PRODUCTION OF NORMAL OFFSPRING FROM MOUSE OOCYTES INJECTED WITH SPERMATOZOA CRYOPRESERVED WITH OR WITHOUT CRYOPROTECTION

Epididymal mouse spermatozoa were suspended in various physiological s olutions (CZB, PBS or isotonic saline) with or without 18% (w/v) raffi nose before cooling to -20 degrees, -50 degrees or -196 degrees C and storage for 1-28 days. After thawing, a few spermatozoa frozen with ra ffinose were partially motile (about 2%) but in all other treatments t hey were immotile and diagnosed as 'dead' by staining that differentia tes between live and dead spermatozoa. Almost all oocytes injected wit h sperm heads (nuclei) from spermatozoa frozen with and without raffin ose were fertilized normally (95-100%) and developed to the two-cell s tage (89-100%). No differences were found between the physiological me dia. The majority of oocytes fertilized with spermatozoa frozen in CZB medium developed to blastocysts (80-94%) but development was signific antly reduced after fertilization with spermatozoa frozen in PBS and i sotonic saline especially in the absence of raffinose (69 and 70% vers us 51 and 50%). Normal fertile offspring were obtained in all treatmen ts but there were significantly fewer offspring with spermatozoa store d at -196 degrees C in isotonic saline with or without raffinose and C ZB with raffinose. Testicular spermatozoa were extremely sensitive to cryodamage: about 50% frozen to -196 degrees 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 s permatozoa developed to the two-cell stage but development to blastocy sts was reduced significantly compared with that of controls especiall y those without raffinose. The data indicate that cryopreservation of sperm nuclei requires less stringent conditions than those for the ret ention of normal physiological function of intact spermatozoa. Motilit y and plasma membrane integrity are not essential for fertilization an d the production of live offspring when nuclei of nonviable spermatozo a are injected into oocytes.