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Inhibition of Ovulation in the Domestic Hen by Intrauterine Insemination
Inhibition of Ovulation in the Domestic Hen by Intrauterine Insemination L. W. BOBR,* P. E. LAKE,** F. W. LORENZ,*** F. X. OGASAWARA*** AND H. KRZANOWSKA**** University of California, Davis, California, and The A.R.C. Poultry Research Centre, Edinburgh, Scotland (Received for publication September 28, 1964)
659
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 1, 2015
ARTIFICIAL insemination (A.I.) di- in guiding it around the vaginal convolu•**• rectly into the uterus has resulted in tions, and to reduce the danger of inadverfertility under circumstances when none tently penetrating the vaginal wall. At could be obtained with intravaginal A.I. least one, and sometimes several, minutes Polge (1951) obtained no fertility follow- were required to push it gently around the ing intravaginal A.I. with semen containing anterior sigmoid flexure of the vagina. Use 15% glycerol, but Allen and Bobr (1955) of the finger made this manipulation easier obtained some fertility when it was depos- and faster, but at the expense of greater ited directly in the uterus. Ogasawara et dilatation of the sphincter. The uterovagial. (1962) obtained fertility with the nal junction was always firmly closed, but semen of two low-fecundity cocks only by could be penetrated after firm but gentle intrauterine A.I. Intrauterine A.I. has been pressure was exerted for some 10 or 15 secused extensively in studies of behavior of onds, when the sphincter would relax and spermatozoa in the oviduct (Bobr et al., allow entry. 1964a, b), but its usefulness in research A number of separate sets of observahas been limited, and any potential practi- tions were made on egg production followcal applications curtailed, by the tendency ing uterovaginal penetration. A group of of birds to pause after using this tech- 37 Small Line Brown Leghorns at Edinnique. burgh (Blyth, 1954) were inseminated This paper places on record a number of using the finger method and compared with observations on the effect of intrauterine 30 hens in which intravaginal A.I. was perA.I. on subsequent egg production and formed just distal to the uterovaginal junccomments on the conditions that affect it. tion. No disturbance of egg production ocIntrauterine A.I. involves penetration of curred in the latter group, but egg producthe uterovaginal junction. Allen and Bobr tion in 30 out of the 37 experimental hens (1955) accomplished this with a 10.5 cm. was adversely affected. Eight laid a softlong glass cannula and they suggested pre- shelled egg within a few hours; they and liminary penetration with the forefinger to the other 22 then ceased to lay. Four guide the cannula. For our work the can- affected hens commenced to lay after nula was modified; it had a slight bend lapses of 13 to 17 days and the eggs were and a small plastic ball near the end to aid fertile. Three which resumed lay after 20 to 22 days produced only infertile eggs. Addresses: * C.S.I.R.O. Poultry Research Cen- The remaining 23 hens were still out of lay tre, Werribee, Victoria, Australia; ** A.R.C. Poul30 days after the manipulation. A similar try Research Centre, West Mains Road, Edintrial was afterwards made with 25 White burgh 9, Scotland; ***University of California, Leghorns at Davis with the cannula Davis, California; **** Jagiellonian University, Cracow, Poland. technique; 11 birds were undisturbed, 11
660
BOBE, LAKE, LORENZ, OGASAWARA AND KRZANOWSKA
TABLE 1.-
in column (d) show a highly significant increase in pauses among the treated hens X2 = 27.68; p < 0.001). The data in columns (e) and (f) show significant increases (X2 = 14.43 and 25.28, respectively; p < 0.001) also in the number of soft-shelled eggs laid both immediately after manipulation and at variable intervals during the subsequent 10-day period. In the affected hens which were autopsied there was either extensive atresia in the ovary or a normal hierarchy of growing follicles, the largest of which had simply failed to ovulate. The hens were less affected than those in Edinburgh. It is possible that, since the latter birds were small, penetration into the uterus constituted a greater nonspecific stress. Also, the Brown Leghorn hens were manipulated during their declining reproductive period when they may have been expecially susceptible to stress. In this connection, Rothchild and Fraps (1945) have shown that surgical operations caused more disturbance to reproductive activity in birds laying less intensively. Another method of intrauterine A.I. was attempted and, in contrast to the abovementioned results, reproductive disturbances were negligible. The method involved prolonged abdominal pressure on the hen lying on its right side; pressure, assisted by the left fore and middle fingers, was exerted to evert the oviduct, and was continued until a change in appearance of
-The effect on egg production of penetrating the vagina per vaginam with the forefinger and cannula. Hens
Treatment
Control Uterus-penetrated
Number of hens
(a) 87 38
Percent of hens with 2day pause prior to treatment (b) 6.9 0
Percent of hens pausing within 3 days after treatment 2-day duration
Longer duration
(c) 4.6 13.2
(d) 4.6 44.7
junction of While Leghorn "normal" cycles Soft-shelled eggs laid Percent of Percent of eggs in oviduct ovulations ocat treatment curring on day of treatment (e)
0 26.7
(f) 0 42. i
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 1, 2015
produced 1 to 4 soft-shelled eggs and 6 of the latter plus the remaining 3 birds paused for varying periods. This manipulation was repeated three weeks later on 23 hens then in production; 9 produced softshelled eggs and 5 of these plus 3 others paused. Meanwhile, only 2 pauses occurred among 25 control birds subjected to intravaginal A.I. Two weeks following the second A.I. all birds were everted for examination of the vaginal mucosa. Those subjected to uterine penetration had characteristically altered appearances suggestive of trauma, very likely due to inexperience of the operators with the technique. Subsequent experiments were performed only after some skill had been gained through practice. An experiment (the results of which are given in Table 1) was later carried out using only birds with "normal" laying cycles. Normal cycles were defined as closed clutches of at least two eggs, no more than a single 2-day pause during the week preceding the treatment, and not in a pause of more than two days duration on the treatment day. A number of birds were autopsied a week after treatment to determine the condition of the ovaries; the remainder were allowed to recover from any effects of the treatment undisturbed. From an examination of the results in Table 1 it is clear that a disturbance of egg-laying again occurred in several birds after intrauterine penetration. The results
661
INHIBITION OF OVULATION
ACKNOWLEDGEMENTS
This investigation was supported in part by a National Science Foundation Grant
(G-9837) and a Public Health Service training grant (GM-646). One of us (P. E. L.) held a Lalor Foundation Fellowship. REFERENCES Allen, T. E., and L. W. Bobr, 1955. The fertility of fowl spermatozoa in glycerol diluents after intrauterine insemination. Poultry Sci. 34: 11671169. Blyth, J. S. S., 1954. Notes on the Poultry Research Centre flock of Brown Leghorns. World's Poultry Sci. J. 10: 140-143. Bobr, L. W., F. W. Lorenz and F. X. Ogasawara, 1964a. Distribution of spermatozoa in the oviduct and fertility in domestic birds. I. Residence sites of spermatozoa in fowl oviducts. J. Reprol. Fertil. 8: 39-47. Bobr, L. W., F. X. Ogasawara and F. W. Lorenz, 1964b. Distribution of spermatozoa in the oviduct and fertility in domestic birds. II. Transport of spermatozoa in the fowl oviduct. J. Reprod. Fertil. 8: 49-58. Huston, T. M., and A. V. Nalbandov, 1953. Neurohumoral control of the pituitary in the fowl. Endocrinology 52: 149-156. Lake, P. E., and A. B. Gilbert, 1964. The effect on egg production of a foreign object in the lower oviduct regions of the domestic hen. Res. Vet. Sci. 5: 39-45. Ogasawara, F. X., F. W. Lorenz and L. W. Bobr, 1962. Intrauterine insemination of lowfecundity cock semen. Poultry Sci. 4 1 : 1671. Polge, G, 1951. Functional survival of fowl spermatozoa after freezing at — 79 °C. Nature, 167: 949-950. Rothchild, I., and R. M. Fraps, 1945. The relation between ovulation frequency and the incidence of follicular atresia following surgical operations in the domestic hen. Endocrinology, 37: 415-430. Sykes, A. H., 1953. Premature oviposition in the hen. Nature, 172: 1098-1099. Sykes, A. H., 1955. The effect of adrenaline on oviduct motility and egg production in the fowl. Poultry Sci. 34: 622-628. van Tienhoven, A., 1953. Further study on the neurogenic blockage of LH release in the hen. Anat. Rec. 115: 374.
JUNE 8-11. ROYAL S( CIETY OF CANADA ANNUAL MEETING, VANCOUVER, B.C.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 1, 2015
the mucosa indicated exposure of the uterovaginal junction. Occasionally a small amount of clear fluid was exuded from the exposed orifice. The cannula was carefully inserted through the exposed junction with a twisting motion. Among 51 hens so manipulated and inseminated through the dilated orifice, only 2 short pauses occurred. The results obtained after intrauterine insemination provide further evidence implicating the nervous system in normal functioning of the ovary and oviduct of the hen. Previously Sykes (1955) found that adrenaline disturbed egg production, and Sykes (1953), van Tienhoven (1953), Huston and Nalbandov (1953), and Lake and Gilbert (1964) demonstrated that irritations applied to various parts of the oviduct had adverse effects on oviduct function and possibly ovulation. The physiological mechanisms involved in the adverse reactions to intrauterine penetration under some circumstances have yet to be elucidated. It would appear that when the uterovaginal junction is probed per vaginam egg production is only affected when it is forcibly penetrated, because manipulation without penetration had no effect. It is possible that a traumatic reflex is set up by abnormal stimulation of the uterovaginal sphincter. When the entire vagina was everted by prolonged abdominal pressure—to expose the uterine mucosa, negligible effects on egg-laying were produced by placing an object in the uterus. Of course, during natural oviposition, the uterovaginal junction is dilated far more than by the technique herein described.
659
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 1, 2015
ARTIFICIAL insemination (A.I.) di- in guiding it around the vaginal convolu•**• rectly into the uterus has resulted in tions, and to reduce the danger of inadverfertility under circumstances when none tently penetrating the vaginal wall. At could be obtained with intravaginal A.I. least one, and sometimes several, minutes Polge (1951) obtained no fertility follow- were required to push it gently around the ing intravaginal A.I. with semen containing anterior sigmoid flexure of the vagina. Use 15% glycerol, but Allen and Bobr (1955) of the finger made this manipulation easier obtained some fertility when it was depos- and faster, but at the expense of greater ited directly in the uterus. Ogasawara et dilatation of the sphincter. The uterovagial. (1962) obtained fertility with the nal junction was always firmly closed, but semen of two low-fecundity cocks only by could be penetrated after firm but gentle intrauterine A.I. Intrauterine A.I. has been pressure was exerted for some 10 or 15 secused extensively in studies of behavior of onds, when the sphincter would relax and spermatozoa in the oviduct (Bobr et al., allow entry. 1964a, b), but its usefulness in research A number of separate sets of observahas been limited, and any potential practi- tions were made on egg production followcal applications curtailed, by the tendency ing uterovaginal penetration. A group of of birds to pause after using this tech- 37 Small Line Brown Leghorns at Edinnique. burgh (Blyth, 1954) were inseminated This paper places on record a number of using the finger method and compared with observations on the effect of intrauterine 30 hens in which intravaginal A.I. was perA.I. on subsequent egg production and formed just distal to the uterovaginal junccomments on the conditions that affect it. tion. No disturbance of egg production ocIntrauterine A.I. involves penetration of curred in the latter group, but egg producthe uterovaginal junction. Allen and Bobr tion in 30 out of the 37 experimental hens (1955) accomplished this with a 10.5 cm. was adversely affected. Eight laid a softlong glass cannula and they suggested pre- shelled egg within a few hours; they and liminary penetration with the forefinger to the other 22 then ceased to lay. Four guide the cannula. For our work the can- affected hens commenced to lay after nula was modified; it had a slight bend lapses of 13 to 17 days and the eggs were and a small plastic ball near the end to aid fertile. Three which resumed lay after 20 to 22 days produced only infertile eggs. Addresses: * C.S.I.R.O. Poultry Research Cen- The remaining 23 hens were still out of lay tre, Werribee, Victoria, Australia; ** A.R.C. Poul30 days after the manipulation. A similar try Research Centre, West Mains Road, Edintrial was afterwards made with 25 White burgh 9, Scotland; ***University of California, Leghorns at Davis with the cannula Davis, California; **** Jagiellonian University, Cracow, Poland. technique; 11 birds were undisturbed, 11
660
BOBE, LAKE, LORENZ, OGASAWARA AND KRZANOWSKA
TABLE 1.-
in column (d) show a highly significant increase in pauses among the treated hens X2 = 27.68; p < 0.001). The data in columns (e) and (f) show significant increases (X2 = 14.43 and 25.28, respectively; p < 0.001) also in the number of soft-shelled eggs laid both immediately after manipulation and at variable intervals during the subsequent 10-day period. In the affected hens which were autopsied there was either extensive atresia in the ovary or a normal hierarchy of growing follicles, the largest of which had simply failed to ovulate. The hens were less affected than those in Edinburgh. It is possible that, since the latter birds were small, penetration into the uterus constituted a greater nonspecific stress. Also, the Brown Leghorn hens were manipulated during their declining reproductive period when they may have been expecially susceptible to stress. In this connection, Rothchild and Fraps (1945) have shown that surgical operations caused more disturbance to reproductive activity in birds laying less intensively. Another method of intrauterine A.I. was attempted and, in contrast to the abovementioned results, reproductive disturbances were negligible. The method involved prolonged abdominal pressure on the hen lying on its right side; pressure, assisted by the left fore and middle fingers, was exerted to evert the oviduct, and was continued until a change in appearance of
-The effect on egg production of penetrating the vagina per vaginam with the forefinger and cannula. Hens
Treatment
Control Uterus-penetrated
Number of hens
(a) 87 38
Percent of hens with 2day pause prior to treatment (b) 6.9 0
Percent of hens pausing within 3 days after treatment 2-day duration
Longer duration
(c) 4.6 13.2
(d) 4.6 44.7
junction of While Leghorn "normal" cycles Soft-shelled eggs laid Percent of Percent of eggs in oviduct ovulations ocat treatment curring on day of treatment (e)
0 26.7
(f) 0 42. i
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 1, 2015
produced 1 to 4 soft-shelled eggs and 6 of the latter plus the remaining 3 birds paused for varying periods. This manipulation was repeated three weeks later on 23 hens then in production; 9 produced softshelled eggs and 5 of these plus 3 others paused. Meanwhile, only 2 pauses occurred among 25 control birds subjected to intravaginal A.I. Two weeks following the second A.I. all birds were everted for examination of the vaginal mucosa. Those subjected to uterine penetration had characteristically altered appearances suggestive of trauma, very likely due to inexperience of the operators with the technique. Subsequent experiments were performed only after some skill had been gained through practice. An experiment (the results of which are given in Table 1) was later carried out using only birds with "normal" laying cycles. Normal cycles were defined as closed clutches of at least two eggs, no more than a single 2-day pause during the week preceding the treatment, and not in a pause of more than two days duration on the treatment day. A number of birds were autopsied a week after treatment to determine the condition of the ovaries; the remainder were allowed to recover from any effects of the treatment undisturbed. From an examination of the results in Table 1 it is clear that a disturbance of egg-laying again occurred in several birds after intrauterine penetration. The results
661
INHIBITION OF OVULATION
ACKNOWLEDGEMENTS
This investigation was supported in part by a National Science Foundation Grant
(G-9837) and a Public Health Service training grant (GM-646). One of us (P. E. L.) held a Lalor Foundation Fellowship. REFERENCES Allen, T. E., and L. W. Bobr, 1955. The fertility of fowl spermatozoa in glycerol diluents after intrauterine insemination. Poultry Sci. 34: 11671169. Blyth, J. S. S., 1954. Notes on the Poultry Research Centre flock of Brown Leghorns. World's Poultry Sci. J. 10: 140-143. Bobr, L. W., F. W. Lorenz and F. X. Ogasawara, 1964a. Distribution of spermatozoa in the oviduct and fertility in domestic birds. I. Residence sites of spermatozoa in fowl oviducts. J. Reprol. Fertil. 8: 39-47. Bobr, L. W., F. X. Ogasawara and F. W. Lorenz, 1964b. Distribution of spermatozoa in the oviduct and fertility in domestic birds. II. Transport of spermatozoa in the fowl oviduct. J. Reprod. Fertil. 8: 49-58. Huston, T. M., and A. V. Nalbandov, 1953. Neurohumoral control of the pituitary in the fowl. Endocrinology 52: 149-156. Lake, P. E., and A. B. Gilbert, 1964. The effect on egg production of a foreign object in the lower oviduct regions of the domestic hen. Res. Vet. Sci. 5: 39-45. Ogasawara, F. X., F. W. Lorenz and L. W. Bobr, 1962. Intrauterine insemination of lowfecundity cock semen. Poultry Sci. 4 1 : 1671. Polge, G, 1951. Functional survival of fowl spermatozoa after freezing at — 79 °C. Nature, 167: 949-950. Rothchild, I., and R. M. Fraps, 1945. The relation between ovulation frequency and the incidence of follicular atresia following surgical operations in the domestic hen. Endocrinology, 37: 415-430. Sykes, A. H., 1953. Premature oviposition in the hen. Nature, 172: 1098-1099. Sykes, A. H., 1955. The effect of adrenaline on oviduct motility and egg production in the fowl. Poultry Sci. 34: 622-628. van Tienhoven, A., 1953. Further study on the neurogenic blockage of LH release in the hen. Anat. Rec. 115: 374.
JUNE 8-11. ROYAL S( CIETY OF CANADA ANNUAL MEETING, VANCOUVER, B.C.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 1, 2015
the mucosa indicated exposure of the uterovaginal junction. Occasionally a small amount of clear fluid was exuded from the exposed orifice. The cannula was carefully inserted through the exposed junction with a twisting motion. Among 51 hens so manipulated and inseminated through the dilated orifice, only 2 short pauses occurred. The results obtained after intrauterine insemination provide further evidence implicating the nervous system in normal functioning of the ovary and oviduct of the hen. Previously Sykes (1955) found that adrenaline disturbed egg production, and Sykes (1953), van Tienhoven (1953), Huston and Nalbandov (1953), and Lake and Gilbert (1964) demonstrated that irritations applied to various parts of the oviduct had adverse effects on oviduct function and possibly ovulation. The physiological mechanisms involved in the adverse reactions to intrauterine penetration under some circumstances have yet to be elucidated. It would appear that when the uterovaginal junction is probed per vaginam egg production is only affected when it is forcibly penetrated, because manipulation without penetration had no effect. It is possible that a traumatic reflex is set up by abnormal stimulation of the uterovaginal sphincter. When the entire vagina was everted by prolonged abdominal pressure—to expose the uterine mucosa, negligible effects on egg-laying were produced by placing an object in the uterus. Of course, during natural oviposition, the uterovaginal junction is dilated far more than by the technique herein described.