306 THE EFFECT OF BOVIPURE GRADIENT ON BISON SPERM CRYOPRESERVATION

Abstract

Endemic tuberculosis and brucellosis threaten Canada’s wood bison population. For the purposes of developing a procedure to harvest pathogen-free sperm from bison, a sperm separation and purification product, BoviPure, was tested to determine its effect on bison sperm viability before and after cryopreservation. BoviPure (NidaCon International AB, Mölndal, Sweden) is a density centrifugation gradient system that contains trypsin and is designed to separate motile from non-motile sperm and remove infectious pathogens. Spermatozoa were expelled from the caudal epididymis of bison testicles collected at slaughter and diluted in 3 to 5 mL of TCM-199 medium (Gibco, Burlington, Ontario, Canada). After 1 h of incubation at 37°C, 1 to 1.5 mL of diluted sample (approximately 300 × 10⁶ sperm) was placed in (1) a BoviPure gradient containing trypsin (n = 18 samples), (2) a BoviPure gradient without trypsin (n = 10 samples), or 3) left untreated (control; n = 20). Gradients were centrifuged at 300g for 20 min. The sperm pellet was resuspended in 5 mL of TCM-199 medium and recentrifuged at 500 g for 10 min. Total and progressive motility were estimated using computer-assisted sperm analysis (CASA). Gradient-treated and untreated sperm samples were diluted in an egg yolk extender (Triladyl; Minitube Canada, Ingersoll, Ontario, Canada), placed in 0.5-mL straws (50-200 × 10⁶ spermatozoa per straw), frozen, and stored in liquid nitrogen for a minimum of 24 h. Semen straws were thawed by plunging in a 37°C water bath for 30 s, and motility measurements were taken immediately by CASA. Total and progressive motility were compared among groups by analysis of variance. Before cryopreservation, total motility and progressive motility were not affected by gradient processing. Total motility was 85±5.1%, 80±4.8%, and 77 ± 3.7% for untreated, BoviPure with trypsin, and BoviPure without trypsin, respectively (mean ± SEM), and progressive motility was 74 ± 2.7%, 77 ± 3.8%, and 68 ± 4.7%, respectively. After freezing and thawing, total motility decreased compared to unfrozen samples (P < 0.0001) but was not different among groups (37 ± 3.6%, 33 ± 3.7%, and 29 ± 4.8% for untreated, BoviPure with trypsin, and BoviPure without trypsin, respectively). Similarly, progressive motility decreased compared to unfrozen samples (P < 0.0001) but was not different among groups (26 ± 3.6%, 21 ± 3.7%, and 14.5 ± 4.9% for untreated, BoviPure with trypsin, and BoviPure without trypsin, respectively). We conclude that the BoviPure gradient with or without trypsin does not influence total motility or progressive motility of bison sperm either before or after cryopreservation and has potential as a method of harvesting pathogen-free sperm from wild animals of unknown disease status.

This study was supported by Canadian Adaptation and Rural Development in Saskatchewan.