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).     

Fertilization and embryo recovery rates in superovulated chios ewes after laparoscopic intrauterine insemination.

Forty superovulated dairy ewes of the Greek Chios breed were used in an experiment to evaluate the efficiency of laparoscopic intrauterine insemination on fertilization and embryo recovery rates as well as embryo quality. Estrus was synchronized by intravaginal progestagen impregnated sponges and superovulation was induced by administration of 8.8 mg o-FSH i.m. following a standard 8 dose protocol. A small volume (0.3 mL) of diluted fresh ram semen was deposited in each uterine horn 24 to 28 h after onset of the estrus by a laparoscopic technique. The animals were allocated randomly into two groups (Group A and B) of 20 animals each. In Group A, embryos were recovered 18 to 24 h after the intrauterine insemination and in Group B on Day 6. The average number of corpora lutea was 12.8 +/- 1.2 and 11.5 +/- 1.1 (+/- SEM); the overall embryo recovery was 66.4% and 57% and the percentage of recovered fertilized ova was 81% and 82.8% in Groups A and B, respectively. More fertilized ova were collected per ewe from Group A (P < or = 0.1). Results indicated that in Chios breed, superovulation using homologous FSH combined with laparoscopic AI leads to good ovarian response with satisfactory results in fertilization, embryo recovery and quality of embryos. This could lead to improved and more efficient methods for obtaining large numbers of high quality oocytes and embryos for embryo transfer programs which could contribute to genetic improvement and increase of the population size.     

Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro

Ram spermatozoa are most susceptible to damage during freezing between the temperatures of -10 degrees C and -25 degrees C. The objectives of the present study were to examine how freezing rate through this critical temperature zone affected the fertility of spermatozoa as assessed in vivo and in vitro. Semen from six adult rams was frozen at two different rates ("fast": 5 degrees C/min from +5 to -25 degrees C; "slow": 0.5 degrees C/min from +5 to -25 degrees C). In Experiment 1, semen from the fast and slow treatments was used to fertilize ovine oocytes that had been matured in vitro. Semen from the fast treatment yielded a higher cleavage rate (57% vs. 26%; P<0.001) and more blastocysts per oocyte (28% vs. 13%, P<0. 001) than slow-frozen. No correlation was found between fertilizing ability and viability as assessed by fluorescent probes. Experiment 2 was designed to establish the conception rates following both cervical and intrauterine insemination of frozen-thawed semen from the same bank of semen as used in Experiment 1. Ewes were superovulated with FSH and inseminated by laparoscopy with frozen semen. A significant difference was found in the number of fertilized ova following embryo recovery (81.4% vs. 39.3%; P<0.001). In a further study, 119 mature cull ewes were inseminated following a 12-day synchronization treatment with frozen semen by either intrauterine (laparoscopic) or cervical insemination. Insemination with fast-frozen semen resulted in a significantly higher pregnancy rate (P<0.05) irrespective of method of insemination. The data show that freezing rate affects the proportion of spermatozoa that retain their fertilizing ability post-thawing. However, once fertilization has occurred, development to the blastocyst stage is independent of freezing rate.     

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.     

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.     

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.     

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.     

Transferable embryo recovery rates following different insemination schedules in superovulated beef cattle

The objective of this study was to evaluate the transferable embryo recovery rates from superovulated donor cattle after different artificial insemination (AI) schedules. Sixty mixed-breed crossbred females were administered follicle stimulating hormone (FSH) and prostaglandin F(2)alpha (PGF(2)alpha) to induce a superovulatory response. At standing estrus, donor females were randomly allotted to one of five treatment groups for AI. Donors were inseminated with two units of high-quality or low-quality frozen semen at 12, 24, 36, or 48 h after the onset of estrus in treatment Groups I, II, III, and IV, respectively, or inseminated with two units at 12, 24, 36, and 48 h (eight units/donor) in control Group V. Donor females inseminated once at either 12 or 24 h after the onset of estrus did not differ from donors inseminated in Group V in overall fertilization and transferable embryo recovery rates. The highest fertilization rate (89.5%) and transferable embryo recovery rate (74.9%) per donor resulted when AI was performed with high-quality semen at 24 h after the onset of estrus. These findings indicate that repeated insemination of superovulated beef cattle is not necessary to attain optimal fertilization rates and production of transferable quality embryos in beef cattle.     

Fertility of ewes following artificial insemination with semen frozen in pellets or straws, a preliminary report

In two trials involving the artificial insemination of 194 ewes, the fertility of ram semen was examined following freezing, either in pellet form or in straws, and after storage in a chilled state (15 degrees C) for up to 16 hours. Estrus was synchronized in ewes by intravaginal sponge (MAP) treatment for 14 days. At sponge removal 600 IU PMSG was injected and the ewes received two inseminations 50 and 60 hours later. Fertility was assessed at lambing. In trial 1, the mean lambing rate of 52% (16 31 ) for semen frozen in pellets was higher than 29% (9 31 ) for semen frozen in straws but this difference was not significant. In trial 2, ewes inseminated with chilled semen and semen frozen in pellets had lambing rates of 83% (44 53 ) and 55% (44 79 ) respectively (P<0.001).     

Fertility of ram semen frozen in Bioexcell and used for cervical artificial insemination

The current use of ingredients of animal origin, such as egg yolk, in semen extenders presents a risk of microbial contamination, and has led to the search for alternatives. Such an extender is commercially available for bull semen (Bioexcell), IMV, L'Aigle, France), and it has previously been tested in vitro for freezing ram semen, with satisfactory results. The aim of the present study was to compare the fertility results of ewes in Uruguay, after cervical insemination with ram semen that was frozen in Bioexcell versus semen frozen in a conventional milk-egg yolk extender (control). Semen from five Corriedale rams was frozen, using a split sample design, in either milk-egg yolk or Bioexcell extender, using a two-step extension method. The sperm parameters assessed after thawing were subjective motility, membrane integrity (SYBR-14/PI), and capacitation status (CTC). Thawed semen was inseminated intracervically once during spontaneous estrus in 970 Corriedale ewes that grazed in natural pastures, under extensive management conditions. Fertility was recorded as nonreturn rates at 21 days (NRR-21) and 36 days (NRR-36) after artificial insemination (AI), as well as pregnancy rate (PR-US, diagnosed ultrasonographically 50 days after AI of the last ewe). Subjective motility was slightly higher in Bioexcell than in the milk extender (47 vs. 46.5%; NS), as was membrane integrity (38 vs. 37.7%; NS) and the percentage of uncapacitated spermatozoa (28.5 vs. 26.3%; NS). There were no statistically significant differences in fertility rates found between Bioexcell and the control extender: NRR-21 (35.9 vs. 33.2%), NRR-36 (34.8 vs. 32.6%), and PR-US (28.4 vs. 27.2%). In conclusion, Bioexcell appears to be an alternative to the conventional milk-egg yolk extender for freezing ram semen, and provides similar fertility results after cervical AI under extensive management conditions. Thus, Bioexcell, containing no additives of animal origin, can offer a safer alternative when frozen semen is used for introducing new genetic material into a flock or a country.     

Fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without Equex STM paste

Twenty-five bitches were artificially inseminated with semen that was frozen-thawed using an egg yolk-Tris-glucose-citrate extender containing 5% glycerol with, or without the addition of 0.5% Equex STM Paste. Semen was collected on 2 occasions from 11 dogs, pooled, and evaluated for sperm motility, morphology and plasma membrane integrity. Each pool was then divided in 2 parts, diluted with 1 of the 2 extenders, and frozen in 0.5-mL straws. In the bitches, plasma progesterone was assayed daily during late proestrus and estrus. Artificial insemination (AI) was performed twice on Days 3 and 5 after the estimated LH peak. For each insemination, 200x10(6) spermatozoa were used. Ten bitches were inseminated with semen frozen without Equex: In 5 females, semen was deposited transcervically into the uterus with the aid of a fiberoptic endoscope and a urethral catheter, while the remaining 5 bitches were inseminated in the cranial vagina using a Norwegian catheter. Fifteen bitches were inseminated with semen frozen-thawed with Equex: Two groups of 5 bitches were inseminated according to the techniques described above, while 5 bitches were inseminated vaginally using the Osiris catheter. Pregnancy was diagnosed and the number of fetuses counted by ultrasound examination. Post-thaw, spermatozoa frozen with Equex tended to have higher total and progressive motility and to survive longer in vitro than when the extender without Equex was used. Spermatozoal concentration, age of the bitches, duration of heat and estrus, and progesterone concentration at LH peak and at the first and second AI did not differ among the 5 groups. The overall pregnancy rate of 84% (21/25) was close to what can be expected from well controlled natural matings. For both freezing extenders tested, 5/5 bitches were pregnant after uterine deposition of semen and 4/5 were pregnant when semen was deposited in the anterior vagina using the Norwegian catheter. With the Osiris catheter, 3/5 inseminations resulted in a pregnancy. No significant differences in pregnancy rate or number of fetuses were found between groups, site of deposition or freezing extender.     

Effects of single deep insemination on transferable embryo recovery rates in superovulated dairy cows

Thirty superovulated Friesian lactating cows were randomly assigned to two groups. Group I donors were inseminated with one unit of semen deposited into the uterine body at 8, 20 and 32 h after the onset of estrus. Group II donors were inseminated with one unit of semen deposited deep into the uterine horns at 15 h after the onset of estrus. Neither the mean rates of fertilized ova nor the mean rates of transferable embryos were different between treatments (P > 0.05).     

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.     

Fertility in the ewe following cervical insemination with fresh or frozen-thawed semen at a natural or synchronised oestrus

Artificial insemination (AI) in sheep is currently limited by the poor fertility obtained following non-surgical intracervical insemination of frozen-thawed semen. An exception to this general finding is the non-return rate of around 58% reported for large scale on-farm AI in Norway. The objective of the present study was to determine if similar results could be obtained under Irish conditions. Comparisons were made between semen collected, and frozen, from rams in Norway (NOR) and Ireland (IRL). The effects of synchronisation and inseminator were also examined. Parous ewes (n=297) of various breed types were inseminated to a natural (N) or synchronised (S) oestrus with either fresh (from Irish rams) or frozen-thawed (IRL and NOR) semen. Ewes were randomly assigned, within breed, to the following treatment groups: (i) Fresh-N: n=28, (ii) Fresh-S: n=30, (iii) IRL-N: n=62, (iv) IRL-S: n=50, (v) NOR-N: n=68, (vi) NOR-S: n=59. Within each group, ewes were inseminated by an experienced Norwegian or by an Irish inseminator. Pregnancy rate did not differ significantly between ewes inseminated to a natural or synchronised oestrus nor between Norwegian and Irish frozen semen. The proportion of ewes pregnant after insemination with fresh semen was 0.82 and 0.70 (treatments i and ii) compared with 0.40, 0.52, 0.34 and 0.37 (treatments (iii)-(vi)) for frozen semen (P<0.001). Corresponding litter sizes (+/-S.E.), adjusted for ovulation rate, were 2.9+/-0.22, 3.3+/-0.23, 2.2+/-0.21, 1.7+/-0.21, 2.2+/-0.21 and 2.1+/-0.21 (fresh versus frozen; P<0.001). There was an interaction between semen type (fresh or frozen) and oestrus type (N or S) for litter size due to an increased adverse effect of frozen semen on litter size in synchronised ewes (P<0.05). Pregnancy rate was significantly influenced by breed of ewe (P<0.01) and inseminator (P<0.05). These results suggest that ewe breed may be a critical determinant of the potential for the exploitation of cervical insemination of frozen-thawed semen in sheep breeding programmes.     

Estrus synchronisation and fertility in gilts using a synthetic progestagen (Allyl Trenbolone) and inseminated with fresh stored or frozen semen

An orally active synthetic progestagen was administered at two dosage levels to synchronise estrus in gilts. Fertility following insemination with either fresh stored or frozen semen was determined by examining surgically recovered ova for cleavage, and numbers of spermatozoa attached to the zona pellucida, or enumeration of embryos in gilts slaughtered 30 days post insemination. There was no significant difference (P>0.05) between treated and control groups in the duration of estrus or in fertility as determined by cleavage of ova. A significantly (P<0.001) shorter interval to estrus and better synchronisation was obtained with both treatment groups than with the control group. The mean interval from the end of treatment to the onset of estrus for the untreated controls and the treated groups receiving 12.5 and 15 mg compound per day was 11.25 +/- 10.4 SD; 5.6 +/- 0.52 SD and 7.3 +/- 5.3 SD. Fresh semen yielded significantly (P<0.01) more cleaved ova than frozen semen.     

The effect of DMA level on morphology and fertilising ability of Japanese quail (Coturnix japonica) spermatozoa

The effect of different levels (2, 4 or 6%) of DMA (dimethylacetamide) on the morphology and fertilising ability of unfrozen quail spermatozoa was evaluated. Semen was collected from 72 males kept individually in cages and randomly divided into four groups: Group I--control -- fresh undiluted semen (12 males) and three experimental groups (20 males each) - semen diluted 1:1 with Lake's extender and supplemented with 2% (Group II), 4% (Group III) or 6% (Group IV) of DMA (final concentration). Sperm morphology was evaluated at each step of semen preparation, i.e. in fresh and diluted semen, semen supplemented with DMA and semen that remained after insemination. For fertility tests, 36 females were divided into four groups (nine females each). Females in the control group were inseminated with 10 microl of fresh semen, in the experimental groups with 40 microl of diluted semen. Each stage of quail semen treatment had a deleterious effect on sperm morphology. The highest percentage of morphologically normal cells in semen evaluated after insemination, was observed in samples with 2% DMA, and the lowest--in samples with 6% DMA. Semen dilution and DMA addition significantly affected the fertilising potency of spermatozoa. Fertility of eggs collected from the control group (71.5% on average) was significantly higher (P相似文献   

The effect of time of intrauterine insemination on the development and viability of embryos collected from superovulated ewes

Eighteen Border Leicester x Scottish Blackface ewes, primed with 300 mg progesterone (12 d) and superovulated with decreasing doses (6, 5, 3 and 2 mg) of porcine FSH, were inseminated with fresh semen, using laparoscopic intrauterine procedures at 48 (Group E) or 60 h (Group L) after exogenous progesterone removal. Five days after insemination, embryos were collected and classified on the basis of their morphological development. During the subsequent 3 d of in vitro culture (38.5 degrees C; 5% CO2) the embryos were evaluated at 24-h intervals. After 72 h, the embryos were individually fixed (24 h) and stained with aceto-orcein and the nuclei were then counted to provide an objective index of cell proliferation and development. Mean (+/-SEM) ovulation rates for the 2 groups (9.2+/-1.5 and 7.1+/-1.2, respectively) and the corresponding percentages (53 vs 59) of embryos collected by laparoscopy were unaffected by insemination time. All donors yielded fertilized ova, but whereas all Group-E donors yielded 1 or more viable embryos (i.e., >32 cells), only 5 Group-L ewes yielded viable embryos (P<0.10). At collection, the percentages of embryos at the morula stage of development were 98 (Group E n = 44) and 39 (Group L n = 38; P<0.001). Few of the remaining ova (Group E = 0% Group L = 8%) were at the 1-cell stage of development when collected, indicating that retarded development post fertilization, not fertilization failure, was the principal consequence of delayed insemination. The percentages of embryos that continued to develop during in vitro culture were 91 and 37 for Groups E and L, respectively (P<0.001), and all of these reached the blastocyst stage. Of these blastocysts, 75 and 50% in Groups E and L hatched in vitro (P<0.10), with mean (+/-SEM) nuclei counts of 148+/-22.7 and 76+/-13.8 (P<0.02), respectively. In conclusion, while delayed intrauterine insemination did not affect the efficiency of ovum collection, it caused a major reduction in the yield of embryos that were capable of developing during in vitro culture. However, fertilization failure accounted for only 13% of the loss in viability following late insemination.     

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 repeatability of superovulatory response and embryo recovery in sheep

Over an 8-year period, a total of 328 Scottish Blackface donor ewes were involved in a MOET program. They were synchronized with fluorogestone acetate sponges and superovulated with ovine FSH. After the onset of estrus, ewes were hand-mated and laparoscopic artificial insemination was performed with fresh semen 44-46 h after sponge removal. Embryos were recovered semi-laparoscopically on either Day 5 or Day 6 after insemination. Of the 328 donor ewes used, 222 ewes were supervoulated only once, while the remaining ewes were superovulated either twice (73 ewes), 3 times (26 ewes) or 4 times (7 ewes) at yearly intervals to generate a maximum of 474 records for subsequent analysis. There was no significant change in either mean ovulation rate or the mean number of embryos recovered per donor ewe at successive treatments. However, significant (P < 0.05 at least) effects of both year and donor ewe age existed for superovulatory response and number of embryos recovered, though only the effect of year was significant (P < 0.001) for percentage embryo recovery. Repeatability was significant (P < 0.05 at least) for both superovulatory response (r = 0.55, s.e. 0.055) and number of embryos recovered (r = 0.38, SE 0.074), but not for percentage embryo recovery (r = 0.04, SE 0.102).