Proteome of cat semen obtained after urethral catheterization Marzena Mogielnicka-Brzozowska a, * , Sylwia Prochowska b , Wojciech Ni _ za  nski b , Mariusz A. Bromke c , Jerzy Wi  sniewski c , Beata Olejnik d , Anna Kuzborska a , Leyland Fraser a , Piotr Mlynarz e ,Wladyslaw Kordan a a Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-957, Olsztyn, Poland b Department of Reproduction and Clinic of Farm Animals, Wroclaw University of Environmental and Life Sciences, pl. Grunwaldzki 49, 50-366, Wroclaw, Poland c Department of Medical Biochemistry, Medical University of Wroclaw, Chalubi  nskiego 10, 50-368, Wroclaw, Poland d Department of Chemistry, Wroclaw University of Technology, 50-370, Wroclaw, Poland e Department of Chemistry and Immunochemistry, Medical University of Wroclaw, Bujwida 44a, 50-345, Wroclaw, Poland article info Article history: Received 22 February 2019 Received in revised form 3 September 2019 Accepted 3 September 2019 Available online 5 September 2019 Keywords: Proteins Seminal plasma Spermatozoa Proteomics Cat abstract The binding of seminal plasma (SP) proteins by spermatozoa plays an important role in the regulation of sperm epididymal maturation, motility gaining in female reproductive tracts and sperm-egg interaction. The aim of the study was to analyze the SP and sperm extracts proteome of cat (Felis catus) semen. The seminal plasma and spermatozoa were obtained by urethra catheterization from 10 male cats. Proteins were extracted using RIPA buffer and separated by electrophoresis (SDS-PAGE). The gels were analyzed using MultiAnalyst software. The proteins were subsequently analyzed using NanoUPLC-Q-TOF/MS. UniProt database-supported identification resulted in 106 proteins identified in the cat SP and 98 pro- teins in the extracts of spermatozoa. Based on a gene ontology analysis, dominant molecular functions of feline SP proteins were binding, catalytic, and antioxidant activity (56%, 33%, and 11% of cases, respec- tively). The molecular functions of sperm extracts proteins were mainly involved in catalytic activity (41%) and binding (23%). The proteins present in both, the SP and spermatozoa's extracts, were: serum albumin (ALB), semenogelin 2 (SEMG 2), clusterin (CLU), lactoferrin (LTF), prostatic acid phosphatase (ACPP), prolactin inducible protein (PIP), negative elongation factor E (NELF-E) and ectonucleotide pyrophosphatase (ENPP3). Protein-protein interactions analysis showed significant connection for 12 proteins in the cat semen. The seminal plasma proteins which, with high probability score, participate in important metabolic pathways are: glutathione peroxidases (GPx5 and 6), prostatic acid phosphatase (ACPP), b-hexosaminidase (HEXB), polymeric immunoglobulin receptor (pIgR) and serpin family F member 1 (SERPINF1). For sperm protein extracts it were: pyruvate dehydrogenase (PDHB), succinate- CoA-ligase (SUCLA2), malate dehydrogenase (MDH2), ATP synthase F1 subunit alpha (ATP5F1A) and tubulin beta (TUBB). © 2019 Published by Elsevier Inc. 1. Introduction The proteome, a full set of cell, tissue or organism proteins, provides information on the final effects of gene expression in a given area at certain time. Genes are only a potential that can be revealed or not, depending on the environmental factors. Research on the semen proteome (seminal plasma and spermatozoa) is necessary to understand how external factors affect the expression of proteins associated with the regulation of fertilization-related processes [1]. Proteomic studies contribute to the explanation of the role of individual proteins at various stages of epididymal sperm maturation, capacitation, acrosomal reaction and sperm-egg cell fusion [2,3]. The knowledge of the function of the proteins gives us potential possibilities of using them as markers of biological value in reproductive processes or future constituents of contra- ceptives [3,4]. The search for marker proteins associated with the process of sperm preservation is of great importance. It allows to identify males or semen samples with greater or lesser usefulness * Corresponding author. E-mail address: mmog@uwm.edu.pl (M. Mogielnicka-Brzozowska). Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com https://doi.org/10.1016/j.theriogenology.2019.09.003 0093-691X/© 2019 Published by Elsevier Inc. Theriogenology 141 (2020) 68e81