Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection

Abstract

Epididymal mouse spermatozoa were suspended in various physiological solutions (CZB, PBS or isotonic saline) with or without 18% (w/v) raffinose before cooling to -20°, -50°or -196°C and storage for 1-28 days. After thawing, a few spermatozoa frozen with raffinose were partially motile (about 2%) but in all other treatments they were immotile and diagnosed as 'dead' by staining that differentiates between live and dead spermatozoa. Almost all oocytes injected with sperm heads (nuclei) from spermatozoa frozen with and without raffinose were fertilized normally (95-100%) and developed to the two-cell stage (89-100%). No differences were found between the physiological media. The majority of oocytes fertilized with spermatozoa frozen in CZB medium developed to blastocysts (80-94%) but development was significantly reduced after fertilization with spermatozoa frozen in PBS and isotonic saline especially in the absence of raffinose (69 and 70% versus 51 and 50%). Normal fertile offspring were obtained in all treatments but there were significantly fewer offspring with spermatozoa stored at -196°C in isotonic saline with or without raffinose and CZB with raffinose. Testicular spermatozoa were extremely sensitive to cryodamage: about 50% frozen to -196°C in CZB with or without raffinose disintegrated after thawing. Almost 100% of oocytes injected with sperm heads from intact (at light microscope level) testicular spermatozoa developed to the two-cell stage but development to blastocysts was reduced significantly compared with that of controls especially those without raffinose. The data indicate that cryopreservation of sperm nuclei requires less stringent conditions than those for the retention of normal physiological function of intact spermatozoa. Motility and plasma membrane integrity are not essential for fertilization and the production of live offspring when nuclei of nonviable spermatozoa are injected into oocytes.