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Some Characteristics of Eggs from Estrogen-Fed Pullets1
Some Characteristics of Eggs from Estrogen-Fed Pullets1 JOHN L. ADAMS, L. E. CASIDA AND W. H. MCGIBBON Departments of Poultry Husbandry and Genetics, University of Wisconsin, Madison (Received for publication December 11, 1950)
1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. (Department of Genetics contribution No. 443). We are indebted to Dr. C. W. Sondern of White Laboratories, Inc., Newark, New Jersey, for the synthetic hormones used in this study.
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abnormalities of shape, shell and internal structure and the percent of thick albumen in the eggs from estrogen-treated pullets. EXPERIMENTAL Single Comb White Leghorn pullets were selected," caged and fed as described in a previous report (Adams et al., 1950). Treatment levels of dienestrol diacetate and dianisylhexene (abbreviated herein as D.D. and D.H. respectively) were as follows: Experiment 1. Four groups of six pullets each: Control (no estrogen), dienestrol diacetate as . 0 1 % of the ration, and dianisylhexene as . 0 1 % and as .02% of the ration by weight. Experiment 2. Seven groups of six pullets each: Controls, (one lot of no estrogen), three lots receiving dienestrol diacetate and three dianisylhexene. The different lots receiving each estrogen were fed the hormone at levels of .01%, .02% and .04% of the ration by weight respectively. The alternate eggs of each hen were "broken out" immediately on the day laid and the internal observations were made. A two man team was utilized in order to avoid bias; one member read all observations on the egg and the other member recorded the data. The treatment of the hen to which each egg belonged was known only to the recorder. The unbroken eggs (alternate eggs of each hen)
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T J O T H natural and synthetic estrogens *-* are known to have marked effects on the oviducts of immature female fowl, Juhn and Gustavson (}928), Asmundson et al. (1936), Munroand Kosin (1943) and Herrick (1944). Some of the changes which take place in the female with the onset of laying are believed to be caused by estrogen i.e., rise in blood fat, hypertrophy of the oviduct and vent, and a rise in blood calcium. Previous reports by the present authors (1950) have indicated that exogenous estrogen supplied orally may affect ovulation and calcium metabolism. Very little has been published regarding the effects of gonadal hormones upon the eggs laid by treated hens. Asmundson (1931) injected estrogen into Single Comb White Leghorn pullets and Barred Plymouth Rock hens without apparent effect on the weights of eggs laid. The above author weighed yolk, albumen and shell separately but did not detect any effects on these parts in the small numbers examined. In view of the more recent evidence cited above, it is believed that more critical data are needed on the eggs from estrogen-treated hens. The present paper deals with egg weight, yolk weight,
CHARACTERISTICS OF EGGS FROM ESTROGEN-FED BIRDS
Yolk weights. After the albumen had been removed, the yolks were transferred to a damp paper towel and rolled to remove albumen; they were then weighed to the nearest tenth of a gram. Ridged and lopsided eggs, body checks, cracks and flat areas. Eggs were recorded as "ridged" if any slight ridges were evident. "Lopsided" referred to an asymmetrical condition. Eggs with "body checks" were those that appeared to have been cracked in the uterus before calcification was completed and then recalcified before oviposition. "Cracks" were observed by candling. The term "flat areas" as used herein refers to eggs with definite thin shelled areas imprinted
on the side of the egg as if something had been in contact with the egg in the uterus and had interfered with the shell deposition and the natural contour of the egg in that area. No observations were made on ridged, lop-sided or flat areas in Experiment 2. Double yolks. These were determined on all incubated eggs by candling and on broken-out eggs by inspection at time of breaking. Soft shells. These were definitely pliable to the touch and were recorded at the time of gathering and usually were found in the dropping pans beneath the hens in the laying cages. Blood, meat spots, mottled yolks. In eggs which were broken for examination, specks of blood on the yolk and clots of blood in the albumen were recorded with no distinction being made as to size or location; in eggs which were incubated, only "spots" which appeared definitely red when candled were included. Meat spots detected by candling or found inside the egg were designated as inclusions which were not red in color. Mottled yolks were observed under a strong light immediately after breaking the eggs and before any albumen was removed. No attempt was made to classify the incubated eggs. Shell thickness. Each egg when gathered was marked on the large end with the hen number and the date. Shell from the large end of each egg examined internally was stripped of its membranes and allowed to dry. Four samples were broken from each shell and the thickness measured with a dial indicator micrometer which was read to one quarter of one thousandth of an inch. Care was taken in sampling to take pieces of the shell at about the same distance from the apex of the large end and to distribute sampled areas evenly around the circumference.
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were incubated in settings made weekly. Results on fertility and hatchability of eggs and upon growth, viability and sexual maturity of chicks are reported elsewhere. The procedures in gathering the data were as follows: Egg weights. All eggs were weighed to the nearest gram. Percent thick albumen. This was obtained by breaking the egg into a Petri dish and drawing off the thin albumen by means of a 50 ml. volumetric pipette. The outer thin albumen was drawn off by using the small end of the pipette, the thick albumen envelope was then punctured and the inner thin albumen was removed. The thin albumen thus obtained was then emptied into a graduated cylinder and the thick albumen removed by reversing the pipette and using the larger tip. The thick albumen was then expelled into a second graduated cylinder and the small amount of thin albumen left with the yolk in the Petri dish was removed by using the small tip of the pipette and added to the thin albumen. The two graduated cylinders were then read to the nearest 0.5 ml.
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J. L. ADAMS, L. E. CASIDA AND W. H.
MCGIBBON
TABLE 1.—The weights of eggs and yolks of eggs from estrogen-fed pullets (Experiment 1) Treatment
No. of eggs laid
No. eggs examined internally
Egg wt. (grams)
Yolk wt. (grams)
Control . 0 1 % dienestrol diacetate . 0 1 % dianisylhexene .02% dianisylhexene
389 384 291 207
194 195 148 104
51.8 52.8 53.4 52.5
14.0 14.4 14.6 14.0
50.8 54.0 52.5 50.1 52.3 50.4 50.7
14.7 15.6 14.8 14.3 15.2 14.8 14.6
(Experiment 2) 269 298 328 183 196 216 113
RESULTS AND DISCUSSION
Egg weights and yolk weights. There were no significant differences in egg weights or yolk weights when analyzed by analysis of variance (Snedecor, 1946) Table 1. It has been postulated in a previous paper that estrogens probably reduce ovulation rate by suppressing the production or release of follicle stimulating hormone (FSH) by the pituitary. An all-or-none situation for the individual ovum is indicated; apparently if enough FSH is liberated by the pituitary for production of ova, they develop normally insofar as weight is concerned. Ridged, asymmetrical eggs, flat areas. These abnormalities are presumed to originate in the isthmus or uterus of the bird. The laying of ridged and lop-sided or asymmetrical eggs seemed to be a TABLE 2 —-The
134 147 159 90 96 105 56
characteristic, of the individual hen and although the means appear to be somewhat different, the variation between individuals within treatments was so large that there was no statistically significant difference between treatments (Table 2). The occurrence of flat areas seems to be closely related to the laying of eggs with double yolks and both conditions are apparently caused by decreased intervals between ovulations. The exact mechanism of ovulation in the domestic fowl is not understood but various hormones are known to affect it. Anterior pituitary extracts (Koch, 1934), chorionic gonadotropins (Fraps, Riley and Olsen, 1942) and progesterone (Fraps and Dury, 1943) have been shown to cause ovulation whereas synthetic estrogen has been shown to inhibit ovulation when fed in
effects of various levels of estrogens on egg shape
%
Treatment
No. eggs
% Ridged
% Asymmetrical
Flat areas
Control . 0 1 % dienestrol diacetate . 0 1 % dianisylhexene .02% dianisylhexene
389 384 291 207
5.2 9.0 11.4 12.4
.85 1.64 6.26 6.15
0.2 0.0 0.0 4.8*
* Denotes significance at the .05 level.
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Control . 0 1 % dienestrol diacetate .02% dienestrol diacetate .04% dienestrol diacetate' . 0 1 % dianisylhexene .02% dianisylhexene .04% dianisylhexene
CHARACTERISTICS OF EGGS FROM ESTROGEN-FED BIRDS
TABLE 3.—Effects
ovulation. Since no disturbance of follicle stimulating hormone is indicated by egg weight and yolk weight, it is possible that modification in the production or release of luteinizing hormone was responsible for double yolks by irregular ovulation. Most of the double yolks laid by hens occur when they are just getting into production as pullets (Conrad and Warren, 1940). The lack of effect by estrogen in Experiment 2 and the lower incidence of double yolks may be due to the fact that the pullets in Experiment 2 were about a month older than those in Experiment 1 when treatment started. Blood and meat spots. According to Nalbandov and Card (1944), most blood included within eggs is the result of preovulatory intrafollicular hemorrhages; also meat spots are bleached and degenerated blood spots and have the same origin. A terrific rise in blood-fat content in hens is known to occur under estrogenic treatment ("unpublished data, Adams et al.). Zondek and Marx (1939) have reported fat thrombosis in the pulmonary
of various levels revealed by internal examination of. eggs from treated, pullets Average percent of eggs per hen
Treatment
No. eggs examined
Double
yolks
1
-o, j Blood
Meat spots '
0.0 4.5* 2.3 3.8**
9.58 5.00 7.37 8.62
.28 .30 1.80 1.40 4.50 .30 4.70
2.14 1.20 5.52 .63 3.34 .86 12.70
Mottled yolks
% Thick albumen
(Experiment 1) Control . 0 1 % dienestrol diacetate . 0 1 % dianisylhexene .02% dianisylhexene
194 195 148 104
.73 1.90 1.82 7.30**
10.8 26.6* 27.3* 46.0**
51.2 52.7 54.2 55.5
62.8 57.4 60.2 52.6 52.9 54.7 50.9
55.3 53.8 55.6 52.6 53.8 55.7 55.1
(Experiment 2) Control . 0 1 % dienestrol diacetate .02% dienestrol diacetate .04% dienestrol diacetate . 0 1 % dianisylhexene .02% dianisylhexene .04% dianisylhexene
134 147 159 90 96 105 56
* Denotes significance at the .05 level. ** Denotes significance at the .01 level.
.41 .00 .79 .35 .69 .00 .98
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amounts of .04% of the ration (Adams et al., 1950). It was observed that when eggs were laid with "flat areas" they were usually followed or preceded by soft or thin shelled eggs. This condition and its cause has been described by Scott (1940). The .02% D.H. group of Experiment 1 had significantly more flat areas than the controls but none was found in the .01% D.D. or D.H. groups. Double yolks. Curtis (1915) studied the production of double yolked eggs and found that about thirty-six % of all double yolks were preceded by a normal egg on the day before. She found that the yolks might have albumen envelopes in common or separate. Conrad and Warren (1940) attributed sixy-five % of double yolks to simultaneous ovulation, twentyfive % to premature ovulation and ten % to delayed ovulation. Judging by the increase in numbers of flat areas described in the preceding paragraph and the increase in double yolks (Table 3), estrogen fed to pullets just after coming into production seems to upset regularity of
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J. L. ADAMS, L. E. CASIDA AND W. H.
Mottled yolks. A mottled condition in fresh eggs has been described b y Schaible et al. (1935), and attributed to a nonuniformity in the peripheral light yolk layer. If estrogen in some way influences time of ovulation, it seems possible t h a t some yolks might be ovulated before the peripheral layer of light yolk is complete thus resulting in a mottled yolk. I n Experiment 1 only cursory examination of yolks was made and only specimens having large and easily apparent spots were recorded. The results showed a marked and highly significant increase in this condition in treated groups. I n Experiment 2 interest in the character having been increased b y the results of Experiment 1, a very critical inspection of each broken out yolk was made and very slight spots recorded. Consequently, the incidence of mottled yolks increased markedly in the later trial. Unfortunately the degree of mottling of individual eggs was
not recorded b u t it is the opinion of the authors t h a t there is an increased occurrence of the severely mottled yolks as a result of estrogenic t r e a t m e n t . There were no significant differences in the numbers of mottled yolks due to treatment in Experiment 2. Percent of thick albumen. The percentage of the total albumen which is thick, has long been of interest to poultry investigators because of its association with egg quality. Hoist and Almquist (1931) found the average percent thick albumen to be sixty-two. Individual hens were relatively constant in the expression of this characteristic. Estrogen did not produce any a p preciable effects on the % thick albumen of fresh eggs laid by treated hens (Table 3). Individual hens were relatively constant in the amount of thick albumen found in their eggs but variation between hens within treatments was great. Shell thickness, body checks, soft shells and cracked eggs. Previous work on the effects of estrogens on the mature male fowl (Zondek and Marx, 1939) and the immature female fowl (Fleischmann and Fried, 1945, and many others) has shown t h a t estrogen produced hypercalcemia. I t has been noted t h a t calcium rises (Russell et al., 1930) upon the development of ova in the follicles of the pullet. Since any effect on calcium metabolism might be reflected in egg shell thickness, measurements were made on all shells of eggs which were examined for internal characters. There is a fairly high correlation ( + . 6 3 3 ) between shell thickness and breaking strength and a correlation of + .835 between thickness and puncturing strength (Lund et al., 1937). The manner in which the eggs became cracked in the present experiment was by the drop of six inches or less to the wire floor of the cages
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blood vessels accompanied by acute dyspneic symptoms. I t is interesting to speculate on what must be the situation in the follicle of the laying hen under conditions of acute hyperlipemia. I t seems plausible t h a t the clogging of the follicular capillaries which may occur might in some way be responsible for an increased number of intrafollicular hemorrhages which ultimately result in blood in the egg. The . 0 1 % D . D . and .02% D . H . groups of Experiment 1 had more blood spots than the controls (Table 3) and the trend is the same in Experiment 2 b u t the differences are not significant in the latter experiment. No difference in the incidence of meat spots was observed. Since work by Nalbandov and Card (1944) has indicated t h a t meat spots are older than blood spots, the estrogen may act late or near time of ovulation so t h a t though blood spots may be increased, there is no effect on meat spots.
MCGIBBON
699
CHARACTERISTICS or EGGS FROM ESTROGEN-FED BIRDS
Thayer et al. (1945) have reported that the bones of the wing and leg were more
fragile in broilers which had been fed dianisylhexene. This seems to indicate that though hypercalcemia occurs as a result of estrogenic treatment, the additional calcium may not be available for shell or bone formation to the extent that it is normally and that the high level of calcium in the blood is maintained at the expense of shell and bones. For some unknown reason the birds of Experiment 2 consumed more oyster shell than did those in the first experiment and laid somewhat thicker shelled eggs with fewer cracked or soft shelled. Since the first and second experiments were composed of birds predominantly from two different strains perhaps inherited differences in shell thickness demonstrated by Taylor and Lerner (1938) is one of the reasons for the inconsistency. SUMMARY
The eggs from Single Comb White Leghorn pullets which had been fed levels of dianisylhexene and dienestrol diacetate ranging from .01% to were examined.
TABLE 4.—The effects of various levels of estrogens on the shells of eggs from treated pullets
Treatment
No. eggs examined
%
Cracks
Body checks
Soft shells
Shell thickness (.001 inch)
(Experiment 1) Control . 0 1 % dienestrol diacetate . 0 1 % dianisylhexene .02% dianisylhexene
194 195 148 104
.48 .79 5.35 10.62**
0.0 0.0 1.8 4.7
.50 .98 4.80 8.25**
13.4 12.9* 12.7 12.9
0.00 0.00 2.30 0.00 1.20 0.00 0.00
14.1 14.1 13.2 13.3 13.8 13.6 14.4
(Experiment 2) Control . 0 1 % dienestrol diacetate . 02% dienestrol diacetate .04% dienestrol diacetate . 0 1 % dianisylhexene .02% dianisylhexene .04% dianisylhexene
134 147 159 90 96 105 56
* Denotes significance at the .05 level. ** Denotes significance at the .01 level.
0.0 0.0 10.70 .34 .69 .29 .35
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at oviposition. In many cases the imprint of a wire could be clearly seen in the shell. This manner of breaking would more neatly approximate the method used by Lund and co-workers to determine puncturing strength than that used to determine breaking strength where pressure was applied to a greater area of the shell. It cannot be assumed that the lesser thickness is entirely responsible for the larger numbers of cracked eggs however, because it will be noted that the .01 D.D. group laid significantly thinner shelled eggs than the controls (Table 4) and yet the difference in numbers of cracked eggs was not significant. The trend in shell thickness was generally downward in the other treated groups of both experiments though no other group differed significantly from its control. The number of cracked eggs was increased significantly in the .02 D.H. group of Experiment 1 and body checks also seemed to be increased in .02 D.H. but the difference is not significant.
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J. L. ADAMS, L. E. CASIDA AND W. H. MCGIBBON
REFERENCES Adams, John L., W. H. McGibbon and L. E. Casida, 1950. The effect of orally administered synthetic estrogens on Single Comb White Leghorn pullets. Poultry Sci'. 29:666-671. Asmundson, V. S., 1931. Effect of hormone on the formation of the hen's egg. Poultry Sci. 10: 157165. Asmundson, V. S., C. A. Gunn and A. A. Klose, 1936. Some responses of the immature female fowl to injections of mare gonadotropic hormone and oestrin. Poultry Sci. 16: 194-206. Conrad, R. M., and D. C. Warren, 1940. The production of double yolked eggs in the fowl. Poultry Sci. 19: 9-17. Curtis, M. R., 1915. Relation of simultaneous ovulation to production of double yolked eggs. J. Agric. Res. 3:375-386. Fleischmann, W., and I. A. Fried, 1945. Studies on the mechanism of the hypercholesteremia and hypercalcemia induced by estrogen in immature chicks. Endocrinology 36: 406-415. Fraps, R. M., G. M. Riley and M. W. Olsen, 1942. Time required for induction of ovulation following intravenous injection of hormone preparations in fowl. Proc. Soc. Exp. Biol. Med. 50: 313-317.
Fraps, R. M., and A. Dury, 1943. Occurrence of premature ovulation in the domestic fowl following administration of progesterone. Proc. Soc. Exp. Biol. Med. 52: 346-349. Herrick, E. H., 1944. Some influences of stilbestrol, estrone and testosterone propionate on the genital tract of young female fowl. Poultry Sci. 23:65-66. Hoist, W. F., and H. J. Almquist, 1931. Measurement of deterioration in the stored hen's egg. Hilgardia 6: 49-60. Koch, W., 1934. tj'ber den einfluss von prolan auf die legtatigkeit der vogel. Klin. Wochenschr. 13: 1647-1648. Juhn, M., and R. O. Gustavson, 1928. Demonstration of the positive action of the placental hormone on the oviduct of the fowl. Anat. Rec. 4 1 : 41H12. Lund, W. A., Victor Heiman and L. A. Wilhelm, 1937. The relationship between egg shell thickness and strength. Poultry Sci. 17: 373-376. Munro, S. S., and I. L. Kosin, 1943. Dramatic response of the chick oviduct to estrogen. Poultry Sci. 22:330-331. Nalbandov, A. V., and L. E. Card, 1944. The problem of blood clots and meat spots in chicken eggs. Poultry Sci. 23: 170-180. Russell, W. C , C.-H. Howard and A. F. Hess, 1930. The relationship in the hen between the development of ova, blood calcium and the anti-rachitic factors. Science 72: 506-507. Schaible, P. J., J. A. Davidson and J. M. Moore, 1935. The egg yolk surface in fresh eggs. Poultry Sci. 15:298-303. Scott, H. M., 1940. A note on abnormal shape of egg. Amer. Nat. 74: 185-188. Snedecor, O. W., 1946. Statistical methods. Iowa State College Press, the Collegiate Press Inc., Ames, Iowa. Taylor, L. W., and I. M. Lerner, 1938. Inheritance of eggshell thickness in White Leghorn pullets. J. Agr. Res. 58:383-396. Thayer, Rollin H., R. G. Jaap and Robt. Penquite, 1945. Fattening chickens by feeding estrogens. Poultry Sci. 24: 483-495. Zondek, B., and L. Marx, 1939. Lipemia and calcemia induced by diethylstilbestrol. Nature 143: 378-379.
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Egg weight, yolk weight, occurrence of misshapen eggs, body checks, meat spots and percent thick albumen were not affected significantly by estrogenic treatment. The incidence of cracked eggs, soft shells, double yolks, blood spots and mottled yolks were all significantly increased and shell thickness decreased in the first experiment. A second experiment using a predominantly different strain of birds with lower egg production and treatment beginning at a later age did not show any significant effects although the trend in some cases supports the results in the first experiment.
1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. (Department of Genetics contribution No. 443). We are indebted to Dr. C. W. Sondern of White Laboratories, Inc., Newark, New Jersey, for the synthetic hormones used in this study.
694
abnormalities of shape, shell and internal structure and the percent of thick albumen in the eggs from estrogen-treated pullets. EXPERIMENTAL Single Comb White Leghorn pullets were selected," caged and fed as described in a previous report (Adams et al., 1950). Treatment levels of dienestrol diacetate and dianisylhexene (abbreviated herein as D.D. and D.H. respectively) were as follows: Experiment 1. Four groups of six pullets each: Control (no estrogen), dienestrol diacetate as . 0 1 % of the ration, and dianisylhexene as . 0 1 % and as .02% of the ration by weight. Experiment 2. Seven groups of six pullets each: Controls, (one lot of no estrogen), three lots receiving dienestrol diacetate and three dianisylhexene. The different lots receiving each estrogen were fed the hormone at levels of .01%, .02% and .04% of the ration by weight respectively. The alternate eggs of each hen were "broken out" immediately on the day laid and the internal observations were made. A two man team was utilized in order to avoid bias; one member read all observations on the egg and the other member recorded the data. The treatment of the hen to which each egg belonged was known only to the recorder. The unbroken eggs (alternate eggs of each hen)
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 19, 2015
T J O T H natural and synthetic estrogens *-* are known to have marked effects on the oviducts of immature female fowl, Juhn and Gustavson (}928), Asmundson et al. (1936), Munroand Kosin (1943) and Herrick (1944). Some of the changes which take place in the female with the onset of laying are believed to be caused by estrogen i.e., rise in blood fat, hypertrophy of the oviduct and vent, and a rise in blood calcium. Previous reports by the present authors (1950) have indicated that exogenous estrogen supplied orally may affect ovulation and calcium metabolism. Very little has been published regarding the effects of gonadal hormones upon the eggs laid by treated hens. Asmundson (1931) injected estrogen into Single Comb White Leghorn pullets and Barred Plymouth Rock hens without apparent effect on the weights of eggs laid. The above author weighed yolk, albumen and shell separately but did not detect any effects on these parts in the small numbers examined. In view of the more recent evidence cited above, it is believed that more critical data are needed on the eggs from estrogen-treated hens. The present paper deals with egg weight, yolk weight,
CHARACTERISTICS OF EGGS FROM ESTROGEN-FED BIRDS
Yolk weights. After the albumen had been removed, the yolks were transferred to a damp paper towel and rolled to remove albumen; they were then weighed to the nearest tenth of a gram. Ridged and lopsided eggs, body checks, cracks and flat areas. Eggs were recorded as "ridged" if any slight ridges were evident. "Lopsided" referred to an asymmetrical condition. Eggs with "body checks" were those that appeared to have been cracked in the uterus before calcification was completed and then recalcified before oviposition. "Cracks" were observed by candling. The term "flat areas" as used herein refers to eggs with definite thin shelled areas imprinted
on the side of the egg as if something had been in contact with the egg in the uterus and had interfered with the shell deposition and the natural contour of the egg in that area. No observations were made on ridged, lop-sided or flat areas in Experiment 2. Double yolks. These were determined on all incubated eggs by candling and on broken-out eggs by inspection at time of breaking. Soft shells. These were definitely pliable to the touch and were recorded at the time of gathering and usually were found in the dropping pans beneath the hens in the laying cages. Blood, meat spots, mottled yolks. In eggs which were broken for examination, specks of blood on the yolk and clots of blood in the albumen were recorded with no distinction being made as to size or location; in eggs which were incubated, only "spots" which appeared definitely red when candled were included. Meat spots detected by candling or found inside the egg were designated as inclusions which were not red in color. Mottled yolks were observed under a strong light immediately after breaking the eggs and before any albumen was removed. No attempt was made to classify the incubated eggs. Shell thickness. Each egg when gathered was marked on the large end with the hen number and the date. Shell from the large end of each egg examined internally was stripped of its membranes and allowed to dry. Four samples were broken from each shell and the thickness measured with a dial indicator micrometer which was read to one quarter of one thousandth of an inch. Care was taken in sampling to take pieces of the shell at about the same distance from the apex of the large end and to distribute sampled areas evenly around the circumference.
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 19, 2015
were incubated in settings made weekly. Results on fertility and hatchability of eggs and upon growth, viability and sexual maturity of chicks are reported elsewhere. The procedures in gathering the data were as follows: Egg weights. All eggs were weighed to the nearest gram. Percent thick albumen. This was obtained by breaking the egg into a Petri dish and drawing off the thin albumen by means of a 50 ml. volumetric pipette. The outer thin albumen was drawn off by using the small end of the pipette, the thick albumen envelope was then punctured and the inner thin albumen was removed. The thin albumen thus obtained was then emptied into a graduated cylinder and the thick albumen removed by reversing the pipette and using the larger tip. The thick albumen was then expelled into a second graduated cylinder and the small amount of thin albumen left with the yolk in the Petri dish was removed by using the small tip of the pipette and added to the thin albumen. The two graduated cylinders were then read to the nearest 0.5 ml.
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J. L. ADAMS, L. E. CASIDA AND W. H.
MCGIBBON
TABLE 1.—The weights of eggs and yolks of eggs from estrogen-fed pullets (Experiment 1) Treatment
No. of eggs laid
No. eggs examined internally
Egg wt. (grams)
Yolk wt. (grams)
Control . 0 1 % dienestrol diacetate . 0 1 % dianisylhexene .02% dianisylhexene
389 384 291 207
194 195 148 104
51.8 52.8 53.4 52.5
14.0 14.4 14.6 14.0
50.8 54.0 52.5 50.1 52.3 50.4 50.7
14.7 15.6 14.8 14.3 15.2 14.8 14.6
(Experiment 2) 269 298 328 183 196 216 113
RESULTS AND DISCUSSION
Egg weights and yolk weights. There were no significant differences in egg weights or yolk weights when analyzed by analysis of variance (Snedecor, 1946) Table 1. It has been postulated in a previous paper that estrogens probably reduce ovulation rate by suppressing the production or release of follicle stimulating hormone (FSH) by the pituitary. An all-or-none situation for the individual ovum is indicated; apparently if enough FSH is liberated by the pituitary for production of ova, they develop normally insofar as weight is concerned. Ridged, asymmetrical eggs, flat areas. These abnormalities are presumed to originate in the isthmus or uterus of the bird. The laying of ridged and lop-sided or asymmetrical eggs seemed to be a TABLE 2 —-The
134 147 159 90 96 105 56
characteristic, of the individual hen and although the means appear to be somewhat different, the variation between individuals within treatments was so large that there was no statistically significant difference between treatments (Table 2). The occurrence of flat areas seems to be closely related to the laying of eggs with double yolks and both conditions are apparently caused by decreased intervals between ovulations. The exact mechanism of ovulation in the domestic fowl is not understood but various hormones are known to affect it. Anterior pituitary extracts (Koch, 1934), chorionic gonadotropins (Fraps, Riley and Olsen, 1942) and progesterone (Fraps and Dury, 1943) have been shown to cause ovulation whereas synthetic estrogen has been shown to inhibit ovulation when fed in
effects of various levels of estrogens on egg shape
%
Treatment
No. eggs
% Ridged
% Asymmetrical
Flat areas
Control . 0 1 % dienestrol diacetate . 0 1 % dianisylhexene .02% dianisylhexene
389 384 291 207
5.2 9.0 11.4 12.4
.85 1.64 6.26 6.15
0.2 0.0 0.0 4.8*
* Denotes significance at the .05 level.
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Control . 0 1 % dienestrol diacetate .02% dienestrol diacetate .04% dienestrol diacetate' . 0 1 % dianisylhexene .02% dianisylhexene .04% dianisylhexene
CHARACTERISTICS OF EGGS FROM ESTROGEN-FED BIRDS
TABLE 3.—Effects
ovulation. Since no disturbance of follicle stimulating hormone is indicated by egg weight and yolk weight, it is possible that modification in the production or release of luteinizing hormone was responsible for double yolks by irregular ovulation. Most of the double yolks laid by hens occur when they are just getting into production as pullets (Conrad and Warren, 1940). The lack of effect by estrogen in Experiment 2 and the lower incidence of double yolks may be due to the fact that the pullets in Experiment 2 were about a month older than those in Experiment 1 when treatment started. Blood and meat spots. According to Nalbandov and Card (1944), most blood included within eggs is the result of preovulatory intrafollicular hemorrhages; also meat spots are bleached and degenerated blood spots and have the same origin. A terrific rise in blood-fat content in hens is known to occur under estrogenic treatment ("unpublished data, Adams et al.). Zondek and Marx (1939) have reported fat thrombosis in the pulmonary
of various levels revealed by internal examination of. eggs from treated, pullets Average percent of eggs per hen
Treatment
No. eggs examined
Double
yolks
1
-o, j Blood
Meat spots '
0.0 4.5* 2.3 3.8**
9.58 5.00 7.37 8.62
.28 .30 1.80 1.40 4.50 .30 4.70
2.14 1.20 5.52 .63 3.34 .86 12.70
Mottled yolks
% Thick albumen
(Experiment 1) Control . 0 1 % dienestrol diacetate . 0 1 % dianisylhexene .02% dianisylhexene
194 195 148 104
.73 1.90 1.82 7.30**
10.8 26.6* 27.3* 46.0**
51.2 52.7 54.2 55.5
62.8 57.4 60.2 52.6 52.9 54.7 50.9
55.3 53.8 55.6 52.6 53.8 55.7 55.1
(Experiment 2) Control . 0 1 % dienestrol diacetate .02% dienestrol diacetate .04% dienestrol diacetate . 0 1 % dianisylhexene .02% dianisylhexene .04% dianisylhexene
134 147 159 90 96 105 56
* Denotes significance at the .05 level. ** Denotes significance at the .01 level.
.41 .00 .79 .35 .69 .00 .98
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amounts of .04% of the ration (Adams et al., 1950). It was observed that when eggs were laid with "flat areas" they were usually followed or preceded by soft or thin shelled eggs. This condition and its cause has been described by Scott (1940). The .02% D.H. group of Experiment 1 had significantly more flat areas than the controls but none was found in the .01% D.D. or D.H. groups. Double yolks. Curtis (1915) studied the production of double yolked eggs and found that about thirty-six % of all double yolks were preceded by a normal egg on the day before. She found that the yolks might have albumen envelopes in common or separate. Conrad and Warren (1940) attributed sixy-five % of double yolks to simultaneous ovulation, twentyfive % to premature ovulation and ten % to delayed ovulation. Judging by the increase in numbers of flat areas described in the preceding paragraph and the increase in double yolks (Table 3), estrogen fed to pullets just after coming into production seems to upset regularity of
697
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J. L. ADAMS, L. E. CASIDA AND W. H.
Mottled yolks. A mottled condition in fresh eggs has been described b y Schaible et al. (1935), and attributed to a nonuniformity in the peripheral light yolk layer. If estrogen in some way influences time of ovulation, it seems possible t h a t some yolks might be ovulated before the peripheral layer of light yolk is complete thus resulting in a mottled yolk. I n Experiment 1 only cursory examination of yolks was made and only specimens having large and easily apparent spots were recorded. The results showed a marked and highly significant increase in this condition in treated groups. I n Experiment 2 interest in the character having been increased b y the results of Experiment 1, a very critical inspection of each broken out yolk was made and very slight spots recorded. Consequently, the incidence of mottled yolks increased markedly in the later trial. Unfortunately the degree of mottling of individual eggs was
not recorded b u t it is the opinion of the authors t h a t there is an increased occurrence of the severely mottled yolks as a result of estrogenic t r e a t m e n t . There were no significant differences in the numbers of mottled yolks due to treatment in Experiment 2. Percent of thick albumen. The percentage of the total albumen which is thick, has long been of interest to poultry investigators because of its association with egg quality. Hoist and Almquist (1931) found the average percent thick albumen to be sixty-two. Individual hens were relatively constant in the expression of this characteristic. Estrogen did not produce any a p preciable effects on the % thick albumen of fresh eggs laid by treated hens (Table 3). Individual hens were relatively constant in the amount of thick albumen found in their eggs but variation between hens within treatments was great. Shell thickness, body checks, soft shells and cracked eggs. Previous work on the effects of estrogens on the mature male fowl (Zondek and Marx, 1939) and the immature female fowl (Fleischmann and Fried, 1945, and many others) has shown t h a t estrogen produced hypercalcemia. I t has been noted t h a t calcium rises (Russell et al., 1930) upon the development of ova in the follicles of the pullet. Since any effect on calcium metabolism might be reflected in egg shell thickness, measurements were made on all shells of eggs which were examined for internal characters. There is a fairly high correlation ( + . 6 3 3 ) between shell thickness and breaking strength and a correlation of + .835 between thickness and puncturing strength (Lund et al., 1937). The manner in which the eggs became cracked in the present experiment was by the drop of six inches or less to the wire floor of the cages
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blood vessels accompanied by acute dyspneic symptoms. I t is interesting to speculate on what must be the situation in the follicle of the laying hen under conditions of acute hyperlipemia. I t seems plausible t h a t the clogging of the follicular capillaries which may occur might in some way be responsible for an increased number of intrafollicular hemorrhages which ultimately result in blood in the egg. The . 0 1 % D . D . and .02% D . H . groups of Experiment 1 had more blood spots than the controls (Table 3) and the trend is the same in Experiment 2 b u t the differences are not significant in the latter experiment. No difference in the incidence of meat spots was observed. Since work by Nalbandov and Card (1944) has indicated t h a t meat spots are older than blood spots, the estrogen may act late or near time of ovulation so t h a t though blood spots may be increased, there is no effect on meat spots.
MCGIBBON
699
CHARACTERISTICS or EGGS FROM ESTROGEN-FED BIRDS
Thayer et al. (1945) have reported that the bones of the wing and leg were more
fragile in broilers which had been fed dianisylhexene. This seems to indicate that though hypercalcemia occurs as a result of estrogenic treatment, the additional calcium may not be available for shell or bone formation to the extent that it is normally and that the high level of calcium in the blood is maintained at the expense of shell and bones. For some unknown reason the birds of Experiment 2 consumed more oyster shell than did those in the first experiment and laid somewhat thicker shelled eggs with fewer cracked or soft shelled. Since the first and second experiments were composed of birds predominantly from two different strains perhaps inherited differences in shell thickness demonstrated by Taylor and Lerner (1938) is one of the reasons for the inconsistency. SUMMARY
The eggs from Single Comb White Leghorn pullets which had been fed levels of dianisylhexene and dienestrol diacetate ranging from .01% to were examined.
TABLE 4.—The effects of various levels of estrogens on the shells of eggs from treated pullets
Treatment
No. eggs examined
%
Cracks
Body checks
Soft shells
Shell thickness (.001 inch)
(Experiment 1) Control . 0 1 % dienestrol diacetate . 0 1 % dianisylhexene .02% dianisylhexene
194 195 148 104
.48 .79 5.35 10.62**
0.0 0.0 1.8 4.7
.50 .98 4.80 8.25**
13.4 12.9* 12.7 12.9
0.00 0.00 2.30 0.00 1.20 0.00 0.00
14.1 14.1 13.2 13.3 13.8 13.6 14.4
(Experiment 2) Control . 0 1 % dienestrol diacetate . 02% dienestrol diacetate .04% dienestrol diacetate . 0 1 % dianisylhexene .02% dianisylhexene .04% dianisylhexene
134 147 159 90 96 105 56
* Denotes significance at the .05 level. ** Denotes significance at the .01 level.
0.0 0.0 10.70 .34 .69 .29 .35
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at oviposition. In many cases the imprint of a wire could be clearly seen in the shell. This manner of breaking would more neatly approximate the method used by Lund and co-workers to determine puncturing strength than that used to determine breaking strength where pressure was applied to a greater area of the shell. It cannot be assumed that the lesser thickness is entirely responsible for the larger numbers of cracked eggs however, because it will be noted that the .01 D.D. group laid significantly thinner shelled eggs than the controls (Table 4) and yet the difference in numbers of cracked eggs was not significant. The trend in shell thickness was generally downward in the other treated groups of both experiments though no other group differed significantly from its control. The number of cracked eggs was increased significantly in the .02 D.H. group of Experiment 1 and body checks also seemed to be increased in .02 D.H. but the difference is not significant.
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J. L. ADAMS, L. E. CASIDA AND W. H. MCGIBBON
REFERENCES Adams, John L., W. H. McGibbon and L. E. Casida, 1950. The effect of orally administered synthetic estrogens on Single Comb White Leghorn pullets. Poultry Sci'. 29:666-671. Asmundson, V. S., 1931. Effect of hormone on the formation of the hen's egg. Poultry Sci. 10: 157165. Asmundson, V. S., C. A. Gunn and A. A. Klose, 1936. Some responses of the immature female fowl to injections of mare gonadotropic hormone and oestrin. Poultry Sci. 16: 194-206. Conrad, R. M., and D. C. Warren, 1940. The production of double yolked eggs in the fowl. Poultry Sci. 19: 9-17. Curtis, M. R., 1915. Relation of simultaneous ovulation to production of double yolked eggs. J. Agric. Res. 3:375-386. Fleischmann, W., and I. A. Fried, 1945. Studies on the mechanism of the hypercholesteremia and hypercalcemia induced by estrogen in immature chicks. Endocrinology 36: 406-415. Fraps, R. M., G. M. Riley and M. W. Olsen, 1942. Time required for induction of ovulation following intravenous injection of hormone preparations in fowl. Proc. Soc. Exp. Biol. Med. 50: 313-317.
Fraps, R. M., and A. Dury, 1943. Occurrence of premature ovulation in the domestic fowl following administration of progesterone. Proc. Soc. Exp. Biol. Med. 52: 346-349. Herrick, E. H., 1944. Some influences of stilbestrol, estrone and testosterone propionate on the genital tract of young female fowl. Poultry Sci. 23:65-66. Hoist, W. F., and H. J. Almquist, 1931. Measurement of deterioration in the stored hen's egg. Hilgardia 6: 49-60. Koch, W., 1934. tj'ber den einfluss von prolan auf die legtatigkeit der vogel. Klin. Wochenschr. 13: 1647-1648. Juhn, M., and R. O. Gustavson, 1928. Demonstration of the positive action of the placental hormone on the oviduct of the fowl. Anat. Rec. 4 1 : 41H12. Lund, W. A., Victor Heiman and L. A. Wilhelm, 1937. The relationship between egg shell thickness and strength. Poultry Sci. 17: 373-376. Munro, S. S., and I. L. Kosin, 1943. Dramatic response of the chick oviduct to estrogen. Poultry Sci. 22:330-331. Nalbandov, A. V., and L. E. Card, 1944. The problem of blood clots and meat spots in chicken eggs. Poultry Sci. 23: 170-180. Russell, W. C , C.-H. Howard and A. F. Hess, 1930. The relationship in the hen between the development of ova, blood calcium and the anti-rachitic factors. Science 72: 506-507. Schaible, P. J., J. A. Davidson and J. M. Moore, 1935. The egg yolk surface in fresh eggs. Poultry Sci. 15:298-303. Scott, H. M., 1940. A note on abnormal shape of egg. Amer. Nat. 74: 185-188. Snedecor, O. W., 1946. Statistical methods. Iowa State College Press, the Collegiate Press Inc., Ames, Iowa. Taylor, L. W., and I. M. Lerner, 1938. Inheritance of eggshell thickness in White Leghorn pullets. J. Agr. Res. 58:383-396. Thayer, Rollin H., R. G. Jaap and Robt. Penquite, 1945. Fattening chickens by feeding estrogens. Poultry Sci. 24: 483-495. Zondek, B., and L. Marx, 1939. Lipemia and calcemia induced by diethylstilbestrol. Nature 143: 378-379.
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Egg weight, yolk weight, occurrence of misshapen eggs, body checks, meat spots and percent thick albumen were not affected significantly by estrogenic treatment. The incidence of cracked eggs, soft shells, double yolks, blood spots and mottled yolks were all significantly increased and shell thickness decreased in the first experiment. A second experiment using a predominantly different strain of birds with lower egg production and treatment beginning at a later age did not show any significant effects although the trend in some cases supports the results in the first experiment.