208 IN VITRO FERTILIZATION UNDER SIMULATED STRESS AND SUBSEQUENT IN VITRO EMBRYO PRODUCTION IN THE PIG

Abstract

Fertilization is a crucial step for successful reproduction and can be negatively influenced by stressful situations. It is generally accepted that stress affects reproduction, altering the endocrine profile of the female. An altered hormonal environment where the oocyte is developing could affect critical processes such as fertilization. Using a mixed in vivo–in vitro system, we assessed the ability of the oocyte to undergo fertilization and early development after exposure to blood plasma from sows that had experienced simulated stress through repeated injections of adrenocorticotropic hormone (ACTH) before ovulation (known concentrations of cortisol and reproductive hormones as well as exact ovulation time assessed by ultrasonography). Oocytes (n = 926, 7 replicates) collected from abattoir ovaries were matured in TCM-199 with BSA supplemented with hormones (10 IE mL^–1 of pregnant mare serum gonadotropin and 5 IE mL^–1 of hCG) and insulin-transferrin-selenium (5 μL mL^–1) for 24 h, followed by 22 h without supplements. During IVF, gametes were exposed to 10% of pooled plasma (n = 3 per treatment) collected approximately 1 h before ovulation from ACTH-treated sows (A group), nontreated control sows (C group), or media with BSA (B group) for 24 h. Fresh semen was added at 5 × 10⁵ cells mL^–1. Afterward, the remaining cumulus cells and sperm were removed from oocytes by vortexing (1 min), and presumptive zygotes were placed in culture medium (porcine zygote medium). Cleavage rate was assessed at 48 h post-insemination (hpi) and the embryos (n = 433, 7 replicates) were cultured up to Day 7 and stained with Hoechst 33342 (10 μg mL^–1) to count the total number of nuclei. In addition, non-cleaved oocytes were stained at 48 hpi with Hoechst to assess sperm-zona binding. Binding to the zona was assessed only in oocytes found to be matured. Statistical analysis was done using Kruskal-Wallis ANOVA and the Mann-Whitney U test. The number of spermatozoa bound to the zona pellucida was higher in the B group, and binding was notably negatively affected in the ACTH group (0.43 ± 0.18, 35.93 ± 2.50, and 3.44 ± 1.04 for the A, B, and C group, respectively; P < 0.001). Cleavage rate (over total number of presumptive zygotes) in the A group (30.71 ± 3.76%) was significantly lower than in the control groups (59.93 ± 4.0 and 52.2 ± 5.31% for the B and C group, respectively; P < 0.01). Blastocyst rate expressed over the total number of embryos was reduced in the A group (9.40 ± 5.20%) compared with the controls (27.10 ± 5.79 and 25.66 ± 5.28% in the B and C group, respectively; P < 0.05). However, no differences were found in the total number of nuclei in the blastocysts. The results suggest that fertilization is a sensitive event that could be negatively influenced by stress, subsequently affecting early embryo development. A reduced number of spermatozoa attached to the zona and a lower number of embryos and lower blastocyst development were observed in the simulated-stress group. Further studies would help to elucidate which (in the oocyte, spermatozoon, or both) mechanisms are being affected by ACTH-simulated stress around fertilization. Data are expressed as mean ± SEM.