Equine Spermatozoa Selection by Magnetic Activation for Use in Assisted Reproduction.

Abstract

This study aimed to select high-quality spermatozoa by sperm separation by magnetic activation of the fresh equine semen, compared to density gradient centrifugation and evaluating cell quality after selection. The semen of 10 stallions was collected by the artificial vagina technique. The samples analyzed were: (1) fresh semen; (2) density gradient centrifugation (DGC); (3) separation by magnetic activation (MASS) (nonapoptotic portion NAP); (4) separation by MASS (apoptotic portion-APT). Was analyzed: motility (light microscopy), concentration (Neubauer chamber), semen morphology (humid chamber in phase contrast), and supravital test (eosin/nigrosine). In DGC, 20×106 spermatozoa were used in the gradient of Percoll at 90% and 45% (400 μL each), centrifugation at 900 G/5 min, the pellet was diluted in HEPES. In MASS, 10×106 spermatozoa were diluted in 1.5 mL of HEPES, centrifugation at 300 G/10 min, pellet was resuspended in 150 μL of HEPES with 20 μL of nanoparticles bound to annexin V, incubation for 15 minutes and filtered in the magnetic separation column. The nonapoptotic fraction was collected directly and the apoptotic fraction after removal the column from the magnet and adding 300 μL of HEPES. The total abnormalities were 43.2% ± 2.78%, with the DGC and MASS being effective in reducing sperm abnormality by 15.6% ± 2.10% and 24.30% ± 1.63%, respectively, like the observed for the number of cells with intact membranes (50% lower in the APT portion). This nanotechnological method is efficient in producing high-quality semen samples for assisted reproduction procedures.