Document Type : Original Article


1 Department of Animal Sciences, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

2 Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil


Previous studies have reported many discrepancies about the best type and concentration of cryoprotective agents (CPAs) and biological variability among various pre-antral follicle classes after cryopreservation of ovarian tissue. The aim of this study was to investigate the impacts of some synthetic polymers on histological characteristics of different types of pre-antral follicles after bovine ovarian tissue vitrification. From each bovine ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control group) or submitted to vitrification (sucrose, X-1000, Z-1000 and polyvinylpyrrolidone groups), either followed by in vitro culture for 1 or 5 days. In this case, although, the addition of X-1000 resulted in greater percentages of normal follicles for almost all pre-antral follicle classes compared to those of other groups, there are some exceptions. These results indicate that the inclusion of polyvinylpyrrolidone in the freezing media can improve the morphology of the post-warmed transitional follicles and cultured primordial follicles on day five more than other CPAs. According to the results of this study, it can be concluded that although ovarian tissue cryopreservation is often performed to preserve the primordial follicles, by choosing the best combination of permeating and non-permeating CPAs (synthetic polymers), more advanced stages of bovine pre-antral follicles, transitional, primary and secondary follicles, may also survive the cryopreservation process.


Main Subjects


    1. Aubard Y, Poirot C, Piver P, et al. Are there indications for ovarian tissue cryopreservation? Fertil Steril 2001; 76 (2): 414-415.
    2. Wallin A, Ghahremani M, Dahm-Kähler P, et al. Viability and function of the cryopreserved whole ovary: In vitro studies in the sheep. Hum Reprod 2009; 24 (7):1684-1694.
    3. Arav A, Gavish Z, Elami A, et al. Ovarian function 6 years after cryopreservation and transplantation of whole sheep ovaries. Reprod Biomed Online 2010; 20 (1): 48-52.
    4. Shaw JM OA, Trounson A. Cryopreservation of oocytes and embryos. In: Gardner DK, Simon C (Eds). Handbook of in vitro fertilization. 2nd ed. Boca Raton, USA: CRC Press 2000; 373-412.
    5. Cleary M, Snow M, Paris M, et al. Cryopreservation of mouse ovarian tissue following prolonged exposure to an ischemic environment. Cryobiology 2001; 42 (2):121-133.
    6. Smith GD, Serafini PC, Fioravanti J, et al. Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertil Steril 2010; 94 (6): 2088-2095.
    7. Ubaldi F, Anniballo R, Romano S, et al. Cumulative ongoing pregnancy rate achieved with oocyte vitrification and cleavage stage transfer without embryo selection in a standard infertility program. Hum Reprod 2010; 25 (5): 1199-1205.
    8. Kagawa N, Silber S, Kuwayama M. Successful vitrification of bovine and human ovarian tissue. Reprod Biomed Online 2009; 18 (4): 568-577.
    9. Carvalho AA, Faustino LR, Silva CM, et al. Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue. Theriogenology 2011; 76 (5): 933-941.
    10. Ksiazkiewicz LK. Recent achievements in in vitro culture and preservation of ovarian follicles in mammals. Reprod Biol 2006; 6 (1): 3-16.
    11. Santos R, Amorim C, Cecconi S, et al. Cryopreservation of ovarian tissue: an emerging technology for female germline preservation of endangered species and breeds. Anim Reprod Sci 2010; 122 (3): 151-163.
    12. Hovatta I, Tennant RS, Helton R, et al. Glyoxalase 1 and glutathione reductase 1 regulate anxiety in mice. Nature 2005; 438 (7068):662-666.
    13. Santos RR, Van Haeften T, Roelen BA, et al. Osmotic tolerance and freezability of isolated caprine early-staged follicles. Cell Tissue Res 2008; 333 (2): 323-331.
    14. Barrett SL, Shea LD, Woodruff TK. Noninvasive index of cryorecovery and growth potential for human follicles in vitro. Biol Reprod 2010; 82 (6): 1180-1189.
    15. Ting AY, Yeoman RR, Lawson MS, et al. Synthetic polymers improve vitrification outcomes of macaque ovarian tissue as assessed by histological integrity and the in vitro development of secondary follicles. Cryobiology 2012; 65 (1):1-11
    16. Santos R, Van den Hurk R, Rodrigues A, et al. Viability of oocytes and granulosa cells from cryopreserved ovine ovarian primordial, primary and secondary follicles. Small Rumin Res 2011; 99 (2-3): 203-207.
    17. Santos RR, 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 2007; 327(1): 167-176.
    18. Keros V, Xella S, Hultenby K, et al. Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue. Hum Reprod 2009; 24 (7): 1670-1683.
    19. Wildt DE, Wemmer C. Sex and wildlife: The role of reproductive science in conservation. Biodivers Conserv 1999; 8 (7): 965-976.
    20. Amorim CA, Curaba M, Van Langendonckt A, et al. Vitrification as an alternative means of cryopreserving ovarian tissue. Reprod Biomed Online 2011; 23 (2): 160-186.
    21. Badrzadeh H, Najmabadi S, Paymani R, et al. Super cool X-1000 and Super cool Z-1000, two ice blockers, and their effect on vitrification/warming of mouse embryos. Eur J Obstet Gynecol Reprod Biol 2010; 151 (1): 70-71.
    22. Deller RC, Vatish M, Mitchell DA, et al. Synthetic polymers enable non-vitreous cellular cryo-preservation by reducing ice crystal growth during warming. Nat Commun 2014; 5: 3244.
    23. Mitchell DE, Cameron NR, Gibson MI. Rational, yet simple, design and synthesis of an antifreeze-protein inspired polymer for cellular cryopreservation. Chem Commun (Camb) 2015; 51 (65): 12977-12980.
    24. Fahy GM, Wowk B, Wu J, et al. Cryopreservation of organs by vitrification: Perspectives and recent advances. Cryobiology 2004; 48 (2): 157-178.
    25. Zhou XH, Wu YJ, Shi J, et al. Cryopreservation of human ovarian tissue: comparison of novel direct cover vitrification and conventional vitrification. Cryobiology 2010; 60(2): 101-105.
    26. Marco-Jimenez F, Jimenez-Trigos E, Lavara R, et al. Generation of live offspring from vitrified embryos with synthetic polymers SuperCool X-1000 and SuperCool Z-1000. Cryo Letters 2014; 35 (4): 286-292.
    27. de Graaf IA, Draaisma AL, Schoeman O, et al. Cryo-preservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification. Cryobiology 2007; 54 (1): 1-12.
    28. Wowk B, Fahy GM. Inhibition of bacterial ice nucleation by polyglycerol polymers. Cryobiology 2002; 44 (1): 14-23.
    29. Taylor MJ, Song YC, Brockbank KG. Vitrification in tissue preservation: New developments. In: BJ Fuller, N Lane, EE Benson (Eds). Life in the frozen state. Boca Raton, FL USA; CRC Press 2004: 604-641.
    30. Wowk B, Leitl E, Kersh K, et al. Molecular mechanisms of ice control by polyvinyl alcohol. Cryobiology 2002; 41 (4): 352-353.
    31. Carvalho AA, Faustino LR, Silva CM, et al. Novel wide-capacity method for vitrification of caprine ovaries: Ovarian tissue cryosystem (OTC). Anim Reprod Sci 2013; 138 (3): 220-227.
    32. Sales AD, Duarte AB, Santos RR, et al. Modulation of aquaporins 3 and 9 after exposure of ovine ovarian tissue to cryoprotectants followed by in vitro culture. Cell Tissue Res 2016; 365 (2): 415-424.
    33. Castro SV, Carvalho AA, Silva CM, et al. Fresh and vitrified bovine preantral follicles have different nutritional requirements during in vitro culture. Cell Tissue Bank 2014; 15 (4): 591-601.
    34. McLaughlin M, Telfer EE. Oocyte development in bovine primordial follicles is promoted by activin and FSH within a two-step serum-free culture system. Reproduction 2010; 139 (6): 971-978.
    35. Pinto LC, Santos RR, Faustino LR, et al. Quantification of dimethyl sulfoxide perfusion in sheep ovarian tissue: A predictive parameter for follicular survival to cryo-preservation. Biopreserv Biobank 2008; 6 (4): 269-276.
    36. Wowk B, Leitl E, Rasch CM, et al. Vitrification enhancement by synthetic ice blocking agents. Cryobiology 2000; 40 (3): 228-236.
    37. Wowk B. Anomalous high activity of a subfraction of polyvinyl alcohol ice blocker. Cryobiology; 2005; 50 (3): 325-331.
    38. Hashimoto S, Suzuki N, Yamanaka M, et al. Effects of vitrification solutions and equilibration times on the morphology of cynomolgus ovarian tissues. Reprod Biomed Online 2010; 21 (4): 501-509.
    39. Shaw JM, Kuleshova LL, MacFarlane DR, et al. Vitrification properties of solutions of ethylene glycol in saline containing PVP, Ficoll, or dextran. Cryobiology 1997; 35 (3): 219-229.
    40. Leibo S, Oda K. High survival of mouse zygotes and embryos cooled rapidly or slowly in ethylene-glycol plus polyvinylpyrrolidone. Cryo Letters 1993; 14 (3): 133-144.
    41. Whittingham DG. Survival of mouse embryos after freezing and warming. Nature. 1971; 233 (5315):125-126.
    42. Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to-196 degreesand-269 degrees C. Science 1972; 178 (4059): 411-414.
    43. Titterington JL, Robinson J, Killick SR, et al. Synthetic and biological macromolecules: protection of mouse embryos during cryopreservation by vitrification. Hum Reprod 1995; 10 (3): 649-653.
    44. Fuller BJ. Cryoprotectants: The essential antifreezes to protect life in the frozen state. Cryo Letters 2004; 25 (6): 375-388.
    45. Amorim CA, Jacobs S, Devireddy RV, et al. Successful vitrification and autografting of baboon (Papio anubis) ovarian tissue. Hum Reprod 2013; 28 (8): 2146-2156.
    46. Isachenko V, Isachenko E, Rahimi G, et al. Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen: Negative effect of disaccharides in vitrification solution. Cryo Letters 2002; 23 (5): 333-344.
    47. Rahimi G, Isachenko E, Isachenko V, et al. Comparison of necrosis in human ovarian tissue after conventional slow freezing or vitrification and transplantation in ovariectomized SCID mice. Reprod Biomed Online 2004; 9 (2): 187-193.
    48. Rahimi G, Isachenko V, Todorov P, et al. Apoptosis in human ovarian tissue after conventional freezing or vitrification and xenotransplantation. Cryo Letters 2009; 30 (4): 300-309.