32 Vitrification of in vitro-matured bovine oocytes in triacetate cellulose hollow fibres

Abstract

A prospective method of vitrification in triacetate cellulose hollow fibres (HF) introduced by Matsunari et al. (2012 J. Reprod. Dev. 58, 599-608) allowed significant simplification and standardization of vitrification/warming procedures and was successfully used for group cryopreservation of various pre-implantation mammalian embryos. The goal of the current work was to evaluate the effectiveness of the HF vitrification method for cryopreservation of in vitro-matured bovine oocytes. The base medium for all the vitrification and rewarming solutions was calcium-free TBP-like protein-HEPES supplied with 20% of fetal bovine serum. Groups of 15 morphologically normal in vitro-matured bovine oocytes were equilibrated with 3% (vol/vol) ethylene glycol for 15 min, loaded into HF, and transferred into vitrification solution containing 30% ethylene glycol and either 0.5 M (Group 1) or 1.0 M (Group 2) sucrose. Hollow fibres were incubated for either 60 s (Group 1) or 30 s (Group 2) and immediately plunged into LN. Rewarming was conducted at 39°C. Oocytes within HF were placed in decreasing concentrations of sucrose solutions to remove cryoprotectants. Then, oocytes were subjected to IVF. Non-vitrified denuded oocytes were used as a control. Survival rates were evaluated at 21 h post-rewarming. Part of the presumptive zygotes were fixed and stained with acetolacmoid for fertilization rate. The remaining zygotes were cultured for 10 days. Developmental rates were evaluated at 44 h and 7 and 10 days post-IVF. All results are presented as mean percentage ± standard deviation. Data were analysed using Mann-Whitney U test. Significance was set at P < 0.05. Survival rate was significantly lower in Group 1 (79.0 ± 8.0%) and Group 2 (75.0 ± 5.0%) compared with the control group (97.0 ± 4.0%). Fertilization rate in Group 1 differed significantly from the control (80.5 ± 18.3% v. 95.5 ± 9.1%). Cleavage rates in Groups 1 and 2 did not differ significantly from the control (42.5 ± 15.7% v. 60.7 ± 11.1% v. 63.0 ± 15.8%, respectively). Blastocyst yields at 7 days post-IVF were 0.9 ± 2.3 (1/116) and 9.6 ± 5.4% (6/65) in Groups 1 and 2, respectively. The former was significantly lower than in the control group (17.0 ± 10.3%, 23/154). It should be noted that hatching in the control group started at 8 days post-IVF and was delayed in Groups 1 and 2 for at least 24 h. Day 10 blastocyst yields were 3.0 ± 3.3 (P < 0.05), 20.9 ± 13.8, and 30.4 ± 9.6% in Groups 1 and 2 and the control group, respectively. All obtained Day 10 blastocysts (3/116) in Group 1 hatched. Hatching rate in Group 2 was significantly lower than in the control group. Both Groups 1 and 2 showed relatively high survival and fertilization rates, but embryo development rates in both groups had a tendency to be lower than in the control. However, the obtained results indicate that the modifications of the protocol may increase the effectiveness of HF vitrification. The HF vitrification method remains a prospective option for simultaneous cryopreservation of a group of bovine oocytes.