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Effect of cryopreservation on the cellular integrity of equine embryos

Faculty of Veterinary Medicine, Department of Equine Sciences, Utrecht University, Yalelaan 12, 3584 CM, Utrecht, The Netherlands

Correspondence should be addressed to T Tharasanit; Email: T.Tharasanit{at}vet.uu.nl

Horse embryos are rarely cryopreserved in practice because expanded blastocysts tolerate freezing poorly, and the embryo begins expanding very soon after entering the uterine cavity. This study examined the effects of freezing on cytoskeleton integrity, and investigated whether cell damage could be reduced using trypsin to thin the blastocyst capsule or cytochalasin-B (cyto-B) to stabilise the cytoskeleton. Sixty-nine embryos were recovered 7 days after ovulation and equilibrated in 10% glycerol, with or without pretreatment with 0.2% trypsin or 7.5 µg/ml cyto-B. Forty-two of the embryos were frozen; the rest were used to determine whether pre-freezing treatment alone caused cell damage. Subsequently, embryos were stained with 4',6-diamidino-2-phenylindole dihydrochloride, to identify dead cells, and fluorescently labelled phalloidin, to assess cytoskeleton quality. Without freezing, none of the treatments affected cell viability. And although Cyto-B altered actin distribution, the cytoskeleton returned to normal during a 4-h culture. Following cryopreservation, the percentage of dead cells (11.1 ± 1.3%) did not differ between treatments (P > 0.05), but significantly fewer cells died in small (300 µm) than in large embryos when neither pretreatment was used (P > 0.05); the effect of embryo size was, however, not significant after pretreatment with trypsin or cyto-B, and trypsin improved the likelihood of an intact cytoskeleton post thaw. However, trypsin treatment also resulted in a ‘sticky’ capsule that complicated embryo handling, and cyto-B-induced actin-depolymerisation was not reversed during a 6-h post-thaw incubation. Thus, while trypsin pretreatment improved cytoskeleton preservation and both trypsin and cyto-B may reduce cell death during cryopreservation of large embryos, both treatments induced other changes likely to compromise embryo survival.