Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

In livestock, in vitro embryo production systems can be developed and sustained
thanks to the large number of ovaries and oocytes that can be easily obtained from
a slaughterhouse. Adult ovaries always bear several antral follicles, while in prepubertal
donors the maximal numbers of oocytes are available at 4 weeks of age, when
ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered
good donors, even if the developmental competence of prepubertal oocytes is lower
compared to their adult counterpart.
Basic research and commercial applications would be boosted by the possibility of
successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal
donors. The vitrification of oocyte collected from prepubertal donors would also allow
shortening the generation interval and thus increasing the genetic gain in breeding
programs. However, the loss of developmental potential after cryopreservation makes
mammalian oocytes probably one of the most difficult cell types to cryopreserve.
Among the available cryopreservation techniques, vitrification is widely applied to
animal and human oocytes. Despite recent advancements in the technique, exposures
to high concentrations of cryoprotective agents as well as chilling injury and osmotic
stress still induce several structural and molecular alterations and reduce the
developmental potential of mammalian oocytes. Here, we describe a protocol for the
vitrification of sheep oocytes collected from juvenile and adult donors and matured in
vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in
vitro maturation to vitrification, warming and post-warming incubation period. Oocytes
vitrified at the MII stage can indeed be fertilized following warming, but they need
extra time prior to fertilization to restore damage due to cryopreservation procedures
and to increase their developmental potential. Thus, post-warming culture conditions
and timing are crucial steps for the restoration of oocyte developmental potential,
especially when oocyte are collected from juvenile donors.