13 REACTIVE OXYGEN SPECIES IN STORED STALLION SEMEN

2013 ◽  
Vol 25 (1) ◽  
pp. 153
Author(s):  
J. M. Morrell ◽  
A. Lundgren ◽  
P. Humblot ◽  
A. Johannisson
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Flow Cytometry ◽  
Sperm Motility ◽  
Chromatin Structure ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Pregnancy Rates ◽  
Oxygen Species ◽  
Progressive Motility

The quality of cooled semen doses for AI varies considerably between stallions. One of the factors affecting sperm quality may be the content of reactive oxygen species (ROS), which are known to affect fertility in some species. The objective of this study was to measure the ROS content of cooled stored stallion semen doses for AI as part of an evaluation of sperm quality and to correlate it with pregnancy rates. Ejaculates (3 per stallion) were collected from 14 stallions at a commercial stud farm and were extended in INRA 96 (IMV Technologies) as standard cooled semen doses for AI. After transporting the semen doses overnight to the laboratory at the Swedish University of Agricultural Sciences (Uppsala, Sweden) in an insulated container with a cold pack, aliquots were evaluated for sperm quality and ROS content by staining with hydroethidine and dihydrodichlorofluorescein diacetate and measuring fluorescence by flow cytometry (Guthrie and Welch 2006 J. Anim. Sci. 84, 2089–2100). Seven sperm sub-populations were quantified: superoxide positive or negative, living (S+L and S–L, respectively); superoxide positive, dead (S+D); and hydrogen peroxide positive or negative, living or dead (P+L, P–L, P+D, and P–D, respectively). Sperm motility was measured by computer-assisted sperm motility analysis (Sperm Vision, Minitube), membrane integrity by flow cytometry, and chromatin structure using the sperm chromatin structure assay (all assays described in Morrell et al. 2011 Theriogenology 76, 1424–1432). Pregnancy rates following AI with cooled semen doses from the same stallions were available later in the year. The effect of stallion was tested by ANOVA. Correlations were calculated between the proportions of sperm stained for S or P and other sperm quality parameters and also with pregnancy rates (SAS version 9.1, SAS Institute Inc., Cary, NC, USA). The effect of stallion was significant on all variables measured (P < 0.01). There were no significant correlations between percentages of S+L or P+L spermatozoa and progressive motility, membrane integrity, or DNA fragmentation index (%DFI). There was a trend for the proportion of P-L spermatozoa to be correlated with progressive motility (r = 0.51; P < 0.07) whereas the proportions of S+D and P–D were negatively correlated with progressive motility (r = –0.66 and –0.61; P < 0.02). The proportion of S+D and chromatin damage were negatively correlated (r = –0.54; P < 0.05). There was a trend for the proportions of S+D and P+D to be negatively related to the overall pregnancy rate (r = –0.52; P < 0.07, and r = –0.58; P < 0.05, respectively). The proportions of superoxide and hydrogen peroxide-containing live spermatozoa were not correlated to stallion fertility or other parameters of semen quality in stored semen samples. In contrast, a significant negative relationship was found between the proportion of dead spermatozoa producing superoxide radicals and progressive motility, and between dead spermatozoa producing superoxide radicals and chromatin damage. However, many factors contribute to sperm quality and fertility, with many interactions between them. More work is needed to unravel these different effects and determine which factors can be used to predict stallion fertility.

scholarly journals CYTOPROTECTIVE EFFECT OF ETHANOL FRACTION OF VERNONIA AMYGDALINA DEL. LEAVES AGAINST THE VERO CELLS

2018 ◽  
Vol 11 (13) ◽  
pp. 146
Author(s):  
Safriana Safriana ◽  
Rosidah Rosidah ◽  
Poppy Anjelisa Zaitun Hasibuan ◽  
Denny Satria
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Flow Cytometry ◽  
Viable Cell ◽  
Vero Cells ◽  
Oxygen Species ◽  
Vernonia Amygdalina ◽  
Cytoprotective Effect ◽  
Cytoprotective Activity ◽  
Ethanol Fraction

Objectives: The objective of this study is to assess the cytoprotective effect of ethanol fraction (EtF) of Vernonia amygdalina Del. leaves EtF against Vero cells which induced by hydrogen peroxide (H2O2).Methods: Cytoprotective effects of EtF were analyzed by 3-(4,5-dimethylthiazolyl-2)2,5-dipheniltetrazolium bromide to Vero cells which induced by 0.8 mM H2O2, apoptosis was determined by flow cytometry, and reactive oxygen species (ROS) expression was analyzed by immunocytochemistry.Results: EtF was showed the largest percentage viable cell (78.75±2.51%) at 50 μg/mL. In the analysis of apoptosis by flow cytometry was showed the percentage of viable cell count of 59.56% and EtF was decreased the expression of ROS.Conclusion: EtF has cytoprotective activity toward Vero cells induced by 0.8 mM H2O2.

scholarly journals Single Layer Centrifugation with Androcoll-P Can Be Scaled-Up to Process Larger Volumes of Boar Semen

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Marjet van Wienen ◽  
Anders Johannisson ◽  
Margareta Wallgren ◽  
Joyce Parlevliet ◽  
Jane M. Morrell
Keyword(s):  
Reactive Oxygen Species ◽  
Sperm Quality ◽  
Scale Up ◽  
Membrane Integrity ◽  
Single Layer ◽  
Ros Production ◽  
Oxygen Species ◽  
Normal Morphology ◽  
Preliminary Step ◽  
Boar Semen

The objective of this study was to scale-up the procedure for Single Layer Centrifugation (SLC) through AndrocollTM-P, as a preliminary step towords processing the whole ejaculate. The first experiment compared Single Layer Centrifugation using 4.5 mL and 15 mL extended ejaculate (SLC-4.5 and SLC-15, resp.), assessing sperm quality by objective motility analysis, morphology, viability, and the production of reactive oxygen species (ROS). In the second experiment, SLC-4.5 was compared to Single Layer Centrifugation with 25 mL extended ejaculate (SLC-25) using motility analysis and morphology. In both experiments, normal morphology and linear motility were significantly higher in the SLC-selected samples than in the uncentrifuged controls (P<.001), whereas total motility and membrane integrity were unchanged. Although ROS production was higher in the SLC-selected samples than in the controls (P<.01), this might have been due to the presence of antioxidants in seminal plasma in the latter. In conclusion, there was no difference in sperm quality between SLC-4.5 and SLC-15 samples, or between SLC-4.5 and SLC-25 samples, indicating that the SLC method can be scaled-up successfully.

34 OVINE SPERM IS HIGHLY SUSCEPTIBLE TO ATTACK BY HYDROGEN PEROXIDE

2010 ◽  
Vol 22 (1) ◽  
pp. 175
Author(s):  
E. G. A. Perez ◽  
M. Nichi ◽  
F. A. Oliveira Neto ◽  
R. O. C. Silva ◽  
A. Dalmazzo ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Lipid Peroxidation ◽  
Hydroxyl Radical ◽  
Ferrous Sulfate ◽  
Present Experiment ◽  
Membrane Integrity ◽  
Oxygen Species ◽  
Mitochondrial Potential ◽  
Ram Sperm

Ram sperm membrane displays a particular lipid composition, especially regarding the high quantity of polyunsaturated cholesterol. This trait improves membrane fluidity; however, the spermatozoa become more susceptible to the attack of reactive oxygen species (ROS), which may lead to structural and functional damage, impairment or even impeded fecundity. The aim of the present experiment was to study the resistance of ovine spermatozoa to different ROS. Sperm samples from 4 rams were collected using an artificial vagina. Sperm samples were then incubated (1 h, 37°C) with four ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produces hydroxyl radical), and malondialdehyde (MDA, lipid peroxidation product). Samples were analysed using the 3-3′ diamino benzidine (DAB) stain as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity; the simple stain (fast green/Bengal rose) as an index of acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Results showed that acrosome and membrane integrity as well as mitochondrial potential were highly impaired by hydrogen peroxide, which was not the case for the other ROS (Table 1). Surprisingly, TBARS production was higher in samples incubated with ascorbate and ferrous sulfate (hydroxyl radical). Furthermore, sperm showing impaired mitochondrial potential were negatively correlated with membrane and acrosome integrities (r = -0.83, P < 0.0001 and r = -0.62, P = 0.01, respectively). Results of the present experiment suggest that semen of rams is extremely susceptible to attack by hydrogen peroxide. However, the mechanism by which this substance impairs sperm quality apparently does not involve oxidative stress, because no increase in TBARS was observed. Despite the necessity of further studies to investigate how hydrogen peroxide negatively influences sperm function, the use of catalase and glutathione peroxidase, important hydrogen peroxide scavengers, appears to be an alternative to improve the quality of ram sperm. Table 1.Effect of different reactive oxygen species in semen of rams The authors thank Nutricell for the media used in the experiment and CAPES for financial support.

Effect of adding heterologous versus homologous bovine seminal plasma prior to cryopreservation on bull sperm quality after thawing

Zygote ◽  
2018 ◽  
Vol 26 (5) ◽  
pp. 388-394
Author(s):  
Thanapol Nongbua ◽  
Essraa M Al-Essawe ◽  
Anders Edman ◽  
Anders Johannisson ◽  
Jane M Morrell
Keyword(s):  
Reactive Oxygen Species ◽  
Seminal Plasma ◽  
Deleterious Effect ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Reactive Oxygen ◽  
Low Fertility ◽  
Computer Assisted ◽  
High Fertility ◽  
Oxygen Species

SummaryThe aim of this study was to investigate the effect of adding homologous or heterologous bovine seminal plasma (SP) to SP-free sperm samples before freezing on sperm quality after thawing. Ejaculates from bulls of known fertility were used as a source of SP. The SP was removed from further aliquots of the same ejaculates by colloid centrifugation to create SP-free sperm samples; the resuspended sperm pellets were treated with homologous or heterologous SP from high or low fertility bulls at 0%, 1% or 5% before freezing. After thawing, sperm quality was evaluated by computer-assisted sperm analysis and flow cytometry for membrane integrity, reactive oxygen species, chromatin structure, mitochondrial membrane potential and protein tyrosine phosphorylation. Data were analysed using Proc MIXED, SAS®. Post-hoc comparisons were adjusted for multiplicity using Tukey’s method. The addition of SP resulted in significant differences in sperm quality, namely velocity class A, Velocity Straight Line (VSL), Velocity Average Path (VAP), Velocity Curved Line (VCL), Amplitude of Lateral Head Displacement (ALH), Hyperactive (HYP), reactive oxygen species (ROS) production and % DNA fragmentation index (DFI) (P<0.05 for each). Although adding 5% homologous SP from high fertility bulls was beneficial to sperm kinematics, 5% heterologous SP from high fertility bulls had a deleterious effect on chromatin integrity and on sperm velocity. In conclusion, adding SP may have either a beneficial effect or a deleterious effect depending on the individuals involved. It might be feasible to use this method to improve sperm quality in some circumstances.

scholarly journals Improved Post-Thaw Quality of Canine Semen after Treatment with Exosomes from Conditioned Medium of Adipose-Derived Mesenchymal Stem Cells

Animals ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 865 ◽  
Author(s):  
Ahmad Qamar ◽  
Xun Fang ◽  
Min Kim ◽  
Jongki Cho
Keyword(s):  
Reactive Oxygen Species ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Group 2 ◽  
And Function

Freezing decreases sperm quality, ultimately affecting fertilizing ability. The repair of freeze-damaged sperm is considered crucial for improving post-thaw viability and fertility. We investigated the effects of exosomes derived from canine adipose-derived mesenchymal stem cells on dog sperm structure and function during cryopreservation. The pooled ejaculate was diluted with buffer, without (Control), or with exosomal proteins (25, 50, or 100 µg/mL). Using fresh semen, the determined optimal exosomal protein concentration was 50 µg/mL (Group 2) which was used in further experiments. Post-thaw sperm treated with exosomes were superior to control (p < 0.05) in terms of motility (56.8 ± 0.3% vs. 47.2 ± 0.3%), live sperm percentage (55.9 ± 0.4% vs. 45.4 ± 0.4%), membrane integrity (55.6 ± 0.5% vs. 47.8 ± 0.3%), and acrosome integrity (60.4 ± 1.1% vs. 48.6 ± 0.4%). Moreover, expression of genes related to the repair of the plasma membrane (ANX 1, FN 1, and DYSF), and chromatin material (H3, and HMGB 1) was statistically higher in exosome-treated sperm than control, but the expression of the mitochondrial reactive oxygen species modulator 1 gene was significantly higher in control. Therefore, exosomal treatment may improve the quality of post-thaw dog semen through initiating damaged sperm repair and decreasing reactive oxygen species production.

scholarly journals CYTOPROTECTIVE EFFECT OF ETHANOL FRACTION OF VERNONIA AMYGDALINA DEL. LEAVES AGAINST THE VERO CELLS

2018 ◽  
Vol 11 (13) ◽  
pp. 214
Author(s):  
Safriana Safriana ◽  
Rosidah Rosidah ◽  
Poppy Anjelisa Zaitun Hasibuan ◽  
Denny Satria
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Flow Cytometry ◽  
Viable Cell ◽  
Vero Cells ◽  
Oxygen Species ◽  
Vernonia Amygdalina ◽  
Cytoprotective Effect ◽  
Cytoprotective Activity ◽  
Ethanol Fraction

  Objectives: The objective of this study is to assess the cytoprotective effect of ethanol fraction (EtF) of Vernonia amygdalina Del. leaves EtF against Vero cells which induced by hydrogen peroxide (H2O2).Methods: Cytoprotective effects of EtF were analyzed by 3-(4,5-dimethylthiazolyl-2)2,5-dipheniltetrazolium bromide to Vero cells which induced by 0.8 mM H2O2, apoptosis was determined by flow cytometry, and reactive oxygen species (ROS) expression was analyzed by immunocytochemistry.Results: EtF was showed the largest percentage viable cell (78.75±2.51%) at 50 μg/mL. In the analysis of apoptosis by flow cytometry was showed the percentage of viable cell count of 59.56% and EtF was decreased the expression of ROS.Conclusion: EtF has cytoprotective activity toward Vero cells induced by 0.8 mM H2O2.

A compensatory increase in trehalose synthesis in response to desiccation stress in Saccharomyces cerevisiae cells lacking the heat shock protein Hsp12p

2008 ◽  
Vol 54 (7) ◽  
pp. 559-568 ◽  
Author(s):  
Vanessa J. Shamrock ◽  
George G. Lindsey
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Flow Cytometry ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Integrity ◽  
Oxygen Species ◽  
Wild Type ◽  
Plasma Membrane Integrity ◽  
Trehalose Synthesis

The effect of HSP12 deletion on the response of yeast to desiccation was investigated. The Δhsp12 strain was found to be more desiccation tolerant than the wild-type strain. Furthermore, the increased intracellular trehalose levels in the Δhsp12 strain suggested that this strain compensated for the lack of Hsp12p synthesis by increasing trehalose synthesis, which facilitated increased desiccation tolerance. Results obtained from flow cytometry using the membrane exclusion dye propidium iodide suggested that Hsp12p helped maintain plasma membrane integrity during desiccation. Analysis of the oxidative loads experienced by the wild-type and Δhsp12 strains showed that during mid-exponential phase, the increased trehalose levels present in the Δhsp12 cells resulted in increased protection of these cells against reactive oxygen species compared with wild-type cells. During stationary phase, lower levels of reactive oxygen species reduction by reduced glutathione was enhanced in the wild-type strain, which displayed lower intracellular trehalose concentrations. Comparison of the tolerance of the wild-type and Δhsp12 strains with applied oxidative stress showed that the Δhsp12 strain was more tolerant to exogenously applied H2O2, which we attributed to the higher intracellular trehalose concentration. Flow cytometry demonstrated that Hsp12p played a role in maintaining plasma membrane integrity during applied oxidative stress.

scholarly journals Influence of Seasons and Extenders on Quality and Freezability of Gir Bull Semen under Middle Gujarat Climate

2018 ◽  
Vol 13 (03) ◽  
Author(s):  
D. V. Chaudhari ◽  
J. A. Patel ◽  
K. K. Hadiya ◽  
A. J. Dhami
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Winter Season ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Summer Season ◽  
Progressive Motility ◽  
Bull Semen

            The study was conducted to evaluate the seasonal influence (peak winter and summer) and the efficacy of three extenders (egg yolk based TFYG extender and egg yolk free soya bean based commercial extenders Optixcell and Andromed) on quality and freezability of Gir bull semen in Middle Gujarat. Semen ejaculates (6/bull/season, total36) revealed mean ejaculate volume 6.49±0.30 ml, sperm concentration1212.36±58.10 million/ml, progressive motility 74.17±0.78 %, live sperm 81.39±0.80 %, abnormal sperm 7.36±0.31 %, and sperm with intact plasma membrane 81.31±0.98 % and intact acrosome 94.81±0.24 %. Only the progressive sperm motility was significantly (P<0.05) higher(76.39±0.97 % vs. 71.94±1.00 %) with lesser sperm abnormality(6.17±0.37 % vs. 8.56±0.30 %) during winter than in summer. Semen samples split diluted with TFYG, Optixcell and Andromed extenders recorded the overall mean values of progressive sperm motility, livability, abnormality, plasma membrane integrity and acrosomal integrity during winter season as 77.87±0.51, 77.50±0.45, 5.56±0.20,76.02±0.81 and 94.35±0.29 on dilution; 72.41±0.51, 70.50±0.64, 5.96±0.26, 71.20±0.79 and 93.09±0.32 at pre-freeze stage; 41.30 ±0.94, 50.28±1.03, 9.15±0.31, 29.89±0.40 and 90.65±0.40 at post-thaw stage, respectively. The respective values in summer season were 72.13±0.60, 75.50±0.60, 7.48±0.25, 75.61 ±0.55 and 94.09±0.30 on dilution; 65.46±0.66, 69.41±1.05, 8.89±0.28, 69.70±0.66 and 92.63 ±0.33 at pre-freeze stage; 31.48±0.52, 45.09±0.85, 13.48±0.33, 26.85±0.71 and91.26±0.38 at post-thaw stage.  The overall mean sperm post-thaw motility/longevity at 0, 30, 60 and 120 min of incubation at 37°C was 41.20±1.51, 35.19±1.47, 28.80±1.75 and 17.50±1.47 % during winter season and 31.57±0.89, 26.20±0.77, 20.37±0.83 and 13.80±0.77% in summer season, respectively. The initial quality as well as freezability of semen in terms of motile, live, normal and HOS reactive sperm including post thaw longevity were better in winter season than in summer season. Further, the values of all the five semen quality parameters studied were comparatively better in Optixcell than TFYG and Andromed extenders with significant differences only in sperm progressive motility in both the seasons.The season x extender interaction was not significant for any of the sperm quality parameters studied.

scholarly journals Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation

Reproduction ◽  
2016 ◽  
Vol 152 (6) ◽  
pp. 683-694 ◽  
Author(s):  
M Plaza Davila ◽  
P Martin Muñoz ◽  
J M Gallardo Bolaños ◽  
T A E Stout ◽  
B M Gadella ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Oxidative Phosphorylation ◽  
Sperm Motility ◽  
Mitochondrial Respiration ◽  
Membrane Integrity ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Atp Content ◽  
Oligomycin A ◽  
Mitochondrial Uncouplers

To investigate the hypothesis that oxidative phosphorylation is a major source of ATP to fuel stallion sperm motility, oxidative phosphorylation was suppressed using the mitochondrial uncouplers CCCP and 2,4,-dinitrophenol (DNP) and by inhibiting mitochondrial respiration at complex IV using sodium cyanide or at the level of ATP synthase using oligomycin-A. As mitochondrial dysfunction may also lead to oxidative stress, production of reactive oxygen species was monitored simultaneously. All inhibitors reduced ATP content, but oligomycin-A did so most profoundly. Oligomycin-A and CCCP also significantly reduced mitochondrial membrane potential. Sperm motility almost completely ceased after the inhibition of mitochondrial respiration and both percentage of motile sperm and sperm velocity were reduced in the presence of mitochondrial uncouplers. Inhibition of ATP synthesis resulted in the loss of sperm membrane integrity and increased the production of reactive oxygen species by degenerating sperm. Inhibition of glycolysis by deoxyglucose led to reduced sperm velocities and reduced ATP content, but not to loss of membrane integrity. These results suggest that, in contrast to many other mammalian species, stallion spermatozoa rely primarily on oxidative phosphorylation to generate the energy required for instance to maintain a functional Na+/K+ gradient, which is dependent on an Na+-K+ antiporter ATPase, which relates directly to the noted membrane integrity loss. Under aerobic conditions, however, glycolysis also provides the energy required for sperm motility.

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