30 EFFECT OF THE REPEATED USE OF OPEN SYSTEM VITRIFICATION DEVICES ON MII STAGE AND CLEAVAGE RATES OF BOVINE CUMULUS-OOCYTE COMPLEXES

Abstract

Vitrification of mammalian gametes and embryos has become the cryopreservation tool of choice in research and commercial clinical programs because of its high efficiency. Vitrification relies upon high cooling rates. In this regard the use of open system vitrification devices (OSVD) provides the highest cooling-warming rates. A limiting factor of vitrification research in domestic animals is the high cost of OSVD. Reuse of these devices could be a viable alternative for cost reduction in vitrification research projects. The objective of this study was to evaluate the effect of the repeated use of OSVD on the developmental competence of bovine cumulus-oocyte complexes (COC). A 6-treatment factorial arrangement was evaluated, where factor A was number of uses of OSVD (new, one use, and two uses) and factor B was COC type (immature, mature). Cumulus-oocyte complexes were obtained by ovum pickup from crossbred nonlactating beef cows. The vitrification procedure consisted of exposure of COC to 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 9 min. Afterward, COC were exposed to 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.5 M sucrose and loaded into the tip of the OSVD (Cryolock®) with minimal volume (<1 μL) to be immediately plunged into LN within 60 s. Three to four COC were loaded per OSVD. For warming, the COC were exposed to 0.5-M (37.5°C) and 0.25-M sucrose for 2.5 min each. The base medium for all solutions was D-PBS plus 20% fetal bovine serum. A total of 266 COC were used in the study collected during 6 days (repetitions). Cumulus-oocytes complexes obtained per day were divided for immediate vitrification or for a 22-h maturation period (84.5% MII stage rate) and then vitrified. In each case COC were randomly assigned to treatments (numbers of use of OSVD) before vitrification. From each treatment warmed oocytes were divided for assessment of maturation and cleavage rate evaluation. To assess MII stage rate, COC were exposed to Hoechst 33342 (3.5 μg mL^–1) for 15 min at 37.5°C and then observed under an epifluorescence microscope. A standard bovine IVF protocol was used, and cleavage rate was evaluated at 42 h following fertilization. Results of the experiment were tested by chi-square test of independence or Fisher’s exact test when required (P < 0.05). The MII stage rates (mean ± standard error) for immature vitrified COC were 82 ± 2.3, 78.2 ± 5.8, and 75 ± 5.3, and for mature vitrified COC were 69.3 ± 4.3, 88.2 ± 4.1, 96 ± 2.1, for new, one-use, and two-uses OSVD, respectively. The cleavage rates for immature vitrified COC were 31.6 ± 4.8, 38.8 ± 3.4, and 37.7 ± 1.1, and for mature vitrified COC were 31.7 ± 4.8, 34.4 ± 4.7, and 33.3 ± 7.1, for new, one-use, and two-uses OSVD, respectively. No differences in cleavage rate were found when comparing immature vitrified COC with mature vitrified COC (36.1 ± 1.7 v. 33.1 ± 3.2; P > 0.05). Results of the experiment shown that no differences were detected, and similar results in terms of maturation and cleavage rate were obtained when reusing OSVD.