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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., Competing Interests: The authors declare that the research was caonducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xiong, Hong, Li, Gao, Wang, Liu, Zhao, Li and Qian.)

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., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The conventional storage temperature of 16-18 °C provides optimal conditions for the preservation of boar sperm quality, which are extremely cold sensitive cells. On the other hand, however, it requires the addition of antibiotics to inhibit bacterial growth. Rising numbers of antibiotic resistant bacteria call for alternatives to this conventional storing method. As potential alternative, three different bacteriocin candidates with known bacteriolytic activity against E. coli were examined on possible negative effects concerning the sperm quality and on their impact on bacterial growth of E. coli ILSH 02692 in BTS-extended semen w/o antibiotics. Although the lower concentrations (0.01 and 0.25%) of all bacteriocins did not show any impact on the quality of the semen, the higher concentrations (0.5 and 1.0%) of two bacteriocins led to a significant (P < 0.05) reduction in several sperm quality characteristics. The bacteriocin 860/1c after AMS/dialysis did not affect the sperm quality in any of the tested concentrations and in all tested extenders (BTS, MIII, Androstar Premium and Androhep all w/o antibiotics) at 16 °C as well as at 6 °C. This bacteriocin reduced growth of E. coli ILSH 02692 in BTS-extended semen by 50% compared to the control w/o bacteriocin. Furthermore, a preliminary insemination trial indicated no impact of the selected bacteriocin on fertility. These promising results show that the application of bacteriocins in liquid-preserved semen is a feasible possibility in the future., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Antibiotic agents such as gentamicin represent essential components of semen extenders in order to reduce bacterial contamination. But antibiotic resistance increases and AI centers start utilizing antibiotic agents which are more potent. Therefore, a shift to preventing bacterial contamination has to take place. In this study, we could demonstrate that hygiene is a tool capable of reducing bacterial load. In order to analyze 1434 extended semen samples and nine specially established hygienic critical control points (HCCPs, n = 828), 92 quality control audits have been carried out in a time period from 2012 until 2019 in 28 European AI centers. The results show the process of introducing a basic hygienic standard in audit 1 (2012/2013) and 2 (2014/2015) and the resulting achievements by means of improved hygienic conditions in audit 3 (2016/2017) and 4 (2018/2019). Within the scope of audit 1, 19% of the semen samples were contaminated with bacteria (cutoff ≥100 colony-forming units/mL). Audit 2 showed a bacterial load of 13.6% whereas during audit 3 and 4 very low bacterial contamination rates were recorded (4.5 and 5.5%, respectively). In the same manner, analysis of hygiene at different CCPs during semen production showed a decrease in all average HCCP-scores (score 1-6) comparing audit 4 to 1. By regression analysis we could show a significant audit-dependent association of the bacterial contamination in semen samples and hygiene of HCCPs. Furthermore, analysis of the odds ratio (OR) reveals that the bacterial contamination of certain HCCPs poses an increased risk of receiving bacterially contaminated semen samples (filling machine: OR = 3.02, P = 0.06; extender: OR = 8.97, P < 0.001; inner face of dilution tank lids: OR = 3.14, P = 0.09). Around 60% of the variance of the bacterial contamination in semen samples could be explained by hygienic conditions at different control points and their interaction with audit period and AI center. Antimicrobial agents are essential to protect human and animal health but excessive or inappropriate use can lead to the emergence of resistant bacteria. As shown in our study, hygiene management can significantly reduce bacterial contamination and is therefore capable of preventing antibiotic resistance., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The aim of the present study was to characterize the lysozyme concentration and the bacterial killing activity (BKA) of boar seminal plasma against E. coli and S. aureus in 119 fertile Pietrain boars (aged: 18.1 ± 10.5 months). Lysozyme concentration was 2.4 ± 1.2 μg/ml in seminal plasma. More than 80% of the samples (97 of 119) showed BKA against E. coli or S. aureus or both bacterial strains: Group 1 (BKA against E. coli and S. aureus, n = 38), Group 2 (BKA against E. coli, n = 13), Group 3 (BKA against S. aureus, n = 46), and Group 4 (no BKA, n = 22). Boars with BKA against E. coli (Group 1 plus 2) were older (P <  0.001) than boars with BKA against S. aureus only or without BKA. Thermo-resistance of spermatozoa was lowest in boars without BKA (P =  0.002). Lysozyme concentration was higher in boars with BKA against S. aureus only compared to boars with BKA against both bacterial species (P =  0.005) and boars with BKA against E. coli only (P =  0.047). In Group 2, the ratio of morphologically normal spermatozoa was lower than in all other groups (P <  0.001) and mitochondrial activity of spermatozoa was lower compared to Group 3 (P =  0.023). The results suggest an age related variance of BKA against E. coli in boar semen. BKA is related to different sperm quality characteristics. Further research is necessary to discover the molecular components, which are responsible for BKA of boar seminal plasma., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The processing of ejaculates is a fundamental step for the fertilizing capacity of boar spermatozoa. The aim of the present study was to identify factors that affect quality of boar semen doses. The production process during 1 day of semen processing in 26 European boar studs was monitored. In each boar stud, nine to 19 randomly selected ejaculates from 372 Pietrain boars were analyzed for sperm motility, acrosome and plasma membrane integrity, mitochondrial activity and thermo-resistance (TRT). Each ejaculate was monitored for production time and temperature for each step in semen processing using the special programmed software SEQU (version 1.7, Minitüb, Tiefenbach, Germany). The dilution of ejaculates with a short-term extender was completed in one step in 10 AI centers (n = 135 ejaculates), in two steps in 11 AI centers (n = 158 ejaculates) and in three steps in five AI centers (n = 79 ejaculates). Results indicated there was a greater semen quality with one-step isothermal dilution compared with the multi-step dilution of AI semen doses (total motility TRT d7: 71.1 ± 19.2%, 64.6 ± 20.0%, 47.1 ± 27.1%; one-step compared with two-step compared with the three-step dilution; P < .05). There was a marked advantage when using the one-step isothermal dilution regarding time management, preservation suitability, stability and stress resistance. One-step dilution caused significant lower holding times of raw ejaculates and reduced the possible risk of making mistakes due to a lower number of processing steps. These results lead to refined recommendations for boar semen processing., (Copyright © 2018 Elsevier B.V. All rights reserved.)