The aim of this study was to evaluate the protective efficiency of cholesterol on sheep oocytes exposed
to cold stress or vitrification. Cholesterol loaded methyl-ß-cyclodextrin (CLC) (2mg/mL) was added to
the maturation media (TCM-199) of the treatment groups for 2, 4 or 24 hours. Following 24 hour
maturation period, oocytes were exposed to 30 min. cold shock at +4°C.
Oocytes (N= 1404) from all groups were evaluated for their viability, nuclear maturity and
chromosomal dispersion by staining with Trypan Blue and Hoechst stains respectively. The results
showed that no significant differences between all groups in regarding to oocyte viability or ooplasm
integrity, but the oocytes exposed to cold stress without pre-incubation with CLC had a
significant high chromatin dispersion and lower maturation rates when compared with other groups
(p < 0.05). Oocytes incubated for 4h with CLC had a significant high maturation rates compared to
the remain treatment groups (p < 0.05).
In vitrification experiment, oocytes (N= 759) were matured for 20 h and then incubated with 2mg/mL
CLC for 2h. Oocytes were vitrified by 0.25 ml straws or open pulled straws (OPS). The final vitrification
solution composed of 20% EG, 20% DMSO and 0.5 M sucrose. Two to 4 weeks vitrified oocytes were
warmed and lift for another 2 hours in maturation medium to complete their maturation period up to 24
To evaluate the osmotic stress of vitrification media; a portion of oocytes (N= 465) were exposed to the
same procedure of vitrification and warming but without immersing in liquid nitrogen. Results verify that
there were no negative effects of vitrification media on oocytes viability and nuclear maturity.
Vitrified-warmed oocytes were stained and evaluated similarly as the first phase experiment. Results
showed that oocytes matured in CLC supplemented medium had a significant (p < 0.05) higher viability
rates than oocytes matured in CLC-free medium. Ooplasm integrity rates were increased significantly (p
< 0.05) in oocytes vitrified by OPS. Oocyte maturity and chromosome integrity rates were also increased
significantly (p < 0.05) in oocytes pre incubated with CLC and vitrified by both straw or OPS.
We conclude that CLC can indirectly improved nuclear maturation by reducing chromosome dispersion
after cold stress or vitrification by its protective ability on oocyte plasma membrane, no significant
differences between oocyte packaging devices in regard to post vitrification results; however, more
studies are required to increase oocytes nuclear integrity by using cytoskeleton protecting particles beside