Protein content of equine seminal plasma and sperm plasma membrane in subfertile stallions P.N. Guasti * , F.F. Souza, C. Scott, F.P. Hartwig, P.M. Papa, G.A.M. dos Santos, C.P. Freitas-Dellaqua, F.O. Papa Department of Animal Reproduction, College of Veterinary Medicine and Animal Science, Unesp, Botucatu, Brazil 1. Introduction Subfertility or infertility in breeding stallions contrib- utes to poor reproductive efficiency and low conception rates. Alterations at the molecular level in spermatozoa and the seminal plasma may contribute to stallion reproductive disorders. It is known that the seminal plasma proteins are involved in the process of fertilization [1] as the proteins in the sperm plasma membrane, these are important for sperm adhesion in formation of the sperm reservoir in the female tract and in sperm-egg binding [3]. The objective of the present study was to correlate the total protein content of equine seminal plasma and sperm plasma membrane with fertility. 2. Materials and methods Three ejaculates from 9 subfertile stallions (<40% conception rate) and from 10 fertile stallions (>60% conception rate) were collected. The conception data from each stallion were provided by the respective breeding stations, which housed the stallions and were based on the number of recovered embryos/number of inseminations. A minimum of 20 different donor mares were inseminated by each stallion during the 2012 breeding season. The ejacu- lates were diluted in a skim milk based extender (BotuSe- men, Botupharma, Botucatu, SP, Brazil) to a concentration of 80 x 10 6 / mL total motile sperm and immediately eval- uated by CASA for total motility (TM), progressive motility (PM) and rapid sperm (RAP). Another aliquot (10 mL) of fresh, raw semen was separated for seminal plasma and membrane protein analysis. For the quantification of sem- inal plasma proteins, 5 mL of fresh semen was submitted to centrifugation at 600xg for 10 min, at room temperature, and the supernatant was frozen in liquid nitrogen. After thawing, the sample was centrifuged at 15,000 x g for 60 min at 4  C to remove debris and collect the supernatant, and total protein from seminal plasma was quantified. For membrane protein analysis, 5 mL of raw semen was added to a protease inhibitor cocktail (S8820, Sigma, St. Louis, USA) and 0.1 mg/mL PMSF (P7626, Sigma, St. Louis, USA) and then washed three times at 700 x g for 5 min at 4  C. Membrane proteins were extracted from sperm pellet with 0.5% (v/v) Nonidet P-40 (74385, NonidetÔ P 40, BioXtra, Sigma, St. Louis, USA) and with a sonicator (Digital Sonifier, Model 450, Branson Ultrasonics Corporation, Connecticut, USA), using 3.0 mm probe, 20% amplitude in 10 steps of 30 seconds in an ice bath, shut off 1 minute between the steps. After sonication, samples were centrifuged for 10,000xg for 30 min at 4  C and the supernatant containing the solubi- lized plasma membrane proteins was collected. Seminal plasma and plasma membrane proteins were quantified by bicinchoninic acid (BCA) assay. For flow cytometry analysis, semen samples from each ejaculate were diluted in modi- fied Tyrode’s medium at concentration of 25 x 10 6 sperm/ mL. The assessment of plasma membrane was performed using a 200-mL aliquot of semen to 2 mL of Hoechst 33342 (H33342; 40 mg/mL, Molecular Probes, Life Technologies do Brasil Ltda, São Paulo, Brazil), 3 mL of Propidium Iodide (0.5 mg/mL, L0770, Sigma-Aldrich, St. Louis, USA) and 10 mL of FITC-PSA (100 mg/mL, L-0770, Sigma-Aldrich, St. Louis, MO, USA). Samples were incubated at 37  C for 15 min in the dark. For evaluation of DNA fragmentation, a 100-mL aliquot was added to 400 mL of acid-detergent solution (0.08 N HCl, 0.15 M NaCl, 0.1% Triton x100; pH 1.4). Exactly 30 sec later, 1.2 mL of acridine orange (AO) staining solution (0.037 M citric acid, 0.126 M Na 2 HPO 4 , 0.0011 M dissodium EDTA, 0.15 M NaCl; pH 6.0, 4uC) containing 6 mg/mL electro- phoretically purified AO was added. Samples were analyzed after 30 sec in the dark. A total of 10,000 gated- events were analyzed per sample by flow cytometry (BD * Presenting author Contents lists available at ScienceDirect Journal of Equine Veterinary Science journal homepage: www.j-evs.com Journal of Equine Veterinary Science 34 (2014) 84