MOUSE SPERM CRYOPRESERVATION: A BIOTECHNOLOGICAL NEED TO ACOMPLISH THE BIOETHICAL PRINCIPLE OF THE 3R

NAVARRO C; KLINSKY LAHOZ OG; SARTOR T; FURLÁN M; WETTEN PA; MICHAUT MA

Mendoza

Congreso; XL Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2022

Sociedad de Biología de Cuyo

Sperm cryopreservation is an important technique for maintaining valuable genetic resources in biomedical research and wildlife. Research projects on reproductive biology use the mouse as an experimental model due to its short reproductive cycle. However, maintaining mice strain in an animal facility has the disadvantage of occupying large spaces with high economic and human costs. Thus, mouse sperm cryopreservation becomes a biotechnological need to reduce the drawbacks of this model and to accomplish the bioethical principle of the 3R: Replacement, Reduction and Refinement. Although the cold has been used as a method of preservation since ancient times, it was only in 1990 that the ideal conditions to cryopreserve mouse sperm with a relatively simple cryoprotectant were found. This cryoprotectant solution (CPS) is composed by raffinose and skim milk. The aim of this work was to set up the sperm cryopreservation from CF-1 mouse strain using this CPS. The sperm were collected from caudae epididymides of 3-5 month old male mice. After killing each mouse, both epididymides were removed and placed in a 35-mm sterile dish containing CPS (18% raffinose and 3% skim milk) equilibrated at 37 °C. Each cauda epididymis was cut three times and sperm were allowed to disperse for 3 min in the CPS solution. Immediately, 0.25 ul straws were loaded and rapidly cooled in liquid nitrogen vapor for 10 minutes. Finally, straws were plunged directly into liquid nitrogen for storage. To evaluate the effect of cryopreservation on sperm motility, frozen samples were rapidly thawed by transferring them from liquid nitrogen into a 37 °C water bath for 10 min. After 30 minutes of recovering in HTF medium supplemented with 0.5% BSA, in situ and progressive motility were evaluated for each sample. Results shown that this protocol yielded 3-6 % and 8-14 % of mouse sperm with in situ and progressive motiliy, respectively. These results demonstrate that we have setted up the mouse sperm cryopreservation protocol to reduce the breeding and slaughter of CF-1 mice males.