77 FREEZING OF MOUSE SPERM BY THREE DIFFERENT CRYOPROTECTANTS

Abstract

The cryopreservation of sperm has contributed greatly to animal breeding and reproduction. This study was designed to examine the effect of raffinose, sucrose, and trehalose as cryoprotectants for freezing of mouse sperm. The cryoprotectant solution (CPA) consisting of 3% skim milk (Skim Milk dehydrated, Bacto, Difco, Seoul, Korea) as buffer or extender was prepared and supplemented with 0.3 M raffinose (D[+]raffinose pentahydrate, Sigma) or sucrose or trehalose as non-permeating cryoprotectants. Sperm samples for cryopreservation were collected from caudae epididymides and vas deferens of males of four mouse strains (ICR, FVB, C57BL/6, and CBA). Sperm samples from individual males were aliquoted in cryotubes, placed immediately in the vapor phase of a liquid nitrogen storage container for 10 min, and then plunged directly in liquid nitrogen. For thawing, frozen cryotubes were removed from liquid nitrogen and placed directly into a water bath kept at 37°C for approximately 2 min until the ice melted. Survival of mouse sperm was measured by vital staining. Survival rates of spermatozoa frozen and thawed in freezing solution supplemented with raffinose were higher (ICR: 51.0%, FVB: 27.6%, C57BL/6: 25.7%, and CBA: 23.3%) than those supplemented with sucrose (35.8, 19.6, 12.3, and 19.7%) or trehalose (16.0, 25.4, 25.3, and 24.7%), all respectively. Furthermore, in vitro fertilization of mouse oocytes with sperm frozen in raffinose gave cleavage rates of 61.1 (FVB: 217/355 eggs), 59.4 (C57BL/6: 165/278 eggs), and 57.1% (CBA: 144/252 eggs), respectively. These cleaved embryos (FVB: 163, C57BL/6: 137, and CBA: 134) were transferred to 7, 5, and 6 pseudopregnant females, respectively. A total of 4, 3, and 4 females in the respective strains became pregnant and delivered 17 (10.4%), 17 (12.4%), and 18 (13.4%) offspring, respectively. Our results suggest that raffinose is a good cryoprotectant for freezing of sperm for production of inbred mice.