287 DEVELOPMENT OF DOMESTIC CAT EMBRYOS GENERATED BY INTRACYTOPLASMIC SPERM INJECTION EXPOSED TO IONOMYCIN ACTIVATION AND DIFFERENT CULTURE CONDITIONS

Abstract

Assisted reproduction techniques in the domestic cat could be successfully applied in endangered wild felids for sustaining genetic biodiversity. One technique with great potential is intracytoplasmic sperm injection (ICSI). The objective of this study was to evaluate the development of domestic cat embryos after ICSI exposed to ionomycin activation, using a free-radical reducer (insulin-transferrin-selenium, ITS) in oocyte maturation and low oxygen tension in culture. Domestic cat ovaries were recovered from cats subjected to ovariectomy and transported to the laboratory within 2 h. They were washed in TALP-H and the oocytes were released from the follicle by repeatedly puncturing and scraping the ovaries. The cumulus–oocyte-complexes selected were in vitro matured in TCM 199 containing 1 IU mL^–1 HCG, 10 ng mL^–1 ECG, 2.2 mM calcium lactate, 0.3 mM pyruvate, 0.3% BSA, and 3% antibiotic-antimycotic. Matured oocytes were denuded of cumulus cells after 22 h of culture and individually injected with a frozen–thawed epididymal spermatozoon. After ICSI, one group of presumptive zygotes was immediately cultured (ICSI-control group) and another group was activated by incubation with 5 μM ionomycin in TALP-H for 4 min before culture (ICSI-Io group). In both experiments culture conditions were 5% CO₂ in air at 39°C. A third group was matured using ITS supplementation, activated with ionomycin after ICSI and then cultured in 5% O₂, 5% CO₂, and 90% N₂ at 39° (ITS-ICSI-Io-O₂ group). Control SHAM groups were done for each ICSI treatment: SHAM-control, SHAM-Io, and ITS-SHAM-Io-O₂ groups. The culture medium used was SOF and all treatments were analysed by Fisher test (P < 0.05). Non statistical differences were observed among the three ICSI groups in cleavage (day 2), 61.2% (n = 85); 70.7% (n = 58), and 65.5% (n = 58), neither in blastocyst rates (day 8) 10.6%, 10.3%, and 17.2%, for ICSI-control, ICSI-Io, and ITS-ICSI-Io-O₂, respectively. In SHAM controls, cleavage rate was low 38.3% (n = 60) and no blastocysts were obtained when no ITS supplementation nor artificial activation was applied (SHAM-control group). However, cleavage improved to 70% (n = 20) and 81% (n = 37) in SHAM-Io and ITS-SHAM-Io-O ₂ groups, respectively. Moreover, no statistical differences were observed in these both groups respect to ICSI groups in cleavage neither in blastocyst rates (0% and 5.4%). Our results showed that ITS supplementation during maturation, ionomycin exposure after ICSI, and low oxygen tension in culture did not improve embryo development after ICSI, though allowed parthenogenetic progression to blastocyst stage after SHAM. In conclusion, ICSI without external assistance is a good reproductive technique to be applied in the domestic cat, potentially applicable in wild cat conservation.