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).

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.

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.

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.

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.

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