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P A A Góes,
M Nichi,
R O C Silva,
E G A Perez,
A Dalmazzo, J R C Gurgel,
C C Rocha,
R Simões,
M A Peres,
M E O A Assumpção,
R C Barnabe,
V H Barnabe
[show abstract]
[hide abstract]
ABSTRACT: Semen quality after cryopreservation is one of the main limiting factors for the success of artificial insemination in goats. Previous studies indicate that cryo-injuries may be related to the oxidative stress which is caused by the reactive oxygen species (ROS) and leads to structural and functional damages to the sperm. The understanding of sperm oxidative mechanisms in goats may provide information on possible treatments to improve semen quality post cryopreservation. The aim of the present study was to verify the resistance of cryopreserved goat spermatozoa to different reactive oxygen species. Semen samples from 5 adult goats were collected and cryopreserved (Botubov(®), Biotech Ltda.). After thawing, samples were washed twice with PBS and incubated (1h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4mM), ascorbate and ferrous sulfate (4mM; produces hydroxyl radical) with and without the addition of seminal plasma. Samples were analysed for motility using computer-assisted sperm analysis (CASA); the 3-3' diaminobenzidine 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; sperm chromatin structure assay as an index of DNA fragmentation; and the measurement of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P<0.05). Results showed that cryopreserved goat sperm after thawing is highly susceptible to the hydroxyl radical. No differences were found on CASA variables between the different ROS. On the other hand, lipid peroxidation and DNA fragmentation were higher for samples treated with hydroxyl radical when compared to samples treated with the other ROS. Furthermore, sperm showing low mitochondrial activity were lower also for samples treated with hydroxyl radical. Negative correlations were found between lipid peroxidation, and most of the variables evaluated by the CASA. A positive correlation was found between the percentage of sperm showing low mitochondrial potential and DNA fragmentation, indicating that impaired mitochondrial activity may be related to an increase on DNA fragmentation. Previous studies indicate that fresh goat semen is highly susceptible to the attack of hydrogen peroxide. We observed that after thawing there is a shift towards a higher susceptibility to the hydroxyl radical. This may indicate that seminal plasma in goats may be an important source of hydroxyl radical scavengers and that, due to the dilution of the seminal plasma with the extender, such antioxidant protection may be impaired. Therefore, an alternative to improve semen quality in cryopreserved goat semen would be the treatment with hydroxyl radical scavengers such as vitamins E and C, reduced glutathione, and other non-enzymatic antioxidants.
Reproduction Fertility and Development 01/2011; 23(1):143. · 2.11 Impact Factor
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R O C Silva,
M Nichi,
E G A Perez,
P A A Góes,
A Dalmazzo, J R C Gurgel,
C C Rocha,
R Simões,
M A Peres,
M E O A Assumpção,
R C Barnabe,
V H Barnabe
[show abstract]
[hide abstract]
ABSTRACT: Semen cryopreservation is extremely important to the use of reproductive biotechnologies in goats. However, studies indicate that cryopreservation may lead to increased oxidative stress which may cause structural damage to biomolecules, DNA, lipids, carbohydrates, and proteins, as well as other cellular components. Previous studies performed by our group indicate fresh goat sperm is highly susceptible to the attack of hydrogen peroxide. Therefore, the treatment with hydrogen peroxide scavengers would be an alternative to improve post-thaw sperm quality in goats. The aim of the present study was to evaluate the efficiency of catalase, an important hydrogen peroxide scavenger, to improve post-thaw quality in cryopreserved goat semen samples. Semen samples from 5 adult goats were collected and cryopreserved (Botu-Bov(®), Biotech Ltda.). After thawing, samples were incubated (2h, 37°C) with 0, 60, 120, and 240UImL(-1) of catalase. Samples were then analysed for motility using the computer assisted sperm analysis (CASA); the 3-3' diaminobenzidine 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; sperm chromatin structure assay, as an index of DNA fragmentation; and the measurement of thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P<0.05). Results showed that catalase treatment after thawing played a role on improving mitochondrial activity. Samples treated with 240UImL(-1) showed lower percentage of sperm showing low mitochondrial activity when compared with samples treated with 0 and 120UImL(-1) of catalase (6.5±2.3, 17.2±3.5, and 10.0±1.3%, respectively). However, no effect of catalase was observed on any of the other variables studied. Results indicate that catalase, despite its beneficial effect on mitochondrial activity, does not influence positively on sperm quality after thawing. A hypothesis to explain such results would be that because of seminal plasma dilution with the extender, the antioxidants were also diluted. Therefore, the antioxidant protection would be impaired and the most deleterious reactive oxygen species, as observed in fresh semen, would also be different depending on the semen extender used because sperm are extremely dependent on the extracellular environment due to the reduced cytoplasm and the high content of polyunsaturated fatty acids in the membrane. A study performed by our group confirms such a hypothesis. Possibly, the treatment with catalase would be more effective if performed before cryopreservation. Also, it is possible that the use of different antioxidants would provide better results.
Reproduction Fertility and Development 01/2011; 23(1):151. · 2.11 Impact Factor
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A Dalmazzo,
P A A Góes,
M Nichi,
R O C Silva, J R C Gurgel,
E G A Perez,
C C Rocha,
R Simões,
M A Peres,
M E O A Assumpção,
R C Barnabe,
V H Barnabe
[show abstract]
[hide abstract]
ABSTRACT: Due to the importance of dogs to humans, there is increasing interest in breeders in the use of reproductive biotechnologies. However, most of the biotechnologies would require the removal or dilution of the seminal plasma, which is known to exert both beneficial and deleterious effects on sperm quality. One of the beneficial effects of seminal plasma would be the antioxidant protection because sperm are particularly susceptible to oxidative stress, mainly due to the reduced cytoplasm and the high content of polyunsaturated fatty acids in their membrane. An alternative to overcome the injuries caused by oxidative stress is the antioxidant treatment, which requires the identification of those reactive oxygen species (ROS) that are the most deleterious. The aim of this study was to identify the most harmful ROS to dog semen. Semen samples from 6 adult dogs were collected and centrifuged. Seminal plasma (SP) was removed and samples were incubated (1h, 37°C) with 4 ROS-inducing mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4mM), ascorbate and ferrous sulfate (4mM; produces hydroxyl radical) alone or with additional SP. Samples were analysed for motility by computer assisted sperm analysis (CASA). The 3-3' diaminobenzidine stain was used 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, sperm chromatin structure assay (SCSA) as an index of DNA fragmentation, and measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P<0.05). Results showed that dog sperm is differentially modulated depending on the presence of SP. In addition, damage to the different sperm structures depended on the different ROS. Samples incubated with SP showed no differences concerning TBARS (1233 in SP, 1260 in Tris; P=0.99). On the other hand, samples incubated without SP showed higher lipid peroxidation when treated with hydroxyl radical compared with the other ROS. Furthermore, although hydroxyl radical mostly altered mitochondrial activity in samples incubated with SP (DAB IV=4.3%; P<0.05 against all other ROS), the most significant ROS in samples incubated without SP was hydrogen peroxide (DAB IV=4.7%; P<0.05 against all other ROS). Superoxide anion was less harmful to acrosome integrity in samples incubated with SP and to motility in samples incubated without SP. The present results suggest that seminal plasma may play an important role in the susceptibility of dog sperm to oxidative stress. Moreover, the results indicate that different sperm compartments are susceptible to different ROS. It is concluded that the quality of frozen-thawed dog semen may be improved by treating with a combination of different antioxidants to destroy the chain reaction causing the oxidative stress.
Reproduction Fertility and Development 01/2011; 23(1):215. · 2.11 Impact Factor
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J R C Gurgel,
M Nichi,
E G A Perez,
P A A Góes,
A Dalmazzo,
R O C Silva,
C C Rocha,
R Simões,
M A Peres,
M E O A Assumpção,
V H Barnabe,
R C Barnabe
[show abstract]
[hide abstract]
ABSTRACT: Mangalarga, due to its marching abilities, is the mostly widespread and numerous equine breed in Brazil. Furthermore, previous studies indicate that the semen of these horses is particularly susceptible to cryo-injuries. Therefore, the use of chilled semen is crucial when employing reproductive biotechnologies. However, previous studies indicate that chilled semen is highly impaired by the oxidative stress, which is caused by reactive oxygen species (ROS). An alternative to overcome the injuries caused by oxidative stress is antioxidant treatment, which requires the identification of those ROS that are the most deleterious. The aim of this study was to identify the most harmful ROS to Mangalarga sperm. Semen samples from 4 horses were collected, mixed with chilling media (Equimix(®), Nutricell) and transported to the laboratory at 15°C. Samples were then incubated (1h, 37°C) with 4 ROS inducing mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4mM), ascorbate and ferrous sulfate (4mM; produces hydroxyl radical). Samples were analysed for motility using computer assisted sperm analysis (CASA). The 3-3' diaminobenzidine stain was used 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, sperm chromatin structure assay (SCSA) as an index of DNA fragmentation, and the measurement of thiobarbituric acid reactive substances (TBARS) an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P<0.05). Results showed that Mangalarga sperm is highly susceptible to the hydroxyl radical. Samples treated with this ROS showed a lower percentage of sperm with high mitochondrial activity then samples treated with hydrogen peroxide (24.6±5.9 v. 43.7±6.8%, respectively). Similarly, lipid peroxidation (TBARS) was higher in samples treated with hydroxyl radical when compared with those treated with both superoxide anion and hydrogen peroxide (2037.7±154.8, 681.2±170.1, and 789.4±124.5 ng/10(6) sperm). In addition, for all variables analysed using CASA except for ALH and BCF, samples treated with hydroxyl radical showed decreased quality when compared with the other samples. A positive correlation was found between TBARS and mitochondrial activity, indicating that the higher the sperm susceptibility of sperm against oxidative stress, the lower the mitochondrial activity. Level of TBARS also correlated negatively with most of the variables evaluated by CASA. The present results suggest that Mangalarga sperm is highly susceptible to the hydroxyl radical, a mechanism apparently related to the mitochondrial activity. Therefore, an alternative to overcome the deleterious influence of oxidative stress in semen of Mangalarga stallions would be the treatment with hydroxyl radical scavengers such as vitamins C and E, reduced glutathione, and other nonenzymatic antioxidants.
Reproduction Fertility and Development 01/2011; 23(1):216. · 2.11 Impact Factor
