ARTIFICIAL INSEMINATION IN FARM ANIMALS - Phenix-Vet

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202Artificial Insemination in Farm Animalsby greater proportion of chlortetracycline fluorescent pattern “B” due to freezing thawing inbull (Cormier et al, 1997), boar (Maxwell, and Johnson, 1997), equine spermatozoa (Thomas,et al, 2006), and in buffalo bull semen (Kadirvel et al, 2009). Impaired sperm membranefunction due to cryopreservation inevitably diminishes the successful union of the oocyteand spermatozoa during in vivo fertilization. The structural reorganization of the spermhead plasma membranes after cryopreservation appears to disrupt the ability of the spermto interact normally with cells of the female genital tract (Lessard et al, 2000; Medeiros et al,2002; Watson, 2000). Poorly motile spermatozoa are also less likely to arrive at the site offertilization in vivo or to penetrate the zona. Moreover, the proportion of motile spermpopulation itself is adversely affected by cryopreservation (Cerolini et al, 2001; Gillan et al,2004). Reduced sperm binding is likely a result of membrane injury, possibly by structuraldamage to the sperm receptors or by incomplete receptor aggregation.4. Evaluation of post-thaw semen qualityThe semen quality and its relationship with fertility have great importance in animalproduction. Hence, in vitro tests are frequently applied to determine the quality of semenfor its approval and use in both AI and other biotechnology procedures. Conventionallaboratory tests for assessment of semen quality include light microscopic study ofspermatozoal morphology, and estimation of spermatozoal motility which in turnencompass percentages of motile and progressively motile sperm; velocity of spermatozoalmovement; and longevity following in vitro storage. Other features of semen quality includeconcentration, volume, detection of the presence of urine, blood, or potentially pathogenicbacteria and functional integrity tests. The choice of adequate parameters by reproducible,fast and sensitive methods is of increasing concern. This is because the predictive value ofthe standard seminal parameters is limited or insufficient for the identification of subfertileindividuals (Clement, 2001; Love et al, 2000).The nucleus, acrosome, the flagellum, mitochondria, and the plasma membrane are the mostimportant regions of the spermatozoa that need to be assessed during postthaw semenevaluation. A series of laboratory tests devised to evaluate these various compartments willaid in the improved localization of spermatozoal dysfunction thereby improving thepredictive values of laboratory-based semen evaluation to a relatively more accurate level.More specific techniques such as testing the mitochondrial function, flagellar substructure,and plasma membrane integrity are already available (Graham and Moce, 2005; Gravance etal, 2001; Thomas et al, 1998). A variety of laboratory procedures used today in theassessment of the integrity of the plasma membrane are important components of thepostthaw semen evaluation. Among them is the evaluation of the ability of spermatozoa toexclude extracellular dyes, such as eosin, which are non permeable when the membrane isintact. Another approach is the hypo-osmotic swelling test (HOST) in which thespermatozoa are exposed to a hypotonic media (50 to 100-mOsm range) to test theirosmoregulatory function (Davies-Morel, 1999; Neild et al, 1999). Dyes that can traverse themembrane and those that are membrane-impermeable can be combined in a solution beforespermatozoal exposure to provide a more accurate reflection of membrane integrity. Forinstance, fluorescent plasma-membrane dyes can be combined with mitochondrial dyes oracrosomal dyes to provide more thorough coverage of the functional regions in the assay(Garner et al, 1994; Kavak et al, 2003; Love et al, 2003). More recently, a computer assistedsemen analysis (CASA) is in use and gives extensive information about the kinetic property
Effect of Cryopreservation on Sperm Quality and Fertility 203of the ejaculate based on measurements of the individual sperm cells. Using CASA, motilityand movement characteristics of spermatozoa have been correlated to in vivo fertility. Still,CASA-assessed motility is done on a rather limited number of spermatozoa and ispredisposed to a certain degree of human bias.An understanding of how molecular and ultra-structural basis of spermatozoal function,spermatozoa-oviductal interactions, and gamete engagement are influenced bycryopreservation will undoubtedly lead to many practical applications in semen evaluation.From this may arise possibilities for detailed laboratory tests to assess spermatozoalfunction, to introduce improved methods of semen preservation, options for applications ofassisted reproductive technologies and even treatment options for subfertile animals. Thechromatin structure assay (SCSA) tests, for instance, is a flow cytometric procedure that usesthe metachromatic fluorochrome to test the denaturability of spermatozoal chromatin that isnot normally monitored by conventional methods in various species (Evenson et al, 1995;Love 2005; Makhlouf and Niederberger, 2006). Chromatin susceptibility to denaturation iscorrelated with the level of actual DNA strand breaks and might be indicative of geneticallydefective spermatozoa (Evenson et al, 1995). Several types of defective sperm organelles andDNA can be detected in large number of sperm by immunochemical assays and flowcytometry. Microarray profiling of sperm mRNA has been shown to indicate geneexpression associated with both fertile and infertile males (Evenson et al, 2002; Thomas et al,1998). More importantly, spermatozoa affected by such damage might show no apparentlydetectable alteration in motility or membrane integrity, but may induce embryonic failureafter fertilization (Fatehi et al, 2006). Further DNA damages are not evident until the time offertilization making the chromatin defect clinically significant as it represents a potentialnon repairable defect. This becomes quite important clinically, because affectedspermatozoa in an ejaculate may not be impaired from fertilization, and hence performingrepeated insemination may not increase pregnancy rate.The search for the identification of biochemical markers of spermatozoal function is stillongoing. Such finding will improve the efficiency of laboratory-based detection of infertilityinduced by the process of freezing/thawing by targeting specific cellular components.However, incorporation of detailed tests such as SCSA for semen evaluation should notreplace or reduce the value of the conventional methods of spermatozoal motility ormorphology tests. More recently, much attention has been given to the test of capacitationprocess, as an immediate precursor to fertilization. However, on the path to fertilizationthere are many preliminary steps prior to capacitation leading to the need for spermevaluation involving tests of spermatozoal response to particular environmental conditionsrelated to the overall fertilization process (Petrunkina et al, 2007).5. Fertility of cryopreserved spermatozoaThe process of cryopreservation represents an artificial interruption of the progress of thespermatozoon towards post-ejaculation maturation and fertilization. Even with the bestpreservation techniques to date, cryopreservation process still causes harmful damage to thespermatozoa. As it has been discussed earlier in this chapter, cryopreservation affectsfertility by virtue of its effect on sperm membranes, cytoskeleton, motile apparatus andnucleus, and cell metabolism. Moreover, freezing and subsequent thawing proceduresrender the remaining surviving spermatozoa physiologically different from spermatozoabefore cryopreservation. Sprmatozoa become very sensitive to any form of stress in their
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VIContentsChapter 9Evaluation of a
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ARTIFICIAL INSEMINATION IN FARM ANIMALS - Phenix-Vet

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