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.

The advent of nanotechnology in the field of animal reproduction has led to the development of safer and more efficient therapies. The use of nanotechnology allows us to avoid the detrimental effects of certain traditional antioxidants, such as Vitamin E. Its hydrophobic nature makes mandatory the use of organic solvents, which are toxic to sperm cells. This study aims to evaluate the efficiency of vitamin E nanoemulsions (NE) on ram (Ovis aries) spermatozoa. For this purpose, the effect of three NE concentrations (6, 12, and 24 mM) were assessed on sperm of 10 mature rams of the Manchega breed. Sperm samples were collected by artificial vagina, pooled, and diluted in Bovine Gamete Medium. The samples were stored at 37 °C and assessed at 0, 4, 8, and 24 h under oxidative stress conditions (100 µM Fe²⁺/ascorbate). Motility (CASA), viability (YO-PRO/IP), acrosomal integrity (PNA-FITC/IP), mitochondrial membrane potential (Mitotracker Deep Red 633), lipoperoxidation (C₁₁ BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA^®®) were assessed. A linear mixed-effects models were used to analyze the effects of time, NE, and oxidant (fixed factors) on sperm parameters, and a random effect on the male was also included in the model with Tukey’s post hoc test. Protection of ram spermatozoa with NE resulted in a more vigorous motility under oxidative stress conditions with respect Control and Free vitamin E, while preventing the deleterious effects of oxidative stress coming from the production of free radicals and lipid peroxidation. These results ascertain the high relevance of the use of delivery systems for sperm physiology preservation in the context of assisted reproduction techniques.

Oocyte quality is crucial for subsequent embryo development and so it is a major challenge in assisted reproductive technologies. The aim of the present work was to evaluate the morphometric parameters of oocytes (experiment 1) and the relative gene expression of oocytes and cumulus cells (CCs) (experiment 2) as biomarkers of oocyte quality after individually culturing them (one oocyte or embryo/drop). In experiment 1, individually matured oocytes were measured and classified into small, intermediate, and large oocytes after a cluster analysis, based on total diameter (with zona pellucida, ZP), oocyte diameter (without ZP), and ZP thickness. These oocytes were individually fertilized in vitro and cultured. The embryo development was evaluated up to the blastocyst stage. According to the total diameter, oocyte diameter, and ZP thickness, the blastocyst rate decreased in the small oocytes group (3.1 ± 3.1, 14.1 ± 9.4, and 26.7 ± 3.9, respectively) compared to the intermediate (29.4 ± 5.2, 30.5 ± 10.1, and 28.6 ± 9.6, respectively) and large oocytes groups (54.2 ± 13.5, 44.4 ± 3.9, and 67.6 ± 12.4, respectively). In addition, the probability of reaching the blastocyst stage was positively related to the total diameter (p < 0.001), oocyte diameter (p < 0.05), and ZP thickness (p < 0.001). Furthermore, the relative gene expression of BAX, BCL2, GDF9, and GJA1 was lower in oocytes classified as large. In experiment 2, the mRNA transcript relative abundance pattern of genes in CCs was evaluated according to oocyte total diameter and developmental stage reached. CCs from oocytes classified as large and oocytes capable of developing to the blastocyst stage had a lower relative expression of BAX, STAR, and PTGS2, while a higher expression of HAS2 and SDC2 transcript was observed for those oocytes. In conclusion, oocyte morphometric parameters and gene expression analysis in oocytes and CCs provide methods for the identification of the most competent oocytes for assisted reproductive technologies in sheep.

Sperm cryopreservation is a complex process that needs to be adapted to wild and domestic avian species to ensure proper efficiency. Because of its accessibility, the peregrine falcon may be used as a good model for studying other raptor species. To find the most optimal cryopreservation protocol for peregrine falcon ejaculates, sperm parameters such as motility, viability, DNA fragmentation, acrosome integrity, and mitochondrial activity were analyzed under different conditions by varying the freezing method (slow freezing in straws vs. ultrarapid freezing in pellets), thawing conditions (37 °C for 30 s vs. 5 °C for 1 min), type of cryoprotectant (DMA vs. DMSO), and concentration of DMSO (4% vs. 8%). Results show that slow cryopreservation in straws yielded greater percentages (p < 0.05) of motile spermatozoa (22.5% ± 4.4% vs. 0.0% ± 4.1%), viable spermatozoa with intact acrosomes (84.6% ± 4.3% vs. 77.4% ± 4.3%), and spermatozoa with active mitochondria (41.0% ± 6.7% vs.12.8% ± 6.7%), compared with those obtained by the ultrarapid freezing in pellets. However, no differences were found between different thawing conditions. Moreover, all sperm motility parameters were greater (p < 0.05) when DMSO was used during freezing compared with DMA, although the use of 3% and 8% DMSO produced similar results. In conclusion, these results represent important progress in the study of falcon semen cryopreservation protocol, highlighting the crucial steps of the process and the most suitable conditions.

Conventional DNA analysis techniques can hardly detect DNA damage in ruminant spermatozoa due to high DNA compaction in these cells. Furthermore, these techniques cannot discriminate whether the damage is due to oxidative stress. The main purpose of this study was to evaluate the efficacy of two techniques for determining DNA damage in ovine sperm when the source of that damage is oxidative stress. Semen samples from twenty Manchega rams (Ovis aries) were collected and cryopreserved. After thawing, the samples were subjected to different levels of oxidative stress, and DNA oxidation was quantified using an 8-hydroxy-2′-deoxyguanosine (8-OHdG) immunodetection assay and Sperm Chromatin Structure Assay (SCSA^®). For this purpose, we evaluated five different concentrations of an oxidation solution (H₂O₂/FeSO₄•7H₂O) on ram sperm DNA. Our study with the 8-OHdG immunodetection assay shows that there are higher values for DNA oxidation in samples that were subjected to the highest oxidative stress (8 M H₂O₂/800 µM FeSO₄•7H₂O) and those that were not exposed to high oxidative stress, but these differences were not significant (p ≥ 0.05). The two SCSA^® parameters considered, DNA fragmentation index (DFI %) and high DNA stainability (HDS %), showed significant differences between samples that were subjected to high concentrations of the oxidation agent and those that were not (p < 0.05). We can conclude that the 8-OHdG immunodetection assay and SCSA^® detect DNA damage caused by oxidative stress in ovine sperm under high oxidative conditions; SCSA^® is a more straightforward method with more accurate results. For these reasons, an oxidative-stress-specific assay such as 8-OHdG immunodetection is not needed to measure DNA damage caused by oxidative stress in ram sperm samples.