Boar sperm are prone to functional deterioration during incubation, which is commonly associated with reduced motility, impaired mitochondrial function, and insufficient adenosine triphosphate (ATP) production. This study investigated whether glycine supplementation improves boar sperm progressive and linear motility by enhancing mitochondrial energy metabolism through the 5-aminolevulinic acid (5-ALA)/heme-associated mitochondrial pathway. Boar sperm samples were incubated with different concentrations of glycine (0, 0.1, 1, and 10 mM) at 37 °C for 6 h, and sperm motility was assessed at 0, 1, 3, and 6 h. Mitochondrial membrane potential (MMP), ATP levels, 5-ALA levels, heme levels, and mitochondrial complex IV activity were also evaluated. Compared with the control group, addition of 1 mM glycine significantly increased sperm total motility, progressive motility, average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), and linearity (LIN) after 3 h of incubation (P < 0.05), whereas 10 mM glycine treatment reduced sperm motility. Glycine supplementation also significantly enhanced MMP, ATP levels, 5-ALA levels, heme levels, and complex IV activity (P < 0.05). To further evaluate the involvement of the 5-ALA/heme-associated mitochondrial pathway, exogenous 5-ALA supplementation and pharmacological inhibition of 5-aminolevulinate synthase (ALAS) and 5-aminolevulinate dehydratase (ALAD) were performed. Exogenous 5-ALA reproduced several glycine-induced improvements in sperm motility and mitochondrial energy metabolism, whereas addition of ALAS or ALAD inhibitors attenuated the beneficial effects of glycine on sperm motility, ATP levels, and heme levels (P < 0.05). In addition, artificial insemination with glycine-supplemented semen significantly increased total born litter size without significantly affecting sow pregnancy rate. These findings demonstrate that glycine improves boar sperm progressive and linear motility by enhancing mitochondrial energy metabolism. Therefore, glycine may serve as a promising metabolic additive for improving boar sperm quality and reproductive performance.

Oxidative damage is closely associated with the gradual decline in boar sperm quality during liquid storage at 17 °C. To determine whether L-histidine (L-His) could limit this deterioration, semen was diluted with an extender containing 0, 1, 10, 100, or 1000 μM L-His. Motility and acrosomal integrity were measured throughout storage. On day 7, mitochondrial membrane potential (MMP) and ATP content were determined together with intracellular probe oxidation, membrane lipid oxidation, and Annexin V-FITC/PI staining patterns. The stored sperm were subsequently exposed to capacitating conditions and tested for their ability to bind to oviductal explants. The most favorable responses were observed with 100 μM L-His. Compared with untreated semen, this group retained higher motility and acrosomal integrity and showed higher MMP and ATP content (p < 0.05). It also exhibited less intracellular probe oxidation and membrane lipid oxidation, together with lower proportions of Annexin V-positive and membrane-compromised sperm. Following capacitation induction, sperm stored with 100 μM L-His showed increased tyrosine phosphorylation and higher proportions of capacitated sperm. Their binding index to oviductal explants was also higher than that of the control group. These findings indicate that supplementation with 100 μM L-His can improve the preservation quality of boar sperm during extended liquid storage at 17 °C and maintain functional characteristics of stored sperm, providing new insights into the potential use of L-His as a semen extender supplement for prolonged storage.

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.

Boar sperm are highly susceptible to oxidative stress during in vitro storage, and the inclusion of antioxidants in semen extenders has become an essential strategy to mitigate oxidative damage. This study evaluated the effects of succinic acid (SA) supplementation on boar semen quality and reproductive performance. In Experiment 1, boar semen was diluted in Modena extender supplemented with 1.5, 3.0, or 4.5 mM SA and stored at 17 °C. The addition of 3.0 mM SA significantly improved sperm motility and the proportion of viable sperm with an intact acrosome (p < 0.05). Notably, it enhanced mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content, glutathione (GSH) concentration, and glutathione peroxidase (GPx) activity, while markedly reducing lipid peroxidation (LPO), malondialdehyde (MDA) levels, and mitochondrial reactive oxygen species (ROS) production (p < 0.05). In Experiment 2, oxidative stress was induced using 100 μmol/L hydrogen peroxide (H2O2) to further assess the cytoprotective effect of SA. Supplementation with 3.0 mM SA effectively counteracted H2O2-induced reductions in sperm motility and acrosome integrity in viable sperm and restored MMP, ATP content, GSH levels, and GPx activity (p < 0.05), and significantly decreased ROS and MDA levels (p < 0.05). In Experiment 3, boar semen stored for five days was used for artificial insemination to determine the impact of SA supplementation on fertility outcomes. Inclusion of SA in the extender resulted in a significant increase in both the total number of piglets born and the number of live piglets born per litter (p < 0.05). Taken together, these results demonstrate that supplementation of boar semen extenders with 3.0 mM SA provides robust protection against oxidative stress, preserves mitochondrial function, and improves overall sperm quality during storage at 17 °C, and importantly, these cellular benefits translate into enhanced reproductive performance following artificial insemination.

Cryopreservation of sperm is an essential approach for the preservation and utilization of poultry genetic resources. However, during the freezing process, drake sperm often suffers structural damage caused by low-temperature stress and physical injury, which impairs mitochondrial function and markedly reduces post-thaw sperm quality and fertilizing ability. In this present study, different concentrations of 5-aminolevulinic acid (5-ALA; 0, 100, 250, 500, and 1000 μM) were supplemented into the freezing extender, and several key parameters of post-thaw drake sperm were evaluated to investigate the potential role of 5-ALA in semen cryopreservation. The results demonstrated that supplementation with 500 μM 5-ALA significantly improved post-thaw sperm motility, acrosome integrity, and viability compared to the control group (P < 0.05). Further analyses revealed that treatment with 500 μM 5-ALA significantly increased the heme levels in sperm (P < 0.05), enhanced the activity of mitochondrial complex IV, and elevated both mitochondrial membrane potential (MMP) and adenosine triphosphate ATP levels (P < 0.05), indicating that 5-ALA facilitated mitochondrial energy metabolism. In addition, 500 μM 5-ALA treatment significantly reduced sperm reactive oxygen species (ROS) levels, lipid peroxidation (LPO) content, apoptosis levels and DNA fragmentation (P < 0.05). This indicates that 5-ALA can reduce the damage caused by oxidative stress during freezing, inhibit LPO reactions, indirectly protect the integrity of sperm membrane and nucleus, and thus maintain the survival ability of sperm. Taken together, these findings indicate that supplementation with an appropriate concentration of 5-ALA effectively supports the maintenance and recovery of mitochondrial function. Thereby improving the efficiency of the electron transport chain and enhancing ATP production to meet the energy demands of sperm. Moreover, by alleviating the damage caused by oxidative stress to sperm, the integrity of sperm structure has been stabilized, and ultimately contributes to the enhancement of post-thaw sperm motility.

Due to oxidative damage and mitochondrial dysfunction, boar semen cryopreservation remains a significant challenge. This study investigates the effects of pyrroloquinoline quinone (PQQ), a mitochondrial-targeted antioxidant, on the post-thaw boar sperm quality during cryopreservation. Boar semen was diluted in a freezing extender containing different concentrations of PQQ (0, 10, 100, 1000, 10,000 nM). After freezing-thawing, the sperm motility, viability, acrosome integrity, mitochondrial activity, adenosine triphosphate (ATP) levels, DNA integrity, malondialdehyde (MDA) levels, reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity, mitochondrial transcription proteins levels, and fertilization capacity were assessed. The results show that 1000 nM PQQ supplementation to the freezing extender significantly enhanced post-thaw sperm motility, viability, and acrosome integrity compared to the control (p < 0.05). Additionally, 1000 nM PQQ increased mitochondrial membrane potential (MMP) and ATP levels, while decreasing MDA and mitochondrial ROS levels, and reducing DNA damage (p < 0.05). Furthermore, the levels of mitochondrial-encoded proteins were significantly elevated in the 1000 nM PQQ group compared to the control (p < 0.05). Interestingly, sperm in the 1000 nM PQQ group showed a higher binding rate to oviductal epithelial cells and the zona pellucida (ZP), indicating higher fertilization potential. These findings suggest that the use of mitochondria-target antioxidant, PQQ, can improve post-thaw boar sperm quality and fertilization via its capacity to reduce oxidative stress and protect mitochondrial function.

Cryopreservation of semen is crucial for preserving genetic resources and maintaining biodiversity. However, the unique structural characteristics of avian sperm make them particularly susceptible to cryoinjury, often resulting in impaired motility following thawing. Currently, research on drake semen cryopreservation remains limited. In this study, an integrated proteomic and untargeted metabolomic analysis was employed to investigate the molecular mechanisms underlying cryodamage-induced impairment of drake sperm motility. The findings revealed that glycolytic enzymes and peroxisomes play essential roles in maintaining sperm function during cryopreservation. Proteomic and metabolomic profiling of fresh (FS) and frozen-thawed (FT) semen samples from drakes identified a total of 6,946 proteins and 1,568 metabolites. Comparative analysis showed that, relative to FS samples, the FT group exhibited an upregulation of 94 proteins and 80 metabolites, and a downregulation of 259 proteins and 138 metabolites. The findings of this indicate that several key antioxidant enzymes were significantly downregulated in the FT samples, suggesting a lack of capacity to eliminate excessive reactive oxygen species (ROS) generated during the freezing process. This imbalance likely contributes to oxidative stress and apoptosis in sperm cells. Additionally, suppressing the glycolytic pathway during freezing reduces ATP production, further compromising sperm motility. These results demonstrate that oxidative stress and energy metabolism disruption are major contributors to cryoinjury in drake sperm, providing potential targets for improving cryopreservation outcomes in drake species.

Goat artificial insemination has become an indispensable tool for improving reproductive outcomes by enabling more precise control over genetic transmission [...]

Mitochondrial dysfunction causes an increase in oxidative stress and depletion in ATP production. Studies have shown that mitochonic acid 5 (MA-5) improves cellular mitochondrial metabolism and ATP production. To this end, varying concentrations of MA-5 (0.01, 0.1, 1, and 10 nM) were added to a BTS extender, and thereafter, sperm motility and morphological parameters were assessed. Results indicate that adding 1 nM MA-5 significantly enhanced boar sperm motility and progressive motility, with higher values observed at the 1- and 2-h time points compared to the control. Sperm membrane and acrosome integrity values between the control and treatment groups were not different, except for 10 nM MA-5, which caused a reduction in membrane integrity. Treatment with MA-5, specifically 1 nM MA-5, also significantly boosted mitochondrial membrane potential and ATP content at the 2-h incubation point. In addition, MA-5 treatments stabilized mitochondrial transcription and translation processes, maintained overall sperm functionality, increased NADPH dehydrogenase subunits 1 (MT-ND1) and NADPH dehydrogenase subunits 6 (MT-ND6) protein and gene expression, and affected mitochondrial transcription factor A (TFAM) levels. However, after 4 h of incubation with MA-5, a decline in sperm quality parameters and an increase in ROS levels were observed. Interestingly, adding 10 nM pyrroloquinoline quinone (PQQ) and MA-5 to the extender restored mitochondrial function by enhancing mitochondrial potential and ATP content after 4 h of incubation. Overall, treatment with 1 nM MA-5 can help maintain sperm quality and mitochondrial metabolism during incubation (up to 2 h) at 37oC. Moreover, addition of a combination of MA-5 and 10 nM PQQ to the boar sperm extender is crucial for maintaining high-quality reproduction after 4 h of incubation. Consequently, MA-5 and PQQ play a role in maintaining sperm quality and mitochondrial function and can help in boar sperm preservation and artificial insemination (AI) practices.

5-Aminolevulinic acid (5-ALA) is a crucial metabolic intermediate that affect mitochondrial function in somatic cells. However, the mechanisms by which 5-ALA regulates boar reproductive performance remain unclear. The effect of dietary supplementation of 5-ALA on boar semen quality and reproductive performance were investigated in this study. Forty-five boars were randomly assigned to a control group and four 5-ALA-treatment groups (125, 250, 500, and 1000 mg/kg/d). After nine weeks of treatment, serum and semen samples were collected from the boars for analysis. Results showed that the supplementation of 5-ALA to boar diet significantly (p < 0.05) increased semen volume, total sperm count, and sperm motility while reducing the proportion of abnormality (p < 0.05). Specifically, 250 mg/kg/d 5-ALA treatment significantly (p < 0.05) reduced sperm DNA oxidative damage and elevated serum testosterone levels. Metabolomic analysis revealed that 250 mg/kg/d 5-ALA supplementation significantly increased (p < 0.05) the levels of tricarboxylic acid (TCA) cycle intermediates such as malate and isocitrate, while significantly (p < 0.05) reducing harmful metabolites such as N-acetyl phenylalanine that negatively impact sperm quality and fertility. Notably, 250 mg/kg/d 5-ALA supplementation upregulated the protein levels of mitochondrial oxidative phosphorylation (OXPHOS) complex subunits (NDUFB8, SDHB, UQCRC2, MTCO2, and ATP5A1) and improved sperm mitochondrial membrane potential, adenosine triphosphate (ATP) levels, and complex IV activity (p < 0.05). Regarding reproductive performance, compared to the control, the percentage of piglets born alive increased by 2.97 %, the farrowing rate improved by 10.59 %, and the occurrence of mummified fetuses decreased by 1.07 % in the 5-ALA-treated groups (p < 0.05). Additionally, 250 mg/kg/d 5-ALA had a long-term beneficial advantage on boar semen quality parameters. Accordingly, dietary supplementation of 5-ALA, specifically, 250 mg/kg/d 5-ALA improved boar semen quality by enhancing seminal plasma metabolome, sperm mitochondrial function and overall reproductive performance.

Mitochondrial energy metabolism is fundamental to sperm function, and fatty acid β-oxidation is an important pathway for adenosine triphosphate (ATP) production. Riboflavin, a precursor of key flavin cofactors, plays a critical role in regulating β-oxidation and supports multiple physiological processes. This study aimed to determine whether adding riboflavin to semen dilution media could enhance goat sperm motility and to elucidate the underlying metabolic mechanisms. Goat semen was diluted in tris-citrate-glucose (TCG) medium containing 0, 5, 10, 15, and 20 μM riboflavin and incubated at 37 °C, after which sperm motility, acrosome integrity, mitochondrial membrane potential, ATP levels, malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) activities, and the NADH/NAD+ were evaluated. The localization and expression of the β-oxidation enzymes carnitine palmitoyltransferase 1 (CPT1) and extremely long chain acyl-CoA dehydrogenase (ACADVL) were examined, and CPT1 activity was quantified. The results showed that CPT1 and ACADVL were present in goat sperm, and that 10 μM riboflavin significantly increased sperm motility, acrosome integrity, mitochondrial activity, ATP levels, and the activities of MDH, SDH, and CPT1, while also elevating NADH/NAD+ levels (p < 0.05). Notably, these enhancements were suppressed by 100 μM etomoxir, a mitochondrial β-oxidation inhibitor, which reduced total motility, ATP Levels, and CPT1 activity after riboflavin supplementation (p < 0.05). These findings indicate that goat sperm at least partly rely on mitochondrial β-oxidation for ATP generation and that riboflavin supplementation enhances mitochondrial metabolism, thereby improving sperm quality.

Nicotinamide mononucleotide (NMN), a key precursor of nicotinamide adenine dinucleotide (NAD+), plays a central role in cellular energy metabolism. This study investigated the effect of NMN supplementation on boar sperm quality during liquid storage. Semen samples diluted with Modena extender containing 0 to 80 μM NMN were stored at 17 °C for 7 days. Results demonstrate that supplementation with 20 μM NMN significantly improved sperm motility, acrosome integrity, and mitochondrial activity compared with the control group, accompanied by markedly elevated intracellular NAD+ and ATP level (p < 0.05). Also, Western blot analysis confirmed the presence of nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3) in boar sperm. Furthermore, sperm treated with 20 μM NMN exhibited a higher level of protein tyrosine phosphorylation and an increased capacitation rate following storage. Tissue explant assays further revealed a significant increase in the number of sperm attached to oviductal epithelial fragments, indicating enhanced sperm–oviduct interactions. The present findings demonstrate that 20 μM NMN supplementation effectively preserves the metabolic activity and functional competence of boar sperm during liquid storage. It provides a promising strategy for improving boar semen preservation.

This study aimed to investigate the effects of ellagic acid (EA), an antioxidant, on goat sperm quality after freezing and thawing. Goat semen was frozen using Tris-citric acid-glucose (TCG) extender containing 0, 1.25, 2.5, 5, and 10 μM of EA. Egg yolk represented 20 % (v/v) and glycerol represented 5 % (v/v) of the extender's final concentration. Goat sperm post-thaw motility, acrosome integrity, plasma membrane integrity, mitochondrial activity, ATP content, NADH/NAD+ levels, and NADH-CoQ activity were evaluated. Moreover, to elucidate how EA enhanced the goat sperm characteristics, the post-thaw sperm mitochondrial reactive oxygen species (ROS) level, malondialdehyde (MDA) level, oxidative DNA damage, apoptosis, levels of NADH dehydrogenase 1 (MT-ND1) and NADH dehydrogenase 6 (MT-ND6) proteins, and the 4-hydroxynonenal (4-HNE) level were also measured after thawing. The results demonstrated that motility, plasma membrane integrity, and acrosome integrity rates were enhanced in the group treated with 5 μM of EA compared to the other concentrations (0 μM, 1.25 μM, 2.5 μM, 5, and 10 μM). Moreover, mitochondrial activity and ATP content were notably superior in the 5 μM EA group compared to all other treatment groups, along with a considerable decrease in ROS and MDA levels. The 4-HNE level and oxidative DNA damage in sperm were also reduced by EA supplementation. Additionally, it was found that EA (5 μM) significantly (p < 0.05) decreased sperm apoptosis levels. Furthermore, the addition of 5 μM EA maintained the post-thaw sperm MT-ND1 and MT-ND6 levels and reduced the negative impact of ROS on MT-ND1 and MT-ND6, thereby sustaining mitochondrial function for ATP generation. These results suggest that ellagic acid supplementation could maintain goat post-thaw sperm quality by reducing ROS damage and maintaining mitochondrial function for ATP generation. Antioxidant treatments, such as ellagic acid are a useful tool for maintaining frozen-thawed sperm quality.

Due to the current limitations of boar semen cryopreservation systems, the effective restoration of sperm quality following thawing remains a significant challenge. This study investigates whether post-thaw boar sperm can uptake exogenous long-chain fatty acids (LCFAs) and utilize them for ATP generation, thereby sustaining linear motility and enhancing sperm vitality. Boar semen was diluted in extender solutions supplemented with varying concentrations of a lipid mixture (0, 0.01 %, 0.1 %, and 1 % LM). Following cryopreservation and subsequent thawing, key sperm parameters were assessed, including motility, viability, acrosome integrity, mitochondrial membrane potential (MMP), and ATP levels, as well as the enzymatic activities of malate dehydrogenase (MDH), succinate dehydrogenase (SDH), and carnitine palmitoyltransferase-1 (CPT-1). Compared to the control group, supplementation with 0.1 % LM significantly enhanced post-thaw sperm motility and improved viability and acrosomal integrity (P < 0.05). This concentration also led to increased MMP, elevated ATP levels, as well as enhanced activities of MDH, SDH, and CPT1 (P < 0.05). Furthermore, to further verify that post-thaw boar sperm utilize LCFAs to generate ATP through mitochondrial β-oxidation. It was found that adding 100 μM mitochondrial β-oxidation inhibitor etomoxir to the extender, significantly reduced post-thaw boar sperm progressive motility, MMP, ATP levels, and CPT1 activity (P < 0.05), thereby attenuating the beneficial effects of exogenous LCFAs supplementation. These findings suggest that post-thaw boar sperm are capable of assimilating exogenous LFCAs, which subsequently promote mitochondrial β-oxidation, increasing TCA cycle activity and ATP production, therefore improving the quality of post-thaw boar sperm.

Succinylation is a recently identified post-translational modification (PTM) that modulates enzyme activity and metabolism. However, how it regulates metabolic reprogramming in sperm remains unclear. In this study, we show that succinylation of pyruvate kinase M2 (PKM2) promotes oxidative phosphorylation (OXPHOS) and linear motility in boar sperm. Under low glucose (LG) extender, sperm linear motility was significantly enhanced, mitochondrial activity increased, glycolysis was inhibited, and the pentose phosphate pathway (PPP) was promoted. PKM2 interacted with voltage-dependent anion channel 3 (VDAC3), increasing mitochondrial permeability. Supplementing high glucose (HG) extender with succinic acid (SA) enhanced these effects. In contrast, the addition of resveratrol (RES) to LG extender, significantly reduced progressive motility (PM), decreased mitochondrial activity, and promoted the glycolysis pathway. Additionally, LG extender facilitated PKM2 succinylation and mitochondrial translocation. These findings demonstrate that PKM2 succinylation reprograms glucose metabolism from glycolysis to PPP, enhancing ATP production and supporting sustained linear motility, providing insights into optimizing sperm preservation.

Semen preservation involves lengthening sperm’s fertile lifespan without any detrimental effects on its biochemical, functional, and ultrastructural properties. Liquid storage at 4 °C is a ram sperm preservation method. However, this method of storage causes irreversible damage due to cold shocks, osmotic stresses, oxidative stresses, and reductions in sperm metabolism. The present study aims to investigate whether the supplementation of mitochonic acid 5 (MA-5) in a sperm extender could improve chilled ram sperm quality and elucidate its mechanism of action. Ram sperm were diluted with a tris-citrate-glucose extender containing different concentrations of MA-5 (0, 0.1, 1, 10, and 100 nM) and stored at 4 °C for up to 48 h. Sperm motility, membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) level, ATP content, and the expression of NADPH dehydrogenase subunits 1 (MT-ND1) and NADPH dehydrogenase subunits 6 (MT-ND6) were evaluated. It was observed that compared to the control, the 10 nM MA-5 treatment significantly (p < 0.05) increased total motility (82 ± 3.5% vs. 76 ± 5.9%), progressive motility (67.6 ± 8.2% vs. 51 ± 8.3%), and other parameters (straight-line velocity (VSL), average path velocity (VAP), and curvilinear velocity (VCL)). In addition, 10 nM MA-5 supplementation also improved ram sperm membrane integrity and acrosomal integrity as well increased mitochondrial membrane potential (51.1 ± 0.7% vs. 37.7 ± 1.3%), reduced ROS levels, and elevated adenosine triphosphate (ATP) contents. Furthermore, a Western blot analysis demonstrated that the addition of MA-5 significantly (p < 0.05) increased the expression of MT-ND1 and MT-ND6 proteins in ram sperm, with the 10 nM MA-5 treatment resulting in the highest expression level. These results suggest that MA-5 improves ram sperm quality by maintaining high sperm mitochondrial function during liquid storage at 4 °C.

This investigation aimed to study the effects of varying light exposure durations on ram sperm. A total of 25 rams were randomly divided into five groups. The control group was exposed to light durations of 12 h, while the experimental groups were exposed to light durations of 14, 16, 18, and 20 h. After three months of rearing, semen was collected from each ram four times using the artificial vagina method. The sperm motility parameters, sperm abnormality, sperm concentration, acrosome integrity, membrane integrity, semen volume, and total sperm number were measured. Thereafter, the metabolome, amino acid level, testosterone content, plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, and sperm antioxidant capacity were measured. The results showed that the sperm motility, sperm concentration, ejaculation volume, total sperm number, acrosome integrity, and membrane integrity in the 16 h light group were significantly improved compared to the control (p < 0.05), meanwhile the sperm abnormality was decreased. Moreover, we found 345 different metabolites between the control and 16 h light group. Among these, 273 were upregulated and 72 were downregulated. Furthermore, the amino acid content of the seminal plasma in the 16 h light group was significantly increased (p < 0.05) compared to the control. Interestingly, the seminal plasma testosterone content and the levels of FSH and LH in the serum in the 16 h light group were significantly increased (p < 0.05) compared to the control. In terms of the sperm antioxidant capacity, it was observed that the CAT activity was the highest in the group exposed to 16 h of light and decreased at 18 h of light exposure when compared to the control group; however, the CAT activity at 20 h was not different from the control. Additionally, within the 14 to 18 h light exposure range, prolonged light exposure increased the GSH content (p < 0.05), whereas 20 h of light exposure reduced the GSH content. The MDA levels decreased with prolonged light exposure, reaching the lowest point at 16 h (p < 0.05), but increased again at 20 h of light exposure. KEGG analysis indicated that the differential metabolites were mainly involved in metabolic and synthetic activities. Based on the results of this study, we can conclude that the artificial extension of the light duration for 16 h has a positive effect on ram sperm quality.

Artificial insemination (AI) with liquid-preserved semen has recently become common in pig breeding. The semen doses are produced in a centralized manner at the boar stud and then subsequently distributed and transported to sow farms. However, vibration emissions during transportation by logistic vehicles may adversely affect the quality of boar sperm. Therefore, this study aimed to explore the impact of vibration-induced emissions on sperm quality and function under simulated transportation conditions. Each time, ejaculates from all 15 boars were collected and then pooled together to minimize individual variations, and the sample was split using an extender for dilution. Different rotational speeds (0 rpm, 80 rpm, 140 rpm, 200 rpm) were utilized to simulate varying intensities of vibration exposure using an orbital shaker, considering different transportation times (0 h, 3 h, and 6 h). Subsequently, evaluations were conducted regarding sperm motility, plasma membrane integrity, acrosome integrity, mitochondrial function, adenosine triphosphate (ATP) levels, mitochondrial reactive oxygen species (ROS) levels, pH, glycolytic pathway enzyme activities, and capacitation following exposure to vibration emissions. Both vibration time and intensity impact sperm motility, plasma membrane integrity, and acrosomal integrity. Vibration exposure significantly reduced sperm ATP levels, mitochondrial membrane potential, and the levels of mitochondria-encoded proteins (MT-ND1, MT-ND6) (p < 0.05). After vibration emission treatment, the pH value and mitochondrial ROS levels significantly increased (p < 0.05). Inhibition of sperm glycolysis was observed, with reduced activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH), along with decreased lactate levels (p < 0.05). Additionally, sperm tyrosine phosphorylation levels were significantly reduced by vibration emissions compared to the control group (p < 0.05). After the vibration emission treatment, the number of sperm bound to each square millimeter of oviduct explants decreased significantly compared to the control group (p < 0.05). Similarly, compared to the control group, using semen subjected to vibration stress for AI results in significantly reduced pregnancy rates, total born litter size, live-born litter size, and healthy born litter size (p < 0.05).

Objective: The present study aimed to investigate the effect of β-nicotinamide mononucleotide (NMN) supplementation on ram sperm quality during storage at 4°C in vitro.Methods: Tris-citric acid-glucose solution containing different doses of NMN (0, 30, 60, 90, and 120 μM) was used to dilute semen collected from rams and it was stored at 4°C. Sperm motility, plasma membrane integrity as well as acrosome integrity were evaluated at 0, 24, and 48 h time points after storage at 4°C. In addition, sperm mitochondrial activity, lipid peroxidation (LPO), malondialdehyde (MDA) content, reactive oxygen species (ROS) content, glutathione (GSH) content, superoxide dismutase (SOD) activity, and apoptosis were measured at 48 h time point after storage at 4°C.Results: Results demonstrate that the values obtained for sperm motility, acrosome integrity, and plasma membrane integrity in the NMN treatments were significantly higher than control (p<0.05). The addition of 60 μM NMN significantly improved ram sperm mitochondrial activity and reduced LPO, MDA content, and ROS content compared to control (p<0.05). Interestingly, sperm GSH content and SOD activity for the 60 μM NMN treatment were much higher than those observed for control. NMN treatment also decreased the level of Cleaved-Caspase 3, Cleaved-Caspase 9, and Bax while increasing Bcl-2 level in sperm at 48 h time point after storage at 4°C.Conclusion: Ram sperm quality can be maintained during storage at 4°C with the addition of NMN at 60 μM to the semen extender. NMN also reduces oxidative stress and apoptosis. Overall, these findings suggest that NMN is efficient in improving the viability of ram sperm during storage at 4°C in vitro.

Sperm motility is an important factor in the migration of sperm from the uterus to the oviduct. During sperm preservation in vitro, sperm generates excessive ROS that damages its function. This study aims to investigate whether the addition of pyrroloquinoline quinone (PQQ) to the diluted medium could improve chilled ram sperm quality, and then elucidates the mechanism. Ram semen was diluted with Tris-citric acid-glucose (TCG) medium containing different doses of PQQ (0 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM), and stored at 4 °C. Sperm motility patterns, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) levels, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and ATP levels were measured after preservation. Furthermore, the expressions of NADH dehydrogenase 1 (MT-ND1) and NADH dehydrogenase 6 (MT-ND6) in sperm were also detected by western blotting. In addition, sperm capacitation and the ability of sperm to bind to the zona pellucina were also evaluated. It was observed that the addition of PQQ significantly (p < 0.05) improved ram sperm motility, membrane integrity, and acrosome integrity during preservation. The percentage of sperm with high mitochondrial membrane potential in the PQQ treatment group was much higher than that in the control. In addition, supplementation of PQQ also decreased the sperm MDA and ROS levels, while increasing ATP levels. Interestingly, the levels of MT-ND1 and MT-ND6 protein in sperm treated with PQQ were also higher than that of the control. Furthermore, the addition of 100 nM PQQ to the medium decreased ROS damage in MT-ND1 and MT-ND6 proteins. The addition of 100 nM PQQ significantly (p < 0.05) increased protein tyrosine phosphorylation in ram sperm after induced capacitation. Furthermore, the value of the sperm–zona pellucida binding capacity in the 100 nM PQQ treatment group was also much higher than that of the control. Overall, during chilled ram- sperm preservation, PQQ protected ram sperm quality by quenching the ROS levels to reduce ROS damage and maintain sperm mitochondrial function, and preserved the sperm’s high ability of fertilization.