Wake respirometry allows breath-by-breath assessment of ventilation and CO2 production in unrestrained animals.

Quantifying stress and energetic responses in animals are major challenges, as existing methods lack temporal resolution and elevate animal stress. We propose "wake respirometry," a new method of quantifying fine-scale changes in CO2 production in unrestrained animals, using a nondispersive infrared CO2 sensor positioned downwind of the animal, i.e., in its wake. We parameterize the dispersion of CO2 in wakes using known CO2 flow rates and wind speeds. Tests with three bird species in a wind tunnel demonstrated that the system can resolve breath-by-breath changes in CO2 concentration, with clear exhalation signatures increasing in period and integral with body size. Changes in physiological state were detectable following handling, flight, and exposure to a perceived threat. We discuss the potential of wake respirometry to quantify stress and respiratory patterns in wild animals and provide suggestions for estimating behavior-specific metabolic rates via full integration of CO2 production across the wake.

Highlighting when animals expend excessive energy for travel using dynamic body acceleration.

Travel represents a major cost for many animals so there should be selection pressure for it to be efficient - at minimum cost. However, animals sometimes exceed minimum travel costs for reasons that must be correspondingly important. We use Dynamic Body Acceleration (DBA), an acceleration-based metric, as a proxy for movement-based power, in tandem with vertical velocity (rate of change in depth) in a shark (Rhincodon typus) to derive the minimum estimated power required to swim at defined vertical velocities. We show how subtraction of measured DBA from the estimated minimum power for any given vertical velocity provides a "proxy for power above minimum" metric (PPAmin), highlighting when these animals travel above minimum power. We suggest that the adoption of this metric across species has value in identifying where and when animals are subject to compelling conditions that lead them to deviate from ostensibly judicious energy expenditure.

Ecological inference using data from accelerometers needs careful protocols.

Accelerometers in animal-attached tags are powerful tools in behavioural ecology, they can be used to determine behaviour and provide proxies for movement-based energy expenditure. Researchers are collecting and archiving data across systems, seasons and device types. However, using data repositories to draw ecological inference requires a good understanding of the error introduced according to sensor type and position on the study animal and protocols for error assessment and minimisation.Using laboratory trials, we examine the absolute accuracy of tri-axial accelerometers and determine how inaccuracies impact measurements of dynamic body acceleration (DBA), a proxy for energy expenditure, in human participants. We then examine how tag type and placement affect the acceleration signal in birds, using pigeons Columba livia flying in a wind tunnel, with tags mounted simultaneously in two positions, and back- and tail-mounted tags deployed on wild kittiwakes Rissa tridactyla. Finally, we present a case study where two generations of tag were deployed using different attachment procedures on red-tailed tropicbirds Phaethon rubricauda foraging in different seasons.Bench tests showed that individual acceleration axes required a two-level correction to eliminate measurement error. This resulted in DBA differences of up to 5% between calibrated and uncalibrated tags for humans walking at a range of speeds. Device position was associated with greater variation in DBA, with upper and lower back-mounted tags varying by 9% in pigeons, and tail- and back-mounted tags varying by 13% in kittiwakes. The tropicbird study highlighted the difficulties of attributing changes in signal amplitude to a single factor when confounding influences tend to covary, as DBA varied by 25% between seasons.Accelerometer accuracy, tag placement and attachment critically affect the signal amplitude and thereby the ability of the system to detect biologically meaningful phenomena. We propose a simple method to calibrate accelerometers that can be executed under field conditions. This should be used prior to deployments and archived with resulting data. We also suggest a way that researchers can assess accuracy in previously collected data, and caution that variable tag placement and attachment can increase sensor noise and even generate trends that have no biological meaning.

Animal lifestyle affects acceptable mass limits for attached tags.

Animal-attached devices have transformed our understanding of vertebrate ecology. To minimize any associated harm, researchers have long advocated that tag masses should not exceed 3% of carrier body mass. However, this ignores tag forces resulting from animal movement. Using data from collar-attached accelerometers on 10 diverse free-ranging terrestrial species from koalas to cheetahs, we detail a tag-based acceleration method to clarify acceptable tag mass limits. We quantify animal athleticism in terms of fractions of animal movement time devoted to different collar-recorded accelerations and convert those accelerations to forces (acceleration × tag mass) to allow derivation of any defined force limits for specified fractions of any animal's active time. Specifying that tags should exert forces that are less than 3% of the gravitational force exerted on the animal's body for 95% of the time led to corrected tag masses that should constitute between 1.6% and 2.98% of carrier mass, depending on athleticism. Strikingly, in four carnivore species encompassing two orders of magnitude in mass (ca 2-200 kg), forces exerted by '3%' tags were equivalent to 4-19% of carrier body mass during moving, with a maximum of 54% in a hunting cheetah. This fundamentally changes how acceptable tag mass limits should be determined by ethics bodies, irrespective of the force and time limits specified.

Scaling of axial muscle architecture in juvenile Alligator mississippiensis reveals an enhanced performance capacity of accessory breathing mechanisms.

Quantitative functional anatomy of amniote thoracic and abdominal regions is crucial to understanding constraints on and adaptations for facilitating simultaneous breathing and locomotion. Crocodilians have diverse locomotor modes and variable breathing mechanics facilitated by basal and derived (accessory) muscles. However, the inherent flexibility of these systems is not well studied, and the functional specialisation of the crocodilian trunk is yet to be investigated. Increases in body size and trunk stiffness would be expected to cause a disproportionate increase in muscle force demands and therefore constrain the basal costal aspiration mechanism, necessitating changes in respiratory mechanics. Here, we describe the anatomy of the trunk muscles, their properties that determine muscle performance (mass, length and physiological cross-sectional area [PCSA]) and investigate their scaling in juvenile Alligator mississippiensis spanning an order of magnitude in body mass (359 g-5.5 kg). Comparatively, the expiratory muscles (transversus abdominis, rectus abdominis, iliocostalis), which compress the trunk, have greater relative PCSA being specialised for greater force-generating capacity, while the inspiratory muscles (diaphragmaticus, truncocaudalis ischiotruncus, ischiopubis), which create negative internal pressure, have greater relative fascicle lengths, being adapted for greater working range and contraction velocity. Fascicle lengths of the accessory diaphragmaticus scaled with positive allometry in the alligators examined, enhancing contractile capacity, in line with this muscle's ability to modulate both tidal volume and breathing frequency in response to energetic demand during terrestrial locomotion. The iliocostalis, an accessory expiratory muscle, also demonstrated positive allometry in fascicle lengths and mass. All accessory muscles of the infrapubic abdominal wall demonstrated positive allometry in PCSA, which would enhance their force-generating capacity. Conversely, the basal tetrapod expiratory pump (transversus abdominis) scaled isometrically, which may indicate a decreased reliance on this muscle with ontogeny. Collectively, these findings would support existing anecdotal evidence that crocodilians shift their breathing mechanics as they increase in size. Furthermore, the functional specialisation of the diaphragmaticus and compliance of the body wall in the lumbar region against which it works may contribute to low-cost breathing in crocodilians.

Turning turtle: scaling relationships and self-righting ability in Chelydra serpentina.

Testudines are susceptible to inversion and self-righting using their necks, limbs or both, to generate enough mechanical force to flip over. We investigated how shell morphology, neck length and self-righting biomechanics scale with body mass during ontogeny in Chelydra serpentina, which uses neck-powered self-righting. We found that younger turtles flipped over twice as fast as older individuals. A simple geometric model predicted the relationships of shell shape and self-righting time with body mass. Conversely, neck force, power output and kinetic energy increase with body mass at rates greater than predicted. These findings were correlated with relatively longer necks in younger turtles than would be predicted by geometric similarity. Therefore, younger turtles self-right with lower biomechanical costs than predicted by simple scaling theory. Considering younger turtles are more prone to inverting and their shells offer less protection, faster and less costly self-righting would be advantageous in overcoming the detriments of inversion.

Inferring cost of transport from whole-body kinematics in three sympatric turtle species with different locomotor habits.

Chelonians are mechanically unusual vertebrates as an exoskeleton limits their body wall mobility. They generally move slowly on land and have aquatic or semi-aquatic lifestyles. Somewhat surprisingly, the limited experimental work that has been done suggests that their energetic cost of transport (CoT) are relatively low. This study examines the mechanical evidence for CoT in three turtle species that have differing degrees of terrestrial activity. Our results show that Apolone travels faster than the other two species, and that Chelydra has higher levels of yaw. All the species show poor mean levels of energy recovery, and, whilst there is considerable variation, never show the high levels of energy recovery seen in cursorial quadrupeds. The mean mechanical CoT is 2 to 4 times higher than is generally seen in terrestrial animals. We therefore find no mechanical support for a low CoT in these species. This study illustrates the need for research on a wider range of chelonians to discover whether there are indeed general trends in mechanical and metabolic energy costs.

A novel accessory respiratory muscle in the American alligator ( Alligator mississippiensis).

The muscles that effect lung ventilation are key to understanding the evolutionary constraints on animal form and function. Here, through electromyography, we demonstrate a newly discovered respiratory function for the iliocostalis muscle in the American alligator ( Alligator mississippiensis). The iliocostalis is active during expiration when breathing on land at 28°C and this activity is mediated through the uncinate processes on the vertebral ribs. There was also an increase in muscle activity during the forced expirations of alarm distress vocalizations. Interestingly, we did not find any respiratory activity in the iliocostalis when the alligators were breathing with their body submerged in water at 18°C, which resulted in a reduced breathing frequency. The iliocostalis is an accessory breathing muscle that alligators are able to recruit in to assist expiration under certain conditions.

Locomotion pattern and foot pressure adjustments during gentle turns in healthy subjects.

People suffering from locomotor impairment find turning manoeuvres more challenging than straight-ahead walking. Turning manoeuvres are estimated to comprise a substantial proportion of steps taken daily, yet research has predominantly focused on straight-line walking, meaning that the basic kinetic, kinematic and foot pressure adaptations required for turning are not as well understood. We investigated how healthy subjects adapt their locomotion patterns to accommodate walking along a gently curved trajectory (radius 2.75m). Twenty healthy adult participants performed walking tasks at self-selected speeds along straight and curved pathways. For the first time for this mode of turning, plantar pressures were recorded using insole foot pressure sensors while participants' movements were simultaneously tracked using marker-based 3D motion capture. During the steady-state strides at the apex of the turn, the mean operating point of the inside ankle shifted by 1 degree towards dorsiflexion and that for the outside ankle shifted towards plantarflexion. The largest change in relative joint angle range was an increase in hip rotation in the inside leg (>60%). In addition, the inside foot was subject to a prolonged stance phase and a 10% increase in vertical force in the posteromedial section of the foot compared to straight-line walking. Most of the mechanical change required was therefore generated by the inside leg with hip rotation being a major driver of the gentle turn. This study provides new insight into healthy gait during gentle turns and may help us to understand the mechanics behind some forms of impairment.

Differential sex-specific walking kinematics in leghorn chickens (Gallus gallus domesticus) selectively bred for different body size.

The differing limb dynamics and postures of small and large terrestrial animals may be mechanisms for minimising metabolic costs under scale-dependent muscle force, work and power demands; however, empirical evidence for this is lacking. Leghorn chickens (Gallus gallus domesticus) are highly dimorphic: males have greater body mass and relative muscle mass than females, which are permanently gravid and have greater relative intestinal mass. Furthermore, leghorns are selected for standard (large) and bantam (small) varieties and the former are sexually dimorphic in posture, with females having a more upright limb. Here, high-speed videography and morphological measurements were used to examine the walking gaits of leghorn chickens of the two varieties and sexes. Hindlimb skeletal elements were geometrically similar among the bird groups, yet the bird groups did not move with dynamic similarity. In agreement with the interspecific scaling of relative duty factor (DF, the proportion of a stride period with ground contact for any given foot) with body mass, bantams walked with greater DF than standards, and females walked with greater DF than males. Greater DF in females than in males was achieved via variety-specific kinematic mechanisms, associated with the presence/absence of postural dimorphism. Females may require greater DF in order to reduce peak muscle forces and minimise power demands associated with lower muscle to reproductive tissue mass ratios and smaller body size. Furthermore, a more upright posture observed in the standard, but not bantam, females, may relate to minimising the work demands of being larger and having proportionally larger reproductive tissue volume. Lower DF in males relative to females may also be a work-minimising strategy and/or due to greater limb inertia (as a result of greater pelvic limb muscle mass) prolonging the swing phase.

Variety, sex and ontogenetic differences in the pelvic limb muscle architectural properties of leghorn chickens (Gallus gallus domesticus) and their links with locomotor performance.

Leghorn (layer) chickens (Gallus gallus domesticus) differ in locomotor morphology and performance due to artificial selection for standard (large) and bantam (small) varieties, sexual dimorphisms and ontogenetic stage. Here, the hind limb skeletal muscle architectural properties of mature and juvenile standard breeds and mature bantams are compared and linked to measures of locomotor performance. Mature males possessed greater relative muscle physiological cross-sectional areas (PCSAs) than their conspecific females, indicative of greater force-generating capacity, and in line with their greater maximum sustainable speeds compared with females. Furthermore, some of the relative fascicle lengths of the pennate muscles were greater in mature males than in mature females, which may permit greater muscle contractibility. Immature standard leghorns, however, did not share the same dimorphisms as their mature forms. The differences in architectural properties between immature and mature standard males indicate that with the onset of male sexual maturity, concomitant with increasing muscle mass in males, the relative fascicle lengths of pennate muscles and the relative PCSAs of the parallel-fibred muscles also increase. The age-related differences in standard breed male muscle architecture are linked to the presence and absence of sex differences in maximum aerobic speeds. Males of bantam and standard varieties shared similar muscle proportions (% body mass), but exhibited intrinsic muscle differences with a tendency for greater force-generating capabilities in bantams and greater contractile capabilities in standards. The metabolic costs associated with the longer fascicle lengths, together with more crouched limbs in standard than in bantam males may explain the lack of allometry in the minimum metabolic cost of transport between these birds of different size.

Sex differences in gait utilization and energy metabolism during terrestrial locomotion in two varieties of chicken (Gallus gallus domesticus) selected for different body size.

In leghorn chickens (Gallus gallus domesticus) of standard breed (large) and bantam (small) varieties, artificial selection has led to females being permanently gravid and sexual selection has led to male-biased size dimorphism. Using respirometry, videography and morphological measurements, sex and variety differences in metabolic cost of locomotion, gait utilisation and maximum sustainable speed (Umax) were investigated during treadmill locomotion. Males were capable of greater Umax than females and used a grounded running gait at high speeds, which was only observed in a few bantam females and no standard breed females. Body mass accounted for variation in the incremental increase in metabolic power with speed between the varieties, but not the sexes. For the first time in an avian species, a greater mass-specific incremental cost of locomotion, and minimum measured cost of transport (CoTmin) were found in males than in females. Furthermore, in both varieties, the female CoTmin was lower than predicted from interspecific allometry. Even when compared at equivalent speeds (using Froude number), CoT decreased more rapidly in females than in males. These trends were common to both varieties despite a more upright limb in females than in males in the standard breed, and a lack of dimorphism in posture in the bantam variety. Females may possess compensatory adaptations for metabolic efficiency during gravidity (e.g. in muscle specialization/posture/kinematics). Furthermore, the elevated power at faster speeds in males may be linked to their muscle properties being suited to inter-male aggressive combat.

Intraspecific scaling of the minimum metabolic cost of transport in leghorn chickens (Gallus gallus domesticus): links with limb kinematics, morphometrics and posture.

The minimum metabolic cost of transport (CoTmin; J kg(-1) m(-1)) scales negatively with increasing body mass (∝Mb (-1/3)) across species from a wide range of taxa associated with marked differences in body plan. At the intraspecific level, or between closely related species, however, CoTmin does not always scale with Mb. Similarity in physiology, dynamics of movement, skeletal geometry and posture between closely related individuals is thought to be responsible for this phenomenon, despite the fact that energetic, kinematic and morphometric data are rarely collected together. We examined the relationship between these integrated components of locomotion in leghorn chickens (Gallus gallus domesticus) selectively bred for large and bantam (miniature) varieties. Interspecific allometry predicts a CoTmin ∼16% greater in bantams compared with the larger variety. However, despite 38% and 23% differences in Mb and leg length, respectively, the two varieties shared an identical walking CoTmin, independent of speed and equal to the allometric prediction derived from interspecific data for the larger variety. Furthermore, the two varieties moved with dynamic similarity and shared geometrically similar appendicular and axial skeletons. Hip height, however, did not scale geometrically and the smaller variety had more erect limbs, contrary to interspecific scaling trends. The lower than predicted CoTmin in bantams for their Mb was associated with both the more erect posture and a lower cost per stride (J kg(-1) stride(-1)). Therefore, our findings are consistent with the notion that a more erect limb is associated with a lower CoTmin and with the previous assumption that similarity in skeletal shape, inherently linked to walking dynamics, is associated with similarity in CoTmin.

The influence of load carrying on the energetics and kinematics of terrestrial locomotion in a diving bird.

The application of artificial loads to mammals and birds has been used to provide insight into the mechanics and energetic cost of terrestrial locomotion. However, only two species of bird have previously been used in loading experiments, the cursorial guinea fowl (Numida meleagris) and the locomotor-generalist barnacle goose (Branta leucopsis). Here, using respirometry and treadmill locomotion, we investigate the energetic cost of carrying trunk loads in a diving bird, the tufted duck (Aythya fuligula). Attachment of back loads equivalent to 10% and 20% of body mass increased the metabolic rate during locomotion (7.94% and 15.92%, respectively) while sternal loads of 5% and 10% had a greater proportional effect than the back loads (metabolic rate increased by 7.19% and 13.99%, respectively). No effect on locomotor kinematics was detected during any load carrying experiments. These results concur with previous reports of load carrying economy in birds, in that there is a less than proportional relationship between increasing load and metabolic rate (found previously in guinea fowl), while application of sternal loads causes an approximate doubling of metabolic rate compared to back loads (reported in an earlier study of barnacle geese). The increase in cost when carrying sternal loads may result from having to move this extra mass dorso-ventrally during respiration. Disparity in load carrying economy between species may arise from anatomical and physiological adaptations to different forms of locomotion, such as the varying uncinate process morphology and hindlimb tendon development in goose, guinea fowl and duck.