Structural Changes in Cattle Immature Oocytes Subjected to Slow
Freezing and Vitrification
H. Wahid*, M. Thein1, E.A. El-Hafez2, M.O.
Abas3, K. Mohd Azam4, O. Fauziah5,
Y. Rosnina and H. Hajarian6
Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400
Serdang, Selangor, Malaysia; 1Department of Surgery and
Reproduction, Faculty of Veterinary Science, Yezin, Nay Pyi Taw,
Myanmar; 2Faculty
of Veterinary Medicine, Assiut University, Egypt; 3Agro-Biotechnology
Institute, Ministry of Science, Technology and Innovation,
Aras 3-7, Blok C4, Parcel C, 62662 Putrajaya,
Malaysia; 4Faculty
of Veterinary Medicine, Universiti Malaysia Kelantan,
Karung Berkunci 36, Pengkalan Chepa, 16100 Kota Bharu, Kelantan,
Malaysia;
5Faculty
of Medicine and Health Sciences, Universiti Putra Malaysia, 43400
Serdang, Selangor, Malaysia; 6Department of Animal
Science, Faculty of Agriculture, Razi University, Iran.
Abstract
This study was conducted to evaluate
the effect of different cryopreservation methods (slow-freezing and
vitrification) on structural changes of bovine immature oocytes.
Bovine ovaries were collected from
local abattoirs. Cumulus-oocyte-complexes (COCs) were retrieved using
aspiration method from 2-6 mm follicles. In Experiment 1, selected oocytes were
randomly divided into 4 treatment groups namely freezing solution-exposed,
frozen-thawed, vitrification solution-exposed and vitrified-thawed and then
oocytes abnormalities were examined under a stereomicroscope.
In Experiment 2, oocytes were randomly allocated to the same grouping as
experiment 1 plus control group. Following freezing or vitrification, all
oocytes were
fixed in glutaraldehyde
and processed for transmission electron microscopy. In experiment 1, there was a
higher incidence of abnormalities in the frozen-thawed and vitrified-warmed
oocytes compared to those in freezing solution and vitrification
solution-exposed groups (P<0.05). In experiment 2, there were marked alterations
in the perivitelline space, microvilli and vesicles of frozen-thawed and
vitrified-warmed oocytes characterized by loss of elasticity and integrity of
cytoplasmic processes and microvilli following cooling and warming. In
conclusion,
ethylene glycol-based freezing and vitrification solutions are suitable choices
for cryopreservation of immature oocytes and most organelles are able to retain
their normal morphology following cryopreservation and thawing processes.