Application of embryo biopsy and sex determination via polymerase chain reaction in a commercial equine embryo transfer program in Argentina.

Embryo biopsy for fetal sexing has clinical application, but few reports are available of its use within an active embryo transfer program. We evaluated results on biopsy of 459 embryos over one breeding season. There were no significant differences in pregnancy rate between biopsied and non-biopsied embryos (72% vs 73%) or for biopsied embryos recovered at the centre (73%) compared with those shipped overnight (72%). However, the pregnancy rate decreased significantly in shipped embryos biopsied ≥20h after collection. Overall, 86% of biopsies provided a sex diagnosis. The likelihood of a positive genomic (g) DNA result was significantly higher for biopsies from large blastocysts (96%) than from smaller embryos (70-85%). In total, 38% of biopsies were positive for Y chromosome DNA (Y-DNA) and were diagnosed as male. Subsequently, 95% of Y-DNA-positive embryos were confirmed as male and 78% of Y-DNA-negative embryos were confirmed as female. The accuracy of prediction of female (Y-DNA negative) was significantly higher when the biopsy sample was probed for Y-DNA only compared with probing for both gDNA and Y-DNA. We estimate that by transferring only Y-DNA-negative embryos, 3% of potential female pregnancies may have been lost, and production of male pregnancies was reduced by 72%.

Successful cryopreservation of expanded equine blastocysts.

Effective cryopreservation of expanded equine blastocysts (> 300 µm in diameter) has been difficult, perhaps due to the volume of blastocoele fluid or the presence of the equine embryonic capsule. Recently, we reported normal viability of equine embryos after trophoblast biopsy, which resulted in blastocyst collapse. The present study addressed the effect of biopsy and resultant breach of the capsule and blastocyst collapse on survival of expanded equine blastocysts after vitrification. First, non-biopsied, small embryos (< 300 µm) were vitrified in fine-diameter microloader pipette tips using dimethylsulfoxide-containing medium (DM) or ethylene glycol-containing medium (EG). A third group was vitrified with EG, but was warmed using sucrose (EG/s). Embryos in the DM and EG/s treatments grew in culture after vitrification, and established pregnancies after transfer (3 of 12 and 3 of 6, respectively). Expanded blastocysts 300-730 µm in diameter were then biopsied and vitrified; rates of normal pregnancy (detection of embryonic heartbeat) after warming and transfer were 2 of 16 (13%) and 6 of 13 (46%) for DM and EG/s treatments, respectively (P = 0.05). Within the EG/s treatment, it appeared that greater loss of blastocoele fluid after biopsy was associated with higher survival. Therefore, an altered ("Central") biopsy technique was used to aspirate blastocoele fluid, followed by vitrification in EG/s. Pregnancy rates were 1 of 8 (13%) for embryos cultured after warming and 4 of 7 (57%) for embryos transferred immediately after warming (P = 0.1). Finally, expanded blastocysts 407 to 565 µm in diameter were biopsied from the periphery, and blastocoele fluid was removed with gentle suction. After vitrification with EG/s, this resulted in a rate of normal pregnancy of 5 of 7 (71%). These findings demonstrated that blastocoele collapse and vitrification in fine-diameter pipettes allowed successful cryopreservation of expanded equine blastocysts.

Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis.

The equine embryo possesses a capsule that is considered essential for its survival. We assessed viability after breaching the capsule of early (Day 6) and expanded (Day 7 and 8) equine blastocysts by micromanipulation. The capsule was penetrated using a Piezo drill, and trophoblast biopsy samples were obtained for genetic analysis. Pregnancy rates for Day-6 embryos, which had intact zonae pellucidae at the time of recovery, were 3/3 for those biopsied immediately after recovery and 2/3 for those biopsied after being shipped overnight under warm (∼28 °C) conditions. The pregnancy rates for encapsulated Day-7 expanded blastocysts were 5/6 for those biopsied immediately and 5/6 for those biopsied after being shipped overnight warm. Two of four encapsulated Day-8 blastocysts, 790 and 1350 µm in diameter, established normal pregnancies after biopsy. Nine mares were allowed to maintain pregnancy, and they gave birth to nine normal foals. Biopsied cells from eight embryos that produced foals were subjected to whole-genome amplification. Sex was successfully determined from amplified DNA in 8/8 embryos. Identification of disease-causing mutations matched in the analyses of 6/6 samples for the sodium channel, voltage-gated, type IV, alpha subunit (SCN4A) gene and in 6/7 samples for the peptidylprolyl isomerase B (PPIB) gene, in embryo-foal pairs. Thus, the capsule of the equine embryo can be breached without impairing viability. Further work is needed to determine whether this breach is transient or permanent. These findings are relevant to the understanding of equine embryo development and to the establishment of methods for micromanipulation and embryo cryopreservation in this species.

Effect of administration of prostaglandin F2 alpha on embryo recovery from the uterus on day 5 after ovulation in mares.

Ten mares were used to investigate the effect of administration of prostaglandin F2 alpha on uterine tubal motility, as reflected by embryo recovery from the uterus 5 days after ovulation (day 0). Mares were assigned to 3 groups: group A, uterine flush for embryo recovery on day 7; group B, uterine flush for embryo recovery on day 5; and group C, uterine flush for embryo recovery on day 5, after treatment with prostaglandin F2 alpha (10 mg, IM) on day 3. Each mare was assigned to each group once. Embryo recovery rates for the 3 groups were: A, 6 of 10; B, 2 of 8; and C, 0 of 10. The embryo recovery rate for group C was significantly lower (P less than 0.01) than that for group A. Embryo recovery rate for group B was not significantly different from group A or group C. Administration of prostaglandin on day 3 did not increase embryo recovery rate from the uterus on day 5. Additionally, the 25% embryo recovery rate (2 of 8) for group B mares suggested an earlier time for entry of the embryo into the uterus than has previously been reported.

Cervical hyperplasia with prolapse in a mare.

Cervical hyperplasia with prolapse through the vulvar lips was documented in a mare. Postmortem examination indicated that the mass originated from the cervical wall. The surface of the prolapsed mass had histologic features of normal cervix. Cervical hyperplasia can be considered in a list of differential diagnoses in cases of prolapse of the internal genitalia in mares.

Establishment of pregnancy after embryo transfer in mares with gonadal dysgenesis.

Embryo transfer was performed in three mares with gonadal dysgenesis. Karyotypes of the mares were as follows: Mare 1, 63,XX, 64,XX, 65,XX; Mare 2, 63,X; and Mare 3, 65,XXX. The mares were administered progesterone in oil, 300 mg intramuscularly daily, starting 1 or 2 days after donor mare ovulation. Embryos were transferred on day 7 after donor ovulation. Mare 1 became pregnant after the first embryo transfer and had a normally developing fetus on necropsy on day 45 of gestation. Mare 3 became pregnant after the third embryo transfer, but the embryo was lost between day 14 and day 18 of gestation. Mare 2 received embryos on six occasions without maintaining pregnancy after transfer. Mares with gonadal dysgenesis treated with progesterone can establish and maintain pregnancy after embryo transfer, but there may be differences in this capability among mares, possibly related to the cause of the gonadal dysgenesis.