Ultrasound-assisted deep eutectic solvents extraction of polysaccharides from Loquat leaf: Process optimization and bioactivity study.

Loquat leaves are the by-product of loquat fruit production. Polysaccharides are one of the main active ingredients in loquat leaves. In this study, polysaccharides were extracted from loquat leaves by ultrasonic-assisted deep eutectic solvents (DESs) extraction method. Further, the extracted crude loquat leaf polysaccharides (CLLP) were purified and separated via S-8 resin and DEAE-52 cellulose column chromatography, respectively. Additionally, the effects of polysaccharides on activity of sperm in boar semen preserved in medium at 17 °C, were evaluated preliminarily. DES, composed of choline chloride/ethylene glycol (1:6, molar ratio), was proved to be the suitable solvent for LLP extraction. The optimized extraction conditions were water content 44 %, liquid-solid ratio 1:29 (g/g), extraction temperature 61 °C and extraction time 98 min. Under these conditions, the LLP yield was 57.82 ± 1.50 mg/g. A homogeneous polysaccharide (LLP1-2, Mw: 2.17 × 104 Da) was isolated from CLLP. Its total sugar, uronic acid and protein contents were 76.31 ± 1.25 %, 14.19 ± 0.67 % and 3.28 ± 0.42 %, respectively. Further, 800 µg/mL LLP1-2 could effectively enhance the antioxidant activity of sperm. This study laid a foundation for DESs and column chromatography in the field of polysaccharide extraction and separation, proving that LLP can be used as a natural antioxidant for sperm preservation.

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.

The Adaptation Time to the Extender as a Crucial Step for an Accurate Evaluation of Ram Sperm Quality during the Liquid Storage.

Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation. This study aimed to determine: (1) the best time to assess ram sperm quality, and (2) the factor responsible for the altered state of ram sperm during the first hours of liquid storage. In Experiment 1, ejaculated sperm were diluted and assessed for sperm motility and functionality at four preservation times: 0, 3, 6, and 24 h as sperm damage control. Both sperm motility and functionality improved after 6 h. Experiment 2 investigated the factor responsible for sperm quality change by testing the interactions of seminal plasma and extender with sperm from epididymides independently and in combination. The evaluation of sperm was performed as in Experiment 1. Sperm in groups containing the extender showed altered motility at 0 and 24 h, and lower functionality at 0 h. Thus, we could assume that extender addition initially alters ram sperm, causing sublethal damage that is reversible after 3 to 6 h of semen preservation. In conclusion, ram sperm require an adaptation time of 3 to 6 h to the extender before an accurate quality assessment can be conducted. This has practical implications for reproduction centers, enabling better workflow organization and optimal expression of ram sperm attributes when cervical artificial insemination is routinely performed.

Heat stress and ram semen production and preservation: Exploring impacts and effective strategies.

As global warming persists, heat stress (HS) continues to affect animals, particularly those raised in extensive systems such as sheep. As a result, there is a growing body of research investigating the physiological and biological consequences of HS on these animals. Recent studies have specifically examined the effects of climate change, global warming, and HS on gametes. Heat stress has been shown to affect ram semen production, resulting in decreased sperm quality and volume in both fresh and stored samples. This is attributed to the effect of heat on hormone production in the testicles, which is critical for successful spermatogenesis. Such effects can have significant consequences on the fertility of female sheep, which could affect the farmers' revenue. Therefore, farmers and researchers are utilizing various strategies and laboratory techniques to mitigate these negative effects. This review aims to comprehensively evaluate the impact of HS on ram semen production and conservation and analyze the different mitigation strategies at various levels, including management and nutritional interventions. The findings of this review will serve as a critical foundation for the development of targeted interventions and sustainable practices in sheep farming, ensuring resilient and profitable operations in the face of ongoing global climate challenges.

Roles of Y-27632 on sheep sperm metabolism.

To investigate the effect of Y-27632 on low-temperature metabolism of sheep sperm, different concentrations of Y-27632 were added to sheep semen at 4 °C in this experiment to detect indicators such as sperm motility, plasma membrane, acrosome, antioxidant performance, mitochondrial membrane potential (MMP), and metabolomics. The results showed that the addition of 20 µM Y-27632 significantly increased sperm motility, plasma membrane integrity rate, acrosome integrity rate, antioxidant capacity, MMP level, significantly increased sperm adenosine triphosphate (ATP) and total cholesterol content, and significantly reduced sperm Ca2+ content. In metabolomics analysis, compared with the control group, the 20 µM Y-27632 group screened 20 differential metabolites, mainly involved in five metabolic pathways, with the most significant difference in Histidine metabolism (P = 0.001). The results confirmed that Y-27632 significantly improved the quality of sheep sperm preservation under low-temperature conditions.

Sheep semen preservation and artificial insemination is an important reproductive technology that supports the large-scale and intensive development of the sheep farming industry. Under low-temperature condition, sperm metabolic activity slows down or pauses, energy consumption decreases, thereby prolonging sperm preservation time and motility. During the process of sperm preservation, sperm are susceptible to cold shock damage, which affects the quality of sperm preservation. Y-27632 is a rho-associated cooled-coil kinase (ROCK) inhibitor that competes with ATP to inhibit the kinase activity of ROCK-I and ROCK-II. However, the study of Y-27632 used in sheep semen preservation and its protective mechanism is less. In this study, we used the ROCK inhibitor Y-27632 and the ROCK activator arachidonic acid (AA) for low-temperature preservation of sheep semen and related metabolic regulation mechanisms. This experiment confirmed that Y-27632 played a significant protective role by regulating sperm metabolism and protecting sperm plasma membrane in sheep.

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.

Semen quality and sperm DNA fragmentation in cancer patients undergoing sperm cryopreservation.

PURPOSE: We compared semen quality and sperm DNA fragmentation in cancer patients who underwent sperm banking and controls who underwent sperm cryopreservation for assisted reproductive technology (ART). MATERIALS AND METHODS: A total of 132 men, 65 cancer patients and 67 controls, were prospectively enrolled and performed sperm cryopreservation for fertility preservation from May 2019 to February 2021. Sperm quality was determined by measuring semen volume, sperm concentration, sperm motility, and sperm DNA fragmentation index (DFI). Sperm quality and sperm DFI were compared in cancer patients and controls. RESULTS: The major cancers of the 65 cancer patients were leukemia (26.2%), testicular cancer (23.1%), and lymphoma (20.0%). Sperm concentration, sperm total motility, and sperm progressive motility were significantly lower in cancer patients than in controls. Sperm DFI was significantly higher in cancer patients than in controls (24.32%±15.69% vs. 19.11%±11.63%; p=0.033). After excluding 8 cancer patients who received chemotherapy before sperm banking, sperm concentration, sperm total motility, and sperm progressive motility were significantly lower in cancer patients than in controls, but there was no significant difference in sperm DFI for cancer patients and controls (23.14%±12.79% vs. 19.11%±11.63%; p=0.069). CONCLUSIONS: Sperm quality was lower in cancer patients than in controls. There was no difference in the sperm DFI of cancer patients prior to chemotherapy and men presenting for sperm cryopreservation for ART. We recommend that all men who are planning cancer therapy should be offered sperm banking prior to gonadotoxic chemotherapy as a standard of fertility preservation.

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.

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.

Dual use of breeding stallions is possible without affecting the sperm quality.

Artificial insemination (AI) is commonly used in the equine industry to enhance the genetic value in breeding programs and to effectively utilize ejaculates. Many stallions are used as breeding stallions as well as in high-level sports competitions to improve their market value. The goal of the present study was to investigate whether this dual use of stallions influences the animals´ stress levels and/or the quality of their ejaculates. For this purpose, 18 stallions were grouped into two categories: breeding stallions with (BSC = breeding stallion competition), and breeding stallions without secondary use in competitions (BS = breeding stallion). Two ejaculates were collected at a one-week interval and analysed with an extended spectrum of spermatological methods. Furthermore, saliva, as well as seminal plasma samples were taken, and the concentration of cortisol therein was determined. Additionally, dehydroepiandrosterone (DHEA) and the cortisol/DHEA ratio were analysed and calculated for seminal plasma. After statistical analysis of the correlations and interdependences between the two groups, the results showed that the BSC group had significantly higher saliva cortisol levels (p = .027) and tendentially higher DHEA concentrations in their seminal plasma (p = .056). No difference between BS and BSC could be found in regard to the sperm quality parameters and the cortisol concentration in seminal plasma samples. It can be concluded that while active participation in competitions represents a stress factor, the dual use of stallions in breeding programs and sports competitions is possible without negative effects on their sperm quality.

Update on artificial insemination: Semen, techniques, and sow fertility.

Over the years, reproductive efficiency in the swine industry has focused on reducing the sperm cell number required per sow. Recent advances have included the identification of subfertile boars, new studies in extended semen quality control, new catheters and cannulas for intrauterine artificial insemination (AI), and fixed-time AI under commercial use. Therefore, it is essential to link field demands with scientific studies. In this review, we intend to discuss the current status of porcine AI, pointing out challenges and opportunities to improve reproductive efficiency.

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.

Reproductive efficiency of sows inseminated at single dose fixed time with refrigerated, cryopreserved and encapsulated spermatozoa.

The aim was to assess the reproductive efficiency of different techniques used to preserve spermatozoa in artificial insemination semen doses (AI-doses) by evaluating refrigeration at 15°C, cryopreservation and encapsulation. Forty-two hyperprolific sows were treated with buserelin and inseminated once at a single fixed time. The fertility rate, embryonic vesicles viability and the early embryonic mortality (arrested conceptuses) evaluated post-mortem at 24th day of pregnancy, were analysed in order to assess the effectiveness of each proposed technique. Results show an overall reduction on fertility using the three proposal sperm preservation techniques (69.27%, 60.00% and 78.75% for refrigerated, frozen-thawed and encapsulated AI-doses, respectively). Total number of embryonic vesicles was very similar among the three treatments; yet, the number of viable vesicles was numerically different among groups, and thus, embryonic viability was 79.25%, 80.0% and 87.15% for refrigerated, frozen-thawed and encapsulated AI-doses, respectively.

Assessment of Chilling Injury in Boar Spermatozoa by Kinematic Patterns and Competitive Sperm-Oviduct Binding In Vitro.

Sensitive detection of chilling injury in boar spermatozoa is required to evaluate novel hypothermic preservation concepts. The study's aim was to examine whether analyses of motility patterns and sperm binding in a competitive oviduct explant assay (cOEA) sensitively detect chilling-induced alterations in sperm function. Semen samples (n = seven boars) were split into four subsamples by dilution either in Beltsville Thawing Solution (BTS) or Androstar® Plus and stored at 5 °C or 17 °C. Storage temperature had a significant effect on the distribution of spermatozoa in seven major kinematic clusters. The effect size of chilling at 5 °C as estimated by Cramer's V was higher (p < 0.05) in the BTS medium (0.21) compared to AndroStar® Plus (0.11). Spermatozoa extended in Androstar® Plus had higher relative binding capacity compared to sperm in BTS (p < 0.05). Binding indices correlated with the percentage of viable, acrosome-intact (r = 0.62) and motile spermatozoa (r = 0.72, both p < 0.001). The cluster size of sperm with slow, vigorous movement was negatively correlated with sperm-oviduct binding (r = −0.43, p < 0.05). In conclusion, the cluster analysis of sperm kinematics and competitive sperm oviduct binding in vitro present meaningful biological tests to assess novel concepts for hypothermic semen preservation.

Factors Affecting the Survival of Ram Spermatozoa during Liquid Storage and Options for Improvement.

Semen preservation is an essential component of reproductive technologies, as it promotes genetic gain and long-distance semen transport and multiplies the number of ewes able to be inseminated per single ejaculate. However, the reduced temperature during cold storage at 5 or 15 °C inflicts sub-lethal damage to spermatozoa, compromising sperm quality and the success of artificial breeding. New and emerging research in various species has reported the advantages of storing spermatozoa at higher temperatures, such as 23 °C; however, this topic has not been thoroughly investigated for ram spermatozoa. Despite the success of storing spermatozoa at 23 °C, sperm quality can be compromised by the damaging effects of lipid peroxidation, more commonly when metabolism is left unaltered during 23 °C storage. Additionally, given the biosafety concern surrounding the international transport of egg-yolk-containing extenders, further investigation is critical to assess the preservation ability of synthetic extenders and whether pro-survival factors could be supplemented to maximise sperm survival during storage at 23 °C.

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.

Development of a new antimicrobial concept for boar semen preservation based on bacteriocins.

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.