Moreover, the heterogeneous nature of the ejaculate is not disclosed in classical flow cytometry analysis based in manual gating. 2015, Pena 2016 a), this therefore demands the interpretation of the results due to the multidimensional nature of the data gathered, compared with the traditional bi-dimensional dot plots or uni-dimensional histograms. Recent developments in flow cytometry applied to sperm analysis are increasing the number of parameters that can be assessed simultaneously in a single assay ( Gallardo Bolanos et al. We aimed to focus our study on the surviving population of spermatozoa, to identify how characteristics related to apoptosis or capacitation change during this process in the same ejaculates following a split sample design. However, there is a lack of detailed studies analyzing both types of changes simultaneously. 2012), while spermptosis implies caspase activation, loss of mitochondrial membrane potential, and transposition of phosphatidylserine (PS) to the outer leaflet of the membrane ( Ortega-Ferrusola et al. Capacitation is characterized by changes in sperm membrane fluidity, overall increase in tyrosine phosphorylation, and decrease in intracellular sodium ( Escoffier et al. 2008), or spermptosis as it has been recently termed ( Gallardo Bolanos et al. 2009) or changes that resemble apoptosis ( Ortega-Ferrusola et al. The reduced lifespan of the thawed sperm has been attributed to either changes similar to capacitation ( Cormier et al. This intensive management increases the costs related to insemination precluding a wider use of frozen-thawed semen. To obtain acceptable fertility, intensive management of the mares is required to perform AI close enough to ovulation as the surviving spermatozoa have reduced lifespan in the mare’s reproductive tract. In spite of equine male gametes trade constituting a major component of the equine industry, cryopreservation of stallion spermatozoa, as an indispensable tool for the commerce of frozen-thawed semen doses, still has numerous drawbacks ( Pena et al. For the first time advanced computational techniques were applied to the analysis of spermatozoa, and these techniques were able to disclose relevant information of the ejaculate that remained hidden using conventional flow cytometry. Average values of markers of capacitative changes were not affected by cryopreservation however, the analysis of the phenotype of individual spermatozoa using computational flow cytometry revealed the presence of subpopulations of spermatozoa experiencing capacitative changes. Most of the changes induced by cryopreservation were apoptotic, with increase in caspase 3 activation ( P < 0.01), PS translocation to the outer membrane ( P < 0.001), loss of mitochondrial membrane potential ( P < 0.05), and increase in intracellular Na+ ( P < 0.01). Conventional and computational flow cytometry using nonlinear dimensionally reduction techniques ( t-distributed stochastic neighbor embedding (t-SNE)) and automatic classification of cellular expression by nonlinear stochastic embedding (ACCENSE) were used. Markers of changes resembling capacitation were membrane fluidity, tyrosine phosphorylation, and intracellular sodium. Apoptotic markers evaluated were caspase 3 activity, externalization of phosphatidylserine (PS), and mitochondrial membrane potential. In order to improve our knowledge in this particular point, we studied in raw and frozen-thawed samples apoptotic and capacitative markers using a wide battery of test based in flow cytometry. However, there is a lack of studies investigating both phenomena simultaneously. The reduced lifespan of cryopreserved spermatozoa in the mare reproductive tract has been attributed to both capacitative and apoptotic changes.
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