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.

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.

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.

Growth Dynamic and Threshold Values for Spermicidal Effects of Multidrug-Resistant Bacteria in Extended Boar Semen.

The aim of this study was first to examine the prevalence of bacteria-associated loss of sperm quality in samples from insemination centers during a seven-year semen monitoring program and, second, to investigate the growth dynamic of four different multidrug-resistant bacterial species and their impact on sperm quality during semen storage. A reduced sperm quality associated with bacterial contamination was found in 0.5% of 3219 of the samples from insemination centers. In samples spiked with Serratia marcescens and Klebsiella oxytoca, bacterial growth by six log levels was seen during storage at 17 °C, causing loss of sperm motility, membrane integrity, membrane fluidity, and mitochondrial membrane potential at >107 CFU/mL (p < 0.05). Storage at 5 °C in the Androstar Premium extender efficiently inhibited their growth. Achromobacter xylosoxidans and Burkholderia cepacia showed limited growth up to two log levels at 17 °C and did not impair sperm quality. In conclusion, spermatozoa tolerate moderate loads of multidrug-resistant bacteria, and hypothermic, antibiotic-free semen storage effectively limits bacterial growth. The constant use of antibiotics in semen extenders should be reconsidered.

Compensability of an enhanced incidence of spermatozoa with cytoplasmic droplets in boar semen for use in artificial insemination: a single cell approach.

This single cell study aimed to clarify whether an elevated incidence of sperm with a retained cytoplasmic droplet (CD) can be compensated by a higher sperm number in boar semen doses to maintain fertility. Cluster analysis of motile spermatozoa (ten boars) revealed that spermatozoa with a CD are underrepresented in the fast, linearly moving sperm cohort compared to morphologically normal sperm. Nonetheless, the response to the motility stimulator procaine was barely affected in spermatozoa with distal CD (Cramer's V = 0.14), but moderately affected in sperm with proximal CD (V = 0.28). Viability was lower in sperm with distal CD (p < 0.05) but not with proximal CD compared to normal sperm during 168 h storage of extended semen samples (n = 11) and subsequent thermic stress. Morphologically normal sperm from normospermic samples (n = 10) or samples with a high incidence (≥ 15%) of sperm with CD (n = 9) had similar motility patterns and responses to procaine. The origin of morphologically normal sperm had no effect on sperm viability (p > 0.05; n = 26). In conclusion, a moderately enhanced prevalence of sperm with CD seems to be compensable by an increase in sperm numbers in boar semen doses.

Studying the Interaction of Neutrophils and Glaesserella Parasuis Indicates a Serotype Independent Benefit from Degradation of NETs.

Glaesserella (G.) parasuis is one of the most important porcine pathogens causing Glaesser's disease. Neutrophil granulocytes are the major counteracting cell type of the innate immune system, which contribute to the host defense by phagocytosis or the formation of neutrophil extracellular traps (NETs). Recently, NET-formation has been shown to facilitate the survival of bacteria from the Pasteurellaceae family. However, the interaction of NETs and G. parasuis is unclear so far. In this study, we investigated the interplay of three G. parasuis serotypes with porcine neutrophils. The production of reactive oxygen species by neutrophils after G. parasuis infection varied slightly among the serotypes but was generally low and not significantly influenced by the serotypes. Interestingly, we detected that independent of the serotype of G. parasuis, NET formation in neutrophils was induced to a small but significant extent. This phenomenon occurred despite the ability of G. parasuis to release nucleases, which can degrade NETs. Furthermore, the growth of Glaesserella was enhanced by external DNases and degraded NETs. This indicates that Glaesserella takes up degraded NET components, supplying them with nicotinamide adenine dinucleotide (NAD), as this benefit was diminished by inhibiting the 5'-nucleotidase, which metabolizes NAD. Our results indicate a serotype-independent interaction of Glaesserella with neutrophils by inducing NET-formation and benefiting from DNA degradation.

In vitro storage of boar spermatozoa increases the demand of adenosine triphosphate for reactivation of motility.

BACKGROUND: Prolonging the shelf-life of liquid-preserved semen without compromising its fertilizing capacity may increase the efficiency of artificial insemination in pigs. Many fertilization-relevant processes are adenosine triphosphate dependent. The impact of semen storage and rewarming to body temperature on the energy status of spermatozoa is as yet unknown. OBJECTIVES: To investigate the energy status of boar spermatozoa during storage and subsequent rewarming and to reveal the potential role of mitochondrial function for reactivation and maintenance of sperm motility. MATERIALS AND METHODS: Extended semen samples (n = 7 boars) were used. Spermatozoa were challenged by storage at 17°C for 7 days and incubation at 38°C for 180 min. The adenosine triphosphate concentration and energy charge in semen samples and lactate concentration in the extracellular medium were assessed. Viability and mitochondrial activity were determined by flow cytometry, and clustered single-cell analysis of motility parameters was performed. RESULTS: The energy status was not affected by semen storage (p > 0.05). Rewarming resulted in a net reduction in adenosine triphosphate concentration, which increased with storage time (maximum Day 5: -24.2 ± 10.3%) but was not accompanied by a loss in viability, motility, or mitochondrial activity. Blocking glycolysis with 2-deoxy-d-glucose prevented the re-establishment of motility and mitochondrial activity after rewarming. Mitochondrial activity gradually subsided in virtually all spermatozoa during incubation at 38°C, while adenosine triphosphate and energy charge remained high. Concomitantly, extracellular lactate levels rose, and sperm populations with lower velocity, increased linearity, and low lateral head displacement grew larger. Size changes for major sperm subpopulations correlated with the percentage of viable spermatozoa with high mitochondrial activity (r = 0.44-0.70 for individual subpopulations, p < 0.01). CONCLUSION: Storage of boar spermatozoa increases the demand of adenosine triphosphate for reactivation of spermatozoa towards fast, non-linear, and hyperactivation-like motility patterns upon rewarming. Maintenance of glycolysis seems to be decisive for sperm function after long-term storage in vitro.

Antimicrobially Active Semen Extenders Allow the Reduction of Antibiotic Use in Pig Insemination.

Antibiotic use in semen extenders for livestock may contribute to the development and spreading of multi-drug resistance. Antimicrobial control in semen doses for artificial insemination of pigs is indispensable due to the relatively high storage temperature (17 °C). The objectives of this study were first, to examine whether the antimicrobial capacity differs between antibiotic-free extenders and second, to determine whether an antimicrobial active extender provides the possibility to reduce antibiotics. Antibiotic-free semen extenders Beltsville Thawing Solution (BTS) and Androstar Premium were inoculated at 103 to 104 CFU/mL with four pure bacterial strains isolated from boar ejaculates or a mixture thereof, and then stored for 144 h at 17 °C. Bacterial counts after aerobic culture decreased in BTS up to one log level and decreased in Androstar Premium by 2 to 3.5 log levels (p < 0.05). In semen samples from nine boars stored in the inoculated Androstar Premium extender containing half of the standard concentration of gentamicin, bacteria counts were below 101 CFU/mL. Likewise, half of the standard dose of apramycin and ampicillin was fully antimicrobially active and sperm quality was maintained. In conclusion, semen extenders with intrinsic antimicrobial activity allow a reduction in antibiotic use in pig insemination.

A High Incidence of Sperm with Cytoplasmic Droplets Affects the Response to Bicarbonate in Preserved Boar Semen.

Retained cytoplasmic droplets (CD) are the most frequent sperm abnormality in boar semen. A high incidence of CD is associated with subfertility, but the underlaying reasons are not well understood. The storage of extended semen might augment the adverse effects of CD on essential steps towards fertilization, such as capacitation. The aim of this study was to examine whether the enhanced presence of CD in boar semen influences sperm's response to the capacitation stimulus bicarbonate during long-term semen storage. Extended semen samples (n = 78) from 13 artificial insemination centers were analyzed using a flow cytometric calcium influx assay. Samples with >15% of CD showed a reduced specific response to bicarbonate and a higher non-specific destabilization after storage for 96 h and subsequent incubation at 38 °C in three variants of Tyrode's medium (p < 0.05). The size of the bicarbonate-responsive sperm population was inversely correlated with the presence of CD-bearing sperm (r = -0.61, p < 0.01). Samples with ≤15% and samples with >15% of CD did not differ in motility or viability and acrosome integrity during semen storage. In conclusion, incomplete epididymal sperm maturation impairs the in vitro capacitation ability and promotes sperm destabilization in stored boar semen.

Factors influencing the response of spermatozoa to agitation stress: Implications for transport of extended boar semen.

The shipping of liquid preserved semen is common practice in animal breeding and prior to cryopreservation for gene banking. Vibration emissions during transport may be harmful to spermatozoa. Therefore, strategies to minimize agitation-induced sperm injury are needed. The aim was to examine whether the type of semen extender, time after semen processing and the temperature in simulated transport conditions influence the response of boar spermatozoa to agitation stress. In Experiment 1, boar semen samples (n = 16) extended in Beltsville Thawing Solution (BTS) or Androstar Plus (APL) medium were filled in 90 mL tubes and shaken for 4 h at 200 rpm either at 22 °C or 17 °C. Samples were then stored at 17 °C for 144 h. In Experiment 2, semen samples (n = 11) extended in Androstar Premium were shaken either directly after filling at 22 °C or 20 h later after cooling to 5 °C. Samples were stored at 5 °C for 144 h. In Experiment 1, sperm motility and viability were lower (p < 0.05) in the shaken samples compared to the controls. The temperature, extender and the storage length had no effect on the agitation-induced loss of sperm quality. Sperm quality traits were higher in samples stored in APL compared to BTS. In Experiment 2, sperm motility at 24 h was reduced (p < 0.05) in those samples shaken at 22 °C but not at 5 °C. Sperm viability, membrane fluidity and mitochondrial membrane potential were not affected in either of the treatment groups. Extended boar semen designed for 17 °C storage and shipped on the day of collection is sensitive to agitation stress. In contrast, spermatozoa slowly cooled to 5 °C and shaken 20 h after processing are more resistant to agitation-induced shear forces and interfacial phenomena.

Assessment of chilling injury in hypothermic stored boar spermatozoa by multicolor flow cytometry.

Hypothermic storage of boar semen may allow antibiotic-free semen preservation but is limited due to chilling sensitivity of boar spermatozoa. Progress in this area requires sensitive tools to detect chilling injury. Therefore, multiparameter flow cytometry panels were evaluated to ascertain whether they are useful tools for identifying sublethal damage of sperm function at a single cell level, thus considering the high intrinsic sperm heterogeneity in a sample. The first fluorochrome panel consisted of Hoechst 33342 to identify DNA-containing events, Yo-Pro 1 to detect viability, merocyanine 540 to describe membrane fluidity, and PNA-Alexa Fluor™ 647 to identify acrosomic integrity. The second fluorochrome panel consisted of SiR700-DNA to identify DNA-containing events, JC-1 to characterize the mitochondrial transmembrane potential (MMP), and Calbryte 630 to assess the intracellular calcium level. Extended boar semen was stored either at 17°C (control) or 5°C (chilled). It is shown that chilling increased membrane fluidity in the viable (Yo-Pro 1 negative) sperm population at 24 h (p < 0.05). At 144 h, the viable, acrosomic intact sperm population with low membrane fluidity was similar for both storage temperatures. Moreover, chilling reduced the main sperm population with high MMP, medium fluorescence for JC-1 monomer and low intracellular calcium level (p < 0.05). However, after in vitro sperm capacitation, this population did not differ between the two storage temperatures. Exemplary computational data visualization in t-distributed stochastic neighbor embedding (t-SNE) maps and moving radar plots revealed similar subpopulations as identified by three-dimensional stacked bar charts. In conclusion, sperm surviving an initial chilling injury withstand long-term storage and respond in a similar manner to capacitation conditions as sperm stored conventionally at 17°C. Multicolor flow cytometry is a valuable tool for detecting chilling-induced alterations of cell function in sperm subpopulations.

Tolerance of Stored Boar Spermatozoa to Autologous Seminal Plasma: A Proteomic and Lipidomic Approach.

Long-term exposure of liquid preserved boar spermatozoa to seminal plasma (SP) can cause dramatic sperm injury. This study examined whether boar specificity exists in the sensitivity of spermatozoa to SP and whether correspondent biomarkers can be identified. Consecutive ejaculates (n = 4-5) collected from 19 boars were centrifuged, diluted with a pH-stablising extender with 10% (v/v) autologous SP and evaluated by computer-assisted semen analysis and flow cytometry. Up until 144 h storage, four boars showed consistently high sperm motility, viability and mitochondria activity, and one boar showed consistently low values. Intra-boar variability was high in the other boars. Screening of SP (n = 12 samples) for protein markers using mass spectrometry identified three protein candidates of which the granulin precursor, legumain and AWN were 0.5 to 0.9 log2-fold less abundant (p < 0.05) in SP-resistant compared to SP-sensitive samples. Lipidome analysis by mass spectrometry revealed 568 lipids showing no difference between the SP-groups. The most abundant lipids were cholesterol (42,442 pmol), followed by phosphatidylserine (20,956 pmol) and ether-linked phosphatidylethanolamine (13,039 pmol). In conclusion, three candidate proteins were identified which might be indicative of SP-tolerance of sperm during long-term storage. Noteworthy, a first lipidomic profile of boar SP is presented.

Determination of a cooling-rate frame for antibiotic-free preservation of boar semen at 5°C.

Hypothermic storage of boar semen provides the possibility to omit antibiotics from semen extenders so long as sperm quality is maintained and bacterial growth prevented. The objective of this study was to determine an optimal cooling-rate frame for boar semen preserved at 5°C in an antibiotic-free extender. Semen from eight boars extended in AndroStar® Premium was cooled from 30°C to 5°C using seven different cooling rates, ranging initially from 0.01 to 0.36°C min-1 and reaching 5°C between 2 h and 24 h after dilution. Sperm motility, membrane integrity, membrane fluidity, mitochondrial membrane potential and the response to the capacitation stimulus bicarbonate remained at a high level for 144 h at 5°C when the semen was initially cooled in a cooling-rate frame ranging from 0.01 to 0.09°C min­1 in the temperature zone from 30 to 25°C, followed by 0.02 to 0.06°C min-1 to 10°C and 0.01 to 0.02°C min­1 to the final storage temperature. A cooling rate of 0.07°C min-1 in the temperature zone from 30 to 10°C led to a reduced response to bicarbonate (P < 0.01) and fast cooling to 5°C within 1 h with a cooling rate of 0.31°C min-1 resulted in lower values (P > 0.05) of all sperm parameters. In a further experiment, slow cooling with a holding time of 6 h at 22°C induced after 6 h storage a temporary increase in Escherichia coli of 0.5 × 103 to 2.4 × 103 CFU mL-1 in the sperm-free inoculated extender. Overall, the load of mesophilic bacteria in the stored semen was below 6 × 103 CFU mL-1, a level that is not regarded as critical for sperm quality. In conclusion, appropriate cooling protocols were established for the antibiotic-free storage of boar semen at 5°C, allowing the application of hypothermic preservation in research and in artificial insemination.

The role of seminal plasma in the liquid storage of spermatozoa.

Ongoing progress in proteomic characterization of seminal plasma has stimulated research on the identification of biomarkers for male fertility and sperm preservability. So far, many studies have evaluated the benefits of reconstituting cryopreserved or sex-sorted semen with seminal plasma. Less information is available about the effect of remaining or added seminal plasma in liquid preserved semen. The interaction between seminal plasma and spermatozoa is species -specific, and within species often complex and ambiguous. This article aims to review the action of seminal plasma on sperm function in preserved semen with a focus on liquid storage. Effects of seminal plasma on sperm traits during in vitro storage are summarized for males from four domestic farm animals, namely the bull, ram, boar and stallion. Special emphasis is placed on the effect of seminal plasma on long-term stored boar semen, including novel data demonstrating the attenuating effect of protective extender on the adverse effect of seminal plasma in some boars.

Sperm function in vitro and fertility after antibiotic-free, hypothermic storage of liquid preserved boar semen.

The role of antibiotics (AB) in semen extenders as a potential contribution to the global antimicrobial resistance threat is emerging. Here, we establish an AB-free hypothermic preservation strategy for boar semen and investigate its impact on sperm function, microbial load and fertility after artificial insemination (AI). Spermatozoa (12 boars) preserved in AB-free AndroStar Premium extender at 5 °C maintained high motility, membrane integrity, and a low DNA-fragmentation index throughout 72 h storage and results did not significantly differ from controls stored at 17 °C in extender containing AB (p = 0.072). Likewise, kinetic response of spermatoza to the capacitation stimulus bicarbonate during 180 min incubation in Tyrode's medium did not differ from 17 °C-controls. In a competitive sperm oviduct binding assay, binding indices did not differ between semen stored for 72 h AB-free at 5 °C and 17 °C-controls (n = 6 boars). Bacterial load < 103 CFU/ml after 72 h was measured in 88.9% of samples stored at 5 °C AB-free compared to 97.2% in 17 °C-controls (n = 36 semen pools, 23 boars). Fertility traits of 817 females did not differ significantly between the two semen groups (p > 0.05). In conclusion, a hypothermic semen preservation strategy is presented which offers antibiotic-free storage of boar semen doses.