323 INTERACTIONS OF OXYGEN TENSION, GLUCOSE CONCENTRATION, AND HEAT SHOCK ON MATURATION OF BOVINE OOCYTES

In many cell types, oxygen tension and glucose concentration affect the magnitude of heat shock effects. Experiments were performed to (1) develop oocyte maturation protocols under high (20.95%) and low (5%) oxygen tensions, and (2) test the hypothesis that low oxygen tension and high glucose concentration reduce the magnitude of the deleterious effects of heat shock on in vitro maturation of bovine oocytes. In Experiment 1, oocytes were matured in tissue culture medium-199 (TCM-199) or synthetic oviduct fluid (SOF) containing 5.6 or 20 mM glucose, under either high or low oxygen. Fertilization was performed under high oxygen and embryos were cultured in KSOM-BE2 under low oxygen. Data were analyzed by least squares analysis of variance using the GLM procedure of SAS (SAS Institute, Inc., Cary, NC, USA), with maturation medium, glucose concentration, and oxygen tension as main effects. Oxygen concentration affected percent blastocyst formation when TCM-199 was used (greater for high oxygen than low oxygen) but not when SOF was used (medium � oxygen, P < 0.05). Similarly, glucose had no effect on percent blastocyst formation under high oxygen but high glucose was superior under low oxygen (glucose � oxygen, P < 0.05). Therefore, SOF with either 5.6 or 20 mM glucose was used to study the effect of heat shock on maturation under high and low oxygen in Experiment 2. Maturation was at 38.5�C for 22 h (control) or 41�C for 12 h and 38.5�C for 10 h (heat shock). Fertilization was performed at 38.5�C under high oxygen and embryos were cultured in KSOM-BE2 under low oxygen at 38.5�C. Data were analyzed as described above with glucose concentration, temperature and oxygen tension as main effects. The experiment was replicated nine times with a total of 3215 embryos. Heat shock decreased the cleavage rate slightly regardless of the maturation conditions (69 � 1% vs. 66 � 1%, P < 0.01). For oocytes matured in 5.6 mM glucose, percent blastocyst formation was greater for high oxygen while there was no effect of oxygen tension for oocytes matured in 20 mM glucose (glucose � oxygen, P < 0.01). Heat shock decreased the percent blastocyst formation under all maturation conditions except for the group matured under high oxygen and high glucose (temperature � oxygen � medium, P = 0.07; temperature � oxygen for oocytes under 5% oxygen; P < 0.05). The percent blastocyst formation for control and heat-shocked oocytes was 25.9% vs. 22.5% (low oxygen-5.6 mM glucose), 41.6% vs. 34.9% (low-20 mM), 41.7% vs. 35.0% (high-5.6 mM), and 37.6% vs. 37.5% (high-20 mM) (SEM = 2.0%). In conclusion, heat shock during maturation under an oxygen tension (5% oxygen) that approaches physiological conditions reduces the percent blastocyst formation. Moreover, a high glucose concentration protects oocytes from heat shock under high oxygen. This work was supported by USDA NRICGP 2002-35203-12664 and CAPES #134202-9.