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Technological Advancements in Semen Evaluation of Stallions
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Conventional laboratory tests for assessment of spermatozoal quality have included light microscopic evaluation of spermatozoal morphologic characteristics and estimation of spermatozoal motility (including percentages of motile and progressively motile sperm; velocity of spermatozoal movement; and longevity of spermatozoal motility following in-vitro storage). While other features of semen quality are also considered, including spermatozoal concentration, semen volume, and presence of blood, urine, or potentially pathogenic bacteria, the basic features of this examination process date back several decades. The value of the examination is predicated, to a large extent, on reliable equipment and personnel with good observational power. Even when these stipulations are in effect, the predictive value of the examination can be limited. The same holds true for spermatozoa of other mammalian species. As such, expanded analysis of equine spermatozoal populations might improve the predictive value of the testing process. In fact, additional tests shown to be of diagnostic value are presently incorporated into the spermatozoal examination process within some reference laboratories today.
One of these tests is the Sperm Chromatin Structure Assay (SCSA). This assay, introduced by Evenson in 1980, has been applied to spermatozoa from a number of species, including horses. The SCSA tests a compartment of spermatozoa that is not monitored by conventional methods, i.e., nuclear chromatin. The SCSA is a flow cytometric procedure that utilizes the metachromatic fluorochrome, acridine orange, and tests the denaturability of spermatozoal chromatin challenged with acid treatment. The literature provides variable results regarding the relationship of stallion spermatozoal chromatin denaturation to the extent of disulfide bonding within and between protamine molecules; however, chromatin susceptibility to denaturation is correlated with the level of actual DNA strand breaks. The DNA strand breaks can be associated with a myriad of factors, including idiopathic apoptosis, oxidative stress, heat stress, radiation injury, or protamine deficiency, and may involve double- stranded or single stranded DNA fragmentation or oxidized nucleosides. Such lesions could create genetically defective spermatozoa, leading to germ-line mutations. Interestingly, spermatozoa affected by such damage may appear to be normal, based on laboratory parameters such as spermatozoal motility and membrane integrity, but may induce post-fertilization embryonic failure. Owing to the highly condensed nature of the spermatozoal chromatin, mature spermatozoa are known to be transcriptionally inactive, so it is logical that DNA damage might not be expressed until mitosis occurs at the time of spermatozoon-oocyte fusion. This becomes quite important clinically as it represents a potential noncompensible defect, i.e., affected spermatozoa in an ejaculate may not be impaired for fertilization, so increasing the insemination number will not increase pregnancy rate. Ejaculated spermatozoa are known to retain a cohort of cytoplasmic mRNAs and as well as translational ability, so it is also possible that fragmentation of mRNA could have a negative impact on some other spermatozoal functions leading to reduced fertilization potential. Assays other than the SCSA are available to measure spermatozoal DNA fragmentation/chromatin disruption, including a TdT-mediated-dUTP nick end labeling (TUNEL) assay, an in-situ nick translation (NT) assay, a sperm chromatin dispersion (SCD) assay, and an electrophoresis-based Comet Assay. While these assays have not been used to the same extent as the SCSA in the equine arena, they are commonly applied in the human field. An immunofluorescence assay has also been developed for evaluation of human spermatozoal protamine levels, and a similar assay for equine spermatozoa could have diagnostic value. [...]
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