34 Effect of polysaccharide from Flammulina velutipes on the vitrification of bovine oocytes

Abstract

The developmental competence of oocytes after cryopreservation is compromised by the physical injury due to the ice crystallisation. Recent studies have reported that polysaccharide (xylomannan) derived from the mycelium and fruit body of the basidiomycete Flammulina velutipes inhibits the ice recrystallisation in the cryopreserved Chinese hamster ovary cells. In this study, we aimed to clarify the effect of xylomannan from Flammulina velutipes on the developmental competence of bovine vitrified oocytes. Bovine ovaries were obtained from a local abattoir, and cumulus-oocyte complexes (COCs) were aspirated from follicles (2-6 mm in diameter) using a 19-gauge needle attached to a syringe. The COCs were matured for 22 h in tissue culture medium-199 supplemented with 5% fetal bovine serum (FBS), 0.02 IU mL⁻¹ FSH, and 10 μg mL⁻¹ gentamycin. After maturation, COCs were incubated in base solution (BS: 10% FBS-tissue culture medium-199, control group; n = 149) or BS supplemented with 100 μg mL⁻¹ xylomannan (xylomannan group; n = 175) for 1 h before vitrification. All vitrification procedures were performed at room temperature. The COCs were equilibrated in BS with 3% ethylene glycol for 12 min and then in vitrification solution (BS with 30% ethylene glycol, 1.0 M sucrose) for 1 min. The COCs were loaded on a Cryotop (Kitazato) and transferred into liquid nitrogen. The warming procedure was performed on a warm plate (42°C). The COCs were placed into BS supplemented with 0.5, 0.25, 0.125, and 0 M sucrose for 5 min each. After washing with IVF100 solution (Research Institute for the Functional Peptide), COCs were applied for IVF. The viability of putative zygotes was morphologically evaluated following IVF, and ones that survived were cultured in CR1aa supplemented with 5% FBS. The cleavage pattern was evaluated at 28 h after IVF as follows: embryos with blastomeres of the same size without fragmentation were classified as normal cleavage; embryos with 2 blastomeres and several small fragments, direct cleavage from the 1-cell stage to 3 or 4 blastomeres, or 2 blastomeres of different size were classified as abnormal cleavage. The rates of cleavage and blastocyst formation were calculated on 2 and 8 days after culture, respectively. Total cell number and apoptosis of blastocysts were measured by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. All data were obtained from more than four replicates. Viability and in vitro development data were analysed using the chi-squared test. Total cell number and apoptosis data were analysed by a Student's t-test. Although no significant differences in viability, cleavage pattern, and cleavage rate (85.8 vs. 80.3%, 17.2 vs. 14.8%, and 35.4 vs. 36.7%, respectively) were observed, the developmental rate to blastocysts in the xylomannan group was significantly higher than that in the control group (68.6 vs. 42.2%; P < 0.01). The present results suggest that co-incubation with xylomannan before vitrification is an effective method to improve the vitrification outcome in bovine oocytes.