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

J. Baumber, A. Vo, K. Sabeur, B. A. Ball

Research output: Contribution to journalArticlepeer-review

75 Scopus citations


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.

Original languageEnglish
Pages (from-to)1025-1033
Number of pages9
Issue number3
StatePublished - 2002

Bibliographical note

Funding Information:
This research was supported by the John P. Hughes Endowment and by the Center for Equine Health with funds provided by the Oak Tree Racing Association, the State of California pari-mutual fund, and contributions by private donors. The authors thank Dr. Jan Roser of the Department of Animal Science, UC-Davis for use of some of the stallions in this study.


  • Equine
  • Hydrogen peroxide
  • Leukocytes
  • Oxidative stress
  • Spermatozoa

ASJC Scopus subject areas

  • Small Animals
  • Food Animals
  • Animal Science and Zoology
  • Equine


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