158 SUCCESSFUL TRANSFER OF FRESH AND VITRIFIED BIOPSIED EQUINE EMBRYOS

Abstract

In horses, determination of certain genetic traits/alleles (e.g. HYPP, SCID, sex, or color) before transfer of embryo often would be advantageous due to the costs of these pregnancies. An attractive option is pre-implantation genetic diagnosis, but to date, few biopsied equine embryos have resulted in pregnancies. In the current experiment, 10 embryos ranging in diameter from 160-575 μm were biopsied. To obtain embryos, donor mares were monitored using transrectal ultrasonography. When a follicle >35 mm in diameter was observed, 2500 IU of hCG or 1.5 mg of deslorelin acetate was administered, and mares were inseminated daily until ovulation was detected. Embryos were recovered nonsurgically on Days 6.5-7 (Day 0 = ovulation). Trophoblast biopsies were collected in a 30-μL droplet of Syngro Holding Media (Bioniche, Belleville, Ontario, Canada) using a Piezo drill and beveled injection pipette (7-10 μm outer diameter). Biopsy samples ranged from 4-10 cells by visual assessment. After removal of the embryo, the droplet containing the biopsied cells was moved into an Eppendorf tube and centrifuged at 11 000g for 10 min. Supernatant was removed leaving 5 μL of sample, which was snap frozen for later genetic testing. Embryos were immediately transferred nonsurgically into the uteri of recipient mares synchronized to ovulate 0 to 2 days after the donor. Pregnancy examinations were performed via ultrasonography on Days 11, 12, 14, 16, 20, and 25. Pregnancies were confirmed on Day 16 for 6 of the 10 transfers. Five embryos (175-240 μm) had confirmed heartbeats on Day 25; one recipient mare lost her pregnancy (160 μm) between Days 16 and 20. The two largest embryos, 390 and 575 μm, did not result in pregnancies. As a preliminary experiment, 7 embryos (150-300 μm) were vitrified according to Eldridge-Panuska et al. (2005) after biopsying and later warmed and transferred directly. Three (all 200 μm) Day 16 pregnancies resulted from the transfer of vitrified, biopsied embryos. Two pregnancies were maintained to a heartbeat; one pregnancy resulted in the formation of an empty trophoblastic vesicle. All pregnancies were terminated on or after Day 25 to collect embryos for further genetic testing. For pre-implantation genetic testing, a duplex-nested PCR was developed for amplification of the DNA from the biopsied cells using primers for sex chromosome-linked zinc finger protein genes (ZFX/ZFY; 445 bp), and 2 pairs of primers for equine-specific sex-determining region on the Y-chromosome (SRY; 217 bp, 121 bp). Experiments on XX and XY genomic DNA from white blood cells revealed accurate genetic testing on as little as 9 pg of DNA, which equals 1 cell. Preliminary results revealed successful sex genotyping on biopsied material, and current experiments are underway to confirm and expand on these results.