85 The effects of heat exposure on the growth and developmental competence of oocytes derived from early antral follicles in dairy cows

Abstract

Summer heat stress in dairy cows impairs the developmental competence of oocytes from antral follicles (2–8mm) which are used in conventional IVM and IVF systems. Moreover, summer heat stress is considered to impair the oocyte competence derived from smaller follicles; therefore, the impairment of oocyte competence possibly continues into the cooler autumn season. To investigate the thermosensitivity of early antral follicles (<1mm), we evaluated the effects of heat exposure on the growth and developmental competence of oocytes using invitro culture of oocyte–cumulus-granulosa complexes (OCGCs) derived from early antral follicles. OCGCs (n=315) were collected from early antral follicles (0.5–1mm) and cultured for 12 days. OCGCs in the heat shock group were cultured using a temperature cycle of 38.5°C for 5h, 39.5°C for 5h, 40.5°C for 5h, and 39.5°C for 9h, whereas those in the control group were cultured at a constant temperature of 38.5°C for 24h. The diameters of oocytes were measured before culture. Half of the culture medium was replaced every 4 days. Oestradiol-17β (E2) and progesterone (P4) production during the first, second, and third 4-day periods were measured by enzyme immunoassay; the viability of OCGCs was evaluated based on their morphology. Oocytes that survived after 12 days of culture (n=191) were subjected to IVM (38.5°C, 22h); their diameter and nuclear status were evaluated. Some oocytes (n=71) were subjected to IVF (38.5°C, 18h) and embryo culture (39.0°C, 150h). Cleavage and blastocyst rates were evaluated at 48h and 168h after IVF. Effects of treatment groups and culture periods on E2 and P4 production and diameters of oocytes were evaluated by two-way ANOVA followed by Tukey-Kramer or Student’s t-test. The viability of OCGCs, nuclear maturation, cleavage and blastocyst rates between two groups were compared by the chi-squared or Fisher’s exact test. E2 and P4 production and the viability of OCGCs were not different between the 2 groups. Although mean oocyte diameters before culture did not differ between the 2 groups, the mean diameters after IVM were significantly smaller in the heat shock group (108.0µm, n=56) than in the control group (111.7µm, n=61; P<0.05). The nuclear maturation rate in the heat shock group (36.4%, n=55) was significantly lower than in the control group (60.3%, n=58; P<0.05). Cleavage rates were similar between the control (54.5%, n=33) and heat shock groups (45.7%, n=35). However, no oocytes developed to blastocysts in the heat shock group (0%, n=35), whereas 30.3% (n=33) oocytes developed to blastocysts (cell number±s.d.; 92.4±28.4) in the control group (P<0.05). These findings suggest that summer heat stress in dairy cows impairs the growth, nuclear maturation, and developmental competence of oocytes derived from early antral follicles. This experimental model could be used to explore the mechanisms by which heat stress subsequently impairs oocyte competence during the cooler autumn season.