Abstract
The aim of this study was to develop a new container for cryopreservation of a limited number of spermatozoa. To evaluate the efficacy and safety of this new container, we performed preclinical evaluations using human sperm or mouse oocytes and sperm. First, using human sperm that was frozen and then thawed, we demonstrated that the sperm recovery rate using the new container was 96.7% (58/60), which was significantly higher (P < 0.05) than the recovery rate of 21.2% (11/52) when using the Cryotop®. Sperm motility rates were 19.2% (10/52) using the Cryotop® and 35.0% (21/60) using the new container. Second, murine epididymal spermatozoa were divided into three groups: fresh spermatozoa, spermatozoa frozen using a straw, and spermatozoa frozen using the new container. Sperm motility, sperm membrane and DNA integrity, in vitro development of fertilized eggs, and offspring development after embryo transfer were assessed. The motility of freeze-thawed sperm was lower in spermatozoa that were frozen using the new container than in fresh spermatozoa or those that were frozen using a straw. After intracytoplasmic sperm injection, the survival rate was 96.7% (145/150), the 2-cell development rate was 90.3% (131/145), and the blastocyst development rate was 77.2% (112/145), when using the new container. There were no differences in the sperm membrane, DNA integrity, or in the embryo development rates to the blastocyst stage among the different frozen groups. Six offspring were derived from spermatozoa freeze-thawed in the new container, and they developed normally. Thus, the new container allows easy handling of a small number of sperms and minimizes sperm loss during cryopreservation.