Sperm Motility Test Research Articles

Protective Effects of Tigernut (Cyperus esculentus) on Bisphenol A- Induced Testicular Toxicity in Wistar Rats

Bisphenol A (BPA) have been reported to induced reprotoxicity in rats. This study was conducted to find out the ameliorative properties of aqueous extract of Cyperus esculentus (tigernut) on BPA induced testicular toxicity in Wistar rats. Methods: Twenty male rats were divided randomly into 4 groups (n=5): group A: (Control); group B: Bisphenol A (BPA) (25 mg/kg b.w/day); group C: tigernut (200 mg/kg b.w); group D: (25 mg/kg of BPA+ 200 mg/kg of tigernut extract. 25 mg/kg of BPA was dissolved in 0.2 ml of olive oil as vehicle and administration was given by oral gavage for 4 weeks. The body weights were measured. Blood were collected for the testosterone (T) and luteinizing hormone (LH) assays; the epididymis were processed for sperm count, sperm motility, sperm viability and sperm abnormality test; while the testes were harvested for histology. Results: There was a significantly (p < 0.05) decreased in body weight; reduced (sperm count, motility, viability, serum testosterone and luteinizing hormone) in BPA compared with control group. These parameters however increased significantly (p < 0.05) in tigernut (200mg) and BPA + tigernut (200mg) compared with BPA. Also, histological examination showed widened interstitial spaces, some distorted seminiferous tubules, degeneration of basement membrane, scanty Leydig cells, fewer spermatozoa and vacuolation While BPA + tigernut (group D); showed improved testicular architecture (preserved interstitial spaces and interstitial cells. restoration of the loss of the basement membrane and closely packed seminiferous tubules with well-arranged germinal epithelium. Supplementation with tigernuts following BPA administration produces a reversal of the deleterious effect of BPA on the testis.

Comparison of deep-litter bedding materials and analysis of semen traits in Piétrain boars: A randomized controlled field study

External factors can affect reproductive traits of breeding boars and especially the sensitive process of spermatogenesis. The aim of this study was to investigate probable influences of bedding materials (chipsy wood shavings (CWS), hemp straw (HS), linen straw (LS), spelt husks (SH), and regional wood shavings (RWS)) on semen traits of 40 randomly selected Piétrain boars (8 boars per group, age: 2.35 ± 1.23 years). After a six-week adaptation period, 40 fresh semen samples were collected weekly for four weeks and diluted in BTS (4 consecutive ejaculates per boar, 32 samples per group, 160 samples in total). Semen samples were analyzed using an extended range of spermatological methods (e.g., computer-assisted sperm analysis and flow cytometry). Generalized linear mixed models for each sperm parameter as well as the area under the curve for total sperm motility and thermo-resistance test were calculated. Materials LS and SH exceeded the standard maximum level for pesticide residues (VO (EG) No. 396/2005). Materials HS and LS presented the highest water-binding capacity of 413 % and 357 %, respectively, while SH showed the lowest value of 250 %. There were no significant (P > 0.05) differences between groups in any sperm characteristic, therefore indicating that bedding material had no influence on sperm quality. For most semen traits, however, we found significant (P ≤ 0.001) differences between sampling weeks. Based on pesticide results, we suggest CWS, RWS, or HS as possible bedding materials for pig production farms in the future. Furthermore, we strongly recommend a quality analysis of any new bedding material before use in swine husbandry.

Drug Release, Stability And Efficiency Of Vitamin E Loaded In Liposomes For Bovine Sperm Protection In Cryopreservation Medium

BACKGROUND:It is known that a considerable number of drugs in clinical use or under development are water-insoluble drugs with poor bioavailability. The liposomal delivery system has drawn attention as one of the noteworthy approaches to increase both dissolution and absorption because of its biocompatibility and ability to encapsulate hydrophobic molecules in the lipid domain. However, several drawbacks have been reported, the most common is liposome structural instability .OBJECTIVE:To encapsulate alpha tocopherol into liposomes, to determine the new formulation stability and to study the drug-release of alpha tocopherol into the sperm cryopreservation medium.MATERIALS AND METHODS:The liposomes prepared by an ethanol injection method were characterized for size stability, alpha tocopherol release and sperm motility tests.RESULTS:The prepared unilamellar vesicles had both narrow size distribution (around 99 nm) and a good physical and chemical stability at 4°C during 12 months. The liposomes did not release the vitamin E immediately, but retained the protectant for 24 hours, probably due to the rigidity of the liposomal fence which was reinforced by adding cholesterol. Then, all vitamin E molecules were released by 48 hours. Release was potentially by Fickian diffusion probably by the creation of mini-ducts due to both agitation and fence hydration. Moreover, semen motility treated with vitamin E liposome preparations was significantly improved compared to all other treatments (including commonly used sperm conservation media).CONCLUSION :The stable vitamin E liposomes formulated in this work are a promising alternative for semen cryopreservation protection.

Web- and Artificial Intelligence-Based Image Recognition For Sperm Motility Analysis: Verification Study.

BackgroundHuman sperm quality fluctuates over time. Therefore, it is crucial for couples preparing for natural pregnancy to monitor sperm motility.ObjectiveThis study verified the performance of an artificial intelligence–based image recognition and cloud computing sperm motility testing system (Bemaner, Createcare) composed of microscope and microfluidic modules and designed to adapt to different types of smartphones.MethodsSperm videos were captured and uploaded to the cloud with an app. Analysis of sperm motility was performed by an artificial intelligence–based image recognition algorithm then results were displayed. According to the number of motile sperm in the vision field, 47 (deidentified) videos of sperm were scored using 6 grades (0-5) by a male-fertility expert with 10 years of experience. Pearson product-moment correlation was calculated between the grades and the results (concentration of total sperm, concentration of motile sperm, and motility percentage) computed by the system.ResultsGood correlation was demonstrated between the grades and results computed by the system for concentration of total sperm (r=0.65, P<.001), concentration of motile sperm (r=0.84, P<.001), and motility percentage (r=0.90, P<.001).ConclusionsThis smartphone-based sperm motility test (Bemaner) accurately measures motility-related parameters and could potentially be applied toward the following fields: male infertility detection, sperm quality test during preparation for pregnancy, and infertility treatment monitoring. With frequent at-home testing, more data can be collected to help make clinical decisions and to conduct epidemiological research.

At-home sperm testing for epidemiologic studies: Evaluation of the Trak male fertility testing system in an internet-based preconception cohort.

Semen quality assessment in population-based epidemiologic studies presents logistical and financial challenges due to reliance on centralised laboratory semen analysis. The Trak Male Fertility Testing System is an FDA-cleared and validated at-home test for sperm concentration and semen volume, with a research use only sperm motility test. Here we evaluate the Trak System's overall utility among men participating in Pregnancy Study Online (PRESTO), a web-based study of North American couples planning pregnancy. US male participants aged ≥21years with ≤6months of pregnancy attempt time at study enrolment were invited to participate in the semen testing substudy after completing their baseline questionnaire. Consenting participants received a Trak Engine (battery-powered centrifuge) and two test kits. Participants shared their test results via smartphone images uploaded to online questionnaires. Data were then linked with covariate data from the baseline questionnaire. Of the 688 men invited to participate, 373 (54%) provided consent and 271 (73%) completed at least one semen test result. The distributions of semen volume, sperm concentration, motile sperm concentration, total sperm count, and total motile sperm count were similar to 2010 World Health Organization (WHO) semen parameter data of men in the general population. The overall usability score for the Trak System was 1.4 on a 5-point Likert scale (1=Very Easy, 5=Difficult), and 92% of participants believed they performed the test correctly and received an accurate result. Lastly, men with higher motile sperm count were more likely to report feeling "at ease" or "excited" following testing, while men with low motile sperm count were more likely to report feeling "concerned" or "frustrated." Overall, 91% of men reported they would like to test again. The Trak System provides a simple and potentially cost-effective means of measuring important semen parameters and may be useful in population-based epidemiologic fertility studies.

Effect of supplementing vitamin E analogues on post-thaw semen parameters and fertility in chicken

ABSTRACT 1. The effect of supplementing water-soluble vitamin E analogues 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) and butylated hydroxy toluene (BHT) was studied in two separate experiments. 2. In the first experiment, trolox was supplemented at 0.2 mM, 0.4 mM and 0.8 mM concentrations along with N-methylacetamide (MA; 12% final concentration) and semen was cryopreserved in 0.5 ml French straws. Different semen parameters and fertility were assessed from post-thaw samples. 3. Sperm motility, live sperm, and mitochondrial activity were significantly lower (P < 0.05) in cryopreserved semen. Lipid peroxidation (LPO) was significantly higher (P < 0.05) in cryopreserved semen that was reduced by trolox supplementation. The treatment containing trolox at 0.2 mM concentration produced significantly higher (P < 0.05) fertility compared to unsupplemented cryopreservation treatment. 4. In the second experiment, BHT was supplemented at 0.25 mM, 0.5 mM, and 1 mM concentrations along with MA during semen cryopreservation. 5. Sperm motility, live sperm and MTT dye reduction test were significantly lower (P < 0.05) in cryopreserved semen. These parameters declined with increasing BHT concentration. Abnormal sperm was significantly higher (P < 0.05) in the BHT supplemented treatments. The sperm chromatin dispersion (SCD) test was significantly higher (P < 0.05) in cryopreserved samples and was highest in samples supplemented with 0.5 mM and 1 mM BHT. The percentage fertility was significantly lower (P < 0.05) in cryopreserved semen and BHT supplementation did not improve fertility. 6. In conclusion, trolox supplementation at 0.2 mM concentration during semen cryopreservation improved fertility, whereas BHT supplementation resulted in a decline in post-thaw semen parameters.

Automated semen analysis by SQA Vision® versus the manual approach-A prospective double-blind study.

Next to clinical investigations, the evaluation of male fertility relies mainly on detailed sperm analyses, for example, cell counting, motility, cell morphology and vitality testing. The manual creation of a spermiogram is time- and material-consuming. Therefore, reliable high-throughput systems that may be substituted for manual methods are urgently needed. The present study aimed to compare conventional sperm analysis performed as per WHO 5th guidelines and semen analysis performed with the SQA Vision® machine. SQA Vision® is a commercial device for automated sperm analysis. Data obtained independently by both methods were compared by statistical analyses using Bland-Altman plots and Passing-Bablok regression analyses. The analyses revealed that the results for sperm concentration and total motility were comparable. The agreement for progressive motility was poor, and there were clear deviations in the determination of normal sperm morphology. Passing-Bablok regression analyses and the consideration of the 95% confidence intervals pointed out systematic and proportional differences between the manual semen analysis and the automated approach.

Antioxidants protect proteins' anchorage to the bilayer by improving plasma membrane integrity of ram spermatozoa during liquid preservation in a soya lecithin-based diluent.

Antioxidants are known to prevent the reactive oxygen species (ROS)-mediated peroxidative damage to the membrane lipids during hypothermic storage of mammalian spermatozoa. We hypothesized here that ROS also affect the lipid-protein interactions, thereby diminishing the membrane's integrity and proteins' anchorage to the bilayer. Antioxidants prevent these damages by scavenging the ROS. Ejaculates from Patanwadi rams were pooled after subjective evaluation and centrifuged using Percoll® . Sperm pellet was resuspended in soya lecithin-Tris-fructose diluent (400×106 cells/ml) containing either antioxidants (100IU/ml catalase+10mM reduced glutathione) or no antioxidant. Aliquots were chilled to 5°C in a cabinet and stored in a refrigerator at 3-5°C for 72hr. Sperm motility, viability, lipid peroxidation (LPO) and hypo-osmotic swelling test (HOST) were performed at 0, 24, 48 and 72hr. Sperm proteins extracted with 0.5% Triton X-100 were resolved by SDS-PAGE and quantified using Quantity One software (Bio-Rad, USA). The rapid motility, linearity and straight-line velocity (VSL) were found significantly (p<.05) higher in the antioxidant-treated group compared to the control at 48hr of storage. Sperm viability was found comparable between the groups. Higher HOST response and lower LPO were found in the antioxidant-treated sample compared to the control both at 48 and at 72hr. Overall, the proteins P1 (106.09kDa), P2 (87.00kDa) and P4 (51.14kDa) were lower (p<.05) in the sperm extract of antioxidant-treated group compared to the control. The content of P4 (51.14kDa) in sperm extract was found to increase (p<.05) earlier (48 vs. 72hr) in the control group compared to the antioxidant-treated group. Altogether, the results suggested that antioxidants reduced LPO in spermatozoa, resulting in higher sperm motility, plasma membrane integrity and protection of proteins' anchorage to the plasma membrane at 48 and 72hr of storage.

Effect of different extenders for preservation of ram semen at 4°C

The aim of this study was to investigate the effects of different extenders viz. Tris citric acid fructose egg yolk (TCFEY), Tris citric acid glucose egg yolk (TCGEY), Egg yolk citrate fructose (EYCF) and Egg yolk citrate glucose (EYCG) on the quality of ram spermatozoa during preservation at 4°C. Semen samples showing more than 3+ mass motility and 70% progressive motility were pooled and subsequently divided into four aliquots. Each aliquot was extended separately in four different extenders viz. TCFEY, TCGEY, EYCF and EYCG and stored at 4°C up to 72h. The quality of spermatozoa on the basis of percentage of sperm motility, live sperm, morphological abnormalities, intact acrosome and hypoosmotic swelling test (HOST) reacted spermatozoa was evaluated immediately after extension in particular extenders (0 h), 24 h, 48 h and 72 h after preservation at 4°C. The percent sperm motility was significantly (P&lt;0.01) higher for TCFEY and TCGEY than EYCF and EYCG at 72 h of preservation at 4°C. The percent HOST reacted spermatozoa and intact acrosomes were significantly (P&lt;0.01) higher and morphological abnormalities were significantly (P&lt;0.01) lower for Tris based fructose extender than other three extenders at 72 h at 4°C. In conclusion, Tris citric acid fructose egg yolk (TCFEY) was found the best in maintaining the quality of ejaculated ram spermatozoa during preservation for 72 h at 4°C.

Addition of antioxidants improves quality of ram spermatozoa during preservation at 4 °C

The aim of this study was to investigate the effects of different antioxidants on the quality of ram spermatozoa. For this purpose, ejaculates were collected from six adult healthy rams and extended with Tris egg yolk fructose extender without (Control) or with different antioxidants like ascorbic acid (4.5mg/ml), butylated hydroxytoluene (BHT, 1mM) and taurine (25mM) and stored up to 72h at 4°C. The quality of spermatozoa on the basis of percentage of sperm motility, live sperm, morphological abnormalities, intact acrosome and hypoosmotic swelling test (HOST) reacted spermatozoa was evaluated at 0, 24, 48 and 72h of preservation at 4°C. The percent sperm motility was significantly (P<0.01) higher in BHT and taurine and significantly (P<0.01) lower in ascorbic acid than the control at 72h of preservation. The percent live sperm did not differ significantly (P>0.05) amongst the extenders containing different antioxidants and the control. The morphological abnormalities were significantly (P<0.01) lower in BHT and taurine than ascorbic acid and control during preservation up to 72h at 4°C. The percent HOST reacted spermatozoa was significantly (P<0.01) higher for BHT and significantly lower (P<0.01) for ascorbic acid than control. The percent intact acrosome was significantly (P<0.01) higher in all the extenders containing antioxidants than control. In conclusion, BHT and Taurine were found to improve the quality of ejaculated ram spermatozoa during preservation for 72h at 4°C.

Incidence of Sperm Surface Autoantibodies and Relationship with Routine Semen Parameters and Sperm Kinematics.

Antisperm antibodies (ASA) are associated with male subfertility. However, results on sperm surface autoantibodies are controversial, the relationship between ASA and semen parameters (WHO, 2010) is unknown, and data on ASA and sperm kinematics are scarce. A retrospective study carried out in men undergoing routine semen analysis (WHO 2010), ASA evaluation (direct SpermMAR(™) (IgG) test), and computer-assisted sperm analysis (CASA). A 2.6% and a 5.9% incidence of ASA-positive cases were found (cut-off 50% and 10%, respectively; n = 7492). ASA-positive samples had lower (P < 0.0001) sperm concentration, count, motility, and hypo-osmotic swelling (HOS) test score. HOS results did not correlate with sperm vitality in normozoospermic samples with high ASA levels. In unselected samples, ASA-positive samples (cut-off 50%) showed decreased sperm kinematics (VSL, VAP, LIN, ALH, STR, BCF, WOB), but in normozoospermic samples, ASA-positive and ASA-negative subgroups had similar CASA results. ASA evaluation is highly relevant in full semen assessment.

Effect of washing on the post-thaw quality of cryopreserved ram epididymal spermatozoa

Aim:The aim of the study was to evaluate the effect of washing on the post-thaw quality of ram cauda epididymal spermatozoa (P1: Unwashed, P2: Washed).Materials and Methods:Fresh testicles of adult healthy slaughtered rams were collected and transported to the laboratory in an ice chest, where they were weighed, and cauda epididymides were separated. These cauda epididymides were used for recovery of spermatozoa in tris-citric acid fructose buffer by incision method. Spermatozoa samples showing ≥70% progressive motility were pooled and processed further. The mean values (±standard error) of various parameters such as the percentage of sperm motility, live sperm, intact acrosome, and hypo-osmotic swelling test (HOST) reacted spermatozoa were recorded.Results:In this experiment, the percent sperm motility, live spermatozoa, and intact acrosome both at pre-freeze and post-thaw were higher (p>0.05) in P1 than P2. However, the post-thaw percent HOST reacted spermatozoa was slightly higher (p>0.05) for P2 than P1.Conclusion:Washing of cauda epididymal spermatozoa has no significant adverse effect on the quality during cryopreservation. Therefore, this processing method can be applied wherever necessary before the extension of the recovered spermatozoa sample in different ram extenders.

Reduction of fertility in male mice immunised with pSG.SS.C3d3.YL.Bin1b recombinant vaccine

Objectives To study the anti-fertility effect of a DNA vaccine using Bin1b as the target antigen in male mice.Methods A novel recombinant eukaryotic vector containing a fusion gene sequence of mouse Bin1b in tandem with three copies of C3d fragment (C3d3) was used to construct pSG.SS.C3d3.YL.Bin1b. The correct expression of the Bin1b-C3d3 protein was confirmed in transfected HEK293 cells by indirect immunofluorescence and western blot analysis. The fertility of immunised mice was determined by a mating experiment and sperm motility test. Anti-Bin1b antibody titres in sera were examined by ELISA assays. Binding activity of C3d3 fragment of the fusion protein was verified in C3d receptor-expressing Raji cells and flow cytometric analysis.Results Immunisation of pSG.SS.C3d3.YL.Bin1b recombinant DNA vaccine significantly decreased sperm motility and compromised fertility in male mice. ELISA results showed that the titres of anti-Bin1b IgG in sera of immunised mice increased markedly with the immunisation process. Further, the anti-fertility effect of pSG.SS.C3d3.YL.Bin1b was significantly better than that of pSG.SS.YL.Bin1b DNA vaccine and generated higher titres of anti-Bin1b antibody.Conclusions Our results show that recombinant DNA vaccine targeting Bin1b can markedly reduce fertility in male mice, providing an alternative approach for birth control.Chinese 摘要目的研究以Bin1b作为雄性小鼠靶抗原的DNA疫苗的抗生育作用。方法使用包含小鼠Bin1b与三拷贝C3d片段（C3d3）融合基因序列的新型重组真核载体构建pSG.SS.C3d3.YL.Bin1b。间接免疫荧光和蛋白印迹分析证实Bin1b-C3d3蛋白在转染HEK293细胞中正确表达。交配试验和精子运动试验来确定免疫小鼠的生育能力。ELISA 检测血清中的抗Bin1b抗体滴度。C3d受体表达Raji细胞和流式细胞分析验证融合蛋白中C3d3片段的结合活性。结果免疫接种pSG.SS.C3d3.YL.Bin1b重组NDZ疫苗显著降低雄性小鼠的精子运动力，损害其生育能力。ELISA 结果表明，免疫过程中雄性小鼠抗Bin1b抗体IgG的血清滴度显著降低。而且，pSG.SS.C3d3.YL.Bin1b产生较高的抗Bin1b抗体滴度，其抗生育效应明显优于pSG.SS.YL.Bin1b DNA疫苗的抗生育效应。结论我们的研究结果表明，以Bin1b为靶抗原的重组DNA疫苗能够显著降低雄性小鼠的生育能力，为生育控制提供了一种新的方法。关键词抗生育能力，Bin1b，C3d，重组载体疫苗

Aged men share the sperm protein PATE1 defect with young asthenozoospermia patients.

Does a defect in the human sperm-located protein prostate and testis expressed 1 (PATE1) exist in both aged men and young asthenozoospermia patients? A defect in sperm PATE1 exists in both aged men and young asthenozoospermia patients, and an antibody against PATE1 can decrease human sperm motility and zona-free hamster oocyte penetration. Both aged men and young asthenozoospermia patients have poor sperm quality. The PATE1 protein seems to mediate sperm-egg interactions; however, the mechanisms are still unknown. This was a case-control study including 60 young fathers (aged 28-32 years) and 60 aged fathers (68-72 years old) who donated semen by masturbation after 7 days of sexual abstinence. Comparative sperm proteome analysis from the young fathers and aged fathers was performed to discover key proteins. The target protein PATE1 was chosen and validated by western blotting and immunohistochemistry. Quantitative assessment of sperm PATE1 protein was performed on sperm from 60 young fathers, 60 aged fathers and 110 young asthenozoospermia patients. Furthermore, an antibody against PATE1 assay was used to test whether PATE1 participated in sperm motility and penetration of zona-free hamster egg. Samples were pooled and separated by two-dimensional gel electrophoresis followed by identification by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Western blotting and immunohistochemistry were used to validate the confidence of proteomic data. Sperm immunofluorescence quantification experiments disclosed whether the aged men indeed shared the same PATE1 defect with 110 young asthenozoospermia patients. The sperm motility test and penetration of zona-free hamster egg assay were performed for PATE1. Twenty-two sperm proteins with significant differential expression between young adults and aged men were identified (P < 0.05, mean ratio >1.5), including 13 proteins with decreased expressions with aging. Based on bioinformatics, PATE1 was chosen for further study, and exhibited similar changes in expression level and localization on sperm from aged men and young asthenozoospermia patients. Antibody blocking revealed that PATE1 was involved in sperm-egg penetration and sperm motility. Before any clinical application of PATE1 as a biomarker for the diagnosis of male infertility, more cases should be used to evaluate confidence in this approach. This study revealed a common molecular basis underlying the decline in sperm quality in the natural aging process and in young men with asthenozoospermia. The data should greatly contribute to the development of molecular evaluation of sperm quality, and the diagnosis and treatment of asthenozoospermia. This work was supported by grants from the National Natural Science Foundation of China (NO. 81300533, 81370013 and 81000277) and Shandong Provincial Natural Science Foundation, China (ZR2013HQ002, ZR2014HQ068). The authors declare no competing financial interests.

Effect of different extenders on ram sperm traits during storage

The aim of the study was to test commercial extenders used for short-term and long-term sperm preservation. Semen was collected in the reproduction season, i.e. from June to December. The ejaculates were obtained from single services and the routine analysis of the semen was performed immediately after the collection. The examination included semen volume, colour and texture, sperm concentration and motility, ejaculate turbulence and percentage of sperm with abnormal morphology. The semen was diluted with an extender in the ratio of 1:4. The processed semen was transported in an insulated container at 16–18 °C to the laboratory and stored in a stationary thermostat under the same temperature. Sperm motility tests were performed 24, 48, 72 and 96 hours after the placement in to thermostat. Ejaculates diluted with Ovipro, Optidyl, Triladyl and Andromed CSS gave very good results of viability (81.23 %–83.41 %) after 24 hours of storage. After 48 hours, Ovipro, Andromed, Optidyl and Triladyl gave values above 75 %. The Triladyl extender proved to be a good stabilizing agent, showing consistent results during a long-term storage. It was chosen as a control one for overall assessment. Other preservation media did not show any improving or worsening effects. The extender Ovipro showed a high motility effect in the first 48 hours only, and hence it appears to be the best solution for the short-term preservation.

Total reactive antioxidant potential and DNA fragmentation index as fertility sperm parameters

There is a growing evidence that oxidative stress play a major role in the etiology of defective sperm function including impaired morphology, motility, metabolism and fertility. The aim of the present study was to examine: 1/ total reactive antioxidant potential (TRAP) in seminal plasma; 2/ sperm DNA fragmentation index (DFI), 3/ sperm morphology and motility and 4/ cellular membrane integrity (hypoosmotic swelling test: HOS test) in patients attending in vitro fertilization/intracytoplasmatic sperm injection ( IVF/ICSI) program. According to the DFI value, the men were divided into: group 1 with DFI ≤15% (n=38) and group 2 with DFI ≥15% (n=37). Significant differences between the two groups were found in TRAP, sperm motility, morphology and concentration as well as HOS test scores. In group 1, DFI was negatively correlated with sperm motility and HOS test scores (p<0.05). The sperm morphology was positively correlated with sperm motility and HOS test scores in both groups. There was no correlation between TRAP and sperm chromatin fragmentation. Our results suggest that seminal plasma TRAP level may be a DFI independent parameter of sperm fertility.

Demonstration of the effect of epidermal growth factor on ram sperm parameters using two fluorescent assays

ABSTRACT: The goal of this study was to examine the effect of epidermal growth factor (EGF) on sperm viability using two fluorescent techniques and to analyze the obtained results in relation to sperm motility, determined by subjective estimation. Fresh ram semen diluted in a Biladyl commercial extender was cooling stored (at 4 &amp;deg;C in a fridge) for four days in the presence of EGF at doses of 0, 10, 100, 200 or 400 ng/ml. Thereafter, sperm samples were analyzed for progressive motility (Motility test) and membrane integrity using two fluorescent techniques: SYBR-14/PI (Method 1) or PI/DAPI (Method 2). Application of Method 1 did not detect an effect of EGF at any concentration on sperm membrane integrity. A positive effect of EGF (200 ng/ml) on sperm membrane integrity was found using Method 2 of staining, and this result was confirmed by the sperm motility test, which demonstrated an EGF-stimulating effect (200 or 400 ng/ml) on a percentage of progressively moving spermatozoa. Strong positive correlations between Methods 1 and 2 (r = 0.785), Method 1 and Motility (r = 0.803), Method 2 and Motility (r = 0.699), as well as between both techniques taken together and the Motility test (r = 0.853) were found. Regression analysis confirmed that Method 2 was more exact than Method 1, and the results obtained with Method 2 are comparable with those of the Motility test. Dependence of the viability or motility on EGF concentrations (linear regression function) was significant only for Method 2 or the Motility test. The obtained results suggest a stimulating effect of EGF (at higher concentrations) on ram sperm functions (viability/membrane integrity and motility). Furthermore, they indicate substantial differences between two fluorescent techniques in the determination of sperm membrane integrity. Only the data obtained using PI/DAPI were confirmed by a functional Motility test. These findings suggest that the technique chosen for analysis of sperm viability can influence the conclusion concerning the effects of the treatment on sperm function.

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.

Effect of antioxidants on microscopic semen parameters, lipid peroxidation and antioxidant activities in Angora goat semen following cryopreservation

The aim of this study was to determine the effects of the antioxidants glutamine and hyaluronan and the inclusion of different levels on microscopic semen parameters, lipid peroxidation and the antioxidant activities following the freeze–thawing of Angora goat semen. Ejaculates collected from three Angora goat bucks, were evaluated and pooled at 37 °C. The semen samples which were diluted with a Tris-based extender containing additives including glutamine (2.5; 5 mM) and hyaluronan (500; 1000 μl/ml), and an extender containing no antioxidants (control) were cooled to 5 °C and frozen in 0.25 ml French straws and stored in liquid nitrogen. Frozen straws were thawed individually (37 °C) for 20 s in a water bath for microscopic evaluation. Freezing extenders supplemented with 2.5 and 5 mM glutamine led to higher sperm motility and hypo-osmotic swelling test (HOST) values, compared to the control ( P < 0.05) following the freeze–thawing process. The addition of 500 μl/ml hyaluronan resulted in a higher HOST percentage, compared to the addition of 1000 μl/ml hyaluronan and the control ( P < 0.001). No significant difference was recorded in the percentage acrosome and total sperm abnormalities, following supplementation with antioxidants. The addition of antioxidants did not prevent malondialdehyde (MDA) formation, compared to the controls. Antioxidant treatment however decreased ( P < 0.01) the superoxide dismutase (SOD) activity. The maintenance of catalase (CAT) activity was demonstrated to be insignificant following addition of antioxidants. Further studies are required to obtain more repeatable results regarding the characterization of the enzymatic and non-enzymatic antioxidant systems in cryopreserved goat sperm.

Influence of Temperature during Glycerol Addition and Post‐thaw Dilution on the Quality of Canine Frozen Semen

Contents The aim of the present study was to compare the influence of room temperature (27 degrees C) and 4 degrees C during glycerol addition on canine semen cryopreservation and verify the effect of different post-thawing dilutions on canine semen. Ten ejaculates from five stud dogs were collected by digital manipulation. Semen samples were evaluated and further divided into two aliquots. The first aliquot was extended in Tris-egg yolk-glycerol at 27 degrees C and the second one received glycerol at 4 degrees C. Samples were frozen and stored in liquid nitrogen. After 1 week, samples were thawed and submitted to evaluations of progressive sperm motility, morphology, acrosomal integrity, hypo-osmotic swelling (HOST) and thermoresistance tests. For thermoresistance test, aliquots were divided in two portions: one portion was kept undiluted (1 : 0) and the other one was diluted in a 1 : 4 ratio (one part semen to four parts extender). No differences were observed between temperatures for glycerol addition regarding seminal parameters evaluated. Furthermore, post-thawing dilutions demonstrated similar effect on canine semen longevity. Correlations among post-thaw sperm motility and HOST and results from thermoresistance test were observed for both temperatures for glycerol addition. In conclusion, glycerol could be added to canine semen at room temperature (27 degrees C) or at 4 degrees C. Moreover, there is no need to extend canine semen after thawing for the thermoresistance test, but if we need to increase the inseminating volume for artificial inseminations, the addition of extender will not damage the semen.