89 HIGH PREGNANCY RATES AFTER TRANSFER OF LARGE EQUINE BLASTOCYSTS COLLAPSED VIA MICROMANIPULATION BEFORE VITRIFICATION

Abstract

Whereas small equine embryos may be successfully cryopreserved, pregnancy rates after cryopreservation of large equine blastocysts (>300 μm) are low. This has been attributed to their large diameter, failure of cyoprotectants to diffuse into the blastocoele, and/or impedance from the equine embryonic capsule. Previously, we successfully biopsied the trophoblast of equine in vivo-recovered expanded blastocysts (>300 μm; Choi et al. 2009 Reprod. Fertil. Dev. 21, 166-167). In that study, we observed embryonic growth in vitro after warming of a blastocyst vitrified immediately after biopsy. An efficient vitrification method was recently described for ferret embryos, which, like horse embryos, contain a large amount of lipid. This method utilizes very small (250 μm inner diameter) pipettes (Femtotips, Eppendorf; Sun et al. 2008 Biol. Reprod. 79, 832-840). We utilized this technique, with minor modifications, to vitrify equine embryos. In Study 1, the vitrification procedure was performed on equine in vitro-produced blastocysts (<200 μm diameter). All 11 embryos vitrified with the procedure and subsequently warmed grew in culture, showing an increase in diameter over the first 24 h, followed by protrusion of embryonic tissue through holes in the zona pellucida. In Study 2, 10 in vivo-recovered expanded blastocysts, >300 μm diameter, were biopsied using a Piezo drill, causing blastocoele collapse, and were then immediately vitrified. Three of these vitrified blastocysts (346, 399, and 514 μm in diameter) were warmed and transferred to culture; all 3 expanded within 8 h and subsequently proliferated through holes in the embryonic capsule. Seven vitrified in vivo-recovered blastocysts were warmed at the laboratory, shipped to a transfer facility, and transferred transcervically to recipient mares. Five out of 7 transferred blastocysts (71%) resulted in pregnancy at Day 14. Diameters of these embryos before biopsy and vitrification were 360, 398, 415, 434, and 599 μm. The 2 biopsied and vitrified embryos that did not result in pregnancy after transfer were originally 376 and 515 μm in diameter. To our knowledge, this represents the first report of normal (>50%) pregnancy rates achieved after transfer of cryopreserved expanded equine blastocysts. Our findings suggest that biopsy before cryopreservation, which punctures the embryonic capsule and causes blastocoele collapse, combined with vitrification in a small-diameter pipette, may provide an efficient procedure for cryopreservation of large equine blastocysts. Further studies are needed to explore the different factors associated with success of this procedure. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.