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Simple Summary  Spermatozoa go through diverse changes to achieve their fertilizing potential (capacitation) and develop a specific motility pattern (hyperactivation). However, to ensure a greater reproductive success, not all the spermatozoa present in an ejaculate react equally or at the same time. Therefore, a comparative analysis was performed in the present study to improve our current understanding about how cryopreservation may affect the heterogeneous nature of fresh ejaculates during these two events. Among the four motile sperm subpopulations identified in fresh and frozen-thawed ram semen, one of them developed a hyperactivated-like movement and was the main group involve in those changes associated with sperm capacitation based on the marked increase and the positive correlation with mitochondrial activity and tyrosine phosphorylation, two relevant parameters that usually increase during capacitation. In addition, cryopreservation altered the distribution of the motile sperm subpopulations. Although the subpopulation with hyperactivated-like movement increased at the beginning of incubation in frozen-thawed samples, this subpopulation together with the subpopulation of rapid and progressive spermatozoa were replaced after a prolonged incubation by the subpopulation of slow spermatozoa with the lowest mitochondrial activity, which clearly indicate the reduction in sperm quality. These findings will aid to optimize the current cryopreservation and in vitro fertilization protocols.    Abstract  The heterogeneous nature of ejaculates highlights the relevance of studying the behavior of different sperm subpopulations. Changes in sperm motility and the increase in tyrosine phosphorylation are key events that usually occur during capacitation and can be modified by the cryopreservation process. However, the relationship between both events remains poorly defined throughout capacitation in the different sperm subpopulations. Fresh and frozen-thawed spermatozoa were incubated in capacitating (CAP) and non-capacitating (NC) media up to 240 min. Sperm kinematics, tyrosine phosphorylation and mitochondrial activity were measured by the CASA system and imaging flow cytometry. Four motile sperm subpopulations (SP) were identified in fresh and frozen-thawed ram semen after the cluster analysis. Incubation under CAP conditions over time led to greater changes in the percentage of spermatozoa included in each subpopulation compared to NC conditions, being different between fresh and frozen-thawed spermatozoa. The SP1, characterized by slow spermatozoa, progressively increased after 15 min in frozen-thawed samples incubated in both media but not in fresh ones. The SP4, characterized by fast and non-linear spermatozoa, showed a marked increase during CAP, but not under NC conditions, occurring more rapidly in frozen-thawed spermatozoa. This subpopulation (SP4) was also the only one positively and strongly correlated with mitochondrial activity and all phosphorylated sperm regions during capacitation, either in fresh or frozen-thawed samples. Our results indicated that in vitro capacitation induced significant changes in the distribution of motile sperm subpopulations, being affected by cryopreservation. Notwithstanding, the subpopulation which probably represents hyperactivated-like spermatozoa (SP4) also increased in frozen-thawed samples, occurring faster and simultaneously to the increment of mitochondrial activity and tyrosine phosphorylation of different sperm regions.

Impact of Cryopreservation on Motile Subpopulations and Tyrosine-Phosphorylated Regions of Ram Spermatozoa during Capacitating Conditions