Spindle configuration and developmental competence of in vitro-matured bovine oocytes exposed to NaCl or sucrose prior to Cryotop vitrification
Núria Arcarons A , Roser Morató B , Jozé F. W. Spricigo C , Marcia A. M. M. Ferraz D and Teresa Mogas A E
+ Author Affiliations
- Author Affiliations
A Department of Animal Medicine and Surgery, University Autonomous of Barcelona, Campus UAB, 08193 Cerdanyola del Vallès, Spain.
B Department of Biology, Universitat de Girona, Campus de Montilivi, 17071 Girona, Spain.
C School of Agriculture and Veterinary Medicine, University of Brasilia, CEP 70910-900, Brasília-DF, Brazil.
D Department of Farm Animal Health, University of Utrecht, 3584 CM Utrecht, The Netherlands.
E Corresponding autor. Email: teresa.mogas@uab.cat
Reproduction, Fertility and Development 28(10) 1560-1569 https://doi.org/10.1071/RD14516
Submitted: 28 December 2014 Accepted: 26 February 2015 Published: 22 April 2015
Abstract
In the present study we examined whether exposure to high concentrations of NaCl or sucrose before vitrification improves the cryotolerance of in vitro-matured bovine oocytes. In Experiment 1, oocytes were exposed to different concentrations of NaCl (375–1517 mOsm) or sucrose (375–812 mOsm) for 1 h. On the basis of the results of this experiment, in Experiment 2 oocytes were exposed to 0.25% NaCl (375 mOsmol) or 2.77% sucrose (375 mOsmol) solution, vitrified and warmed. Microtubule and chromosome configurations were examined by immunocytochemistry. In Experiment 3, in vitro embryo development was assessed after vitrification of oocytes with or without 2.77% sucrose (375 mOsmol) pretreatment. There was a similar percentage of oocytes showing normal spindle configurations in the sucrose-pretreated and control groups. Higher rates of abnormal spindles were found in groups treated with NaCl or sucrose solutions with >375 mOsmol. After vitrification and warming, a significantly higher percentage of oocytes with normal chromosome configurations was recorded for oocytes exposed to 375 mOsmol sucrose solution before vitrification compared with the control vitrified oocytes. However, these percentages were significantly lower than those recorded in untreated controls. Cleavage and blastocyst rates were higher in non-vitrified than vitrified oocytes. In conclusion, pretreatment with 375 mOsmol NaCl or sucrose solution had no adverse effects on the spindle status of vitrified–warmed cow oocytes. However, sucrose pretreatment offered no benefits for embryo development.
Additional keywords: blastocyst, chromosomes, embryo development, microtubule configuration, osmotic stress.
References
Agca, Y., Liu, J., Rutledge, J. J., Critser, E. S., and Critser, J. K. (2000). Effect of osmotic stress on the developmental competence of germinal vesicle and metaphase II stage bovine cumulus oocyte complexes and its relevance to cryopreservation. Mol. Reprod. Dev. 55, 212–219.| Effect of osmotic stress on the developmental competence of germinal vesicle and metaphase II stage bovine cumulus oocyte complexes and its relevance to cryopreservation.Crossref | GoogleScholarGoogle Scholar | 10618661PubMed |
Albarracín, J. L., Morató, R., Rojas, C., and Mogas, T. (2005). Effects of vitrification in open pulled straws on the cytology of in vitro matured prepubertal and adult bovine oocytes. Theriogenology 63, 890–901.
| Effects of vitrification in open pulled straws on the cytology of in vitro matured prepubertal and adult bovine oocytes.Crossref | GoogleScholarGoogle Scholar | 15629805PubMed |
Arav, A., Zeron, Y., Leslie, S. B., Behboodi, E., Anderson, G. B., and Crowe, J. H. (1996). Phase transition temperature and chilling sensitivity of bovine oocytes. Cryobiology 33, 589–599.
| Phase transition temperature and chilling sensitivity of bovine oocytes.Crossref | GoogleScholarGoogle Scholar | 8975686PubMed |
Asgari, V., Hosseini, S. M., Ostadhosseini, S., Hajian, M., and Nasr-Esfahani, M. H. (2011). Time dependent effect of post warming interval on microtubule organization, meiotic status, and parthenogenetic activation of vitrified in vitro matured sheep oocytes. Theriogenology 75, 904–910.
| Time dependent effect of post warming interval on microtubule organization, meiotic status, and parthenogenetic activation of vitrified in vitro matured sheep oocytes.Crossref | GoogleScholarGoogle Scholar | 21167581PubMed |
Attanasio, L., Boccia, L., Vajta, G., Kuwayama, M., Campanile, G., Zicarelli, L., Neglia, G., and Gasparrini, B. (2010). Cryotop vitrification of buffalo (Bubalus bubalis) in vitro matured oocytes: effects of cryoprotectant concentrations and warming procedures. Reprod. Domest. Anim. 45, 997–1002.
| Cryotop vitrification of buffalo (Bubalus bubalis) in vitro matured oocytes: effects of cryoprotectant concentrations and warming procedures.Crossref | GoogleScholarGoogle Scholar | 19473307PubMed |
Boiso, I., Martí, M., Santaló, J., Ponsá, M., Barri, P. N., and Veiga, A. (2002). A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage. Hum. Reprod. 17, 1885–1891.
| A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage.Crossref | GoogleScholarGoogle Scholar | 12093855PubMed |
Checura, C. M., and Seidel, G. E. (2007). Effect of macromolecules in solutions for vitrification of mature bovine oocytes. Theriogenology 67, 919–930.
| Effect of macromolecules in solutions for vitrification of mature bovine oocytes.Crossref | GoogleScholarGoogle Scholar | 17175017PubMed |
Cobo, A., Garcia-Velasco, J. A., Domingo, J., Remohí, J., and Pellicer, A. (2013). Is vitrification of oocytes useful for fertility preservation for age-related fertility decline and in cancer patients? Fertil. Steril. 99, 1485–1495.
| Is vitrification of oocytes useful for fertility preservation for age-related fertility decline and in cancer patients?Crossref | GoogleScholarGoogle Scholar | 23541405PubMed |
Csermely, P., Schnaider, T., Soti, C., Prohaszka, Z., and Nardai, G. (1998). The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol. Ther. 79, 129–168.
| The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review.Crossref | GoogleScholarGoogle Scholar | 9749880PubMed |
Du, Y., Lin, L., Schmidt, M., Bogh, I. B., Kragh, P. M., Sorensen, C. B., Li, J., Purup, S., Pribenszky, C., Molnar, M., Kuwayama, M., Zhang, X., Yang, H., Bolund, L., and Vajta, G. (2008a). High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival. Cloning Stem Cells 10, 325–330.
| High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival.Crossref | GoogleScholarGoogle Scholar | 18479211PubMed |
Du, Y., Pribenszky, C. S., Molnar, M., Zhang, X., Yang, H., Kuwayama, M., Pedersen, A. M., Villemoes, K., Bolund, L., and Vajta, G. (2008b). High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification. Reproduction 135, 13–17.
| High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification.Crossref | GoogleScholarGoogle Scholar | 18159079PubMed |
Geraci, F., Turturici, G., Galli, D., Cossu, G., Giudice, G., and Sconzo, G. (2006). Stress response in mesoangioblast stem cells. Cell Death Differ. 13, 1057–1063.
| Stress response in mesoangioblast stem cells.Crossref | GoogleScholarGoogle Scholar | 16282982PubMed |
Gook, D. A., Osborn, S. M., and Johnston, W. I. (1995). Parthenogenetic activation of human oocytes following cryopreservation using 1,2-propanediol. Hum. Reprod. 10, 654–658.
| 7782448PubMed |
Holm, P., Booth, P. J., Schmidt, M. H., Greve, T., and Callesen, H. (1999). High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. Theriogenology 52, 683–700.
| High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins.Crossref | GoogleScholarGoogle Scholar | 10734366PubMed |
Horvath, G., and Seidel, G. E. (2006). Vitrification of bovine oocytes after treatment with cholesterol-loaded methyl-beta-cyclodextrin. Theriogenology 66, 1026–1033.
| Vitrification of bovine oocytes after treatment with cholesterol-loaded methyl-beta-cyclodextrin.Crossref | GoogleScholarGoogle Scholar | 16620935PubMed |
Horvath, G., and Seidel, G. E. (2008). Use of fetuin before and during vitrification of bovine oocytes. Reprod. Domest. Anim. 43, 333–338.
| Use of fetuin before and during vitrification of bovine oocytes.Crossref | GoogleScholarGoogle Scholar | 18069945PubMed |
Hyttel, P., Vajta, G., and Callesen, H. (2000). Vitrification of bovine oocytes with the open pulled straw method: ultrastructural consequences. Mol. Reprod. Dev. 56, 80–88.
| Vitrification of bovine oocytes with the open pulled straw method: ultrastructural consequences.Crossref | GoogleScholarGoogle Scholar | 10737970PubMed |
Isachenko, V., Alabart, J. L., Michelmann, H. W., Bezugly, N., Isachenko, E., Soler, C., and Nawroth, F. (2001). Ultrastructure of centrifuged bovine oocyte–cumulus complexes after pre-treatment with cytoskeletal relaxant. Anat. Histol. Embryol. 30, 163–167.
| Ultrastructure of centrifuged bovine oocyte–cumulus complexes after pre-treatment with cytoskeletal relaxant.Crossref | GoogleScholarGoogle Scholar | 11447941PubMed |
Kawarsky, S. J., and King, W. A. (2001). Expression and localisation of heat shock protein 70 in cultured bovine oocytes and embryos. Zygote 9, 39–50.
| Expression and localisation of heat shock protein 70 in cultured bovine oocytes and embryos.Crossref | GoogleScholarGoogle Scholar | 11273032PubMed |
Kultz, D. (2003). Evolution of the cellular stress proteome: from monophyletic origin to ubiquitous function. J. Exp. Biol. 206, 3119–3124.
| Evolution of the cellular stress proteome: from monophyletic origin to ubiquitous function.Crossref | GoogleScholarGoogle Scholar | 12909693PubMed |
Kuwayama, M., Vajta, G., Kato, O., and Leibo, S. P. (2005). Highly efficient vitrification method for cryopreservation of human oocytes. Reprod. Biomed. Online 11, 300–308.
| Highly efficient vitrification method for cryopreservation of human oocytes.Crossref | GoogleScholarGoogle Scholar | 16176668PubMed |
Larman, M. G., Katz-Jaffe, M. G., Sheehan, C. B., and Gardner, D. K. (2007a). 1,2-Propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology. Hum. Reprod. 22, 250–259.
| 1,2-Propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology.Crossref | GoogleScholarGoogle Scholar | 16905767PubMed |
Larman, M. G., Minasi, M. G., Rienzi, L., and Gardner, D. K. (2007b). Maintenance of the meiotic spindle during vitrification in human and mouse oocytes. Reprod. Biomed. Online 15, 692–700.
| Maintenance of the meiotic spindle during vitrification in human and mouse oocytes.Crossref | GoogleScholarGoogle Scholar | 18062868PubMed |
Leibo, S. P. (2008). Cryopreservation of oocytes and embryos: optimization by theoretical versus empirical analysis. Theriogenology 69, 37–47.
| Cryopreservation of oocytes and embryos: optimization by theoretical versus empirical analysis.Crossref | GoogleScholarGoogle Scholar | 18023472PubMed |
Lin, L., Du, Y., Liu, Y., Kragh, P. M., Li, J., Purup, S., Kuwayama, M., Zhang, X., Yang, H., Bolund, L., and Vajta, G. (2009a). Elevated NaCl concentration improves cryotolerance and developmental competence of porcine oocytes. Reprod. Biomed. Online 18, 360–366.
| Elevated NaCl concentration improves cryotolerance and developmental competence of porcine oocytes.Crossref | GoogleScholarGoogle Scholar | 19298735PubMed |
Lin, L., Kragh, P. M., Purup, S., Kuwayama, M., Du, Y., Zhang, X., Yang, H., Bolund, L., Callesen, H., and Vajta, G. (2009b). Osmotic stress induced by sodium chloride, sucrose or trehalose improves cryotolerance and developmental competence of porcine oocytes. Reprod. Fertil. Dev. 21, 338–344.
| Osmotic stress induced by sodium chloride, sucrose or trehalose improves cryotolerance and developmental competence of porcine oocytes.Crossref | GoogleScholarGoogle Scholar | 19210925PubMed |
Liu, R. H., Sun, Q. Y., Li, Y. H., Jiao, L. H., and Wang, W. H. (2003). Effects of cooling on meiotic spindle structure and chromosome alignment within in vitro matured porcine oocytes. Mol. Reprod. Dev. 65, 212–218.
| Effects of cooling on meiotic spindle structure and chromosome alignment within in vitro matured porcine oocytes.Crossref | GoogleScholarGoogle Scholar | 12704733PubMed |
Magnusson, V., Feitosa, W. B., Goissis, M. D., Yamada, C., Tavares, L. M., D’Avila Assumpcao, M. E., and Visintin, J. A. (2008). Bovine oocyte vitrification: effect of ethylene glycol concentrations and meiotic stages. Anim. Reprod. Sci. 106, 265–273.
| Bovine oocyte vitrification: effect of ethylene glycol concentrations and meiotic stages.Crossref | GoogleScholarGoogle Scholar | 17686591PubMed |
McEvoy, T. G., Coull, G. D., Broadbent, P. J., Hutchinson, J. S., and Speake, B. K. (2000). Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida. J. Reprod. Fertil. 118, 163–170.
| 10793638PubMed |
Men, H., Monson, R. L., Parrish, J. J., and Rutledge, J. J. (2003a). Degeneration of cryopreserved bovine oocytes via apoptosis during subsequent culture. Cryobiology 47, 73–81.
| Degeneration of cryopreserved bovine oocytes via apoptosis during subsequent culture.Crossref | GoogleScholarGoogle Scholar | 12963414PubMed |
Men, H., Monson, R. L., Parrish, J. J., and Rutledge, J. J. (2003b). Detection of DNA damage in bovine metaphase II oocytes resulting from cryopreservation. Mol. Reprod. Dev. 64, 245–250.
| Detection of DNA damage in bovine metaphase II oocytes resulting from cryopreservation.Crossref | GoogleScholarGoogle Scholar | 12506358PubMed |
Morató, R., Izquierdo, D., Albarracin, J. L., Anguita, B., Palomo, M. J., Jiménez-Macedo, A. R., Paramio, M. T., and Mogas, T. (2008a). Effects of pre-treating in vitro-matured bovine oocytes with the cytoskeleton stabilizing agent taxol prior to vitrification. Mol. Reprod. Dev. 75, 191–201.
| Effects of pre-treating in vitro-matured bovine oocytes with the cytoskeleton stabilizing agent taxol prior to vitrification.Crossref | GoogleScholarGoogle Scholar | 17474095PubMed |
Morató, R., Izquierdo, D., Paramio, M. T., and Mogas, T. (2008b). Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development. Cryobiology 57, 137–141.
| Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development.Crossref | GoogleScholarGoogle Scholar | 18680737PubMed |
Morató, R., Izquierdo, D., Paramio, M. T., and Mogas, T. (2008c). Embryo development and structural analysis of in vitro matured bovine oocytes vitrified in flexipet denuding pipettes. Theriogenology 70, 1536–1543.
| Embryo development and structural analysis of in vitro matured bovine oocytes vitrified in flexipet denuding pipettes.Crossref | GoogleScholarGoogle Scholar | 18755504PubMed |
Mullen, S. F., Agca, Y., Broermann, D. C., Jenkins, C. L., Johnson, C. A., and Critser, J. K. (2004). The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation. Hum. Reprod. 19, 1148–1154.
| The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation.Crossref | GoogleScholarGoogle Scholar | 15070883PubMed |
Mullen, S. F., Rosenbaum, M., and Critser, J. K. (2007). The effect of osmotic stress on the cell volume, metaphase II spindle and developmental potential of in vitro matured porcine oocytes. Cryobiology 54, 281–289.
| The effect of osmotic stress on the cell volume, metaphase II spindle and developmental potential of in vitro matured porcine oocytes.Crossref | GoogleScholarGoogle Scholar | 17485076PubMed |
Otoi, T., Yamamoto, K., Koyama, N., Tachikawa, S., Murakami, M., Kikkawa, Y., and Suzuki, T. (1997). Cryopreservation of mature bovine oocytes following centrifugation treatment. Cryobiology 34, 36–41.
| Cryopreservation of mature bovine oocytes following centrifugation treatment.Crossref | GoogleScholarGoogle Scholar | 9028915PubMed |
Papis, K., Shimizu, M., and Izaike, Y. (2000). Factors affecting the survivability of bovine oocytes vitrified in droplets. Theriogenology 54, 651–658.
| Factors affecting the survivability of bovine oocytes vitrified in droplets.Crossref | GoogleScholarGoogle Scholar | 11101028PubMed |
Prentice-Biensch, J. R., Singh, J., Mapletoft, R. J., and Anzar, M. (2012). Vitrification of immature bovine cumulus–oocyte complexes: effects of cryoprotectants, the vitrification procedure and warming time on cleavage and embryo development. Reprod. Biol. Endocrinol. 10, 73.
| 22954348PubMed |
Pribenszky, C., Vajta, G., Molnar, M., Du, Y., Lin, L., Bolund, L., and Yovich, J. (2010). Stress for stress tolerance? A fundamentally new approach in mammalian embryology. Biol. Reprod. 83, 690–697.
| Stress for stress tolerance? A fundamentally new approach in mammalian embryology.Crossref | GoogleScholarGoogle Scholar | 20554920PubMed |
Pribenszky, C., Lin, L., Du, Y., Losonczi, E., Dinnyes, A., and Vajta, G. (2012). Controlled stress improves oocyte performance–cell preconditioning in assisted reproduction. Reprod. Domest. Anim. 47, 197–206.
| Controlled stress improves oocyte performance–cell preconditioning in assisted reproduction.Crossref | GoogleScholarGoogle Scholar | 22827371PubMed |
Rall, W. F., and Fahy, G. M. (1985). Ice-free cryopreservation of mouse embryos at –196 degrees C by vitrification. Nature 313, 573–575.
| Ice-free cryopreservation of mouse embryos at –196 degrees C by vitrification.Crossref | GoogleScholarGoogle Scholar | 3969158PubMed |
Rho, G. J., Kim, S., Yoo, J. G., Balasubramanian, S., Lee, H. J., and Choe, S. Y. (2002). Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes. Mol. Reprod. Dev. 63, 464–470.
| Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes.Crossref | GoogleScholarGoogle Scholar | 12412049PubMed |
Rizos, D., Ward, F., Boland, M. P., and Lonergan, P. (2001). Effect of culture system on the yield and quality of bovine blastocysts as assessed by survival after vitrification. Theriogenology 56, 1–16.
| Effect of culture system on the yield and quality of bovine blastocysts as assessed by survival after vitrification.Crossref | GoogleScholarGoogle Scholar | 11467505PubMed |
Rojas, C., Palomo, M. J., Albarracin, J. L., and Mogas, T. (2004). Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments. Cryobiology 49, 211–220.
| Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments.Crossref | GoogleScholarGoogle Scholar | 15615607PubMed |
Ruffing, N. A., Steponkus, P. L., Pitt, R. E., and Parks, J. E. (1993). Osmometric behavior, hydraulic conductivity, and incidence of intracellular ice formation in bovine oocytes at different developmental stages. Cryobiology 30, 562–580.
| Osmometric behavior, hydraulic conductivity, and incidence of intracellular ice formation in bovine oocytes at different developmental stages.Crossref | GoogleScholarGoogle Scholar | 8306705PubMed |
Seidel, G. E. (2006). Modifying oocytes and embryos to improve their cryopreservation. Theriogenology 65, 228–235.
| Modifying oocytes and embryos to improve their cryopreservation.Crossref | GoogleScholarGoogle Scholar | 16263160PubMed |
Silva, R. C., Bao, S. N., Jivago, J. L., and Lucci, C. M. (2011). Ultrastructural characterization of porcine oocytes and adjacent follicular cells during follicle development: lipid component evolution. Theriogenology 76, 1647–1657.
| Ultrastructural characterization of porcine oocytes and adjacent follicular cells during follicle development: lipid component evolution.Crossref | GoogleScholarGoogle Scholar | 21835450PubMed |
Somfai, T., Ozawa, M., Noguchi, J., Kaneko, H., Kuriani Karja, N. W., Farhudin, M., Dinnyes, A., Nagai, T., and Kikuchi, K. (2007). Developmental competence of in vitro-fertilized porcine oocytes after in vitro maturation and solid surface vitrification: effect of cryopreservation on oocyte antioxidative system and cell cycle stage. Cryobiology 55, 115–126.
| Developmental competence of in vitro-fertilized porcine oocytes after in vitro maturation and solid surface vitrification: effect of cryopreservation on oocyte antioxidative system and cell cycle stage.Crossref | GoogleScholarGoogle Scholar | 17681290PubMed |
Sripunya, N., Somfai, T., Inaba, Y., Nagai, T., Imai, K., and Parnpai, R. (2010). A comparison of cryotop and solid surface vitrification methods for the cryopreservation of in vitro matured bovine oocytes. J. Reprod. Dev. 56, 176–181.
| A comparison of cryotop and solid surface vitrification methods for the cryopreservation of in vitro matured bovine oocytes.Crossref | GoogleScholarGoogle Scholar | 19815985PubMed |
Succu, S., Leoni, G. G., Berlinguer, F., Madeddu, M., Bebbere, D., Mossa, F., Bogliolo, L., Ledda, S., and Naitana, S. (2007). Effect of vitrification solutions and cooling upon in vitro matured prepubertal ovine oocytes. Theriogenology 68, 107–114.
| Effect of vitrification solutions and cooling upon in vitro matured prepubertal ovine oocytes.Crossref | GoogleScholarGoogle Scholar | 17537497PubMed |
Tian, S.-J., Yan, C.-L., Yang, H.-X., Zhou, G.-B., Yang, Z.-Q., and Zhu, S.-E. (2007). Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration. Anim. Reprod. Sci. 101, 365–371.
| Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration.Crossref | GoogleScholarGoogle Scholar | 17293065PubMed |
Toner, M., Cravalho, E. G., and Karel, M. (1993). Cellular response of mouse oocytes to freezing stress: prediction of intracellular ice formation. J. Biomech. Eng. 115, 169–174.
| Cellular response of mouse oocytes to freezing stress: prediction of intracellular ice formation.Crossref | GoogleScholarGoogle Scholar | 8326722PubMed |
Zhou, X. L., Al Naib, A., Sun, D. W., and Lonergan, P. (2010). Bovine oocyte vitrification using the Cryotop method: effect of cumulus cells and vitrification protocol on survival and subsequent development. Cryobiology 61, 66–72.
| Bovine oocyte vitrification using the Cryotop method: effect of cumulus cells and vitrification protocol on survival and subsequent development.Crossref | GoogleScholarGoogle Scholar | 20510225PubMed |
