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 × 10⁶sperm/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 log₁₀increase in total bacterial count (p = 0.026) andStaphylococcus 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), while the total bacterial count was lower (1.9 ± 0.1 versus 3.9 ± 0.1 log₁₀, respectively;p < 0.001). Without antibiotic supplementation, the total numbers of bacteria counted on days 2 and 3 of storage were higher than those determined on days 0 and 1 (p < 0.001). Differences in semen quality were detected on days 2 and 3 between the NO-ANTIBIOTIC and ANTIBIOTIC groups in high-viability semen (p < 0.05). However, no differences in sperm quality between the NO-ANTIBIOTIC and ANTIBIOTIC groups were detected in the low-viability semen on each storage day (p > 0.05). On the last day of preservation,Globicatella sanguinis (57.2%),Delftia acidovorans (18.9%)andMicrococcus 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.

The present study was carried out to determine the seminal microbiota of boars and their correlation with sperm quality. A total of 17 ejaculates were collected from 17 Duroc boars and were classified according to sperm quality into two groups: low-quality (n = 8) and high-quality (n = 9). Each ejaculate was subjected to (i) semen evaluation, (ii) bacterial culture and MALDI-TOF identification, and (iii) 16S rRNA gene sequencing and bioinformatic analyses. No difference in the total bacterial count, alpha diversity, and beta diversity between the high-quality group and the low-quality group was detected (p > 0.05). While Globicatella sanguinis was negatively correlated with sperm quality (p < 0.05), Delftia acidovorans was positively correlated with sperm quality (p < 0.05). Lactobacillales (25.2%; LB) and Enterobacterales (10.3%; EB) were the most dominant bacteria and negatively correlated: EB = 507.3 − 0.5 × LB, R² = 0.24, p < 0.001. Moreover, the abundance of Escherichia-shigella was negatively correlated with LB (r = −0.754, p < 0.001) and positively correlated with Proteus (r = 0.533, p < 0.05). Alysiella was positively correlated with Lactobacillus (r = 0.485, p < 0.05), Prevotella (r = 0.622, p < 0.01), and Staphylococcus (r = 0.489, p < 0.05). In conclusion, seminal microbiota is significantly associated with boar semen qualities. The distributions of the most dominant bacterial genera, the differences in the abundance of small subset microbes, and their correlation appear to have far more impact than the overall seminal bacterial content (e.g., total bacterial count, alpha diversity, and beta diversity) on sperm quality.

Background/Objectives: Alternatives to antibiotics in semen extenders used for pig breeding are needed. Cold storage of boar semen is possible in certain extenders; however, bacteria can still survive. Colloid centrifugation can remove bacteria; therefore, a combination of colloid centrifugation and cold storage on the bacterial load and sperm quality of boar semen was investigated. Results: The sperm quality in the original samples were similar in samples with and without antibiotics, respectively. At Day 4, the bacterial count was not different between controls stored at 17 °C with antibiotics and SLC cold-stored samples without antibiotics. The proportion of dead sperm increased in controls at 16–18 °C (28.12 ± 21.72% vs. 32.88 ± 20.94%; p < 0.05), and DNA fragmentation increased from 1.5 ± 1.02% at Day 0 to 6.1 ± 3.64% at Day 4 (p < 0.01), whereas it did not deteriorate in the cold-stored samples (Day 4: %DFI 2.00 ± 1.65% and 1.90 ± 1.05% for SLC samples with and without antibiotics, respectively). Methods: Aliquots of boar ejaculates were extended in Androstar Premium with and without antibiotics. The samples without antibiotics were processed by Single Layer Centrifugation (SLC) through Porcicoll; sperm pellets were resuspended in AndroStar Premium, with or without antibiotics, cooled slowly, and stored for 4 days at 4 °C. Controls (aliquots extended in AndroStar Premium with antibiotics) were stored at 17 °C. The bacterial load and sperm quality were assessed in the original samples and after 4 days of storage. Conclusions: Single Layer Centrifugation combined with cold storage is an effective method for reducing the bacterial load in boar semen, with or without antibiotics.

The aim of the present study was to investigate the effect of adding ethyl lauroyl arginate (ELA), encapsulated in organic nanoparticles, to a semen extender without antibiotics on sperm quality and bacterial load during short-term storage. Three ejaculates from Duroc boars were divided into four equal portions and diluted with Beltsville Thawing Solution to produce semen doses containing 3 billion spermatozoa in 80 ml. The extended semen did not include antibiotics but instead incorporated different concentrations of ELA nanoparticles into the extenders: 0 ppm, 5 ppm, 10 ppm, and 15 ppm. The semen doses were stored for 4 days, with daily evaluations of semen quality and bacterial counts conducted on days 0, 1, 2, and 3. During storage, sperm motility, viability, acrosome integrity, sperm membrane integrity, mitochondrial activity, and total bacterial count (CFU/ml, log10) were evaluated. Total sperm motility and motion parameters, including straight-line velocity (VSL, µm/sec), curvilinear velocity (VCL, µm/sec), and average path velocity (VAP, µm/sec), were assessed using a computer-assisted sperm analyzer (CASA). During the 3 days of semen preservation, the bacterial load in extended boar semen without ELA (0 ppm group) tended to be higher than in semen extended with 5 ppm ELA (3.9 vs. 3.4 CFU/ml, P = 0.096) and was significantly higher than in semen extended with 10 ppm (3.3 CFU/ml, P = 0.045) or 15 ppm ELA (3.2 CFU/ml, P = 0.010). No significant differences were observed in total sperm motility, VSL, sperm viability, or sperm membrane integrity between the 0 ppm and 5 ppm ELA groups (P > 0.05). However, total sperm motility in boar semen without ELA supplementation (69.6 ± 6.7%) was higher than in semen extended with 10 ppm (58.6 ± 6.7%, P = 0.031) or 15 ppm ELA (55.1 ± 6.7%, P = 0.006). In addition, sperm plasma membrane integrity in the 0 or 5 ppm ELA treatments was higher than in semen supplemented with either 10 ppm or 15 ppm ELA (P < 0.05). No differences in sperm acrosome integrity were found across the four groups (P > 0.05). In conclusion, our findings suggest an alternative antimicrobial agent for reducing antibiotic use in the boar artificial insemination industry. Encapsulated in organic nanoparticles at concentrations of 10 to 15 ppm, ELA effectively controlled bacterial growth during short-term storage without the need for antibiotics but negatively affected some sperm quality parameters. Therefore, using 5 ppm of ELA encapsulated in organic nanoparticles is recommended, as it reduces the bacterial load from 3.9 to 3.4 log10 CFU/ml without adversely impacting sperm characteristics during short-term storage, making it a viable option for antibiotic-free semen extenders.