Viability and ultrastructure of cryopreserved equine embryos collected from two-year-old fillies

Abstract

The objectives of this study were to: 1) perform repeated embryo recoveries and evaluate subsequent fertility of two-year-old fillies; 2) develop and test the efficacy of a cryopreservation protocol for equine embryos using pregnancy following non-surgical transfer as the criterion, and 3) evaluate via transmission electron microscopy the possible ultrastructural changes in cryopreserved equine embryos. During a two-year study thirty-nine two-year-old fillies were evaluated as embryo donors. Eight fillies which had undergone a mean of 12 collection attempts each during 1984 were bred during the second year. Five of the eight conceived during the first estrous cycle and the remaining three conceived during the second cycle. Embryos were collected approximately d 6 post-ovulation for evaluation of a cryopreservation protocol. Following freezing and thawing, embryos were transferred non-surgically to recipients which were maintained on exogenous progestin therapy until pregnancy determination by ultrasonography. Three mares (30%) became pregnant and one was allowed to complete gestation. Electron microscopy was used to elucidate the ultrastructural changes which may cause frozen-thawed embryos, which appear viable post-thaw, to be unable to continue development following transfer. Intercellular bridges and tight junctions were evident between inner cell mass cells (ICM) of the equine embryos. Trophoblast (TB) cells had developed junctional complexes by the early blastocyst stage. The mitochondria of the frozen-thawed embryos appeared to have changed morphologically. Specifically, the cristae thickened and lost the distinct lamellar appearance. Also some of the ICM mitochondria had distinct blebbing of the outer membranes. The membrane thickening was also observed in some of the glycerol-treated embryos but the extent of damage did not appear to be as great. The mitochondria of the TB cells did not display these same morphological changes. The stage of development at which embryos are cryopreserved and(or) the possible ability of the embryonic TB cells to exclude cryoprotectants from the blastocoele may help explain why frozen-thawed embryos consistently produce lower pregnancy rates as compared to embryos which were transferred soon after recovery from the donor mare.