Abstract
Markers of freezability allow the selection of ejaculates of good freezability. So far, most investigations were conducted in boars, bulls, rams and horses, with high economic interests triggering the efforts. The progress in dogs is comparably slow. A critical evaluation of the methods requires consideration of practicability, with most labs not even possessing a computer assisted sperm analyser (CASA); furthermore, small canine ejaculates mostly do not allow the use of large semen volumes. In dogs, modern markers of freezability no longer assess single membrane constituents or seminal plasma components but comprise tests of cell functionality and adaptability, energy metabolism, cluster analyses of kinetic and morphometric parameters, as well as DNA intactness. Identification of the most efficient combination of tests seems useful. At present, examination by CASA combined with cluster analysis of kinetic subgroups, JC-1 staining and COMET assay or staining with toluidine blue seem most appropriate; however, cell volumetry and other functional tests deserve better attention. A better understanding of spermatozoa energy metabolism might reveal new markers. This review focuses on the requirements and markers of freezability of canine semen, highlighting potential future candidates.
Highlights
The freezability of a canine spermatozoa is determined by the complex interplay of sperm membrane and seminal plasma, diluent and cooling-freezing-thawing protocols [1–6], and it is influenced by further parameters such as age [7]
Even though former studies did not provide satisfying results [13], we previously showed that kinematic parameters objectively measured by use of computer assisted sperm analyser (CASA), such as progressive motility (P), velocity curvilinear (VCL), mean coefficient (STR) and linear coefficient (LIN), are useful for the prediction of post-thaw sperm quality
Cell volumetry requires an electric field multi-channel cell counting system that recognizes the changes in the electric resistance caused by cells as the voltage changes; the latter are related to cell volume, as greater cells cause greater pulses
Summary
Cryopreservation of canine spermatozoa requires high adaptability of the cell to changing osmolarity and temperature. The membrane lipids change toward the crystalline phase with lateral segregation, lipid peroxidation, loss of lipids and formation of reactive oxygen species [38] This leads to membrane destabilization and may cause membrane damage, especially when the cooling rates during freezing are too high or too low [39–41]. Cryodamage, which is comparable among species, causes, among others, a severe loss of essential membrane proteins and receptors, degenerative acrosome exocytodue to ice crystal formation, concentration of solutes, electrolytes and cell dehydration. Increased efflux and loss ofmotility, potassium further in membrane fluidity/integrity and in ioncholesterol channels, reduction inasperm disruption decrease the fertilizing ability of the cell by causing preterm capacitation and acrosome of disulfide bridges between cysteine radicals of protamines and DNA fragmentation [25]. Increased cholesterol efflux and a loss of potassium further decrease the fertilizing ability of the cell by causing preterm capacitation and acrosome reaction [39,40]
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