91 TREATMENT OF GOAT SPERM WITH CATALASE TO IMPROVE POST-THAW QUALITY

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 (2 h, 37°C) with 0, 60, 120, and 240 UI mL^–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 240 UI mL^–1 showed lower percentage of sperm showing low mitochondrial activity when compared with samples treated with 0 and 120 UI mL^–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.