Generation of reactive oxygen species by equine neutrophils and their effect on motility of equine spermatozoa

Contaminating leukocytes in the ejaculate are an important source of reactive oxygen species (ROS) in human semen. When present in sufficient numbers, they can have a detrimental influence on sperm function in humans. Unfortunately, there is little published information regarding the importance of leukocytes in stallion semen. The objectives of this study were to determine the production of hydrogen peroxide (H₂O₂) by activated equine neutrophils and to examine the effect of this ROS production on equine sperm motility in vitro. Motile equine spermatozoa (two ejaculates each from four stallions) and peripheral blood neutrophils were isolated on discontinuous Percoll gradients, washed and resuspended in a modified Tyrode's medium. Spermatozoa (25 × 10⁶/ml) were incubated for 30 min at 38°C with neutrophils(0,0.5 × 10⁶, 1 × 10⁶, 5 × 10⁶ and 10 × 10⁶/ml) activated by either the protein kinase C agonist, 12-myristate, 13-acetate phorbol ester (PMA; 100 nM) or the leukocyte chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP; 0.1 mM). Sperm motility was determined by computer-assisted semen analysis (CASA) at time 0 min (T0) and time 30 min (T30), and H₂O₂ was measured at T30 with the Amplex Red™ assay kit. At T30, there was a significant (P < 0.01) increase in H₂O₂ with the addition of 5 × 10 and 10 × 10⁶ neutrophils/ml activated by FMLP (0.76 ± 0.3 and 0.99 ± 0.4 μM, respectively, versus 0.0024 ± 0.002 μM in sperm alone), and this increase was associated with a significant (P < 0.001) decrease in total motility (52 ± 5.1 and 48 ± 6.0%, respectively, versus 80 ± 4.7% in sperm alone). At T30, there was also a significant (P < 0.001) increase in H₂O₂ with the addition of 5 × 10⁶ and 10 × 10⁶ neutrophils/ml activated by PMA (1.88 ± 0.2 and 2.07 ± 0.3 μM, respectively, versus 0.0009 ± 0.0006 μM in sperm alone). The results of this study demonstrate that 5 × 10⁶ activated neutrophils/ml are sufficient to impair equine sperm motility in vitro.