CASA System Research Articles

Biogenic and risk elements in wild boar testes and relation to spermatozoa motility

Environmental pollution results in serious health hazards to animals, reflected in biogenic and risk element concentrations in animal tissues. The objective of this study was to examinate concentration of selected elements in testes, and epididymal spermatozoa motility of wild boars. Wild boars were hunted in region Žuhračka - Levice, Slovak Republic. Testes were removed postmortem, spermatozoa were collected from cauda epididymis and assessed by CASA system. Elements concentrations were measured by ICP and by CV-AAS. Spermatozoa motility was 44.29% and progressive motility 18.47%. Concentration of elements in testes was in following order: K > Na > Mg > Ca > Fe > Zn > Al > Cu > Se > Mn > As > Cr > Pb > Mo > Sr > Ni > Ba > Cd > Li > Hg. The most notable correlations indicate association between Se and total spermatozoa motility, as well as with progressive motility, furthermore between As and velocity curved line and beat cross frequency. A high positive significant correlation was found between mercury and beat cross frequency. The data may serve as a fine control indicator to detect potentially toxic elements accumulated from polluted environment that can affect reproduction of wild animals.

Effect of Probiotics on Sperm Quality in the Adult Mouse.

The administration of probiotics for the treatment of different diseases has gained interest in recent years. However, few studies have evaluated their effects on reproductive traits. The objective of this study was to examine the effect of two mixtures of probiotics, a commercial probiotic (Vivomixx®) and a mix of Lacticaseibacillus rhamnosus GG and Faecalibacterium duncaniae A2-165, on sperm quality in a mouse model. Adult male mice (8months old) were used for two experimental and one control groups (n = 5 each). The probiotics or physiological serum (control) was administered orally, twice a week, during 5weeks. Sperm were collected from the cauda epididymis, and their total number, motility, kinematics, morphology, and acrosome integrity were assessed in recently collected samples and after a 60-min in vitro incubation. Results showed a higher percentage of normal sperm in both experimental groups, with fewer head abnormalities than in the control. Differences were found among groups in the morphometry of sperm heads, being more elongated in mice treated with probiotics. Sperm from probiotic-treated mice showed similar total motility when compared to the controls, although the proportion of progressively moving sperm and their vigor of motility were lower. Sperm swimming descriptors were measured with a CASA system. Velocity parameters were similar among groups whereas linearity was higher in mice treated with the commercial probiotic. These results suggest that the administration of probiotics may increase the proportion of sperm with normal morphology and lead to modifications in sperm head shape that may enhance sperm swimming. Studies using a longer administration period would be useful in further characterizing the effect of these probiotic mixtures on sperm quality and fertilization capacity.

Melatonin production improves Senegalese sole sperm motility at night, but fails as a supplement during cryopreservation

Melatonin is a powerful antioxidant present in fish seminal plasma. This study aimed to understand melatonin's endogenous and exogenous effects on first-generation Senegalese sole sperm quality for sperm management applications. In the first experiment, samples were collected at mid-light (ML) and mid-dark (MD) daytimes, to evaluate the effects on sperm motility. In a second experiment, using confocal microscopy and melatonin-FITC, spermatozoa permeability to melatonin was evaluated and, after showing that it enters the nucleus and mitochondria by passive diffusion, exogenous melatonin toxicity and antioxidant potential during a cryopreservation assay were performed. The toxicity assay tested different melatonin concentrations (0.01, 0.1, 1, and 10 mM) and exposure times (3, 5, 15 and 30 min), and sperm motility parameters were measured (TM, PM, VCL, VSL, LIN) using CASA system. The best conditions (0.1 and 10 mM) were selected for the cryopreservation assay, and a set of post-thaw sperm quality analyses were performed (motility, viability, reactive oxygen species, lipid peroxidation, and DNA fragmentation). The motility analyzed at ML and MD showed significant differences in all parameters, mainly on velocities (VCL, VSL, VAP), that were significantly higher at MD. Supplemented melatonin did not influence spermatozoa motility, MDA content or DNA fragmentation, although a lower percentage of viable cells was obtained on the 10 mM treatment. Altogether, Senegalese sole spermatozoa motility was enhanced at night, putatively by endogenous melatonin through direct or indirect mechanisms, whereas supplemented melatonin did not confer extra protection during cryopreservation.

Effect of Thawing Procedure and Thermo-Resistance Test on Sperm Motility and Kinematics Patterns in Two Bovine Breeds.

This investigation aimed to analyze the effect that thawing time and temperature in combination with a termo-resistance test had on straws from dairy bulls used for artificial insemination (AI) on semen motility and kinematic variables measured with CASA systems. Eight animals of Holstein and Jersey breeds were used, and nine frozen-thawed semen doses per animal were analyzed for each breed. Three temperatures (35, 37, and 40 °C) and three thawing times (35, 40, and 45 s) were evaluated using a factorial design. Motility and kinematic patterns were analyzed using CASA-mot (Computer-Assisted Semen Analysis of motility) technology at different post-thawing times (0.5, 1, and 2 h). Sperm motility in Jersey bulls was higher (p < 0.05) than in Holstein ones (64.52 ± 1.45% and 53.10 ± 1.40%, respectively). The same effect was seen with progressive motility among the two breeds (Jersey: 45.29 ± 1.00%; Holstein: 36.30 ± 0.98%, p < 0.05). The Jersey breed presented higher values (p < 0.05) of curvilinear velocity (VCL), rectilinear velocity (VSL), average velocity (VAP), linearity on forward progression (LIN), and wobble (WOB). The Holstein breed showed a lower mean value (p < 0.05) of the beat-cross frequency (BCF) compared to the Jersey breed, thus suggesting an effect on VCL and VAP. During the post-thaw period, a gradual increase in VCL was observed at 2 h. VSL and VAP showed a decrease (p < 0.05) as the post-thaw period was prolonged. The study showed differences in sperm quality between Holstein and Jersey breeds, influenced by cryopreservation, thawing, and post-thawing incubation. Thawing at 37 °C for 30 s was considered optimal in relation to sperm motility. In addition, a decrease in sperm quality was observed as post-thawing time increased.

Boar Sperm Performance in Polystyrene Transport Cool Box Under Various Storage Conditions and Holding Times

When transporting highly valuable boar semen samples over long distances, it is crucial to ensure that boar semen is stored ideally at approximately 15 – 17°C to minimize the impact of cold shock and oxidative damage on sperm quality. This study evaluated the performance of a commercially available semen and embryo polystyrene transport cool box, BotuFLEX®, in preserving the quality of extended boar semen under different storage conditions and holding times. Boar ejaculates were extended at 1:1 or 1:3 using a commercial mid-term extender, stored in a BotuFLEX® semen cool box, and examined 24 vs. 48 h thereafter (Experiment 1; n = 12), or 24 h after storage in air-conditioned room vs. ambient room (Experiment 2; n = 9) conditions. Comprehensive sperm motility using the Sperm Class Analyzer® CASA system revealed no significant difference in either total and progressively motile sperm, and the combined progressivity and velocity characteristics across different treatments. Almost similar results were observed on sperm vitality using the standard supravital staining technique except that percent live spermatozoa were higher at 24 h of storage using the 1:1 dilution.

P-058 The role of sperm selection in improving embryo euploidy rate: a comparison between Swim-up and Microfluidic Sperm Selection

Abstract Study question Does Microfluidic Sperm Selection (MSS) improve the blastocysts’ development rate and the euploidy rate compared to Swim-up sperm selection? Summary answer MSS doesn’t improve the rate of blastocyst development and euploidy compared to Swim-up. Furthermore, in women age ≥ 37 years D6 aneuploid blastocyst rate increases What is known already Sperm defects have been shown to have a negative impact on embryo quality and development. MSS was introduced as an alternative to traditional centrifugation-based sperm preparation techniques to efficiently isolate highly motile and healthy spermatozoa. Centrifugation has been suggested to compromise membrane integrity, motility, mitochondrial energy production, and DNA structure. Less DNA fragmentation and ROS formation was observed in spermatozoa isolated with MSS compared to standard techniques. Our study aims to analyze whether MSS also improves biological outcomes. Study design, size, duration In a prospective study of 51 ICSI/PGT-A cycles performed between September 2022 and November 2023, freshly ejaculated semen sample was divided into two aliquots and processed by Swim-up and MSS. Split sperm sample was used to inject siblings’ mature oocytes (249 vs 274 oocytes in Swim-up vs MSS groups). Inclusion criteria: retrieved MII oocytes ≥8, oligoteratozoospermic and normozoospermic semen. Results were analyzed according to women’s age &amp;lt;37 years (21 cycles) and ≥37 years (30 cycles). Participants/materials, setting, methods Spermatozoa were selected using either conventional Swim-up or a microfluidics device (ZyMōtTM). Post treatment sperm parameters (sperm count; total sperm motility; progressive motility and hyperactivated motility) were examined for both. Embryo culture was performed in Irvine Continuous Culture-NX media until day5 or day6 and biopsy was performed at the blastocyst stage by next generation sequencing. Primary endpoint was fertilization rate, blastocyst development and usable blastocysts rate. Chi-square analysis was performed and p &amp;lt; 0.05 was considered significant Main results and the role of chance There is no significant differences overall between Swim-up and MSS in fertilization rate (73% vs 75%), blastocyst development rate (47% vs 54%) and ploidy rate: aneuploid (47% vs 50%), usable blastocyst (i.e. euploid+mosaics) (53% vs 50%). When considering female age, no significant difference is observed in the primary endpoints in the &amp;lt;37 years group. However, in women aged ≥37 years, the rate of blastocyst development is similar in the two sperm preparation groups, but a significant difference is observed in the timing of blastocyst development: The percentage of blastocysts formed on day 6 in the MSS group is significantly higher than in the Swim-up group (43% vs. 19% p &amp;lt; 0.05). In addition, the aneuploidy rate of blastocysts on day 6 is also significantly higher than in the Swim-up group (79% vs. 50% respectively, p &amp;lt; 0.05). The concentration of sperm recovered post-treatment was similar in Swim-up vs MSS (23 x106 vs 19 x106); total motility and progressive motility were similar in Swim-up compared to MSS (92.7% vs. 95.3% and 85% vs. 87% respectively). Only hyperactivated motility assessed with the SCASCOPE CASA System (Microptic) was significantly higher in MSS vs Swim-up 41% vs 4% respectively (p &amp;lt; 0.0005). Limitations, reasons for caution Our study included couples with different infertility diagnosis and semen parameters. Further studies with an increased number of patients may provide more comprehensive data to better evaluate biological and clinical outcomes to eventually suggest the use of MSS in laboratory routines Wider implications of the findings Recently, some studies reported that microfluidic-sorted sperm showed significantly less ROS formation and DNA fragmentation compared to those treated with conventional Swim-up method. Nevertheless, our data does not currently show the expected increase in embryo euploidy rate to justify the routine use of these expensive devices in male infertility treatment Trial registration number non applicabile

P-040 The characteristic of human seminal plasma extracellular vesicle (SPEVs) profiles with different sperm features in a large cohort of 309 Taiwanese men

Abstract Study question To investigate human seminal plasma extracellular vesicle profiles in different sperm characteristics and their potential clinical application. Summary answer Normozoospermic semen has significantly higher SPEV concentration than asthenozoospermic or oligozoospermic samples. Moreover, SPEV concentration was highly correlated with total motile sperm count. What is known already Seminal plasma extracellular vesicles (SPEVs) are cell-derived membrane structures that carry proteins, nucleic acid, and lipids, which can modify the constitution and biological function of sperm. However, the characteristics of SPEVs in different sperm features remained unclear. Study design, size, duration From January 2021 to September 2023, 309 men who visited the NCKUH andrology clinic agreed to collect semen samples for this study, which the Institutional Review Board has approved. Participants/materials, setting, methods Semen parameters were measured by the X1 CASA system (Bonraybio, Taiwan). SPEVs were isolated by size exclusion chromatography column and ExoQuick™. Next, the SPEVs’ concentration and size distribution were performed with nanoparticle tracking analysis. The Kruskal-Wallis test calculated comparison in multiple groups, and Dunn’s multiple comparison tests accessed group-group comparison. The Pearson correlation coefficient r of exosome concentration with individual sperm parameters was calculated. A p-value less than 0.05 was set as statistical significance. Main results and the role of chance Among them, 59 semen samples were presented with normozoospermia, 36 were oligozoospermia, 73 were asthenozoospermia, and 141 samples had no sperm in semen. Among these 141 azoospermic men, 89 men are characterized by non-obstructive azoospermia (NOA), while the other 52 men had a vasectomy. Our data showed that normozoospermic semen has significantly higher SPEV concentration than asthenozoospermic or oligozoospermic semen samples (26 x109 [IQR 17.25 x109-56.25 x109] versus 12 x109 [IQR 5.6 x109-19.5 x109] versus 15.5 x109 [IQR 8.575 x109-26.75 x109], p = 0.0001). Nevertheless, azoospermic semen has the lowest SPEV concentrations. The concentration of SPEVs from infertile men diagnosed with NOA is slightly lower than vasectomized men (3.9 x109 [IQR 1.875 x109-7.675 x109] versus 5.4 x109 [IQR 2.9 x109-8 x109], p = 0.0001). The Pearson r coefficient of SPEV concentration with sperm concentration, progressive motility, and total motile sperm count was 0.238, 0.303, and 0.451, respectively. (P &amp;lt; 0.0001) Limitations, reasons for caution Because our data were collected from Taiwanese men, it is not yet known whether SPEV profiles differ among races. Wider implications of the findings Human SPEV concentration and its bio-content may be a potential non-invasive biomarker to predict the male reproductive outcome. Trial registration number not applicable

Optimizing extenders for short-term storage of Sterlet (Acipenser ruthenus) sperm

Considering endangered fish, storage of sterlet (Acipenser ruthenus) gamete is crucial, and investigating the most suitable extender for short-term storage of sperm is necessary. Therefore, the present study aimed to optimize suitable extenders for short-term storage of sterlet spermatozoa and determine the effects of storage periods and different extenders on spermatozoa motility parameters. The sperm of five mature males with good spermatozoa motility (∼80 %) were diluted with 20 different extenders having various Na+ and K+ concentrations and osmolality. Spermatozoa motility performances were assessed using a CASA system within 144 h storage at 0–2 °C. Undiluted sperm was used as a control. Seminal plasma Na+, K+, Ca2+ and Cl- concentrations were 17.56±10.12, 2.48±1.14, 0.11±0.06, and 6.20±3.42 mmol/L with osmolality of 35.60±20.70 mOsm/kg. A two-way ANOVA showed significant effects of storage time, extender, and their interaction on spermatozoa motility, curvilinear velocity (VCL), and straight-line velocity (VSL) (p<0.001). The model was decomposed into one-way ANOVA to study the effects of storage time and extenders. Excluding 1 h post-storage, spermatozoa motility, VCL, and VSL were lower in the non-diluted sperm than those diluted with E18-E20 at 24 h post-storage (p<0.05). The osmolality of these extenders was respectively 46, 55, and 62 mOsmol/kg and contained 1 mM KCl. Increasing the extender KCl concentrations to >3 mM resulted in lower spermatozoa motility performance. When the effects of storage time were studied, spermatozoa motility, VCL, and VSL in non-diluted sperm and those diluted with E18-E20 were decreased at 24–48 h and 72–96 h post-storage, respectively. This study shows that extenders with higher osmolality (39–62 mOsm/kg) and lower K+ (1 mmol/L) are the most suitable for storing sterlet sperm for a short period.

The neonicotinoid, imidacloprid, disrupt the chicken sperm quality through calcium efflux

Imidacloprid (IMI), an insecticide from the neonicotinoid group widely used in agriculture, has drawn attention due to its potential harmful effects on non-target species, including bird populations. In the present work, we investigated the effect of IMI on avian semen by in vitro exposure of rooster spermatozoa to this pesticide. The semen was collected twice a week. Samples collected on one day were pooled and incubated with the following IMI concentrations: 0 mM, 0.5 mM, 5 mM, 10 mM, and 50 mM at 36°C for 3 h. Comprehensive semen analysis was carried out after 1 h and 3 h of incubation, evaluating sperm motility parameters with the CASA system and using flow cytometry to assess membrane integrity, mitochondrial activity, acrosome integrity, chromatin structure, intracellular calcium level and apoptosis markers such as: early apoptosis and caspase activation and lipid peroxidation. The results of the first experiment suggest that low concentrations of IMI have a different effect on sperm motility compared to higher concentrations. In IMI samples, we also observed a lower percentage of cells with a high level of calcium ions compared to the control, and a lower level of lipid peroxidation. We concluded that IMI may act as a blocker of calcium channels, preventing the influx of these ions into the cell. To confirm this mechanism, we conducted a second experiment with calcium channel blockers: SNX 325, MRS-1845, and Nifedipine. The results of this experiment confirmed that the mechanism of action of IMI largely relies on the blockade of calcium channels in rooster sperm. Blocking the influx of calcium ions into the cell prevents the formation of Ca²⁺-dependent pores, thereby preventing an increase in cell membrane permeability, ultimately blocking early apoptosis and lipid peroxidation in chicken spermatozoa.

238 Effect of electronically controlled floor cooling pad on semen performance of Duroc boars during the summer heat stress

Abstract The objective of the study was to evaluate the effects of using electronically controlled floor cooling pad (ECP) on boar semen production during the summer season in a boar stud in Scottsville, Kentucky. Sixty boars (526.26 ± 188.69 d old) were placed in two rows of pens (2.06 x 0.66 m) in a 350 head tunnel ventilated boar barn and were randomly assigned to either CONTROL (n = 30; no ECP) or PAD (n = 30) treatments. The ECP had 4.25 L cold water flushed through a group of 5 pads connected to each other, when temperature sensors reached 27°C or every 4 mins for sensors placed on 5th pad and 24°C or every 4 mins for sensors placed on 1st pad. Boars experienced natural summer heat stress (daily temperature, humidity and dewpoint: Mean = 24.03°C, 87.9%, 21.7°C, hourly mean high = 28.9°C, 83.4%, 25.8°C) for a period of 30 d from July 15, 2023 to August 14, 2023 wherein the cooling pad remained on and were flushing based on the set points mentioned above. Semen was collected from each boar at least once each week and semen samples were diluted in a commercial semen extender and analyzed for motility using a CASA system, volume using a gram scale, and concentration using a spermiodensimeter. A semen sample was also stained using Hancock stain and evaluated at 100x oil immersion to assess sperm morphology (100 sperm cells analyzed as normal or having retained cytoplasmic droplets, distal midpiece reflex or other head/tail abnormalities). Total sperm production, percentage of morphologically normal sperm, percentage of motile sperm were calculated and used to determine whether the ejaculate met threshold for use in an artificial insemination program or whether the ejaculate was trashed. Data were evaluated using PROC MIXED for repeated measures in SAS (v.9.4) with the main effects of treatment and week, using the age of the boar, and the number of days between collections as covariates, and boar nested within treatment as a random effect. There were no differences between CONTROL and PAD boars for semen production measures (P &amp;gt; 0.05). PAD boars had greater morphologically normal sperm compared with CONTROL boars using Hancock stain (P = 0.0362); however, this effect was lost when the percentage of total normal sperm for the entire ejaculate was calculated (P = 0.56). Overall, maintaining boars on floor cooling pads during the summer months had minimal impacts on semen production and reduced the presence of morphologically abnormal sperm.

Cold-induced damage in boar sperm cooled to 5°C in Lactose Egg Yolk (LEY) medium with different Glycerol concentrations

Boar sperm is highly susceptible to cold damage. When temperature drops to 5°C, the plasmatic membrane is destabilized. The freezing process causes a reduction of the fertility window because frozen/thawed boar sperm has less survivability. The aim of this work was to analyze the effect on sperm characteristics and response to capacitation stimuli of cooling to 5°C using a controlled protocol. Also, we evaluated if the addition of Glycerol 2% or 3% at 5°C was able to modify these parameters. For this purpose, we assessed motility, plasmatic membrane integrity and acrosomal membrane status. Capacitation was induced using Tyrode´s capacitating medium (TCM) and assessed by chlortetracycline stain and induction of acrosomal reaction with Progesterone. Motility patterns were analyzed using a CASA system. These tests were performed at three different points of the freezing curve: 37°C; 17°C and 5°C. Response to TCM vs TBM was only significant at 37°C. While at 37°C and 17°C capacitated sperm was below 20%, at 5°C reached 50% both in the TBM and TCM. CASA analysis showed that spermatozoa exposed to TCM had higher LIN and WOB than those in TBM. All parameters were similar in the Glycerol concentrations studied. These results suggest that the chilling process may be causing an effect similar to cryocapacitation along the cooling curve, starting subtle at 17°C and reaching 50% of the sperm population at 5°C, being independent of Glycerol concentration.

Effect of Extender Supplementation With Caffeine on Arabian Stallion’s Semen Quality After Freezing

In the current study, we aimed to investigate the effect of adding different levels of caffeine to the diluent on the Arabian stallion&amp;rsquo;s sperm quality post cryopreservation. Semen cryopreservation is an essential part of artificial insemination in Arabian horses. In most of cases, the stallion selection is based on desirable phenotypic and physical traits rather than on fertility. This approach may have resulted in the propagation of low-fertility genotypes that are unable to withstand the various stresses associated with cryopreservation such as freezing-thawing stress, oxidative stress, and osmotic stress. Caffeine has been shown to act as a phosphodiesterase inhibitor which may increase the level of cAMP in sperm and subsequently, increase the formation of adenosine triphosphate, which is important for sperm motility. Because it contains xanthine and theobromine, caffeine has antioxidant properties that increase sperm motility and their fertilizability. Five healthy stallions aged 4 to 10 years have been used for semen collection. Semen collections has been done twice weekly by artificial vagina. The ejaculates were evaluated for general progressive motility and sperm concentration. Sperm concentration and motility were determined using the CASA system (ISAS program, Prosser R+D, Paterna, Valencia, Spain). Samples with a minimum concentration of 200 &amp;times; 106 sperm/ml and motility &amp;gt; 60% used for this study. The filtered semen of each ejaculate was diluted (1:1) and divided into 4 aliquots. The aliquots were centrifuged at 800 g for 10 minutes, the seminal plasma was removed, and each sample was re-suspended with FH-20 (0) without any supplementation as a control or with caffeine at a concentration of 0.5 mM, 1 mM, 3 mM or 5 mM. The final semen concentration after dilution was 200 &amp;times; 106 sperm/ml. All samples were assessed before freezing in liquid nitrogen. The frozen thawed semen was assessed for general and progressive motility and then evaluated using the ISAS program and assessed for plasma membrane integrity, morphology defects, acrosome integrity, and viability. Descriptive analyses were determined for the evaluated variables. Statistical comparisons between groups were performed with one-way analysis of variance (ANOVA) (p &amp;lt; 0.05). The addition of 1 mM caffeine to the extender gave better results in total motility (TM), progressive motility (PM) and vitality. However, all studied caffeine concentrations had good effects on straight line velocity (VSL), straightness (STR), linearity (LIN), wobble (WOB) and HOST with no effects on acrosome integrity. It could be concluded that caffeine has a beneficial effect on Arabian stallion semen cryopreservation when added at a concentration of 1 mM to the extender.

Kinematic and Morphometric Assessment of Fresh Semen, before, during and after Mating Period in Brahman Bulls.

The objective of the present study was to determine the effects that the reproductive season has on the motility, kinematics, morphology, and sperm morphometry of Brahman bulls evaluated with a commercial CASA system. The experiment was carried out at the Costa Rica Institute of Technology from March to August 2021. A total of eight Brahman bulls were used. A total of 28 ejaculates were collected in the pre-mating period (PMP), during it (DMP), and after it (AMP) using an electroejaculator. The sperm concentration was measured with the Accuread photometer. The motility was measured using a Spermtrack® counting chamber. The analyses were performed with the CASA-Mot ISAS®v1 system. The morphology was analyzed using a microscope with a negative phase contrast objective. Morphometry was evaluated with the CASA-Morph. The sperm concentration did not present differences between the PMP and AMP; however, it was significantly higher than DMP (p > 0.05). Regarding the progressiveness variables, linearity on forward progression (LIN), straightness (STR), and wobble (WOB) were higher (p < 0.05) DMP. A kinematic principal component analysis grouped all the variables into three factors and an effect on the reproductive period was found (p < 0.05) in the parameters of the head and middle part of the sperm, such as width and perimeter, which were greater in the PMP. The length of the sperm head in the PMP and DMP did not show differences; however, both were larger (p < 0.05) than AMP. The insertion distance of the middle piece of the sperm was significantly greater than DMP. Finally, the PMP contained cells with a larger insertion angle (p < 0.05) than AMP. These findings are important to understand the implications of reproductive status on sperm quality and to consider them in andrological evaluations.

Cryopreserving Rabbit Semen: Impact of Varying Sperm Concentrations on Quality and the Standardization of Protocol.

This study aimed to investigate the impact of sperm concentrations on the in vitro quality of cryopreserved rabbit semen. The semen pools (n = 8, from 80 donors) were split into five aliquots with final sperm concentrations of 15, 25, 35, 55, and 75 × 106 per straw. The sperm motility parameters (CASA system) and membrane integrity (flow cytometric analysis) were both evaluated at various stages of the cryopreservation process: fresh semen dilution, cooling, equilibration, and immediately after and 30 min post-thawing. The results indicated the significant influence of the sperm concentration on the total motility (TM) and progressive motility (PM), with a consistent decline in all sperm variables over the time points. Notably, the semen with a final concentration of 15 × 106 exhibited a higher TM and PM after cooling and equilibration. The post-thawing quality (TM, PM) was higher (p < 0.05) in the mid-range sperm concentrations of 25 × 106 (49.9% and 19.7%) and 35 × 106 (46.2% and 19.7%) compared to the other concentrations. This study demonstrated that the sperm concentration per straw played a significant role in specific phases of the cryopreservation process. These findings contribute valuable insights for refining and standardizing the cryopreservation protocol for rabbit semen, emphasizing the importance of the sperm concentration.

A comparison of the biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state at 5 °C

The biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state were compared. Spermatozoa were collected from the epididymides harvested post-mortem. In the first variant, spermatozoa were diluted in two extenders (Bovidyl® and Salomon's), and were stored at 5 °C for up to 144 h. In the second variant, spermatozoa were stored in the epididymides at 5 °C for up to 144 h, and then diluted in the same extenders. Biological properties were evaluated after 0, 48, 96, and 144 h of storage. Sperm motility parameters were determined in the CASA system. Plasma and acrosomal membrane integrity, mitochondrial activity, apoptotic changes, and DNA integrity were assessed by the fluorescence method. Most variables were significantly influenced by the storage method and time. At 144 h, differences (P ≤ 0.05) in sperm parameters were observed between storage variants. Total motility (TMOT), plasma membrane integrity, and mitochondrial activity decreased below 50% of baseline values in the spermatozoa stored in the epididymides, but remained above 70% of baseline values in the spermatozoa stored in a liquid state. The compared storage variants did not differ in TMOT, mitochondrial activity, or the percentage of viable spermatozoa without apoptotic-like changes up to 96 h of storage, regardless of the applied extender. The earliest significant changes were noted in sperm motility parameters. In conclusion, European red deer spermatozoa can be stored in the epididymides at 5 °C for up to 96 h, but their biological parameters are more effectively preserved during liquid storage.

Flow cytometry and its use in modern human and veterinary andrology

Fertility of a male is sometimes difficult to assess and results obtained using regular sperm analysis methods are often inconclusive. Flow cytometry was proven to generate crucial information, that allows specialists to conclude upon the reproductive capacity of an individual. Together with computer assisted sperm analysis CASA systems, flow cytometers allow an in-depth analysis of fresh, chilled or frozen/thawed semen, which is currently essential for research purposes, but also for diagnosis of various male-related fertility disorders, both in veterinary and human medicine. Several parameters can be assessed using this method, such as sperm viability, acrosome integrity, mitochondrial activity, concentration and total sperm number, DNA fragmentation, capacitation, oxidative stress, etc. This paper provides a rapid reference to specialists involved in semen analysis by flow cytometry, regarding semen sample preparation, instrument setup, and evaluation of the most important sperm parameters (viability, acrosome reaction and mitochondrial activity).

In Vitro and In Vivo Effect of Oxytetracycline on Sperm Parameters In Breeding Rooster

BACKGROUND: Some antimicrobials could adversely affect sperm quality during sperm cryopreservation and antibiotic treatment with subsequent effects on fertility outputs. To our knowledge, no similar studies have been conducted on breeding roosters, especially for oxytetracycline (OTC). OBJECTIVE: To investigate both in vitro and in vivo impact of oxytetracycline on sperm parameters in breeding roosters. METHODS: Sperm motility parameters were objectively analyzed using the CASA system including total motility (TM %), progressive motility (PM %), all sperm velocities, the sperm count, and cell viability during 9 days of in vivo treatment. In the in vitro investigation, the pooled sperm was diluted and divided into a control aliquot (diluted in 0.9% NaCl) and treated samples. Motility parameters were assessed after 0, 1, 2, 3, 4, 5, and 6 hours of storage at 37ºC. In the in vivo study, 1 g/L of OTC was administrated to five individuals for nine consecutive days. Fresh semen samples were analyzed at T0 (before treatment) and after 6 (T6) and 9 days (T9) of treatment. RESULTS: OTC caused significant impairment of sperm quality in vivo. A drastic reduction in sperm concentration, viability, TM, PM, and all kinematic parameters was observed after 6 days of treatment. However, at day 9 sperm quality had improved to be nearly similar to T0. In vitro, OTC induced similar sperm impairment on all sperm motility parameters. CONCLUSION: Oxytetracycline exhibited negative effects on rooster sperm both in vivo and in vitro and appears consequently not suitable in cryopreservation extenders.

The protective effect of endogenous melatonin on gilthead seabream sperm during cryopreservation

Cryopreservation is widely used for artificial reproduction techniques (ART) but requires good gamete quality to succeed. Considering the endogenously produced melatonin by the fish and its protective role in the organism, the objective of this experiment was to search for the best moment of the day to collect gilthead seabream (Sparus aurata) sperm with better quality and, at the same time, to test the potential protective effect of the exogenous melatonin during sperm freezing and thawing processes. Fish were first sampled every 6 h, corresponding to the beginning of the light period (6 h), mid-light (12h), beginning of the dark period (18 h), and mid-dark (24 h) to determine melatonin concentration in blood plasma by radioimmunoassay (RIA). The analysis showed higher values at 24 h (244 pg/mL) compared with 12 h (99 pg/mL), which allowed the selection of those moments for the next experiment. After, fish were sampled for sperm collection at mid-light (ML, 12 h) and mid-dark (MD, 24 h), and fresh sperm was used to assess gamete quality and for the cryopreservation assay. Sperm samples were cryopreserved to test supplementation of different melatonin concentrations (0.001 mM, 0.01 mM and 0.1 mM) together with a control group without added melatonin. Gamete quality was assessed through spermatozoa concentration and motility (CASA system), cell viability (PI/SYBR-green) and DNA fragmentation (Comet assay). Despite cell viability that was higher at ML, most of the fresh sperm motility parameters did not differ between ML and MD, only linearity (LIN) was enhanced at MD. Nevertheless, in cryopreserved samples, total motility (TM) was significantly higher at MD in all melatonin treatments, control, and fresh samples, revealing an endogenous night-effect. Moreover, spermatozoa concentration was also higher at MD (28.9 × 109/mL) than at ML (20.7 × 109/mL). Supplemented melatonin did not confer extra protection to gilthead seabream sperm during cryopreservation since the tested concentrations did not differ between the control in any sperm quality test. It is here suggested that endogenously produced melatonin may contribute to the improvement of some gamete quality parameters at mid-dark, allowing the aquaculture sector to select better sperm quality in a non-invasive way by choosing it as the best moment of the day for sperm collection.

Comparison of two pharmacological semen collection times with α2-adrenergic agonist in domestic cats.

The use of α2-adrenergic agonists in association with urethral catheterization has been used as a technique for pharmacological semen collection in cats. The mechanism of action of this drug is the stimulation of adrenoreceptors in the vas deferens, which results in ejaculation. While medetomidine is the α2-agonist most commonly used in studies, ejaculation with the use of dexmedetomidine associated with ketamine has been effective, but with variable results. Therefore, further studies regarding the methodology of use are required to obtain better seminal quality. This study aimed to compare two pharmacological semen collection times after the association of dexmedetomidine (30 μg/kg, IM; Dormitor®, Zoetis), ketamine (5 mg/kg, IM; ketamine, Vetnil) and urethral catheterization using a tomcat probe (0.8 mm × 1.00 mm × 11 cm). The collections were divided into two experimental groups: G10 (N = 8; urethral catheterization after 10 min of anaesthesia) and G15 (N = 8; urethral catheterization after 15 min of anaesthesia). The ejaculates were evaluated for ejaculate volume, sperm concentration, morphology and kinetics using the CASA system. To compare the groups, the t-test and the Mann-Whitney U-test were used with a significance level of 5%. It was identified that ejaculate volume (G10: 22.62 ± 2.13 vs. G15: 26.81 ± 1.55; p < .001) and sperm concentration (G10: 48.10 × 106 ± 17.84 vs. G15: 90.18 × 106 ± 19.35; p < .001) was higher in G15 than in G10 and had a lower percentage of minor defects than G10 (G10: 3.12 ± 2.41 vs. G15: 1.00 ± 1.19; p = .043). Regarding the kinetic parameters, the results of G15 were better for total motility-TM (G10: 67.00 ± 10.33 vs. G15: 81.87 ± 7.99; p = .006) and faster cells-RAPID: (G10: 55.00 ± 16.63 vs. G15: 74.25 ± 11.94; p = .019); whereas a higher proportion of cells with slow speed-SLOW were seen in G10 (G10: 31.00 ± 12.07 vs. 17.12 ± 7.53; p = .015). Based on these findings, we suggest that collection via urethral catheterization should be performed 15 min after the application of ketamine-associated dexmedetomidine to obtain a better-quality ejaculate.

Comparative evaluation of Duragen® and skim milk extenders to enhance ram semen quality and fertility: A promising alternative in ovine artificial insemination.

This study aimed to investigate the effectiveness of Duragen® and skimmed milk (SM) extenders on the quality parameters, bacterial load and fertilization ability of stored ram semen. A total of 50 ejaculates from Sardi rams (n = 5) aged 2.5-3 years, were collected and stored in Duragen® and SM at 15°C. The motilities and velocity parameters generated by the CASA system were then evaluated at 0, 8 and 24 h of storage. Afterward, bacterial loads of sperm extended in Duragen® and SM were determined at 0, 5 and 24 h of incubation. In addition, ewes (n = 100) aged 2 years, have been chosen in the same herd. The selected ewes were then synchronized and inseminated using semen extended in Duragen® and SM and stored for 5 h at 15°C. The results revealed that total and progressive motilities, straight velocity (VSL), straightness (SRT), lateral head displacement (ALH) and beat cross frequency (BCF) were not affected by the extender type after 24 h of storage (p > .05). However, curvilinear velocity (VCL), velocity average path (VAP), linearity (LIN) and wobble (WOB) showed higher values in Duragen® compared with SM extender after 24 h of storage (p < .05). Bacterial loads were observed mainly in sperm stored in SM at 5 h (183 UFC/mL) and at 24 h (357 UFC/mL) of incubation. However, the only case showing a bacterial load in Duragen® is when the storage time attains 24 h (199 UFC/mL). Concerning fertility, sperm diluted in both extenders resulting in high fertility rates which reaches 66% and 73% for Duragen® and SM, respectively, with no statistical difference (p > .05). In summary, Duragen® extender decreased bacterial load in stored semen and maintained high ram sperm quality and fertility. These findings suggest that Duragen® extender could be used as SM alternative in ovine artificial insemination (OAI).