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The porcine bulbourethral glands produce a gel-type secretion. Although the role of these contributions to reproductive success remains murky, the bulbourethral glands are major accessory sex glands in this species. Isometric growth in the early neonatal interval is followed by allometric growth in the late juvenile interval (6 to 11 weeks of age), while circulating endogenous steroids are low. The rate of allometric growth increases during the peripuberal interval (16 to 20 weeks of age) when systemic testosterone is relatively high. Gene expression for androgen receptor (AR) and for the steroid 5 alpha-reductase 2 (SRD5A2) enzyme that synthesizes the more potent androgen dihydrotestosterone from its precursor was evaluated by qPCR analyses of bulbourethral gland tissue. Tissues were collected from control boars (2 weeks to 40 weeks of age) and from littermates of these boars treated with letrozole to suppress endogenous estrogen synthesis. Gene expression for these two key proteins in androgen signaling was quite low during the initial allometric growth in the late juvenile and prepuberal intervals, suggesting that this initial growth was not primarily stimulated by androgens. These observations are consistent with a more direct estrogen-mediated inhibition of growth via GPER previously proposed, with the sensitivity extending into the late juvenile interval when estrogens as well as androgens are normally relatively low.

The present experiment was carried out to investigate the role of Oxyrase in preserving the in vitro quality, redox status and in vivo fertility of crossbred boar spermatozoa. A total of 24 ejaculates from 6 crossbred (n = 4 from each boar) boars were collected and extended in Beltsville Thawing Solution (BTS) in 1:2 ratio and divided into three aliquots. The first aliquot served as a control (without Oxyrase). Rest of the two aliquots were supplemented with 0.125 (T1) and 0.25 IU/mL Oxyrase (T2). Semen samples were preserved at 15°C for 5 days and kinematics of spermatozoa by CASA, semen quality parameters and oxidative stress status were evaluated at 0, 72 and 120 h of storage. The findings of studies revealed that supplementation of Oxyrase at 0.25 IU/mL resulted in higher (p < 0.05) total motility, progressive motility, plasma membrane integrity, acrosome integrity and functional integrity of plasma membrane at 72 and 120 h in comparison to the control group. Mitochondrial membrane potential (MMP) was higher (p < 0.05) at 72 and 120 h, whereas higher (p < 0.05) DNA integrity was observed at 120 h in T2. The lipid peroxidation (LPO) was lower (p < 0.05) and superoxide dismutase activity (SOD) and total antioxidant capacity (TAC) were higher (p < 0.05) in the T2 group at 120 h as compared to control. In vivo fertility trials indicated a higher (p < 0.05) litter size in T2 in comparison to other groups. The study concluded that the inclusion of Oxyrase at 0.25 IU/mL in the extender protects the crossbred boar spermatozoa against oxidative damage and improves the in vivo fertility. [ABSTRACT FROM AUTHOR]

Nanotechnology and its applications have advanced significantly in recent decades, contributing to various fields, including reproduction. This study introduces a novel method to label porcine oocytes with nanoparticles (NPs) bound to oviductin (OVGP1, Ov) for use in Assisted Reproductive Technologies (ARTs). Despite promising developments, concerns about NP toxicity in gametes necessitate thorough investigation. This research aims to assess the impact of functionalized NPs (NPOv) on sperm functionality. Boar sperm were co‐incubated with NPOv for 0, 0.5 and 1 h in two media: BTS (semen dilution and conservation) and TALP (sperm capacitation and in vitro fertilization‐IVF). Sperm quality parameters (viability, motility and kinematics) showed no significant differences in TALP medium (p >.05). In BTS, although some kinetic parameters were altered, motility, progressive motility and viability remained unaffected (p >.05). Additionally, NPs presence on the zona pellucida (ZP) of oocytes did not affect sperm attachment (p >.05). In conclusion, in vitro exposure of boar sperm to OVGP1‐functionalized NPs in IVF medium or attached to the ZP surface of matured oocytes does not impair sperm functionality, including their binding ability to the ZP. [ABSTRACT FROM AUTHOR]

This study investigates the impact of bovine serum albumin (BSA) inclusion in the thawing extender on boar sperm quality. Thawing protocols in sperm cryopreservation are vital, yet underexplored. It has been determined that BSA can interact with membranes, stabilizing them and preventing damage during the freezing process, for this reason it could also have a beneficial effect during thawing. Our study explores modifications in the conventional Beltsville thawing solution, incorporating BSA at different concentrations (0.005, 0.01, 0.025 and 0.050 g/mL), and evaluating the sperm quality after up to 150 min post‐thawing incubation (37°C). All BSA concentrations preserved plasma membrane and acrosome integrity, relative to control (BSA absence). In addition, 0.025 g/mL BSA group preserves acrosome integrity over time without compromising motility or kinetic parameters. Our study suggests that BSA inclusion in thawing extenders should be considered as an additive for improving post‐thaw boar sperm quality. [ABSTRACT FROM AUTHOR]

Simple Summary: Liquid-preserved boar semen is widely used in the pig breeding industry. Preservation and transportation can affect the fertilizing ability of sperm, reducing the efficiency of artificial insemination. Betaine, a natural plant extract, has been shown to benefit male fertility. However, it remains unclear whether it exerts a protective effect on liquid boar sperm during preservation and transportation. The results showed that the addition of appropriate amounts of betaine to boar semen extenders effectively mitigated the adverse effects of road transport and room temperature storage on sperm quality. Specifically, betaine supplementation showed significant protective effects against temperature fluctuation and vibration stress that inevitably occurred during transportation. The increased antioxidant capacity of boar sperm may underlie its beneficial effects. These findings offer a theoretical foundation for the use of betaine in artificial insemination procedures. Boar semen is commonly used in artificial insemination (AI) for pig breeding, but its quality can be negatively affected by liquid preservation and transportation, leading to reduced fertility rates. Vibration and temperature fluctuations are critical factors that significantly impact semen quality during storage and transportation, influencing the success rate of AI procedures. Betaine, a naturally occurring compound known for its role in maintaining male fertility, demonstrates potential for improving the preservation and transportation of liquid-preserved boar sperm. The present study demonstrated that betaine supplementation in the semen extenders at 0.5 mg/mL had a significant protective effect on boar sperm motility during storage at 17 °C for 3 to 5 days. During road transportation, 2.5 mg/mL betaine showed significant protective effects on boar sperm progressive motility, while 0.4 mg/mL betaine notably improved boar sperm mitochondrial activity and antioxidant capacity, and reduced lipid peroxidation damage. Simulation models also demonstrated that betaine supplementation increased the proportion of sperm displaying progressive motility and possessing intact acrosomes, regardless of the storage temperature (17 °C or 25 °C), and effectively mitigated the damage caused by vibration at a speed of 200 r/min. Overall, supplementing liquid-preserved boar semen extenders with betaine shows promise in mitigating damage to sperm quality during storage and transportation. [ABSTRACT FROM AUTHOR]

Background Objective Materials and methods Results Discussion and conclusions Boar semen quality emphasizes three major criteria: sperm concentration, motility, and morphology. Methods to analyze concentration and motility quickly and objectively readily exist, but few exist for analyzing morphology outside of subjective manual counting. Other vital factors for fertilization, like acrosome health, lack efficient detection methods due to limitations in detection by the human eye and costly biomarker analysis, which is rarely used in semen diagnostics.To overcome these challenges, we propose a novel approach integrating deep‐learning technology with high‐throughput image‐based flow cytometry (IBFC) for objective and accurate analysis of both morphology and label‐free acrosome health of thousands of individual spermatozoa at once, as opposed to manually counting on a microscope slide.Images of 10,000 spermatozoa were captured using an IBFC and manually annotated based on the primary morphological defect or acrosome health status for the training of the convolutional neural network (CNN). The CNN used these images to train and then applied that training to unannotated images to predict the model accuracy.Using the CNNs, high F1 scores of 96.73%, 98.55%, and 99.31% for 20x, 40x, and 60x magnifications, respectively, for morphological classification were attained. Additionally, the model demonstrates an F1 score of 99.8% in detecting subtle acrosome health variations at the 60x magnification.We have established an integrated approach to rapidly collect and classify morphological defects and acrosome health status, without the use of manual counting or biomarker labeling. Our study underscores the potential of artificial intelligence in semen diagnostics, reducing technician variability, streamlining assays, and facilitating the development of additional label‐free detection methods. This innovative approach addresses the barriers hindering biomarker adoption in semen analysis, offering a promising avenue for enhancing reproductive health assessments. [ABSTRACT FROM AUTHOR]

Single Layer Centrifugation (SLC) through a low density colloid offers an alternative solution to antibiotic use in boar semen extenders, with lower costs compared to high density colloids. The aim of this study was to explore the reproductive performance of sows when using SLC-prepared semen doses without antibiotics, employing low density Porcicoll to prepare semen doses for artificial insemination in a commercial swine herd in Thailand. Ejaculates were divided into two equal parts to create insemination doses, with each dose containing 3000 × 106 sperm/80 ml for intra-uterine insemination in individual sows. The sows were inseminated twice, with the interval between the two inseminations ranging from 8 to 16 h. The CONTROL group consisted of 206 semen doses treated with antibiotics, prepared for insemination in 103 sows, while the SLC group comprised 194 SLC-prepared semen doses without antibiotics for inseminating 97 sows. Fertility and fecundity traits, including non-return rate, conception rate, farrowing rate, and litter traits (i.e., the total number of piglets born per litter, number of piglets born alive per litter, number of stillborn piglets, and number of mummified fetuses), were compared between groups. Furthermore, data on piglet characteristics, including live-born and stillborn piglets (i.e., the prevalence of stillbirth (yes, no), birth weight, crown-rump length, body mass index (BMI), and ponderal index (PI)), were determined. No significant differences in non-return rate (75.7 % vs. 77.3 %), conception rate (73.8 % vs. 73.2 %), and farrowing rate (71.8 % vs. 73.2 %) were observed between the CONTROL and SLC groups, respectively (P > 0.05). Nevertheless, the total number of piglets born per litter in the SLC group was higher than in the CONTROL group (14.6 ± 0.9 vs. 12.3 ± 0.6, respectively, P = 0.049). Interestingly, the prevalence of stillbirth in the SLC group was lower than in the CONTROL group (6.2 % vs. 11.6 %, respectively, P < 0.001). Moreover, the newborn piglets in the SLC group exhibited higher birth weight and BMI compared to those in the CONTROL group (1.36 ± 0.03 vs. 1.26 ± 0.02 kg, P = 0.005, and 18.3 ± 0.3 vs. 17.3 ± 0.2 kg/m2, P = 0.003). In conclusion, employing sperm doses after SLC through a low density colloid in artificial insemination within a commercial breeding operation did not have a detrimental impact on either fertility or fecundity traits but showed potential benefits in increasing the total number of piglets born per litter. Moreover, improvements were observed in the birth weight and body indexes of piglets, and the percentage of stillbirths was reduced. Our findings introduce new possibilities for antibiotic alternatives in semen extenders to reduce the risk of antimicrobial resistance in the swine industry. Additionally, they provide compelling reproductive outcomes supporting the integration of SLC-prepared semen doses into artificial insemination practices. • Single Layer Centrifugation is an alternative solution to antibiotic use in boar semen extenders. • Farrowing rate did not differ between control and SLC groups. • The total born in the SLC group was higher than in the control group. • The prevalence of stillbirth in the SLC group was lower than in the control group. • Newborn piglets in the SLC group had higher birth weight and BMI than control group. [ABSTRACT FROM AUTHOR]

Melatonin, a hormone synthesized in various tissues, plays a crucial role in modulating sperm characteristics, yet its protective function on boar sperm remains poorly understood. This study aimed to investigate the expression and localization of melatonin-related proteins (AANAT, ASMT, MT1, MT2, and NQO2) in pig tissues, assess the impact of melatonin on pig sperm motility parameters and quality, and elucidate the underlying molecular mechanisms. Our results revealed widespread expression of AANAT, ASMT, MT1, MT2, and NQO2 proteins in pig tissues, particularly in the testis. Specific localization patterns were observed in Leydig cells, reproductive epithelium, and columnar epithelium cells in the testis and cauda epididymis. Additionally, melatonin membrane receptors MT1 and MT2 were detected in boar sperm. Melatonin treatment significantly enhanced boar sperm motility parameters and quality, particularly with 10 nM melatonin treatment. Inhibition of the MT1 receptor, but not the MT2 receptor, resulted in decreased sperm motility, highlighting the pivotal role of the MT1 receptor in mediating melatonin's effects on boar sperm. Metabolomic analysis revealed significant alterations in sperm metabolites following melatonin supplementation, particularly in amino acid metabolism. Overall, our findings provide comprehensive insights into melatonin's mechanisms in improving boar sperm quality, suggesting its potential as a therapeutic agent for enhancing male fertility. • Comprehensive study shows melatonin boosts boar sperm motility and quality, mainly via the MT1 receptor. • Melatonin-related proteins in pig tissues suggest roles in testicular physiology and sperm protection. • Melatonin alters seminal plasma metabolites, showing diverse impacts on sperm function and metabolism. [ABSTRACT FROM AUTHOR]

Objective: This study investigated the efficacy of different concentrations of cholesterolloaded cyclodextrin (CLC) on cryopreservation in boar sperm quality. Methods: In this study, we treated boar sperm with different concentrations of CLC before freezing and analyzed the sperm cholesterol concentration, plasma membrane, acrosome integrity rate and total motility rate before and after freeze-thawing. We also investigated the levels of reactive oxygen species (ROS), malondialdehyde (MDA), adenosine triphosphate (ATP), and structural- and oxidative-damage related proteins in all groups after thawing. Results: The results revealed that the cholesterol concentration of the CLC-treated groups was higher than that of the control group, both before freezing and after thawing (p<0.05). The plasma membrane integrity rate, acrosome integrity rate, and total motility rate of sperm were also enhanced after thawing in the CLC-treated group (all p<0.05). Moreover, ROS and MDA production and ATP loss were reduced in CLC-treated sperm during freezing and thawing (p<0.05). Finally, CLC pretreatment partially prevented the consumption of various proteins involved in metabolism including capping actin protein of muscle Z-line subunit beta (CAPZB), heat shock protein 90 alpha family class A member 1 (HSP90AA1) and phosphoglycerate mutase 2 (PGAM2) (p<0.05). Conclusion: The CLC treatment increased cholesterol concentration and decreased structural injury and oxidative damage during boar sperm freezing and thawing, improving the efficacy of sperm cryopreservation in boar. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance is an increasing challenge in semen preservation of breeding animals, especially in the porcine species. Bacteria are a natural component of semen, and their growth should be inhibited to protect sperm fertilizing capacity and the female's health. In pig breeding, where semen is routinely stored at 17°C in the liquid state, alternatives to conventional antibiotics are urgently needed. Photodynamic inactivation (PDI) of bacteria is a well-established tool in medicine and the food industry but this technology has not been widely adopted in semen preservation. The specific challenge in this setting is to selectively inactivate bacteria while maintaining sperm integrity and functionality. The aim of this study was to test the principle of PDI in liquid stored boar semen using the photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) and a white light LED-setup. In the first step, photophysical experiments comprising singlet oxygen phosphorescence kinetics of TMPyP and determination of the photosensitizer triplet time revealed a sufficiently high production of reactive singlet oxygen in the Androstar Premium semen extender, whereas seminal plasma acted as strong quencher. In vitro experiments with extended boar semen showed that the established PDI protocol preserves sperm motility, membrane integrity, DNA integrity, and mitochondrial activity while efficiently reducing the bacteria below the detection limit. A proof-of-concept insemination study confirmed the in vivo fertility of semen after photodynamic treatment. In conclusion, using the PDI approach, an innovative tool was established that efficiently controls bacteria growth in extended boar and maintains sperm fertility. This could be a promising contribution to the One Health concept with the potential to reduce antimicrobial resistance in animal husbandry. [ABSTRACT FROM AUTHOR]

Simple Summary: Artificial insemination is a common breeding technique employed in the contemporary livestock industry. However, the efficacy of this technique is contingent upon the quality of the sperm utilized. Several factors influence the quality of sperm during liquid storage. It is, therefore, essential to select an effective physiological regulator in order to maintain sperm quality during this process. This study aimed to evaluate the expression of MTFMT in different motile sperm and to investigate the effects of the addition of amino acids and methionine on sperm quality during liquid storage of boar sperm. The results demonstrated that MTFMT was highly expressed in high-motility sperm. Methionine could act as a substrate for protein translation, affecting mitochondrial function by regulating mitochondrial protein translation and directly influencing boar. Boar sperm quality serves as an important indicator of reproductive efficiency, playing a direct role in enhancing the output of livestock production. It has been demonstrated that mitochondrial protein translation is present in sperm and plays a crucial role in regulating sperm motility, capacitation and in vitro fertilization rate. The present study aimed to determine whether methionine supplementation enhances mitochondrial translation in boar sperm, thereby improving sperm quality. The results showed a significant elevation in the abundance of mitochondrial methionyl-tRNA formyltransferase (MTFMT), a crucial enzyme for mitochondrial protein translation, and mitochondrial DNA-encoded cytochrome c oxidase subunit 1 (COX1) in boar sperm exhibiting high motility. Both amino acids and methionine supplementation significantly enhanced boar sperm motility during storage. Moreover, methionine supplementation mitigates the loss of acrosomal integrity, enhances the expression of COX1, and boosts mitochondrial activity. Furthermore, the positive impact of methionine was negated in the presence of the mitochondrial translation inhibitor chloramphenicol. Together, these findings suggest that boar sperm may utilize methionine as a protein translation substrate to enhance sperm motility by stimulating mitochondrial protein translation. The supplementation of methionine may enhance the quality of boar sperm, thereby providing guidance for the optimization of diluent formulations for liquid storage and the identification of physiological regulators that regulate sperm motility. [ABSTRACT FROM AUTHOR]

Heat stress reduces the number of Sertoli cells, which is closely related to an imbalanced redox status. Glutamate functions to maintain the equilibrium of redox homeostasis. However, the role of glutamate in heat treated Sertoli cells remains unclear. Herein, Sertoli cells from 3-week-old piglets were treated at 44 °C for 30 min (heat stress). Glutamate levels increased significantly following heat stress treatment, followed by a gradual decrease during recovery, while glutathione (GSH) showed a gradual increase. The addition of exogenous glutamate (700 μM) to Sertoli cells before heat stress significantly reduced the heat stress-induced apoptosis rate, mediated by enhanced levels of antioxidant substances (superoxide dismutase (SOD), total antioxidant capacity (TAC), and GSH) and reduced levels of oxidative substances (reactive oxygen species (ROS) and malondialdehyde (MDA)). Glutamate addition to Sertoli cells before heat stress upregulated the levels of glutamate-cysteine ligase, modifier subunit (Gclm), glutathione synthetase (Gss), thioredoxin (Trx1) and B-cell leukemia/lymphoma 2 (Bcl-2), and the ratio of phosphorylated Akt (protein kinase B)/total Akt. However, it decreased the levels of Bcl2-associated X protein (Bax) and cleaved-caspase 3. Addition of the inhibitor of glutaminase (Gls1), Bptes (Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, 30 μM)to Sertoli cells before heat stress reversed these effects. These results inferred that glutamate rescued heat stress-induced apoptosis in Sertoli cells by enhancing activity of antioxidant enzymes and activating the Trx1-Akt pathway. Thus, glutamate supplementation might represent a novel strategy to alleviate the negative effect of heat stress. • Heat treatment could cause apoptosis and oxidate stress of boar SCs. • Exogenous glutamate reduced apoptosis by enhancing the levels of antioxidant substances. • Glutamate improves oxidation resistance by regulating the expression of Gclm/Gss to increase GSH. • Protective effects of glutamate are mediated through the Trx1-Akt pathway. [ABSTRACT FROM AUTHOR]

Protein glycosylation is a post-translational modification involved in wide range of biological processes. In mammalian spermatozoa this modification has been identified in numerous proteins, and membrane glycoproteins are involved in the fertilization process. The objective of the present study was to identify changes in protein glycosylation after acrosome reaction (AR) induction using the 4-Br-A23187 ionophore. Our results showed that treatment with 10 μM of 4-Br-A23187 for 20 min significantly increased the percentage of live acrosome-reacted spermatozoa compared to the control (69.8 ± 0.8 vs. 6.4 ± 0.5; mean % ± SEM, respectively). Also, we observed an increase in 32 kDa tyrosine-phosphorylated protein (p32) and a decrease in serine/threonine phosphorylation of the protein kinase A substrates (phospho-PKA-substrates) after ionophore treatment. Furthermore, changes in glycosylated proteins following AR induction were analyzed using different HRP-conjugated lectins (GNA, DSA, and SNA), revealing changes in mannose and sialic acid residues. Proteomic analysis of isolated proteins using GNA lectin revealed that 50 proteins exhibited significantly different abundance (q-value < 0.01). Subsequent analysis using Uniprot database identified 39 downregulated and 11 upregulated proteins in the presence of 4-Br-A23187. Notably, six of these proteins were classified as transmembrane proteins, namely LRRC37A/B like protein 1 C-terminal domain-containing protein, Membrane metalloendopeptidase like 1, VWFA domain-containing protein, Syndecan, Membrane spanning 4-domains A14 and Serine protease 54. This study shows a novel protocol to induce acrosome reaction in boar spermatozoa and identifies new transmembrane proteins containing mannose residues. Further work is needed to elucidate the role of these proteins in sperm-oocyte fusion. • Incubation with 4-Br-A23187 for 20 min induces acrosome reaction in boar spermatozoa. • Glycosylated protein profile changes after acrosome reaction induction. • 50 proteins containing mannose residues showed regulation after acrosome reaction. • 6 transmembrane proteins containing mannose residues were regulated. [ABSTRACT FROM AUTHOR]

We evaluated the quality and fertilizing ability of frozen–thawed porcine sperm that were selected using a commercially available device (MIGLIS, Menicon Life Science) consisting of three parts: an outer lid, an inner lid, and a tube. Firstly, to determine an adequate concentration of caffeine for separation, frozen–thawed sperm were incubated with different concentrations of caffeine (0, 1, 2.5, 5, and 10 mM) in a MIGLIS device. To determine the appropriate incubation time for separating sperm in the MIGLIS device, frozen–thawed sperm were incubated with 2.5 mM caffeine for 5, 10, 15, or 20 min. To evaluate the fertilization and embryo development of oocytes fertilized with frozen–thawed sperm separated into two regions (outer and inner) in the MIGLIS device, the separated sperm from the three boars was used to fertilize in vitro‐matured oocytes and cultured in vitro for 7 days. Sperm quality parameters of sperm collected from the inner tube after incubation with 2.5 mM caffeine were superior to sperm incubated without caffeine. Moreover, sperm collected from the inner tube after incubation for 10 min had a higher progressive motility. The rate of blastocyst produced from spermatozoa collected from the inner tube after incubation with 2.5 mM caffeine for 10 min significantly increased compared to that produced from spermatozoa from the outer tube, regardless of the boar. In conclusion, sperm sorting using the MIGLIS device may be useful for separating high‐quality sperm after incubation with 2.5 mM caffeine for 10 min to improve blastocyst formation. [ABSTRACT FROM AUTHOR]

In this study, we investigated the correlation between the composition and function of the gut microbiota and the semen quality of Rongchang boars. Significant differences in gut microbial composition between boars with high (group H) and low (group L) semen utilization rates were identified through 16S rRNA gene sequencing, with 18 differential microbes observed at the genus level. Boars with lower semen utilization rates exhibited a higher relative abundance of Treponema, suggesting its potential role in reducing semen quality. Conversely, boars with higher semen utilization rates showed increased relative abundances of Terrisporobacter, Turicibacter, Stenotrophomonas, Clostridium sensu stricto 3, and Bifidobacterium, with Stenotrophomonas and Clostridium sensu stricto 3 showing a significant positive correlation with semen utilization rates. The metabolomic analyses revealed higher levels of gluconolactone, D-ribose, and 4-pyridoxic acid in the H group, with 4 pyridoxic acid and D-ribose showing a significant positive correlation with Terrisporobacter and Clostridium sensu stricto 3, respectively. In contrast, the L group showed elevated levels of D-erythrose-4-phosphate, which correlated negatively with Bifidobacterium and Clostridium sensu stricto 3. These differential metabolites were enriched in the pentose phosphate pathway, vitamin B6 metabolism, and antifolate resistance, potentially influencing semen quality. These findings provide new insights into the complex interplay between the gut microbiota and boar reproductive health and may offer important information for the discovery of disease biomarkers and reproductive health management. [ABSTRACT FROM AUTHOR]

This study aims to see the effectiveness of Yorkshire boar semen natural modification of the natural diluent of green guava filtrate (Syzygiumaqueum). Semen is stored per 72 hours from a 3-year-old sow. Macro and microscopic evaluation of semen includes Volume, Colour, Consistency, pH, Odor and motility, concentration, viability, abnormality and normality of spermatozoa. The semen was diluted with palm fruit juice and added green guava filtrate (Syzygiumaqueum) in tiers: 100μL, 200μL, 300μL, 400 μL and 500 μL. The results showed that the modification of palm fruit juice diluent with the addition of 200 μL of green guava filtrate (Syzygiumaqueum) in Yorkshire boar semen was able to maintain the quality of spermatozoa up to 22 hours of storage. [ABSTRACT FROM AUTHOR]

Simple Summary: Liquid nitrogen (LN) is usually used for semen cryopreservation. The most significant point is that it has no time or space restrictions, maximizing the use of high-quality males for breeding. Dry ice is also one of the earliest media used in animal semen cryopreservation. The efficiency of dry ice cryopreservation is lower than that of LN. However, compared with LN, dry ice has an advantage in transportation, as it permits express delivery and can be carried on trains and high-speed railways. Therefore, using dry ice as a cold source for preserving and transporting ultralow-temperature-preserved semen liquid has received extensive attention from scholars. It has been demonstrated in frozen semen from mice and cattle that dry ice can be used as a reliable cold source for transportation. For this reason, we simulated a dry ice transportation environment by placing ultralow-temperature-frozen semen in a foam box with sufficient dry ice to maintain it for one week. We found that one week of storage in dry ice did not cause significant damage to porcine spermatozoa. The effects on the semen were essentially the same as those of LN storage when the dry-ice-preserved spermatozoa were transferred to LN again after one week. To address the safety problems posed by the transportation of boar semen using LN, this study was conducted on the short-term storage of frozen boar semen in dry ice (−79 °C). Boar semen frozen in LN was transferred to dry ice, kept for 1 day, 3 days, 5 days, 7 days, or 8 days, and then moved back to LN. The quality of frozen semen stored in LN or dry ice was determined to evaluate the feasibility of short-distance transportation with dry ice. The results showed that 60 °C for 8 s was the best condition for thawing frozen semen stored in dry ice. No significant differences in spermatozoa motility, plasma membrane integrity, or acrosome integrity were observed in semen after short-term storage in dry ice compared to LN (p > 0.05). There were no significant changes in antioxidant properties between storage groups either (p > 0.05). In conclusion, dry ice could be used as a cold source for the short-term transportation of frozen boar semen for at least 7 days, without affecting sperm motility, morphological integrity, or antioxidant indices. [ABSTRACT FROM AUTHOR]

A total of 125 animals were examined to check the validity of the estimation of the carcass composition of boars and immuno castrates using AutoFOM III (AF3) and the German reference formula for lean meat content based on females and surgical castrates. These animals represented four sex categories, gilts (W), surgical castrates (K), immunological castrates (IM) and boars (ME) originating from two different genetic backgrounds (Duroc and Pietrain sire breeds). Manual dissection was used as reference method. In addition, 32 of the 125 animals were fully dissected into tissue components according to the EU reference method. The percentage of total carcass lean meat was estimated with sufficient accuracy regardless of genetics or category. However, weight estimation of primal cuts by AF3 alone showed in some cases high deviations. An average overestimation of 8.4% in boneless loin weights was found for animals with Duroc sires. In Pietrain, an average overestimation of 5.3% was found for the weight of boneless ham. The AF3 weight estimation of all cuts for ME showed the highest deviations, closely followed by the cut weights from IM. Particularly striking was the extent of the overestimation for boneless loin with an average of 6.3% for ME and 5.6% for IM. [ABSTRACT FROM AUTHOR]

Simple Summary: Boar sperm is highly sensitive to reactive oxygen species (ROS) during the freezing–thawing process. κ-Carrageenan is a sulfated polysaccharide from seaweed reported to possess substantial antioxidant activities. However, the influence of κ-Carrageenan on porcine sperm cryo-survival remains to be clarified. Therefore, different concentrations of 0 (control), 0.2, 0.4, 0.6, and 0.8 mg/mL κ-Carrageenan were added to the freezing diluent. Sperm kinematics were assessed (CASA software) immediately after thawing (AT) and post-incubation for 120 min. Viability, acrosome integrity, lipid peroxidation, and intracellular caspase activities were measured AT using SYBR-14/PI, PSA/FITC, the MDA assay, and a commercial kit, respectively. The results indicated that the addition of κ-Carrageenan to the extender could enhance the quality of frozen–thawed (FT) boar sperm through its influence on membrane stability, mitochondrial potential, and reducing oxidative stress. Furthermore, 0.2 mg/mL κ-Carrageenan was revealed to be the optimal concentration for FT sperm quality. κ-Carrageenan is a sulfated polysaccharide from red seaweed with substantial antioxidant activities. This study aimed to investigate the effect of κ-Carrageenan treatment on frozen–thawed (FT) porcine semen quality. Therefore, the spermatozoa were diluted and cryopreserved in a freezing extender supplemented with 0 (control), 0.2, 0.4, 0.6, and 0.8 mg/mL κ-Carrageenan. Sperm kinematics were assessed immediately after thawing (AT) and post-incubation for 120 min. The viability, acrosome integrity, lipid peroxidation, mitochondrial membrane potential (MMP), and intracellular caspase activity were measured AT. The results indicated that 0.2 mg/mL κ-Carrageenan increased total and progressive motility AT and post-incubation for 120 min (p < 0.05). Moreover, the viable sperm percentage and MMP after 0.2 mg/mL treatment were higher than those after control and other κ-Carrageenan concentration treatments. The proportion of acrosome-intact spermatozoa was significantly higher after 0.2 and 0.4 mg/mL κ-Carrageenan treatment than that after control and other κ-Carrageenan concentration treatments. The intracellular caspase activity was not significantly different among the experimental groups. However, the MDA concentration after 0.2 mg/mL κ-Carrageenan treatment was lower (p < 0.05) than that after the control treatment. Taken together, adding κ-Carrageenan to the porcine semen freezing extender improved the FT sperm quality mainly by influencing membrane stability and protecting against oxidative stress. [ABSTRACT FROM AUTHOR]

Sperm quality is an important indicator to evaluate the reproduction ability of animals. Nicotinamide mononucleotide (NMN) participates in cell energy metabolism and reduces cell oxidative stress. However, the effect and regulatory mechanism of NMN on porcine sperm quality are still unknown. Here, 32 Landrace boars were randomly assigned to four groups (n = 8) and fed with different levels of NMN (0, 8, 16 or 32 mg/kg/d) for 9 weeks, and then serum and semen samples of the boars were collected to investigate the function and molecular mechanism of NMN in sperm quality. The results showed that the dietary NMN supplementation significantly increased sperm volume, density and motility (p < 0.05). Interestingly, NMN apparently improved the antioxidative indexes and increased the levels of testosterone (p < 0.05) in serum. Furthermore, NMN upregulated the protein levels of sirtuin 3 (SIRT3), antioxidation and oxidative phosphorylation (OXPHOS), but downregulated the protein levels of apoptosis in semen. Mechanically, NMN protected sperm from H2O2-induced oxidative stress and apoptosis through SIRT3 deacetylation. Importantly, the SIRT3-specific inhibitor 3-TYP attenuated the antioxidation and antiapoptosis of NMN in sperm. Therefore, NMN exerts antioxidation and antiapoptosis to improve boar sperm quality via the SIRT3 signaling pathway. Our findings suggest that NMN is a novel potential boar antioxidative feed additive to produce high-quality porcine semen. [ABSTRACT FROM AUTHOR]

The elimination of ejaculates and males with low fertility despite good sperm motility and morphology is crucial to maintain high pregnancy rates after artificial insemination (AI) in farm animals. The ability of sperm to survive in the female tract is particularly crucial in pigs due to the large variation in the timing between AI and ovulation and the high number of oocytes to fertilise. The objective of this study was to characterise a new in vitro model of oviduct sperm reservoir using porcine oviduct epithelial spheroids (OES) and to assess the variability in sperm binding to OES among gilts, boars and their ejaculates. Isthmic mucosa fragments were collected from gilt oviducts at a slaughterhouse, and after 48 h of culture, the OES that had spontaneously formed were sorted according to their vesicle shape and size (150–200 μm in diameter) for characterisation and sperm binding assays. The OES contained viable, cytokeratin-positive and vimentin-negative cells, of which 36.4 ± 2.0% were multiciliated. The average proportion of multiciliated cells per OES did not change among culture replicates. After co-incubation with boar fresh semen, only sperm of normal morphology were found to bind, by their head, to cilia of OES. The density of sperm bound to the OES surface increased linearly with sperm concentration. The bound sperm density on OES was used to assess the binding capacity of fresh ejaculates collected from Pietrain boars. For a given ejaculate, the bound sperm density did not vary among pools of OES female donors. The analysis of five successive ejaculates from nine boars indicated significant differences in bound sperm densities on the OES among individual boars and their ejaculates (P < 0.01). There was no correlation between the sperm bound density and sperm parameters measured by computer-assisted sperm analysis or the initial dilution of the ejaculate. In conclusion, the OES characterised in this study offered physiological conditions to study sperm binding to the isthmic reservoir and evidenced that sperm from different ejaculates and different boars vary in their ability to bind to these oviduct spheroids despite homogeneous motility and morphology. • Isthmus epithelial spheroids provided a standardised tool for sperm binding assays. • Binding capacity of boar fresh semen to spheroids did not change among gilt donors. • A considerable variation in sperm binding to spheroids was observed among boars. • A high variation in binding was also observed among ejaculates from individual boars. • This variability could not be explained by sperm parameters measured by CASA. [ABSTRACT FROM AUTHOR]

High sperm cryotolerance is crucial to the successful cryopreservation of boar sperm. Evaluating the cryotolerance of boar sperm by using a rapid and convenient technique can enhance the commercial viability of these sperm. This study investigated the correlation between sperm parameters for three sample subsets--fresh sperm, sperm with H2O2-induced oxidative damage (hereinafter referred to as H2O2-induced sperm), and frozen-thawed sperm--to identify the potential of these correlations to predict cryotolerance. A total of 64 sperm samples were obtained from 64 Duroc boars. The sperm parameters of the three subsets, where the frozen-thawed sperm were analysed at 30 or 180 min after thawing, were determined, and the coefficients of correlation between these parameters were calculated. The results indicated that H2O2-induced oxidative stress resulted in decreases in various sperm parameters--including total motility (TM), viability (VIA), mitochondrial membrane potential (MMP), and live sperm with MMP (LMP)--but increased their coefficients of variation. Receiver operating characteristic (ROC) curve analysis revealed that the kinematic parameters of the H2O2-induced sperm effectively predicted those of the frozen-thawed boar sperm at 30 min after thawing; the corresponding area under the ROC curve (AUC) was 0.8667 for TM and 0.8733 for progressive motility in the H2O2-induced sperm. For measurement at 180 min after thawing, the sperm membrane and mitochondrial parameters of the H2O2-induced sperm effectively predicted the LMP of the frozen-thawed boar sperm; the corresponding AUC was 0.8489 for VIA, 0.8289 for MMP, and 0.8444 for LMP. To our knowledge, this is the first study to directly establish a strong correlation between post-thaw boar sperm quality and H2O2-induced oxidative stress before freezing. Our proposed technique can serve as a valuable reference for the development of practical applications aimed at enhancing techniques for cryopreserving boar sperm. [ABSTRACT FROM AUTHOR]

We aimed to investigate the impact of processing boar spermatozoa with phosphate-buffered saline (PBS) at 4 ˚C on acrosomal integrity and increase in 32 kDa tyrosine-phosphorylated protein (p32). Following cooled PBS washing, we observed a significant increase in p32 levels and in the proportion of dead spermatozoa with compromised acrosomal integrity compared to sperm washing using PBS at room temperature. Interestingly, this increase in p32 was effectively inhibited when cooled PBS was supplemented with 1 mM AEBSF, a serine protease inhibitor. Our findings suggest that the increase of p32 in response to cooled PBS washing in boar spermatozoa is associated with enhanced protease activity in dead spermatozoa. [ABSTRACT FROM AUTHOR]

Editors have struggled to reconcile the two appearances of the term knot in the boar chase of Sir Gawain and the Green Knight (lines 1431 and 1434) with the competing demands of intelligible syntax and etymological sense. Most opt for the sense “rocky hill” for both, but this forces other terms in the passage, namely umbekesten and even hem (“them”) into the status as very unlikely hapaxes. This essay advocates acceptance of the necessity of emendation at line 1434 as by far the least intrusive solution, and proposes the new possibility of covert, “hiding place, thicket.” [ABSTRACT FROM AUTHOR]

Hypothermic liquid storage at 4–5 °C has emerged as a novel approach for preserving boar semen, offering innovative possibilities for semen preservation. However, this method also presents challenges, including cold shock and excessive reactive oxygen species (ROS) production. Therefore, reducing oxidative damage induced by low temperatures becomes essential while supplementing appropriate protectants. In this study, we investigated the efficacy of Bovine Serum Albumin (BSA) compared to Polyvinylpyrrolidone (PVP) and Skim Milk Powder (SMP) in maintaining boar sperm motility and progressive motility using computer-assisted sperm analysis (CASA). Among the tested concentrations, 4 g/L of BSA exhibited the best protective effect. Subsequently, we supplemented different concentrations of l -cysteine (LC) and N-acetyl- l -cysteine (NAC) as additives in the presence of BSA as a protectant. Our results demonstrated that 1 mmol/L of LC and 0.5 mmol/L of NAC exhibited superior protection of sperm quality compared to other concentrations. Furthermore, the 1 mmol/L LC and 0.5 mmol/L NAC groups showed significantly improved plasma membrane integrity and acrosome integrity compared to the control group. These groups also exhibited enhanced antioxidant capacity, evidenced by increased mitochondrial membrane potential (MMP), ATP production, total superoxide dismutase (T-SOD) activity, total antioxidant capacity (T-AOC), glutathione (GSH), glutathione peroxidase (GSH-PX), and GPX-4 levels. Additionally, they demonstrated decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as reduced oxidized glutathione (GSSG) and glutathione reductase (GR) levels. Furthermore, LC and NAC treatment enhanced AMP-activated protein kinase (AMPK) phosphorylation. However, inhibiting AMPK using compound C did not inhibit the protective effects of LC and NAC on low-temperature preserved boar sperm. These findings suggest that 4 g/L BSA can serve as an effective protectant for hypothermic liquid storage of boar semen. Additionally, LC and NAC supplementation reduces oxidative damage by enhancing antioxidant capacity rather than through AMPK-mediated ATP supplementation. These results contribute to advancing the application of LC and NAC in hypothermic liquid storage of boar semen. • 4 g/L BSA can serve as an effective cryoprotectant for hypothermic liquid storage of boar semen. • LC and NAC supplementation reduces oxidative damage through enhancing antioxidant capacity. • Inhibiting AMPK did not inhibit the protective effects of LC and NAC on low-temperature preserved boar sperm. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance is an increasing challenge in semen preservation of breeding animals, especially in the porcine species. Bacteria are a natural component of semen, and their growth should be inhibited to protect sperm fertilizing capacity and the female’s health. In pig breeding, where semen is routinely stored at 17°C in the liquid state, alternatives to conventional antibiotics are urgently needed. Photodynamic inactivation (PDI) of bacteria is a well-established tool in medicine and the food industry but this technology has not been widely adopted in semen preservation. The specific challenge in this setting is to selectively inactivate bacteria while maintaining sperm integrity and functionality. The aim of this study was to test the principle of PDI in liquid stored boar semen using the photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) and a white light LED-setup. In the first step, photophysical experiments comprising singlet oxygen phosphorescence kinetics of TMPyP and determination of the photosensitizer triplet time revealed a sufficiently high production of reactive singlet oxygen in the Androstar Premium semen extender, whereas seminal plasma acted as strong quencher. In vitro experiments with extended boar semen showed that the established PDI protocol preserves sperm motility, membrane integrity, DNA integrity, and mitochondrial activity while efficiently reducing the bacteria below the detection limit. A proof-of-concept insemination study confirmed the in vivo fertility of semen after photodynamic treatment. In conclusion, using the PDI approach, an innovative tool was established that efficiently controls bacteria growth in extended boar and maintains sperm fertility. This could be a promising contribution to the One Health concept with the potential to reduce antimicrobial resistance in animal husbandry.

Objective This study investigated the efficacy of different concentrations of gelatin supplementation in long-term semen extender on boar semen quality during storage for 10 days at 17°C. Additionally, oxytocin was added to stored semen to enhance fertility. Methods In Experiment 1, boar semen was collected, diluted with gelatin at concentrations between 0% and 2.5% (w/v) and mixed with a semen extender. Then, it was kept in a refrigerator at 17°C and stored for 10 days. In Experiment 2, the sperm quality was examined after adding 0, 5, and 10 IU oxytocin per artificial insemination dose to the most effective semen extender from Experiment 1 and placing it in a refrigerator at 17°C for 10 days. In Experiment 3, the fertility potential in terms of non-return rate and litter size was determined using the most effective solid-stored semen supplemented with oxytocin. Results The results indicated that sperm quality decreased with increasing storage time (p0.05). The non-return rate and litter size after insemination with semen supplemented with 1.5% gelatin and 10 IU of oxytocin after 8 to 10 days of storage were comparable to those of the control group (p>0.05). Conclusion A semen extender as a solid medium supplemented with 1.5% gelatin successfully preserved boar semen for a long storage duration. Treatment with oxytocin did not affect sperm quality. In addition, the fertility capacity using 1.5% gelatin with 10 IU oxytocin and stored for 8 to 10 days was acceptable and comparable to that of short-term storage.

The primary objective of this research was to evaluate boar ejaculate variability and the occurrence of anomalies in spermatozoa, considering climatic factors during spermatogenesis breed, and utilization frequency. This study involved 17 boars (n=129 ejaculates) and fertility testing was conducted during the most critical period of the year, from August to October. The observed sperm characteristics included: ejaculate volume (VOL), sperm concentration (CON, spermatozoa/ml), total number and number of functional spermatozoa (NT, NF), percentage of sperm motility in the native ejaculate and after dilution (MOTN, MOTD), number of produced doses (NPD), percentage of dead and live spermatozoa (PM, PZ), and sperm anomalies. The assessment of the effect was performed using a General Linear Model procedure. The breed did not influence sperm variability, while the frequency of boar utilization impacted on the occurrence of secondary anomalies. The determined regression coefficient indicated that extending the interval by one day increased PPPK by 0.340-0.348%. The maximum daily temperature during semen collection (model 1) and the value of the TH index during semen collection (model 3) influenced ejaculate volume. An increase of one ℃ in temperature, or one unit in THI value, led to a (p

Mesenchymal stem cells (MSCs) have been known to mankind since the mid-20th century. The comprehensive study revealed their high biologically active potential. Capacity of forming several types of body tissues was demonstrated. The stem cells, like any other cells, exert their effect on surrounding cells and tissues by secreting extracellular vesicles. The extracellular vesicles of the stem cells possess biological activity of parent cells. Taking into account the regenerative potential of the mesenchymal stem cells, they are currently used in medicine, and also in veterinary medicine for treatment of various injuries of the companion animals. Effect of the mesenchymal stem cells on boar and rat sperm cells during 12-hour storage was studied. The study results demonstrated that during 12 hours of coincubation, the porcine MSCs contributed to the survival of the boar sperm cells and maintenance of their motility at 60–80% (depending on the solvent) as compared to the controls. Such a significant effect was not however observed during coincubation of the rat sperm cells with rat MSCs. But it should be noted that before the3rdhour of coincubation, the experimental sperm motility was higher than that of the control. By hour5 of the observation, this difference was leveled. The rat and boar sperm cells are likely to have different physiological characteristics, which were reflected in the results obtained. Therefore, possibility of using the MSCs for the storage and cryopreservation of the semen of some animals was demonstrated, but this requires further research.

The purpose of the research is localization and morphometry of sarcocysts in animals.Materials and methods. Samples of striated muscle tissue of cows and wild boars were studied to determine the infection intensity and prevalence and determine morphometric parameters of cysts. Pathological material was collected to 10 % buffered formalin solution. For histological examination, muscle tissue samples were taken from the abdominal wall and cardiac muscle. To process tissue samples, paraffin coating was used in semi-automatic equipment from Thermo Scientific. Histological specimens were stained with hematoxylin and eosin and by Mallouri. The specimen histoarchitecture was assessed using an Axio A1.0 microscope; photographs were taken with AxioVision.Results and discussion. The sarcocyst infection prevalence was 60% in cattle and 100% in the boars, with the infection intensity of 1-2 and 2-4 sarcocysts in one FOV, respectively. It was found that the cyst formed a capsule in infected boars with a thickness of 2.6±0.28 μm, while it was practically invisible in cattle, and grouped loose connective tissue fibers were observed around the cyst. The infection intensity can be assessed as low in the studied animals; however, even single parasite cysts entered the human body contribute to sarcocystosis.

This study aimed to investigate whether the intensity of lighting may affect Duroc boars’ semen parameters in different seasons and in different periods from the beginning of the use of boars for semen collection. A total of 6879 ejaculates were collected from 21 boars and kept under natural low light intensity and daylight length conditions, and 3141 ejaculates from 10 boars were kept under high light intensity (190-210 lx) and natural daylight length conditions. Experimental groups were divided according to the intensity of lighting (low, high), season (winter, spring, summer, fall), and the period from the beginning of the use of boars for semen collection (0-1 year, 1.1-2 years, 2.1-3 years). Selected parameters were analyzed: semen volume, sperm concentration, total number of sperms, sperm motility, percentage of sperms with an abnormal morphology and number of insemination doses from one ejaculate. Semen volume, sperm concentration, and rate of sperms with an abnormal morphology were associated with the two-way interaction between light intensity and season (P < 0.0001), and between light intensity and period from the first semen collection (P < 0.0001). The total number of sperms and number of insemination doses from one ejaculate were associated with the two-way interaction between light intensity and season (P < 0.0001). Sperm motility was associated with the two-way interaction between light intensity and season (P < 0.0001), and between light intensity and period from the first semen collection (P < 0.01). In conclusion, the intensity of lighting can affect Duroc boars’ semen parameters in different seasons and different periods from the beginning of the use of boars for semen collection.

Boar semen is commonly used in artificial insemination (AI) for pig breeding, but its quality can be negatively affected by liquid preservation and transportation, leading to reduced fertility rates. Vibration and temperature fluctuations are critical factors that significantly impact semen quality during storage and transportation, influencing the success rate of AI procedures. Betaine, a naturally occurring compound known for its role in maintaining male fertility, demonstrates potential for improving the preservation and transportation of liquid-preserved boar sperm. The present study demonstrated that betaine supplementation in the semen extenders at 0.5 mg/mL had a significant protective effect on boar sperm motility during storage at 17 °C for 3 to 5 days. During road transportation, 2.5 mg/mL betaine showed significant protective effects on boar sperm progressive motility, while 0.4 mg/mL betaine notably improved boar sperm mitochondrial activity and antioxidant capacity, and reduced lipid peroxidation damage. Simulation models also demonstrated that betaine supplementation increased the proportion of sperm displaying progressive motility and possessing intact acrosomes, regardless of the storage temperature (17 °C or 25 °C), and effectively mitigated the damage caused by vibration at a speed of 200 r/min. Overall, supplementing liquid-preserved boar semen extenders with betaine shows promise in mitigating damage to sperm quality during storage and transportation.

Semen proteins play an important role in male reproductive performance and sperm fertilization ability and can be used as potential biomarkers to evaluate male fertility. The role of cysteine-rich secretory protein 3 (CRISP3) in male reproduction remains unknown. This study aimed to investigate the role of CRISP3 in the reproductive performance of boars. Our results showed that the CRISP3 protein content was significantly and positively correlated with boar fertility, sow delivery rate, and litter size. CRISP3 is highly expressed in the bulbourethral gland of adult boars and is enriched in the seminal plasma. It is localized in the post-acrosomal region of the sperm head and migrates to the anterior end of the tail after capacitation. The CRISP3 recombinant protein did not affect sperm motility and cleavage rate, but it significantly downregulated the mRNA expression of inflammatory factors IL-α, IL-1β, and IL-6 and the protein expression of IL-α and IL-6 in lipopolysaccharide (LPS)-induced RAW264.7 cells, indicating that CRISP3 has an immunomodulatory function. In conclusion, our study suggests that semen CRISP3 protein levels positively correlate with reproductive performance, which may be achieved by regulating immune responses in the female reproductive tract. [ABSTRACT FROM AUTHOR]

Chromatin status is critical for sperm fertility and reflects spermatogenic success. We tested a multivariate approach for studying pig sperm chromatin structure to capture its complexity with a set of quick and simple techniques, going beyond the usual assessment of DNA damage. Sperm doses from 36 boars (3 ejaculates/boar) were stored at 17 °C and analyzed on days 0 and 11. Analyses were: CASA (motility) and flow cytometry to assess sperm functionality and chromatin structure by SCSA (%DFI, DNA fragmentation; %HDS, chromatin maturity), monobromobimane (mBBr, tiol status/disulfide bridges between protamines), chromomycin A3 (CMA3, protamination), and 8-hydroxy-2′-deoxyguanosine (8-oxo-dG, DNA oxidative damage). Data were analyzed using linear models for the effects of boar and storage, correlations, and multivariate analysis as hierarchical clustering and principal component analysis (PCA). Storage reduced sperm quality parameters, mainly motility, with no critical oxidative stress increases, while chromatin status worsened slightly (%DFI and 8-oxo-dG increased while mBBr MFI—median fluorescence intensity—and disulfide bridge levels decreased). Boar significantly affected most chromatin variables except CMA3; storage also affected most variables except %HDS. At day 0, sperm chromatin variables clustered closely, except for CMA3, and %HDS and 8-oxo-dG correlated with many variables (notably, mBBr). After storage, the relation between %HDS and 8-oxo-dG remained, but correlations among other variables disappeared, and mBBr variables clustered separately. The PCA suggested a considerable influence of mBBr on sample variance, especially regarding storage, with SCSA and 8-oxo-dG affecting between-sample variability. Overall, CMA3 was the least informative, in contrast with results in other species. The combination of DNA fragmentation, DNA oxidation, chromatin compaction, and tiol status seems a good candidate for obtaining a complete picture of pig sperm nucleus status. It raises many questions for future molecular studies and deserves further research to establish its usefulness as a fertility predictor in multivariate models. The usefulness of CMA3 should be clarified. [ABSTRACT FROM AUTHOR]

Boar fertility is a key determinant of the production efficiency of the whole pig breeding industry and boar sperm motility is the seminal parameter with the greatest impact on the fecundity of a sow. Exosomes are small, extracellular vesicles found in many body fluids. Seminal plasma exosomes, which are secreted by the epididymis, prostate, seminal vesicles, and testes, contain a large number of miRNAs, the types and levels of which can reflect the physiological state of source cells. It has been shown that the expression profile of seminal plasma exosomal miRNA differs between low-motility semen and normal semen. The aim of this study was to investigate the relationship between semen motility and exosomal miRNA profiles to obtain information that would allow to predict boar fertility, as well as contribute to the understanding of the mechanisms by which exosomal miRNAs regulate semen motility. Three high-motility (semen motility >90 %) and three low-motility (semen motility <80 %) semen samples were collected from Landrace and Yorkshire boars, respectively, and seminal plasma exosomes were extracted by ultracentrifugation. Exosome characterization was performed using transmission electron microscopy, NTA, and Western blot. The expression profiles of exosomal miRNAs associated with semen motility in the two boar breeds were subsequently determined by small RNA sequencing. The results showed that 297 known miRNAs and 295 novel RNAs were co-expressed in the four groups. Notably, six miRNAs (ssc-miR-122–5p, ssc-miR-486, ssc-miR-451, ssc-miR-345–3p, ssc-miR-362, and ssc-miR-500–5p) were found to be differentially expressed in both boar breeds. Enrichment analysis of the target genes of the differentially expressed miRNAs showed that they were mainly involved in biological processes such as regulation of transcription from RNA polymerase II promoter, regulation of gene expression, and intracellular signal transduction and signaling pathways such as the PI3K-Akt, MAPK, and Ras signaling pathways. The six differentially expressed miRNAs identified in this study have significant potential as noninvasive markers of boar semen motility. Meanwhile, the results of the enrichment analysis provide novel insights into the mechanisms underlying the regulation of semen motility. • Exosomal miRNAs related to sperm motility identified in Landrace and Yorkshire. • miRNA profiles in exosomes from distinct semen motility levels differed. • Higher miRNA expression in low motility group compared to high motility group. • Common detection of 6 miRNAs in the differential analysis of Landrace and Yorkshire. • The targets of DE miRNAs are involved in RNA transcription, gene regulation, etc. [ABSTRACT FROM AUTHOR]

Nobiletin (NOB) is a bioflavonoid compound isolated from citrus fruit peels. The present study aimed to elucidate whether NOB facilitates the porcine sperm cryosurvival and embryo development after in vitro fertilization (IVF). To this end, spermatozoa were diluted and cryopreserved in a freezing extender supplemented with 0 (control), 50, 100, 150, and 200 μM Nobiletin. The kinematic patterns of frozen-thawed (FT) sperm were assessed after 30 and 90 min incubation using a Sperm Class Analyzer (SCA). Viability, acrosome integrity, and mitochondrial membrane potential (MMP) were measured by fluorescence microscopy 30 min after thawing using SYBR-14/PI, PSA/FITC, and R123/PI, respectively. Lipid peroxidation was determined using MDA assay after incubation for 90 min. The addition of 100 μM and 150 μM NOB to the extender significantly improved sperm progressive motility, and acrosome integrity compared to the control group (P < 0.05). The proportion of viable spermatozoa was significantly higher in the 150 μM NOB group. MDA levels were less in 50 μM and 150 μM NOB treated groups compared to the control. In addition, IVF with FT sperm was used to assess the embryo developmental competence. Treatment with 150 μM NOB before cryopreservation increased the cleavage and blastocyst formation rates compared to the control group. Furthermore, the relative expression of POU5F1 and AMPK , genes related to pluripotency and cell differentiation were significantly upregulated in embryos resulting from NOB-treated sperm compared to the control group. These results suggest that Nobiletin is a functionally novel phytochemical to mitigate oxidative stress during the freezing-thawing of porcine spermatozoa as reflected by improved FT sperm quality and IVF outcome. • Supplementation with Nobiletin (NOB) improved porcine sperm cryo-survival. • NOB improves sperm freezability by lowering the lipid peroxidation of sperm cells. • NOB addition to the freezing extender enhanced the embryo development. • NOB affects the expression of cell differentiation and pluripotency genes in embryos. [ABSTRACT FROM AUTHOR]

Spermatogenesis is a delicate and complex biological process in which spermatogonial stem cells continue to proliferate and differentiate into mature spermatozoa, maintaining sperm production in male mammals throughout the lifetime. To study the molecular mechanism of spermatogenesis, researchers had to isolate different germ cell subpopulations for in vitro culture and characterization. However, due to the existence of several stages of germ cells and a variety of populations of somatic cells in the testis of male mammals, it is a challenge for us to obtain high-purity germ cell subpopulations for further research. Here, we optimized the STA-PUT device and successfully applied it to isolate and purify spermatogonia populations in piglets, and multiple germ cell populations at different developmental stages in testes of adult mice and boars. This work provides a simple platform for germ cell fractionation to facilitate the molecular mechanistic study of animal spermatogenesis in vitro. • We established an optimized velocity sedimentation STA-PUT device for cell fractionation. • The STA-PUT device was applied to isolate multiple germ cell populations in mice and pigs. • The STA-PUT device can isolate spermatogonia, spermatocytes, and spermatids in a high purity and a sufficient number. [ABSTRACT FROM AUTHOR]

[Objectives]The objective of this experiment was to study the microbial composition, testosterone and endotoxin content of feces, from boars with different semen quality, and analyze the correlation between semen quality and fecal microbiota, testosterone and endotoxin. It will provide a basis for improving semen quality by regulating gut microbiota in pig production. [Methods]According to semen quality parameters of all boars in the three months before the experiment, 33 York boars of similar age(479.33±2.85)and body condition were selected and divided into low semen quality group(LSQ, n=12)and high semen quality group(HSQ, n=21). Fresh feces of experimental boars were collected to detect the content of testosterone and endotoxin, and the microbial composition of feces was analyzed by 16S ribosomal RNA gene sequencing technique. [Results]The fecal testosterone level of boars in the HSQ group was significantly higher than that in the LSQ group(P<0.01), and there was no significant difference in endotoxin levels. There were 3 differential bacteria on family level and 5 differential bacteria on genera level in the boar fecal microbiota of the HSQ group and LSQ group. At the family level, Eubacterium_coprostanoligenes_group was significantly enriched in the LSQ group(P<0.05), Atopobiaceae and Akkermansiaceae were significantly enriched in the HSQ group(P<0.05); at the genus level, g_norank_f_Eubacterium_coprostanoligenes_group and Faecalibaculum were significantly enriched in the LSQ group(P<0.05)and g_norank_f_Atopobiaceae, Akkermansia and Clostridium_sensu_stricto_6 were significantly enriched in the HSQ group(P<0.05). There was a significant negative correlation between Eubacterium_coprostanoligenes_group and sperm motility and sperm density(P<0.01), and there was a negative correlation between Eubacterium_coprostanoligenes_group and fecal testosterone levels(P=0.11); there was a significant positive correlation between Atopobiaceae and sperm motility, sperm density and fecal testosterone levels(P<0.05); Akkermansiaceae significantly positively correlated with sperm motility(P<0.01)and sperm density(P<0.05), and positively correlated with fecal testosterone levels(P=0.09). [Conclusions]The results showed that there were differences in the fecal testosterone level and microbial composition of boars with different semen quality, and there were different degrees of correlations between boar fecal microbial structure and semen quality, fecal testosterone levels. The results provide a new theoretical support for improving boar semen quality by regulating gut microbiota. [ABSTRACT FROM AUTHOR]

Artificial insemination (AI) with liquid-stored semen is the most prevalent and efficient assisted reproduction technique in the modern pork industry. Pyruvate dehydrogenase complex component X (PDHX) was demonstrated to be associated with sperm metabolism and affected the boar sperm viability, motility, and fertility. Pyruvate Dehydrogenase Kinases (PDKs) are the key metabolic enzymes that regulate pyruvate dehydrogenase complex (PDHC) activity and also the conversion from glycolysis to oxidative phosphorylation. In the present study, two PDK inhibitors, Dichloroacetate (DCA) and Phenylbutyrate (4-PBA), were added to an extender and investigated to determine their regulatory roles in liquid-stored boar sperm at 17 °C. The results indicated that PDK1 and PDK3 were predominantly located at the head and flagella of the boar sperm. The addition of 2 mM DCA and 0.5 mM 4-PBA significantly enhanced the sperm motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and ATP content. In addition, DCA and 4-PBA exerted their effects by inhibiting PDK1 and PDK3, respectively. In conclusion, DCA and 4-PBA were found to regulate the boar sperm metabolic activities via PDK1 and PDK3. These both can improve the quality parameters of liquid-stored boar sperm, which will help to improve and optimize liquid-stored boar semen after their addition in the extender. [ABSTRACT FROM AUTHOR]

Background and Aim: Seasonal changes, especially temperature and photoperiod, are well-known determining factors of swine reproductive capacity, but the underlying mechanisms remain unknown. This study aimed to investigate the effect of age and seasonal variations on boar seminal plasma steroids (dehydroepiandrosterone [DHEA], cortisol [CORT], and testosterone [TEST]) over 1 year. Materials and Methods: Four commercial hybrid adult boars (Large White × Duroc), aged between 12 and 44 months, were repeatedly evaluated at the Department of Veterinary Medical Sciences of the University of Bologna. Daily temperature and light hours relating to the collection date were considered for each observation within the four astronomical seasons: Winter, spring, summer, and autumn. Hormones were quantified using radioimmunoassay. The association between seasonal factors and hormone concentrations was evaluated using linear regression models. Univariate models were estimated for each hormone to assess the influence of the independent variables; two multivariate models were assessed to evaluate the effect of temperature and daylight hours, including boar and season factors. Results: Age significantly affected all analyzed hormones (CORT p < 0.0001; DHEA p < 0.0001; and TEST p < 0.0001). The highest average levels were found for each hormone during summertime, suggesting a positive correlation between steroid concentrations with temperature and light hours. Conclusion: The results of this study support the hypothesis that the increase in external temperature and light hours is somehow associated with higher levels of steroid concentrations in the seminal plasma of in-housed boars. These findings may help further investigate seasonal fluctuations in reproductive outcomes, which are well-known for porcine species.

Boar sperm quality serves as an important indicator of reproductive efficiency, playing a direct role in enhancing the output of livestock production. It has been demonstrated that mitochondrial protein translation is present in sperm and plays a crucial role in regulating sperm motility, capacitation and in vitro fertilization rate. The present study aimed to determine whether methionine supplementation enhances mitochondrial translation in boar sperm, thereby improving sperm quality. The results showed a significant elevation in the abundance of mitochondrial methionyl-tRNA formyltransferase (MTFMT), a crucial enzyme for mitochondrial protein translation, and mitochondrial DNA-encoded cytochrome c oxidase subunit 1 (COX1) in boar sperm exhibiting high motility. Both amino acids and methionine supplementation significantly enhanced boar sperm motility during storage. Moreover, methionine supplementation mitigates the loss of acrosomal integrity, enhances the expression of COX1, and boosts mitochondrial activity. Furthermore, the positive impact of methionine was negated in the presence of the mitochondrial translation inhibitor chloramphenicol. Together, these findings suggest that boar sperm may utilize methionine as a protein translation substrate to enhance sperm motility by stimulating mitochondrial protein translation. The supplementation of methionine may enhance the quality of boar sperm, thereby providing guidance for the optimization of diluent formulations for liquid storage and the identification of physiological regulators that regulate sperm motility.

Abstract Background Scrotal swelling is a clinical situation which can be caused by different aetiologies. In this case report, we describe a multi-week episode of unilateral and bilateral scrotal swelling in boars at an Austrian boar stud and its diagnostic work-up. Case presentation In the summer of 2020, the herd veterinarian of an Austrian boar stud reported that over a period of six weeks, five out of 70 boars presented with unilateral severe swelling of the left scrotum and three out of 70 boars with bilateral severe swelling of the left and moderate swelling of the right scrotum, respectively. A complete history was obtained and an on-site evaluation of the facility was done. Five boars were necropsied, and a variety of samples harvested for further diagnostic investigations. Infectious differential diagnoses associated with unilateral swelling of the scrotum or the testis were excluded through serological and tissue testing. In three of the five boars, histopathology revealed complete acute haemorrhagic necrosis of the left testis concurrent with strongly congested blood vessels. Review of the collected information with a group of experts in the field of boar stud management resulted with consensus that, most likely, trauma was the etiologic event causing the clinical signs and pathology. Coincident with discussion of implementing video recording cameras in the boar housing area, no further clinical cases followed. As this case occurred during the first lockdown of the COVID-19 pandemic, we propose that the distress and travelling restrictions may have contributed to frustration among boar stud workers, which was consequently expressed as misbehaviour against boars. Conclusions Once all known infectious causes of unilateral swelling of the scrotum were excluded, a critical diagnostic work-up focused on non-infectious causes. Non-infectious causes, such as trauma, need to be carefully evaluated, as it may also include human misbehaviour against boars. Summarizing all findings of this case report, the authors hypothesize that a blunt trauma was the reason for the series of mainly unilateral swelling of the scrota of boars.

Background and Aim: Seasonal changes, especially temperature and photoperiod, are well-known determining factors of swine reproductive capacity, but the underlying mechanisms remain unknown. This study aimed to investigate the effect of age and seasonal variations on boar seminal plasma steroids (dehydroepiandrosterone [DHEA], cortisol [CORT], and testosterone [TEST]) over 1 year. Materials and Methods: Four commercial hybrid adult boars (Large White × Duroc), aged between 12 and 44 months, were repeatedly evaluated at the Department of Veterinary Medical Sciences of the University of Bologna. Daily temperature and light hours relating to the collection date were considered for each observation within the four astronomical seasons: Winter, spring, summer, and autumn. Hormones were quantified using radioimmunoassay. The association between seasonal factors and hormone concentrations was evaluated using linear regression models. Univariate models were estimated for each hormone to assess the influence of the independent variables; two multivariate models were assessed to evaluate the effect of temperature and daylight hours, including boar and season factors. Results: Age significantly affected all analyzed hormones (CORT p < 0.0001; DHEA p < 0.0001; and TEST p < 0.0001). The highest average levels were found for each hormone during summertime, suggesting a positive correlation between steroid concentrations with temperature and light hours. Conclusion: The results of this study support the hypothesis that the increase in external temperature and light hours is somehow associated with higher levels of steroid concentrations in the seminal plasma of in-housed boars. These findings may help further investigate seasonal fluctuations in reproductive outcomes, which are well-known for porcine species. [ABSTRACT FROM AUTHOR]

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Simple Summary: Cryopreservation protocol induces reactive oxygen species formation and leads to a decrease in post-thawed sperm quality and fertilizing capacity. We investigated the effect of resveratrol and its optimal concentration on the quality of post-thawed boar semen and the level of malondialdehyde (MDA). Our investigations showed that the supplement of 50–100 µM resveratrol enhanced the quality of the post-thawed semen and decreased the MDA level. However, the maximum concentration of resveratrol (250 µM) tended to decrease sperm motility, sperm viability, intact acrosomes, and mitochondrial membrane potential. The protective or cytotoxic effect of resveratrol on the quality of frozen boar sperm depends on the concentration of resveratrol and extender composition as well as limiting antioxidant uptake capacity in sperm. This study aimed to determine the effect of resveratrol and its optimal concentration on the quality of frozen-thawed (FT) boar sperm. Semen ejaculates were obtained from 13 Duroc boars aged between 1.5 and 3 years. The sperm sample was separated into 7 groups based on the concentrations of resveratrol in the freezing extender, which were 0 (control), 25, 50, 75, 100, 125, and 250 µM, respectively. The sperm was frozen using liquid nitrogen vapor and thawed at 50 °C for 12 s. After thawing, total motility, progressive motility, viability, intact acrosomes, mitochondrial membrane potential and level of MDA were assessed. The supplementation of 50–100 µM resveratrol improved the sperm motility and viability of FT sperm in comparison to the control group (p < 0.05). Furthermore, the 50 µM resveratrol group was significantly more protective than the control group in terms of intact acrosome, mitochondrial membrane potential, and level of MDA (p < 0.05). Nonetheless, the detrimental effect of resveratrol was found at a concentration of 250 µM. In conclusion, the addition of 50–100 µM resveratrol to a freezing extender is the optimal concentration for enhancing the quality of cryopreserved boar sperm. [ABSTRACT FROM AUTHOR]

The intestinal microbiota is required for maintaining the development and health of the host. However, the gut microbiota contributing to the regulation of the growth performance and health of Duroc and Yorkshire boars remains largely unknown. In this study, we first evaluated the difference in the growth performance between Duroc and Yorkshire boars when their body weight reached 100 kg. Relative to Duroc boars, Yorkshire boars weighed 100 kg at a younger age and exhibited a significantly lower backfat thickness and eye muscle area. Microbial analysis of the fecal samples revealed a marked difference in gut microbiota composition between the two pig models and remarkably increased α-diversity in Yorkshire boars compared to Duroc boars. Further analysis indicated that Bacteroidota, Prevotellaceae, and Ruminococcaceae might be associated with the growth performance and lean meat rate of Yorkshire boars. Taken together, these results highlight that Yorkshire boars exhibit a faster growth rate and higher lean meat rate compared to Duroc boars, and these differences may be attributed to the influence of the gut microbiota, thereby providing valuable insight into optimizing pig breeding systems and selecting terminal paternal sires to enhance overall productivity and quality. [ABSTRACT FROM AUTHOR]

This study examined the effects of ergothioneine (EGT) supplementation as an antioxidant on the quality of boar spermatozoa when using liquid and frozen preservation methods. In the first experiment, boar semen was preserved in an extender supplemented with 0, 50, 100 and 200 µM EGT, at 15 °C, part of the samples for one and another part for three weeks. In comparison with the control (without EGT), EGT supplementation at 100 µM significantly increased the percentage of total motility of spermatozoa that were preserved as a liquid both for one and three weeks (P < 0.05). EGT supplementation did not affect the quality of preserved spermatozoa, irrespective of the EGT concentration. In the second experiment, semen was frozen and thawed in the freezing extender supplemented with 0, 50, 100 and 200 µM EGT. In comparison with the control, the 100 µM EGT supplementation significantly increased the percentages of total and progressive motility of frozen-thawed spermatozoa (P < 0.05). EGT (100 µM) supplementation did not affect the viability, the plasma membrane integrity, or the acrosomal integrity of frozen-thawed spermatozoa. These findings indicate that supplementing extenders with 100 µM EGT may improve the motility of boar sperm in both liquid and freezing preservation methods. [ABSTRACT FROM AUTHOR]

Abstract Background In tropical environments, boar semen is prepared either from a boar on the same farm as the sow herd or collected in semen collection centers and then transported to other farms. Thus, the semen doses can be used for artificial insemination either immediately or preserved for 2–3 days. The present study investigated the bacteriospermia and its antimicrobial resistance in relation to boar sperm quality during short-term storage in semen extender with or without antibiotics in Thailand. M&M In total, 20 Duroc ejaculates were collected. Each ejaculate was diluted in Beltsville Thawing Solution extender either with 0.25 g of gentamicin per liter (ANTIBIOTIC) or without gentamicin (NO-ANITIBIOTIC) to create semen doses containing 3,000 × 106 sperm/100 mL. These were stored at 17 °C for 4 days. Semen characteristics and total bacterial count (CFU per mL, log10) were measured after collection and during storage. Results Sperm viability was decreased by 6.4% for every 1.0 log10 increase in total bacterial count (p = 0.026) and Staphylococcus spp. were the most frequently isolated across ejaculates. Throughout the 4 days of storage, sperm motility, viability and acrosome integrity in the ANTIBIOTIC group were higher than those in the NO-ANTIBIOTIC group (p 0.05). On the last day of preservation, Globicatella sanguinis (57.2%), Delftia acidovorans (18.9%) and Micrococcus spp. (5.9%) remained as the top three most abundant contaminants in the semen with antibiotic. Conclusion Our findings contribute new insights toward reducing antibiotics as well as rational antibiotic use in the boar AI industry. The growth of bacteria was significantly greater only after 2 days of preservation in the semen without antibiotic. For semen doses diluted from highly viable ejaculates, it is possible to store for 2 days without any antibiotic supplementation. Moreover, bacterial counts increased at the end of storage in the presence of gentamycin, suggesting the loss of bacteriostatic properties of gentamicin to the growth of bacteria during storage.

Fertile sperm production is crucial to the reproductive efficiency of swine operations; therefore, many factors impair quality semen production including hormonal imbalance and altered immune function. Toll-like receptors (TLRs) are a family of pattern recognition receptors which recognize and protect their host against pathogenic microorganisms through the regulation of inflammatory pathways. Recent data from our lab demonstrated greater expression of TLR2 mRNAs in the testes compared with other reproductive tissues (e.g., epididymis, seminal vesicles, and prostate) of pre-pubertal boars, suggesting TLR2 may aid early-in-life testes function or development. Expression of TLRs in various tissues has been shown to be influenced by hormones in rodents and humans. Dihydrotestosterone (DHT) is a sex steroid hormone and androgen that has a role in sexual development of males. However, little research has related TLRs with hormone concentration, which may help give insight to their physiological significance, particularly in the pre-pubertal male. Therefore, the objective of this project was to characterize relationships between expression of extracellular TLRs (TLR2, TLR4, TLR5 and TLR6) in the reproductive tissues (testes, epididymis, seminal vesicles, and prostate) with DHT concentrations in pre-pubertal boars. Intact reproductive tracts of pre-pubertal crossbred boars (Duroc x Landrace x Yorkshire; n = 4) were harvested at 2 mo of age and tissues were stored at –80°C for RT-qPCR analysis. From each boar, blood was collected postmortem and subsequent plasma samples stored at –20°C. Concentration of DHT in boar plasma was quantified using a competitive ELISA (MyBioSource, San Diego, CA) according to the manufacturer’s instructions. Expression of TLR2, TLR4, TLR5, and TLR6 mRNAs in the pre-pubertal boar tissues were quantified by RT-qPCR. Correlations of DHT concentration and TLR expression were calculated using Pearson Correlation within the PROC CORR function in SAS 9.4. The mean concentration of DHT was 9.32 ng/mL (range of 4.52 to 14.80 ng/mL). In the seminal vesicles, DHT concentration was significantly correlated with expression of both TLR2 (r = 0.95; P = 0.05) and TLR4 (r = 0.96; P = 0.04). In the testes, there was a tendency for a positive correlation between DHT concentration and expression of TLR2 mRNA (r = 0.92; P = 0.08). However, there were no correlations between DHT concentration and expression of any TLRs in the pre-pubertal boar epididymis or prostate (P > 0.10). These results suggest a relationship between androgens and TLR expression, particularly TLR2 and TLR4 mRNAs, in specific reproductive tissues of the pre-pubertal boar. Additional work will investigate TLR expression and androgen concentration in the pubertal boar. [ABSTRACT FROM AUTHOR]