175 EFFECTS OF GLUCURONIC ACID AND N-ACETYL-D-GLUCOSAMINE SUPPLEMENTATION ON THE PERIVITELLINE SPACE DURING THE IN VITRO MATURATION OF PORCINE OOCYTES

Abstract

Pig oocytes fertilized in vitro experience high polyspermic penetration rates due to inadequate cortical granule exocytosis into reduced perivitelline space (PVS) thickness. The objective of this study was to minimize polyspermic penetration by increasing the PVS thickness through supplementation of its hyaluronic acid components, glucuronic acid (GA), and N-acetyl-d-glucosamine (GlcNAc) during maturation. Oocytes were supplemented during the first 24 h or second 24 h of maturation with 0.01 mM GA and 0.01 mM GlcNAc and then evaluated for nuclear maturation (n = 200), PVS thickness (n = 245), and the amount of hyaluronic acid (n = 245) present. The PVS thickness was determined at the equatorial plane of the oocyte using a micrometer. Hyaluronic acid concentrations were determined using an enzyme-linked immunosorbent assay method. Oocytes (n = 800) were fertilized using frozen-thawed semen and evaluated for fertilization characteristics and subsequent embryonic development at 48 and 144 h for cleavage and blastocyst formation, respectively. The PVS thickness was significantly thicker (P < 0.05) with oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation (3.20 ± 0.29) and all of maturation (2.78 ± 0.21) compared with no supplementation (2.22 ± 0.13) and supplementation during only the second half of maturation (2.02 ± 0.16). The amount of hyaluronic acid present at 24 h of maturation was significantly greater (P < 0.05) in oocytes supplemented with the PVS components (2.03 ± 0.07 pg/oocyte) compared with no supplementation (0.21 ± 0.02 pg/oocyte). At the end of maturation, oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the entire maturation had significantly greater (P < 0.05) amounts of hyaluronic acid present (4.16 ± 0.19 pg/oocyte) compared with all other groups. There was no significant difference in penetration rates between the groups. Polyspermic penetration was significantly less (P < 0.05) in oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation compared with no supplementation or supplementation during only the second half of maturation. Oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation (87.36 ± 4.01) compared with no supplementation (76.47 ± 5.67) or supplementation during only the second half of maturation (80.23 ± 3.21) had significantly higher percentages (P < 0.05) of male pronuclear formation by 12 h after IVF. Supplementing 0.01 mM GA and 0.01 mM GlcNAc during maturation significantly increased (P < 0.05) the percentage of cleaved embryos by 48 h after IVF and the percentage of those reaching the blastocyst stage by 144 h after IVF compared with those that were not supplemented during maturation (55.00 ± 6.43, 20.00 ± 6.16). There were no significant differences between the supplementation treatment groups at 48 or 144 h after IVF. These results indicate that supplementing GA and GlcNAc to the media during maturation, specifically during the first 24 h, decreases polyspermic penetration by increasing PVS thickness, hyaluronic acid amount, and male pronuclear formation, which improves subsequent embryonic development.