Vitrification of Immature Swine Oocytes by the SSV Technique using Different Concentration of Cryoprotector and Exposure Time
Keywords:
cryoprotectant, ethylene glycol, oocyte, swine, vitrificationAbstract
The present study was design to establish the optimal vitrification protocol for the immature swine oocytes using different concentration of ethylene glycol (40, 45 and 50%) in the vitrification media and different exposure time (30, 40, 50 and 60 seconds). Vitrification was performed by the SSV method in a stepwise manner. After thawing morphological and viability assessments were done. The results shows that, once the concentration of the EG and the exposure time are increased, the percentage of morphologically normal oocytes after freezing/thawing is decreasing. Meanwhile the rate of viable oocytes is increasing once the concentration of EG reaches 45% in the VM and no longer exposing the eggs then 40 seconds. It is not justified the increasing of it in the VM over 45%. At 50%, the cryoprotectant has no longer a beneficial effect in the vitrification process. High concentrations lead to cell toxicity and cell death. An acceptable vitrification protocol can be obtained only with repeated exploratory regarding to both cryoprotectant concentration and exposure time.
References
Somfai, T., Dinnyes, A., Sage, D., Marosan, M, Carnwath, J. W., Ozawa, M., Kikuchi, K., Niemann, H., Development to the blastocyst stage of parthenogenetically activated in vitro matured porcine oocytes after solid surface vitrification (SSV), Theriogenology, 2006, 66, 415-422
Gabor Vajta, Vitrification of the oocytes and embryos of domestic animals, Animal Reproduction Science, 2000, 60-61, 357-364
Rojas, C., Palomo, M. J., Albarracin, J. L., Mogas, T., Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments, Cryobiology, 2004, 49, 211–20
Ghetler, Y., Yavin, S., Shalgi, R., Arav, A., The effect of chilling on membrane lipid phase transition in human oocytes and zygotes, Hum Reprod., 2005, 20, 3385–9.
Azambuja, R. M., Kraemer, D. C., Westhusin, M. E., Effect of low temperatures on in-vitro matured bovine oocytes, Theriogenology, 1998, 49, 1155–64
Fujihira, T., Nagai, H., Fukui, Y., Relationship between equilibration times and the presence of cumulus cells, and effect of taxol treatment for vitrification of in vitro matured porcine oocytes, Cryobiology, 2005, 51, 339–43
Nagashima, H., Cameron, R. D. A., Kuwayama, M., Young, M., Beebe, L., Blackshaw, A.W., Nottle, M. B., Survival of porcine delipidated oocytes and embryos after cryopreservation by freezing or vitrification, Journal of Reprod Dev., 1999, 45, 167–76
Mazur, P., The role of intracellular freezing in the death of cells cooled at supraoptimal rates, Cryobiology, 1977, 14, 251–72
Fahy, G. M., MacFarlane, D. R, Angell, C. A, Meryman, H. T., Vitrification as an approach to cryopreservation, Cryobiology ,1984, 21, 407–26
Paynter, S., A rational approach to oocyte cryopreservation, Reprod Biomed Online, 2005, 10, 578–586
Fuller, B., Paynter, S., Fundamentals of cryobiology in reproductive medicine, Reprod. Biomed Online, 2004, 9, 680–91
Dinnyes, A., Dai, Y., Jiang, S., Yang, X., High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer, Biol Reprod., 2000, 63, 513–8
Begin, I., Bhatia, B., Baldassare, H., Dinnyes, A., Keefer, C. L., Cryopreservation of goat oocytes and in vitro derived 2–4 cell embryos using the cryoloop (CLV) and solid-surface vitrification (SSV) methods, Theriogenology, 2003, 59, 1839–50
Nagashima, H., Cameron, R. D. A., Kuwayama, M., Young, M., Beebe, L., Blackshaw, A. W., et al. Survival of porcine delipated oocytes and embryos after cryopreservation by freezing or vitrification, J Reprod Dev., 1999, 45, 167–76
Arav, A., Rubinsky, B., Fletcher, G., Seren, E., Cryogenic protection of oocytes with antifreeze proteins, Mol Reprod Dev, 1993, 36, 488–93
Cean, A., Pacala, N., Boleman, A., Fast freezing of mouse embryos in classic straws with one equilibration step, using two vitrification media, Journal of Biotechnology, 2010, 150 (Suppl. 1), 127, doi:10.1016/j.jbiotec.2010.08.331
