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Participation of the Ovarian Follicle in Control of Time of Oviposition in the Domestic Fowl
Participation of the Ovarian Follicle in Control of Time of Oviposition in the Domestic Fowl KOUSAKU TANAKA AND TADASHI NAKADA
Faculty of Agriculture, Kyushu University, Fukuoka-shi 812, Japan (Received for publication March 1, 1974)
POULTRY SCIENCE 53: 2120-2125, 1974
INTRODUCTION
I
T is well known that there is a high degree of regularity in the time interval between which the successive eggs in a clutch are laid by the hen (Atwood, 1929; Heywang, 1938). Since a close relation between the time of oviposition and a succeeding ovulation in a given clutch has been reported by Warren and Scott (1935) and Phillips and Warren (1937), the time of ovulation can be predicted within rather narrow limits from the individual hen's record of lay. Riddle (1921) and Burrows and Byerly (1942) demonstrated that the administration of posterior pituitary preparations to the hen caused a premature expulsion of a uterine egg. Oxytocin and vasotocin were found in the posterior pituitary of the hen (Munsick et al., 1960) and from in vitro and in vivo studies, vasotocin was found to be the most potent oxytocic substance (Munsick et al., 1960; Heller and Pickering, 1961; Rzasa and Ewy, 1970). Tanaka and Nakajo (1960, 1962) and Opel (1966) reported that posterior pituitary hormones were depleted shortly before oviposition, while blood levels of oxytocic activity were highest during oviposition (Douglas and Sturkie, 1964; Sturkie and Lin, 1966) and high
within 2 minutes after oviposition (Opel, 1966). Rothchild and Fraps (1944a) showed that removal of the most recently ruptured follicle led to a retarded oviposition and suggested that the time of normal lay of the hen's egg appeared to be under the immediate control of the ruptured follicle from which that egg had originated. Studies on the relation between light-dark rhythms and time of oviposition of eggs, indicated that an extraovarian, light-sensitive agent was involved in the process of oviposition (Rothchild and Fraps, 1944b). Shirley and Nalbandov (1956) found that while removal of the posterior lobe of the pituitary caused an immediate cessation of ovulation and lay, lobectomized hens returned to the normal production of eggs within 30 to 40 days. Opel (1965) reported that removal of the posterior lobe of the pituitary on the day before the terminal oviposition of the 2-egg sequence did not prevent or delay oviposition of the egg, nor did it prevent ovipositions induced by a subsequent piqure of the preoptic brain. He suggested that the posterior lobe of the pituitary was not essential for oviposition in the hen. Normal
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ABSTRACT Removal of the largest follicle or the yolk of this follicle 2 to 8 hours after ovulation of the oviducal egg invariably led to a delayed oviposition of a few hours while a similar delay was observed in two-thirds of the hens following removal of the second largest follicle. About seventy percent of the hens subjected to removal of the ovarian remnants of the most recently ruptured follicle, or of these remnants together with the largest follicle, held their eggs over 7 hours beyond the expected time of lay. The intraperitoneal administration of a homogenate made from ovarian remnants of ruptured follicles, or the intravenous administration of an extract made from these remnants caused premature ovipositions. It is suggested, therefore, that the ovarian remnants of the most recently ruptured follicle pay an important role in controlling oviposition, presumably through secretion of an aqueous hormone or hormone-like substance.
OVARIAN FOLLICLE AND OVOPOSITION
oviposition thus appears to be accomplished by a co-ordination of more than one mechanism. No single mechanism may be absolutely necessary since others may bring about expulsion of the egg. Present experiments were designed to determine the role of the ovarian follicle in oviposition and whether the ruptured follicle contained any biologically active substance influencing oviposition.
Birds belonging to a commercial hybrid strain bred for egg laying were used. They were maintained in individual laying cages and were exposed to a 14-hour photoperiod. Time of oviposition was recorded from 8:00 A.M. through 4:00 P.M. All hens selected for testing were producing clutches of more than 5 eggs with a lapse of but a single day between clutches. An automatic oviposition recorder was utilized to check the time of oviposition accurately. Removal of the maturing and ruptured follicles was performed on follicles other than the first and terminal follicles of the clutch except where otherwise stated. Operative details on removal of the follicle were essentially similar to those described by Rothchild and Fraps (1944a). Removal of the yolk of the largest follicle due to ovulate next was performed by making a small slit along the stigma and the yolk material was almost completely taken out through a glass pipette coupled with a vacuum pump. As a control (sham-operation) a similar operation was performed, the follicles being handled but not removed. All surgical operations were carried out in conscious hens and the interval from ovulation of the oviducal egg to operation varied from 2 to 8 hours. For the preparation of follicular homogenates, the most recently ruptured follicles, aged 1 to 6 hours after ovulation, were collected from 72 good layers and kept in a freezer until used. The pooled follicles were
thawed, washed with water, cut into small pieces, and then homogenized with 10 ml. of 0.9% saline solution. About 4.5 g. of the homogenate containing 3.2 to 3.6 g. of the ruptured follicle tissue (approximately equivalent to 8 ruptured follicles) were placed into the abdominal cavity by an enema-syringe just before closing the incision through which both the largest and most recently ruptured follicles had been excised. An homogenate of 30 g. of the hen's breast muscle was also prepared and was administered by the same procedure as described above. The amount of this homogenate given to a hen was about 4.5 g. which contained 3.2 to 3.6 g. of the muscle tissue. Acetone-treated chicken anterior pituitary powder (CAP) suspended in a saline solution (2 mg./ml.), and estradiol dipropionate singly and in combination with testosterone were injected intramuscularly just after removal of both the largest and most recently ruptured follicles. The treatments were carried out during a period when a soft-shelled egg was present in the uterus. For the extraction of follicular tissue, 72 ruptured follicles, aged 1 to 6 hours after ovulation, were washed with water, cut into small pieces and lyophilized. The dried tissue was mixed with 200 ml. acetone at 5 ° C , allowed to stand for a time at 5° C. and the acetone was then discarded. This process was repeated three times. The residue was then dried under reduced pressure at room temperature to remove the remaining acetone, and was then powdered. This material was mixed with 90 ml. of saline solution, stirred for 10 minutes, followed by centrifugation at 10,000 r.p.m. for 30 minutes at 0° C. The supernatant was set aside. The sediment was resuspended in 45 ml. of saline solution and centrifuged as above. The two supernatants were pooled and the sediment was suspended in 27 ml. of saline solution. A dose of 13 ml. of supernatant was injected intravenously with or without heparin sodium (4 mg.) to an intact hen when a soft-shelled egg was
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MATERIALS AND METHODS
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present in the uterus. A suspension of the sediment fraction was injected intraperitoneally with an enema-syringe through a small incision (approximately 2 cm. long) made on the left abdominal wall. The amount of the suspension injected was 3.2 to 3.5 ml. per hen. An intravenous injection of 13 ml. of saline solution served as a control. RESULTS
TABLE 1.—The effect of removing maturing follicle and yolk of largest follicle on retention of the oviducal egg at time of operation Hens showing delay
Follicle removed F,' Yolk of F , F/ Sham-operation
No. hens operated
No. hens laid at expected time
9 13 15 7
0 0 5 6
No. hens (2.5-5 hr.)
Interval from ovulation to oviposition (hr.)
No. hens laid soft egg
9 12 10 1
28.1 ± 0.6° 27.4 ± 0.5 27.2 ± 0.7 27.0
0 1 0 0
"The largest follicle or most maturing follicle. b The second largest follicle in order of recency of origin. 'Standard deviation of mean.
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As shown in Table 1, all hens subjected to removal of the largest follicle held their eggs over 2.5 hours but not more than five hours beyond the expected time of oviposition. Removal of the yolk of the largest follicle caused a similar delay in oviposition in all but one hen which laid prematurely. Five out of 15 hens subjected to removal of the second largest follicle laid at the expected time, while 10 hens held their eggs for a few hours. Post-mortem examination revealed that the largest follicle in all of these 10 hens, due to ovulate on the day following the operation, had not ovulated on the expected day, but skipped for 1 day without follicular regression. The average intervals between ovulation and oviposition were 28.1,27.4 and 27.2 hours after removal of the largest follicle, the yolk of this follicle or the second largest follicle, respectively. The interval in the former treatment was significantly longer than those of the other two. Six out of 7
sham-operated hens laid at the expected time, and one hen showed a delayed oviposition coincident with a delayed ovulation. The results obtained from removal of the most recently ruptured follicle, or of this follicle together with the largest follicle are summarized in Table 2. Nine out of 20 hens laid at the expected time following removal of the most recently ruptured follicle. Two hens held their eggs for 2.5 and 3 hours, 5 for 7 to 17 hours and 4 over 18 hours. In this treatment it was found that 14 hens laid their eggs in association with ovulation, and in 6 hens ovulation had occurred prior to oviposition. When the most recently ruptured terminal follicle of the clutch was excised, all 11 hens held their eggs more than 13 hours beyond the expected time of oviposition. Oviposition of these eggs was found to have occurred in conjunction with ovulation. When both the largest and most recently ruptured follicles were removed, 2 hens held their eggs for 2.5 hours, 2 for 11 and 13 hours, and 9 over 18 hours. A trial was made to determine whether the exogenous administration of the ruptured follicle, CAP or steroid hormones could eliminate the adverse effect of removing both the largest and most recently ruptured follicles on oviposition. As shown in Table 3, none of the hens so treated laid at the expected
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OVARIAN FOLLICLE AND OVIPOSITION
TABLE 2.- -The effect of removing largest and most recently ruptured follicles on retention of the
oviducal egg at time of operation
No. hens Follicle removed MR a Mr-Ct b Mr and F, c
No. hens operated
Laid at expected time
2.5-3 hr
7-17 hr.
18 hr.-3 days
20 11 13
9 0 0
2 0 2
5 6 2
4 5 9
Retaining egg (range)
a The b The c
most recently ruptured follicle. most recently ruptured terminal follicle of the clutch. The largest follicle or most maturing follicle.
No. hens Dose per hen Materials Mr a 3.2-3.6 g. 3.2-3.6 g. Breast muscle CAP 2mg. 1 mg. Estradiol dipropionate Estradiol dipropionate 1 mg. each Testosterone
Routes of dose i.p.» i.p. i.m. c i.m. l.m.
No. hens treated 9 8 5 5 5
Laid at expected time 0 0 0 0 0
Laid soft egg 8 0 0 0 0
Retaining egg (range) 2.5-5 hr. 7 h r .-2 days 1 0 7 1 5 0 3 2 5 0
"The most recently ruptured follicle aged 1 to 6 hours after ovulation. b Intraperitoneal administration. c Intramuscular administration.
times. However, the intraperitoneal administration of homogenized ruptured follicles, which had been collected one to six hours after ovulation, caused premature ovipositions in 8 out of the 9 hens treated. Of the 8 hens which laid prematurely, seven expelled soft-shelled eggs within 40 minutes after the treatment and one hen within 2 hours of treatment. In sharp contrast, such a striking effect was not observed in any of the hens when an homogenate of the hen's breast muscle was administered. Intramuscular injection of CAP, estradiol dipropionate singly or in combination with testosterone, also failed to cause a premature expulsion of the egg. The egg production of these hens also failed to return to normal.
Attempts were made to extract an oviposition-inducing factor from the ruptured follicles. After several preliminary experiments, it was found that the active fraction for inducing oviposition could be extracted with a saline solution. The results are summarized in Table 4. Four intact hens with a soft-shelled egg in the uterus were injected intravenously with 13 ml. of the extract. One out of the 4 hens expelled a soft-shelled egg within 10 minutes but the remaining three hens died with tetanic spasms during or immediately after the injection. Since it was considered that the reaction might be attributed to an intravenous coagulation of the blood, caused by a common reaction between the blood and the crude tissue extract, 4 mg. heparin
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TABLE 3.—The effect of administration of different tissues, CAP and steroid hormones following removal of both largest and most recently ruptured follicles on oviposition of the uterine egg at time of treatment
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K. TANAKA AND T. NAKADA
TABLE 4.—The effect of administration of fractionated ruptured follicles on oviposition of the uterine egg at time of treatment
Dose per hen (ml.) 13
sodium was added to a dose of the extract. As expected, all of the 5 hens so treated survived following an intravenous injection of the heparinized extract and expelled softshelled eggs within 15 minutes. Intraperitoneal injections of the sediment fraction suspended in a saline solution also caused premature ovipositions in 5 out of the 9 hens within about 40 minutes after the treatment, while the remaining 4 hens laid at the expected time. All control hens laid normally. DISCUSSION It is evident that interruption of ovulation by removal of the largest follicle or the yolk of this follicle invariable leads to a delayed oviposition of several hours. A similar delay in oviposition was observed in hens which showed a delayed ovulation of the largest follicle because of removal of the second largest follicle. Such delays in oviposition of only a few hours is comparable to that found with the terminal egg of the clutch. On the other hand, 9 out of the 20 hens subjected to removal of the most recently ruptured follicle laid at the expected time, in close association with normal ovulations. "Except for two hens which showed minor delays, all of the remaining hens held their eggs more than seven hours beyond the expected time of oviposition. However, the
Laid soft egg 1
0 4 10
5 5 0
absence of a normally due ovulation together with removal of the most recently ruptured follicle, led to delayed ovipositions far beyond the expected time in a high percentage of hens so treated. This generally agreed with the conclusion of Rothchild and Fraps (1944a). Since the ruptured follicles were first treated with acetone, after which the oviposition inducing principle was extracted with a saline solution, it is unlikely that the substance is a steroid or a lipid. On the other hand, the present process for extraction of the ruptured follicles should be improved upon, since the residue fraction exhibited considerable activity for inducing premature ovipositions. However, the detail role of and the identification of this hormone-like factor remains to be investigated. REFERENCES Atwood, H., 1929. Observations concerning the time factor in egg production. Poultry Sci. 8: 137-140. Burrows, W. H., and T. C. Byerly, 1942. Premature expulsion of eggs by hens following injection of whole posterior pituitary preparations. Poultry Sci. 21:416-421. Douglas, D. S., and P. D. Sturkie, 1964. Plasma levels of antidiuretic hormone during oviposition in the hen. Fed. Proc. 23: 150. Heller, H., and B. T. Pickering, 1961. Neurohypophysial hormones of non-mammalian vertebrates. J. Physiol. Lond. 155:98-114.
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Routes No. hens Fraction Died of dose treated Supernatant i.v. b 4 3d Supernatant with 0 heparin 13 i.v. 5 0 Sediment suspension 3.2-3.5 9 i.p. c 0 Vehicle control 2 10 13 i.v. a 0.9% saline solution. b Intravenous injection. c Intraperitoneal injection. d The hens died with tetanic spasms during or immediately after injection.
No. hens Laid at expected time 0
OVARIAN FOLLICLE AND OVDOSITION
between light-dark rhythms and hour of lay of eggs experimentally retained in the hen. Endocrinology, 35: 355-362. Rzasa, J., and Z. Ewy, 1970. Effect of vasotocin and oxytocin on oviposition in the hen. J. Reprod. Fert. 21: 549-550. Shirley, H. V., Jr., and A. V. Nalbandov, 1956. Effects of neurohypophysectomy in domestic chickens. Endocrinology, 58: 477-483. Sturkie, P. D. and Y. Lin, 1966. Release of vasotocin and oviposition in the hen. J. Endocr. 35: 325-326. Tanaka, K., and S. Nakajo, 1960. Oxytocin in the neurohypophysis of the laying hen. Nature, 187: 245. Tanaka, K., and S. Nakajo, 1962. Participation of neurohypophysial hormone in oviposition in the hen. Endocrinology, 70: 453-458. Warren, D. C , and H. M. Scott, 1935. The time factor in egg formation. Poultry Sci. 14: 195-207.
Studies on a Possible Interaction Between Riboflavin and Vitamin B 12 as it Affects Hatchability of the Hen's Egg P . J . TUITE' AND R . E . AUSTIC Department of Poultry Science, Cornell University, Ithaca, New York 14850 (Received for publication March 4, 1974)
ABSTRACT Experiments were conducted using Single Comb White Leghorn chickens to investigate a possible interaction between the requirements for riboflavin and vitamin B 12 for hatchability and to determine how important such an interaction might be in altering the requirements for one or both of these vitamins. The vitamin B 12 requirement for optimum hatchability was found to be 3.45-3.60 pig. /kg. of diet, and the requirement for dietary riboflavin, approximately 2.75 mg./kg. of diet. Dietary riboflavin at deficient or practical levels of feeding was found not to affect the vitamin B12 concentration in egg yolk, or the vitamin B a requirement for optimum hatchability. Although a slight sparing effect of vitamin B 12 upon the riboflavin requirement was suggested by the present studies, the effect was not statistically significant (p < .05). High levels of dietary vitamin B 12 exerted a striking negative effect on riboflavin concentrations in egg yolk (p < .05). Egg production and food consumption were not affected by dietary riboflavin levels ranging from 1.61 to 5.51 mg./kg. of diet or vitamin B12 ranging from 1.0 to 11.0 p.g./kg. of diet. However, optimum egg size was achieved only when the highest level of vitamin B 12 was fed. POULTRY SCIENCE 53: 2125-2136, 1974
INTRODUCTION HE need for adequate riboflavin and vitamin B 12 in the diet of breeding chickens for normal hatchability has long been recognized [refer to Beer (1969) and Lan-
T
1. Present address: Department of Agriculture and Fisheries, Dublin 2, Ireland.
dauer (1967) for extensive reviews]. Evidence for an interaction between vitamin B 12 and riboflavin has been presented by a number of investigators. Research on this subject was spawned by the discovery that 5,6-dimethylbenzimidazole was a degradation product of vitamin B n (Folkers, 1950). Since this compound is an integral part of the riboflavin molecule, several studies were
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Heywang, B. W., 1938. The time factor in egg production. Poultry Sci. 17: 240-247. Munsick, R. A., W. H. Sawyer and H. B. van Dyke, 1960. Endocrinology, 66: 860-871. Opel, H., 1965. Oviposition in chickens after removal of the posterior lobe of the pituitary by an improved method. Endocrinology, 76: 673-677. Opel, H., 1966. Release of oviposition-inducing factor from the median eminence-pituitary stalk region in neural lobectomized hens. Anat. Rec. 154: 396. Phillips, R. E., and D. C. Warren, 1937. Observations concerning the mechanics of ovulation in the fowl. J. Exp. Zool. 76: 117-136. Riddle, O., 1921. A simple method of obtaining premature eggs from birds. Science, 54: 664-666. Rothchild, I., and R. M. Fraps, 1944 a. On the function of the ruptured ovarian follicle of the domestic fowl. Proc. Soc. Exp. Biol. Med. 56: 79-82. Rothchild, I., and R. M. Fraps, 1944 b. Relation
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Faculty of Agriculture, Kyushu University, Fukuoka-shi 812, Japan (Received for publication March 1, 1974)
POULTRY SCIENCE 53: 2120-2125, 1974
INTRODUCTION
I
T is well known that there is a high degree of regularity in the time interval between which the successive eggs in a clutch are laid by the hen (Atwood, 1929; Heywang, 1938). Since a close relation between the time of oviposition and a succeeding ovulation in a given clutch has been reported by Warren and Scott (1935) and Phillips and Warren (1937), the time of ovulation can be predicted within rather narrow limits from the individual hen's record of lay. Riddle (1921) and Burrows and Byerly (1942) demonstrated that the administration of posterior pituitary preparations to the hen caused a premature expulsion of a uterine egg. Oxytocin and vasotocin were found in the posterior pituitary of the hen (Munsick et al., 1960) and from in vitro and in vivo studies, vasotocin was found to be the most potent oxytocic substance (Munsick et al., 1960; Heller and Pickering, 1961; Rzasa and Ewy, 1970). Tanaka and Nakajo (1960, 1962) and Opel (1966) reported that posterior pituitary hormones were depleted shortly before oviposition, while blood levels of oxytocic activity were highest during oviposition (Douglas and Sturkie, 1964; Sturkie and Lin, 1966) and high
within 2 minutes after oviposition (Opel, 1966). Rothchild and Fraps (1944a) showed that removal of the most recently ruptured follicle led to a retarded oviposition and suggested that the time of normal lay of the hen's egg appeared to be under the immediate control of the ruptured follicle from which that egg had originated. Studies on the relation between light-dark rhythms and time of oviposition of eggs, indicated that an extraovarian, light-sensitive agent was involved in the process of oviposition (Rothchild and Fraps, 1944b). Shirley and Nalbandov (1956) found that while removal of the posterior lobe of the pituitary caused an immediate cessation of ovulation and lay, lobectomized hens returned to the normal production of eggs within 30 to 40 days. Opel (1965) reported that removal of the posterior lobe of the pituitary on the day before the terminal oviposition of the 2-egg sequence did not prevent or delay oviposition of the egg, nor did it prevent ovipositions induced by a subsequent piqure of the preoptic brain. He suggested that the posterior lobe of the pituitary was not essential for oviposition in the hen. Normal
2120
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ABSTRACT Removal of the largest follicle or the yolk of this follicle 2 to 8 hours after ovulation of the oviducal egg invariably led to a delayed oviposition of a few hours while a similar delay was observed in two-thirds of the hens following removal of the second largest follicle. About seventy percent of the hens subjected to removal of the ovarian remnants of the most recently ruptured follicle, or of these remnants together with the largest follicle, held their eggs over 7 hours beyond the expected time of lay. The intraperitoneal administration of a homogenate made from ovarian remnants of ruptured follicles, or the intravenous administration of an extract made from these remnants caused premature ovipositions. It is suggested, therefore, that the ovarian remnants of the most recently ruptured follicle pay an important role in controlling oviposition, presumably through secretion of an aqueous hormone or hormone-like substance.
OVARIAN FOLLICLE AND OVOPOSITION
oviposition thus appears to be accomplished by a co-ordination of more than one mechanism. No single mechanism may be absolutely necessary since others may bring about expulsion of the egg. Present experiments were designed to determine the role of the ovarian follicle in oviposition and whether the ruptured follicle contained any biologically active substance influencing oviposition.
Birds belonging to a commercial hybrid strain bred for egg laying were used. They were maintained in individual laying cages and were exposed to a 14-hour photoperiod. Time of oviposition was recorded from 8:00 A.M. through 4:00 P.M. All hens selected for testing were producing clutches of more than 5 eggs with a lapse of but a single day between clutches. An automatic oviposition recorder was utilized to check the time of oviposition accurately. Removal of the maturing and ruptured follicles was performed on follicles other than the first and terminal follicles of the clutch except where otherwise stated. Operative details on removal of the follicle were essentially similar to those described by Rothchild and Fraps (1944a). Removal of the yolk of the largest follicle due to ovulate next was performed by making a small slit along the stigma and the yolk material was almost completely taken out through a glass pipette coupled with a vacuum pump. As a control (sham-operation) a similar operation was performed, the follicles being handled but not removed. All surgical operations were carried out in conscious hens and the interval from ovulation of the oviducal egg to operation varied from 2 to 8 hours. For the preparation of follicular homogenates, the most recently ruptured follicles, aged 1 to 6 hours after ovulation, were collected from 72 good layers and kept in a freezer until used. The pooled follicles were
thawed, washed with water, cut into small pieces, and then homogenized with 10 ml. of 0.9% saline solution. About 4.5 g. of the homogenate containing 3.2 to 3.6 g. of the ruptured follicle tissue (approximately equivalent to 8 ruptured follicles) were placed into the abdominal cavity by an enema-syringe just before closing the incision through which both the largest and most recently ruptured follicles had been excised. An homogenate of 30 g. of the hen's breast muscle was also prepared and was administered by the same procedure as described above. The amount of this homogenate given to a hen was about 4.5 g. which contained 3.2 to 3.6 g. of the muscle tissue. Acetone-treated chicken anterior pituitary powder (CAP) suspended in a saline solution (2 mg./ml.), and estradiol dipropionate singly and in combination with testosterone were injected intramuscularly just after removal of both the largest and most recently ruptured follicles. The treatments were carried out during a period when a soft-shelled egg was present in the uterus. For the extraction of follicular tissue, 72 ruptured follicles, aged 1 to 6 hours after ovulation, were washed with water, cut into small pieces and lyophilized. The dried tissue was mixed with 200 ml. acetone at 5 ° C , allowed to stand for a time at 5° C. and the acetone was then discarded. This process was repeated three times. The residue was then dried under reduced pressure at room temperature to remove the remaining acetone, and was then powdered. This material was mixed with 90 ml. of saline solution, stirred for 10 minutes, followed by centrifugation at 10,000 r.p.m. for 30 minutes at 0° C. The supernatant was set aside. The sediment was resuspended in 45 ml. of saline solution and centrifuged as above. The two supernatants were pooled and the sediment was suspended in 27 ml. of saline solution. A dose of 13 ml. of supernatant was injected intravenously with or without heparin sodium (4 mg.) to an intact hen when a soft-shelled egg was
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MATERIALS AND METHODS
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present in the uterus. A suspension of the sediment fraction was injected intraperitoneally with an enema-syringe through a small incision (approximately 2 cm. long) made on the left abdominal wall. The amount of the suspension injected was 3.2 to 3.5 ml. per hen. An intravenous injection of 13 ml. of saline solution served as a control. RESULTS
TABLE 1.—The effect of removing maturing follicle and yolk of largest follicle on retention of the oviducal egg at time of operation Hens showing delay
Follicle removed F,' Yolk of F , F/ Sham-operation
No. hens operated
No. hens laid at expected time
9 13 15 7
0 0 5 6
No. hens (2.5-5 hr.)
Interval from ovulation to oviposition (hr.)
No. hens laid soft egg
9 12 10 1
28.1 ± 0.6° 27.4 ± 0.5 27.2 ± 0.7 27.0
0 1 0 0
"The largest follicle or most maturing follicle. b The second largest follicle in order of recency of origin. 'Standard deviation of mean.
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As shown in Table 1, all hens subjected to removal of the largest follicle held their eggs over 2.5 hours but not more than five hours beyond the expected time of oviposition. Removal of the yolk of the largest follicle caused a similar delay in oviposition in all but one hen which laid prematurely. Five out of 15 hens subjected to removal of the second largest follicle laid at the expected time, while 10 hens held their eggs for a few hours. Post-mortem examination revealed that the largest follicle in all of these 10 hens, due to ovulate on the day following the operation, had not ovulated on the expected day, but skipped for 1 day without follicular regression. The average intervals between ovulation and oviposition were 28.1,27.4 and 27.2 hours after removal of the largest follicle, the yolk of this follicle or the second largest follicle, respectively. The interval in the former treatment was significantly longer than those of the other two. Six out of 7
sham-operated hens laid at the expected time, and one hen showed a delayed oviposition coincident with a delayed ovulation. The results obtained from removal of the most recently ruptured follicle, or of this follicle together with the largest follicle are summarized in Table 2. Nine out of 20 hens laid at the expected time following removal of the most recently ruptured follicle. Two hens held their eggs for 2.5 and 3 hours, 5 for 7 to 17 hours and 4 over 18 hours. In this treatment it was found that 14 hens laid their eggs in association with ovulation, and in 6 hens ovulation had occurred prior to oviposition. When the most recently ruptured terminal follicle of the clutch was excised, all 11 hens held their eggs more than 13 hours beyond the expected time of oviposition. Oviposition of these eggs was found to have occurred in conjunction with ovulation. When both the largest and most recently ruptured follicles were removed, 2 hens held their eggs for 2.5 hours, 2 for 11 and 13 hours, and 9 over 18 hours. A trial was made to determine whether the exogenous administration of the ruptured follicle, CAP or steroid hormones could eliminate the adverse effect of removing both the largest and most recently ruptured follicles on oviposition. As shown in Table 3, none of the hens so treated laid at the expected
2123
OVARIAN FOLLICLE AND OVIPOSITION
TABLE 2.- -The effect of removing largest and most recently ruptured follicles on retention of the
oviducal egg at time of operation
No. hens Follicle removed MR a Mr-Ct b Mr and F, c
No. hens operated
Laid at expected time
2.5-3 hr
7-17 hr.
18 hr.-3 days
20 11 13
9 0 0
2 0 2
5 6 2
4 5 9
Retaining egg (range)
a The b The c
most recently ruptured follicle. most recently ruptured terminal follicle of the clutch. The largest follicle or most maturing follicle.
No. hens Dose per hen Materials Mr a 3.2-3.6 g. 3.2-3.6 g. Breast muscle CAP 2mg. 1 mg. Estradiol dipropionate Estradiol dipropionate 1 mg. each Testosterone
Routes of dose i.p.» i.p. i.m. c i.m. l.m.
No. hens treated 9 8 5 5 5
Laid at expected time 0 0 0 0 0
Laid soft egg 8 0 0 0 0
Retaining egg (range) 2.5-5 hr. 7 h r .-2 days 1 0 7 1 5 0 3 2 5 0
"The most recently ruptured follicle aged 1 to 6 hours after ovulation. b Intraperitoneal administration. c Intramuscular administration.
times. However, the intraperitoneal administration of homogenized ruptured follicles, which had been collected one to six hours after ovulation, caused premature ovipositions in 8 out of the 9 hens treated. Of the 8 hens which laid prematurely, seven expelled soft-shelled eggs within 40 minutes after the treatment and one hen within 2 hours of treatment. In sharp contrast, such a striking effect was not observed in any of the hens when an homogenate of the hen's breast muscle was administered. Intramuscular injection of CAP, estradiol dipropionate singly or in combination with testosterone, also failed to cause a premature expulsion of the egg. The egg production of these hens also failed to return to normal.
Attempts were made to extract an oviposition-inducing factor from the ruptured follicles. After several preliminary experiments, it was found that the active fraction for inducing oviposition could be extracted with a saline solution. The results are summarized in Table 4. Four intact hens with a soft-shelled egg in the uterus were injected intravenously with 13 ml. of the extract. One out of the 4 hens expelled a soft-shelled egg within 10 minutes but the remaining three hens died with tetanic spasms during or immediately after the injection. Since it was considered that the reaction might be attributed to an intravenous coagulation of the blood, caused by a common reaction between the blood and the crude tissue extract, 4 mg. heparin
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TABLE 3.—The effect of administration of different tissues, CAP and steroid hormones following removal of both largest and most recently ruptured follicles on oviposition of the uterine egg at time of treatment
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TABLE 4.—The effect of administration of fractionated ruptured follicles on oviposition of the uterine egg at time of treatment
Dose per hen (ml.) 13
sodium was added to a dose of the extract. As expected, all of the 5 hens so treated survived following an intravenous injection of the heparinized extract and expelled softshelled eggs within 15 minutes. Intraperitoneal injections of the sediment fraction suspended in a saline solution also caused premature ovipositions in 5 out of the 9 hens within about 40 minutes after the treatment, while the remaining 4 hens laid at the expected time. All control hens laid normally. DISCUSSION It is evident that interruption of ovulation by removal of the largest follicle or the yolk of this follicle invariable leads to a delayed oviposition of several hours. A similar delay in oviposition was observed in hens which showed a delayed ovulation of the largest follicle because of removal of the second largest follicle. Such delays in oviposition of only a few hours is comparable to that found with the terminal egg of the clutch. On the other hand, 9 out of the 20 hens subjected to removal of the most recently ruptured follicle laid at the expected time, in close association with normal ovulations. "Except for two hens which showed minor delays, all of the remaining hens held their eggs more than seven hours beyond the expected time of oviposition. However, the
Laid soft egg 1
0 4 10
5 5 0
absence of a normally due ovulation together with removal of the most recently ruptured follicle, led to delayed ovipositions far beyond the expected time in a high percentage of hens so treated. This generally agreed with the conclusion of Rothchild and Fraps (1944a). Since the ruptured follicles were first treated with acetone, after which the oviposition inducing principle was extracted with a saline solution, it is unlikely that the substance is a steroid or a lipid. On the other hand, the present process for extraction of the ruptured follicles should be improved upon, since the residue fraction exhibited considerable activity for inducing premature ovipositions. However, the detail role of and the identification of this hormone-like factor remains to be investigated. REFERENCES Atwood, H., 1929. Observations concerning the time factor in egg production. Poultry Sci. 8: 137-140. Burrows, W. H., and T. C. Byerly, 1942. Premature expulsion of eggs by hens following injection of whole posterior pituitary preparations. Poultry Sci. 21:416-421. Douglas, D. S., and P. D. Sturkie, 1964. Plasma levels of antidiuretic hormone during oviposition in the hen. Fed. Proc. 23: 150. Heller, H., and B. T. Pickering, 1961. Neurohypophysial hormones of non-mammalian vertebrates. J. Physiol. Lond. 155:98-114.
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Routes No. hens Fraction Died of dose treated Supernatant i.v. b 4 3d Supernatant with 0 heparin 13 i.v. 5 0 Sediment suspension 3.2-3.5 9 i.p. c 0 Vehicle control 2 10 13 i.v. a 0.9% saline solution. b Intravenous injection. c Intraperitoneal injection. d The hens died with tetanic spasms during or immediately after injection.
No. hens Laid at expected time 0
OVARIAN FOLLICLE AND OVDOSITION
between light-dark rhythms and hour of lay of eggs experimentally retained in the hen. Endocrinology, 35: 355-362. Rzasa, J., and Z. Ewy, 1970. Effect of vasotocin and oxytocin on oviposition in the hen. J. Reprod. Fert. 21: 549-550. Shirley, H. V., Jr., and A. V. Nalbandov, 1956. Effects of neurohypophysectomy in domestic chickens. Endocrinology, 58: 477-483. Sturkie, P. D. and Y. Lin, 1966. Release of vasotocin and oviposition in the hen. J. Endocr. 35: 325-326. Tanaka, K., and S. Nakajo, 1960. Oxytocin in the neurohypophysis of the laying hen. Nature, 187: 245. Tanaka, K., and S. Nakajo, 1962. Participation of neurohypophysial hormone in oviposition in the hen. Endocrinology, 70: 453-458. Warren, D. C , and H. M. Scott, 1935. The time factor in egg formation. Poultry Sci. 14: 195-207.
Studies on a Possible Interaction Between Riboflavin and Vitamin B 12 as it Affects Hatchability of the Hen's Egg P . J . TUITE' AND R . E . AUSTIC Department of Poultry Science, Cornell University, Ithaca, New York 14850 (Received for publication March 4, 1974)
ABSTRACT Experiments were conducted using Single Comb White Leghorn chickens to investigate a possible interaction between the requirements for riboflavin and vitamin B 12 for hatchability and to determine how important such an interaction might be in altering the requirements for one or both of these vitamins. The vitamin B 12 requirement for optimum hatchability was found to be 3.45-3.60 pig. /kg. of diet, and the requirement for dietary riboflavin, approximately 2.75 mg./kg. of diet. Dietary riboflavin at deficient or practical levels of feeding was found not to affect the vitamin B12 concentration in egg yolk, or the vitamin B a requirement for optimum hatchability. Although a slight sparing effect of vitamin B 12 upon the riboflavin requirement was suggested by the present studies, the effect was not statistically significant (p < .05). High levels of dietary vitamin B 12 exerted a striking negative effect on riboflavin concentrations in egg yolk (p < .05). Egg production and food consumption were not affected by dietary riboflavin levels ranging from 1.61 to 5.51 mg./kg. of diet or vitamin B12 ranging from 1.0 to 11.0 p.g./kg. of diet. However, optimum egg size was achieved only when the highest level of vitamin B 12 was fed. POULTRY SCIENCE 53: 2125-2136, 1974
INTRODUCTION HE need for adequate riboflavin and vitamin B 12 in the diet of breeding chickens for normal hatchability has long been recognized [refer to Beer (1969) and Lan-
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1. Present address: Department of Agriculture and Fisheries, Dublin 2, Ireland.
dauer (1967) for extensive reviews]. Evidence for an interaction between vitamin B 12 and riboflavin has been presented by a number of investigators. Research on this subject was spawned by the discovery that 5,6-dimethylbenzimidazole was a degradation product of vitamin B n (Folkers, 1950). Since this compound is an integral part of the riboflavin molecule, several studies were
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Heywang, B. W., 1938. The time factor in egg production. Poultry Sci. 17: 240-247. Munsick, R. A., W. H. Sawyer and H. B. van Dyke, 1960. Endocrinology, 66: 860-871. Opel, H., 1965. Oviposition in chickens after removal of the posterior lobe of the pituitary by an improved method. Endocrinology, 76: 673-677. Opel, H., 1966. Release of oviposition-inducing factor from the median eminence-pituitary stalk region in neural lobectomized hens. Anat. Rec. 154: 396. Phillips, R. E., and D. C. Warren, 1937. Observations concerning the mechanics of ovulation in the fowl. J. Exp. Zool. 76: 117-136. Riddle, O., 1921. A simple method of obtaining premature eggs from birds. Science, 54: 664-666. Rothchild, I., and R. M. Fraps, 1944 a. On the function of the ruptured ovarian follicle of the domestic fowl. Proc. Soc. Exp. Biol. Med. 56: 79-82. Rothchild, I., and R. M. Fraps, 1944 b. Relation
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