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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-1of 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-1showed lower percentage of sperm showing low mitochondrial activity when compared with samples treated with 0 and 120UImL-1of 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. Thanks to Nutricell for the media used and CAPES for financial support. [ABSTRACT FROM AUTHOR]

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/106sperm). 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. The authors acknowledge Nutricell for the media used and CAPES for financial support. [ABSTRACT FROM AUTHOR]

Cryopreservation of equine semen has been widely studied by several research groups because of the large breed and individual variation in sperm freezability. A key factor in sperm cryopreservation is the high incidence of oxidative stress, an imbalance between reactive oxygen species (ROS) and antioxidant protection, which impairs sperm functionality by attacking plasma membrane, acrosome, mitochondria, and DNA. In order to study the resistance of equine spermatozoa to different reactive oxygen species (ROS), sperm samples from 4 Mangalarga stallions were collected using an artificial vagina. Samples were cryopreserved in extenders containing dimethylformamide (DMF) or methylformamide (MF). After thawing and washing, sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produced hidroxyl radical), and malondialdehyde (MDA, lipid peroxidation product). Samples were evaluated using the 3-3′ diamino benzidine (DAB) stain, as an indicator of mitochondrial activity; the eosin nigrosin staining, to evaluate plasma membrane integrity; the simple stain (fast green/Bengal rose), to assess acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS), a lipid peroxidation product. Statistical analysis was performed using the Student t-test and LSD test. Results showed that sperm mitochondrial potential of frozen-thawed samples in MF was highly susceptible to the attack of hydroxyl radical and hydrogen peroxide. No effect of ROS was observed on membrane and acrosome integrity. On the other hand, samples cryopreserved in DMF showed no differences in susceptibility to ROS. When evaluating the main effects of different extenders, results showed a higher protective effect of the MF extender on acrosome integrity and mitochondrial potential (MF: 12.1 ± 2.2 and 7.8 ± 2.3% v.DMF: 3.4 ± 0.7 and 1.1 ± 0.7%, respectively, P< 0.05). However, a negative effect of MF extender was observed regarding the percentage of sperm showing intact membrane and TBARS content (MF: 2.0 ± 0.8% and 517 ± 115 ng/106sperm v.DMF: 20.6 ± 1.7% and 118 ± 44 ng/106sperm, respectively, P< 0.05). A strong negative correlation was found between TBARS and plasma membrane integrity (r = -0.88; P= 0.004) for samples cryopreserved in DMF, whereas a positive correlation was found between TBARS and sperm with full mitochondrial potential (r = 0.73; P= 0.04). Results of the present study indicate that DMF may play a role in the protection of sperm against the attack of ROS. However, such action is apparently limited to the plasma membrane. On the other hand, the MF-supplemented extender exerts an intracellular protection. Therefore, the antioxidant therapy, especially hydrogen peroxide and hydroxyl radical scavengers, may be an alternative to improve the post-thaw quality of MF-supplemented cryopreserved semen in stallions, by increasing extracellular antioxidant capacity. The authors thank Nutricell for financial support and the media used in the present experiment. [ABSTRACT FROM AUTHOR]