In vitro effects of nonylphenol on motility,mitochondrial, acrosomal and chromatin integrity of ram and boar spermatozoa

The objective of this study was to determine the effects of nonylphenol (NP) on viability of ram and boar sperm in vitro. Ram or boar spermatozoa were exposed to 1, 10, 100, 250 and 500 μg NP ml^?1 for 1, 2, 3 or 4 h. Computer‐assisted sperm motility analysis (CASA) system was used to evaluate sperm motility characteristics. Flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity, while epifluorescent microscopy was used to determine sperm acrosomal status. Exposure of both species spermatozoa to 250 and 500 μg NP ml^?1 was detrimental to progressive motility (P < 0.05), and its adverse effect was significant at lower (100 μg NP ml^?1) concentration (P < 0.05). The percentages of ram and boar spermatozoa with high MMP declined drastically after exposures to ≥250 μg ml^?1 NP (P < 0.05). Unlike chromatin integrity, which did not appear to be altered by NP exposure, there were dose‐dependent NP effects (P < 0.05) on acrosomal integrity of both species at as low as 1 μg ml^?1 NP for boar spermatozoa and 10 μg ml^?1 NP for ram spermatozoa. These data show adverse effects of NP on ram and boar spermatozoa and thus its potential harmful effects on male reproduction as NP is found in fruits, vegetables, human milk, fish and livestock products.     

l‐Cysteine improves antioxidant enzyme activity,post‐thaw quality and fertility of Nili‐Ravi buffalo (Bubalus bubalis) bull spermatozoa

The effects of l ‐cysteine in extender on antioxidant enzymes profile during cryopreservation, post‐thaw quality parameters and in vivo fertility of Nili‐Ravi buffalo bull spermatozoa were studied. Semen samples from 4 buffalo bulls were diluted in Tris–citric acid‐based extender having different concentrations of l ‐cysteine (0.0, 0.5, 1.0, 2.0 and 3.0 mm ) and frozen in 0.5‐ml French straws. The antioxidative enzymes [catalase, super oxide dismutase and total glutathione (peroxidase and reductase)] were significantly higher (P < 0.05) at pre‐freezing and post‐thawing in extender containing 2.0 mm l ‐cysteine as compared to other groups. Post‐thaw total motility (%), progressive motility (%), rapid velocity (%), average path velocity (μm s^?1), straight line velocity (μm s^?1), curvilinear velocity (μm s^?1), beat cross frequency (Hz), viable spermatozoa with intact plasmalemma (%), acrosome and DNA integrity (%) were higher with the addition of 2.0 mm l ‐cysteine as compared to other groups (P < 0.05). The fertility rates (59 versus 43%) were higher (P < 0.05) in buffaloes inseminated with doses containing 2.0 mm of l ‐cysteine than in the control. In conclusion, the addition of 2.0 mm l ‐cysteine in extender improved the antioxidant enzymes profile, post‐thaw quality and in vivo fertility of Nili‐Ravi buffalo bull spermatozoa.     

Effect of Eurycoma longifolia Jack (Tongkat ali) extract on human spermatozoa in vitro

Eurycoma longifolia (Tongkat ali; TA) is a Malaysian shrub used to treat various illnesses including male infertility. Considering that TA is used to improve male fertility and no report regarding its safety has been published, this study investigated the effects of TA extract on various sperm functions. Semen samples of 27 patients and 13 donors were divided into two groups, washed and swim‐up spermatozoa, and incubated with different concentrations of TA (1, 10, 20, 100, 2000 μg ml^?1) for 1 h at 37 °C. A sample without addition of TA served as control. For washed spermatozoa, significant dose‐dependent trends were found for vitality, total motility, acrosome reaction and reactive oxygen species‐positive spermatozoa. However, these trends were only significant if the highest concentrations were included in the calculation. Contrary, the increase in the percentage of acrosome‐reacted spermatozoa with increasing TA concentrations is very significant (P < 0.0001), and a significant difference (P = 0.0069) to the control could even be recorded at 20 μg TA per ml. For swim‐up spermatozoa, no trend could be observed. Results indicate that the TA extract has no deleterious effects on sperm functions at therapeutically used concentrations (<2.5 μg ml^?1). However, at very high concentrations, TA may have harmful effects in vitro.     

Antioxidant effect of hydroxytyrosol on human sperm quality during in vitro incubation

The aim of the study was to evaluate the antioxidant potential of hydroxytyrosol (HT) on human sperm quality during incubation in vitro. Semen samples collected from men attending the Laboratory of Histology‐Embryology of Sfax Faculty of Medicine (Tunisia) for infertility investigations were evaluated for initial sperm parameters. Only normal selected ejaculates (n = 15) were centrifuged and incubated further with or without HT (200ug ml^?1) at room temperature for 45 min. After incubation, sperm motility and viability, DNA oxidation and reactive oxygen species (ROS) production were assessed. The results showed that centrifugation significantly influenced sperm motility and viability. The supplementation of HT in incubating media improved (P = 0.01) significantly sperm viability and decreased sperm DNA oxidation (P < 0.001) and ROS levels (P = 0.03) following centrifugation. It can be concluded that supplementation of HT might be helpful to maintain the human spermatozoon after centrifugation.     

Catalase improves motility,vitality and DNA integrity of cryopreserved human spermatozoa

Cryopreservation of human spermatozoa offers a pre‐therapeutic possibility of preserving progenity in patients with testicular tumours. We aimed to investigate effects of cryopreservation and addition of catalase on sperm motility, vitality and DNA integrity in fresh and swim‐up spermatozoa. Semen samples were collected from 50 fertile men. Each sample was divided into two parts. First part was subdivided into two equal aliquots: both cryopreserved with and without catalase. The second part was subdivided into two equal aliquots: both processed by swim up and then cryopreserved with or without catalase. Semen analyses, sperm vitality and sperm DNA integrity were performed. Sperm concentration showed significant decrease while percentage of progressive motility, sperm vitality and % of DNA damage showed significant increase in processed and cryopreserved processed samples when compared with fresh and cryopreserved fresh samples. There was no significant difference in sperm concentration while there was significant increase in % of progressive motility and sperm vitality and % of DNA damage showed significant decrease in samples with catalase when compared with samples without catalase (either fresh or processed). Catalase supplementation (fresh and processed) during cryopreservation results in better post‐thawing percentage of progressive motility and percentage of sperm vitality and improved DNA integrity.     

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.     

Ultrastructure evaluation of goat spermatozoa after freezing in a skim milk‐based extender with Trolox supplementation

The aim of this work was to evaluate the in vitro effect of adding Trolox in freezing extender for goat semen. Ejaculates from five bucks were evaluated, and when approved, the samples were pooled, diluted according to experimental groups [Trolox 0 (control), 30, 60 and 120 nmol ml^?1] and frozen in an automated system. Thawed samples (37 °C/30 s) were evaluated for plasma membrane (PMi) and acrosome integrity (Aci), mitochondrial membrane potential (MMP) and sperm kinematics by CASA system. Spermatozoa ultrastructure was evaluated in fresh and post‐thawed semen. No significant difference (P > 0.05) was observed among control and Trolox groups in the analyses of PMi, Aci, MMP and CASA in goat spermatozoa after thawing. Samples of 60 and 120 nmol ml^?1 Trolox groups had a higher percentage of cells that had intact plasma membranes in spermatozoa head than in the other groups, although they did not differ (P > 0.05) before being frozen. A higher percentage (P < 0.05) of spermatozoa with intact mitochondria was observed in fresh semen, control and Trolox 60 nmol ml^?1 groups than in the other groups. Addition of Trolox to skim milk extender at 60 nmol ml^?1 ultrastructurally preserves the plasma membrane and mitochondrial sheath integrity in goat spermatozoa 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.     

Antioxidant effect of lemon balm (Melissa officinalis) and mate tea (Ilex paraguensys) on quality,lipid peroxidation and DNA oxidation of cryopreserved boar epididymal spermatozoa

In this study, we investigated the protective ability of the addition of two antioxidant herb extracts, mate tea and lemon balm, on boar epididymal frozen–thawed spermatozoa quality. Testes from mature boars were collected at local slaughterhouse, and sperm samples from epididymis were recovered by flushing. Spermatozoa were cryopreserved in lactose–egg yolk buffer supplemented with various concentrations of lemon balm and mate tea (0, 2.5, 5 and 10 g l^?1) using the straw‐freezing procedure. Motion parameters, acrosome and plasma membrane integrity, lipoperoxidation levels and DNA oxidative damage (8‐hydroxy‐2′‐deoxyguanosine base lesion) were evaluated. There were no differences among experimental groups with regard to motility characteristics, viability, acrosome and plasma membrane integrity; however, the highest concentration of lemon balm produced significant (P < 0.05) improvement in curvilinear trajectory, straightness and amplitude of lateral head displacement after thawing. The supplementation of freezing extender with mate tea and lemon balm reduced sperm lipid membrane peroxidation, and only mate tea protected DNA against oxidative damage during cryopreservation at 120 min post‐thawing (P < 0.05). Mate tea experimental extender at concentration of 10 g l^?1 showed the lowest percentage of sperm oxidised DNA and malondialdehyde generation; thus, mate tea is a potential candidate such as antioxidant compound on boar sperm cryopreservation medium.     

Comparison of cryopreserved human sperm from solid surface vitrification and standard vapor freezing method: on motility, morphology, vitality and DNA integrity

Solid surface vitrificaition (SSV) is a cryoperservative method that has been used in the cryopreservation of oocytes, and embryos. Here, we report an application of the SSV in the cryopreservation of human spermatozoa. We compared the SSV with a standard freezing method in terms of sperm motility, morphology, vitality and DNA integrity. Sperm motility was determined by computer assisted semen analysis, morphology and vitality were determined by eosin-methylene blue staining, and DNA integrity was determined by a TUNEL assay. We found that while both cryopreservative methods produced spermatozoa with comparable vitality and motility, the SSV gave slightly, but significantly fewer sperm with DNA damage, and loose tail. We concluded that, a cryopreservation of human spermatozoa by SSV is feasible and provides a quick and practical way to preserve human spermatozoa with a comparable, if not better, quality of the preserved spermatozoa to the standard freezing method.     

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 new approach for cryoprotectant‐free freezing of goat epididymal spermatozoa

A cryoprotectant‐free method was successfully used for rapid freezing of goat epididymal spermatozoa. Lowering sperm volume may increase the temperature exchange rate and improve the freezing output of spermatozoa. The aim of this study was to compare two different packaging types [0.25 ml French straws (FS) and 96‐well immune plate (WIP)] for rapid freezing of goat epididymal spermatozoa. Eleven pairs of the goat testes were transferred to the laboratory; cauda epididymidides were dissected and sliced in TRIS‐BSA solution for 15 min and temperature 33–35 °C. Sperm concentration was adjusted to 20 × 10⁶ ml^?1, and the suspension was subjected to rapid freezing within FS or WIP. The volume of spermatozoa in WIP method was set at 25 μl. Sperm motility, viability and abnormalities, and sperm DNA integrity were compared between two devices. The results showed similar effectiveness of WIP and FS on post‐thaw sperm parameters. In conclusion, for cryoprotectant‐free rapid freezing of goat epididymal spermatozoa, it is recommended to use WIP instead of French 0.25 ml straws.     

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.     

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.     

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 metabolic and antioxidant supplementation on sperm parameters in oligo‐astheno‐teratozoospermia,with and without varicocele: A double‐blind placebo‐controlled study

Since sperm require high energy levels to perform their specialised function, it is vital that essential nutrients are available for spermatozoa when they develop, capacitate and acquire motility. However, they are vulnerable to a lack of energy and excess amounts of reactive oxygen species, which can impair sperm function, lead to immotility, acrosomal reaction impairment, DNA fragmentation and cell death. This monocentric, randomised, double‐blind, placebo‐controlled trial investigated the effect of 6 months of supplementation with l ‐carnitine, acetyl‐l ‐carnitine and other micronutrients on sperm quality in 104 subjects with oligo‐ and/or astheno‐ and/or teratozoospermia with or without varicocele. In 94 patients who completed the study, sperm concentration was significantly increased in supplemented patients compared to the placebo (p = .0186). Total sperm count also increased significantly (p = .0117) in the supplemented group as compared to the placebo group. Both, progressive and total motility were higher in supplemented patients (p = .0088 and p = .0120, respectively). Although pregnancy rate was not an endpoint of the study, of the 12 pregnancies that occurred during the follow‐up, 10 were reported in the supplementation group. In general, all these changes were more evident in varicocele patients. In conclusion, supplementation with metabolic and antioxidant compounds could be efficacious when included in strategies to improve fertility.     

Gradient sperm selection for reproductive techniques in cattle: Is Isolate a suitable replacement for Percoll?

In assisted reproductive techniques, it is essential to perform a sperm selection to obtain spermatozoa with high motility and membrane integrity for in vitro fertilisation (IVF) and high‐DNA integrity for intracytoplasmic sperm injection (ICSI). In this study, we evaluated whether Isolate ^® was a suitable substitute for Percoll ^® for assisted reproductive techniques. Commercial cryopreserved bovine semen was used after selection in both gradients, and plasma and acrosome membrane integrity, reactive oxygen species (ROS) levels, DNA integrity and mitochondrial membrane potential (ΔΨm) were assessed by flow cytometry. Motility parameters were also evaluated by CASA system. A similar percentage of spermatozoa with intact plasma membrane, acrosome integrity and high ΔΨm was observed in both sperm selection methods, but only Percoll ^® showed higher percentage of spermatozoa with intact plasma and acrosome membrane compared to the post‐thawing group. No differences were observed in the motility, ROS, DNA fragmentation and on the in vitro embryo production in all experimental groups. In conclusion, the selection of bovine spermatozoa with Isolate ^® generates spermatozoa with similar quality parameters and embryonic development compared to Percoll ^® providing a suitable alternative sperm selection method for assisted reproductive techniques in this species.     

Short‐term storage of salmonids semen in a sodium alginate‐based extender

Short‐term storage of semen is a useful strategy for preservation of fish spermatozoa. However, there is a significantly decrease on sperm function mainly due to oxidative stress. In this way, sodium alginate plays an important role as free radical scavenger compound. Accordingly, the aim of our study was to analyse the effect of a sodium alginate‐based extender on sperm function in the short‐term storage of salmonids semen. Samples of Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss were stored in Storfish^® (Ext‐C) and Storfish^® supplemented with sodium alginate (Ext‐A) during 10 days at 4°C. After storage, motility, viability, mitochondrial membrane potential (ΔΨmit), superoxide anion (O₂^?) level and DNA fragmentation (DNA Frag) were assessed. Ext‐A had positive effect in preservation of sperm motility, viability, ΔΨmit, O₂^? level and DNA integrity in the three species analysed compared to control samples. In Ext‐A, the spermatozoa of S. salar and O. mykiss showed significantly higher motility, viability and ΔΨmit than O. kisutch. However, O. kisutch and O. mykiss had significantly lower O₂^? level than S. salar, and DNA fragmentation in O. kisutch and S. salar was significantly lower than in samples of O. mykiss (p < 0.05). Dilution of salmonids semen in a sodium alginate‐based extender is effective for protecting sperm quality during 10 days of short‐term storage.     

Sperm abnormalities induced by pre‐pubertal exposure to cyclophosphamide are effectively mitigated by Moringa oleifera leaf extract

Moringa oleifera L. is a medicinal plant with potential antioxidant property. This study was aimed at investigating the chemoprotective effect of Moringa oleifera leaf extract (MOE) on cyclophosphamide (CP)‐induced testicular toxicity. Two‐week‐old male Swiss albino mice were intraperitoneally injected with phosphate‐buffered saline, 50 mg kg^?1 of CP and 25 mg kg^?1 of MOE. In combination treatment, mice were injected with 25 mg kg^?1 of MOE 24 h prior to CP injection, 24 h prior and post‐CP injection and 24 h post‐CP injection for 5 consecutive days (10 mg kg^?1). Six weeks later, mice were sacrificed to assess epididymal sperm parameters. MOE alone did not have any significant effect on sperm parameters. However, acute injection of CP resulted in significant decline in motility (P < 0.001), increase in head abnormality (P < 0.01) and DNA damage (P < 0.05). Combining MOE with CP increased the sperm density, motility and reduced head defect and DNA damage, irrespective of the schedule and dosage of MOE. Administration of MOE prior to CP significantly elevated the level of superoxide dismutase and catalase with concomitant decrease in lipid peroxidation in the testicular tissue. In conclusion, MOE may have potential benefit in reducing the loss of male gonadal function following chemotherapy.     

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.