Optimization of cryopreservation of Arabian stallion sperm using dimethylformamide, glycerol, and different freezing protocols

This study evaluated the effect of penetrating cryoprotectant agents (CPA) and the cryosurvival of three freezing protocols on the kinematics and integrity of membranes of frozen-thawed stallion sperm. Twenty-four ejaculates of four adult Arabian horses were collected in six weekly sessions (six ejaculates/horse). Each ejaculate was divided into two aliquots. With the first aliquot, three CPA treatments were conformed: 5% glycerol (GLY), 5% dimethylformamide (DMF), and 3%–3% DMF–GLY combination, and the sperm samples were frozen exposing them to liquid nitrogen (LN2) vapors. The second aliquot was diluted with freezing medium plus 5% DMF and the sperm samples were frozen in three freezing protocols: (P1) Styrofoam cryo-box (30 × 29 × 31 cm of length, width, and height, respectively) with two ramps (at 17 and 7 cm above LN2); (P2) freezing unit® (Minitüb, Germany); and (P3) programmable TK 4000-freezer® (Compacta, Brazil). The DMF-GLY combination and DMF yielded higher (p<.05) post-thaw values than the GLY regarding the motility (SM: 54.2±2.25 and 50.2±1.80 vs. 41.4±2.35%, respectively), curvilinear velocity (VCL: 58.0±1.71 and 54.0±1.58 vs. 42.3±1.60 µm/s), and the proportion of sperm with intact plasma and intact acrosome (IPIA: 58.0±1.11 and 52.6±0.99 vs. 42.5±1.07%). Furthermore, the P1 protocol produced a similar (p>.05) post-thaw SM, VCL, and IPIA than the other protocols. Indeed, the P1 and P3 protocols yielded lower proportion (p<.05) of sperm with damaged plasma and damaged acrosome than the P2 protocol after thawing (3.7±0.18 and 3.1±0.18 vs. 6.1±0.44%, respectively). In conclusion, the addition of DMF or combined with GLY to freezing medium, and the freezing with Styrofoam cryo-box with two ramps increase the cryosurvival of Arabian stallion spermatozoa.