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.     

Embryo survival and conceptus growth after reciprocal embryo transfer between Chinese Meishan and Landrace x Large White gilts

Embryos were transferred between Meishan and Landrace x Large White (control) gilts on Day 4 or 5 to establish approximately equal numbers of all four possible combinations of donor breed and recipient breed. The breed of the donor gilt significantly (P less than 0.01) affected embryo survival with 44.5% of transferred Meishan embryos and 69.6% of transferred control embryos surviving to Day 30 +/- 1. There was no influence of the breed of the recipient gilt on the proportion of embryos which survived. These differences in embryo survival between the two breeds could not be explained by differences in (1) the number of embryos transferred, (2) the stage of development of the embryos transferred, (3) the interval between ovulation and transfer or (4) the degree of asynchrony between donor and recipient gilt. On Day 30 +/- 1 embryos from control donors developed into longer fetuses (P less than 0.01) with larger allantoic sacs (P less than 0.05) than did embryos from Meishan donors. Fetuses in control recipients were longer (P less than 0.01), heavier (P less than 0.001) and had larger allantoic sacs (P less than 0.05) than fetuses occupying Meishan uteri. The interaction between breed of donor gilt and breed of recipient gilt did not significantly affect conceptus growth. These results suggest that Meishan pig embryos may be less tolerant to routine embryo transfer procedures than those of control gilts, that the genotype of the dam does not affect the proportion of embryos surviving to Day 30 +/- 1, and that both fetal and maternal factors affect conceptus growth.     

Factors affecting the survival of sheep embryos after transfer within a MOET program

Multiple ovulation and embryo transfer (MOET) has the potential to increase the rate of genetic improvement in sheep. However, better realization of this potential requires maximum survival rates of transferred embryos of high genetic merit after transfer into recipient ewes. These studies were therefore conducted to investigate the effect of both embryonic and recipient ewe factors on the survival rate of transferred embryos. Survival rate was similar after transfer of morula or blastocyst stage embryos, and these were higher (P<0.05) than for very early morulae and early morulae. Advanced embryos (Day 5 blastocyst) had an advantage (P<0.05) in survival rate over retarded embryos (Day 6 morula). Grades 1 and 2 embryos survived significantly (P<0.05) better than Grades 3 or 4 embryos. There was no difference in embryo survival rate following transfer to recipients with different numbers of corpora lutea. In general, age or parity of recipient ewes did not affect embryo survival rate, although a higher (P<0.05) embryo survival rate was observed for yearling recipients. Buserelin (GnRH agonist) treatment of recipient ewes 5 or 6 days after transfer of embryos (Day 12 of the cycle) did not improve embryo survival rate. These results confirm that both embryonic and recipient factors can play an important role in the success of a MOET program in sheep.     

Embryo survival in pseudopregnant and in pregnant but genetically semi-sterile recipients after nonsurgical embryo transfer in the mouse

A new nonsurgical embryo transfer technique was used in the mouse that yielded survival rates of between 40 and 70% depending on embryo stage and, possibly, on the degree of synchrony between the embryo and recipient. Three variables were tested using this embryo transfer technique: a) pseudopregnant recipients vs pregnant but genetically semi-sterile recipients, b) embryos resulting from superovulation vs embryos from natural ovulation, and c) 12-hour vs 24-hour asynchrony between donors and recipients. None of these variables significantly affected the pregnancy rate or the percentage of transferred embryos developing to term. The pregnancy rates were between 77 and 90% in 6 experimental groups of 8 to 13 females. Survival rates were between 41 and 63% when all recipients were considered and between 53 and 68% when only the pregnant recipients were included. The embryo transfer procedure influenced litter size composition of the endogenous conceptuses of the semi-sterile recipients. Too many females were devoid of these. Recipients of expanded blastocysts had significantly better transfer results than recipients that also received morulae and early blastocysts. It was concluded that the transfer success rates were influenced by the recipients and possibly by their preparation for transfer.     

Superovulation treatments and embryo transfer in Angora goats

A high incidence of early luteal regression after PMSG superovulation was associated with low recovery of embryos from reproductive tracts of Angora goats flushed later than Day 5 after onset of oestrus. Embryos were successfully recovered (mean 7.9/female) by flushing on Days 2-5. Mean ovulation rate after an FSH regimen (16.1 +/- 0.8) was significantly higher than that after a single injection of PMSG (10.8 +/- 1.2). Fertilization rate and survival of embryos following transfer to naturally synchronized recipient feral goats did not differ between the two gonadotrophin regimens: the mean number of kids born to 47 donors treated with FSH (7.5 +/- 0.6) was significantly greater than that to 28 donors treated with PMSG (4.8 +/- 0.6). Irrespective of hormonal treatment, the numbers of embryos recovered and of kids born were correlated with ovulation rate (r = 0.82, P less than 0.001 for both). Embryo survival was influenced by ovulation rate in recipients, with 52%, 63% and 75% of transferred embryos being carried to term by recipients with 1,2 and 3 CL, respectively (P less than 0.01). More embryos survived (65%) when 2 embryos were transferred to each recipient than when 1 (51%) or 3 (48%) were transferred. In recipients receiving 2 embryos, survival was significantly improved by transfer of both embryos to the same oviduct (70%) than when one was transferred to each oviduct (62%). The percentage survival of embryos was optimal when oestrus of recipients was synchronized within +/- 1 day of oestrus in donors.     

Donor and recipient ewe factors affecting in vitro development and post-transfer survival of cultured sheep embryos

Donor and recipient factors were assessed during development of embryos following superovulation, collection at the pronuclear and two-cell stage, culture in Synthetic Oviduct Fluid medium for 5 days and twin transfer into synchronised recipients to elucidate what factors affect embryo development and post-transfer survival. In particular, the administration of exogenous progesterone to recipients using an intravaginal CIDR^TM device immediately following embryo transfer was investigated.

From 138 embryos collected from 30 donor ewes, 75% (103) were of transferable quality following culture, of which 100 were transferred to 50 recipients. There was significant variation (P < 0.001) in embryo development to the blastocyst stage between different donor ewes, but this was not related to the donor ovulation rate. At ultrasound sonography (approximately Day 60 of pregnancy), 58% of recipients were pregnant and 42% embryos had survived. Donor ovulation rate was related to embryo survival (P < 0.05) after transfer; the survival rate of embryos from ewes with high ovulation rates was lower than that of embryos from ewes with low ovulation rates. Exogenous progesterone supplementation following transfer did not affect embryo survival, rate of embryo development or plasma progesterone levels. In general, the results from this study suggest that factors other than efficacy of embryo culture can affect the outcome of embryo survival following transfer and that, where possible, these factors should be considered and balanced in experimental designs.     

Donor category and seasonal climate associated with embryo production and survival in multiple ovulation and embryo transfer programs in Holstein cattle

The present study investigated the effect of Holstein donor category (cows vs. heifers) and climate variation (hot vs. cooler season) on the efficiency of in vivo embryo production programs as well as embryo survival after transferred to Holstein recipient cows. A total of 1562 multiple ovulation (MO) procedures (cows: n = 609, and heifers: n = 953) and 4076 embryo transfers (ETs) performed in two dairy herds were evaluated. Donor cows had greater number of CLs (10.6 ± 0.6 vs. 7.5 ± 0.4; P < 0.0001) and ova/embryos recovered (7.6 ± 0.6 vs. 4.6 ± 0.4; P < 0.0001) compared with donor heifers. However, fertilization rate (47.9 vs. 82.4%; P < 0.0001) and proportion of transferable embryos (31.5 vs. 67.4%; P < 0.0001) were lower in donor cows than heifers, respectively. Regardless of donor category, the proportion of freezable embryos was less (P < 0.001) during hot season than in cooler season (21.4 vs. 32.8%). However, greater decline in the proportion of freezable embryos during the hot season was observed in cows (21.7 vs. 10.7%) compared with heifers (46.2 vs. 38.1%; P = 0.01). In contrast, the season on which the embryo was produced (hot or cool) did not affect pregnancy rate on Day 31 (30.5 vs. 31.7%; P = 0.45) and 45 (25.3 vs. 25.1%; P = 0.64) of pregnancy. Regardless of the season in which the embryos were produced, embryonic survival after transferring embryos retrieved from donor cows was greater on Days 31 (36.0 vs. 30.7%; P = 0.001) and 45 (28.3 vs. 23.1%; P = 0.001) of pregnancy when compared with embryos from donor heifers. In conclusion, MO embryo production efficiency decreased during the hot seasons both in cows and heifers; however, the decline was more pronounced in donor cows. Regardless of the embryo source, similar pregnancy rate was observed in the recipient that received embryos produced during the hot and cooler seasons. Curiously, embryos originating from donor cows had higher embryonic survival when transferred to recipient cows than embryos originating from heifers.     

Failure to transmit scrapie infection by transferring preimplantation embryos from naturally infected donor sheep

The objective of the study was to examine whether or not the preimplantation embryo can act as a carrier of classic scrapie infection. The study was carried out on quarantined premises with sheep of highly susceptible scrapie genotypes. Uninfected embryos, collected from New Zealand–derived Suffolk ewes, were surgically transferred into recipient ewes that were also of New Zealand origin. Seventeen negative control lambs were born on the study premises from these embryo transfers. Thirty-nine experimental lambs were from embryos collected from naturally infected donor ewes. The experimental lambs were also born on the study premises after their surgical transfer into recipient ewes of New Zealand origin. These embryos had been collected from donor ewes in a scrapie-infected flock where the ewes were clinically sick with scrapie or developed clinical scrapie after embryo collection. All lambs were confirmed as scrapie susceptible of the ARQ/ARQ genotype. Twenty-eight experimental animals survived to the end point of the study at 5 yr of age with a mean survival of 1579 d. In the negative control group, 12 of 17 sheep survived to 5 yr of age with a mean survival of 1508 d. Postmortem examinations were carried out on all animals derived by embryo transfer, and in none was histologic or immunohistochemical evidence of scrapie found. In contrast, in the originating flock the majority of scrapie cases occurred in ARQ/ARQ genotyped animals where a 56% mortality from scrapie had been recorded in animals of this genotype. Thus, the study provides no evidence for transmission of scrapie and reinforces published evidence that vertical transmission of scrapie may be circumvented by embryo transfer procedures.     

Improvement of the nuclear transfer efficiency by using the same genetic background of recipient oocytes as the somatic donor cells in goats

We have compared the effect of the genetic background of recipient oocytes on the in vitro and in vivo development of nuclear transfer reconstructed embryos in goats. Adult fibroblast cells from Boer goats were used as donor cells, and recipient oocytes were obtained from Boer goats and Boer cross-breeds (Boer♂×Huanghuai♀). Nuclear transfer reconstructed embryos were cultured in vitro, or transferred into recipient goats. The mitochondrial origin of 2 cloned Boer goats was investigated by analysing the D-loop region based on polymorphisms via DNA sequencing. There was no significant difference in the fusion rate and cleavage rate of reconstructed embryos (P>0.05), when using Boer and cross-breeding goat oocytes as recipient cytoplast respectively. However, in vitro morula development of reconstructed embryos from Boer oocytes was significantly higher than that of cross-breeding embryos (34.1% versus 19.1%, P<0.05). There was no significant difference in the rate of pregnancy and foetus loss between the 2 breeds. However, the live-birth rate was significantly higher with Boer goat oocyte recipients than the cross-breeds (3.1% versus 0.8%, P<0.05). Mitochondrial analysis showed that the 2 cloned goats were similar to their respective oocyte donor goats, and significantly different from the nucleus donor. In conclusion, genetic background of recipient oocytes affected in vitro and in vivo development of reconstructed embryos, with the homologous background of cytoplast and nuclear donor benefiting development of reconstructed embryos. The mitochondrial origin of the 2 cloned Boer goats came from recipient oocytes, not donors.     

Bovine embryo morphology and evaluation

The following paper briefly reviews the morphology of the bovine embryo and presents a retrospective analysis of bovine embryo transfer results accumulated from April to December of 1982 at a commercial embryo transfer center. Of particular interests were bovine embryo morphology, assessment of embryo quality, and recipient-donor, recipient-embryo synchrony requirements. Embryos were recovered from superovulated donors five to nine days after estrus (estrus = day O). All embryos were individually examined at 200X for cell stage of development and embryo quality. Embryos were nonsurgically transferred to recipients that were within two days of estrous cycle synchrony with the donor. Attempts were made to synchronize estimated developmental age of embryos to the day of the recipient cycle. A high degree of variability was observed in morphological development and embryo quality within and among donors. Embryo recovery in individual donors resulted in a wide range of embryonic cell stages, often differing in estimated developmental ages from 24 to 48 hours. A total of 783 embryos were transferred, resulting in 308 pregnancies. Stage of embryonic development (16-cell through hatched blastocyst) had little effect on pregnancy rates. Embryo quality was a more accurate predictor of success. Embryos of excellent, good, fair and poor categories resulted in 45%, 44%, 27% and 20% pregnancy rates, respectively. Recipient-donor estrous cycle synchrony of two days in either direction did not significantly alter pregnancy rates. However, 88% of 258 pregnancies (584 total transfers) occurred with a +/-1 day recipient-embryo synchrony compared to 74% based on +/-1 day recipient-donor cycle synchrony (P<0.001). Results suggest that transfer of bovine embryos based on synchrony between day of recipient cycle and state of embryonic development provides higher pregnancy rates than transfers based on recipient-donor cycle synchrony.     

A procedure for successful nonsurgical embryo transfer in swine

The current study was undertaken to develop a successful procedure for the nonsurgical transfer of pig embryos. A total of 663 embryos were surgically collected on Day 4 or 5 from 55 donors, of which 542 embryos of acceptable quality were nonsurgically transferred to 46 recipients. Nonsurgical recipient gilts were sedated 15 min prior to transfer with 20 mg im acepromazine maleate. A disposable insemination spirette with an attached 3-way stopcock was manipulated into the cervix of each gilt. Embryos were expelled from a tomcat catheter into the spirette, and 10 to 12 ml of Whitten's medium were used to flush embryos through the spirette into the reproductive tract. Sixteen (34.8%) recipient gilts did not return to estrus before Day 36, and 10 (21.7%) gilts farrowed with an average litter size of 4.3 +/- 0.7. Embryos were collected from an additional 20 donors and were surgically transferred to an additional 19 recipients. Surgical transfers conducted at the same time as the nonsurgical transfers resulted in 12 (63.2%) gilts farrowing and 7.1 +/- 0.6 pigs were born per litter. In conclusion, a procedure has been developed for nonsurgical transfer of swine embryos which simplifies the process of embryo transfer and which may increase the potential for utilization of embryo transfer technologies by swine producers.     

Transfer of vitrified blastocysts from one or two superovulated Large White Hyperprolific donors to Meishan recipients: reproductive parameters at Day 30 of pregnancy

The present study was designed to determine the effect of pooling embryos from two donors on the reproductive success of transfer of vitrified/warmed porcine blastocysts. Intact blastocysts were collected from superovulated Large White Hyperprolific gilts (n = 24) on Days 5-5.5 after artificial insemination. Embryos were recovered by flushing the uterine horns, and unhatched blastocysts were selected. Vitrification and warming were performed as described by Berthelot et al. [Cryobiology 41(2000) 116]. To evaluate in vitro development, 37 vitrified/warmed blastocysts were cultured, non-vitrified embryos (n = 48) were used as controls. Embryo transfers were conducted in asynchronous (-24 h) Meishan gilts (n = 20). Twenty vitrified/warmed blastocysts were surgically transferred into one uterine horn. Ten recipients received embryos from one donor (Group 1) and the other 10 transfers were performed with mixed embryos from two donors (Group 2). Pregnancy was assessed ultrasonographically at Day 25 after estrus and recipients were slaughtered at Day 30 after transfer. In vitro survival rate of the vitrified/warmed blastocysts was lower (P < 0.01) than that from control embryos (73.0% versus 93.7%). The pregnancy rate for Group 1 (70%) was not different (P > 0.05) than that from Group 2 (90%). No significant differences were detected between Groups 1 and 2 for in vivo embryo development (number fetuses/transferred embryos in pregnant recipients) or in vivo embryo survival (number viable fetuses/transferred embryos in pregnant recipients). However, the in vivo efficiency (number viable fetuses/total transferred embryos) was higher (P < 0.05) when transfers were performed with embryos from two donors (19.5% versus 30.5%). These results indicate that pooling embryos from two donors increases the in vivo efficiency after transfer of vitrified/warmed porcine blastocysts.     

Successful direct transfer of vitrified sheep embryos

The use of a simple cryopreservation method, adapted to direct transfer of thawed embryos may help to reduce the costs of embryo transfer in sheep and increase the use of this technique genetic improvement of this species. Two experiments were made to test a vitrification method that is easy to apply in field conditions. All embryos were collected at Day 7 of the estrous cycle of FSH-stimulated donor ewes and were assessed morphologically, washed in modified PBS and incubated for 5 min in 10% glycerol, for 5 min in 10% glycerol and 20% ethylene glycol and were transferred into the vitrification solution (25% glycerol and 25% ethylene glycol). All solutions were based on mPBS. Embryos were loaded in straws (1 cm central part, the remaining parts being filled with 0.8 M galactose in mPBS) and plunged into liquid N2 within 30 sec of contact with the vitrification solution. The straws were thawed (10 sec at 20 degrees C) and the embryos were either transferred directly or after 5 min of incubation in the content of the straw (followed by washing in PBS) into the uterus of a recipient ewe. In Trial 1, the pregnancy rates at term (72 vs. 72%) as well as the embryo survival rates (60 vs 50% respectively) were not different between fresh (n = 48 embryos) and vitrified (n = 50) embryos. In a second trial no difference was observed between vitrified embryos transferred after in vitro removal of the cryoprotectant (n = 86 embryos) or directly after thawing (n = 72) both in terms of lambing rate (67 vs. 75%, respectively) and embryo survival rate (lambs born/embryos transferred; 49 vs. 53%). This method of sheep embryo cryopreservation provided high pregnancy and embryo survival, even after direct transfer of the embryos.     

Factors affecting pregnancy rates and early embryonic death after equine embryo transfer

In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantly higher for recipients that had excellent to good uterine tone or were graded as "acceptable" during a pretransfer examination, usually performed 5 d after ovulation, versus recipients that had fair to poor uterine tone or were graded "marginally acceptable." Embryonic factors that significantly affected pregnancy rates were morphology grade, diameter and stage of development. The incidence of early embryonic death was 15.5% (65 of 419) from Days 12 to 50. Embryo loss rates were significantly higher in recipients used 7 or 9 d vs 5 or 6 d after ovulation. Embryos with minor morphological changes (Grade 2) resulted in more (P<0.05) embryo death than embryos with no morphological abnormalities (Grade 1). Between Days 12 and 50, the highest incidence of embryo death occurred during the interval from Days 17 to 25 of gestation. Embryonic vesicles that were imaged with ultrasound during the first pregnancy exam (5 d after transfer) resulted in significantly fewer embryonic deaths than vesicles not imaged until subsequent exams. In the present study, embryo morphology was predictive of the potential for an embryo to result in a viable pregnancy. Delayed development of the embryo upon collection from the donor or delayed development of the embryonic vesicle within the recipient's uterus was associated with a higher incidence of pregnancy failure. Recipient selection (age, day after ovulation, quality on Day 5) significantly affected pregnancy and embryo loss rates.     

Non-surgical embryo transfer in cattle

Embryos collected surgically from donors superovulated with PMSG and synchronized with either prostaglandin F(2)alpha or progestagen impregnated sponges were transferred non-surgically to prostaglandin or progestagen synchronized recipients. One embryo was transferred to the uterine horn ipsilateral to the corpus luteum either through a flexible catheter introduced through a steel tube and passed to the uterine tip, or through a Cassou inseminating gun passed approximately 6 cm into the horn. Of 16 recipients receiving 5 or 6 day old embryos through the catheter (1976), 6 (38%) were palpated pregnant at 42 days and 4 (25%) subsequently calved. Of 16 recipients receiving 7 or 8 day old embryos through the straw and 16 through the catheter (1977), 10 (63%) and 3 (19%), respectively, were palpated pregnant (P<0.05) and 8 (50%) and 3 (19%), respectively, had normal embryos at slaughter 4 to 29 days after palpation (P reverse similar0.10 ). Forty 7 to 9 day old embryos were transferred through the straw in 1978. Eighteen (45%) of the recipients were palpated pregnant and 16 (40%) had normal embryos at slaughter 98 to 168 days after palpation. The success of the transfers in 1978 was affected by embryo quality [good vs poor embryos; 64% vs 22% recipients pregnant (P<0.01) and 59% vs 17% embryos surviving to slaughter (P<0.05)]. Also, in 1978, pregnancy rate was affected by the time taken to transfer the embryo with the highest rate achieved with the fastest transfers (P<0.10, b = -0.47). Injection of Indomethacin near the time of transfer, synchronization between donor and recipient onset of estrus and embryo age did not affect pregnancy rates. The pregnancy rate achieved after the transfer of good quality embryos by the straw technique was equal to that expected from surgical techniques.     

Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program

In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to −6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), −1 (68%), −2 (63%), −3 (66%), −4 (76%), −5 (61%), and −6 (27%). The −6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to −5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season.     

Transfer of superovulated sheep embryos obtained with different FSH-P

Embryo transfer is one way of accelerating genetic improvement in sheep. One of the main obstacles has been the production of good-quality embryos. The use of progestagens and the stimulation of ovulation with follicle stimulating hormone pituitary extract (FSH-P) has permitted the superovulation of donor and recipient ewes and the synchronization of their cycles. The injection of 16 mg FSH-P at the end of progestin treatment gave means of 9 +/- 1.5, 12 +/- 1.5, and 19.5 +/- 2.6 corpora lutea per ewes in the Préalpes, Lacaune, and Romanov x Préalpes breeds respectively (this last breed is particularly prolific). Twenty Préalpes donor ewes produced 133 embryos that were recovered surgically at Day 6 of gestation; of these, 99 morulae were transferable. Forty-five morulae transferred surgically into 24 Préalpes recipient ewes yielded 16 pregnant ewes and 27 lambs (1.7 per ewe). Twenty-two Lacaune ewes yielded 204 embryos, of which 152 morulae were transferable. Of 76 recipients, 58 became pregnant and gave birth to 97 lambs (1.7 per ewe). During anoestrus, the mean ovulation rate decreased from 11.2 to 8.4; 40.6% of the embryos recovered were of transferable quality versus 74.5% during the normal breeding season. An improved superovulation technique, based on the use of FSH-P with a known follicle stimulating hormone to luteinizing hormonal (FSH/LH) ratio, provided us with good-quality embryos. This treatment must be adapted to the season.     

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.     

Preventing experimental vertical transmission of scrapie by embryo transfer

This study investigated whether the transmission of naturally occurring scrapie in sheep can be prevented using embryo transfer. Embryos were collected from 38 donor ewes in a Suffolk sheep flock with a high incidence of naturally occurring scrapie, treated with a sanitary procedure (embryo washing) recommended by the International Embryo Transfer Society and then transferred to 58 scrapie-free recipient ewes. Ninety-four offspring were produced. None of the offspring or the recipient ewes developed scrapie. Furthermore, offspring derived from embryos collected from donor ewes bred to the immunohistochemically positive ram did not develop scrapie. We conclude that scrapie was not transmitted to offspring via the embryo nor was the infective agent transmitted to recipient ewes during embryo transfer procedures.     

Nuclear transfer in the bovine embryo: a comparison of 5-day, 6-day, frozen-thawed, and nuclear transfer donor embryos

Micromanipulation and electrofusion were utilized for nuclear transfer in bovine embryos. Embryonic blastomeres from 5-day (estrus = day 0), 6-day, frozen-thawed 5-day, and first-generation nuclear transfer embryos (embryos were themselves a product of nuclear transfer with the original donor being a 5-day embryo) were transferred into bisected bovine oocytes by electrofusion. The percentage of donor cells fusing with the recipient oocytes was compared between different types of donor embryos. The percentage of embryos developing normally into morula or blastocysts following 6 days culture in the sheep oviduct was also recorded and compared between different donor embryo types. No significant differences were found between donor blastomeres for the percent successfully fused to oocytes: 5-day, 294 of 513 (57.3%); 6-day, 252 of 405 (62.2%); frozen-thawed 5-day, 111 of 144 (77.1%); nuclear transfer, 142 of 223 (63.7%); or the percent developing normally following nuclear transfer: 5-day, 92 of 444 (20.7%); 6-day, 84 of 357 (23.5%); frozen-thawed 5-day, 32 of 127 (25.2%); nuclear transfer, 31 of 199 (15.6%). These data suggest that a variety of donor embryos can successfully be utilized for bovine embryo cloning. Also, development of blastomeres from frozen-thawed 5-day donors and from donors that are themselves the product of nuclear transfer suggest that the production of multiple identical offspring is possible by frozen storage of seed stock and serial recloning.