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.

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.

The effect of subchronic oral exposure to zearalenone on hematologic and biochemical analytes, and the blood redox status of adult rabbit bucks.

BACKGROUND: Zearalenone (ZEN) is a mycoestrogen with a ubiquitous presence in animal feeds, which also has hematotoxic, hepatotoxic, nephrotoxic, and immunotoxic properties. However, there is a paucity of literature that discusses the effects of ZEN on rabbits. OBJECTIVES: The aim of this study was to evaluate the effect of a prolonged, low-level (50 µg ZEN/kg body weight) exposure on the clinicopathologic and redox status analytes of rabbit bucks. METHODS: Ten adult bucks were included in the study. Each underwent a 7-week control period, followed by a 7-week exposure period. Water or ZEN solutions were daily administered orally (0.5 mL) during the control and exposure periods, respectively. Blood samples were collected weekly for Complete Blood Counts, serum biochemical analyte and reactive oxygen metabolite (ROM) measurements. Data were analyzed using a mixed model, and the level of significance was set at a P of <0.05. RESULTS: During the ZEN exposure period, significant increases were noted in the red blood cell distribution width (RDW) and mean platelet volumes (MPVs), as well as in the white blood cell, monocyte, and eosinophil counts. Significant increases were observed in aspartate aminotransferase and total bilirubin, whereas urea, creatinine, glucose, total calcium, sodium, and potassium concentrations were significantly decreased. The ROM concentrations did not differ significantly between the control and ZEN exposure periods. CONCLUSIONS: Under the present experimental conditions, ZEN affected some of the clinicopathologic analytes of adult rabbit bucks; these changes were mostly indicative of mild hepatocellular damage and dysfunction, inflammatory and/or allergic responses, and renal tubular damage. A ZEN dose of 50 µg/kg body weight did not seem to affect the blood redox status of bucks, as evaluated by the ROM concentrations.

Effect of subchronic oral exposure to zearalenone on the reproductive system of rabbit bucks.

OBJECTIVE To determine the effect of subchronic oral exposure to zearalenone (ZEA) at a daily dose of 50 µg of ZEA/kg of body weight (an environmentally relevant concentration) on the reproductive system of rabbit bucks. ANIMALS 8 healthy sexually mature New Zealand White rabbits. PROCEDURES During the experimental period (March to June), each rabbit underwent a 7-week control protocol and then a 7-week treatment protocol. Water (0.5 mL) or ZEA solution (50 µg/kg [0.5 mL]) was administered orally once daily during the control and treatment period, respectively; ejaculates were collected weekly. Studied end points included semen quality variables (spermatozoa kinetics, morphology, viability, and DNA fragmentation), serum testosterone concentration, and results of histologic examination of the testes and epididymides following euthanasia at the end of the experimental period. RESULTS Treatment with ZEA solution resulted in significant increases in spermatozoa beat-cross frequency, in the percentages of spermatozoa with head and midpiece abnormalities, and in the percentages of DNA-fragmented spermatozoa, compared with effects of the control treatment. Serum testosterone concentration, other spermatozoa velocity variables, and percentages of progressive and total motility, rapidly or slowly moving spermatozoa, and live spermatozoa did not differ significantly between the 2 periods. Histologic examination revealed no patterns of abnormal findings in the testes and epididymides. CONCLUSIONS AND CLINICAL RELEVANCE Oral treatment with ZEA solution at an enviromentally relevant concentration caused minor interference with rabbit bucks' sperm quality. Although mostly considered mild, the sperm quality changes warrant further investigation in terms of fertilizing capacity impairment.

The effect of antioxidant agents' addition and freezing method on quality parameters of frozen thawed ram semen.

The aim of this study was to evaluate the effect of antioxidant agents and freezing methods on the ability of ram sperm to preserve its post-thaw quality characteristics. Six Chios rams were subjected to 52 weekly semen collections. Each ram was used as semen donor for freezing experiments once every 2 weeks. Equal number of good quality spermatozoa from each ejaculate (concentration ≥1 × 109 spermatozoa/ml, motility ≥70%, motility score ≥3.5) were pooled. Three equal aliquots of the pooled sample were diluted using three different fractions of a milk-based and glycerol extender (control, quercetin-enriched, α-tocopherol-enriched). Three freezing methods were applied (slow and fast freezing rate in a programmable freezer, vapors of liquid nitrogen) in every aliquot. Sperm aliquots were tested before freezing, immediately after thawing and after 3 h of incubation at 37 °C. Sperm motility (%) was evaluated microscopically. The percentage of membrane and acrosome-intact spermatozoa (IL%) as well as the percentage of membrane-intact and acrosome-reacted spermatozoa (ARL%) were determined by eosin-nigrosin stain. Furthermore, the percentage of hypo-osmotic swelling (HOS) test-positive spermatozoa was estimated. The results revealed no beneficial effect of the antioxidant treatment on the parameters of post-thaw semen (P > 0.05). However, the slow freezing rate method was more beneficial regarding motility, IL, ARL and HOS-positive spermatozoa compared to the other methods. In conclusion, the antioxidant agents used in this study failed to protect sperm against cryopreservation stress; however, the choice of the appropriate freezing method could contribute to the improvement of post-thaw ram sperm quality.

Age-related changes in quality and fertility of porcine semen.

The aim of this study was to investigate the effect of boar age on quality traits and fertility of liquid-stored semen. Boars were allocated into 3 age groups: 7-10 months (young), 18-33 months (mature), 51-61 months (old). Ejaculates of > 200x10(6) sperm/ml and 85% total motile sperm were extended to 30x10(6) sperm/ml, stored at 17-18 °C and used within 12-24 h for artificial insemination (AI) of 2062 multiparous sows. After 24 h of storage, aliquots of diluted semen were assessed for sperm progressive motility (SPM), incidence of sperm chromatin instability (SCI), proportion of live morphologically normal sperm (LMNS) and head morphometry of LMNS. The results showed that young boars had higher percentages of SCI and lower proportions of LMNS than those of the mature (p < 0.05) and old (p < 0.001) boars, respectively. Sperm head dimensions of young and old boars were greater (p < 0.03-0.001) than those of mature boars. The farrowing rate of young boars (65%) was significantly lower (p < 0.001; χ2= 30-61) than those of the mature (87.2%) and old (84.7%) boars. The relationship between sperm head dimensions and boar fertility was non-significant. In conclusion, boar age is an important physiological factor contributing to the success of swine AI.

Age-related changes in quality and fertility of porcine semen

The aim of this study was to investigate the effect of boar age on quality traits and fertility of liquid-stored semen. Boars were allocated into 3 age groups: 7-10 months (young), 18-33 months (mature), 51-61 months (old). Ejaculates of > 200x10(6) sperm/ml and 85% total motile sperm were extended to 30x10(6) sperm/ml, stored at 17-18 °C and used within 12-24 h for artificial insemination (AI) of 2062 multiparous sows. After 24 h of storage, aliquots of diluted semen were assessed for sperm progressive motility (SPM), incidence of sperm chromatin instability (SCI), proportion of live morphologically normal sperm (LMNS) and head morphometry of LMNS. The results showed that young boars had higher percentages of SCI and lower proportions of LMNS than those of the mature (p < 0.05) and old (p < 0.001) boars, respectively. Sperm head dimensions of young and old boars were greater (p < 0.03-0.001) than those of mature boars. The farrowing rate of young boars (65%) was significantly lower (p < 0.001; χ2= 30-61) than those of the mature (87.2%) and old (84.7%) boars. The relationship between sperm head dimensions and boar fertility was non-significant. In conclusion, boar age is an important physiological factor contributing to the success of swine AI.

Evaluation of zearalenone and alpha-zearalenol toxicity on boar sperm DNA integrity.

This study aimed at investigating the in vitro effects of zearalenone (zen) and alpha-zearalenol (alpha-zen) on motility and nuclear chromatin integrity (NCI) of boar spermatozoa. Mycotoxins were tested, at levels ranging from 10 to 30 microg ml(-1) of diluted semen. Four boars were used for semen collection (eight replicates per boar, four per mycotoxin). After the addition of zen or alpha-zen, semen samples were incubated for 4 h at 38.5 degrees C, 5% CO(2) and 96% humidified air. Motility and NCI were assessed at 0 and 4 h of incubation. No significant differences were noticed in motility among the experimental groups (P > 0.05) for all tested boars. Chromatin instability was significantly higher (P < 0.05) in spermatozoa of only one boar treated with zen and alpha-zen independently of the dose. In conclusion, under our experimental conditions, zen and alpha-zen did not affect the motility of boar sperm, whereas the effects of these toxins on sperm NCI were individual-dependent.