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.

Mammalian sperm must undergo a set of structural and functional changes collectively termed as capacitation to ensure a successful oocyte fertilization. However, capacitation can be compromised by cryopreservation procedures, which alter the proteome and longevity of sperm. To date, how the protein changes induced by cryopreservation could affect the acquisition of sperm fertilizing potential remains unexplored. The present study investigated the protein profile of ram sperm during in vitro capacitation before and after cryopreservation to elucidate the impact of cryopreservation on sperm capacitation at a molecular level. Fresh and cryopreserved ram sperm were incubated under capacitating (CAP) and non-capacitating (NC) conditions for 240 min. The sperm proteome of these four treatments was analyzed and compared at different incubation times using reverse phase liquid chromatography coupled to mass spectrometry (RP-LC-MS/MS). The comparison between fresh and cryopreserved sperm suggested that cryopreservation facilitated an apoptosis-stress response and redox process, while the comparison between sperm incubated in CAP and NC conditions showed that capacitation increased those biological processes associated with signaling, metabolism, motility, and reproductive processes. In addition, 14 proteins related to mitochondrial activity, sperm motility, oocyte recognition, signaling, spermatogenesis, and the apoptosis-stress response underwent significant changes in abundance over time when fresh and cryopreserved sperm incubated in CAP and NC conditions were compared. Our results indicate that disturbances in a ram sperm proteome after cryopreservation may alter the quality of sperm and its specific machinery to sustain capacitation under in vitro conditions.

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Background Sperm chromatin structure provides valuable information for the prediction of male fertility and can be altered during different procedures. Previous studies have shown that sperm chromatin condensation decreased during in vitro capacitation. Moreover, cryopreservation can affect sperm DNA integrity and chromatin compaction. Objectives This study aimed to investigate dynamic modifications produced in the chromatin structure of ram spermatozoa during in vitro capacitation before and after cryopreservation. Materials and methods Chromatin decondensation (AB+), DNA methylation, DNA fragmentation index (%DFI) and high DNA stainability (HDS) were evaluated in fresh and frozen‐thawed ram spermatozoa incubated under capacitating (CAP) conditions at 1, 5, 15, 30, 60, 120, 180 and 240 minutes and under non‐capacitating (NC) conditions at 0, 15 and 240 minutes. Results Incubation in NC conditions did not induce significant changes in chromatin condensation (P > .05; AB + and HDS). However, incubation of fresh and cryopreserved ram spermatozoa under CAP conditions significantly increased chromatin decondensation (P < .05), reaching the highest percentage of AB + and HDS from 180 to 240 minutes in fresh samples and from 5 to 30 minutes in cryopreserved samples. Both variables (HDS and AB+) were positively correlated with tyrosine phosphorylation, total motility, progressive motility, curvilinear velocity and amplitude of lateral head displacement, as well as between them under CAP conditions in fresh and cryopreserved spermatozoa. DNA methylation significantly increased in cryopreserved spermatozoa (P < .05), but only after extended incubation under CAP conditions (60‐240 minutes), while the %DFI, albeit higher in cryopreserved samples, remained constant under CAP and NC conditions in both types of sample (P > .05). Discussion and conclusions Our results suggest that sperm chromatin condensation decreased progressively during in vitro capacitation of ram spermatozoa, while sperm DNA integrity remained intact. Such changes in chromatin condensation appeared faster after sperm cryopreservation.