Cryopreservation of Adult Bovine Testicular Tissue for Spermatogonia Enrichment

To develop a procedure for cryopreservation of adult bovine testis tissue, the effects of dimethyl sulphoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), and their concentrations (v/v), as well as different thawing temperatures, on the cell viability of bovine testis tissue after freezing/thawing were examined. The highest testicular cell viabilities came from the media containing DMSO (85.3 ± 1.2%), PG (82 ± 1.0%) and EG (83.4 ± 1.0%) at 10% concentration respectively. Using 10% DMSO gave significantly higher spermatogonia percentage (61.1 ± 1.2%, P < 0.001) than processing with 10% PG (54.3 ± 0.6%) or 10% EG (55 ± 1.8%) after differential plating. Thawing in water bath of 37 or 97-100°C also provided significantly higher viabilities (85.1 ± 1.0, 85 ± 1.0%, P < 0.01, respectively) and spermatogonia percentages (56.6 ± 2.0, 56.6 ± 2.6%, P < 0.01, respectively) than that thawing at 4°C (23.4 ± 0.8% for total viability, 8.97 ± 1.0% for spermatogonia percentage). Collectively, 10% DMSO and thawing in 37-100°C water baths were appropriate for the cryopreservation of bovine testicular tissue and subsequent spermatogonia enrichment.