title: Bovine sperm separation by Swim-up and density gradients (Percoll and BoviPure): Effect on sperm quality, function and gene expression.
abstract: 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 (ΔYm; 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 ΔΨm 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.
authors.affiliation: Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine Universidad de La Frontera, Temuco, Chile; Department of Animal Production, Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Temuco, Chile.
authors.forename: María Elena
authors.initials: ME
authors.lastname: Arias
authors.affiliation: Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine Universidad de La Frontera, Temuco, Chile.
authors.forename: Katherine
authors.initials: K
authors.lastname: Andara
authors.affiliation: Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine Universidad de La Frontera, Temuco, Chile.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Briones
authors.affiliation: Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine Universidad de La Frontera, Temuco, Chile; Department of Agricultural Sciences and Natural Resources, Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Temuco, Chile. Electronic address: rfelmerd@gmail.com.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Felmer
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D002499:Centrifugation, Density Gradient; D004249:DNA Damage; D008297:Male; D053078:Membrane Potential, Mitochondrial; D011205:Povidone; D017382:Reactive Oxygen Species; D055101:Semen Analysis; D012662:Semen Preservation / Q000379:methods; D012821:Silanes; D017640:Silicates; D012822:Silicon Dioxide; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology*
chemical_list: C509975:BoviPure; D017382:Reactive Oxygen Species; D012821:Silanes; D017640:Silicates; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone
keywords: BoviPure; Bovine sperm; Gene expression; Percoll; Sperm quality; Swim-up