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Influence of Diethylstilbestrol on the Turkey With Special Reference to Histological Changes in the Aorta1
Influence of Diethylstilbestrol on the Turkey With Special Reference to Histological Changes in the Aorta 1 L. M. KRISTA, 2 J. H. SAUTTER AND P. E. WAIBEL Departments of Animal Science, and Veterinary Pathology, University of Minnesota, St. Paul, Minnesota 55101 (Received for publication June 6, 1969)
INTRODUCTION
T
HE use of diethylstilbestrol (DES) presently is not permissible in the United States as an aid to increasing body weight gains or improving carcass quality in poultry. However, this compound represents a valuable tool for the study of spontaneous dissecting aneurysm (aortic rupture) in turkeys and other metabolic and physiological changes in the bird. DES implantations have been reported by Krista et al. (1961, 1965), Beall et al. (1963), and Simpson and Harms (1965) to increase the incidence of aortic rupture in turkeys. Krista et al. (1965) and Simpson and Harms (1965) reported a concomitant decrease in blood pressure with an increase in aortic rupture. The mechanism of DES producing an increase in aortic rupture has not been elucidated. As reported by Krista et al. (1965), simultaneous injections of testosterone with DES reduced the incidence of aortic rupture. Simpson et al. (1964) noted that additional dietary sodium chloride enhanced the effect of DES with respect to the incidence of aortic rupture.
Considerable experimental data are available pertaining to the influence of lipid and/or cholesterol on aortic lesions in the chick (Katz and Pick, 1961). In1 Published as Paper No. 6957 Scientific Journal Series of the Minnesota Agricultural Experiment Station. This investigation supported by grant HE-04177 from National Heart Institute, U.S.P.H.S. 2 Present address: Department of Anatomy and Histology, Auburn University, Auburn, Alabama.
creasing the blood lipid and/or cholesterol levels increased the lipid and/or cholesterol levels in atherosclerotic plaques of chickens (Lindsay et al., 1955). Krista et al. (1965) fed a high level of animal fat and cholesterol to growing turkeys and obtained no increase in incidence of aortic rupture. However, administration of DES increased blood lipid levels (Simpson et al., 1964) and incidence of aortic rupture. DES implantation increased lipid deposits at the site of aortic plaques (Ball, 1964) and increased smooth muscle cell degeneration in plaques (Kurtz, 1966). Kurtz (1969) also demonstrated ulcer formation at plaque sites due to DES. Atherosclerotic plaques are commonly observed in the abdominal aorta of the turkey and increase in size with age. Such plaques have been associated with aortic rupture (Carnahgan, 1955; Gibson and de Gruchy, 1955) because of the structural changes which they produce in the aorta. DES represents a potent physiological stimulus to the turkey in terms of growth, blood pressure, sexual characteristics, and aortic integrity. It seemed important to study the influence of graded and frequently administered doses of DES on these characters as contrasted to the usual high level and infrequent pellet method of administration. Such a study is described herein. METHODS Broad White male turkey poults (commercial strain) were obtained at one day
1961
1962
L. M . K R I S T A , J. H . SAUTTER AND P . E .
TABLE 1.—Effect
Level of DES mg./kg. — body wt. 0 2 10 50 1 2 3 4
of DES on body weight, indirect systolic blood pressure, aortic rupture, and tissue analysis1
Age in weeks 6 kg. 1.66a 1.59a 1.65a 1.49b
WAIBEL
Blood pressure2
Aortic rupture
Liver weight3'4
12 kg. 5.52a 5.32a 4.71b 3.06c
Muscle content 4 Fat
mm. Hg 240a 237a 194b 172c
/o 5.00a 6.25a 20.00b 5.00a
%
1.11a 1.44b 2.14c 2.60d
Moisture
%
3.40a 5.00b 6.16b 5.12b
% 74.9a 73.2b 72.0b 72.2b
Multiple range test. Data significantly different (P<.05) if followed by uncommon letter. Recorded at eight weeks of age. Liver weight expressed as percentage of body weight. Determined at termination of experiment (16 weeks of age).
of age and were brooded in conventional batteries with raised wire-mesh floors and contact heaters for a seven-day pre-experimental period. The birds were then separated into groups a t five gm. intervals and then distributed uniformly into eight experimental pens (40 poults/pen) and wingbanded. T h e birds were maintained in brooders for two additional weeks and then transferred to floor pens (1.8 X 2.7 m.) with wood shavings litter. Feed and water were given ad libitum. T h e diets used are described b y Krista et al. (1969) (diet #3). D E S injections were made at two week intervals starting at two weeks of age and continuing until 14 weeks of age. Each bird received 0, 2, 10, or 50 mg. of D E S / k g . of body weight at each injection. Doses were prepared b y mixing a proprietary paste product 3 with similar proportions of the stated carrier ingredients (rosin, propylene glycol, and isopropanol). Controls received a placebo of the carrier. Individual body weight and observational data were obtained a t two week intervals. Indirect systolic blood pressure measurements were recorded at eight weeks of age as described b y Krista et al. (1963). 3 Caponade (60 mg. diethylstilbestrol/cc), Vineland Poultry Laboratories, Vineland, New Jersey 08360.
At two week intervals two poults were randomly selected from each pen and sacrificed. Blood plasma samples were obtained b u t were useless for analysis following mechanical failure of a freezer unit. The aortas were placed in buffered 10 percent formalin for morphological studies. At 16 weeks of age muscle from the right leg was removed, ground, frozen, and later analyzed for moisture and fat content (A.O.A.C, 1950). Typical testes were photographed and liver weights recorded at 16 weeks of age. RESULTS AND DISCUSSION The influence of D E S on experimental measurements is presented in Table 1. Body weight changes b y two week intervals are presented in Fig. 1. No marked effect due to D E S treatment was observed until six weeks of age at which time the poults on the high level (50 mg./kg.) of D E S t r e a t m e n t weighed approximately 9 percent less than the control or lower D E S groups (significance: P < . 0 5 ) . At the highest level of D E S , strutting was noted one week after the first injection. At six weeks of age the high D E S birds showed a deterioration of gross physical characteristics and all birds receiving D E S t r e a t m e n t showed increased libido as demonstrated b y strutting and caruncle involvement. Upon sacrifice a t 16 weeks of
DlETHYLSTILBESTROL AND TURKEYS
1963
age, observations indicated depression of testes size (Fig. 2) and increased body and liver fat (Fig. 3) due to D E S . Similar results are reported by Simpson el al. (1965). Significantly lower systolic arterial blood pressures were observed in the medium and high D E S groups than the control or low D E S groups at eight weeks of age. The medium and high D E S groups were significantly different from each other. At this age, growth was not depressed b u t arterial blood pressure was significantly reduced by D E S (10 m g . / kg.) treatment. T h e two mg. level of D E S neither depressed growth nor altered blood pressure. The highest incidence of mortality due to aortic rupture was observed in the medium D E S group. T h e low mortality rate observed in the high D E S group may
Diathyl»tilb«»trol (mo/kg body weight)
FIG. 2. Testes photographed at 16 weeks of age. DES treatment levels of 0, 2, 10, and 50 mg./kg. body weight are represented as A, B, C, and D, respectively.
2 10 50
» A g . In
FIG. 1. The effect of diethylstilbestrol injection on body weight.
be explained by the decreased growth rate, reduced blood pressure, and general debility including rectal prolapse, poor conformation, leg deformity, and decreased activity. All groups receiving D E S treatment displayed clinical and physiological changes which were manifested by changes in liver weight and deposition of body fat. Percent liver weights were significantly increased with increasing treatment levels of D E S (Table 1 and Fig. 3). The groups receiving D E S treatment had significantly more muscle fat than control birds. T h e level of fat deposited may have been associated with the level of D E S although the differences among D E S groups were not significant. Regarding
morphological
changes in
1964
L. M. K R I S T A , J. H. SAUTTER AND P . E. W A I B E L
FIG. 3. Display of carcasses sacrificed at 16 weeks of age. Note the difference in body fat (abdominal) and liver size and color (Left to right: 50, 10, 2, and 0 mg. DES/kg. body weight).
the aorta due to graded levels of D E S , similar lesions were observed in all tissues examined b u t tissues from D E S treated birds showed exaggerated changes. In the experimental turkeys, the aortic plaques were larger and the intimal proliferation more marked than in the control birds. Also, increased numbers of vacuoles were observed in the treated birds. Ruptures in the aorta occurred anterior to the emergence of the sciatic arteries. Examination of the areas revealed uneven growth of intimal structure to almost papillary proportions and very thin tunica media (Fig. 4). Microscopic examination of the ruptured and the deformed aortas revealed a few foci of cysts or vacuoles (Fig. 5) containing a proteinaceous granular material which gave the staining reaction of mucopolysaccharide b u t with very little fat and no cholesterol. Oil Red 0 positive material was located
usually at the base of plaques with an occasional focus in the media, and small droplets (Fig. 6) were located intracellular^' in the media. When present, fat was in insignificant amounts in cells and not associated with the mechanism of rupture. The tissue in association with developing ruptures appeared to be fragmented, degenerated, disorganized, and lacking in normal appearing intracellular substance. The vacuoles or clear spaces resembled lipid but failed to stain with Oil Red (). Some stained a pale blue with alcian blue indicating the presence of mucopolysaccharide. T h e large numbers of vacuoles in the endothelial cells (Figs. 5, 7, and 8) formed an extremely porous and apparently weak barrier to the pulsation of the blood under pressure in the aorta. T h e increased number of vacuoles or clear areas and the
DlETHYLSTILBESTROL AND TURKEYS
1965
FIG. 4. Cross section of aorta just anterior to the sciatic arteries. Note the polypoid structures and uneven-disorganized wall (16 weeks of age), Movat stain.
tendency for the internal elastic membrane (Figs. 5 and 8) to break, fragment, and completely disappear in many instances were prominent features in the D E S treated turkeys. When the latter occurred, myointimal or muscle cells proliferated and became arranged vertically or at right angles to the lumen, thus affording little or no resistance to subsequent distortion and splitting. This peculiar arrangement was noted in layers throughout the tunica media. The incidence of plaques and plaque size in the experimental groups increased up to 16 weeks of age when the experiment was terminated. Compared with the controls, the aortic wall (Fig. 4) was
increasingly distorted with grossly observable thin areas. Although D E S has been reported (Simpson and Harms, 1965) to increase the amount of lipid in the cells of the intimal layer in the plaques, and in the tunica media, large amounts of lipid and cystic degeneration were not observed in the present study. As indicated b y stains, there was very little fat in the cells of the internal layer, b u t cystic degeneration represented b y vacuoles (Figs, 5, 7, and 8) was frequent in the treated birds. Foci of fat in plaques and media were infrequent b u t cystic or clear vacuoles were common. A typical amount and distribution of lipid present in a portion of a plaque in a
1966
L. M. K R I S T A , J. H. SAUTTER AND P . E. W A I B E L
FIG. 5. Vacuoles or cystic degeneration in intima. Note fine granular material (arrow) in the smaller spaces. Stains for fat were negative. Note ruptured internal elastic membrane 200X, Movat stain.
14 week old bird is illustrated in Fig. 6. An interesting feature of the developing vessel wall in association with the early or small plaque was the reorientation of the muscle cells (Fig. 7) from a usual parallel position to vertical or right angles to the lumen. T h e appearance of the muscle or myointimal cells was suggestive of a connection between two or more cells as described b y Dewey and Barr (1962) which they term a "nexus." The cells appeared to be firmly implanted in (Fig. 7) and associated with the elastic lamina while the end toward the lumen blended into the internal layer of cells which were usually a developing plaque. These muscle cells so arranged have varying numbers of vacuoles (Fig. 7) which were
mostly negative to fat stains and alcian blue. Certainly, these structural changes result in altered metabolic and physical function. SUMMARY
Physiological and metabolic changes were induced b y D E S treatment. Even though distinct morphological differences are h a r d to establish, the levels of D E S could be related tochanges in body weight, blood pressure, carcass composition, genera] conformation, and secondary sex characteristics. The lowest level of D E S did not influence body weight, blood pressure, or aortic rupture, b u t an increase in body fat and liver size was observed. T h e medium
DlETHYLSTILBESTROL AND TURKEYS
level of DES resulted in a significant reduction in weight gain at 12 weeks of age, a significant decrease in blood pressure at eight weeks of age, and a significant increase in aortic rupture, plaque formation, and degeneration. The highest level of DES had an obvious toxic effect on the birds as indicated by a debilitated appearance and depressed weight gains by six weeks of age. Morphological changes due to DES included increased reorientation of fibers (presumably smooth muscle cells) from a parallel position to a radial position. This reorientation of fibers and subsequent thinning of the aortic wall and degeneration of the elastic tissue of the media are factors that may lead to the rupture of the wall. Plaque size increased with age. Many of the vacuoles observed did not stain with Oil Red 0. Dosage of DES represents an important variable in relation to results ob-
';>::V* I
FIG. 6. Droplets of fat dispersed throughout a portion of a plaque. Oil Red 0 stain 110X.
1967
FIG. 7. Vertical arrangement of myointimal cells under thickened cystic intima 160X, Movat stain.
tained and should be carefully considered in experimental work. ACKNOWLEDGMENT The following firms generously provided materials for this study: Abbott Laboratories, No. Chicago, 111.; American Cyanamid Co., Princeton, N. J.; Commercial Solvents, Corp., Terre Haute, Ind.; Distillation Products Industries, Rochester, N. Y.; E. I. du Pont de Nemours and Co., Western Springs, 111.; Hoffmann-LaRoche, Inc., Nutley, N. J.; Merck and Co., Rah way, N. J.; Nopco
riG. 8. Cystic endothelial cells, disintegrating elastica, and disorganized ground substance 160X, Movat stain.
1968
L. M. KRISTA, J. H. SAUTTER AND P. E. WAIBEL
Chemical Co., Newark, N. J.; and Van Hoven Co., Inc., So. St. Paul, Minnesota. REFERENCES Association of Official Agricultural Chemists. 1950. Official Methods of Analysis, 7th ed. p. 359. George Banta Publishing Company. Ball, R. A., 1964. A study of vascular diseases in the turkey. Ph.D. Thesis, University of Minnesota, St. Paul, Minnesota. Beall, C. W., C. F. Simpson, W. R. Prichard and R. H. Harms, 1963. Aortic rupture in turkeys induced by diethylstilbestrol. Proc. Soc. Exp. Biol. Med. 113: 442-43. Carnaghan, R. B. A., 1955. Atheroma of the aorta associated with dissecting aneurysms in turkeys. Vet. Rec. 67: 568-569. Dewey, M. M., and L. Barr, 1962. Intercellular connection between smooth muscle cells: The nexus. Science, 137: 670-672. Gibson, E. A., and P. H. de Gruchy, 1955. Aortic rupture in turkeys subsequent to dissecting aneurysm. Vet. Rec. 67: 650-654. Katz, L. N., and R. Pick, 1961. Morphological aspects of atherosclerosis in the chick. Conn. Med. J. 25: 84-87. Krista, L. M., P. E. Waibel, R. E. Burger, R. A. Ball and J. H. Sautter, 1961. Influence of dietary and hormonal alterations during a natural outbreak of dissecting aneurysm in turkeys. Poultry Sci. 40: 1421. Krista, L. M., R. E. Burger and P. E. Waibel, 1963. Blood pressure and heart rate in the turkey as measured by the indirect method and their modifications by pharmacological agents. Poultry Sci. 42: 646-652.
Krista, L. M., P. E. Waibel and R. E. Burger, 1965The influence of dietary alterations, hormones, and blood pressure on the incidence of dissecting aneurysms in the turkey. Poultry Sci. 44: 15-22. Krista, L. M., P. E. Waibel, J. H. Sautter and R. N. Shoffner, 1969. Aortic rupture, body weight, and blood pressure in the turkey as influenced by strain, dietary fat, Aeta-aminopropionitrile fumarate, and diethylstilbestrol. Poultry Sci. 48: 1954-1960. Kurtz, H. J., 1966. A study of factors influencing aortic dissecting aneurysms. Ph.D. Thesis, University of Minnesota, St. Paul, Minnesota. Kurtz, H. J., 1969. Aortic plaque degeneration as a cause of aortic rupture in turkeys. Am. J. Vet. Res. 30: 251-259. Lindsay, S., C. W. Nichols and I. L. Chairkoff, 1955. Aortic lesions induced in the bird by diethylstilbestrol injections and cholesterol feeding. A.M.A. Archives Path. 59: 173-184. McSherry, J. B., A. E. Ferguson and J. Ballantyne, 1954. A dissecting aneurysm in internal hemorrhage in turkeys. J. Am. Vet. Med. Assoc. 124: 279-283. Simpson, C. F., R. H. Harms and F. C. Neal, 1964. Influence of high dietary sodium chloride on aortic rupture in turkeys induced by diethylstilbestrol. Proc. Soc. Exp. Biol. Med. 116: 334-336. Simpson, C. F., and R. H. Harms, 1965. Relationship of age and sex of turkeys to aortic ruptures induced by diethylstilbestrol. Proc. Soc. Exp. Biol. Med. 119: 509-512. Simpson, C. F., R. H. Harms and H. R. Wilson, 1965. Alteration of sex characteristics of turkey poults with diethylstilbestrol. Proc. Soc. Exp. Biol. Med. 119: 435-438.
Egg Quality in Gifu City, Japan A. W. BRANT, 1 S. SENDA, T. TAKAHASHI AND T. NAKAMURA Department of Poultry and Animal Sciences, Gifu University, Kagamihara, Gifu (Received for publication June 7, 1969)
INTRODUCTION
T
HE last 10 years have witnessed a very rapid rise in egg production in Japan. In 1957 there were an estimated 30 1 Sabbatical leave, University Davis, California 95616.
of
California,
million laying hens. In 1967 this number had risen to 90 million. The Ministry of Agriculture and Forestry of the Japanese government estimated the 1967 per capita intake of eggs per day was 27.5 grams. This would be the equivalent of approx-
INTRODUCTION
T
HE use of diethylstilbestrol (DES) presently is not permissible in the United States as an aid to increasing body weight gains or improving carcass quality in poultry. However, this compound represents a valuable tool for the study of spontaneous dissecting aneurysm (aortic rupture) in turkeys and other metabolic and physiological changes in the bird. DES implantations have been reported by Krista et al. (1961, 1965), Beall et al. (1963), and Simpson and Harms (1965) to increase the incidence of aortic rupture in turkeys. Krista et al. (1965) and Simpson and Harms (1965) reported a concomitant decrease in blood pressure with an increase in aortic rupture. The mechanism of DES producing an increase in aortic rupture has not been elucidated. As reported by Krista et al. (1965), simultaneous injections of testosterone with DES reduced the incidence of aortic rupture. Simpson et al. (1964) noted that additional dietary sodium chloride enhanced the effect of DES with respect to the incidence of aortic rupture.
Considerable experimental data are available pertaining to the influence of lipid and/or cholesterol on aortic lesions in the chick (Katz and Pick, 1961). In1 Published as Paper No. 6957 Scientific Journal Series of the Minnesota Agricultural Experiment Station. This investigation supported by grant HE-04177 from National Heart Institute, U.S.P.H.S. 2 Present address: Department of Anatomy and Histology, Auburn University, Auburn, Alabama.
creasing the blood lipid and/or cholesterol levels increased the lipid and/or cholesterol levels in atherosclerotic plaques of chickens (Lindsay et al., 1955). Krista et al. (1965) fed a high level of animal fat and cholesterol to growing turkeys and obtained no increase in incidence of aortic rupture. However, administration of DES increased blood lipid levels (Simpson et al., 1964) and incidence of aortic rupture. DES implantation increased lipid deposits at the site of aortic plaques (Ball, 1964) and increased smooth muscle cell degeneration in plaques (Kurtz, 1966). Kurtz (1969) also demonstrated ulcer formation at plaque sites due to DES. Atherosclerotic plaques are commonly observed in the abdominal aorta of the turkey and increase in size with age. Such plaques have been associated with aortic rupture (Carnahgan, 1955; Gibson and de Gruchy, 1955) because of the structural changes which they produce in the aorta. DES represents a potent physiological stimulus to the turkey in terms of growth, blood pressure, sexual characteristics, and aortic integrity. It seemed important to study the influence of graded and frequently administered doses of DES on these characters as contrasted to the usual high level and infrequent pellet method of administration. Such a study is described herein. METHODS Broad White male turkey poults (commercial strain) were obtained at one day
1961
1962
L. M . K R I S T A , J. H . SAUTTER AND P . E .
TABLE 1.—Effect
Level of DES mg./kg. — body wt. 0 2 10 50 1 2 3 4
of DES on body weight, indirect systolic blood pressure, aortic rupture, and tissue analysis1
Age in weeks 6 kg. 1.66a 1.59a 1.65a 1.49b
WAIBEL
Blood pressure2
Aortic rupture
Liver weight3'4
12 kg. 5.52a 5.32a 4.71b 3.06c
Muscle content 4 Fat
mm. Hg 240a 237a 194b 172c
/o 5.00a 6.25a 20.00b 5.00a
%
1.11a 1.44b 2.14c 2.60d
Moisture
%
3.40a 5.00b 6.16b 5.12b
% 74.9a 73.2b 72.0b 72.2b
Multiple range test. Data significantly different (P<.05) if followed by uncommon letter. Recorded at eight weeks of age. Liver weight expressed as percentage of body weight. Determined at termination of experiment (16 weeks of age).
of age and were brooded in conventional batteries with raised wire-mesh floors and contact heaters for a seven-day pre-experimental period. The birds were then separated into groups a t five gm. intervals and then distributed uniformly into eight experimental pens (40 poults/pen) and wingbanded. T h e birds were maintained in brooders for two additional weeks and then transferred to floor pens (1.8 X 2.7 m.) with wood shavings litter. Feed and water were given ad libitum. T h e diets used are described b y Krista et al. (1969) (diet #3). D E S injections were made at two week intervals starting at two weeks of age and continuing until 14 weeks of age. Each bird received 0, 2, 10, or 50 mg. of D E S / k g . of body weight at each injection. Doses were prepared b y mixing a proprietary paste product 3 with similar proportions of the stated carrier ingredients (rosin, propylene glycol, and isopropanol). Controls received a placebo of the carrier. Individual body weight and observational data were obtained a t two week intervals. Indirect systolic blood pressure measurements were recorded at eight weeks of age as described b y Krista et al. (1963). 3 Caponade (60 mg. diethylstilbestrol/cc), Vineland Poultry Laboratories, Vineland, New Jersey 08360.
At two week intervals two poults were randomly selected from each pen and sacrificed. Blood plasma samples were obtained b u t were useless for analysis following mechanical failure of a freezer unit. The aortas were placed in buffered 10 percent formalin for morphological studies. At 16 weeks of age muscle from the right leg was removed, ground, frozen, and later analyzed for moisture and fat content (A.O.A.C, 1950). Typical testes were photographed and liver weights recorded at 16 weeks of age. RESULTS AND DISCUSSION The influence of D E S on experimental measurements is presented in Table 1. Body weight changes b y two week intervals are presented in Fig. 1. No marked effect due to D E S treatment was observed until six weeks of age at which time the poults on the high level (50 mg./kg.) of D E S t r e a t m e n t weighed approximately 9 percent less than the control or lower D E S groups (significance: P < . 0 5 ) . At the highest level of D E S , strutting was noted one week after the first injection. At six weeks of age the high D E S birds showed a deterioration of gross physical characteristics and all birds receiving D E S t r e a t m e n t showed increased libido as demonstrated b y strutting and caruncle involvement. Upon sacrifice a t 16 weeks of
DlETHYLSTILBESTROL AND TURKEYS
1963
age, observations indicated depression of testes size (Fig. 2) and increased body and liver fat (Fig. 3) due to D E S . Similar results are reported by Simpson el al. (1965). Significantly lower systolic arterial blood pressures were observed in the medium and high D E S groups than the control or low D E S groups at eight weeks of age. The medium and high D E S groups were significantly different from each other. At this age, growth was not depressed b u t arterial blood pressure was significantly reduced by D E S (10 m g . / kg.) treatment. T h e two mg. level of D E S neither depressed growth nor altered blood pressure. The highest incidence of mortality due to aortic rupture was observed in the medium D E S group. T h e low mortality rate observed in the high D E S group may
Diathyl»tilb«»trol (mo/kg body weight)
FIG. 2. Testes photographed at 16 weeks of age. DES treatment levels of 0, 2, 10, and 50 mg./kg. body weight are represented as A, B, C, and D, respectively.
2 10 50
» A g . In
FIG. 1. The effect of diethylstilbestrol injection on body weight.
be explained by the decreased growth rate, reduced blood pressure, and general debility including rectal prolapse, poor conformation, leg deformity, and decreased activity. All groups receiving D E S treatment displayed clinical and physiological changes which were manifested by changes in liver weight and deposition of body fat. Percent liver weights were significantly increased with increasing treatment levels of D E S (Table 1 and Fig. 3). The groups receiving D E S treatment had significantly more muscle fat than control birds. T h e level of fat deposited may have been associated with the level of D E S although the differences among D E S groups were not significant. Regarding
morphological
changes in
1964
L. M. K R I S T A , J. H. SAUTTER AND P . E. W A I B E L
FIG. 3. Display of carcasses sacrificed at 16 weeks of age. Note the difference in body fat (abdominal) and liver size and color (Left to right: 50, 10, 2, and 0 mg. DES/kg. body weight).
the aorta due to graded levels of D E S , similar lesions were observed in all tissues examined b u t tissues from D E S treated birds showed exaggerated changes. In the experimental turkeys, the aortic plaques were larger and the intimal proliferation more marked than in the control birds. Also, increased numbers of vacuoles were observed in the treated birds. Ruptures in the aorta occurred anterior to the emergence of the sciatic arteries. Examination of the areas revealed uneven growth of intimal structure to almost papillary proportions and very thin tunica media (Fig. 4). Microscopic examination of the ruptured and the deformed aortas revealed a few foci of cysts or vacuoles (Fig. 5) containing a proteinaceous granular material which gave the staining reaction of mucopolysaccharide b u t with very little fat and no cholesterol. Oil Red 0 positive material was located
usually at the base of plaques with an occasional focus in the media, and small droplets (Fig. 6) were located intracellular^' in the media. When present, fat was in insignificant amounts in cells and not associated with the mechanism of rupture. The tissue in association with developing ruptures appeared to be fragmented, degenerated, disorganized, and lacking in normal appearing intracellular substance. The vacuoles or clear spaces resembled lipid but failed to stain with Oil Red (). Some stained a pale blue with alcian blue indicating the presence of mucopolysaccharide. T h e large numbers of vacuoles in the endothelial cells (Figs. 5, 7, and 8) formed an extremely porous and apparently weak barrier to the pulsation of the blood under pressure in the aorta. T h e increased number of vacuoles or clear areas and the
DlETHYLSTILBESTROL AND TURKEYS
1965
FIG. 4. Cross section of aorta just anterior to the sciatic arteries. Note the polypoid structures and uneven-disorganized wall (16 weeks of age), Movat stain.
tendency for the internal elastic membrane (Figs. 5 and 8) to break, fragment, and completely disappear in many instances were prominent features in the D E S treated turkeys. When the latter occurred, myointimal or muscle cells proliferated and became arranged vertically or at right angles to the lumen, thus affording little or no resistance to subsequent distortion and splitting. This peculiar arrangement was noted in layers throughout the tunica media. The incidence of plaques and plaque size in the experimental groups increased up to 16 weeks of age when the experiment was terminated. Compared with the controls, the aortic wall (Fig. 4) was
increasingly distorted with grossly observable thin areas. Although D E S has been reported (Simpson and Harms, 1965) to increase the amount of lipid in the cells of the intimal layer in the plaques, and in the tunica media, large amounts of lipid and cystic degeneration were not observed in the present study. As indicated b y stains, there was very little fat in the cells of the internal layer, b u t cystic degeneration represented b y vacuoles (Figs, 5, 7, and 8) was frequent in the treated birds. Foci of fat in plaques and media were infrequent b u t cystic or clear vacuoles were common. A typical amount and distribution of lipid present in a portion of a plaque in a
1966
L. M. K R I S T A , J. H. SAUTTER AND P . E. W A I B E L
FIG. 5. Vacuoles or cystic degeneration in intima. Note fine granular material (arrow) in the smaller spaces. Stains for fat were negative. Note ruptured internal elastic membrane 200X, Movat stain.
14 week old bird is illustrated in Fig. 6. An interesting feature of the developing vessel wall in association with the early or small plaque was the reorientation of the muscle cells (Fig. 7) from a usual parallel position to vertical or right angles to the lumen. T h e appearance of the muscle or myointimal cells was suggestive of a connection between two or more cells as described b y Dewey and Barr (1962) which they term a "nexus." The cells appeared to be firmly implanted in (Fig. 7) and associated with the elastic lamina while the end toward the lumen blended into the internal layer of cells which were usually a developing plaque. These muscle cells so arranged have varying numbers of vacuoles (Fig. 7) which were
mostly negative to fat stains and alcian blue. Certainly, these structural changes result in altered metabolic and physical function. SUMMARY
Physiological and metabolic changes were induced b y D E S treatment. Even though distinct morphological differences are h a r d to establish, the levels of D E S could be related tochanges in body weight, blood pressure, carcass composition, genera] conformation, and secondary sex characteristics. The lowest level of D E S did not influence body weight, blood pressure, or aortic rupture, b u t an increase in body fat and liver size was observed. T h e medium
DlETHYLSTILBESTROL AND TURKEYS
level of DES resulted in a significant reduction in weight gain at 12 weeks of age, a significant decrease in blood pressure at eight weeks of age, and a significant increase in aortic rupture, plaque formation, and degeneration. The highest level of DES had an obvious toxic effect on the birds as indicated by a debilitated appearance and depressed weight gains by six weeks of age. Morphological changes due to DES included increased reorientation of fibers (presumably smooth muscle cells) from a parallel position to a radial position. This reorientation of fibers and subsequent thinning of the aortic wall and degeneration of the elastic tissue of the media are factors that may lead to the rupture of the wall. Plaque size increased with age. Many of the vacuoles observed did not stain with Oil Red 0. Dosage of DES represents an important variable in relation to results ob-
';>::V* I
FIG. 6. Droplets of fat dispersed throughout a portion of a plaque. Oil Red 0 stain 110X.
1967
FIG. 7. Vertical arrangement of myointimal cells under thickened cystic intima 160X, Movat stain.
tained and should be carefully considered in experimental work. ACKNOWLEDGMENT The following firms generously provided materials for this study: Abbott Laboratories, No. Chicago, 111.; American Cyanamid Co., Princeton, N. J.; Commercial Solvents, Corp., Terre Haute, Ind.; Distillation Products Industries, Rochester, N. Y.; E. I. du Pont de Nemours and Co., Western Springs, 111.; Hoffmann-LaRoche, Inc., Nutley, N. J.; Merck and Co., Rah way, N. J.; Nopco
riG. 8. Cystic endothelial cells, disintegrating elastica, and disorganized ground substance 160X, Movat stain.
1968
L. M. KRISTA, J. H. SAUTTER AND P. E. WAIBEL
Chemical Co., Newark, N. J.; and Van Hoven Co., Inc., So. St. Paul, Minnesota. REFERENCES Association of Official Agricultural Chemists. 1950. Official Methods of Analysis, 7th ed. p. 359. George Banta Publishing Company. Ball, R. A., 1964. A study of vascular diseases in the turkey. Ph.D. Thesis, University of Minnesota, St. Paul, Minnesota. Beall, C. W., C. F. Simpson, W. R. Prichard and R. H. Harms, 1963. Aortic rupture in turkeys induced by diethylstilbestrol. Proc. Soc. Exp. Biol. Med. 113: 442-43. Carnaghan, R. B. A., 1955. Atheroma of the aorta associated with dissecting aneurysms in turkeys. Vet. Rec. 67: 568-569. Dewey, M. M., and L. Barr, 1962. Intercellular connection between smooth muscle cells: The nexus. Science, 137: 670-672. Gibson, E. A., and P. H. de Gruchy, 1955. Aortic rupture in turkeys subsequent to dissecting aneurysm. Vet. Rec. 67: 650-654. Katz, L. N., and R. Pick, 1961. Morphological aspects of atherosclerosis in the chick. Conn. Med. J. 25: 84-87. Krista, L. M., P. E. Waibel, R. E. Burger, R. A. Ball and J. H. Sautter, 1961. Influence of dietary and hormonal alterations during a natural outbreak of dissecting aneurysm in turkeys. Poultry Sci. 40: 1421. Krista, L. M., R. E. Burger and P. E. Waibel, 1963. Blood pressure and heart rate in the turkey as measured by the indirect method and their modifications by pharmacological agents. Poultry Sci. 42: 646-652.
Krista, L. M., P. E. Waibel and R. E. Burger, 1965The influence of dietary alterations, hormones, and blood pressure on the incidence of dissecting aneurysms in the turkey. Poultry Sci. 44: 15-22. Krista, L. M., P. E. Waibel, J. H. Sautter and R. N. Shoffner, 1969. Aortic rupture, body weight, and blood pressure in the turkey as influenced by strain, dietary fat, Aeta-aminopropionitrile fumarate, and diethylstilbestrol. Poultry Sci. 48: 1954-1960. Kurtz, H. J., 1966. A study of factors influencing aortic dissecting aneurysms. Ph.D. Thesis, University of Minnesota, St. Paul, Minnesota. Kurtz, H. J., 1969. Aortic plaque degeneration as a cause of aortic rupture in turkeys. Am. J. Vet. Res. 30: 251-259. Lindsay, S., C. W. Nichols and I. L. Chairkoff, 1955. Aortic lesions induced in the bird by diethylstilbestrol injections and cholesterol feeding. A.M.A. Archives Path. 59: 173-184. McSherry, J. B., A. E. Ferguson and J. Ballantyne, 1954. A dissecting aneurysm in internal hemorrhage in turkeys. J. Am. Vet. Med. Assoc. 124: 279-283. Simpson, C. F., R. H. Harms and F. C. Neal, 1964. Influence of high dietary sodium chloride on aortic rupture in turkeys induced by diethylstilbestrol. Proc. Soc. Exp. Biol. Med. 116: 334-336. Simpson, C. F., and R. H. Harms, 1965. Relationship of age and sex of turkeys to aortic ruptures induced by diethylstilbestrol. Proc. Soc. Exp. Biol. Med. 119: 509-512. Simpson, C. F., R. H. Harms and H. R. Wilson, 1965. Alteration of sex characteristics of turkey poults with diethylstilbestrol. Proc. Soc. Exp. Biol. Med. 119: 435-438.
Egg Quality in Gifu City, Japan A. W. BRANT, 1 S. SENDA, T. TAKAHASHI AND T. NAKAMURA Department of Poultry and Animal Sciences, Gifu University, Kagamihara, Gifu (Received for publication June 7, 1969)
INTRODUCTION
T
HE last 10 years have witnessed a very rapid rise in egg production in Japan. In 1957 there were an estimated 30 1 Sabbatical leave, University Davis, California 95616.
of
California,
million laying hens. In 1967 this number had risen to 90 million. The Ministry of Agriculture and Forestry of the Japanese government estimated the 1967 per capita intake of eggs per day was 27.5 grams. This would be the equivalent of approx-