74 MECHANICAL DELIPIDATION IMPROVES CRYOSURVIVAL AND IN VITRO DEVELOPMENT OF VITRIFIED CAT OOCYTES

Abstract

Although considerable progress has been made in the development of successful methods for cryopreservation of embryos, oocytes are much less cryotolerant. There appears to be an inverse relationship between cryosurvival and intracellular lipid levels. For example, cat oocytes, which appear microscopically as coffee-coloured, nearly opaque spheres due to their high lipid content, are extremely sensitive to cryopreservation. Oocyte delipidation thus represents a potential approach to improving cryosurvival. The objectives of the present study were to examine 1) the effects of calcium (Ca²⁺, 0 v. 10 nM), FBS (0 v. 10%), and cytochalasin B (CB, 7.5 v. 20.0 μg mL^–1) during mechanical delipidation by high-speed centrifugation on in vitro development of IVM cat oocytes, and 2) the influence of centrifugation, degree of lipid polarization (partial v. full), and co-culture with cat fetal fibroblasts (CFF) on in vitro development of vitrified IVM cat oocytes. In Experiment 1, oocytes were randomly allocated to each centrifugation medium and centrifuged at 12 000 × g for 20 min. Oocytes were then fertilized with epididymal sperm (motile sperm mL^–1) and cultured until Day 8 (Pope et al. 2006 Theriogenology 66, 59–71). In Experiment 2, oocytes were centrifuged with the optimal centrifugation medium obtained in experiment 1, allocated to each treatment and vitrified in a solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose (2008 Reprod. Fertil. Dev. 20, 188). Liquified oocytes were fertilized and cultured until Day 8. In both experiments, cleavage and degeneration rates were determined on Day 2 and blastocyst development on Day 8. Data were analysed by 2-way ANOVA and chi-square tests. In Experiment 1, of 939 oocytes that were centrifuged and fertilized, 40% of those treated in 0 nM Ca²⁺ cleaved and 22% developed into blastocysts, v. 33 and 6%, respectively, in 10 nM Ca²⁺ (P < 0.05). The respective cleavage and degeneration frequencies for oocytes treated in 10% FBS were 43 and 19% v. 19 and 3% in 0% FBS (P < 0.05). Cleavage and blastocyst development after treatment with 7.5 and 20.0 μg mL^–1 CB were 36 and 15% v. 42 and 22%, respectively. In Experiment 2, 493 oocytes were vitrified/liquified and fertilized. The degeneration, cleavage, and blastocyst rates of non-centrifuged oocytes were 49, 21, and 0% v. 31 (P < 0.05), 38 (P < 0.05), and 7%, respectively, of centrifuged oocytes. Of centrifuged oocytes with partially extruded lipids, 34% degenerated, 34% cleaved, and 4% developed into blastocysts v. 29, 42, and 10%, respectively, of oocytes with fully extruded lipids. Degeneration, cleavage and blastocyst rates of co-cultured v. control oocytes were 18, 36, and 10%, v. 26 (P < 0.05), 34, and 3%, respectively. In summary, cryotolerance of domestic cat oocytes to vitrification was 1) affected by their lipid content, and 2) improved by mechanical reduction of intracellular lipids. When oocytes were fully delipidated in Ca²⁺-free medium containing 10% FBS and 20.0 μg mL^–1 CB before vitrification and co-cultured after IVF with CFF, blastocyst development was similar to that of control, non-vitrified oocytes.