Abstract. The rabbit is considered to be a valuable laboratory animal. We compared glycerol, lactamide, acetamide, and dimethylsulfoxide (DMSO) as cryoprotectants in egg-yolk diluent of ejaculated Japanese white rabbit spermatozoa for improvement of sperm cryopreservation methods. Rabbit semen was frozen with 1.0 M glycerol, lactamide, acetamide, or DMSO in plastic straws. Forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rate of forward progressive motile spermatozoa in lactamide (37.8 ± 3.0%) was significantly (P<0.05) higher than in glycerol (17.0 ± 3.3%). In addition, the rates of sperm plasma membrane integrity in lactamide and acetamide (35.9 ± 3.3% and 30.2 ± 3.0%, respectively) were significantly (P<0.05) higher than in glycerol (17.0 ± 2.6%). The results indicate that 1.0 M lactamide and acetamide have higher cryoprotective effects than 1.0 M glycerol for cryopreservation of Japanese white rabbit spermatozoa. Key words: Acetamide, Cryopreservation, Glycerol, Lactamide, Rabbit spermatozoa (J. Reprod. Dev. 52: [511][512][513][514][515][516] 2006) he rabbit is considered to be a valuable laboratory animal for research in teratology, immunology, microbiology, and medical and life sciences. In addition, many valuable mutants [1,2] including transgenic [3][4][5][6], have been established in the rabbit. Therefore, a need has been recognized for reliable methods to bank rabbit genetic resources efficiently in the form of cryopreserved spermatozoa. Glycerol has been extensively used as a cryoprotectant for various cells, including mammalian spermatozoa and embryos, since Polge et al. [7] reported its cryoprotective properties. In early research on freezing of rabbit spermatozoa, spermatozoa frozen with glycerol resulted in extremely low fertility [8][9][10][11]. After 1980, studies employing acetamide [12][13][14][15] or DMSO [12,[16][17][18][19][20][21][22] as a cryoprotectant have been encouraging. H o w e v e r , t h e r e i s a l i t t l e i n f o r m a t i o n o n cryoprotectants when freezing Japanese white rabbit spermatozoa.In the present study, we compared 1.0 M glycerol, lactamide, acetamide, and DMSO as cryoprotectants in egg-yolk diluent for ejaculated spermatozoa of the Japanese white rabbit. Forward progressive motility and plasma membrane