Computer-assisted Semen Analysis Research Articles

Effects of Extenders Supplementation with Gum Arabic and Antioxidants on Ram Spermatozoa Quality after Cryopreservation

Semen cryopreservation is very important in animal agriculture to maximize the number of daughters of genetically superior males and to distribute the cryopreserved semen of good males all over the world. However, the freezing process generates some damage to sperm that reduce their fertilizing ability after thawing. Moreover, egg yolk, which is the most common animal-origin cryoprotectant used in semen dilution, is considered a source of biosecurity risk. In the current study, we aimed to compare the replacement of egg yolk in the extender by gum arabic (5%) along with supplementation with antioxidant cysteine or ascorbic acid on semen quality and freezability in Noemi rams in vitro. Semen from six rams were collected with an artificial vagina two times per week. Semen evaluation parameters such as color, volume, pH, general motility, percentage motility, concentration and cell viability ratio were assessed. Spermatozoa motility and concentration were estimated with the computer-assisted semen analysis system. The semen samples were frozen using a Tris extender containing either 15% egg yolk or 5% gum arabic. For antioxidant-supplemented extenders, cysteine or ascorbic acid was dissolved at concentrations of 0.10, 0.50 or 1.0 mM in egg yolk or gum arabic extender. The semen from each ejaculate of each ram were resuspended with a specific extender with glycerol (5%); the final volume after dilution was 1 mL semen to 4 mL extender. The samples were then cooled to 4 °C for 120 min, loaded into 0.5 mL straws and frozen in liquid nitrogen for 7 days. Supplementation of gum arabic or egg yolk extenders for ram semen with antioxidants such as cysteine or ascorbic acid has beneficial effects on semen quality after cold storage or cryopreservation. However, supplementation of a 5% gum arabic extender with cysteine at 0.5 or 1 mM concentration or ascorbic acid at 0.5 mM concentration improved the quality of spermatozoa postcryopreservation. It could be concluded that gum arabic is a good alternative for egg yolk in Noemi ram semen extenders. Antioxidants are necessary to support the addition of gum arabic to the extender to help the ram spermatozoa to survive freezing-thawing and oxidative stresses.

Curcumin Attenuates Damage to Rooster Spermatozoa Exposed to Selected Uropathogens.

Artificial insemination, as an essential pillar of the modern poultry industry, primarily depends on the quality of semen collected from stud roosters. Since the collection and storage of ejaculates is not a sterile process, antimicrobial agents have become essential supplements to semen extenders. While the use of traditional antibiotics has been challenged because of rising bacterial resistance, natural biomolecules represent an appealing alternative because of their antibacterial and antioxidant properties. As such, this study strived to compare the effects of 50 μmol/L curcumin (CUR) with 31.2 µg/mL kanamycin (KAN) as a conventional antibiotic on rooster sperm quality in the presence of Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa. Changes in sperm structural integrity and functional activity were monitored at 2 and 24 h of culture. Computer-assisted semen analysis revealed significant sperm motility preservation following treatment with KAN, particularly in the case of Salmonella enterica and Pseudomonas aeruginosa (p < 0.001) after 24 h. On the other hand, CUR was more effective in opposing ROS overproduction by all bacteria (p < 0.05), as determined by luminol-based luminometry, and maintained sperm mitochondrial activity (p < 0.001 in the case of Salmonella enterica; p < 0.05 with respect to Escherichia coli and Pseudomonas aeruginosa), as assessed by the fluorometric JC-1 assay. The TUNEL assay revealed that CUR readily preserved the DNA integrity of rooster sperm exposed to Salmonella enterica (p < 0.01) and Escherichia coli (p < 0.001). The bacteriological analysis showed higher efficiency of KAN in preventing the growth of all selected bacterial species (p < 0.0001) as opposed to CUR. In conclusion, CUR provided protection to rooster spermatozoa against alterations caused by uropathogens, most likely through its antioxidant activity. Hence, CUR supplementation to poultry semen extenders in combination with properly selected antibacterial substances may become an interesting strategy in the management of bacterial contamination during semen storage.

Melatonin improves post-thaw sperm quality after mild testicular heat stress in rams.

The objective was to determine effects of slow-release melatonin on post-thaw sperm quality in rams exposed to mild testicular heat stress (HS; scrotal neck insulation). Twelve yearling Dorset rams were randomly and equally allocated to receive either 36 mg melatonin in 1ml corn oil or 1ml corn oil injected subcutaneously (SQ); 15 day later, all rams had HS for 96 h (start of HS=start of Week 0). Semen was collected before HS and once weekly from Weeks 1 to 7, extended in Steridyl CSS One Step, held at 5°C for ~3h, loaded into 0.5ml straws, held 5cm above liquid nitrogen for 10min and then plunged. Computer assisted semen analysis (CASA) was conducted on frozen-thawed sperm. There were group and week effects for total and progressive motility (p< .001), plus group and week effects and group*week interactions (p< .001) for post-thaw total abnormalities, acrosome integrity, post-thaw sperm DNA fragmentation index (DFI) and high mitochondrial membrane potential (HMMP). Post-thaw sperm total and progressive motility, acrosome integrity and HMMP were higher (p< .05) in melatonin versus control groups from Weeks 1 to 7, and the melatonin group reached baseline level (pre-heat stress) at Week 7 (75.79 ± 0.96, 65.48 ± 1.51, 75.00 ± 0.89 and 67.00 ± 1.06, respectively; mean ± SEM). Conversely, post-thaw sperm total abnormalities and DFI were lower (p< .05) in melatonin versus control, and both reached baseline at Week 7 in the melatonin group (26.00 ± 0.57 and 5.66 ± 0.17, respectively). Coiled tails, distal midpiece reflexes, distal cytoplasmic droplets, ruffled acrosomes, bowed midpieces, pyriform heads and knobbed acrosomes were the most common abnormalities in both groups, with lower percentages in melatonin-treated rams. Results supported our hypothesis that HS reduces post-thaw sperm quality, and that melatonin lessens those reductions, manifested by significantly better total and progressive motility, acrosome integrity and HMMP, and fewer sperm total abnormalities and DFI.

Inhibition of HSP70 during prolonged liquid storage at 17 °C increases mitochondrial membrane potential and calcium levels

Serine phosphorylation of heat shock protein 70 (HSP70/HSPA1A) is known to be involved in sperm cryotolerance. This study aimed to determine whether HSP70 is implicated in the resilience of pig semen to prolonged storage at 17 °C. For this purpose, HSP70 was blocked with various concentrations of YM-1 (0, 0.05, 0.1 and 0.2 µM), an allosteric inhibitor of this chaperone, and samples were stored for 21 d. At various time points (0, 4, 10, 14, and 21 d), sperm motility and kinetic parameters by computer-assisted semen analysis, and viability (SYBR14/PI), mitochondrial membrane potential (JC1) and intracellular levels of calcium (FLUO3), total ROS (H2DCFDA) and superoxides (HE) through flow cytometry were assessed. No significant differences in sperm motility parameters were observed, except a reduction of VSL in the treatment containing 0.2 µM YM-1 at day 14 (P<0.05). Although no YM-1 concentration affected viability and intracellular concentrations of total ROS and superoxides, inhibiting HSP70 with YM-1, regardless of inhibitor concentration, increased intracellular calcium concentrations at day 10 compared to the control (P<0.05). Moreover, the treatment containing 0.05 µM YM-1 had higher JC1agg/JC1mon ratios than the control at day 10 (P<0.05). In conclusion, although inhibition of HSP70 with YM-1 did not affect pig sperm survival during long-term storage at 17 °C, it increased mitochondrial activity and intracellular calcium concentrations. This work was supported by the Ministry of Science and Innovation, Spain (PID2020-113320RB-I00) and the Regional Government of Catalonia, Spain (2017-SGR-1229).

THE DEPENDENCE OF SPERM PROGRESSIVE MOTILITY AND SEMINAL PLASMA BIOCHEMICAL COMPOSITION ON BACTERIAL LOAD IN DUROC BOAR SEMEN

Interactions between spermatozoa and bacterial species in ejaculates comes along with deleterious effects on the structure and function of male gametes. However, the effect of bacterial load on the seminal plasma remains unexplored. Therefore, this study was focused on the identification of bacterial species present in Duroc boar semen (n=18), quantification of the bacterial load (CFU) and alterations to the biochemical parameters of seminal plasma. The computer-assisted semen analysis (CASA) analysed the progressive motility (PRO; motility≥25µm/s) of spermatozoa, their concentration and kinematic parameters. The MALDI Biotyper was used for the bacterial characterization of fresh ejaculates. Seminal plasma was separated and biochemical analysis was carried out using the Randox RX Monza analyzer. Calcium (Ca), magnesium (Mg), total proteins (TP), aspartate transaminase (AST), alanine transaminase (ALT), cholesterol (CHOL), bilirubin (BR), albumins (ALB) and uric acid (UA) were subjected to the Pearson correlation with CFU and PRO. The bacterial identification showed the prevalence of Escherichia coli and Pseudomonas aeruginosa. The correlation analysis showed that the progressive motility of spermatozoa was strongly dependent (-0.799; P&lt;0.01) on the seminal bacterial load. Moreover, a negative significant correlation (-0.810; P&lt;0.001) was detected between CFU and Mg concentration. Also ALT (-0.647; P&lt;0.01) and ALB (-0.484; P&lt;0.05) were negatively affected by the bacterial load. Altogether, the presence of bacterial species may adversely affect the progressive motility as well as the biochemical composition of seminal plasma and thereby create unfavorable conditions for the sperm survival. Further analyses are necessary to reveal interactions between semen and bacteria.

Does Antioxidant Mitoquinone (MitoQ) Ameliorate Oxidative Stress in Frozen-Thawed Rooster Sperm?

In this study, we aimed to determine the benefit of mitoquinone (MitoQ) in rooster semen extenders on sperm quality, motility parameters, antioxidant capacities, and apoptotic changes in post-thawed rooster semen. A total of 85 ejaculates from 18 roosters were collected and then divided into five equal aliquots and cryopreserved in extenders with 1.0% soy lecithin nanoparticles that contained various concentrations of MitoQ (0 nM (M0), 50 nM (M50), 100 nM (M100), 150 nM (M150), and 200 nM (M200)). By using a computer-assisted semen analyzer, sperm motility parameters were assessed after freeze thawing. The M150 group had significantly higher percentages of total motility, progressive motility, viability, acrosome membrane integrity, and mitochondrial activity than the other groups (p &lt; 0.05). Compared to other groups, M100 and M150 groups produced a higher percentage of plasma membrane integrity and ATP contents (p &lt; 0.05). Additionally, the lowest levels of ROS and MDA in spermatozoa were observed in M150 group (p &lt; 0.05), whereas the highest levels of ROS and MDA were observed in sperm in the controls or the M200 group (p &lt; 0.05). Significantly higher values of SOD, GPx, and Cas-3 were found in the M150 group compared to other groups (p &lt; 0.05). Overall, these results demonstrate that MitoQ at 150 nM not only ameliorates post-thawed sperm quality and motility parameters by restoring ATP levels and preventing membrane damage, but also improves redox balance and antiapoptotic activities.

Effects of extender filtration and egg yolk concentration on canine semen cryopreservation.

The semen cooling and freezing extenders commonly contain the chicken egg yolk (EY) as the main sperm cryoprotectant. Besides its advantages, the EY has large lipoprotein granules that cause several physical and biological interferences. The previous studies have proposed several methods to resolve the problems with the EY-based semen extenders, including mechanical agitation, EY fractionation, replacing the EY with purified EY LDL, and ultrasonication. In the current research, we aimed to evaluate the syringe filtration (220 nm) of an EY-based canine semen freezing extender as a simple and cheap method to remove the EY granules. We also studied the possibility of re-aggregation of EY granules after cooling, freeze/thawing, and lyophilization/rehydration of the filtered extenders. Additionally, we compared the effects of the filtration on lipid profile, turbidity, EY particle size distribution, and osmolality of the EY-based extenders. Next, we examined the effects of filtered extenders containing different levels of EY (5%, 10%, 15%, 20%, and 25%) versus the control extender (20% EY, unfiltered) on post-thaw sperm quality traits. We collected the semen samples from seven clinically healthy mixed-breed adult dogs and pooled them for sperm freezing procedures. Samplings were repeated at least five times, independently. Our results indicated that the syringe filtration could remove the large EY particles and reduce the extender turbidity without affecting the lipid profile of the whole extender solution. The filtered extender supplemented with 25% (v/v) EY led to the best post-thaw canine spermatozoa quality markers. The frozen-thawed spermatozoa evaluations included motility parameters (computer-assisted semen analysis system), membrane and acrosome integrity (hypo-osmotic swelling test, chlortetracycline binding assay), DNA fragmentation (sperm chromatin dispersion assay), membrane lipid peroxidation (MDA levels), apoptosis (Annexin V/propidium iodide assay), and fertility-associated sperm mRNA transcript abundance (protamine 2 and 3). In conclusion, the syringe filtration of the EY-based semen extenders was a simple and cheap method that could effectively remove large EY lipoprotein granules and possibly prevent EY-origin bacterial contamination of the final extender solution. The EY at 25% (v/v) concentration in the filtered extenders resulted in the highest canine spermatozoa cryo-tolerance.

An assessment tool for computer-assisted semen analysis (CASA) algorithms

Computer-Assisted Semen Analysis (CASA) enables reliable analysis of semen images, and is designed to process large number of images with high consistency, accuracy, and repeatability. Design and testing of CASA algorithms can be accelerated greatly if reliable simulations of semen images under a variety of conditions and sample quality modes are available. Using life-like simulation of semen images can quantify the performance of existing and proposed CASA algorithms, since the parameters of the simulated image are known and controllable. We present simulation models for sperm cell image and swimming modes observed in real 2D (top-down) images of sperm cells in laboratory specimen. The models simulate human sperm using four (4) types of swimming, namely linear mean, circular, hyperactive, and immotile (or dead). The simulation models are used in studying algorithms for segmentation, localization, and tracking of sperm cells. Several segmentation and localization algorithms were tested under varying levels of noise, and then compared using precision, recall, and the optimal subpattern assignment (OSPA) metric. Images of real human semen sample were used to validate the segmentation and localization observations obtained from simulations. An example is given of sperm cell tracking on simulated semen images of cells using the different tracking algorithms (nearest neighbor (NN), global nearest neighbor (GNN), probabilistic data association filter (PDAF), and joint probabilistic data association filter (JPDAF)). Tracking performance was evaluated through multi-object tracking precision (MOTP) and multi-object tracking accuracy (MOTA). Simulation models enable objective assessments of semen image processing algorithms. We demonstrate the use of a new simulation tool to assess and compare segmentation, localization, and tracking methods. The simulation software allows testing along a large spectrum of parameter values that control the appearance and behavior of simulated semen images. Users can generate scenarios of different characteristics and assess the effectiveness of different CASA algorithms in these environments. The simulation was used to assess and compare algorithms for segmentation and tracking of sperm cells in semen images.

A Method of Digital Washing of Fresh Semen Samples

Background and Aim: Semen analysis is of great importance in the evaluation and treatment of male reproductive pathology [1]. Semen analysis may be performed on fresh/unwashed or washed semen samples. The semen washing process eliminates undesirable cells and particles from a fresh specimen. Computer-Assisted Semen Analysis (CASA) systems have shown to work extremely well, particularly on washed debris-free semen samples [2]. However, analyzing fresh samples could be problematic, especially in the presence of non-sperm cells.

Effects of dietary carnitine supplementation on semen output and quality of boars

Carnitine is an amino acid derivative that performs the functions of increasing energy production as well as acting as an antioxidant for sperm cells. This study was conducted to investigate the effects of the inclusion of carnitine in boar diets on semen output and quality. Sixty-four purebred and hybrid boars at a commercial boar stud were blocked by age and semen quality and randomly allotted to receive a daily 30 g top-dress of either soybean meal (CON) or soybean meal and 625 mg of L-Carnitine (CARN). Supplementation lasted for 12 weeks from May to July 2021 during which weekly semen collection was performed. Semen was evaluated in the stud for concentration and motility parameters using computer-assisted semen analysis (CASA). Samples were shipped to Purdue University for detailed morphology, viability, and CASA analysis performed in samples stored at 17 °C for 5 days. PROC Mixed (SAS v 9.4) was used to analyze data, with boar nested within treatment used in repeated measures analysis. Semen quality estimates from the week before supplementation were used as covariates in the statistical model. Tukey-Kramer adjustment was used for means separation. Carnitine supplementation had no effects on total sperm produced (P = 0.35). Percentage of motile sperm cells (P = 0.63), morphologically normal sperm (P = 0.42), viable sperm (P = 0.43), or sperm with normal acrosomes (P = 0.61) in the ejaculates were not different among treatments. Sperm kinematics in CARN ejaculates tended to have greater straight-line velocity and distance (P = 0.06 and P = 0.07, respectively). There were several interactions of treatment and day of storage for the kinematic parameters. However, these interactions do not show observable trends for CARN to improve or depress sperm function. Overall, the inclusion of 625 mg/d of carnitine in the diet of boars for 12 weeks had no effects on sperm output or quality with minor changes to sperm cell kinematics.

PSI-1 An Evaluation of Functionality of Cryopreserved Sperm Cells Treated with Heifer-Plus™

Abstract The commercial beef cattle industry produces calves for replacement or slaughter. Female replacements are important to advance herd genetics, therefore, a tool to influence sex ratios may enhance reproductive efficiency. A commercial semen sexing kit, Heifer-Plus™, used in conjunction with artificial insemination, may alter the sex ratio of offspring by increasing motility of the X chromosome-bearing sperm. The aim of this research was to assess the methodology of Heifer-Plus™ through evaluation of motion patterns and kinematic parameters in conventional semen straws. Four bulls representing Angus, Wagyu, and Holstein breeds were utilized. Six straws of cryopreserved semen from each bull were thawed in a water bath (37.5°C for 35 sec). Three straws from each bull were pooled and treated with Heifer-Plus™ and 3 straws were pooled as the no-treatment control. Post treatment, all samples were aliquoted in microcentrifuge tubes and Computer Assisted Semen Analysis (CASA) measurements of motility and kinematic factors were assessed. Semen samples were held at 37.5°C for incubation for three 15-min periods (15-mins, 30-mins, and 45-mins) measuring thermoresistance and longevity of spermatozoa. After incubation, 80 µL of each semen sample were observed on CASA. Data were analyzed using ANOVA method in GraphPad Prism and P-values ≤ 0.05 were considered significant. No effect of treatment on motility parameters or rapid velocity were measured, however there was a decrease in total motility (P=0.0045), progressive motility (P=0.0115) and rapid velocity (P =0.0099) across time intervals. Heifer-Plus™ treated cells had higher incidence of static velocity compared to control (P = .0038). Semen samples were clustered into subpopulation threshold-based on sperm average path velocity (VAP) and linearity (LIN) at each time point. Spermatozoa treated with Heifer-Plus™ consistently exhibited a high velocity and variable linearity therefore were categorized consistently into two categories at each time point.

Linseed oil in boar's diet improved in vivo fertility and antioxidant status.

The reactive oxygen species (ROS) which are produced during storage of boar semen are causing oxidative stress and leads to poor fertility. Also, tropical and sub-tropical weather condition adversely impacts the physicomorphological quality and fertility of boar sperm. The aim of this study was to examine the effects of feeding linseed oil to boar on its seminal attributes, sperm kinetics, biomarkers of antioxidant, fatty acid profile of seminal plasma (SP) and sperm and in vivo fertility. Six Hampshire crossbreed boars were fed with 90 ml linseed oil (LIN) whereas six Hampshire crossbreed boars were fed 90 ml canola oil (CON) for 16 weeks. Sperm quality was evaluated (60 ejaculates for each group; a total of 120 ejaculates) for motility, livability, abnormal morphology, acrosomal membrane integrity, hypo-osmotic swelling test (HOST) and sperm kinetic parameters by computer assisted semen analysis (CASA) at 0h and at 72 h of storage at 17°C. Biomarkers of antioxidant (glutathione peroxidase; GPx, catalase; CAT, total antioxidant capacity; TAC) and malondialdehyde (MDA) were measured in SP and serum. Gas chromatography-mass spectrometry (GC-MS) was used for the estimation of fatty acid composition of SP and sperm. Boars fed with linseed oil had higher semen volume (p < .01) and more total sperm numbers (p < .01). Feeding linseed oil to boar enhanced seminal attributes (p < .05) at 0h as well as at 72 h of storage. Linseed oil feeding (p < .01) improved biomarkers of antioxidants and significantly (p < .01) lowered the lipid peroxidation in serum and SP. Linseed oil feeding (p < .05) increased the proportion of alpha linolenic (ALA), arachidonic and docosahexaenoic (DHA) fatty acids in SP. The ratio of n-6 to n-3 fatty acids in sperm increased significantly (p < .01) in treatment group. Farrowing rate was significantly (p < .05) higher in treatment group. In conclusion, feeding linseed oil to boar improved the in vivo fertility, enhanced antioxidant capacity and increased the DHA content of SP and sperm.

Effect of Moringa oleifera seed extract on antimicrobial activity and in vitro fertilization ability of cryopreserved ram semen.

Cryopreservation has adverse effects on the post-thaw sperm quality due to oxidative stress and the presence of bacteria. To minimize such effects, plant extracts have been included in the composition of the semen diluents. The objective of the present study was to evaluate the antimicrobial and antioxidant effects of Moringa oleifera seed extract (MOSE) on cryopreserved ram semen, as well as its impact on in vitro fertilization. Semen from six hair rams was treated with five treatments before cryopreservation: Control (without any antibiotic), Standard (conventional antibiotic), 1.0, 10.0, and 50.0mg/ml of MOSE. Post-thawing sperm characteristics were evaluated by the computer-assisted semen analysis. Antimicrobial activity was assessed by counting colony-forming units (CFU) and the antioxidant capacity by the ferric reducing antioxidant power method. A heterologous in vitro fertilization technique was implemented to measure the fertilization rate. Progressive and rapid motility, membrane and acrosome integrity, and active mitochondria were higher (p < .05) in the 10.0mg/ml treatment compared with Standard after thawing. All M. oleifera treatments showed inhibition of CFU. The antioxidant capacity of M. oleifera seed extract was higher in the 10.0 and 50.0mg/ml treatments. Fertilization rate (cleavage percentage) was higher (p < .05) in the 10.0mg/ml (82.9 ± 10.0) and Control (82.5 ± 9.9) treatments compared with Standard (73.7 ± 9.1). The addition of 10.0mg/ml of MOSE to ram semen inhibits the development of microorganisms and improves sperm characteristics and the in vitro fertility of the semen.

Age-Dependent Variations in Functional Quality and Proteomic Characteristics of Canine (Canis lupus familiaris) Epididymal Spermatozoa.

Increased male age is associated with a significant reduction in semen quality. Little is known about the sperm proteome changes resulting from the aging process. This study aimed to investigate the relationship between the functional quality and proteome of epididymal spermatozoa of dogs that were differing in age. The study was conducted on 30 male dogs that were divided into three age groups. G1—12 to 41 months old, G2—42 to 77 months old, and G3—78 to 132 months old. The sperm samples were assessed using a computer-assisted semen analysis (CASA). The epididymal sperm proteins were analyzed using gel electrophoresis (SDS-PAGE), nano-liquid chromatography coupled to quadrupole time of flight mass spectrometry (NanoUPLC-Q-TOF/MS) and bioinformatic tools. The sperm quality parameters were significantly lower in older dogs. NanoUPLC-Q-TOF/MS identification resulted in 865 proteins that were found in the G1, 472 in G2, and 435 in G3. There were seven proteins that were present in all three age groups, and four of them (ACTB, CE10, NPC2, CRISP2) showed significant changes among the studied groups. Age-dependent variations were detected in the sperm proteome composition and were related to important metabolite pathways, which might suggest that several proteins are implicated in sperm maturation and could be potential aging biomarkers.

The influence of Percoll® density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa

BackgroundThe wisent (Bison bonasus) is a species that has undergone a population bottleneck. Homozygosity is prevalent within the population and may have a negative impact on semen quality in wisent bulls. Semen samples containing a large amount of functionally and morphologically impaired or dead spermatozoa have lower tolerance for cryopreservation process. Such samples are prone to involve damage acrosomes, to produce and release reactive oxygen which negatively affects proper function of spermatozoas. It is a good practice to select intact and viable gametes before subjecting the sample to cryopreservation to improve the efficiency of this process. The aim of this study was to assess the ability of Percoll® density gradient centrifugation in order to improve the quality of wisent spermatozoa after cryopreservation. Spermatozoa samples were analysed with computer-assisted semen analysis system and flow cytometry.ResultsPercoll® density gradient centrifugation resulted in increased percentage of motile spermatozoa, higher proportion of spermatozoa with normal morphology and proper functionality but also in a significant reduction of the total number of gametes. Nevertheless, the concentration of frozen spermatoza was still sufficient for obtaining a few complete insemination doses suggested for cattle from each epididymis.ConclusionsWhile creating a high-quality genetic reserve, for in vitro fertilisation purposes, eliminating detritus and improving the overall quality of samples is more important than total number of spermatozoa. For these reasons, the achievement of higher post thaw quality of spermatozoa justifies the purification of samples by centrifugation in a Percoll® density gradient prior to the cryopreservation process.

The sirtuin 1 activator YK 3-237 stimulates capacitation-related events in human spermatozoa

Research questionDoes sirtuin-1 (SIRT1) have a role in the human spermatozoa capacitation process? DesignHuman spermatozoa were incubated for 6 h in a capacitating medium in presence or absence of the specific SIRT1 activator, YK 3-237. Several sperm parameters were determined by flow cytometry: viability, acrosome reaction and mitochondria membrane status. Sperm motility was determined objectively by computer-assisted semen analysis. Sperm capacitation status was evaluated by the extent of protein tyrosine phosphorylation and by the percentage of spermatozoa with the acrosome reacted by a calcium ionophore challenge. ResultsSIRT1 was detected in the connecting piece of human spermatozoa where a lysine acetylation pattern was mainly found along the sperm tail. SIRT1 activation accelerates the occurrence of a phenotype associated with human sperm capacitation, with no differences seen in the lysine acetylation pattern. After 1 h of co-incubation of YK 3-237 with human spermatozoa, tyrosine phosphorylation levels were comparable to control levels after 6 h of incubation in capacitating conditions. In addition, the activator improved sperm responsiveness to a Ca2+ ionophore (A23187) challenge determined by an increase in acrosome-reacted spermatozoa (P = 0.025). Importantly, sperm viability and mitochondrial activity-related parameters assessed by flow cytometry were not affected by YK 3-237. ConclusionYK 3-237 induces capacitation-related events in human spermatozoa such an increase of tyrosine phosphorylation levels and acrosome-reacted spermatozoa after the ionophore challenge. Together, these results show that YK 3-237 affects human spermatozoa capacitation-related events by a mechanism independent of protein lysine acetylation but dependent on bicarbonate and calcium.

Computer aided semen analysis (CASA) to determine the quality and fertility of frozen thawed sumba ongole sperm supplemented with amino acids

&lt;p&gt;&lt;strong&gt;Objective&lt;/strong&gt;: The objective of this research was to investigate the quality and reproductive potential of frozen-thawed Sumba Ongole (SO) bull spermatozoa supplemented with specified amino acids using computer-assisted semen analysis (CASA).&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods&lt;/strong&gt;: A total of 18 ejaculates were collected once a week from three Sumba Ongole bulls and diluted in tris-citric-fructose-egg yolk (TCFY) extender and various concentrations of selected amino acids. The post-thaw spermatozoa were examined for motility, head behavior, and swimming pattern using CASA technique. Data were analysis using SPSS 24 ebvaluated by ANOVA.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results&lt;/strong&gt;: Total motility (72.56%±3.20) and progressive motility (71.11%±3.31) the addition of Glutamine 5% significantly different (p&amp;lt;0.05) than other treatments. The addition of 7mM cysteine resulted in significantly (p&amp;lt;0.05) in velocity average path (VAP), velocity curve line (VCL), velocity straight line(VSL) parameters also have higher in distance average path (DAP), distance curve line (DCL), dan distance straight line (DSL) parameters significantly (p&amp;lt;0,05). &lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusion&lt;/strong&gt;: In conclusion, the introduction of CASA systems enabled quantification of individual spermatozoa motility patterns and CASA colud be used to estimate the fertilizing capacity of sperm based on the characteristics of sperm velocity.&lt;/p&gt;

Influence of Fat-Soluble Vitamin Intramuscular Supplementation on Kinematic and Morphometric Sperm Parameters of Boar Ejaculates.

Ejaculate quality can be regarded as multifactorial, with nutrition being a factor that could directly influence sperm parameters. The present study aimed to evaluate seminal quality associated with seasonal fat-soluble vitamin supplementation of boars. Seven sexually mature boars were randomly allotted to one of the three groups, and fed one of the three supplementary diets for 32 weeks: (1) control treatment (COD), without supplementation of fat-soluble vitamins, (2) treatment containing 100% fat-soluble vitamin supplementation administered intramuscularly, which was based on fat soluble vitamin supplementation (A, D3, E) (FVD1), and (3) treatment containing 50% of fat-soluble vitamin supplementation (FVD). Semen was collected at 7-day intervals. Semen samples were analyzed to assess several sperm parameters using the Computer-Assisted Semen Analysis (CASA) ISAS®v1 system. Results showed that groups receiving FVD1 and FVD supplementation had an increased semen volume. The percentages of motile and progressively motile sperm were increased by FVD1 treatment. A statistically significant interaction between treatment and season was found in the percentage of motility and progressive motility (p < 0.05). Sperm concentrations showed significant differences (p < 0.05) between treatments. Velocity variables (VSL, VCL, and VAP) were higher (p < 0.05) in boars that received fat-soluble vitamin supplementation in comparison to controls receiving no supplementation. The FVD1 treatment presented spermatozoa with greater head size and more elongated heads (p < 0.05). Overall, the utilization of dietary fat-soluble vitamin supplementation significantly improved the semen quality of boar ejaculates. This highlights the importance of fat-soluble vitamin supplementation in sexually active boars.

P-038 Comparison of three methods of semen analysis: A novel at-home sperm test kit, a computer-assisted assessment and an embryologist

Study questionHow does computer-assisted semen analysis (CASA) (Lenshooke, LOGIXX) and at-home sperm testing (ExSeed) compare with manual methodology assessment of male fertility based on WHO criteria (5thEdition); a three-method comparison study.Summary answerAll methods showed good agreement for concentration and limited agreement for motility highlighting the need for further development of alternatives to manual assessment.What is known alreadySeveral studies have shown good correlation between CASA systems and laboratory-based manual semen analysis, but only a few have carried out a three-way comparison study. One of which showed positive correlation with motility and concentration between a smartphone-based semen analysis and laboratory-based CASA system and positive correlation between concentration and motility between the smartphone-based CASA system and microscopic-based results. The at-home sperm test kit may play a role in motivating infertile males to visit clinics for early diagnosis and also reducing the need for multiple visits to the fertility clinic for repeat semen analyses.Study design, size, durationFifty patients (between 29 and 56 years) attending a fertility clinic were included in the study between September 2021 to December 2021. Semen samples were split into 3 aliquots and evaluated using manual semen assessment (MSA) according to the WHO 5th Edition (2010) guidelines, the ExSeed Home Sperm Test (HST) and the LensHooke® X1 PRO Semen Quality Analyzer (CASA).Participants/materials, setting, methodsThe semen samples were collected from fifty participants at CARE Fertility Tunbridge Wells. The samples were placed in an incubator at 37°C for 30 minutes to liquefy. After liquefaction, sperm concentration, total motility, total motile sperm count (TMSC) and normal morphology were evaluated. Spearman’s Rank correlations (>0.7) and Chi-squared tests were used and the p-value < 0.05 was considered as statistically significant.Main results and the role of chanceAll variables were highly significantly (p < 0.0002) positively correlated between all 3 methods.The greatest correlations were obtained for sperm concentration (CASA/HST: r = 0.826; MSA/HST: r = 0.870; MSA/CASA: r = 0.871) and TMSC (MSA/CASA: p = 0.792; CASA/HST: r = 0.800; MSA/HST: r = 0.854). Correlations for motility were markedly lower (MSA/HST: r = 0.611; CASA/HST: r = 0.717; MSA/LCASA: r = 0.750). The lowest correlation was found for morphology (MSA/CASA: r = 0.500). The HST device does not determine morphology.As compared to MSA, using the HST device agreement for normal or low sperm concentration (≥15 × 106/mL or < 15 × 106/mL respectively) was identified in 84.3% of the cases, whereas 94.0% with CASA. The agreement between CASA and HST was 82.0%. For total motility ≥ or < 40%, the agreements were 68.0% (CASA/HST), 56.9% (MSA/HST) and 82.0% (MSA/CASA), respectively.For the identification of patients with normal morphology >4%, the agreement between MSA and CASA was 30%.Limitations, reasons for cautionThe small sample size was 50 cases. The home testing device does not assess morphology. Furthermore, it is difficult to ascertain whether a consumer would carry out the analysis with this device with the same accuracy as an embryologist.Wider implications of the findingsThe need for repeated semen analyses, the effect of the COVID-19 pandemic and the discomfort some patients feel in a clinical setting, necessitate the need for evaluation of novel semen analysis approaches. These emerging technologies have potential to be more patient friendly, convenient and efficient than standard semen assessment methods.Trial registration numbernot applicable

P-188 The effect of follicular fluid extracellular vesicles on motility and hyperactivation of human spermatozoa and differential miRNA levels depend on the woman's age

Abstract Study question How does the woman’s reproductive age affect the ability of follicular fluid (FF) extracellular vesicles (EV) to change the motility and hyperactivation of spermatozoa and follicular fluid microRNA profiles? Summary answer The effect of FF EVs on sperm motility and hyperactivation decreases with a woman's age. MiR-134-5p, miR-21-5p expression levels increase in older age group. What is known already Aging reduces human fertility. FF is an important factor in attracting and activating spermatozoa in the oviduct during oocyte fertilization, enhancing the process of sperm capacitation and acrosomal reaction in various mammalian species, including humans. EVs from seminal plasma and oviduct fluid carry proteins and miRNAs playing a vital role in a multi-step process including sperm motility, capacitation, acrosomal reaction, further fertilization. Results of our previous experiments showed that FF EVs from young women significantly improve the indices of sperm motility and hyperactivation. Several studies have isolated some age associated differentially expressed miRNAs (hsa-miR-424, hsa-miR-21-5p, hsa-miR-134, hsa-miR-190b, hsa-miR-99b -3p). Study design, size, duration FF EVs were obtained by sequential centrifugation at different rotational speeds, frozen at -80 °C. The sperm fraction was isolated, washed by differential centrifugation in a density gradient, suspended in the saline to a concentration of 106/ml and incubated with EVs (1:2) at 37 °C in CO2-incubator for 1 h. Samples were centrifuged and fixed in 2.5% glutaraldehyde in 0.1 M buffer for TEM. Sperm motility were assessed using CASA. MicroRNAs isolation (miRNeasy Serum/Plasma Kit (Qiagen)). Participants/materials, setting, methods All study participants signed a voluntary informed consent for the use of biological samples for research purposes. Sperm samples were isolated from seminal fluid (n = 18) aged 28-36 years without severe pathozoospermia. FF EVs were obtained from young (n = 4) aged 27-31 (EVs+) and older patients (n = 4) aged 41-46 with several IVF attempts (EVs-). The methods used in this work include sperm and EVs isolation, incubation, transmission electron microscopy, RT-PCR, statistical data analysis, computer-assisted semen analysis (CASA). Main results and the role of chance Our results showed that sperm incubation with EVs+ led to a significant increase in the number of progressively motile spermatozoa (paired Student's t test, p &amp;lt; 0.001), indicators of general mobility (p = 0.05) and hyperactivation of spermatozoa after 1 hour. Sperm incubation with EVs- slightly changed these parameters compared to the control (p = 0.171). Results from TEM showed that EVs- bind to spermatozoa worse than EVs+ after incubation. EVs- bind predominantly to the lateral membrane of the spermatozoon, rather than to the acrosomal region, compare to EVs+, which may reflect changes in the functional composition of FF EVs in older patients group and target areas of interaction with spermatozoa. This hypothesis is also supported by slight improvements in progressive sperm motility after incubation. Age-related changes are reflected in energy, metabolic and other important biological processes, which leads to a change in the composition and functions of FF (decrease in progesterone, glucose, increase in lactate and pyruvate, increase in reactive oxygen species) and affect the functional role of FF EVs. MiR-134-5p (p = 0.008), miR-21-5p (p = 0.008) expression levels were statistically significantly higher in EVs- group compared with EV + group with a successful first attempt. The expression of other microRNAs analyzed did not differ between groups. Limitations, reasons for caution Small amount of data. This study does not characterize isolated FF EVs in any way (specific markers, EVs concentration, size). To discuss the possible mechanisms underlying the effects of FF EVs of different age groups on sperm characteristics, it is necessary to perform metabolomics analyses of FF EVs. Wider implications of the findings Further studies of the influence of FF EVs on sperm motility may help to improve of ART outcomes, associated with male and female infertility by improving sperm morphofunctional characteristics and increasing their fertilizing ability. The solutions to this problem could be the use of FF EVs from young fertile donors. Trial registration number not applicable