Closed system for bovine oocyte vitrification

Abstract

The aim of our study was to develop a vitrification carrier for bovine oocyte cryopreservation. The carrier was to be cheap enough, elementary in its construction and meet contemporary requirements for a safe closed system. In a closed system, a cell is prevented from direct exposure to liquid nitrogen, thus minimizing the risk of cross-contamination. Furthermore, two questions regarding the proper vitrification technique were resolved: if it is necessary to partially denude the oocytes before the vitrification process or whether intact cumulus oocyte complexes should be frozen; and if it is more advantageous to preheat the vitrification solutions to female body temperature (39 °C) or to keep them at room temperature. Our results show that it is better to partially denude the oocytes prior to vitrification because cryopreserved intact cumulus oocyte complexes often proved dark, non-homogeneous or fragmented cytoplasm after warming, with many of them having visibly widened perivitelline spaces or fractured zonae pellucidae as a result of extensive damage during vitrification. Consequently, intact cumulus oocyte complexes showed significantly lower numbers of cleavage stage embryos on Day 3 compared to partially denuded oocytes (7.4% and 26%, respectively). On the other hand, the survival rate and following development of fertilized oocytes in preheated vitrification solution were equal to results reached at room temperature conditions. In conclusion, results achieved with the newly developed carrier were comparable to previously published studies and therefore they could be recommended for common use.

Embryo morphokinetics derived from fresh and vitrified bovine oocytes predict blastocyst development and nuclear abnormalities

“…By leaving only corona radiata cells, some layers of cumulus cells are maintained, which are necessary during maturation and fertilization, while the vitrification procedure is facilitated and the cooling rate increased due to the consistent, small sizes of the COCs 78,[81][82][83][84] . However, in the present study, partial removal of cumulus cells resulted in slower cleavage and impaired developmental potential of the fresh oocytes too, which is in accordance with previous findings [78][79][80] . During fertilization, cumulus cells attract, trap, select spermatozoa, induce changes in sperm physiology, and prevent premature hardening of the zona pellucida, allowing fertilization and increasing fertility [85][86][87][88] .…”

“…Partial denudation of oocytes has been proposed for oocyte vitrification, since full cumulus enclosed oocytes exhibit lower maturation after vitrification, probably because of reduced permeability to cryoprotectants [78][79][80] and higher volumes of vitrification solution surrounding large cumulus oocyte complexes (COCs) 76 . By leaving only corona radiata cells, some layers of cumulus cells are maintained, which are necessary during maturation and fertilization, while the vitrification procedure is facilitated and the cooling rate increased due to the consistent, small sizes of the COCs 78,[81][82][83][84] .…”