Vitrification of Immature Bovine Oocytes Using Two Protocols Containing Ice Blockers: Effects on Oocyte Quality.

Objectives

This study compared the synthetic polymer (SP) and the antifreeze protein type 3 (AFP3) protocols for the vitrification of bovine cumulus-oocyte complexes (COCs).

Methods

Fresh bovine COCs were subjected to in vitro maturation (IVM) for 24 hours, while other COCs were vitrified using the SP or AFP protocols. After vitrification and warming, the COCs were subjected to IVM for 24 hours. Both fresh and vitrified COCs were analyzed for chromatin status, mitochondrial activity, reactive oxygen species levels, integrity of TZPs, DNA damage, and the expression of MPS1, BUB1, MAD1, CX43, and ZP3.

Results

The metaphase II (MII) rates of COCs vitrified with SPp (38%) were significantly higher than those vitrified with AFP3p (10%) (p < 0.05). The fluorescence intensity for CM-H2DCFDA (30 ± 3.2) and nitrite/nitrate levels (10.6 ± 1.6) were higher in AFP3p COCs (p < 0.05). The transzonal projections (TZPs) of SPp COCs were intact and showed less DNA damage (25 ± 1.15) compared with those of AFP3p (43 ± 3.9) COCs (p < 0.05). The expression of the MPS1 (SPp 0.3 ± 0.4; AFP3p 0.07 ± 0.06) and BUB1 (SPp: 0.2 ± 0.4; AFP3p 0.005 ± 0.005) genes was higher in vitrified COCs compared with fresh control COCs (0.001 ± 0.0006; 0.001 ± 4.0) (p < 0.05). On the other hand, the MAD1, CX43, and ZP3 genes were expressed only in fresh oocytes.

Conclusion

Under the conditions tested, SPp was the most suitable protocol for vitrifying bovine COCs, guaranteeing good MII rates, maintaining TZP integrity and reducing DNA damage.