Original article Bovine sperm separation by Swim-up and density gradients (Percoll and BoviPure): Effect on sperm quality, function and gene expression María Elena Arias a,b , Katherine Andara a , Evelyn Briones a , Ricardo Felmer a,c, * a Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine Universidad de La Frontera, Temuco, Chile b Department of Animal Production, Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Temuco, Chile c Department of Agricultural Sciences and Natural Resources, Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Temuco, Chile A R T I C L E I N F O Article history: Received 23 January 2017 Received in revised form 9 March 2017 Accepted 12 March 2017 Available online xxx Keywords: BoviPure Bovine sperm Gene expression Percoll Sperm quality Swim-up A B S T R A C T This study assesses the effect of bovine sperm (obtained from three bulls) separation using density gradients (Percoll and BoviPure) and Swim-up on sperm function and gene expression. Sperm evaluations included the plasma membrane integrity (SYBR14/PI), acrosomal integrity (PNA-FITC/PI), oxidative stress (ROS; CH2FDDA), DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (DYm; TMRM) using flow cytometry. Sperm motility was evaluated by computer-assisted sperm analysis (CASA) and gene expression using RT-qPCR. The results showed that separation by Percoll achieves a higher proportion of sperm with intact plasma and acrosomal membranes (89.8 and 87.5%, respectively) than the unseparated control (70.3 and 62.4%, respectively), as well as by Swim-up (74.9 and 63.3%, respectively) and BoviPure (83.3 and 80.4%, respectively). No differences were observed in the proportion of spermatozoa with high DCm between Percoll and BoviPure (84.3% and 83.5%, respectively), which were higher than Swim-up and the unseparated control (72.8% and 43.8%, respectively). The ROS levels were higher in the spermatozoa separated by Percoll and no differences were observed in the sperm DNA integrity between all groups. The motility analysis showed that the separation methods improve (p < 0.05) total and progressive motility compared to the control, with Percoll proving the most efficient in this regard. Finally, the gene expression analysis of leptin (LEP), aromatase cytochrome P450 (CYP19) and protamine I (PRM1), after validation of 6 reference genes, showed no differences between groups. In conclusion, bovine sperm separation using density gradient improves the parameters of motility and sperm function without affecting the gene expression. © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved. 1. Introduction Assisted reproduction techniques (ARTs) in humans and animals depend on developing methods that can efficiently separate the motile sperm fraction from the other semen components. Selecting good quality, high density sperm that maintain their fertilization potential is a key factor for the success of ARTs, since dead and abnormal sperm exert toxic and lithic effects on normal sperm and thus have a negative impact on fertility [1]. Sperm selection methods mainly separate the motile from the immotile sperm, eliminate the seminal plasma, diluents and cryoprotectants [2,3], and significantly improved sperm quality, thereby increasing the progressive motility and normal morphology. The first method described for separating spermato- zoa consisted only of washing the sperm cells by centrifugation. Later, Swim-up was described and more sophisticated sperm separation methods were designed that obtained a high number of motile spermatozoa to protect sperm function and reduce environmental effects such as reactive oxygen species (ROS). These methods include: Swim-up with Hyaluronic acid [4], Sephadex columns, glass wool filtration, migration/sedimentation and centrifugation in density gradients, among others (reviewed by Henkel and Schill [5]). Percoll is a commercial medium used to separate cells and subcellular particles by means of density gradients, and it has been widely used in the separation of animal spermatozoa and other type of cells. Percoll is composed of colloidal silica particles coated with non-dialyzable polyvinylpyrrolidone (PVP). Centrifugation in Percoll density gradient separates the spermatozoa according to * Corresponding author at: Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Montevideo 0870, P.O. Box 54-D, Temuco, Chile. E-mail address: rfelmerd@gmail.com (R. Felmer). http://dx.doi.org/10.1016/j.repbio.2017.03.002 1642-431X/© 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved. Reproductive Biology xxx (2016) xxx–xxx G Model REPBIO 226 No. of Pages 7 Please cite this article in press as: M.E. Arias, et al., Bovine sperm separation by Swim-up and density gradients (Percoll and BoviPure): Effect on sperm quality, function and gene expression, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.03.002 Contents lists available at ScienceDirect Reproductive Biology journal homepage: www.elsevier.com/locate/repbio