Evaluation of the Freeze-dried Human gamete DNA integrity by the "Comet Assay”
Freeze-drying; sperm damage; fertilization
Freeze-drying is a method of preservation of sperm that is raising great interest due to the advantages such as shipping cost in transport and storage. This process, although widely studied, still has some disadvantages such as non-recovery of sperm motility. The aim of this work was to maintain human sperm DNA integrity after freeze-drying process. For this were tested five sugars (D-glucose, D-lactose and D-maltose, D-mannitol and D-sorbitol, at a 0.2mol/L, 0.4mol/L, 0.5 mol/L and 0.6 mol/L). The Comet Assay was used to detect the rate of DNA sperm damage recovered after freeze-drying. The cryoprotectants used in this experiment were unable to preserve sperm motility, and observed that loss even after the thawing process. This process, although widely studied, still has some disadvantages such as non-recovery of sperm motility. However they were able to keep 62.30 ± 13.76% of the plasma membrane when fully preserved with 0.6M mannitol. The medium that best preserved the DNA was 0.2M mannitol, 0.6M mannitol and 0,6 M sorbitol, with only 4% of DNA damaged. These results suggest a good protection to the DNA human spermatozoa subjected to freeze drying, but still required additional tests to evaluate fertilization and studies to ensure a viable offspring.