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Fertility studies in young and mature merino ewes
THERIOGENOLOGY FERTILITY STUDIES IN YOUNG AND MATURE MERINO EWES 2.
SPERM TRANSPORT A.B. SELAIVE-VILLARROEL* and J.P. KENNEDY
School of Wool and Pastoral Sciences The University of New South Wales Kensington, NSW, Australia, 2033 Received for publication: May 10, 1983 Accepted: September 15, 1983 ABSTRACT Samples of cervical mucus were collected from young and mature ewes at different stages of oestrue. The migration rate of spermatozoa (time taken to travel 410 mm) was not different in samples of mucus from young and mature ewes. Spermatozoa were unable to penetrate the mucus in 7/27 samples, five of which were collected about 16 hours after onset of oestrus. Migration rate in samples collected between 10 and 16 hours after onset of oestrus was significantly slower than in samples collected in the first five hours of oestrus. The transport of spermatozoa in young and mature ewes was also studied by observing distribution of spermatozoa in the female genital tract 26 to 27 hours after hand-mating. There were no significant differences between young and mature ewes either in mean sperm count or in variation of mean sperm numbers in all sections of the tract. INTRODUCTION Published studies on effects of age on rate of sperm transport suggest lower efficiency in young ewes than mature ewes, and this could be a cause of the higher incidence of failure to lamb in young ewes. To explain the low sperm numbers in the oviducts in association with low cervical sperm counts, it has been suggested (1) that some characteristic of the cervical mucus secretion of young ewes adversely affects the entry of sperm into and through the cervix. The aim of this study was to evaluate cervical factors in relation to fertility of young and mature ewes. The in vitro migration rate of spermatozoa in cervical mucus from different stages of oestrus and the in vivo transport of spermatozoa in young and mature ewes were studied. MATERIALS AND METHODS Experiment 1: In Vitro Sperm Migration in Cervical Mucus Twenty-seven samples of fresh mucus were collected from five young ewes (about 1.5 years old) and ten mature ewes (four to six years old). Mucus was collected once or twice from each ewe at different times during natural oestrus, as detected by aproned rams. Using a vaginal speculum, mucus was aspirated by a pipette from the cervical OS or * Present address: EMBRAPA, Caixa Postal 242, Bage-RS. BRAZIL 96.400
NOVEMBER
1983 VOL. 20 NO. 5
543
THERIOGENOLOGY the anterior part of the vagina and used immediately after collection. Semen of high quality, in terms of wave motion, motility and concentration of normal sperm, was collected by artificial vagina from two rams. The semen was used within 15 minutes of being collected and sperm migration was observed by the method of Tampion and Gibbons (2). After the mucus was collected, each ewe was placed with an aproned ram to ensure that she was in oestrus. Experiment 2: Sperm Transport in the Female Genital Tract Sixteen maiden ewes aged 1.5 years (young ewes) and 13 parous ewes aged between 3.5 to 7 years (mature ewes) were hand-mated 2.5 to 4.5 hours after being detected in oestrus. The mob was yarded each day at 0600 and 1800 h, and ewes selected for the experiment were those that were first recorded as marked at 0600 h. Each ewe was served once by one of the five rams used during the experiment. Rams were selected from a group of 12 on the basis of similarity of sperm numbers per ejaculate and libido. In an attempt to maintain constant numbers of spermatozoa in the ejaculate, all rams served three times daily, either in hand-service or into an artificial vagina during the experiment and for 12 days beforehand. Completion of ejaculation was judged by observation of introduction of the penis, pelvic thrust and behaviour after demounting (3). At the end of the experiment each ram had served the same number of young and mature ewes. Ewes were slaughtered 26 to 27 hours after mating; the reproductive tract was removed, the utero-tubal and utero-cervical junctions wereliga ted and the caudal end of the cervix was clamped. The oviducts, uterus and cervix were flushed with saline (4); then the length of the cervix was measured and opened lengthwise and divided into caudal, mid- and cranial sections. Each section was dissected into four longitudinal strips which were covered with 20 ml of saline in a vial. All collections were frozen until thawing and counting of spermatozoa. We estimated the number of spermatozoa in flushings by methods similar to those described by Quinlivan and Robinson (5). Before estimating sperm numbers in the cervix, the cervical strips were thawed and stored overnight at 4OC to loosen spermatozoa remaining in the cervical crypts. The saline solutions containing the strips were vigorously shaken before removing the strips and pooling the solutions with flushings from the cervix. Statistical Analysis Individual sperm counts from each genital tract segment of young and mature ewes were transformed to logarithms (plus 1 to allow for zero value) because the means for untransformed counts were proportional to the standard deviations. The means of the logarithmic transformed data of each segment were tested between ages by t-test. The coefficient of variation of the mean sperm numbers from each segment of the genital tract was calculated and compared between age groups of ewes. The relationship between oviductal and cervical sperm numbers in both groups of ewes was estimated, after logarithmic transformation, by correlation coefficient and by simple linear regressions. The correlation between length of the cervix and cervical sperm numbers was also estimated.
544
NOVEMBER
1983VOL.20N0.5
THERIOGENOLOGY RESULTS Experiment 1 From a total of 27 samples tested, spermatozoa were unable to penetrate the cervical mucus in seven samples (25.9%), five of which were collected about 16 hours after the onset of oestrus. The migration rate of spermatozoa was estimated in 20 samples, each estimate being based on 10 spermatozoa. Mean speeds in samples collected from young and mature ewes at different times of oestrus are shown in Table 1. Because some ewes had two mucus collections at different times of oestrus while mucus was collected only once in others, results were separately analysed. The separate estimates were then averaged and standard errors for pooled estimates were calculated. TABLE 1 SPERM MIGRATION IN CERVICAL MUCUS COLLECTED FROM YOUNG AND MATURE EWES UP TO 5 HOURS AND BETWEEN 10 TO 16 HOURS AFTER ONSET OF OESTRUS
up to 5 hr
Between lo-16 hr
up to 5 hr
i
Between lo-16 hr
up to 5 hr
Between lo-16 hr
Young
4
3
1
1
7.44kO.76 10.76k1.51
Mature
I
6
0
5*
8.2OCO.80
9.81k1.04
* 16 hours There was no age-by-time interaction in time taken to travel 41Omm. The difference between the time effects at the two ages was 1.88 2 1.38 seconds. The difference between ages was 1.25 + 1.29 seconds and was not significant, but the difference between times for combined ages was 1.98 C 0.76 seconds (P < 0.05). Experiment 2 The mean liveweights of the young and mature ewes were 38.9 ?r 0.9kg and 45.3 + 1.5 kg, respectively (P < 0.001). The means of the sperm count in different segments of the genital tract of young and mature ewes are given in Table 2. The t-tests of the logarithmic means from each segment indicated no significant difference between age groups. There was significant correlation between the numbers of spermatozoa in the cervix and in the oviducts of young (r = 0.60, P < 0.05) and mature ewes (r = 0.87, ? < 0.01). Variances of mean numbers of
NOVEMBER
1983 VOL. 20 NO. 5
545
THERIOGENOLOGY spermatozoa found in young and older ewes were tested for equality in the three segments of the tract and were found to be homogeneous. The correlations between the length of the cervix and the logarithmic transformed cervical sperm counts were not significantly different between ages (young ewes, r = 0.06; mature ewes, r = 0.30). TABLE 2 MEAN NUMBERS (LogI,,? S.E.) OF SPERMATOZOA IN THE REPRODUCTIVE TRACT OF YOUNG AND MATURE EWES 26 TO 27 HOURS AFTER HAND-MATING
Age of Iwes
No. of Ewes
MEAN NUMBER OF SPERM (+ S.E.) Cervix
Uterus
Loglo
Loglo
Log10
Oviducts
Young
16
6.5375 f0.5409
5.2896 kO.3993
3.1830 kO.2594
Mature
13
6.3505 20.7083
5.9200 to.4402
3.0074 to.3626
DISCUSSION An earlier explanation (1) of low sperm numbers in the cervix and oviducts of young ewes was that some characteristic of the cervical mucus of young ewes adversely affects the entry of sperm into and through the cervix. Inthis study the swimming rate of spermatozoa was similar in the cervical mucus of young and mature ewes. In both age groups, however, sperm penetration of cervical mucus became difficult 16 hours after the onset of oestrus, when the mucus was becoming thicker. This negative effect of time in oestrus on sperm migration may be more marked in young than mature ewes because of their shorter oestrus and earlier decline of mucus production (6), which could make the cervical mucus impenetrable to sperm more promptly than in mature ewes. No significant differences were found between young and matureewes, either in mean sperm counts or in variation of mean sperm numbers, in all sections of the genital tract. The regression analysis indicated that the sperm numbers in the oviducts were associated with the numbers of spermatozoa present in the cervix of both young and mature ewes. The absence of any difference between age groups in the regression coefficients of sperm numbers in oviducts and cervix shows that the efficiency of sperm transport from the cervix to the oviducts did not differ with age. Also, there was no significant variability between young and mature ewes in number of spermatozoa (x2 = 1.19, P < 0.05) recovered from the oviducts 24 hours after service. These results do not agree with a similar study (1) in which sperm transport from the cervix to the uterus was less efficient in young ewes, but no difference was found between
546
NOVEMBER
1983VOL.20NO.5
THERIOCENOLOGY ages in the transport of spermatozoa from the uterus to the oviducts. Since muscular activity is important in sperm transport through the uterus, the uterine sperm population is expected to be more variable than in the other sections of the genital tract. Thus, any correlation between the uterine sperm numbers and those in either lower or upper parts of the genital tract is not very significant in the estimation of the efficiency of sperm transport. In view of the shorter cervix in young ewes (6), a lower population of spermatozoa in their oviducts might be expected, but the observed lack of relationship between cervical sperm numbers and length of cervix suggests this is not a limiting factor in sperm transport in either young or mature ewes. If there is a lower efficiency of sperm transport in young ewes, it should have appeared in this study since the ewes wexe inseminated with a smaller number of sperm than that reported by other workers. From these observations, it seems that from an adequate cervical sperm reservoir, the efficiency of sperm transport to the site of fertilization is similar in young and mature ewes; however, because of the shorter oestrus and lower mucus production in young ewes, the cervical mucus may become impenetrable to sperm earlier in oestrus in young than in mature ewes. REFERENCES 1.
Kennedy, T.G. Some aspects of fertility in the young ewe and in the mouse. Ph.D. Thesis, University of New South Wales (1969).
2.
Tampion, D. and Gibbons, R.A. Orientation of spermatozoa in mucus of the cervix uteri. Nature, London -194: 381 (1962).
3.
Banks, E.M. Some aspects of sexual behaviour in domestic sheep ovis dries. Behaviour -23: 294-279 (1964).
4.
Allison, A.J. A comparison of the transport of spermatozoa in spayed and entire ewes. J. Reprod. Fertil. -31: 415-423 (1972).
5.
Quinlivan, T.D. and Robinson, T.J. The number of spermatozoa in the fallopian tubes of ewes at intervals after artificial insemination following withdrawal of SC-9880-impregnated intravaginal sponges. In The Control of the Ovarian Cycle in Sheep. Ed. T.J. Robinson. Sydney University Press, Sydney, 1967, pp. 177-194.
6.
Selaive-Villarroel, A.B. and Kennedy, J.P. Fertility studies in young and mature Merino ewes. 1. Cervical Mucus Production Theriogenology 20: 537-541 (1983).
NOVEMBER
1983 VOL. 20 NO. 5
547
SPERM TRANSPORT A.B. SELAIVE-VILLARROEL* and J.P. KENNEDY
School of Wool and Pastoral Sciences The University of New South Wales Kensington, NSW, Australia, 2033 Received for publication: May 10, 1983 Accepted: September 15, 1983 ABSTRACT Samples of cervical mucus were collected from young and mature ewes at different stages of oestrue. The migration rate of spermatozoa (time taken to travel 410 mm) was not different in samples of mucus from young and mature ewes. Spermatozoa were unable to penetrate the mucus in 7/27 samples, five of which were collected about 16 hours after onset of oestrus. Migration rate in samples collected between 10 and 16 hours after onset of oestrus was significantly slower than in samples collected in the first five hours of oestrus. The transport of spermatozoa in young and mature ewes was also studied by observing distribution of spermatozoa in the female genital tract 26 to 27 hours after hand-mating. There were no significant differences between young and mature ewes either in mean sperm count or in variation of mean sperm numbers in all sections of the tract. INTRODUCTION Published studies on effects of age on rate of sperm transport suggest lower efficiency in young ewes than mature ewes, and this could be a cause of the higher incidence of failure to lamb in young ewes. To explain the low sperm numbers in the oviducts in association with low cervical sperm counts, it has been suggested (1) that some characteristic of the cervical mucus secretion of young ewes adversely affects the entry of sperm into and through the cervix. The aim of this study was to evaluate cervical factors in relation to fertility of young and mature ewes. The in vitro migration rate of spermatozoa in cervical mucus from different stages of oestrus and the in vivo transport of spermatozoa in young and mature ewes were studied. MATERIALS AND METHODS Experiment 1: In Vitro Sperm Migration in Cervical Mucus Twenty-seven samples of fresh mucus were collected from five young ewes (about 1.5 years old) and ten mature ewes (four to six years old). Mucus was collected once or twice from each ewe at different times during natural oestrus, as detected by aproned rams. Using a vaginal speculum, mucus was aspirated by a pipette from the cervical OS or * Present address: EMBRAPA, Caixa Postal 242, Bage-RS. BRAZIL 96.400
NOVEMBER
1983 VOL. 20 NO. 5
543
THERIOGENOLOGY the anterior part of the vagina and used immediately after collection. Semen of high quality, in terms of wave motion, motility and concentration of normal sperm, was collected by artificial vagina from two rams. The semen was used within 15 minutes of being collected and sperm migration was observed by the method of Tampion and Gibbons (2). After the mucus was collected, each ewe was placed with an aproned ram to ensure that she was in oestrus. Experiment 2: Sperm Transport in the Female Genital Tract Sixteen maiden ewes aged 1.5 years (young ewes) and 13 parous ewes aged between 3.5 to 7 years (mature ewes) were hand-mated 2.5 to 4.5 hours after being detected in oestrus. The mob was yarded each day at 0600 and 1800 h, and ewes selected for the experiment were those that were first recorded as marked at 0600 h. Each ewe was served once by one of the five rams used during the experiment. Rams were selected from a group of 12 on the basis of similarity of sperm numbers per ejaculate and libido. In an attempt to maintain constant numbers of spermatozoa in the ejaculate, all rams served three times daily, either in hand-service or into an artificial vagina during the experiment and for 12 days beforehand. Completion of ejaculation was judged by observation of introduction of the penis, pelvic thrust and behaviour after demounting (3). At the end of the experiment each ram had served the same number of young and mature ewes. Ewes were slaughtered 26 to 27 hours after mating; the reproductive tract was removed, the utero-tubal and utero-cervical junctions wereliga ted and the caudal end of the cervix was clamped. The oviducts, uterus and cervix were flushed with saline (4); then the length of the cervix was measured and opened lengthwise and divided into caudal, mid- and cranial sections. Each section was dissected into four longitudinal strips which were covered with 20 ml of saline in a vial. All collections were frozen until thawing and counting of spermatozoa. We estimated the number of spermatozoa in flushings by methods similar to those described by Quinlivan and Robinson (5). Before estimating sperm numbers in the cervix, the cervical strips were thawed and stored overnight at 4OC to loosen spermatozoa remaining in the cervical crypts. The saline solutions containing the strips were vigorously shaken before removing the strips and pooling the solutions with flushings from the cervix. Statistical Analysis Individual sperm counts from each genital tract segment of young and mature ewes were transformed to logarithms (plus 1 to allow for zero value) because the means for untransformed counts were proportional to the standard deviations. The means of the logarithmic transformed data of each segment were tested between ages by t-test. The coefficient of variation of the mean sperm numbers from each segment of the genital tract was calculated and compared between age groups of ewes. The relationship between oviductal and cervical sperm numbers in both groups of ewes was estimated, after logarithmic transformation, by correlation coefficient and by simple linear regressions. The correlation between length of the cervix and cervical sperm numbers was also estimated.
544
NOVEMBER
1983VOL.20N0.5
THERIOGENOLOGY RESULTS Experiment 1 From a total of 27 samples tested, spermatozoa were unable to penetrate the cervical mucus in seven samples (25.9%), five of which were collected about 16 hours after the onset of oestrus. The migration rate of spermatozoa was estimated in 20 samples, each estimate being based on 10 spermatozoa. Mean speeds in samples collected from young and mature ewes at different times of oestrus are shown in Table 1. Because some ewes had two mucus collections at different times of oestrus while mucus was collected only once in others, results were separately analysed. The separate estimates were then averaged and standard errors for pooled estimates were calculated. TABLE 1 SPERM MIGRATION IN CERVICAL MUCUS COLLECTED FROM YOUNG AND MATURE EWES UP TO 5 HOURS AND BETWEEN 10 TO 16 HOURS AFTER ONSET OF OESTRUS
up to 5 hr
Between lo-16 hr
up to 5 hr
i
Between lo-16 hr
up to 5 hr
Between lo-16 hr
Young
4
3
1
1
7.44kO.76 10.76k1.51
Mature
I
6
0
5*
8.2OCO.80
9.81k1.04
* 16 hours There was no age-by-time interaction in time taken to travel 41Omm. The difference between the time effects at the two ages was 1.88 2 1.38 seconds. The difference between ages was 1.25 + 1.29 seconds and was not significant, but the difference between times for combined ages was 1.98 C 0.76 seconds (P < 0.05). Experiment 2 The mean liveweights of the young and mature ewes were 38.9 ?r 0.9kg and 45.3 + 1.5 kg, respectively (P < 0.001). The means of the sperm count in different segments of the genital tract of young and mature ewes are given in Table 2. The t-tests of the logarithmic means from each segment indicated no significant difference between age groups. There was significant correlation between the numbers of spermatozoa in the cervix and in the oviducts of young (r = 0.60, P < 0.05) and mature ewes (r = 0.87, ? < 0.01). Variances of mean numbers of
NOVEMBER
1983 VOL. 20 NO. 5
545
THERIOGENOLOGY spermatozoa found in young and older ewes were tested for equality in the three segments of the tract and were found to be homogeneous. The correlations between the length of the cervix and the logarithmic transformed cervical sperm counts were not significantly different between ages (young ewes, r = 0.06; mature ewes, r = 0.30). TABLE 2 MEAN NUMBERS (LogI,,? S.E.) OF SPERMATOZOA IN THE REPRODUCTIVE TRACT OF YOUNG AND MATURE EWES 26 TO 27 HOURS AFTER HAND-MATING
Age of Iwes
No. of Ewes
MEAN NUMBER OF SPERM (+ S.E.) Cervix
Uterus
Loglo
Loglo
Log10
Oviducts
Young
16
6.5375 f0.5409
5.2896 kO.3993
3.1830 kO.2594
Mature
13
6.3505 20.7083
5.9200 to.4402
3.0074 to.3626
DISCUSSION An earlier explanation (1) of low sperm numbers in the cervix and oviducts of young ewes was that some characteristic of the cervical mucus of young ewes adversely affects the entry of sperm into and through the cervix. Inthis study the swimming rate of spermatozoa was similar in the cervical mucus of young and mature ewes. In both age groups, however, sperm penetration of cervical mucus became difficult 16 hours after the onset of oestrus, when the mucus was becoming thicker. This negative effect of time in oestrus on sperm migration may be more marked in young than mature ewes because of their shorter oestrus and earlier decline of mucus production (6), which could make the cervical mucus impenetrable to sperm more promptly than in mature ewes. No significant differences were found between young and matureewes, either in mean sperm counts or in variation of mean sperm numbers, in all sections of the genital tract. The regression analysis indicated that the sperm numbers in the oviducts were associated with the numbers of spermatozoa present in the cervix of both young and mature ewes. The absence of any difference between age groups in the regression coefficients of sperm numbers in oviducts and cervix shows that the efficiency of sperm transport from the cervix to the oviducts did not differ with age. Also, there was no significant variability between young and mature ewes in number of spermatozoa (x2 = 1.19, P < 0.05) recovered from the oviducts 24 hours after service. These results do not agree with a similar study (1) in which sperm transport from the cervix to the uterus was less efficient in young ewes, but no difference was found between
546
NOVEMBER
1983VOL.20NO.5
THERIOCENOLOGY ages in the transport of spermatozoa from the uterus to the oviducts. Since muscular activity is important in sperm transport through the uterus, the uterine sperm population is expected to be more variable than in the other sections of the genital tract. Thus, any correlation between the uterine sperm numbers and those in either lower or upper parts of the genital tract is not very significant in the estimation of the efficiency of sperm transport. In view of the shorter cervix in young ewes (6), a lower population of spermatozoa in their oviducts might be expected, but the observed lack of relationship between cervical sperm numbers and length of cervix suggests this is not a limiting factor in sperm transport in either young or mature ewes. If there is a lower efficiency of sperm transport in young ewes, it should have appeared in this study since the ewes wexe inseminated with a smaller number of sperm than that reported by other workers. From these observations, it seems that from an adequate cervical sperm reservoir, the efficiency of sperm transport to the site of fertilization is similar in young and mature ewes; however, because of the shorter oestrus and lower mucus production in young ewes, the cervical mucus may become impenetrable to sperm earlier in oestrus in young than in mature ewes. REFERENCES 1.
Kennedy, T.G. Some aspects of fertility in the young ewe and in the mouse. Ph.D. Thesis, University of New South Wales (1969).
2.
Tampion, D. and Gibbons, R.A. Orientation of spermatozoa in mucus of the cervix uteri. Nature, London -194: 381 (1962).
3.
Banks, E.M. Some aspects of sexual behaviour in domestic sheep ovis dries. Behaviour -23: 294-279 (1964).
4.
Allison, A.J. A comparison of the transport of spermatozoa in spayed and entire ewes. J. Reprod. Fertil. -31: 415-423 (1972).
5.
Quinlivan, T.D. and Robinson, T.J. The number of spermatozoa in the fallopian tubes of ewes at intervals after artificial insemination following withdrawal of SC-9880-impregnated intravaginal sponges. In The Control of the Ovarian Cycle in Sheep. Ed. T.J. Robinson. Sydney University Press, Sydney, 1967, pp. 177-194.
6.
Selaive-Villarroel, A.B. and Kennedy, J.P. Fertility studies in young and mature Merino ewes. 1. Cervical Mucus Production Theriogenology 20: 537-541 (1983).
NOVEMBER
1983 VOL. 20 NO. 5
547