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).     

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.     

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.     

The effects of the prostaglandin E analogue Misoprostol and follicle-stimulating hormone on cervical penetrability in ewes during the peri-ovulatory period

Two experiments in parous Welsh Mountain ewes determined the pattern of natural cervical relaxation over the peri-ovulatory period and investigated FSH and Misoprostol as cervical relaxants to facilitate transcervical passage of an insemination pipette into the uterine cavity. Following synchronisation of oestrus using progestagen sponges and PMSG (500 IU) the depth of cervical penetration was determined using a modified cattle insemination pipette as a measuring device. Penetration of the cervix was least at the time of sponge removal and increased to a maximum at 72 h after sponge removal and then declined. Intra-cervical administrations of either ovine FSH (Ovagen; 2mg) or Misoprostol (1mg; a Prostaglandin E(1) analogue) facilitated cervical penetration. Ovagen given 24h after sponge removal allowed transcervical intrauterine penetration in 100% of ewes at 54 and 60 h after sponge removal while Misoprostol given 48 h after sponge removal allowed trans-cervical penetration in 100% of ewes at 54 h. A combination of Ovagen and Misoprostol was as effective but not more so than Ovagen or Misoprostol alone. These results show that there is natural relaxation of the cervix at oestrus and that maximum relaxation occurs 72 h after sponge removal, which is too late for the correct timing of insemination. The intra-cervical administration of FSH or Misoprostol enhanced relaxation of the cervix and both were able to relax the cervix to allow intrauterine penetration 54 h after sponge removal, the optimum time for insemination. The results also show that FSH is biologically active after intracervical, topical application.     

The evaluation of a laparoscopic insemination technique in ewes

Following synchronisation of oestrus using FGA and PMSG, ewes were inseminated by either the conventional cervical (CC) method or directly into the uterus by laparoscopy (LI). The CC method was carried out either at 48 and 60 hours following progestagen withdrawal with 480 x 10(6) spermatozoa per inseminate or once only at 56 hours with 600 x 10(6) spermatozoa. The laparoscopic method was performed at 52 hr using 48 x 10(6) spermatozoa per ewe. In the first two trials eggs were recovered at laparotomy. The egg recovery rate was significantly lower (P<0.05) for those ewes which had been inseminated by the LI method (74%) compared with those inseminated by the CC method (85%); fertilization rates were not significantly different (92% and 89% respectively). In the third trial 20 ewes were bled to determine their periovulatory LH concentrations and the timing of peak LH concentrations correlated with the outcome of each insemination. Ewes inseminated using laparoscopy did not conceive when their LH surge occurred >58 hr after progestagen withdrawal. In this and in the final experiment, the combined pregnancy rates and litter sizes (assessed radiographically) were 67% (n = 51) and 2.21 (n = 34) for the CC method and 75% (n = 48) and 1.97 (n = 36) for the LI method (P>0.05).     

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.     

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.     

Factors influencing the success of transcervical insemination in Merino ewes

The experiments described examined the effects of a number of factors on the level of uterine insemination achieved in Merino ewes by a transcervical insemination technique (Guelph system for transcervical artificial insemination; GST-AI). Cervical penetration rate is an important limitation to the use of such methods in Merinos. Simulated insemination was performed to estimate the proportion of ewes in which a pipette could be passed through the cervix to the uterus. In Experiment 1, cervical penetration rate (n = 14 to 30) was unaffected by an increase in postpartum interval at AI from 12 to 26 wk. The results of cervical penetration for individual ewes were found to be repeatable (P < 0.05). Experiment 2 (197 ewes) revealed a clear effect of ewe parity on penetration rates in hormonally synchronized ewes during the nonbreeding season (P < 0.05). In Experiment 3, estrus synchronization using progestagen (n = 51) or prostaglandin (n = 50) did not affect penetration rate. The penetration rate was slightly higher in the naturally cycling ewes, but the difference was not significant. Comparison of ewes from Experiments 2 and 3 suggests the possibility of a major effect of stage of the breeding season on the penetration rate (P < 0.05). It is concluded that ewe selection and management techniques may be used to increase the proportion of transcervical insemination attempts resulting in uterine insemination. However, fertility testing will be required to determine whether such improvements translate into correspondingly increased pregnancy rates.     

The use of synthetic gonadotropin releasing hormone treatment in the collection of sheep embryos

Gonadotropin releasing hormone (GnRH) treatment was examined as a means of improving the efficacy of embryo collection in the sheep following intrauterine insemination of frozen-thawed semen. In summary, treatment consistently improved fertilization rates and the number of fertilized ova collected per ewe was enhanced compared with untreated ewes. The yield of fertilized ova in ewes treated with follicle stimulating hormone (FSH) was maximized by administering GnRH 36 h after progestagen treatment; 24 h was the preferred time in ewes treated with pregnant mare serum gonadotropin (PMSG). There was a significant (P < 0.001) increase in the percentage of unfertilized ova in the former treatment when GnRH was given at 24 h. An examination of the time of insemination (0, 6, 12 and 18 h before the median time of ovulation) indicated that fertilization rates were highest when insemination occurred at 6 h in both GnRH-treated ewes and in untreated ewes. In GnRH-treated ewes, the recovery of ova was lowest when insemination occurred at the time of ovulation. The number of motile frozen-thawed spermatozoa required for fertilization following treatment was estimated to be approximately 20 x 10(6) per uterine horn. GnRH-treatment also improved the yield of fertilized ova in sheep that were naturally mated, although this yield was lower than that obtained with intrauterine insemination of frozen-thawed semen. It is concluded that fertilization failure, a major problem in sheep embryo collection, can be eliminated through judicious use of GnRH treatment and properly timed intrauterine insemination.     

Transcervical artificial insemination in sheep: effects of a new transcervical artificial insemination instrument and traversing the cervix on pregnancy and lambing rates

Cervical anatomy limits the use of transcervical intrauterine artificial insemination (TC AI) in sheep. We have developed an instrument to cope atraumatically with the cervix; although this instrument has not affected fertilization rate or pregnancy rate through Day 3, the effects on sperm transport and pregnancy after Day 3 are not known. The objective of the present study was to determine whether our TC AI instrument affected sperm transport, pregnancy rates, or lambing rate. In Experiment 1, ewes were assigned to two treatments: TC AI using the new TC AI instrument (n=10) or AI via laparotomy using a laparoscopic AI instrument (n=10). Twenty hours after artificial insemination, the uterine horns and oviducts were recovered and flushed to collect spermatozoa. Sperm transport did not differ (P>0.05) between the two treatments. In Experiment 2, ewes were assigned to three treatments: TC AI using the new TC AI instrument+sham intrauterine AI via laparotomy (n=29); sham TC AI+intrauterine AI via laparotomy using a laparoscopic AI instrument (n=29); and sham TC AI+intrauterine AI via laparotomy using the new TC AI instrument (n=30). On Day 14 after AI, uteri were collected and flushed to recover blastocysts. Transcervical deposition of semen reduced (P<0.05) Day 14 pregnancy rate (17.2% versus 61%), but intrauterine deposition of semen using the TC AI instrument via midventral laparotomy increased (P<0.05) Day 14 pregnancy rate (76.6% versus 44.8%). In Experiment 3, ewes were assigned to two treatments: sham cervical manipulation (n=40) or cervical manipulation to mimic TC AI (n=40). Immediately after treatment, each ewe was mated with a ram and watched until the ram mounted and ejaculated into the ewe. Treatment did not affect Day 30 or 50 pregnancy rate (67.5 and 66.2%, respectively), determined ultrasonically, or lambing rate (62.5%). The differences between Days 30 and 50 pregnancy rates and lambing rate were not significant. In Experiment 4, ewes were assigned to two treatments: TC AI (n=99) or laparoscopic AI (n=99). Transcervical AI reduced (P<0.01) Day 30 (TC AI versus laparoscopic AI; 5.0% versus 46.0%) and Day 50 pregnancy rates (4.0% versus 41.0%), determined ultrasonically, and lambing rate (4.0% versus 41.0%). Although the TC AI procedure significantly reduced pregnancy and lambing rates, large numbers of spermatozoa deposited at natural insemination seemed to compensate. Because our TC AI procedure has all but eliminated any visual evidence of trauma, and because the procedure does not seem to affect sperm transport or embryonal survival until Day 3, we speculate that cervical manipulation associated with TC AI may activate pathways that interrupt pregnancy between Days 3 and 14.     

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.     

Investigations on spermicidal activity in the sheep vagina.

This study was conducted to elucidate some of the effects of a synthetic progestagen and natural ovarian hormones on spermicidal activity in the sheep vagina. In the first experiment, parous ewes were treated for 17 days either intravaginally with medroxyprogesterone acetate (MAP) or subcutaneously with progesterone. They were inseminated artificially either on the last day of progestagen treatment or during estrus after progestagen withdrawal. Their vulvovaginal junctions were ligated to prevent the loss of sperm cells by drainage to the exterior. Untreated control ewes were inseminated during either estrus or the luteal phase of the estrous cycle. The ewes were killed 22 hr. after insemination, their vaginas flushed, and intact sperm cells and tailless sperm heads counted. In the second and third experiments, some of the ewes were bilaterally ovariectomized and inseminated several weeks later. Other ewes were ovariectomized and given subcutaneous injections of estradiol, progesterone, or both hormones.In the first experiment, most sperm cells were recovered intact from estrous or luteal phase control ewes. The intravaginal administration of MAP increased both the breakage of sperm cells into heads and tails and the disappearance of sperm cells. The spermicidal effects of MAP were just as great in ewes inseminated on the last day of treatment. as in those inseminated during the ensuing estrus; these results indicated that the peak estrogen secretion that occurs near the beginning of estrus was not necessary for the intensification of spermicidal activity.In the second experiment, ovariectomized ewes were compared to estrous and luteal phase ewes in regard to vaginal spermicidal activity. Sperm breakage and disappearance occurred least in estrous ewes, to a somewhat greater degree in luteal phase ewes, and to the greatest extent in ovariectomized ewes. The results suggested that endogenous ovarian hormones, particularly those in estrous ewes, suppress spermicidal mechanisms in the vagina.In the third experiment, the administration of estradiol and progesterone to ovariectomized ewes prevented the increase in sperm cell disappearance. Neither hormone alone prevented the increase.     

Fertility and sperm transport in Merino ewes at the first oestrus following embryonic death.

The embryos of ewes were killed with colchicine on Day 17 of gestation and the ewes were mated at the subsequent oestrus. Fertility was reduced at this mating, and fewer spermatozoa were found in the uterus and oviducts than in control animals. The total number of spermatozoa in the cervix and their distribution between the lumen and walls of the cervix were not altered, but the linear distribution along the cervical walls was changed. The density of the reamining spermatozoa in the control animals after flushing the cervix showed a progressive decrease from the posterior to the anterior segments. This did not occur in the untreated ewes. It seems likely that impaired sperm transport contributed to the lowered fertility.     

The pattern of cervical penetration and the effect of topical treatment with prostaglandin and/or FSH and oxytocin on the depth of cervical penetration in the ewe during the peri-ovulatory period

Artificial insemination in sheep has two major limiting factors: the poor quality of frozen-thawed ram semen and the convoluted anatomy of the sheep cervix that does not allow transcervical passage of an inseminating catheter. It has been demonstrated that in the ewe during estrus, there is a degree of cervical relaxation mediated by ovarian and possibly gonadotrohic hormones, and we set out to investigate factors that might enhance cervical relaxation. Five experiments were conducted on ewes of different breeds to determine: 1) the pattern of cervical penetration during the periovulatory period in ewes of several breeds (Welsh Mountain, Île-de-France, Vendéenne, Romanov and Sarda); 2) the effect of the “ram effect” a socio-sexual stimulus, on cervical penetration; and 3) the effects of the intracervical administration of follicle-stimulating hormone (FSH), oxytocin and a prostaglandin E agonist (misoprostol) on the depth of cervical penetration during the periovulatory period. The results showed that during the periovulatory period in all breeds examined, there was increased penetration of the cervical canal (P < 0.05) by an inseminating catheter. Cervical penetration increased to a maximum 54 h after the removal of progestagen sponges and then gradually declined. Furthermore, the depth of cervical penetration but not its pattern, was affected (P < 0.05) by the breed of ewe. The maximum depth of cervical penetration was lower (P < 0.05) in the Vendéenne breed compared to the Île-de-France and Romanov breeds, which did not differ from one another. In the presence of rams, the depth of cervical penetration was increased at 48 and 54 h after removal of sponges (P < 0.05) and reduced at 72 h (P < 0.05). The local administration of hormones FSH, misoprostol (a PGE agonist) and oxytocin alone and in various combinations did not have any significant effect on the depth of cervical penetration during the periovulatory period. In conclusion, the natural relaxation of the cervix observed in ewes of several breeds occurs at a time during estrus, 54 h after the removal of progestagen sponges, which is the most suitable for artificial insemination. The effect was enhanced by the presence of a ram but not by the local intracervical administration of FSH, misoprostol and oxytocin even though oxytocin and PGE₂ are involved in cervical function. The time of maximum cervical penetration in the preovulatory period (54 h) coincides with high LH and estradiol concentrations suggesting they might be responsible for the relaxation of the cervix probably through an oxytocin-PGE mediated pathway.     

Reproduction in mice: Spermatozoa as factors in the development and implantation of embryos

The effects on mouse embryo development in vivo of varying the numbers of spermatozoa used in artificial inseminations was studied. The two criteria used in the evaluation of the progress of embryo development were 1) ability to reach the two-cell stage and 2) success of development from the two-cell stage through implantation. A 44% reduction in the yield of two-cell embryos and a 67% reduction in the number of implants was observed when C3HeB/FeJ females were inseminated with one-twentieth the number of spermatozoa estimated to be present in a typical ejaculate. The reduction in the yield of two-cell embryos was substantially reversed by a second insemination of a large number of heat-inactivated spermatozoa 12 hr after the first insemination. The sperm-dependent reduction in development from the two-cell stage through implantation was prevented only by normal viable (unheated) spermatozoa. These results were rationalized by the hypothesis that in female C3HeB/FeJ mice spermatozoa serve physiological functions beyond the fertilization of ova and that spermatozoa may act to foster early embryo development through modulation of the environments embryos experience as they move through the reproductive tract.     

Fertilization and ovum recovery rates in superovulated ewes following cervical insemination or laparoscopic intrauterine insemination at different times after progestagen withdrawal and in one or both uterine horns

In Exp. 1, 40 ewes were used in a 2 x 2 factorial design to investigate the effects of intrauterine versus cervical insemination and superovulation using pig FSH or PMSG and GnRH on egg recovery and fertilization rate. Cervical inseminations were carried out at 48 and 60 h (N = 20 ewes) and intrauterine insemination at 52 h (N = 20 ewes) after progestagen pessary withdrawal. Eggs were recovered on Day 3 of the oestrous cycle. Ovulation, egg recovery and fertilization rates were independent of the type of superovulatory hormone used. Fertilization rate was high irrespective of insemination site but intrauterine insemination at 52 h was associated with a significant (P less than 0.01) decrease in egg recovery of over 40% compared with cervically inseminated ewes. In Exp. 2 ewes were inseminated at 36 (N = 5), 48 (N = 6) or 60 (N = 6) h after pessary withdrawal to determine the optimum intrauterine insemination time to maximize both fertilization rate and egg recovery. Egg recovery per ewe flushed was 23, 59 and 67% after intrauterine insemination at 36, 48 and 60 h respectively. Correspondingly, 0, 85 and 100% of the eggs recovered were fertilized. The results of Exps 1 and 2 suggest that when intrauterine insemination occurs before or during ovulation it interferes with oocyte collection by the fimbria. In Exp. 3 egg recovery and fertilization rates were determined after cervical insemination at 48 and 60 h (N = 8) or intrauterine insemination at 48 (N = 9) or 60 (N = 8) h after progestagen withdrawal. Ewes in the last two groups were subdivided and inseminated unilaterally or bilaterally. Egg recovery was high after cervical insemination (95%) but only 36% of these eggs were fertilized. Unilateral intrauterine insemination was as effective as bilateral in ensuring high fertilization rates (100 versus 97%). Intrauterine insemination at 48 h compared with 60 h resulted in a significantly lower (P less than 0.05) percentage of eggs recovered (42 versus 90% respectively). However, reducing the degree of interference by adopting unilateral rather than bilateral insemination did not alleviate the detrimental effects of the 48-h insemination time on egg recovery. From these results we advocate the adoption of intrauterine insemination at 60 h after progestagen withdrawal to maximize fertilization rate and egg recovery in superovulated ewes.     

Intrauterine insemination of sows with reduced sperm numbers: results of a commercially based field trial

Artificial insemination (AI) in pigs requires 2-3 billion spermatozoa to achieve consistently high fertility with current practice of inseminating into the posterior region of the cervix. We have investigated the potential advantages of inseminating through the cervix into the caudal region of the uterus using lower sperm numbers. Total sperm doses from 22 boars of 3, 2 or 1 billion spermatozoa were packaged in 80 ml volumes in X-Cell extender in gene-flat-pack (Cochette) bags. A novel inseminating device, the Deepgoldenpig, was employed which permits the ready introduction of a narrow catheter through the cervix into the uterus without traumatic injury to the mucosa. This was compared with the standard Goldenpig device that allows semen to be deposited in the posterior folds of the cervix. Sows of two different genotypes and of parities ranging from 2 to 11 were used. They were selected solely on the basis of a weaning to estrus interval of 4-6 days. Two inseminations, with a 24 h interval between them, were carried out on each sow. Pregnancy was determined at 35 days by ultrasound scan, and farrowing and litter size recorded. Pregnancy and farrowing data were very similar. The standard inseminating device produced farrowing rates of 91.1, 91.8 and 65.8% for insemination with 3, 2 and 1 billion spermatozoa, whereas the deep insemination device gave rates of 90.5, 90.5 and 86.9%. Only the 1 billion dose with the standard device was significantly different from the high dose control (P < 0.001). Similarly, the mean litter sizes with the standard device were 12.5, 12.6 and 10.3 and with the deep insemination device 12.3, 12.3 and 12.1. Only the 1 billion dose with the standard device was significantly lower (P < 0.001). None of the covariates differed significantly and there were no significant interactions with treatment. We conclude that transcervical insemination in the sow is simple, effective and safe, and allows the sperm dose to be reduced to 1 billion spermatozoa.     

Birth of offspring of pre-determined sex after artificial insemination of frozen-thawed, sex-sorted and re-frozen-thawed ram spermatozoa

The fertility of ram spermatozoa cryopreserved prior to, and following, sex-sorting by flow cytometry was assessed after insemination of mature Merino ewes at a synchronised estrus. Ewes were inseminated with spermatozoa from three rams, split into four treatment groups: 50 x 10(6) motile non-sorted, frozen-thawed (Control50), 15 x 10(6) motile non-sorted, frozen-thawed (Control15), 15 x 10(6) motile sex-sorted, frozen-thawed (SF15) or 15 x 10(6) motile frozen-thawed, sex-sorted, re-frozen-thawed (FSF15) ram spermatozoa. Separation of SF15 and FSF15 treatments into X- and Y-chromosome-bearing populations was achieved using a high-speed sperm sorter. The percentage of ewes lambing after insemination was similar for Control15 (36/74; 48.6%), SF15 (35/76; 46.1%) and FSF15 (26/72; 36.1%) groups (P>0.05). A higher percentage of ewes produced lambs in the Control50 (38/70; 54.3%) than the FSF15 group (P<0.05). Fifty-one of the 55 (92.7%) lambs derived from fresh, sex-sorted frozen-thawed spermatozoa were of the predicted sex, as were 41/43 (95.3%) lambs derived from frozen-thawed, sex-sorted, re-frozen-thawed spermatozoa. This study demonstrated for the first time in any species that frozen-thawed spermatozoa, after sex-sorting and a second cryopreservation step, are capable of producing offspring of the predicted sex following artificial insemination.     

The effect of timing of laparoscopic insemination in superovulated ewes, with or without sedation, on the recovery of embryos, their stage of development and subsequent viability

Twenty ewes were used as donors in a 2x2 factorial design experiment to investigate the effects of two different insemination times (48 vs 60 h after pessary withdrawal), with or without sedation, on the ovum recovery rate 5 d after insemination, the proportion of transferable embryos recovered, and the subsequent survival rate of embryos transferred to recipients. The ovum recovery rate following intauterine insemination at 48 h after progestagen pessary withdrawal was 63.8 and 53.4% for sedated and nonsedated control ewes, respectively. Following intrauterine insemination at 60 h the corresponding values for sedated and control ewes were 72.6 and 73.9%, respectively. The proportion of transferable quality embryos recovered was not affected by sedation but was improved by insemination at 48 h rather than 60 h after pessary withdrawal (100 vs 35.4%). Embryo survival following laparoscopic transfer to recipients from donor ewes inseminated at 48 h, with or without sedation was 38.8% (7 18 ) and 50% (7 14 ), respectively. Following intrauterine insemination of the donors at 60 h, the survival rate in recipients was reduced for embryos transferred from both the sedated and control ewes to 6.25% (1 16 ) and 36.3% (4 11 ). It is concluded that delaying the timing of intrauterine insemination relative to pessary withdrawal and the use of acepromazine maleate as a sedative at the time of insemination are deleterious to embryo development and subsequent viability.     

Embryo production from superovulated sheep inseminated with sex-sorted ram spermatozoa

An experiment was undertaken to assess the fertilizing capacity of sex-sorted, frozen-thawed ram spermatozoa, artificially inseminated into superovulated ewes, and the quality and survivability of the resultant pre-sexed embryos. Synchronized (intravaginal progestagen pessary and GnRH) donors were superovulated using PMSG and repeat ovarian stimulation with FSH before insemination. Ewes (n=67) were inseminated with either 30x10(6) or 15x10(6) motile non-sorted (control) or 15x10(6) motile sex-sorted (sorted) frozen-thawed spermatozoa (control: C30 or C15; sorted: S15, respectively) and the resultant embryos transferred immediately into synchronized recipients (n=160). The percentage of transferable embryos, pregnancy rate and embryo survival were similar (P>0.05) across all treatments. Oocyte cleavage rate was higher for ewes inseminated with S15 (172/230; 74.8%; P<0.05) than for C15 (97/151; 64.2%) or C30 (89/141; 63.1%) spermatozoa. Of the lambs resulting from embryos produced with sex-sorted spermatozoa, 86/93 (92.5%) were born of the predicted sex. This study demonstrated for the first time that pre-sexed offspring derived from superovulated sheep can be produced following transfer of embryos. Furthermore, sex-sorting by flow cytometry did not compromise the in vivo fertilizing capacity of ram spermatozoa in superovulated sheep, nor did it affect the quality or survivability of the resultant embryos.