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.     

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).     

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).     

Insemination time and dilution rate of cooled and chilled ram semen affects fertility

Adult Merino ewes (n=448) were apportioned into two groups and inseminated with: extended at 30 degrees C with skim milk and stored for 6h at 15 degrees C (cooled semen) or extended with skim milk-citrate trisodium with egg yolk and stored for 24h at 5 degrees C (chilled semen). Each group was further subdivided according to the time of cervical insemination at 42, 46 and 50h after pessary (MAP-60 mg) removal and according to the dilution of the semen (120 x 10(6) spermatozoa in 0.05, 0.1 and 0.2 ml). The pregnancy rate after insemination with cooled semen was 50% better than that after chilled semen (56.7 vs. 37.5%; P<0.001). Pregnancy rate was not affected by the volume of insemination; however, there was a tendency of increased lambing rate with an insemination dose of 0.1 cc (1:2, dilution), especially when the ewes were inseminated with cooled semen. The effect of time on insemination was significant only in ewes inseminated with chilled semen at 5 degrees C (P<0.01). Insemination carried out 46 h after pessary removal resulted in higher pregnancy and lambing rate (36.5, 31.1; 52.0, 45.3; and 24.0, 20.0 at 42, 46 and 50h, respectively). Pregnancy of ewes inseminated with chilled semen at 46 h after pessary removal was similar to that obtained using cooled semen (52.0 vs. 56.7%). From this study, it is concluded that advancing the time of insemination with chilled semen at 5 degrees C improves pregnancy and that the lambing obtained under these conditions is similar to the one obtained with cooled semen.     

Artificial insemination of ewes with frozen-thawed semen at a synchronized oestrus. 1. Effect of time of onset of oestrus, ovulation and insemination on fertility

In three experiments, the onset of oestrus, time of ovulation and lambing after intrauterine insemination with frozen-thawed semen were examined following synchronisation of oestrus using intravaginal progestagen-impregnated sponges (inserted for 12 days) and an injection of PMSG at sponge removal.

The number (and percentage) of ewes detected in oestrus 12, 24, 36, 48, 60 and 72 h after sponge removal was 1 (0.3), 2 (0.6), 17 (5.2), 120 (36.7), 65 (20.0) and 10 (3.1) respectively. One hundred and twelve ewes (34.3%) remained unmarked. Egg fertilisation rates were not different between ewes irrespective of time of onset of oestrus or whether or not ewes were marked.

The median time of ovulation with respect to sponge removal (with 95% fiducial limits) for ewes joined with vasectomised rams (10:1) at spronge removal (teased ewes) was 55.8 h (54.61–57.09) and for unteased ewes 59.7 h (58.27–61.12).

In the third experiment, a total of 394 ewes were inseminated by laparoscopy with frozen-thawed semen. The percentage of ewes lambing and lambs born per ewe inseminated, and number of lambs born per ewe lambing for inseminations 48, 60, 72 and 78 h after sponge removal were 45.9, 57.7 and 1.25; 55.1, 72.0 and 1.31; 57.4, 80.9 and 1.41; and 39.3, 60.7 and 1.54, and for 59 control ewes receiving fresh semen by cervical insemination 47.5, 69.5 and 1.46 respectively. The lambing data after insemination with frozen semen was not different to that of the controls. The percentage of ewes lambing and lambs born per ewe inseminated increased with time of insemination at 48, 60 and 72 h (linear, P < 0.01) but was lower for inseminations at 78 h after sponge removal. Number of lambs born per ewe lambing increased with time of insemination after sponge removal (linear, P < 0.05).     

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.     

Ovarian response in ewes following horse anterior pituitary extract and progestagen treatment

Sixty adult ewes were treated intravaginally for 12 days with one of three progestagen treatments: (1) 500 mg progesterone pessary, (2) 30 mg flurogestone acetate pessary, (3) 12% (w/w) progesterone in a Controlled Internal Drug Release (CIDR) device. On days 10, 11 and 12 of progestagen treatment ewes received either 60, 45 and 30 mg; 45, 45 and 45 mg or 30, 45 and 60 mg of horse anterior pituitary extract (HAP), respectively. Progestagen treatment was terminated on day 12 at the time of the last HAP treatment. Ewes were bred by artificial insemination at 8- and 16-h intervals during oestrus, which was checked by using vasectomized rams. Ovarian response and fertility were determined at laparotomy 8 days after progestagen withdrawal. Progestagen treatment significantly increased (P<0.05) ovulation rate, with a mean of 9.1, 12.0 and 6.6 ovulations from treatments (1), (2) and (3), respectively. The method of HAP administration had no significant effect on ovulation rate, with a mean of 9.8, 10.0 and 7.8 after either decreasing, constant or increasing levels during treatment. There was no difference in the fertilization rate following flurogestone acetate (45.7%) or CIDR (47.0%), but a lower rate resulted after the progesterone pessary (30.0%) (P<0.025). More eggs were recovered following local than following general anaesthesia (P<0.05). Results indicate that cervical insemination does not give a satisfactory yield of fertilized eggs following hormonal inductions of superovulation.     

Ram-induced ovulation to improve artificial insemination efficiency with frozen semen in sheep.

Ram effect, defined as shortening of seasonal anestrus in ewes by exposure to the ram, is now well recognized but the underlying mechanisms are still unclear. Little information also exists whether the ram is able to influence the estrus cycle and ovulation. Three experiments were conducted to investigate endocrine response, time of ovulation and pregnancy rate of ewes in proestrus, exposed to the ram (treated) or an adult ewe (control). In the first experiment, ewes (n = 20) were treated with fluorgestone acetate pessaries for 12 days and were given eCG and cloprostenol one day before withdrawal of pessaries. On the day after removal of the pessaries ewes in the treated group (n = 10) were exposed to the ram and those in the control group (n = 10) were exposed to an adult ewe. Blood samples were taken for LH assay every 20 min from 2 h before to 24 h after ram exposure. In the second experiment, ewes (n = 120) were induced into proestrus and on the day after removal of the pessaries were exposed to either a ram (n = 60) or a ewe (n = 60) as described above and were laparoscoped 50, 60 or 70 h after pessary withdrawal (n = 20 at each time interval). In the third experiment ewes (n = 90) were induced and exposed to the ram (n = 45) or an adult ewe (n = 45) and inseminated via a laparoscope whit frozen-thawed semen at 50 or 60 h after pessary removal, respectively. Exposure to the ram was followed in 2 h by a marked rise in LH, equivalent to a preovulatory surge in duration and amplitude. It was also followed by concentrated ovulation within 25 to 30 h and by an increased pregnancy rate in exposed ewes (73.3 vs. 53.3%).     

A large-scale program in laparoscopic intrauterine insemination with frozen-thawed semen in Australian Merino sheep in Argentine Patagonia

This report shows the results of a large-scale laparoscopic intrauterine insemination program on a flock of Australian Merino sheep in Argentine Patagonia. The study was carried out on a total of 1824 ewes (3-to-7-yr-old) and 480 ewe hoggets (19-20 months old) on 2 farms in the southeastern region of Santa Cruz Province, in April and May 1996. The animals, divided into 15 groups, were synchronized with vaginal sponges containing 60 mg medroxyprogesterone acetate for 14 d and injected with 200 IU PMSG upon sponge removal. Estrus was screened every 12 h by means of vasectomized marker rams. The animals were inseminated laparoscopically by the intrauterine route using 2 schemes: 1) at a fixed time (12 h) after estrus detection, or 2) at a fixed time (60 h) after sponge removal irrespective of estrus. Pregnancy was determined at 30 d by transrectal ultrasound imaging. The results showed that 1) the onset of estrus occurs most often between 24 and 48 h after sponge removal, 2) ewe hoggets undergo estrus significantly earlier than sexually mature ewes, 3) in those animals showing estrus, there appears to be no relationship between fertility (as assessed by pregnancy outcome) and time of estrus, 4) there is a significant association between the percentage of estrus occurrence and pregnancy rate, 5) fertility is significantly higher in ewes than in hoggets, 6) for practical purposes insemination at a fixed time after the onset of estrus has no advantage over that of to insemination at a fixed time after sponge removal. It is concluded that large-scale laparoscopic intrauterine insemination can be successfully applied in Australian Merino ewes and ewe hoggets in low-productivity areas such as that of Argentine Patagonia and that estrus detection is unnecessary when insemination is performed at 60 h after sponge removal.     

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.     

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.     

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.     

A new strategy for superior reproductive performance of ewes bred out-of-season utilizing progestagen supplement prior to withdrawal of intravaginal pessaries

Two experiments were conducted to examine the effect of progestagen supplement 24h prior to intravaginal pessary withdrawal on reproductive performance of seasonal anestrous ewes. Ewes in each experiment were allocated to treatment and control and all were induced to estrus using either intravaginal MAP (Exp. 1; n=24) or CIDR-G (Exp. 2; n=28) pessaries for 12 days. Half of the ewes in each experiment were supplemented 24h before withdrawal of pessaries with either 10mg oral MAP tablets (Exp. 1) or 25mg i.m. progesterone (P(4)) administration (Exp. 2; P(4)-supplement-treated group). Fertile rams were allowed with the ewes at sponge removal (Day 0, 0h) and estrus was monitored at 6-h intervals for 3 days. Blood samples were collected for measurements of P(4) (Exp. 1 and Exp. 2) and LH (Exp. 2). In both experiments, the percent of ewes in estrus was greater (P<0.05) and intervals to estrus were longer (P<0.05) in progestagen-supplement-treated than control ewes. In Exp. 2, the occurrence and magnitude of LH surges were greater (P<0.01) and intervals to onset of LH surge were longer (P<0.01) in P(4)-supplement-treated than control ewes. In Exp. 2, P(4) supplement elevated P(4) levels from 1.8+/-0.1ng/mL on Day -1 to 4.2+/-0.3 on Day 0 (P<0.001). Following pessaries removal, P(4) concentrations fell to basal values on Day 1 in both groups and remained low until Day 5. Then, P(4) concentrations increased and remained elevated through Day 19 in all (100%) progestagen-supplement-treated in Exp. 1 (12/12) and Exp. 2 (14/14) and in only 5/12 (41.7%) and 6/14 (42.9%) control ewes, respectively. These ewes were confirmed pregnant by ultrasonography and lambed on Day 149.2+/-0.2 following Day 0. In conclusion, progestagen supplement 24h prior to removal of pessary can be used successfully to improve reproductive performance of ewes bred out-of-season.     

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.     

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.     

Successful uterine insemination of fallow deer with fresh and frozen semen

Six fallow does were inseminated directly into the uterine horns 72 h (three does) or 78 h (three does) after the removal of progestagen intravaginal sponges. Three does were inseminated with fresh (two at 72 h and one at 78 h) or frozen-thawed (one at 72 h and two at 78 h) semen. The semen used had been collected by electroejaculation and had been stored for 2 yr in liquid nitrogen in a Tris, citric acid, glycerol diluent containing 2.25% egg yolk. Three does each produced a live fawn to insemination and all does had been inseminated 72 h after removal of sponges; two with fresh semen and one with frozen semen. The remaining three does failed to conceive to insemination, but did produce fawns to mating at a subsequent estrus.     

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.     

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.     

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.     

Successful pregnancies after transfer of embryos recovered from ewes induced to ovulate 24-29 days post partum

After lambing in late November, oestrus and ovulation were induced by using a CIDR device and PMSG in early weaned (N = 13) or lactating (N = 14) Border Leicester x Scottish Blackface ewes between 23 and 29 days after parturition. Ewes were intrauterine inseminated under laparoscopic visualization 54-55 h after CIDR-device withdrawal and eggs recovered on Day 3 of the cycle. Ovum recovery and fertilization rates were higher in lactating than in early weaned ewes, with fertilization being achieved as early as 24 days post partum in both groups. Of the 7 early weaned and 11 lactating ewes yielding eggs, fertilization occurred in 4 and 7 ewes respectively. A total of 20 embryos were transferred to the normal uterine environment of 15 recipient ewes in which the interval from parturition was greater than 150 days. Pregnancies were successfully established in 9 recipient ewes, resulting in the birth of 10 viable lambs. Prolactin concentrations were significantly higher (P less than 0.001) in lactating than in early weaned ewes throughout the study. Nevertheless, normal luteal function (as assessed by daily progesterone concentrations) was exhibited by 12 of 14 lactating and 8 of 13 early weaned ewes. Two post-partum donors in which the corpora lutea completely failed to secrete progesterone yielded fertilized eggs which developed to term when transferred to a normal uterine environment. The results show that sheep oocytes can be fertilized using laparoscopic intrauterine insemination as early as 24 days after parturition and that the resulting embryos are viable when recovered on Day 3 after oestrus and transferred to a normal uterine environment.