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In vitro growth (IVG) culture of bovine oocyte-cumulus-granulosa complexes (OCGCs) is generally carried out for 12 or 14 days using conventional gas impermeable culture devices. The culture duration may be longer compared to follicular development in vivo. During follicular development, follicles receive oxygen from micro vessels; however, oxygen supply is limited under the culture using conventional gas impermeable devices. The purpose of this study was to investigate the effect of increasing dissolved oxygen availability using a gas permeable (GP) culture device with or without antioxidant (astaxanthin, Ax) supplementation on 8-day IVG culture systems for bovine OCGCs derived from early antral follicles. We cultured OCGCs in GP, GP supplemented with Ax (GP + Ax), and a conventional gas impermeable device (control) for 8 or 12 days. OCGC viability were significantly higher when cultured for 8 days than 12 days (p < 0.001) in all culture condition, but significant difference was not observed between groups (p > 0.05). Antrum formation rates of OCGCs were higher after 12 days than 8 days of culture in all culture condition (p < 0.001) and were significantly higher in the control than GP groups regardless of Ax supplementation (p < 0.05). Oocyte diameters were similar among day-8 GP + Ax, day-8 control and day-12 control groups (p > 0.05). Nuclear maturation rates of oocytes grown in vitro for 8 days were significantly higher in the GP + Ax group than in the control and the GP groups (p < 0.05) and similar to oocytes grown for 12 days regardless of the culture conditions (p > 0.05). The generation of reactive oxygen species in OCGCs on day 8 of IVG culture was significantly lower in the GP + Ax group than those of the GP and control groups (p < 0.05). IVG oocytes after eight days of culture developed into blastocysts, and the cleavage and blastocyst rates were similar in all treatment groups. However, in vivo -grown oocytes had significantly higher (p < 0.05) cleavage and blastocyst rates than the IVG oocytes in all groups. The present study demonstrates that increased oxygen availability using a GP culture device with Ax supplementation promotes oocyte growth and maturation competence but inhibits proliferation of granulosa cells and antrum formation compared with a conventional gas impermeable culture device, and that OCGCs can attain developmental competence after 8 days of IVG culture. • Gas permeable culture device with astaxanthin supplementation promotes bovine oocyte growth and maturation competence. • Longer than 8-day IVG culture reduces the viability of oocyte-cumulus-granulosa complexes regardless of culture conditions. • Blastocyst can be produced by oocytes derived from 8-day in vitro growth culture. [ABSTRACT FROM AUTHOR]

Theca cells (TCs) are essential to folliculogenesis by contributing to steroidogenesis. However, the in vitro growth (IVG) of oocytes co-cultured with TCs has not yet been examined. In the present study, we investigated the feasibility of the IVG of bovine oocyte-cumulus-granulosa cell complexes (OCGCs) co-cultured with TCs and the developmental competence of co-cultured oocytes. OCGCs and TCs were co-cultured without steroid hormone addition for 12 days. Steroidogenesis, the viability of OCGCs, and TC numbers during co-culture were assessed every 4 days. After IVG, oocytes were matured and the nuclear status was evaluated. Some oocytes were inseminated and cultured to examine blastocyst development. During the co-culture, androstenedione production by TCs was only observed during the first 4 days (1.1 ng/well) while estradiol-17β was continuously produced, peaking during the second 4 days (0.5 ng/well). The number of TCs decreased to ∼60% of the seeding number (4.0 × 104 cells/well) during the first 4 days, and was maintained thereafter. The majority of co-cultured OCGCs (82.7%) survived after 12-day IVG. Only a few OCGCs (6.2%) survived in the OCGC culture without TCs (p < 0.01); however, the addition of androstenedione to the culture medium markedly improved survivability to 80.1%, which was similar to that in the co-culture with TCs. In the subsequent development of oocytes derived from the co-culture, 58.3% reached metaphase II stage, 58.7% cleaved, and 17.3% developed to blastocysts, which were similar values to those of oocytes cultured with the addition of androstenedione. In conclusion, TC-produced androgen contributes to OCGC growth and the acquisition of subsequent embryonic developmental competence. • Theca cells can produce androstenedione and support oocyte development in vitro. • However, theca cells showed quick spontaneous luteinization. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein-4 (BMP-4) and FSH play important regulatory roles in follicular growth and steroidogenesis in vivo . The purpose of this study was to investigate the effects of BMP-4 and FSH on in vitro growth (IVG) and steroidogenesis of bovine oocyte-cumulus-granulosa complexes (OCGCs). We cultured OCGCs collected from early antral follicles (0.5–1 mm) in medium without BMP-4 and FSH for 4 days and investigated the appearance of OCGCs and their steroidogenesis. During the first 4 days of IVG, morphologically normal OCGCs produced more estradiol-17β (E 2 ), but less progesterone (P 4 ). Morphologically normal OCGCs were subjected to an additional culture in medium supplemented with BMP-4 (0, 10, and 50 ng/mL) and FSH (0 and 0.5 ng/mL) until day 12. We examined the viability and steroidogenesis of OCGCs after 8 and 12 days of culture. Oocyte growth, characteristics of granulosa cells, and the maturational competence of oocytes were also investigated. On day 8, the viability of OCGCs cultured without FSH was higher in the 10 ng/mL BMP-4 group than in the 50 ng/mL BMP-4 group (P < 0.05). No significant difference was observed in the viability of groups cultured with FSH, regardless of the addition of BMP-4, and FSH improved the viability of 50 ng/mL BMP-4 group similar to 10 ng/mL BMP-4 group. The total number of granulosa cells was larger in 10 ng/mL BMP-4 group cultured with FSH than in 50 ng/mL BMP-4 group cultured with FSH on day 8 (P < 0.05). E 2 production decreased from days 8–12, and P 4 production increased throughout IVG culture, regardless of the addition of BMP-4 and FSH (P < 0.05). No significant differences in E 2 production were observed between groups from days 4–8, regardless of whether BMP-4 was added without FSH; however, E 2 production in the group cultured with 50 ng/mL BMP-4 was suppressed by FSH. BMP-4 suppressed E 2 production from days 8–12, regardless of whether FSH was added. The group cultured with 10 ng/mL BMP-4 without FSH showed the lowest P 4 production among all groups for all culture periods. OCGCs that produced mature oocytes tended to secrete more E 2 and less P 4 than OCGCs that produced immature oocytes. In conclusion, until day 8 of the IVG culture, P 4 production by OCGCs was suppressed by the addition of 10 ng/mL BMP-4 in the absence of FSH, without inhibiting E 2 production. These conditions appear to mimic growing follicles until day 8 and mimic degenerating follicles from days 8–12 of culture. [ABSTRACT FROM AUTHOR]