Trends and challenges in liquid-preserved boar semen production: From boar to product.

Boar semen production plays a pivotal role in modern swine breeding programmes, influencing the genetic progress and overall efficiency of the pork industry. This review explores the current challenges and emerging trends in liquid-preserved boar semen production, addressing key issues that impact the quality and quantity of boar semen. Advances in new reproductive technologies, boar selection, housing, semen processing, storage and transport, and the need for sustainable practices including the use of artificial intelligence are discussed to provide a comprehensive overview of the field.

Effect of Antimicrobial Peptide BiF2_5K7K on Contaminated Bacteria Isolated from Boar Semen and Semen Qualities during Preservation and Subsequent Fertility Test on Pig Farm.

The purpose of this study was to determine the impact of an antimicrobial peptide, BiF2_5K7K, on semen quality and bacterial contamination in boar semen doses used for artificial insemination. A key factor affecting semen quality and farm production is bacterial contamination in semen doses. Using antibiotics in a semen extender seems to be the best solution for minimizing bacterial growth during semen preservation. However, concern regarding antibiotic-resistant microorganisms has grown globally. As a result, antimicrobial peptides have emerged as interesting alternative antimicrobial agents to replace the current antibiotics used in semen extenders. BiF2_5K7K is an antimicrobial peptide that can inhibit Gram-negative and Gram-positive bacteria isolated from boar semen and sow vaginal discharge. In this study, ten fresh boar semen samples were collected and diluted with one of two types of semen extender: with (positive control) or without (negative control) an antibiotic (i.e., gentamicin). The semen extender without an antibiotic contained antimicrobial peptide BiF2_5K7K at different concentrations (15.625, 31.25, 62.5, and 125 µg/mL). The samples were stored at 18 °C until use. Semen quality parameters were assessed on days 0, 1, 3, and 5, and the total bacterial count was also evaluated at 0, 24, 36, 48, and 72 h after storage. A fertility test on a pig farm was also performed via sow insemination with a commercial extender plus BiF2_5K7K at a concentration of 31.25 µg/mL. No significant difference was found in terms of semen quality on days 0 or 1. On days 3 and 5, the total motility, progressive motility, and viability remained normal in the 15.625 and 31.25 µg/mL groups. However, the sperm parameters decreased starting on day 3 for the 125 µg/mL group and on day 5 for the 62.5 µg/mL group. For total bacterial count at 0, 24, 36, 48, and 72 h, the lowest bacterial count was found in the positive control group, and the highest bacterial count was found in the negative control group compared with the other groups. Comparing antimicrobial peptide groups from 0 to 48 h, the lowest bacterial count was found in the 125 µg/mL group, and the highest bacterial count was found in the 15.625 µg/mL group. For the fertility test, artificial insemination was conducted by using a commercial extender plus BiF2_5K7K at a concentration of 31.25 µg/mL. The results showed a superior pregnancy rate, farrowing rate, and total number of piglets born compared with artificial insemination conducted using a commercial extender plus antibiotic. In conclusion, BiF2_5K7K can inhibit bacterial growth in extended boar semen for 24 h, and thereafter, the bacterial count slightly increases. However, the increase in the number of bacterial counts from days 0 to 3 had no negative effect on sperm quality in the positive control, 15.625, or 31.25 µg/mL groups. This indicates that BiF2_5K7K might be an antimicrobial peptide candidate with potential for use as an alternative antimicrobial agent to replace the conventional antibiotic used in boar semen extenders.

Genome-Wide Association Study Reveals Novel Candidate Genes Influencing Semen Traits in Landrace Pigs.

Artificial insemination plays a crucial role in pig production, particularly in enhancing the genetic potential of elite boars. To accelerate genetic progress for semen traits in pigs, it is vital to understand and identify the underlying genetic markers associated with desirable traits. Herein, we genotyped 1238 Landrace boars with GeneSeek Porcine SNP50 K Bead chip and conducted genome-wide association studies to identify genetic regions and candidate genes associated with 12 semen traits. Our study identified 38 SNPs associated with the analyzed 12 semen traits. Furthermore, we identified several promising candidate genes, including HIBADH, DLG1, MED1, APAF1, MGST3, MTG2, and ZP4. These candidate genes have the potential function to facilitate the breeding of boars with improved semen traits. By further investigating and understanding the roles of these genes, we can develop more effective breeding strategies that contribute to the overall enhancement of pig production. The results of our study provide valuable insights for the pig-breeding industry and support ongoing research efforts to optimize genetic selection for superior semen traits.

Establishment of a Suitable Diagnostic Workflow to Ensure Sensitive Detection of African Swine Fever Virus Genome in Porcine Semen.

The rapid spread of African swine fever virus (ASFV), causing severe and often lethal disease in domestic pigs and Eurasian wild boar, continues to be a threat to pig populations and dependent industries. Despite scientific achievements that have deepened our understanding of ASFV pathogenesis, alternative transmission routes for ASFV remain to be elucidated. We previously demonstrated the efficient transmission of ASFV from infected boars to naïve recipient gilts via artificial insemination, thereby highlighting the importance of surveillance of boar semen prior to its shipment. Since the accurate and reliable detection of even low amounts of ASFV in boar semen is key to disease prevention and control, we established a suitable diagnostic workflow to efficiently detect the ASFV genome in boar semen. Here, we assessed the sensitivity of various routine nucleic acid extraction kits as well as qPCR protocols in detecting the ASFV genome in the blood and semen of infected boars. The feasibility of the respective kits and methods for future use in boar studs was also considered. Variability in sensitivity mostly concerned samples with low to very low amounts of the ASFV genome. Ultimately, we defined a well-suited workflow for precisely detecting the ASFV genome in boar semen as early as 2 days post ASFV infection.

Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation.

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.

The Effects of Different Antimicrobial Peptides (A-11 and AP19) on Isolated Bacteria from Fresh Boar Semen and Semen Quality during Storage at 18 °C.

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.

Proteomic Analysis of Differences in the Freezability of Porcine Sperm Identifies α-Amylase As a Key Protein.

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.

Boar semen storage at 5 °C for the reduction of antibiotic use in pig insemination: Pathways from science into practice.

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.

Boar semen cryopreservation: State of the art, and international trade vision.

Biosecurity is a major concern in the global pig production. The separation in time of semen collection, processing and insemination in the pig farm is a few days for chilled semen but it can be indefinite when using cryopreserved semen. Field fertility results of boar cryopreserved semen are close to chilled semen, which makes it a valuable resource for the establishment of semen genebanks, long-distance semen trade, and the implementation of other technologies such as the sex-sorted semen. But cryopreserved semen is far from being routine in pig farms. The most recent research efforts to facilitate its implementation include the use of additives before freezing, or in the thawing extender. Long-term preserved semen trade is a biosecurity challenge. To harmonize international trade of germplasm, the World Organization of Animal Health (WOAH) established a regulatory framework for all member countries. The present paper aims to review the latest advances of boar semen cryopreservation with special focus on the benefits of its inclusion as a routine tool in the pig industry. We also review recently reported field fertility results of cryopreserved semen, its international trade compared to chilled semen, and the regulatory framework involved. Boar cryopreserved semen is a valuable tool to control biosecurity risk, implement other technologies, and facilitate international trade. Research already demonstrated good field fertility results, but it still represents less than 0.1 % of the international trade. As boar cryopreserved semen gets closer to implementation, the correspondent authorities are reviewing the trade rules.

Enrichment of thawed boar spermatozoa with an intact membrane using Magnetic Activated Cell Sorting.

Not all boar sperm samples survive cryopreservation well. A method of eliminating damaged sperm might enable more cryopreserved boar semen to be used for pig breeding. In this study we investigated the use of Magnetic Activated Cell sorting (MACS) to eliminate damaged sperm from thawed boar semen samples. The thawed samples were mixed with Dead cell removal particles and were applied to the column in a SuperMACS II. Different fractions were collected: Original sample (O), Flow-through (FT), and Eluate (E). Sperm membrane integrity, mitochondrial membrane potential and reactive oxygen species were evaluated by flow cytometry after staining with SYBR 14 and propidium iodide, or 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide, or hydroethidine and dichlorodihydrofluorescein diacetate, respectively. The FT samples had increased membrane integrity, a greater proportion of sperm with high mitochondrial membrane potential and a greater proportion of sperm negative for hydrogen peroxide than O samples (P<0.0001), which in turn had increased membrane integrity than E samples (P <0.0001). However, differences were seen between boars. The FT samples had increased values of live, superoxide positive sperm than O samples (P <0.0001) and O samples had greater values than E samples (P <0.0001), while there was no effect of boar. Sperm quality was best in the FT fraction, comprising approximately 32% of the sperm sample. In conclusion, although there were differences between boars, MACS separation can improve sperm quality in thawed semen samples. It would be interesting to see if this improvement is reflected in fertility outcomes.

Selection and direct biomarkers of reproductive capacity of breeding boars.

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.

Modern feeding strategies for breeding boars.

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.

Effects of vibrations during boar semen transport: Low-temperature transport as a new management tool.

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.

Storage of boar semen at 17°C without conventional antibiotics in an extender containing an organic bactericidal substance.

Introduction: 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. Methods: 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. Results: 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. Conclusion: 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.

Comparison of deep-litter bedding materials and analysis of semen traits in Piétrain boars: A randomized controlled field study.

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.

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.

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.

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.

Storage of Extended Boar Semen at 5 °C Inhibits Growth of Multi-Drug Resistant Serratia marcescens and Klebsiella oxytoca while Maintaining High Sperm Quality.

Multi-drug antibiotic resistance of Serratia (S.) marcescens and Klebsiella (K.) oxytoca in boar semen is an emerging threat to pig reproduction and the environment. The aim of this study is to examine the efficiency of a novel hypothermic preservation method to inhibit the growth of these bacterial species in extended boar semen and to maintain the sperm quality. The semen samples extended in an antibiotic-free Androstar Premium extender were spiked with ~102 CFU/mL of S. marcescens or K.oxytoca. Storage at 5 °C for 144 h inhibited the growth of both bacterial species and maintained the sperm quality, whereas bacterial counts increased to more than 1010 CFU/mL in the 17 °C samples used as positive controls. This was accompanied by an increase in the sperm agglutination and the loss of motility and membrane integrity. We conclude that hypothermic storage is a promising tool to combat resistant bacteria in boar semen and to contribute to the One Health approach.

Low-density colloid centrifugation removes bacteria from boar semen doses after spiking with selected species.

Single-layer centrifugation (SLC) with a low-density colloid is an efficient method for removing contaminating microorganisms from boar semen while recovering most spermatozoa from the original sample. This study tested the performance of this technique, using 50-ml tubes, by spiking commercial semen doses prepared without antibiotics with selected bacterial species followed by storage at 17 °C. The doses were spiked up to 102/ml CFU (colony forming units) of the bacteria Burkholderia ambifaria, Pseudomonas aeruginosa, and Staphylococcus simulans. The semen was processed by SLC (15 ml of sample and 15 ml of colloid) with the colloid Porcicoll at 20% (P20) and 30% (P30), with a spiked control (CTL) and an unspiked control (CTL0), analyzing microbiology and sperm quality on days 0, 3 and 7. SLC completely removed B. ambifaria and S. simulans, considerably reducing P. aeruginosa and overall contamination (especially P30, ∼104 CFU/ml of total contamination on day 7, median). Sperm viability was lower in P20 and P30 samples at day 0, with higher cytoplasmic ROS. Still, results were similar in all groups on day 3 and reversed on day 7, indicating a protective effect of SLC (possibly directly by removal of damaged sperm and indirectly because of lower bacterial contamination). Sperm chromatin was affected by the treatment (lower DNA fragmentation and chromatin decondensation) and storage (higher overall condensation on day 7 as per chromomycin A3 and monobromobimane staining). In conclusion, SLC with low-density colloids can remove most bacteria in a controlled contamination design while potentially improving sperm quality and long-term storage at practical temperatures.