Boar Semen Research Articles

Screening for atypical porcine pestivirus in Swedish boar semen used for artificial insemination and a characterisation of the seminal RNA microbiome including the virome

BackgroundThis study aimed to characterise the RNA microbiome, including the virome of extended semen from Swedish breeding boars, with particular focus on Atypical porcine pestivirus (APPV). This neurotropic virus, associated with congenital tremor type A-II in piglets, was recently demonstrated to induce the disease through insemination with semen from infected boars.ResultsFrom 124 Artificial Insemination (AI) doses from Swedish breeding boars, APPV was detected in one dose in addition to a sparse seminal RNA virome, characterised by retroviruses, phages, and some fecal-associated contaminants. The detected seminal microbiome was large and characterized by Gram-negative bacteria from the phylum Proteobacteria, mainly consisting of apathogenic or opportunistic bacteria. The proportion of bacteria with a pathogenic potential was low, and no antimicrobial resistance genes (ARGs) were detected in the datasets.ConclusionOverall, the results indicate a good health status among Swedish breeding boars. The detection of APPV in semen raises the question of whether routine screening for APPV in breeding boars should be instigated.

Effect of BTS, GEPS and MODENA Extender on DNA Integrity and Relative Expression of HSP70 and Cas3 Gene in HD-K75 Boar Semen

Background: The present study compared the quality characteristics of boar semen diluted with three extenders at different hours of preservation. Methods: A total of 24 ejaculates comprising 6 ejaculates from each of four boars of 10-12 months age maintained under uniform management conditions are being selected for the present study. Semen was collected once weekly from four clinically healthy HD-K75 boars by simple fist method. The ejaculated semen was extended in BTS, GEPS and MODENA extender and maintained at 15°C upto 120 hours of preservation. The DNA integrity and the mean fold change in relative expression of HSP70 and Cas3 gene in BTS, GEPS and MODENA extender is also being studied. Result: The percentage of DNA integrity revealed that no DNA damaged spermatozoa were observed in MODENA extender from 0 to 120 hours of preservation. The relative expression of HSP70 gene elucidated significant difference (P less than 0.01) at 24 hours in three extenders. The relative expression of Cas3 gene in BTS, GEPS and MODENA extender differed significantly (P less than 0.01) at 0, 72 and 96 hours of preservation. The overall expression (fold change) of apoptotic gene was found to be higher in BTS extender followed by MODENA and GEPS.

Effect of L-proline on sperm quality during cryopreservation of boar semen

L-proline has been reported to be useful in semen cryopreservation. However, its use has rarely been reported in the freezing of boar semen. The objective of this study was to evaluate the effects of different concentrations of L-proline (0, 10, 30, 50, and 90 mM) on the quality of boar semen after freezing and thawing. Semen samples from boars (n = 6) were frozen using freezing extenders with added concentrations of L-proline. Total sperm motility, progressive motility, survival time at 37 °C, acrosome integrity, mitochondrial activity, DNA integrity, the content of the lipid peroxidation product malondialdehyde (MDA), total antioxidant capacity (T-AOC) and, expression levels of apoptosis protein (cleaved caspase 3 and Bax) were evaluated after thawing. The results showed that total sperm viability (73.96% vs. 63.58%) and progressive motility (56.88% vs. 47.26%) after thawing were significantly higher in the 10 mM L-proline treatment group than in the control group. The survival time at 37 °C and the total motility of sperm in the 10 mM group within one hour after thawing were significantly higher than in the control group. Acrosome integrity and mitochondrial activity of sperm in the 10 mM group were significantly higher than those in the control, 50 mM, and 90 mM groups. The DNA integrity rate in the 10 mM group was significantly higher than in the control group. The L-proline treatment did not affect sperm MDA content or T-AOC. The expression levels of apoptosis protein (cleaved caspase 3 and Bax) in the 10 mM L-proline supplemented group were lower than those in the control group. In conclusion, the freezing extender containing 10 mM L-proline improved semen quality after freezing and thawing and thus would be a useful reagent for boar semen cryopreservation.

The multidrug-resistant Pseudomonas fluorescens strain: a hidden threat in boar semen preservation.

Although the bacterial composition of boar ejaculate has been extensively studied, the bacterial composition of extended boar semen is often overlooked, despite the potential risks these microorganisms may pose to the long-term preservation of extended boar semen at 15-17°C. In this study, we characterized the bacterial community composition of extended semen and discovered that Pseudomonas spp. was the dominant flora. The dominant strains were further isolated and identified as a potential new species in the Pseudomonas fluorescens group and named GXZC strain, which had adverse effects on sperm quality and was better adapted to growth at 17°C. Antimicrobial susceptibility testing showed that the GXZC strain was resistant to all commonly used veterinary antibiotics. Whole-genome sequencing (WGS) and genome annotation revealed the large genetic structure and function [7,253,751 base pairs and 6,790 coding sequences (CDSs)]. Comparative genomic analysis with the closest type strains showed that the GXZC strain predicted more diversity of intrinsic and acquired resistance genes to multi-antimicrobial agents. Taken together, our study highlights a problem associated with the long-term storage of extended boar semen caused by a P. fluorescens group strain with unique biological characteristics. It is essential to develop a new antibacterial solution for the long-term preservation of boar semen.

Palm Kernel Meal Protein Hydrolysates Enhance Post-Thawed Boar Sperm Quality.

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.

Comparative efficacy of certain extenders on preservation of liquid boar semen

This study was carried out to compare the efficacy of certain liquid semen extenders for boar semen preservation. Semen ejaculates (n=30) from three boars (one ejaculate/boar/week) stationed at University pig farm, Ludhiana were collected using dummy sow and extended (1:4) in lactose-sodium salt of EDTA-Egg yolk (LSEEY); glucose sodium salt of EDTA-potassium sodium tartrate-sodium citrate (GEPS); and Modena (MOD). The extended semen was kept at 17°C up to 120 h in a BOD incubator. The semen was assessed for various sperm characteristics and lipid peroxidation at 0, 24, 48, 72, 96 and 120 h of preservation. The results revealed significantly higher percentage of sperm motility, viability, plasma membrane integrity and acrosome integrity in semen extended with MOD thanin GEPS and LSEEY at different preservation times. The mean percentage of in vitro capacitation/acrosome reacted spermatozoa was significantly higher in MOD as compared to GEPS and LSEEY at 96 h and 120 h of storage period. In all the extenders, the mean percentage of most sperm attributes decreased progressively as the storage period increased. Correspondingly, the MDA levels were lower in MOD than in their contemporary extenders at all hours of preservation. In conclusion, Modena exhibited improved sperm parameters and reduced oxidative stress for liquid preservation of boar semen.

Effect of certain extenders on semen quality of boars during preservation at 17°C

The present study was designed to determine the effect of three selected extenders on liquid preservation of boar semen at 17°C. Ejaculates (30) were collected from three boars (one ejaculate/boar/week) housed at University pig farm, Ludhiana using dummy sow. Only ejaculates having >70% initial progressive motility were extended in Beltsville Thawing Solution (BTS), Safe Cell (SFC) and Tris-Egg Yolk (TEY) extenders in the ratio 1:4 and preserved in a BOD incubator at 17°C for 120 h. The semen was evaluated for different sperm attributes and lipid peroxidation (MDA) at 0, 24, 48, 72, 96 and 120 h of preservation. The mean percentage of sperm motility, viability,plasma membrane integrity and acrosome integrity were significantly higher in BTS as compared to SFC and TEY at different hours of storage period. At 120 h of preservation, highest percentage of in vitro capacitation/acrosome reaction was seen in semen stored in BTS followed by SFC and lowest in TEY after 6 h of incubation. Eventually, significantly lower levels of MDA were noticed in semen extended in BTS than in their contemporary extenders (SFC and TEY) at 96 and 120 h of storage period. The mean percentage of most sperm parameters decreased gradually from day of collection (0 h) up till 120 h in all the extenders. In conclusion, Beltsville Thawing Solution was better than Safe Cell and Tris-Egg Yolk extenders in liquid preservation of boar semen at 17oC owing to improved spermcharacteristics and reduced oxidative stress.

Effects of gelatin and oxytocin supplementation in a long-term semen extender on boar semen quality and fertility potential.

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. 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. The results indicated that sperm quality decreased with increasing storage time (p<0.05). The sperm quality in terms of total motility, progressive motility, and viable sperm with intact acrosomes and high mitochondrial potential was the highest with 1.5% gelatin supplementation (p<0.001) on all days of storage. Treatment with oxytocin did not affect sperm quality (p>0.05). 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). 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.

Antioxidant Effects of Myo-Inositol Improve the Function and Fertility of Cryopreserved Boar Semen.

During cryopreservation, sperm undergoes structural and molecular changes such as ice crystal formation, DNA fragmentation, and reactive oxygen species (ROS) production, leading to decreased sperm quality after thawing. Antioxidants play a crucial role in preventing these damages, both in vivo and in vitro. One potent antioxidant is myo-inositol, known for its protective effects on sperm against ROS. This study aimed to investigate the protective effect of myo-inositol on cryopreserved boar semen. The semen was diluted, cooled, and cryopreserved using a BF5 extender. It was then divided into five groups: control and different concentrations of myo-inositol (0.5, 1, 1.5, and 2 mg/mL). The post-thaw evaluation included assessments of motility, viability, acrosome integrity, mitochondrial membrane potential (MMP), caspase activity, gene expression, ROS levels, apoptosis, and IVF with treated semen. Results showed that myo-inositol at 0.5 mg/mL improved motility, acrosome integrity, and fertilization ability. It also reduced the expression of pro-apoptotic genes and increased SMCP expression. Lower concentrations also demonstrated improved viability and reduced apoptosis and ROS levels. In conclusion, myo-inositol treatment during cryopreservation improved sperm quality, reduced apoptosis and ROS levels, and enhanced fertility rates in boar semen.

Low-density lipoproteins, resveratrol and quercetin as alternative additives to improve boar semen cooling.

Low-density lipoproteins (LDL), quercetin (Q) and resveratrol (R) have been used for sperm preservation to improve sperm quality in different species. To evaluate the effects of LDL, Q and R during the cooling of boar semen. Fifteen boar semen samples were diluted in a BTS extender supplemented with the treatments: LDL at 6%, Q at 10 μM (Q10), 30 μM (Q30) and 50 μM (Q50), or R at 10 μM (R10), 30 μM (R30) and 50 μM. A control without supplementation was included. The semen was stored by cooling at 16°C for 96 h. Every 24 h, sperm motility and kinetics were evaluated using a computer-assisted sperm analyzer (IVOS). At 24 and 96 h of cooling, functional membrane integrity and mitochondrial membrane potential (ΔΨM) of sperm were evaluated by the hypoosmotic swelling test (HOST) an flow cytometry with JC-1 probe, respectively, LDL improved progressive motility of sperm during cooling. Likewise, LDL increased average path velocity (VAP) and straight-line velocity (VSL) and/or curvilinear velocity (VCL) during the first 48 h of cooling. The use of Q between 10 and 30 μM caused a reduction in total motility, progressive motility and amplitude of the lateral head displacement during the entire cooling period, as well as a decrease in VAP, VSL and VCL at 96 h of cooling. LDL, Q10, Q30 and Q50 modulated mitochondrial activity by reducing high-ΔΨM sperm at 0 and 96 h of cooling. During the cooling of the boar semen prior to artificial insemination, the parameters of sperm quality that could influence fertility decrease; however, the inclusion of antioxidants and additives that protect the plasma membrane, such as LDL, could mitigate the damaging effects on spermatozoa. It is concluded that LDL can improve the motility and kinetics of boar semen during cooling while it could modulating the sperm's mitochondrial activity. On the contrary, Q could alter the motility and kinetics of boar sperm during the cooling period.

Selenium Nanoparticles as Potential Antioxidants to Improve Semen Quality in Boars.

Selenium is an essential compound which can influence the fertility of boars by a greater margin. In past decades, research was mainly focused on a bioavailability of various selenium forms and the effect on semen quality. Recently, nanotechnology has expanded the possibilities of selenium supplementation research. Twenty-one Duroc boars (three groups with seven boars each) were included in this experiment with the first group being a control group with no selenium supplementation, and the second group being supplemented with 0.3 mg Se/kg of selenium in inorganic form of Na2SeO3. The third group was supplemented with selenium nanoparticles (100 nm) at the same dose as that of the second group. The experiment lasted for 126 days (three spermatogenesis cycles of boars) and the antioxidant parameters of boar semen were analysed at 42, 84 and 126 days, respectively. The antioxidant parameters (DPPH, FRAP, DMPD, GSH, GSSG) were not influenced by both Se2NO3 and selenium nanoparticle supplementation during this experiment. At the end of the monitored period, significantly higher (p < 0.004) antioxidant readings were observed by using the ABTS method but not the DPPH, DMPD and FRAP methods on the supplemented groups compared to the control. Moreover, selenium-nanoparticle-supplemented groups showed elevated glutathione peroxidase activity in the seminal fluid (p < 0.008). However, the selenium nanoparticle supplementation has not shown an improving effect on sperm quality. This could be considered as a safe alternative to inorganic selenium as well as having a potential to enhance the antioxidant properties of the semen of boars.

Myo-inositol improves the viability of boar sperm during liquid storage.

Liquid preservation of boar semen is a highly preferred method for semen preservation in pig production. However, oxidative stress is the main challenge during the liquid preservation of boar semen in a time dependent manner. Therefore, supplementation of sperm with antioxidants during storage to protect them from oxidative stress has been the focus of recent research. Myo-inositol (Myo-Ins), the most active form of inositol, which belongs to the vitamin (Vit.) (B1 group has been shown to improve semen quality) (1). This study aimed to investigate whether Myo-Ins supplementation protects boar sperm in liquid preservation against oxidative stress and determine the appropriate concentration of Myo-Ins to be used in this regard. Boar sperm was diluted with a semen extender with different concentrations of Myo-Ins (2, 4, 6, and 8 mg/mL) depending on the previous studies (1, 24). Sperm motility and viability, plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), semen time survival, and gene expression were measured and analyzed on days 0, 1, 3, 5, and 7 for the different samples. Different concentrations of Myo-Ins exerted different protective effects on the boar sperm quality. The addition of 2 mg/mL Myo-Ins resulted in higher sperm motility and viability, plasma membrane and acrosome integrity, MMP, and effective survival time. Investigation of mRNA expression patterns via qRT-PCR suggested that the 2 mg/mL Myo-Ins sample had increased expression of antioxidative genes. The addition of Myo-Ins to semen extender improved the boar semen quality by decreasing the effects of oxidative stress during liquid preservation at 17°C. Additionally, 2 mg/mL is the optimum inclusion concentration of Myo-Ins for semen preservation.

Analysis of changes in the morphological structures of sperm during preservation of liquid boar semen in two different seasons of the year

Animal fertility is the result of a combination of genetic, physiological and environmental factors. Assessment of semen quality plays a key role in determining the reproductive performance of boars. The aim of the study was to determine the effect of two seasons of the year on the morphology, morphometry, cell membrane integrity and mitochondrial activity of sperm during storage of liquid boar semen at 17 °C. The study was carried out using 20 boars. Four ejaculates were collected from each boar in each of two seasons of the year. All tests were carried out five times: at 1, 24, 48, 96 and 168 h during storage. The results showed that sperm in ejaculates collected in summer are more sensitive to storage conditions than those from ejaculates obtained in winter. The percentage of sperm with morphological defects was shown to increase with the storage time of the diluted ejaculates, particularly between 96 and 168 h of preservation. In summer, the percentage of sperm with an intact cell membrane and the percentage with high mitochondrial membrane potential are much lower than in winter, at every hour of semen preservation. In the case of boars used for artificial insemination, it is worth taking into account the season when the semen is collected, especially if it is to be stored in liquid form. Assessment of sperm cell structures during storage of liquid semen should be implemented at insemination stations and should be carried out more often in summer.

Evaluation of Boar’s Semen Production and Characteristics for Artificial Insemination

The objective of this work was to evaluate the production and characteristics of boar semen from different genetic lines used for artificial insemination, for which 19 boars were used: LM100 (n=4); LM200 (n=2); LM300 (n=2); LTP27 (n=4); LTH1 (n=2); LTB1 (n=3) and LTB37 (n=2) for two and a half years; the variables evaluated were Ejaculate Volume, Motility %, Temperature, Sperm Concentration, Collection frequency, Season of the year, Age and Number of doses. The general averages of the results obtained were: semen volume 263.64 ± 3.59, Motility 89.99 ± 0.20, Ejaculate temperature 37.12 ± 0.03, Dose x ejaculate 19.43 ± 0.36, Winter season, semen volume 263.69 ± 3.59, Spring Motility 90.81 ± 0.43, Autumn temperature of the Ejaculate 37.12 ± 2.74, Spring number of doses (300 x 109) ; 19.43 ± .35, in relation to the Genetic Lines in which there was a significant difference with respect to the evaluated variables were in Volume it was LTP-27 with 328.74 ± 7.97 mL (P &lt;0.05), in % Motility it was LTH-1, 91.81 ± 0.52 (P&lt;0.05), in Ejaculate Temperature The LTB-1 with 37.70 ± .09 (P&gt;0.05), in dose was the LTP-27, 23.15 ± 0.64 (P&lt;0.05), the behavior by season of the year and your variables.

Sperm concentration of boar semen doses and sperm quality: Novel perspectives based on the extender type and sperm resilience

This study evaluated the effect of sperm concentration of boar semen doses on their capacity to maintain its motility over the thermo-resistance test (TRT; sperm resilience) and verified if the extender type (short or long-term) could influence this effect. Thirty ejaculates from five crossbred mature PIC® boars were used, and a factorial design was followed to produce semen doses with 1.5 billion cells in 45 or 90 mL, using Beltsville Thawing Solution (BTS) or Androstar® Plus (APlus). Then, low-concentration doses (16.7 × 106 cells/mL in 90 mL) and higher-concentration doses (33.3 × 106 cells/mL in 45 mL) with BTS or APlus were produced and stored at 17 °C for 168 h. At 72 h, during the TRT, the low-concentration doses (16.7 × 106 cells/mL) lost three-fold less motility than doses with 33.3 × 106 cells/mL (P < 0.01), regardless of the extender type (11. 5% vs. 30.5% of initial motility, respectively). Similar results were found when the TRT was carried out at 168 h, with low-concentration doses losing two-fold less motility (11.4%) than highly concentrated doses (25.9%; P < 0.01). No sperm concentration effect was observed on membrane integrity or mitochondrial membrane potential (P ≥ 0.23). The osmolarity was not affected by the sperm concentration (P = 0.56), only by the extender and the storage time (P < 0.01). In conclusion, the sperm concentration effect on sperm quality was not influenced by extender type, and the data suggest that a low concentration of semen doses positively affects sperm resilience.

Influence of age, breed, and season on the quality of boar semen stored at low-temperature

In the face of antimicrobial resistance, antibiotic-free, low-temperature storage of boar semen has been well-researched in recent years and promising results have been obtained. With the prospect of establishing this new preservation method in practice, it is important to evaluate a range of factors, possibly influencing the general and/or boar individual preservation suitability for 5 °C storage. The present study aimed to evaluate the influence of boar age (<18 months (n = 29) vs. 18–36 months (n = 68) vs. >36 months (n = 56)), breed (Pietrain (n = 104) vs. Duroc (n = 49)), as well as the influence of season (summer (n = 73) vs. winter (n = 80)) on the quality of boar semen preserved in antibiotic-free Androstar® Premium extender. AI doses were stored at 5 °C after cooling according to an established cooling protocol. In total, 153 ejaculates were analyzed throughout two identical experimental runs in summer and in winter, and the boars were divided into the corresponding sub-groups based on their age and breed. The application of a general linear model (GLM) and subsequent Bonferroni-corrected post hoc tests did not reveal any significant differences in the quality of semen stored at 5 °C between the different age groups. Regarding the season, a difference was found in the progressive motility (PM) at two out of seven analysis time points (P ≤ 0.01), however, this difference in PM was also present in fresh semen (P < 0.001). Most significant differences were found when comparing the two breeds. At six out of seven analysis time points, PM of Durocs was significantly lower than PM of Pietrains. Again, this difference in PM was also recognizable in fresh semen (P < 0.001). No differences were found in plasma membrane and acrosome integrity examined by flow cytometry. In conclusion, our study confirms the feasibility of 5 °C storage of boar semen under production conditions regardless of boar age. While season and breed have an influence on boar semen stored at 5 °C, these differences are not primarily caused by storage temperature, as they were already apparent in fresh semen.

Porcine reproductive and respiratory syndrome virus regulates lipid droplet accumulation in lipid metabolic pathways to promote viral replication

Porcine reproductive and respiratory syndrome (PRRS) is a severe respiratory disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) that can lead to the abortion of pregnant sows and decreased boar semen quality. However, the mechanisms of PRRSV replication in the host have not yet been fully elucidated. As lipid metabolism and lipid droplets (LDs) have been reported to play important roles in the replication of various viruses, we aimed to explore the mechanisms through which LDs affect PRRSV replication. Laser confocal and transmission electron microscopy revealed that PRRSV infection promoted intracellular LD accumulation, which was significantly reduced by treatment with the NF-κB signaling pathway inhibitors BAY11–7082 and metformin hydrochloride (MH). In addition, treatment with a DGAT1 inhibitor significantly reduced the protein expression of Phosphorylated NF-ΚB P65and PIκB and the transcription of IL-1β and IL-8 in the NF-κB signaling pathway. Furthermore, we showed that the reduction of the NF-κB signaling pathway and LDs significantly reduced PRRSV replication. Together, the findings of this study suggest a novel mechanism through which PRRSV regulates the NF-κB signaling pathway to increase LD accumulation and promote viral replication. Moreover, we demonstrated that both BAY11–7082 and MH can reduce PRRSV replication by reducing the NF-κB signaling pathway and LD accumulation. This study lays a theoretical foundation for research on the mechanism of PRRS prevention and control, as well as the research and development of antiviral drugs.

Colloid centrifugation as an alternative to antibiotics in stallion semen preparation

Antibiotics are added to semen extenders to inhibit bacterial growth and maintain sperm quality in semen doses during storage and transportation. The low concentration of antibiotics used, however, could contribute to increased antimicrobial resistance. Recent studies with boar semen suggest that centrifugation through a low density colloid could separate the spermatozoa from most of the bacteria. Such methods can only be recommended for stallion semen if they are without detrimental effects on sperm characteristics. Therefore, the purpose of this pilot study was to investigate if semen processing by single layer centrifugation through a low density colloid reduces the bacterial load of stallion semen without impairing sperm characteristics. Six healthy and fertile stallions were available at the Centre for Artificial Insemination of the University of Veterinary Medicine Vienna. One ejaculate from each stallion was collected and divided into three parts. Each part was either extended to 15 mL (100 million/mL) and processed by single layer centrifugation with 15 mL of 25% low density colloid (SLC), extended with Equiplus without antibiotics (Minitube, Tiefenbach, Germany), or kept as unprocessed control (raw semen). Aliquots from all samples were sent to the Institute of Microbiology for bacteriologicalexamination where they were serially diluted, plated onto blood agar plates, and incubated under aerobic and anaerobic conditions. The number of bacterial colony forming units (cfu/mL) was determined after 4 days of incubation. Sperm motility and viability in SLC and extended samples were evaluated by computer assisted sperm analysis (CASA; SpermVision, Minitube). The SLC and extended samples were stored at 5°C and re-evaluated after 24 h. For statistical analysis, the mean number of bacterial cfus and sperm characteristics were compared among treatments by one-way ANOVA and Tukey's test, with significance set at p < 0.05. Overall sperm yield after SLC was 76.6% (±16.4). Bacterial cfus were lower in SLC samples and extended samples compared to raw semen (4.5 × 106 cfu/mL, (14.5 × 106cfu/mL, and 8.3 × 106, respectively; p= 0.017, and p = 0.038, respectively). Sperm motility was higher in SLC samples at both time points (93.8% at 0 h, p = 0.001; 87.7% at 24h, p = 0.007) than in extended semen at 24 h (56.3%). Sperm viability was higher in the SLC at both time points (88.4% at 0 h, p = 0.0002; 86.4% at 24h, p = 0.002) than in extended semen at 24 h (73.2%). In conclusion, bacterial load, and sperm characteristics, were improved in SLC compared to extended stallion semen. An extended investigation is warranted.

Effects of Melatonin and Silymarin on Reactive Oxygen Species, Nitric Oxide Production, and Sperm Viability and Motility during Sperm Freezing in Pigs.

Sperm during the freezing and thawing process is damaged by oxidative stress. Thus, its antioxidant scavenger is essential for sperm survival and death in frozen-thawed semen. We used melatonin and silymarin in experiments after the dose-dependent experiment. Our study aimed to identify the effect of melatonin and silymarin on the motility and viability of sperm, reactive oxygen species (ROS), and nitric oxide (NO) production in frozen-thawed boar semen. Melatonin and silymarin were treated alone and cotreated in the fresh boar semen. Boar semen was collected using the gloved-hand method from ten crossbred pigs, and samples were used in the experiments. We evaluated sperm viability using SYBR-14 and PI kit, and ROS and NO production were detected by DCF-DA and DAF-2, respectively. The sperm motility was not significantly different between non-treatment and treatment. ROS and NO production in frozen-thawed sperm were decreased by melatonin and silymarin. Moreover, silymarin significantly reduced NO production more than melatonin. Melatonin and silymarin enhanced the viability of sperm. We suggest that melatonin and silymarin are essential antioxidants in semen cryopreservation for protecting sperm damage and maintaining sperm viability. Melatonin and silymarin may be useful antioxidants in freezing boar sperm.

SPAG6 c.900 T>C affects boar semen quality and blood-testis barrier function by creating a new splice acceptor site.

Sperm associated antigen 6 (SPAG6) acts as a scaffolding protein in the center of the flagellar axoneme and has an impact on the maturation of the motility of mammalian sperm flagella and the maintenance of sperm structure. In our previous research, SPAG6 c.900 T>C in exon 7 and exon 7 skipped transcript was identified by analyzing RNA-seq data of testicular tissues from 60 day (sexually immature) and 180 day (sexually mature) Large White boars. Herein, we found porcine SPAG6 c.900 T>C to be associated with semen quality traits in Duroc, Large White and Landrace pigs. SPAG6 c.900 C can generate a new splice acceptor site, inhibit the occurrence of SPAG6 exon 7 skipping to a certain extent, thereby promote the growth of Sertoli cells and maintain the normal blood-testis barrier function. This study provides new insights into the molecular regulation of spermatogenesis and a new genetic marker for the improvement of semen quality in pigs.