188 Improvement of bovine oocyte maturation in vitro through cytokine supplementation

Abstract

Oocyte competence is one of the key factors determining the proportion of embryos that develop to the blastocyst stage. There is vast evidence that IVM oocytes exhibit less developmental potential than their in vivo counterparts. Here, we tested whether supplementation of three cytokines [FGF2 (40 ng mL⁻¹), LIF (20 ng mL⁻¹), and IGF1 (20 ng mL⁻¹), termed FLI] improved oocyte maturation, and as a consequence, preimplantation development of bovine embryos in vitro. In the first experiment, cumulus-oocyte complexes (COCs) were collected from abattoir-derived ovaries and placed in maturation medium, ± FLI, for 18 to 22 h. At the end of maturation, COCs were fertilized with sperm from a single Holstein bull known to have high fertility. After an 18- to 20-h fertilization period, putative zygotes were cultured in synthetic oviductal fluid for 8 days. The number of embryos that underwent at least one cellular division (cleavage) and the number of embryos that developed to the blastocyst stage was recorded on Days 3 and 8 after insemination, respectively. The COCs supplemented with FLI (n = 554) and controls (n = 534) were evaluated across 5 replicates. There was no difference in the cleavage rate (P > 0.05) between the two treatments. Development to the blastocyst stage was higher (P = 0.05) for FLI-treated COCs (34.9% ± 1.96) than for the control group (23.9% ± 1.96). In a second experiment, COCs (n = 204) supplemented ± FLI were collected and fixed at 6, 12, 18, and 24 h after placement in maturation medium. The number of transzonal projections in the COCs was determined by localization of actin filaments by using confocal microscopy. Data were analysed by ANOVA using the GLM procedure of SAS software (version 9.4; SAS Institute Inc.). The model included treatment, time, and the interaction of treatment × time as fixed effects. There was no difference (P > 0.05) in the number of transzonal projections at 6 h (166.3 ± 8.6 vs. 143.9 ± 8.8) and 12 h (107.8 ± 8.4 vs. 128.3 ± 7.7) between FLI-treated and control COCs. However, FLI-treated COCs had fewer (P < 0.05) transzonal projections at 18 h (67.9 ± 7.8 vs. 100.1 ± 7.7) and 24 h (56.4 ± 7.2 vs. 80.6 ± 7.4) compared with the controls. There was a significant treatment × time interaction (P = 0.006). In a third experiment, we tested whether the timing of transzonal projection disassociation affected lipid accumulation in the embryo. Blastocysts (n = 59) on Day 8 produced from COCs matured ± FLI were collected and lipid content was determined by using Nile Red staining. There was no difference (P > 0.05) in lipid content between treatments. Thus, supplementation of maturation medium with FLI accelerates the disassociation of transzonal projections in COCs and improves subsequent embryonic development to the blastocyst stage while having no detectable effect on lipid content. Further research is necessary to understand how these cytokines modulate IVM of bovine oocytes.

This project was supported by Food for the 21st Century and the Clifton Murphy scholarship fund.