New assays for detection and localization of endogenous lipid peroxidation products in living boar sperm after BTS dilution or after freeze–thawing Jos F. Brouwers a , Patricia F.N. Silva a,b , Barend M. Gadella a,b, * a Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht, The Netherlands b Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Abstract Reactive oxygen species have been implicated in sperm aberrations causing multiple pathologies including sub- and infertility. Freeze/thawing of sperm samples is routinely performed in the cattle breeding industries for semen storage prior to artificial insemination but unusual in porcine breeding industries as semen dilution and storage at 17 ˚ C is sufficient for artificial insemination within 2–3 days. However, longer semen storage requires cryopreservation of boar semen. Freeze/thawing procedures induce sperm damage and induce reactive oxygen species in mammalian sperm and boar sperm seems to be more vulnerable for this than bull sperm. We developed a new method to detect reactive oxygen species induced damage at the level of the sperm plasma membrane in bull sperm. Lipid peroxidation in freshly stored and frozen/thawed sperm cells was assessed by mass spectrometric analysis of the main endogenous lipid classes, phospha- tidylcholine and cholesterol and by fluorescence techniques using the lipid peroxidation reporter probe C11-BODIPY 581/591 . Peroxidation as reported by the fluorescent probe, clearly corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of lipid peroxidation. This allowed us, for the first time, to correlate endogenous lipid peroxidation with localization of this process in the living sperm cells. Cytoplas- matic droplets in incompletely matured sperm cells were intensely peroxidized. Furthermore, lipid www.journals.elsevierhealth.com/periodicals/the Theriogenology 63 (2005) 458–469 * Corresponding author. Tel.: +31 30 2535383; fax: +31 30 2535492. E-mail address: b.gadella@vet.uu.nl (B.M. Gadella). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.09.046