Good quality bovine cumulus-oocyte complexes were subjected to a vitrification program for study of in vitro developmental competence. 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 plunged in liquid nitrogen using 0.25 ml straw. Thawing was made with step wise dilution method. Post thaw normal vitrified and non vitrified oocytes were subjected to IVM and IVF. Post thaw survivality of vitrified oocyte 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 oocyte was 49.46% as compared to non vitrified (63.11%). Similarly blastocyst formation of vitrified oocytes was 21.74% as compared to 32.47% in non vitrified. Vitrification of immature bovine oocyte using 7.5% EG+ 7.5% DMSO for equilibration and 15%EG +15%DMSO+0.6 M sucrose as vitrification solution yielded acceptable in-vitro fertilization and blastocyst formation rate.
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Keywords: oocytes, vitrification, in-vitro maturation, in-vitro fertilization
Oocyte cryopreservation is paramount importance for assisted reproductive technologies (ART). The difficulties associated with oocyte cryopreservation are mostly related to the special structure and sensitivity of this cell. Differences observed in plasma membrane permeability to water and cryoprotectants, compared with later stages of development, makes oocyte survival rate very low with standard slow freezing protocols. Novel approaches to slow freezing have been introduced with improved oocyte survival rate and subsequent embryo development. As an alternative to slow freezing, vitrification procedure has been suggested for oocyte (and embryo) cryopreservation by different investigators. The recent improvements in this approach, including increased cooling and warming rates using very small volumes and decreasing toxicity by reducing cryoprotectants concentrations, have allowed very high results in terms of oocyte survival, fertilization, embryo development rates . The difference in efficacy between slow freezing and vitrification procedures may be related to the fact that vitrification has a lower impact on oocyte physiology as compared with slow freezing . Although oocyte competence with the vitrification procedure appears to have improved, further studies are needed to determine the efficacy of this approach.
In order to evaluate the developmental competence of a vitrification approach for bovine oocyte cryopreservation a study was thus designed .Oocyte fertilization rate after thawing were evaluated as primary outcomes. Secondary outcomes were maturation rate and embryo development.
Materials and Methods
Oocyte recovery: Indigenous cattle ovaries were collected from slaughter house and within 11/2 - 2 hours processed as per routine standard protocol. Oocytes were aspirated from 3 to 8mm ovarian follicles with medium containing TCM-199 and supplemented with 200mM L-glutamine solution, 0.4% BSA and antibiotics. The cumulus-oocyte complexes (COCs) were categorised and morphologically evaluated under stereo zoom microscope . Homogenous and compact COCs were washed four times in holding media (Modified TCM-199, 200mM L-glutamine solution, 10% FBS, 0.8M Sodium pyruvate and 50Î¼g/ml Gentamicin and 50Î¼M Cysteamine) by gentle pipetting and were subjected to cryopreservation by vitrification. Vitrification and thawing: Two vitrification solutions were prepared in media consisting of TCM-199 with 10% FBS. Vitrification solution I (VS I) consisted of 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) and vitrification solution II (VS II) consisted of 15 % EG + 15% DMSO + 0.6M sucrose. Manipulation of oocytes and vitrification process were performed at room temperature (22-250C). The immature bovine oocytes with cumulus cells (COCs) were exposed to VS I for equilibration upto 3 minutes followed by 25-30 seconds in VS II. The oocytes in VS II were immediately loaded to a 0.25 ml French straw preloaded with 0.6 M sucrose in holding medium with air gap in between and plunged into Liquid Nitrogen (LN2). The straws were stored for a period of 7 days and then thawed in 37oC water bath for 30 seconds. After immersion in the water bath, oocytes were gradually rehydrated in sucrose solution. Oocytes were expelled into the holding medium with 0.6M of sucrose and held for 1 minute. Oocytes were then transferred successively into a holding medium with 0.6, 0.3 and 0.15 M of sucrose for one minute in each solution. At the end of the last rehydration, oocytes were washed successively (three times) in holding medium. Post thaw vitrified oocytes were then examined for their morphological integrity . Briefly, the recovery rate was defined as the number of oocytes counted after the end of rehydration, in relation to the total of oocytes vitrified. Oocytes with fractured zona pellucida and with loss of the cytoplasmic contents were discarded. The remaining oocytes were subjected to IVM. Freshly collected COCs were separately used for in-vitro maturation and kept as control.
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In vitro maturation: The fresh or post thaw vitrified normal oocytes were matured in Modified TCM-199, 200mM L-glutamine solution, 10% FBS, 0.8M Sodium pyruvate and 50Î¼g/ml Gentamicin and 50Î¼M Cysteamine supplemented with p-FSH (5Âµg/ml), 10% v/v Follicular fluid, 1Î¼g/ml 17-Î² estradiol at 38.5oC in a humidified atmosphere of 5% CO2 for 24 hours. For confirmation of maturation after 24 hours the oocytes were evaluated for morphological change and in-vitro maturation performance under stereo zoom microscope. The oocytes were defined as morphologically normal if it possessed an intact zona pellucida and plasma membrane and homogenous cytoplasm. In-vitro maturation performance was confirmed on the basis of cumulus cell expansion and nuclear maturation .
In vitro fertilization: For in-vitro fertilization, frozen bovine semen (2straws each) was prepared for capacitation with swim-up technique using B.O. washing medium . After washing spermatozoa were added to B.O. fertilization medium to final concentration 2x106 sperm/ml. In-vitro matured oocytes of both the vitrified and non vitrified groups were co-incubated with spermatozoa in B.O. fertilization medium at 38.50C, 5% CO2 in air and saturated humidity for 20-22 hr. The presumptive zygotes were washed through in vitro culture (IVC) medium consisting of mCR2aa with 5%FBS 2% essential amino acids (v/v), 1 % non essential amino acids (v/v), 1% Î±- glutamic acid, 0.3% BSA and 0.05 Âµg/ml, Gentamicin sulphate. The zygotes were transferred into IVC droplets under mineral oil and incubated at 38.50C under 5% CO2 saturated humidity. After 48hrs culture the cleavage up to (2-8 cell) were recorded. Subsequently culture was continued for further development. Embryos in each group were observed under a microscope every 24hrs following insemination. Development to 2 cell stage was assessed at 48 hrs after insemination. The cleaved embryos were cultured for additional 7 days to evaluate the ability to develop into blastocyst. About one-half of the culture medium was replaced with fresh medium every 48 hrs after insemination. The experiment was replicated ten (10) times on different dates.
Statistical analysis: The data were complied and analysed using 't-test' as per standard procedure.
Results and Discussion
In the present study a total of 259 good qualities bovine COCs were the subject of the experiment. Of which 129 COCs were subjected to vitrification and their performance in respect of post thaw survivality and in-vitro maturation on the basis of cumulus cell expansion were more than 80%. In comparison the non vitrified COCs group, the study demonstrated 93.85% in-vitro maturation performances (Table 1). The vitrification thus makes minimal affect on the survival rates and the ability of the oocyte to mature in vitro in the present study. High proportions of bovine COCs retain their post-thaw morphology after a short exposure to high concentration of permeating cryoprotectants namely EG and DMSO with sucrose using 0.25 ml straw for freezing. Previous studies on vitrified cattle oocytes had recorded the morphological survival rates between 65 to 94% [6, 7]. The chilling sensitivity of mammalian oocyte is well documented and consequently success to cryopreservation is variable. Cryopreservation involves controlling many variables, such as cryoprotectants (CP) used, the method for adding and removing CPs and cooling and thawing rate. Each of these factors being a possible source of cell damage . The maturation rate depends on oocyte quality, sufficiency and efficacy of the media and optimization of incubation period. Cooling Germinal Vesicle (GV) stage bovine oocyte had no effect on the nuclear maturation or fertilization. The nuclear material is membrane bound; there is very low risk of any chromosomal damage as compared to that with dividing cells such as within an embryo or maturing oocytes. Hurtt et al.  compared viability of immature and mature bovine oocytes vitrified in Ethylene Glycol based solution and recorded 60% & 70% nuclear and cytoplasmic maturation rate. Faster membrane penetration of ethylene glycol make it as an ideal cryoprotectant combine with non penetrating cryoprotectant like sucrose that act as a stabilizer, minimizing the affect of high concentration of ethylene glycol .
The subsequent in vitro fertilization performance of vitrified post thaw in vitro matured oocytes in the present study was recorded as 49.46% with 21.74% of blastocyst formation. The results are in agreement with the findings of Vajta et al  and Papis et al  using vitrified matured bovine oocytes. The improved success rate was believed to be attributed to the increased cooling rate during oocyte vitrification and step wise dilution method. Hong et al  reported more of the vitrified oocytes developed to the two cell (71-100%), four cell (71-93%), eight cell (46-71%) and blastocyst (23-36%) stages after thawing using the four step method with 2.5 minutes interval. Step wise dilution might be beneficial because it decreases the severe osmotic shock that occurs during thawing. There have been several earlier reports in mammalian species that oocytes at the GV stage had lower cryopreservation capacity than oocytes at the metaphase I through metaphase II stages [14, 15]. However, notably vitrification program could lessen the difference in cryopreservation capacity of different maturational stages [16, 17].
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Table-1. Effect of cryoprotectants exposed / vitrified immature oocytes on survivality and
No. of COCs
Post thaw survivability /
Maturation performance on the basis of cumulus expansion
** significant at p<0.01
Table- 2. In-vitro fertilization performances of fresh and vitrified bovine immature oocytes.
No. of oocytes
In vitro fertilization
2-4 cell %
8-16 cell %
* Significant at p<0.05
Vitrification of immature bovine oocyte using 7.5% EG+ 7.5% DMSO for equilibration and 15%EG +15%DMSO+0.6 M sucrose as vitrification solution yielded acceptable in-vitro fertilization and blastocyst formation rate.
Authors are thankful to Department of Biotechnology (DBT), Govt. of India for their financial support towards smooth running of the research.
Authors declare that they have no competing interest.