Search Results (38)

Cryopreservation of equine semen remains challenging due to pronounced individual variability in cryotolerance. Because freezing induces oxidative stress and spermatozoa are particularly susceptible to such damage, this study aimed to comparatively evaluate the effects of natural extracts from nutraceutical compounds with high antioxidant activity, specifically matcha, spirulina, and horseradish, as well as quercetin, a well-known antioxidant molecule. These compounds were added to the freezing extender, and semen from 12 Salernitano stallions (48 ejaculates in total) was analyzed. Several parameters were assessed, including sperm kinetics, bioenergetics, oxidative and nitrosative stress markers, and the sperm DNA fragmentation index, both before and after cryopreservation. Neither the natural extracts nor quercetin significantly improved sperm freezability, likely due to the high degree of inter-individual variability. Stallion age also had a significant effect on nearly all the parameters evaluated, although no significant interactions were observed between age and treatment for any of the sperm quality traits. In conclusion, supplementation of the freezing extender with matcha, spirulina, horseradish extracts, or quercetin did not significantly enhance stallion semen cryopreservation outcomes. Conversely, stallion age and individual variability had a marked effect on sperm cryotolerance, highlighting the need for customized and holistic strategies to optimize cryotolerance in individual stallions. Full article

Consisting of phospholipids, sperm membranes surround the head and tail, playing essential roles in maintaining cellular structural integrity and functions. Their characteristics directly influence sperm fertility and cryopreservation outcomes. This minireview provides a summary of how sperm fertility and freezability are affected by the characteristics of its cell membranes. The primary emphasis is on the molecular and cellular anatomy as well as the physiology of sperm membranes and their attributes associated with fertility determinants or biomarkers for fertility and freezability. It also explores how this knowledge can guide the development of extenders to improve sperm freezability and enhance reproductive technologies in mammals. By providing integrity, fluidity, and selective permeability, the membranes play vitally important roles in sperm motility, which is required for successful fertilization. Cryopreservation, which involves freezing and thawing of sperm for storage or ART, alters the integrity and functionality of the sperm membranes. Sperm freezability, its viability following freezing and thawing, is influenced by several properties of the sperm cell membranes, such as lipid composition, cholesterol content, and structures and functions of the membrane proteins. This review provides concise information about the nature of sperm membranes. It highlights the importance of understanding specific biophysical and biochemical features, including lipid composition, protein distribution, and membrane phase behavior. Particular attention is given to parameters such as the cholesterol–phospholipid ratio and membrane phase transition temperature (Tm). A deeper understanding of these factors can contribute to the identification of reliable fertility biomarkers and the optimization of cryopreservation techniques used in ART and animal breeding programs. Furthermore, this review underscores the need for comprehensive investigations into the molecular and cellular architecture of sperm cells. Such studies are essential for advancing both fundamental and applied aspects of reproductive biology in food-producing animals, endangered species, and humans. Full article

Semen cryopreservation is a crucial technology for enhancing reproductive efficiency in livestock production; however, oxidative stress-induced sperm damage during the freeze–thaw process remains a significant challenge. In this study, metabolomics was used to analyze the differences in metabolites in semen from Qinchuan cattle with different freezing tolerance, and to screen out the candidate markers of sperm freezing tolerance. The metabolomics results indicate that a total of 264 differential metabolites were identified, and KEGG pathway annotation revealed that amino acid metabolism (15.07%) were prominently represented, and L-glutamine (L-Gln) showed a particularly high abundance in high freezability group (HFG) compared to the low freezability group (LFG). Further experiments demonstrated that L-glutamine supplementation significantly improved post-thaw sperm motility, plasma membrane integrity, and acrosomal integrity (p < 0.05). It also enhanced sperm antioxidant capacity by increasing the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), while reducing malondialdehyde (MDA) content (p < 0.05). Additionally, L-Gln maintained mitochondrial function and energy homeostasis by elevating mitochondrial membrane potential (MMP) and promoting AMPK phosphorylation (p < 0.05). These results indicate that L-glutamine alleviates oxidative damage during cryopreservation and enhances semen freeze tolerance. Full article

This study evaluated the repeatability of selected sperm parameters in Salernitano stallions housed on the same farm. Semen was collected weekly for four weeks, and sperm kinetics, mitochondrial activity, and oxidative/nitrosative status were assessed before and after freezing the sperm with HF-20 and INRA Freeze. Pre-freezing, significant individual variability was observed, with low repeatability for semen volume (r = 0.32), total motility (r = 0.38), curvilinear velocity (r = 0.32), and lipoperoxidation (r = 0.36). Post-thaw, sperm frozen with INRA Freeze showed significant inter-stallion differences and low-to-moderate repeatability across kinematic parameters, mitochondrial membrane potential, nitric oxide, and lipoperoxidation, whereas those frozen with HF-20 showed repeatability only for progressive motility and intracellular H₂O₂. An assessment of freezability revealed significant inter-stallion variability and low-to-moderate repeatability for most kinematic traits in sperm frozen with INRA Freeze. Age influenced specific parameters in both fresh and frozen–thawed semen. Kinematic traits were strongly intercorrelated and associated with mitochondrial activity, as well as with lipoperoxidation, the latter being significantly related to H₂O₂ and nitric oxide levels. Although the overall post-thaw differences between extenders were not statistically significant, INRA Freeze enabled clearer discrimination among stallions. The generally low-to-moderate repeatability observed in this study suggests that extender choice can influence cryopreservation outcomes, and supports the need for tailored protocols. Full article

Subfertile boars often go undetected until they cause significant reproductive losses. Current semen quality assessments are limited in their ability to predict fertility, highlighting the need for complementary biomarkers. This study explored whether semen freezability could serve as an indirect indicator of boar fertility. Eighteen boars were classified based on historical fertility records and semen freezability, assessed by post-thaw quality. Fresh and post-thaw semen samples were analyzed using the CASA system and fluorescence microscopy. High-fertility boars showed significantly better motility and functional sperm parameters in fresh semen compared to low-fertility boars. However, these differences were mostly lost after cryopreservation. Conversely, boars with good freezability had consistently better post-thaw semen quality, though this did not correlate directly with higher fertility outcomes. Notably, a combined analysis revealed that boars with both high fertility and poor freezability had the lowest post-thaw semen quality. This suggests that cryopreservation may expose hidden sperm defects not detectable in fresh semen. Total motility was the only parameter associated with both fertility and freezability. In conclusion, while freezability alone may not directly predict fertility, it may help identify low-performing males. The combined assessment of fresh semen motility and freezability could support more effective boar selection strategies. Full article

Sperm freezability exhibits marked individual variability, yet the mechanisms remain unclear. Using bulls as the experimental model, we integrated proteomic (sperm) and metabolomic (seminal plasma) analyses of high-freezability (HF) and control (CF) bulls to identify key biomarkers associated with sperm freezability. Post-thaw motility and membrane integrity were significantly higher in HF bulls (p < 0.05). Sperm proteome analysis revealed upregulated antioxidant proteins (PRDX2, GSTM4), heat shock proteins (HSP70, HSP90), and key enzymes in arginine and proline metabolism (PRODH, LAP3). Seminal plasma metabolomics revealed elevated spermine in HF bulls. Meanwhile, we found that spermine abundance was positively correlated with post-thaw motility, as well as with the expression levels of both PRODH and LAP3 (r > 0.6, p < 0.05). Functional validation demonstrated that 200 μM spermine supplementation in cryopreservation extenders enhanced post-thaw motility, kinematic parameters (VAP, VSL, VCL), membrane integrity, and acrosome integrity (p < 0.05). Concurrently, spermine enhanced antioxidant enzyme (SOD, CAT, GSH-Px) activity and reduced ROS and MDA levels (p < 0.05). Our study reveals a spermine-driven antioxidant network coordinating sperm–seminal plasma synergy during cryopreservation, offering novel strategies for semen freezing optimization. Full article

Semen cryopreservation is a crucial technology in the artificial insemination of livestock and poultry. It not only contributes to the conservation of germplasm resources but also facilitates the cross-regional exchange of high-quality breeding stock. In this study, 165 Duroc boars were selected for genome-wide genotyping, and the sperm freezing/thawing motility ratio (sperm recovery rate) was used as phenotypic data for genome-wide association analysis (GWAS). Considerable individual variations in sperm recovery rates (SRRs) were detected, and the sperm structure after cyropreservation was significantly better in highly freeze-tolerant individuals compared to non-freeze-tolerant ones. The heritability of the SRR was calculated and found to be 0.199 ± 0.158, representing low heritability. Through GWAS, eight single-nucleotide polymorphism (SNP) loci and four candidate genes (SLC10A6, MYRF, GGA1, and UTRN) were identified as being significantly associated with sperm freezing tolerance. Moreover, the dominant genotypes of four SNPs were finally determined to be valuable for identifying individuals with high sperm freezing tolerance. This study reveals the heritability of the sperm recovery rate and identifies molecular markers associated with sperm freezing tolerance in Duroc boars, which is of great significance for accelerating boar genetic improvement and enhancing the economic efficiency of pig breeding industry. Full article

The main objective of this study was to investigate boar-to-boar variations in the quality characteristics of sperm from the sperm-rich fractions (SRFs) and whole ejaculates (WEs) following freezing–thawing. Several sperm attributes, such as motility patterns analyzed by the computer-assisted sperm analysis (CASA) system, mitochondrial function, membrane integrity, and DNA fragmentation were used to compare the cryo-survival of sperm from SRFs and WEs from boars with good and poor semen freezability (GSF and PSF, respectively). In this study, boars with post-thaw total motility (TMOT) more than 30% (>30%) were classified as having GSF, while those with post-thaw TMOT less than 30% (<30%) were classified as having PSF. Principal component analysis 1 (PCA1), which is the main component of the sample variation, explained approximately 75% of the variance between the GSF and PSF groups, reaffirming the reliability of post-thaw TMOT as a reliable criterion used to classify the animals. Most of the post-thaw sperm parameters of the SRFs and WEs were positively correlated. Furthermore, scatter plot analyses show stronger relationships between the analyzed post-thaw parameters of the frozen–thawed (FT) sperm of SRFs than those of WEs. Individual boar variations or the sperm source had marked effects on the quality characteristics of FT sperm. The higher TMOT, velocity straight line (VSL), and velocity average path (VAP) of FT sperm were more enhanced in the SRFs compared with the WEs of the PSF group. Furthermore, the mitochondrial function, membrane integrity, and DNA fragmentation of FT sperm were markedly higher in the SRFs than in the WEs, particularly for the poor freezability boars. We suggest that the freezability potential of sperm of the GSF group does not differ significantly between the SRFs and WEs, reaffirming that boar variability is an important factor that affects the cryo-survival of sperm. Full article

Background: Seminal plasma is an important component of semen and has a significant effect on sperm function. However, the relationship between seminal plasma and sperm freezing capacity has not been fully studied. Purpose: Exploring metabolites and proteins related to the boar sperm freezing capacity in seminal plasma, by metabolomic and proteomic approaches, and directly verifying the protective effect of seminal plasma on the cryopreservation of boar sperm using high and low freezability seminal plasma as base freezing extender. Methods: Semen samples were collected from 30 different boars, 11 high and 11 low freezing-resistant boars were selected after freezing 2~4 times, and seminal plasma was selected at the same time. Sperm motility and movement parameters were analyzed using a CASA system. Reproductive hormones (Testosterone, progesterone, estradiol, prolactin, prostaglandin F2α, luteinoid hormone) in seminal plasma were detected by ELISA. Analysis of proteins and metabolites in high and low freezing-resistant seminal plasma by proteomics and metabolomics techniques. Results: The six reproductive hormones tested were not significantly associated with sperm freezing resistance. A total of 13 differentially expressed metabolites (DEMs) and 38 differentially expressed proteins (DEPs) were identified, while a total of 348 metabolites and 1000 proteins were identified. These DEMs were related to energy metabolism, drugs, or environmental pollutants, while the DEPs were mainly involved in the cytoskeletal dynamics and cell adhesion processes. There were 33 metabolites and 70 proteins significantly associated with mean progress motility (PM) at 10 min and 2 h after thawing. The 70 related proteins were associated with cell division and cycle regulation in gene ontology (GO) terms, as well as KEGG pathways, thermogeneration, and pyruvate metabolism. Using highly freezable boar SP as a base freezing extender made no difference from using lowly freezable boar SP, and both were not as good as the commercial control. Conclusion: There were significant differences in seminal plasma with different freezability, but the similarity was much greater than the difference. The protection effect of seminal plasma is not remarkable, and it does not exhibit superior cryoprotective properties compared to commercial semen cryoelongators. Significance: This study provides a deeper understanding of how seminal plasma composition affects sperm freezabilty. It provides potential biomarkers and targets for improving sperm cryopreservation techniques. Full article

In mammals, the pineal hormone melatonin is the most powerful pacemaker of the master circadian clock and is responsible for reproduction in seasonal breeders. It is also well known that melatonin and its metabolites play antioxidant roles in many tissues, including reproductive cells. Melatonin synthesis and secretion from the pineal gland occurs during scotophase (the dark phase during a day–night cycle), while its inhibition is observed during photophase (period of light during a day–night cycle). Short-day breeders, such as goats, are stimulated to breed in a manner dependent on high endogenous levels of melatonin. This hormone can be synthesized in various extra-pineal tissues, such as retina, gastrointestinal tract, ovaries, and testis, with its main function being as a local antioxidant, given that melatonin and its metabolites are potent scavengers of reactive oxygen and nitrogen species. Moreover, it has been reported that some functions of melatonin can be exerted through plasma membrane and intracellular receptors expressed in the male reproductive system, including germ cells, immature and mature spermatozoa. It has been shown that melatonin may enhance gamete cryosurvival mainly by its addition into the media and/or in exogenous melatonin treatments in several species. In the present review, the physiological effects of endogenous melatonin in mammals are described, with a deeper focus on caprine reproduction. Additionally, results from recent investigations on the roles of exogenous melatonin aimed at improving the reproductive efficiency of goat bucks are discussed. There are contradictory findings and a limited amount of research available in the field of goat sperm cryopreservation associated with the use of melatonin. Understanding and improving goat reproduction and production is essential for many marginalized human populations around the world who directly depend on goats to maintain and improve their lifestyle. Full article

This work was aimed to identify androgen receptors (AR) in the spermatozoa of wild and domestic ruminants and to assess the effect of testosterone on sperm localization of aquaporin-3 (AQP3) and cryopreservation process. Sperm samples from wild species were incubated with testosterone (T group), 1,3-propanediol (PDO group), phloretin (PHL group), PDO+T group, PHL+T group. Western blot identified the presence of AR as a single band of about 48 KDa. Immunolabelling of AR was located in the equatorial segment of the sperm head. In mouflons, the cryoresistance ratio for acrosome integrity was lower (p < 0.05) in the PHL+T than in Control and T groups. In ibexes, the cryoresistance ratio for acrosome integrity was lower (p < 0.05) in the PHL+T, PHL, and T group than in the Control group; the cryoresistance ratios for sperm kinematic variables were lower (p < 0.05) in PDO+T than in Control. No changes were found among treatments in the proportion of spermatozoa showing AQP3 in the different membrane domains after incubation and thawing in both mouflon and ibex. In conclusion, testosterone negatively affected sperm cryoresistance expressed as acrosome integrity, enhancing the effects of the AQP blocker PHL. Our findings provide a sound knowledge of the molecular mechanisms that explain the seasonal variation in sperm freezability from ruminants. Full article

Cryopreservation poses significant challenges to the preservation of sperm integrity and function, particularly in small ruminants where cryodamage is pronounced. This review explores the molecular mechanisms underlying sperm cryodamage and strategies for improving cryopreservation outcomes, with a focus on the role of antioxidants. Cryopreservation-induced alterations in proteins and RNA transcripts critical for sperm function, including motility, capacitation, fertilization, and embryo development, are discussed. Proteomic, transcriptomic, and epigenomic advancements have provided valuable insights into these mechanisms, offering potential biomarkers for predicting sperm freezability and enhancing cryopreservation strategies. Combining technologies such as mass spectrometry and flow cytometry allows for a comprehensive understanding of molecular and cellular changes induced by the freezing–thawing process. However, challenges remain in optimizing cryoprotectant formulations and antioxidant supplementation to improve post-thaw sperm fertility. Further research is needed to explore a wider range of novel cryoprotectants, antioxidants, and proteins for cryopreservation media, as well as to validate their efficacy in enhancing sperm viability and function. Additionally, investigations into the effects of cryopreservation on RNA transcripts and epigenetic factors in small ruminant species are warranted to advance our understanding of sperm preservation. Overall, this review highlights the importance of antioxidants in mitigating cryodamage and underscores the need for continued research to refine cryopreservation protocols and improve reproductive outcomes in small ruminants. Full article

Cryopreservation is a stressful process for sperm, as it is associated with an increased production of reactive oxygen species (ROS). Elevated ROS levels, which create an imbalance with antioxidant capacity, may result in membrane lipid peroxidation (LPO), protein damage and DNA fragmentation. This study aimed to determine whether the membrane LPO and DNA fragmentation of frozen–thawed horse sperm relies upon antioxidant activity, including enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and paraoxonase type 1 (PON1)); non-enzymatic antioxidant capacity (Trolox-equivalent antioxidant capacity (TEAC), plasma ferric reducing antioxidant capacity (FRAP) and cupric reducing antioxidant capacity (CUPRAC)); and the oxidative stress index (OSI) of their seminal plasma (SP). Based on total motility and plasma membrane integrity (SYBR14⁺/PI⁻) after thawing, ejaculates were hierarchically (p < 0.001) clustered into two groups of good- (GFEs) and poor-(PFEs) freezability ejaculates. LPO and DNA fragmentation (global DNA breaks) were higher (p < 0.05) in the PFE group than in the GFE group, with LPO and DNA fragmentation (global DNA breaks) after thawing showing a positive relationship (p < 0.05) with SP OSI levels and ROS production. In addition, sperm motility and membrane integrity after thawing were negatively (p < 0.05) correlated with the activity levels of SP antioxidants (PON1 and TEAC). The present results indicate that LPO and DNA fragmentation in frozen–thawed horse sperm vary between ejaculates. These differences could result from variations in the activity of antioxidants (PON1 and TEAC) and the balance between the oxidant and antioxidant components present in the SP. Full article

Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics used in semen extenders, since the volume of frozen semen is smaller than when cooled semen is inseminated. However, there is considerable variability in the cryosurvival of spermatozoa from different stallions, leading to the classification of stallions as good or bad freezers. Improvements could be made at the level of stallion nutrition, the semen collection regimen, the extender, the removal of seminal plasma, and the cooling protocol, among others. Stallion sperm membranes are highly susceptible to lipid peroxidation, but research on antioxidants has failed to identify an additive that would benefit all stallions. In the future, biomarkers for sperm freezability could be used as an aid in identifying suitable ejaculates for cryopreservation. Full article

The results of artificial insemination (AI) are adversely affected by changes in sperm motility and function throughout the cryopreservation procedure. The proteome alterations of frozen–thawed spermatozoa with various levels of freezability in dairy goats, however, remain largely unknown. To discover differentially expressed proteins (DEPs) and their roles in dairy goat sperm with high or low freezability (HF or LF), we conducted 4D-DIA quantitative proteomics analysis, the results of which are presented in this work. Additionally, we explored the underlying processes that may lead to the variations in sperm freezing resistance. A total of 263 DEPs (Fold Change > 2.0, p-value < 0.05) were identified between the HF group and LF group in frozen–thawed dairy goat spermatozoa. In our Gene Ontology (GO) enrichment analysis, the DEPs were mostly associated with the regulation of biological processes, metabolic processes, and responses to stress and cellular component biogenesis. Our Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis also revealed that the DEPs were predominantly engaged in oxidative phosphorylation, N-Glycan biosythesis, and cysteine and methionien metabolism. A protein–protein interaction (PPI) network analysis revealed 14 potential proteins (NUDFB8, SDHC, PDIA4, HSPB1, etc.) that might influence the freezability of dairy goat sperm. These findings shed light on the processes underlying alterations in the proteome and sperm freezability, aiding further research on sperm cryopreservation. Full article