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).

The parameters of sperm apoptosis and capacitation during liquid storage at 17°C can indicate the quality of pig sperm and the potential development of early embryos. However, the effect of kojic acid (KA) on semen preservation and its mechanism has not been fully understood. In this study, we discovered that adding KA to the diluent improved the antioxidant capacity of sperm mitochondria, maintained the normal structure of sperm mitochondria, and reduced sperm apoptosis. Western blot analysis revealed that KA prevented the release of Cytochrome c from mitochondria to the cytoplasm, reduced the expression of pro-apoptosis proteins cleaved Caspase-3 and cleaved Caspase-9, and increased the expression of the antiapoptosis protein Bcl-XL. Furthermore, KA also enhanced the motility parameters, oxidative phosphorylation level, adenosine triphosphate level, and protein tyrosine phosphorylation of capacitated sperm, while preserving the acrosome integrity and plasma membrane integrity of capacitated sperm. In conclusion, this study offers new insights into the molecular mechanism of how KA inhibits porcine sperm apoptosis and improves capacitated sperm parameters. Additionally, it suggests that KA can serve as an alternative to antibiotics.

The supplementation of sperm culture media with serum is quite common, and improves both sperm survival and motility. However, the link between serum and sperm remains poorly understood. The present study is the first investigation of the effects on sperm quality and function of endogenous porcine serum exosomes in medium used for culturing boar sperm. Scanning electron microscopy (SEM) confirmed that serum-derived exosomes from both castrated boars (cbsExos) and sows (ssExos) exhibited typical nanostructural morphology and expressed CD63, CD9, and Alix, as shown by Western blotting. At 17 °C, the progressive motility and membrane integrity of sperm were significantly increased after incubation of fresh boar semen for 7 days with cbsExos-4 (8 × 1010 particles/mL) or ssExos-16 (32 × 1010 particles/mL). Moreover, cbsExos-4 and ssExos-16 were found to be effective sperm additives, improving mitochondrial transmembrane potential (ΔΨm) and adenosine triphosphate (ATP) content, total antioxidant activity (T-AOC), superoxide dismutase (SOD) activity, and glutathione peroxidase (GPx) activity while reducing reactive oxygen species (ROS) levels, and malondialdehyde (MDA) content following preservation at 17 °C after a 5-day incubation. Both fluorescence and SEM showed that the serum exosomes bound directly to the sperm membrane, suggesting an interaction that could influence sperm-zona pellucida binding. Overall, this study provides new insights into the potential benefits of adding cbsExos and ssExos to enhance the quality of boar sperm during ambient temperature preservation, which may lead to advancements in sperm preservation strategies.

BACKGROUND: Boar sperm are highly susceptible to specific conditions during cryopreservation, leading to a significant decrease in their fertilizing potential due to damage to their membranes. Camellia oil, known for its fatty acids with antioxidant and biological properties, has not been previously explored for the cryopreservation of boar semen. This study aimed to examine the effects of camellia oil on post-thawed boar sperm quality. Boar semen ejaculates (n = 9) were collected and divided into six equal aliquots based on camellia oil concentrations (0, 0.5, 1, 1.5, 2 and 2.5% v/v) in the freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm morphology by scanning electron microscope, sperm motility using a computer-assisted sperm analyzer, sperm viability, acrosome integrity, mitochondrial function, MDA level and total antioxidant capacity.
RESULTS: The results demonstrated that the supplementation of 1.5% (v/v) camellia oil showed superior post-thaw sperm qualities such as improved sperm morphology, motility, acrosome integrity and mitochondrial function by 14.3%, 14.3% and 11.7%, respectively, when compared to the control group. Camellia oil at a concentration of 1.5% (v/v) showed the lowest level of MDA (18.3 ± 2.1 µmol/L) compared to the other groups.
CONCLUSIONS: In conclusion, adding 1.5% (v/v) camellia oil in the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in a higher post-thawed sperm quality.

Understanding the liquid preservation ability of boar sperm is pivotal for efficient management and breeding of livestock. Although sperm proteins play an important role in semen quality and freezability, how the levels of protein change in boar sperm with different liquid preservation abilities at 17 °C remains unclear. In this study, two groups of boar sperm with extreme difference in liquid preservation ability, namely the good preservation ability (GPA) and the poor preservation ability (PPA) groups, were selected by evaluating sperm motility parameters on the 7th day of liquid preservation at 17 °C. Quantitative proteomics based on tandem mass tag (TMT) labeling was used, sperm proteomic characteristics from two groups were analyzed, and potentially key proteins related to the fluid preservation ability of sperm were identified. A total of 187 differentially expressed proteins (DEPs) were identified among 2791 quantified proteins, including 85 upregulated, and 102 downregulated proteins. Further, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the DEPs revealed that they were enriched in GO terms associated with response to oxidative stress, enzyme activity related to oxidative stress or redox reactions, and several metabolic activities. The significant KEGG pathways included peroxisome, metabolic pathways, selenocompound metabolism, and collection duct acid secretion. In addition, analysis of protein-protein interactions further identified 8 proteins that could be used as biomarker candidates, including GPX5, GLRX, ENO4, QPCT, BBS7, OXSR1, DHRS4 and AP2S1, which may play an essential role in indicating the liquid preservation ability of boar sperm. These findings in this study provide new insights into the underlying molecular mechanisms of the liquid preservation ability of boar sperm. Moreover, the selected candidate proteins can serve as a reference for evaluating sperm quality or preservation ability in boars and their application in related biotechnologies.

Semen delivery practice is crucial to the efficiency of artificial insemination using high-quality boar sperm. The present study aimed to evaluate the effect of a common semen delivery method, a Styrofoam box, under elevated temperatures on boar sperm quality and functionality and to investigate the underlying molecular responses of sperm to the temperature rise. Three pooled semen samples from 10 Duroc boars (3 ejaculates per boar) were used in this study. Each pooled semen sample was divided into two aliquots. One aliquot was stored at a constant 17 °C as the control group. Another one was packaged in a well-sealed Styrofoam box and placed in an incubator at 37 °C for 24 h to simulate semen delivery on hot summer days and subsequently transferred to a refrigerator at 17 °C for 3 days. The semen temperature was continuously monitored. The semen temperature was 17 °C at 0 h of storage and reached 20 °C at 5 h, 30 °C at 14 h, and 37 °C at 24 h. For each time point, sperm quality and functionality, apoptotic changes, expression levels of phosphorylated AMPK, and heat shock proteins HSP70 and HSP90 were determined by CASA, flow cytometry, and Western blotting. The results showed that elevated temperature during delivery significantly deteriorated boar sperm quality and functionality after 14 h of delivery. Storage back to 17 °C did not recover sperm motility. An increased temperature during delivery apparently promoted the conversion of sperm early apoptosis to late apoptosis, showing a significant increase in the expression levels of Bax and Caspase 3. The levels of phosphorylated AMPK were greatly induced by the temperature rise to 20 °C during delivery but reduced thereafter. With the temperature elevation, expression levels of HSP70 and HSP90 were notably increased. Our results indicate that a temperature increase during semen delivery greatly damages sperm quality and functionality by promoting sperm apoptosis. HSP70 and HSP90 could participate in boar sperm resistance to temperature changes by being associated with AMPK activation and anti-apoptotic processes.

Boar sperm is sensitive to particular conditions during cryopreservation, resulting in an extreme reduction in fertilizing ability due to damage to the sperm membranes. PKMPH contains bioactive peptides that have antioxidant and antimicrobial activities. There is no information on the use of palm-kernel-meal-derived bioactive peptides for boar semen cryopreservation. This study aimed to examine the effects of bioactive peptides from PKMPH on post-thawed boar sperm quality. Boar semen ejaculates (n = 17) were collected and divided into six equal aliquots based on PKMPH concentrations (0, 1.25, 2.5, 5, 10, and 15 µg/mL) in a freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, the frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm motility using a computer-assisted sperm analyzer and for sperm viability, acrosome integrity, mitochondrial function, and lipid peroxidation by measuring the level of malondialdehyde (MDA). The results demonstrate that the supplementation of PKMPH with 2.5 µg/mL afforded superior post-thawed sperm qualities, such as increased total motility, viability, acrosome integrity, and mitochondrial function by 10.7%, 12.3%, 18.3%, and 12.7%, respectively, when compared to the control group. PKMPH at a concentration of 2.5 µg/mL showed the lowest level of MDA (40.6 ± 2.0 µMol/L) compared to the other groups. In conclusion, adding PKMPH peptides at 2.5 µg/mL to the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in higher post-thawed sperm quality.

The slow freezing of boar sperm is the only way to preserve genetic material for extended periods; this can be achieved with exposure to liquid nitrogen vapors (conventional) or by using automated freezing equipment. The aim was to compare the effect of both techniques on post-thaw functionality. Boar sperm devoid of seminal plasma and resuspended in lactose-egg yolk-glycerol medium were cryopreserved. Conventional: straws were exposed to LN2 vapors; automated: using a drop curve of -39.82 °C·min-1 for 113 s from -5 to -80 °C during the critical period; and subsequent immersion in NL2. Cell viability, cholesterol flow, mitochondrial membrane potential (MMP), lipid peroxidation, peroxynitrite, superoxide anion levels, phosphatidylserine translocation, and caspase activation were evaluated by flow cytometry. In addition, total motility (TM) and progressive motility (PM) were determined by the SCA system immediately (T0), 60 (T60), and 120 min (T120) post-thawing. Automated freezing significantly reduces cholesterol flow and free radical and lipid peroxidation levels, making it possible to preserve motility for 120 min of incubation. At the same time, viability, acrosome integrity, MMP, and caspase activation did not differ from the conventional technique. In conclusion, controlling the temperature drop curve using automated freezing equipment reduces oxidative/nitrosative stress, preserving membrane fluidity and sperm motility.

Ejaculated boar spermatozoa can be liquid preserved for several days and be easily activated to produce physiological changes. One of the major changes is acrosome exocytosis that is physiologically related to capacitation. Glycolysis and reactive oxygen species (ROS) were studied regarding several boar sperm functions, but data available about their effect on boar sperm acrosome exocytosis are scarce. The objective of this work was to evaluate the effect of glucose and ROS on boar sperm acrosome exocytosis. We evaluated acrosome exocytosis by progesterone induction of capacitated sperm and assess viability, kinematics parameters, ROS levels, ATP content and Protein Kinase A activity in media with or without glucose and hydrogen peroxide or potassium chromate, as source of ROS. Our results show that glucose has no effect on acrosome exocytosis and also, it is not necessary for boar sperm capacitation, although it has a positive effect in the presence of ROS. On the other hand, ROS effects are related to spontaneous acrosome reaction. We conclude that glycolysis may function as a metabolic pathway that provides sustain but is not directly involved in boar sperm acrosome exocytosis and capacitation. Also, ROS do not promote capacitation in boar sperm, but affect spontaneous acrosome exocytosis.

Boar sperm are less resistant to the dramatic environmental changes that occur during in vitro preservation. Spermidine has various physiological functions including the anti-oxidative effect. The main objective of this study was to clarify whether spermidine could protect boar sperm from the attack of reactive oxygen species under cryopreservation treatment. We set the concentrations of spermidine at 0, 2, 4, 6, and 8 mmol/L and evaluated the effects of spermidine on sperm motility, viability, malformation rates, kinetic parameters, membrane integrity, mitochondrial activity, DNA integrity, H2 O2 content, malondialdehyde content, total antioxidant capacity, and antioxidant enzyme activity. Finally, the effects of spermidine on the sperm fertility were assessed by artificial insemination. The results showed that spermidine improved various physiological parameters of sperm in a dose-dependent manner. The quality and antioxidant capacity of sperm cryopreserved with 6 mmol/L spermidine were significantly less reduced (P < 0.05), and the contents of malformation rate, H2 O2 , and malondialdehyde content were significantly decreased (P < 0.05). The significant increase in the number of litters indicated the possibility that spermidine had important practical value in pig reproduction (P < 0.05). Therefore, the addition of appropriate concentrations of spermidine to cryopreservation extenders may effectively improve the quality of boar sperm for in vitro preservation.