Comparison of permeating and nonpermeating cryoprotectants for mouse spermcryopreservation

Mouse sperm has proven to be more difficult to cryopreserve than sperm of o ther mammalian species. Published reports show that only three cryoprotecta nt agents (CPAs), alone or combined, have been studied: glycerol and dimeth yl sulfoxide (DMSO). as permeating agents, and raffinose. as a nonpermeatin g agent. To date. the most consistent results for mouse sperm cryopreservat ion have been achieved by use of raffinose/skim milk as cryoprotectant with rapid cooling at 20 degreesC per minute. In this study. we compared the cr yoprotection provided by permeating (glycerol. formamide. propanediol. DMSO . adonitol) or nonpermeating (lactose. raffinose, sucrose. trehalose. D-man nitol) compounds for freezing mouse sperm. Different solutions were made us ing 3% skim milk solution as the buffer or extender in which all different cryoprotectant agents were dissolved at a concentration of 0.3 M. with a fi nal osmolality of approx. 400 mOsm. Sperm samples from CB6FI (hybrid) and C 57BL/6J (inbred) mice collected directly Into each CPA were frozen/thawed u nder identical conditions. After thawing and CPA elimination (centrifugatio n) raffinose (59%). trehalose (61%). and sucrose (61%) sustained the best m otility (P = < 0.1) of the nonpermeating agents. whereas the best of the pe rmeating agents was DMSO (42%). Membrane integrity was analyzed and showed that the simple exposure (perfreeze) to sugars was less harmful than the ex posure to glycols. Coincidentally. sperm frozen in trehalose (41%). raffino se (40.5%), and sucrose (37.5%) were the samples less injured among all dif ferent postthawed CPA tested. The in vitro fertilization results demonstrat ed that hybrid mouse spermatozoa frozen with sugars (lactose 80%, raffinose 80%. trehalose 79% of two-cell embryos production) were more fertile than those frozen with glycols (glycerol 11%). (C) 2001 Academic Press.