Maintenance of Bovine Oocytes in Meiotic Arrest and Subsequent Development In Vitro: A Comparative Evaluation of Antral Follicle Culture with Other Methods¹

^c Roslin Institute, Roslin, Midlothian EH25 9PS, Scotland, United Kingdom ^d Centre for Tropical Veterinary Medicine, University of Edinburgh, Easter Bush, Midlothian EH25 9QR, Scotland, United Kingdom ^e PPL Therapeutics, Roslin, Midlothian, EH25 9PP, Scotland, United Kingdom

The frequency of development of bovine embryos produced by maturation, fertilization, and culture in vitro is lower than that observed in vivo. One factor that may affect both the frequency of development and the quality of the embryos produced is the developmental competence of the oocyte. In current in vitro production systems, oocyte maturation, characterized by the resumption of meiosis, occurs after oocyte aspiration from the follicle. The developmental competence of individual oocytes may be improved by inducing maturation after culturing under conditions that inhibit the resumption of meiosis. In order to test this hypothesis, a system has been established in which intact antral follicles (3–8 mm in diameter) are cultured in vitro. During this period the oocytes are maintained at the germinal vesicle (GV) stage under the inhibitory effects of the follicle. Culture of intact antral follicles was compared with two other "physiological" methods for the maintenance of GV arrest: oocytes were cultured attached to a small part of the follicle wall or within hemisections of follicles. It was found that 96.8% of oocytes recovered from intact antral follicles—as compared to 24.6% attached to a small part of the follicle wall and 62.7% within hemisections of follicles—were maintained at the GV stage after 24-h culture. The effects on GV arrest and subsequent maturation of the oocytes were evaluated after longer periods of antral follicle culture (2, 4, and 7 days). As the culture period increased, the number of GV-arrested oocytes decreased; the maximum percentage of GV arrest was observed after 24-h culture. The majority of these oocytes matured to metaphase II. A comparison of blastocyst production was made after fertilization and subsequent development of oocytes obtained following follicle culture and of control oocytes aspirated directly from antral follicles. The cleavage rate and percentage of blastocyst production in these two groups were 54.6 ± 13.9%, 48.4 ± 8.4% and 68.6 ± 8.6%, 32.8 ± 10.8%, respectively. Statistical analysis showed significant differences in both cleavage rate and blastocyst production between these two groups. Total cell numbers in the control group were 144.6 ± 7.28 and 152.0 ± 25.8 after follicle culture. It is concluded that culture of intact antral follicles for 24 h is an alternative method for the maintenance of bovine oocytes in meiotic arrest and that these oocytes acquire a greater developmental competence in vitro.

¹ This work was supported by Ministry of Agriculture, Fisheries and Food (MAFF grant #DS0206). A.F. was funded by a Ph.D. studentship grant from Ministry of Culture and Higher Education, Islamic Republic of Iran.

² Correspondence. FAX: 0131 440 0434; akbar.fouladi{at}bbsrc.ac.uk

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