Search Results (131)

Several factors, including semen quality, can influence fertilisation success. Poor semen parameters may necessitate more frequent inseminations or the removal of males with consistently low fertility. This study evaluated turkey ejaculates (n = 37) with good fertility (GF) and impaired fertility (IF). The analyses included sperm motility parameters (total motility—TMOT, progressive motility—PMOT, curvilinear velocity—VCL, straight-line velocity—VSL, average path velocity—VAP, linearity—LIN, straightness—STR, amplitude of lateral head displacement—ALH, and beat cross frequency—BCF), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and nitric oxide (NO) production, as well as enzymatic and biochemical assays of semen, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) activities, glutathione (GSH) content, malondialdehyde (MDA) levels, and zinc (Zn²⁺) concentration. In parallel, the proteomes of seminal plasma and spermatozoa were separated using SDS- and Tricine-PAGE, and selected proteins were identified by nano LC-MS/MS. Spermatozoa derived from IF ejaculates exhibited significantly reduced TMOT (p = 0.002), VCL (p = 0.028), and PMI (p = 0.000), accompanied by elevated STR (p = 0.000) and NO production (p = 0.044). In the seminal plasma of IF males, a significant decrease was noted in SOD (p = 0.000) and GPx (p = 0.001) activities, whereas CAT activity was markedly higher (p = 0.014). Seminal fluid from IF ejaculates was also characterised by increased GSH (p = 0.014) and MDA (p = 0.014) concentrations, accompanied by reduced Zn²⁺ content (p = 0.014). In contrast, IF spermatozoa exhibited elevated SOD activity (p = 0.001), but reduced GPx (p = 0.000) and CAT (p = 0.012) activities. Sperm cells from IF ejaculates also had lower GSH levels (p = 0.000), higher MDA concentrations (p = 0.000), and increased Zn²⁺ content (p = 0.018) compared with those from GF ejaculates. A proteomic analysis revealed differences in fertility-associated proteins: peroxiredoxin 6 (PRDX6) was detected exclusively in GF semen, whereas alpha-enolase (ENO1), fatty acid-binding protein (FABP7), cytoplasmic aspartate aminotransferase (GOT1), and L-lactate dehydrogenase B (LDHB) were detected only in IF semen. Overall, the results demonstrate that both semen parameters and proteome composition may potentially affect the fertilisation outcomes in turkeys. Full article

The Zi goose is an excellent local goose breed in China, characterized by strong tolerance to roughage, high cold resistance, and high egg production. Anthocyanins are natural water-soluble pigments with numerous biological functions such as growth promotion, antioxidant activity, and immune regulation. However, there are few reports on whether anthocyanins have an improving effect on Zi goose semen. This study aimed to explore the effects of anthocyanins on semen quality, semen antioxidant capacity, and sperm apoptosis of Zi geese, so as to provide references for the large-scale breeding and industrial utilization of Zi geese. Sixty 12-month-old Zi geese were selected for the experiment, and their semen was collected by the massage method. Semen diluent containing different concentrations of anthocyanins was added to the mixed semen, which was then stored at 37 °C for detections. The results showed that the sperm survival rate of Zi geese was the highest when 30 mg/L of anthocyanins was added to the diluent. Compared with the control group, the anthocyanin group showed significantly higher sperm survival rate, sperm motility, plasma membrane integrity rate, acrosome integrity rate, mitochondrial activity, and DNA integrity (p < 0.05), while the sperm mortality rate was significantly decreased (p < 0.05). There were no significant differences in semen pH value, sperm density, and sperm abnormality rate (p > 0.05). The contents of superoxide dismutase and glutathione peroxidase were significantly increased (p < 0.05), while the levels of reactive oxygen species and malondialdehyde were significantly decreased (p < 0.05) in the anthocyanin group. Anthocyanins significantly increased the mRNA and protein expression of Bcl-2 (p < 0.05), and significantly decreased the mRNA and protein expression of Bax, Caspase-3, and P53 (p < 0.05). In conclusion, adding 30 mg/L anthocyanins to semen diluent can improve semen quality in Zi geese. Full article

Ultrasonic treatment significantly improves the emulsifying properties of chicken egg yolk. This advancement not only provides a novel approach for enhancing the physical stability of yolk-based cryodiluents, but also holds promising implications for optimizing the cryopreservation efficacy of boar semen. This study evaluated the effects of conventional egg yolk (CON) and ultrasonicated egg yolk (UT-CEY) on boar semen cryopreservation. Semen samples were cryopreserved using standard straw freezing methods, with post-thaw sperm quality parameters assessed. Results demonstrated that UT-CEY significantly reduced yolk particle size (p < 0.01), improved emulsion stability (p < 0.01), and decreased creaming index (p < 0.05). Additionally, UT-CEY enhanced total motility, progressive motility, straight-line velocity (VSL), and plasma membrane integrity (p < 0.01), along with acrosome integrity (p < 0.05) compared to CON. Furthermore, catalase (CAT) and superoxide dismutase (SOD) activities were elevated in UT-CEY (p < 0.01), while reactive oxygen species (ROS) fluorescence intensity showed no significant difference (p >0.05). Gene expression analysis revealed upregulated Bcl-2, CAT (p < 0.01), and SOD₂ (p < 0.05) in UT-CEY. In conclusion, ultrasonicated egg yolk diluent improves boar semen cryopreservation efficiency and post-thaw sperm quality. 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

The evaluation of sperm proteins has emerged as a promising approach to predicting semen quality across animal species. This study investigated the relationship between post-thaw concentrations of precursor A-kinase anchor protein 4 (proAKAP4) and objective sperm quality parameters in goats. Semen was collected from 16 adult goats (Boer, n = 8; Anglo-Nubian, n = 8) and frozen using a standardized protocol with OptiXcell (IMV Technologies, l′Aigle, France) extender (n = 5). After thawing, proAKAP4 concentrations were measured with an enzyme-linked immunosorbent assay (ELISA), while sperm motility and kinematics were assessed with computer-assisted analysis (CASA), and viability, plasma membrane integrity, acrosome integrity, and mitochondrial activity were evaluated using flow cytometry. Samples were grouped according to low, medium, or high proAKAP4 levels for comparison, and correlations with sperm parameters were examined. The results showed that semen with higher proAKAP4 concentrations had significantly greater total and progressive motility, more favorable kinematic values, and improved viability, plasma membrane integrity, and mitochondrial function (p < 0.05), whereas acrosome integrity was not influenced (p > 0.05). The average post-thaw proAKAP4 concentration was 38.66 ± 1.11 ng/10⁶ sperm, and no differences were observed between Boer and Anglo-Nubian breeds (p > 0.05). These findings indicate that proAKAP4 is strongly associated with multiple sperm functional traits and may serve as a reliable biomarker for assessing post-thaw semen quality in goats. Full article

HP, as an isotropic physical stress, has been widely applied in cell biology and reproductive research to simulate the effects of environmental pressure on cellular functions. In this study, the elastic silicone membrane of a novel bionic insemination catheter was employed as the pressure medium, with semen perfused into a sealed silicone chamber. As the silicone membrane underwent controlled deformation, the liquid inside the chamber generated a nearly uniform isotropic pressure, thereby maintaining spermatozoa in a stable HP environment. Boar sperm are susceptible to physiological and functional damage under HP stress, which can impair fertilization capacity. This study aimed to investigate the effects of TMAO, BET, or their combination on the quality of semen from eight Landrace boars under HP during storage at 17 °C (experiment repeated three times). Semen was collected using the manual collection method and treated with different concentrations of TMAO or BET. Sperm motility parameters were assessed using a CASA system to determine the optimal concentrations. Subsequently, experimental groups were established: the fresh group, HP control group, T group (optimal TMAO), B group (optimal BET), and H group (optimal TMAO + BET). The results showed that the optimal concentrations were 8 mmol/L for TMAO and 20 mmol/L for BET. Compared with the HP control group, the T, B, and H groups showed significantly improved sperm viability, mitochondrial membrane potential (MMP), and plasma membrane integrity (p < 0.05), and significantly reduced DFI, ROS, MDA, and NO contents (p < 0.05), while acrosome integrity showed no significant differences (p > 0.05). Additionally, the B group showed significantly increased T-AOC (p < 0.05). Non-targeted lipidomic analysis revealed 49 differential lipids in the T group, 262 in the B group, and 269 in the H group compared with the HP control. These differential lipids were mainly associated with PC, AcCa, and sphingolipid signaling pathways, with key sphingolipid pathway lipids including Cer, SM, and DG. These findings indicate that BET and TMAO + BET improve HP-induced sperm damage by modulating the sphingolipid signaling pathway and maintaining PC and AcCa levels, whereas TMAO alone may exert protective effects through additional mechanisms. In conclusion, TMAO, BET, or their combination effectively mitigates the detrimental effects of HP on boar sperm. Full article

This research explored the effects of different concentrations of p-coumaric acid (PCA) on the quality of frozen-thawed boar semen. Boar sperm samples were pre-treated with different concentrations of PCA (0, 30, 60, 90, 120 μg/mL) prior to the freezing process. Subsequently, multiple parameters were analyzed post-freeze-thawing, including sperm morphological and kinetic characteristics, acrosome and membrane integrity, mitochondrial function, DNA integrity, antioxidant enzyme activities, the expression levels of the BCL-2, BAX, and Caspase-3 proteins, the in vitro fertilization rate of porcine oocytes, and the embryo cleavage rate. The findings indicated that, compared with the control group, the addition of 90 μg/mL PCA led to significant improvements in several key aspects. Sperm motility, average path velocity, straight-line velocity, curvilinear velocity, and beat cross frequency were all notably enhanced. Moreover, parameters related to sperm quality, such as acrosome integrity, plasma membrane integrity, mitochondrial activity, and DNA integrity, also showed significant increases (all p < 0.05). In terms of antioxidant capacity, the 90 μg/mL PCA treatment significantly elevated the total antioxidant capacity, as well as the activities of superoxide dismutase, glutathione peroxidase, and catalase. Simultaneously, it caused a significant reduction in the contents of malondialdehyde and hydrogen peroxide (p < 0.05). Regarding protein expression, the addition of 90 μg/mL PCA significantly upregulated the expression level of the BCL-2 protein, while downregulating the relative expression levels of BAX and Caspase-3 (p < 0.05). Additionally, this concentration of PCA significantly improved the in vitro fertilization rate of porcine oocytes and the embryo cleavage rate (p < 0.05). In conclusion, incorporating PCA into the semen extender can potentially be advantageous for the cryopreservation of boar sperm, with 90 μg/mL being the optimal concentration. Full article

Mammalian sperm has a high metabolic activity and primarily relies on glycolysis and mitochondrial oxidative phosphorylation for energy supply. It has been reported that imiquimod (R837), a specific ligand of TLR7 protein, reduces the motility of sperm. However, the specific molecular mechanism by which TLR7 modulates boar sperm is unclear. In this study, the effect of R837, a Toll-like receptor 7 (TLR7) agonist, on boar sperm motility was investigated. Sperm samples were incubated with varying concentrations of R837 (0 to 0.8 µM) at different time points (30, 60, and 90 min). Findings reveal for the first time that TLR7 protein, a key component of the immune system’s Toll-like receptors, is predominantly localized in the middle section of the boar sperm tail, with a smaller concentration observed in the neck. Also, immunofluorescence (IF) revealed that approximately half of the boar sperm sample expressed TLR7. Furthermore, the TLR7 agonist influenced glycolytic hexokinase activity and mitochondrial function via the PI3K-GSK3β signaling pathway. It also selectively inhibited motility in the lower-layer sperm, while motility in the upper-layer sperm remained unaffected. Additionally, this study determined that incubation conditions for boar sperm with 0.2 μM R837 at 37 °C for 60 min yielded the most pronounced inhibition of forward motility in the lower layer of sperm, without compromising the integrity of the acrosome or plasma membrane. The present study reveals the crucial role of R837 in boar sperm motility and highlights TLR7 as an important protein that regulates boar sperm energy metabolism. Full article

Semen cryopreservation is associated with sperm vulnerability to oxidative stress and ice crystal-induced damage, adversely affecting in vitro fertilization (IVF) success. This study aimed to investigate the effects of freezing diluent supplemented with antioxidant limonin (Lim), myo-inositol (MYO), and the ice crystal formation inhibitor L-proline (LP) through sperm motility, morphological integrity, and antioxidant capacity. The Lim (150 mM), MYO (90 mM), and LP (100 mM) significantly ameliorated the quality of post-thaw sperm in Debao boar, and combined treatment of these agents significantly enhanced sperm motility, structural integrity, and antioxidant capacity compared with individual agents (p < 0.05). Notably, the combined use of these agents reduced glycerol concentration in the freezing diluent from 3% to 2%. Meanwhile, the integrity of the sperm plasma membrane, acrosome membrane, and mitochondrial membrane potential was significantly improved (p < 0.05), and the result of IVF revealed the total cell count of the blastocysts was also greater in the 2% glycerol group (p < 0.05). In conclusion, the newly developed freezing diluent for semen, by adding Lim (150 mM), MYO (90 mM), and LP (100 mM), can enhance the quality of frozen–thawed Debao boar sperm and reduce the concentration of glycerol from 3% to 2% as high concentrations of glycerol can impair the quality of thawed sperm and affect in vitro fertilization outcomes. In conclusion, the improved dilution solution formulated demonstrated efficacy in enhancing the quality of porcine spermatozoa following cryopreservation and subsequent thawing. Full article

The development of biometric techniques in domestic animals has greatly advanced scientific practices in wildlife research. The association between seminal characteristics and body and testicular biometry enables the selection of suitable breeders, though appropriate measurement techniques are required. The present study assessed differences among conventional methods and formulas for estimating testicular parameters. Testicular length, width, and thickness were measured using three methods in 13 adult male domestic cats. Testicular area, volume, and weight were estimated, from which the gonadosomatic index (GSI) was calculated. Sperm were collected using an alpha-2 adrenergic agonist and urethral catheterization, and characterized in terms of volume, vigor, total motility, progressive motility, concentration, plasma membrane integrity, and morphology. The three methods were consistent in terms of testicular area, volume, weight, and GSI. Moderate positive correlations were observed for testicular weight (r = 0.61, p < 0.05) and GSI (r = 0.58, p < 0.05). Testicular parameters showed strong positive correlations among each other (r > 0.80, p < 0.05). We observed a moderate positive correlation between head length and progressive motility (r = 0.65, p < 0.05). In conclusion, all testicular measurement and estimation techniques showed comparable performance. Therefore, testicular biometry is useful for selecting breeding males in feline conservation programs, wherein larger body biometrics are related to improved seminal and reproductive parameters. Full article

Semen collection is an important component of conservation and animal husbandry. Semen quality is generally improved using voluntary collection methods, particularly artificial vaginas (AVs). Most commercially available AVs are tube-shaped with few species-specific design augmentations. As genitalia are highly variable across taxa, incorporating species-specific genital morphologies into AV designs may enhance collected semen quality. We compared dolphin semen quality using: (1) silicone bioinspired artificial vaginas (BAVs) that reflect the internal shape of dolphin vaginas, and (2) manual stimulation. Sperm motility and kinematic parameters of five bottlenose dolphins (Tursiops sp.) were assessed using computer-aided sperm analysis (CASA). Sperm collected using BAVs showed non-significant increases in median progressive and rapid motility, and increases in median and mean linear motility, supporting a sexual selection functional hypothesis for the biodiverse vaginal folds unique to whales, dolphins, and porpoises. Sperm concentration decreased with BAV collection, while no consistent trends were detected in volume, pH, velocity, or plasma membrane integrity. Modifications to AVs for other species that incorporate genital morphologies may also optimize collected semen quality for application to artificial insemination. 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

This study optimized the cryopreservation protocol for cashmere goat semen by testing centrifugation speeds (750, 1000, 1250, 1500 rpm) for seminal plasma removal and L-proline concentrations (10, 30, 50 mmol/L) in a freezing extender. Semen from six 3-year-old breeding bucks of Inner Mongolia cashmere goats was evaluated post-thaw in terms of motility, membrane integrity, antioxidant capacity, and artificial insemination (AI) outcomes (n = 130 does). The results demonstrated that the group that underwent centrifugation at 1250 rpm saw significantly improved sperm motility (p < 0.05), curvilinear velocity (VCL, p < 0.05), and straight-line velocity (VSL, p < 0.05) compared to the other groups. The addition of 30 mmol/L L-proline further enhanced post-thaw sperm motility (p < 0.05), plasma membrane integrity (p < 0.05), and acrosome integrity (p < 0.05), while significantly reducing reactive oxygen species (ROS, p < 0.05) and malondialdehyde (MDA, p < 0.05) levels. This group also exhibited the highest antioxidant capacity, as indicated by elevated levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (p < 0.05). AI trials revealed that semen treated with 1250 rpm centrifugation and 30 mmol/L L-proline achieved the highest kidding rate (56.82%), significantly outperforming the control group (37.21%, p < 0.05). Meanwhile, no significant differences were observed in prolificacy or offspring sex ratio (p > 0.05). In conclusion, this study demonstrates that combining 1250 rpm centrifugation for seminal plasma removal with the addition of 30 mmol/L L-proline to the freezing extender significantly improves the quality of cryopreserved cashmere goat semen and enhances AI outcomes. 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 quality is adversely affected by cryopreservation due to the increased production of reactive oxygen species, which affects the integrity of sperm membranes, motility, and DNA fragmentation. Three methods for removing seminal plasma, washing (centrifuging extended semen at 800× g for 10 min) and Single Layer Centrifugation with high or low density Equicoll, were used to prepare 29 ejaculates from ten stallions for freezing. Sperm quality parameters (kinematics, plasma membrane integrity, superoxide and hydrogen peroxide production, mitochondrial membrane potential, and DNA fragmentation) were evaluated before and after freezing using kinematic and flow cytometric analysis. The parameters for fresh samples were within the normal range for stallion semen but were lower after thawing. There were few differences between the three preparation methods. Interestingly, DNA fragmentation was affected most by the sperm preparation method, being lowest for SLC through high density Equicoll, although SLC through low density Equicoll was effective for some stallions. Some differences were observed in the proportions of live or dead spermatozoa positive for hydrogen peroxide. In conclusion, all of these methods would be suitable for the preparation of semen prior to cryopreservation, but Single Layer Centrifugation through high density Equicoll was the most effective in removing spermatozoa with damaged DNA. Full article