It takes several hours for mammalian sperm to migrate from the ejaculation or insemination site to the fertilization site in the female reproductive tract in which glucose, amino acids, and fatty acids are regarded as the primary substrates for ATP generation. The present study was designed to investigate whether oleic acid and palmitic acid were beneficial to boar sperm in vitro; and if yes, to elucidate the mechanism that regulates sperm motility. Therefore, the levels of oleic acid and palmitic acid, motility, membrane integrity, acrosome integrity, and apoptosis of sperm were evaluated. Moreover, the enzymes involved in mitochondrial β-oxidation (CPT1: carnitine palmitoyltransferase 1; ACADVL: long-chain acyl-coenzyme A dehydrogenase) were detected with immunofluorescence and Western blotting. Consequently, the ATP content and the activities of CPT1, ACADVL, malate dehydrogenase (MDH), and succinate dehydrogenase (SDH) were also measured. We observed that CPT1 and ACADVL were expressed in boar sperm and localized in the midpiece. The levels of oleic acid and palmitic acid were decreased during storage at 17 °C. The addition of oleic acid and palmitic acid significantly increased sperm motility, progressive motility, straight-line velocity (VSL), membrane integrity, and acrosome integrity with a simultaneous decrease in sperm apoptosis after seven days during storage. When sperm were incubated with oleic acid and palmitic acid at 37 °C for 3 h, the activities of CPT1 and ACADVL, the ATP level, the mitochondrial membrane potential, the activities of MDH and SDH, as well as sperm motility patterns were significantly increased compared to the control (p < 0.05). Moreover, the addition of etomoxir to the diluted medium in the presence of either oleic acid or palmitic acid and the positive effects of oleic acid and palmitic acid were counteracted. Together, these data suggest that boar sperm might utilize oleic acid and palmitic acid as energy substrates for ATP production via β-oxidation. The addition of these acids could improve sperm quality.

Sperm cryopreservation contributes to the extensive utilization of artificial insemination (AI) in the daily livestock industry. However, due to the presence of few sperm with good biological function in post-thaw goat sperm, its use has been limited for AI purposes. Hence, its improvement has been the focus of many research studies. This study aimed to investigate the effects of proline supplementation of the freezing medium on goat sperm. The goat semen was cryopreserved with freezing medium supplementation of different concentrations of proline (0, 0.5, 1, 2 and 4 mM). The post-thaw sperm motility patterns, membrane integrity, acrosome integrity, lipid peroxidation (LPO) levels, malondialdehyde (MDA) levels, total antioxidant capacity (T-AOC), proline dehydrogenase (PRODH) activity, superoxide dis-mutase (SOD) activity, glutathione (GSH) levels and GSH/GSSG were evaluated. Likewise, the expression and immunofluorescent localization of PRODH in post-thaw goat sperm was also detected. It was observed that addition of 2 mM proline to the freezing medium significantly enhanced post-thaw goat sperm total motility, progressive motility, straight-linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), straightness (STR), linearity (LIN), membrane integrity and acrosome integrity. Interestingly, PRODH was expressed in post-thaw goat sperm, especially in the post-acrosome and sperm tail. Addition of 2 mM proline also significantly increased the post-thaw sperm PRODH activity compared to the control. Moreover, post-thaw goat sperm LPO levels and MDA levels were reduced by supplementation of 2 mM proline. Furthermore, compared to the control, the values of post-thaw goat sperm T-AOC, SOD activity, GSH level and GSH/GSSG were also significantly increased in 2 mM proline treatment. Reduction of post-thaw goat sperm apoptosis in 2 mM proline treatment was also observed as the levels of Caspase3 and Caspase9 were decreased by the supplementation with 2 mM proline. These observations suggest that the addition of 2 mM proline to the freezing medium increased post-thaw goat sperm quality by reducing oxidative stress during cryopreservation. These findings also provide novel insights into the use of proline as an efficient additive to enhance post-thaw goat sperm quality during cryopreservation.

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.

Carboxylated ε-poly-l-lysine (CPLL), a novel cryoprotectant, can protect the sperm membranes by inhibiting ice crystal formation during the cryopreservation process. The present study was conducted to investigate the consequence of CPLL supplementation on the post-thaw quality of cryopreserved goat sperm. For this, different doses (0, 0.5%, 1%, 1.5%, and 2%; v/v) of CPLL were added to the cryopreservation medium, and the motility, membrane and acrosome integrity, mitochondrial membrane potential (MMP), ATP level, ROS production, anti-oxidant defense system, malondialdehyde (MDA) level, and apoptosis in post-thaw sperm were evaluated. It was observed that the addition of 1% CPLL significantly (p < 0.05) increased the total motility, membrane integrity, acrosome integrity, and catalase (CAT) activity of post-thaw sperm compared to those of control and other CPLL doses. The ATP content was observed significantly (p < 0.05) higher in 0.5% and 1% CPLL, however, the SOD activity and progressive motility were significantly (p < 0.05) increased by adding CPLL at 1% and 1.5% level. Moreover, the addition of CPLL at 1% dose not only showed a lower percentage of apoptosis, but also significantly (p < 0.05) increased the MMP while reducing ROS production and MDA levels compared to those of other CPLL doses and/or control. Therefore, it is clear that the supplementation of 1% CPLL can remarkably improve the post-thaw goat sperm motility, membrane and acrosome integrity, antioxidant abundance, mitochondrial potentials, and ATP supply by protecting the sperm from cryodamage and undergoing apoptosis. These findings will provide novel insights into sperm cryobiology.