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.

Effects of long-term ethanol storage on muscle architecture.

Muscle excursion and force potential can be estimated from architectural variables, including mass, volume, fascicle length, and density. These have been collected from fresh specimens, preserved specimens, and sometimes mixed samples of both. However, preservation alters the gross morphology of muscles. This study aims to quantify the effects of long-term storage on myological properties across a sample of fresh and ethanol preserved Mus musculus specimens ranging in storage time from 16 to 130 years. Masses, volumes, and densities of biceps femoris, quadriceps femoris, and triceps surae were measured, and histological cross-sections of some specimens were used to evaluate the microscale effects of long-term fluid preservation. For the remainder of the sample, chemically dissected fascicle lengths were measured to evaluate the fixation effects on the linear dimensions of muscle architecture. Relative muscle mass, volume, fascicle length, average fiber area, and density, and percent fiber area were regressed against years stored in ethanol. Muscle size dropped steeply between fresh and stored samples, ultimately decreasing by 62 and 60%, respectively. These losses correlate with histologically measured shrinking of average muscle fiber area. Density of stored specimens plateaued 5% below that of fresh ones. Although muscles lost mass and volume during ethanol storage, fascicle lengths did not shorten significantly (presumably because they were preserved attached on either end to bone). This study demonstrates that muscle mass, volume, and density of specimens stored long-term in ethanol should be corrected by factors of 2.64, 2.49, and 1.054 respectively for comparability to fresh specimens.

Masticatory muscle architectural correlates of dietary diversity in Canidae, Ursidae, and across the order Carnivora.

Carnivorans represent extreme ecomorphological diversity, encompassing remarkable variation in form, habitat, and diet. The relationship between the masticatory musculature and dietary ecology has been explored in a number of carnivoran lineages, including felids and the superfamily Musteloidea. In this study, we present novel architectural data on two additional carnivoran families-Ursidae and Canidae-and supplement these previous studies with additional felid, musteloid, herpestid, hyaenid, and viverrid taxa (a total of 53 species across 10 families). Gross dissection data were collected following a standardized protocol-sharp dissection followed by chemical digestion. Summed jaw adductor forces were also transformed into bite force estimates (BF) using osteologically calculated leverages. All data were linearized, log-transformed, and size-adjusted using two proxies for each taxon-body mass (BM) and cranial geometric mean-to assess relative scaling trends. These architectural data were then analyzed in the context of dietary ecology to examine the impact of dietary size (DS) and dietary mechanical properties (DMP). Muscle mass, physiological cross-sectional area, and BF scaled with isometry or positive allometry in all cases, whereas fascicle lengths (FLs) scaled with isometry or negative allometry. With respect to diet, BM-adjusted FLs were strongly correlated with DS in musteloids, but not in any other lineage. The relationship between size-adjusted BF and DMP was also significant within musteloids, and across the sample as a whole, but not within other individual lineages. This interfamilial trend may reflect the increased morphological and dietary diversity of musteloids relative to other carnivoran groups.

Myological variation in the forearm anatomy of Callitrichidae and Lemuridae.

The anatomy of the primate forearm is frequently investigated in terms of locomotor mode, substrate use, and manual dexterity. Such studies typically rely upon broad, interspecific samples for which one or two representative taxa are used to characterize the anatomy of their genus or family. To interpret variation between distantly related taxa, however, it is necessary to contextualize these differences by quantifying variation at lower hierarchical levels, that is, more fine-grained representation within specific genera or families. In this study, we present a focused evaluation of the variation in muscle organization, integration, and architecture within two speciose primate families: the Callitrichidae and Lemuridae. We demonstrate that, within each lineage, several muscle functional groups exhibit substantial variation in muscle organization. Most notably, the digital extensors appear highly variable (particularly among callitrichids), with many unique configurations represented. In terms of architectural variables, both families are more conservative, with the exception of the genus Callimico-for which an increase is observed in forearm muscle mass and strength. We suggest this reflects the increased use of vertical climbing and trunk-to-trunk leaping within this genus relative to the more typically fine-branch substrate use of the other callitrichids. Overall, these data emphasize the underappreciated variation in forearm myology and suggest that overly generalized typification of a taxon's anatomy may conceal significant intraspecific and intrageneric variation therein. Thus, considerations of adaptation within the forearm musculature should endeavor to consider the full range of anatomical variation when making comparisons between multiple taxa within an evolutionary context.

Anatomical and ontogenetic influences on muscle density.

Physiological cross-sectional area (PCSA), an important biomechanical variable, is an estimate of a muscle's contractile force potential and is derived from dividing muscle mass by the product of a muscle's average fascicle length and a theoretical constant representing the density of mammalian skeletal muscle. This density constant is usually taken from experimental studies of small samples of several model taxa using tissues collected predominantly from the lower limbs of adult animals. The generalized application of this constant to broader analyses of mammalian myology assumes that muscle density (1) is consistent across anatomical regions and (2) is unaffected by the aging process. To investigate the validity of these assumptions, we studied muscles of rabbits (Oryctolagus cuniculus) in the largest sample heretofore investigated explicitly for these variables, and we did so from numerous anatomical regions and from three different age-cohorts. Differences in muscle density and histology as a consequence of age and anatomical region were evaluated using Tukey's HSD tests. Overall, we observed that older individuals tend to have denser muscles than younger individuals. Our findings also demonstrated significant differences in muscle density between anatomic regions within the older cohorts, though none in the youngest cohort. Approximately 50% of the variation in muscle density can be explained histologically by the average muscle fiber area and the average percent fiber area. That is, muscles with larger average fiber areas and a higher proportion of fiber area tend to be denser. Importantly, using the age and region dependent measurements of muscle density that we provide may increase the accuracy of PCSA estimations. Although we found statistically significant differences related to ontogeny and anatomical region, if density cannot be measured directly, the specific values presented herein should be used to improve accuracy. If a single muscle density constant that has been better validated than the ones presented in the previous literature is preferred, then 1.0558 and 1.0502 g/cm3 would be reasonable constants to use across all adult and juvenile muscles respectively.