Ribogospod. nauka Ukr., 2020; 3(53): 69-79
DOI: https://doi.org/10.15407/fsu2020.03.069
УДК [597-1.044:621.59]:597.554.3 

Membrane permeability of Prussian carp (Carassius auratus Linnaeus, 1758) spermatozoa for water and cryoprotectants molecules

A. Puhovkin, This email address is being protected from spambots. You need JavaScript enabled to view it. , Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine, Kharkiv
K. Mikson, This email address is being protected from spambots. You need JavaScript enabled to view it. , Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine, Kharkiv

Purpose. Determination of permeability parameters of Prussian carp (Carassius auratus Linnaeus, 1758) spermatozoa membranes for water and cryoprotectants molecules as an important stage in the development of a protocol for their cryopreservation by vitrification.

Methodology. Osmotic response of Prussian carp spermatozoa was studied using photoelectric colorimeter KF-77 (Poland) equipped with a magnetic mixer and thermostated cuvette compartment according to our technique. To determine the permeability of plasma membranes of fish spermatozoa to cryoprotectant molecules, they were incubated in the solutions of ethyleneglycol (EG), 1,2-propanediol (1,2-PD), methanol (Met) of different concentrations, or a mixture of these cryoprotectants prepared with  isotonic 0.12 M NaCl aqueous solution. Permeability coefficients of spermatozoa plasma membranes for either water (Lp) or cryoprotectant (Кp) molecules were determined by fitting the experimental dependences of relative cell volumes on time and solving theoretical model equations. The activation energy (Еа) of substance transfer through cell membranes was calculated from lnLp(1/T) or lnKp(1/T), the slope of which was equal Еа/R according to the Arrhenius equation, where R was the universal gas constant.

Findings.  It was found that the permeability of Prussian carp spermatozoa membranes to water molecules at 20°С was 3,53±0,18  10-14  m3/Ns, and a decrease in membrane permeability of Prussian carp spermatozoa within the range of 30–18°C was characterized by the activation energy of 48±4 kJ/mol. A decrease in membrane permeability of Prussian carp spermatozoa for cryopotectants within the range of 30–18°C was characterized by the activation energy of 82±5 kJ/mol for ethyleneglycole, 99±7 for 1,2-propanediol and 84±6 for the mixture. This fact indicates that the molecules of the studied substances penetrate into the spermatozoon via passive diffusion through the lipid bilayer. The data obtained can be used to determine the optimal regime of spermatozoa cryopreservation for cyprinids.

Originality. For the first time, the coefficients of the membranes permeability of Prussian carp spermatozoa to water molecules and cryoprotectants (ethyleneglycol, methanol, 1,2-propanediol) and the activation energy of the these molecules transfer through the membranes were determined.

Practical value. The results of the study are used in the development of media and regimes of cryopreservation of freshwater fish spermatozoa.

Key words: spermatozoa, Prussian carp (Carassius auratus Linnaeus, 1758), membrane permeability, cryoprotectants, activation energy.


  1. Cabrita, E., Sarasquete, C., Martínez-Páramo, S., Robles, V., Beirão, J., Pérez-Cerezales, S., & Herráez, M. P. (2010). Cryopreservation of fish sperm: applications and perspectives. J. Appl. Ichthyol., 26, 623-635. https://doi.org/10.1111/j.1439-0426.2010.01556.x 
  2. Kopeika, E., Kopeika, J., & Zhang, T. (2007). Cryopreservation of fish sperm. Cryopreservation and freeze-drying protocols. Totowa, New Jersey: Humana Press, 203-217. https://doi.org/10.1007/978-1-59745-362-2_14 
  3. Bobe, J., & Labbé, C. (2010). Egg and sperm quality in fish. General and Comparative Endocrinology, 165, 535-548. https://doi.org/10.1016/j.ygcen.2009.02.011 
  4. Xin, M., Siddique, M. A. M., Dzyuba, B., Cuevas-Uribe, R., Shaliutina-Kolešová, A., & Linhart, O. (2017). Progress and challenges of fish sperm vitrification: A mini review. Theriogenology, 98, 16-22. https://doi.org/10.1016/j.theriogenology.2017.04.043 
  5. Kása, E., Lujić, J., Marinović, Z., Kollár, T., Bernáth, G., Bokor, Z., Urbányi, B., Lefler, K.K., Jesenšek, D., & Horváth, Á. (2018). Development of sperm vitrification protocols for two endangered salmonid species: the Adriatic grayling, Thymallus thymallus, and the marble trout, Salmo marmoratus. Fish Physiology and Biochemistry, 44, 1499-1507. https://doi.org/10.1007/s10695-018-0516-y 
  6. Kása, E., Bernáth, G., Kollár, T., Zarski, D., Lujić, J., Marinović, Z., & Horváth, A. (2017). Development of sperm vitrification protocols for freshwater fish (Eurasian perch, Perca fluviatilis) and marine fish (European eel, Anguilla anguilla). General and Comparative Endocrinology, 245, 102-107. https://doi.org/10.1016/j.ygcen.2016.05.010 
  7. Cuevas-Uribe, R., Hu., E., Daniels, H., Gill, A.O., & Tiersch, T.R. (2017). Vitrification as an Alternative Approach for Sperm Cryopreservation in Marine Fishes. North American journal of aquaculture, 79(2), 187-196. https://doi.org/10.1080/15222055.2017.1281855 
  8. Kutluyer, F., Öğretmen, F., & İnanan, B.E. (2016). Cryopreservation of Goldfish (Carassius Auratus) Spermatozoa: Effects of Extender Supplemented with Taurine on Sperm Motility and DNA Damage. CryoLetters, 37(1), 41-46.
  9. Kutluyer, F., Öğretmen, F., & İnanan, B.E. (2015). Effects of Semen Extender Supplemented with Lmethionine and Packaging Methods (Straws and Pellets) on Post-Thaw Goldfish (Carassius Auratus) Sperm Quality and DNA Damage. CryoLetters, 36(5), 336-343.
  10. Taghizadeh, V., Imanpoor, M.R., & Sadeghi, A. (2013). Effect of Extenders and Different Concentrations of Methanol on Motility Parameters of Goldfish (Carassius auratus gibelio) Spermatozoa after Short-Term Storage. World Journal of Fish and Marine Sciences, 5(5), 492-496.
  11. Chantzaropoulos, A., Nathanailides, C., Kokokiris, L., Barbouti, A., & Zhang, T. (2015). A brief exposure to low pH prior to refrigerated storage reduces the motility and viability of goldsh sperm (Carassius auratus, Linnaeus, 1758). Journal of Applied Ichthyology, 31(S1), 89-93. https://doi.org/10.1111/jai.12735 
  12. Cabrita, E., Alvarez, R., Anel, E., & Herraez, M. P. (1999). The hypoosmotic swelling test performed with coulter counter: a method to assay functional integrity of sperm membrane in rainbow trout. Animal Reproduction Science, 55, 279-287. https://doi.org/10.1016/S0378-4320(99)00014-7 
  13. Petrunkina, A. M. (2007). Fundamental aspects of gamete cryobiology. Journal of Reproductive Medicine and Endocrinology, 4, 78-91.
  14. Pinisetty, D., Huang, C., & Dong, Q., et al. (2005). Subzero water permeability parameters and optimal freezing rates for sperm cells of the southern platyfish, Xiphophorus maculates. Cryobiology, 50, 250-263. https://doi.org/10.1016/j.cryobiol.2005.02.003 
  15. Hagedorn, M., Ricker, J., McCarthy, M., et al. (2009). Biophysics of zebrafish (Danio rerio) sperm. Cryobiology, 58, 12-19. https://doi.org/10.1016/j.cryobiol.2008.09.013 
  16. Puhovkin, A. Y., Kopeika, E. F., Nardid, O. A., & Cherkashina, Y. O. (2014). Investigation of membrane permeability of carp spermatozoa for water molecules. Biofizika, 59(3), 481-487. https://doi.org/10.1134/S000635091403018X 
  17. Puhovkin, A. Y., Kononenko, I. S., Cherepnin, V. O., Hrytsyniak, I. I., & Kopeika, E. F. (2016). Permeability of sterlet sperm membranes (Acipenser ruthenus L., 1758) for water molecules. Fisheries Science of Ukraine, 1, 70-77. https://doi.org/10.15407/fsu2016.01.070 
  18. Puhovkin, A. Y., Kopeika, E. F., Mikson, K. B., Cherepnin, V. O., & Hrytsyniak, I. I. (2016). A study of the osmotic sensitivity of pike (Esox lucius, L., 1758) spermatozoa for the optimization of their cryopreservation. Fisheries Science of Ukraine, 4, 103-112. https://doi.org/10.15407/fsu2016.04.103 
  19. Puhovkin, A. Y., & Kopeika, E. F. (2015). Investigation of water and cryoprotectants molecules transfer through common carp (Cyprinus carpio, L.) spermatozoa membranes (CRYO conference of the Society for Cryobiology, Ostrava, Czech Republic, 2015). Cryobiology, 71(3), 567. https://doi.org/10.1016/j.cryobiol.2015.10.129 
  20. Fauvel, C., Suquet, M., & Cosson, J. (2010). Evaluation of fish sperm quality. Appl. Ichthyol, 26, 636-643. https://doi.org/10.1111/j.1439-0426.2010.01529.x  
  21. Puhovkin, A. Y., Kopeika, E. F., Hordiienko, Y. O., & Nardid, O. A. (2014). Sposib vyznachennia pronyknosti membran spermatozoidiv koropa do molekul vody. Patent of Ukraine. №104809.
  22. Puhovkin, A. Y., & Kopeika, E. F. (2016). Plasma Membrane Permeability of Carp (Cyprinus carpio, L., 1758) Spermatozoa for Water and Cryoprotectants Molecules at Different Stages of Cryopreservation. Problems of Cryobiology and Cryomedicine, 26(4), 340-348. https://doi.org/10.15407/cryo26.04.340 
  23. Kononenko, I. S., Puhovkin, A. Y., Kononenko, R. V. Cherepnin, V. O., Butskyi, K. I.,  & Kopeika, E. F. (2017). Optimization of the conditions of starlet (Acipenser ruthenus, L., 1758) sperm cryopreservation for egg fertilization in fish farm conditions. Fisheries Science of Ukraine, 3(41), 83-97. https://doi.org/10.15407/fsu2017.03.083 
  24. Fürböck, S., Lahnsteiner, F., & Patzner, R. A. (2009). A fine structural review on the spermatozoa of Cyprinidae with attention to their phylogenetic implications. Histol. Histopathol., 24(10), 1233-1244.