Skip to main content Accessibility help

Quality of Pinzgau bull spermatozoa following different periods of cryostorage

  • P. Chrenek (a1) (a2), E. Spaleková (a3), L. Olexikova (a3), A. Makarevich (a3) and E. Kubovicova (a3)...


The aim of this work was to examine the influence of cryostorage duration of Pinzgau bull's insemination doses (IDs) on some sperm traits. The IDs were frozen by a slow freezing method and stored in liquid nitrogen for different periods: less than 8 years (group 1), 8–13 years (group 2) and 14–18 years (group 3). Motility (CASA), pathological sperm rate (Giemsa staining), apoptotic (Yo-Pro-1-positive) and necrotic (propidium iodide-positive) cell occurrence and fertilizing ability (penetration/fertilization test) of spermatozoa were evaluated post-thaw. The average post-thaw sperm motility in all examined groups was over 40%. No significant influence of storage length either on the sperm total motility or progressive movement was revealed. In each tested group the average rate of malformed spermatozoa did not exceed 20%. No effect of cryostorage length on the occurrence of apoptotic or necrotic sperm was noted. Similarly, penetrating/fertilizing ability of sperm did not differ among the groups, excepting differences in the rate of pronuclei (PN) formation. In group 1, 72.9% of eggs showed two visible PN following 20 h incubation with sperm, whilst in groups 2 and 3 only 67 and 54.5% of zygotes, respectively, had both PN at this time. These results revealed no influence of storage time on the bull spermatozoa in all parameters excepting the rate of PN formation. As high inter-male variability was observed in the susceptibility of bull sperm to cryostorage, individual differences should be taken into account when semen from individual bulls is to be stored for a long time.


Corresponding author

All correspondence to: Peter Chrenek. National Agricultural and Food Centre, Research Institute for Animal Production Nitra, Slovak Republic. E-mail:


Hide All
Bailey, J.L., Bilodeau, J.F. & Cormier, N. (2000). Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J. Androl. 21, 17.
Barbas, J.P. & Mascarenhas, R.D. (2009). Cryopreservation of domestic animal sperm cells. Cell Tissue Bank 10, 4962.
Brito, L.F., Silva, A.E., Rodrigues, L.H., Vieira, F.V., Deragon, L.A. & Kastelic, J.P. (2002). Effects of environmental factors, age and genotype on sperm production and semen quality in Bos indicus and Bos taurus AI bulls in Brazil. Anim. Reprod. Sci. 70 (3–4), 181–90.
Cavalcanti, M., Steilmann, C., Failing, K., Bergmanm, K.S., Weidner, W. & Steger, K. (2011). Apoptotic gene expression in potentially fertile and subfertile men. Mol. Hum. Reprod. 17, 415–20.
Clarke, G.N., Liu, D.Y. & Baker, H.W. (2006). Recovery of human sperm motility and ability to interact with the human zona pellucida after more than 28 years of storage in liquid nitrogen. Fertil. Steril. 86 (3), 721–2.
Cormier, N. & Bailey, J.L. (2003). A differential mechanism is involved during heparin and cryopreservation induced capacitation of bovine sperm. Biol. Reprod. 69, 177–85.
Donnelly, E.T., Steele, E.K., McClure, N. & Lewis, S.E. (2001). Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum. Reprod. 16, 1191–9.
Edelstein, A., Yavetz, H., Kleiman, S.E., Hauser, R., Amnon, B., Paz, G. & Yogev, L. (2008). Effect of long-term storage on deoxyribonucleic acid damage and motility of sperm bank donor specimens. Fertil. Steril. 90, 1327–30.
Feldschuh, J., Brassel, J., Durso, N. & Levine, A. (2005). Successful sperm storage for 28 years. Fertil. Steril. 84, 1017–e3.
Fraser, L., Strzeżek, J. & Kordan, W. (2014). Post-thaw sperm characteristics following long-term storage of boar semen in liquid nitrogen. Anim. Reprod. Sci. 147, 119–27.
Freneau, G.E., Chenoweth, P.J., Ellis, R. & Rupp, G. (2010). Sperm morphology of beef bulls evaluated by two different methods. Anim. Reprod. Sci. 118 (2–4), 176–81.
García-Herreros, M., Barón, F.J., Aparicio, I.M., Santos, A.J., García-Marín, L.J. & Gil, M.C. (2008). Morphometric changes in boar spermatozoa induced by cryopreservation. Int. J. Androl. 31, 490–8.
Hamamah, S., Royère, D., Nicolle, J.C., Paquignon, M. & Lansac, J. (1990). Effects of freezing−thawing on the spermatozoon nucleus: a comparative chromatin cytophotometric study in the porcine and human species. Reprod. Nutr. Develop. 30, 5964.
Hammadeh, M.E., Askari, A.S., Georg, T., Rosenbaum, P. & Schmidt, W. (1999). Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Int. J. Androl. 22, 155–62.
Haugan, T., Gröhn, Y.T., Kommisurd, E., Ropstad, E. & Reksen, O. (2007). Effects of sperm concentration at semen collection and storage period of frozen semen on dairy cow conception. Anim. Reprod. Sci. 97, 111.
Hidalgo, M., Rodríguez, I. & Dorado, J. (2006). Influence of staining and sampling procedures on goat sperm morphometry using the Sperm Class Analyzer. Theriogenology. 66, 9961003.
Kadirvel, G., Periasamy, S. & Kumar, S. (2012). Effect of cryopreservation on apoptotic-like events and its relationship with cryocapacitation of buffalo (Bubalus bubalis) sperm. Reprod. Dom. Anim. 47, 143–50.
Kadlecik, O., Kasarda, R. & Hetényi, L. (2004). Genetic gain, increase in inbreeding rate and generation interval in alternatives of Pinzgau breeding program. Czech J. Anim. Sci. 49 (12), 524–31.
Kouba, A.J., Lloyd, R.E., Houck, M.L., Silla, A.J., Calatayud, N., Trudeau, V.L., Clulow, J., Molonia, F., Langhorne, C., Vance, C., Arregui, L., Germano, J., Lermen, D. & Della Togna, G. (2013). Emerging trends for biobanking amphibian genetic resources: The hope, reality and challenges for the next decade. Biol. Conserv. 164, 1021.
Kreysing, U., Nagai, T. & Niemann, H. (1997). Male-dependent variability of fertilization and embryo development in two bovine in vitro fertilization systems and the effects of casein phosphopeptides (CPPs). Reprod. Fertil. Dev. 9, 465–74.
Leibo, S.P., Semple, M.E. & Kroetsch, T.G. (1994). In vitro fertilization of oocytes by 37-year-old cryopreserved bovine spermatozoa. Theriogenology 42, 1257–62.
Lessard, C., Parent, S., Leclerc, P., Bailey, J.L. & Sullivan, R. (2000). Cryopreservation alters the levels of the bull sperm surface protein P25b. J. Androl. 21, 700–7.
Loomis, P.R. & Graham, J.K. (2008). Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim. Reprod. Sci. 105 (1–2), 119–28.
Medeiros, C.M., Forell, F., Oliveira, A.T. & Rodrigues, J.L. (2002). Current status of sperm cryopreservation: why isn't better. Theriogenology 57, 327–44.
O'Connell, M., McClure, N. & Lewis, S.E. (2002). The effects of cryopreservation on sperm morphology, motility, and mitochondrial function. Hum. Reprod. 17, 704–9.
Ozkavukcu, S., Erdemli, E., Isik, A., Oztuna, D. & Karahuseyinoglu, S. (2008). Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. J. Assist. Reprod. Genet. 25, 403–11.
Royere, D., Hamamah, S., Nicolle, J.C. & Lansac, J. (1991). Chromatin alterations induced by freeze-thawing influence the fertilizing ability of human sperm. Int. J. Androl. 14, 328–32.
Sailer, B.L., Jost, L.K. & Evenson, D.P. (1996). Bull sperm head morphometry related to abnormal chromatin structure and fertility. Cytometry 24, 167–73.
Saint Jalme, M., Lecoq, R., Seigneurin, F., Blesbois, E. & Plouzeau, E. (2003). Cryopreservation of semen from endangered pheasants: the first step towards a cryobank for endangered avian species. Theriogenology 59 (3–4), 875–88.
Salamon, S. & Maxwell, W.M.C. (2000). Storage of ram semen. Anim. Reprod. Sci. 62, 77111.
Söderquist, L., Janson, L., Larsson, K. & Einarsson, S. (1991). Sperm morphology and fertility in A.I. bulls. Zentralbl. Veterinarmed. A. 38, 534–43.
Walters, A.H., Saacke, R.G., Pearson, R.E. & Gwazdauskas, F.C. (2005). The incidence of apoptosis after IVF with morphologically abnormal bovine spermatozoa. Theriogenology 64, 1404–21.
Wass, J.A. (2009). SigmaPlot 11: Now with total sigmaStat integration. Sci. Comput. 26, 21–3.
Yogev, L., Kleiman, S.E., Shabtai, E., Botchan, A., Paz, G., Ron Hauser, R., Lehavi, O., Yavetz, H. & Ronni Gamzu, R. (2010). Long-term cryostorage of sperm in a human sperm bank does not damage progressive motility concentration. Hum. Reprod. 25, 1097–103.



Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed