Advertisement

Electric ultrafreezer (− 150 °C) as an alternative for zebrafish sperm cryopreservation and storage

  • Patrícia Diogo
  • Gil Martins
  • Isa Quinzico
  • Rita Nogueira
  • Paulo J. Gavaia
  • Elsa Cabrita
Article

Abstract

Zebrafish sperm cryopreservation is a fundamental methodology to manage and back-up valuable genetic resources like transgenic and mutant strains. Cryopreservation usually requires liquid nitrogen for storage, which is expensive and hazardous. Our objective was to evaluate if electric ultrafreezers (− 150 °C) are a viable alternative for zebrafish sperm storage. Zebrafish sperm was cryopreserved in the same conditions (− 20 °C/min), stored either in liquid nitrogen or in an ultrafreezer, and thawed after 1 week, 1 month, and 3 months. Sperm motility, membrane integrity, and fertilization ability were assessed. There were no significant differences in motility and hatching rate throughout storage time. Additionally, we aimed at understanding if cryopreservation directly in an ultrafreezer (− 66 °C/min) could improve post-thaw sperm quality. Freezing at − 20 °C/min was performed as before, and compared to samples cryopreserved with a fast cooling rate by placing directly in an ultrafreezer (− 66 °C/min). Sperm quality was assessed according to motility, viability, DNA fragmentation, and apoptosis (annexin V). The − 66 °C/min cooling rate showed significantly higher membrane and DNA integrity, and lower number of cells in late apoptosis in comparison to the other treatments. This study showed that zebrafish sperm cryopreservation and storage in an ultrafreezer system is possible and a fast cooling rate directly in ultrafreezer improves post-thaw sperm quality.

Keywords

Storage Ultrafreezer Zebrafish sperm Cryopreservation Cooling rate 

Notes

Acknowledgments

Patricia Diogo acknowledges the financial support from the Portuguese Foundation for Science and Technology (FCT) through the doctoral grant SFRH/BD/97466/2013. This work was partly founded by the FCT and the European Commission (ERDF-COMPETE) through PEst-C/MAR/LA0015/2011 project and by the FCT through UID/Multi/04326/2013 project. The authors acknowledge the support of Ana Marreiros for the statistical analysis and Marco Tarasco for technical support during the samplings.

Supplementary material

10695_2018_500_Fig8_ESM.gif (14 kb)
Online Resource 1 (GIF 14 kb)
10695_2018_500_MOESM1_ESM.tiff (216 kb)
High Resolution (TIFF 215 kb)

References

  1. Álamo D, Batista M, González F, Rodríguez N, Cruz G, Cabrera F, Gracia A (2005) Cryopreservation of semen in the dog: use of ultra-freezers of -152°C as a viable alternative to liquid nitrogen. Theriogenology 63(1):72–82.  https://doi.org/10.1016/j.theriogenology.2004.03.016 CrossRefPubMedGoogle Scholar
  2. Asturiano JF, Riesco MF, Martins G, Vílchez MC, Pérez L, Gavaia PJ, Cabrita E (2015) Cryopreservation of zebrafish sperm, first trials and results. 5th International Workshop on the Biology of Fish Gametes, Ancona, ItalyGoogle Scholar
  3. Batista M, Álamo D, González F, Cruz MG, Gracia A (2006) Influence of the freezing technique (nitrogen liquid vs ultrafreezer of −152°C) and male-to-male variation over the semen quality in Canarian mastiff breed dogs. Reprod Domest Anim 41(5):423–428.  https://doi.org/10.1111/j.1439-0531.2006.00687.x CrossRefPubMedGoogle Scholar
  4. Batista M, Niño T, Álamo D, Castro N, Santana M, González F, Cabrera F, Gracia A (2009) Successful artificial insemination using semen frozen and stored by an ultrafreezer in the Majorera goat breed. Theriogenology 71(8):1307–1315.  https://doi.org/10.1016/j.theriogenology.2008.12.024 CrossRefPubMedGoogle Scholar
  5. Bernáth G, Bokor Z, Kása E, Várkonyi L, Hegyi Á, Kollár T, Urbányi B, Żarski D, Radóczi JI, Horváth Á (2015) Comparison of two different methods in the cryopreservation of Eurasian perch (Perca fluviatilis) sperm. Cryobiology 70(1):76–78.  https://doi.org/10.1016/j.cryobiol.2014.12.003 CrossRefPubMedGoogle Scholar
  6. Bernáth G, Żarski D, Kása E, Staszny Á, Várkonyi L, Kollár T, Hegyi Á, Bokor Z, Urbányi B, Horváth Á (2016) Improvement of common carp (Cyprinus carpio) sperm cryopreservation using a programmable freezer. Gen Comp Endocrinol 237:78–88.  https://doi.org/10.1016/j.ygcen.2016.08.013 CrossRefPubMedGoogle Scholar
  7. Bobe J, Labbé C (2010) Egg and sperm quality in fish. Gen Comp Endocrinol 165(3):535–548.  https://doi.org/10.1016/j.ygcen.2009.02.011 CrossRefPubMedGoogle Scholar
  8. Cabrita E, Robles V, Cuñado S, Wallace JC, Sarasquete C, Herráez MP (2005) Evaluation of gilthead sea bream, Sparus aurata, sperm quality after cryopreservation in 5ml macrotubes. Cryobiology 50:273–284.  https://doi.org/10.1016/j.cryobiol.2005.02.005 CrossRefPubMedGoogle Scholar
  9. Cabrita E, Robles V, Herráez P (2009) Sperm quality assessment. In: Cabrita E, Robles V, Herráez P (eds) Methods in reproductive aquaculture: marine and freshwater species. CRC press, Boca Raton, pp 93–148Google Scholar
  10. Cabrita E, Sarasquete C, Martínez-Páramo S, Robles V, Beirão J, Pérez-Cerezales S, Herráez MP (2010) Cryopreservation of fish sperm: applications and perspectives. J Appl Ichthyol 26(5):623–635.  https://doi.org/10.1111/j.1439-0426.2010.01556.x CrossRefGoogle Scholar
  11. Carmichael C, Westerfield M, Varga ZM (2009) Cryopreservation and in vitro fertilization at the zebrafish international resource center. Methods Mol Biol 546:45–65.  https://doi.org/10.1007/978-1-60327-977-2_4 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Daly J, Tiersch TR (2012) Sources of variation in flow cytometric analysis of aquatic species sperm: the effect of cryoprotectants on flow cytometry scatter plots and subsequent population gating. Aquaculture 370-371:179–188.  https://doi.org/10.1016/j.aquaculture.2012.09.024 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Desrosiers P, Légaré C, Leclerc P, Sullivan R (2006) Membranous and structural damage that occur during cryopreservation of human sperm may be time-related events. Fertil Steril 85(6):1744–1752.  https://doi.org/10.1016/j.fertnstert.2005.11.046 CrossRefPubMedGoogle Scholar
  14. Diogo P, Martins G, Gavaia P, Pinto W, Dias J, Cancela L, Martínez-Páramo S (2015) Assessment of nutritional supplementation in phospholipids on the reproductive performance of zebrafish, Danio rerio (Hamilton, 1822). J Appl Ichthyol 31:31(S1):3–31(S1):9.  https://doi.org/10.1111/jai.12733 CrossRefGoogle Scholar
  15. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516.  https://doi.org/10.1080/01926230701320337 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Esteves-Ferreira AA, Corrêa DM, Carneiro APS, Rosa RM, Loterio R, Araújo WL (2013) Comparative evaluation of different preservation methods for cyanobacterial strains. J Appl Phycol 25(4):919–929.  https://doi.org/10.1007/s10811-012-9927-9 CrossRefGoogle Scholar
  17. Figueroa E, Valdebenito I, Farias JG (2016) Technologies used in the study of sperm function in cryopreserved fish spermatozoa. Aquac Res 47(6):1691–1705.  https://doi.org/10.1111/are.12630 CrossRefGoogle Scholar
  18. Fuller BJ (2004) Cryoprotectants: the essential antifreezes to protect life in the frozen state. Cryo Lett 25(6):375–388Google Scholar
  19. Grout BW, Morris GJ (2009) Contaminated liquid nitrogen vapour as a risk factor in pathogen transfer. Theriogenology 71(7):1079–1082.  https://doi.org/10.1016/j.theriogenology.2008.12.011 CrossRefPubMedGoogle Scholar
  20. Hagedorn M, Carter VL (2011) Zebrafish reproduction: revisiting in vitro fertilization to increase sperm cryopreservation success. PLoS One 6(6):e21059.  https://doi.org/10.1371/journal.pone.0021059 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hagedorn M, McCarthy M, Carter VL, Meyers SA (2012) Oxidative stress in zebrafish (Danio rerio) sperm. PLoS One 7(6):e39397.  https://doi.org/10.1371/journal.pone.0039397 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Harvey B, Kelley RN, Ashwood-Smith MJ (1982) Cryopreservation of zebrafish spermatozoa using methanol. Can J Zool 60(70):1867–1870.  https://doi.org/10.1139/z82-242 CrossRefGoogle Scholar
  23. Jing R, Huang C, Bai C, Tanguay R, Dong Q (2009) Optimization of activation, collection, dilution, and storage methods for zebrafish sperm. Aquaculture 290:165–171.  https://doi.org/10.1016/j.aquaculture.2009.02.027 CrossRefGoogle Scholar
  24. Kopeika J, Kopeika E, Zhang T, Rawson DM, Holt WV (2004) Effect of DNA repair inhibitor (3-aminobenzamide) on genetic stability of loach (Misgurnus fossilis) embryos derived from cryopreserved sperm. Theriogenology 61:1661–1673.  https://doi.org/10.1016/j.theriogenology.2003.09.010 CrossRefPubMedGoogle Scholar
  25. Larman MG, Hashimoto S, Morimoto Y, Gardner DK (2014) Cryopreservation in ART and concerns with contamination during cryobanking. Reprod Med Biol 13(3):107–117.  https://doi.org/10.1007/s12522-014-0176-2 CrossRefGoogle Scholar
  26. Lessard C, Parent S, Leclerc P, Bailey JL, Sullivan R (2000) Cryopreservation alters the levels of the bull sperm surface protein P25b. J Androl 21:700–707.  https://doi.org/10.1002/j.1939-4640.2000.tb02138.x PubMedGoogle Scholar
  27. Liu J, Zhou Y, Qi X, Chen J, Chen W, Qiu G, Wu Z, Wu N (2017) CRISPR/Cas9 in zebrafish: an efficient combination for human genetic diseases modeling. Hum Genet 136(1):1–12.  https://doi.org/10.1007/s00439-016-1739-6 CrossRefPubMedGoogle Scholar
  28. Mazur P (1984) Freezing of living cells: mechanisms and implications. Am J Phys 247(3 Pt 1):C125–C142CrossRefGoogle Scholar
  29. Medrano A, Cabrera F, González F, Batista M, Gracia A (2002) Is sperm cryopreservation at −150 degree C a feasible alternative? Cryo Lett 23(3):167–172Google Scholar
  30. Pérez-Cerezales S, Martínez-Páramo S, Beirão J, Herráez MP (2010) Fertilization capacity with rainbow trout DNA-damaged sperm and embryo developmental success. Reproduction 139(6):989–997.  https://doi.org/10.1530/REP-10-0037 CrossRefPubMedGoogle Scholar
  31. Polak R, Pitombo RNM (2011) Care during freeze-drying of bovine pericardium tissue to be used as a biomaterial: a comparative study. Cryobiology 63(2):61–66.  https://doi.org/10.1016/j.cryobiol.2011.05.001 CrossRefPubMedGoogle Scholar
  32. Reinardy HC, Skippins E, Henry TB, Jha AN (2013) Assessment of DNA damage in sperm after repeated non-invasive sampling in zebrafish Danio rerio. J Fish Biol 82(3):1074–1081.  https://doi.org/10.1111/jfb.12042 CrossRefPubMedGoogle Scholar
  33. Robles V, Cabrita E, Herráez MP (2009) Germplasm cryobanking in zebrafish and other aquarium model species. Zebrafish 6(3):281–293.  https://doi.org/10.1089/zeb.2009.0592 CrossRefPubMedGoogle Scholar
  34. Rurangwa E, Volckaert FA, Huyskens G, Kime DE, Ollevier F (2001) Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African catfish (Clarias gariepinus). Theriogenology 55(3):751–769CrossRefPubMedGoogle Scholar
  35. Suster ML, Kikuta H, Urasaki A, Asakawa K, Kawakami K (2009) Transgenesis in zebrafish with the tol2 transposon system. Methods Mol Biol 561:41–63.  https://doi.org/10.1007/978-1-60327-019-9_3 CrossRefPubMedGoogle Scholar
  36. Tiersch TR, Yang H, Jenkins JA, Dong Q (2007) Sperm cryopreservation in fish and shellfish. Soc Reprod Fertil Suppl 65:493–508PubMedGoogle Scholar
  37. Tsai S, Lin C (2012) Advantages and applications of cryopreservation in fisheries science. Braz Arch Biol Technol 55(3):425–434.  https://doi.org/10.1590/S1516-89132012000300014 CrossRefGoogle Scholar
  38. Wang G, Kang N, Gong H, Luo Y, Bai C, Chen Y, Ji X, Huang C, Dong Q (2015) Upregulation of uncoupling protein Ucp2 through acute cold exposure increases post-thaw sperm quality in zebrafish. Cryobiology 71(3):464–471.  https://doi.org/10.1016/j.cryobiol.2015.08.016 CrossRefPubMedGoogle Scholar
  39. Ward JHJ (1963) Hierarchical grouping to optimize an objective function. J Am Stat Assoc 58:236–244CrossRefGoogle Scholar
  40. Westerfield M (2000) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 4th edn. Univ of Oregon Press, OregonGoogle Scholar
  41. Woelders H, Matthijs A, Engel B (1997) Effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing. Cryobiology 35(2):93–105.  https://doi.org/10.1006/cryo.1997.2028 CrossRefPubMedGoogle Scholar
  42. Yang H, Carmichael C, Varga ZM, Tiersch TR (2007) Development of a simplified and standardized protocol with potential for high-throughput for sperm cryopreservation in zebrafish Danio rerio. Theriogenology 68(2):128–136.  https://doi.org/10.1016/j.theriogenology.2007.02.015 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Yang H, Daly J, Carmichael C, Matthews J, Varga ZM, Tiersch T (2016) A procedure-spanning analysis of plasma membrane integrity for assessment of cell viability in sperm cryopreservation of zebrafish Danio rerio. Zebrafish 13(2):144–151.  https://doi.org/10.1089/zeb.2015.1176 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Yavaş K, Daşkin A (2012) Effect of alternative cryopreservation procedures on bull semen. Ankara Üniv Vet Fak Derg 59:231–234CrossRefGoogle Scholar
  45. Yuan Y, Yang Y, Tian Y, Park J, Dai A, Roberts RM, Liu Y, Han X (2016) Efficient long-term cryopreservation of pluripotent stem cells at −80 °C. Sci Rep 6:34476.  https://doi.org/10.1038/srep34476 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Patrícia Diogo
    • 1
    • 2
  • Gil Martins
    • 1
    • 2
  • Isa Quinzico
    • 1
  • Rita Nogueira
    • 1
  • Paulo J. Gavaia
    • 2
    • 3
  • Elsa Cabrita
    • 1
    • 2
  1. 1.Faculty of Sciences and TechnologyUniversity of AlgarveFaroPortugal
  2. 2.Centre of Marine SciencesUniversity of AlgarveFaroPortugal
  3. 3.Department of Biomedical Sciences and MedicineUniversity of AlgarveFaroPortugal

Personalised recommendations