163 DOMESTIC CAT (FELIS CATUS) EMBRYO CRYOPRESERVATION: SLOW-COOLING VERSUS VITRIFICATION

Abstract

A variety of small domestic or nondomestic felid species have been produced from cryopreserved embryos using slow-cooling (controlled rate) cryopreservation methods (Pope et al. 2006 Theriogenology 66, 59–71; 1518–1524). However, in our laboratory these methods have not been as successful as vitrification in freezing in vitro-derived embryos of larger felid species such as tigers, Panthera tigris spp. (Crichton et al. 2000 Theriogenology 53, 238). The objective of this study was to examine the effects of slow-cooling cryopreservation v. vitrification methods on the developmental competence of in vitro-produced 2- to 4-celled domestic cat embryos as compared to non-frozen controls. Mature tomcat testicles and queen ovaries were collected from local veterinary clinics. Epididymal sperm were processed using one of three methods according to availability and time of use: 1) freshly washed if used the same day for IVF, 2) stored in cooled, non-electrolyte medium (Pope et al. 2000 ICAR Proc. 2, 191) if used within one week, or 3) frozen according to Tebet et al. (2006 Theriogenology 66, 1629–1632). Batches of oocytes were inseminated with the same pool and type of processed sperm per replicate. All in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) procedures followed those previously reported (Pope et al. 2000 Theriogenology 53, 163–174). After 48 h in IVC, 2- to 4-celled embryos were equally divided into the three groups: 1) control, non-frozen; 2) slow-cooling cryopreservation using propanediol and sucrose; and 3) vitrification using DMSO, ethylene glycol and sucrose. The cryopreservation/thawing procedure followed that of Gomez et al. (2003) and the vitrification/warming procedure followed that of Vajta et al. (2000 Anim. Reprod. Sci. 60, 357–364). Results were recorded as the percentages of blastocyst formations at Day 8 of IVC and were compared between groups using chi-square analysis. In March 2000 to 2001, 806 feline oocytes were recovered from 101 ovaries and 286 blastocysts were obtained on Day 8 IVC in seven replicates. There were no significant differences in the numbers of degenerated embryos (2- to 8-celled) or morulae in all three groups (P > 0.05). There were more blastocysts that developed in the non-frozen control group (45/97; 46.4%), but this was not statistically different (P > 0.05) from the cryopreserved group (37/91; 40.7%). However, there were significantly more blastocysts (P < 0.01) produced in the non-frozen control group than the vitrified group (28/98, 28.6%). There were no differences between the cryopreserved and vitrified groups (P > 0.05). In conclusion, this study verifies previous reports that slow, or controlled-rate cryopreservation is effective for domestic cat embryos. Vitrification is less effective, as compared to non-frozen control embryos, but this may be improved with modified protocols.