Cervical dilation with exogenous oxytocin does not affect sperm movement into the oviducts in ewes

Exogenous oxytocin aids in the transcervical passage of an AI gun into the uterus of ewes, and it may be an effective adjunct to sheep AI procedures. However, the effects of oxytocin on sperm transport and fertility are unclear. Thus, experiments were conducted to evaluate the effects of oxytocin on variables that may affect fertility. In Experiment 1, five ewes/group received intravenous injections of 0, 50, 100, 200 or 400 USP units of oxytocin. Oxytocin enhanced (P < 0.001) uterine entry; the rates were 0% for control, 60% for the 50- and 100-unit doses, and 100% for the 200- and 400-unit doses. In Experiment 2, five ewes/group received intravenous injections of 0, 50, 100, 200, or 400 USP units of oxytocin, and the effect on uterine contractions was observed with a laparoscope. Oxytocin induced myometrial tetany within 2 min. The dose affected (P < 0.05) the duration of tetany, which was 0, 21, 27, 29, and 41 min for the 0-, 50-, 100-, 200- and 400-unit doses, respectively. In Experiment 3, either 0 or 200 USP units of oxytocin were injected intravenously 52 h after removal of progestogen pessaries from 20 ewes. Ewes were inseminated laparoscopically 10 min later with fresh, extended semen (500 × 10⁶ sperm cells) into the right uterine horn. Ewes were slaughtered 20 h after AI, and the numbers of spermatozoa were determined. Oxytocin did not affect (P > 0.05) the movement of spermatozoa throughout the uterus and into both oviducts. In summary, oxytocin induced myometrial tetany and permitted the passage of the tip of an AI gun into the uterus. However, oxytocin did not disrupt sperm transport to the oviducts. We conclude that oxytocin-induced cervical dilation may be a useful adjunct to transcervical intrauterine AI procedures for sheep.     

Differences between Belclare and Suffolk ewes in fertilization rate, embryo quality and accessory sperm number after cervical or laparoscopic artificial insemination

Ewe breed has been shown to have a major effect on pregnancy rates following cervical AI using frozen-thawed semen. The main objective of this study was to examine the differences between purebred Belclare and Suffolk ewes (multiparous) in fertilization rate, number of accessory sperm and stage of embryo development on day 6 after cervical or laparoscopic AI with frozen-thawed semen. In experiment 1, Belclare and Suffolk ewes were synchronized for 12 days and were either cervically inseminated (year 1: n=28 and 31; year 2: n=16 and 15, respectively) or laparoscopically inseminated (year 2: n=13 and 14). In experiment 2, superovulated Belclare (n=4) and Suffolk (n=13) ewes were laparoscopically inseminated. All ewes were slaughtered 6 days after AI; oocytes/embryos were recovered, morphologically graded and stained to assess the number of cells and accessory spermatozoa. Data from both experiments were combined for statistical analysis. The proportion of ewes with fertilized oocytes was significantly higher following laparoscopic AI compared with cervical AI (54% versus 19%). More Belclare than Suffolk ewes yielded fertilized oocyte(s) after cervical AI (34% versus 10%, P<0.02) but there was no difference after laparoscopic AI (62% versus 60%). From the ewes that yielded at least one fertilized oocyte the proportion of Belclare ewes with embryos at the morula/blastocyst stage was significantly greater than for Suffolk ewes (94% versus 59%, P<0.02). A higher proportion of Belclare than Suffolk ewes had evidence of sperm reaching the site of fertilization following cervical AI (39% versus 15%, P<0.02) but there was no difference after laparoscopic AI (62% versus 64%, P>0.8). Amongst the ewes with evidence of sperm at the site of fertilization, laparoscopic AI resulted in a higher number of sperm per oocyte/embryo or per ewe than cervical AI (P<0.01). These results suggested that the difference in pregnancy rate between Suffolk and Belclare ewes following cervical AI was due to: (i) sperm traversing the cervix and uterus in a higher proportion of Belclare than Suffolk ewes, leading to a higher incidence of fertilization and (ii) the lower developmental competence of fertilized oocytes from Suffolk ewes.     

Transcervical artificial insemination of Australian Merino ewes with frozen-thawed semen

We compared conventional methods for laparoscopic and cervical artificial insemination (AI) to a transcervical AI procedure (Guelph System for Transcervical AI; GST-AI) for use with frozen semen in Merino ewes. The GST-AI procedure was performed by an experienced operator in Experiment 1 (771 ewes) and by 2 inexperienced operators in Experiment 2 (555 ewes). In Experiment 1, intrauterine insemination by GST-AI was achieved in 76% of the ewes. The pregnancy rate at Day 70 for ewes inseminated by laparoscopy (48%, 120 251 ) was higher (P<0.01) than for ewes inseminated by either intrauterine GST-AI (32%, 64 201 ) or cervical AI (9%, 24 256 ). The overall (intrauterine and intracervical) pregnancy rate for GST-AI was 26% (68 264 ) and was unaffected by depth of insemination within the cervix. Pregnancy rates were unaffected by ram or day of insemination. In Experiment 2, the operators achieved intrauterine inseminations by GST-AI in 43% (78 182 ) of the ewes, with a significant operator effect (P<0.01) on depth of cervical penetration. The pregnancy rate to intrauterine GST-AI (40%, 31 78 ) did not differ from that to laparoscopic insemination. The total pregnancy rate for GST-AI in Experiment 2 (19%, 34 182 ) was lower (P<0.05) than that for laparoscopic AI (39%, 72 187 ) but superior (P<0.05) to that for cervical AI (1%, 1 186 ). The GST-AI pregnancy rates were affected by depth of AI (P<0.01) and by operator (P<0.05). It is concluded that GST-AI is superior to cervical AI, and may have application in Merinos if cervical penetration rates can be improved.     

Development of a new transcervical artificial insemination method for sheep: effects of a new transcervical artificial insemination catheter and traversing the cervix on semen quality and fertility

The difficulty of traversing the cervix severely limits transcervical artificial insemination (TC AI) in sheep. Cervical trauma and poorly designed instruments can reduce fertility after AI. To overcome problems associated with TC AI, we developed a new TC AI catheter. Three bench experiments were conducted to determine the effects of the new TC AI catheter on semen quality independent of the effects of moving the catheter through the cervix. In each of the three bench experiments, the standard laparoscopic instrument for intrauterine AI in sheep was used as the control for the TC AI catheter. In Experiment 1, the total volume of semen extender expelled and void volumes for both types of AI instrument (TC versus laparoscopic) were determined. In Experiment 2, the effects of each type of AI instrument (TC versus laparoscopic) on semen quality, estimated as percentage motility and percentage forward progressive motility, of frozen-thawed semen was determined. In Experiment 3, the effects of both types of AI instrument (TC versus laparoscopic) on number of spermatozoa expelled was determined. The type of AI instrument affected neither semen quality nor the number of spermatozoa expelled. However, void volume differed (P < 0.01) between the two instruments. After differences in void volume were taken into account, an in vivo experiment was conducted to determine whether using our new TC AI catheter for TC or surgical intrauterine AI affected fertilization and pregnancy rates. For this, ewes were assigned to one of three treatments: (1) TC AI using the new TC AI catheter + sham AI via laparotomy (n = 9); (2) sham TC AI + AI via laparotomy using a laparoscopic AI instrument (n = 8); and (3) sham TC AI + AI via laparotomy using the new TC Al catheter (n = 10). To synchronize estrus, progestogenated pessaries were inserted and left in place for 12 days. On Day 5 after pessary insertion, PGF2alpha (15 mg) was given i.m. At pessary removal, 400 IU of eCG were administered i.m. Ewes were inseminated 48-52 h after pessary removal using fresh diluted semen (200 x 10(6) to 350 x 10(6) spermatozoa per 0.2 ml) pooled from the same four rams each day during the experiment. At 72 h after AI, uteri were collected postmortem and flushed. Oocytes and embryos were recovered and evaluated. Treatments did not affect (P > 0.01) ovum and embryo recovery rate (mean = 87.3%), fertilization rate (59.3%), or Day 3 pregnancy rate (mean = 66.6%). We conclude from these data that the use of our new TC AI catheter for TC AI or intrauterine AI should not impair the success of AI in sheep.     

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.     

Lambing rates and litter sizes following intrauterine or cervical insemination of frozen/thawed semen with or without oxytocin administration

Intrauterine insemination by laparoscopy is required to achieve acceptable lambing rates in ewes when using frozen semen but the procedure has evoked welfare concerns. Oxytocin has been used to dilate the cervix as a means of accessing the uterus during conventional cervical insemination, but its effect on fertility is not well documented. Three hundred crossbred ewes were synchronised in estrus and randomly allocated to one of three insemination procedures using frozen/thawed semen containing 400 x 10(6)/ml progressively motile sperm: single cervical (0.2 ml), multiple cervical (4 x 0.05 ml) or laparoscopic (0.05 ml per uterine horn). The effects of each insemination procedure on lambing rate (percentage of treated ewes lambing) and litter size (lambs per ewe lambing) were tested with and without oxytocin (10 IU given i.m.) prior to fixed-time insemination. Oxytocin did not permit complete cervical penetration in any ewes and neither lambing rate nor litter size was influenced by the number of inseminations. Lambing percentages were 69 and 42 (P < 0.01) for the laparoscopic and cervical insemination methods, respectively, and oxytocin reduced these to 58 (NS) and 10 (P < 0.001) percent, respectively. Corresponding litter sizes for ewes not receiving oxytocin were 1.91 and 1.51 and for those receiving oxytocin, 1.83 and 1.41 (laparoscopic versus cervical, P < 0.02). Thus, in the absence of complete cervical penetration at insemination, 10 IU oxytocin decreased the number of ewes lambing but had no effect on their litter size.     

Fertilization rates in superovulating cows after deposition of semen on the infundibulum, near the uterotubal junction or after insemination with high numbers of sperm

Three experiments were conducted with 105 superovulating Holstein dairy cows in attempts to improve the fertilization rate. Cows were superovulated with follicle-stimulating hormone (FSH) and time of estrus was regulated with prostaglandin F(2)alpha (PGF(2)alpha). Semen was deposited on each infundibulum through a laparoscope inserted through the flank (Experiment 1) or near the uterotubal junctions through flexible tubing passed through the cervix and uterine horns (Experiment 2). In the third experiment, high numbers of sperm in fresh semen were deposited in the uterus. Cows were necropsied and ova were recovered and examined about 3.5 d after the beginning of estrus. Deposition of 0.5 ml of frozen-thawed semen on each infundibulum (Experiment 1) reduced both ovum recovery and fertilization. In ten cows inseminated on the infundibulum, ova representing 43% of ovulation points were recovered and 9% of these recovered ova were fertilized. In ten control cows, ova representing 80% of ovulation points were recovered and 62% of them were fertilized. In a 2 x 2 experiment with 36 superovulating cows (Experiment 2), 1 ml of diluted fresh or frozen semen was deposited either near the uterotubal junction or in the uterine body. The overall fertilization rate was 61%, with no significant effect of site of semen deposition or type of semen used. In Experiment 3, 2 or 3 ml of neat semen (average of 4.4 billion sperm) was deposited in the uterus of 12 cows; 183 of 197 intact ova (93%) were fertilized. In 56 control cows inseminated with 0.5 to 1.5 ml of frozen diluted semen (average of 70 million sperm), 502 of 947 intact ova were fertilized (53%, P<0.001). Insemination with high numbers of fresh sperm overcame problems of sperm loss or sperm transport and improved the fertilization rate.     

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).     

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.     

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen-thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5+/-5.4 x 10(6) sperm (range, 6.8-22 x 10(6)) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 x 10(6) sperm, with 70+/-5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17beta and progesterone were determined in most queens on the day of AI and again 30-40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P=0.58); overall 33% (5/15) of the queens became pregnant. For frozen-thawed semen, AI was consistently done 28h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P=0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen-thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.     

How the FSH/LH ratio and dose numbers in the p-FSH administration treatment regimen, and insemination schedule affect superovulatory response in ewes

We wished to evaluate the effects of FSH/LH ratio and number of doses of p-FSH during a superovulatory treatment on ovulation rate and embryo production (Experiment I). In Experiment II, we studied the efficacy of fertilization after various insemination schedules in superovulated donors. In Experiment I estrus was synchronized in 40 ewes (FGA, for 9 days plus PGF2alpha on Day 7) and the ewes were randomly assigned to four treatment groups as follows (n = 10 ewes each): Group A: four p-FSH doses with the FSH/LH ratio held constant (1.6); Group B: four p-FSH doses with the FSH/LH ratio decreasing (FSH/LH 1.6-1.0-0.6-0.3); Group C: eight p-FSH doses with the FSH/LH ratio held constant (1.6); Group D: eight p-FSH doses and FSH/LH ratio decreasing (1.6-1.6, 1.0-1.0, 0.6-0.6, 0.3-0.3). p-FSH administrations were performed twice daily 12 h apart. The ewes were mated at the onset of estrus and again after 12 and 24 h; then, one ram per four ewes was maintained with the ewes for two additional days. Ovarian response and embryo production were assessed on Day 7 after estrus. Experiment II. Three groups (n = 10 each) of superovulated ewes were inseminated as follows: Group M: mated at onset of estrus; Group AI: artificial insemination 30 h after onset of estrus; M + AI) mating at onset of estrus and intrauterine AI performed 30 h from estrus with fresh semen. Results of Experiment I showed that treatment (D) improved (P < 0.05) ovulatory response in comparison to Groups (C) and (A). The fertilization rate was lower (P < 0.01) in Group D) than Group (A). Also the proportion of transferable embryos was lower in Group (D) in comparison to all the other treatments (P < 0.01). Group A gave the best production of embryos (7.3/ewe; 89.0% transferable). In Experiment II, combined mating plus AI improved fertilization rate (80.3%) compared to both mating (P < 0.01) and AI (P < 0.02) alone.     

Effect of insemination dose and site on uterine inflammatory response of mares

It is unclear whether AI of mares deep into the uterine horn causes more or less inflammation of the endometrium than conventional AI. Thus, we compared uterine inflammatory reactions of mares inseminated with two different doses of frozen-thawed semen into the tip of the uterine horn (UH) ipsilateral to the preovulatory follicle with those of mares inseminated into the uterine body (UB). Thirty-two mares were assigned to one of four groups (eight mares/group): UB20=AI into UB, 20 x 10(6)sperm/0.5 mL; UB200=AI into UB, 200 x 10(6)sperm/0.5 mL; UH20=AI into UH, 20 x 10(6)sperm/0.5 mL; UH200=AI into UH, 200 x 10(6)sperm/0.5 mL, and inseminated 24 h after hCG administration. Before and 24 h after AI, they were examined with ultrasonography for the presence of intrauterine fluid. At 24 h, uterine fluid samples were obtained first by absorbing fluid into a tampon and then by uterine lavage. Uterine fluid was examined for polymorphonuclear leukocytes (PMN) and bacteriology, and frozen for lysozyme and TIC (trypsin-inhibitor capacity) assays. Only three mares conceived, one in each of the following groups: UB200, UH20, and UH200. Mares in the UH20 group accumulated less intrauterine fluid (p<0.05) than those in the other groups, which had similar amounts. No significant differences in PMN numbers were detected in either tampon or lavage fluid. Enzyme levels between groups did not differ statistically, except for TIC, which was lowest in the UH200 group. Thus, deep uterine horn AI caused no greater inflammation or irritation than uterine body AI in normal mares 24 h after insemination.     

Intrauterine insemination enhances fertility of frozen semen in superovulated ewes

Superovulated ewes were inseminated with fresh or frozen semen in a factorial experiment which compared two techniques of artificial insemination; i.e. conventional cervical deposition and intrauterine deposition at laparoscopy. Similar fertilization rates resulted from insemination with fresh semen at cervical (81% of ova from 11/11 ewes) and intrauterine (83% of ova from 10/12 ewes) sites. These results approached those observed in a naturally-mated group (95% of ova from 5/5 ewes). In ewes inseminated with frozen semen, fertilization rate was markedly reduced (P less than 0.05) after cervical insemination (11% of ova from 3/11 ewes) and partly restored (P less than 0.05) after intrauterine insemination (50% of ova from 8/11 ewes).     

Post-thaw survival of ram spermatozoa and fertility after insemination as affected by prefreezing sperm concentration and extender composition

A study was conducted to investigate the effects of prefreezing sperm concentration using two extenders on post-thaw survival and acrosomal status of ram spermatozoa (Experiment 1) and fertility after intrauterine insemination with differing doses of semen (Experiment 2). In autumn (Northern hemisphere), semen was collected by artificial vagina from 8 adult Leccese rams and ejaculates of good quality semen were pooled. Two extender systems for cryopreservation were considered, one based on milk-lactose egg yolk (Milk-LY) and the other based on tris-fructose egg yolk (Tris-FY). Experiment 1 (2 x 6 factorial scheme) examined the in vitro characteristics of spermatozoa in relation to the Milk-LY and Tris-FY extenders and six prefreezing sperm concentrations (50, 100, 200, 400, 500 and 800 x 10(6) spermatozoa/mL). Experiment 2 (2 x 4 factorial) evaluated the influence of the Milk-LY vs Tris-FY extenders and four doses (20, 40, 80 and 160 x 10(6) spermatozoa/0.25 mL) corresponding to prefreezing spermatozoa concentrations of 100, 200, 400 and 800 x 10(6) spermatozoa/mL, on fertility of ewes inseminated in uterus by laparoscope. Prefreezing sperm concentration influenced (P < 0.01) freezability of spermatozoa and affected negatively all the in vitro parameters at 800 x 10(6) spermatozoa/mL. Overall, Milk-LY tended to ensure higher viability and acrosomal integrity of spermatozoa after thawing at the intermediate sperm densities (range 100 to 500 x 10(6) spermatozoa/mL). At 500 x 10(6) spermatozoa/mL concentration corresponded the best condition for survival of spermatozoa (71.2%), acrosome integrity (71.5%) and acrosomal loss (6.0%). At the lowest sperm concentration (50 x 10(6) spermatozoa/mL), Tris-FY resulted in a higher survival rate than Milk-LY (61.3%, P < 0.05) and lower acrosomal loss (9.7%, P < 0.05). Milk-LY supported spermatozoa motility better than Tris-FY after incubation at sperm concentration between 50 and 400 x 10(6) spermatozoa/mL (0.05 > P < 0.01). Semen doses of 20 to 40 x 10(6) spermatozoa/ewe provided satisfactory fertility rates (64 to 81%). The increase of inseminate doses to 160 x 10(6) spermatozoa/ewe failed to improve fertility, actually tending to decrease lambing rates.     

Cervical versus intrauterine insemination of ewes using fresh or frozen semen diluted with aloe vera gel

This study was conducted at Belen de Escobar, Argentina, in March and April 1987. Experimental work on synchronization of estrus, deep-freeze conservation of ram semen and small fertility trials involving cervical and intrauterine (i.u.) insemination methods was undertaken. A total of 80 Corriedale ewes were used in seven insemination trials. Insemination trials were grouped into two experimental groups for comparison of 1) frozen semen diluted with an experimental extender and a control diluent inseminated cervically or i.u. in synchronized/superovulated ewes and 2) cervical insemination of fresh diluted or frozen semen in ewes inseminated at natural estrus or in ewes that were synchronized/superovulated. An overall ovulation rate of 8.7 +/- 0.5 was obtained by using a superovulatory regimen consisting of 3 mg Norgestomet implants and a total dose of 18 mg follicle stimulating hormone-pituitary (FSH-P). Numbers of ova recovered per ewe following superovulation ranged from 4.3 to 5.4. In experimental Group I, fertilization rates improved when laparoscopic intrauterine AI was used compared with cervical insemination (P<0.05). Fertility rates of i.u. and cervical insemination of frozen semen diluted with the experimental extender showed satisfactory fertilizing capacity. In experimental Group II, a lower number of fertilized ova were recovered from ewes inseminated with frozen semen (P<0.02), irrespective of their estrus manipulation.     

Ewe breed differences in fertility after cervical AI with frozen-thawed semen and associated differences in sperm penetration and physicochemical properties of cervical mucus

The objective of this study was to examine sperm penetration through cervical mucus and associated physicochemical properties of cervical mucus from Belclare and Suffolk ewes - two breeds with divergent pregnancy rate following cervical AI using frozen-thawed semen. In Experiment 1, sperm penetration through cervical mucus was assessed in 15 Belclare and 15 Suffolk ewes at 30, 48 and 57h post sponge removal. In Experiment 2, rheological properties of mucus from 17 Belclare and 19 Suffolk ewes at 48 and 57h post sponge removal were determined. In Experiment 3, 20 Belclare and 20 Suffolk ewes were used to assess mucus ferning and pH collected at 42, 48, 57 and 65h post sponge removal. In Experiment 1, a higher number of sperm penetrated cervical mucus from Belclare ewes at 48h, reflected by a breed by time interaction (P=0.05). In Experiment 2, mucus from Suffolk ewes tended to have higher elastic and complex moduli than that from Belclare ewes (P=0.06) regardless of time of collection. There was no effect of ewe breed on the viscous modulus. In Experiment 3, there was a significant effect of time post sponge removal on ferning (P<0.01), but there was no effect of breed. There was no effect of time or breed on mucus pH. It is concluded that breed differences in the rheological properties of cervical mucus affect the ability of sperm to swim through cervical mucus and this may explain breed differences in fertility observed after cervical AI using frozen-thawed semen.     

Reproductive response of ewes synchronized with different lengths of MGA treatments in intrauterine insemination program

A total of 415 fat tailed ewes were randomly assigned to two groups to assess the effect of duration of melengestrol acetate (MGA) (9 versus 12d) administration on reproductive parameters associated with laparoscopic artificial insemination. At the end of MGA treatment, ewes in each group were subdivided and inseminated with one of two different insemination doses (10×10(7) or 20×10(7) sperm per 0.5 ml insemination dose) of fresh diluted semen. Inseminations were carried out 11-18 h after first detected estrus. Ewes were screened for their return to oestrus from 10 to 21 days post AI and inseminated at their returned oestrus. Pregnancy diagnosis was done from approximately 55 days after insemination in both synchronized and return estrus. For short (9-day) and long (12-day) term MGA treated groups, estrus rates were 62% versus 89% (P<0.0001), respectively. Ewes (n=115) that returned to estrus were inseminated (7-11h after estrus detection) with fresh diluted semen at different doses (20×10(7) or 40×10(7) or 60×10(7) sperm per 0.5 ml insemination dose). Pregnancy rates were 41% and 44% for short term and long term MGA treated ewes, respectively. Pregnancy rate of ewes which returned to oestrus was 53.4%. There was a significant (P<0.05) increase in pregnancy rates (38-52% for 11-16 h; 63% for 17-18 h) when insemination was held at 17-18 h after first detected estrus following MGA treatments. Pregnancy rates were found to be similar in ewes inseminated with 10×10(7) (36%) or 20×10(7) (47%) motile spermatozoa at first AI, and 20×10(7) (44%) or 40×10(7) (59%) or 60×10(7)(48%) at second AI. It was concluded that short term MGA treated ewes were recorded with lower estrus rates but was similar to pregnancy rates with long term MGA treatment. Acceptable pregnancy rates were achieved in MGA induced estrus when insemination is conducted at 17-18 h after estrus onset and with 20×10(7) sperm per insemination dose.     

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.     

The effect of seminal sperm concentration on sperm transport in the reproductive tract of female rabbits treated with progesterone or diethylstilbestrol

Forty-two mature Baladi female rabbits were used in a randomized 3x2 factorial experiment to determine the effects of three treatments (control, progesterone injection: 2 mg/doe and DES injection: 0.1 mg/doe) and two semen sperm cell concentrations (1x10(6) and 60x10(6) sperm/0.25 ml semen on sperm transport and distribution in the female reproductive tract. The injections were given for three consecutive days after which rabbits were injected with 5 IU HCG and inseminated with 0.25 ml semen. The does were sacrificed 10 hrs after insemination and the sperm were recovered and counted from the oviducts, uterine horns, cervices and vagina. Total spermatozoa recovered was high when rabbits were inseminated with 60x10(6) sperm as compared to those inseminated with 1x10(6) sperm. When rabbits were injected with progesterone or DES, the number of sperm recovered relative to the total number of sperm inseminated was high in rabbits inseminated with 1x10(6) sperm, in comparison to those inseminated with 6x10(6) sperm. The number of sperm recovered was highest from cervix which was followed by vagina, uterus and oviducts. DES increased the number of the total sperm recovered while progesterone decreased the number as compared to control. This trend was also observed within the different segments of the reproductive tract and with groups inseminated with 1x10(6) or 60x10(6) sperm/0.25 ml semen. The effect of DES was more obvious with does inseminated with low sperm numbers. Significant correlation coefficients were found between the sperm numbers recovered in the uterus and oviducts and in the cervix and uterus of all groups of rabbits.     

Birth of pouch young after artificial insemination in the tammar wallaby (Macropus eugenii)

Timing of artificial insemination (AI) in marsupials is critical because fertilization must occur before mucin coats the oocyte during passage through the oviduct. In this study, timing and the site of insemination were examined to develop AI in the tammar wallaby (Macropus eugenii). Birth and postpartum (p.p.) estrus was synchronized in 46 females. Epididymal spermatozoa (n=4) or semen collected by electroejaculation (n=42) were inseminated early (4-21 h p.p.) into the urogenital sinus (n=7), the anterior vaginal culs de sac (n=7), the uterus by transcervical catheter (n=5), or the uterus by injection (intrauterine artificial insemination, IUAI) (n=5). A further 16 females were inseminated late (19-48 h p.p.) by IUAI. All females were monitored for birth. A third group of six females was inseminated late (21-54 h p.p.) by IUAI and 0.4-6.6 h later, sperm had reached the oviduct in all animals. In total, an oocyte to which spermatozoa were attached was recovered and two young were born after IUAI using epididymal (n=1) or electroejaculated (n=2) spermatozoa, but no young resulted from insemination at other sites. Two females were successfully inseminated at 43 and 47 h p.p., later than most other animals, and the third was inseminated much earlier (18 h p.p.) but with highly motile spermatozoa. These young represent the first macropodids born by AI and the first marsupials conceived using epididymal spermatozoa.