Available at www.veterinaryworld.org/Vol.6/Oct-2013/7.pdf RESEARCH ARTICLE Open Access In vitro blastocyst development of post-thaw vitrified bovine oocytes

Aim: To evaluate the developmental competence of post-thaw vitrified bovine cumulus-oocyte complexes (COCs) in vitro. Materials and Methods: A total of 129 COCs were cryopreserved using vitrification solution comprising of 15% ethylene glycol (EG) + 15% dimethyl sulfoxide (DMSO) + 0.6 M sucrose in medium TCM-199 with 10% FBS. Immediately, within a minute they are plunged into liquid nitrogen using 0.25 ml straws. Thawing was made with a step wise dilution method. Postthaw normal vitrified and non-vitrified oocytes were subjected to in vitro maturation and in vitro fertilization. Results: Post-thaw survival percentage of vitrified oocytes was 88.37% and maturation performance of vitrified oocytes on the basis of cumulus expansion was 81.58% as compared to non-vitrified control 93.85%. The in vitro fertilization performance of vitrified oocytes was 49.46% as compared to the non-vitrified ones (63.11%). Similarly, blastocyst formation of vitrified oocytes was 21.74% as compared to 32.47% in non-vitrified oocytes. Conclusion: Vitrification of immature bovine oocytes using 7.5% EG + 7.5% DMSO for equilibration and 15% EG +15% DMSO + 0.6 M sucrose as vitrification solution yielded better in vitro fertilization and blastocyst formation rate.


Introduction
competence with vitrification procedure seems to improve, additional experiments are required to ascertain its Retrieval of a higher number of competent efficacy in oocytes towards its developmental compeoocytes for in vitro maturation and in vitro fertilization tence in vitro.(IVM -IVF) to obtain superior transferable bovine Hence the present study was conducted to embryos coupled with development of freezing technique evaluate the developmental competence of bovine through vitrification will entail more productivity from immature oocytes post vitrification. the non descript animals.Oocyte cryopreservation is of paramount importance for assisted reproductive

technologies (ART). Unique characteristics of
Oocyte recovery: Cattle ovaries were collected from mammalian oocytes in respect of permeability of 1 slaughter house and within 1 / -2 hours, they are 2 cryoprotectants and water through plasma membrane processed as per the routine standard protocols.Oocytes make them vulnerable for conventional freezing protocols were aspirated from 3 to 8mm ovarian follicles with as compared to embryo freezing.insemination.Development to 2 cell stage was having fragmented zona pellucida and absence of assessed at 48 hrs after insemination and subsequently cytoplasmic contents were not considered.The cultured for 7 days to evaluate the blastocysts remaining morphologically normal post thaw oocytes formation.Medium was replaced with fresh medium were taken for IVM.Freshly collected COCs were after every 48 hrs of culture.The study was carried out separately used for in vitro maturation and kept as on different days with replicates of 10. control.
Statistical analysis: The data were compiled and the In vitro maturation: The fresh or post-thaw vitrified performance of maturation and in vitro fertilization normal oocytes were matured in Modified TCM-199, between vitrified and non vitrified groups were 200mM L-glutamine solution, 10% FBS, 0.8M compared by chi-square test.Sodium pyruvate and 50µg/ml Gentamicin and 50µM

Results and Discussion
Cysteamine supplemented with p-FSH (5µg/ml), 10% o v/v follicular fluid, 1µg/ml 17-β estradiol at 38.5 C in a In the present study, a total of 259 good quality humidified atmosphere of 5% CO for 24 hours.For 2 bovine COCs were the subject of the experiment.Of confirmation of maturation after 24 hours the oocytes which, 129 COCs were subjected to vitrification and were evaluated for morphological changes and in vitro the rest were non-vitrified and served as control.The maturation performance under stereo zoom microscope.
performance in respect of post-thaw survival rates and The oocytes with an intact zona pellucida, plasma in vitro maturation on the basis of cumulus cell membrane and homogenous cytoplasm were considered expansion was more than 80% respectively.In as morphologically normal in the study.In vitro comparison the non vitrified COCs group, the study maturation performance was assessed on the basis of demonstrated 93.85% in vitro maturation performances expansion of cumulus cells surrounding the homo-(Table -1).The vitrification thus had minimal effect on genous oocytes [5].
the survival rates and the ability of the oocyte to mature in vitro in the present study.High proportions of bovine In-vitro fertilization: For in vitro fertilization, frozen COCs retain their post-thaw morphology after a short bovine semen (2 straws each) was prepared for exposure to high concentration of permeating capacitation with swim-up technique using B.O.

Table - 1
. Effect of cryoprotectants exposed / vitrified immature oocytes on survival percentagr and maturation in vitro

of COCs Post thaw survivability / recovery performance Maturation performance on the basis of cumulus expansion
Values in maturation performance column with different superscripts differ significantly.Chi square test (P<0.05)