129 Embryo Production from Fully Grown and Growing Stage Oocytes in Japanese Black Calves

Abstract

Before fattening of Japanese Black female calves, the ovaries are sometimes removed and discarded. Production of embryos from the oocytes residing in such ovaries is beneficial for the rescue of genetic resources. The aim of this study was to establish an embryo production system using oocytes collected from the ovaries of calves just before fattening and to investigate the correlation between the developmental competence of oocytes and the onset of puberty. Ovaries were collected from Japanese Black calves (9.5 ± 0.1 months old, n = 30, 3 replicates) in a fattening farm and separated according to the presence or absence of corpus luteum as the indicator of puberty (CL+ and CL– groups, respectively). Immature fully grown oocytes (IM oocytes), ~120 μm in diameter, were aspirated from follicles of 2 to 6 mm in diameter (CL+; n = 132, CL–; n = 41) and cultured for 22 to 23 h for maturation (IVM). After in vitro fertilization (IVF) for 6 h (designated Day 0), the oocytes were cultured for 9 days (in vitro culture, IVC) (Matoba et al. 2014 J. Dairy Sci. 97, 743-753). Growing oocytes, ~100 μm in diameter, were also collected by dissecting the follicles smaller than 1 mm in diameter. The growing oocytes were cultured for 14 days on membrane inserts for in vitro growth (IVG) (Hirao et al. 2013 Biol. Reprod. 89, 1-11). Then, IVG oocytes (CL+; n = 29, CL–; n = 32) were subjected to IVM, IVF, and IVC. Presumptive zygotes were cultured individually in microwells in culture dishes. Mitochondrial DNA (mtDNA) copy numbers (COX1 gene) of oocytes were examined (Takeda et al. 2010 Mitochondrion 10, 137-142). A comparison was made between the oocytes derived from calf ovaries and those of oocytes collected from cow ovaries by transvaginal ovum pick-up or by aspiration of the ovaries obtained at a local slaughterhouse. In IM oocytes, the rate of embryos developed to the blastocyst stage on Day 7 to 9 was higher in the CL+ group than in the CL– group (38.9 ± 2.6 v. 10.0 ± 7.1%, respectively; P < 0.05, t-test). However, IVG oocytes were compared, there was no significant difference in the blastocyst formation rate between the CL+ or CL– groups (34.7 ± 5.2 v. 19.8 ± 10.1%, respectively). The mtDNA copy numbers of matured oocytes were similar between IM and IVG oocytes irrespective of the maturity of the donor animals. In conclusion, we demonstrated the possibility of embryo production by IVM/IVF/IVC using fully grown and growing oocytes that are present in the ovaries of calves before fattening. Puberty positively affected the developmental competence of IM oocytes but the effect was not significant in IVG oocytes. Utilisation of both fully grown oocytes and growing oocytes may double the chance of rescuing genetic resources of high-breeding-value calves.

This study was partly supported by grants from the Ito Foundation. We thank staff at Mie-Katoubokujou for allowing access to calves’ ovaries.