Chapter 15 Sperm Cryopreservation of Aquatic Species

  • Ákos HorváthEmail author
  • Béla Urbányi


Cryopreservation of fish sperm is feasible, and methods have been developed for several hundred species. The procedure includes the collection of sperm and assessment of its quality, dilution in suitable extenders, addition of cryoprotectants, loading into freezing devices, cooling, storage, thawing, and finally post-thaw use of sperm. An alternative to traditional freezing methods is the vitrification of sperm which is promising primarily in smaller model fish species. A wide variety of protocols are available in the literature; however, in spite of the significant progress made by the scientific community, commercial application of fish sperm cryopreservation is still very limited.


Sperm Quality Freezing Cryopreservation Vitrification 



This research was supported by the Higher Education Institutional Excellence Program (1783-3/2018/FEKUTSRAT) awarded by the Ministry of Human Capacities within the framework of water related researches of Szent István University as well as the EFOP-3.6.3-VEKOP-16-2017-00008 project co-financed by the European Union and the European Social Fund. Parts of this chapter are translations from the Hungarian language of the dissertation by Á. Horváth submitted to the Hungarian Academy of Sciences for the title of Doctor of the Hungarisn Academy of Sciences.


  1. Asturiano JF, Marco-Jiménez F, Peñaranda DS, Garzón DL, Pérez L, Vicente JS, Jover M (2007) Effect of sperm cryopreservation on the European eel sperm viability and spermatozoa morphology. Reprod Domest Anim 42:162–166. Scholar
  2. Asturiano JF, Cabrita E, Horváth Á (2017) Progress, challenges and perspectives on fish gamete cryopreservation: a mini-review. Gen Comp Endocrinol 245:69–76. Scholar
  3. Babiak I, Glogowski J, Goryczko K, Dobosz S, Kuzminski H, Strzezek J, Demianowicz W (2001) Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved spermatozoa. Theriogenology 56:177–192. Scholar
  4. Beirão J, Cabrita E, Pérez-Cerezales S, Martínez-Páramo S, Herráez MP (2011) Effect of cryopreservation on fish sperm subpopulations. Cryobiology 62:22–31. Scholar
  5. Bernáth G, Bokor Z, Żarski D, Várkonyi L, Hegyi Á, Urbányi B, Radóczi Ifj J, Horváth Á (2016) Commercial-scale out-of-season cryopreservation of Eurasian perch (Perca fluviatilis) sperm and its application for fertilization. Anim Reprod Sci 170:170–177. Scholar
  6. Billard R, Cosson MP (1992) Some problems related to the assessment of sperm motility in freshwater fish. J Exp Zool 261:122–131. Scholar
  7. Billard R, Cosson J, Crim LW (1993) Motility of fresh and aged halibut sperm. Aquat Living Resour 6:67–75. Scholar
  8. Blaxter JHS (1953) Sperm storage and cross-fertilization of spring and autumn spawning herring. Nature 172:1189–1190CrossRefGoogle Scholar
  9. Blom E (1950) A one-minute live-dead sperm stain by means of eosin-nigrosin. Fertil Steril 1:176–177. Scholar
  10. Boryshpolets S, Dzyuba B, Stejskal V, Linhart O (2009) Dynamics of ATP and movement of Eurasian perch (Perca fluviatilis L.) sperm in conditions of decreasing osmolality. Theriogenology 72:851–859CrossRefGoogle Scholar
  11. Boryshpolets S, Sochorová D, Rodina M, Linhart O, Dzyuba B (2017) Cryopreservation of Carp (Cyprinus carpio L.) sperm: impact of seeding and freezing rates on post–thaw outputs. Biopreserv Biobank 15:234–240. Scholar
  12. Cabrita E, Robles V, Alvarez R, Herráez MP (2001) Cryopreservation of rainbow trout sperm in large volume straws: application to large scale fertilization. Aquaculture 201:301–314CrossRefGoogle Scholar
  13. Cabrita E, Robles V, Rebordinos L, Sarasquete C, Herráez MP (2005) Evaluation of DNA damage in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) cryopreserved sperm. Cryobiology 50:144–153. Scholar
  14. Cabrita E, Ma S, Diogo P, Martínez-Páramo S, Sarasquete C, Dinis MT (2011) The influence of certain aminoacids and vitamins on post-thaw fish sperm motility, viability and DNA fragmentation. Anim Reprod Sci 125:189–195. Scholar
  15. Chao N-H, Chao W-C, Liu K-C, Liao I-C (1987) The properties of tilapia sperm and its cryopreservation. J Fish Biol 30:107–118. Scholar
  16. Ciereszko A, Dabrowski K (1993) Estimation of sperm concentration of rainbow trout, whitefish and yellow perch using a spectrophotometric technique. Aquaculture 109:367–373. Scholar
  17. Ciereszko A, Dietrich GJ, Nynca J, Dobosz S, Zalewski T (2014) Cryopreservation of rainbow trout semen using a glucose-methanol extender. Aquaculture 420–421:275–281. Scholar
  18. Conget P, Fernández M, Herrera G, Minguell JJ (1996) Cryopreservation of rainbow trout (Oncorhynchus mykiss) spermatozoa using programmable freezing. Aquaculture 143:319–329CrossRefGoogle Scholar
  19. Cuevas-Uribe R, Leibo SP, Daly J, Tiersch TR (2011a) Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling. Cryobiology 63:186–197. Scholar
  20. Cuevas-Uribe R, Yang H, Daly J, Savage MG, Walter RB, Tiersch TR (2011b) Production of F1 offspring with vitrified sperm from a live-bearing fish, the green swordtail Xiphophorus hellerii. Zebrafish 8:167–179. Scholar
  21. Cuevas-Uribe R, Chesney EJ, Daly J, Tiersch TR (2015) Vitrification of sperm from marine fish: effect on motility and membrane integrity. Aquacult Res 46:1770–1784. Scholar
  22. Denniston RS, Michelet S, Bondioli KR, Godke RA (2011) Principles of embryo cryopreservation. In: Tiersch TR, Green CC (eds) Cryopreservation in aquatic species, 2nd edn. World Aquaculture Society, Baton Rouge, pp 274–290Google Scholar
  23. Diogo P, Martins G, Quinzico I, Nogueira R, Gavaia PJ, Cabrita E (2018) Electric ultrafreezer (- 150 °C) as an alternative for zebrafish sperm cryopreservation and storage. Fish Physiol Biochem 44(6):1443–1455. Scholar
  24. Fahy GM, MacFarlane DR, Angell CA, Meryman HT (1984) Vitrification as an approach to cryopreservation. Cryobiology 21:126–407Google Scholar
  25. Fauvel C, Savoye O, Dreanno C, Cosson J, Suquet M (1999) Characteristics of sperm of captive seabass in relation to its fertilization potential. J Fish Biol 54:356–369. Scholar
  26. Fauvel C, Suquet M, Cosson J (2010) Evaluation of fish sperm quality. J Appl Ichthyol 26:636–643CrossRefGoogle Scholar
  27. Figueroa E, Risopatrón J, Sánchez R, Isachenko E, Merino O, Isachenko V, Valdebenito I (2013) Spermatozoa vitrification of sex-reversed rainbow trout (Oncorhynchus mykiss): effect of seminal plasma on physiological parameters. Aquaculture 372–375:119–126. Scholar
  28. Flajšhans M, Cosson J, Rodina M, Linhart O (2004) The application of image cytometry to viability assessment in dual fluorescence-stained fish spermatozoa. Cell Biol Int 28:955–959. Scholar
  29. Gallego V, Asturiano JF (2018a) Sperm motility in fish: technical applications and perspectives through CASA-Mot systems. Reprod Fertil Dev 30:820–832. Scholar
  30. Gallego V, Asturiano JF (2018b) Fish sperm motility assessment as a tool for aquaculture research: a historical approach. Rev Aquacult 11(3):697–724. Scholar
  31. Glenn DW III, Lang RP, Tiersch TR (2011) Evaluation of extenders for refrigerated storage of koi carp and goldfish sperm. In: Tiersch TR, Green CC (eds) Cryopreservation in aquatic species, 2nd edn. World Aquaculture Society, Baton Rouge, pp 107–124Google Scholar
  32. Gwo J-C (1994) Cryopreservation of yellowfin seabream (Acanthopagrus latus) spermatozoa (Teleost, Perciformes, Sparidae). Theriogenology 41:989–1004. Scholar
  33. Harvey B, Kelley RN, Ashwood-Smith MJ (1982) Cryopreservation of zebra fish spermatozoa using methanol. Can J Zool 60:1867–1870CrossRefGoogle Scholar
  34. Hirsh AG, Williams RJ, Meryman HT (1985) A novel method of natural cryoprotection. Plant Physiol 79:41–56CrossRefGoogle Scholar
  35. Horokhovatskyi Y, Rodina M, Asyabar HD, Boryshpolets S (2017) Consequences of uncontrolled cooling during sterlet (Acipenser ruthenus) sperm cryopreservation on post-thaw motility and fertilizing ability. Theriogenology 95:89–95. Scholar
  36. Horváth Á, Urbányi B (2000) The effect of cryoprotectants on the motility and fertilizing capacity of cryopreserved African catfish Clarias gariepinus (Burchell 1822) sperm. Aquacult Res 31:317–324. Scholar
  37. Horváth Á, Miskolczi E, Urbányi B (2003) Cryopreservation of common carp sperm. Aquat Living Resour 16:457–460. Scholar
  38. Horváth Á, Miskolczi E, Mihálffy S, Ősz K, Szabó K, Urbányi B (2007) Cryopreservation of common carp (Cyprinus carpio) sperm in 1.2 and 5 ml straws and occurrence of haploids among larvae produced with cryopreserved sperm. Cryobiology 54:251–257. Scholar
  39. Horváth Á, Urbányi B, Wang C, Onders RJ, Mims SD (2010) Cryopreservation of paddlefish sperm in 5-mL straws. J Appl Icthyology 26:715–719. Scholar
  40. Hu E, Liao TW, Tiersch TR (2013) A quality assurance initiative for commercial-scale production in high-throughput cryopreservation of blue catfish sperm. Cryobiology 67:214–224. Scholar
  41. Jähnichen H, Warnecke D, Trölsch E, Kohlman K, Bergler H, Pluta HJ (1999) Motility and fertilizing capability of cryopreserved Acipenser ruthenus L. sperm. J Appl Ichthyol 15:204–206. Scholar
  42. Judycka S, Cejko BI, Dryl K, Dobosz S, Grudniewska J, Kowalski RK (2016) The effect of supplementation of a trehalose-based extender with KCl on rainbow trout (Oncorhynchus mykiss) sperm freezability and post-thaw motility. Aquaculture 465:303–310. Scholar
  43. Judycka S, Nynca J, Liszewska E, Dobosz S, Grudniewska J, Ciereszko A (2018) Optimal sperm concentration in straws and final glucose concentration in extender are crucial for improving the cryopreservation protocol of salmonid spermatozoa. Aquaculture 486:90–97. Scholar
  44. Kása E, Bernáth G, Kollár T, Żarski D, Lujić J, Marinović Z, Bokor Z, Hegyi Á, Vílchez MC, Morini M, Peñaranda DS, Pérez L, Asturiano JF, Horváth Á (2017) Development of sperm vitrification protocols for freshwater fish (Eurasian perch, Perca fluviatilis) and marine fish (European eel, Anguilla anguilla). Gen Comp Endocrinol 245:102–107. Scholar
  45. Kurokura H, Hirano R, Tomita M, Iwahashi M (1984) Cryopreservation of carp sperm. Aquaculture 37:267–273. Scholar
  46. Lahnsteiner F (2011) Cryopreservation protocols for sperm of salmonid fishes. In: Tiersch TR, Green CC (eds) Cryopreservation in aquatic species, 2nd edn. World Aquaculture Society, Baton Rouge, pp 409–420Google Scholar
  47. Lahnsteiner F, Patzner RA, Weismann T (1993) Energy resources of spermatozoa of the rainbow trout Oncorhynchus mykiss (Pisces, Teleostei). Reprod Nutr Dev 33:349–360CrossRefGoogle Scholar
  48. Lahnsteiner F, Berger B, Weismann T, Patzner R (1996) The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation. J Appl Ichthyol 12:99–106. Scholar
  49. Lahnsteiner F, Weismann T, Patzner RA (1997) Methanol as cryoprotectant and the suitability of 1.2 ml and 5 ml straws for cryopreservation of semen from salmonid fishes. Aquacult Res 28:471–479. Scholar
  50. Legendre M, Billard R (1980) Cryopreservation of rainbow trout sperm by deep-freezing. Reprod Nutr Dev 20:1859–1868. Scholar
  51. Legendre M, Linhart O, Billard R (1996) Spawning and management of gametes, fertilized eggs and embryos in Siluroidei. Aquat Living Resour 9:59–80. Scholar
  52. Leibo SP, McGrath JJ, Cravalho EG (1978) Microscopic observation of intracellular ice formation in unfertilized mouse ova as a function of cooling rate. Cryobiology 15:257–271. Scholar
  53. Matthews JL, Murphy JM, Carmichael C, Yang H, Tiersch TR, Westerfield M, Varga ZM (2018) Changes to extender, cryoprotective medium, and in vitro fertilization improve zebrafish sperm cryopreservation. Zebrafish 15:279–290. Scholar
  54. Mazur P (1970) Cryobiology: the freezing of biological systems. Science (80- ) 168:939–949CrossRefGoogle Scholar
  55. Mazur P (1984) Freezing of living cells: mechanisms and implications. Am J Physiol 247:C125–C142CrossRefGoogle Scholar
  56. Mazur P (1990) Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos. Cell Biophys 17:53–92. Scholar
  57. Mazur P, Seki S (2011) Survival of mouse oocytes after being cooled in a vitrification solution to -196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: a new paradigm for cryopreservation by vitrification. Cryobiology 62:1–7. Scholar
  58. Mazur P, Leibo SP, Chu EHY (1972) A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells. Exp Cell Res 71:345–355. Scholar
  59. McNiven MA, Gallant RK, Richardson GF (1993) Dimethyl-acetamide as a cryoprotectant for rainbow trout spermatozoa. Theriogenology 40:943–948CrossRefGoogle Scholar
  60. Merino O, Sánchez R, Risopatrón J, Isachenko E, Katkov II, Figueroa E, Valdebenito I, Mallmann P, Isachenko V (2012) Cryoprotectant-free vitrification of fish (Oncorhynchus mykiss) spermatozoa: first report. Andrologia 44(Suppl 1):390–395. Scholar
  61. Morris JP, Berghmans S, Zahrieh D, Neuberg DS, Kanki JP, Look AT (2003) Zebrafish sperm cryopreservation with N,N-dimethylacetamide. Biotechniques 35:956–968CrossRefGoogle Scholar
  62. Mounib MS (1978) Cryogenic preservation of fish and mammalian spermatozoa. J Reprod Fertil 53:13–18. Scholar
  63. Mounib MS, Hwang PC, Idler DR (1968) Cryogenic preservation of Atlantic cod (Gadus morhua) sperm. J Fish Res Board Can 25:2623–2632. Scholar
  64. Nynca J, Judycka S, Liszewska E, Dobosz S, Ciereszko A (2017) Standardization of spermatozoa concentration for cryopreservation of rainbow trout semen using a glucose-methanol extender. Aquaculture 477:23–27. Scholar
  65. Ogier De Baulny B, Le Vern Y, Kerboeuf D, Maisse G (1997) Flow cytometric evaluation of mitochondrial activity and membrane integrity in fresh and cryopreserved rainbow trout (Oncorhynchus mykiss) spermatozoa. Cryobiology 34:141–149CrossRefGoogle Scholar
  66. Peñaranda DS, Pérez L, Gallego V, Jover M, Asturiano JF (2009) Improvement of European eel sperm cryopreservation method by preventing spermatozoa movement activation caused by cryoprotectants. Cryobiology 59:119–126CrossRefGoogle Scholar
  67. Perchec G, Jeulin C, Cosson J, André F, Billard R (1995) Relationship between sperm ATP content and motility of carp spermatozoa. J Cell Sci 108:747–753PubMedGoogle Scholar
  68. Polge C, Smith AU, Parkes AS (1949) Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666CrossRefGoogle Scholar
  69. Riesco MF, Raposo C, Engrola S, Martínez-Páramo S, Mira S, Cunha ME, Cabrita E (2017) Improvement of the cryopreservation protocols for the dusky grouper, Epinephelus marginatus. Aquaculture 470:207–213. Scholar
  70. Sarvi K, Niksirat H, Amiri BM, Mirtorabi SM, Rafiee G, Bakhtiyari M (2006) Cryopreservation of semen from the endangered Caspian brown trout (Salmo trutta caspius). Aquaculture 256:564–569CrossRefGoogle Scholar
  71. Schuster TG, Keller LM, Dunn RL, Ohl DA, Smith GD (2003) Ultra-rapid freezing of very low numbers of sperm using cryoloops. Hum Reprod 18:788–795. Scholar
  72. Segovia M, Jenkins JA, Paniagua-Chavez C, Tiersch TR (2000) Flow cytometric evaluation of antibiotic effects on viability and mitochondrial function of refrigerated spermatozoa of nile tilapia. Theriogenology 53:1489–1499. Scholar
  73. Seki S, Jin B, Mazur P (2014) Extreme rapid warming yields high functional survivals of vitrified 8-cell mouse embryos even when suspended in a half-strength vitrification solution and cooled at moderate rates to -196°C. Cryobiology 68:71–78. Scholar
  74. Stewart DL (1951) Storage of bull spermatozoa at low temperatures. Vet Rec 63:65–66Google Scholar
  75. Stoss J, Holtz W (1983) Cryopreservation of rainbow trout (Salmo gairdneri) sperm: IV. The effect of DMSO concentration and equilibration time on sperm survival, sucrose and KCl as extender components and the osmolality of the thawing solution. Aquaculture 32:321–330. Scholar
  76. Suquet M, Dreanno C, Fauvel C, Cosson J, Billard R (2000) Cryopreservation of sperm in marine fish. Aquacult Res 31:231–243. Scholar
  77. Tiersch TR, Yang H, Hu E (2011) Outlook for development of high-throughput cryopreservation for small-bodied biomedical model fishes. Comp Biochem Physiol C Toxicol Pharmacol 154:76–81. Scholar
  78. Viveiros ATM, Godinho HP (2009) Sperm quality and cryopreservation of Brazilian freshwater fish species: a review. Fish Physiol Biochem 35:137–150. Scholar
  79. Viveiros ATM, Nascimento AF, Orfão LH, Isaú ZA (2010) Motility and fertility of the subtropical freshwater fish streaked prochilod (Prochilodus lineatus) sperm cryopreserved in powdered coconut water. Theriogenology 74:551–556. Scholar
  80. Viveiros ATM, Orfão LH, Nascimento AF, Corrêa FM, Caneppele D (2012) Effects of extenders, cryoprotectants and freezing methods on sperm quality of the threatened Brazilian freshwater fish pirapitinga-do-sul Brycon opalinus (Characiformes). Theriogenology 78:361–368. Scholar
  81. Viveiros ATM, Nascimento AF, Leal MC, Gonçalves ACS, Orfão LH, Cosson J (2014) Methyl glycol, methanol and DMSO effects on post-thaw motility, velocities, membrane integrity and mitochondrial function of Brycon orbignyanus and Prochilodus lineatus (Characiformes) sperm. Fish Physiol Biochem 41:193–201. Scholar
  82. Warnecke D, Pluta HJ (2003) Motility and fertilizing capacity of frozen/thawed common carp (Cyprinus carpio L.) sperm using dimethyl-acetamide as the main cryoprotectant. Aquaculture 215:167–185. Scholar
  83. WHO (2010) WHO laboratory manual for the examination and processing of human semen, 5th edn. World Health Organization, GenevaGoogle Scholar
  84. Wilson-Leedy JG, Ingermann RL (2007) Development of a novel CASA system based on open source software for characterization of zebrafish sperm motility parameters. Theriogenology 67:661–672CrossRefGoogle Scholar
  85. Young WP, Frenyea K, Wheeler PA, Thorgaard GH (2009) No increase in developmental deformities or fluctuating asymmetry in rainbow trout (Oncorhynchus mykiss) produced with cryopreserved sperm. Aquaculture 289:13–18CrossRefGoogle Scholar
  86. Zilli L, Schiavone R, Zonno V, Storelli C, Vilella S (2003) Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation. Cryobiology 47:227–235CrossRefGoogle Scholar
  87. Zilli L, Schiavone R, Zonno V, Storelli C, Vilella S (2004) Adenosine triphosphate concentration and β-d-glucuronidase activity as indicators of sea bass semen quality. Biol Reprod 70:1679–1684. Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2020

Authors and Affiliations

  1. 1.Department of AquacultureSzent István UniversityGödöllőHungary

Personalised recommendations