Effect of mitochondrial calcium uniporter blocking on human spermatozoa

Calcium (Ca²⁺) regulates a number of essential processes in spermatozoa. Ca²⁺ is taken up by mitochondria via the mitochondrial calcium uniporter (mCU). Oxygen‐bridged dinuclear ruthenium amine complex (Ru360) has been used to study mCU because it is a potent and specific inhibitor of this channel. In bovine spermatozoa, it has been demonstrated that mitochondrial calcium uptake inhibition adversely affects the capacitation process. It has been demonstrated in human spermatozoa that mCU blocking, through Ru360, prevents apoptosis; however, the contribution of the mCU to normal human sperm function has not been studied. Therefore, the aim of this study was to evaluate the effect of mCU blocking on human sperm function. Spermatozoa obtained from apparently healthy donors were incubated with 5 and 10 μm Ru360 for 4 h at 37 °C. Viability was assessed using propidium iodide staining; motility was determined by computer‐aided sperm analysis, adenosine triphosphate (ATP) levels using a luminescence‐based method, mitochondrial membrane potential (ΔΨm) using JC‐1 staining and reactive oxygen species (ROS) production using dihydroethidium dye. Our results show that mCU blocking significantly reduced total sperm motility and ATP levels without affecting sperm viability, ΔΨm and ROS production. In conclusion, mCU contributes to the maintenance of sperm motility and ATP levels in human spermatozoa.     

LED‐based red light photostimulation improves short‐term response of cooled boar semen exposed to thermal stress at 37°C

Pre‐treatment of boar semen with a red light photostimulation procedure increases its “in vivo” fertilising ability. However, “in vitro” conducted studies shown contradictory results regarding the ability of photostimulated spermatozoa to react against strong stress and to achieve the capacitation status. The aim here was to determine the effect of photostimulation on the response to short‐term moderate thermal stress of boar semen. Boar semen was exposed to red LED light regime emitting a 620–630 nm during 10 min of light, 10 min of rest and 10 min of light after 3 hr since semen was collected. An aliquot without photostimulation was included as a control. After the photostimulation, the sperm cells were incubated for 15 min at 37°C. Afterwards, motility, viability, intracellular Ca²⁺ level and production of reactive oxygen species (ROS) and peroxynitrite were analysed. The results showed that the photostimulated group maintained total motility throughout the time, whereas a significant decrease in total motility was observed in the nonphotostimulated control group. Furthermore, for kinetic parameters of motility, a significant increase was observed in LIN, STR and WOB in photostimulated spermatozoa. Peroxynitrite production was significantly increased in the photostimulated spermatozoa, whereas viability, ROS production and intracellular Ca²⁺ levels were not affected by photostimulation. In conclusion, photostimulation of commercial boar semen has a positive effect on motility of spermatozoa subjected to a short‐term moderate thermal stress, which was concomitant with an increase in peroxynitrite production.     

In vitro effect of gold and silver nanoparticles on human spermatozoa

The cytotoxicity of Au/Ag nanoparticles (NPs) on human spermatozoa was investigated in vitro. Semen from donors were incubated (37 °C, 60′–120′) with 30, 60, 125, 250 and 500 μM Au/Ag‐NPs. Sperm motility was evaluated following WHO guidelines; sperm viability was assessed with eosin Y test. Au‐NPs were characterised and localised with field emission gun‐based scanning transmission electron microscope/energy dispersive spectroscopy and transmission electron microscopy. Both tested NPs exerted a significant dose‐dependent effect on motility and viability of human spermatozoa (P < 0.001). Ag‐NPs seem to show a slightly elevated toxicity although not significant (P > 0.05). Au‐NPs were localised in spermatozoa, whereas Ag‐NPs were undetectable. In conclusion, Au‐NPs and Ag‐NPs do not appear to be harmful for human spermatozoa up to high concentrations (250–500 μM) that are probably difficult to reach in vivo. It is mandatory to explore the genotoxic effect of NPs in germ cells.     

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.     

Cryoprotectant‐free ultra‐rapid freezing of human spermatozoa in cryogenic vials

The objective of this study was to investigate the effect of ultra‐rapid freezing (direct immersion in liquid nitrogen) on human spermatozoa in cryogenic vials (≥0.5 ml) at different concentrations of sucrose. After swim‐up, the sperm suspensions (N = 58) were diluted with sperm preparation medium and divided into six aliquots: swim‐up (fresh), conventional freezing group (slow freezing) and four ultra‐rapid freezing groups containing sucrose at different concentrations (0.15 m , 0.20 m , 0.25 m and 0.30 m ). Sperm motility, progressive motility, plasma membrane integrity, DNA stability and acrosome integrity of fresh and cooled‐warmed spermatozoa were analysed. The progressive motility, plasma membrane and acrosome integrity of spermatozoa in the 0.20 m sucrose group were significantly higher than those of the slow freezing group (47.5 ± 6.8% versus 36.4 ± 8.7%, 73.2 ± 6.9% versus 63.9 ± 6.3%, 53.7 ± 10.0% versus 35.9 ± 9.7% respectively, P < 0.05). However, no differences were found in sperm motility or DNA stability (58.5 ± 6.3% versus 54.2 ± 5.3%, 90.1 ± 2.8% versus 87.2 ± 4.7%, P > 0.05 respectively) between the 0.20 m sucrose and the slow freezing group. No differences were found between the ultra‐rapid and slow freezing group at the other concentrations of sucrose. Our findings suggest that the method of ultra‐rapid freezing of human spermatozoa in cryogenic vials with a solution containing 0.20 m sucrose results in recovery of spermatozoon of superior qualities. In contrast to slow freezing, the ultra‐rapid freezing technique of human spermatozoa seems to reduce cryoinjuries and maintain important physiological characteristics of the spermatozoa after warming.     

Alpha‐lipoic acid minimises reactive oxygen species‐induced damages during sperm processing

Shearing forces during sperm preparation for assisted reproduction techniques may lead to excessive production of reactive oxygen species (ROS) which may have an unpleasant effect on embryonic development. In the current study, we assessed the effect of alpha‐lipoic acid (ALA) on ROS‐induced damages during sperm preparation process. Semen samples were collected from 15 normozoospermic men. Each semen sample was divided into two parts; one part was washed and centrifuged with sperm washing medium plus 0.02 mM ALA. Then, sperm pellet was diluted and incubated for 1 hr at 37°C in sperm washing media in the absence (ALA?) or presence of 0.02 mM ALA (ALA+). The second part was washed and centrifuged with sperm washing media in the absence of ALA, and then, sperm pellet was incubated for 1 hr at 37°C in sperm washing media in the absence (ALA?) or presence of 0.02 mM ALA (ALA+). Sperm viability, motility, intracellular oxidative stress and DNA fragmentation were assessed by eosin‐nigrosin, computer‐assisted sperm analysis system, H₂DCFDA staining and acridine orange staining respectively. Our results showed that addition of ALA as a fat‐ and water‐soluble antioxidant to sperm washing media maintains sperm viability and motility by reduction in ROS production and can also protect sperm DNA integrity.     

Protective effect of the superoxide dismutase mimetic MnTBAP during sperm vitrification process

Sperm cryopreservation is widely used in assisted reproduction and male infertility therapy; however, it induces oxidative stress affecting sperm quality. This work evaluated the effect of the antioxidant MnTBAP during vitrification steps in human spermatozoa. First, the effect of MnTBAP on viability and ROS production was evaluated. Then, the spermatozoa were vitrified in straws with the vitrification, warming and post-warming incubation media separately supplemented with MnTBAP. An untreated control was included. The sperm viability, ROS production, total and progressive motility were evaluated. The results showed that the direct exposure of spermatozoa to MnTBAP significantly decreases the ROS levels in comparison with the untreated control without affecting the viability. The supplementation of the vitrification medium with MnTBAP did not affect the parameters analysed. However, the supplementation of the warming and incubation post-warming media resulted in a decrease in ROS production and maintained viability and motility for 4 hr after warming with concentrations up to 100 μM of MnTBAP. Higher concentrations of MnTBAP caused a decrease in total motility. In conclusion, the use of MnTBAP during the warming or post-warming incubation media has beneficial effect decreasing ROS levels and maintaining the viability and motility during the vitrification procedure.     

Comparison and evaluation of capacitation and acrosomal reaction in freeze‐thawed human ejaculated spermatozoa treated with L‐carnitine and pentoxifylline

Cryopreservation is used to preserve the spermatozoa; however, it leads to a reduction in sperm quality. L‐carnitine (LC) influences sperm motility and preserves the sperm membrane and DNA integrity. The objectives of this study were to evaluate the protective effects of LC on the membrane integrity of normal human spermatozoa and compare it with pentoxifylline (PT) during cryopreservation. Thirty normal semen samples, prepared by swim‐up procedure, were divided into three aliquots: a control without any treatment and two experimental aliquots that were incubated in PT or LC for 30 min. All aliquots were cryopreserved and thawed after 48 hr. To evaluate the percentages of intact, acrosomal‐reacted and capacitated spermatozoa, lectin histochemistry and flow cytometry were performed by wheat germ agglutinin, peanut agglutinin and Con A. Statistical analyses were performed using ANOVA. LC supplementation elevated the percentage of noncapacitated spermatozoa compared with control and PT‐treated samples and the percentages of acrosomal intact spermatozoa compared with PT‐treated samples. PT pre‐treatment improved the motility but not membrane integrity. LC supplementation reduced the percentages of acrosomal‐reacted spermatozoa compared with the control and PT‐treated samples. Although LC did not improve motility, it protected the plasma membrane and acrosomal integrity. Therefore, LC may be the superior choice compared to PT for maintaining the sperm integrity.     

Effects of H2O2 exposure on human sperm motility parameters,reactive oxygen species levels and nitric oxide levels

Research has revealed that reactive oxygen species (ROS) negatively affect sperm function, both in vivo and in vitro. Sperm preparation techniques for assisted reproductive technologies (ART) are potential causes for additional ROS production. This study aimed to correlate the concentration of exogenous H₂O₂ with sperm motility parameters and intracellular ROS and nitric oxide (NO) levels to reiterate the importance of minimising ROS levels in ART. Human spermatozoa from 10 donors were incubated and exposed to different exogenous H₂O₂ concentrations (0, 2.5, 7.5 and 15 μm ). Subsequently, motility was determined using computer‐aided semen analysis, while ROS (2,7‐dichlorofluorescin diacetate) and NO (diaminofluorescein‐2/diacetate) were analysed using fluorescence‐activated cell sorting. Results showed that H₂O₂ did affect the sperm parameters. Exogenous H₂O₂ was detrimental to motility and resulted in a significant increase in overall ROS and NO levels. A significant increase in static cells was seen as well. It is important to elucidate the mechanisms between intracellular ROS levels with sperm motility parameters. While this experiment demonstrated a need to reduce exogenous ROS levels during ART, it did not illustrate the cause and effect relationship of intracellular ROS and NO levels with sperm motility. Further research needs to be conducted to define a pathological level of ROS.     

N‐acetyl‐L‐cysteine pre‐treatment protects cryopreserved bovine spermatozoa from reactive oxygen species without compromising the in vitro developmental potential of intracytoplasmic sperm injection embryos

Excess of reactive oxygen species (ROS) on in vitro embryo production systems negatively affects the quality and developmental potential of embryos, as result of a decreased sperm quality and increased DNA fragmentation. This issue is of major importance in assisted fertilisation procedures such as intracytoplasmic sperm injection (ICSI), because this technique does not allow the natural selection of competent spermatozoa, and therefore, DNA‐damaged spermatozoa might be used to fertilise an egg. The aim of this study was to investigate a new strategy to prevent the potential deleterious effect of ROS on cryopreserved bovine spermatozoa. We evaluated the effect of a sperm pre‐treatment with different concentrations of N‐acetyl‐L‐cysteine (NAC) on ROS production, viability and DNA fragmentation and assessed the effect of this treatment on the in vitro developmental potential and quality of embryos generated by ICSI. The results show a strong scavenging effect of 1 and 10 mm NAC after exposure of spermatozoa to a ROS inducer, without compromising the viability and DNA integrity. Importantly, in vitro developmental potential and quality of embryos generated by ICSI with spermatozoa treated with NAC were not affected, confirming the feasibility of using this treatment before an ICSI cycle.     

Detection of a mutation in the intron of Sperm‐specific glyceraldehyde‐3‐phosphate dehydrogenase gene in patients with fibrous sheath dysplasia of the sperm flagellum

The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibres of the sperm flagellum. Dysplasia of the fibrous sheath (DFS) is a defect of spermatozoa observed in severe asthenozoospermic patients and characterised by morphologically abnormal flagella with distorted fibrous sheaths. Sperm‐specific glyceraldehyde‐3‐phosphate dehydrogenase (GAPDS) is a glycolytic enzyme that is tightly associated with the fibrous sheath of the sperm flagellum. The enzymatic activity of GAPDS was investigated in sperm samples of seven patients with DFS and compared to that of normal spermatozoa (n = 10). The difference in GAPDS activity in DFS and normal spermatozoa was statistically significant (0.19 ± 0.11 and 0.75 ± 0.11 μmol NADH per min per mg protein respectively). Immunochemical staining revealed irregular distribution of GAPDS in the flagellum of DFS spermatozoa. Other five samples with typical alterations in the fibrous sheath were assayed for mutations within human GAPDS gene. In all five cases, a replacement of guanine by adenine was revealed in the intron region between the sixth and the seventh exons of GAPDS. It is assumed that the deficiency in GAPDS observed in most DFS sperm samples is ascribable to a disorder in the regulation of GAPDS expression caused by the mutation in the intron region of GAPDS gene.     

The in vitro effects of melatonin on human sperm function and its scavenging activities on NO and ROS

Various systems of antioxidants exist endogenously in the body to help protect it against free radical damage by scavenging excessive ROS and RNS. Melatonin, a hormone secreted by the pineal gland, and responsible for controlling the circadian rhythm, is one such endogenous antioxidant. Melatonin has been reported to be present in human seminal fluid, but its antioxidant activities in semen are rather contradictory. This study aimed at establishing the effects of melatonin treatment on human spermatozoa. Spermatozoa were incubated with 2 mm melatonin (120 min, 37 °C, 5% CO₂) after which motility parameters were measured by computer aided motility analysis, while cell viability (PI), intracellular NO (DAF‐2/DA) and ROS (DCFH‐DA) were assessed using flow cytometry. In vitro melatonin treated samples (n = 12) showed a significantly higher percentage of motile, progressive motile and rapid cells, while simultaneously reducing the number of nonviable spermatozoa when compared with the control. Endogenous NO was significantly decreased, but no effect was observed on ROS levels. From these results, it can be concluded that melatonin was able to directly or indirectly scavenge NO, as indicated by the reduction in 4,5‐diaminofluorescein‐2/diacetate fluorescence. Future studies will indicate whether melatonin treatment during sperm preparation techniques could protect spermatozoa from excessive NO production.     

In vitro effects of nicotine on human spermatozoa

Washed human spermatozoa from 12 normozoospermic donors were treated with different concentrations of nicotine 0.1, 1.0, 5.0 and 10.0 mm and were compared to spermatozoa suspended in nutrient medium only (control). Computer‐aided sperm analysis was used to assess sperm kinematic properties after 30, 60, 120 and 180 min of incubation. Viability was assessed by means of a dye exclusion staining technique (eosin/nigrosin), while acrosome‐reacted cells were identified under a fluorescent microscope using fluorescein isothiocyanate–Pisum sativum agglutinin as a probe. Nicotine significantly reduced total motility, progressive motility, curvilinear velocity, amplitude of lateral head displacement, beat cross‐frequency, viability and caused spontaneous acrosome reaction at concentrations of ≥5.0 mm after 2 and 3 h of exposure. Nicotine concentrations of 0.1 and 1.0 mm had no significant effect (P < 0.05) on spermatozoa except that 1.0 mm significantly decreased (P < 0.05) sperm progressive motility at 2 and 3 h of incubation as well as viability after 3 h of incubation. This study concludes that the occurrence of high levels of nicotine in the body and seminal fluid might adversely affect fertilisation capacity of human spermatozoa through a mechanism that involves decreased motility, viability and premature induction of the acrosome reaction.     

Antioxidant effects of the essential oil of Syzygium aromaticum on bovine epididymal spermatozoa

Focusing on its application in reproductive biotechnology, we evaluated the effects of the essential oil of Syzygium aromaticum (EOSA) on bovine epididymal sperm quality variables, including morphology, membrane functional integrity, membrane structural integrity, mitochondrial activity, metabolic activity, motility and oxidative stress by reactive oxygen species (ROS) levels. Bovine spermatozoa from eight males were incubated into the following groups: EOSA0 (without EOSA), EOSA10 (10 μg/ml of EOSA), EOSA15 (15 μg/ml of EOSA) and EOSA20 (20 μg/ml of EOSA); the incubation time with and without the EOSA was 1 or 6 hr. None of the sperm quality variables presented difference among the EOSA concentrations. However, the incubation time had a significant effect on the membrane functional integrity, membrane structural integrity, mitochondrial activity, progressive motility and some kinetic parameters. The effect of interaction among EOSA and incubation time was significant only on ROS levels. Spermatozoa incubated in the presence of 15 μg/ml of the EOSA for 1 hr had significantly reduced ROS levels compared with all other groups in the same time. In conclusion, the EOSA at a concentration of 15 µg/ml has antioxidant effects and protects bovine epididymal spermatozoa; hence, the EOSA may potentially be used in the field of reproductive biotechnology.     

Comparison of Tris egg yolk‐based,Triladyl® and Optixell® extender on post‐thaw quality,Kinematics and in vivo fertility of Nili Ravi Buffalo (Bubalus bubalis) bull spermatozoa

Our objectives were to ascertain the comparison of Tris egg yolk‐based, Triladyl ^® and Optixell ^® extender on postthaw quality, CASA parameters and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen samples (n = 35) from five bulls were diluted in Tris egg yolk‐based, Triladyl ^® , Optixell ^® extender and frozen in 0.50 ml French straws. Postthaw sperm CASA motility (%) was higher (p < 0.05) in Optixell extender as compared to Triladyl and Tris egg yolk‐based extender. Although sperm progressive motility (%), morphology (%), average path velocity (μm/s), straight line velocity (μm/s), curvilinear velocity (μm/s), amplitude of lateral head displacement (μm), beat cross‐frequency (Hz), straightness (%), length of curvilinear path (μm), length of average path (μm), intact plasma and acrosome membrane (%), and DNA integrity (%) were higher (p < 0.05) in spermatozoa cryopreserved in Optixell ^® extender as compared to Tris egg yolk‐based and Triladyl ^® extender. The fertility rates (68.18%, 45.45%, 55.4%) were higher (p < 0.05) in buffaloes inseminated with semen doses frozen in Optixell extender than the Tris egg yolk‐based and Triladyl ^® extender respectively. It is concluded that Optixell ^® extender improves postthaw semen quality and fertility in Nili Ravi buffaloes.     

Double‐blind,randomised, placebo‐controlled trial on the effect of L‐carnitine and L‐acetylcarnitine on sperm parameters in men with idiopathic oligoasthenozoospermia

Carnitine is essential for energy metabolism and spermatozoa maturation. Combining L‐carnitine and L‐acetylcarnitine with micronutrients has been investigated as a treatment for infertility in men. We evaluated the effects of a therapeutic formulation, Proxeed Plus, on sperm parameters in oligoasthenozoospermic men. This prospective, randomised, double‐blind, placebo‐controlled clinical trial involved 175 males (19–44 years) with idiopathic oligoasthenozoospermia who failed to impregnate their partners (12 months). Males received Proxeed Plus or placebo for 3 and 6 months. Sperm volume, progressive motility and vitality significantly (p < 0.001) improved after 6 months compared to baseline. Sperm DNA fragmentation index significantly decreased compared to baseline (p < 0.001) and the 3‐month therapy (p = 0.014) in treated men. Increased seminal carnitine and α‐glucosidase concentration also positively correlated with improved progressive motility. Decreased DNA fragmentation index was the good predictor of progressive sperm motility >10%, and simultaneous measurement of changes in sperm vitality and DNA fragmentation index gave the highest probability of sperm motility 10% (AUC = 0.924; 95% CI = 0.852–0.996; p < 0.001). Logistic regression analyses revealed DNA fragmentation index decrease as the only independent predictor of sperm motility 10% (OR = 1.106; p = 0.034). We have demonstrated the beneficial effects of carnitine derivatives on progressive motility, vitality and sperm DNA fragmentation. Combining metabolic and micronutritive factors is beneficial for male infertility.     

A novel combination of silane‐coated silica colloid with hybrid RNA extraction protocol and RNA enrichment for downstream applications of spermatozoal RNA

Spermatozoa are specialised cells with low RNA content as compared to somatic cells. The suitable sperm RNA extraction and enrichment protocols for downstream applications are available for human, cattle, stallion and mouse but not for buffalo spermatozoa. Therefore, the present work was conducted to find out suitable colloidal solution for sperm purification and appropriate protocol for sperm RNA extraction and enrichment/amplification of RNA. For purification, we used PVP‐coated silica colloidal solution (PVP‐Si), silane‐coated silica colloidal solution (Silane‐Si) and iodixanol. Sperm recovery rate, total sperm motility and progressive sperm motility were significantly improved after separation by Silane‐Si and iodixanol compared to PVA‐Si method. The combined guanidinium thiocyanate–phenol–chloroform (GTPC) with silica matrix (SM)‐based RNA extraction yielded more quantity of RNA in compared to individual method. The hybrid of SM and GTPC into a single protocol yielded 360–450 ng RNA from 30 million buffalo spermatozoa. For the first time, we adopted new way to enrich sperm RNA that increased the RNA concentration 4–5 times that was sufficient for downstream applications. The linear amplification of sperm RNA increased RNA concentration around 27–45 times. In summary, Silane‐Si colloid for sperm separation, hybrid SM and GTPC protocol for sperm RNA extraction followed by enrichment or amplification of RNA was found suitable for high‐throughput analyses of buffalo sperm RNA.     

Effect of anion channel blockers on l‐arginine action in spermatozoa from asthenospermic men

In earlier studies, we have established that l‐ arginine enhances motility and metabolic rate in spermatozoa of goat, bull and mouse. In the present study this work was extended to human sperm cells obtained from the semen samples of asthenospermic patients, which are characterised by low motility. The metabolic rate was followed by monitoring the glucose consumption (1‐¹³C glucose as substrate) and the production of lactate in sperm cells, using ¹³C NMR. The stimulatory effect of l‐ arginine was neutralised on adding an NO‐synthase inhibitor like N^ω‐nitro‐l‐ arginine methyl ester. On the other hand, the inactive d ‐enantiomorph did not affect the stimulatory effect of l‐ arginine. This strongly suggests that l‐ arginine acts through the NO signal pathway. We also demonstrated that the stimulatory effect of l‐ arginine was inhibited in the presence of anion channel inhibitors like 4‐acetamido‐4′‐isothiocyanostilbene‐2,2′‐disulphonic acid, 2,4‐dinitrophenol and carbonyl cyanide m‐chlorophenylhydrazone. Furthermore, bicarbonate supplementation was found to be essential for the action of l‐ arginine. These observations indicate that l‐ arginine induces NO synthesis and stimulates motility and metabolism only when an active anion transport system is present.     

Effect of brain‐derived neurotrophic factor on sperm function,oxidative stress and membrane integrity in human

Oxidative stress has negative impacts on the clinical outcomes of assisted reproduction techniques. The brain‐derived neurotrophic factor (BDNF) promotes the viability of nerve cells and is known to decrease oxidative stress and apoptosis in different cells. The aim of this study was to evaluate the effect of BDNF treatment on human sperm functions that are known to be essential for fertilisation. Our findings showed that treatment of human spermatozoa with 0.133 nM BDNF significantly increased the percentages of both total (P = 0.001) and progressive (P < 0.01) motile sperm cells compared to those observed in the nontreated (control) group. We also showed that the mean fluorescence intensity of DCFH‐DA, as an indicator of intracellular reactive oxygen species, was significantly lower (P < 0.05) in spermatozoa treated with BDNF compared to the control group. Treatment of spermatozoa with BDNF significantly decreased the percentages of both dead (P = 0.001) and apoptotic‐like sperm cells (P < 0.05) compared to the control group. On the other hand, BDNF treatment significantly increased the percentage of viable sperm cells compared to the control (P = 0.001). In conclusion, BDNF has protective effects against oxidative stress in spermatozoa and could improve sperm functions that are essential for sperm–egg fusion and subsequent fertilisation.     

Effect of cysteine and glutamine added to extender on post‐thaw sperm functional parameters of buffalo bull

Amino acids seem to be crucial components for semen freezing extender due to antioxidant properties. Therefore, this study aimed to assess motility parameters, membrane integrity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage to detect the optimum concentrations of cysteine and glutamine for buffalo semen cryopreservation. Twenty ejaculates of four buffalo bulls were diluted in tris‐egg yolk extender and divided into seven equal groups consisting of cysteine (5, 7.5 and 10 mmol), glutamine (10, 15 and 20 mmol) and no additive. Supplementation of 5 and 7.5 mmol cysteine and 15 mmol glutamine in cryopreservation extender significantly increased post‐thaw motility and plasma membrane integrity of spermatozoa with significant reduction in intracellular ROS when compared with control groups (P < 0.05). Cysteine at 7.5 mmol concentration elevated progressive motility and MMP, compared with control (P < 0.05). No significant differences were observed for motion patterns and DNA damage of frozen–thawed buffalo spermatozoa in extender containing amino acids. The findings of this study showed that supplementation of 7.5 mmol cysteine and 15 mmol glutamine in semen cryopreservation extender has more potential to decrease intracellular ROS, and subsequently elevate motility and membrane integrity of buffalo frozen–thawed spermatozoa.