In vitro development and DNA fragmentation of domestic cat ICSI embryos subjected to antioxidant conditions and ionomycin activation

MORO LN; CANEL N; SALAMONE D

Vancouver

Otro; International Congress on Animal Reproduction (ICAR); 2012

ICSI has gained importance to support feline reproduction and preserve existing genetic biodiversity when poor seminal quality is observed. The objective of this study was to determine the best conditions to generate ICSI embryos in the domestic cat, as a model of wild felids. Ovaries were recovered from cats subjected to ovariectomy. Cumulus-oocyte-Complexes of good quality were in vitro matured in standard maturation medium (SMM): TCM 199 containing 1 IU/ml HCG, 10 ng/ml ECG, 2.2 mM calcium lactate, 0.3 mM pyruvate, 0.3% BSA and 3% antibiotic-antimycotic; or SMM supplemented with 1 ll/ml of insulin, transferrin and selenium (ITS, a free radical reducer). In the first experiment, we compared in vitro development of ICSI embryos after oocyte maturation in SMM and zygotes cultured in atmospheric oxygen tension (21% O2), respect to maturation with ITS supplementation and low oxygen tension in culture (5% O2). Moreover the effect of ionomycin activation after injection (Io) was evaluated. Control SHAM groups were included (Table 1). In vitro embryo development was compared by non-parametric Fisher exact test (p £ 0.05). Cleavage rates in ICSI groups increased when Io was employed but blastocyst rates were higher when ITS–5% O2 was used, regardless Io exposure. Karyotype analyses of cleaved embryos from ICSI–2 and ICSI–4 groups were done to assess if the increment in cleavage, when Io was employed, was a result of parthenogenetic activation instead of sperm fertilization. Nevertheless, we confirmed that nearly 70% of cleaved embryos were diploid in both groups. In the second experiment we determined total cell number and DNA fragmentation by TUNEL assay in ICSI blastocysts from all treatments, after 7 (Bd7) and 8 (Bd8) days of in vitro culture. Differences in total cell number were analyzed using one-way ANOVA and the proportion of fragmented nuclei over total cell number was analyzed by the Difference of proportions test. The ICSI–4 group showed the least amount of cells after 7 and 8 days, respect to the other groups which did not differ among them. Moreover, proportion of TUNEL+ cells increased in ICSI–2 and ICSI–4 Bd8, respect to Bd7 (55.5% and 37.4% for Bd7 vs. 89.9% and 67.6% for Bd8; p £ 0.05), in contrast to the other two groups that remained the same proportion of fragmented cells. We conclude that chemical activation enhances cleavage but not blastocyst formation, and that antioxidant conditions improve embryo development in vitro but increase DNA fragmentation of Bd8. Therefore, we consider that the best conditions to generate ICSI embryos in the cat are ITS–5% O2, but embryo transfers and pregnancy rates are needed to assess this assumption. Key Words: Cat, ICSI, DNA fragmentation, oxygen tension, embryos