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.

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.

Total Eclipse of the Zoo: Animal Behavior during a Total Solar Eclipse.

The infrequency of a total solar eclipse renders the event novel to those animals that experience its effects and, consequently, may induce anomalous behavioral responses. However, historical information on the responses of animals to eclipses is scant and often conflicting. In this study, we qualitatively document the responses of 17 vertebrate taxa (including mammals, birds, and reptiles) to the 2017 total solar eclipse as it passed over Riverbanks Zoo and Garden in Columbia, South Carolina. In the days leading up to the eclipse, several focal teams, each consisting of researchers, animal keepers, and student/zoo volunteers conducted baseline observations using a combination of continuous ad libitum and scan sampling of each animal during closely matched seasonal conditions. These same focal teams used the same protocol to observe the animals in the hours preceding, during, and immediately following the eclipse. Additionally, for one species-siamangs (Symphalangus syndactylus)-live video/audio capture was also employed throughout observations to capture behavior during vocalizations for subsequent quantitative analysis. Behavioral responses were classified into one or more of four overarching behavioral categories: normal (baseline), evening, apparent anxiety, and novel. Thirteen of seventeen observed taxa exhibited behaviors during the eclipse that differed from all other observation times, with the majority (8) of these animals engaging in behaviors associated with their evening or nighttime routines. The second predominant behavior was apparent anxiety, documented in five genera: baboons (Papio hamadryas), gorillas (Gorilla gorilla gorilla), giraffes (Giraffa cf. camelopardalis), flamingos (Phoenicopterus ruber), and lorikeets (Trichoglossus moluccanus and Trichoglossus haematodus). Novel behaviors characterized by an increase in otherwise nearly sedentary activity were observed only in the reptiles, the Galapagos tortoise (Chelonoidis nigra) and the Komodo dragon (Varanus komodoensis). While the anthropogenic influences on animal behaviors-particularly those relating to anxiety-cannot be discounted, these observations provide novel insight into the observed responses of a diverse vertebrate sample during a unique meteorological stimulus, insights that supplement the rare observations of behavior during this phenomenon for contextualizing future studies.

The Forearm Musculature of the Gray Mouse Lemur (Microcebus murinus): An Ontogenetic Study.

Although studies have sought to characterize variation in forearm muscular anatomy across the primate order, none have attempted to quantify ontogenetic changes in forearm myology within a single taxon. Herein, we present muscle architecture data for the forearm musculature (flexors and extensors of the wrist and digits) of Microcebus murinus, a small Lemuroid that has been the focus of several developmental studies. A quadratic curvilinear model described ontogenetic changes in muscle mass and fascicle length; however, fascicle lengths reached peak levels at an earlier age and showed a stronger decline during senescence. Conversely, physiological cross-sectional area followed a more linear trend, increasing steadily throughout life. As previous studies into the functional role of the primate forelimb emphasize the importance of long muscle fascicles within arboreal taxa in order to maximize mobility and flexibility, the early attainment of peak fascicle lengths may consequently reflect the importance of agility within this mobile and highly arboreal species. Similarly, observed myological trends in forearm strength are supported by previous in vivo data on grip strength within M. murinus in which senescent individuals showed no decline in forearm force relative to prime age individuals. This trend is interpreted to reflect compensation for the previously reported decline in hind limb grip strength in the hind limb with age, such that older individuals are able to maintain arboreal stability. Interestingly, the ontogenetic trajectory of each architectural variable mirrored previous observations of the masticatory musculature in M. murinus, suggesting that ontogenetic trends are relatively conserved between anatomical regions. Anat Rec, 303:1354-1363, 2020. © 2019 American Association for Anatomy.

The Ontogeny of Masticatory Muscle Architecture in Microcebus murinus.

The masticatory apparatus has been the focus of many studies in comparative anatomy-especially analyses of skulls and teeth, but also of the mandibular adductor muscles which are responsible for the production of bite force and the movements of the mandible during food processing and transport. The fiber architecture of these muscles has been correlated to specific diets (e.g., prey size in felids) and modes of foraging (e.g., tree gouging in marmosets). Despite the well-elucidated functional implications of this architecture, little is known about its ontogeny. To characterize age-related myological changes, we studied the masticatory muscles in a large (n = 33) intraspecific sample of a small, Malagasy primate, Microcebus murinus including neonatal through geriatric individuals. We removed each of the mandibular adductors and recorded its mass as well as other linear measurements. We then chemically dissected each muscle to study its architecture-fascicle length and physiological cross-sectional area (PCSA) which relate to stretch (gape) and force capabilities, respectively. We observed PCSA and muscle mass to increase rapidly and plateau in adulthood through senescence. Fascicle lengths remained relatively constant once maximal length was reached, which occurred early in life, suggesting that subsequent changes in PCSA are driven by changes in muscle mass. Quadratic curvilinear models of each of the architectural variables of all adductors combined as well as individual muscles regressed against age were all significant. Anat Rec, 303:1364-1373, 2020. © 2019 American Association for Anatomy.

Scaling of Anatomically Derived Maximal Bite Force in Primates.

By combining muscle architectural data with biomechanical variables relating to the jaw, we produce anatomically derived maximum bite force estimations for 23 species of catarrhine and platyrrhine primates. We investigate how bite force scales across the sample as a whole (and within each parvorder) relative to two size proxies, body mass and cranial geometric mean, and the effect of diet upon bite force. Bite force is estimated at three representative bite points along the dental row: the first maxillary incisor, canine, and third-most mesial paracone. We modeled bite force by combining calculated physiological cross-sectional area of the jaw adductors from Hartstone-Rose et al. [Anat Rec 301 (2018) 311-324] with osteological measurements of lever- and load-arm lengths from the same specimens [Hartstone-Rose et al., Anat Rec 295 (2012) 1336-1351]. Bite force scales with positive allometry relative to cranial geometric mean across our entire sample and tends toward positive allometry relative to body mass. Bite force tends toward positive allometry within platyrrhines but scales isometrically within catarrhines. There was no statistically significant scaling difference with diet. Our findings imply an absence of a dietary signal in the scaling of bite force, a result that differs from the scaling of physiological cross-sectional area alone. That is, although previous studies have found a dietary signal in the muscle fiber architecture in these species, when these are combined with their leverages, that signal is undetectable. On the parvorder level, our data also demonstrate that the platyrrhine masticatory system appears more mechanically advantageous than that of catarrhines. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:2026-2035, 2020. © 2019 American Association for Anatomy.

Extraordinary grip strength and specialized myology in the hyper-derived hand of Perodicticus potto?

Previous behavioral reports of the African lorisid, Perodicticus potto, have speculated that these animals have an extraordinary grip strength. This ability is hypothesized to be facilitated by a range of anatomical features within the forelimb, ranging from the presence of a retia mirabilia in its wrist to the hyper-abduction of its pollex. Despite numerous behavioral reports, however, this claim of extraordinary grip strength has not been empirically substantiated. This study quantifies the physiological cross-sectional area of the digital flexor muscles within P. potto. These data are compared with a broad primate sample, including several similarly sized strepsirrhines. Contrary to expectation, we found that P. potto actually has relatively below-average digital flexor PCSA. However, we identified other myological characteristics in the upper limb of P. potto that were unexpected, including the largest brachioradialis muscle (an elbow flexor) among our primate sample, and - despite P. potto having only a vestigial second digit - an independent digital extensor indicis that is absent in almost a quarter of our primate sample.