Cryoprotectant-free vitrification of llama spermatozoa: cryoloop vs sphere method, warmed rapidly or ultra-rapidly

Our objective was to determine the effect of vitrification using both cryoloop and sphere methods, combined with two warming protocols, on the percentage of motility, membrane integrity and DNA fragmentation/ condensation of llama spermatozoa. Raw semen (n = 5; r = 3) was incubated with collagenase (0.1 % in Tyrode's Albumin Lactate Pyruvate, TALP, solution), centrifuged and diluted in TALP with or without 5% dimethylformamide (DMF). Two spermatozoa concentrations were used: 20 x 106 sperm/mL (cryoloops) and 5 x 106 sperm/mL (spheres). Two warming procedures were evaluated: a rapid method (30 s at 37 degrees C) and an ultra-rapid method (7 s at 75 degrees C, followed by 30 s at 37 degrees C). Percentages of sperm total motility, viability, membrane function, chromatin condensation and chromatin fragmentation were evaluated before and after vitrification and analyzed using Friedman's test. The only seminal parameters maintained following vitrification were: 1) chromatin condensation, in all evaluated protocols and 2) DNA integrity, with the spheres method using both warming protocols without cryoprotectants and warming rapidly with cryoprotectants. Spermatozoa vitrification is a simple and inexpensive cryopreservation method that was able to preserve llama sperm chromatin condensation and integrity, especially using the spheres method without cryoprotectants combined with warming at 37 degrees C for 30 s. However it failed to preserve spermatozoa motility, membranes and viability.