**5. Acknowledgments**

This work was supported by the Polish National Science Centre, grant no N N311 530040.

## **6. References**

564 Current Frontiers in Cryobiology

The reduced binding ability of the frozen-thawed spermatozoa might be due to the higher proportion of acrosome reacted and damaged spermatozoa, after cryopreservation and thawing. Moreover, impaired receptor-ligand interaction in frozen-thawed spermatozoa could be caused by cryoelution of an "essential ligand" from the sperm surface that has been

Oocyte penetration assays (OPT) involve multiple sperm penetrations of each oocyte and permit the observation of pronuclear development (Yanagimachi et al., 1976). The application of the zona-free hamster oocyte assay has been used to assess the fertility of men (Freeman et al., 2001; ) and domestic animals (Cormier et al., 1997; De los Reyes et al., 2009; Hewitt & England, 1997; Maxwell et al., 1996;). The OPT is a less time-consuming technique than in vitro fertilization (IVF) test, because oocytes can be immatured, and after evaluation are not further subjected to development. In this assay spermatozoa presented in the pervitelline space and ooplasm of the oocytes are observed under fluorescent microscopy using Hoechst 33258, PI or light microscopy (aceto-orcein) (Hay et al., 1997; Hewitt &

All changes in cryopreserved spermatozoa described in the above sections may affect the final percentage of fertilized oocytes, and also the time course of sperm penetration through the oocyte envelop, as reported previously in frozen-thawed ram and bovine sperm (Cormier et al., 1997; Maxwell et al., 1996). Nevertheless, the previous study has indicated that the major ability of cryopreserved sperm to penetrate oocytes occurs at the 1st hour of co-culture (Cormier et al., 1997; De los Reyes et al., 2009). This finding indicates that these sperm can undergo the events associated with fertilization earlier or faster than fresh sperm

Because the efficiency of oocyte penetration is a result of sperm–oocyte interaction, variation in oocyte properties are likely to produce large diversity in this assay results. However, this can be reduced with the use of a large number of oocytes (Lucas et al., 2003). However, in dogs in vitro maturation (IVM) of oocytes and IVF is difficult to achieve. Nevertheless capacitated dog spermatozoa are able to penetrate immature oocytes, inducing chromatin decondensation and resumption of meiosis (Luvoni et al., 2005; Hay et al., 1994; Sain-Dizier et al., 2001). Thus, in dogs both, immature or mature oocytes, may be use for this test.

For many years, scientists have made every endeavour to develop laboratory assays that precisely estimate the fertilizing capacity of semen. Laboratory semen appraisals can be classified in several ways. Nevertheless, an important factor for a laboratory analysis to be useful, it must be objective, repeatable, accurate and as far as possible, rapid. Among others, there can be distinguished one major division into conventional methods and advanced techniques of sperm assessment. It is little questionable, whether the subjective assessment of parameters related to the functional and morphological characteristics of spermatozoa, would increase the predictability of the fertilizing potential of cryopreserved semen. However, conventional methods for sperm evaluation in connection with the more objective computer-assisted sperm analyzers, flow cytometry and in vitro fertilization tests, have

described in human (Amann et al., 1999).

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