The reduced lifespan of cryopreserved spermatozoa in the mare reproductive tract has been attributed to both capacitative and apoptotic changes. However, there is a lack of studies investigating both phenomena simultaneously. 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. Apoptotic markers evaluated were caspase 3 activity, externalization of phosphatidylserine (PS), and mitochondrial membrane potential. Markers of changes resembling capacitation were membrane fluidity, tyrosine phosphorylation, and intracellular sodium. 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. 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). 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. 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.

Testicular function is particularly susceptible to vascular insult, resulting in a negative impact on sperm production and quality of the ejaculate. A prompt diagnosis of testicular dysfunction enables implementation of appropriate treatment, hence improving fertility forecasts for stallions. The present research aims to: (1) assess if Doppler ultrasonography is a good tool to diagnose stallions with testicular dysfunction; (2) to study the relationship between Doppler parameters of the testicular artery and those of sperm quality assessed by flow cytometry and (3) to establish cut off values to differentiate fertile stallions from those with pathologies causing testicular dysfunction. A total of 10 stallions (n: 7 healthy stallions and n: 3 sub-fertile stallions) were used in this study. Two ejaculates per stallion were collected and preserved at 5°C in a commercial extender. The semen was evaluated at T0, T24 and T48h by flow cytometry. Integrity and viability of sperm (YoPro^®-1/EthD-1), mitochondrial activity (MitoTracker^® Deep Red FM) and the DNA fragmentation index (Sperm Chromatin Structure Assay) were assessed. Doppler parameters were measured at three different locations on the testicular artery (Supratesticular artery (SA); Capsular artery (CA) and Intratesticular artery (IA)). The Doppler parameters calculated were: Resistive Index (RI), Pulsatility Index (PI), Peak Systolic Velocity (PSV), End Diastolic Velocity (EDV), Time Average Maximum Velocity (TAMV), Total Arterial Blood Flow (TABF) and TABF rate. The capsular artery was the most reliable location to carry out spectral Doppler assessment, since blood flow parameters of this artery were most closely correlated with sperm quality parameters. Significant differences in all the Doppler parameters studied were observed between fertile and subfertile stallions (p ≤ 0.05). The principal components analysis assay determined that fertile stallions are characterized by high EDV, TAMV, TABF and TABF rate values (high vascular perfusion). In contrast, subfertile stallions tend to present high values of PI and RI (high vascular resistance). The ROC curves revealed that the best Doppler parameters to predict sperm quality in stallions were: Doppler velocities (PSV, EDV and TAMV), the diameter of the capsular artery and TABF parameters (tissue perfusion parameters). Cut off values were established using a Youden´s Index to identify fertile stallions from stallions with testicular dysfunction. Spectral Doppler ultrasound is a good predictive tool for sperm quality since correlations were determined among Doppler parameters and markers of sperm quality. Doppler ultrasonography could be a valuable diagnostic tool for use by clinical practitioners for the diagnosis of stallions with testicular dysfunction and could be a viable alternative to invasive procedures traditionally used for diagnosis of sub-fertility disorders.

To improve artificial insemination protocols in ovine species it is crucial to optimize sperm quality evaluation after preservation technologies. Emerging technologies based on novel biomolecules and related to redox balance and proteins involved in sperm motility such as ProAKAP4 could be successfully applied in ram sperm evaluation. In this work, a multiparametric analysis of fresh, cooled, and cryopreserved ram sperm was performed at different complexity levels. Samples were evaluated in terms of motility (total motility, progressive motility, and curvilinear velocity), viability, apoptosis, content of reactive oxygen species, oxidation‒reduction potential, and ProAKAP4 expression and concentration. As expected, cryopreserved samples showed a significant decrease of sperm quality (p < 0.05), evidencing different freezability classes among samples that were detected by ProAKAP4 analyses. However, in cooled sperm no differences were found concerning motility, viability, apoptosis, ROS content, and redox balance compared to fresh sperm that could explain the reported decrease in fertility rates. However, although the proportion of sperm ProAKAP4 positive-cells remained unaltered in cooled sperm compared to fresh control, the concentration of this protein significantly decreased (p < 0.05) in cooled samples. This altered protein level could contribute to the decrease in fertility rates of cooled samples detected by some authors. More importantly, ProAKAP4 can be established as a promising diagnostic parameter of sperm quality allowing us to optimize sperm conservation protocols and finally improve artificial insemination in ovine species.

The optimization of sperm cryopreservation protocols in ram is a feasible tool to reinforce artificial insemination technologies considering the desirable application of sperm by vaginal/cervical or transcervical deposition. Cryopreservation provokes different types of damage on spermatozoa and many of these detrimental effects are triggered by redox deregulation. For this reason, the antioxidant supplementation in sperm cryopreservation protocols to decrease reactive oxygen species (ROS) levels and to equilibrate redox status has been widely employed in different species. Despite this, more fertility trials are necessary to provide the definitive tool to ensure the antioxidant effectiveness on sperm quality. For this reason, in this work, we performed a multiparametric analysis of some previously tested antioxidants (crocin, GSH and Trolox) on ram sperm cryopreservation from field trials to sperm quality analyses focused on new strategies to measure redox balance. Attending to fertility trial, Trolox supplementation registered an improvement concerning to fertility (when we considered high fertility males) and multiple lambing frequency and other complementary and descriptive data related to lambing performance such as prolificacy and fecundity. This positive effect was more evident in multiple lambing frequency when we considered low fertility males than in global male analysis. In vitro analyses of sperm quality confirmed in vivo trials registering a positive effect on sperm viability and redox balance. In this study, we provided the definitive evidence that the role of trolox on redox balance maintenance has a direct effect on fertility parameters, such as prolificacy. The effectiveness of antioxidant treatments was tested, for the first time in ovine species, using an integrative and multiparametric approach combining in vivo and in vitro analyses and novel approaches, such as RedoxSYS. These types of strategies should be applied to improve sperm conservation methods and optimize AI technologies upgrading the correlation between in vitro and in vivo analyses.

Sperm cryopreservation has become an indispensable tool in biology. Initially, studies were aimed towards the development of efficient freezing protocols in different species that would allow for an efficient storage of semen samples for long periods of time, ensuring its viability. Nowadays, it is widely known that an important individual component exists in the cryoresistance of semen, and efforts are aimed at identifying those sperm characteristics that may allow us to predict this cryoresistance. This knowledge would lead, ultimately, to the design of optimized freezing protocols for the sperm characteristics of each male. We have evaluated the changes that occur in the sperm head dimensions throughout the cryopreservation process. We have found three different patterns of response, each of one related to a different sperm quality at thawing. We have been able to characterize males based on these patterns. For each male, its pattern remained constant among different ejaculates. This latter would imply that males always respond in the same way to freezing, giving even more importance to this sperm feature. Changes in the sperm head during cryopreservation process have resulted useful to identify the ability of semen of males for freezing. We suggest that analyses of these response patterns would represent an important tool to characterize the cryoresistance of males when implemented within breeding programs. We also propose follow-up experiments to examine the outcomes of the use of different freezing protocols depending on the pattern of response of males.