Environment

Susceptibility of Bovine Germinal Vesicle-Stage Oocytes from Antral Follicles to Direct Effects of Heat Stress In Vitro

Abstract
Delineation of maternal versus direct effects of heat stress to reduce development at the germinal vesicle (GV)-stage has proven challenging as oocytes spontaneously resumes meiosis after removal from antral follicles. The use of S-roscovitine (inhibitor of p34^cdc2/cyclin B kinase)to hold bovine oocytes at the GV-stage, without compromising early embryo development, was previously validated in our laboratory. The objective of this study was to assess direct effects of an elevated temperature commonly seen in heat-stressed dairy cows on cumulus oocyte complexes (COC) held at the GV-stage using 50 µM S-roscovitine. During roscovitine culture, GV-stage COC (3-8 mm antral follicles) were cultured at 38.5 or 41°C. Thereafter, oocytes were removed from roscovitine medium and allowed to undergo in vitro maturation, fertilization, and culture. Zona pellucida hardening (solubility to 0.5% pronase), nuclear stage (Hoechst 33342), cortical granule type (lens culinaris agglutinin-fluorescein isothiocyanate), and early embryo development were evaluated. Culture of GV-stage COC at 41°C increased the proportion that had type III cortical granules and reduced the proportion that progressed to metaphase II after in vitro maturation. Effects of 41°C on zona pellucida hardening, fertilization (penetration, sperm per oocyte, pronuclear formation, monospermic and putative embryos) and cleavage of putative zygotes were not noted. However, culture of GV-stage COC at 41°C for 6 h decreased the proportion of 8-16 cell embryos while 41°C for 12 h reduced blastocyst development. In summary, antral follicle COC are susceptible to direct effects of elevated body temperature, which may account in part, for reduced fertility in heat-stressed cows.

Key words: Environment • Gamete Biology • Meiosis • Oocyte development • Stress

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