Melhoria do desenvolvimento e da crioresistência de embriões bovinos produzidos in vitro com adição de moduladores da apoptose celular e da síntese lipídica

Abstract

The production of bovine embryos in vitro is deficient, due to aspects such as suboptimal conditions where they develop, which induces apoptosis, leading to low cryotolerance. The aim of this study was to modulate the activation of apoptosis and lipid synthesis through activation of proliferation receptors (PPARs) to diminish cell apoptosis and increase cryotolerance. In experiment 1, day 1 presumptive zygotes were allocated at random for embryo culture (control group n = 609, DHA group n = 611, and L-165041 (selective agonist of PPARD) group n = 608). The cleavage rate was evaluated on day 2 (D2) and blastocyst production on day 7 (D7). To evaluate the pre-vitrification apoptosis rate, embryos were fixed in D7 for TUNEL assay. Lipid accumulation was analyzed by the Sudan Black technique in embryos fixed in D7. In experiment 2, blastocysts were vitrified and devitrified (control group n = 98, DHA group = 76, L-165041 group = 111) to evaluate the eclosion rate. The embryos that ecloded were frozen to analyze the lipid profile by the mass spectrometry technique (MALDI-MS). The post-vitrification apoptosis rate was analyzed by TUNEL assay. In experiment 1, blastocyst production in D7 was less in the DHA group than production in the control and L-165041 groups (P < 0.05). However, the proportion of MCI cells was greater, and the total apoptosis and MCI rates were lower in the L-165041 group compared to the control and DHA groups (P < 0.05). The DHA group had a lower proportion of MCI and higher total apoptosis and MCI rates compared to the control and L-165041 groups (P < 0.05). In the DHA group, the mitochondria exhibited signs of cell stress, just as various cells exhibited intense vacuolization. The total apoptosis rate and inner cell mass was reduced (P<0.05) in the L-165041 group compared to the DHA and control group. In experiment 2, the eclosion rates from 36 h to 72 h after devitrification were greater (P < 0.05) in the L- 165041 group compared to the control group, and from 48 h on compared to the DHA group. A relative abundance of protonated phosphatidylcholine [PC (34:2) + H] + and oxidized PC [PC (36:1) + H] + in the L-165041 group was found compared to the control group. The DHA group had relative abundance (P <0.05) of protonated PC (32:0) compared to the control group. In conclusion, the addition of L-165041 in the embryonic culture decreases pre- and post-vitrification apoptosis. Our study indicates that the addition of 1 µM of L-165041 in the culture medium increases pre-vitrification cell proliferation and decreases pre- and postvitrification apoptosis, and also increases cryotolerance. In contrast, addition of DHA decreases embryonic development, and its use in in vitro production of bovine embryos under the conditions of this study is not recommended.