346 BENEFICIAL EFFECTS OF ACETYL-l-CARNITINE TREATMENT DURING IVM ON POST-FERTILIZATION DEVELOPMENT OF BOVINE OOCYTES IN VITRO

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The lower competence of in vitro-matured oocytes for post-fertilization development is attributed to the lack of physiological factors in in vitro maturation (IVM) that regulate maturation events which occur exclusively in the cytoplasm of oocytes. It has been found recently that mitochondrial function plays an important role in regulation of oocyte developmental competence via metabolic regulation of energy production. Acetyl-l-carnitine (ALC) is known to enhance fatty acid oxidation and energy production in the mitochondria, and to exert enhancing effects on cellular proliferation and survival. In this experiment, we examined the effects of ALC on IVM and post-fertilization development of bovine oocytes. Cumulus-oocyte complexs (COCs) were aspirated from 2-5 mm follicles of ovaries from a slaughterhouse. COCs were cultured in IVM medium (mSOFaa+estradiol+hCG+BSA) with or without ALC (10 mM) for 24 h at 39�C under 5% CO2 in air, and then fertilized according to the conventional method. After 6 h of insemination, presumptive zygotes were freed from cumulus cells by repeated pipetting and cultured in mSOFaa with 1% FCS at 39�C under 5% CO2, 5% O2, and 90% N2. At 48 h post-fertilization, the rates of cleaved embryos were assessed. The cleaved embryos were transferred to mSOFaa with 5% FCS and cultured for additional 6 days at 39�C under 5% CO2, 5% O2, and 90% N2. The percentages of embryos developing to the blastcyst stage were assessed on Days 6, 7, and 8 (fertilization = Day 0), and the data were analyzed for statistically significant differences with the t-test. For examinination of mitochondrial organization in oocytes at different maturation stages, oocytes were stained for active mitochondria with MitoRed (1 �M in IVM medium for 2 h at 37�). When COCs were matured in medium without (control) or with ALC, although the rates of post-fertilization cleavage of oocytes were 60% to 70% despite the presence or absence of ALC, ALC significantly (P < 0.05) increased the rates of cleaved embryos forming blastcysts on Days 6, 7, and 8 (30%, 36%, 40%) compared with those in the control (13%, 21%, 34%). We next examined effects of ALC treatment during IVM on active mitochondria distribution in oocytes. In 75% of immature oocytes, active mitochondria localized in the periphery of the oocytes (peripheral type). After 24 h of IVM without ALC, while 17% of oocytes remained in a peripheral type, 44% showed some migration of active mitochondria toward the center of the oocytes (semiperipheral type) and 39% presented a diffused distribution of active mitochondria in the whole oocyte cytoplasm (diffused type). On the other hand, in ALC treated oocytes, 60% of the oocytes presented a diffused type, 25% exhibited a semiperipheral type, and 15% had still maintained a peripheral distribution. These results provide the first evidence that ALC treatment during IVM of bovine oocytes enhances their post-fertilization development to the blastcyst stage and enhances the frequency of oocytes that exhibit an extensive relocation (diffused type) of active mitochondria to the inner oocyte cytoplasm.