The effect of oestrogen administered during the progestational phase of the cycle on transport of spermatozoa in ewes.

Two split-plot factorial experiments are described, the first with 72 entire cyclic ewes and the second with 80. The pattern of transport of spermatozoa through the reproductive tract was studied, following treatments with progestagen and oestrogen or with oestrogen alone during 2 weeks preceding insemination. A daily dose of 25 mug oestradiol-17 beta administered to ewes for 14 days preceeding oestrus had a deleterious effect on the passage of spermatozoa through the cervix into the uterus within the first 2 hr after insemination. The numbers of spermatozoa recoverable from the cranial region of the cervix 2 hr after insemination appeared to be related to the numbers in the oviducts at 24 hr. These numbers were related to fertility data from an earlier experiment using similar treatments. The data for log numbers of spermatozoa recoverable from the cervix formed a near-normal distribution and so were suitable for formal statistical analysis. There was an interaction between progestagen and oestrogen influence before mating on the pattern of sperm transport through the cervix.     

Persistent infertility in ewes after prolonged exposure to oestradiol-17 beta

Merino ewes were treated with implants which released 300 micrograms oestradiol-17 beta per day or 5 mg progesterone per day, or both, for 9 months (Months 1-9), and after an 11-month intermission were treated again for 6 months (Months 20-26). Ewes were run with rams at Months 16, 28 and 40. Fertility was not affected by the first exposure period, but the second exposure to oestradiol reduced the fertility of ewes at both subsequent mating periods. Affected ewes returned to service more frequently (P less than 0.01) and were less likely to conceive (P less than 0.05). After mating, a normal population of spermatozoa was established in the caudal cervix, but transport through the cervix was impaired in affected ewes and there were fewer spermatozoa (P less than 0.01) in the cranial cervix. In affected ewes, the spinnbarkeit of cervical mucus was reduced (P less than 0.05), and the histological appearance of the cervix changed, looking like that of the uterus. Treatment with progesterone did not affect fertility, cervical mucus or sperm transport, but diminished the histological abnormalities produced by oestradiol (P less than 0.05). These results show that oestradiol-17 beta given after puberty can cause the same kind of permanent sexual transdifferentiation that is produced by the oestrogenic isoflavones in ewes with clover disease. The results suggest that this change may require more than a single exposure to oestrogen.     

The passage of spermatozoa through the cervix of ovariectomized ewes treated with progesterone and oestrogen.

Thirty spayed ewes were used in a 2 x 3 split-plot factorial experiment (n = 5) in which oestrus was induced with 30 or 90 mug oestradiol benzoate (OB) following a period of progesterone priming. They were inseminated 36 hr after oestrogen and the numbers of spermatozoa in the caudal, mid- and cranial regions of the cervix (sub-plots) were estimated 1, 12 and 24 hr later. At each interval of time and for each region of the cervix, fewer spermatozoa were recovered from the ewes treated with 30 mug OB than from those treated with 90 mug (P less than 0-05).     

Fertility studies in young and mature merino ewes 2. Sperm transport

Samples of cervical mucus were collected from young and mature ewes at different stages of oestrus. 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.     

Plasma progesterone concentrations, ovarian and endocrinological response and sperm transport in ewes with synchronized oestrus

Two experiments involving 24 and 54 Australian Merino ewes were conducted in which the establishment of a cervical population of spermatozoa and several endocrinological events were studied after several regimens for the synchronization of oestrus. Intravaginal sponges impregnated with 500 mg (Exp. 1) or 200, 400 or 600 mg (Exp. 2) progesterone resulted in the maintenance of plasma progesterone concentrations of 1.5-4.9 ng/ml over a 12-day insertion period compared with 1.9-6.9 ng/ml during dioestrus in control ewes. In Exp. 1 basal concentrations of less than or equal to 0.25 ng/ml plasma were attained by 4 h after sponge withdrawal and this decline was much more rapid than in normal luteolysis. This was associated with fewer spermatozoa recovered from the cervix 2 h after insemination, and PMSG had no significant effect. In Exp. 2 injection of a supplementary dose of progesterone at sponge withdrawal resulted in a rapid increase in plasma progesterone concentrations followed by an equally rapid decrease and an attenuation of the rise in plasma oestradiol-17 beta, the LH surge, and the onset of oestrus. The numbers of spermatozoa recovered 4 h after insemination were not increased, and PMSG had no significant effect. Two factors were significant, namely the dose of progesterone in the sponge (600 mg greater than 400 or 200 mg, P less than 0.05) and stage of oestrus when inseminated (mid- or late oestrus greater than early). The data demonstrated that an adequate dose of progesterone/progestagen incorporated into intravaginal sponges and accurate timing of insemination relative to the LH surge are the most important factors involved in penetration of the cervix by spermatozoa.     

Further development of a transcervical technique for artificial insemination in sheep using previously frozen semen

A transcervical technique (the Guelph System for transcervical AI) was used to inseminate 2060 ewes on 65 farms (average 31 ewes, range 5 to 107) in Ontario, Canada, from October 1990 to September 1992, using previously frozen semen. Estrus was synchronized using progestagen pessaries and PMSG with median inseminations done at 54 h from pessary removal. Maiden ewes were not included. Only ewes in which the cervix could be penetrated were inseminated with 150 million spermatozoa per insemination. A total of 1809 were penetrated and inseminated (penetration rate 87.8%). Success of penetration increased from 76.3% in the first 500 ewes to 97.9% in the last 500 (P=0.01). Cervical penetration was more successful in ewes in the accelerated lambing program (92.3%, average 3.1 mo since the previous lambing) than those in the annual lambing program (82.4%, average 7.0 mo since the previous lambing; P=0.06). The lambing rate for ewes bred during the combined traditional breeding seasons (Fall of 1990, 1991, 1992) was 50.7% compared to 24.4% for ewes bred at other periods (P=0.00001). The average time required for handling and insemination decreased from 8.62 min in the first 500 ewes to 3.62 min in the last 500 ewes. The Guelph System for Transcervical AI was found to be successful for cervical penetration in most ewes. Penetration success was affected by period since the last lambing and by inseminator experience. The lambing rate was higher for ewes bred during the traditional Fall breeding seasons than during other times of the year.     

Single layer colloid centrifugation technique improves motility,viability and chromatin integrity of ram spermatozoa after thawing

The cell membrane of ram spermatozoa is more sensitive to the freezing process than in other species due to its composition. As a result, the quality and viability of frozen thawed ram spermatozoa are often poor, which together with the specific structure of the ewe's cervix are the main reasons for lower fertility in ewes after intracervical insemination. In the present study we investigated the effects of semen centrifugation through a single layer of a species-specific colloid (Androcoll-O) on post-thaw quality of ram spermatozoa. Motility, viability and morphology were analysed 0, 6, 12 and 24 h after thawing. DNA fragmentation index (%DFI) of the samples was assessed 0 h after thawing, by SCSA™. Membrane and acrosome integrity of spermatozoa were analysed by Sybr-14/PI/PNA test 0 h after thawing.The proportion of motile spermatozoa was significantly higher in SLC – selected samples in comparison to control (not SLC – selected) samples at 0, 6, 12 (P < 0.001) and 24 h (P < 0.05). The proportion of viable spermatozoa was also significantly higher in SLC - selected samples in comparison to control samples at all times (P < 0.001). The proportion of abnormal acrosomes and morphologically abnormal spermatozoa (MAS) were significantly lower in SLC – selected samples compared to control samples at all times (P < 0.001). Analysis of chromatin stability revealed significantly lower %DFI values in SLC – selected samples compared to control samples (P < 0.001). The SYBR-14/PI/PNA test also revealed significantly better values in SLC – selected compared to control samples (P < 0.05). In conclusion, single layer colloid centrifugation significantly improved post-thaw quality and longevity of ram spermatozoa, making it suitable for artificial insemination initiatives.     

Uterine presensitization and embryo survival and growth in Booroola Merino x South Australian Merino ewes

The fertility enhancing effects of semen were examined following the intra-uterine insemination of killed spermatozoa plus seminal plasma 17 d prior to insemination with viable spermatozoa. Three experiments were conducted: two on 1.5-yr old and 2.5 to 5.5 yr-old Booroola Merino x South Australian Merino ewes in 1986 and one on 1.5 yr-old ewes in 1987. Differences between treatment and control groups for the percentage of ewes exhibiting estrus by Days 21 and 35 following fertile insemination, the percentage of ewes with viable embryos at Day 35, the number and weight of viable embryos per ewe, the nubmer of caruncular implantation sites and the progesterone level were not statistically significant (P>0.05). There were no statistically significant treatment by experiment interactions for any of the variables examined. Inflammation and edema of the endometrial tissue was not observed following the presensitization treatment.     

The number of spermatozoa required by naturally mated ewes and the ability of rams to meet these requirements

The fertility of naturally mated ewes was compared with the number of spermatozoa deposited, and the number of times they were mated. The number of spermatozoa received was estimated from ejaculates flushed directly from the vagina of naturally mated ewes.In one experiment, maximum fertility was achieved with as few as 140 × 10⁶ spermatozoa. The percentage of pregnant ewes was similar in ewes mated once or more than once (68.4% vs. 72.5%). Similar results were obtained in one test of a further experiment but in a second test fertility was higher in ewes that were mated more than once. If this effect was due to the extra spermatozoa received, then ewes required 500 × 10⁶ spermatozoa to achieve maximum fertility. Half of the ewes were mated at their first oestrus after treatment with progestagens in the second experiment. The fertility of these ewes was similar to that of the remaining ewes, which were mated at natural oestrus.The mean number of spermatozoa ejaculated by individual rams varied from 140 × 10⁶ to 1050 × 10⁶, following depletion of the epididymal reserves of spermatozoa. The rams ejaculated an average of 9.1 × 10⁶ spermatozoa per gram of testis per day in the first experiment. The rams mated an average of 10.9 times per day in the first experiment, 6.9 and 6.1 times per day, respectively, for the first and second tests in the second experiment. The number of times that rams mated was highly correlated with the number of ewes with which they mated in the second experiment.     

Cervical dilation, conception rate, and concentrations of progesterone and estradiol-17B in postpartum ewes treated with porcine relaxin

Experiments were conducted to determine the effects of porcine relaxin (pRLX) on cervical dilation and conception rates in postpartum ewes. In Experiment 1, ewes received medroxyprogesterone acetate (MAP) sponge on day 16 (day 0 = lambing) and 750 IU pregnant mare serum gonadotropin (PMSG) at sponge removal on day 30. Control ewes received saline and relaxin-treated (RLX) ewes received 0.5 mg pRLX (>/= 3000 U/mg) i.m. at 24 h and 1.0 mg pRLX at 36 h after PMSG. All ewes were inseminated (Al) at 55 h after PMSG with 0.4 ml fresh semen. The proportion of RLX treated ewes (6 6 ) in which the cervix was penetrated was greater (P < 0.05) than in Control ewes (0 5 ). However, ova recovery rate was lower (P < 0.05) for RLX ewes (1 6 ) than for control ewes (5 5 ). In Experiment 2, ewes between Days 90 to 120 post partum received MAP sponges for a period of 8 d and 750 IU PMSG at sponge removal. Control ewes (n = 9) received saline; RLX-1 ewes (n = 8) received 0.5 mg pRLX at 24 h and an additional 0.5 mg pRLX at 36 h after PMSG; and RLX-1.5 ewes (n = 9) received 0.5 mg pRLX at 24 h and an additional 1.0 mg pRLX at 36 h after PMSG. Ewes were mated to rams at estrus, and cervical dilation was checked at 55 h after PMSG. The cervix could not be penetrated in any of the ewes. Conception rates on Day 26 were 66, 56 and 63% for control, RLX-1 and RLX-1.5 groups, respectively. These results demonstrate that the effect of relaxin on cervical dilation and conception rate is dependent upon the postpartum stage of the ewes.     

Lambing rates and litter size following carazolol administration prior to insemination in Kivircik ewes

The effect of carazolol on the ease of penetrating the cervix during artificial insemination, lambing rate and litter size was studied using 1.5–4.0-year old Kivircik ewes in an incomplete 3 × 2 × 2 experimental design. All of the ewes in this study were synchronized for oestrus by insertion of a progesterone impregnated vaginal sponge for 12 days and administration of 400 IU PMSG at sponge withdrawal. Three methods of service were compared: natural service, artificial insemination (AI) with fresh semen, or AI with frozen semen. Two times of insemination (fixed time AI versus AI at observed oestrus) were compared on the fresh and frozen AI treatments. The absence (control) or use of carazolol (carazolol; 0.5 mg/ewe i.m. 30 min before mating) was the third factor in the design and penetration of the cervix by the insemination pipette was assessed as shallow (<10 mm), middle (10–20 mm) or deep (>20 mm). Natural service ewes were only mated at observed oestrus. Consequently, the factorial design was incomplete and there were a total of 10 treatments each represented by 30 ewes. Natural service resulted in a significantly (P < 0.05) higher lambing rate and litter size (86%; 2.0 ± 0.05 lambs/ewe) than AI using fresh (65%; 1.6 ± 0.1 lambs/ewe) or frozen (40%; 1.4 ± 0.14 lambs/ewe) semen. For AI animals the lambing rate and litter size were not significantly different when service was at a fixed time (50%; 1.5 ± 0.12 lambs/ewe) or at observed oestrus (56%; 1.5 ± 0.12 lambs/ewe). Carazolol did not permit complete cervical penetration in any ewe. Deep penetration of the cervix at AI was achieved in 33% of untreated (control) and 48% of carazolol treated ewes (P < 0.05). However, the proportion of ewes in which penetration of the cervix and semen deposition was greater than shallow was similar for control (82%) and carazolol (85%), and lambing rate and litter size were similar for both treatments. Over the three service methods, the lambing rate was 56% for control and 63% for carazolol (NS) and litter size was similar for both treatments. It was concluded that the carazolol treatment used prior to natural mating or AI in this experiment did not improve lambing rate or litter size in Kivircik ewes.     

Time required for spermatozoa to remain in the vagina of the ewe to ensure conception

Oestrous ewes (N = 202) were inseminated with 0.1 ml of semen containing 500 X 10(6) motile spermatozoa and the spermatozoa were flushed from their vagina either immediately or 0.25, 0.5, 1, 2, 4 and 8 h after insemination. Pregnancy was determined by returns to service and laparoscopy. Some ewes became pregnant (10.71%) after spermatozoa had been flushed from the vagina only seconds after insemination and about 40% of ewes became pregnant after spermatozoa had been in the vagina for 15 min. Maximum conception (55%) was achieved when spermatozoa had been in the vagina for at least 2 h. It was concluded that the losses of spermatozoa that occur from the vagina will not influence the chance of a ewe conceiving because sufficient spermatozoa to ensure a normal conception move up the reproductive tract before large losses from the vagina take effect.     

Fertility studies in young and mature merino ewes 1. Cervical mucus production

The production of cervical mucus was determined at six-hour intervals during oestrus in young (1 (1 2 ) years old) and mature (3 to 7 years old) Merino ewes. The length of the cervix and the length of the cervical epithelium was also measured in similar 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 12 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.     

Causes for decreased fertility in out-of-season mated ewes

The interval from onset of estrus to preovulatory luteinizing hormone (LH) release, conception and fertilization rates, and number of accessory spermatozoa per ovum at 48 hr postmating in untreated cyclic ewes and in progestogen-pregnant mare serum gonadotropin (PMSG) treated, anestrous ewes were compared in efforts to identify sources of lowered fertility for matings induced in anestrous ewes with exogenous hormones. Blood samples for LH determination were collected at 0, 2, 4, 6, 8, 10, 12, and 24 hr after the onset of estrus. Conception and fertilization failure rates were determined at 48 hr, 12 days, or parturition. The progestogen-PMSG treated ewes had a shorter interval from onset of estrus to preovulatory LH release, lower conception rates, and fewer accessory spermatozoa than cyclic ewes had. Conception failure, rather than embryonic mortality, was the major cause of reduced fertility for the out-of-season mated ewes and apparently resulted from insufficient viable spermatozoa in the oviducts to fertilize the ova.     

Enzyme activities and protein and carbohydrate concentrations in cervical secretions at dioestrus in normal ewes and ewes with permanent phytooestrogenic infertility

Cervical secretions of clover-affected and control ewes in the luteal phase of the ovarian cycle were obtained by flushing the anterior vagina. The flushings were analysed for proteins, carbohydrates and enzyme activities, and were found to be similar to the secretions of the normal ovine uterus. There was significantly more protein, carbohydrate and acid-soluble glycoprotein but less alkaline phosphatase, N-acetylglycosidases (EC 3.2.1.30 and 3.2.1.53) and ribonuclease I in the vaginal flushings of clover-affected ewes. The observed changes were not due to more inflammation in the cervix of clover-affected ewes as there were fewer bacteria, leukocytes and epithelial cells and no elevation of lysozomal enzyme activities in their flushings. It is suggested that the cervix of the clover-affected ewe behaves as though under a stronger than normal oestrogenic stimulation during dioestrus.     

Differential transport of spermatozoa into the two sides of the genital tract of a monovular marsupial, the tammar wallaby (Macropus eugenii)

Ovulation in the tammar wallaby alternates between the ovaries. The genital duct of each side enters the median vaginal culs-de-sac separately. Post-partum oestrus occurred 0.4 days after birth and ovulation 1 day later. After a single copulation spermatozoa were found in both cervical canals at 0.5 h and extended to the oviduct on the non-parturient side only by 8 h. Very few spermatozoa were found in sections of the post-partum uterus or its associated oviduct at any time. Spermatozoa were recovered by flushing from both sides but the numbers were 2-20 times greater in the non-parturient than in the post-partum side: the greatest difference occurred in the cervical canals 2-5 h after copulation. In females which had undergone a previous infertile cycle, spermatozoa were abundant in both cervices and both uteri. It is concluded that the differential distribution of spermatozoa in post-partum animals was (1) due to failure of transport in the recently pregnant side of the tract, rather than attraction of spermatozoa to the ovulation side, and (2) established at the cervix which, on the ovulation side, provides a reservoir of spermatozoa for 24 h after copulation.     

A preliminary study on the usefulness of huIL-8 in cervical relaxation of the ewe for artificial insemination and for embryo transfer

The efficacy of using human interleukin 8 (huIL-8) as an agent for inducing cervical relaxation in estrous and diestrous sheep was assessed in a small pilot study. Multiparous, estrus-synchronized ewes were treated for either 2 or 5 consecutive days with vaginal suppositories with or without 5 micrograms cytokine. Cervical penetration with an insemination instrument was then assessed in vivo. After euthanasia, physical, histological and enzymological properties of the cervix were examined. Treatment of diestrous sheep with huIL-8 did not result in recruitment of neutrophils into the cervix. Treatment of estrous sheep with huIL-8 usually led to neutrophil recruitment to the cervix and to either full or partial penetration of the cervix. However, some animals receiving placebo treatment had neutrophil infiltration of both the vagina and cervix and, in one of these, partial penetration of the cervix was also achieved. Thus, treatment with IL-8 as the sole agent in the vaginal suppository was not sufficient to relax the cervix of the nonpregnant ewe in this study.     

Investigations on the detrimental effect of prostaglandin F2α-regulated estrus on the number and condition of sperm in the reproductive tract of the ewe

Ewes in the luteal phase of the estrous cycle were treated with prostaglandin F₂α (PGF), mated to rams at the ensuing estrus 2 days later, and necropsied at 2 or 23 hr after mating. At 2 hr after mating, ewes in PGF-regulated estrus had significantly fewer sperm in the middle and anterior one-thirds of the cervix and in the uterus than did ewes mated during natural estrus. At 23 hr, soon after ovulation, significantly fewer ewes in PGF-regulated estrus had sperm in the oviducts than did ewes in natural estrus.In Experiment 2, ewes in PGF-regulated or natural estrus were laparotomized, inseminated by deposition of semen in the uterine lumen, and necropsied 2 or 23 hr later. Intrauterine insemination prevented most of the reduction in sperm numbers in the reproductive tract at PGF-regulated estrus.In Experiment 3, ewes in PGF-regulated or natural estrus were either mated to rams or inseminated in the uterine lumen and necropsied 2 hr later. Sperm were recovered from three segments of the cervix and were counted and evaluated for motility, response to live-dead staining, and acrosomal morphology. Intrauterine insemination again reduced the detrimental effect of PGF-regulated estrus on sperm numbers. However, the percentages of sperm recovered from the cervix that were motile, live, and had normal acrosomes were much lower in ewes in PGF-regulated estrus than in ewes in natural estrus. Compared with natural mating, intrauterine insemination reduced but did not eliminate the detrimental effects of PGF-regulated estrus on the viability and morphology of sperm. Regulating estrus with PGF resulted in damage to sperm in the cervix regardless of whether sperm reached the cervix from the vagina or from the uterus.     

Changed control of cervical secretion from infertile ewes previously exposed to oestrogenic clover pasture

The amount of cervical mucus recovered from control ovariectomized ewes increased with increasing doses of oestradiol benzoate (OB), while the maximum Spinnbarkeit of mucus occurred at an intermediate dose of OB. Neither the amount nor the Spinnbarkeit of mucus varied with the dose of OB in ewes with permanent infertility caused by grazing oestrogenic pasture (clover-affected ewes). Furthermore, the increase in Spinnbarkeit of cervical mucus seen in normal ewes treated over a 3-day period with OB or with implants containing oestradiol did not occur in affected ewes. In control ewes treated repeatedly with OB, production of mucus declined within 5 days, but no change in secretion was detectable in clover-affected ewes. Therefore, neither the amount nor the duration of oestrogenic stimulation affected the cervical mucus in ewes with clover disease. Affected ewes produced more mucus than did controls in the absence of oestrogenic stimulation. It is concluded that the relatively normal volume of mucus in affected ewes treated with OB results largely from autonomous production. The Spinnbarkeit does not increase in these ewes because the ability of the cervix to respond to oestrogen is impaired.     

The anatomy of the sheep cervix and its influence on the transcervical passage of an inseminating pipette into the uterine lumen

The anatomy of the sheep cervix is highly variable between animals and may explain the differing success of transcervical AI between individuals. This study aims to quantify the variation in cervical morphology between ewes and investigate the relationship between cervical anatomy and cervical penetration. Two series of reproductive tracts were collected. Series A: 132 adult anoestrous ewes, and series B: 165 cycling adult ewes and ewe lambs which were identified as luteal or non-luteal based on the presence of a corpus luteum. The morphology of the cervical external os was classified as slit, papilla, duckbill, flap or rose. An inseminating pipette was inserted into the lumen and the depth of penetration recorded. The cervix was opened longitudinally, its length recorded, the number of cervical rings counted and the arrangement of those rings graded. The maximum depth of cervical penetration was affected by cervical grade (series A: P=0.021; series B: P=0.037) and the stage of the oestrous cycle (P=0.008). Grade 1 cervices were more penetrable than grade 2, with grade 3 the least penetrable and non-luteal cervices could be penetrated further than luteal cervices. The distribution of os types differed with age, with rose types more common in adult ewes, and papilla os types more common in ewe lambs. These results indicate that the depth of cervical penetration is affected by the anatomy of the cervical lumen. Cervices with a less convoluted lumen (grade 1) were more penetrable. Non-luteal cervices are likely to have higher oestradiol concentrations than luteal, stimulating cervical relaxation and enabling deeper penetration. The difference in os types with age may be contributable to a morphological alteration at parturition.