Otimização do sistema de produção in vitro de embriões caprinos utilizando retinóides e fator de crescimento

Abstract

The aim of this study, divided into two trials was to evaluate the beneficial effect of retinoids (retinol - RT; retinoic acid - RA) and growth factor (IGF-I) on in vitro production of goat embryos, and to evaluate apoptosis through the activity of enzymes of group II caspases and DNA fragmentation by TUNEL assay. In the first experiment, divided into three groups (CG, GRT and GAR), was evaluated apoptosis of goat oocytes and embryos undergo maturation in vitro in culture medium with or without RT and HR. The second experiment was divided into two treatments, the treatment was first investigated the efficiency of both retinoids distributed in oocytes in TCM 199, and the second treatment was investigated the efficacy of RT, RA, or IGF-I of zygotes in KSOM medium using the same means in conjunction with the monolayer of oviduct cells (MCO). A total 4320-cumulus-oocyte complex obtained by aspiration from follicles (2-6 mm in diameter) ovarian animals slaughtered in abattoirs were selected for IVM and incubated in TCM 199 at 39 º C in air with 5% CO2 for 24 hours. Thereafter, the oocytes were processed through mDM and exposed to sperm for 18 hours. Presumptive zygotes were placed in drops of KSOM medium enriched with RT, RA, or IGF-I. At the end of the 10th day of co-culture blastocysts were prepared for the location of characteristic changes of apoptosis using the reagent PhiPhiLux-G1D2 and solutions of 4% paraformaldehyde and Phosphate-Buffered Saline + 1 mg / ml polyvinylpyrrolidone . In the first experiment, the activity of caspase enzymes did not differ (P> 0.05) between oocytes in the CG (7,20±0,91), GRT (6,60±0,68) and the GAR (7,30±0,67) and between the GRT and the GAR. The oocytes and blastocysts positive for TUNEL assay were higher (P <0.05), respectively, in the CG (8,20±0,78; 8,70±1,05) than in GRT (5,60±0,52; 4,80±0,51 ) and the GAR (6,40±0,69; 5,40±0,69), no difference (P> 0.05) between the GRT and the GAR. Zygotes GC had lower (P <0.05) capacity development to the blastocyst stage (5.32 ± 0.81) than those of the GRT (7.94 ± 0.93) and GAR (7.36 ± 1.02), no difference (P> 0.05) between the GRT and the GAR.In the second experiment, the results showed that the groups of oocytes and embryos treated only with retinoids produced fewer blastocysts (P <0.05) than in groups of oocytes and embryos treated with retinoids associated with IGF-I. As to the blastocyst positive for apoptosis, is assessed by the activity of enzymes caspazes or DNA fragmentation, there was no difference (P> 0.05) between groups. It is concluded that the addition of retinoids to oocytes maturation medium reduced apoptosis and cell association of retinoids with IGF-I is beneficial and can be recommended to enhance in vitro production of goat embryos.