heat on body weight change (Table 29) was highly significant (P < 0.0005) after one week. Birds fed all treatments in the heated environment lost weight during the first week. There was no significant interaction between diet and temperature environment for any of the traits measured during any part of the test. Thus, the two environments were pooled to determine differences between treatments. 61 a.mm m.om N.Ho N.NN umummm m H.oo m.Hs m.mo a.NN smummea: m A.mo m.Ho m.No m.¢N cmummm a m.No m.No N.mo m.No assumes: a N.Hn H.mo H.mo a.mo assume m «.wo m.mo m.mo «.Hm pmummSpD m o.mm N.No o.eo m.Nm . sauna: N a.qo m.Ne m.No N.HN amumoac: N N.am m.am o.ao o.H~ sauna: H N.No N.No m.mm o.qo umummna: H m N H axm-mum H3-N umHn mxooz H> ucoawuomxm you Ammpncmn unmouomv cowuosvoum mwm .NN ounmfi .Amo.o v m muocov mumuuma Hamam mnfio.o v m ouooob mumuumfi Hmuwamo .ucmuomwflp mfiucmowwwcwflm mum muouuoa uconoMMHm wcfi>m£ woman .Hm>uoucH mafia co>ww ham uomr 62 wk ammo ammo cow mm am nmN woummm n ma as ea Na mm as mm emummnas m . . . . mumm No maom «mama awooH so am nNN a m e moH moH qu NoH am «OH mm assumes: a we HN ow mm «m ca Hm amummm m m m U m N am a m.aHoH n maqu s 50H HNH oHH moH snowman: m NN mama maNa mom «a no flask emummm N NHH aoH qHH oHH mHH HHH so sausage: N so unmao ammk UBNN NN Na «Na emummm, H Ha ca Na Na moH qu NoH emummaasw H u m w w n o a m e m N H uoHa mxooz ¥H> ucoawuonxm now Ammp\oufin\mawv oxmuow room .mm oHan 63 .Amo.o v av samummmHe sHuamonHame mum mhmuumH UCOHmMWHU Snug» muQHQ k. «H + mm - N + a m + «H + was - emummm n HN - «H + 0H + «H + NN + om + NN - smummzao m mm + mH - NH + m + oH - om - flNoH. smummm a H - H - N + N m - m + me - assumes: a om - an + m - NH + oH - NH + swam . smummz m NN - NN - N - q + aH + NH + 0 assumes: m oq - HH + oH - m HN - mH + mama - emummm N NN - HN + wH + H NH + NH + «H - assumes: N a + 0 mm - o + a - o - mHN - cmuamm H Hm - oH - N + AN mN + HN + NH + emummsc: H NH - a N a N o a n e m H umHa mxomz #H> ucmefiuomxm Ga Amawv mowamzo uamfimz hpom .mN m~AMH 64 Table 30. Average liver scores for Experiment VI _.__.‘__ Weeks Diet 1 2 3 4 8 12 1 Unheated 2.93 2.75 3.08 2.50 2.88 2.28 l Heated 2.67 3.33 3.25 2.94 2.93 2.70 2 Unheated 2.06 2.63 2.06 2 Heated 1.86 2.00 2.12 3 Unheated 2.81 3.00 2.93 3 Heated 2.94 2.75 3.83 4 Unheated 1.75 1.81 1.31 4 Heated 1.63 1.31 1.71 5 Unheated 1.38 1.38 1.36 5 Heated 1.75 1.50 1.31 65 Birds fed the low-energy diet (diet 4) lost significantly more weight (P < 0.05) than birds remaining on the basal diet during the first week. This weight loss was due to inadequate feed intake since fat supplemen- tation (diet 5) of the low-energy diet prevented a portion of the weight loss. Feed intake was significantly (P < 0.0005) depressed by the high temperature environment during the first week of the test (Table 28). This accounts for the body weight loss in the heated environment during this period. Birds fed diets 4 and 5 consumed significantly less feed during the first week. Apparently density of the ration governed feed intake during this period since birds fed diets 4 and 5 consumed the same amount of feed and their energy content is quite different. During the second week of the test, birds on all treatments ad- justed their feed intake toward the energy content of the diet. There was no significant diet effect during this period. However, the high temperature continued to significantly depress (P < 0.0005) feed intake. The increase in feed intake among birds fed the lower energy diets in the unheated environment and among birds of all treatments in the high temperature environment resulted in a gain in body weight for most treatments. However, birds fed the lownenergy diet (diet 4) in the heated environment continued to lose weight. Since birds fed most of the treatments gained weight during the second week of the test, the effect of high temperature was less depressing (P < 0.05) on body weight change. A significant drop (P < 0.05) in egg production was observed during the third week of the test due to the high temperature environ- ment. Apparently, by drawing on body stores and increasing feed intake, 66 the birds were able to withstand the heat stress during the first two weeks of the test. As the experiment progressed, the effect of heat became more pronounced as far as egg production was concerned. As the experiment progressed, birds fed the lower energy diets increased their feed intake to more nearly reflect the energy content of the diet. By the fourth week of the test, the feed intake of birds fed diet 2 was significantly greater (P < 0 05) than those from diet 1. Likewise, birds fed diet 4 consumed significantly more feed than those from diet 5 during this period. These differences were further magni- fied during the fifth and sixth weeks of the test. Average liver scores of birds fed diet 1 that were sacrificed after 1, 2 and 3 weeks on test indicated that fatty liver was present in both temperature environments (see Table 30). Apparently, since more birds died due to fatty liver in the high temperature environment, laying hens with fatty livers are less able to withstand high tempera- ture stress. When birds from all treatments were sacrificed after four weeks on test, the low-energy diet (diet 4) had once again corrected the fatty livers. Interestingly, this low—energy diet supplemented with 12 percent animal fat (diet 5) had also corrected the fatty livers. This further substantiated the results of Experiment IV that energy is not the primary cause of fatty livers. Wheat bran also corrected the fatty livers to an extent, but less so than the wheat bran and fish meal supplementation used in Experiment V. In this experiment, the wheat bran was more effective in the heated environment. One of the differences, in addition to energy content, between the basal diet and the low-energy diet was the phosphorus content. In 67 diet 3 of this experiment, the phosphorus content was increased to equal that of the low-energy diet. This increase in phosphorus content had no effect in reducing the incidence of fatty livers. This experiment indicates that there is/are some factor(s) in the low-energy diet that corrects fatty livers. Wheat bran is less effective, but the low-energy diet also contains ten percent wheat middlings. Since wheat bran is low in energy and bulky, a reduction in energy intake results when it is substituted for corn in a ration. The reduction of energy intake when wheat bran is used makes it difficult to assess the value of this ingredient in correcting fatty livers. Experiment VII was a preliminary attempt in this respect. EXPERIMENT VII 332229259 Three groups of six birds that had been fed the basal diet in the unheated environment of Experiment VI were available for this pre- liminary study. A water extract of wheat bran was prepared as follows: 4 kilograms of wheat bran was soaked with constant stirring in 40 liters of distilled water for 7 hours. This material was allowed to set for 30 minutes and then most of the filtrate was drawn off by siphon. The remaining material was filtered through 8 layers of cheesecloth and this filtrate added to that previously siphoned. The residue was air— dried in thin layers with a circulating fan for 36 hours. The filtrate was dried in shallow trays onto corn in a hot—air oven at 500 C for 16 hours. Both products were reground after drying. This procedure was repeated to obtain enough material for this experiment. Approximately 65 percent of the wheat bran remained in the residue after extraction. This does not take into account any losses when transferring the product, but these losses were considered small. The residue was included in the ration at ten percent of the ration based on this yield. This extraction was considered incomplete and the extract was added to the basal diet at twice the level of wheat bran. (The water extract of 300 grams of wheat bran was added per kilogram of feed.) These materials were fed for 28 days and the results are pre- sented in Table 31. 68 69 mm.~ an n mooH m.mo mad osnfimmu cmun ummLB + Hmmmm m N¢.H no + moofi N.uo ow Homuuxm smug omens + Hmmmm N ON.m N + quN 0.0m eHH H HawaHHmaxm .umHe Hammm H muoom owcmco HmwuficH .voua wwm Ammv\vuwn\mawv ucmaoaamdm uofio um>HH unmouom mxmucw noon owmuo>< Amawv .uz zbom HH> ucoEHuonxm cw pouooHHoo mumm .Hm mHan 70 Results and Discussion: The average liver scores were reduced by both the extract and residue of wheat bran. It would appear that there is/are some factor(s) in wheat bran responsible for the correction of the fatty liver. How— ever, it should be pointed out that the birds fed the wheat bran ex- tract were much smaller than the other two groups. Unfortunately, this was not observed at the start of the test. To help substantiate this effect of the wheat bran extract, the liver scores of all birds fed the basal diet in Experiment VI that weighed between 1550 and 1750 grams were averaged. There were 17 such birds in Experiment VI and their average body weight and liver scores were 1630 and 2.88, respec— tively. This would indicate that birds of this size had fatty liver in Experiment VI. Due to the small number of birds in this experiment (6 per treatment), this experiment does not definitely prove that wheat bran contains factor(s) that correct fatty liver. However, the results are suggestive of the value of further work in this area. GENERAL DISCUSSION The basal diet used in these experiments was of the high-energy, commercial-type and most of this energy (75% corn and 0.5% fat) was supplied by carbohydrate sources. This level of energy was accomplished by maximum utilization of corn and minimum use of fibrous ingredients in the diet. Methionine was considered a borderline deficiency in this diet but methionine supplementation in Experiment II did not correct the fatty liver. The results of Experiment I and II demonstrated that fatty liver is much more prevalent in cages than in floor birds. The difference in opportunity for exercise between cage and floor birds might allow energy used by floor birds for this purpose to be unused and reflected as fat in cage birds. Another possibility could be that floor birds obtain some factor(s) from the litter that is beneficial in preventing fatty livers. After sacrificing the birds used in these experiments, it would appear that fatty liver mortality occurs as a result of rupture of the liver capsule causing massive bleeding into the abdominal cavity. How- ever, some birds survived these liver ruptures as evidenced by blood in the abdominal cavity as well as abdominal wall discoloration of some birds in these studies. Apparently, if the liver capsule did not rupture, most birds survive these hemorrhages. Interestingly, most non-layers did not have fatty livers. This indicates that hormonal balance is involved in this condition. 71 72 The results of Experiment III indicated that development of fatty livers could be prevented by use of a specific low—energy, high- fiber diet. This diet was much lower in energy content than the basal diet used to create fatty livers. Hill et 31. (1956) found that in- creasing the energy level of the diet by the use of fat reduced the feed requirement by a rate of two percent for each one percent of added fat. Body weight was maintained at a higher level by high- energy diets. Birds fed the basal diet in Experiment III gained more weight than those fed the low-energy, high—fiber diet and also developed fatty livers. Yet, in Experiment IV where varying energy and protein levels were fed, birds from all diets had some fatty livers, although the incidence was higher in the higher-energy diets. This indicated that energy level of the diet has some effect on the incidence of fatty livers, but other factors are also involved. Experiment V demonstrated that choline does not correct fatty livers or its effect is very erratic. This is in agreement with re- ports by Ringer and Sheppard (1963), Fisher and Weiss (1956), and Quisenberry (1967). The failure of aureomycin and furazolidone to affect fatty livers discounts the theory of low—level infection. Michigan State University Poultry Pathologists have not detected any disease organisms related to the fatty liver condition. This is in agreement with the report of Texas veterinarians in which unsuccessful attempts were made to transmit the condition. In Experiment V, wheat bran and fish meal were demonstrated to correct the fatty liver condition. This effect was probably due to the wheat bran as evidenced by Experiment VI. Wheat bran is a low—energy 73 material as compared to corn and is a good source of B—vitamins and trace minerals. However, the high—energy basal diet was considered well-fortified with vitamins and a multiple B-vitamin supplementation had no effect on fatty livers in Experiment V. It could be postulated that the higher calcium level in present- day laying rations increases the requirement for certain trace minerals. This possibility was not investigated in this study. However, the low- energy diet used in Experiment III contains about the same level of calcium as the basal diet. In most of these experiments, mortality due to fatty liver was noted during periods of hot weather. Experiment VI demonstrated that more fatty liver mortality does occur during hot weather, but the actual incidence of fatty livers in living birds was just as high in a cool environment (10 — 15.5° C). Mueller (1961) reported that continuous exposure to temperature of 32° C reduced egg production 28 percent, and feed intake 32 percent. In a later report, Mueller (1967) found that supplemental methionine had no effect on these traits at 32° C. Experiment VI demonstrated the effects of high environmental temperatures (32° C) on egg production and feed intake. Yet, this depressed feed intake and lower egg production seemed to be unrelated to the occurrence of fatty livers. When birds were switched to the low-energy diet described in Experiment III, a weight loss occurred. This weight loss was largely prevented by the addition of 12 percent fat to this diet in Experiment VI. Even with 12 percent added fat, this diet still corrected fatty livers. This suggests that dietary fat is not the source of liver fat accumulation and agrees with the work of Harper_gt El. (1954) with rats. 74 The results of Experiment VII suggest that there is some water soluble factor(s) in wheat bran that helps to correct fatty livers. If so, the properties of this material(s) may be similar to that re- ported by Thornton and MePherron (1962) in brewers' dried grains. It should be mentioned that fatty liver studies with laying hens are difficult and time-consuming. It takes several weeks or even months to develop the condition and, since no tests are available, some birds must be sacrificed to determine the incidence of fatty livers. Corrective treatments employed should not be so drastic as to stop egg production, since most non-layers do not have fatty livers. As men- tioned earlier, this indicates that hormonal balance is involved in this condition also. SUMMARY In this study, a commercially available, high-energy diet that had experienced some fatty liver problems in field conditions was used. Fatty liver was much more prevalent in birds in cages than those in floor pens. Methionine failed to correct the condition, but a specific diet low in energy, and high in fiber was very effective in preventing or correcting the fatty liver syndrome. In one experiment, energy and protein levels were varied. The incidence of fatty liver was increased as the energy content of the diet was increased. However, some fatty livers occurred among all treatments in this experiment. A higher protein level in the diet was without apparent effect. In another experiment, fatty livers were corrected by either switching birds to a low-energy, high-fiber diet or by substituting 15 percent wheat bran and 3 percent fish meal for corn. Supplemental choline, vitamin B12 and vitamin E gave very erratic results. In some birds fed these vitamins, there were no fatty livers while other birds had very fatty livers with hemorrhages present.- There was no effect in this experiment from supplemental aureomycin or furazolidone or from a multiple B-vitamin supplement. An experiment in which some birds were held in an artificially heated environment (32° C) during a lZ—week experiment indicated that mortality from fatty livers was increased during the high temperatures. However, similar birds in a cool environment had the same degree of 75 76 fatty livers but mortality was lower. The low-energy diet effectively corrected fatty livers in this experiment and wheat bran had a similar but less effective action. A water extract of wheat bran was effective in connecting fatty livers in a small preliminary test, but the residue was more effective. This suggests that some factor(s) in wheat bran was partially respon- sible for correction of fatty livers. That the fatty liver syndrome is a nutritional problem is sug- gested by the fact that fatty livers were induced and/or corrected by diet manipulation. Non—laying birds in these experiments generally had normal appearing livers, implicating hormonal balance as a factor in this condition. The results of this study help to explain the failure of some research workers to create the fatty liver syndrome. Mest previous attempts had utilized high-fat diets, while a high-energy, high- carbohydrate diet was used in this study. In this study, fatty livers were produced in birds in cages using a high—energy diet in which the energy was supplied primarily from carbohydrate sources. Birds in floor pens had a much lower in- cidence of fatty livers when fed the same diet. A specific low—energy, high—fiber diet was very effective in preventing fatty livers from developing and corrected the fatty livers once they had been produced. Fifteen percent wheat bran was also effective in this respect, but required a longer period of time for correction of fatty livers than the low—energy,lhigh—fiber diet. IIIERAIURE CITED Abels, Jules C., C. W. Kupel, G. T. Pack, and C. P. Rhoads, 1943. Metabolic studies in patients with cancer of gastro-intestinal tract. XV. Lipotropic properties of inositol. Proc. Soc. Exptl. Biol. Med. 54: 157-158. Amdur, M. 0., L. C. Norris and G. F. Heuser, 1946. The lipotropic action of manganese. J. Biol. Chem. 164: 783-784. Baxter, J. H. and H. Campbell, 1952. Effects of aureomycin on renal lesions, liver lipid, and tissue choline in choline deficiency. Proc. Soc. Exptl. Biol. Med. 80. 415-419. Best, C. H., J. M. Hershey and M. E, Huntsman, 1932. The effect of lecithine on fat deposition in the liver of the normal rat. J. Physiol. 75: 56-66. Best, C. H. and M. E. Huntsman, 1932a. The effects of the components of lecithine upon deposition of fat in the liver. J. Physiol. 75: 405-412. Couch, J. R., 1956. Fatty livers in laying hens -- A condition which may occur as a result of increased strain. Feedstuffs 28(47): 46-54. Couch, J. R., 1964. The fatty liver syndrome in laying hens. Research Digest 11(5): 1-2. Di Luzio, N. R. and D. B. Zilversmit, 1956. Effect of choline, heparin and aureomycin on fatty livers of dogs. Proc. Soc. Exptl. Biol. Med. 91: 338-341. Donaldson, W. E. and C. D. Gordon, 1960. The effect of 3% added animal fat on laying hen performance. Poultry Sci. 39: 583-587. Donaldson, W. E. and R. I. Millar, 1958. Observations on added animal fat in laying rations. Poultry Sci. 37; 1199 Abstragt. Donovan, G. A. and L. S. Balloun, 1955. Effect of dietary protein and energy on chick liver fat accumulation. Proc. Soc. Exptl. Biol. Med. 90: 692-694. Duncan, D. B., 1955. MUltiple range and multiple F tests. Biometrics 11: 1-42. 77 78 Fisher, H. and H. S. Weiss, 1956. Observations on the fatty liver syndrome of laying hens. Feedstuffs 28(51): 16. Gavin, G. and E. W. McHenry, 1941. Inositol: A lipotrophic factor. J. Biol. Chem. 139: 485. Gyorgy, P., J. Stokes, Jr., H- Goldblatt and H. Popper, 1951. Anti- microbial agents in the prevention of dietary hepatic injury (necrosis, cirrhosis) in rats. J. Exptl. Med. 93: 513—522. Harper, A. E., W. J. Monson, D. A. Benton, M. E. Ulinje and C. A. Elvehjem, 1954. Factors other than choline which affect the deposition of liver fat. J. Biol. Chem. 206: 151-158. Hawkins, R. A. and P. J. Heald, 1966. Lipid metabolism and the laying hen. IV. The synthesis of triglycerides by slices of avian liver in vitro. Biochem. Biophys. Acta 116: 41-55. Heald, P. J., 1963. The metabolism of carbohydrate by liver of the domestic fowl. Biochem. J. 86. 103-110. Heald, P. J. and K. A. Rookledge, 1964. Effect of gonadal hormones, gonadotrophins and thyroxine on plasma free fatty acids in the domestic fowl. J. Endocrinol. 30: 115-130. Hershey, J. M., 1930. Substitution of lecithin for raw pancreas in the diet of the depancreatized dog. Am. J. Physiol. 93: 657-658. Hill, F. W., D. L. Anderson and L. M. Dansky, 1956. Studies of the energy requirements of chickens. III. The effect of dietary energy level on the rate and gross efficiency of egg production. Poultry Sci. 35: 54-59. Leach, R. M., Jr., E. Ceballos and M. C. Nesheim, 1966. Recent studies on choline requirements of chicks and hens. Proc. Cornell Nutrition Conf. 69-76. Leevy, C. M., 1962. Fatty liver: A study of 270 patients with biopsy proven fatty liver and a review of the literature. Medicine 41: 249-276. MCDaniel, A. H., J. D. Price, J. H. Quisenberry, B. L. Reid and J. R. Couch, 1957. Effect of energy and protein level on caged layers. Poultry Sci. 36: 850-854. MeDaniel, A. H., J. H. Quisenberry, B. L. Reid and J. R. Couch, 1959. The effect of dietary fat, caloric intake and protein level on caged layers. Poultry Sci. 38: 213-219. March, B. E. and J. Biely, 1962. The effect of dietary fat level on the rate of mortality in caged layers. Poultry Sci. 41: 9-12. 79 March, B. E. and J. Biely, 1963. The effects of dietary fat and energy levels on the performance of caged laying birds. Poultry Sci. 42: 20-24. Mueller, W. J., 1961. The effect of constant and flucuating environ— mental temperatures on the biological performance of laying pullets. Poultry Sci. 40: 1562-1571. Mueller, W. J., 1967. The effect of two levels of methionine on the biological performance of laying pullets in controlled environ- ments. Poultry Sci. 46: 82-88. Popper, H., and F. Schaffner, 1957. _Liver: Structure and Function. McGraw-Hill Co., New York. Quisenberry, J. H., 1966. Research reports on factors that influence laying hen performance. Feedstuffs 38(20): 28—34. Quisenberry, J. H., L. A. Young and P. V. L. N. Murthy, 1967. The fatty liver syndrome in commercial layers. Abstracts of papers to be presented at the 56th Annual Meeting of the Poultry Science Ass'n. pp. 84-85, Ringer, R. K. and C. C. Sheppard, 1963. Report of fatty liver syndrome in a Michigan caged layer operation. Mich. Agr. Expt. Sta. Quart. Bull. 45(3); 426-427. Rutenburg, A. M., E. Sonnenblick, I. Koven, H. A. Aprahamian, L. Reiner and J. Fine, 1957. The role of intestinal bacteria in the development of dietary cirrhosis in rats. J. Exper. Med. 106: 1-13. Shils, M. E., I. Friedland and W. B. Stewart, 1954. Rapid development of portal fatty liver in rats consuming various plant materials. Proc. Soc. Exptl. Biol. MEd. 87; 473-476. Shils, M. E. and W. B. Stewart, 1954a. Development of portal fatty liver in rats on corn diets: Response to lipotropic agents. Proc. Soc. Exptl. Biol. Med. 85: 298-303. Shils, M. E. and W. B. Stewart, 1954b. Preventive influence of certain amino acids on experimental fatty liver of portal type. Proc. Soc. Exptl. Biol. Med. 87: 629—631. Singal, S. A., V. P. Sydenstricker and J. M. Littlejohn, 1949. The lipotropic action of threonine. Fed. Proc. 8: 251. Snedecor, G. W., 1956. Statistical Methods Applied to Experiments in Agriculture and Biology, 5th ed., Iowa State College Press, Ames, Iowa. Sunde, M. L., 1966. Nutritional factors associated with fatty livers. Proc. Minnesota Nutrition Conf. pp. 85-94. 80 Summers, J. D., W. F. Pepper and J. R. Cavers, 1966. Poultry feed formulas, pp. 51-52. Ontario Agricultural College, Guelph, Ont., Canada. Sutton, J. B., M. W. Pasvogel, A- R- Kemmerer and M. G. vavich, 1957. The influence of choline and methionine on the deposition of fat in the liver of the mature laying hen. Poultry Sci. 36: 1161. Abstract. Thornton, P. A. and T. A. MCPherron, 1962. Controlling body weight and liver lipid accumulation in the chicken with dietary brewers' dried grains. Fed. Proc. 21: 397. Treat, C. M., B. L. Reed, R. E. Davies and J. R. Couch, 1960. Effect of animal fat and mixtures of animal and vegetable fats containing varying amounts of free fatty acids on performance of caged layers. Poultry Sci. 39; 1550-1555. Turk, D. E., H. R. Bird and M. I. Sunde, 1958. Effect of fats on replacement pullets and laying hens. Poultry Sci. 37: 1249. .éEEEEEEEB Vennart, G. P., V. P. Perna and W. B. Stewart, 1958. Fatty liver of portal type: Cured by lysine, plus tryptophan. J. Nutr. 64: 635-638. Weiss, H. S. and H. Fisher, 1957. Plasma lipid and organ changes associated with the feeding of animal fat to laying chickens. J. Nutr. 61: 267-280. Yoshida, A. and A. E. Harper, 1960. Effect of threonine and choline deficiencies on the metabolism of C14 labeled acetate and palmitate in the intact rat. J. Biol. Chem. 235: 2586-2589. Yoshida, A., K. Askida and A. E. Harper, 1961. Prevention of fatty liver due to theronine deficiency by moderate caloric restriction. Nature 189: 917-918. 81 .somo mqun mN mo meme 0 mo mwmuo>m cm mH uoHp comm * ame «mNH amNH w¢NH mNNH QHNH aon «moH omoH mnoH q uan qme mmmH oomH mamH onH mmoH mmoH mqu «on m wmmH «mmH ooNH nmmH «mmH ommH moNH mqu «moH amoH N onH wqu ame HmmH wGNH «MNH moNH muoH mmoH mooH H A.mawv uzmHmB anon mwmum>< moH ma moH «OH ooH HHH HHH oHH on HoH a 50H ooH noH ooH HHH NHH mHH oHH moH HoH m HoH mm «OH OCH mHH mHH ¢HH nHH MHH om N HoH mo NoH «OH noH NHH qHH mHH noH mo H Ammv\UpHn\mamuwv omadmcoo room m.¢c m.mo o.wo 0-0m w.mo H.Hn m.0n o.HN ¢.¢m o.on q w.o~ a.Hm o.Hm o.¢> o.on N.Hm m.no m.no ¢.Hn s.qn m m.Ho N.©o o.oo m.Nn m.mm H.Hm o.m~ H.mn n.0n m.Nm N H.mo m.no m.no H.oo a.Hm N.Nm m.Nm N.Nm m.Nn n.mm H Azmv-Cm£ «NV eoHuosmoua wwo ommum>< 0H m w m o m a m N H «usoaumona mpoHHma xom3a¢ monuoa zmvuwN he H ucmaHuoaxm aH wouooHHoo mumv mo mumaasm .H Emma 3389. 82 Appendix Table 2. Summary of data collected in Experiment II by 28-day periods* 28—day periods Diet 1 2 3 4 5 6 7 Percent egg production (hen day) 5 77.5 75.8 73.6 68.6 62.0 56.4 42.9 6 74.7 72.8 69.8 64.1 64.0 64.1 53.6 7 76.6 77.6 72.2 70.2 64.5 63.7 60.4 8 75.5 71.2 69.3 69.7 73.0 66.3 59.0 9 78.2 75.6 70.7 71.3 69.3 63.8 55.3 Feed consumption (gms/bird/day) 5 114 119 115 107 110 103 93 6 112 119 120 107 119 112 100 7 113 115 114 110 108 105 100 8 113 113 117 108 114 99 92 9 109 115 112 110 113 103 93 Average body weight (gms) Init. wt; 5‘ul966 1943 1979 1998 1998 2016 1998 2007 6 1938 1947 1993 2043 2070 2116 2070 2106 7 1907 1902 1934 1957 1988 2075 2057 2116 8 1893 1934 1970 2025 2034 2061 2011 2070 9 1879 1875 1938 1979 1966 2047 2011 2052 * Each diet is an average of four groups of eight birds. 83 Appendix Table 3. Summary of data collected during the first 12 weeks in Experiment IV by 2-week periods Periods Pre— Diet exp. 1 2 3 4 5 6 Percent egg production 1 84.8 80.6 81.7 81.2 80.8 79.5 79.5 2 86.4 82.6 84.8 81.7 82.1 76.3 78.1 3 87.1 87.5 85.7 84.4 84.4 80.8 80.0 4 87.1 80.4 80.8 77.6 75.7 79.1 77.1 5 85.0 82.1 83.0 79.5 78.1 77.4 80.6 6 84.4 81.3 81.7 78.5 77.2 79.5 76.8 Body weight (gms) l 1733 1686 1710 1719 1731 1707 1719 2 1738 1709 1724 1721 1756 1719 1741 3 1745 .1735 1768 1774 1795 1764 1782 4 1740 1716 1735 1748 1774 1730 1755 5 1740 1774 1779 1795 1842 1796 1825 6 1745 1807 1838 1857 .1901 1854 1862 Gms. feed/bird/day 1 99 116 114 115 105 115 2 100 108 108 109 104 112 3 98 102 99 104 94 103 4 98 106 106 110 101 109 5 102 104 97 98 95 104 6 101 100 94 95 86 94 Metabolizable Calories/bird/day l 245 288 282 284 259 284 2 271 293 292 296 282 304 3 292 306 298 311 282 309 4 266 288 288 298 274 296 5 303 309 291 292 282 310 6 315 314 293 297 269 293 Gms. protein/bird/day 1 14.9 17.5 17.1 17.2 15.7 17.2 2 15.0 16.2 16.1 17.3 15.5 16.8 3 14.6 15.3 14.9 15.6 .14.1 15.5 4 17.7 19.1 19.1 19.8 18.2 19.7 5 18.3 18.7 17.5 17.6 17.0 18.7 6 18.2 18.1 16.9 17.1 15.5 16.9