Acta Univ. Agric. Silvic. Mendelianae Brun. 2023, 71(1), 35-42 | DOI: 10.11118/actaun.2023.003

Effect of Different Equilibration Times and Freezing Rates on Bull Spermatozoa Cryotolerance

Jan Pytlík1, Jaromír Ducháček1, Radim Codl1, Filipp Georgijevič Savvulidi1, Marek Vrhel1, Luděk Stádník1
1 Department of Animal Science, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, 165 00 Prague 6-Suchdol, Czech Republic

Sperm cryopreservation is a complex process consisting of several steps, the details of which are not yet universally agreed upon. The objective of this study was to evaluate the effects of different equilibration times and freezing rates on post-thaw resilience and functional integrity. Collected semen from five Holstein bulls (30 samples in total) were split into 4 aliquots to produce frozen straws after 2 different equilibration times (2 h and 4 h) and freezing rates (2-phase and 3-phase). After thawing, the sperm characteristics were measured by flow cytometry. A linear model procedure was used to determine the effects of bull, equilibration time, freezing rate and interaction between them, on sperm variables. The evaluated sperm characteristics included spermatozoa with intact plasma and acrosomal membrane (PMAI), PMAI with high mitochondrial membrane potential (HMMP), spermatozoa with plasma membrane damage (PMD), and spermatozoa with acrosome damage (AD) were analysed immediately, 1 h and 2 h after thawing. In conclusion, the extension of the equilibration time from to 4 h in combination with 2-phase freezing rate led to a significant improvement in all measured parameters of frozen-thawed spermatozoa (P < 0.01).

Keywords: equilibration, freezing rate, cryopreservation, thawing, bull, spermatozoa

Received: December 8, 2022; Revised: December 8, 2022; Accepted: February 15, 2023; Published: March 1, 2023  Show citation

ACS AIP APA ASA Harvard Chicago IEEE ISO690 MLA NLM Turabian Vancouver
Pytlík, J., Ducháček, J., Codl, R., Georgijevič Savvulidi, F., Vrhel, M., & Stádník, L. (2023). Effect of Different Equilibration Times and Freezing Rates on Bull Spermatozoa Cryotolerance. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, 71(1), 35-42. doi: 10.11118/actaun.2023.003
Share...
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)
    • Open full article

    References

    1. AHMAD, M., NASRULLAH, R. and AHMAD, N. 2015. Effect of cooling rate and equilibration time on pre-freeze and posthaw survival of buck sperm. Cryobiology, 70(3): 233-238. DOI: https://doi.org/10.1016/j.cryobiol.2015.03.002 Go to original source...
    2. ANSARI, M. S., RAKHA, B. A., MALIK, M. F., ANDRABI, S. M. H., ULLAH, N., IQBAL, R., HOLT, W. V. and AKHTER, S. 2014. Effect of cysteine addition to the freezing extender on the progressive motility, viability, plasma membrane and DNA integrity of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa. Journal of Applied Animal Research, 44(1): 36-41. DOI: https://doi.org/10.1080/09712119.2014.987292 Go to original source...
    3. ANZAR, M., KROETSCH, T. and BOSWALL, L. 2011. Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes. Animal Reproduction Science, 126(1-2): 23-31. DOI: https://doi.org/10.1016/j.anireprosci.2011.04.018 Go to original source...
    4. BARBAS, J. P. and MASCARENHAS, R. D. 2009. Cryopreservation of domestic animal sperm cells. Cell Tissue Bank, 10(1): 49-62. DOI: https://doi.org/10.1007/s10561-008-9081-4 Go to original source...
    5. BELALA, R., BRIAND-AMIRAT, L., VINCIGUERRA, L., TAINTURIER, D., KAIDI, R., THORIN, C., MICHAUD, S. ANTON, M. and BENCHARIF, D. 2016. Effect of equilibration time on the motility and functional integrity of canine spermatozoa frozen in three different extenders. Research in Veterinary Science, 106: 66-73. DOI: https://doi.org/10.1016/j.rvsc.2016.03.010 Go to original source...
    6. BERAN, J., ©IMONÍK, O., STÁDNÍK, L., RAJMON, R., DUCHÁČEK, J., KREJCÁRKOVÁ TURNEROVÁ, A., DOLE®ALOVÁ, M. and ©ICHTAŘ, J. 2013. Effect of bull, diluter and LDL-cholesterol concentration on spermatozoa resistance against cold shock. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, 61(6): 1575-1581. DOI: https://doi.org/10.11118/actaun201361061575 Go to original source...
    7. BUCHER, K., MALAMA, E., SIUDA, M., JANETT, F. and BOLLWEIN, H. 2019. Multicolor flow cytometric analysis of cryopreserved bovine sperm: A tool for the evaluation of bull fertility. Journal of Dairy Science, 102(12): 11652-11669. DOI: https://doi.org/10.3168/jds.2019-16572 Go to original source...
    8. CLULOW, J. R., MANSFIELD, L. J., MORRIS, L. H. A., EVANS, G. and MAXWELL, W. M. C. 2008. A comparison between freezing methods for the cryopreservation of stallion spermatozoa. Animal Reproduction Science, 108(3-4): 298-308. DOI: https://doi.org/10.1016/j.anireprosci.2007.08.014 Go to original source...
    9. DALAL, J., KUMAR, A., DUTT, R., SINGH, G. and CHANDOLIA, R. K. 2018. Different Cooling Rates for Cryopreservation of Semen in Various Livestock Species: A Review. International Journal of Current Microbiology and Applied Sciences, 7(8): 1903-1911. DOI: https://doi.org/10.20546/ijcmas.2018.708.219 Go to original source...
    10. DALAL, J., KUMAR, A., KUMAR, P., HONPARKHE, M., MALIK, V. S., SINGHAL, S., KAUR, S. and BRAR, P. S. 2018. Improvement in cryosurvival of buffalo bull (Bubalus bubalis) sperm by altering freezing rate within critical temperature range. Asian Pacific Journal of Reproduction, 7(2): 72-78. DOI: https://doi.org/10.4103/2305-0500.228016 Go to original source...
    11. DIAS, E. A. R., CAMPANHOLI, S. P., ROSSI, G. F., DELL´AQUA, C. P. F., DELL´AQUA JR., J. A., PAPA, F. O., ZORZETTO, M. F., PAZ, C. C. P., OLIVEIRA, L. Z., MERCADANTE, M. E. Z. and MONTEIRO, F. M. 2018. Evaluation of cooling and freezing systems of bovine semen. Animal Reproduction Science, 195: 102-111. DOI: https://doi.org/10.1016/j.anireprosci.2018.05.012 Go to original source...
    12. DOLE®ALOVÁ, M., STÁDNÍK, L., BINIOVÁ, Z., DUCHÁČEK, J. and STUPKA, R. 2016. Equilibration and freezing interactions affecting bull sperm characteristics after thawing. Czech Journal of Animal Science, 61(11): 515-525. DOI: http://dx.doi.org/10.17221/23/2016-CJAS Go to original source...
    13. FLEISCH, A., MALAMA, E., WITSCHI, U., LEIDING, C., SIUDA, M., JANETT, F. and BOLLWEIN, H. 2017. Effects of an extension of the equilibration period up to 96 hours on the characteristics of cryopreserved bull semen. Theriogenology, 89: 255-262. DOI: https://doi.org/10.1016/j.theriogenology.2016.10.018 Go to original source...
    14. FOOT, R. H. and KAPROTH, M. T. 2002. Large batch freezing of bull semen: effect of time of freezing and fructose on fertility. Journal of Dairy Science, 85(2): 453-456. DOI: https://doi.org/10.3168/jds.s0022-0302(02)74094-0 Go to original source...
    15. GILLIS, A. B., GUY, E. L., KOUBA, A. J., ALLEN, P. J., MARCEC-GREAVES, R. M. and VANCE, C. K. 2020. The effect of trehalose and freezing rate in cryopreservation of caudate spermatozoa. Cryobiology, 97: 259. DOI: https://doi.org/10.1016/j.cryobiol.2020.10.044 Go to original source...
    16. GÜRLER, H., MALAMA, E., HEPPELMANN, M., CALISICI, O., LEIDING, C., KASTELIC, J. O. and BOLLWEIN, H. 2016. Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm. Theriogenology, 86(2): 562-571. DOI: https://doi.org/10.1016/j.theriogenology.2016.02.007 Go to original source...
    17. LAYEK, S. S., MOHANTY, T. K., KUMARESAN, A. and PARKS, J. E. 2016. Cryopreservation of bull semen: evolution from egg yolk based to soybean based extenders. Animal Reproduction Science, 172: 1-9. DOI: https://doi.org/10.1016/j.anireprosci.2016.04.013 Go to original source...
    18. LEITE, T. G., FILHO, V. R. V., ARRUDA, R. P., ANDRADE, A. F. C., EMERICK, L. L., ZAFFALON, F. G., MARTINS, J. A. M. and ANDRADE, V. J. 2016. Effects of extender and equilibration time on post-thaw motility and membrane integrity of cryopreserved Gyr bull semen evaluated by CASA and flow cytometry. Animal Reproduction Science, 120(1-4): 31-38. DOI: https://doi.org/10.1016/j.anireprosci.2010.04.005 Go to original source...
    19. LÓPEZ-URUEṄA, E., ALVAREZ, M., GOMES-ALVES, S., MARTÍNEZ-RODRÍGUEZ, C., BORRAGAN, S., ANEL-LÓPEZ, L., DE PAZ, L. and ANEL, L. 2014. Tolerance of brown bear spermatozoa to conditions of pre-freezing cooling rate and equilibration time. Theriogenology, 81(9): 1229-1238. DOI: https://doi.org/10.1016/j.theriogenology.2014.02.004 Go to original source...
    20. KHALIL, W. A., EL-HARAIRY, M. A., ZEIDAN, A. E. B., HASSAN, M. A. E. and MOHEY-ELSAEED, O. 2018. Evaluation of bull spermatozoa during and after cryopreservation: Structural and ultrastructural insights. International Journal of Veterinary Science and Medicine, 6(sup1): S49-S56. DOI: https://doi.org/10.1016/j.ijvsm.2017.11.001 Go to original source...
    21. MALO, C., CRICHTON, E. G. and SKIDOMORE, J. A. 2017. Optimization of the cryopreservation of dromedary camel semen: Cryoprotectants and their concentration and equilibration times. Cryobiology, 74: 141-147. DOI: https://doi.org/10.1016/j.cryobiol.2016.11.001 Go to original source...
    22. MICHEL, G., CHAIGNEAU, A., GUYONNET, B. 2016. Effects of equilibration time on post thaw motility and fertility of bull semen. Animal Reproduction Science, 169, 102. https://doi.org/10.1016/j.anireprosci.2016.03.026 Go to original source...
    23. MOHAMMED, A. A., AHMED, W. M. M. 2017: Pure Egyptian Cattle Bulls Show bot Individual Variation and Different Interaction with Extender in the Post-Thawing Sperm Parameters. Andrology, 6(2): 193. DOI: 10.4172/2167-0250.1000193 Go to original source...
    24. MUIṄO, R., FERNANDEZ, M. and PEṄA, A. 2007. Post-thaw survival and longevity of bull spermatozoa frozen with an egg yolk-based or two egg yolk-free extenders after an equilibration period of 18 h. Reproduction in Domestic Animals, 42(3): 305-311. DOI: https://doi.org/10.1111/j.1439-0531.2006.00784.x Go to original source...
    25. MURPHY, E. M., EIVERS, B., O´MEARA, C. M., LONERGAN, P. and FAIR, S. 2018. Effect of increasing equilibration time of diluted bull semen up to 72 h prior to freezing on sperm quality parameters and calving rate following artificial insemination. Theriogenology, 108: 217-222. DOI: https://doi.org/10.1016/j.theriogenology.2017.11.034 Go to original source...
    26. LOOMIS, P. R. and GRAHAM, J. K. 2008. Commercial semen freezing: Individual male variation in cryosurvial and the response of stallion sperm to customized freezing protocols. Animal Reproduction Science, 105(1-2): 119-128. DOI: https://doi.org/10.1016/j.anireprosci.2007.11.010 Go to original source...
    27. PINI, T., LEAHY, T. and DE GRAAF, S. P. 2018. Sublethal sperm freezing damage: Manifestations and solutions. Theriogenology, 118: 172-181. DOI: https://doi.org/10.1016/j.theriogenology.2018.06.006 Go to original source...
    28. PYTLÍK, J., SAVVULIDI, F. G., DUCHÁČEK, J., CODL, R., VRHEL, M., NAGY, S. and STÁDNÍK, L. 2022. Effect of extender on the quality and incubation resilience of cryopreserved Holstein bull semen. Czech Journal of Animal Science, 67(3): 75-86. DOI: https://doi.org/10.17221/196/2021-CJAS Go to original source...
    29. RAJANI, K. P., BALAGANUR, K., KUMAR, D. and SINGH, R. 2020. Pre-freezing equilibration for 22 h improves post-thaw sperm fuctions in cryopreserved ram semen by reducing cholesterol efflux. Cryobiology, 96: 76-84. DOI: https://doi.org/10.1016/j.cryobiol.2020.07.013 Go to original source...
    30. RASTEGARNIA, A., SHAHVERDI, A., TOPRAGGALEH, T. R. and SHAFIEPOUR, V. 2014. In vitro comparison of soybean lecithin-based extenders for cryopreservation of buffalo (Bubalus bubalis) semen. Comparaticve Clinical Pathology, 23: 893-900. DOI: https://doi.org/10.1007/s00580-013-1708-6 Go to original source...
    31. RUBIO-GUILLÉN, J., GONZÁLEZ, D., GARDE, J. J., ESTESO, M. C., FERNÁNDEZ-SANTOS, M. R., RODRIGUEZ-GÍL, J. E., MADRID-BURY, N. and QUINTERO-MORENO, A. 2007. Effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Reproduction in Domestic Animals, 42(4): 354-357. DOI: https://doi.org/10.1111/j.1439-0531.2006.00788.x Go to original source...
    32. SELEM, E., BROEKHUIJSE, M. L. W. J., CHEVRIER, L., CAMUGLI, S., SCHMITT, E., SCHIBLER, L. and KOENEN, E. P. C. 2015. Use of combinations of in vitro quality assessments to predict fertility of bovine semen. Theriogenology, 84(9): 1447-1454. DOI: https://doi.org/10.1016/j.theriogenology.2015.07.035 Go to original source...
    33. SCHÄFER, J., WABERSKI, D., JUNG, M. and SCHULZE, M. 2017. Impact of holding and equilibration time on post-thaw quality of shipped boar semen. Animal Reproduction Science, 187: 109-115. DOI: https://doi.org/10.1016/j.anireprosci.2017.10.014 Go to original source...
    34. SHAH, S. A. H., ANDRABI, S. M. H. and QURESHI, I. Z. 2016. Effect of equilibration times, freezing and thawing rates on post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa. Andrology, 4(5): 972-976. DOI: https://doi.org/10.1111/andr.12214 Go to original source...
    35. SHAHVERDI, A., RASTEGARNIA, A. and TOPAGGALEH, T. R. 2014. Effect of extender and equilibration time on post thaw motility and chromatin structure of buffalo bull (Bubalus bubalis) spermatozoa. Cell Journal, 16(3): 279-288.
    36. SIEME, H., OLDENHOF, H. and WOLKERS, W. F. 2016. Mode of action of cryoprotectants for sperm preservation. Animal Reproduction Science, 169: 2-5. DOI: https://doi.org/10.1016/j.anireprosci.2016.02.004 Go to original source...
    37. SINGH, A. K., KUMAR., A., HOMPARKHE, M., KAUR., S., KAUR, H., GHUMAN, S. P. S. and BRAR, P. S. 2018. Comparison of in vitro and in vivo fertilizing potential of buffalo bull semen frozen in egg yolk-, soya bean lecithin-, and liposome-based extenders. Reproduction in Domestic Animals, 53(1): 195-202. DOI: https://doi.org/10.1111/rda.13092 Go to original source...
    38. TIRPAN, M. B., OLĞAÇ, K. T., GÜRLER, H. and KAYA, U. 2017. Effects of Different Equilibration Conditions on cryopreserved Bovine Sperm Quality. Kocatepe Veterinary Journal, 10(2): 57-62.
    39. UGUR, M. G., ABDELRAHMAN S. A., EVANS, H. C., GILMORE, A. A., HITIT, M., ARIFIANTINI, R. I., PURWANTARA, B., KAYA, A. and MEMILI, E. 2019. Advances in cryopreservation of bull sperm. Frontiers in Veterinary, 27(6): 268. DOI: https://doi.org/10.3389/fvets.2019.00268 Go to original source...
    40. VARELA, E., ROJAS, M. and RESTREPO, G. 2020. Membrane stability and mitochondrial activity of bovine sperm frozen with low-density lipoproteins and trehalose. Reproduction in Domestic Animals, 55(2): 146-153. DOI: https://doi.org/10.1111/rda.13599 Go to original source...
    41. YANG, D. H., STANDLEY, N. T. and XU, Z. Z. 2018. Application of liquid semen technology under the seasonal dairy production system in New Zealand. Animal Reproduction Science, 194: 2-10. DOI: https://doi.org/10.1016/j.anireprosci.2018.01.004 Go to original source...
    42. YOSHIDA, M. 2000. Conservation of sperms: current status and new trends. Animal Reproduction Science, 60-61: 349-355. DOI: https://doi.org/10.1016/S0378-4320(00)00125-1 Go to original source...

    This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY NC ND 4.0), which permits non-comercial use, distribution, and reproduction in any medium, provided the original publication is properly cited. No use, distribution or reproduction is permitted which does not comply with these terms.


    Return to the content