Percentage Of Total Motility Research Articles

Effect of Skim Nanoparticles on the Motility and Kinematics of Simmental Cattle Frozen Semen

Nanotechnology has positively impacted the development of extenders used in the cryopreservation of spermatozoa in livestock. The inclusion of nano skim in the extender is expected to preserve semen quality after thawing. This study aimed to determine the optimal concentration of nano skim. Various concentrations of nano skim (0%, 1.66%, 3.33%, 5%, 6.66%, 8.33%, and 10%) with the addition of 10% nano egg yolk in each extender were compared. Fresh semen samples were collected from three three-year-old Simmental bulls using an artificial vagina. The study parameters included motility (total motility, progressive motility) and kinematics (velocity curve linear (VCL), velocity straight linear (VSL), velocity average pathway (VAP), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and wobble (WOB). A completely randomized design was employed, and data were analyzed using analysis of variance (ANOVA). Fresh semen dilution results indicated that only the control group (0%) and the nano skim group with concentrations of 6.66% and 8.33% were eligible for further processing into frozen semen. The results showed no significant difference between the three treatment groups on sperm motility and kinematics of Simmental cattle after thawing (p0.05). The control group exhibited the highest percentage of total motility, VCL, VAP, and ALH values, while the 6.66% nano skim group outperformed the 8.33% nano skim group. The highest percentage of progressive motility and VSL value was observed in the 6.66% nano-skim group compared to the 8.33% nano-skim and control groups. The highest values of BCF, WOB, LIN, and STR was observed in the nano-skim group at 8.33% compared to the nano-skim group at 6.66% and control. The study concluded that 6.66% nano skim was the optimal concentration to maintain the quality of frozen semen of Simmental cattle.

Effects of hydroxytyrosol on post-thaw quality of rooster sperm.

Semen cryopreservation is one of the most important reproduction techniques in the livestock and poultry industry. Cryopreservation induces cold stress, generating reactive oxygen species (ROS) and oxidative stress causing structural and biochemical damages in sperm. In this study, we evaluated the effects of the hydroxytyrosol (HT), as an antioxidant, at the concentrations of 0, 25, 50, and 100 μg/mL on post-thaw semen quality metrics in rooster. Semen samples were collected twice a week from 10 roosters (29 weeks), processed and frozen according to experimental groups. Different quality parameters, including total motility, progressive motility, viability, morphology, membrane integrity, and malondialdehyde were measured after thawing. Results showed that 25 and 50 μg/mL of HT produced the highest percentage of total motility (51.01 ± 2.19 and 50.15 ± 2.19, respectively) and progressive motility (35.74 ± 1.34 and 35.15 ± 1.34, respectively), membrane integrity (48.00 ± 2.18 and 46.75 ± 2.18, respectively) as well as viability (53.00 ± 2.17 and 52.50 ± 2.17, respectively) compared with the other groups (p < .05). The group with 25 μg/mL of HT showed the lowest significant (p < .05) MDA concentration (1.81 ± 0.25). Our results showed that the effect of HT was not dose-dependent and optimum concentration of HT could improve functional parameters of rooster sperm after freezing-thawing. These findings suggest that HT may have protective effects on the rooster sperm during the freezing-thawing process.

(300) Systematic Review and Meta-analysis of Seasonal Variation on Human Semen Quality in the United States

Abstract Introduction Different environmental exposures such as daylight hours and temperature influence spermatogenesis, which vary across seasons. However, studies on the variation of semen analysis across different seasons reported inconsistent results. Objective To investigate the seasonality of human semen parameters in the United States through a systematic review and meta-analysis. Methods We systematically searched PubMed, Scopus, and Web of Science for studies on seasonal variation of human semen parameters in the United States from conception to December 11, 2022, without language restriction, and also scrutinized the reference list of included studies for any additional related study. We determined the pooled standardized mean difference (SMD) and its 95% confidence interval (95%CI) between seasons using the random effect model in a case of substantial heterogeneity (I2 index≥50%) and the fixed-effect model in a case of low heterogeneity (I2 index&amp;lt;50%) in STATA version 17. The methodological quality was checked using Newcastle–Ottawa Quality Assessment Scale (NOS). Results Our search yielded 548 studies. After screening, 7 studies on 131,306 semen analyses were included for meta-analysis. All included studies had high methodological quality (NOS ≥ 7). Figure 1 shows the pooled SMD of semen parameters between seasons. Semen analysis in winter had a higher volume than those in spring (p-value=0.010; I2=4.63%). Winter had better semen quality than summer in terms of concentration (p-value=0.014; I2=0%) and total sperm count (p-value=0.044; I2=47.80%). Semen quality was also better in winter than in fall, in terms of volume (p-value=0.001; I2=0%), concentration (p-value=0.016; I2=23.68%), and total sperm count (p-value=0.022; I2=13.95%). Spring had better semen quality than summer in terms of concentration (p-value&amp;lt;0.001; I2=33.74%) and percent of normal morphology (p-value&amp;lt;0.001; I2=0%), and also had higher concentration than fall (p-value&amp;lt;0.001; I2=42.82%). Semen analysis in summer had a lower percentage of total motility than those in fall (p-value=0.045; I2=0%). Conclusions Semen parameters follow a seasonal pattern, with winter and spring showing better sperm quality regarding concentration and total sperm count than other seasons. Disclosure No.

Effects of ergothioneine supplementation on the quality of liquid-preserved and frozen-thawed boar semen.

This study examined the effects of ergothioneine (EGT) supplementation as an antioxidant on the quality of boar spermatozoa when using liquid and frozen preservation methods. In the first experiment, boar semen was preserved in an extender supplemented with 0, 50, 100 and 200 µM EGT, at15 °C, part of the samples for one and another part for three weeks. In comparison with the control (without EGT), EGT supplementation at 100 µM significantly increased the percentage of total motility of spermatozoa that were preserved as a liquid both for one and three weeks (P < 0.05). EGT supplementation did not affect the quality of preserved spermatozoa, irrespective of the EGT concentration. In the second experiment, semen was frozen and thawed in the freezing extender supplemented with 0,50, 100 and 200 µM EGT. In comparison with the control, the 100 µM EGT supplementation significantly increased the percentages of total and progressive motility of frozen-thawed spermatozoa (P < 0.05). EGT (100 µM) supplementation did not affect the viability, the plasma membrane integrity, or the acrosomal integrity of frozen-thawed spermatozoa. These findings indicate that supplementing extenders with 100 µM EGT may improve the motility of boar sperm in both liquid and freezing preservation methods.

Smoking Induces a Decline in Semen Quality and the Activation of Stress Response Pathways in Sperm.

Male infertility is a prevalent concern affecting couples worldwide. While genetic factors, hormonal imbalances, and reproductive system defects play significant roles, emerging evidence suggests that lifestyle choices also profoundly impact male fertility. This study aimed to explore the effects of several lifestyle factors, including tobacco and alcohol consumption, physical activity, and dietary habits, on semen quality parameters and molecular biomarkers. Thirty healthy male volunteers were recruited in the Urology service at Hospital Infante D. Pedro, Aveiro, Portugal. Participants completed lifestyle questionnaires and provided semen samples, which were analyzed according to the World Health Organization criteria by experienced technicians. We also analyzed the expression levels of antioxidant enzymes and heat-shock response-related proteins to explore the activation of signaling pathways involved in stress response within sperm cells. Our results revealed that tobacco consumption reduced semen volume and total sperm count. Although the changes in the percentage of total motility and normal morphology in the smokers' group did not reach statistical significance, a slight decrease was observed. Moreover, we identified for the first time a significant association between tobacco consumption and increased levels of heat shock protein 27 (HSP27) and phosphorylated HSP27 (p-HSP27) in sperm cells, indicating the potential detrimental effects of tobacco on the reproductive system. This study highlights that lifestyle factors reduce semen quality, possibly by inducing stress in sperm, raising awareness about the effects of these risk factors among populations at risk of male infertility.

Impact of DNA methylation of the human mesoderm-specific transcript (MEST) on male infertility

Male infertility accounts for nearly 40%–50% of all infertile cases. One of the most prevalent disorders detected in infertile men is errors in the MEST differentially methylated region (DMR), which has been correlated with poor sperm indexes. The aim of our study was to characterize the methylation pattern of the MEST gene, along with assessing seminal factors and chromatin condensation in sperm samples from both infertile patients and fertile cases, all of whom were candidates for intracytoplasmic sperm injection. We collected forty-five semen specimens from men undergoing routine spermiogram analysis at the Infertility Treatment Center. The specimens consisted of 15 samples of normospermia as the control group, 15 individuals of asthenospermia, and 15 individuals of oligoasthenoteratospermia as the cases group. Standard semen analysis and the chromatin quality and sperm maturity tests using aniline blue staining were performed. The DNA from spermatozoa was extracted and treated with a sodium bisulfite–based procedure. The methylation measure was done quantitatively at the DMRs of the MEST gene by quantitative methylation-specific polymerase chain reaction (qMSP). The mean percentages of total motility, progression, and morphology in normospermia were significantly higher than oligoasthenoteratospermia and asthenospermia, and they were substantially higher in asthenospermia compared to oligoasthenoteratospermia (P ≤ 0.05). The mean percentages of histone transition abnormality and MEST methylation in oligoasthenoteratospermia were significantly higher than asthenospermia and normospermia (P ≤ 0.05). A negative correlation existed between the histone transition abnormality and MEST methylation with sperm parameters. In conclusion, chromatin integrity, sperm maturity, and MEST methylation may be considered important predictors for addressing male factor infertility. Therefore, we suggest that male infertility may be linked to methylation of the imprinted genes.

Dietary Moringa oleifera leaves to male rainbow trout (Oncorhynchus mykiss) broodstock: Effects on sperm quality and reproductive performance

Moringa oleifera leaves (MOL), a superfood rich in protein and vitamins, is native to the Indian subcontinent. Moringa were added to the broodstock trout diet to examine their influence on the reproductive performance of male broodstock rainbow trout (Oncorhynchus mykiss). Females were fed with a commercial broodstock trout diet without any inclusion of moringa leaves. In 12 circular fiberglass tanks, 36 male rainbow trout aged 3+ (1786.36 ± 35.36 g; 56.08 ± 0.94 cm) were divided into four groups (n = 9) (control, MOL-1, MOL-2, and MOL-3) with three parallels each. Fourteen weeks of feeding trials were performed where powdered moringa leaf was used in the diets at various levels: 0% (control), 4% (MOL-1), 8% (MOL-2), and 16% (MOL-3). Sperm quality parameters and fertilization success were investigated. Sperm motility parameters were examined at 10.5 °C using a computer-aided sperm analysis (CASA) system, and all samples were analyzed on the same day. Sperm volume and seminal plasma osmolality did not differ significantly across groups (p > 0.05). Spermatozoa concentration (6.71 ± 0.25, 7.54 ± 0.31, 8.44 ± 0.55, and 7.66 ± 0.35 × 109 cells ml−1 in control, MOL-1, MOL-2, and MOL-3 groups, respectively), spermatocrit ratio (18 ± 2.55%, 25 ± 3.16%, 38 ± 6.44%, and 28 ± 5.15%), and motility duration differed significantly (p < 0.05) among groups. Total motility percentage did not change, but progressive motility (74.72 ± 5.94%, 88.52 ± 4.35%, 89.36 ± 1.76%, and 90.47 ± 2.67%) was considerably higher in all three moringa groups than in control one (p < 0.05). Average Path Velocity (VAP), Straight Line Velocity (VSL), and Curvilinear Velocity (VCL) significantly differ between control and treatment groups (p < 0.05). The fertilization rate in the MOL-2 group was similar to the control and MOL-3 groups, while significant differences were observed from the eyeing stage, with the highest result observed in the MOL-2 group (p < 0.05). Hatching rates were also highest in the MOL-2 group (81.65 ± 0.49%), followed by MOL-3 (79.08 ± 1.27%) and MOL-1 (74.40 ± 2.32%), with the control group having the lowest hatching rate (74.24 ± 0.98%) (p < 0.05). Progressive motility, VAP, VSL, and VCL have significantly enhanced in the moringa-treated groups than the control one, hence boosting the fertilization success. The present study revealed that moringa leaves in the diet improve the sperm quality parameters and enhance the reproductive performance in broodstock male rainbow trout (Oncorhynchus mykiss). Therefore, 8% moringa leaf can be recommended as a supplement diet for male rainbow trout broodstock.

Effects of curcumin supplementation on quality of porcine spermatozoa irradiated with ultraviolet-C at 228 nm during liquid preservation.

It is important to prevent microbial contamination during liquid preservation of semen in pigs. We examined the effects of curcumin supplementation on the quality of porcine spermatozoa irradiated with ultraviolet-C (UV-C) at 228 nm. UV-C is used to disinfect gases and solid surfaces. In the first experiment, porcine semen was preserved with 0, 10, 25, 50 or 100 μM curcumin under UV-C irradiation at 228 nm for 7 days at 15°C. The irradiation did not affect the motility and viability of preserved spermatozoa but decreased the percentage of plasma membrane integrity of spermatozoa. Curcumin supplementation at 25 μM significantly improved the plasma membrane and acrosome integrity of irradiated spermatozoa compared with spermatozoa preserved without curcumin (p < .05). In the second experiment, semen was preserved with or without 25 μM curcumin with UV-C irradiation at 228 or 260 nm for 3 days at 15°C. Curcumin supplementation increased the percentages of total motility, sperm viability and plasma membrane integrity of preserved spermatozoa at both irradiation wavelengths (p < .05). All quality parameters of 260 nm irradiated spermatozoa decreased compared to those of the other groups, irrespective of curcumin supplementation. The collective findings indicate that porcine spermatozoa can retain their viability even after continuous long-duration irradiation with 228 nm UV-C. Curcumin supplementation improves the quality of UV-C irradiated spermatozoa during semen preservation.

Sperm cryopreservation in Windsnyer boars; principles, technique, and updated outcomes.

The domestic pig breeds are in danger of extinction whereas the erosion of their gene pool is a serious concern because they significantly contribute to the rich biodiversity. Overall aim of this study was to determine the protocol for preserving the semen of the Windsnyer boars for conservation. A total of 18 ejaculates (6 replications/boar) were collected from three Windsnyer boars of proven fertility with the use of hand-gloved approach method, twice per week. Boars semen were pooled and extended with Beltsville Thawing Solution [(BTS) IMV Technologies, France], held at 18°C for 3 hours and centrifuged. The sperm pellet was re-suspended with Fraction A (20% egg yolk + BTS) and cooled at 5°C for 1 hour. Following cooling, semen was divided and diluted into different cryoprotectants (ethylene glycol, glycerol, propanediol, ethylene glycol + glycerol + propanediol) at equal contribution to make the total concentrations [4, 8, 12 and 16% and the 0% (control; without cryoprotectant)] and loaded into 0.25 mL straws. Two cryopreservation methods (liquid nitrogen vapour and controlled rated) were used to cryopreserve the semen straws. Semen straws were thawed at different temperatures (5, 18, 37 and 40°C) and evaluated for sperm motility, viability, and morphology traits. Post-thawed sperm total motility (36.0±5.3) and live normal sperm (49.5±8.3) percentages were recorded to be higher in the treatment supplemented with 16% glycerol (P<0.05). The highest sperm total motility percentage was recorded at 40°C (26.8±3.2) thawing temperature for liquid nitrogen vapour treatment (P<0.05). In conclusion, 16% glycerol was found to be the suitable cryoprotectant concentration for semen cryopreserved with liquid nitrogen vapour method and thawed at 40°C.

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.

Spirulina Supplementation to the Semen Extender Influences the Quality and Antioxidant Parameters of Chilled or Cryopreserved Arabian Stallion Spermatozoa

The present study attempted to investigate the effect of various concentrations of spirulina platensis additions to the semen extender on Arabian stallion spermatozoa quality. Semen samples were collected with artificial vagina from five fertile stallions and diluted with an extender containing spirulina (2, 4, 6, and 8 mg/100 mL) or without spirulina (control). Aliquots of diluted semen were cooled (5°C, 90 minutes) and frozen (-196°C, 7 days), then physical traits of thawed spermatozoa were examined. Furthermore, antioxidant activities of superoxide dismutase (SOD), glutathione reductase (GSR), total antioxidant capacity (TAC), and malondialdehyde (MDA) were measured in post-cooling and freezing spermatozoa. The results indicated that spirulina supplemented with the extender had no effect (P >.05) on sperm quality parameters and antioxidant activities after cooling. However, adding 6 mg spirulina/100 mL to the freezing extender improved (P < .05) the speed parameters and total motility percentage of frozen/thawed spermatozoa. Besides, supplementation of freezing extender with the previous level increased (P < .05) TAC, SOD and GSR concentrations/activities (0.86 ± 0.32 mM/L, 323.70 ± 12.81 U/mL, and 38.65 ± 1.90 U/mL, respectively) compared with the control (0.70 ± 0.25 mM/L, 165.80 ± 8.12 U/mL, and 25.70 ± 1.83 U/mL, respectively). While, lipid peroxidation of the frozen-thawed semen was reduced (P < .05, 17.97 ± 1.30 µmol/ L) compared with the control (29.39 ± 1.89 µmol/ L). Accordingly, the present results revealed that additions of 6 mg spirulina/100 mL to the freezing extender improved semen quality and reduced cryodamage of the Arabian stallion spermatozoa.

A new fluorescent method to determine honey bee sperm motility parameters with computer-aided sperm analysis

Honey bees are a keystone species, playing an important role in the food cycle. Improving honey bee reproduction will aid in the replacement of lost colonies. Fertility potential and reproductive health are dependent on semen and sperm quality. Current data on drone semen parameters are limited to semen volume, sperm concentration, sperm viability, and the assignment of sperm motility grade scores. The assessment of drone sperm motility is of importance to determine fertility potential and colony health. This study aimed to establish a quantitative and a qualitative method to measure drone sperm quality, and the fertility potential of Apis mellifera capensis subspecies of South Africa. Firstly, an improved five-point semi-quantitative manual motility index score was used to classify drone sperm motility. Secondly, it was possible to accurately analyse drone sperm motility qualitatively using a fluorescent technique in conjunction with a computer-aided sperm analysis (CASA) system. Manual motility index scores corresponded with total motility percentages as determined by using a CASA system. Furthermore, low values for motility kinematic parameters, particularly velocity parameters, were obtained in samples with both low motility index scores and low total motility percentages. Additionally, total sperm motility percentage and velocity parameters positively correlated with sperm total progressivity. This study provides in-depth data on honey bee drone sperm motility and motility kinematic parameters, which can serve as a reference for future studies on honey bee sperm and possibly related species.

The replacement of fresh egg yolk by lyophilized egg yolk in Tris-base extender in cryopreserved Boer and Saanen semen.

Egg yolk is a common cryoprotectant that can be used as a semen extender to protect the spermatozoa from damage during cryopreservation. Therefore, the aim of this study was to investigate the efficacy of fresh and lyophilized egg yolk, as a Tris-base extender, on the quality of cryopreserved goat semen. Semen from 10 rams of two different breeds (Boer and Saanen) was collected using an artificial vagina. Each ejaculate sample was divided into four equal aliquots, which contained 20% of the fresh egg yolk (a control group), and then 10%, 15%, and 20% of the lyophilized egg yolk as a Tris-base extender. Sperm motility and kinetic parameters were determined using a computer-assisted semen analyser. The results showed that the addition of 20% of the fresh egg yolk in Tris-base extender exhibited significantly higher progressive motility, progressive fast motility, distance curve line, and beat-cross frequency parameters in the post-thaw Boer and Saanen goat sperm when compared with the addition of 10%, 15%, and 20% of the lyophilized egg yolk. The percentage of total motility and immotile parameters in the post-thaw Boer and Saanen goat sperm were not significantly different between the control and 10%, 15% as well as 20% of the lyophilized egg yolk groups. Moreover, the percentage of viability parameter in the Boer and Saanen goat sperm was not significantly different between the control and 10% of the lyophilized egg yolk group but showed significant difference between the control group and 15% and 20% of the lyophilized egg yolk groups. Furthermore, the interaction between the two breeds was significantly different in terms of head activity and straightness parameter. In conclusion, the treatment with 20% of fresh egg yolk in Tris-base extender is superior to the lyophilized egg yolk. However, an addition of 10% of the lyophilized egg yolk in Tris-base extender presented the percentage of total motility and viability parameters showing no difference with 20% of fresh egg yolk. Therefore, 10% of the lyophilized egg yolk in Tris-base extender provided detail of the lyophilized egg yolk protocol in cryopreserved goat semen as an example of an alternative extender to 20% of fresh egg yolk for situations where an animal's origin represents a microbiological risk.

Impact of Seminal Plasma Antioxidants on Donkey Sperm Cryotolerance.

This study investigated whether the activities of the antioxidant components of donkey seminal plasma (SP)—both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant capacity (TEAC))—and oxidative stress index (OSI) are related to sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected and split into two aliquots. The first one was used for measuring the activities levels of enzymatic and non-enzymatic antioxidants and OSI in SP, whereas the other aliquot was cryopreserved. Before cryopreservation, sperm quality parameters (concentration, motility, and viability) were evaluated. After thawing, sperm motility, plasma membrane integrity, lipid disorder, mitochondrial membrane potential, reactive oxygen species (ROS), and calcium intracellular levels were also determined. Based on the percentages of total motility (TM) and of sperm with an intact plasma membrane (SYBR14+/PI−) after thawing, samples were classified as good-freezability (GFE) or poor-freezability (PFE) ejaculates through cluster analyses. The SP activity levels of enzymatic (SOD and PON1) and non-enzymatic antioxidants (CUPRAC, FRAP, and TEAC) were higher (p < 0.05) in GFE than in PFE, whereas SP-OSI was higher (p < 0.05) in PFE than in GFE. In addition, the activity levels of SOD, PON1, GPX, CUPRAC, FRAP, and TEAC were positively (p < 0.05) related to post-thaw sperm motility and plasma membrane integrity and negatively to intracellular ROS levels. The SP-OSI was negatively correlated (p < 0.05) to post-thaw sperm quality parameters and positively to intracellular ROS levels. It can thus be concluded that donkey SP antioxidants are related to sperm cryotolerance and that measurements of antioxidants PON1, SOD, CUPRAC, FRAP, and TEAC, as well as SP-OSI, could be used as markers of sperm cryotolerance. Further research addressing the relationship of these antioxidants and SP-OSI with sperm cryotolerance and their potential use as freezing markers is warranted.

Effects of tris (hydroxymethyl) aminomethane and egg yolk on the cryopreservation of buck semen.

This study was designed to examine the effects of various concentrations of tris (hydroxymethyl) aminomethane (tris) and egg yolk on the quality of cryopreserved buck sperm. The collected semen samples were pooled, washed, and diluted into five different freezing extender groups, viz., extender I (tris 0% + egg yolk 0%), extender II (tris 1.41% + egg yolk 4%), extender III (tris 2.41% + egg yolk 8%), extender IV (tris 3.41% + egg yolk 16%), and extender V (tris 4.41% + egg yolk 24%). The sperm parameter of the five groups of extenders was evaluated after equilibration and cryopreservation. The results showed that extenders II-V provided significantly higher semen progressive motility and total motility percentages than extender I after equilibration (p < 0.05). The higher percentages of semen progressive motility, total motility, viability, and plasma membrane integrity (by both HOST under light microscopy and stain after HOST under light microscopy) were found in the sperm cryopreserved with extender IV than extender I, extender II, and extender III groups after thawing (p < 0.05). In addition, semen progressive motility, total motility, and viability were not further increased, or plasma membrane integrity (by both HOST tests) was decreased by the addition of tris and egg yolk (extender V) after cryopreservation (p < 0.05). In conclusion, our result indicates that the following washing, the supplementation of tris (3.41% + egg yolk 16%) on the freezing extender are suitable for improving the semen quality of buck after freezing and thawing.

Male Reproductive Tract Involvement and Sperm Parameters in SARS-CoV-2 Patients: A Systematic Review and Meta-Analysis.

There is a growing concern regarding the impact of SARS-CoV-2 infection on the male reproductive tract due to ACE2 receptor expression, however, its impact remains unclear. We performed this review to evaluate whether SARS-CoV-2 infection affects the male reproductive system. We conducted a search in the Embase, Scopus, and MEDLINE databases, adhering to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guideline. Eligible studies comprised articles reporting viral RNA presence in semen, sperm parameters, and orchitis or orchiepididymitis occurrence in SARS-CoV-2 patients. Observational studies' quality was determined using the Newcastle-Ottawa Scale (NOS). Case reports were assessed using the Joanna Briggs Institute (JBI)'s checklist. A total of 32 relevant articles were included. Viral RNA was found in 7% of infected patients' semen (95% CI, -0.01 to 0.15) from 3 studies. There were also only 7% of patients with orchitis or orchiepididymitis clinical manifestations (95% CI, 0.05-0.10). The semen volume and concentration were 2.34 mL (95% CI, 1.87-2.81) and 51.73 million/mL (95% CI, 31.60-71.85). The progressive and total motility percentages were 36.11% (95% CI, 28.87-43.35) and 43.07% (95% CI, 28.57-57.57), respectively. The morphology was 6.03% (95% CI, -1.05 to 13.10). There is a difference in semen volume between moderate and severe infections (MD, 0.52; 95% CI, 0.27-0.76; p<0.0001) and concentration between mild and moderate (MD, 18.74; 95% CI, 1.02-36.46; p=0.04), mild and severe (MD, 43.50; 95% CI, 13.86-73.14; p=0.004), as well as moderate and severe (MD, 22.25; 95% CI, 9.33-35.17; p=0.0007). SARS-CoV-2 infection may result in decreased sperm concentration in severe cases and the mechanism relates to potential reproductive tract inflammation. The absence of large viral RNA detection in the semen indicates a systemic effect, although this is largely unproven.

Effects of enzymatic and non-enzymatic antioxidants in diluents on cryopreserved bull epididymal sperm

Objective: To evaluate the supplementation effects of vitamin E, vitamin C, superoxide dismutase (SOD), and catalase to diluents on bull cryopreserved epididymal sperm. Methods: Sperm were retrieved from 20 bull testes and were then supplemented with 0.1 mM vitamin E, 5.0 mM vitamin C, 100.0 IU/mL SOD, and 100.0 μg/mL catalase alone, or in a combination. The control treatment contained no addition. After supplementation, samples were frozen and stored in liquid nitrogen. The sperm parameters including motility, progressive motility, viability, acrosome integrity, plasma membrane integrity, kinematics and DNA damage were evaluated following the thawing process. Results: Vitamin E alone significantly increased the parameters of acrosome and membrane integrity compared to the control treatment (P&lt;0.05). While compared to the control treatment, vitamin C had no improvement effect on sperm characteristics except for membrane integrity. Treatment of vitamin E+vitamin C had a significant improvement in total motility, progressive motility, viability, membrane and acrosome integrity compared to the control and other treatments (P&lt;0.05). Compared to the control treatment, addition of SOD or catalase alone significantly improved the percentages of total motility, progressive motility, viability, membrane and acrosome integrity (P&lt;0.05). Furthermore, SOD+catalase significantly increased total motility, progressive motility, viability, acrosome and membrane integrity characteristics compared to the catalase treatment (P&lt;0.05). Vitamin E alone, vitamin E+vitamin C, and SOD in diluents decreased DNA damages and thereby improved the rate of intact sperm heads. Conclusions: Addition of 100.0 IU/mL SOD alone and 0.1 mM vitamin E+5.0 mM vitamin C, and also 5.0 mM vitamin C+100 μg/mL catalase in a combination improves the quality of cryopreserved bull epididymal sperm and could be used for cryopreservation.

Preconditioning of bull semen with sub-lethal oxidative stress before cryopreservation: Possible mechanism of mitochondrial uncoupling protein 2

Preconditioning of sperm using sub-lethal oxidative stress before cryopreservation is an innovative approach that can improve sperm cryo-survival. Mitochondrial uncoupling proteins (UCPs) are critical in reducing ROS level during stress conditions. The aim of the current study was to investigate whether mild sub-lethal stress induced by low concentrations of nitric oxide and hydrogen peroxide has a protective effect on quality parameters of post–thaw bull semen through modulations of mitochondrial uncoupling protein 2 (UCP2) expression. Semen samples were collected from 6 mature Holstein bulls, then mixed and divided into 8 aliquots: fresh, frozen control and frozen groups treated with NO: 0.1 (NO-0.1), 1(NO-1), 10 μM (NO-10), and H2O2: 0.1(H2O2-0.1), 1(H2O2-1) and 10μM (H2O2-10). A significantly higher percentage of total motility, progressive motility and viability was observed in NO-1 and H2O2-10 compared to the other frozen groups (P < 0.05). Sperm exposed to 1 μM NO and 10μM H2O2 showed significantly increased percentages of mitochondria activity and membrane integrity (P < 0.05). Moreover, the lowest percentage of apoptotic percentage was observed in the NO-1 and H2O2-10 in comparison to the other frozen groups. In addition, the expression level of UCP2 was higher in the NO-1 and H2O2-10 compared to the other groups (P < 0.05). It can be concluded that stress preconditioning of bull sperm before cryopreservation can increase UCP2 expression of sperm, that can play a protective role against cryoinjury after thawing.

Seminal plasma proteins as potential biomarkers for sperm motility and velocities

Seminal plasma proteins have important roles in sperm functionality, and different mechanisms including micro-vesicle transport of proteins are involved in the regulation of sperm biology. Due to the role of seminal plasma, we hypothesized that specific proteins present in seminal plasma may be used as discriminant variables with potential to identify stallions producing different quality ejaculates; 10 fertile stallions, with different motility and velocity values (although within normal ranges) were used in this study. Motilities and velocities were studied using computer assisted sperm analysis (CASA), while protein composition of the seminal plasma was studied using UHPLC-MS/MS. Specific proteins were more abundant in samples with poorer percentages of total motility, average path velocity and circular velocity, and were: Secreted phosphoprotein 1, Fructose-bisphosphate aldolase (p = 1,95E-09; q = 0.0005) and Malate dehydrogenase 1 (p = 1,41E-11; q = 0.002), to the contrary samples with better straight-line velocity values were enriched in Glutathione peroxidase (p=0.00013; q=0.04) and Triosephosphate isomerase (p=0.00015; q=0.04).

P–098 Use of Dimethylxanthine Theophylline in surgical retrieved sperms that do not recover motility after thawing

Abstract Study question Can the use of Theophylline recover motility of frozen surgically retrieved sperms in case of absence of motility after thawing? Summary answer Theophylline allows to recover motility of thawed surgically retrieved sperms. The utilization of sperms with or without pharmacological activation gives comparable clinical outcomes. What is known already Testicular sperm motility is usually poor. A method is needed to detect viable sperm for ICSI when motility is totally absent after freezing/thawing. Hypo-osmotic swelling test, mechanical touch technique, laser-assisted immotile sperm selection, birefringence-polarization microscopy and exposure to pharmacological stimulation are techniques used for this purpose. Among pharmacological agents Dimethylxanthine Theophylline is a phosphodiesterase inibitor that improves sperm motility by promoting an increase in intracellular cyclic AMP levels. Few studies report that it is efficient for recovery of sperm motility in cases of thawed testicular and retrograde ejaculation samples improving reproductive outcomes. Study design, size, duration Retrospective analysis of sixty frozen surgical sperm cycles (45 patients) utilized from February 2018 to November 2020. After thawing, samples were divided in two Groups according to motility recovery. Group A: presence of motility, Group B: absence of motility. Group B was treated with Theophilline and motility was re-assessed after incubation. Activated sperms were utilized for ICSI when available. Sperm motility recovery, fertilization, pregnancy rate/transfer, implantation and miscarriage rate were evaluated in both Groups. Participants/materials, setting, methods Surgical specimens were treated and concentrated in SpermRinse™ Medium (Vitrolife) and then cryopreservated in nitrogen vapor in TEST Yolk Buffer (Irvine Scientific). After thawing, only samples with no motility recovery were treated with a brief incubation in Theophylline (GM501 SpermMobil, Gynemed) and washed in Polyvinylpyrrolidone (ICSITM Vitrolife) before injection. ICSI was performed in all cases approximately 4–5 hours after sperm thawing. After fertilization check, transfer was scheduled in day 2. Main results and the role of chance Women’s age Group A (34,39±2,29 M±SD) and group B (35,87±4,34 M±SD) and men’s age Group A (37,31±5,12 M±SD) and group B (40,89±8.15 M±SD) were not significantly different (P= .328 and P=.218) respectively. Group A: 13/60 cycles (21.7%) (9 patients). Pre freezing and post thawing total motility percentage were 34.0±19.0 (M±SD) and 13.5±15.6 (M±SD) respectively (39.8% recovery). Group B: 47/60 cycles (78.3%) (36 patients). Pre freezing total motility percentage was 5.3±8.5 (M±SD) and no motility was recovered post thawing (0%). After treatment with Theophylline total motility was 1.8±1.8 (M±SD) (33.5% recovery). Motile sperms were utilized in all cases except from two in the Group B. Number of injected oocytes was 2.8±1.1 (M±SD) in Group A and 4.3±3.1 (M±SD) in Group B (P=.004) respectively. Fertilisation rate (63.1% and 45.4%, P=.066), Number of embryos transferred (1.8±0.7 M±SD and 1.6±0.7 M±SD, P=.271), Pregnancy rate/Transfer (54.5% and 37.1%, P=.502), Implantation rate (30.0% and 27.8%, P=.919) and Miscarriage rate (33.3% and 30.7%, P=.675) were not statistically significant between Group A and B respectively. In the two cases of group B injected with immotile sperm, fertilization rate was 0% (0/3) and 50% (2/4). Limitations, reasons for caution A larger study is needed to investigate the recovery of sperms motility (and/or their activation) and clinical outcomes, in particular referring to the origin of sampling (epididymal aspirate and testicular tissue) and type of azoospermia (obstructive and non-obstructive). Wider implications of the findings: Theophylline is an effective tool for sperm motility recovery after thawing allowing to inject viable sperm and facilitating laboratory handling. Trial registration number Not applicable