The interaction of in vivo muscle operating lengths and passive stiffness in rat hindlimbs.

The operating length of a muscle is a key determinant of its ability to produce force in vivo. Muscles that operate near the peak of their force-length relationship will generate higher forces whereas muscle operating at relatively short length may be safe from sudden lengthening perturbations and subsequent damage. At longer lengths, passive mechanical properties have the potential to contribute to force or constrain operating length with stiffer muscle-tendon units theoretically being restricted to shorter lengths. Connective tissues typically increase in density during aging, thus increasing passive muscle stiffness and potentially limiting the operating lengths of muscle during locomotion. Here, we compare in vivo and in situ muscle strain from the medial gastrocnemius in young (7 months old) and aged (30-32 months old) rats presumed to have varying passive tissue stiffness to test the hypothesis that stiffer muscles operate at shorter lengths relative to their force-length relationship. We measured in vivo muscle operating length during voluntary locomotion on inclines and flat trackways and characterized the muscle force-length relationship of the medial gastrocnemius using fluoromicrometry. Although no age-related results were evident, rats of both age groups demonstrated a clear relationship between passive stiffness and in vivo operating length, such that shorter operating lengths were significantly correlated with greater passive stiffness. Our results suggest that increased passive stiffness may restrict muscles to operating lengths shorter than optimal lengths, potentially limiting force capacity during locomotion.

Examining Cultural Structures and Functions in Biology.

Scientific culture and structure organize biological sciences in many ways. We make choices concerning the systems and questions we study. Our research then amplifies these choices into factors that influence the directions of future research by shaping our hypotheses, data analyses, interpretation, publication venues, and dissemination via other methods. But our choices are shaped by more than objective curiosity-we are influenced by cultural paradigms reinforced by societal upbringing and scientific indoctrination during training. This extends to the systems and data that we consider to be ethically obtainable or available for study, and who is considered qualified to do research, ask questions, and communicate about research. It is also influenced by the profitability of concepts like open-access-a system designed to improve equity, but which enacts gatekeeping in unintended but foreseeable ways. Creating truly integrative biology programs will require more than intentionally developing departments or institutes that allow overlapping expertise in two or more subfields of biology. Interdisciplinary work requires the expertise of large and diverse teams of scientists working together-this is impossible without an authentic commitment to addressing, not denying, racism when practiced by individuals, institutions, and cultural aspects of academic science. We have identified starting points for remedying how our field has discouraged and caused harm, but we acknowledge there is a long path forward. This path must be paved with field-wide solutions and institutional buy-in: our solutions must match the scale of the problem. Together, we can integrate-not reintegrate-the nuances of biology into our field.

Effects of selective breeding for high voluntary wheel-running behavior on femoral nutrient canal size and abundance in house mice.

Bone modeling and remodeling are aerobic processes that entail relatively high oxygen demands. Long bones receive oxygenated blood from nutrient arteries, epiphyseal-metaphyseal arteries, and periosteal arteries, with the nutrient artery supplying the bulk of total blood volume in mammals (~ 50-70%). Estimates of blood flow into these bones can be made from the dimensions of the nutrient canal, through which nutrient arteries pass. Unfortunately, measuring these canal dimensions non-invasively (i.e. without physical sectioning) is difficult, and thus researchers have relied on more readily visible skeletal proxies. Specifically, the size of the nutrient artery has been estimated from dimensions (e.g. minimum diameters) of the periosteal (external) opening of the nutrient canal. This approach has also been utilized by some comparative morphologists and paleontologists, as the opening of a nutrient canal is present long after the vascular soft tissue has degenerated. The literature on nutrient arteries and canals is sparse, with most studies consisting of anatomical descriptions from surgical proceedings, and only a few investigating the links between nutrient canal morphology and physiology or behavior. The primary objective of this study was to evaluate femur nutrient canal morphology in mice with known physiological and behavioral differences; specifically, mice from an artificial selection experiment for high voluntary wheel-running behavior. Mice from four replicate high runner (HR) lines are known to differ from four non-selected control (C) lines in both locomotor and metabolic activity, with HR mice having increased voluntary wheel-running behavior and maximal aerobic capacity (VO2 max) during forced treadmill exercise. Femora from adult mice (average age 7.5 months) of the 11th generation of this selection experiment were µCT-scanned and three-dimensional virtual reconstructions of nutrient canals were measured for minimum cross-sectional area as a skeletal proxy of blood flow. Gross observations revealed that nutrient canals varied far more in number and shape than prior descriptions would indicate, regardless of sex or genetic background (i.e. HR vs. C lines). Canals adopted non-linear shapes and paths as they traversed from the periosteal to endosteal borders through the cortex, occasionally even branching within the cortical bone. Additionally, mice from both HR and C lines averaged more than four nutrient canals per femur, in contrast to the one to two nutrient canals described for femora from rats, pigs, and humans in prior literature. Mice from HR lines had significantly larger total nutrient canal area than C lines, which was the result not of an increase in the number of nutrient canals, but rather an increase in their average cross-section size. This study demonstrates that mice with an evolutionary history of increased locomotor activity and maximal aerobic metabolic rate have a concomitant increase in the size of their femoral nutrient canals. Although the primary determinant of nutrient canal size is currently not well understood, the present results bolster use of nutrient canal size as a skeletal indicator of aerobically supported levels of physical activity in comparative studies.

Crouching to fit in: the energetic cost of locomotion in tunnels.

Animals that are specialized for a particular habitat or mode of locomotion often demonstrate locomotor efficiency in a focal environment when compared to a generalist species. However, measurements of these focal habitats or behaviors are often difficult or impossible to do in the field. In this study, the energetics and kinematics of simulated tunnel locomotion by two unrelated semi-fossorial mammals, the ferret and degu, were analyzed using open-flow respirometry and digital video. Animals were trained to move inside of normal (unconstrained, overground locomotion) and height-decreased (simulated tunnel, adjusted to tolerance limits for each species) Plexiglas chambers that were mounted flush onto a treadmill. Both absolute and relative tunnel performance differed between the species; ferrets tolerated a tunnel height that forced them to crouch at nearly 25% lower hip height than in an unconstrained condition, whereas degus would not perform on the treadmill past a ∼9% reduction in hip height. Both ferrets and degus exhibited significantly higher metabolic rates and cost of transport (CoT) values when moving in the tunnel condition relative to overground locomotion. When comparing CoT values across small (<10 kg) mammals, ferrets demonstrated a lower than predicted metabolic cost during both tunnel and terrestrial locomotion, whereas degus were very close to the line of best fit. Although tunnel locomotion requires a more striking change in posture for ferrets, ferrets are more efficient locomotors in both conditions than mammals of similar mass.

Trackways Produced by Lungfish During Terrestrial Locomotion.

Some primarily aquatic vertebrates make brief forays onto land, creating traces as they do. A lack of studies on aquatic trackmakers raises the possibility that such traces may be ignored or misidentified in the fossil record. Several terrestrial Actinopterygian and Sarcopterygian species have previously been proposed as possible models for ancestral tetrapod locomotion, despite extant fishes being quite distinct from Devonian fishes, both morphologically and phylogenetically. Although locomotion has been well-studied in some of these taxa, trackway production has not. We recorded terrestrial locomotion of a 35 cm African lungfish (Protopterus annectens; Dipnoi: Sarcopterygii) on compliant sediment. Terrestrial movement in the lungfish is accomplished by planting the head and then pivoting the trunk. Impressions are formed where the head impacts the substrate, while the body and fins produce few traces. The head leaves a series of alternating left-right impressions, where each impact can appear as two separate semi-circular impressions created by the upper and lower jaws, bearing some similarity to fossil traces interpreted as footprints. Further studies of trackways of extant terrestrial fishes are necessary to understand the behavioural repertoire that may be represented in the fossil track record.

Fluoromicrometry: A Method for Measuring Muscle Length Dynamics with Biplanar Videofluoroscopy.

Accurate measurements of muscle length changes are essential for understanding the biomechanics of musculoskeletal systems, and can provide insights into muscular work, force, and power. Muscle length has typically been measured in vivo using sonomicrometry, a method that measures distances by sending and receiving sound pulses between piezoelectric crystals. Here, we evaluate an alternative method, fluoromicrometry, which measures muscle length changes over time by tracking the three-dimensional positions of implanted, radio-opaque markers via biplanar videofluoroscopy. To determine the accuracy and precision of fluoromicrometry, we simultaneously measured length changes of an isolated muscle, the frog sartorius, in an in vitro setup using both fluoromicrometry and a servomotor. For fluoromicrometry to perfectly match the results of the servomotor, the relationship between the two measurements should be linear, with a slope of 1. Measurements of muscle shortening from fluoromicrometry and the motor were compared across 11 isotonic contractions. The precision of fluoromicrometry was ±0.09 mm, measured as the root mean square error of the regression of fluoromicrometry versus servomotor muscle lengths. Fluoromicrometry was also accurate: the mean slope of the fluoromicrometry-servomotor regressions did not differ significantly from the ideal line once off-axis motion was removed. Thus, fluoromicrometry provides a useful alternative for measuring muscle length, especially in studies of live animals, as it permits long-term marker implantation, wireless data collection, and increased spatial sampling. Fluoromicrometry can also be used with X-Ray Reconstruction of Moving Morphology to simultaneously measure muscle shortening and skeletal kinematics, providing a potent new tool for biomechanics research.

Lungfish axial muscle function and the vertebrate water to land transition.

The role of axial form and function during the vertebrate water to land transition is poorly understood, in part because patterns of axial movement lack morphological correlates. The few studies available from elongate, semi-aquatic vertebrates suggest that moving on land may be powered simply from modifications of generalized swimming axial motor patterns and kinematics. Lungfish are an ideal group to study the role of axial function in terrestrial locomotion as they are the sister taxon to tetrapods and regularly move on land. Here we use electromyography and high-speed video to test whether lungfish moving on land use axial muscles similar to undulatory swimming or demonstrate novelty. We compared terrestrial lungfish data to data from lungfish swimming in different viscosities as well as to salamander locomotion. The terrestrial locomotion of lungfish involved substantial activity in the trunk muscles but almost no tail activity. Unlike other elongate vertebrates, lungfish moved on land with a standing wave pattern of axial muscle activity that closely resembled the pattern observed in terrestrially locomoting salamanders. The similarity in axial motor pattern in salamanders and lungfish suggests that some aspects of neuromuscular control for the axial movements involved in terrestrial locomotion were present before derived appendicular structures.

Ontogenetic scaling of fore- and hind limb posture in wild chacma baboons (Papio hamadryas ursinus).

Large-scale interspecific studies of mammals ranging between 0.04-280 kg have shown that larger animals walk with more extended limb joints. Within a taxon or clade, however, the relationship between body size and joint posture is less straightforward. Factors that may affect the lack of congruence between broad and narrow phylogenetic analyses of limb kinematics include limited sampling of (1) ranges of body size, and/or (2) numbers of individuals. Unfortunately, both issues are inherent in laboratory-based or zoo locomotion research. In this study, we examined the relationship between body mass and elbow and knee joint angles (our proxies of fore- and hind limb posture, respectively) in a cross-sectional ontogenetic sample of wild chacma baboons (Papio hamadryas ursinus) habituated in the De Hoop Nature Reserve, South Africa. Videos were obtained from 33 individuals of known age (12 to ≥ 108 months) and body mass (2-29.5 kg) during walking trials. Results show that older, heavier baboons walk with significantly more extended knee joints but not elbow joints. This pattern is consistent when examining only males, but not within the female sample. Heavier, older baboons also display significantly less variation in their hind limb posture compared to lighter, young animals. Thus, within this ontogenetic sample of a single primate species spanning an order of magnitude in body mass, hind limb posture exhibited a postural scaling phenomenon while the forelimbs did not. These findings may further help explain 1) why younger mammals (including baboons) tend to have relatively stronger bones than adults, and 2) why humeri appear relatively weaker than femora (in at least baboons). Finally, this study demonstrates how field-acquired kinematics can help answer fundamental biomechanical questions usually addressed only in animal gait laboratories.

Effects of early-stage aging on locomotor dynamics and hindlimb muscle force production in the rat.

Attenuation of locomotor function is common in many species of animals as they age. Dysfunctions may emerge from a constellation of age-related impairments, including increased joint stiffness, reduced ability to repair muscle tissue, and decreasing fine motor control capabilities. Any or all of these factors may contribute to gait abnormalities and substantially limit an animal's speed and mobility. In this study we examined the effects of aging on whole-animal locomotor performance and hindlimb muscle mechanics in young adult rats aged 6-8 months and 'early aged' 24-month-old rats (Rattus norvegicus, Fischer 344 × Brown Norway crosses). Analyses of gaits and kinematics demonstrated that aged rats moved significantly more slowly, sustained longer hindlimb support durations, moved with a greater proportion of asymmetrical gaits, were more plantigrade, and moved with a more kyphotic spinal posture than the young rats. Additionally, the external mechanical energy profiles of the aged animals were variable across trials, whereas the younger rats moved predominantly with bouncing mechanics. In situ analyses of the ankle extensor/plantar flexor muscle group (soleus, plantaris, and medial and lateral gastrocnemii) revealed reduced maximum force generation with aging, despite minimal changes in muscle mass. The weakened muscles were implicated in the degradation of hindfoot posture, as well as variability in center-of-mass mechanics. These results demonstrate that the early stages of aging have consequences for whole-body performance, even before age-related loss of muscle mass begins.

A comparison of epigean and subterranean locomotion in the domestic ferret (Mustela putorius furo: Mustelidae: Carnivora).

Burrows are used by many mammals to escape predation, cache food and for parturition. Although the construction of burrows has been studied in some taxa, the locomotion while inside of them has received scant attention. In this study we collected simultaneous video and force data to characterize gaits, kinematics, peak ground reaction forces (GRFs) and external mechanical energy profiles in the domestic ferret, an animal that displays the typical morphology and behaviors associated with subterranean adaptations in mustelines. We compared kinematics and kinetics between locomotion in two experimental conditions: subterranean, simulated by a Plexiglass tunnel designed such that the ferrets' peak back height was reduced by 40% and hip height by 25%, and epigean, or unconstrained overground. Despite the large change in posture, a striking number of gait and force variables were not statistically different between experimental conditions. In both subterranean and epigean conditions, the ferrets in our study traveled at similar average velocities (approximately 0.8 m s(-1)), preferred to use a lateral-sequence diagonal-couplet gait, and were more likely to demonstrate the in-phase fluctuations of external mechanical energy indicative of running mechanics (68% of all trials). The ferrets conformed to gait and mechanical patterns seen in a variety of other small (<1 kg) mammals rather than being unique, despite the divergent morphology of mustelines. Our results demonstrated biodynamically similar locomotion in both epigean and subterranean conditions and support the hypothesis that ferrets possess adaptations for tunnel locomotion.

The effects of viscosity on the axial motor pattern and kinematics of the African lungfish (Protopterus annectens) during lateral undulatory swimming.

Separate studies of terrestrial and aquatic locomotion are abundant, but research addressing locomotion in transitional environments (e.g. mud) is scant. The African lungfish (Protopterus annectens) moves in a gradation of water to mud conditions during seasonal droughts, and breathes air. Thus, the lungfish was an ideal organism for our study to determine the effects of a wide range of viscosities on lateral undulatory swimming and to simulate some of the muddy conditions early tetrapods may have encountered. Regardless of viscosity, several aspects of lungfish swimming were similar to those of other swimming vertebrates including: posteriorly propagated muscle activity that was unilateral and alternated between the left and right sides at each longitudinal location, and posterior increases in the amount of bending, the amplitude of muscle activity and the timing differences between muscle activity and bending. With increased viscosity (1-1000 cSt), significant increases occurred in the amount of lateral bending of the vertebral column and the amplitude of muscle activity, particularly in the most anterior sites, but the distance the fish traveled per tail beat decreased. The magnitude of the phase shift between EMG onset relative to bending increased by as much as 13% of a cycle with increased viscosity, so that the muscles were increasingly active during lengthening rather than shortening. Therefore, with increased viscosity the relationship between axial muscle activity and bending in the lungfish became more dissimilar rather than converging on the motor pattern used by other ectothermic vertebrates when undulating in fully terrestrial environments.