Search Results (32)

Cryopreservation is a key tool in assisted reproduction, but it often compromises post-thaw sperm quality due to cryodamage. Optimizing the initial cooling phase, specifically from room temperature to 5 °C, is a critical determinant of successful outcomes. This study aimed to evaluate the impact of different pre-freeze cooling rates on stallion sperm quality using a novel, precision cooling device. Semen samples from five healthy stallions were divided into three groups and cooled at distinct rates: Slow (0.3 °C/min), Moderate (1 °C/min), and Fast (approximately 30 °C/min). Sperm motility parameters were assessed using a Computer-Assisted Sperm Analyzer (CASA) before freezing and after thawing. Additionally, sperm integrity and physiological parameters, including viability, acrosomal integrity, Reactive Oxygen Species (ROS) expression, and mitochondrial membrane potential, were assessed by flow cytometry post-thaw. The analysis of post-thaw kinematics revealed a significant interaction between the cooling rate and processing stage (post-cooling vs. post-thaw). The Fast-cooling protocol resulted in higher post-thaw total motility (51.8%) compared to the Slow protocol (45.01%). Crucially, no significant differences were detected among cooling rates for the critical parameter of progressive motility or curvilinear velocity (VCL). Circle motility had higher values in the Fast-cooling group compared to the Slow group. Cell viability demonstrated a tendency (p = 0.08), where the Slow cooling group exhibited higher mean values (65.59%) compared to the Fast group (61.67%). Comprehensive flow cytometry assessments of other cellular integrity markers, including acrosomal integrity, mitochondrial function (MMP), and ROS expression, were statistically equivalent across all cooling rates (p > 0.05). The results confirm that this fast pre-freeze cooling rate, integrated within the highly controlled environment of Directional Freezing technology, successfully preserved essential sperm function and structure. Critically, the demonstrated functional equivalence in progressive motility validates the Fast protocol as an efficacious strategy to increase the efficiency and adaptability of equine semen cryopreservation protocols for commercial utilization. Full article

The silver pomfret (Pampus argenteus), widely distributed across the Indo-West Pacific and prevalent in China’s coastal waters, has experienced significant resource decline due to anthropogenic impacts such as habitat alteration and overfishing, which disrupt its natural reproduction and growth. Cryopreservation technology overcomes spatiotemporal constraints by enabling the long-term storage of high-quality sperm for future use. This study optimized cryopreservation protocols for silver pomfret sperm, evaluation key parameters including extenders, cryoprotectants, dilution ratios, cooling heights, and thawing temperatures. Sperm quality was assessed post thaw via enzyme activity assays and electron microscopy. Results demonstrated that modified plaice Ringer solution (MPRS) extender yielded the highest post-thaw motility (95.98 ± 1.59)%. The optimal cryopreservation conditions for silver pomfret sperm were established as follows: MPRS diluent, 20% EG, a 1:6 dilution ratio, a 7 cm cooling height, and a 28 °C thawing temperature. This protocol yielded post-thaw sperm with motility and motion parameters most closely resembling those of fresh sperm. Ultrastructural observations and enzyme activity assays, however, confirmed that cryopreservation induced sublethal damage, including significant reduction in ATPase activity, as well as structural anomalies such as head deformation, membrane damage, and organelle disarray. This work establishes a foundational cryopreservation protocol, providing critical tools for conserving the genetic resources of this declining species and supporting sustainable aquaculture and wild population restoration efforts. Full article

The physiological functions of mammalian sperm, such as motility, hyperactivation, and capacitation, require substantial energy. This study investigates the effects of two classic cryopreservation extenders—TCG (tris-citrate-glucose) and LEY (lactose-egg yolk)—on the energy metabolism of frozen–thawed boar semen. By comparing the quality indicators, key metabolite levels, and the activities of critical enzymes involved in glycolysis and the tricarboxylic acid cycle, we aim to understand how these different semen extenders influence the spermatozoa vitality of frozen–thawed boar semen. Following thawing, the LEY-cryopreserved sperm demonstrated significantly elevated motility parameters (viability, VCL, VSL, and VAP) and enhanced plasma membrane and acrosomal integrity compared with the TCG group (p < 0.05), though both cryopreserved groups exhibited significantly reduced performance relative to fresh semen controls. Cryopreservation markedly reduced intracellular adenosine triphosphate (ATP), pyruvate, and acetyl coenzyme A (A-CoA) levels (fresh > LEY > TCG; p < 0.05). The LEY-preserved spermatozoa retained higher activities of glycolysis-related enzymes (phosphofructokinase, PFK; pyruvate kinase, PK) compared with the TCG group, which, in turn, showed elevated lactate dehydrogenase (LDH) activity. Critically, TCG-suppressed pyruvate dehydrogenase (PDH) activity (p < 0.05) coincided with diminished A-CoA, indicating impaired mitochondrial oxidative phosphorylation. These results demonstrate LEY’s superior preservation of motility and membrane stability but highlight cryodamage-induced energy metabolism dysregulation, particularly TCG’s disruption of the glycolysis–TCA cycle coordination essential for spermatozoa function. In conclusion, the choice of semen extender has a significant impact on the energy metabolism and overall quality of frozen–thawed semen, highlighting the importance of optimizing cryopreservation protocols for improved spermatozoa viability and functionality. Full article

Sperm cryopreservation is a fundamental reproductive biotechnology, enabling the long-term storage of genetic material and supporting assisted reproduction programs. Despite its widespread application, the process is associated with significant limitations due to the cryo-induced cellular damage that occurs during freezing and thawing. These injuries primarily affect the plasma membrane, nuclear DNA, and motility, thereby compromising the fertilizing potential of spermatozoa. Furthermore, interspecies variability in terms of cryo-sensitivity poses a major challenge to the development of standardized cryopreservation protocols. Recent advances have focused on mitigating cryodamage through the use of various strategies. The inclusion of antioxidants in cryopreservation media has proven effective in reducing oxidative stress, thereby enhancing cellular protection. Similarly, the addition of lipid-based supplements contributes to membrane stabilization, improving post-thaw sperm viability and functionality. Moreover, the application of omics technologies, such as transcriptomics and proteomics, has facilitated a deeper understanding of molecular damage and protective responses, paving the way for the development of tailored, species-specific protocols. These integrated approaches optimize cryopreservation conditions, maximizing post-thaw survival and the fertilizing capacity of sperm. Enhancing cryopreservation techniques not only improves the outcomes of assisted reproductive technologies, but also plays a crucial role in the conservation of genetically valuable livestock species. In conclusion, the integration of biotechnological and molecular tools holds significant promise for overcoming the current limitations and advancing the efficacy of sperm cryopreservation. Full article

Ferroptosis is implicated in cryodamage to sheep sperm, potentially due to glutathione peroxidase 4 (GPX4) degradation during freezing; however, the pathway underlying GPX4 degradation remains unclear. In this study, a comparison of cryoprotective effects between the autophagy inhibitor chloroquine (CQ) and the ubiquitination inhibitor MG132 revealed that 5 μM CQ treatment significantly enhanced the motility (p < 0.01) and sperm plasma membrane integrity rate (p < 0.01) of frozen–thawed sperm; no protective effects were observed in any MG132 treatment group. Mechanistic analysis indicated that CQ treatment substantially restored GPX4 protein expression (p < 0.01), and concurrently reduced lipid peroxidation (p < 0.01) and free iron ion accumulation (p < 0.01), in frozen–thawed sperm. These findings suggest that GPX4 degradation during cryopreservation occurs via the autophagy pathway. This study established a ferroptosis–GPX4–autophagy axis during sheep sperm cryopreservation and identified autophagy-mediated GPX4 loss as a potential target for enhancing sperm cryoprotection. Full article

Cryopreservation can adversely affect sperm motility, structural integrity, and fertilization ability. This study investigated the effects of MitoQ and antifreeze protein III (AFP III) on frozen–thawed semen from eight adult dogs using a Tris–fructose extender. Ejaculates were divided and diluted with a standard Tris–fructose–egg yolk extender containing MitoQ (200 nM/mL) and AFP III (0.75, 1.0, 2.0 µg/mL), individually or combined. Post-thaw, samples were evaluated for motility, viability, membrane and acrosome integrity, lipid peroxidation, apoptosis indicators, mitochondrial function, and reactive oxygen species (ROS-H₂O₂). The results showed significant (p < 0.05) improvements in motility rate, progressive motility, VAP, VSL, VCL, ALH, and BCF with MitoQ or AFP alone. AFP III (0.75, 1.0 µg/mL) showed higher values than controls (p > 0.05), while MitoQ alone showed no significant effect. Viability and acrosome integrity improved with AFP III. Membrane integrity and lipid peroxidation were better in 0.75 and 1.0 µg/mL AFP III groups. ROS-H₂O₂ levels and mitochondrial membrane potential were unaffected except at 1.0 µg/mL AFP III. The phosphatidylserine translocation assay showed no significant differences in dead sperm between controls and individual treatments, but significant differences occurred with combined MitoQ/AFP III. In conclusion, AFP III and MitoQ in diluents protect canine sperm cells from cryodamage. Full article

There are many applications of soybean lecithin (SL) and cholesterol-loaded cyclodextrin (CLC) in sperm freezing processes. To the best of our knowledge, there have been few cases of the combined use of SL and CLC in freezing rooster semen. We investigated the effects of CLC, SL, and their combination on rooster sperm cryodamage. Three experiments were conducted: experiment 1, SL (0.5%, 1%, 1.5%, 2.0%); experiment 2, CLC (1.25 mg, 2.5 mg, 5 mg, 10 mg, 15 mg); and experiment 3, CLC + SL (2.5 mg + 0.25%, 2.5 mg + 0.5%, 2.5 mg + 1%, 2.5 mg + 1.5%). Semen samples were cryopreserved using a programmed cryostat, followed by the determination of post-thaw sperm quality, antioxidant indices, and hatching. The results showed that the combination of 2.5 mg CLC + 0.5% SL had the most significant synergistic effect on cryodamage, and the viability (56.69%), motility (54.35%), mitochondrial activity (54.23%), plasma membrane integrity (53.52%), acrosome integrity (54.71%), and antioxidant activity (MDA concentration: 5.65 nmol/mL; SOD activity: 152.73 U/mL) were significantly greater than those of the other combinations (p < 0.05). Nevertheless, the combined CLC and SL addition group did not substantially increase the fertilization and hatching rates of frozen semen compared with the addition of 2.5 mg CLC. In conclusion, the addition of 2.5 mg CLC and 2.5 mg CLC + 0.5% SL enhanced the quality and fertility of frozen rooster sperm. Full article

Background/Objectives: Semen cryopreservation is routinely performed in fertility clinics for a variety of reasons, including fertility preservation and storage of donor sperm, yet the freeze–thaw process leads to cellular damage via ice crystal formation, osmotic shock, and supraphysiological levels of oxidative stress. Sperm resistance to damage during the freeze–thaw process varies widely, yet the intrinsic factors associated with sperm cryotolerance are largely unknown. The study aimed to investigate whether poor chromatin condensation renders sperm vulnerable to DNA fragmentation and cell death induced by the freeze–thaw process. Methods: Participants (n = 51) from the general community who met the inclusion criteria collected a semen sample after 3–8 days of abstinence. Neat semen samples underwent traditional semen analysis, aniline blue (AB)-eosin staining for chromatin condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay for DNA fragmentation, and the Annexin V assay for apoptosis/necrosis, prior to being cryopreserved using the liquid nitrogen vapour method and stored at −196 °C. Stored samples were later thawed at room temperature and processed using density gradient centrifugation. Motile sperm concentration, DNA fragmentation and apoptosis/necrosis were analysed in post-thaw samples. Results: As indicated by a significant interaction effect in linear mixed models, an increased proportion of AB-positive sperm in the pre-freeze sample exacerbated the adverse effect of freezing on sperm DNA fragmentation (p = 0.004), late apoptosis (p = 0.007), and necrosis (p = 0.007). AB-staining was positively correlated with all three parameters in the post-thaw sample (all r_s ≥ 0.424, all p < 0.01) and remained significant after adjusting for neat sperm concentration (all partial r_s ≥ 0.493, all p < 0.01). Similarly, AB-staining was significantly correlated with the percentage point change in sperm DNA fragmentation (r_s = 0.366, p = 0.014) and necrosis (r_s = 0.403, p = 0.009), both of which remained significant after adjusting for neat sperm concentration (both partial r_s ≥ 0.404, both p < 0.01), and borderline significantly correlated with percentage point change in late apoptosis (r_s = 0.307, p = 0.051). Conclusions: Sperm with poorly condensed chromatin may be more susceptible to cellular damage during the freeze–thaw process, independent of pre-freeze sperm concentration. These findings may help to explain the intrinsic variation in sperm resistance to cryodamage within and between individuals that is poorly understood. 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

Orange Bombax ceiba (B. ceiba) is an indigenous plant, and its stamen is an important ingredient in traditional Lanna food. There are limitations in scientific reports on the effects of the biological activities of B. ceiba stamens on the male reproductive system. This study aims to investigate the phytochemical compounds of the orange B. ceiba stamen and its potential effect on the antioxidant properties and quality of cattle sperm treated with Fe. The orange BUE had the highest total phenolics, total tannins, total monomeric anthocyanins, and maximal antioxidant potential. The orange BAE had the highest concentration of total flavonoids. LC-QTOF/MS showed that the orange BUE contained the highest number of phytochemical compounds related to male reproductive enhancement. The orange BUE enhanced sperm motility, and both the orange BUE and the BAE enhanced sperm viability and normal sperm morphology via free radical scavenging. It might be suggested that B. ceiba stamens have benefits for sperm preservation, sperm quality, and increasing the economic value of local plants, and that they may be developed and used to guard against oxidative stress from cryodamage induced by frozen semen technology. Full article

High mortalities and highly variable results during the subsequent development of post-thaw larvae have been widely considered as key issues restricting the application of cryopreservation techniques to support genetic improvement programs and hatchery production in farmed marine bivalve species. To date, few studies have been undertaken to investigate the effects of cryodamage at the molecular level in bivalves. This study is the first to evaluate the effect of larval cryopreservation on the epigenetics of the resultant progenies of the Pacific oyster Crassostrea gigas. The results show that the level of DNA methylation was significantly (p < 0.05) higher and lower than that of the control when the trochophore larvae were revived and when they developed to D-stage larvae (day 1 post-fertilization), respectively, but the level returned to the control level from day 8 post-fertilization onwards. The expression of the epigenetic regulator genes DNMT3b, MeCP2, JmjCA, KDM2 and OSA changed significantly (p < 0.05) when the trochophore larvae were thawed, and then they reverted to the control levels at the D- and later larval developmental stages. However, the expression of other epigenetic regulator genes, namely, MBD2, DNMT1, CXXC1 and JmjD6, did not change at any post-thaw larval developmental stage. For the newly thawed trochophore larvae, the amount of methylated H3K4Me1 and H3K27Me1 significantly changed, and the expression of all Jumonji orthologs, except that of Jumonji5, significantly (p < 0.05) decreased. These epigenetic results agree with the data collected on larval performances (e.g., survival rate), suggesting that the effect period of the published cryopreservation technique on post-thaw larvae is short in C. gigas. Full article

To preserve male fertility after diagnosis of any kind of cancer, a prompt assessment of the semen quality and an appropriate semen cryopreservation must be performed before radio-chemotherapy starts. The present work aims to evaluate the semen parameters at diagnosis of different cancer patients before cryopreservation and after thawing. Testicular tumors and lymphomas are among the most common cancers in younger patients, and while chemotherapy significantly increases patients’ survival, it can epigenetically alter the semen fluid, resulting in temporary or permanent infertility. We analyzed data from the database of the Gamete Cryopreservation Center (Annunziata Hospital, CS; Italy) in the period of 2011–2020 from a cohort of 254 cancer patients aged 18–56 years. The evaluation was performed in a blind manner and anonymously recovered; the main parameters referring to semen quality were assessed in accordance with the WHO guidelines and decision limits (6th edition; 2021). The cancer types were as follows: testis cancers (TC; n = 135; 53.1%), hematological cancers (HC; n = 76; 29.9%), and other types of cancer (OC; n = 43; 17%). Comparing TC vs. HC (P₁) and vs. OC (P₂), TC had the worst semen quality: sperm number/mL (P₁ = 0.0014; P₂ = 0.004), total motility (P₁ = 0.02; P₂ = 0.07), progressive motility (P₁ = 0.04; P₂ = 0.05), viability (P₁ = 0.01; P₂ = 0.02), and percentage of atypical morphology (P₁ = 0.05; P₂ = 0.03). After semen thawing, viability and progressive motility recovery lowered, accounting for 46.82% and 16.75%, respectively, in the whole cohort; similarly, in the subgroups ascribed to TC, they showed the lowest recovery. Strong correlation existed between pre- and post-cryopreservation viability and progressive motility in the whole cohort (p < 0.001) and in the TC subgroup (p < 0.05). All cancer subgroups, to significantly different extents, had semen findings below the WHO reference values, suggesting diverse sperm susceptibilities to different cancers and cryodamage. Cancer and associated treatments epigenetically affect patients’ semen quality, meaning cryopreservation should be considered a useful personalized prerogative for any kind of cancer in a timely manner. Full article

Background: Sperm cryopreservation is recommended to preserve male fertility for cancer patients or other medical conditions at risk of sperm decline. Whether motility and viability recovery rates vary depending on the medical conditions requiring cryopreservation is poorly known. We report here on the 24-year experience of our semen bank. Methods: Motility and viability recovery rates were evaluated in 1973 collections from patients with various medical conditions and 67 collections from donors, and the results were related to basal semen quality. Results: Motility and viability recovery were highly related to basal semen quality and varied between cancer and non-cancer conditions, independently of the duration of cryopreservation and patient age. In samples with a sperm number below 2 × 10⁶/mL, recovery rates approximated to zero. The highest recovery rates were found in donor collections. Cut-off values for the recovery of at least 1% motile spermatozoa were established based on initial semen quality. Conclusions: Our results indicate that the occurrence of any pathological or medical condition resulted in lower recovery rates with respect to donors, indicating that intrinsic sperm characteristics drive susceptibility to cryodamage. Established cut-off values for motility recovery can be useful for patient counseling as well as for ART laboratories to decide the type of procedure. Full article

Cryodamage affects the normal physiological functions and survivability of boar sperm during cryopreservation. Lysine acetylation is thought to be an important regulatory mechanism in sperm functions. However, little is known about protein acetylation and its effects on cryotolerance or cryodamage in boar sperm. In this study, the characterization and protein acetylation dynamics of boar sperm during cryopreservation were determined using liquid chromatography–mass spectrometry (LC-MS). A total of 1440 proteins were identified out of 4705 modified proteins, and 2764 quantifiable sites were elucidated. Among the differentially modified sites, 1252 were found to be upregulated compared to 172 downregulated sites in fresh and frozen sperms. Gene ontology indicated that these differentially modified proteins are involved in metabolic processes and catalytic and antioxidant activities, which are involved in pyruvate metabolism, phosphorylation and lysine degradation. In addition, the present study demonstrated that the mRNA and protein expressions of SIRT5, IDH2, MDH2 and LDHC, associated with sperm quality parameters, are downregulated after cryopreservation. In conclusion, cryopreservation induces the acetylation and deacetylation of energy metabolism-related proteins, which may contribute to the post-thawed boar sperm quality parameters. Full article

The cryopreservation of human spermatozoa has been an option for patients undergoing chemo or radiotherapies since the late 1950s. Presently, there are different techniques for the cryopreservation of spermatozoa. The most commonly used techniques are programmable slow freezing and freezing on liquid nitrogen vapors, while the use of vitrification is still not accepted as clinically relevant. Although there have been many improvements, the ideal technique for achieving better post-thaw sperm quality continues to be a mystery. A major obstacle during cryopreservation is the formation of intracellular ice crystals. Cryodamage generated by cryopreservation causes structural and molecular alterations in spermatozoa. Injuries can happen because of oxidative stress, temperature stress, and osmotic stress, which then result in changes in the plasma membrane fluidity, motility, viability, and DNA integrity of the spermatozoa. To prevent cryodamage as much as possible, cryoprotectants are added, and in some clinical trial cases, even antioxidants that may improve post-thaw sperm quality are added. This review discusses cryopreservation techniques, cryodamage on molecular and structural levels, and cryoprotectants. It provides a comparison of cryopreservation techniques and describes recent advances in those techniques. Full article