73 VITRIFICATION OF BOVINE OOCYTES: EFFECT OF PACKAGING AND EQUILIBRATION TIME ON NUCLEAR MATURATION

Abstract

The conservation of female animal genetics is challenging because of the scarcity of oocytes and their sensitivity to cryopreservation techniques. During slow, controlled freezing procedures, intracellular ice crystallization often leads to cell damage. Vitrification as an alternate method of cryopreservation exposes cells to a higher concentration of cryoprotectants with an ultra-rapid cooling rate, leading them to an ice-crystal-free, solid glasslike structure. The vitrification procedure has been used successfully for the cryopreservation of embryos and other body tissues, but very few reports of successful oocyte cryopreservation exist because of their complex structure. The present study was designed to compare two packaging methods (Cryotop v. 0.25-mL straw) and two equilibration times (10 v. 0 min) for vitrification of bovine oocytes. COC were aspirated from follicles <8 mm in diameter on bovine ovaries collected from a slaughterhouse. COC with ≥3 layers of cumulus cells and a uniform cytoplasm were selected, washed in Dulbecco’s phosphate-buffered saline (DPBS) + 5% calf serum (CS), and divided into five equal groups. In the control group, the COC were washed in TCM-199 + 5% CS and matured in vitro in TCM-199 containing 5% CS, 5 μg mL^–1 of LH, 0.5 μg mL^–1 of FSH, and 0.05 μg mL^–1 of gentamicin at 38.5°C, 5% CO₂, and high humidity for 22 h. In the treatment groups, half the COC were equilibrated with vitrification solution 1 [VS1: TCM-199, 7.5% ethylene glycol (EG), 7.5% DMSO, and 20% CS] for 10 min. After equilibration, COC were exposed to vitrification solution 2 (VS2: TCM-199, 15% EG, 15% DMSO, 20% CS, and 17.1% sucrose) for 30 s. The remaining half of the COC were directly exposed to VS2 without equilibration in VS1. Groups of five equilibrated or nonequilibrated COC were either loaded in a 0.25-mL straw or placed on Cryotop and plunged in liquid nitrogen. The COC were thawed by immersing straws and Cryotops into 37°C thawing solution (TCM-199, 20% CS, and 17.1% sucrose) for 1 min, and were washed and matured in vitro, as described above. After maturation, the COC were denuded using 0.3% hyaluronidase in Ca-Mg free DPBS and mounted on slides. The oocytes were fixed in ethanol:acetic acid (3:1) for 24 h, stained with aceto-orcein for 20 min, and evaluated for stage of maturation. The data (maturation rates) were analyzed using chi-square analysis. In the control group, 61% (n = 54) of the oocytes reached the metaphase-II (M-II) stage. In the treatment groups, more (P < 0.001) oocytes vitrified on Cryotops reached the M-II stage than those vitrified in straws (23.4%; n = 107 v. 9.4%; n = 116). The effect of equilibration time was not significant (P > 0.05) in either packaging method. In conclusion, vitrification of bovine oocytes using the Cryotop method provides an alternative for the cryopreservation of bovine oocytes. Moreover, bovine oocytes can be successfully vitrified without equilibration.

This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.