Effect of short‐term semen storage in salmon (Oncorhynchus mykiss) on sperm functional parameters evaluated by flow cytometry

The short‐term storage of salmonid semen is a viable method for in vitro fertilisation. Previous studies have found that short‐term storage affects sperm motility, compromising quality and fertilising capacity. However, the functional characteristics of the spermatozoa of O. mykiss during storage time and its relation to the spawning period are little known. This study was designed to evaluate the effects of in vitro short‐term storage on sperm functional parameters in O. mykiss, determined by flow cytometry. Semen samples of the first spawning – undiluted (SSD) and diluted (SD) (Storfish^® 1 : 2v/v; IMV AI solutions, France) – were stored at 4 °C for 14 days. Motility, viability (PMI: plasma membrane integrity) and mitochondrial membrane potential (ΔΨM) were assessed. On the fifth day of storage, spermatozoa showed a motility >70% (SSD: 78.3% versus SD 85.0%), PMI (81.5% SSD/87.2% SD) and ΔΨM (72.5% SSD/SD 80.0%) (P < 0.05). However, a significant decline in the percentage of all functional parameters (P < 0.05) was observed after 5 days of storage for all samples of both undiluted (SSD) and diluted semen. In conclusion, the results here provide new data on O. mykiss sperm quality with respect to in vitro short‐term storage evaluated by flow cytometry.     

Cryoprotective effect of l‐carnitine on motility,vitality and DNA oxidation of human spermatozoa

Successful cryopreservation for human spermatozoa markedly influences the reproductive outcomes of assisted reproductive technologies. But in spite of its usefulness, cryopreservation significantly decreases sperm quality. l ‐carnitine has been found to improve the quality of spermatozoa in selected cases with male infertility. Here, we examined the efficacy of l ‐carnitine in improving sperm motility and vitality and reducing sperm DNA oxidation during cryopreservation. Semen samples from infertile patients (n = 22) were collected and analysed. Cryopreservation medium supplemented with l ‐carnitine was mixed with the semen at a ratio of 1 : 1 (v/v). The final l ‐carnitine concentration in each cryovial was 0.5 mg ml^?1 per 5 × 10⁶ cell ml^?1. Controls were cryopreserved without addition of l ‐carnitine. After 24 h of cryopreservation, thawed sperm samples were analysed for motility, vitality and DNA oxidation. Sperm vitality was assessed by the eosin–nigrosin test, while sperm DNA oxidation was measured by flow cytometry. Addition of l ‐carnitine significantly improved sperm motility and vitality (P < 0.05) compared with the control. The flow cytometry experiment showed no statistical difference (P > 0.05) in the levels of DNA oxidation between samples and controls. In conclusion, l ‐carnitine improves human sperm motility and vitality, but has no effect on sperm DNA oxidation after cryopreservation.     

Effect of l‐arginine addition on long‐term storability of ram semen

This study was conducted to investigate the effect of l ‐arginine addition on long‐term storability of ram semen. Six Akkaraman rams were used as material. Semen samples were collected. Pooled samples were diluted and were divided into six equal aliquots. While aliquot 1 was kept as control, the stock solutions including 0.1, 0.5, 1, 5 and 10 mm l ‐arginine were added to other aliquots. All aliquots were routinely frozen in 0.25‐ml straws at ?130°C liquid nitrogen vapour and stored in liquid nitrogen ?196°C until being analysed. The equilibrated and thawed sperm motility, membrane integrity and arginase activity were evaluated. While the 10 mm l ‐arginine supplementation significantly (p < .001) decreased equilibrated sperm motility, the 5 mm significantly (p < .05) increased the membrane integrity and arginase activity in comparison with the control group. The motility (p < .001) and membrane integrity (p < .01) were determined to be highest in 0.5 mm group, while significant reductions were observed in motility (p < .001) of 10 mm group and arginase activity (p < .05) of 1, 10 mm groups as compared to the control group. It was concluded that in vitro addition of 0.5 mm l ‐arginine to ram semen may be useful, but 10 mm may be harmful to spermatozoa quality during long‐term storage.     

Semen quality in adult male survivors 5 years after the 2008 Wenchuan earthquake

The influence of the Wenchuan earthquake on semen quality of adult male survivors is unclear. We investigated the semen quality included 673 male survivors from the worse‐affected counties in the earthquake between Aug 2008 and July 2013. Semen parameters including pH, volume, concentration, motility and morphology were measured according to the World Health Organization (WHO) criteria. Kruskal–Wallis analysis of variance was used to examine the statistical differences between years, and a logistic regression was used to analyse the impacts caused by earthquake on the changes of semen quality. We found the medians (5th and 95th) were 2.5 ml (0.6–5.5) for semen volume, 59.0 × 10⁶ ml^?1 [(13.0–133.0)] × 10⁶ ml^?1 for semen concentration, 46% (13–64%) for sperm progressive motility and 3.0% (0–17.5%) for normal morphology for adult male survivors. Semen concentration, the percentage of sperm progressive motility, total motility and sperm normal morphology were all decreased in the first 3 years, and the differences among years 1, 2 and 3 were significant except the percentage of sperm progressive motility (P < 0.05). The casualties and heavy housing damage caused by earthquake had a negative effect on semen quality. The main findings will provide further diagnosis and therapy basis of male fertility by data, for affected populations in the earthquake.     

Lycopene and resveratrol improve post‐thaw bull sperm parameters: sperm motility,mitochondrial activity and DNA integrity

We focussed on evaluating the protective effect of lycopene and resveratrol on post‐thaw bull sperm and oxidative stress parameters. Nine ejaculates for each bull were used in the study. Each ejaculate, splitted into three equal aliquots and diluted at 37 °C with base extenders containing lycopene (1 × 10^?3 g ml^?1) and resveratrol (1 mm ), and no antioxidant (control), was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. The supplementation of the semen extender with lycopene and resveratrol increased the percentages of post‐thawed computer‐assisted sperm analysis (CASA) motility (55.8 ± 3.8 and 61.9 ± 4.0%) and progressive motility (38 ± 2.4 and 37 ± 8.8), compared with the controls (50.7 ± 2.65 and 33.3 ± 3.74%, respectively, P < 0.05). Resveratrol provided a higher ALH (4.3 ± 0.1), in comparison with the control (3.9 ± 0.3, P < 0.05). The supplementation of the semen extender with lycopene and resveratrol produced a higher mitochondrial activity (24.6 ± 2.9 and 30.1 ± 6.5% respectively), compared with that of the control (11.8 ± 9.5%, P < 0.05). It was determined that both antioxidants resulted in a lower percentage of sperm with damaged DNA than that of the control (P < 0.05). Sperm motion characteristics except for ALH, acrosome integrity, sperm viability and oxidative stress parameters were not affected by the adding of lycopene and resveratrol.     

Semen quality evaluation in a cohort of 28213 adult males from Sichuan area of south‐west China

The trends in semen quality are conflicting. Although many previous surveys on semen quality indicated a decline, the trends in semen quality in Sichuan area of south‐west China are not clear. We analysed the semen parameters in a cohort of 28 213 adult males close to general population in Sichuan between July 2007 and June 2012, and investigated the changes on semen quality. The semen parameters including pH, volume, concentration, motility, morphology were measured according to the World Health Organization (WHO) criteria. Kruskal–Wallis analysis of variance was used to examine the statistical differences of semen quality between groups. We found that the medians (5th and 95th percentiles) were 2.4 ml (1.0–5.0) for semen volume, 62.0 × 10⁶ ml^?1 (15.0–142.0) for semen concentration, 39% (18–60%) for sperm progressive motility and 10.5% (1.0–34.5%) for normal morphology. In these 5 years, sperm concentration and the percentage of sperm normal morphology were decreased from 66.0 × 10⁶ ml^?1 to 49.0 × 10⁶ ml^?1 and from 13.5% to 4.5%, respectively; among different reproductive history groups, sperm concentration and the percentage of sperm normal morphology were also decreased in these 5 years. And the incidence of azoospermia was increasing. These may imply that there is a decline in semen quality of adult males in Sichuan area.     

Relationship of spermatozoal DNA fragmentation with semen quality in varicocele‐positive men

The aim of the study was to assess the semen quality and levels of spermatozoal nuclear DNA fragmentation in subfertile subjects clinically diagnosed with varicocele, subfertile subjects without varicocele and healthy fertile controls. Semen samples were obtained from 302 subjects. Of them, 115 were healthy fertile controls having normal semen characteristics, 121 subfertile men diagnosed with varicocele, both, clinically and on ultrasonography, while 66 subjects were subfertile with no varicocele. Spermatozoal concentration, percentage motility, morphology and DNA fragmentation were measured. In the study population, deterioration in semen quality‐decreased spermatozoal concentration, percentage motility and normal morphology was seen in subfertile subjects, especially with varicocele. Highest spermatozoal DNA fragmentation was observed in varicocele‐positive subjects as compared with varicocele‐negative subjects and healthy fertile controls. Significant negative correlation was seen between spermatozoal DNA fragmentation and concentration (r = ?0.310), motility (r = ?0.328) normal morphology, WHO method (r = ?0.221) and Tygerberg strict criteria (r = ?0.180) in the varicocele‐positive subfertile subjects. In conclusion, this study suggests existence of a negative relationship between spermatozoal DNA fragmentation and semen quality in varicocele‐positive subfertile subjects.     

Anti‐oxidative Status and Semen Quality during Cooled Storage in Stallions

Activity of the anti‐oxidative enzymes glutathione peroxidase (GSH‐Px), superoxide dismutase (SOD) and catalase (CAT), content of thiobarbituric acid reactive substances (TBARS) and SH‐groups were determined in native stallion semen (n = 8 stallions). Semen was then diluted in Kenney extender, EquiPro^® extender either with or without addition of N‐acetyl cysteine or phosphate‐buffered saline (PBS) and stored for 72 h at 5°C. Correlations between initial activity of enzymes and development of semen motility and membrane integrity were calculated. Activities of GSH‐Px, SOD and CAT immediately after semen collections were 10.0 ± 0.6 picokatals, 0.40 ± 0.03 SOD units and 0.70 ± 0.05 nanokatals/10⁶ spermatozoa respectively. TBARS content was 0.06 ± 0.01 nmol and SH‐group content 1.7 ± 0.5 mmol/10⁶ spermatozoa. The loss of motile spermatozoa during storage did not differ between extenders. N‐acetyl cysteine had no effect on semen motility and membrane integrity. The loss in membrane‐intact spermatozoa was highest (P < 0.05) in semen diluted in PBS. Motility and membrane integrity after addition of extender were positively correlated with GSH‐Px and CAT, indicating that anti‐oxidative mechanisms contribute to the initial high percentage of motile and membrane‐intact spermatozoa. However, in these samples the decrease in semen quality was most pronounced. No correlations existed between initial activity of anti‐oxidative enzymes, peroxidation products and semen quality during storage. This indicates that once extender has been added, peroxidative damage to sperm membranes is not the predominant cause of losses in semen quality.     

Effect of the age of broodstock males on sperm function during cold storage in the trout (Oncorhynchus mykiss)

The knowledge of sperm quality in the broodstock males of different ages is a prerequisite to identify the reproductive ability of cultivated fish for the hatchery management. Thus, in this work, we analysed sperm function of the semen stored of broodstock males of rainbow trout (Oncorhychus mykiss) in different reproductive ages (2, 3 and 4 years old). Sperm samples of each reproductive age were stored in Storfish^® during 10 days at 4°C, and then, motility, viability, mitochondrial function (MMP), superoxide anion () level and DNA fragmentation (DNA_frag) were assessed. The results demonstrated that sperm function parameters were affected significantly by the age of the males and the time of storage. Motility, viability and MMP significantly decreased, and DNA_frag and level increased with the age increment and the time of storage. In conclusion, sperm quality of 2 and 3 years old were superior to those of 4 years old, based on higher quality of various sperm functions such as motility, viability, MMP, DNA integrity and level during short‐term storage. This information must be considered for optimum utilization of broodstock males in aquaculture.     

Utilisation of sperm‐binding assay combined with computer‐assisted sperm analysis to evaluate frozen‐thawed bull semen

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap‐freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm² of membrane was assessed by conventional microscopy (CM) and computer‐assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r² = 0.91 and CASA: r² = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm‐egg‐binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.     

The micronutrient supplements,zinc sulphate and folic acid,did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men

We investigated the effects of folic acid and zinc sulphate supplementation on the improvement of sperm function in subfertile oligoasthenoteratozoospermic (OAT) men. Eighty‐three OAT men participated in a 16‐week intervention randomised, double‐blind clinical trial with daily treatment of folic acid (5 mg day^?1) and zinc sulphate (220 mg day^?1), or placebo. Before and after treatment, semen and blood samples were obtained for determining sperm concentration, motility, and morphology, sperm viability, sperm mitochondrial function, sperm chromatin status using toluidine blue, aniline blue, acridine orange and chromomycin A₃ staining; and semen and blood folate, zinc, B₁₂, total antioxidant capacity ( TAC) and malondialdehyde (MDA) concentrations. Sperm concentration (×10⁶ ml^?1) increased in subfertile men receiving the combined treatment of folic acid and zinc sulphate and also in the group receiving only folic acid treatment; however, it was not statistically significant (P = 0.056 and P = 0.05, respectively). Sperm chromatin integrity (%) increased significantly in subfertile men receiving only zinc sulphate treatment (P = 0.048). However, this improvement in sperm quality was not significant after adjusting placebo effect. This study showed that zinc sulphate and folic acid supplementation did not ameliorate sperm quality in infertile men with severely compromised sperm parameters, OAT. Male infertility is a multifactorial disorder, and also nutritional factors play an important role in results of administration of supplementation on sperm parameters. However, these results should be confirmed by multiple studies in larger populations of OAT men.     

Relationship between nuclear DNA fragmentation,mitochondrial DNA damage and standard sperm parameters in spermatozoa of fertile and sub‐fertile men before and after freeze‐thawing procedure

The aim of this study was to assess the stability of nuclear and mitochondrial DNA (n‐DNA and mt‐DNA) of spermatozoa under freeze‐thawing and to find out the correlation between them and their association with standard sperm parameters. Forty‐three semen samples were collected from fertile (G.1; n = 29) and sub‐fertile (G.2; n = 14). N‐DNA fragmentation was determined by TUNEL assay and mt‐DNA using caspase 3 staining. Each semen sample was frozen at ?196°C by the programmed freezer. Freeze‐thawing decrease vitality, total motility and membrane integrity from (43.02 ± 22.74%; 31.63 ± 18.15%; 51.5 ± 24.82%) to (22.71 ± 17.3%; 9.21 ± 6.61%; 34.64 ± 19.92% respectively [p < .001]). G.1 native spermatozoa stained positive with TUNEL and caspase 3 were (14.85 ± 17.6% and 5.8 ± 11.59%) and increased after freeze‐thawing to 27.54 ± 19.74% (p = .004) and 7.3 ± 6.13% (p = .01) respectively. In G.2, TUNEL and caspase 3 were (19.84 ± 17.52% and 7.53 ± 8.56%) and increased to (29.48 ± 16.97% [p = .03] and 10.21 ± 11.73%). In conclusion, freeze‐thawing process affects not only semen parameters but also n‐DNA and mt‐DNA. Therefore, n‐DNA and mt‐DNA could be used as sensitive parameters for assessment of the cryodamage of human spermatozoa.     

Deep‐Freezing of Boar Semen in Plastic Film ‘Cochettes’

The motility and membrane integrity of spermatozoa from nine boars frozen with a programmable freezing machine in plastic bags, ‘cochettes’^®, and in ‘maxi‐straws’, in total doses of 5 × 10⁹ spermatozoa/5 ml with glycerol (3 %) used as cryoprotectant, were assessed after thawing. A computer‐based cell motion analyser was used to evaluate sperm motility, while the integrity of the plasmalemma was assessed with fluorescent supravital dyes (C‐FDA/PI). The fertilizing capacity of the semen frozen in the two containers was investigated by inseminating (AI) gilts. Pregnancy was monitored by Doppler‐ultrasound, and the numbers of corpora lutea and viable embryos counted at slaughter, between days 30 and 38 after AI. The cochettes sustained the overall procedure of freezing/thawing (FT), with 30 min post‐thaw (PT) sperm motility being significantly higher than for straws, 46.9 vs. 39.5 %. The only significant difference in motility patterns detected when comparing the packages was a higher sperm velocity (VCL) in cochettes at 30 min PT. However, percentages of FT‐spermatozoa with intact membranes, detected with the supravital probes, were higher in maxi‐straws than in cochettes, 46.8 vs. 43.0 % (P < 0.05). There were no significant differences found in fertilizing capacity between spermatozoa frozen in maxi‐straws and those frozen in cochettes. The results indicate that although the deep‐freezing of AI‐doses of boar semen in large plastic bags is feasible, problems such as their inconvenient size for storage and inconsistent thawing must be solved before this type of container can be used for the commercial cryopreservation of boar semen.     

Saturated,omega‐6 and omega‐3 dietary fatty acid effects on the characteristics of fresh,frozen–thawed semen and blood parameters in rams

The aim of this study was to investigate the effects of several dietary fatty acids (FAs) on semen quality and blood parameters in rams. We gave diet‐supplemented treatments (35 g day^?1 ram^?1) by C16:0 (palm oil), C18:2 [sunflower oil (SO)] and an n‐3 source [fish oil (FO)] to 12 rams, who were fed for 15 weeks during their breeding season. Semen was collected once per week. Semen samples were extended with Tris‐based cryoprotective diluents, then cooled to 5 °C and stored in liquid nitrogen. Positive responses were seen with FO after 4 weeks. The mean prefreezing semen characteristics improved with the intake of FO (P < 0.05). Interestingly, maximum sperm output in FO was achieved 7.5 × 10⁹ when compared to palm oil 5.3 × 10⁹. Rams that received FO had the highest total testosterone concentrations (11.3 ng ml^?1 for FO, 10.8 ng ml^?1 for SO and 10.2 ng ml^?1 for palm oil) during the experiment (P < 0.05). FO also improved the rams' sperm characteristics after thawing (P < 0.05). Although C16:0 is a major saturated FA in ram sperm and all rams have been fed isoenergetic rations, the unique FAs of FO improved fresh semen quality and freezing ability compared to other oils.     

Effect of Cissampelos capensis rhizome extract on human spermatozoa in vitro

Cissampelos capensis is commonly known by the Afrikaans name ‘dawidjies’ or ‘dawidjieswortel’. C. capensis is the most important and best‐known medicinal plant of the family Menispermaceae used by the Khoisan and other rural people in the western regions of South Africa. Among numerous other ailments, it is traditionally taken to treat male fertility problems. Yet, no studies have investigated the effects of this plant or its extracts on human spermatozoa. The aim of study was to investigate the effects of C. capensis extracts on sperm function. A total of 77 semen samples were collected. Spermatozoa were washed with HTF‐BSA medium and incubated with different concentrations of C. capensis (0, 0.05, 0.5, 5, 50, 200 μg ml^?1) for 1 h at 37 °C. Sperm motility, vitality, acrosome reaction, reactive oxygen species (ROS), capacitation, Annexin V binding, DNA fragmentation and mitochondrial membrane potential (Δψ_m) were determined. While viability, Annexin V positivity and Δψ_m were not affected, the percentages of ROS‐positive, TUNEL‐positive, capacitated and hyperactivated spermatozoa increased significantly and dose‐dependently. It is concluded that the alkaloids present in the extract of C. capansis rhizomes triggered sperm intrinsic superoxide production leading to sperm capacitation and DNA fragmentation.     

Cumene hydroperoxide induced changes in oxidation–reduction potential in fresh and frozen seminal ejaculates

Oxidation–reduction potential (ORP) is a newer integrated measure of the balance between total oxidants (reactive oxygen species—ROS) and reductants (antioxidants) that reflects oxidative stress in a biological system. This study measures ORP and evaluates the effect of exogenous induction of oxidative stress by cumene hydroperoxide (CH) on ORP in fresh and frozen semen using the MiOXSYS Analyzer. Semen samples from healthy donors (n = 20) were collected and evaluated for sperm parameters. All samples were then flash‐frozen at ?80°C. Oxidative stress was induced by CH (5 and 50 μmoles/ml). Static ORP (sORP—(mV/10⁶ sperm/ml) and capacity ORP (cORP—μC/10⁶ sperm/ml) were measured in all samples before and after freezing. All values are reported as mean ± SEM. Both 5 and 50 μmoles/ml of CH resulted in a significant decline in per cent motility compared to control in pre‐freeze semen samples. The increase in both pre‐freeze and post‐thaw semen samples for sORP was higher in the controls than with 50 μmoles/ml of CH. The change from pre‐freeze to post‐thaw cORP was comparable. The system is a simple, sensitive and portable tool to measure the seminal ORP and its dynamic impact on sperm parameters in both fresh and frozen semen specimens.     

Effect of acute tadalafil on sperm motility and acrosome reaction: in vitro and in vivo studies

Effects of acute tadalafil on sperm motility and acrosome reaction were investigated, both in vitro and in vivo. Twenty asthenozoospermic and 20 normozoospermic patients as control were randomly enrolled. For in vitro part, 0.5 ml tadalafil solutions with different concentrations were added (0.2, 0.1, 0.05 and 0.025 μg ml^?1, respectively) into semen samples. In both groups, samples treated with 0.2 μg ml^?1 tadalafil had significant increase in sperm motility after 2 h incubation. For in vivo part, oral administration of tadalafil (20 mg) or sildenafil (100 mg) was given. In both groups, computer‐assisted semen analysis parameters showed no significant difference. After the administration of tadalafil (2 h) and sildenafil (1 h), there was no significant difference observed in premature acrosome reaction incidence rate. Taking both in vitro and in vivo results into consideration, acute on‐demand administration of tadalafil would have no adverse effect on semen parameters.     

Hypo‐osmotic test in cat spermatozoa

The hypo‐osmotic (HOS) test has been used in other species as an indicator of the fertilising capacity of spermatozoa. The aims of this study were to assess the response of domestic cat spermatozoa to the hypo‐osmotic test, to determine the type of solution, concentration and time of incubation needed to obtain a maximum percentage of swelling, to correlate the selected combination with the percentages of progressive motility and to evaluate whether dilution of the ejaculate alters the results. Incubation for 30 and 45 min in solutions of fructose and of citrate of 50 and 100 mOsmol kg^?1 was evaluated. The highest percentage of swelling was obtained using the 50 mOsmol kg^?1 solution, and no significant differences were observed between the times of exposure to the solutions. A positive correlation was observed between the percentage of individual progressive motility and the percentage of sperm swelling in a 50 mOsmol kg^?1 fructose solution, with no significant differences being observed between raw and diluted semen samples. The results of this study suggest that the HOS test could be useful for evaluating membrane function in domestic cat spermatozoa, both in raw semen and in samples diluted in the EZ Mixin^® commercial extender, and thus could be incorporated into routine semen evaluation protocols.     

Survival of buffalo bull spermatozoa: effect on structure and function due to alpha‐lipoic acid and cholesterol‐loaded cyclodextrin

Sperm survival depending upon integral membranes and function is imperative for fertilization. This study was designed to augment survival of buffalo spermatozoa using alpha‐lipoic acid (ALA) and cholesterol‐loaded cyclodextrin (CLC) during cryopreservation. Semen was frozen using 0, 0.5, 1, 1.5, 2 and 2.5 mmol L^?1 ALA (experiment 1) and ALA or CLC separately or together (experiment 2). Semen was assessed for post‐thaw motility, plasma membrane integrity (PMI), intact acrosome and plasma membrane (IACR‐IPM) and DNA integrity at 0, 1.5, 3 and 4.5 hr of incubation. In experiment 1, use of 0.5 mmol L^?1 ALA enhanced the sperm cryosurvival and post‐thaw longevity than other groups up to 4.5 hr of incubation, and this concentration of ALA was used in second experiment with CLC. The results revealed higher (p < .05) sperm survival function and time of sperm attributes due to use of ALA than CLC and control. However, the sperm quality did not improve (p > .05) when ALA was combined with CLC. In conclusion, survival of buffalo bull spermatozoa during freeze‐thawing and post‐thaw incubation can be enhanced more with ALA than CLC or control, followed by CLC than control. However, there is no synergistic effect on survival of buffalo bull spermatozoa due to ALA and CLC.     

Sperm motility and DNA integrity affected by different g‐forces in the preparation of sperm in urine specimens

Retrograde ejaculation, a common type of anejaculation, is attributable to many causes, some of which can be treated with medication and some of which cannot. For infertility treatment, sperm must be collected from the urine of the patients. Our study attempts to ascertain the effects of different g‐forces on sperm motility, morphology and DNA integrity in sperm preparation by the Sil‐Select? density gradient method of isolating sperm from urine specimens. Forty‐seven semen samples with normal semen analyses according to World Health Organisation (WHO) 1999 criteria were included in this study. Semen samples of 1 ml were mixed with 20 ml alkalinised normal urine and then divided equally into tubes A and B. The two samples were prepared by the Sil‐Select? density gradient centrifugation method at 350 g (tube A) and at 700 g (tube B). Total motile sperm after centrifugation at 700 g was significantly higher than after centrifugation at 350 g [6.7 (0.4–23.0) million versus 3.1 (0.1–13.7) million] (P < 0.001). There was no significant difference between the either the percentage of sperm with normal morphology or with DNA damage between centrifugation at 350 g and 700 g (P > 0.05), although centrifugation at 700 g achieves a higher number of total motile sperm compared with Sil‐Select? sperm preparation at 350 g centrifugation.