Resumo Objetivou-se avaliar a infuência do estresse térmico em oócitos utilizados na produção in vitro de embriões (PIV) bovinos da raça Holandesa de alta produtividade no dia da aspiração folicular (OPU; 0), 30, 60 e 90 dias antes da OPU. A partir da temperatura média no dia 0 e aos 30, 60 e 90 dias anteriores, foram classificados nos grupos conforto (CT; até 15°C) e estresse por calor (ET -acima de 15°C). Observou-se infuência negativa em oócitos e embriões viáveis (total e grau I). A submissão ao estresse térmico nos períodos de 30 e 60 dias anteriores à OPU resultou em menor produção de oócitos viáveis (P=0,0028; P=0,0092, respectivamente). Sob estresse, no dia da OPU (ET-OPU), as vacas não apresentaram redução na quantidade de oócitos viáveis (P=0,5497) e não houve infuência da temperatura para o grupo estressado 90 dias antes da OPU (P=0,8287). Para embriões totais, a diferença ocorreu apenas no grupo ET-30 (P=0,0317), onde os grupos ET-OPU, ET-60, ET-90 apresentaram, respectivamente, P=0,1987, P=0,0596 e P=0,4580. Em relação à produção de embriões grau 1, não houve diferença para os grupos ET-OPU (P=0,2291) e ET-90 (P=0,2868), porém houve redução para ET-30 (P=0,0143) e ET- 60 (P=0,0253). Em resumo, o estresse por calor teve impacto negativo quando ocorreu 30 ou 60 dias antes da aspiração folicular. Além disso, 30 dias parece ser o período de maior suscetibilidade e que causa os maiores efeitos deletérios na viabilidade oocitária e na PIV.

Abstract The objective of this study was to evaluate the influence of thermal shock on oocytes used in the production of in vitro embryos (IVP) of high productivity Holstein cows on the day of follicular aspiration (OPU; 0), 30, 60 and 90 days before the OPU. From the mean temperature on day 0 and on the previous 30, 60 and 90 days, they were classifed into comfort group (TC; up to 15°C) and heat stress (HS; above 15°C) groups.Anegative influence was observed on oocytes and viable embryos (total and grade I). The heat stress in the periods of 30 and 60 days prior to OPU resulted in lower production of viable oocytes (P=0.0028; P=0.0092, respectively). Under stress, on the day of OPU (HS-OPU), cows showed no reduction in the amount of viable oocytes (P=0.5497) and there was no influence of temperature for the group stressed 90 days before OPU (P=0.8287). For total embryos, the difference occurred only in the HS-30 group (P=0.0317), where the groups HS-OPU, HS-60, HS-90 presented, respectively, P=0. 1987, P=0.0596 and P=0.4580. Regarding the production of embryos of grade 1, there was no difference for the groups HS-OPU (P=0.2291) and HS-90 (P=0.2868), but there was a reduction for HS-30 (P=0.0143) and HS-60 (P=0.0253). In summary, heat stress had a negative impact when it occurred 30 or 60 days before follicular aspiration. In addition, 30 days seems to be the period of more susceptibility and that causes the greatest deleterious effects on oocyte viability and IVP.

Abstract Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.

This study evaluated the influence of follicular fluid (FF) added to the maturation medium on the quality of bovine embryos produced in vitro. In the first experiment, oocytes were matured in media containing five different FF concentrations with different maturation times and classified according to meiotic progression and migration of cortical granules. In the second experiment, oocytes matured in the same media were fertilized at three different maturation times; thereafter, cleavage and blastocyst rates were evaluated. In the third experiment, oocytes were matured in media containing three different FF concentrations at two different maturation times, and embryo quality, inferred by the ratio of inner cell mass and trophectoderm cells compared with total cell number, was evaluated. Higher FF concentration (75 - 100% FF) slowed meiotic progression and CG migration (control - 78.13% vs. treated - 52.58% and control - 52.7% vs. treated - 11.59%, respectively, at 24 h of maturation). Also, FF at concentration of 75% or 100% had a negative influence on cleavage and blastocyst rates (control - 90.13% vs. treated - 82.64% and control - 35.73% vs. treated - 11.57%, respectively, at 24 h of maturation). The 50% FF resulted in embryos with increased inner cell mass numbers (control - 29.91 vs. treated - 35.49, at 24 h of maturation) and total cell numbers (control - 109.53 vs. treated - 120.67, at 26 h of maturation). Even though higher concentration of FF added to the maturation medium reduced embryonic development rates, in lower concentrations, FF slowed the meiotic progression and migration of CG and contributed to increases in inner cell mass number. Thus, FF added to the maturation medium enhances the number of cells in bovine embryos produced in vitro, especially for inner cell mass.