In vivo oocytes grow and mature in ovarian follicles whereas oocytes are matured in vitro in plastic culture dishes with a hard surface. In vivo oocytes show a superior developmental ability to in vitro counterparts, indicating suboptimal environments of in vitro culture. This study aimed to evaluate the influence of an agarose matrix as a culture substrate during in vitro maturation (IVM) on the development of pig oocytes derived from small antral follicles (SAFs). Cumulusoocyte complexes (COCs) retrieved from SAFs were grown in a plastic culture dish without an agarose matrix and then cultured for maturation in a plastic dish coated without (control) or with a 1% or 2% (w/v) agarose hydrogel. Then, the effect of the soft agarose matrix on oocyte maturation and embryonic development was assessed by analyzing intra-oocyte contents of glutathione (GSH) and reactive oxygen species (ROS), expression of VEGFA, HIF1A , and PFKP genes, and blastocyst formation after parthenogenesis. IVM of pig COCs on a 1% (w/v) agarose matrix showed a significantly higher blastocyst formation, intra-oocyte GSH contents, and transcript abundance of VEGFA. Moreover, a significantly lower intra-oocyte ROS content was detected in oocytes matured on the 1% and 2% (w/v) agarose matrices than in control. Our results demonstrated that IVM of SAFs-derived pig oocytes on a soft agarose matrix enhanced developmental ability by improving the cytoplasmic maturation of oocytes through redox balancing and regulation of gene expression.

This study was conducted to evaluate the effects of insulin and epidermal growth factor (EGF) in a in vitro growth (IVG) medium on oocyte growth, in vitro maturation (IVM) and embryonic development of pig oocytes derived from small antral follicles (SAF) less than 3 mm in diameter. SAF oocytes were cultured for 2 days to induce IVG in alpha-minimal essential medium supplemented with 1 mM dbcAMP and 15% (v/v) fetal bovine serum. After IVG culture, oocyte maturation was induced by culturing IVG oocytes in IVM medium for 44 h. IVM oocytes that extruded the first polar body were selected and induced for parthenogenesis (PA) by applying electric stimulus. SAF oocytes cultured under the insulin treatment showed a significantly increased (P < 0.05) nuclear maturation (73.8%) compared to those cultured with insulin and EGF (59.8%). After PA, the proportions of blastocysts based on the number of metaphase II oocytes were significantly higher (P < 0.05) in oocytes that were cultured for IVG with insulin, EGF, and insulin + EGF (32.4%, 35.2%, and 34.8%, respectively) than in control (22.9%). IVG oocytes treated with insulin showed an increased oocyte diameter (116.3 μm) compared to those treated with insulin and EGF (114.0 μm) (P < 0.05). Intra-oocyte GSH content significantly increased (1.07 pixels/oocyte) by insulin treatment during IVG compared to that of oocytes treated with insulin + EGF (0.78 pixels/oocyte). These results demonstrate that IVG culture of SAF oocytes under insulin or/and EGF treatment supports oocyte maturation and improves embryonic development to the blastocyst stage after PA in pigs.

Oocytes from small antral follicles (< 3 mm in diameter; SAFO) show lower developmental competence compared to those from medium antral follicles (3-8 mm in diameter; MAFO) in pigs. This study was designed to evaluate the effect of various macromolecules such as fetal bovine serum (FBS), porcine follicular fluid (PFF), bovine serum albumin (BSA) and polyvinyl alcohol (PVA) in in vitro growth (IVG) medium on oocyte growth, maturation, and embryonic development after parthenogenesis (PA). The base medium for IVG was α-MEM supplemented with dibutyryl cyclic AMP, pyruvate, kanamycin, hormone. This medium was further supplemented with 10% FBS, 10% PFF, 0.4% BSA, or 0.1% PVA. The in vitro maturation (IVM) medium was medium-199 supplemented with 10% PFF, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. SAFO were cultured for 2 days for IVG and then cultured for 44 h for IVM. After IVG, the mean diameter of SAFO treated with FBS, PVA, and no IVG-MAFO (114.1, 113.0, and 114.8 μm, respectively) was significantly larger (P<0.01) than that of no IVG-SAF (111.8 μm). Oocyte diameter after IVM was greater (P<0.01) in SAFO treated with FBS, BSA and PVA (112.8, 112.9 and 112.6 μm, respectively) than other groups (110.4, 109.6, and 109.8 μm for no IVG-MAFO, no IVG-SAFO and PFF, respectively). Intraoocyte GSH content was not influenced by the macromolecules in IVG medium (0.92, 0.93, 1.05, and 1.12 pixels/oocyte for FBS, PFF, BSA and PVA, respectively). The proportion of oocytes reached the metaphase II stage was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but not different from that of FBS treatment (61.5%). The cumulus expansion score of oocytes after IVG was significantly influenced (P<0.01) by the macromolecules (2.94, 2.24, 1.84, and 1.38 for PFF, FBS, PVA, and BSA treatments, respectively). Blastocyst formation of PA oocytes that were treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) during IVG was higher (P<0.05) than that of BSA-treated oocytes (20.6%) but was not significantly different from that (54.8%) of no IVG-MAFO oocytes. Our results demonstrated that growth, maturation, and embryonic development of SAFO are greatly influenced by macromolecules in IVG medium and that PFF or FBS can be replaced with a chemically defined synthetic macromolecule PVA.