Effects of single layer density gradient centrifugation on cooled-stored stallion semen

Abstract

The objective of this studywas to evaluate effects of a single layer density gradient EquiPure (Nidacom) centrifugation on cooled-stored stallion semen. Each of two ejaculates collected from 10 stallions was divided into three aliquots: control, centrifuged and treatment. In the control group, semen was diluted with a skim milk-based extender (Botu-Semen) at a concentration of 50x106sperm/ml. In centrifuged and treatment groups, samples with 1x109 spermatozoa were diluted with Botu-Semen 1:1 (v:v) and centrifuged at 600xg for 10 minutes. The supernatant of the centrifuged groupwas removed and the pellet resuspended in Botu-Semen to a final concentration of 50x106sperm/mL. In the treatment group, the pellet was resuspended in 5 ml of Botu-Semen. This sample was placed upon 5 ml of density gradient in a 15 ml tube and centrifuged at 400xg for 20 minutes. The pellet was recovered and diluted in Botu-Semen to a final concentration of 50x106 sperm/ml. All samples were cooled in an isothermal box (Botu-Flex) at 5 oC for 24 hours. Semen was evaluated before and after cooling by CASA for total motility (TM), progressivemotility (PM), rapid sperm (RAP); plasmamembrane integrity (PMI) was evaluated by epifluorescence microscopy. All parameters were analyzed by ANOVA, followed by Tukey's test to identify significant differences (P < 0.05). The average recovery rate after centrifugation with the density gradient was 41%. Before cooling, the treatment group was superior in all parameters when compared to centrifuged samples, and demonstrated higher values of PM and RAP compared to control. However, control group showedhigher valuesof TMandPMI than centrifugedgroup. After cooling, treatment group was greater compared to control and centrifuged groups in all parameters evaluated (P < 0.05). In conclusion, the centrifugation with a density gradient was effective in selecting sperm more resistant to cooling at 5oC for 24 hours. This gradient may be an important option to improve the fertility of “bad cooler” stallions.