Antibiotic resistance (AMR) is a major public health concern. Antimicrobial peptides (AMPs) could be an alternative to conventional antibiotics. The purpose of this research was to investigate the antimicrobial ability of the synthetic AMPs (i.e., A-11 and AP19) on the most frequently isolated bacteria in boar semen and their effect on extended boar semen quality during storage. We tested the antimicrobial effect of A-11 and AP19 at different concentrations and compared them with gentamicin for inhibiting the growth of E. coli, Pseudomonas aeruginosa and Proteus mirabilis that were isolated from fresh boar semen. In order to evaluate the effect of AMP on semen qualities on days 0, 1, 3, and 5 after storage at 18 °C, seven fresh boar semen samples were collected, diluted with semen extender with antibiotic (i.e., gentamicin at 200 µg/mL, positive control) or without (negative control), and semen extender contained only A-11 or AP19 at different concentrations (i.e., 62.50, 31.25, and 15.625 µg/mL). The total bacterial count was also measured at 0, 24, 36, 48, and 72 h after storage. Comparable to gentamicin, both A-11 and AP19 inhibited the growth of E. coli, Pseudomonas aeruginosa, and Proteus mirabilis at 62.50, 31.25, and 15.625 µg/mL, respectively. Comparing the total bacterial count at 0, 24, 36, 48 and 72 h after storage, the lowest total bacterial concentration was found in the positive control group (p < 0.05), and an inferior total bacterial concentration was found in the treatment groups than in the negative control. On day 1, there is a lower percentage of all sperm parameters in the AP19 group at a concentration of 62.50 µg/mL compared with the other groups. On day 3, the highest percentage of all sperm parameters was found in the positive control and A-11 at a concentration of 31.25 µg/mL compared with the other groups. The AP19 group at 62.5 µg/mL constantly yielded inferior sperm parameters. On day 5, only A-11 at a concentration of 15.625 µg/mL showed a total motility higher than 70%, which is comparable to the positive control. A-11 and AP19 showed antimicrobial activity against E. coli, Pseudomonas aeruginosa and Proteus mirabilis isolated from boar semen. Considering their effect on semen quality during storage, these antimicrobial peptides are an alternative to conventional antibiotics used in boar semen extenders. Nevertheless, the utilization of these particular antimicrobial peptides relied on the concentration and duration of storage.

To investigate the mechanisms underlying the differences in the freezability of boar semen, Yorkshire boars with freezing-tolerant semen (YT, n = 3), Yorkshire boars with freezing-sensitive semen (YS, n = 3), Landrace boars with freezing-tolerant semen (LT, n = 3), and Landrace boars with freezing-sensitive semen (LS, n = 3) were selected for this study. Their sperm was subjected to protein extraction, followed by data-independent acquisition proteomics and functional bioinformatics analysis. A total of 3042 proteins were identified, of which 2810 were quantified. Some key KEGG pathways were enriched, such as starch and sucrose metabolism, carbohydrate digestion and absorption, mineral absorption, the HIF-1 signaling pathway, and the necroptosis pathways. Through PRM verification, we found that several proteins, such as α-amylase and epididymal sperm-binding protein 1, can be used as molecular markers of the freezing resistance of boar semen. Furthermore, we found that the addition of α-amylase to cryoprotective extender could significantly improve the post-thaw motility and quality of boar semen. In summary, this study revealed some molecular markers and potential molecular pathways contributing to the high or low freezability of boar sperm, identifying α-amylase as a key protein. This study is valuable for optimizing boar semen cryopreservation technology.

BACKGROUND: Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage.
RESULTS: We found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05).
CONCLUSIONS: The protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.

Feeding of breeding boars in artificial insemination (AI) centers is critical to maintaining and improving breeding quality and performance in agriculture. Modern feeding strategies for AI boars are aiming towards maximizing their lifetime semen dose output. Given the high growth potential of modern swine genetics, AI boars should be controlled in their daily gain to reduce stress factors for the locomotion system, final body weights, and improve their survivability but also the ease of handling boars. The feeding program should be designed in such a way that young boars (up to 7 months of age) are limited to a daily gain of 400-600 g. Mature boars should be fed towards a body condition score of \'2\'. Aside from energy intake, protein sources should provide 0.62% SID lysine. As far as minerals and vitamins, special attention should be given to Calcium and Phosphorus as they play a crucial role in bone mineralization. A standardized total tract digestible Calcium-Phosphorus ratio between 1.75:1 and 1.82:1 seems to be most favorable. While certain nutritional requirements are needed to enable the production of ejaculates eligible for AI, considerations should be given to the mitigation of risk factors like mycotoxins, herbicides, and pesticides. Most feed additives and supplements lack consistency in their effect across studies evaluated in this review.

Efficient management of pig reproduction is paramount for the sustainability and productivity of the global pork industry. Modern artificial insemination (AI) breeding programs have greatly benefited from the integration of advanced selection methods and biomarkers to enhance the reproductive performance of boars. While traditional selection methods have relied soley on boar phenotype, such as growth rate and conformation, modern pig breeding has shifted more and more toward molecular and genetic tools, which are still complemented by phenotypic traits. These methods encompass genomics, transcriptomics, and proteomics. Biomarkers serve as critical indicators of boar reproductive capacity. They can help to identify individuals with superior fertility and aid in the early identification of potential fertility issues, allowing for proactive management strategies. This review summarizes current knowledge of various biomarkers associated with semen quality, sperm function, and overall reproductive fitness in boars. Furthermore, we explore advanced technologies and their potential applications in uncovering novel selection methods and biomarkers for predicting boar fertility. A comprehensive understanding of selection criteria and biomarkers governing boar reproductive capacity is essential for developing effective breeding programs to enhance swine reproductive performance.

Storage of boar semen at 5 °C instead of the conventional temperature of 17 °C is an innovative preservation concept. It enhances protection against the growth of bacteria normally occurring in the ejaculates and potential drug-resistant contaminants from the environment. Thereby it allows the reduction or even elimination of antibiotics in porcine semen extenders. The present article reviews the current state of the low-temperature preservation approach of boar semen, with a special focus on antimicrobial efficiency and fertility in field insemination trials. Particularly the role of semen extenders and temperature management for the achievement of high fertility and biosecurity are elucidated. Insemination data of 1,841 sows in there different countries revealed equally high farrowing rates and litter sizes of semen stored at 5 °C compared to the controls stored at 17 °C. Microbiology data obtained from semen doses spiked with multi-drug resistant bacteria showed the efficiency of the cold semen storage for inhibiting the growth of Serratia marcescens, a bacterial species with high sperm-toxicity. Evolving concepts on the physiological role of the male reproductive microbiome for female fertility provides a further argument against the complete eradication of bacteria in the semen dose by antibiotic additives to the extenders. Finally, motivation and practical considerations for the use of the novel preservation tool in artificial insemination of pigs are revealed, which might encourage the transformation towards a sustainable production of boar semen doses following the One Health approach.

This study was carried out to investigate the effect of different concentrations of selenium nanoparticles (Se-NPs) in the Beltsville Thawing Solution (BTS) extender on the semen quality and fertility of Hampshire crossbred pigs. For the study, semen was collected from four boars (10 ejaculates/boar) by the gloved hand method. Each ejaculate was extended @ 1:2 with the BTS extender and split into four aliquots. The control (C) samples were without the supplementation of Se-NPs, whereas the other three were supplemented with 0.5 (T1), 1 (T2), and 2 μg ml-1 of Se-NPs (T3) and stored at 15°C in a BOD incubator. Extended semen was evaluated at 0 (immediately after dilution), 24, 48, 72, and 96 h of storage for sperm motility, live sperm, plasma membrane integrity, acrosome integrity, DNA integrity, and mitochondrial membrane potential (MMP). The mean percentage of sperm motility, live sperm, and sperm with intact plasma membrane and acrosome, and MMPs were significantly (p < 0.01) higher in all treated groups in comparison to control at 24, 48, 72, and 96 h of storage. Sperm with intact DNA in all treated groups increased significantly at 48 (p < 0.05), and 72 and 96 (p < 0.01) h of storage in comparison to the control group. The concentration of 1 μg ml-1 of Se-NPs was found to be the best among other concentrations. In each group, 10 sows were artificially inseminated with the liquid semen preserved for 72 h at 15°C. Supplementation of 1 μg ml-1 of Se-NPs yielded the highest conception rate in comparison to other groups. In conclusion, supplementation of 1 μg ml-1 of Se-NPs in the BTS extender resulted in the best semen quality and conception rate during the short-time liquid preservation of boar semen.

Transport-related vibrations (TV) compromise the quality of conventionally stored (17 °C) boar semen, but knowledge about TV effects after 5 °C transport is insufficient. This study evaluates the effects of TV after novel 5 °C transport compared to a 17 °C control. Ejaculates of 18 fertile Piétrain boars, diluted in a split sample procedure using Androstar Premium® (AP, 5 °C storage) or Beltsville Thawing Solution (BTS, 17 °C storage), were subjected to transport simulation using a laboratory shaker IKA MTS 4. The timing was set according to the respective processing protocols: for 17 °C BTS samples, TV simulation was performed the day of collection, 5 °C AP samples were subjected to TV the day after collection following completion of the established cooling curve to 5 °C. Six samples per ejaculate were exposed to different TV durations (0 h, 3 h, or 6 h) to evaluate the effect on sperm quality (progressive motility (PM), thermo-resistance test (30 and 300 min incubation at 38 °C (TRT30/TRT300)), mitochondrial activity (MITO), plasma membrane and acrosome integrity (PMAI)). Generalized linear mixed models revealed TV (P = 0.021) and storage time (P < 0.001) dependent declines in PM. Direct, pairwise comparisons revealed that 5 °C samples are not affected by TV (P(3 h vs. 6 h transport) = 1.0; P(0 h vs. 6 h transport) = 1.0). They therefore showed superior quality maintenance after TV compared to 17 °C samples (P(3 h vs. 6 h transport) = 0.025; P(0 h vs. 6 h transport) < 0.001). Concluding, low-temperature transport is possible without significant semen quality loss and with better quality maintenance than standard transport.

UNASSIGNED: Facing the global threat of antimicrobial resistance, the reduction of antibiotic use in semen extenders is a main goal in artificial insemination (AI) of pigs. The aim of this study was to investigate the potential of a commercial extender containing an organic bactericidal supplement in the absence of conventional antibiotics to control bacterial growth and to maintain the quality of boar spermatozoa during long-term semen storage for up to 144 h at 17°C.
UNASSIGNED: Semen from 233 boars housed at 16 European AI centers was split and diluted in the long-term extender \"Androstar Plus without antibiotics + organic bactericidal supplement\" (APlus) and in the control extender Beltsville Thawing Solution (BTS) with gentamicin, which is routinely used in many AI centers. Sperm motility was assessed with computer-assisted semen analysis (CASA) and membrane integrity was evaluated with flow cytometry. The number of bacteria was determined by counting colonies on agar plates.
UNASSIGNED: At the end of storage, bacterial counts were ≥ 106 CFU/mL in 10.7% of the APlus and in 0.4% of the BTS samples. At the same time, bacterial counts were only weakly correlated with sperm motility (r = -0.23, p < 0.05), and there was no correlation with sperm membrane integrity (p > 0.05). Among the 12 identified bacterial species in APlus samples, loss of sperm quality was exclusively observed in the presence of >106 CFU/mL Serratia marcescens and Klebsiella oxytoca. Both these bacterial species, despite their known multi-drug resistance and the continuous use of gentamicin in Europe, proved sensitive to this antibiotic, thus indicating an efficient quality assurance program and responsible antibiotic use.
UNASSIGNED: Long-term storage of boar semen at 17°C without conventional antibiotics in an extender containing an organic bactericidal supplement is an option if semen samples are regularly tested for the presence of S. marcescens and K. oxytoca, and the source of contamination is eliminated.

External factors can affect reproductive traits of breeding boars and especially the sensitive process of spermatogenesis. The aim of this study was to investigate probable influences of bedding materials (chipsy wood shavings (CWS), hemp straw (HS), linen straw (LS), spelt husks (SH), and regional wood shavings (RWS)) on semen traits of 40 randomly selected Piétrain boars (8 boars per group, age: 2.35 ± 1.23 years). After a six-week adaptation period, 40 fresh semen samples were collected weekly for four weeks and diluted in BTS (4 consecutive ejaculates per boar, 32 samples per group, 160 samples in total). Semen samples were analyzed using an extended range of spermatological methods (e.g., computer-assisted sperm analysis and flow cytometry). Generalized linear mixed models for each sperm parameter as well as the area under the curve for total sperm motility and thermo-resistance test were calculated. Materials LS and SH exceeded the standard maximum level for pesticide residues (VO (EG) No. 396/2005). Materials HS and LS presented the highest water-binding capacity of 413 % and 357 %, respectively, while SH showed the lowest value of 250 %. There were no significant (P > 0.05) differences between groups in any sperm characteristic, therefore indicating that bedding material had no influence on sperm quality. For most semen traits, however, we found significant (P ≤ 0.001) differences between sampling weeks. Based on pesticide results, we suggest CWS, RWS, or HS as possible bedding materials for pig production farms in the future. Furthermore, we strongly recommend a quality analysis of any new bedding material before use in swine husbandry.