Comparative study of fertility parameters in vitrified human spermatozoa in the presence or absence of EmbryORP®: A novel antioxidant

The cryopreservation of spermatozoa has the main purpose of preserving male fertility. However, current preservation techniques have shown to produce lesions in the structure and alter sperm functions, probably due to the production of reactive oxygen species (ROS) during cryopreservation. To overcome the damage provoked by ROS, we introduced a novel antioxidant called EmbryORP^® in a vitrification protocol and compared eight fertility parameters: motility, viability, morphology, concentration, the semen pH, the oxidation-reduction potential (ORP), the spontaneous acrosomal reaction (AR) and the mitochondrial membrane potential (MMP), in the presence or absence of EmbryORP^®. We analysed 20 samples from healthy human sperm donors and observed that the antioxidant significantly decreased the semen pH as well as the MMP and the ORP affecting the balance of ROS. The antioxidant also lowered the motility and viability of the cells, but preserved the acrosome and sperm morphology in general. We concluded that EmbryORP^® lowered the ORP, but to a suboptimal level that may be harmful to spermatozoa. Despite these results, our work opens new perspectives on how to improve cryopreservation media. Therefore, we recommend exploring the EmbryORP^® potential benefit by reducing its concentration or changing the exposure time during the cryopreservation protocol.     

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.     

Antioxidant effects of penicillamine against in vitro-induced oxidative stress in human spermatozoa

Oxidative stress contributes importantly to the aetiology of male infertility, impairing sperm function. The protective effect of antioxidants on seminal parameters has been established, and the antioxidant penicillamine has shown beneficial effects; however, its protective effect on human spermatozoa exposed to oxidative stress has not been reported. The objective of this work was to evaluate the effect of penicillamine on human spermatozoa exposed in vitro to oxidative stress. First, the effect of penicillamine on spermatozoa from normozoospermic donors was evaluated. Then, the effect of penicillamine on spermatozoa exposed to oxidative stress induced separately by ionomycin and hydrogen peroxide (H₂O₂) was analysed. An untreated control and a control treated only with the oxidative stress inducer were included. Reactive oxygen species (ROS) levels, viability, mitochondrial membrane potential (MMP) and motility were analysed. The results showed that penicillamine, added to the incubation medium, decreased the ROS levels induced by ionomycin and H₂O₂, and this effect was associated with better preservation of MMP, motility, and ATP levels. These results highlight the potential advantages of penicillamine supplementation of sperm culture medium, especially for semen samples with high ROS levels and also in circumstances where laboratory handling can cause an increase in ROS production.     

Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity

The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 °C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 μ m Fe²⁺/1 m m ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA®) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 ± 2.9%) and OXI (11.6 ± 7.6%) ( p  < 0.001). There were no significant effects of OXI on sperm viability, acrosomal status or proportion of abnormal tails. %DFI (spermatozoa with moderate or high DNA Fragmentation Index) was significantly higher on OXI ( p  < 0.001). Catalase prevented DNA fragmentation even in the presence of the oxidant (%DFI: 30.3 ± 0.8% OXI vs. 17.4 ± 0.7% CAT/OXI). We conclude that catalase supplementation after thawing could protect bull spermatozoa against oxidative stress, and it could improve media used for processing thawed spermatozoa.     

Effects of long-term in vitro incubation of human spermatozoa: functional parameters and catalase effect

Prolonged incubation of human spermatozoa can have deleterious effects on sperm function. The aim of this paper was to describe the effects of a prolonged in vitro incubation, under similar conditions to those employed in human assisted reproduction, on various sperm functional parameters, and to investigate the effect of an antioxidant (catalase) on this system. Freshly collected ejaculates from 20 healthy donors were studied. Samples were divided into two aliquots: the first was incubated with Ham's F10 containing 3.5% HAS, and the second was incubated in the same medium plus catalase (100 units ml-1). All experiments were carried out with spermatozoa isolated using the swim-up technique. Spermatozoa recovered from the supernatant after 1 h (T1) of incubation in 5% CO2 in air at 37 degrees C, and after 5 h (T6), 23 h (T24) and 47 h (T48), were evaluated for concentration, motion parameters including hyperactivation (computer-assisted analysis), viability, ATP concentration, reactive oxygen species (ROS) generation, DNA integrity (acridine orange), and acrosome reaction (AR). The major alteration observed in sperm function during the prolonged in vitro incubation was a reduction in the number of motile spermatozoa, together with an impairment in the quality of sperm movement. ROS levels increased with the incubation time. No substantial modifications of sperm viability, chromatin condensation and AR inducibility were observed. The addition of catalase to the medium, while keeping ROS values within baseline levels, did not prevent the loss of motility or the corresponding increase in ATP.     

Porcine sperm vitrification I: cryoloops method

The aims of this study were to evaluate porcine sperm vitrification in cryoloops, with and without two different cryoprotectants and assess two warming procedures. Extended (n = 3; r = 4) and raw (n = 5; r = 2) semen was diluted in media without and with cryoprotectants (4% dimethylformamide and 4% glycerol) to a final concentration of 20 × 10⁶ spermatozoa ml^?1 and vitrified using the cryoloops method. Two warming procedures were evaluated: rapid method (30 s at 37°C) and an ultra‐rapid method (7 s at 75°C, followed by 30 s at 37°C). Total motility (phase contrast), sperm viability (6‐carboxifluorescein diacetate and propidium iodide stain), membrane function (hypo‐osmotic swelling test), acrosome integrity (phase contrast), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated before and after vitrification and analysed using Friedman's test. In all media, the only seminal parameters that were maintained after vitrification were chromatin condensation and integrity. Vitrification of porcine spermatozoon using cryoloops, both in the presence or absence of cryoprotectants and independent of the warming procedure used, permits conservation of sperm chromatin condensation and integrity. It would be interesting to further verify this by producing porcine embryos using vitrified spermatozoon with intracytoplasmic sperm injection.     

Porcine sperm vitrification II: Spheres method

Owing to current problems in boar sperm cryopreservation, this study proposes to evaluate vitrification in spheres as an alternative cryopreservation procedure, comparing the use or not of permeable cryoprotectants and two warming methods. Extended (n = 3; r = 4) and raw (n = 5; r = 2) porcine spermatozoa were diluted in media, in the absence or presence of either 4% dimethylformamide or 4% glycerol, to a final concentration of 5 × 10⁶ spermatozoa/ml and vitrified using the spheres method. Two warming procedures were evaluated: a rapid method (30 s at 37°C) and an ultrarapid method (7 s at 75°C, followed by 30 s at 37°C). Percentages of total motility (phase contrast), membrane function (hypo‐osmotic swelling test), acrosome integrity (phase contrast), sperm viability (6‐carboxyfluorescein diacetate and propidium iodide stain), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated in the samples before and after vitrification. Results, analysed using Friedman's test, suggest that rapid warming of raw porcine spermatozoa vitrified without permeable cryoprotectants may preserve DNA condensation and integrity better than the other processing methods studied in this work. Hence, porcine sperm vitrification using spheres could be used to produce embryos with ICSI to further validate this method.     

Sperm separation in patients with urogenital infections

Summary. A total of 196 patients attending the Center of Dermatology and Andrology, Giessen, Germany, were examined for fertility problems. Polymorphonuclear elastase, reactive oxygen species (ROS) and the number of round and peroxidase-positive cells were investigated in addition to routine semen analysis. The ejaculates were also analysed before and after sperm separation by means of swim-up or glass wool filtration. In 20 cases of leukocytospermia, sperm concentration, motility, viability, production of reactive oxygen species, and the number of peroxidase-positive cells were evaluated before and after glass wool filtration. The results show that ROS production by viable spermatozoa is highly correlated with the concentration of PMN elastase and the number of both peroxidase-positive and round cells. Multiple regression analysis with motility as dependent parameter showed the number of round cells ( n = 91; r = -0.332; P = 0.0030) to be the most important parameter affecting motility, while ROS mainly affects the viability of spermatozoa ( n = 69; r = 0.250; P = 0.0107). In the case of leukocytospermia, glass wool filtration significantly reduced the number of peroxidase-positive cells and ROS production ( P = 0.0098 and P = 0.0005, respectively). Receiver operating characteristic (ROC) curve analysis for ROS production in the ejaculate using a concentration of 1.000 ng ml⁻¹ PMN elastase as decisive parameter resulted in a cut-off value of 49,489.9 counts 10⁻⁷ viable spermatozoa. The statistical parameters were: Sensitivity: 63.2%, specificity: 100%, positive predictive value: 100%, negative predictive value: 36.1%.     

Effects of reactive oxygen species from activated leucocytes on human sperm motility, viability and morphology

The accumulated data suggest that inflammation can increase the level of reactive oxygen species (ROS), which contribute to impaired sperm function and male infertility. Therefore, we propose that inflammation-mediated production of ROS in male and female reproductive tracts hinder sperm fertilisation. To test this hypothesis, phorbol myristate acetate (PMA) with polymorphonuclear leucocytes (PMNs) was applied to generate endogenous ROS. We evaluated the time-dependent effects of ROS on human sperm motility, viability and mitochondrial membrane potential (MMP). The results showed that after treatment with PMA and PMNs, the motility of human spermatozoa significantly decreased to 50% on Day 1 and 15% on Day 4 compared with that of the, respectively, negative controls (P = 0.012). The viability of human spermatozoa decreased on Day 4 of PMA + PMNs treatment (P = 0.028). The MMP of human spermatozoa significantly decreased from Day 2 to Day 4 in the PMA + PMN group compared with that of the controls (P = 0.019). Taken together, the 4-day cultivation approach provided an accurate evaluation of sperm quality, especially sperm motility and MMP. Our findings indicated that endogenous inflammation increased ROS levels, which might induce sperm oxidative damage. Additionally, sperm motility might be one of the earliest and most sensitive indicators of this damage.     

Sperm quality improvement after date seed oil in Vitro supplementation in spontaneous and induced oxidative stress

In vitro supplementation with date seed oil （DSO） can protect spermatozoa against hydrogen peroxide （HiO2）- mediated damage and can improve sperm function, possibly owing to antioxidant properties. We tested the antioxidant effects of DSO on human sperm motility, sperm viability, reacted acrosome and lipid peroxidation assessed in vitro after H202-mediated oxidative damage in spermatozoa. Sixteen patients （mean age： 35 years; range： 25-45 years） referred to the Histology-Embryology Laboratory of the Medicine Faculty of Sfax for semen analysis after 12-24 months of sexual intercourse without conception were selected. After spermiogram, sperm selection by twointerface discontinuous Sill Select gradient was performed, and selected spermatozoa were used in four experimental assays： control; incubation with 100um H2O2; incubation with 0.1% DSO; and co-incubation with 0.1% DSO and 100 um H2O2. Motility and viability were determined using World Health Organization criteria. Acrosome reaction and lipid peroxidation were assessed by staining with fluorescein isothiocyanate-Pisum sativum and spectrophotometric measurement of malondialdehyde, respectively. Results showed that incubation with H2O2 alone led to a significant increase in lipid peroxidation （57.83%, P 〈 0.05） associated with a significant decrease in sperm motility, sperm viability （after 30 min and 24 h） and percentage of reacted acrosome （P 〈 0.05）. Date seed oil im- proved sperm motility after 24 h of incubation （P 〈 0.05） and protected spermatozoa against the deleterious effects of H2O2 on motility, viability, acrosome reaction and lipid peroxidation. We conclude that supplementation with DSO may have a function in antioxidant protection against male infertility.     

异黄酮辅剂对解冻精子特征的影响

本研究评价了使用异黄酮作为辅剂的解冻剂对解冻精子精液参数的影响。我们分析了辅剂在解冻过程中对精子活力、精子获能能力（膜脂功能紊乱）、活性氧生成、精子核浓缩及DNA损害的影响。根据初步的数据,辅剂可能会改善解冻过程,减少细胞损伤。我们已经证实异黄酮在精子解冻过程中有抗氧化作用、能够减少活性氧的生成,有利于精子活力轻微提高,降低膜脂功能紊乱和由冷冻而引起的DNA破坏。结果显示异黄酮作为辅剂有助于提高精子功能,这对于辅助生育中使用解冻精子很有意义。我们的发现有必要进一步研究来确证并评价临床应用的可能性。     

Ideal culture time for improvement in sperm motility from testicular sperm aspirates of men with azoospermia

PURPOSE: The motility of testicular derived spermatozoa reflects viability and predicts success during intracytoplasmic sperm injection. Although improvements in sperm motility are seen after incubation for extended periods, no guidelines suggest duration or media use for optimal improvement in motility. MATERIALS AND METHODS: Between July 1999 and February 2005 testicular aspirations were performed on 95 men with azoospermia, including 51 with obstructive azoospermia and 44 with nonobstructive azoospermia. Sperm motility was determined at initial collection and following incubation for 24 or 48 hours in processing media or Ham's F10 + protein. A mixed regression model controlling for testis side, media and baseline motility was created to analyze the change in motility between 24 and 48 hours. RESULTS: Mean motility improved from 3% to 20% at 24 hours and 25% at 48 hours for OA cases and from 0% to 5% at 24 hours and 11% at 48 hours for nonobstructive azoospermia cases. The improvement in motility from 24 to 48 hours was significant for obstructive azoospermia cases (p = 0.001). While media was a nonsignificant factor in regression models, when patients were grouped into categories of motility change there was a significantly better response to F10 compared to processing media (p = 0.03). CONCLUSIONS: Incubation in processing media or Ham's F10 + albumin media improves sperm motility with significant improvement noted between 24 and 48 hours for obstructive azoospermia cases. Ham's F10 + albumin media may provide extra benefit for cases of nonobstructive azoospermia or nerve injury. These results suggest the ideal timing of oocyte retrieval for intracytoplasmic sperm injection correlates with 48-hour sperm incubation for obstructive azoospermia cases, and 24 hours for nonobstructive azoospermia and nerve injury cases.     

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.     

Monitoring the reactive oxygen species in spermatozoa during liquid storage of boar semen and its correlation with sperm motility,free thiol content and seasonality

Oxidative stress is an important factor affecting the quality of spermatozoa during liquid storage of boar semen; however, monitoring of reactive oxygen species (ROS) that provides direct insight into the oxidative status is not yet attempted. This study aimed to monitor ROS in boar sperm during liquid semen storage to determine its correlation with sperm motility and free thiol (SH) content, and seasonality. Ejaculate was collected from mature Duroc boars in a commercial farm in autumn and spring, diluted in Mulberry III extender, stored at 15°C, and examined daily for sperm ROS level, SH content and motility. The ROS levels in spermatozoa prepared during autumn and spring were constantly low until days 4 and 5 of storage, respectively, which thereafter progressively increased in association with the loss of sperm motility. The increased sperm ROS level correlated with the higher SH level and lower motility, which was accentuated from day 4 of storage and was higher in September, or early autumn. This study indicates that increased sperm ROS levels during liquid storage results in oxidative damage, causing loss of sperm motility, presumably through decreased sperm viability, suggesting that sperm ROS monitoring effectively evaluates the quality of boar semen.     

Human sperm DNA oxidation, motility and viability in the presence of L-carnitine during in vitro incubation and centrifugation

In vitro incubation and centrifugation is known to decrease human sperm quality. In the human body, besides its antioxidant effects, L-carnitine (LC) facilitates the transport of activated fatty acids from the cytosol to the mitochondrial matrix. In this study, we investigated the effect of LC on human sperm motility, viability and DNA oxidation after incubation and centrifugation, following the sperm preparation protocols of assisted reproduction. Normozoospermic semen samples (n = 55) were analysed according to the World Health Organization (WHO) guidelines. LC concentrations that are not toxic to spermatozoa as determined by sperm motility and viability were standardised after 2 and 4 h of incubation at 37 °C. Semen samples to which the optimal LC concentrations were added were also centrifuged for 20 min at 300 g and analysed for sperm motility, viability and DNA oxidation. Sperm motility was improved at 0.5 mg ml(-1) LC after incubation and centrifugation with 5 × 10(6) sperm ml(-1). Higher concentration of LC (50 mg ml(-1)) significantly decreased sperm motility and viability. LC did not alter the baseline of sperm DNA oxidation during both incubation and centrifugation. In conclusion, LC may enhance sperm motility following incubation and centrifugation, while it might not affect sperm viability and DNA oxidation.     

The effect of cysteine and glutamine on human sperm functional parameters during vitrification

Assuming the adverse effects of reactive oxygen species (ROS) on sperm function, this study was conducted to assess the effects of cysteine and glutamine as effective antioxidants on human sperm parameters under vitrification. Twenty normozoospermic samples were used. The samples were subjected to a vitrification process and cysteine (5 and 10 mM) and glutamine (10 and 15 mM). The sperm motility parameters, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI), DNA damage and intracellular ROS damage were assessed for each sample. Statistical analyses showed that motility, mitochondrial membrane potential and DNA damage decreased in the vitrified groups with cysteine 5, 10 mM and glutamine 10, 15 mM separately. Also intracellular ROS increased significantly compared to the fresh group (p < .05). No significant differences were observed for PMI compared with the fresh group (p > .05). Supplementation of cysteine and glutamine in both concentrations separately decreased intracellular ROS and DNA damage of spermatozoa with significant increase in PMI, MMP and progressive motility compared to vitrified control group (p < .05). The results showed no significant effect of a specific concentration in cysteine and glutamine on sperm parameters compared to other concentrations. Both amino acids have the potential to improve the harmful effects of freezing on sperm parameters.     

The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation

The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the 30-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P < .01) increase in hydrogen peroxide production and an associated decrease (P < .01) in motility parameters. Total motility was significantly (P < .01) lower in the presence of X-XO compared with the case of the control (29%+/-9% vs 73%+/-1%, respectively). Catalase, but not SOD, prevented a decline in motility secondary to oxidative stress (71%+/-4% vs 30%+/-3%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the 30-minute incubation (53%+/-3%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.     

Supplementation of IVF medium with melatonin: effect on sperm functionality and in vitro produced bovine embryos

Gamete co‐incubation generates high free radical levels surrounding growing zygotes which may impair subsequent embryo viability. Melatonin eliminates a wide variety of free radicals; hence, we tried to improve in vitro embryo production by adding melatonin to in vitro fertilisation (IVF) media in high (Exp. 1) and low concentrations (Exp. 2), and we evaluated its effect on bull sperm function during IVF co‐incubation time (Exp. 3). In Experiment 1, we supplemented IVF media culture with 0.01, 0.1 and 1 mmol of melatonin, along with a no melatonin control group. In Experiment 2, melatonin levels were reduced to 10, 100 and 1000 nmol, with a no melatonin control group. In Experiment 3, spermatozoa were incubated in IVF media with melatonin (as Exp. 2) and functional parameters were analysed at 0, 4 and 18 h. In Experiment 1, only 1 mmol melatonin showed lesser blastocyst rates than control (C: 23.2 ± 6.7% versus 1 mmol: 2.0 ± 1.7%). In Experiment 2, no statistical differences were found in cleavage percentage, blastocyst percentage and total cell count for any melatonin treatment. In Experiment 3, sperm samples with 1000 nmol melatonin had a significantly higher wobbler (WOB) coefficient, a lower percentage of intact acrosomes, a lower percentage of viable spermatozoa with ROS, greater DNA fragmentation and higher DNA oxidation than controls. Total fluorescence intensity for ROS at 10 nmol melatonin was significantly greater than controls (P < 0.05). IVF media with 1 mmol melatonin is deleterious for embryo development, and in lower concentrations, it modulated sperm functionality, but had no effects on embryo production.     

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.     

Lycopene and male infertility

Excessive amounts of reactive oxygen species （ROS） cause a state of oxidative stress, which result in sperm membrane lipid peroxidation, DNA damage and apoptosis, leading to decreased sperm viability and motility. Elevated levels of ROS are a major cause of idiopathic male factor infertility, which is an increasingly common problem today. Lycopene, the most potent singlet oxygen quencher of all carotenoids, is a possible treatment option for male infertility because of its antioxidant properties. By reacting with and neutralizing free radicals, lycopene could reduce the incidence of oxidative stress and thus, lessen the damage that would otherwise be inflicted on spermatozoa. It is postulated that lycopene may have other beneficial effects via nonoxidative mechanisms in the testis, such as gap junction communication, modulation of gene expression, regulation of the cell cycle and immunoenhancement. Various lycopene supplementation studies conducted on both humans and animals have shown promising results in alleviating male infertility--lipid peroxidation and DNA damage were decreased, while sperm count and viability, and general immunity were increased. Improvement of these parameters indicates a reduction in oxidative stress, and thus the spermatozoa is less vulnerable to oxidative damage, which increases the chances of a normal sperm fertilizing the egg. Human trials have reported improvement in sperm parameters and pregnancy rates with supplementation of 4-8 mg of lycopene daily for 3-12 months. However, further detailed and extensive research is still required to determine the dosage and the usefulness of lycopene as a treatment for male infertility.