**3.3.1 Zona pellucida binding assay**

The assessment of the ability of sperm cells to bind the homologous zona pellucida (ZP) is the useful test for prediction of spermatozoal fertilizing ability (Hermansson et al., 2006). It is assumed that it is reliable test to detect sperm damage at a molecular level, which is not visible by microscopic analysis, because binding is receptor-ligand mediated reaction. The test may be done in two ways: by using intact homologous oocytes (ZP-binding assay, ZBA) and by using bisected hemizonae (hemizona binding assay, HZA) (Kawakami et al., 1998; Rijsselaere et al., 2005). In ZBA spermatozoa are coincubated with oocytes obtained from sliced ovaries. The number of spermatozoa that bound to ZP is counted with contrast-phase microscopy. The disadvantage of ZBA is the fact that the attachment of sperm cells to zona depends on the oocyte. This feature was partly overcome in HZA. Bisected by micromanipulation two parts of ZP are coincubated with spermatozoa. As a result the direct comparison of sperm cells from two origins may be done (Ivanova et al., 1999; Mayenco-Aguirre & Pérez Cortés, 1998).

A sublethal damage that occurres during cryopreservation leads to loss of sperm surface proteins, segregation of membrane proteins, inactivation of membrane-bound enzymes and decreased lateral protein diffusion within the membrane (Watson, 1995). Kadirvel et al. (2011) observed significant reduction of the zona binding ability of cryocapacitated buffalo bulls spermatozoa, and further reduction of binding ability of frozen-thawed spermatozoa, after incubation, in either capacitating, or non capacitating medium. Similar results have been obtained in bulls (Fazeli et al., 1997) and humans (Amann et al., 1999) spermatozoa, with significantly reduced binding ability to the zona pellucida after freezing and thawing.

Methods of Assessment of Cryopreserved Semen 565

enabled researchers to gain accurate information about the morpho-functional status of

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

Agarwal, A.; Ozturk E. & Loughlin, K.R. (1992). Comparison of semen analysis between the

Agarwal, A.; Prabakaran, S.A. & Said T.M. (2005). Prevention of oxidative stress injury to

Aitken, R.J. (1995). Free radicals, lipid peroxidation and sperm function. *Reprod Fertil Dev* 7,

Aitken, R.J.; Harikss, D. & Buckingham, DW. (1993). Analysis of lipid peroxidation

Aitken, R.J.; Wingate, J.K.; De Iuliis, G.N. & McLaughlin, E.A. (2007). Analysis of lipid

Almeida, J. & Ball, B.A. (2005). Effect of α-tocopherol and tocopherol succinate on lipid

Álvarez, M.; García-Macías, V.; Martínez-Pastor, F.; Martínez, F.; Borragán, S.; Mata, M.;

Amann, R.P. (1999). Cryopreservation of sperm. In: *Encyclopedia of Reproduction*, vol. 1.

Amann, R.P.; Shabanowitz, R.B.; Huszar, G. & Broder, S.J. (1999). In vitro sperm-binding

Arruda, R.P.; Ball, B.A.; Gravance, C.G.; Garcia, A.R. & Liu IKM. (2002). Effects of extender

Bailey, J.L. & Buhr, M.M. (1994). Cryopreservation alters the Ca2+ flux of bovine

Bailey, J.L., Bilodeau J.F. & Cormier, N. (2000). Semen cryopreservation in domestic animals:

Ballachey, B.E.; Evenson, D.P. & Saacke, R.G. (1988). The sperm chromatin structure assay.

Benoff, S. (1997). Carbohydrates and fertilization: an overview. *Mol Hum Reprod* 3, 599–637. Bilodeau, J-F., Chatterjee, S.; Sirard, M-A. & Gagnon, C. (2000). Levels of antioxidant

Blesbois, E.; Grasseau, I.; Seigneurin, F.; Mignon-Grasteau, S.; Saint Jalme, M. & Mialon-

peroxidation in human spermatozoa using BODIPY C11. *Mol Hum Reprod* 13, 203-

Garde, J.; Anel, L. & De Paz, P. (2008). Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear (Ursus arctos)

assay to distinguish differences in populations of human sperm or damage to

and cryoprotectants on stallion sperm head morphometry. *Theriogenology* 58, 253–6.

Relationship with alternate tests of semen quality and heterospermic performance

defences are decreased in bovine spermatozoa after a cycle of freezing and

Richard, M.M. (2008). Predictors of success of semen cryopreservation in chickens.

two Hamilton-Thorn semen analysers. *Andrologia* 24, 327–329.

mechanisms in human spermatozoa. *Mol Reprod Dev* 35, 302-315.

peroxidation in equine spermatozoa. *Anim Reprod Sci* 87, 321-337.

sperm resulting from cryopreservation. *J Androl* 20, 648–654.

a damaging and capacitating phenomenon. *J Androl* 21, 1-7.

spermatozoa. *Theriogenology* 70, 1498-1506.

spermatozoa. *Can J Anim Sci* 74, 45–52.

thawing. *Mol Reprod Dev* 55, 282-288.

of bulls. *J Androl* 9, 109-115.

*Theriogenology* 69, 252-261.

Academic Press, Burlington, MA, USA, 773-783.

spermatozoa and mechanisms of sperm cryoinjury.

sperm. *J Androl* 26, 654-60.

**5. Acknowledgments** 

659-668.

211.

**6. References** 

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