Estudos celulares e moleculares visando o aumento da eficiência de biotécnicas reprodutivas em suínos

Abstract

Although greater rates of in vitro embryo production (IVP) were achieved over the last decade, embryo quality and development still need to be improved. High concentrations of intracellular lipids and polyspermia are the main obstacles. This thesis aimed to investigate molecular and cellular approaches to improve the efficiency of some reproductive techniques in swine. The regulation of the expression of steroidogenic enzymes and members of the TGFβ superfamily during folliculogenesis of prepubertal sows was evaluated from the simulation of two endocrine profiles by hormonal induction. The pro-estrus profile was simulated by treatment with eCG, while the pre-ovulatory profile was simulated by eCG treatment, followed by hCG. The control group consisted of females that did not receive treatment. Samples of blood and ovaries from 30 females were collected at the time of slaughter. Relative gene expression was determined by realtime PCR, while hormonal dosages determined by chemiluminescence. Serum progesterone levels were greater in the periovulatory profile than in the other profiles (P<0.01), whereas estradiol levels were not affected (P>0.05). The endocrine profiles did not influence the expression of the BMP15, BMPR1A, BMPR2, FSHR, GDF9, LHCGR and TGFBR1 genes (P>0.05). The pro-estrus profile presented slightly greater relative expression of CYP11A1 (P=0.08) compared to the control, and CYP19A1 (P<0.05) compared to the other groups. In the periovulatory profile, the relative expression of BMPR1B was lower (P<0.05) than in control group. Other experiments were conducted to evaluate the efficiency of inclusion of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the IVM medium, on embryo development after parthenogenetic activation and on the lipid content of pig oocytes and blastocysts matured in vitro. Cleavage rates were lower in media containing 12.5 μM of EPA and 50 μM of DHA, and blastocysts development rates were reduced in media including any concentration of EPA (P<0.05). A reduction in the accumulation of lipid content (P<0.05) in oocytes and embryos cultured for 7 days was observed in the medium containing 50 μM DHA. These studies concluded that the treatment of prepubertal swine females with gonadotrophins was associated with greater expression of CYP19A1 and CYP11A1 and lower expression of BMPR1B, but effects on steroidogenesis were evident only in the periovulatory endocrine profile, which presented increased progesterone levels. There was a deleterious effect of including EPA in the IVM medium for porcine oocytes, but inclusion of 50 μM DHA was related to reduction in the cytoplasmic lipid content both during oocyte maturation and in blastocysts, which may consist of an alternative to enhance the efficiency of IVM in in pigs.