Extended Boar Semen Research Articles

A PCR detection method for discerning Serratia marcescens in extended boar semen

Serratia marcescens is a bacterial contaminant that can be spermicidal when present in extended boar semen that is typically stored prior to breeding use at 15 to 18 °C for several days. This particular contaminant appears to originate from carrier boars, where it resides in the preputial cavity, but has also been shown to then easily contaminate the semen-processing laboratory. Screening for carrier boars to date has been performed through detection of S.marcescens in ejaculates using traditional agar plate culture techniques. These agar growth techniques are labor and time consuming due to the need for sample °titration and temporal growth followed by isolation, leading to delays in identification. The aim of this study was to develop a rapid, sensitive traditional PCR technique that can detect the presence of S.marcescens in extended boar semen. Primers for the detection of S. marcescens 16S rRNA were designed and specificity tested. After PCR optimization, assay sensitivity was evaluated using extended boar semen that was inoculated with various physiological ratios of spermatozoa: S.marcescens (100:1, 50:1, 20:1, 10:1, 8:1, 6:1, 4:1, 2:1. 1:1 and 1:10). Samples, held at 16 °C, were tested every 24 h over a 96 h period, with bacterial DNA extraction performed at each time point using a commercial kit. As a final step, the developed technique was used to screen random samples of extended boar semen for S. marcescens contamination. Results showed that this PCR technique had a sensitivity (90%) and specificity (100%) at detecting S.marcescens in the different inoculated ratios as well as in random, naturally contaminated samples of extended boar semen. In conclusion, this study reports a traditional PCR technique that is effective at rapidly and accurately detecting the presence of S.marcescens in boar extended semen.

The in Vitro Effect of Taurine on Boar Spermatozoa Quality

The aim of this in vitro study was to evaluate the effects of taurine (TAU) supplementation on boar spermatozoa motility, viability, acrosome integrity and morphology. Eighteen boar semen samples were diluted with the Androhep PlusTM extender containing no TAU (control) or supplemented with 1.5 mM, 7 mM, 12.5 mM TAU and cultured at 4 °C for 18 days. Sperm motility was evaluated using the computer-aided sperm analysis (CASA) system. Furthermore, the samples were fixed and assessed for the occurrence of morphological abnormalities using phase contrast microscopy. Fluorescent dyes SYBR – 14 and propidium iodide were used to determine the sperm viability. Acrosome integrity was examined using PNA – Alexa Fluor 647 and flow cytometry. A gradual decrease of the semen quality was detected in all experimental groups over the course of the study. CASA revealed no selective advantage of TAU supplementation on the spermatozoa motility (p > 0.05). TAU administration showed to be ineffective in preserving spermatozoa viability as well as acrosome integrity as measured by flow cytometry (p > 0.05). Under the conditions of this study, no significant positive effect of TAU was recorded following its administration to the Androhep PlusTM boar semen extender with respect to spermatozoa quality.

A mechanism by which Astragalus polysaccharide protects against ROS toxicity through inhibiting the protein dephosphorylation of boar sperm preserved at 4 °C.

Numerous studies have shown that Astragalus polysaccharide (APS) has strong antioxidant effects and high practical value for preserving semen at low temperatures in vitro. However, to date, little attention has been paid to the precise mechanism of APS in sperm preservation at 4 °C. Thus, to gain further insight into the protective effects of APS, the present study was performed to assess the changes in sperm quality parameters, antioxidant capacity, ATP content, and protein phosphorylation levels. Here, we demonstrated that supplementation with APS could effectively preserve boar sperm quality parameters such as sperm motility, acrosome integrity, and mitochondrial membrane potential. Moreover, we found that the positive effects of APS on boar sperm quality were mainly due to the elimination of excessive mitochondrial ROS, the improvement of antioxidant capacities and the enhancement of ATP levels. Interestingly, by conducting a series of studies on protein phosphorylation, we also discovered that APS could protect boar sperm from oxidative stress and energy deficiency through inhibiting the protein dephosphorylation caused by ROS via the cAMP-PKA signaling pathway. To our knowledge, this is the first exploration of the molecular mechanism underlying the protective roles of APS toward ROS toxicity from the perspective of energy metabolism and protein modification. This study comprehensively provides novel insights into the action mechanism of the protective effects of antioxidants on sperm stored at 4 °C and reveals the practical feasibility of using APS as a boar semen extender supplement for assisted reproductive technology.

Potential of Telfairia occidentalis leaf extract as boar semen extender

The preservation of semen for Artificial insemination (AI) required the use of extender, which served as a buffer media to keep the sperm cell in good condition for fertilization. In this study, fluted pumpkin Telfairia occidentalis leaf extract (TOLE) potential as an unconventional boar semen extender was compared to Beltsville thawing solution (BTS), a conventional semen extender over a 48hr storage period. Aqueous solution of TOLE was prepared at three (3) concentration levels (25, 50 and 75%). Fresh ejaculates from boar were extended at (1:4) in TOLE solutions and BTS extender in a completely randomized design; treatments were replicated four (4) times. Extended semen was stored at 170C and evaluated for semen qualities which include pH, dead/live ratio (%) and sperm cell concentration at time intervals (0, 12, 24, 36 and 48) hrs. Extended semen was significantly (P < 0.05) affected by semen extenders and storage periods for pH, and dead/live ratio (%) but sperm cell concentration (103/cm3) not (P > 0.05) affected. The pH values for BTS was significantly (P < 0.05) higher as compared to TOLE extenders. 75% TOLE extender had highest value (43.89%), and BTS had lowest value (12.15%) for dead/live ratio significantly (P < 0.05). The pH and dead/live ratio (%) values increased over storage time in all extender. The optimum performance of TOLE as compared to BTS extender was recorded at 50% concentration of TOLE extender. TOLE showed a potential as boar semen extender, but further studies are required to validate and improve its application.

Hyaluronan improves neither the long-term storage nor the cryosurvival of liquid-stored CD44-bearing AI boar spermatozoa.

Hyaluronan (hyaluronic acid, HA) apparently improves sperm survival in vitro and in vivo (oviduct), maintaining sperm motility and inducing capacitation, but not acrosome exocytosis, either by direct action as a macromolecule or via CD44 membrane receptors. This study explored ejaculated, liquid-extended pig spermatozoa to ascertain (i) the presence (Western blotting) and specific location (immunocytochemistry) of the CD44 receptor, using a specific monoclonal commercial antibody; (ii) whether the CD44 receptor changed location when exposed to bicarbonate, a capacitating trigger, in vitro; and (iii) whether the addition of HA, of molecular size comparable to that produced in the oviduct sperm reservoir (0.0625 to 2.0 mg/ml; 0 HA: control), to semen extenders would improve sperm liquid storage in vitro or cryosurvival post-freezing. Variables tested were sperm velocity and progressive motility (QualispermTM), sperm viability and acrosome status, membrane integrity and early destabilization, mitochondrial activation, and superoxide production (flow cytometry). The CD44 receptor presence in ejaculated, liquid-stored AI boar spermatozoa, as confirmed by a porcine-specific monoclonal antibody, maintained its membrane location under in vitro capacitation-inducing conditions. HA exposure to 24-, 48-, or 72-h liquid-stored (17–20ºC) spermatozoa lowered sperm velocity in membrane-intact spermatozoa, but increased mitochondrial superoxide production. Finally, HA addition during cooling did not improve cryosurvival but did increase mitochondrial activation and membrane destabilization in surviving cells. These results confirm the existence of a CD44 receptor in pig spermatozoa, but the usefulness of adding HA for long-term storage or cryopreservation of liquid-stored, extended boar semen remains in question, thereby warranting further non-empirical analyses of HA-sperm membrane interactions.

Identification of Trueperella abortisuis contamination in extended boar semen

Trueperella abortisuis is a gram-positive bacterium that has been previously identified in aborted porcine feti and placentae located in Asia and Europe. Routine microbiological screening of extended boar semen from a US mid-Atlantic stud identified delayed growth of very small white colonies on both brain-heart infusion and sheep-blood agars after 48 to 72 hours incubation at 37°C under aerobic conditions. Isolate identification was performed using matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry, with T abortisuis identified (MALDI biotyper score = 2.103). After storage at 16°C and 72 hours post collection, total and progressive motility parameters had decreased in extended semen samples positive for T abortisuis. Further work is needed to elucidate the role T abortisuis may play in extended boar semen quality, extended semen longevity, and sow reproductive performance.

Cluster analysis reveals seasonal variation of sperm subpopulations in extended boar semen.

This study aimed to identify motile sperm subpopulations in extended boar semen and to observe the presumptive seasonal variation in their distribution. Data from 4837 boar ejaculates collected over a two-year period were analyzed in terms of kinematic parameters by Computer Assisted Sperm Analysis (CASA). Individual sperm data were used to determine subgroups of motile sperm within the ejaculates using cluster analysis. Four motile sperm subpopulations (SP) were identified, with distinct movement patterns: SP1 sperm with high velocity and high linearity; SP2 sperm with high velocity but low linearity; SP3 sperm with low velocity but high linearity; and SP4 sperm with low velocity and low linearity. SP1 constituted the least overall proportion within the ejaculates (P < 0.05). Season of semen collection significantly influenced the different proportions of sperm subpopulations. Spring was characterized by similar proportions of SP1 and SP4 (NS) and higher proportions of SP3. Summer brought a decrease in both subgroups containing fast sperm (SP1 and SP2) (P < 0.05). During autumn, increases in SP2 and SP4 were recorded. Winter substantially affected the proportions of all sperm subpopulations (P < 0.05) and SP2 became the most represented subgroup, while SP1 (fast and linear) reached its highest proportion compared to other seasons. In conclusion, extended boar semen is structured in distinct motile sperm subpopulations whose proportions vary according to the season of collection. Summer and autumn seem to have a negative impact on the fast and linear subpopulation. Cluster analysis can be useful in revealing differences in semen quality that are not normally detected by classical evaluation based on mean values.

The impact of bacterial contamination of the ejaculate and extender on the quality of swine semen doses

The aim of this study was to verify the influence of the degree of bacterial contamination of boar ejaculate and semen extender on the quality of semen doses. The experiment was conducted in four boar studs, from which raw semen and two semen doses from each ejaculate were collected to evaluate the number of colony-forming units (CFU), pH, sperm morphology and motility. Extender samples were also evaluated for CFU. Ejaculates that had higher levels of contamination ( &gt; 220 CFU mL-1) resulted in semen doses with a greater degree of bacterial contamination but with no reduction in motility or alteration in pH. When the semen doses were classified according to the degree of contamination of the extender, a decrease in motility was observed after 108 and 168 h of storage (P &lt; 0.05) in the group whose extender had ? 14,000 CFU mL-1 versus the group whose extender had ? 330 CFU mL-1. The pH remained stable during 168 h of storage in semen doses with extender that had lower contamination levels, but decreased from 7.2 to 6.0 between 24 and 168 h of storage (P &lt; 0.05) in the group with extender that had higher levels of contamination. A higher number of abnormal acrosomes (P &lt; 0.05) were observed after 168 h of storage in the semen doses whose extender was highly contaminated. The production of semen doses with low bacterial contamination and high sperm cell viability will only be possible with a strict hygienic control in semen processing, primarily with respect to the extender, combined with minimal contamination during collection.

Effect of the Administration of PGF2α Analogue to Extended Boar Semen on Sperm Motility, Morphology and Kinematic Parameters

Abstract The addition of prostaglandin F2α (PGF2α) to boar semen prior to insemination improves the conception and farrowing rates in sows. It is accepted that this is due to increased myometrial contractility, which improves the spermatozoa movement. However, there are limited data about the effect of the exogenous PGF2α analogs on sperm motility parameters and morphology. The aim of the current study was to define if there are changes in motility, morphology and kinematic parameters of spermatozoa on 1st and 24th hour after addition of PGF2α analogue to extended boar semen. A total of 18 ejaculates, obtained from clinically healthy boars were diluted 1:3 in semen extender, and each of them was separate into four aliquots, 50 ml each. PGF2α was added to 3 of them in concentrations of 6, 12 and 25 μg/ml, and the fourth served as untreated control. The motility, kinematic parameters and morphology of spermatozoa were evaluated on 1st and 24th hours after addition of PGF2α. There was no significant difference in sperm morphology, total and progressive motility between the untreated and treated groups. There was however a significant decrease in the rapid velocity and some of the kinematic parameters (VCL, VSL and VAP) in the group treated with 25 μg/ml compared to the control at the 1st hour after PGF2α treatment, which (except for the rapid velocity) persisted to the 24th hour. The results indicate that addition of Oestrophan (Bioveta, CZ) to the extended boar semen did not improve the sperm motility, morphology and kinematic parameters of the spermatozoa.

Bovine serum albumin and skim-milk improve boar sperm motility by enhancing energy metabolism and protein modifications during liquid storage at 17 °C

Both bovine serum albumin (BSA) and skim-milk have been reported to improve sperm quality, primarily by enhancing sperm motility, but the underlying molecular mechanism remains unknown. In this study, boar semen samples were collected and diluted with Androstar® Plus extender containing different concentrations (0, 2, 4 g/l) of BSA and skim-milk. On days 0, 3, 5 and 7, the sperm motility parameters were determined using computer-assisted sperm analysis (CASA), and the ATP concentrations, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity and mitochondrial membrane potential were evaluated using commercial kits. The levels of protein phosphorylation, acylation and ubiquitination were analyzed by western blot. The results showed that supplementation with BSA and skim-milk provided higher sperm motility parameters, ATP levels, GAPDH activity and mitochondrial membrane potential than the control group (P < 0.05). Interestingly, we found that the levels of protein phosphorylation, acetylation and succinylation of the spermatozoa in the treated groups were dramatically higher than those in the control group (P < 0.05). Though the protein ubiquitination level had a decreasing trend, the change in ubiquitination modification was not significantly different between the control group and treated groups. Moreover, the changes in protein modifications between the BSA treated group and skim-milk treated group were not distinctly dissimilar. Taken together, these results suggest that BSA and skim-milk had a positive role in the regulation of boar sperm motility by influencing sperm protein modifications changes as well as increasing the GAPDH activity, mitochondrial membrane potential, and intracellular ATP content. This research provides novel insights into the molecular mechanisms underlying BSA and skim-milk protective effects on boar sperm in the male reproductive system and suggests the feasibility of using skim-milk instead of BSA as a boar semen extender supplement.

Long-term liquid storage and reproductive evaluation of an innovative boar semen extender (Formula12®) containing a non-reducing disaccharide and an enzymatic agent

There are no reports of saccharolytic enzymes being used in the preparation of formulations for animal semen extenders. In the present study, the use of an innovative semen extender (Formula12®) in the long-term liquid storage of boar semen at 17°C was evaluated. The formulation included use of a disaccharide (sucrose) as the energy source precursor coupled to an enzymatic agent (invertase). The innovative extender was evaluated and compared in vitro to a commercial extender (Vitasem LD®) for the following variables: Total Motility (TM), Forward Progressive Motility (FPM), sperm morphology, membrane integrity, acrosome integrity, and chromatin instability. Boar sperm diluted in Formula12® and stored for 12 days at 17°C maintained a commercially acceptable FPM (>70%). Using the results from the in vitro study, an AI field trial was performed. A total of 170 females were inseminated (135 with Formula12® and 35 with Vitasem LD®). The pregnancy rates were 97.8% compared with 91.4%, and the farrowing rates were 96.3% compared with 88.6% when Formula12® and Vitasem LD® were used, respectively. The mean number of piglets born/sow were 14.92±0.46 compared with 13.83±0.70, and the number of piglets born alive/sow were 14.07±0.46 compared with 12.12±0.70 (P<0.05). The results obtained in this study demonstrated that use of the innovative concept to provide a precursor of glucose and fructose as energy sources for an enzymatic agent in an extender allowed for meeting the metabolic requirements of boar sperm during storage at 17°C. It is suggested that there was a beneficial effect on fertilizing capacity of boar sperm in the female reproductive tract with use of these technologies.

Dose rates of antimicrobial substances in boar semen preservation-time to establish new protocols.

To achieve a standardized number of spermatozoa in the final AI dose, varying amounts of extender fluid with a fixed concentration of antimicrobial substances are currently added to boar ejaculates. This practice ignores the different degrees of dilution of the antimicrobials in the end product. In calculating the final concentration of gentamicin in AI doses from 27,538 processed boar ejaculates, we demonstrated varying gentamicin concentrations in the resultant extended boar semen samples. The median concentration was 220.37mg/L. In 25 of the samples (0.09%), the gentamicin concentration fell below 5mg/L, which is close to or below the epidemiological cut-off value for many bacteria. We calculated the minimum inhibitory concentration of gentamicin for bacteria isolated from raw and extended ejaculates. Five of the isolates from extended ejaculates exceeded the maximum test concentration of 512mg/L. As a result, we are presenting an alternative method of boar semen preservation whereby a particular combination of gentamicin concentrate and antibiotic-free extender is incorporated that standardizes the antibiotic concentration in the diluted semen. The addition of standardized antibiotic concentrations did not negatively affect sperm quality when compared to the use of ready-to-use extenders. In conclusion, an end volume-based and standardized addition of gentamicin to boar ejaculates can be a helpful alternative to prevent insufficient dosage of antibiotics in liquid preserved boar semen without affecting semen quality.

66 DEVELOPMENTAL HEALTH ASSESSMENT OF OFFSPRING PRODUCED FROM MAGNETIC NANOPARTICLES USING NEAR INFRARED ANALYSIS OF PLASMA

In vitro fertilization is optimized when there is a homogenous population of viable spermatozoa, not subjected toxic waste products of apoptotic cells. In a previous study, we developed a “nanopurification” technique to magnetically target and remove non-viable spermatozoa from a boar insemination dose. Nanopurified semen has successfully been studied with IVF in swine and bovine but lacks health data regarding offspring produced from exposed semen. Developmental health performance in mammals is typically assessed through measurements of immune related biomolecules in plasma (e.g. immunoglobulins), quantifying each variable with a specific analytical assay. Recent developments in aqueous based near infrared spectroscopy (NIR), aquaphotomics, have been shown to distinguish reproductive stages (e.g. oestrus, diestrus) in blood serum. Thus, application of aquaphotomics may be ideal for analysis of offspring resulting from fertilization with nanopurified semen, using serum or plasma. Our study objective was to identify holistic differences in blood plasma by characterising NIR spectral profiles in offspring produced from nanopurified semen. Extended boar semen doses were mixed with or without specific nanoparticles to target non-viable spermatozoa. Semen doses were exposed to an electromagnetic field, noninvasively separating non-viable spermatozoa from the insemination dose. Six gilts were bred with (n = 3) or without (n = 3) nanopurified semen. Following birth and weaning, 20 offspring of equal sexes were randomly selected from control and nanopurified litters (10/group) for growth and developmental measurements up until market weight. Blood plasma was collected from offspring at market weight for NIR analysis. Spectral data were collected with a quartz cuvette and ASD FieldSpec® 3 spectrophotometer (ASD Inc., Boulder, CO, USA). Chemometric analysis (Unscrambler® X version 10.4; CAMO Software, Oslo, Norway) included a Savistsky-Golay 1st and 2nd derivative for detection of distinct spectral features. Principal component analysis and partial least-squares block-discrimination were used to examine treatment effects, in a blind experiment. Plasma spectral profiles from control and nanopurified offspring contained 6 shared peaks at 1360, 1373, 1402, 1404, 1422, and 1428 nm. Principle components 1 and 2 accounted for 96.26% of the total variance, with no separation of principal component analysis scores for plasma spectra between groups. Partial least-squares discriminant analysis metrics (slope = 0.026, SECV = 0.52) and Students t-test showed no significant difference (P = 0.57) between groups. Results indicate blood plasma content is not influenced in nanopurified offspring when compared with the control. In addition, solute NIR has shown to be a valuable promising tool for assessing complex aqueous solutions in swine. Further effects on growth and development from offspring born from nanopurified continue to be investigated. This work was supported by USDA-ARS Biophotonics Initiative grant #58–6402–3-018.

Resveratrol and Epigallocatechin-3-gallate addition to thawed boar sperm improves in vitro fertilization

Thawing is one of the most delicate process after semen cryopreservation as spermatozoa pass from a dormant metabolic stage to a sudden awakening in cellular metabolism. The rapid oxygen utilization leads to an overproduction of reactive oxygen species that can damage sperm cells, thus causing a significant decrease of fertilizing potential of frozen-thawed spermatozoa. Resveratrol (Res) is a natural grape-derived phytoalexin and Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis); both molecules are known to possess high levels of antioxidant activity. The objective of the present study was to assess the effect of different concentrations of Res (0.5, 1 or 2 mM; Experiment 1) or EGCG (25, 50 or 100 μM; Experiment 2) supplementation to thawing boar semen extender on sperm quality parameters (viability and acrosome integrity) and in vitro fertilization (IVF). Semen after thawing and dilution with three volumes of Beltsville Thawing Solution (BTS), was immediately divided in control group without antioxidants addition (CTR) and either Res or EGCG groups. Sperm viability and acrosome integrity were evaluated in CTR, Res or EGCG groups after 1 h of incubation at 37 °C. The addition of different doses of Res or EGCG to thawing extender for 1 h did not induce any effect on boar sperm viability and acrosome integrity. However, both Res and EGCG treated samples exhibited a significantly higher penetration rate compared with CTR when used for IVF. In particular the treatment with all the EGCG concentrations increased the penetration rate (P < 0.01) while only Res 2 mM induced a significant increase of this parameter (P < 0.01). In addition, EGCG 25 and 50 μM supplementation significantly increased total fertilization efficiency as compared to control (EGCG 25 μM: 40.3 ± 8.2 vs 26.8 ± 9.5, P < 0.05; EGCG 50 μM: 40.4 ± 7.8 vs 26.8 ± 9.5, P < 0.01). The same effect was observed with Res 2 mM (51.0 ± 7.6 vs 29.6 ± 11.3, P < 0.01). In conclusion, our results indicate that the addition of different doses of the two antioxidants to thawed spermatozoa for one hour, even if does not exert any effect on sperm viability and acrosome integrity, efficiently improves in vitro penetration rate. Moreover, both molecules (EGCG 25 and 50 μM and Res 2 mM) significantly increases the total efficiency of fertilization.

The Influence of Air Contact on the Quality of Extended Boar Semen

Background: The extensive use of artificial insemination in the swine industry emphasizes the need for distribution of good quality semen doses by the artificial insemination centers. A factor that could modify seminal quality is the contact of extended semen with environmental air. However, only few studies have investigated the effects of air presence on semen parameters. Thus, the aim of the present study was to evaluate the influence of different amounts of air on porcine semen quality during the storage period.Materials, Methods &amp; Results: Four ejaculates from five sexually mature boars were used. Each ejaculate (n = 20) was isothermically diluted (30 x 106/mL) in a BTS-extender and assigned to three groups differing on the amount of air entrapped within the polystyrene tubes (100 mL). In group AIR-0 the tubes were completely filled with extended semen (0% air); in group AIR-25, 75% of the tube volume was filled with extended semen and 25% with air; in group AIR-50, 50% of the tube volume was filled with extended semen. The semen doses were stored at 17.3 ± 0.5ºC for 120 h. The motility parameters were assessed at 24, 72 and 120 h of storage using a CASA system. The evaluation of acrosomal integrity was performed by examining formalin-fixed samples under phase-contrast microscopy (1000 x) at 72 and 120 h of storage. The pH was measured at 24, 48, 72, and 120 h of storage with a digital pH-meter. At 120 h, samples were incubated in waterbath at 38°C for the thermoresistance test and sperm motility was assessed after 30 and 120 min of incubation. For each time analysis, a different sealed tube was used in order to maintain a constant amount of air within the tubes during storage. Data were analyzed as repeated measures using the GLIMMIX procedure of SAS® Software. Boar and week of collection were included as random effect in the model. Groups were compared using the Tukey-Kramer test at a significance level of 5%. The pH was significantly influenced by the amount of air entrapped within the tubes (P &lt; 0.01). Throughout the experiment, the pH of semen doses was higher as the amount of air increased within the tubes. The mean pH for groups AIR-50, AIR-25 and AIR-0 was 7.51 ± 0.06, 7.25 ± 0.06 and 7.07 ± 0.06, respectively. Overall motility was similar among the groups for up to 24 h of storage. At 72 h and 120 h of storage, total, progressive and rapid motility were higher (P &lt; 0.05) in AIR-0 compared to AIR-50. The kinematic parameters DAP and VAP decreased as the volume of air within the tubes increased (P &lt; 0.05). Group AIR-0 exhibited higher values (P &lt; 0.05) for DSL, DCL, VSL, VCL, BCF, and WOB compared to AIR-25 and AIR-50. Furthermore, after the thermoresistance test performed at 120 h of storage, total motility, progressive motility and rapid motility were negatively affected (P &lt; 0.05) by the increasing amounts of air within the tubes. There was no effect of air amount within the semen doses on acrosome integrity (P &gt; 0.05).Discussion: The presence of air within tubes affects the quality of extended semen during storage, resulting in increase of pH and subsequent decrease in motility preservation. This pH-rise could be explained by the loss of CO2 from the diluted semen to the air compartment within the tube. At the same time, it is possible to infer that this event influences the intracellular pH of the spermatozoa, increasing sperm metabolism and, consequently, impairing sperm motility. Therefore, we conclude that avoiding air presence within storage containers is recommended in order to ensure the quality of boar semen doses.

Antimicrobials in Boar Semen Extenders A Risk/Benefit Analysis

Antimicrobial agents are added to semen extenders to control the growth of microbes contaminating semen during collection. However, such a non-therapeutic use of antibiotics is not without problems, since it may contribute to the development of antibiotic resistance. This review analyses the risks and benefits of using antibiotics to control bacteria in boar semen extenders, including understanding how semen becomes contaminated and the effects of these contaminants on sperm quality. In addition, the advantages and disadvantages of using antibiotics to control bacteria will be considered, as well as possible alternatives.

A comparative study of boar semen extenders with different proposed preservation times and their effect on semen quality and fertility

The present study compared the quality characteristics of boar semen diluted with three extenders of different proposed preservation times (short-term, medium-term and long-term). A part of extended semen was used for artificial insemination on the farm (30 sows/extender), while the remaining part was stored for three days (16–18 °C). Stored and used semen was also laboratory assessed at insemination time, on days 1 and 2 after the collection (day 0). The long-term extender was used for a short time, within 2 days from semen collection, with the aim to investigate a possible advantage over the others regarding laboratory or farm fertility indicators at the beginning of the preservation time. Viability, motility, kinetic indicators, morphology and DNA fragmentation were estimated. The results showed reduced viability, higher values for most of the kinetics, and higher immotile spermatozoa from day 1 to day 2 in all extenders; however, the long-term extender was superior compared to the other two on both days. With regard to morphology and chromatin integrity, the percentage of abnormal and fragmented spermatozoa increased on day 2 compared to day 1 for all of the extenders. However, based on the farrowing rate and the number of piglets born alive after the application of conventional artificial insemination within 2 days from semen collection/dilution, it was found that the medium-term diluents were more effective. In conclusion, it seems that the in vivo fertilization process involves more factors than simply the quality of laboratory evaluated sperm indicators, warranting further research.

187 EXPLOITATION OF IN VITRO CAPACITATION FOR NANOPARTICLE INCORPORATION WITHIN MAMMALIAN SPERMATOZOA

Migration and interactions of mammalian gametes occur in deep body tissues after mating, rendering difficult any in situ noninvasive evaluation of their performances with current methods. In our effort to develop an effective and real-time in vivo imaging approach, we have successfully labelled porcine gametes with self-illuminating bioluminescent and red-shifted quantum dot nanoparticles (QD) in our previous studies (Feugang et al. 2012 J. Nanobiotechnol. 10, 45; Feugang et al. 2015, J. Nanobiotechnol. 13, 38). The present effort aimed at investigating whether QD could be incorporated into spermatozoa through induced in vitro capacitation, which increases sperm plasma membrane fluidity. Fresh extended boar semen was placed on top of a Percoll gradient and centrifuged. Purified motile spermatozoa were collected and washed with pre-warmed PBS. Pelleted spermatozoa were resuspended in the modified Tris-buffered medium with BSA fraction-V (1 mg mL–1; modified Tween medium B with milk powder and BSA). Sperm aliquots (108) were supplemented or not (control) with QD only (QD+; 1 nM), QD+caffeine (2 mM), or QD+heparin (10 µg mL–1); with caffeine and heparin being used as routine capacitant agents in fertilization media. All aliquots were incubated at 38.5°C, under 5% CO2 for 0.5, 1, or 3 h. Spermatozoa were then analysed for motility characteristics and imaged for confirmation of QD-sperm interactions (bioluminescence emission) and localization (transmission electron microscope; TEM). Motility data of 5 replicates were analysed with ANOVA-2, and P &lt; 0.05 was set as threshold of significance. Total sperm motility (TSM) significantly improved with the presence of either or both QDs and capacitant agents after 0.5 and 1 h incubations. With exception of the QD+heparin, all other groups had significantly decreased TSM after 3 h of incubation, when compared with TSM at 0.5 and 1 h. Higher proportions of progressive and rapid (≥45 µm s–1) spermatozoa were observed in the presence of both capacitant agents (P &lt; 0.05), and only QD+heparin maintained greater proportions after 3 h. Sperm straight-line velocity significantly increased in the QD+caffeine at 0.5 h and in both QD+caffeine and QD+heparin thereafter. Sperm straightness data were increased by both caffeine and heparin during incubations. Strong bioluminescence signals were observed in spermatozoa incubated with QDs compared to the background signal seen in the control group. The TEM images revealed consistent surface membrane attachment of QDs in all QD+ groups, whereas transmembrane and intra-spermatic localizations were visible in both QD+caffeine and QD+heparin groups. We concluded that supplementations of medium containing QDs with caffeine or heparin allow the crossing of sperm plasma membrane by QD. No toxic effect of QD on sperm motility was observed, which confirmed our previous report using a similar ratio of QDs over spermatozoa. Exploration of efficient incorporation of QD into spermatozoa as a promising approach for noninvasive molecular imaging is still ongoing, as well as further sperm viability assessments. Supported by the NIH grant #5T35OD010432 and USDA-ARS Biophotonics Initiative grant #58–6402–3-0120.

The impact of bacteriospermia on boar sperm storage and reproductive performance

Bacteriospermia is a documented risk to reproductive performance when using extended boar semen for artificial insemination. A substantial list of bacteria have been recovered from boar semen attributed to fecal, preputial, skin, and hair microorganisms, with these and other environmental bacteria from processing areas identified in doses prepared for artificial insemination. Gram-negative bacteria are most commonly recovered from extended doses, including both Enterobacteriaceae and environmental contaminants, such as those that inhabit water purification systems. The method of processing, distributing, and storing fresh liquid boar semen before insemination plays a role in bacterial growth dynamics and the degree to which the bacteria may damage the sperm or affect the sow. Not all bacterial isolates or contamination levels have the same impact on sperm, with multiple factors governing if and when storage longevity will be reduced through sperm-to-sperm agglutination, impaired motility, acrosome disruption, or loss of membrane viability. Suboptimal reproductive performance can occur because of reduced fertilizing capacity of the sperm or induction of a uterine environment hostile to sperm and/or embryonic survival. Effective bacterial control strategies are necessary to minimize the risk of bacteria contaminating extended semen doses, including monitoring programs designed for quick detection and intervention, should the need arise.

Centrifugation stress reduces theresponsiveness of spermatozoa to a capacitation stimulus in invitro-aged semen.

Density gradient centrifugation of semen is commonly used in many assisted reproduction techniques. Although gradients have the potential to isolate and enrich motile and viable spermatozoa, the centrifugation force presents a stress factor to cell organelles and membranes. The objective of the study was to evaluate the impact of density gradient centrifugation stress on sperm capacitation dynamics, cell stability and the ability of spermatozoa to specifically respond to bicarbonate in extended semen undergoing in vitro ageing. Extended boar semen (n=7) was stored for 12, 24, 72 and 120h respectively at 17°C before centrifugation and incubation in variations of an invitro capacitation medium. The number of viable, acrosome intact sperm and motility parameters as assessed by computer-assisted semen analysis did not change during storage. Kinetic changes in viability (plasma membrane integrity) and intracellular calcium levels (calcium influx) during invitro capacitation were assessed after preparation of semen samples with both, a Percoll and a sucrose gradient centrifugation, either only Percoll, only sucrose centrifugation or no centrifugation. Changes in the viable sperm population that could be specifically attributed as a response to either bicarbonate or calcium were determined. In invitro-aged (>12h stored) spermatozoa, centrifugation reduced the proportion of spermatozoa which specifically responded to the capacitating stimulus bicarbonate. Concomitantly, centrifugation increased the proportion of spermatozoa responding to calcium in absence of bicarbonate, thus indicating an increased sensitivity to incubation per se. Absence of centrifugation steps during semen preparation, revealed a highly conserved ability of invitro-aged spermatozoa to specifically respond to bicarbonate. In conclusion, density gradient centrifugation alters the physiological property of spermatozoa for controlled capacitation, which may influence the success rates of centrifuged semen in assisted reproductive technologies and confound interpretation of capacitation assays.