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Vitrification of goat preantral follicles enclosed in ovarian tissue by using conventional and solid-surface vitrification methods

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Abstract

Caprine preantral follicles within ovarian fragments were exposed to or vitrified in the presence of sucrose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), or various combinations thereof. The fragments were cryopreserved by using either a conventional (CV) or a solid-surface vitrification (SSV) protocol, and the cryoprotectants were removed by equilibrating vitrified ovarian fragments in “warming solution” consisting of minimum essential medium and heat-inactivated fetal calf serum (MEM+) followed by washes in MEM+ with or without sucrose. Histological analysis of follicle integrity showed that the percentages of normal follicles in ovarian fragments vitrified in sucrose mixed with EG and/or DMSO (CV method) or mixed with EG or DMSO (SSV method) followed by washes in MEM+ plus sucrose were similar to those of controls (ovarian fragments fixed without previous vitrification). Unlike for MEM+ (supplemented or unsupplemented by sucrose) and DMSO followed by washes in the absence of sucrose, the percentages of normal follicles found after exposure to cryoprotectant did not significantly differ from that found after vitrification, indicating that follicular degeneration was attributable to a toxic effect of cryoprotectants and not to the vitrification procedure. The viability of preantral follicles after the CV and SSV procedures was investigated by using calcein-AM and the ethidium-homodimer as “live” and “dead” markers, respectively. In both tested vitrification procedures, the highest percentages of viable follicles were observed when a mixture of sucrose and EG (70.3% for CV and 72.4% for SSV) was used. Preantral follicles were also vitrified (either by CV or SSV) in sucrose and EG and then cultured for 24 h, after which their viability was compared with that of cultured fresh and uncultured vitrified follicles. The viability of these follicles was maintained after SSV, but not after CV. Thus, the viability of caprine preantral follicles can be best preserved after SSV in a mixture of sucrose and EG, followed by washes in medium containing sucrose.

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Acknowledgements

The authors thank Peter Ursem, Arend Rijneveld, Ineke Daemen, Frans van Kooi, and the technicians of the Department of Pathobiology for their help.

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Authors and Affiliations

  1. Departmant of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

    R. R. Santos & R. Van den Hurk

  2. Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

    T. Tharasanit

  3. Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

    T. Van Haeften

  4. Laboratory of Manipulation of Oocytes Enclosed in Preantral Follicles - LAMOFOPA, Faculty of Veterinary Medicine, Ceará University, Fortaleza, CE, Brazil

    R. R. Santos, J. R. Figueiredo & J. R. V. Silva

  5. P.O. Box 80151, NL-3584 TD, Yalelaan 7, Utrecht, The Netherlands

    R. R. Santos

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  1. R. R. Santos
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  2. T. Tharasanit
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  3. T. Van Haeften
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  4. J. R. Figueiredo
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  5. J. R. V. Silva
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  6. R. Van den Hurk
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Correspondence to R. R. Santos.

Additional information

CAPES/Brazil supported this work. Regiane Rodrigues dos Santos is a recipient of a grant from CAPES/Brazil.

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Santos, R.R., Tharasanit, T., Van Haeften, T. et al. Vitrification of goat preantral follicles enclosed in ovarian tissue by using conventional and solid-surface vitrification methods. Cell Tissue Res 327, 167–176 (2007). https://doi.org/10.1007/s00441-006-0240-2

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  • DOI: https://doi.org/10.1007/s00441-006-0240-2

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