134 VIABILITY OF EQUINE BLASTOCYSTS SUBJECTED TO BIOPSY FOR PRE-IMPLANTATION GENETIC DIAGNOSIS

Abstract

Pre-implantation genetic diagnosis of embryos is commonly performed in humans but has not been developed in the horse. To our knowledge, only one previous report of transfer of equine embryos after biopsy is available (Huhtinen et al. 1997 Theriogenology 48, 361); in that study, three pregnancies were achieved after transfer of 14 embryos. Failure of pregnancy after biopsy of equine embryos has been attributed to difficulty in penetrating the equine embryonic capsule, and possible effects of capsule damage on subsequent embryo survival. Recent developments in intracytoplasmic sperm injection (ICSI) and embryo culture make it possible to produce equine blastocysts in vitro. In vitro-produced equine embryos lack a capsule, but form one when transferred to the uterus. This study was designed to examine the viability of equine blastocysts subjected to biopsy. In Study 1, in vitro- and in vivo-produced embryos were vitrified after biopsy, as would be needed while awaiting results of genetic testing. In vitro-produced blastocysts (n = 5) were obtained from slaughterhouse-derived oocytes that were matured, fertilized by ICSI, and cultured 7 to 8 days. In vivo-recovered blastocysts (n = 9) were obtained by uterine flush of inseminated mares 6.5 days after ovulation. The biopsy procedure was conducted using a Piezo drill in modified PBS without calcium and magnesium. Two to three biopsy manipulations were conducted on each embryo to ensure that an adequate number of cells was obtained. After biopsy, embryos were vitrified as described by Eldridge-Panuska et al. (2005 Theriogenology 63, 1308). Four vitrified in vivo-produced blastocysts were warmed and cultured for 2 days. Of these, two appeared viable, showing a 50- to 100-μm increase in diameter, and two did not grow. The remaining 5 in vivo- and 5 in vitro-produced vitrified blastocysts were shipped to a transfer facility, warmed and transferred transcervically to recipient mares. None of these embryos established pregnancy. In Study 2, three Day-6 in vivo-recovered blastocysts were biopsied then shipped by air (6 h transport time) for transfer to recipient mares. All three embryos established pregnancies and showed a normal heartbeat at 25 days. These results indicate that cells may be obtained from in vitro and in vivo-produced equine blastocysts by biopsy with the Piezo drill. In vivo-produced blastocysts transferred directly after biopsy were viable (3/3). While some blastocysts vitrified after biopsy showed growth in vitro, vitrified embryos did not produce pregnancies after transfer. To our knowledge, this is the first report of normal pregnancy rate after transfer of biopsied equine embryos. Further studies are needed to improve survival of embryos vitrified after biopsy.

This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.