In vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatment in rabbit does

This study aimed to evaluate the in vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatments in rabbit does. Ninety-six nulliparous does were randomly subjected to consecutive superovulation treatments with eCG (20 IU/kg body weight intramuscularly (i.m.), eCG group), FSH (3 x 0.6 mg/doe at 24 h intervals i.m., FSH group), or without superovulation treatment (control group). Does were artificially inseminated 3 days later and ovulation was induced immediately by hCG (75 IU/doe intravenous). Seven experimental groups were differentiated: first FSH and eCG treatment, second FSH and eCG treatment, eCG-interchanged group (does with previous FSH treatment), FSH-interchanged group (does with previous eCG treatments) and control group. Embryos were collected in vivo by laparoscopy 76-80 h post-insemination in the first and second recovery cycles and post mortem in the third recovery cycles. The ovulation rate was significantly higher in does treated with the first-FSH than in those treated with eCG or in control does (25.2+/-2.0 versus 19.2+/-1.4 to 11.0+/-1.5, and 12.2+/-1.2, first-FSH, first-eCG to second-eCG and control groups, respectively, P < 0.05). Significant differences were observed in the total recovery influenced by ovulation rate in each group (20.3+/-2.2 to 9.4+/-1.2, first-FSH to control groups). Embryo donor rate (donor with at least one normal embryo) was similar among groups with an overall of 75.1%. The number of normal embryos recovered per doe with at least one normal embryo increased significantly in relation to ovulation rate (17.7+/-2.2 to 8.41+/-3, first-FSH and control groups). The vitrification of embryos negatively affected their in vitro development to hatched blastocyst in all groups (88.1% versus 48%, P > 0.05). However, after embryo transfer, this negative effect was only observed in superovulated vitrified embryos (16.8 and 12.8% versus 39.4% total born rate from eCG, FSH and control vitrified groups, P < 0.05). Results indicated that the primary treatments with eCG or FSH increased the number of normal embryos recovered per donor doe, but these embryos are more sensitive to vitrification protocols.     

Effect of eCG dose and ovulation induction treatments on embryo recovery and in vitro development post-vitrification in two selected lines of rabbit does

The aim of this work was to evaluate the effect of different doses of eCG administered subcutaneously (0, 50 and 200 IU) and the hormonal induction of ovulation (GnRH or hCG) on embryo recovery and in vitro development of embryos post-vitrification in two selected lines of rabbit does. The two selected lines were line V (selected for the litter size at weaning) and line R (selected for growth rate). Administration of 200 IU of eCG significantly increased ovulation rate (19.2 +/- 1.2 versus 15.5 +/- 1.1 and 12.2 +/- 1.3, and the number of haemorrhagic follicles (13.8+/-1.6 versus 3.8+ /- 1.4 and 3.8 +/- 1.7), but significantly decreased recovery rate (28.8 +/- 6.3 versus 47.7 +/- 5.7 and 48.7 +/- 6.7, 200 IU versus 50 IU and 0 IU eCG, respectively), the number of normal embryos recovered per doe with at least one embryo (5.8 +/- 0.9 versus 8.2 +/- 0.9, 200 IU versus 50 IU eCG doses) and the in vitro development of embryos post-vitrification (51.9% versus 66.1%, 200 IU versus 50 IU eCG doses, respectively). Inducing ovulation with hCG significantly increased ovulation rate when compared with GnRH (17.3 +/- 0.8 versus 13.8+/-1.4), but no significant differences in embryo recovery and embryo development post-vitrification were observed between the two treatments. No significant differences were observed between the two selected lines in ovulation and recovery rates, the number of haemorrhagic follicles and the number of recovered embryos per doe. However, the post-vitrification in vitro rate of development was 59.7% for line R and 51.9% for line V (p < 0.05). It was concluded that the use of 50 IU of eCG subcutaneous with hCG or GnRH prior to embryo cryopreservation programmes in rabbits achieves the best results for embryo recovery, with the best development of recovered embryos post-vitrification.     

Use of fluorogestone acetate after breeding to reduce the effect of premature luteal regression in dairy goats when superovulation is induced with FSH

The aim of the present study was to determine the effect of fluorogestone acetate (FGA) administered after mating, on embryo production in the dairy goat subjected to conventional superovulatory and embryo recovery protocols. Adult does, most of them of the French Alpine breed, were randomly assigned after a FSH-superovulatory estrus and fertile matings to a control group (n=20) or to a treated group (n=20) in which intravaginal sponges impregnated with FGA were inserted after mating and remove before embryo collection (day 6). Blood samples were collected every 12h from days 1 to 7 post-estrus and serum progesterone concentrations were determined. The FGA-group had a lesser percentage of does with normal corpora lutea (CL) and a greater percentage of animals with CL in regression or mixed (normal and in regression) when compared with the control group (13.3 and 64.7%, 53.3 and 23.5%, and 33.3 and 11.8%, respectively; P<0.05). Mean number of normal CL per doe was less and mean number of regressed CL greater in FGA as compared with the control group (4.2 compared with 10.7 and 8.5 compared with 3.6, respectively; P<0.05). There were no differences (P>0.05) in recovery rate, total number of CL, total recovered structures, oocytes and transferable and non-transferable embryos between groups. Serum progesterone concentrations from day 5 to 7 post-estrus were lower (P<0.05) in FGA as compared with the control group. Percentage of does with luteal failure on day 6 post-estrus was greater in FGA as compared with the control group (86.6 compared with 33.3%; P<0.01). When considering only does with luteal failure on day 6 post-estrus, mean total recovered structures, transferable embryos and percentage of does rendering > or =3 transferable embryos were greater in the FGA compared with the control group (6.3 and 1.3 structures, 4.5 and 1.2 embryos, 67 and 17%, respectively; P<0.05). In does not having luteal failure, FGA administration did not appear to affect embryo production or embryo survival. These results indicate that FGA administration after mating improves embryo recovery in dairy goats with luteal failure after superovulatory treatment. However, it also increases the incidence of luteal regression when administered early in the estrous cycle.     

In vivo oocyte recovery and in vitro embryo production from bovine oocyte donors treated with progestagen, oestradiol and FSH

The effect of treatment of donor cattle with progestagen and oestradiol or FSH on in vivo oocyte recovery and in vitro embryo production was studied. Forty-eight beefxFriesian cows formed eight replicates of six treatments in a 2 (no steroid versus steroid)x3 (none, single or multiple dose(s) of FSH) factorial design in which follicles were aspirated once weekly for 3 weeks. Oocytes were graded, washed, matured for 20-24h and then inseminated with frozen/thawed semen from a single sire followed by coculture on granulosa cell monolayers.Treatment with steroid had no significant effect on any follicular, oocyte or embryo production variate other than to reduce the number (P<0.05) and the diameter of large follicles>10mm (P<0.01) present at aspiration. FSH increased numbers of medium (6-10mm) and large follicles (P<0.01) and there was a corresponding decrease in the number of small follicles (2-5mm; P<0. 01). The total number of follicles at aspiration increased from 17. 7+/-1.60 for animals not treated with FSH to 23.6+/-1.97 following multiple dose treatment with FSH (P<0.05). Significantly, more follicles were aspirated following FSH treatment (no FSH 9.7+/-1.09, single dose FSH 13.6+/-1.30, multiple dose FSH 17.3+/-1.52; P<0.05) and numbers of oocytes recovered per cow per week increased (no FSH 4.1+/-0.76, single dose FSH 5.3+/-0.87, multiple dose FSH 5.9+/-0. 94) but the differences were not significant. Significantly, more good oocytes (Category 1) were recovered from animals treated with FSH (P<0.05). There was no overall significant effect of FSH on embryo production rate or the total number of transferable embryos produced but the number of transferable embryos was highest following administration of multiple doses of FSH.In conclusion, progestagen plus oestradiol 17beta treatment did not affect follicle, oocyte and embryo production of oocyte donors aspirated once per week. FSH treatment, however, significantly increased the number of follicles aspirated and Category 1 oocytes recovered. Multiple dose administration of FSH resulted in the production of the highest number of transferable embryos but this effect was not significant.     

The effect of melatonin implants during the seasonal anestrus on embryo production after superovulation in aged high-prolificacy Rasa Aragonesa ewes

The aim of this study was to assess the effect of melatonin implants administered in March on the ovarian cyclicity, ovulatory response and embryo production after repeated superovulation of selected high-prolificacy Rasa Aragonesa aged ewes. During the seasonal anestrus of two consecutive years, 113 superovulatory treatments have been performed. Ewes were treated (M) or not (C) with melatonin implants in March (day 0). All of them received intravaginal progestogen sponges on day 24 (recovery 1) and 80 (recovery 2) after melatonin implants insertion in year 1, and on day 28 and 77 in year 2. The intravaginal sponges were removed after 14 days. Superovulatory treatments consisted of eight doses in decreasing concentrations (2 mL x 2 and 1 mL x 6) of oFSH (Ovagen) administered twice daily starting 72 h before sponge removal. Seven days after the onset of estrus, embryos were recovered by laparotomy. Melatonin increased cyclicity only in recovery 2 year 2 (83% versus 42%; P < 0.05) but not in the other experimental periods. Among the 78% (88) ewes that ovulated and produced functional corpora lutea, melatonin implants tended to improve embryo viability in recovery 2 by increasing the number of blastocysts per superovulatory treatment (2.4 +/- 0.6 versus 1.1 +/- 0.4; P = 0.09), the rate of viability (67 +/- 9% versus 43 +/- 9%; P < 0.05), and freezability (55 +/- 9% versus 33 +/- 8%; P < 0.05). More specifically, melatonin induced a significant reduction of the number and rate of non-viable (degenerate and retarded) embryos in recovery 2 (0.4+/-0.1 embryos versus 1.3 +/- 0.3 embryos and 4 +/- 1% versus 22 +/- 6%, respectively; P < 0.05). Our results demonstrate that melatonin implants in March can improve at medium term (3 months after implantation) the viability of embryos collected from selected high-prolificacy Rasa Aragonesa aged ewes after superovulation.     

Season affects characteristics of the pre-ovulatory LH surge and embryo viability in superovulated ewes

The aim of this study was to determine whether there are seasonal shifts in ovulatory response, and in the viability of ova recovered from superovulated ewes. Fifty mature ewes underwent a standard oestrous synchronisation (CIDR), superovulation (oFSH) and artificial insemination procedure during October (peak breeding season) and April (transition to anoestrus). In each month peripheral LH and progesterone concentrations were measured around the time of ovulation and embryos were recovered, graded and cryopreserved on day 6 after insemination. During the subsequent breeding season, grade 1 and 2 morulae and unexpanded blastocysts were thawed and transferred singly to synchronous recipients (October, n = 40; April, n = 40) or cultured in vitro for 18-20 h (October, n = 107; April, n = 98). Following culture, viable embryos were stained to count cell nuclei or assayed to measure their capacity for glucose metabolism ([3H]glucose) and protein synthesis ([35S]methionine). Peak LH concentrations were higher in October than in April (38.2 +/- 3.26 ng ml(-1) versus 25.7 +/- 1.99 ng ml(-1), respectively; P < 0.01) and the pre-ovulatory LH surge was advanced by approximately 3 h (P < 0.05). Progesterone concentrations at CIDR withdrawal were lower in October than in April (3.1 +/- 0.16 ng ml(-1) versus 4.3 +/- 0.19 ng ml(-1), respectively; P < 0.001) but were not different at embryo recovery. Season did not affect the numbers of corpora lutea per ewe or the numbers of ova recovered but the proportion of recovered ova that was unfertilised/degenerate was lower in October than in April (0.43 versus 0.58, respectively; P < 0.001). For embryos containing more than 16 cells, there was no effect of season on the median stage of development or morphological grade. The proportions of October and April embryos that established pregnancy following transfer to recipient ewes were 0.78 and 0.70 (not significantly different), and that were viable after in vitro culture were 0.66 and 0.37 (P < 0.05), respectively. Season did not affect the number of nuclei per viable embryo or the capacity for protein synthesis but the glucose uptake of October embryos was approximately double that of April embryos (3163+/-293.4 dpm versus 1550+/-358.9 dpm, respectively; P < 0.05). Results indicate that during the late compared to peak breeding season, there is an increased incidence of fertilisation failure as a possible consequence of seasonal shifts in LH secretion and (or) associated effects on follicular function. Frozen-thawed embryos produced at contrasting stages of the breeding season are equally viable in vivo but those produced during the late, as opposed to the peak breeding season have lower viability following in vitro culture.     

Improvement in embryo recovery using double uterine flushing

The objective of the present study was to evaluate the effects of double uterine flushing on the recovery of embryos/ova in cattle. Two hundred and ten embryo recovery procedures were conducted using a double uterine flushing method, and the results were compared with 432 conventional single-flushing procedures. Cyclic Limousin (n = 403) and Guzera (n = 239) donor cows received an intravaginal progesterone releasing device and 2 mg of estradiol benzoate on Day 0. Between Days 5 and 9, donors received decreasing doses of FSH, which ranged from 200 to 300 IU (Bos indicus) and 300 to 500 IU (Bos taurus). On the afternoon of Day 7, donors received an injection of 500 microg cloprostenol and progesterone implants were removed 12 h later (morning of Day 8). Artificial insemination was performed between 14 and 26 h after first detection of behavioral estrus. Cows were randomly assigned to have embryos recovered by a double-flushing method (n = 210) or the conventional single-flushing procedure (n = 432). For the double-flushing procedure, after first flushing the whole uterus with 1L of Dubelco's Phosphate Buffered Saline (DPBS), a Foley catheter was positioned in the uterine body to permit refilling of the uterus with fresh DPBS (80-150 mL). The catheter was closed with the plunger of a disposable 5 mL syringe, and the donors were allowed to rest in a holding area for 30 min. Thereafter, a second flush was performed to recover the solution remaining in the uterus. Animals from the control group were subjected to a single uterine flush. From 210 double-flushing procedures, 1409 viable embryos were recovered. In comparison, from 432 cows receiving the single-flushing procedure, 1993 embryos were recovered. Double flushing increased (P < 0.05) the number of embryos recovered per procedure compared to single flushing (6.7 +/- 0.4 versus 4.6 +/- 0.2, respectively; mean +/- S.E.M.). When double flushing was performed, average recovered embryos/ova increased (P < 0.05) from 8.3 +/- 0.4 to 12.7 +/- 0.7 in Limousin and from 7.9 to 11.5 in Guzera. Also, utilization of double flushing increased (P < 0.05) the number of viable embryos from 4.7 +/- 0.3 to 6.9 +/- 0.5 in Limousin and from 4.5 +/- 0.4 to 6.4 +/- 0.7 in Guzera. Mean total embryos/ova was similar (P > 0.05) between the control group and after the first uterine flushing in the double-flushing group; therefore, both flushings were conducted efficiently. In conclusion, double uterine flushing increased embryo recovery in cattle.     

Repeat superovulation, non-surgical embryo recovery, and surgical embryo transfer in transgenic dairy goats

We investigated the capability of repeat superovulation and non-surgical embryo retrieval, coupled with surgical embryo transfer, to expedite the production of transgenic progeny from transgenic founder dairy goat does. In addition, we compared embryo yields, number of embryos transferred per recipient, pregnancy rates, and offspring born during both the traditional (September-December) and non-traditional (January-May) breeding seasons. Although there were no significant differences, there were numerically more transferable embryos recovered per flush (3.5+/-0.9 vs. 2.4+/-0.9 embryos; mean+/-S.E.M.) and increases in both the proportion of recipients that were pregnant (83 vs. 69% pregnant) and offspring born from total embryos transferred (67 vs. 53% offspring) during the traditional versus the non-traditional breeding season. The transfer of one, two or three embryos did not significantly affect the proportion of pregnant recipients during either season. However, there was a difference (P<0.05) in the proportion of offspring produced for one versus two embryo transfers (89 vs. 44% offspring, respectively) during the non-traditional breeding season. Overall, 14 transgenic offspring were produced from 54 total offspring born, and the kidding interval was reduced to <3 months for six of the seven transgenic does. In summary, repeat superovulation and non-surgical embryo retrieval, coupled with surgical embryo transfer, expedited the production of progeny from transgenic founder does.     

Superovulation and embryo recovery from peripubertal Holstein heifers

The use of peripubertal donors in embryo transfer (ET) programs presents significant opportunity to accelerate genetic gain in domestic livestock by reducing the generation interval. These studies were designed to evaluate feasibility of superovulation and embryo recovery in peripubertal heifers (starting at 7.8 months of age), and to determine whether subsequent reproductive and lactational performance of donor heifers were impaired. Study 1 utilized 10 pairs of contemporary full-sibling heifers in which one heifer in each pair was assigned to receive a superovulation regimen and her full-sibling contemporary received placebo. Treated heifers were artificially inseminated at estrus and embryos were flushed transcervically 4-6 days later. Based on recovery of oocytes and/or embryos, 9 of 10 heifers responded to the hormonal regimen and 12 total embryos were recovered. Seven embryos (58%) were transferred into recipients resulting in five pregnancies. Control and treated heifers remained in the herd and were bred at a natural estrus by AI at 15 months of age. Lactation records, i.e., 305 days mature equivalent (305 d ME) were obtained, and all animals were evaluated for udder conformation traits between 32 and 38 months of age. Reproductive traits (age at first calving and days to conception) and lactational traits of heifers subjected to embryo transfer and their non-treated full-siblings did not differ (P > 0.05). Study 2 was conducted to establish the commercial feasibility of hormonally programming peripubertal heifers ranging in age from 7.8 to 9.9; 10 to 11.9; 12 to 13.9 and >/= 14 months. In total, 3982 embryos were recovered from 520 heifers, with 2419 (60.7%) of those categorized as viable (transferable). The number of ova/embryos obtained per flush (5.6 +/- 1.0) and the number of transferable embryos (2.8 +/- 0.5) was reduced (P < 0.05) in heifers of age 7.8-9.9 months compared to all other age groups. There was no difference (P > 0.05) in the number of ova/embryos recovered (7.8 +/- 0.3), or the number of transferable embryos (4.8 +/- 0.2), among heifers that were >/=10 months of age. The number of unfertilized ova did not differ by age, however, more degenerate embryos tended to be recovered from heifers <10 months of age compared to heifers >/=14 months of age. These data indicate that transferable embryos can be safely recovered from heifers beginning at 10 months of age without compromising subsequent reproductive or lactational performance of the donor.     

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).     

Embryo transfer in fallow deer (Dama dama): superovulation, embryo recovery and laparoscopic transfer of fresh and cryopreserved embryos

Multiple ovulation-embryo transfer (MOET) protocols for farmed fallow deer (Dama dama ) were investigated in a series of 3 experiments. A total of 37 donors, of either European (D.d. dama ; n = 30) or Mesopotamian hybrid (D.d. mesopotamica x D.d. dama ; n =7) genotype, each received an intravaginal silastic device containing 0.3 g progesterone (CIDR((R))-type G device) for 14 d and injections of 0.5 units ovine FSH (8 x 0.06 unit injections from Days 10 to 14 of device insertion) and 100 IU PMSG (either with the first or last FSH injection). All donors received laparoscopic intrauterine inseminations of fresh semen (50 x 10(6) spermatozoa) from a Mesopotamian sire 36 h after withdrawal of CIDR((R)) devices. Embryos were recovered by laparotomy on Day 6 (Day 0 = estrus). Mean ovulation rates for the 3 experiments were 8.1, 9.8 and 7.0, with no effect of PMSG timing (P>0.10). However, embryo recovery rates, albeit low throughout the study (29.6%), were significantly improved with later PMSG administration (33.9 vs 20.1%; P<0.05). Hybrid and European donors performed in a similar manner. A range of embryo development stages was recovered throughout the study. In 2 experiments laparoscopic transfer of embryos to 48 recipient does treated previously with intravaginal CIDR((R)) devices for 14 d yielded a total pregnancy rate of 37.5%. In the experiment with fresh embryos, the use of clenbuterol to reduce uterine turgidity resulted in a higher proportion of does conceiving (3 4 ; 75%) compared with that of the untreated does (0 6 , 0%; P<0.05). In the second experiment, in which all the does routinely received clenbuterol, 10 19 (53%) and 5 19 (26%) does conceived following the transfer of fresh and cryopreserved embryos, respectively (P<0.05). While the overall efficiency of the MOET program was low (equivalent of 0.9 to 1.0 surrogate pregnancies per donor), improvements in the recovery rate of transferable embryos have considerable potential for genetic improvement of farm stock and captive propagation of endangered Mesopotamian fallow deer through maternal surrogacy programs.     

Collection and transfer of multiple embryos in the mare

A pituitary extract was used to induce multiple ovulations in mares to determine whether day-7 embryos from multiple ovulators were viable as indicated by their ability to develop when transferred to recipients. There were more ovulations/donor for induced multiple-ovulating mares than for control single-ovulating mares (4.6 +/- 0.5 vs 1.0 +/- 0.0; n=14). The embryo collection rate per ovulation was similar for multiple ovulators (0.6 +/- 0.1 embryos/ovulation) and single ovulators (0.7 +/- 0.1). The embryo collection rate per donor, therefore, was higher (P<0.01) for the multiple ovulators (2.9 +/- 0.7 vs 0.7 +/- 0.1). The transfer success rate per embryo at day 21 was different (P<0.05) among recipients which received an embryo from control single-ovulating donors (7 8 ), multiple ovulators from which a single embryo was recovered (2 2 ), and multiple ovulators from which multiple embryos were recovered (9 19 ). The recipient pregnancy rate/donor at day 21 was 88% (7 8 ) for single-ovulating controls and 138% (11 8 ) for induced multiple ovulators. Results indicate that the survivability of day-7 embryos from multiple-ovulating donors was reduced. However, despite the reduced survival rate/embryo, the number of pregnant recipients/donor was increased by induction of multiple ovulations because of the increased number of embryos available for transfer.     

Repeated superovulation using a simplified FSH/eCG treatment for in vivo embryo production in sheep

This study investigated the efficacy of a simplified repeated superovulation treatment (eCG plus FSH in a single dose, rather than the usual protocol of six decreasing doses of FSH) in the in vivo embryo production in Ojalada donor ewes during the breeding season. In vitro viability after vitrification and warming of embryos recovered from both treatments was also assessed. In addition, the study examined the effects of the concentration of anti-eCG antibodies before each eCG/FSH treatment on in vivo embryo production. Thirty-eight females at the end of their reproductive lives were given the decreasing (n = 19) or simplified (n = 19) superovulatory treatment up to three times at intervals of ≥ 50 d. The onset of estrus was 5 h earlier (P < 0.05) among ewes that received the eCG/FSH protocol (25.2 ± 0.80 h) than it was among those that received the decreasing superovulatory treatment (30.1 ± 1.0 h), but the two treatments did not differ significantly in ovulation rates or the number and viability of embryos recovered. Both of the superovulatory protocols were significantly (P < 0.05 to P < 0.01) less effective after the first application. After three superovulatory treatments, the average number of viable embryos per ewe was 14.1 ± 2.3 and 13.7 ± 2.5 in the decreasing and simplified protocols, respectively. High anti-eCG antibody concentrations just before the superovulatory treatment with eCG/FSH were associated with a significant decrease (P < 0.05) in the rates of fertilization, viability, and freezability, especially in the second and third recoveries. Repeated superovulatory treatments with eCG/FSH can provide an efficient means of producing high quality embryos in the ewes of endangered breeds at the end of their reproductive lives, although further studies are needed to characterize the response associated with high concentrations of anti-eCG antibodies.     

Factors affecting success of embryo collection and transfer in a transgenic goat program

During a goat transgenic program that took place in Israel from July 1995 to February 1996, Saanen (n = 343) and Nubian x Damascus (n = 378) crossbred goats of mixed ages were used as donors (n = 433) and recipients (n = 288). The effects of season, age, number of surgical procedures, previous hormonal treatments and ovulation rate on the number of microinjectable embryos collected were studied. Likewise, the effects of these parameters on the pregnancy rate as well as the number of embryos transplanted, endogenous progesterone concentrations and exogenous progesterone supplementation were studied in recipient does. Following superovulation with ovine follicle stimulating hormone, 85% of the does responded with 13.6 +/- 5.7 (mean +/- S D) ovulations/doe. Age, month and number of previous hormonal treatments significantly affected the ovulation rate. The average recovery rate was 70%, and it was affected only by the ovulation rate. Pronuclei were visualized in about 30% of the flushed embryos (including unfertilized ova), and those were microinjected with human serum albumin gene construct. About 68% of the injected embryos underwent at least one division during an overnight incubation, and those embryos were transferred, giving about 2.0 transferred embryos per ovulated donor. Of the recipients, 86% responded following synchronization with 3.1 +/- 1.6 (mean +/- S D) ovulations per doe. Breed and month had a significant effect on the ovulation rate. Two or three microinjected embryos were transferred to each recipient, resulting in more than a 40% pregnancy rate during September to November. Lower pregnancy rates were obtained before and after that period. By monitoring plasma progesterone concentrations in the recipients it was found that progesterone concentration was correlated with the ovulation rate. However, the pregnancy rate was not affected by progesterone concentration. During January and February, 30 to 50% of the recipients failed to develop functional corpora lutea (CL) following embryo transfer, which explained the lower pregnancy rate in those months. Of the 86 kids born 4 were transgenic.     

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.     

Superovulatory response, embryo quality and fertility after treatment with different gonadotrophins in native cattle

We studied native Mertolengo cattle to evaluate superovulatory (SOV) treatments, subsequent fertility of donors and pregnancy rate of recovered embryos. In Experiment 1 we compared superovulatory response (SR), embryo quality and plasma progesterone (P4) levels between donors treated with eCG (10 cows and 5 heifers) vs. FSH (pure, FSH-1, n=10 cows and crude, FSH-2, n=10 cows), during progestagenic impregnation. We also compared fertilization rates and embryo quality of bred and inseminated eCG and FSH-1 donors. Significantly more viable embryos were yielded by FSH than by eCG treated donors. Less FSH-1 than FSH-2-treated donors showed SR, but the response was identical in responder donors of both groups. Fertilization rates were significantly higher in bred than in inseminated donors. Plasma P4 levels were only significantly different (higher) between responder and non-responder donors on the day of embryo recovery. Experiment 2 compared FSH treatments (FSH-2, crude, n=11 cows and FSH-3, pure, n=10 cows) started at the midluteal phase. The mean number of viable embryos was significantly higher in FSH-3 than in FSH-2 treated donors. Both FSH treatments exerted a similar luteotrophic effect upon injection. The FSH-2 donors treated during the midluteal phase yielded more ova and showed significantly higher plasma P4 levels at all sampling days than those treated during progestagenic impregnation. The pregnancy rates of recipient cows were 67% and 46% for fresh and frozen-thawed embryos respectively. In Experiment 3, the fertility of donors (n=20) after SOV treatments was compared with that of untreated cows (n=40). Time to conception of donors, after mating with a bull 14 days after embryo recovery, was identical to that of control cows. There was some delay to conception in eCG-treated cows, but the difference was not significant. These preliminary results suggest that response to SOV treatments in Mertolengo cattle might be affected by the type of gonadotrophin and by the treatment protocol. The fertility of a traditional breeding season after SOV treatments was not impaired. Cryopreserved embryo banking can be used to preserve the breed.     

Treatment with recombinant equine follicle stimulating hormone (reFSH) followed by recombinant equine luteinizing hormone (reLH) increases embryo recovery in superovulated mares

The dynamics of ovarian follicular development depend on a timely interaction of gonadotropins and gonadal feedback in the mare. The development and efficacy of genetically cloned recombinant equine gonadotropins (reFSH and reLH) increase follicular activity and induce ovulation, respectively, but an optimum embryo recovery regimen in superovulated mares has not been established. The objective of this study was to determine if treatment with reFSH followed by reLH would increase the embryo per ovulation ratio and the number of embryos recovered after superovulation in mares. Sixteen estrous cycling mares of light horse breeds (4-12 years) were randomly assigned to one of two groups: Group 1; reFSH (0.65mg)/PBS (n=8) and Group 2; reFSH (0.65mg)/reLH (1.5mg) (n=8). On the day of a 22-25mm follicle post-ovulation mares were injected IV twice daily with reFSH for 3 days (PGF(2α) given IM on the second day of treatment) and once per day thereafter until a follicle or cohort of follicles reached 29mm after which either PBS or reLH was added and both groups injected IV twice daily until the presence of a 32mm follicles, when reFSH was discontinued. Thereafter, mares were injected three times daily IV with only PBS or reLH until a majority of follicles reached 35-38mm when treatment was discontinued. Mares were given hCG IV (2500IU) to induce ovulation and bred. Embryo recovery was performed on day 8 day post-treatment ovulation. Daily jugular blood samples were collected from the time of first ovulation until 8 days post-treatment ovulation. Blood samples were analyzed for LH, FSH, estradiol, progesterone and inhibin by validated RIA. Duration of treatment to a ≥35mm follicle(s) and number of ovulatory size follicles were similar between reFSH/reLH and reFSH/PBS treated mares. The number of ovulations was greater (P<0.01) in the reFSH/reLH group, while the number of anovulatory follicles was less (P<0.05) compared to the reFSH/PBS group. Number of total embryos recovered were greater in reFSH/reLH mares than in the reFSH/PBS mares (P≤0.01). The embryo per ovulation ratio tended to be greater (P=0.07) in the reFSH/reLH mares. Circulating concentrations of estradiol, inhibin, LH and progesterone were not statistically different between groups. Plasma concentrations of FSH were less (P<0.01) in the reFSH/reLH treated mares on days 0, 1, 4, 6, 7 and 8 post-treatment ovulation. In summary, reFSH with the addition of reLH, which is critical for final follicular and oocyte maturation, was effective in increasing the number of ovulations and embryos recovered, as well as reduce the number of anovulatory follicles, making this a more viable option than treatment with reFSH alone. Further evaluation is needed to determine the dose and regimen of reFSH/reLH to significantly increase the embryo per ovulation ratio.     

Effects of lactation length and an exogenous progesterone and estradiol-17beta regimen during embryo attachment on endogenous steroid concentrations and embryo survival in sows

The hypotheses that short lactation lengths increase embryo mortality by altering endogenous post-weaning steroid concentrations, and that an exogenous steroid regimen during embryo attachment might increase embryo survival were tested using 36 s parity sows assigned randomly to a 2 x 2 factorial. Sows were subjected to either a short lactation (SL, 13.0 days, n = 25) or a long lactation (LL, 31.5 days, n = 11), artificially inseminated at first estrus and treated daily with 2 ml i.m. of either 25 mg progesterone (P4) and 1.25 pg estradiol-17beta (E2) (steroid treatment, ST, n = 17) or the vehicle alone (control treatment, CT, n = 17) on Days 14-20 post-insemination. Blood samples were collected by jugular venipuncture from weaning to 24 days post-insemination on alternate days. Sows subjected to the SL compared to the LL tended to have a longer weaning-to-estrus interval (WEI) (5.3 versus 4.6 days; P < 0. 10), but did not have a significantly reduced conception rate (CR) (71 versus 90%; P > 0.10). The SL and LL sows had a similar ovulation rate (19.9 versus 21.3 corpora lutea, CL; P > 0.05), but SL sows had fewer viable embryos than LL sows (11.5 versus 15.3; P < 0.05) when reproductive tracts were recovered 28-32 days post-insemination. In addition, even after correction for the difference in number of embryos between groups, viable embryos from the SL versus the LL group weighed less (1.63 versus 1.79 g; P < 0.05), had a decreased amnion volume (1.02 versus 1.22 ml; P < 0.05) and apparently produced less estrogens since estrone sulfate concentration was decreased at 24 days post-insemination in SL versus LL sows (4.3 versus 6.3 ng/ml; P < 0.05). Embryo survival (percentage of CL represented by a viable embryo) however, was not different between SL and LL sows (60 versus 74%; P > 0.05) and no differences in post-weaning P4 or E2 concentrations were apparent. Sows that received the ST only tended to have increased P4 concentrations at 16 days post-insemination compared to CT sows and neither the number of viable embryos, nor embryo survival, was increased in ST versus CT sows (14.7 versus 12.2; P > 0.05 and 66 versus 68%; P > 0.05, respectively). These data suggest that short lactations do not increase embryo mortality by inducing aberrant endogenous post-weaning P4 or E2 concentrations. It is unclear whether or not small, repeated doses of exogenous P4 and E2 during attachment can increase embryo survival.     

In vivo oocyte recovery and in vitro embryo production from bovine donors aspirated at different frequencies or following FSH treatment

The effects of frequency of follicular aspiration and treatment of donor cattle with FSH on in vivo oocyte recovery and in vitro embryo production were studied. Simmental heifers (n = 24) formed 8 replicates of 3 treatments in which oocyte donors were aspirated 1) once a week, 2) twice a week, or 3) once a week following treatment with FSH for 3 d prior to aspiration. Oocytes were graded, washed, matured for 20 to 24 h and then inseminated with frozen/thawed semen from a single sire, followed by co-culture on granulosa cell layers. Embryo development was observed until Day 7 after insemination. Significantly fewer follicles per heifer per week were counted (14.7+/-2.3 vs. 27.4+/-3.1 vs. 23.1+/-2.8) and aspirated (12.0+/-2.0 vs. 21.8+/-2.7 vs. 20.1+/-2.6) in heifers on the once-weekly than twice-weekly aspiration treatment (P<0.01) or on the once-weekly aspiration after FSH treatment (P<0.05). There were no significant differences between treatments in the total number of oocytes recovered per week (5.6+/-1.2 vs. 8.9+/-1.5 vs. 6.1+/-1.2), but significantly more oocytes per heifer per week recovered from animals treated with FSH were graded Category 1 (2.8+/-0.4), i.e., >4 layers good cumulus with a clear, even cytoplasm, than from animals aspirated once (0.9+/-0.2; P<0.01) or twice a week (1.5+/-0.3; P<0.05). The number of transferable morulae plus blastocysts produced per heifer per week was higher from animals aspirated twice a week (2.4+/-0.4; P<0.05) or once a week following FSH treatment (2.1+/-0.4; P<0.05) than from animals aspirated once a week without FSH treatment (1.0+/-0.3). In conclusion, FSH treatment of bovine oocyte donors aspirated once a week enabled a similar number of transferable embryos to be produced per donor week as aspiration twice a week without FSH treatment. These 2 treatments produced twice as many transferable embryos per donor week as aspiration once a week without FSH treatment.     

Effects of superovulation, embryo recovery, culture system and embryo transfer on development of rabbit embryos in vivo and in vitro

Uninterrupted development of rabbit embryos in vivo was studied in 7 superovulated and 7 normally ovulating (GnRH-treated) does, while another 7 does were superovulated and 1-cell embryos were collected from them at 19 h after LH to compare development in vivo and in vitro. Embryos from the last group were either cultured in the presence or absence of rabbit oviduct epithelial cells for 65 h in Medium 199, or were immediately transferred to recipients. At 84 h after LH or GnRH, blastomere number, embryo volume and stage of development were assessed for all embryos. Intrazonal embryo volumes were significantly reduced in embryos recovered from superovulated donors. Superovulation also had a negative effect on embryo cell numbers. However, this reduction was more severe in embryos remaining in vivo in superovulated donors until 84 h after LH than it was in embryos transferred to nonsuperovulated recipients at the 1-cell stage (19 h after LH). The embryo recovery procedure apparently caused little harm to the embryos, except that the mucin layer on flushed and immediately transferred embryos was significantly thinner than that of embryos residing continuously in vivo. Co-culture with rabbit oviduct epithelial cells resulted in improved development in vitro, but this development was still significantly retarded compared with embryos developing in vivo.