Comparison of Ham's F10 with CO2 or Hepes buffer for storage of equine embryos at 5 C for 24 H

Forty equine embryos collected 7 d post-ovulation were stored at 5 C for 24 h in one of two culture media (n = 20/group): 1) Ham's F10 + 10% heat-treated fetal calf serum (FCS) buffered by gassing with 5% CO2, 5% O2 and 90% N2 and 2) Ham's F10 + 10% FCS with Hepes buffer (25 mM). Embryos cultured in Ham's F10 + CO2 maintained a better quality score and had a larger average increase in diameter (+34.8 micron) than embryos stored in Hepes buffered Ham's F10 (-10.2 micron). Embryos were transferred surgically into recipient mares that ovulated -3 to +1 d in relation to the donor mare. Twenty embryos cultured in Dulbecco's phosphate buffered saline + 10% FCS and transferred less than 1 h after collection were used as controls. Pregnancy rates were higher (P less than .05) for embryos stored in Ham's F10 + CO2 (70%, 55%) than for embryos stored in Ham's F10 + Hepes (20%, 15%) at 14 and 35 d, respectively. At 14 d, pregnancy rates for control embryos (90%) were similar (P greater than .05) to pregnancy rates for embryos cultured in Ham's F10 + CO2 (70%); however, by 35 d, pregnancy rates were higher (P less than .05) for controls (80%) than for embryos stored in Ham's F10 + CO2 (55%). It was concluded that Ham's F10 + CO2 was superior to Ham's F10 + Hepes for short-term storage of equine embryos at 5 C, and that satisfactory pregnancy rates could be obtained from transfer of embryos stored in Ham's F10 + CO2 at 5 C for 24 h.     

Strategies for Increasing Reproductive Efficiency in a Commercial Embryo Transfer Program With High Performance Donor Mares Under Training

Exercise stress has a negative impact on embryo transfer efficiency (ET). For example, a 34% embryo recovery rate, 43% incidence of poor quality embryos, and a 29% pregnancy rate after transfer have been reported. Administration of nonsteroidal anti-inflammatory drugs (NSAIDs) may reduce the inflammatory response produced after nonsurgical embryo transfer. In addition, progesterone supplementation is commonly administered to some recipient mares to improve uterine conditions before the transfer and to ensure adequate progestational support compatible with pregnancy. The aim of the study was to evaluate embryo recovery rates using BioRelease deslorelin versus hCG and to increase posttransfer pregnancy rates by jointly administering BioRelease progesterone and a NSAID (flunixin or meloxicam) to recipient mares. Seventeen upper-level showjumping mares stabled and in daily training were used as embryo donors. To induce ovulation, 1-mg IM BioRelease deslorelin (BioRelease Technologies, Lexington, KY) was injected in treated cycles (n = 66), or 2500-IU hCG IV (Ovusyn, Syntex, Buenos Aires, Argentina) was given in control cycles (n = 79) when a ≥35 mm follicle was present. Artificial insemination with extended fresh semen (at least 500 × 10⁶ progressively motile sperm) was carried out in both groups immediately after injecting the ovulation induction agent. Day 8 embryos were recovered and nonsurgically transferred using a speculum and a cervical traction forceps. Recipient mares (n = 73) were randomly assigned to one of three groups: Group A received a single injection of 1.5-g IM BioRelease progesterone (Progesterone LA 300, BioRelease Technologies) and 3 IV injections of 0.5 g of flunixin meglumine (Flunix Deltavet, Argentina), one injection administered the day of the transfer and one on each of the next two successive days. Group B received 1.5-g IM BioRelease progesterone and a single dose of 1.5-g IM BioRelease meloxicam (Meloxicam LA, BioRelease Technologies) at the moment of embryo transfer. Group C did not receive any treatment. Pregnancy diagnosis was carried out 7 days after transfer. Results were analyzed using comparisons of proportions. More embryos were recovered per cycle (13% increase) when donor mares in training were induced to ovulate with BioRelease deslorelin (60.6%; 40/66) than with hCG (46.8%; 37 of 79; P < .05). Although both recipient groups given NSAIDs in combination with BioRelease progesterone numerically had higher pregnancy rates (A: 70.8%; 17/24 and B: 75%; 15/20) compared with nontreated control recipients (47.1%; 33/70), pregnancy rates were significantly higher only in recipients given LA meloxicam treatment at the time of transfer (P < .05). The LA meloxicam is released over a 72-hour period making it more practical to use as it requires a single IM injection versus the 3 IV flunixin meglumine injections. Thus, to minimize the effects of exercise stress on ET efficiency, a combination of BioRelease deslorelin to induce ovulation in donors and BioRelease progesterone and LA meloxicam in recipients at the time of transfer may offer an interesting alternative for improving results in commercial ET programs.     

Day of embryo collection, quality and pregnancy rates in cattle

In 1300 donor cows, total embryos increased from 8.5 to 15.3, (P less than 0.001), the mean number of transferable embryos from 3.1 to 6.5 (P = 0.067) and pregnancies increased from 1.3 to 3.3 (P = 0.584) as the day of collection increased from six to 7.5. Most of the embryos recovered on day 6, 7.5 and 8 were morulae, early blastocysts and late blastocysts respectively. Morulae formed the majority of the embryos collected on days 6 and 6.5. Pregnancy rates in early and late blastocysts were highest on days 7 (54.4 and 60.2 per cent) and 7.5 (53.6 and 53.1 per cent, P = 0.009 and 0.004). There were significant differences in pregnancy rates between embryo stages on days 7 and 7.5 (P less than 0.001), embryo grades on days 7, 7.5 and 8 (P less than 0.001), and within embryo grades 1 (P = 0.015) and 3 and 4 (pooled, P = 0.009). On all days combined there were significant differences in pregnancy rates between both embryo stages (P = 0.007) and embryo grades (P less than 0.001). It appears that the concept of embryo fitness may be applied to blastocysts but not to morulae.     

Use of insulin-like growth factor-I during embryo culture and treatment of recipients with gonadotropin-releasing hormone to increase pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed,lactating cows

An experiment was conducted to determine whether pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed cows could be improved by 1) culturing embryos in the presence of IGF-I and 2) treating recipients with GnRH. Lactating Holstein cows (n = 260) were synchronized using a timed ovulation protocol. Embryos were produced in vitro and cultured with or without 100 ng/mL of IGF-I. On d 7 after anticipated ovulation (d 0), a single embryo was transferred to all recipients with a palpable corpus luteum (n = 210). A subset of recipients (n = 164) was injected with either GnRH or placebo on d 11. Plasma progesterone concentrations on d 0 and 7 were used to determine the synchrony of recipients. Pregnancy was diagnosed at d 53 and 81 by rectal palpation. Among all recipients, transfer of IGF-I-treated embryos increased pregnancy rate at d 53 (P < 0.05) and tended to increase pregnancy rate at d 81 (P < 0.06). Calving rate also tended to be higher for recipients that received IGF-I-treated embryos (P < 0.07). Among the subset of synchronized recipients (n = 190), pregnancy rate at d 53 and d 81 and calving rate were higher (P < 0.05) for IGF-I-treated embryos. The GnRH tended to increase pregnancy rate at d 53 for all recipients (P < 0.08) and the subset of synchronized recipients (P < 0.10). There were no effects of GnRH (P > 0.10) for pregnancy rate at d 81 and calving rate. The overall proportion of male calves was 64.3%. There was no effect (P > 0.10) of embryo treatment or GnRH on the birth weight or sex ratio of calves. Results of this experiment indicate that treatment of embryos with IGF-I can improve pregnancy and calving rates following transfer of in vitro-produced embryos. Further research is necessary to determine whether the treatment of recipients with GnRH is a practical approach to increase pregnancy rates following in vitro embryo transfer.     

Increase in calf production by the transfer of bisected bovine embryos

A total of 132 embryos were recovered from 17 superovulated donor cows 7 d after estrus. Seventy-four embryos were selected and assigned to 2 treatment groups. The number of whole embryos that were directly transferred (Group A) and bisected (Group B) were 44 and 30 embryos, respectively. Sixty demi-embryos were produced from 30 morulae to blastocyst-stage embryos that were bisected. One hundred-three embryos, including whole and demi-embryos without zonae pellucidae, were nonsurgically transferred. Only one whole or demi-embryo was transferred to each recipients. The pregnancy rate for whole embryos (A) was 63.6% (28/44), while for demi-embryos (B) it was 74.6% (44/59). There was no significant difference between the pregnancy rates of whole embryos (A) and bisected embryos (B) transferred 7 d after estrus. Forty-three calves including the 14 sets of identical twins were obtained from 30 original embryos (143.3%) using the embryo bisection technique.     

Transfer of pig embryos to different uterine sites.

Embryo transfer in pigs normally involves surgery. In connection with the development of nonsurgical or endoscopic transfer techniques, it is important to know whether the uterine site to which embryos are transferred has an effect on the success rate. In the present investigation, prepubertal donor gilts were treated with 1,500 IU of PMSG and, 72 h later, with 500 IU of hCG. Gilts were artificially inseminated 24 and 36 h after hCG injection. Embryos at the expanded blastocyst stage were collected from donor gilts. Recipient gilts were treated synchronous with the donors, using 1,000 IU of PMSG followed, 72 h later, with 500 IU of hCG. After a maximum of 3 h in vitro, embryos (n = 15 to 20, mean = 17.3) were transferred surgically to the middle of the uterine horn, to the caudal quarter of the uterine horn, or to the uterine body. Recipients were slaughtered between 28 and 34 d after transfer. The pregnancy rate of the recipients was low when the embryos were deposited in the uterine body (12%), compared with the middle (88%) or the caudal quarter of the uterine horn (81%) (P < .01). The corresponding average number of viable fetuses per pregnant recipient was 8.2 in the uterine body, 5.6 in the middle, and 4.5 in the caudal quarter. Average survival rate of embryos after transfer to the middle of the uterine horn was 41% vs 29 and 3% after transfer to the caudal quarter or the uterine body, respectively (P < .01). Hence, the uterine body seems to be an unsuitable site for embryo transfer in pigs. These results may explain the unsatisfactory results achieved with nonsurgical embryo transfer in the past.     

Use of embryo transfer to induce twinning in beef cattle: embryo survival rate, gestation length, birth weight and weaning weight of calves

Experiments were conducted in 1985 and 1986 at the Eastern Ohio Resource Development Center, Belle Valley, to examine the feasibility of using embryo transfer to induce twinning and to examine the influence of twinning on traits of the cow and calf. Embryos were collected from a total of 14 superovulated Angus donors on two dates each in 1985 and 1986 and were transferred to Angus recipients. A total of 124 embryos were transferred to 79 recipients, with 43 (34.7%) calves born alive. Seven of 45 (15.6%) recipients implanted with two embryos produced twins. In no case did both halves of the 15 embryos that were split to produce identical twins and implanted in the same recipient survive to birth. Proportion of calves born alive did not differ among transfer codes 3 (nonsplit embryos from two different donors implanted in separate uterine horns of the same recipient), 6 (nonsplit embryos from one embryo flush implanted in separate uterine horns of the same recipient) and 7 (nonsplit embryos from two different donors implanted in the same uterine horn of one recipient). Surgical transfers tended to result in a higher proportion of embryos surviving to birth (.43 vs .21; P = .16) and a higher twinning rate (.29 vs .04; P = .36) than did nonsurgical transfers. Age of recipient did not influence embryo survival (P = .98) or twinning rate (P = .99). Gestation length was 5 d shorter (P less than .01) for twin calves than for singles. Singles were 9 kg heavier (P less than .01) at birth and 32 kg heavier (P less than .01) at weaning than twins. However, cows raising twins produced 108 kg (51%) more total weaning weight than did cows raising singles.     

Effect of flunixin meglumine treatment during and after embryo transfer on the pregnancy rate in cattle

This study aimed to determine the effect of flunixin meglumine treatment during and after the transfer of in vivo produced embryos to Angus (cows) and Holstein (cows and heifers) breeds of cattle on pregnancy rate. Holstein cows were used as donors in the study. A double dose of prostaglandin F2α was administered to the recipient animals for synchronization. Uterine flushing was performed in donors on day 7 after artificial insemination. A total of 295 transferable embryos were obtained. These embryos were transferred to Angus cows (n = 85), Holstein heifers (n = 80) and Holstein cows (n = 130). After the transfer, these animals were divided into three subgroups. The first subgroup (TI) was administered flunixin meglumine during embryo transfer, and the second subgroup (TII) was administered flunixin meglumine both during embryo transfer and on days 8 and 9 after the transfer. The third subgroup (TIII) was not administered anything and it was considered the control group. Pregnancy examination of the recipients was performed on days 30–35 after the transfer using real-time ultrasonography. The pregnancy rates after embryo transfer were found to be 43.52% in Angus cows, 42.5% in Holstein heifers, and 24.61% in Holstein cows (p < .05). When the animals were not classified according to breed, the pregnancy rates in subgroups TI, TII and TIII were found to be 29.29%, 45.10% and 29.79%, respectively (p < .05). In addition, the pregnancy rates were higher in TII and TIII subgroups of Angus cows and Holstein heifers compared to that of Holstein cows (p < .05). As a result, the pregnancy rates obtained after embryo transfer in Angus cows and Holstein heifers were found to be higher than that in Holstein cows. In addition, it was concluded that the administration of flunixin meglumine during and during/after embryo transfer has a positive effect on pregnancy rates in Angus cows and Holstein heifers.     

Historical Aspects of Equine Embryo Transfer

Early embryo transfer in equids was undertaken simultaneously in the early 1970s in Cambridge, England, and Kyoto, Japan. Both groups achieved limited success when flushing the uterine horn ipsilateral to the side of ovulation but the rates improved markedly when the whole uterus was flushed on realization of the continued movement of the embryo throughout the uterine lumen after day 6. Initial transfers of embryos to recipient mares were carried out surgically, but nonsurgical transfer via the cervix has been used subsequently with increasing success, culminating in pregnancy rates of 75%–90% today. Experimental use of embryo transfer in horses and donkeys demonstrated the unique ability of equids to carry to term a full range of interspecies hybrid conceptuses and extraspecies pregnancies created by embryo transfer. Furthermore, splitting of day 4–8 cell embryos and day 6 compact morulae allowed the creation of genetically identical twin foals. But despite these and other significant advances over the past 45 years, a persisting limitation is the relatively low embryo recovery rates from donor mares treated with exogenous gonadotropins in attempts to induce them to superovulate. This is due to the toughness of the ovarian tunica albuginea which forces ovulation through the ventrally situated ovulation fossa where multiple follicles compete with each other and luteinize before they can ovulate properly.     

Early Pregnancy Diagnosis by Serum Progesterone and Ultrasound in Sheep Carrying Somatic Cell Nuclear Transfer-Derived Pregnancies

Early pregnancy diagnosis and monitoring play an important role following embryo transfer in sheep. The aims of the current study were to investigate (i) the pattern of serum progesterone profiles in sheep carrying somatic cell nuclear transfer (SCNT)‐derived (clone) pregnancies, and (ii) the frequency of pregnancy loss during development following SCNT embryo transfer. Sheep SCNT embryos were made using standard nuclear transfer techniques. Day 7 embryos were surgically transferred to oestrus‐synchronized recipients (n = 27). As a control, normal fertile ewes (n = 12) were bred by natural breeding. Serum was collected from all the ewes on the day of estrus (day 0 sample), 7 days post‐estrus (day 7 sample) and 19 days post‐estrus (day 19 sample) and every 10 days thereafter until lambing or pregnancy loss occurred. Serum progesterone (P4) was assessed using enzyme immunoassay. Pregnancy was confirmed by ultrasound scanning on day 35 of pregnancy followed by subsequent scanning every 10 days. In control ewes, pregnancy rate on day 35 was 83.3% (10/12), whereas in the ewes that received SCNT embryos, it was 22.2% (6/27; p < 0.05). The day 45 pregnancy rate in the control ewes was 83.3%, whereas in the SCNT embryo recipients it was 11.0% (p < 0.05). Hormone analysis revealed that SCNT embryo recipients exhibited a significantly lower P4 profiles at different time points in pregnancy compared to controls (p < 0.05). This study highlights the use of serum progesterone in combination with ultrasound for the investigation of embryo loss and crucial times during development of normal and SCNT embryos in sheep. Further, the serum P4 levels directly reflect the degree of placental development in these two groups.     

Bovine embryo transfer pregnancies. I. Abortion rates and characteristics of calves

Data were obtained from 1,908 pregnancies resulting from bovine embryo transfer procedures. Responses examined included sex ratio, fetal, neonatal and preweaning death losses, birth weight and calving assistance. The sex ratio for 1,751 embryo transfer calves examined was 51.11% males. Cows older than 10 yr that had become repeat breeders produced more (P less than .05) male calves than other donors. Breed of embryo, age and quality of embryos at the time of transfer, embryo storage time from collection to transfer, asynchrony of recipient with donor estrus and number of palpable corpora lutea in superovulated donors were not related to sex ratio (P greater than .05). The abortion rate between 2 and 3 mo of gestation in embryo transfer recipients was 3.15%, and between 3 to 7 mo, 2.14%. Neonatal and preweaning losses for 1,682 calves with complete information were 1) congenital defects, .54%; 2) death due to premature birth (7 to 8 mo of gestation), .18%; 3) dystocia-related deaths, 2.38%; 4) deaths of unknown causes at birth, 2.14%; 5) deaths of unknown causes from 24 h after birth to weaning, 1.43%; 6) deaths due to calfhood diseases, 1.25% and 7) deaths due to environmental factors, 1.13%. Total losses of 2-mo pregnancies due to abortion or death of calves or recipients were 14%. Birth weight of embryo transfer calves changed .29 kg/d of deviation from average gestation length (P less than .005) for pregnancies within breeds. Birth weight was also affected (P less than .005) by donor breed and recipient breed and age. Male calves averaged 2.19 kg heavier (P less than .005) than females. Calving assistance was affected by donor breed; Angus calves required the least assistance (P less than .005). Hereford, Holstein and Limousin calves were similar and intermediate; Simmental calves needed the most calving assistance. Recipient breed and age influenced calving ease, with younger recipients of Angus and Hereford descent requiring more assistance (average calving score, 2.1) than both cow (1.3) and heifer (1.5) recipients of the larger Continental European breeds. Characteristics of 305 non-embryo transfer calves were not significantly different from 185 embryo transfer calves from the same farms. We conclude that embryo transfer calves did not differ from the non-embryo transfer population in any of the characteristics studied.     

Effect of slow cooling end point temperature on survival of frozen bovine embryos

One hundred sixty-two embryos were collected from superovulated crossbred beef cattle 7 to 8 d after the onset of estrus. Embryos were frozen in modified Dulbecco's phosphate-buffered saline supplemented with 20% heat-inactivated fetal calf serum (PBS + FCS) and dimethylsulfoxide (DMSO), which was added in three steps to a final concentration of 1.5 M. Embryos were placed in .25 ml of 1.5 M DMSO in PBS + FCS in 1-ml glass ampules and cooled at 1.0 C/min from ambient temperature to -7 C, seeded and then cooled at .3 C/min to -19, -26, -33, -38, -43, -50 or -57 C before immersion (plunging) in liquid nitrogen. Ampules were thawed in 25 C water, and DMSO was removed in six steps at .25 M increments. 10 min/step. After removal of DMSO, embryos were cultured 24 h in PBS + FCS and then fixed and stained. Just after thawing, embryos for which slow cooling was terminated at -50 C were of lower (P less than .05) morphological quality than other groups. After removal of cryoprotectant, embryos from both the -19 and -50 C treatments had deteriorated more (P less than .05) than had embryos from other treatments. After 24-h culture, embryos slow-cooled to -19, -26 and -50 C had a lower rate of survival (P less than .05) than did embryos from -33, -38, -43 and -57 C temperatures. Embryos slow-cooled to -33, -38 and -43 C showed a higher percentage of healthy nuclei than did embryos slow-cooled to -19, -26 and -50 C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors Affecting Recipients' Pregnancy,Pregnancy Loss,and Foaling Rates in a Commercial Equine Embryo Transfer Program

During 11 breeding seasons, 351 7- to 10-day-old horse embryos were nonsurgically transferred into recipients that ovulated between 3 and 10 days earlier. Pregnancy rates at 14 and 40 days and foaling rates were 77.8% (273/351), 69.2% (243/351), and 64.4% (226/351), respectively. Pregnancy loss between 14 and 40 days was 11% and between 40 days and delivery was 7%. The transfer of quality grade 3 to 4 embryos resulted in a significantly lower pregnancy rate at 14 days compared with the transfer of grade 1 to 2 embryos (46.2% vs. 79%; P < .05). Eight-day-old embryos resulted in significantly lower pregnancy losses than day 9 or 10 embryos, as occurred for embryos between 400 and 1200 μm compared with embryos <400 μm. Embryos recovered from mares >20 years resulted in a significantly higher pregnancy loss rate than those recovered from younger mares. The same happened for embryos coming from mares affected by reproductive pathologies compared with healthy mares performing sport activity. None of the evaluated parameters influenced recipients' foaling rate significantly.     

Embryonic migration relative to maternal recognition of pregnancy in sheep

Twenty-five crossbred ewes were utilized to examine the timing of embryonic migration relative to maternal recognition of pregnancy. These ewes also were utilized to examine whether ovine embryos synthesized estradiol-17 beta in association with embryonic elongation and intrauterine migration. Embryos were flushed on d 11 through 15 from hemiovariectomized ewes. Recovery of embryos from the uterine horn contralateral to the remaining ovary indicated that migration had occurred. Ewes subsequently were returned with rams to determine their interestrous interval. Recovered embryos were classified morphologically, their length determined and individually incubated. Changes in estradiol within the medium were determined after a 6-h incubation. Embryo migration began on d 14 (P less than .05); on consecutive days from 11 through 15, 0, 0, 0, 60 and 100% of ewes examined, respectively, had an embryo in the contralateral horn. Extended estrous cycles (greater than 20 d) were observed in 0, 0, 40, 80 and 100% of ewes examined (P less than .05) following removal of embryos on d 11 through 15 of the cycle. Ovine embryos were longer (P less than .05) on d 14 (4.8 +/- 1.1 cm length, mean +/- SE) compared with d 13 (.2 +/- .1 cm) and increased further (P less than .05) on d 15 (7.8 +/- 1.1 cm). Incidence of intrauterine migration was correlated with embryonic length (r = .83; P less than .01) and estradiol synthesis (r = .77; P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the factors that affect the pregnancy rates during embryo transfer in beef heifers

The aim of this study was to evaluate the effects of the transfer side, transfer location, cervix transfer score, type and diameter of corpus luteum (CL) during embryo transfer on pregnancy rates in beef heifers. Progesterone-based synchronization and superovulation protocol were applied to Simmental cows used as donors (n = 168). Uterine flushings were performed on day 7 following artificial insemination. Obtained Code I (excellent or good) and II (fair) quality embryos were transferred to recipient beef heifers (n = 561). During embryo transfer, side of transfer (right or left), transfer location (the cranial or middle third of uterine horn), cervix transfer score (easy, moderate or difficult) and type (CLa, CLb and CLc) and diameter of CL were determined. Pregnancy rates following the transfer of Code I and II embryos were 44.66% and 33.07%, respectively (p < .05). The rates of pregnancy after transfers to the right and left uterine horn were 37% and 42.2%, respectively (p > .05). The pregnancy rates were 41.2%, 34.9% and 30.3% for cervix transfer scores as easy, moderate and difficult, respectively (p > .05). Pregnancy rates after transfer to the cranial third and middle third were 41.06% and 29.67%, respectively (p < .05). According to types of CL, pregnancy rates were 31.7%, 40.4% and 45.3% for CLa, CLb and CLc, respectively (p < .05). Moreover, it was found that as the CL diameter increased, the pregnancy rates increased. As a result, it was concluded that there was no effect of side of transfer and cervix transfer score, but embryo quality, transfer location, type and diameter of CL had significant effects on the pregnancy rate during embryo transfer in beef heifers.     

Japanese Society for Animal Reproduction: award for outstanding research 2002. The development and prevalence of the transfer technique for frozen-thawed embryos of Japanese black beef cattle in Tochigi Prefecture

The conditions of embryo transfer by the stepwise method, in which frozen-thawed embryos are transferred on day 7 (day 0=onset of estrus), were investigated with the aim of increasing pregnancy rates in frozen-thawed embryo transfer. The use of a vaginal speculum to prevent bacterial infection when passing an embryo transfer gun through the vagina yielded a pregnancy rate equal to or higher than that with application of a sheath cover to the transfer gun. Administration of a sedative, xylazine, to recipient cattle for preventing movement at the time of embryo transfer improved the pregnancy rate. The influence of the time from thawing of frozen embryos to transfer and of the transportation of the recipient by truck upon pregnancy rate was investigated. Embryo transfer within 60 minutes after aspiration into a straw or transportation of the bovine recipient, 1.5 hours each way before and after transfer, had no influence on pregnancy rate. Relations of the embryonic developmental stage and morphological quality after thawing of frozen embryos to pregnancy rate were investigated in recipients of nulliparous Holstein heifers. The pregnancy rate increased as the embryonic developmental stage advanced from compacted morula, early blastocyst, and blastocyst in that order. The pregnancy rate obtained with blastocyst stage embryos was significantly (P<0.05) higher than that with compacted morula stage embryos, and there was no significant difference in pregnancy rates between excellent morphological quality and good morphological quality for compacted morula stage embryos. When correlation of luteal function and pregnancy rate was investigated in bovine recipients, pregnancy rate showed a tendency to increase with increasing blood progesterone (P) concentration on the day before (on day 6 after estrus) and the day of embryo transfer. The pregnancy rate in bovine recipients, which showed a blood P concentration of > or =2.5 ng/ml on the day before embryo transfer, was significantly (P<0.05) higher than that in those with a blood P concentration of <2.5 ng/ml. Pregnancy rate showed a tendency to increase with decreasing blood estradiol-17beta (E2) concentration on the day of embryo transfer. Activation of luteal function by administration of human chorionic gonadotropin (hCG) in cycling cattle was investigated for its effect on increasing pregnancy rate in bovine recipients. A follicle coexisting with cyclic CL ovulated and induced CL formed after injection of hCG 1,500 IU 5 days after ovulation. The blood P concentration was significantly (P<0.05) higher in the administration group than in the control group, and the blood E2 concentration rapidly decreased, showing a lower concentration than in the control group. These results suggest the possibility that the pregnancy rate could be improved by administration of hCG. Pregnancy rate following intramuscular injection hCG 1,500 IU was comparatively investigated in parous Japanese Black beef cattle receiving frozen-thawed embryos 7 days after estrus. Pregnancy rate was 67.5% in the group in which hCG was administered on day 6 after estrus, and was significantly (P<0.05) higher than that in the control group (45.0%) and the group in which hCG was administered on day 1 after estrus (42.5%), revealing that hCG administration facilitated pregnancy. Transfer of frozen-thawed embryos in the blastocyst stage within 60 minutes after the aspiration into a straw, with a vaginal speculum after administration of xylazine is suggested as a way of improving pregnancy rate in bovine recipients with favorable luteal function and in those with luteal function activated by administration of hCG on the day before embryo transfer.     

The transfer of fresh and frozen embryos in an elite swamp buffalo herd

To investigate the development of fresh and frozen swamp buffalo embryos after transfer to synchronized recipients, 14 fresh embryos and 28 frozen embryos, collected from Thai swamp buffalo cows of an elite herd at the Surin Breeding Center, were transferred nonsurgically to 31 synchronized recipients buffalo cows. One fresh embryo was transferred to each of 14 recipients. Twenty eight frozen embryos were transferred to 17 recipients of which 7 cows received I embryo, 9 cows received 2 embryos and 1 cow received 3 embryos. Pregnancy was diagnosed by real time B-mode ultrasonography one month after transfer and confirmed by rectal palpation one and two months later. The pregnant cows were kept under observation until calving. The results of fresh embryo transfer showed that 5/14 (35.7%) were pregnant after 30 days, 4/14 (28.6%) remained pregnant until the 3rd month and 2/14 (14.3%) calved. With the frozen embryos, only one cow which received three embryos became pregnant and remained so for 3 months although the embryo did not survive to full term. The overall pregnancy rate using frozen embryos was 5.9% (1/17). The study demonstrated the possibility of performing embryo transfer in elite buffalo herds for genetic improvement, however the use of frozen embryos needed further investigation.     

Viability of stored equine embryos

Equine embryos were recovered nonsurgically 6.5 d after ovulation (Exp. 1) and those greater than 200 microns were stored in one of three media: 1) Ham's F10 + 10% fetal calf serum (FCS) under 5% CO2, 5% O2 and 90% N2 at 24 C (Ham's F10); 2) Minimal Essential Medium with Hank's balanced salts + 10% FCS in air (MEM) at 24 C or 3) MEM at 5 C n = 10/treatment). Embryos less than or equal to 200 micron (n = 10) were bisected microsurgically; one-half of each embryo was stored in Ham's F10 and the other half in either Dulbecco's phosphate-buffered saline + 10% FCS in air at 24 C (DPBS), or MEM in air at 24 C. At 0, 12 and 24 h, embryos were: 1) measured; 2) assigned a developmental score of 1 to 4 (1 = tight morula, 4 = expanding blastocyst) and 3) assigned a quality score of 1 to 5 (1 = excellent, 5 = degenerate). Whole embryos stored in MEM at 5 C or 24 C did not (P greater than .05) advance in development by 24 h, whereas those stored in Ham's F10 at 24 C were more (P less than .05) advanced (i.e., higher developmental score) by 24 h. From 0 to 24 h, 1 of 10, 6 of 10 and 7 of 10 whole embryos developed when stored in MEM 5 C, MEM 24 C and Ham's F10 24 C, respectively. Embryo quality was better at 24 h (P less than .05) for embryos stored in Ham's F10 at 24 C compared with MEM at 5 C.(ABSTRACT TRUNCATED AT 250 WORDS)     

非繁殖季节胚胎移植受体绵羊妊娠效果影响因素的研究

在非繁殖季节应用CIDR法和Progestagen Sponge法对受体绵羊进行同期发情处理后,进行冷冻胚胎移植,研究不同同期发情处理方法、卵巢状况、胚龄、移植方法和移植时间对冷冻胚胎移植妊娠率的影响,以期为当地实现绵羊胚胎移植产业化提供理论依据和技术参考。结果表明:经同期发情处理后,绵羊在去栓后24~72 h集中发情,在去栓后36~48 h发情最为集中;CIDR法和Progestagen Sponge法的同期发情率分别为91.11%和87.78%(P>0.05),同期发情绵羊移植利用率分别为82.93%和78.48%(P>0.05);单侧卵巢上含有1个和2个黄体其妊娠率分别为46.07%和48.78%(P>0.05);桑椹胚和囊胚的妊娠率分别为49.25%和42.11%(P>0.05);腹腔镜法和手术法的妊娠率分别为45.16%和48.52%(P>0.05);11月和12月进行冷冻胚胎移植的妊娠率分别为50.76%和43.08%(P>0.05),该试验的绵羊冻胚移植妊娠率为46.92%,基本接近国内冻胚移植水平,但低于目前国内羊鲜胚移植妊娠率(50%~60%)。由此可以得出,不同同期发情处理方法、卵巢状况、胚龄、移植方法和移植时间对胚胎移植妊娠率的影响均无显著性差异(P>0.05),大规模群体羊同期发情时,可选价格便宜的Progestagen Sponge法;只要受体绵羊黄体质量好即可移植胚胎;为了减轻受体绵羊的应激反应,降低术后子宫粘连的发病率,可广泛应用腹腔镜法进行胚胎移植;该试验的绵羊冻胚移植妊娠率低于目前国内羊鲜胚移植妊娠率,通过筛选高效低毒的抗冻保护剂,以及提高基层技术人员操作水平,则有望进一步提高绵羊冷冻胚胎移植妊娠率。     

胚胎移植技术的现状与展望

统计与分析了1991－2000年国际胚胎移植状况，结果表明胚胎移植已成为畜牧产业中最活跃的领域之一，胚移比例最大的依然是牛，其它各类的动物胚胎移植，如：绵羊、山羊、猪、马、鹿、美洲驼、羚羊等，也十分活跃。牛的胚胎移植，有几个重要指标分析结果如下：（1）供体牛的品种分布基本相同，肉牛与奶牛所占比例接近，但不同国家之间差别很大；（2）每头供体所得可用胚数在5－6枚之间；（3）鲜胚移植与冻胚移植的比例基本持平，但近年来冻胚移植的比例稍大于鲜胚移植；（4）通过IVF体外方式生产胚胎数目有所增长，目前每年大约在3－4万枚；（5）妊娠率、鲜胚移植妊娠率在60％以上，冻胚移植妊娠率在50％以上，IVF体外方式生产胚胎妊娠率在40％左右；（6）胚胎移植技术的发展状况在全世界分布不均衡，北美和欧洲胚移技术最成熟；（7）胚胎贸易有所增长。最后，对该技术存在的问题与发展前景提出了探讨和展望。