Vitrification of invitro-produced and invivo-recovered equine blastocysts in a clinical program.
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There is a clinical demand for cryopreservation of both invivo-recovered and invitro-produced (IVP) equine embryos. We previously reported successful vitrification of expanded equine blastocysts in fine-diameter microloader pipette tips (MPTs) after blastocoel collapse, in a research setting. Here, we report the results of clinical application of the MPT vitrification technique for both invivo-recovered and IVP blastocysts. Invivo-recovered blastocysts were obtained by referring veterinarians on Days 6 to 8 after ovulation, and shipped 1 to 10hours to the laboratory before vitrification. IVP blastocysts (<300m in diameter) were produced by intracytoplasmic sperm injection and invitro embryo culture. All vitrified-warmed embryos were shipped (0.5-12hours) for transfer to recipient mares. In experiment 1, 47 IVP embryos from our clinical intracytoplasmic sperm injection program were vitrified using the MPT and transferred. The rates of initial pregnancy (59%) and foaling (45%) were equivalent to those for 52 IVP embryos from the same mare aspiration sessions and shipped for the same duration but transferred fresh (75% and 45%, respectively). The pregnancy and foaling rates for invivo-recovered embryos were 76 and 71%, respectively for 17 small blastocysts (<300m in diameter), and 55 and 45%, respectively for 11 large blastocysts (303-608m in diameter, collapsed before vitrification; P>0.1). In experiment 2, the MPT was cut lengthwise to form an open vitrification device, designated "Sujo". Research IVP blastocysts were vitrified at 1, 2, or 3 embryos per Sujo (n=34 embryos), or singly on a commercial open device (Cryolock; n=11). After warming, 97% and 91% of embryos, respectively, grew in culture. Similarly, culture of two invivo-recovered large blastocysts after collapse and vitrification on Sujos both resulted in embryo growth. However, transfer of four invivo-recovered expanded blastocysts after collapse, vitrification on Sujos, and warming resulted in only one foal. These data indicate that vitrification of equine IVP embryos and small invivo-recovered embryos is efficient under clinical conditions. Collapse and vitrification of invivo-recovered large blastocysts in MPT under our clinical conditions resulted in a 45% foaling rate. While numbers are low, use of an open vitrification system did not appear to improve results for these embryos.
