82 VITRIFICATION OF IMMATURE OVINE OOCYTES WITH CRYOLOOP™: EFFECT ON MEIOTIC MATURATION

Abstract

Oocyte cryopreservation will become an important tool for the creation of genetic resources bank in domestic animals. Many problems have been described following cryopreservation of MII oocytes, including spindle disorganization (Ledda et al. 2007 Reprod. Fertil. Dev. 19, 13–23) and loss or clumping of microtubules resulting in some scattered chromosomes (Sathananthan et al. 1988 Hum. Reprod. 3, 968–977). Freezing of immature oocytes at the geminal vesicle stage might circumvent these problems because at this stage the genetic material is contained within the contours of a nuclear envelope (Siqeira-Pyles and Landim-Alvarenga 2005 Anim. Reprod. Sci. 193, 176–182). However, the number of reports relating to cryopreservation of immature oocytes remains low and it has been suggested that immature oocytes are more susceptible to cryoinjuries (Ledda et al. 2007). The aim of the present work was to study the effect of CryoLoop™ (Hampton Research, Aliso Viejo, CA, USA) vitrification on survival and subsequent meiotic maturation of immature ovine oocytes. Cumulus–oocyte complexes (COCs) were isolated from ovaries of slaughtered sheep. COCs were washed three times in basal medium [BM (HEPES–TCM-199 supplemented with 10% fetal bovine serum)], and then exposed to equilibration solution (10% ethylene glycol (EG) and 0.25 m trehalose (T) in BM for 3 min. After that, COCs were exposed to vitrification solution (20% EG and 20% DMSO in BM), loaded into the CryoLoop within 1 min, and immersed in liquid nitrogen. Following cryopreservation, COCs were warmed by exposure of oocytes to (1) 10% EG and 1 mT in BM, (2) 0.5 mT in BM, and (3) BM for 3 min in each solution at 39°C. Then oocytes were matured in vitro for 24 h in maturation medium, as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). The nuclear maturation of oocytes was determined using aceto-orcein staining. Oocytes were classified into four stages: germinal vesicle (GV), metaphase-I (MI), anaphase-I + telophase-I (AI/TI), and metaphase-II (MII). Oocytes at MII stage were considered matured. Data were analyzed using chi-square test. The survival rate of oocytes after vitrification and warming based on oocyte morphology was 72.6% (61/84). Numbers of oocytes remaining at the GV stage were significantly higher in the vitrified group than in the control group (43.4 v. 12.6%, respectively; P < 0.01). Maturation to MII stage was higher (63.2%, 55/87) in the control group than in the vitrified group (43.4%, 23/53); however, the difference was not significant. Also, there were no significant differences between vitrified and control groups in terms of MI and AI/TI (7.5 and 5.7 v. 10.3 and 13.8%, respectively). In conclusion, immature ovine oocytes can be vitrified using CryoLoop with high survival and maturation rates.