Some observations on early embryonic death in mares

Early embryonic death (EED) was studied in 354 pregnant mares during two breeding seasons. Pregnant mares from six independent research projects were examined at 15, 20, 25, 30, 35, 40 and 50 days post-ovulation using a real-time ultrasound scanner. Mares that had previously been treated with anabolic steroids tended (P<0.10) toward a higher incidence of EED than untreated mares (21.1% vs 0%). Of the 59 pregnancies obtained from embryo transfer in 1982, 10 had undergone EED by day 50 (16.9%) compared to 10.1% for embryo transfer recipients in 1983. Overall, there was a 13.3% incidence of EED in embryo transfer recipients. This percentage was nearly identical to 13.4% incidence of EED for mares inseminated with fresh semen. In 1982, mares inseminated with frozen-thawed semen tended (P<0.10) to have a higher incidence of EED compared to those inseminated with fresh semen (34.6% vs 15.8%). However, in 1983 there was no difference (P>0.05) in the incidence of EED between mares inseminated with frozen-thawed and fresh semen (16.7% vs 12.5%). Further, combined over both years, there was no difference (P>0.10) in the incidence of EED (24.2% vs 14.4%). There was a 38.9% incidence of EED in infertile mares.Of the 354 pregnant mares, the overall incidence in EED through day 50 postovulation was 17.3%. The majority of EED (77.1%) occurred prior to day 35 post-ovulation. During the period 15 to 35 days post-ovulation, a greater (P<0.05) incidence of EED occurred between days 15 to 20 (26.2%) and 30 to 35 (29.5%) post-ovulation.     

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.     

Nonsurgical embryo transfer in the common marmoset monkey

Abstract: A technique for nonsurgical embryo transfer in common marmosets was developed. Transfers were either synchronous (ST) or asynchronous (AT). Synchronous transfers (embryo donor and the embryo recipient ovulated on the same day) were performed 5 to 8 days post-ovulation. Asynchronous transfers (embryo donor had ovulated at least 2 days before the embryo recipient) were performed when the recipient was 2 to 4 days post-ovulation (donor was 6 to 8 days post-ovulation). Four pregnancies from nine transfers (44%) were established by AT, and three pregnancies were carried to term. Only 1 of 11 transfers (9%) from ST resulted in a pregnancy, which was lost by Day 40 of gestation. Significantly more infants were born from AT (6 infants from 17 embryos; 35%) than from ST (0 infants from 22 embryos; 0%; p<0.005). This technique allows experimental analysis of primate postimplantation development and provides a tool for conservation of endangered Callithrichid species.     

Commercial aspects of bovine embryo transfer

Superovulated beef cows and heifers were nonsurgically collected 6 to 8 days post estrus. Commercial production results for 1976 through 1978 were 4979 pregnancies from 7814 embryos transferred for an overall pregnancy rate of 63%. In 1978, 519 superovulation procedures averaged 9.95 +/- 8.4 (S.D.) ova collected, 8.2 +/- 7.55 ova fertilized, 5.96 +/- 5.37 embryos transferred and 3.63 +/- 5.37 pregnancies per procedure. Embryos were transferred to recipient cows in estrus 12 hr before the donor (-12) the same time (0) or 12 hr after the donor (+12). The +12 group had a significantly lower pregnancy rate (61%, P<.05) than the 0 group (67%) or -12 group (66%). Transfer of early morula stage embryos resulted in a lower pregnancy rate (61%, P<.05) than late morula (67%) early blastocyst (67%) or late blastocyst (71%) stage embryos. A higher pregnancy rate (P<.05) was obtained with embryos of good morphological quality (71%) than with embryos graded fair and poor (55%). The pregnancy rate for embryos transferred nonsurgically was lower (44%) than the pregnancy rate for embryos transferred surgically during the same time period (66%). Pregnancy rates for three operators performing the nonsurgical transfers were 48%, 53%, 28%. No difference in pregnancy rate was found between embryos cultured 24 hr in BMOC-3 at 37C (62%) and embryos transferred the same day as collection (60%). Pregnancy rates for cultured embryos transferred to recipient cows in estrus 12, 24 or 36 hr after the donor were 68%, 62% and 60%, respectively. Embryos recovered on days 6, 7 and 8 were frozen in 1.5M DMSO and stored in liquid nitrogen several days to several weeks. Of 68 embryos frozen, 34 were viable post thaw. Upon transfer to recipient cows, the 34 viable embryos produced 23 confirmed pregnancies.     

Pregnancy rates in Holstein embryo transfer recipients: Effect of treatment with progesterone or clenbuterol and of natural versus induced cycles

Pregnancy rates were the same (59 and 50%; P > 0.05) in control recipients (n = 22) and recipients treated with 200 mg progesterone 4 h prior to nonsurgical transfer (n = 48) of previously frozen embryos. With small recipients and difficult transfer conditions, the same progesterone supplementation increased pregnancy rates (46 vs 21%; P < 0.05) after transfer of previously frozen embryos. The administration of the β₂-mimetic agent clenbuterol (4-amino- α [(tert. - butylamino) methyl] - 3,5,dichlorobenzyl-alcohol hydrochloride) 30 min prior to nonsurgical transfer resulted in pregnancy rates of 60.0% (n = 25) and 44.2% (n = 52) for Holstein heifers receiving fresh and previously frozen embryos, respectively. Pregnancy rates in untreated controls were 59.9% (n = 142) and 48% (n = 47) for fresh and frozen-thawed embryos. There was no effect (P > 0.05) of spontaneous versus prostaglandin-induced estrus on pregnancy rates for recipients of either fresh or previously frozen embryos.     

Superovulating cows with follicle stimulating hormone and pregnant mare's serum gonadotrophin

A total of 511 embryos was recovered non-surgically from nearly 100 superovulated or untreated donors. Superovulation with FSH-LH resulted in more corpora lutea, recovered ova, and pregnancies (P<.01) than superovulation with PMSG. No differences were observed in numbers of ovulations, embryos recovered, or pregnancies per donor when prostaglandin F_2α was given to donors 2 versus 3 days following gonadotrophin treatment.Pregnancy rates of 12, 31, 58, and 63% were obtained from groups of embryos classified morphologically as poor, fair, good, and excellent (P<.05). Morphologically normal embryos collected and transferred at 5 to 6 days of gestation resulted in more (P<.05) fetuses (75%) than morphologically normal embryos at 8 to 9 days of gestation (56%), but neither was significantly different from morphologically normal embryos at 6.5 to 7.5 days of gestation (61%). There was no difference (P>.05) between pregnancy rates when retarded embryos from untreated donors (12%) were compared to retarded embryos from superovulated donors (22%). However, a higher proportion of morphologically normal embryos from untreated donors developed into fetuses (71%) than did morphologically normal embryos from superovulated donors (59%, P<.05).     

Effects of repeated daily injections of prostaglandin F2α on ovaries in mares

In experiment 1, seven groups of pony mares (2 or 3/group) were given either no injections (controls), or 5(5X) or 10(10X) daily subcutaneous (SC) injections of 1.25 mg PGF_2α beginning on days 1, 7 or 13 post-ovulation. Compared to controls (24.5 days), the interovulatory interval was longer (P<.05) for day 7, 10X (33.5 days) and day 13, 10X mares (49.0 days) but was not different for the remaining groups. In experiment 2, nine groups of pony mares (4/group) were given either no injections (controls) or 1(1X) or 10(10X) daily SC injections of 1.25 mg PGF_2α beginning on day 2 of estrus or on days 1, 7 or 13 post-ovulation. Compared to controls (25.0 days), the interovulatory interval was longer (P<.05) for day 13 post-ovulation, 10X mares (40.0 days) and shorter (P<.05) for day 1 post-ovulation, 10X mares (14.5 days). The interovulatory interval for the remaining groups was not different (P>.05) from that for controls. In day 13 post-ovulation, 10X mares, the longer interovulatory interval did not appear to be related to a depression in either peripheral LH concentration (no effect of treatment on LH) or on follicular development (no effect of treatment on diameter of largest follicle). This suggests that circulating levels of gonadotropins were adequate for ovarian follicular development and ovulation and the effect of repeated daily injections of PGF_2α in preventing ovulation was likely exerted at the ovarian level directly on the follicle.     

Factors affecting pregnancy rate following nonsurgical embryo transfer in buffalo (Bubalus bubalis): a retrospective study

The objectives of this study were to determine the pregnancy rate and factors affecting it following nonsurgical embryo transfer in buffalo. Donor buffalo were superovulated with FSH, and embryos collected nonsurgically were evaluated for stage of development and quality. They were transferred nonsurgically to 91 recipients on Days 5 to 7 of the natural (n = 52) or induced (n = 39) estrus (estrus = Day 0). The overall pregnancy rate of 24/91(26.4%) was higher than in earlier reports for buffalo but was much lower than in cattle. Pregnancy rates were not affected by season (autumn vs winter), side of transfer (right vs left uterine horn), or type of estrus (spontaneous vs induced). The pregnancy rate was high 11/27(40.7%) when donors and recipients were closely synchronized, while it was compromised when recipients were in estrus at +12 h (1/7, 14.3%) and at -12 h (5/27, 18.5%). Asynchrony beyond 12 h on either side resulted into conception failure. The pregnancy rate tended to increase with the increase in CL size of recipients, while stage of embryonic development had no effect. The transfer of an 8-cell embryo with a 16-cell embryo led to the birth of heterosexual twins, indicating that the uterine milieu of Day 5 to 6 recipients may be tolerated by the out-of-phase 8-cell embryo, at least in the presence of a more mature embryo. Embryo quality had the greatest effect on pregnancy rate as it was higher (P < 0.005) after the transfer of Grade I than Grade III embryos (6/10, 60.0% vs 3/36, 13.9%). Assessment of returns to estrus indicated that among nonpregnant recipients, 17/67 (25.4%) embryos never matured sufficiently to prevent luteolysis through maternal recognition of pregnancy (MRP), while 14/67 (20.8%) embryos probably died following MRP. These results indicate that efforts to increase pregnancy rate following embryo transfer in buffalo should include prevention of luteolysis during the first week of transfer and a reduction in the incidence of embryonic mortality.     

Control of estrus in the lactating postpartum mare with fluprostenol (ICI-81,008)

During the 1976 breeding season 68 mares (56 Thoroughbred and 12 Quarter Horse) were allotted to 4 treatment groups. On day 7, 8 or 9 post-ovulation or day 6, 7 or 8 after foal heat each of 28 mares was injected intramuscularly with 250 μg of the prostaglandin analogue ICI-81,008 (generic name fluprostenol). In the second group, 32 mares were bred at foal heat. Group 3 consisted of 9 mares, which were passed at foal heat and bred at the second postpartum estrus. In group four, 12 of the mares which had failed to conceive at foal heat were bred at the second estrus. Twenty-four prostaglandin treated mares returned to estrus 4.2±.4 days post-injection. The interestrual interval was shorter (P<.05) for prostaglandin treated mares (11.4±.6 days) than for previously mated second heat control mares (19.2±2.3). Also, the interval between parturition and second estrus was decreased (P<.05) in treated mares (24.5±.8 days) compared to group three (32.8±2.5 days). Duration of the second postpartum estrus was similar for both treated and control mares. Plasma progesterone levels in prostaglandin treated mares as determined by RIA were 7.5±.5 and 6.9±.5 ng/ml before treatment and at l hour post-injection, then dropped precipitously to near undetectable levels at 48 hours after injection. Pregnancy rate at 45 days was significantly higher (P<.05) in prostaglandin treated mares (78%) as compared to foal heat mares (48%) and mares which were passed at foal heat and bred at the second postpartum estrus (44%).     

Efficiency of superovulation and in vivo embryo production in eFSH-treated donor mares after estrus synchronization with progesterone and estradiol-17β

Reliable methods of regulating estrus and stimulating superovulations in equine embryo transfer programs are desirable. Our objectives were to investigate the efficacy of a progesterone and estradiol-17β (P&E) estrus synchronization regimen in mares with and without subsequent equine follicle-stimulating hormone (eFSH) treatment and to examine the effects of eFSH on folliculogenesis and embryo production. Cycling mares were treated with P&E daily for 10 d. On the final P&E treatment day, prostaglandin F_2α was administered, and mares were randomly assigned to one of two treatment groups (n = 20 mares/group). In both groups, mares were examined daily by transrectal ultrasonography. In the eFSH group, twice-daily eFSH treatments were initiated at follicle diameter 20 to 25 mm and ceased at follicle ≥35 mm; human chorionic gonadotrophin (hCG) was administered after 36 h. In the control group, eFSH treatments were not given, but hCG was administered at follicle ≥35 mm. Mares were inseminated with fresh semen, and embryo recovery attempts were performed 8 d postovulation. Synchrony of ovulations within each group appeared to be similar. Six mares in the eFSH group failed to ovulate. The eFSH treatment resulted in higher (P < 0.05) numbers of preovulatory follicles and ovulations; however, embryo recovery rate did not increase (eFSH 1.0 ± 0.4 vs. control 0.95 ± 0.1 embryos/recovery attempt), and embryo per ovulation rate was significantly lower (36% vs. 73%). The eFSH-treated mares had significantly higher frequency of nonovulatory follicles (28% vs. 0) and higher periovulatory serum concentrations of estradiol-17β. Based on our findings, combined P&E and eFSH regimens cannot be recommended for cycling donor mares.     

Superovulatory responses in FSH- or Pergonal®-treated heifers

Follicle stimulating hormone (FSH) or human menopausal gonadotropin (Pergonal^®) were used in combination with prostaglandin F_2α (injected either intramuscularly or intravenously) to induce superovulation in 32 beef heifers. Two different transfer media (phosphate buffered saline [PBS] + 20% fetal calf serum, or PBS + 1.5% bovine serum albumin) and two different nonsurgical transfer instruments (stainless steel tube with polyethylene tubing and Cassou gun) were used to transfer embryos to recipient cows. There were no significant differences (P>0.10) in the number of corpora lutea, oocyte or embryos recovered, and transferable embryos (good morula and blastocysts) after superovulation with either gonadotropin. More pregnancies (P<0.10) resulted with non-surgical transfer of embryos from FSH- than Pergonal^®-primed donors (29% vs 13%, respectively). However, pregnancy rates were not affected by the two transfer media or transfer instruments used in this study.     

Infection of embryos following insemination of donor mares with equine arteritis virus infective semen

The objective was to evaluate the potential risks associated with embryo transfer from mares bred with equine arteritis virus (EAV) infective semen. Twenty-six mares were embryo donors, whereas 18 unvaccinated and EAV antibody seronegative mares were embryo recipients. Of the 26 donor mares, 15 were unvaccinated and seronegative for antibodies to EAV and 11 were vaccinated for the first time with a commercially available modified live virus vaccine against EVA before breeding and subsequent embryo transfer. All donor mares were bred with EAV-infective semen from a stallion persistently infected with the virus. Twenty-four embryos were recovered 7 d post-ovulation; all were subjected in sequential order to five washes in embryo flush medium, two trypsin treatments, and five additional washes in embryo flush medium (prior to transfer). Twelve and seven embryos (Grades 1 or 2) were transferred from the non-vaccinated and vaccinated donors, respectively, and pregnancy was established in 3 of 12 and 2 of 7. Perhaps trypsin reduced embryo viability and pregnancy rate. The uterine flush fluid of 11 mares (9 of 15 and 2 of 11 from non-vaccinated and vaccinated donor groups, respectively) was positive for EAV by VI (confirmed by real-time RT-PCR); the wash fluid from the embryos of nine of these mares was negative following 10 washes and two trypsin treatments. However, the embryo wash fluid from two mares was still positive for EAV after all 10 washes and the two trypsin treatments, and one embryo was positive for EAV. Two of 18 recipient mares had seroconverted to EAV 28 d after embryo transfer. Virus was not detected in any fetal tissues or fluids harvested after pregnancies were terminated (60 d). In conclusion, we inferred that the washing protocol of 10 washes and two trypsin treatments did not eliminate EAV from all embryos; due to limitations in experimental design, this requires confirmation. Furthermore, there may be a risk of EAV transmission associated with in vivo embryo transfer from a donor mare inseminated with EAV infective semen.     

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.     

Comparison of pregnancy rates from transfer of fresh versus cooled, transported equine embryos

Donor mares of mixed, light-horse breeds, maintained at Colorado State University, provided 104 embryos for immediate transfer (fresh embryos). One hundred and thirty-six additional embryos were collected on various breeding farms in the United States and were shipped to Colorado State University via commercial airlines (cooled embryos). Embryos were harvested 7 d after ovulation, graded, and either transferred into a mare immediately (<1 h) or placed in Ham's F-10 medium plus 10% fetal calf serum in an atmosphere of 5% CO(2), 5% O(2), 90% N(2) and packaged in a passive cooling unit (Equitainer) for shipment to our laboratory. All embryos were measured and graded just prior to surgical transfer via flank incision into synchronized mares. Recipients had ovulated 1 or 2 d before (+1, +2), on the same day as (0), or 1, 2 or 3 d after (-1, -2, -3) the donor mare. Pregnancy of recipients was determined by ultrasonography on 12, 35, and 50 d after ovulation of the donor. Pregnancy rates at 12, 35, and 50 d were similar for fresh (74, 64, 61%) and cooled embryos (80, 67, 66%), respectively. Overall, embryo size affected (P<0.05) pregnancy rates at 12, 35 and 50 d. Embryos of Grade 1 (excellent) or 2 resulted in more pregnancies than those of Grade 3 or 4 (poor) embryos. Embryonic losses between 12 and 35 d or between 35 and 50 d were not altered (P>0.05) by treatment (fresh or cooled) nor by age of the donor mare (P>0.05), but embryonic losses between 12 and 35 d were greater (P<0.06) for embryos stored for >12 h (25%) versus those stored for <12 h (10%). The duration needed for shipment (<12 h or >12 h) of cooled embryos did not alter pregnancy rates at 12 d (P>0.05). Age of donor mare had no effect (P>0.05) upon pregnancy rates of cooled or fresh embryos transferred nor on embryo quality. In summary, equine embryos can be cooled to 5 degrees C and maintained in storage for up to 24 h without decreased fertility, compared with those of embryos transferred in <1 hour.     

One year old fillies can be successfully used as embryo donors

One year old fillies are able to conceive but, usually, not to give birth to a living foal. Although embryo transfer allows the production of foals from mature mares with repeated pregnancy losses, no reports are available on the use of one year old fillies as embryo donors. To evaluate this possibility, eleven 12-16 months old Haflinger mares were inseminated with fresh semen and subjected to embryo recovery. Some of the recovered embryos were non-surgically transferred into synchronized mature recipients. Pregnancies were terminated using PGF2alpha at day 25. Fillies' embryo recovery rate and their recipients' pregnancy rate at day 25 were compared with those achieved in two years old and mature mares of the same breed, subjected to the same management. Embryo recovery rate was 21/44 (47.7%), 12/16 (75%) and 22/26 (84.6%) (P>0.01) for one year old, two years old and mature mares, respectively. Five/7 (71.4%) one year old donors' embryos resulted in a pregnancy after transfer and 4/7 (57.1%) developed until day 25. Significant differences in pregnancy rates after transfer between donors' age groups were not observed; no short term side effects resulted from the use of fillies as embryo donors. This study showed that one year old mares employed as embryo donors produce embryos both morphologically normal and able to develop in recipient mares at least up to day 25 of pregnancy.     

Embryo quality and transcervical technique are not the limiting factors in donkey embryo transfer outcome

Embryo transfer (ET) in the donkey resulted in a very low recipient pregnancy rates. The aim of these studies was to investigate if nonsurgical transfer techniques or donkey embryo quality affect donkey recipient pregnancy failure. In Study 1, the impact of transfer technique was investigated by evaluating if cervical catheterization is associated with prostaglandin release and suppression of luteal function and if donkey recipients would become pregnant after nonsurgical transfer of horse embryos. Four jennies, from 5 to 8 d after ovulation, were submitted to a sham transcervical ET and to evaluation of PGFM and progesterone plasma concentrations. Five 8 d horse embryos were nonsurgically transferred into synchronized donkey recipients (HD). Cervical stimulation caused a transient PGF_2α release in two of four jennies in the absence of a significant decrease in progesterone plasma concentration. All transferred horse embryos resulted in pregnancies in the jenny recipients. In Study 2, donkey embryo viability was investigated by 1.2 meters, 6-diamidino-2-phenylindole (DAPI) staining of 10 embryos and by the transfer of 6 and 12 donkey embryos in synchronized mare (DH) and donkey (DD) recipients, respectively, of known fertility. The estimated proportion of dead cells in DAPI stained embryos was 0.9% (range 0-3.9%) and below what is considered normal (20%) for horse embryos. Three of six and six of 12 of the DH and DD ETs, respectively resulted in pregnancies at 14 and 25 d (50%), a higher pregnancy rate than previously reported after DD ET. The overall results of this study suggest that the transcervical technique for ET and donkey embryo viability are not the reasons for the low pregnancy rates that have previously been described in donkey recipients, and that nonsurgical ET in donkeys can result in acceptable results.     

Embryo transfer in two-year-old donor mares

Embryos were collected from two-year-old donor mares and transferred surgically during 1983 and 1984. The overall embryo recovery rate from two-year-old donors was 36.3%. Over both years, 71.4% of the donors ($\text{30}\text{42}$) provided at least one embryo. There was a trend both years for slightly higher embryo recovery rates prior to August 1 (47.7%), as compared to after August 1 (31.1%). Pregnancy rates in recipient mares after surgical embryo transfer were not affected by month of the breeding season. However, there was a trend for improved pregnancy rates when embryos were transferred after August 1 (87.5%), as compared to before August 1 (70.0%). There was a 8.3% incidence of fetal loss between Days 15 and 35 of gestation in recipients. The incidence of fetal loss between Days 35 and 60 of gestation was 2.7%. Based on these data, the chance of obtaining a pregnancy from a two-year-old mare is 28.2% (36.3% recovery × 77.7% pregnancy rate). Thus embryo transfer may prove to be a beneficial tool for obtaining foals from two-year-old donor mares.     

Effects of cervical dilation and intrauterine infusions on the timing of oviductal transport of equine embryos

Equine embryos spend 5 to 6 d in the oviduct before entering the uterus as expanded blastocysts, and cannot be consistently collected nonsurgically until Day 7. Technologies such as cryopreservation and embryo splitting, which are most successful with embryos at the morula or early blastocyst stage, have not been used in mares because equine morulae and early blastocysts are located in the oviduct and cannot be recovered nonsurgically. These experiments test the hypothesis that transport of equine embryos through the oviduct can be hastened by cervical dilation or by acute, sterile endometritis induced by intrauterine oyster glycogen treatment. Cervical dilation with or without intrauterine infusion of 0.5 ml PBS on Day 4 did not appear to hasten the transport of embryos into the uterus since Day 5 uterine embryo recovery rates were not higher (P > 0.1) for mares with cervical dilation or cervical dilation plus PBS infusion vs mares receiving no treatments (0 of 5 and 0 of 5 vs 0 of 10, respectively). Intrauterine infusions of 40 ml of 1% oyster glycogen or 40 ml of PBS on Day 3 did not appear to hasten the transport of embryos into the uterus since Day 5 uterine embryo recovery rates were not higher (P > 0.1) for oyster glycogen- or PBS-treated vs untreated mares (2 of 12 and 3 of 11 vs 0 of 10, respectively). Cervical and uterine treatments on Day 3 or Day 4 and uterine lavages on Day 5 decreased (P < 0.05) Days 11 to Day 15 pregnancy rates compared with that of untreated mares. Day 11 to Day 15 pregnancy rates were 1 of 5 for mares with Day 4 cervical dilation and Day 5 uterine lavage, 1 of 5 for mares with Day 4 PBS infusion and Day 5 uterine lavage, 2 of 12 for mares with Day 3 oyster glycogen infusion and Day 5 uterine lavage, and 3 of 11 for mares with Day 3 PBS infusion and Day 5 uterine lavage vs 7 of 10 for mares that received no treatment or lavage. Cervical and uterine manipulations on Day 3 or 4 and uterine lavage on Day 5 appeared to decrease pregnancy rates by Days 11 to 15. The results of these experiments do not support the hypothesis that cervical dilation or uterine infusion hasten oviductal transport, since neither cervical manipulation nor transcervical infusion of oyster glycogen or PBS into the uterus significantly hastened the rate of embryo transport into the uterus.     

Effect of exogenous progesterone on pregnancy rates after surgical embryo transfer in mares

A completely randomized experimental design was used to investigate the effect of supplemental progesterone on pregnancy rates of recipient mares. Every other recipient mare received daily 200 mg progesterone in oil beginning the day of surgical embryo transfer and lasting until either Day 120 of pregnancy or until pregnancy failure was confirmed by ultrasound. Progesterone supplementation did not affect pregnancy rate (P > 0.05). Overall, embryos that did not result in pregnancy were of greater mean diameter than embryos that resulted in pregnancy (P < 0.05). Pregnancy rates tended (P < 0.1) to be greater in recipients that were detected to be ovulating the same day or prior to that of the donor and that had been supplemented with progesterone (75 %) as opposed to untreated control mares of the same synchrony group (40 %). Progesterone supplementation did not affect the incidence of embryonic loss; however, there was a slightly higher loss of pregnancies between Day 15 and 30 in treated versus untreated recipients. There was no effect (P > 0.05) of treatment on pregnancy rate for embryos recovered from fertile versus subfertile donor mares. However, overall, there tended (P < 0.1) to be fewer pregnancies with embryos recovered from subfertile (50 %) as compared to fertile donors (75 %). It was concluded that supplemental progesterone at the dosage and frequency described was not beneficial in improving pregnancy rates in cyclic recipient mares after surgical embryo transfer.     

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.