Experimentally Induced "Fatty Liver Syndrome" Condition in Laying Hens } R. H. HARMS, D. A. ROLAND, SR. 2 AND C. F. SIMPSON
Department of Poultry Science, Florida Agricultural
Experiment Station, Gainesville, Florida 32611
(Received for publication July 19, 1976)
POULTRY SCIENCE 56: 517-520,
1977
hens in the same flock. They also observed enlarged combs in the non-laying hens which may have been due to a hormone imbalance. Since it has been impossible to reproduce the condition under experimental conditions it has been very difficult to study. Wolford and Polin (1974) reported hepatic hemorrhages characteristics of "fatty liver-hemorrhagic-syndrome" (FLHS) were induced when hens were force fed in excess of 50% more feed than they would normally consume. They suggested that the forced-feeding procedure could be a technique adaptable for use in studying this condition. Arrington et al. (1967) reported that the feeding of high levels of iodine to laying hens caused a cessation of egg production without regression of the ovary. Therefore, since Harms et al. (1972) had indicated a possible imbalance of hormones with non-laying hens suffering with FLS, the present experiments were conducted to determine the influence of injections of estrogen into hens fed iodine.
INTRODUCTION
T
HE "fatty liver syndrome" (FLS) in laying hens was first reported by Couch (1956), describing field cases submitted to the Texas Diagnostic Laboratory in 1954 and 1955. He reported that the fat content of the liver of laying hens varied greatly reaching levels as high as 70%, and that globules of fat seeped through the liver capsule. Reedy (1968) later reported that FLS was characterized by a decrease in egg production by as much as 50%. These hens were heavier than expected for this stage of production, which was attributed primarily to excessive fat in the abdominal cavity. The liver was enlarged, very fatty, extremely friable and varied in color from tan to pale yellow. Many of the livers contained single or multiple hemorrhages or multiple hematomas. Harms et al. (1972) observed that cholesterol levels were approximately three times as great in non-laying hens suffering from FLS as laying
EXPERIMENTAL PROCEDURE
1. Florida Agr. Exp. Sta. Journal Series no. 6128. 2. Present address: Department of Poultry Science, Auburn University, Auburn, Alabama.
Two experiments were conducted with Babcock B-300 hens approximately 80 weeks 517
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 2, 2015
ABSTRACT Two experiments were conducted with aged laying hens to determine the influence of feeding 5,000 p.p.m. of iodine as potassium iodine (KI) and/or injecting 12 mg. of estradiol upon fat accumulation in the liver and serum cholesterol levels. The KI was fed for 8 days before making liver and blood determinations, and the estradiol was injected 3 days prior to making the determinations. The feeding of KI or injection of estradiol resulted in significantly increased liver weight. When the two treatments were combined a further significant increase in liver weights was obtained. The percent of fat in the liver was significantly increased by the injection of estradiol. However, the feeding of KI in the presence or absence of estradiol did not affect the percentage of fat in the liver. Neither of the treatments significantly affected the fatty acid composition of the liver fat. Feeding of KI or injection of estradiol significantly increased total serum cholesterol levels. When the two treatments were combined a further increase in serum cholesterol level was observed. Histological changes of the livers of hens treated with KI and estradiol were similar to those previously described for the "fatty liver syndrome."
518
R. H. HARMS, D. A. ROLAND, SR. AND C. F. SIMPSON
Hen wt.
Liver wt.
E
(G)
(G)
Liver(% of body wt.)
Liver fat
KI
Liver moisture (%)
+ + 1 1
(%)
+ 1 +1
TABLE 1.—Body weight, liver weight and percent of body weight, percent of fat and moisture in liver' Treatment
1650a 1593ab 1656a 1563b
41.5a 54.0b 53.4b 67.5c
2.54a 3.38b 3.23b 4.17c
11.5a 12.5a 16.0b 11.5a
69.2a 63.2b 64.0b 67.1a
1 Means without common letters are significantly different according to Duncan's multiple range test (1955). KI = Received a diet containing 5000 p.p.m. I as KI. E = Injected with 12 mg. estradiol three days before sacrificing.
TABLE 2.—Composition of liver fat as determined by gas-liquid chromatography
1 + 1+
E
14:0
16:0
16:1
18:0
18:1
18:2
20:4
+ +
2.76 5.46 5.43 2.09
19.43 20.44 23.12 20.72
11.55 10.64 7.39 9.20
11.92 10.98 12.12 10.00
34.31 34.40 38.33 38.12
15.97 14.79 11.54 16.66
4.06 3.29 2.07 3.21
'Carbon chain length: number of double bonds. KI = Received a diet containing 5000 p.p.m. I as KI. E = Injected with 12 mg. estradiol three days before sacrificing. TABLE 3.—Total serum cholesterol from hens receiving four different treatments Serum cholesterol (mg./lOOml.)1 242.8a Control 537.0b Estradiol 752.2c KI 1129.6d KI and estradiol 1 Means without common letters are significantly different according to Duncan's multiple range test (1955). Diet
of age. The hens had been receiving, and were fed during the experimental period a diet containing 16.0% protein, 2853 kilocalories of metabolizable energy per kilogram, 3.00% calcium and 0.75% total phosphorus. Four experimental treatments used were as follows: (1) Control, (2) 5,000 p.p.m. I in the feed as furnished by 6.54 g. of K l / k g . of diet (3) each hen injected subcutaneously with 12 mg. estradiol, and (4) combination of treatment 3 and 4. Three replicates of five individually caged birds were used per treatment in experiment 1, and two replicates of five individually caged birds were used in
experiment 2. The KI was fed for eight days. The estradiol was injected three days prior to termination of the experiment. All the birds were individually weighed at the termination of the experiment. Blood was drawn by anterior heart puncture for total serum cholesterol determination. All birds were then sacrificed and liver weights obtained. The percent fat and moisture in the liver, and fatty acids in the liver was then determined. A portion of liver from 5 birds per treatment group was fixed in 10% neutral formalin, embedded in paraffin, cut at 6 \xm. and stained with hematoxylin and eosin for examination with the light microscope. The fatty acid composition data were obtained by gas-liquid chromatography analysis according to the methods described by Nugura and Edwards (1970). Total serum cholesterol values for 5 birds per treatment group were determined by the procedure outlined by Levine and Zak (1964). Since the treatment x experiment interaction was not significant, the data were pooled for this presentation. Significant differences
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 2, 2015
Fatty acid'
Treatment KI
EXPERIMENTAL "FATTY LIVER SYNDROME" IN LAYING HENS
519
FIG. 2. Liver of hen treated with estradiol. There is a focal area of necrosis (N), and liver cells at the periphery contain lipid (arrows). Hematoxylin and eosin stain, x 80.
FIG. 3. Liver of hen treated with KI and estradiol. There is diffuse necrosis (N) and hepatic cells at the periphery contain lipid (arrows). Hematoxylin and eosin stain, x 80.
x>« , v ' \ « % «i
-
FIG. I. Liver of control hen. The hepatic cells contain droplets of lipid (arrows), and the sinusoids (S) contain blood. Hematoxylin and eosin stain. x 80. between treatment means were determined by Duncan's multiple range tests (1955). RESULTS AND DISCUSSION
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 2, 2015
Feeding 5,000 p.p.m. of iodide or injecting 12 mg. estradiol did not significantly influence body weight (Table 1). However, birds fed KI and injected with estradiol had significantly decreased body weights.
The feeding of KI or injection of estradiol resulted in significantly increased liver weights (Table 2). A further significant increase in liver weights was obtained when the two treatments were combined. Increases in liver weights were evident whether based on an absolute weight or a percentage of the body weight. Injecting hens with estradiol significantly increased liver fat; however, feeding of KI or the combination of KI and estradiol did not influence liver fat. The feeding of KI increased the fat in the liver from 4.77 to 6.75 grams. Injecting the estradiol resulted in 8.54 grams of fat in the liver, and this was slightly reduced to 7.76 when KI was fed along with estradiol injection. Liver moisture was significantly reduced by feeding KI or injecting estradiol (Table 1), however, when the two were combined, liver moisture was not influenced. Neither of the treatments significantly affected the fatty acid composition of the liver fat (Table 2). Total serum cholesterol levels were significantly increased by feeding potassium iodide or injecting the hens with estradiol (Table 3). A further increase occurred with a combination of the treatments.
520
R. H. HARMS, D. A. ROLAND, SR. AND C. F. SIMPSON
Feeding hens a high level of KI and injecting with estradiol produced histological
changes in the liver similar to those previously reported with "fatty liver syndrome." It is suggested that this may be a model for studying the "fatty liver syndrome" in laying hens. REFERENCES Arrington, L. R., R. A. Santa Cruz, R. H. Harms and H. R. Wilson, 1967. Effects of excess dietary iodine upon pullets and laying hens. J. Nutr. 92: 325-330. Couch, J. R., 1956. Fatty livers in laying hens—a condition which may occur as a result of increased strain. Feedstuffs, 28(47): 46. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Levine, J. B., and B. Zak, 1964. Automated determination of serum total cholesterol. Clin. Chim. Acta, 10: 381. Harms, R. H., C. F. Simpson and B. L. Damron, 1972. Some new observations on "fatty liver syndrome" in laying hens. Avian Dis. 16: 1042-1046. Reedy, L. M., 1968. Some clinical observations on the fatty liver syndrome (FLS) in laying hens. Proc. Texas Nutr. Conf. 23: 80. Nugura, D., and H. M. Edwards, 1970. Changes in fatty acid composition of cockerel testes due to age and fat deficiency. J. Nutr. 100: 156-160. Wolford, J. H., and D. Polin, 1974. Induced fatty liver-hemorrhagic syndrome (FLHS) and accumulation of hepatic lipid in force-fed laying chickens. Poultry Sci. 53: 65-74.
NEWS AND NOTES (Continued from page 516) In local community service he has been active in the American Red Cross, Chamber of Commerce, Project Concern, United Fund, and half a dozen other organizations. "Sanford's Morrison Award is, in many ways, as modest as the man himself—an engraved desk pen and credit for purchase of $125 worth of books and educational materials," Heywood said. CALIFORNIA NOTES Dr. A. A. Bickford, avian pathologist, has joined
the University of California Cooperative Extension staff as Veterinarian for Avian Species. He has been associated with the University Cooperative Extension for the last two years while working on a Federal Extension Service project related to the 1971-73 Newcastle disease outbreak. In his new post as Veterinary Specialist for Poultry and Other Avian Species, he will work with County Cooperative staff on field problems, cooperate with the State Department of Food and Agriculture on regulatory matters that affect avian diseases, and will work with faculty in the School of Veterinary Medicine
(Continued on page 562)
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 2, 2015
The histologic appearance of the livers was dependent upon the diet consumed. Livers of hens fed the control diet, or the control diet supplemented with KI had a similar appearance (Fig. 1). It was observed that the majority of the hepatic cells contained lipid droplets of small or moderate size. Such lipid infiltrated hepatic cells were swollen and sinusoids were open and usually contained blood. The hepatic cells of hens injected with estradiol usually contained large lipid droplets which caused pronounced swelling of the involved cells (Fig. 2). Focal areas of necrosis were seen also in liver sections from such treated hens. The most advanced lesions were present in livers of hens treated with both KI and estradiol. In this treatment the hepatic cells were greatly swollen due to the presence of lipid, and, in addition, several areas of diffuse hemorrhagic necrosis were present in the tissue sections (Fig. 3). These histologic observations indicate that feeding birds high levels of KI and injecting estradiol produce a condition similar to FLS. Serum cholesterol levels were elevated and liver damage was found as indicated by histological examination. These conditions are similar for hens with FLS as reported by Harms et al. (1972).
Department of Poultry Science, Florida Agricultural
Experiment Station, Gainesville, Florida 32611
(Received for publication July 19, 1976)
POULTRY SCIENCE 56: 517-520,
1977
hens in the same flock. They also observed enlarged combs in the non-laying hens which may have been due to a hormone imbalance. Since it has been impossible to reproduce the condition under experimental conditions it has been very difficult to study. Wolford and Polin (1974) reported hepatic hemorrhages characteristics of "fatty liver-hemorrhagic-syndrome" (FLHS) were induced when hens were force fed in excess of 50% more feed than they would normally consume. They suggested that the forced-feeding procedure could be a technique adaptable for use in studying this condition. Arrington et al. (1967) reported that the feeding of high levels of iodine to laying hens caused a cessation of egg production without regression of the ovary. Therefore, since Harms et al. (1972) had indicated a possible imbalance of hormones with non-laying hens suffering with FLS, the present experiments were conducted to determine the influence of injections of estrogen into hens fed iodine.
INTRODUCTION
T
HE "fatty liver syndrome" (FLS) in laying hens was first reported by Couch (1956), describing field cases submitted to the Texas Diagnostic Laboratory in 1954 and 1955. He reported that the fat content of the liver of laying hens varied greatly reaching levels as high as 70%, and that globules of fat seeped through the liver capsule. Reedy (1968) later reported that FLS was characterized by a decrease in egg production by as much as 50%. These hens were heavier than expected for this stage of production, which was attributed primarily to excessive fat in the abdominal cavity. The liver was enlarged, very fatty, extremely friable and varied in color from tan to pale yellow. Many of the livers contained single or multiple hemorrhages or multiple hematomas. Harms et al. (1972) observed that cholesterol levels were approximately three times as great in non-laying hens suffering from FLS as laying
EXPERIMENTAL PROCEDURE
1. Florida Agr. Exp. Sta. Journal Series no. 6128. 2. Present address: Department of Poultry Science, Auburn University, Auburn, Alabama.
Two experiments were conducted with Babcock B-300 hens approximately 80 weeks 517
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 2, 2015
ABSTRACT Two experiments were conducted with aged laying hens to determine the influence of feeding 5,000 p.p.m. of iodine as potassium iodine (KI) and/or injecting 12 mg. of estradiol upon fat accumulation in the liver and serum cholesterol levels. The KI was fed for 8 days before making liver and blood determinations, and the estradiol was injected 3 days prior to making the determinations. The feeding of KI or injection of estradiol resulted in significantly increased liver weight. When the two treatments were combined a further significant increase in liver weights was obtained. The percent of fat in the liver was significantly increased by the injection of estradiol. However, the feeding of KI in the presence or absence of estradiol did not affect the percentage of fat in the liver. Neither of the treatments significantly affected the fatty acid composition of the liver fat. Feeding of KI or injection of estradiol significantly increased total serum cholesterol levels. When the two treatments were combined a further increase in serum cholesterol level was observed. Histological changes of the livers of hens treated with KI and estradiol were similar to those previously described for the "fatty liver syndrome."
518
R. H. HARMS, D. A. ROLAND, SR. AND C. F. SIMPSON
Hen wt.
Liver wt.
E
(G)
(G)
Liver(% of body wt.)
Liver fat
KI
Liver moisture (%)
+ + 1 1
(%)
+ 1 +1
TABLE 1.—Body weight, liver weight and percent of body weight, percent of fat and moisture in liver' Treatment
1650a 1593ab 1656a 1563b
41.5a 54.0b 53.4b 67.5c
2.54a 3.38b 3.23b 4.17c
11.5a 12.5a 16.0b 11.5a
69.2a 63.2b 64.0b 67.1a
1 Means without common letters are significantly different according to Duncan's multiple range test (1955). KI = Received a diet containing 5000 p.p.m. I as KI. E = Injected with 12 mg. estradiol three days before sacrificing.
TABLE 2.—Composition of liver fat as determined by gas-liquid chromatography
1 + 1+
E
14:0
16:0
16:1
18:0
18:1
18:2
20:4
+ +
2.76 5.46 5.43 2.09
19.43 20.44 23.12 20.72
11.55 10.64 7.39 9.20
11.92 10.98 12.12 10.00
34.31 34.40 38.33 38.12
15.97 14.79 11.54 16.66
4.06 3.29 2.07 3.21
'Carbon chain length: number of double bonds. KI = Received a diet containing 5000 p.p.m. I as KI. E = Injected with 12 mg. estradiol three days before sacrificing. TABLE 3.—Total serum cholesterol from hens receiving four different treatments Serum cholesterol (mg./lOOml.)1 242.8a Control 537.0b Estradiol 752.2c KI 1129.6d KI and estradiol 1 Means without common letters are significantly different according to Duncan's multiple range test (1955). Diet
of age. The hens had been receiving, and were fed during the experimental period a diet containing 16.0% protein, 2853 kilocalories of metabolizable energy per kilogram, 3.00% calcium and 0.75% total phosphorus. Four experimental treatments used were as follows: (1) Control, (2) 5,000 p.p.m. I in the feed as furnished by 6.54 g. of K l / k g . of diet (3) each hen injected subcutaneously with 12 mg. estradiol, and (4) combination of treatment 3 and 4. Three replicates of five individually caged birds were used per treatment in experiment 1, and two replicates of five individually caged birds were used in
experiment 2. The KI was fed for eight days. The estradiol was injected three days prior to termination of the experiment. All the birds were individually weighed at the termination of the experiment. Blood was drawn by anterior heart puncture for total serum cholesterol determination. All birds were then sacrificed and liver weights obtained. The percent fat and moisture in the liver, and fatty acids in the liver was then determined. A portion of liver from 5 birds per treatment group was fixed in 10% neutral formalin, embedded in paraffin, cut at 6 \xm. and stained with hematoxylin and eosin for examination with the light microscope. The fatty acid composition data were obtained by gas-liquid chromatography analysis according to the methods described by Nugura and Edwards (1970). Total serum cholesterol values for 5 birds per treatment group were determined by the procedure outlined by Levine and Zak (1964). Since the treatment x experiment interaction was not significant, the data were pooled for this presentation. Significant differences
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 2, 2015
Fatty acid'
Treatment KI
EXPERIMENTAL "FATTY LIVER SYNDROME" IN LAYING HENS
519
FIG. 2. Liver of hen treated with estradiol. There is a focal area of necrosis (N), and liver cells at the periphery contain lipid (arrows). Hematoxylin and eosin stain, x 80.
FIG. 3. Liver of hen treated with KI and estradiol. There is diffuse necrosis (N) and hepatic cells at the periphery contain lipid (arrows). Hematoxylin and eosin stain, x 80.
x>« , v ' \ « % «i
-
FIG. I. Liver of control hen. The hepatic cells contain droplets of lipid (arrows), and the sinusoids (S) contain blood. Hematoxylin and eosin stain. x 80. between treatment means were determined by Duncan's multiple range tests (1955). RESULTS AND DISCUSSION
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 2, 2015
Feeding 5,000 p.p.m. of iodide or injecting 12 mg. estradiol did not significantly influence body weight (Table 1). However, birds fed KI and injected with estradiol had significantly decreased body weights.
The feeding of KI or injection of estradiol resulted in significantly increased liver weights (Table 2). A further significant increase in liver weights was obtained when the two treatments were combined. Increases in liver weights were evident whether based on an absolute weight or a percentage of the body weight. Injecting hens with estradiol significantly increased liver fat; however, feeding of KI or the combination of KI and estradiol did not influence liver fat. The feeding of KI increased the fat in the liver from 4.77 to 6.75 grams. Injecting the estradiol resulted in 8.54 grams of fat in the liver, and this was slightly reduced to 7.76 when KI was fed along with estradiol injection. Liver moisture was significantly reduced by feeding KI or injecting estradiol (Table 1), however, when the two were combined, liver moisture was not influenced. Neither of the treatments significantly affected the fatty acid composition of the liver fat (Table 2). Total serum cholesterol levels were significantly increased by feeding potassium iodide or injecting the hens with estradiol (Table 3). A further increase occurred with a combination of the treatments.
520
R. H. HARMS, D. A. ROLAND, SR. AND C. F. SIMPSON
Feeding hens a high level of KI and injecting with estradiol produced histological
changes in the liver similar to those previously reported with "fatty liver syndrome." It is suggested that this may be a model for studying the "fatty liver syndrome" in laying hens. REFERENCES Arrington, L. R., R. A. Santa Cruz, R. H. Harms and H. R. Wilson, 1967. Effects of excess dietary iodine upon pullets and laying hens. J. Nutr. 92: 325-330. Couch, J. R., 1956. Fatty livers in laying hens—a condition which may occur as a result of increased strain. Feedstuffs, 28(47): 46. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Levine, J. B., and B. Zak, 1964. Automated determination of serum total cholesterol. Clin. Chim. Acta, 10: 381. Harms, R. H., C. F. Simpson and B. L. Damron, 1972. Some new observations on "fatty liver syndrome" in laying hens. Avian Dis. 16: 1042-1046. Reedy, L. M., 1968. Some clinical observations on the fatty liver syndrome (FLS) in laying hens. Proc. Texas Nutr. Conf. 23: 80. Nugura, D., and H. M. Edwards, 1970. Changes in fatty acid composition of cockerel testes due to age and fat deficiency. J. Nutr. 100: 156-160. Wolford, J. H., and D. Polin, 1974. Induced fatty liver-hemorrhagic syndrome (FLHS) and accumulation of hepatic lipid in force-fed laying chickens. Poultry Sci. 53: 65-74.
NEWS AND NOTES (Continued from page 516) In local community service he has been active in the American Red Cross, Chamber of Commerce, Project Concern, United Fund, and half a dozen other organizations. "Sanford's Morrison Award is, in many ways, as modest as the man himself—an engraved desk pen and credit for purchase of $125 worth of books and educational materials," Heywood said. CALIFORNIA NOTES Dr. A. A. Bickford, avian pathologist, has joined
the University of California Cooperative Extension staff as Veterinarian for Avian Species. He has been associated with the University Cooperative Extension for the last two years while working on a Federal Extension Service project related to the 1971-73 Newcastle disease outbreak. In his new post as Veterinary Specialist for Poultry and Other Avian Species, he will work with County Cooperative staff on field problems, cooperate with the State Department of Food and Agriculture on regulatory matters that affect avian diseases, and will work with faculty in the School of Veterinary Medicine
(Continued on page 562)
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 2, 2015
The histologic appearance of the livers was dependent upon the diet consumed. Livers of hens fed the control diet, or the control diet supplemented with KI had a similar appearance (Fig. 1). It was observed that the majority of the hepatic cells contained lipid droplets of small or moderate size. Such lipid infiltrated hepatic cells were swollen and sinusoids were open and usually contained blood. The hepatic cells of hens injected with estradiol usually contained large lipid droplets which caused pronounced swelling of the involved cells (Fig. 2). Focal areas of necrosis were seen also in liver sections from such treated hens. The most advanced lesions were present in livers of hens treated with both KI and estradiol. In this treatment the hepatic cells were greatly swollen due to the presence of lipid, and, in addition, several areas of diffuse hemorrhagic necrosis were present in the tissue sections (Fig. 3). These histologic observations indicate that feeding birds high levels of KI and injecting estradiol produce a condition similar to FLS. Serum cholesterol levels were elevated and liver damage was found as indicated by histological examination. These conditions are similar for hens with FLS as reported by Harms et al. (1972).
