Učinak temperature razrjeđivanja dvaju različitih razrjeđivača na kvalitetu sperme nerasta

This study investigated the quality characteristics and the field fertility of boar semen after dilution with OptimIA, a common extender at 23 (n = 20, group A23) or at 30 °C (n = 20, group A30), and after dilution with OptimIA commercial extender at 23 °C (n = 20, group 23A) or with Androhep Plus, a membrane protective extender at 23 °C (n = 20, group 23B). Each sample of extended semen was stored (16-18 °C) and used for a double artificial insemination at 48 h and 72 h after its collection at the farm (n = 30 per group). The semen was assessed in the laboratory (kinetic parameters, morphology and DNA fragmentation) at collection (0 h) and at insemination hours (48 and 72 h). Most of the semen laboratory parameters deteriorated from 48 h to 72 h, regardless of dilution temperature or the use of the protective extender. However, in the special protective extender the percentages of rapid movement spermatozoa, VCL (curvilinear velocity), VAP (average path velocity) and WOB (wobble) did not differ between 48 h and 72 h. A lower farrowing rate was observed in the common extender group at 23 °C, and a lower number of live born piglets in the protective extender group compared to the other two groups. In conclusion, one step dilution of boar semen at 23 °C compared to dilution at 30 °C did not dramatically affect its in vitro quality characteristics after 72 h of storage, although field fertility was negatively influenced. Some of these negative effects can be compensated for by the use of a membrane protective extender.Istražena je kvaliteta i plodnost nerastove sperme u terenskim uvjetima nakon razrjeđivanja s dva različita razrjeđivača: OptimaIA – standardni razrjeđivač na 23 °C (oznaka skupine A23, n = 20) ili na 30 °C (oznaka skupine A30, n = 20) i OptimaIA - komercijalni razrjeđivač na 23 °C (oznaka skupine 23A, n = 20) ili sa zaštitnikom membrane Androhep Plus na 23 °C (oznaka skupine 23B, n = 20). Svaki uzorak razrijeđene sperme bio je pohranjen na 16-18 °C i korišten za dvokratno umjetno osjemenjivanje nakon 48 h i 72 h od prikupljanja na farmi (n = 30 po skupini). Sperma je u laboratoriju ocijenjena (pokazatelji pokretljivosti, morfologija i DNA fragmentacija) prilikom prikupljanja (0 h) i prilikom osjemenjivanja (48 h i 72 h). Većina pokazatelja je, bez obzira na temperature razrjeđivanja ili uporabu zaštitnog razrjeđivača, pokazala pogoršanje u laboratorijskim uvjetima od 48 h na 72 h. Ipak, kod posebnog zaštitnog razrjeđivača, razlike između 48 h i 72 h nisu utvrđene za postotak brzo pokretljivih spermija, valovitost gibanja, prosječnu brzinu i oscilirajuće pokretanje (treperenje). U usporedbi s ostalim dvjema skupinama, niža stopa oprasivosti opažena je kod primjene standardnog razrjeđivača na 23 °C, a niži broj živorođenih odojaka opažen je nakon primjene zaštitnog razrjeđivača. Zaključno, iako postoji negativni utjecaj na plodnost u terenskim uvjetima, nakon 72 sata pohranjivanja, jedan korak razrjeđivanja nerastove sperme na 23 °C, u usporedbi s 30 °C, ne utječe dramatično na njezinu kvalitetu in vitro. Neki od negativnih utjecaja mogu se nadomjestiti uporabom razrjeđivača koji štite membranu spermija

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

Effect of astaxanthin on extended and cryopreserved boar semen

The aim of the study was to examine the effect of astaxanthin on extended and cryopreserved boar semen. The experiment of the study consists of two parts, the experiment A and B. In experiment A, the effect of astaxanthin on extended boar semen was examined. Twenty ejaculates (10 boars; 2ejaculates/boar) were extended in BTS and split in 3 groups: Control (C; no treatment), Solvent (S; semen with DMSO, the diluent of ASX), and Astaxanthin (ASX; semen with ASX at 0.5μΜ). Semen parameters [motility and kinetics evaluated by a CASA system, morphology, viability, functional integrity of sperm plasma membrane by Hypo-Osmotic Swelling Test (HOST), and DNA integrity] were assessed at 0, 24 and 48h of storage at 17o C (trial I), before (0h) and after (1h) sperm thermal resistance assay at 37o C (trial II), and finally before (0h) and after (1h) sperm incubation under in vitro conditions [38,5o C, 5% CO2, maximum humidity (trial III)]. The aim of the experiment B was to test whether overnight preincubation at 17o C of boar semen with astaxanthin prior to freezing (trial I) or overnight preincubation at 17o C of boar semen with astaxanthin prior to freezing, extended of boar semen with cooling extender supplemented with astaxanthin, and ASX supplementation on thawing (trial II) or astaxanthin supplementation only on thawing (trial III) may exert any protective effect against injury related to freezing and thawing. Totally, twenty eight entire ejaculates (4 boars; 7ejaculates/boar) were collected, pooled (4 boars; 1ejaculate/boar), extended in BTS (1:1; v:v) and split in 5 groups for cryopreservation procedure: control (C; no treatment), solvent control (S; semen with DMSO, the diluent of ASX), low astaxanthin (ASX 1; semen with ASX at 0,5μΜ), medium astaxanthin (ASX 2; semen with ASX at 5 μΜ) and high astaxanthin (ASX 3; semen with ASX at 15μΜ). Sperm quality and functionality parameters [motility (CASA) and viability/acrosome integrity, ROS production, lipid peroxidation, apoptosis (flow cytometer analysis)] were evaluated prior to freezing and after thawing (30 and 150 min). The results of the experiment A indicate the positive influence of astaxanthin on boar sperm parameters under storage conditions (17o C), after thermal resistance assay (37o C) and during incubation under in vitro conditions. Also, a mild beneficial effect of ASX on cryopreserved boar semen was observed after its addition on thawing. Further studies regarding in vivo and in vitro fertility should be performed to corroborate the beneficial effect of astaxanthin on boar semen.Σκοπός της διδακτορικής διατριβής ήταν η μελέτη της επίδρασης της φυσικής προέλευσης ασταξανθίνης στο αραιωμένο και κρυοσυντηρημένο σπέρμα του κάπρου. Το πείραμα της παρούσας έρευνας αποτελείται από το δύο πειραματισμούς. Στον πρώτο πειραματισμό μελετήθηκε η επίδραση της προσθήκης ασταξανθίνης στο αραιωμένο σπέρμα του κάπρου υπό συνθήκες συντήρησης σε θερμοκρασία 17ο C για 48 ώρες (δοκιμή Ι), θερμικής καταπόνησης σε θερμοκρασία 37ο C για 1 ώρα (δοκιμή ΙΙ) και επώασης υπό in vitro συνθήκες (38,5ο C, 5% CO2, μέγιστη υγρασία) για 1 ώρα (δοκιμή ΙΙΙ). Συνολικά μελετήθηκαν 20 εκσπερματίσματα (10 κάπροι, 2 εκσπερματίσματα/κάπρο) τα οποία αραιώθηκαν σε BTS και χωρίστηκαν στις ακόλουθες ομάδες: μάρτυρας σπέρματος (Σ), μάρτυρας διαλύτη (αραιωμένο σπέρμα με την προσθήκη DMSO, Δ) και ασταξανθίνη (αραιωμένο σπέρμα με την προσθήκη διαλύματος ασταξανθίνης σε συγκέντρωση 0,5μΜ, ΑΣΤ). Οι παράμετροι του σπέρματος [κινητικότητα και επιμέρους μορφές κίνησης (CASA), μορφολογία, ζωτικότητα, λειτουργική ακεραιότητα της κυτταροπλασματικής μεμβράνης με την εφαρμογή της βιοχημικής δοκιμής αντοχής έναντι της καταπόνησης σε υπό ωσμωτικό διάλυμα (δοκιμή HOST), ακεραιότητα της χρωματίνης του πυρηνικού γενετικού υλικού] εκτιμήθηκαν 0, 24, 48 ώρες υπό συνθήκες συντήρησης σε θερμοκρασία 17ο C, πριν (0 ώρα) και μετά (1 ώρα) την εφαρμογή θερμικής καταπόνησης στους 37ο C και τέλος πριν (0 ώρα) και μετά (1 ώρα) την επώαση του σπέρματος υπό in vitro συνθήκες (38,5ο C, 5% CO2, μέγιστη υγρασία). Στον δεύτερο πειραματισμό της έρευνας μελετήθηκε η επίδραση της ασταξανθίνης στο κρυοσυντηρημένο/αναθερμασμένο σπέρμα του κάπρου με την προσθήκη διαλύματος του αντιοξειδωτικού στο στάδιο της ολονύκτιας επώασης σε θερμοκρασία 17ο C (δοκιμή Ι) ή στο στάδιο της ολονύκτιας επώασης σε θερμοκρασία 17ο C, της αραίωσης με το αραιωτικό ψύξης (17ο C) και της αναθέρμανσης σε θερμοκρασία 37ο C (δοκιμή ΙΙ) ή μόνο στο στάδιο της αναθέρμανσης (δοκιμή ΙΙΙ). Συνολικά μελετήθηκαν 28 εκσπερματίσματα (4 κάπροι, 7 εκσπερματίσματα/κάπρο), τα οποία αραιώνονταν σε BTS (1:1), αναμιγνύονταν (4 εκσπερματίσματα, 1 εκσπερμάτισμα/κάπρο) και διαχωρίζονταν στις ακόλουθες ομάδες: μάρτυρας σπέρματος (αναμεμιγμένο αραιωμένο σπέρμα, Σ), μάρτυρας διαλύτη (αναμεμιγμένο αραιωμένο σπέρμα με την προσθήκη DMSO, Δ), ασταξανθίνη χαμηλής συγκέντρωσης (αναμεμιγμένο αραιωμένο σπέρμα με την προσθήκη διαλύματος ασταξανθίνης 0,5μΜ, ΑΣΤ 1), ασταξανθίνη μέσης συγκέντρωσης (αναμεμιγμένο αραιωμένο σπέρμα με την προσθήκη διαλύματος ασταξανθίνης 5μΜ, ΑΣΤ 2) και ασταξανθίνη υψηλής συγκέντρωσης (αναμεμιγμένο αραιωμένο σπέρμα με την προσθήκη διαλύματος ασταξανθίνης 15μΜ, ΑΣΤ 3). Οι παράμετροι του σπέρματος [κινητικότητα (CASA) και ζωτικότητα/ακεραιότητα της ακροσωμιακής μεμβράνης, ενδοκυτταρικός σχηματισμός ROS, λιπιδική υπεροξείδωση της κυτταροπλασματικής μεμβράνης, απόπτωση (ανάλυση σε κυτταρομετρητή ροής)] αξιολογήθηκαν πριν την κρυοσυντήρηση και σε δύο χρόνους μετά την αναθέρμανση (30 και 150 λεπτά). Σύμφωνα με τα αποτελέσματα των δύο πειραματισμών η προσθήκη διαλύματος ασταξανθίνης είχε θετική επίδραση στις παραμέτρους του αραιωμένου σπέρματος κατά τη συντήρηση σε θερμοκρασία 17ο C, τη θερμική καταπόνηση σε θερμοκρασία 37ο C και κατά την επώαση υπό in vitro συνθήκες. Επίσης, παρατηρήθηκε μία ήπια δράση της ασταξανθίνης στο κρυοσυντηρημένο/ αναθερμασμένο σπέρμα του κάπρου κατά την προσθήκη της μόνο στο στάδιο της αναθέρμανσης. Περαιτέρω μελέτες αναφορικά με την in vivo και in vitro γονιμότητα θα είχαν αξία να πραγματοποιηθούν ώστε να ενισχύσουν την ευεργετική επίδραση της ασταξανθίνης στο σπέρμα του κάπρου

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

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 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

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

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 &mu;M DON, 62.8 &mu;M 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

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

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