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.

Male reproductive success is determined by the ability of males to gain sexual access to females and by their ability to fertilize ova. Among polygyn

Spermatozoa vary enormously in their form and dimensions, both between and within species, yet how this variation translates into fertilizing efficiency is not known. Sperm swimming velocity is a key determinant of male fertilization success, but previous efforts to identity which sperm phenotypic traits are associated with swimming velocity have been unsuccessful. Here, we examine the relationship between the size of several sperm components and sperm swimming velocity in natural populations of red deer (Cervus elaphus hispanicus) where selective pressures to enhance male reproductive success are expected to be strong. Our results show that there is little within-male and considerable between-male variation in sperm dimensions. Spermatozoa with longer midpieces swim more slowly, a finding which does not support the hypothesis that the size of the midpiece determines the amount of energy which is translated into swimming speed. In contrast, spermatozoa with elongated heads, and those in which the relative length of the rest of the flagellum is longer, swim faster. Thus, the hydrodynamic shape of the head and the forces generated by the relative size of the rest of the flagellum seem to be the key determinants of sperm swimming velocity.

Evolutionary theory proposes that exaggerated male traits have evolved via sexual selection, either through female mate choice or male–male competition. While female preferences for ornamented males have been amply demonstrated in other taxa, among mammals sexual characters are commonly regarded as weapons whose main function is to enhance male competitiveness in agonistic encounters. One particularly controversial hypothesis to explain the function of male sexual characters proposes that they advertise male fertility. We test this hypothesis in red deer (Cervus elaphus), a species where sexual characters (antlers) reach an extreme degree of elaboration. We find that a global measure of relative antler size and complexity is associated with relative testes size and sperm velocity. Our results exclude the possibility that condition dependence, age or time of culling, drive these associations. Red deer antlers could signal male fertility to females, the ability to avoid sperm depletion throughout the reproductive season and/or the competitive ability of ejaculates. By contrast, male antlers could also signal to other males not only their competitive ability at the behavioural level (fighting ability) but also at the physiological level (sperm competition).

Antioxidants could improve sperm media, extending the viability of spermatozoa and protecting their DNA. The protective ability of lipoic acid, melatonin, Trolox and crocin was tested on red deer spermatozoa incubated at 37°C. Cryopreserved spermatozoa were thawed and incubated with 1 mM or 0.1 mM of each antioxidant, with or without oxidative stress (100 µM Fe2+). Motility (CASA), viability, mitochondrial membrane potential and acrosomal status were assessed. Lipoperoxidation (malondialdehyde production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were checked at 4 h. Incubation alone increased ROS and decreased motility. Oxidative stress intensified these effects, increasing lipoperoxidation and DNA damage. Lipoic acid had little protective effect, whereas 1 mM melatonin showed limited protection. Trolox lowered ROS and lipoperoxidation both in oxidised and non-oxidised samples. In oxidised samples, Trolox prevented DNA and acrosomal damage, and ameliorated motility. Crocin at 1 mM showed similar results to Trolox, but noticeably stimulated motility and had no effect on lipoperoxidation. In a second experiment, a broader range of crocin and melatonin concentrations were tested, confirming the effects of crocin (positive effects noticeable at 0.5–0.75 mM), but showing an increase in lipoperoxidation at 2 mM. Melatonin was increasingly effective at 2.5 and 5 mM (ROS, lipoperoxidation and DNA status). Crocin seems a promising new antioxidant, but its particular effects on sperm physiology must be further studied, especially the consequences of motility stimulation and confirming its effect on lipoperoxidation. Melatonin might be useful at relatively high concentrations, compared to Trolox.

Efforts to test sex ratio theory have focused mostly on females. However, when males possess traits that could enhance the reproductive success of sons, males would also benefit from the manipulation of the offspring sex ratio. We tested the prediction that more-fertile red deer males produce more sons. Our findings reveal that male fertility is positively related to the proportion of male offspring. We also show that there is a positive correlation between the percentage of morphologically normal spermatozoa (a main determinant of male fertility) and the proportion of male offspring. Thus, males may contribute significantly to biases in sex ratio at birth among mammals, creating the potential for conflicts of interest between males and females.