Cryopreservation of spermatozoa of llama obtained from the vas deferens using three freezing curves

Abstract

The aim of this study was to determine the effect of three freezing curves on the post-thaw viability of spermatozoa collected from the vas deferens of llamas. Six male llamas with surgical diversion of the vas deferens were used. The samples of the six males were mixed for processing (pools) and diluted with Tris-egg yolk. Cooling was carried out to 5 °C where the dilution was completed (dilutor with glycerol) and was maintained for 30 minutes (equilibrium phase). Samples in 0.25 ml straws were subjected to freezing using a temperature drop rate of -20°C/min until reaching -80°C (TI), -100°C (TII) and -120°C (TIII) in 4, 5 and 6 minutes, respectively, to finally store them in liquid nitrogen. The samples were collected for three months (n=19 pools). Total motility (progressive, circular, oscillatory motility), viability and membrane functionality were determined after collection, in the equilibrium phase and upon thawing. A significant decrease (p<0.05) was observed in all the sperm characteristics evaluated in the samples after the equilibrium compared to the samples after collection. A high positive correlation was obtained between viability and total motility (r2=0.78) and in the equilibrium phase between membrane functionality and total motility (r2=0.881). In thawed samples, total motility and viability were significantly higher in frozen samples. with the freezing curve TIII with respect to TI (p=0.041 and p=0.003, respectively). No significant differences in membrane functionality were observed between the three freezing curves (p>0.27). In conclusion, the temperature drop curve down to -120 °C using a rate of -20 °C/min would be the most suitable for cryopreserving llama spermatozoa obtained from the deviation of the vas deferens.