Temperature Elevation during Semen Delivery Deteriorates Boar Sperm Quality by Promoting Apoptosis.

Semen delivery practice is crucial to the efficiency of artificial insemination using high-quality boar sperm. The present study aimed to evaluate the effect of a common semen delivery method, a Styrofoam box, under elevated temperatures on boar sperm quality and functionality and to investigate the underlying molecular responses of sperm to the temperature rise. Three pooled semen samples from 10 Duroc boars (3 ejaculates per boar) were used in this study. Each pooled semen sample was divided into two aliquots. One aliquot was stored at a constant 17 °C as the control group. Another one was packaged in a well-sealed Styrofoam box and placed in an incubator at 37 °C for 24 h to simulate semen delivery on hot summer days and subsequently transferred to a refrigerator at 17 °C for 3 days. The semen temperature was continuously monitored. The semen temperature was 17 °C at 0 h of storage and reached 20 °C at 5 h, 30 °C at 14 h, and 37 °C at 24 h. For each time point, sperm quality and functionality, apoptotic changes, expression levels of phosphorylated AMPK, and heat shock proteins HSP70 and HSP90 were determined by CASA, flow cytometry, and Western blotting. The results showed that elevated temperature during delivery significantly deteriorated boar sperm quality and functionality after 14 h of delivery. Storage back to 17 °C did not recover sperm motility. An increased temperature during delivery apparently promoted the conversion of sperm early apoptosis to late apoptosis, showing a significant increase in the expression levels of Bax and Caspase 3. The levels of phosphorylated AMPK were greatly induced by the temperature rise to 20 °C during delivery but reduced thereafter. With the temperature elevation, expression levels of HSP70 and HSP90 were notably increased. Our results indicate that a temperature increase during semen delivery greatly damages sperm quality and functionality by promoting sperm apoptosis. HSP70 and HSP90 could participate in boar sperm resistance to temperature changes by being associated with AMPK activation and anti-apoptotic processes.

Post-Thaw Parameters of Buck Semen Quality after Soy Lecithin Extender Supplementation with Fumaric Acid.

The supplementation of cryopreservation media with antioxidants improves the post-thaw quality and fertilizing ability of spermatozoa. To maximize the fertility of frozen-thawed buck spermatozoa, further research is required to overcome obstacles that have yielded controversial results and standardize protocols. In the present work, the effect of adding fumaric acid (a well-described antioxidant) to a soy lecithin semen extender on certain quality parameters of spermatozoa following freezing and thawing was examined for the first time. Five sexually mature Skopelos bucks were used, and ejaculates were collected with an artificial vagina. The semen samples (98 samples, five replicates) were diluted (400 × 106 spermatozoa/mL) with OviXcell®, supplemented with fumaric acid (0 mM, 2.15 mM, 10 mM or 30 mM), equilibrated (5 °C; 3 h), packed (0.5 mL straws), frozen and stored (-196 °C) until further processing. After thawing, the spermatozoa total and progressive motility (CASA), viability (eosin-nigrosin), membrane functional integrity (HOST), acrosome integrity (SpermBlue®) and mitochondrial function (Rhodamine-123/SYBR-14/PI) were evaluated. Statistical analysis was performed with one-way ANOVA, followed by Duncan's test; significance was set at 0.05. The addition of 2.15 mM fumaric acid improved (p < 0.05) spermatozoa viability, membrane functional integrity, acrosome integrity and mitochondrial function compared to all other concentrations. The addition of 30 mM fumaric acid decreased (p < 0.05) spermatozoa viability and mitochondrial function compared to all other concentrations. These results indicate a beneficial effect of a 2.15 mM fumaric acid addition to a soy lecithin extender on post-thaw buck spermatozoa quality. Further research is required to evaluate the in vivo fertility of frozen-thawed buck spermatozoa treated with fumaric acid, as well as to elucidate the mechanism of action of fumaric acid in spermatozoa.

Post-Thaw Storage Temperature Influenced Boar Sperm Quality and Lifespan through Apoptosis and Lipid Peroxidation.

Cryopreservation deteriorates boar sperm quality and lifespan, which restricts the use of artificial insemination with frozen-thawed boar semen in field conditions. The objective of this study was to test the effects of post-thaw storage time and temperature on boar sperm survival. Semen ejaculates from five Landrace boars (one ejaculate per boar) were collected and frozen following a 0.5 mL-straw protocol. Straws from the five boars were thawed and diluted 1:1 (v:v) in BTS. The frozen-thawed semen samples were aliquoted into three parts and respectively stored at 5 °C, 17 °C, and 37 °C for up to 6 h. At 0.5, 2, and 6 h of storage, sperm motility, viability, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels and apoptotic changes were measured. Antioxidant and oxidant levels were tested in boar sperm (SPZ) and their surrounding environment (SN) at each timepoint. The results showed significant effects of post-thaw storage time and temperature and an impact on boar sperm quality (total and progressive motility, VCL, viability, acrosome integrity), early and late sperm apoptotic changes, and changes in MDA levels in SPZ and SN. Compared to storage at 5 °C and 37 °C, frozen-thawed semen samples stored at 17 °C displayed better sperm quality, less apoptotic levels, and lower levels of SPZ MDA and SN MDA. Notably, post-thaw storage at 17 °C extended boar sperm lifespan up to 6 h without obvious reduction in sperm quality. In conclusion, storage of frozen-thawed boar semen at 17 °C preserves sperm quality for up to 6 h, which facilitates the use of cryopreserved boar semen for field artificial insemination.

Comparative Effects of Deoxynivalenol, Zearalenone and Its Modified Forms De-Epoxy-Deoxynivalenol and Hydrolyzed Zearalenone on Boar Semen In Vitro.

Deoxynivalenol (DON) and zearalenone (ZEN) are described as detrimental factors to sow and boar fertility. In comparison, literature reports on the impact of modified forms of DON and ZEN, such as de-epoxy-DON (DOM-1) and hydrolyzed ZEN (HZEN), on swine reproduction are scarce. The aim of our study was to compare the effects of DON, DOM-1, ZEN and HZEN on boar semen in vitro. To this end, pooled boar semen ejaculates from two adult boars were treated with either 50.6 µM DON, 62.8 µM ZEN or equimolar concentrations of DOM-1 and HZEN, respectively (dilution volume of v/v 0.7% DMSO in all cases). Effects on semen motility, morphology, viability, hypo-osmotic swelling test reaction and DNA integrity were investigated hourly up to four hours of incubation. DON negatively affected particular parameters evaluated with a computer-assisted sperm analysis system (CASA), such as immotile spermatozoa and progressive motile spermatozoa, whereas those effects were absent in the case of DOM-1 treatment. In contrast to HZEN, ZEN affected almost all CASA parameters. Furthermore, only ZEN decreased the proportion of viable spermatozoa and increased the proportion of spermatozoa with abnormalities. In conclusion, DON and ZEN negatively affected boar semen in vitro, whereas equimolar concentrations of DOM-1 and HZEN did not induce harmful effects.

The Use of Animal's Body, Scrotal Temperature and Motion Monitoring in Evaluating Boar Semen Production Capacity.

Biomedical measurements by specialized technological equipment have been used in farm animals to collect information about nutrition, behavior and welfare. This study investigates the relation of semen quality (CASA analysis, viability, morphology, membrane biochemical activity and DNA fragmentation) with boar behavior during ejaculation. Sensors were placed on the boar's body. Movement features were collected using an inertial measurement unit (IMU), comprising an accelerometer, a gyroscope and a magnetometer. Boar, scrotal and dummy temperatures were measured by an infrared (IR) camera and an IR thermometer, while the face salivation of the boar was recorded by a moisture meter (also based on IR technology). All signals and images were logged on a mobile device (smartphone or tablet) using a Bluetooth connection and then transferred wirelessly to the cloud. The data files were then processed using scripts in MATLAB 2021a (MathWorks, Natick, Massachusetts) to derive the necessary indices. Ninety-four ejaculates from five boars were analyzed in this study. The statistical analysis was performed in the Statistics and Machine Learning Toolbox of MATLAB 2021a using a linear mixed effects model. Significant and strong negative correlations (R2 > 0.5, p ≤ 0.05) were observed between boar, dummy and scrotal temperature with the progressive, rapid and slow movement of spermatozoa, VCL (curvilinear velocity), VSL (straight line velocity) and ALH (amplitude of lateral head displacement) kinematics. The volume of the ejaculate was correlated with the scrotal and dummy temperature. Dummy's temperature was negatively correlated with BCF (beat/cross-frequency), viability and total time of ejaculation, while it was positively correlated with abnormal morphology. Body temperature was negatively correlated with BCF. Positive correlations were noticed between VAP (average path velocity) and total time of ejaculation with body acceleration features, as well as between the overall dynamic body acceleration (ODBA) and total time of ejaculation. In conclusion, the use of biomedical sensors can support the evaluation of boar sperm production capacity, providing valuable information about semen quality.

Varicocele in an Adult Ram: Histopathological Examination and Sperm Quality Evaluation.

Varicocele is a common pathological condition of testis that is related to male fertility problems. A 3-year age Chios ram had an abnormally enlarged scrotal area, was excluded from reproductive duties, and was euthanized with the owners' permission. The main pathological finding was the presence of bilateral multinodular spermatic cord enlargement with laminated vascular thrombi. Histopathological examination revealed commonly mineralized thrombi within the lumen of veins of the pampiniform plexus, inflammation and testicular degeneration. The epididymides were transported to the laboratory and each cauda region was sliced and washed (8 mL water for injection/epididymis), and the epididymal sperm samples were collected. Sperm motility variables (CASA), viability (eosin-nigrosine), morphology (SpermBlue®), and DNA integrity (Acridine Orange Test, AOT) were assessed. The total and progressive motility were low in semen samples of both sides (30.00% and 1.00% vs. 42.60% and 2.50% for left and right epididymis, respectively). Low viability values were observed for both sides (26.00% vs. 23.00% for left and right epididymis, respectively), while sperm morphological abnormalities were within normal limits. No sperm with DNA damage were detected. The results of this case report indicate that varicocele is associated with testis dysfunction and degradation of ram semen quality, mainly affecting motility and kinematics.

Investigating Visual Monitoring of the Scrotum as a Supplementary Tool for Boar Semen Quality Evaluation.

Farm animals behavior research uses video cameras, mainly for visual observation and recording. The purpose of this feasibility study was to enrich the predictable methods of boar semen production capacity by correlating sperm variables with the scrotal contractions (SC) frequency and intensity. A video camera was used to record the reaction of the scrotum during ejaculation. The respective collected ejaculates were evaluated and semen parameters, such as viability, morphology, membranes functional integrity and kinematics, were determined. The camera recorded the scrotal contractions/relaxations and the video was handled by the Image Processing Toolbox of Matlab (Mathworks Inc., Natick, MA, USA). The SC intensity was verified as a percentage change in the scrotum size among the video frames of maximum contraction and relaxation. The archived data from the frames were analyzed statistically, using a linear mixed effects model that involved sperm assessed parameters. Correlations of the SC intensity with the average path velocity, VAP (R2 = 0.591, p = 0.043) and with the percentage of the cytoplasmic droplets (R2 = 0.509, p = 0.036) were noticed. Previous studies reported the positive correlation of VAP with the number of live-born piglets. In conclusion, video monitoring of the boar scrotal function during ejaculation is useful, but more research is needed to establish its appropriateness as a supplementary method for the prognosis of boar ability to produce high-quality semen.

Effect of Boar Sperm Proteins and Quality Changes on Field Fertility.

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.

Efficiency of three boar sperm enrichment techniques.

The objective of the study was to investigate the efficiency of three enrichment methods to separate boar spermatozoa. Twenty-four ejaculates from 12 boars (2 ejaculates/boar) were extended (30 × 106 spermatozoa/mL) in commercial Beltsville Thawing Solution. Each semen sample was processed with glass wool column (GW) and glass beads (GB) filtration and with the single-layer centrifugation (SLC) technique. Semen samples before (control; C) and after treatment were evaluated for sperm CASA motility/kinetics and concentration, viability, morphology and chromatin integrity. Data were analysed with mixed models. The concentration of total and motile spermatozoa was significantly decreased after treatment in groups GW and SLC, but not in group GB. Group GW showed increased values of WOB compared with both groups C and GB. Group GB showed greater values of rapid movement spermatozoa and lower values of slow movement spermatozoa compared with group C. In group SLC, higher values of VSL, LIN and STR were observed compared with group C. In conclusion, all techniques under examination enhanced various CASA variables. Based on our results, the GB method is a promising alternative separation technique for boar sperm and deserves further research regarding swine in vitro fertilization.

Toxic and Microbiological Effects of Iron Oxide and Silver Nanoparticles as Additives on Extended Ram Semen.

The aim of the study was to investigate the effect of iron oxide (Fe) and silver (Ag) nanoparticles (NPs) on ram semen. A skim milk extender without antibiotics was used as a diluent of 21 ejaculates (8 rams; 2-3 ejaculates/ram). The groups of control (C; semen without NPs), Fe NPs (3.072 mg Fe3O4/mL semen), and Ag NPs (2.048 mg Ag-Fe/mL semen) were incubated (15 °C; 30 min), and then a magnetic field was used for NPs' removal. Standard microbiological procedures were performed for all groups. Post-treated samples were stored (15 °C) for 24 h, and sperm variables (kinetics by computer assisted sperm analysis (CASA); viability; morphology; HOST; DNA integrity) were evaluated at 6 and 24 h. Semen data were analyzed by a mixed model for repeated measures and microbiological data with Student's t-test for paired samples. At 6 h of storage, VCL and rapid movement-spermatozoa, and at 24 h, total/progressive motility and amplitude of lateral head displacement (ALH) were significantly decreased in group Ag compared to control. In group Fe, progressive/rapid movement-spermatozoa were significantly lower compared to control after 24 h of storage. Only in group Ag was a significant reduction of total bacterial count revealed. In conclusion, the examined Fe NPs demonstrated slight antibacterial effect, while the examined Ag NPs provided higher antibacterial properties accompanied by cytotoxicity.

Iron Oxide Nanoparticles as an Alternative to Antibiotics Additive on Extended Boar Semen.

This study examined the effect of Fe3O4 nanoparticles on boar semen. Beltsville thawing solution without antibiotics was used to extend ejaculates from 5 boars (4 ejaculates/boar). Semen samples of control group (C) and group with Fe3O4 (Fe; 0.192 mg/mL semen) were incubated under routine boar semen storage temperature (17 °C) for 0.5 h and nanoparticles were removed by a magnetic field. Before and after treatment, aliquots of all groups were cultured using standard microbiological methods. The samples after treatment were stored (17 °C) for 48 h and sperm parameters (computer-assisted sperm analyzer (CASA) variables; morphology; viability; hypo-osmotic swelling test (HOST); DNA integrity) were evaluated at storage times 0, 24, 48 h. Semen data were analyzed by a repeated measures mixed model and microbial data with Student's t-test for paired samples. Regarding CASA parameters, Fe group did not differ from C at any time point. In group C, total motility after 24 h and progressive motility after 48 h of storage decreased significantly compared to 0 h. In group Fe, linearity (LIN) after 48 h and head abnormalities after 24 h of storage increased significantly compared to 0 h. The microbiological results revealed a significant reduction of the bacterial load in group Fe compared to control at both 24 and 48 h. In conclusion, the use of Fe3O4 nanoparticles during semen processing provided a slight anti-microbiological effect with no adverse effects on sperm characteristics.

Individual and Combined In Vitro Effects of Deoxynivalenol and Zearalenone on Boar Semen.

Mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) can negatively affect pig health. However, little is known about their effects on boar semen. We assessed the individual and combined effects of DON and ZEN on boar semen in vitro. In a pretrial, we determined the minimum dose (MiD) of each mycotoxin that induces a significant alteration of sperm progressive motility, as investigated using computer-assisted semen analysis (CASA). In the main trial, the individual and combined effects of each mycotoxin's MiD on sperm motility and kinetics (CASA analysis), morphology (SpermBlue staining), viability (calcein-propidium iodide staining), membrane functional status (hypoosmotic swelling test), and chromatin integrity (acridine orange staining) were analyzed. Pretrial results suggested a MiD of 50.6 µM and 62.8 µM for DON and ZEN, respectively. In the main trial, DON and ZEN administered at MiD significantly affected CASA parameters (e.g., increase of immotile spermatozoa, reduction of progressive motile spermatozoa), decreased sperm viability, and affected sperm morphology (head abnormalities) and membrane functional status. DON and ZEN showed less than additive effects on most parameters tested and a synergistic effect on viability and on two CASA parameters. In conclusion, DON and ZEN showed individual and combined toxic effects on boar semen in vitro.

Method agreement between three different chambers for comparative boar semen computer-assisted sperm analysis.

The computer-assisted sperm analysis (CASA) has become a standard laboratory tool. Although it contributes a lot to the objective sperm motility assessment, its measurements may be affected by many factors. The aim of the study was to evaluate the effect of chamber on boar semen CASA results. Totally, 100 extended (30 × 106  sperm/ml) boar semen samples were analysed by CASA. Each sample was evaluated using Makler, Leja 4 chamber 20 µm and conventional glass slide/coverslip chambers (MC, LC and GSC, respectively). The differences in values between MC and LC and between MC and GSC were significantly positive (higher values for MC compared with LC and GSC) for total motility, progressive, rapid movement, VCL, VSL, VAP, STR and hyperactive, thus indicating a systematic effect. Between LC and GSC, the differences in many parameters (non-progressive, progressive, slow, LIN, STR, hyperactive) were evenly distributed around zero, while in all other parameters the differences were significantly positive (higher values for LC compared with GSC), except for medium movement. Based on the estimated intraclass correlation coefficients, the method agreement between MC and LC and between LC and GSC was overall moderate to good, depending on the parameter; nonetheless, it was poor between MC and GSC. The limits of agreement between methods can vary considerably depending on the parameter and should be considered when comparisons between CASA measurements of different andrology laboratories or studies have to be performed.