69 Cellular differentiation and apoptosis of in vitro-produced Bos indicus embryos is affected by oxidative stress

We aimed to evaluate the effect of oxidative stress (OS) induced by hydrogen peroxide (H₂O₂) in embryonic development. Embryos were produced in vitro using oocytes (n = 3514) recovered from slaughterhouse ovaries. Presumptive zygote drops were treated with 1, 10, 50, or 100 µM H₂O₂ at the beginning of in vitro culture, except for the control group (placebo). Cleavage and blastocyst production were assessed on Days 2.5 and 8, respectively. Obtained expanded blastocysts (Days 7–8, n = 926) were submitted for reactive oxygen species (ROS) abundance determination (using 2,7-dichlorofluorescein), CDX2 immunolocalization (anti-CDX2 primary antibody followed by Alexa Fluor 488–conjugated secondary antibody), and apoptosis (using terminal deoxynucleotidyl transferase dUTP nick end-labeling, TUNEL) assays. Images were captured using Ts2R fluorescence microscope, and ImageJ software was used to count cells (TUNEL, and CDX2 assays) and measure fluorescence intensity (ROS assays). For statistical analysis, data were analyzed with SAS by ANOVA after testing for normality (Shapiro-Wilk). Significance was considered at P < 0.05. Cleavage was reduced in the 100 µM group (29.3 ± 9.3%), but other H₂O₂ treatments (1, 10, and 50 µM) were similar to control (56.6 ± 3.9%, 57.9 ± 4.2%, 41.1 ± 8.6%, and 53.7 ± 6.3%, respectively). The dosages of 50 µM (21.1 ± 4.7) and 100 µM (18.4 ± 7.2) H₂O₂ reduced (P < 0.05) blastocyst production compared with control (32.6 ± 3.5), 1 µM (30.0 ± 3.2), and 10 µM (30.4 ± 2.4). For the remaining experiments only the control, 10 µM, and 50 µM H₂O₂ groups were used. ROS abundance normalized by area and cell number was similar (P > 0.05) between the control and 50 µM groups, whereas the 10 µM treatment showed a reduction (P < 0.05) in ROS. The total number of cells and trophectoderm (TE) cells of 10 µM (95.5 ± 6.9 and 56.3 ± 4.6) was similar (P > 0.05) to the control group (106.4 ± 9.7 and 47.5 ± 4.6) and 50 µM group (122.1 ± 9.7 and 69.6 ± 5.2), respectively, but the 50 µM dose was higher (P < 0.05) than the control. The number of inner cell mass (ICM) in the control (58.9 ± 7.1) was similar (P > 0.5) to the 50 µM treatment (52.4 ± 3.8) and higher than the 10 µM treatment (39.2 ± 4.0). Both 10 and 50 µM H₂O₂ (0.7 ± 0.1 and 0.8 ± 0.1, respectively) had a reduced (P < 0.05) ICM/TE ratio compared with the control (1.3 ± 0.1). The apoptosis in the control (0 ± 0%), 10 µM (4.6 ± 0.8%), and 50 µM (52.7 ± 1.2%) treatments differed (P < 0.05) among each other. Potentially, the presence of cellular antioxidant enzymes (e.g. SOD, CAT, and GPX) may have neutralized ROS levels of 50 µM-derived blastocysts, considering these embryos presented the highest apoptosis ratio and were not accompanied by ROS abundance (similar to control). The preliminary data presented here allow us to conclude that H₂O₂-induced OS influences cellular differentiation and apoptosis of in vitro–produced bovine embryos.

Support for this study was provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001, and FAPESP (grants 2018/21496-8 and 2021/01446-9).