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Maturation competent bovine oocytes can be obtained from early antral ovarian follicles (#490)

Kate B. Carey 1 , Monique H. Rayhanna 1 , Madeleine L.M. Hardy 1 , Nicholas I. Cameron 2 , Wilhelmus T.L. te Brake 2 , Michael B. Morris 1 , Margot L. Day 1
  1. School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Camperdown, New South Wales, Australia
  2. Nbryo Pty Ltd, Brisbane, Queensland, Australia

Background
Assisted reproductive technologies are being increasingly utilised by the agricultural industry to meet the growing demand for animal protein (1). Commercial in vitro production of bovine embryos uses oocytes from antral follicles on the ovary surface (2). However, only ~1-15 surface antral follicles are present per ovary, depending on breed (3). The number of earlier antral follicles is greater with ~250 follicles per ovary and thus are a promising way to increase oocyte yield (4). This study aims to develop a biphasic in vitro maturation (IVM) system that allows oocytes from early antral follicles to successfully mature.

Methods
Early antral, tertiary (< 500 μm) and preovulatory (501-1000 μm), follicles were isolated from fresh bovine ovarian tissue by manual microdissection. Cumulus-oocyte-complexes were cultured in pre-IVM medium for 48 h and in IVM medium for 24 h. Cumulus-oocyte-complexes were stained with DAPI and to visualise α-tubulin and F-actin to determine nuclear and cytoplasmic maturation.

Results
Following culture, the percentage of germinal vesicle (GV) stage oocytes decreased as follicle size increased. The percentage of GV1-GV3 oocytes from intermediate sized (600-800 μm) preovulatory follicles was greater than GV0 oocytes, indicative of chromatin condensation. No distinctive changes in nuclear-to-cytoplasmic ratio, as a potential indicator of GV breakdown, were found. Whilst no differences in α-tubulin distribution across GV stages were observed, the presence of some cytoplasmic network organisation may suggest that preparation for meiotic spindle assembly is occurring. Oocytes from 400-500 μm tertiary follicles began to undergo MI to MII maturation and oocytes from 700-1000 μm preovulatory follicles reached the MII stage.

Conclusion
In conclusion, the isolation of early antral follicles and biphasic IVM of cumulus-oocyte-complexes to produce mature oocytes offers a favourable approach for the in vitro production of bovine embryos to produce large numbers of high-quality livestock.

  1. Viana, JH 2021, ‘2020 Statistics of embryo production and transfer in domestic farm animals’, International Embryo Technology Society, Embryo Technology Newsletter, vol. 39, no. 4.
  2. Hamano, S & Kuwayama, M 1993, ‘In vitro fertilization and development of bovine oocytes recovered from the ovaries of individual donors: A comparison between the cutting and aspiration method’, Theriogenology, vol. 39, no. 3, pp. 703-12, DOI 10.1016/0093-691X(93)90255-4.
  3. Silva‐Santos, KC, Siloto, LS, Santos, GMG, Morotti, F, Marcantonio, TN & Seneda, MM 2014, ‘Comparison of Antral and Preantral Ovarian Follicle Populations Between Bos indicus and Bos indicus‐taurus Cows with High or Low Antral Follicles Counts’, Reproduction in Domestic Animals, vol. 49, no. 1, pp. 48-51, DOI 10.1111/rda.12222.
  4. Erickson, BH 1966, ‘Development and Senescence of the Postnatal Bovine Ovary’, Journal of Animal Science, vol. 25, no. 3, pp. 800-5, DOI 10.2527/jas1966.253800x.