Sperm function in vitro and fertility after antibiotic-free, hypothermic storage of liquid preserved boar semen.
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ABSTRACT:
SUBMITTER: Waberski D
PROVIDER: S-EPMC6791940 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundHypothermic preservation of boar semen is considered a potential method for omitting antibiotics from insemination doses, thereby contributing to the global antibiotic resistance defence strategy. The main challenges are chilling injury to spermatozoa and bacterial growth during semen storage leading to reduced fertility.ObjectivesTo examine chilling injury and the number and type of bacteria in boar semen stored at 5 °C in the absence of antibiotics, and to assess the applicability of hypothermic semen storage under field conditions.Material and methodsBoar ejaculates were extended with AndroStar® Premium, stored at 17 °C with and at 5 °C without antibiotics and tested for functional sperm parameters by flow cytometry. Raw semen and extended samples were investigated bacteriologically. Fertility was evaluated after once-daily inseminations of 194 sows in a field study.ResultsLethal sperm damage assessed by motility and membrane integrity was low throughout storage in both experimental groups. Sublethal chilling effects based on the decrease of viable spermatozoa with low membrane fluidity were higher (P < 0.05) up until 72 h in sperm stored at 5 °C compared to 17 °C but did not differ after 144 h. After 72 h, incubation in capacitating medium for 60 min induced a similar decrease in viable sperm with high mitochondria membrane potential and low cytosolic calcium in both groups. In semen stored at 5 °C, bacteria counts were below 103 CFU/mL and the bacteria spectrum was similar to that of raw semen. In 88% of 34 boars, cooled semen fulfilled the requirements for insemination. Fertility was high and did not differ (P > 0.05) between sow groups inseminated with semen stored antibiotic-free at 5 °C and semen stored at 17 °C with antibiotics.ConclusionDespite subtle chilling effects and low bacterial numbers, antibiotic-free hypothermic storage of boar semen offers the possibility to reduce the use of antibiotics in pig insemination. However, strict sanitary guidelines must be maintained and further evidence of efficiency under field conditions is considered desirable.
| S-EPMC7798330 | biostudies-literature
Project description:Various semen extender formulas are in use to maintain sperm longevity and quality whilst acting against bacterial contamination in liquid sperm preservation. Aminoglycosides are commonly supplemented to aid in the control of bacteria. As bacterial resistance is increasing worldwide, antimicrobial peptides (AMPs) received lively interest as alternatives to overcome multi-drug resistant bacteria. We investigated, whether synthetic cationic AMPs might be a suitable alternative for conventional antibiotics in liquid boar sperm preservation. The antibacterial activity of two cyclic AMPs (c-WWW, c-WFW) and a helical magainin II amide analog (MK5E) was studied in vitro against two Gram-positive and eleven Gram-negative bacteria. Isolates included ATCC reference strains, multi-resistant E. coli and bacteria cultured from boar semen. Using broth microdilution, minimum inhibitory concentrations were determined for all AMPs. All AMPs revealed activity towards the majority of bacteria but not against Proteus spp. (all AMPs) and Staphylococcus aureus ATCC 29213 (MK5E). We could also demonstrate that c-WWW and c-WFW were effective against bacterial growth in liquid preserved boar semen in situ, especially when combined with a small amount of gentamicin. Our results suggest that albeit not offering a complete alternative to traditional antibiotics, the use of AMPs offers a promising solution to decrease the use of conventional antibiotics and thereby limit the selection of multi-resistant strains.
| S-EPMC4141845 | biostudies-literature
Project description:IntroductionLiquid preservation of boar semen is a highly preferred method for semen preservation in pig production. However, oxidative stress is the main challenge during the liquid preservation of boar semen in a time dependent manner. Therefore, supplementation of sperm with antioxidants during storage to protect them from oxidative stress has been the focus of recent research. Myo-inositol (Myo-Ins), the most active form of inositol, which belongs to the vitamin (Vit.) (B1 group has been shown to improve semen quality) (1). This study aimed to investigate whether Myo-Ins supplementation protects boar sperm in liquid preservation against oxidative stress and determine the appropriate concentration of Myo-Ins to be used in this regard.MethodsBoar sperm was diluted with a semen extender with different concentrations of Myo-Ins (2, 4, 6, and 8 mg/mL) depending on the previous studies (1, 24). Sperm motility and viability, plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), semen time survival, and gene expression were measured and analyzed on days 0, 1, 3, 5, and 7 for the different samples.ResultsDifferent concentrations of Myo-Ins exerted different protective effects on the boar sperm quality. The addition of 2 mg/mL Myo-Ins resulted in higher sperm motility and viability, plasma membrane and acrosome integrity, MMP, and effective survival time. Investigation of mRNA expression patterns via qRT-PCR suggested that the 2 mg/mL Myo-Ins sample had increased expression of antioxidative genes.ConclusionThe addition of Myo-Ins to semen extender improved the boar semen quality by decreasing the effects of oxidative stress during liquid preservation at 17°C. Additionally, 2 mg/mL is the optimum inclusion concentration of Myo-Ins for semen preservation.
| S-EPMC10411888 | biostudies-literature
Project description:Rooibos (Aspalathus linearis) is a native shrub from South African fynbos and has become very popular in the last decades for its antioxidant and medicinal attributes. Several studies have shown its beneficial properties in numerous cell lines, but to date, the in vitro effects of rooibos extract on sperm cells are still unknown. In this study, boar semen was supplemented with four concentrations both of fermented and unfermented rooibos extracts during 96 h of liquid storage at 17°C. The effects of rooibos extracts on sperm velocity, membrane integrity, and acrosomal status were evaluated at 2 h, 48 h, and 96 h of semen storage. Overall our results indicate that rooibos extract enhances sperm velocity, protects the acrosome structure, and tends to preserve the membrane integrity during semen storage. Although the unfermented rooibos showed higher total polyphenol content and total antioxidant capacity than the fermented one, the latter had better effects on sperm velocity leading to, for instance, an increase of 30% in the rectilinear velocity (VSL) at 48 h compared to the control group. Taking into account the different storage times, we established a suitable range of extracts concentrations to be used in boar semen. The rooibos extract ought to be considered as a powerful and natural source of antioxidants for the preservation of boar semen.
| S-EPMC5570286 | biostudies-literature
Project description: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.
| S-EPMC8465936 | biostudies-literature
Project description:Antimicrobial resistance is an increasing challenge in semen preservation of breeding animals, especially in the porcine species. Bacteria are a natural component of semen, and their growth should be inhibited to protect sperm fertilizing capacity and the female's health. In pig breeding, where semen is routinely stored at 17°C in the liquid state, alternatives to conventional antibiotics are urgently needed. Photodynamic inactivation (PDI) of bacteria is a well-established tool in medicine and the food industry but this technology has not been widely adopted in semen preservation. The specific challenge in this setting is to selectively inactivate bacteria while maintaining sperm integrity and functionality. The aim of this study was to test the principle of PDI in liquid stored boar semen using the photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) and a white light LED-setup. In the first step, photophysical experiments comprising singlet oxygen phosphorescence kinetics of TMPyP and determination of the photosensitizer triplet time revealed a sufficiently high production of reactive singlet oxygen in the Androstar Premium semen extender, whereas seminal plasma acted as strong quencher. In vitro experiments with extended boar semen showed that the established PDI protocol preserves sperm motility, membrane integrity, DNA integrity, and mitochondrial activity while efficiently reducing the bacteria below the detection limit. A proof-of-concept insemination study confirmed the in vivo fertility of semen after photodynamic treatment. In conclusion, using the PDI approach, an innovative tool was established that efficiently controls bacteria growth in extended boar and maintains sperm fertility. This could be a promising contribution to the One Health concept with the potential to reduce antimicrobial resistance in animal husbandry.
| S-EPMC11335528 | biostudies-literature
Project description: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 min‑1 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 min‑1 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.
| S-EPMC7282664 | biostudies-literature
Project description:The long-term storage of boar sperm presents an ongoing challenge, and the modification of the cryoprotective compounds in semen extenders is crucial for improving cryopreservation's success rate. The aim of our study was to reduce the percentage of glycerol in the extender by elimination or substitution with biocompatible, non-toxic polysaccharides. For boar semen extender improvement, we tested a novel modification with the polysaccharides dextran and pentaisomaltose in combination with unique in silico predictive modeling. We targeted the analysis of in vitro qualitative sperm parameters such as motility, viability, mitochondrial activity, acrosome integrity, and DNA integrity. Non-penetrating polysaccharide-based cryoprotective agents interact with sperm surface proteins such as spermadhesins, which are recognized as fertility markers of boar sperm quality. The in silico docking study showed a moderate binding affinity of dextran and pentaisomaltose toward one specific spermadhesin known as AWN, which is located in the sperm plasma membrane. Pentaisomaltose formed a hydrophobic pocket for the AWN protein, and the higher energy of this protein-ligand complex compared with dextran was calculated. In addition, the root mean square deviation (RMSD) analysis for the molecular dynamics (MD) of both polysaccharides and AWN simulation suggests their interaction was highly stable. The in silico results were supported by in vitro experiments. In the experimental groups where glycerol was partially or entirely substituted, the use of pentaisomaltose resulted in improved sperm mitochondrial activity and DNA integrity after thawing when compared with dextran. In this paper, we demonstrate that pentaisomaltose, previously used for cryopreservation in hematopoietic stem cells, represents a promising compound for the elimination or reduction of glycerol in extenders for boar semen cryopreservation. This novel approach, using in silico computer prediction and in vitro testing, represents a promising technique to help identify new cryoprotectants for use in animal breeding or genetic resource programs.
| S-EPMC8997129 | biostudies-literature
Project description:Artificial insemination (AI) for pigs relies on liquid storage of extended semen at 17°C, which preserves sperm quality and ensures its fertilizing capacity. Routine quality controls include the evaluation of sperm motility, viability and capacitation status. The physiological functions of all these features depend on transmembrane aquaporins (AQPs), proteins playing key roles in osmoadaptation. In this study, we made a relative quantification, using RT-qPCR, of the mRNA of several sperm AQPs in AI-liquid semen doses before and after a 48-hr incubation period, aiming to determine possible quantitative compromising expression changes during the process that could serve as a diagnostic tool. Our results showed a decrease in classical sperm motility variables (total and progressive motility and velocity) and sperm viability after 48-hr storage, whereas capacitation status increased overtime. mRNA expression increased in the orthodox AQP4 and AQP6 after 48-hr incubation, relative to control (0 hr) and 24-hr time-points. Moreover, mRNA expression of aquaglyceroporins AQP3, AQP7 and AQP10 was higher after 48-hr incubation, confirmed by AQP7-protein validation using Western blot. Our results indicate that expression levels of AQPs-mRNA can change in ejaculated pig spermatozoa under conditions of ex-vivo incubation that could modify sperm homeostasis, suggesting it could eventually become a relevant molecular biomarker to assess the efficiency of liquid storage of pig semen.
| S-EPMC9790750 | biostudies-literature
Project description:This study aimed to evaluate boar sperm characteristics and proteins, in relation to their importance regarding in vivo fertility. Sixty-five ejaculates were used and 468 sows (parity ≥ 2) were inseminated. Sperm CASA kinetics, morphology, viability, DNA fragmentation, mitochondrial membrane potential, sperm membrane biochemical activity (HOST) and sperm proteins (Heat Shock Protein 90-HSP90, glutathione peroxidase-5-GPX5, Osteopontin 70-OPN70) were assessed and related to field fertility (number of live-born piglets-NLBP, litter size ≥ 12 piglets-LS, farrowing rate-FR). Statistical analysis was conducted with simple and multiple regression models. Simple regression analysis showed that immotile sperm (IM) significantly affected the NLBP and LS, explaining 6.7% and 6.5% of their variation, respectively. The HOST positive spermatozoa significantly affected the NLBP and LS, explaining 24.5% and 7.8% of their variation, respectively. Similarly, sperm with activated mitochondria significantly affected the NLBP, explaining 13.5% of its variation. Moreover, the OPN70 affected LS and FR, explaining 7.5% and 10.8% of their variation, respectively. Sperm GPX5 protein affected FR, explaining 6.7% of its variation. Multiple regression analysis showed that the combination of IM and/OPN70 explains 13.0% of the variation regarding LS, and the combination of GPX5 and OPN70 explains 13.6% of the variation regarding FR. In conclusion, the estimation of parameters IM, membrane biochemical activity, mitochondrial membrane potential, OPN and GPX5 can provide useful information regarding semen doses for field fertility.
| S-EPMC8234339 | biostudies-literature