In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation

The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham′s F10 medium plus bovine albumin at 37° and 5% CO₂ for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P < 0.05) increase in oxygen‐free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P < 0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity.     

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.     

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.     

Association between body mass index and sperm quality and sperm DNA integrity. A large population study

This study aimed to analyse whether the functional quality of spermatozoa is associated with body mass index (BMI). Semen samples were obtained from 1824 men undergoing fertility evaluation/treatment. Semen analysis was performed using World Health Organization (WHO) criteria, and morphology was evaluated with the motile sperm organelle morphology examination (MSOME). The percentages of sperm DNA fragmentation (using TdT (terminal deoxynucleotidyl transferase)‐mediated dUTP nick‐end labelling (TUNEL) assays), sperm chromatin packaging/underprotamination (using chromomycin A3/CMA₃), mitochondrial damage (using MitoTracker Green) and apoptosis (using annexin V) were also assessed. At least 200 spermatozoa were examined in each evaluation. The following BMI values were used as cut‐off points: ≤24.9 kg/m², 25–29.9 kg/m² (overweight) and ≥30 kg/m² (obese). High BMI negatively affects sperm concentration, vitality, motility and morphology (p < .05). Conversely, high BMI does not seem to be associated with impaired sperm DNA integrity, as assessed by DNA fragmentation, sperm protamination and sperm apoptosis (p > .05). However, increased BMI is associated with increased mitochondrial damage in spermatozoa (p < .05). In conclusion, given the adverse consequences of obesity and the possible effect of male BMI on assisted reproduction technology (ART) outcomes, the benefits of weight reduction should be discussed when counselling couples interested in fertility treatment.     

Effect of Typha capensis (Rohrb.)N.E.Br. rhizome extract F1 fraction on cell viability,apoptosis induction and testosterone production in TM3‐Leydig cells

Typha capensis (Rohrb.)N.E.Br. (bulrush) is used by traditional healers in Southern Africa to treat male reproductive problems. This study aimed at investigating the effects of T. capensis on TM3‐Leydig cells. T. capensis rhizome crude extract obtained from autumn, winter, spring and summer harvest was fractionated using HPLC into four fractions, and TM3‐Leydig cells were incubated with different concentrations of the F1 fraction (0.01, 0.02, 0.1, 1, 10 and 100 μg/ml) for 24, 48 and 96 hr respectively. The following parameters were evaluated: cell morphology, viability (MTT assay), testosterone production (testosterone ELISA test), apoptosis (Annexin V‐Cy3 binding) and DNA fragmentation (TUNEL assay). Results revealed that the summer harvest obtained the highest amount of extract. The F1 fraction of all harvests was the most effective. This fraction significantly enhanced testosterone production in TM3 cells in a dose‐dependent manner with maximum effect at 0.1 μg/ml. At higher concentrations, lower testosterone production was observed. Cell viability including apoptosis was not affected at concentrations used by the traditional healers to treat patients. This study shows that T. capensis enhanced testosterone production and might be useful to treat male infertility and ageing male problems.     

Leucocytes and intrinsic ROS production may be factors compromising sperm chromatin condensation status

Considering that the final protection of the DNA against major assaults in terms of chromatin condensation is finalised in the epididymis, it is not known how sperm production of reactive oxygen species (ROS) and inflammatory processes can contribute to protamine deficiency that is predetermined in the testes. Therefore, this study aimed at investigating relationships between poor chromatin condensation, morphology, ROS production, DNA damage and the impact of the presence of leucocytes. In 70 patients, sperm DNA status was determined using TUNEL and chromomycin A₃ (CMA₃) assays, and ROS‐production by means of dihydroethidine. Morphology was evaluated according to strict criteria. The percentage of CMA₃‐positive spermatozoa and leucocyte concentration (r = 0.178, P = 0.0377) as well as percentage of ROS‐positive spermatozoa (r = 0.3010; P = 0.012) correlated significantly. Particularly, patients with leucocyte counts >0.5 × 10⁶ ml^?1 exhibited higher CMA₃ positivity. No association was found between CMA₃ positivity, TUNEL positivity and sperm morphology. While P‐ (poor prognosis: 0–4% normal morphology) and G‐pattern (good prognosis: 5–14% normal morphology) morphology did not differ regarding chromatin condensation, P‐pattern patients had a significantly higher percentage of DNA fragmentation (P = 0.0323). As oxidative stress is associated with disturbed chromatin condensation, results suggest that the idea that under‐protamination of sperm DNA will automatically lead to DNA fragmentation might have to be revisited.     

A comparative study of two cooling protocols on stallion sperm cryosurvival

There are many protocols for horse sperm cryopreservation, but results are inconsistent; sperm survival after freeze‐thawing is usually poor; in consequence, fertility is low. The objective of this work was to see whether slow cooling before freezing to minus 3 °C instead of +5 °C, the traditional target temperature, could improve horse sperm cryosurvival, capability to carry out capacitation and the acrosome reaction induced by progesterone. Spermatozoa from five stallions were packaged in straws and slowly cooled to +5 °C. Half of the straws were frozen directly and the other half was further cooled to ?3 °C before freezing. Progressive motility, viability, plasma membrane integrity, acrosome integrity and capacitation status were assessed. After thawing, there were no differences between cooling treatments on motility, viability, acrosome integrity and capacitation status; however, there was difference (P < 0.05) regarding plasma membrane integrity. Acrosome integrity decreased as incubation, without or with progesterone (2 μg ml^?1), progressed, but there were no differences between cooling treatments regardless of progesterone. Both capacitated and acrosome‐reacted spermatozoa increased as incubation progressed, but there were no differences between cooling treatments regardless of progesterone. Slow cooling to ?3 °C before freezing did not improve horse sperm cryosurvival or capability to undergo the acrosome reaction.     

Participation of membrane adenylyl cyclase in heparin‐induced capacitation in cryopreserved bovine spermatozoa

The aim of this work was to study the participation of membrane adenylyl cyclase in heparin‐induced capacitation in cryopreserved bovine spermatozoa. Sperm suspensions were incubated in Tyrode's albumin lactate pyruvate medium in the presence of heparin (10 IU ml^?1) or forskolin (1–75 μm ), a well‐known membrane adenylyl cyclase activator. The participation of membrane adenylyl cyclase was confirmed using a specific inhibitor, 2′,5′‐dideoxyadenosine (6–25 μm ). Spermatozoa capacitated with forskolin (25 μm ) were incubated with bovine follicular fluid to evaluate their ability to undergo acrosome reaction. Capacitation percentages were determined by the fluorescence technique with chlortetracycline, and true acrosome reaction was determined by trypan blue and differential interferential contrast. The forskolin concentrations employed had no effect on progressive motility or sperm viability. Capacitation values induced by 25‐μm forskolin treatment (27.80 ± 2.59%) were significantly higher respect to the control (4.80 ± 1.30%). The inhibitor 2′,5′‐dideoxyadenosine prevented forskolin‐induced capacitation and significantly diminished capacitation induced by heparin. Follicular fluid induced physiological acrosome reaction in spermatozoa previously capacitated with 25‐μm forskolin (P < 0.05). Forskolin acts as a capacitation inducer and involves the participation of membrane adenylyl cyclase as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.     

Variations in creatine kinase activity and reactive oxygen species levels are involved in capacitation of bovine spermatozoa

The generation of reactive oxygen species (ROS) is associated with some factors such as oxidative substrate sources, mitochondrial function and NAD(P)H oxidase activity. In bovine spermatozoa, heparin capacitation produces a respiratory burst sensitive to diphenyleneiodonium (DPI). Creatine kinase (CK) is related to extramitochondrial ATP disponibility. Our purpose was to determine the variation in ROS level and its relation with NAD(P)H oxidase sensitive to DPI and CK participation, as factors involved in redox state and energy generation in capacitation. The chlortetracycline technique was used to evaluate capacitation. CK activity and ROS level were measured by spectrophotometry and spectrofluorometry respectively. The capacitation percentage was increased by heparin or quercetin treatment (P < 0.05) and no significant differences in sperm viability were observed. Samples treated with heparin or quercetin maintained the same ROS level as control (238.62 ± 23.47 arbitrary units per 10⁸ spermatozoa) (P > 0.05). CK activity decreased by 50% with heparin or quercetin (P < 0.05). In DPI presence, capacitation was inhibited and differential CK activities and ROS level variations were observed in heparin- or quercetin-treated samples (P < 0.05). In cryopreserved bovine spermatozoa, capacitation requires equilibrium between oxidative damage susceptibility and ROS levels. CK activity is associated with redox state variation and energy sources. In conclusion, capacitation induction depends on NADPH oxidase and the shuttle creatine–creatine phosphate, both sensitive to DPI.     

In vitro equine embryo production using air‐dried spermatozoa,with different activation protocols and culture systems

The aim of this work was to evaluate the use of air‐dried spermatozoa for in vitro production of equine embryos and verify if sperm extract activation and in vivo culture improve in vitro embryo production. Cooled spermatozoa (control) and air‐dried spermatozoa stored for 2, 14 or 28 days were used for ICSI sperm extract, or ionomycin was used for oocyte activation, and embryos were in vitro or in vivo (in mare′s oviduct) cultured for 7 days. With in vitro culture, cleavage rate was higher when activating with sperm extract (P < 0.05). No differences in embryo development were seen between the two activation treatments nor between storage periods (P > 0.05). Blastocysts were obtained with cooled spermatozoa, and morulae were achieved using in vivo culture with 28‐day storage spermatozoa and ionomycin‐activated oocytes. When in vivo culture was performed, sperm DNA fragmentation was assessed using the sperm chromatin dispersion test and did not show statistical correlation with cleavage nor embryo recovery rates. In conclusion, equine embryos can be produced using air‐dried spermatozoa stored for several weeks. Sperm extract activation increased cleavage rates but did not improve embryo development. In vivo culture allowed intrauterine stage embryos to be achieved.     

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.     

Proper ubiquitination effect on the fertilisation outcome post‐ICSI

Ubiquitin is an 8.5‐kDa protein that tags outlived proteins for degradation by the proteasome. It also marks defective spermatozoa during epididymal passage and has been proposed as a biomarker of sperm quality. This study evaluates the relationship between sperm ubiquitination, protamine deficiency, semen parameters and fertilisation rate in infertile individuals undergoing the intracytoplasmic sperm insemination (ICSI) procedure. Semen samples from 73 ICSI candidates were collected and analysed according to World Health Organization criteria. A portion of each sample was evaluated for sperm ubiquitination using the sperm ubiquitin tag immunoassay (SUTI) with flow cytometry, and protamine deficiency by chromomycin A3 (CMA3) staining. In addition, the relationship between the fertilisation rate and sperm ubiquitination was calculated in ICSI candidates. The intensity of ubiquitination showed a significant negative correlation with sperm concentration (r = ?0.255, P = 0.032) and a positive correlation with fertilisation rate (r = 0.384, P = 0.013) post‐ICSI. No correlation was observed between protamine deficiency and the percentage of ubiquitination or ubiquitination intensity. The results of this study suggest that sperm ubiquitination prior to capacitation may be considered as a marker of defective spermatozoon. Spermatozoa that undergo proper ubiquitination may have a higher chance for fertilisation, because they are made redundant by the ubiquitin–proteasome pathway in the epididymis compared to hypo‐ubiquitinated spermatozoa.     

Freeze‐dried stallion spermatozoa: evaluation of two chelating agents and comparative analysis of three sperm DNA damage assays

During the freeze‐drying procedure, sperm DNA might become damaged by both freezing and drying stresses. Sperm DNA status can be detected using well‐established assays; however, most techniques are expensive and involve elaborate protocols and equipment. Indirect assessments can provide alternative strategies. The objective of this study was to compare a simple test of DNA status using Diff‐Quik (DQ) with two established procedures: acridine orange test (AOT) and sperm chromatin dispersion (SCD) on freeze‐dried (FD) stallion spermatozoa. Ejaculated spermatozoa from three stallions were freeze‐dried in basic medium supplemented with two different chelating agents: EGTA or EDTA. After rehydration, the spermatozoa were subjected to DNA damage detection using a SCDt, AOT and DQ stain simultaneously. The results showed that the DNA damage levels in the EGTA group were significantly lower than those in the EDTA group. AOT detected a significantly higher proportion of spermatozoa with fragmented DNA than DQ and SCD. The results of the SCD test and DQ stain exhibited a significant positive correlation for DNA fragmentation (r = 0.528), whereas a negative correlation was observed between SCD, DQ and AOT (r = ?0.134 and r = ?0.332 respectively). The present study shows that both the SCD test and DQ assay are effective methods for detecting FD stallion sperm DNA fragmentation, whereas using of AOT is questionable.     

Spermicidal activity of Indian seaweeds: an in vitro study

Contraceptive properties of seaweeds are still stands as lacuna; in this context, the screening of in vitro male contraceptive properties of crude ethanolic extract of Indian seaweeds against normal human sperm is carried out. In total, twelve seaweeds were screened for in vitro spermicidal activity. Among these twelve seaweeds, Halimeda gracilis showed 100% inhibition of human spermatozoa at 10 mg ml^?1 concentration in 20 s and its EC₅₀ value was 2.05 mg ml^?1 in 20 s. Further, dose‐ and time‐dependent spermicidal assay revealed that the sperm was completely immobilised for 20 s. Plasma membrane of sperm was damaged due to the exposure of H. gracilis extract. MTT assay with H. gracilis extract showed 88.5% of cytotoxic incidence. H. gracilis extract tested for cytotoxicity against Artemia salina recorded LC₅₀ value of 34.8 μg ml^?1. Phytochemical analysis of H. gracilis extract evidenced the presence of alkaloids, flavonoids, proteins and sugars. Results of this study clearly inferred that the synergistic effect of active principles reside within the H. gracilis extract had shown better contraceptive activity.     

Effect of in vitro selenium supplementation on sperm quality in asthenoteratozoospermic men

Sperm DNA damage, excessive oxidative stress and decrease in motility ?may lead to low fertilisation or poor? assisted reproductive techniques outcomes in asthenoteratozoospermic ?men. Selenium was considered as essential element for male reproductive functions. Selenium has important role in enzymatic process for elimination of excessive reactive oxygen species and helps to maintain membrane integrity. The aim of this study was to determine the effect of selenium supplementation on sperm quality, DNA fragmentation, mitochondrial membrane potential and membrane lipid peroxidation during sperm sampling in vitro at different times. In this experimental study, semen samples were collected from 50 asthenoteratozoospermic men. Samples were divided into two groups as control group and test group (incubated with 2 μg/ml selenium at 37°C for 2, 4 and 6 hr). Motility and viability were assessed based on WHO 2010 criteria. Mitochondrial membrane potential, sperm DNA fragmentation and malondialdehyde levels were evaluated in each group. Results revealed that motility, viability and mitochondrial membrane potential were significantly higher in the test group (p < .05). Also malondialdehyde levels were significantly lower in the test group (p < .03). DNA fragmentation significantly decreased in the test group after 6 hr of incubation (p < .02). In conclusion, in vitro selenium supplementation may protect spermatozoa from maltreatment effect of reactive oxygen species (ROS) during sperm sampling via keeping enzymatic and antioxidant process in optimum condition.     

Supplementing oregano essential oil to boar diet with strengthened fish oil: Effects on semen antioxidant status and semen quality parameters

Previous research has shown benefits of dietary fish oil supplementation on semen quality of boars. However, little is known about how antioxidant protects lipid peroxidation on spermatozoa from n‐3 polyunsaturated fatty acid (PUFA) addition. This study evaluated the effect of oregano essential oil (OEO) supplementation on semen antioxidant status and semen quality in boars fed a diet enriched with fish oil. Thirty‐four mature boars of proven fertility, received daily 2.5 kg basal diet top‐dressed with 45 g soybean oil and 15 g fish oil to meet the n‐3 PUFA requirement of spermatozoa, randomly allocated to one of four groups supplemented with 100 mg α‐tocopheryl acetate kg^?1 (control), or 250 or 500 or 750 mg OEO kg^?1 for 16 weeks. Semen was collected at weeks 0, 8, 12 and 16 for measurements of sperm production, motion characteristics, sperm α‐tocopherol content, antioxidant enzyme activities, reactive oxygen species (ROS), DNA damage (8‐hydroxydeoxyguanosine, 8‐OHdG), lipoperoxidation (malondialdehyde, MDA) and seminal total antioxidant capacity (TAC). Sperm production and motion characteristics were similar (p > .05) among groups throughout the experimental week 16, but increased (p < .01) with experimental week. Although higher α‐tocopherol content and superoxide dismutase (SOD) activities were in OEO group spermatozoa, feeding diet with 500 mg/kg OEO resulted in elevation in seminal TAC, decrease in sperm ROS, MDA and 8‐OHdG than control group (p < .05). Overall, these results support the view that oregano essential oil has a positive effect on antioxidant capacity in boar when used fish oil.     

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.     

Oxidative stress‐induced alterations in seminal plasma antioxidants: Is there any association with keap1 gene methylation in human spermatozoa?

Kelch‐like ECH‐associated protein 1 (keap1)‐nuclear factor‐erythroid 2‐related factor 2 (Nrf2) pathway is one of the master regulators of cellular defence against oxidative stress. Epigenetic alterations like hypermethylation of keap1 gene impair keap1‐Nrf2 system in several oxidative stress–associated diseases. The objective of this study was to evaluate the epigenetic status of keap1 in sperm DNA of normozoospermic subjects, having different levels of reactive oxygen species (ROS) in seminal plasma. Semen samples were obtained from 151 apparently healthy male partners of couples who attended the Avicenna infertility clinic. Samples were categorised into four groups according to their ROS levels: group A (n = 39, ROS < 20 RLU/s per 10⁶ spermatozoa), group B (n = 38, 20 ≤ ROS < 40 RLU/s per 10⁶ spermatozoa), group C (n = 31, 40 ≤ ROS < 60 RLU/s per 10⁶ spermatozoa) and group D; (n = 43, ROS ≥ 60 RLU/s per 10⁶ spermatozoa). Keap1 methylation status was assessed using methylation‐specific PCR along with seminal total antioxidant capacity. The results showed no significant alterations in keap1 methylation in any groups, whereas the total antioxidant capacity enhanced with increasing levels of ROS exposure. These results indicate that keap1 was not methylated during ROS elevation and oxidative stress, suggesting that the cells have adopted other mechanisms to elevate antioxidant level.     

Conventional slow freezing cryopreserves mouflon spermatozoa better than vitrification

This work examines the effectiveness of a TCG (Tris, citric acid, glucose, 6% egg yolk and 5% glycerol) and a TEST (TES, Tris, glucose, 6% egg yolk and 5% glycerol) sperm extender in the freezing of mouflon spermatozoa at slow cooling rates, using different pre‐freezing equilibration times (2–3 hr). It also examines the tolerance of mouflon spermatozoa to different concentrations of cryoprotectants (5, 10, 20% glycerol; 5%, 10%, 20% dimethyl sulfoxide; 6% polyvinylpyrrolidone) and/or sucrose (100, 300, 500 mm ). The highest quality (p < .01) thawed spermatozoa were obtained when using the TEST extender and an equilibration time of 3 hr. Sperm motility and membrane integrity were strongly reduced when using rapid freezing rates (60–85°C min^?1), independent of the concentration of cryoprotectants. The lowest sucrose concentration (100 mm ) provided the highest (p < .05) percentage of motile spermatozoa and live spermatozoa with an intact acrosome. Vitrified–warmed sperm variables were at their best when the spermatozoa was diluted in TCG–6% egg yolk + 100 mm sucrose and warmed at 60°C. Slow warming at 37°C strongly reduced (p < .05) sperm motility and viability. However, sperm vitrification returned lower fertility, sperm motility and sperm viability values than conventional sperm freezing.     

Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa

The objective of this study was to examine the effect of reactive oxygen species (ROS) and cryopreservation on DNA fragmentation of equine spermatozoa. In experiment 1, equine spermatozoa were incubated (1 hour, 38 degrees C) according to the following treatments: 1) sperm alone; 2) sperm + xanthine (X, 0.3 mM)-xanthine oxidase (XO, 0.025 U/mL); 3) sperm + X (0.6 mM)-XO (0.05 U/mL); and 4) sperm + X (1 mM)-XO (0.1 U/mL). In experiment 2, spermatozoa were incubated (1 hour, 38 degrees C) with X (1 mM)-XO (0.1 U/mL) and either catalase (200 U/mL), superoxide dismutase (SOD, 200 U/mL), or reduced glutathione (GSH, 10 mM). Following incubation, DNA fragmentation was determined by the single cell gel electrophoresis (comet) assay. In experiment 3, equine spermatozoa were cryopreserved, and DNA fragmentation was determined in fresh, processed, and postthaw sperm samples. In experiment 1, incubation of equine spermatozoa in the presence of ROS, generated by the X-XO system, increased DNA fragmentation (P <.005). In Experiment 2, the increase in DNA fragmentation associated with X-XO treatment was counteracted by the addition of catalase and GSH but not by SOD, suggesting that hydrogen peroxide and not superoxide appears to be the ROS responsible for such damage. In experiment 3, cryopreservation of equine spermatozoa was associated with an increase (P <.01) in DNA fragmentation when compared with fresh or processed samples. This study indicates that ROS and cryopreservation promote DNA fragmentation in equine spermatozoa; the involvement of ROS in cryopreservation-induced DNA damage remains to be determined.