Efficacy of four density gradient separation media to remove erythrocytes and nonviable sperm from canine semen

Abstract

The objective was to compare four commercially available density gradient centrifugation (DGC) media (ISolate [Irvine Scientific; Santa Ana, CA, USA], Percoll [Pharmacia; Uppsala, Sweden], PureCeption [SAGE In-Vitro Fertilization, Inc.; Trumbull, CT, USA], PureSperm 100 [Nidacon International AB; Molndal, Sweden]) for their ability to separate viable, motile sperm from contaminant nonviable (immotile and/or dead) sperm and red blood cells (RBC). Pooled sperm-rich fractions from four healthy dogs were assessed using Spermvison SAR (Minitube of America). For this, 1 mL of the blood/sperm admixture was pipetted over 4 mL of DGC media: 50%/90% ISolate (Irvine Scientific), 45%/90% Percoll (Pharmacia), 40%/80% PureCeption (SAGE In-Vitro Fertilization, Inc.), and 40%/80% PureSperm 100 (Nidacon International AB). After centrifugation, five 1-mL fractions (A, B, C, D, and E) and the sperm pellet (bottom fraction F) were separated. Sperm morphology and red blood cell/sperm ratio (RBC/S) per fraction were determined on stained slides. All DGC media separated RBC from sperm; the highest red blood cell/sperm ratio was present in ISolate (Irvine Scientific) and Percoll (Pharmacia) fraction A (29.4±29.7 and 28.2±20.8, respectively), and in fractions A and B of both PureCeption (SAGE In-Vitro Fertilization, Inc.) (37.0±22.8 and 39.6±24.3, respectively) and PureSperm 100 (Nidacon International AB) (25.2±5.9 and 23.0±3.9, respectively). The fractions with the highest total sperm recovery, motile sperm recovery, as well as overall motility were ISolate (Irvine Scientific) and Percoll (Pharmacia) fraction D (33.9±29.4%; 40.99±27.9%; 71.2±21.8% and 36.4±14.5%; 39.3±15.8%; 88.6±2.3%, respectively), and for PureCeption (SAGE In-Vitro Fertilization, Inc.) and PureSperm 100 (Nidacon International AB), the sperm pellet, fraction F (78.8±28.3%; 88.0±17.4%; 70.2±11.1% and 73.1±21.0%; 75.4±24.6%; 80.6±17.1%, respectively). In the pellet for PureCeption (SAGE In-Vitro Fertilization, Inc.), more sperm and motile sperm were recovered than in ISolate (Irvine Scientific) and Percoll (Pharmacia) fractions D (P<0.0163). Therefore, DGC media should be considered for canine semen purification when contaminated with blood or when separation of motile versus immotile sperm is needed.