title: Generation of reactive oxygen species by equine spermatozoa.
abstract: OBJECTIVE
  To characterize generation of reactive oxygen species (ROS) by equine spermatozoa.
  
  
  METHODS
  Multiple semen samples collected from 9 stallions.
  
  
  METHODS
  Equine spermatozoa were separated from seminal plasma on a discontinuous polyvinylpyrrolidone (PVP)-coated silica gradient and resuspended in a modified Tyrode albumin-lactate-pyruvate medium. Amount of hydrogen peroxide (H2O2) generated was assayed by use of a 1-step fluorometric assay, using 10-acetyl-3,7-dihydroxyphenoxazine as a probe for detection of H2O2 in a microplate assay format. Concentration of H2O2 was determined by use of a fluorescence microplate reader.
  
  
  RESULTS
  Amount of H2O2 generated increased significantly with time and spermatozoa concentration for live and flash-frozen spermatozoa, and amount of H2O2 generated was significantly greater for flash-frozen than for live spermatozoa. Addition of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) significantly increased generation of H2O2 by live and flash-frozen spermatozoa. Addition of a calcium ionophore also significantly increased the amount of H2O2 generated by live spermatozoa but did not have an effect on amount of H2O2 generated by flash-frozen spermatozoa. Abnormal equine spermatozoa generated significantly greater amounts of H2O2 than did normal spermatozoa.
  
  
  CONCLUSIONS
  Equine spermatozoa generate ROS in vitro, possibly via a NADPH-oxidase reaction. Spermatozoa damaged during flash-freezing or morphologically abnormal spermatozoa generated significantly greater amounts of ROS than did live or morphologically normal spermatozoa. Damaged and abnormal spermatozoa generate greater amounts of ROS that may contribute to reduced fertility or problems related to semen preservation.
authors.affiliation: Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis 95616, USA.
authors.forename: B A
authors.initials: BA
authors.lastname: Ball
authors.forename: A T
authors.initials: AT
authors.lastname: Vo
authors.forename: J
authors.initials: J
authors.lastname: Baumber
mesh_terms: D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D002499:Centrifugation, Density Gradient / Q000662:veterinary; D015925:Cryopreservation / Q000662:veterinary; D006736:Horses / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D008857:Microscopy, Interference / Q000662:veterinary; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012662:Semen Preservation / Q000009:adverse effects / Q000662:veterinary; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D007476:Ionophores; D017382:Reactive Oxygen Species; D013481:Superoxides; D000001:Calcimycin; D009249:NADP; D009240:N-Formylmethionine Leucyl-Phenylalanine; D006861:Hydrogen Peroxide; D002374:Catalase; D013755:Tetradecanoylphorbol Acetate