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Fertility studies in young and mature merino ewes
THERIOGENOI,OGY FERTILITY STUDIES IN YOUNG AND MATURE MERINO EWES 1.
CERVICAL MUCUS PRODUCTION 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: Accepted:
May 10, 1983 September 15,
1983
ABSTRACT The production of cervical mucus was determined intervals during oestrus in young (1% years old) and years old) Merino ewes. The length of the cervix and the cervical epithelium was also measured in similar
at six-hour mature (3 to 7 the length of ewes.
Total weight of mucus secreted as well as secretion rate per unit of time was greater in mature ewes than young ewes. Production was maximal during the first six hours of oestrus and declined considerably after i2 hours in young and 18 hours in mature ewes. The cervix was significantly shorter in young ewes while the length of the cervical epithelium was not significantly different in young and mature. It was concluded that lower mucus production in young ewes is not related to lower mucus output per unit area. INTRODUCTION Changes in the quantity or quality of cervical mucus may disturb sperm transport with subsequent fertilization failure (1). Impaired fertility associated with altered mucus production was reported in proqestaqen-synchronized ewes (2) and in ewes following prolonged grazing on oestroqenic pasture (3). It has been proposed that young and mature ewes may differ in the production and/or properties of mucus and that this may partly explain observed differences in their fertility (4). This work tested this hypothesis by (al measuring the cervical mucus production of young and mature Merino ewes and (b) estimating the size of the secretory surface of the cervix. MATERIALS AND METHODS The experiments were conducted at The University of New South
* Present address: EMBRAPA, Caixa Postal 242, Baq&RS.
NOVEMBER
1983 VOL. 20 NO. 5
BRAZIL 96.400
537
THERIOGENOLOGY Wales Field Station at Wellington, New South Wales. Determination of the Amount of Mucus Produced Mucus was collected from 20 nulliparous 1.5-year-old Merino ewes (young ewes) and 20 parous 3- to 7-year-old Merino ewes (mature ewes) during the first two weeks of May. Signs of oestrus were observed hourly from 0630 to 1930 h each day and mucus collection commenced immediately after detection of oestrus. Every six hours during the first 18 hours following the onset of oestrus, and then every three hours, each ewe was tested with an aproned teaser ram for evidence of oestrus. Mucus was collected at six-hour intervals. Two cylindrical cottonwool swabs of known weight, joined by nylon thread 60 cm long, were inserted into the vagina (5). After one hour the swabs were removed and weighed within five minutes. Smaller swabs were used in the young ewes because of their smaller vaginae. Cervical mucus production was estimated on a wet-weight basis. The dry matter content of the mucus was determined by drying the swabs in an oven at 80°C for 24-36 hours followed by 12 hours in a dessicator before weighing again. Differences between groups in mucus Statistical analysis. production were tested using Hotelling's Multivariate T-test. The curve of mucus production from time zero to 30 hours after detection of oestrus was calculated by regression on time. Linear, quadratic and cubic effects for each sheep were estimated (6). The results were compared by t-test and then pooled and tested for significance. Size of Cervical Secretory Surface The size of the secretory surface of the cervix was estimated by measuring the length of the cervix and by estimating the cervical epithelial area. The length of the cervical epithelium in several transverse sections was used as an indicator of epithelial area, this length being related to the degree of folding. The cervices were collected from young and mature ewes slaughtered 24-36 hours after onset of oestrus. Cervices from 16 young and 15 mature ewes were stored at 4°C in 70 per cent alcohol. Each cervix was opened longitudinally through the cervical canal, and the length of the internal surface from the first anterior annular fold to the end of the teat-shaped projection into the vagina was measured. In another group, cervices from 18 young and 17 mature ewes were transected cranially at the first annular fold and caudally at the cervical 05, then placed in Bouin's fixative until processing for the preparation of histological sections. Before processing, the length of the cervices was recorded. The tissue samples were then dehydrated and
538
NOVEMBER
1983 VOL. 20 NO. 5
THERIOGENOLOGY embedded in paraffin wax; transverse cross sections of 10 urnthickness were cut at 500 Ccrn intervals. Sections were stained with haematoxylineosin. Four cross sections per cervix were selected at intervals of about cm from each of eight young and eight mature ewes. Those cut at an oblique angle with respect to the cervical lumen were avoided. Sections were projected onto a table through a photographic enlarger, the outlines were drawn on paper and the total length of cervical epithelium was estimated by calculating the magnification used in projection. 0.75
RESULTS Mucus Production Oestrus was about eight hours longer in mature ewes (26.2 + 1.2) than in young ewes (18.6 * 1.4 ; P < 0.025). Mucus production is reported in Table 1. There was a significant difference in the total weight of mucus produced by mature ewes (5.2 g) compared with young ewes (4.1 g) during the 30 hours following the onset of oestrus. Hotelling's Multivariate T-test gave an F value of 4.67 (6 and 31 degrees of freedom) which was significant (P < 0.05). TABLE 1 THE AMOUNT AND DRY-MATTER CONTENT OF CERVICAL MUCUS PRODUCED BY YOUNG AND MATURE EWES AT DIFFERENT TIMES OF OESTRUS
Time (hr) after onset of oestrus
Young ewes (a)
An-m.lnt (g)
%DM
Mature ewesta)
Amount (9)
%DM
o-1
0.98 * 0.09
2.47
1.14 + 0.12
2.51
6-J
1.07 + 0.14
2.47
1.17 t 0.16
2.99
12 - 13
0.67 + 0.07
3.66
0.98 i 0.16
4.12
18 - 19
0.51 + 0.05
4.03
0.69 ? 0.07
4.92
24 - 25
0.49 ? 0.05
4.79
0.61 2
5.77
30 - 31
0.42 * 0.03
6.50
0.59 * 0.03
0.07
7.25
(a) Data from 20 ewes There was a significant decline in mucus production by ewes of both ages from onset to 30 hours of oestrus. Linear regression of mucus production on time differed significantly between ages. The quadratic and cubic regressions did not differ significantly between ages.
NOVEMBER
1983VOL.20N0.5
539
THERIOGENOLOGY There was a significant relationship between wet and dry weight of mucus in mature ewes (r = 0.461, but not in the young ewes (r = 0.25). Size of Secretory Surface of the Cervix In the first group of young and mature ewes the mean length of the cervix was 4.42 + 0.12 cm and 5.44 ? 0.16 cm, respectively (P < 0.01). The cervices fixed in Bouin's solution had mean lengths of 3.32 + 0.09 cm and 3.76 ?r 0.18 cm for young and mature ewes, respectively (P < 0.05) The mean lengthsofthe cervical epithelium in cross sections of young and mature eweswere95.8 ? 4.5 mm and81.1 f 7.5 mm per section, respectively, and were not significantly different. In analysis of variance in young ewes, the difference between replications for individual animals was not significant, but in mature ewes it was significant at the five Per cent level. DISCUSSION The production of mucus was maximal during the six hours after onset of oestrus and had declined considerably after 12 hours in young ewes and 18 hours in mature ewes. In mature ewes, Smith (1) found that production of mucus reached its peak at onset of oestrus while Smith and Allison (2) reported a curve of mucus production with a peak spanning 12 hours either side of the onset of oestrus. The amount of mucus produced by young ewes remained significantly lower than that of mature ewes during all sample periods. Circulating levels of oestrogen control mucus production (5) and the lower weight of mucus observed in young ewes supports the conclusion (7) that oestrogen concentrations are lower in young ewes. The pattern of mucus production was generally similar in young and mature ewes. Smaller swabs were used in young ewes because of the smaller size of the vaginae. To test the possibility that the smaller swab could not absorb all mucus produced, swabs of both sizes were placed in different containers each with 5.5 ml of mucus. The weight of the mucus used (5.2 g) was larger than that recorded at any collection period. All mucus was absorbed by swabs of both sizes, so it can be assumed that the smaller swabs were able to absorb available mucus at any normal collection period. Differences in mucus production between young and mature ewes cannot be explained by differences in secretory area per unit length since no differences between ages were found in the mean lengths of the cervical epithelium. Only eight animals per group were considered because the results indicated that no significant differences could be expected without using very large numbers of sheep. The smaller length of the entire cervix may be a factor in the lower production of mucus by young ewes. Dry matter contributed little to the total weight of mucus in both. In vitro and in vivo studies show that spermatozoa will have
540
NOVEMBER
1983 VOL. 20 NO. 5
THERIOGENOLOGY optimal conditions for penetration if mucus has a dry matter content of less than five per cent; but if dry matter content is more than seven per cent, penetration will be poor (8). In this study dry matter content approached five per cent 18 hours after onset of oestrus in mature ewes but was still less than five per cent in young ewes at 24 hours after onset of oestrus. Theoretically, less mucus production might lead to lower sperm survival, a smaller sperm reservoir and a decreased protection of sperm cells; however, it is unlikely that the differences in wet weight of mucus observed between young and mature ewes are sufficient to influence fertility. Smith (1) found that the weight of mucus produced in a group of "infertile" ewes (that failed to produce lambs for two consecutive years but had lambed before) was 1.5 times that of a group of "fertile" ewes. The consistency of mucus present at insemination may exert more influence on fertilization than the quantity of mucus produced. The lower weight of mucus produced by young ewes, accompanied by an earlier decline in mucus production after onset of oestrus, suggests that transport of spermatozoa into and through the cervix in some ewes may be adversely affected in late oestrus. REFERENCES 1.
Smith, J.F. Studies on ovine fertility in agricultural regions of Western Australia: Cervical mucus production by fertile and infertile ewes. Aust. J. Agric. Res. 22: 513-519 (1971).
2.
Smith, J.F. and Allison, A.J. The effect of exogenous progestagen on the production of cervical mucus in the ewe. J. Reprod. Fertil. -24: 279-282 (1971).
3.
Lightfoot, R.J., Smith, F.J., Gumming, I-A., Marshall, T., Wroth, R.H. and Hearnshaw, H. Infertility in ewes caused by prolonged grazing on oestrogenic pastures: Oestrus, fertilization and cervical mucus. Aust. J. Biol. Sci. -27: 409-414.
4.
Kennedy, T.G. and Kennedy, J.P. Transport of spermatozoa and apparent fertilization rate in young and mature Merino ewes. Proc. Aust. Sot. Anim. Prod. 2: 176-180 (1972).
5.
Lindsay, D.R. and Francis, C-M. Cervical mucus measurement in ovariectomized ewes as a bioassay of synthetic and phytooestrogens. Aust. J. Agric. Res. -19: 1069-1076 (1968).
6.
Snedecor, G.W. and Cochran, W.G. Statistical Methods. 6th Ed. Iowa State University Press, Ames, Iowa. (1967).
I.
Edgar, D.G. Studies on infertility in ewes. Fertil. 3: SO-54 (1962).
8.
Marcus, C.C. and Marcus, S.L. The cervical factor. In Progress in Infertility. Eds. Behrman, S.J. and Kistner, R.W.,Little Brown and Co., Boston. 1968, pp. 21-62.
NOVEMBER
1983 VOL. 20 NO. 5
J. Reprod.
541
CERVICAL MUCUS PRODUCTION 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: Accepted:
May 10, 1983 September 15,
1983
ABSTRACT The production of cervical mucus was determined intervals during oestrus in young (1% years old) and years old) Merino ewes. The length of the cervix and the cervical epithelium was also measured in similar
at six-hour mature (3 to 7 the length of ewes.
Total weight of mucus secreted as well as secretion rate per unit of time was greater in mature ewes than young ewes. Production was maximal during the first six hours of oestrus and declined considerably after i2 hours in young and 18 hours in mature ewes. The cervix was significantly shorter in young ewes while the length of the cervical epithelium was not significantly different in young and mature. It was concluded that lower mucus production in young ewes is not related to lower mucus output per unit area. INTRODUCTION Changes in the quantity or quality of cervical mucus may disturb sperm transport with subsequent fertilization failure (1). Impaired fertility associated with altered mucus production was reported in proqestaqen-synchronized ewes (2) and in ewes following prolonged grazing on oestroqenic pasture (3). It has been proposed that young and mature ewes may differ in the production and/or properties of mucus and that this may partly explain observed differences in their fertility (4). This work tested this hypothesis by (al measuring the cervical mucus production of young and mature Merino ewes and (b) estimating the size of the secretory surface of the cervix. MATERIALS AND METHODS The experiments were conducted at The University of New South
* Present address: EMBRAPA, Caixa Postal 242, Baq&RS.
NOVEMBER
1983 VOL. 20 NO. 5
BRAZIL 96.400
537
THERIOGENOLOGY Wales Field Station at Wellington, New South Wales. Determination of the Amount of Mucus Produced Mucus was collected from 20 nulliparous 1.5-year-old Merino ewes (young ewes) and 20 parous 3- to 7-year-old Merino ewes (mature ewes) during the first two weeks of May. Signs of oestrus were observed hourly from 0630 to 1930 h each day and mucus collection commenced immediately after detection of oestrus. Every six hours during the first 18 hours following the onset of oestrus, and then every three hours, each ewe was tested with an aproned teaser ram for evidence of oestrus. Mucus was collected at six-hour intervals. Two cylindrical cottonwool swabs of known weight, joined by nylon thread 60 cm long, were inserted into the vagina (5). After one hour the swabs were removed and weighed within five minutes. Smaller swabs were used in the young ewes because of their smaller vaginae. Cervical mucus production was estimated on a wet-weight basis. The dry matter content of the mucus was determined by drying the swabs in an oven at 80°C for 24-36 hours followed by 12 hours in a dessicator before weighing again. Differences between groups in mucus Statistical analysis. production were tested using Hotelling's Multivariate T-test. The curve of mucus production from time zero to 30 hours after detection of oestrus was calculated by regression on time. Linear, quadratic and cubic effects for each sheep were estimated (6). The results were compared by t-test and then pooled and tested for significance. Size of Cervical Secretory Surface The size of the secretory surface of the cervix was estimated by measuring the length of the cervix and by estimating the cervical epithelial area. The length of the cervical epithelium in several transverse sections was used as an indicator of epithelial area, this length being related to the degree of folding. The cervices were collected from young and mature ewes slaughtered 24-36 hours after onset of oestrus. Cervices from 16 young and 15 mature ewes were stored at 4°C in 70 per cent alcohol. Each cervix was opened longitudinally through the cervical canal, and the length of the internal surface from the first anterior annular fold to the end of the teat-shaped projection into the vagina was measured. In another group, cervices from 18 young and 17 mature ewes were transected cranially at the first annular fold and caudally at the cervical 05, then placed in Bouin's fixative until processing for the preparation of histological sections. Before processing, the length of the cervices was recorded. The tissue samples were then dehydrated and
538
NOVEMBER
1983 VOL. 20 NO. 5
THERIOGENOLOGY embedded in paraffin wax; transverse cross sections of 10 urnthickness were cut at 500 Ccrn intervals. Sections were stained with haematoxylineosin. Four cross sections per cervix were selected at intervals of about cm from each of eight young and eight mature ewes. Those cut at an oblique angle with respect to the cervical lumen were avoided. Sections were projected onto a table through a photographic enlarger, the outlines were drawn on paper and the total length of cervical epithelium was estimated by calculating the magnification used in projection. 0.75
RESULTS Mucus Production Oestrus was about eight hours longer in mature ewes (26.2 + 1.2) than in young ewes (18.6 * 1.4 ; P < 0.025). Mucus production is reported in Table 1. There was a significant difference in the total weight of mucus produced by mature ewes (5.2 g) compared with young ewes (4.1 g) during the 30 hours following the onset of oestrus. Hotelling's Multivariate T-test gave an F value of 4.67 (6 and 31 degrees of freedom) which was significant (P < 0.05). TABLE 1 THE AMOUNT AND DRY-MATTER CONTENT OF CERVICAL MUCUS PRODUCED BY YOUNG AND MATURE EWES AT DIFFERENT TIMES OF OESTRUS
Time (hr) after onset of oestrus
Young ewes (a)
An-m.lnt (g)
%DM
Mature ewesta)
Amount (9)
%DM
o-1
0.98 * 0.09
2.47
1.14 + 0.12
2.51
6-J
1.07 + 0.14
2.47
1.17 t 0.16
2.99
12 - 13
0.67 + 0.07
3.66
0.98 i 0.16
4.12
18 - 19
0.51 + 0.05
4.03
0.69 ? 0.07
4.92
24 - 25
0.49 ? 0.05
4.79
0.61 2
5.77
30 - 31
0.42 * 0.03
6.50
0.59 * 0.03
0.07
7.25
(a) Data from 20 ewes There was a significant decline in mucus production by ewes of both ages from onset to 30 hours of oestrus. Linear regression of mucus production on time differed significantly between ages. The quadratic and cubic regressions did not differ significantly between ages.
NOVEMBER
1983VOL.20N0.5
539
THERIOGENOLOGY There was a significant relationship between wet and dry weight of mucus in mature ewes (r = 0.461, but not in the young ewes (r = 0.25). Size of Secretory Surface of the Cervix In the first group of young and mature ewes the mean length of the cervix was 4.42 + 0.12 cm and 5.44 ? 0.16 cm, respectively (P < 0.01). The cervices fixed in Bouin's solution had mean lengths of 3.32 + 0.09 cm and 3.76 ?r 0.18 cm for young and mature ewes, respectively (P < 0.05) The mean lengthsofthe cervical epithelium in cross sections of young and mature eweswere95.8 ? 4.5 mm and81.1 f 7.5 mm per section, respectively, and were not significantly different. In analysis of variance in young ewes, the difference between replications for individual animals was not significant, but in mature ewes it was significant at the five Per cent level. DISCUSSION The production of mucus was maximal during the six hours after onset of oestrus and had declined considerably after 12 hours in young ewes and 18 hours in mature ewes. In mature ewes, Smith (1) found that production of mucus reached its peak at onset of oestrus while Smith and Allison (2) reported a curve of mucus production with a peak spanning 12 hours either side of the onset of oestrus. The amount of mucus produced by young ewes remained significantly lower than that of mature ewes during all sample periods. Circulating levels of oestrogen control mucus production (5) and the lower weight of mucus observed in young ewes supports the conclusion (7) that oestrogen concentrations are lower in young ewes. The pattern of mucus production was generally similar in young and mature ewes. Smaller swabs were used in young ewes because of the smaller size of the vaginae. To test the possibility that the smaller swab could not absorb all mucus produced, swabs of both sizes were placed in different containers each with 5.5 ml of mucus. The weight of the mucus used (5.2 g) was larger than that recorded at any collection period. All mucus was absorbed by swabs of both sizes, so it can be assumed that the smaller swabs were able to absorb available mucus at any normal collection period. Differences in mucus production between young and mature ewes cannot be explained by differences in secretory area per unit length since no differences between ages were found in the mean lengths of the cervical epithelium. Only eight animals per group were considered because the results indicated that no significant differences could be expected without using very large numbers of sheep. The smaller length of the entire cervix may be a factor in the lower production of mucus by young ewes. Dry matter contributed little to the total weight of mucus in both. In vitro and in vivo studies show that spermatozoa will have
540
NOVEMBER
1983 VOL. 20 NO. 5
THERIOGENOLOGY optimal conditions for penetration if mucus has a dry matter content of less than five per cent; but if dry matter content is more than seven per cent, penetration will be poor (8). In this study dry matter content approached five per cent 18 hours after onset of oestrus in mature ewes but was still less than five per cent in young ewes at 24 hours after onset of oestrus. Theoretically, less mucus production might lead to lower sperm survival, a smaller sperm reservoir and a decreased protection of sperm cells; however, it is unlikely that the differences in wet weight of mucus observed between young and mature ewes are sufficient to influence fertility. Smith (1) found that the weight of mucus produced in a group of "infertile" ewes (that failed to produce lambs for two consecutive years but had lambed before) was 1.5 times that of a group of "fertile" ewes. The consistency of mucus present at insemination may exert more influence on fertilization than the quantity of mucus produced. The lower weight of mucus produced by young ewes, accompanied by an earlier decline in mucus production after onset of oestrus, suggests that transport of spermatozoa into and through the cervix in some ewes may be adversely affected in late oestrus. REFERENCES 1.
Smith, J.F. Studies on ovine fertility in agricultural regions of Western Australia: Cervical mucus production by fertile and infertile ewes. Aust. J. Agric. Res. 22: 513-519 (1971).
2.
Smith, J.F. and Allison, A.J. The effect of exogenous progestagen on the production of cervical mucus in the ewe. J. Reprod. Fertil. -24: 279-282 (1971).
3.
Lightfoot, R.J., Smith, F.J., Gumming, I-A., Marshall, T., Wroth, R.H. and Hearnshaw, H. Infertility in ewes caused by prolonged grazing on oestrogenic pastures: Oestrus, fertilization and cervical mucus. Aust. J. Biol. Sci. -27: 409-414.
4.
Kennedy, T.G. and Kennedy, J.P. Transport of spermatozoa and apparent fertilization rate in young and mature Merino ewes. Proc. Aust. Sot. Anim. Prod. 2: 176-180 (1972).
5.
Lindsay, D.R. and Francis, C-M. Cervical mucus measurement in ovariectomized ewes as a bioassay of synthetic and phytooestrogens. Aust. J. Agric. Res. -19: 1069-1076 (1968).
6.
Snedecor, G.W. and Cochran, W.G. Statistical Methods. 6th Ed. Iowa State University Press, Ames, Iowa. (1967).
I.
Edgar, D.G. Studies on infertility in ewes. Fertil. 3: SO-54 (1962).
8.
Marcus, C.C. and Marcus, S.L. The cervical factor. In Progress in Infertility. Eds. Behrman, S.J. and Kistner, R.W.,Little Brown and Co., Boston. 1968, pp. 21-62.
NOVEMBER
1983 VOL. 20 NO. 5
J. Reprod.
541