Sperm cryopreservation represents a powerful tool for livestock breeding. Several efforts have been made to improve the efficiency of sperm cryopreservation in different ruminant species. However, a significant amount of sperm still suffers considerable cryodamage, which may affect sperm quality and fertility. Recently, the use of different “omics” technologies in sperm cryobiology, especially proteomics studies, has led to a better understanding of the molecular modifications induced by sperm cryopreservation, facilitating the identification of different freezability biomarkers and certain proteins that can be added before cryopreservation to enhance sperm cryosurvival. This review provides an updated overview of the molecular mechanisms involved in sperm cryodamage, which are in part responsible for the structural, functional and fertility changes observed in frozen–thawed ruminant sperm. Moreover, the molecular basis of those factors that can affect the sperm freezing resilience of different ruminant species is also discussed as well as the molecular aspects of those novel strategies that have been developed to reduce sperm cryodamage, including new cryoprotectants, antioxidants, proteins, nanoparticles and vitrification.

Fe2+/ascorbate, hydrogen peroxide (H2O2), and hypoxanthine/xanthine oxidase (XOD) are commonly used for inducing oxidative stress on spermatozoa. A comparative study of these agents was carried out on thawed spermatozoa from red deer. First, we tested a high, medium, and low concentration of each agent: 100, 10, and 1 μM Fe2+ (hydroxyl radical generator); 1 mM, 100, and 10 μM H2O2; and 100, 10, and 1 mU/ml XOD (superoxide and H2O2 generator), incubated at 37 °C for 180 min. Intracellular reactive oxygen species (ROS; H2DCFDA) increased with dose and time similarly for the three systems at each concentration level. Motility and mitochondrial membrane potential (Δψm) were considerably decreased by H2O2 (1 mM and 100 μM) and XOD (100 and 10 mU/ml). Only 1 mM H2O2 reduced viability. The antioxidant Trolox (10 μM) reduced intracellular ROS, but could not prevent the H2O2 or XOD effects. In a second experiment, YO-PRO-1 and M540 were used as apoptotic and membrane stability markers respectively. Only H2O2 increased the proportion of apoptotic and membrane-destabilized spermatozoa. Catalase added to XOD prevented Δψm loss, confirming that H2O2 was the causative agent, not superoxide. In a third experiment, caspase activation was tested using the (FAM-VAD-FMK) probe. Viable spermatozoa with activated caspases could be detected in untreated samples, and only H2O2 increased their proportion after 60 min. There were important differences between ROS generators, H2O2 being the most cytotoxic. Although H2O2 and XOD caused Δψm dissipation, this was not reflected in increasing apoptotic markers.

Sperm design and velocity play key roles in influencing sperm performance and, therefore, can determine fertilization success. Several interspecific studies have demonstrated how these features correlate, and it has been hypothesized that selection may drive changes in these sperm traits. Here, we examine the association between sperm design and swimming velocity in a study conducted at an intraspecific level in Iberian red deer (Cervus elaphus hispanicus). We addressed how the structure of different sperm subpopulations, based on sperm morphometry and velocity, are interrelated and, in turn, how they associate with fertility. Our results show that males with high fertility rates have ejaculates with high percentages of spermatozoa exhibiting fast and linear movements and that these are highly correlated with a large proportion of spermatozoa having small and elongated heads. On the other hand, males with low fertility are characterized by a subpopulation structure in which slow and nonlinear as well as small and wide spermatozoa are predominant. These findings provide insight regarding how sperm size and velocity are interrelated and how they both are associated with fertility.