315 REACTIVE OXYGEN SPECIES AND OXIDATIVE STRESS IN CANINE SEMEN FRACTIONS

Abstract

Reactive oxygen species (ROS) are physiologically produced by spermatozoa and leukocytes present in the seminal plasma. In low concentrations, ROS play an important role in sperm function because they are required for sperm fertilizing capacity, mainly during sperm capacitation and hyperactivation (Saleh RA and Agarwal A 2002 J. Androl. 23, 737-752). However, an imbalance between the formation of free radicals and the capacity for defense of the antioxidant mechanisms may lead to cell damage (Rover Jr L et al. 2001 Química Nova 24, 112-119). Spermatozoa are sensitive to lipid peroxidation due to its high content of polyunsaturated fatty acids and low concentrations of protective enzymes (Sharma RK and Agarwa LA 1996 Urology 48, 835-850). The aims of the present study were to compare ROS content among the 3 fractions of canine semen and to correlate these values with sperm variables. Semen samples were collected from 15 healthy dogs of distinct breeds aged 2 to 6 years. Sperm analysis was performed through motility, forward progressive velocity, morphology, and the percentage of viable sperm with the use of the eosin/nigrosin stain. The determination of thiobarbituric acid reactive substances (TBARS) was used to estimate the degree of lipid peroxidation in each sperm fraction. Values were compared using ANOVA and Tukey’s test for multiple comparisons at a significance level of 5%. Pearson correlation was used to calculate the relationship between sperm variables in the second fraction. Sperm motility, velocity, and percentage of viable sperm were within the normal range for canine semen: 84±2%, 3.4±0.1%, and 83 ± 2%, respectively. The sperm-rich fraction presented statistically higher concentrations of TBARS (1474.19 ± 245.78 ng mL^-1) compared to the first and third fractions (579.41 ± 171.23 and 399.62 ± 58.08 ng mL^-1, respectively; P < 0.05), indicating that spermatozoa and epididymal secretions are the main source of free radicals. No statistical correlation among TBARS and sperm motility and velocity were verified. However, a positive correlation was observed between the percentage of sperm proximal droplets and TBARS (r = 0.44, P = 0.7). This result suggests that a high incidence of sperm proximal droplets can enhance ROS formation in seminal plasma. Hence, canine sperm presenting delayed maturation in the epididymes produce higher concentrations of free radicals. In fact, sperm production of ROS occurs mainly by abnormal cells, especially the ones containing cytoplasm residues (Gomez E et al. 1998 Int. J. Androl. 21, 81-94). However, no protective effect of the sperm distal droplets was verified in canine semen as observed elsewhere for the bovine spermatozoa (Nichi M et al. 2007 Theriogenology 67, 334-340). In conclusion, spermatozoa and epididymal fluids are the primordial source of free radicals in canine seminal plasma, mainly when sperm proximal droplets are present.