Effects of freezing and short-term fixation on muscle mass, volume, and density

Preventing postmortem deterioration of soft-tissues is an important requisite of anatomical research. In order to provide corrections for potential myological distortions, this study quantifies the acute effects of freezing, formalin fixation and ethanol storage using muscles from (n = 46) rabbits (Oryctolagus cuniculus). Bilateral dissections of specific muscles were performed and each side was assigned to a different preparation group (fresh, formalin fixation only, fixation followed by short duration ethanol storage, and freezing once or twice). We demonstrate that short-term freezing at -20C and thawing have no significant effect on muscle mass, volume, and density while short-term formalin fixation and ethanol storage significantly reduces mass and volume (density remains relatively constant.) Although freezing may have less of an effect on the gross morphometric characteristics of the musculature than ethanol storage, slow freezing damages muscle microanatomy, and therefore, faster freezing and other modes of preservation such as formalin fixation and ethanol storage may be preferable. Based on our results, we derived the following correction factors for each preparation: the mass of specimens stored in 70% ethanol should be multiplied by 1.69 to approximate fresh muscle mass, and specimens fixed in 10% formalin multiplied by 1.32. Although not significant, specimens frozen-once were slightly less massive and could be multiplied by 1.03 (frozen-twice ×1.09). The volumetric corrections are: ethanol 1.64; 10% formalin 1.32; frozen-once 1.03; frozen-twice 1.10. While the density of ethanol preserved specimens is slightly less than that of fresh ones (correction: 1.03), those preserved in formalin and frozen maintain nearly the same density.