SURGICAL ANATOMY OF CELIOTOMY APPROACHES TO THE STOMACH IN THE COMMON SNAPPING TURTLE (CHELYDRA SERPENTINA).

Entanglement in or ingestion of fishing gear is a common cause of morbidity and mortality in chelonians. Commercial and recreational fishing activities exert bycatch pressures sufficient to cause population declines in the common snapping turtle (Chelydra serpentina) and the alligator snapping turtle (Macrochelys spp.). Gastrotomy for the removal of fish hook foreign bodies from the stomach of freshwater turtles typically requires a plastron osteotomy but in sea turtles has been successfully accomplished via soft tissue approaches. This study compared the anatomy and feasibility of soft tissue surgical approaches to the stomach in the common snapping turtle in both the prefemoral and the axillary regions. Anatomical dissections were performed on cadavers of five adult common snapping turtles, and the surgical anatomy of the left axillary and left prefemoral regions was characterized. The left axillary approach required extensive transection of musculature and provided poor exposure of the coelomic cavity and stomach. In contrast, the left prefemoral approach was simple to perform and provided sufficient access to the stomach for gastrotomy. The prefemoral celiotomy has significant advantages over the axillary celiotomy in the common snapping turtle and should be considered the surgical approach of choice for gastrotomy in this species.

The crouching of the shrew: Mechanical consequences of limb posture in small mammals.

An important trend in the early evolution of mammals was the shift from a sprawling stance, whereby the legs are held in a more abducted position, to a parasagittal one, in which the legs extend more downward. After that transition, many mammals shifted from a crouching stance to a more upright one. It is hypothesized that one consequence of these transitions was a decrease in the total mechanical power required for locomotion, because side-to-side accelerations of the body have become smaller, and thus less costly with changes in limb orientation. To test this hypothesis we compared the kinetics of locomotion in two mammals of body size close to those of early mammals (< 40 g), both with parasagittally oriented limbs: a crouching shrew (Blarina brevicauda; 5 animals, 17 trials) and a more upright vole (Microtus pennsylvanicus; 4 animals, 22 trials). As predicted, voles used less mechanical power per unit body mass to perform steady locomotion than shrews did (P = 0.03). However, while lateral forces were indeed smaller in voles (15.6 ± 2.0% body weight) than in shrews (26.4 ± 10.9%; P = 0.046), the power used to move the body from side-to-side was negligible, making up less than 5% of total power in both shrews and voles. The most power consumed for both species was that used to accelerate the body in the direction of travel, and this was much larger for shrews than for voles (P = 0.01). We conclude that side-to-side accelerations are negligible for small mammals-whether crouching or more upright-compared to their sprawling ancestors, and that a more upright posture further decreases the cost of locomotion compared to crouching by helping to maintain the body's momentum in the direction of travel.

Chemical Prey Luring in Jackson's Chameleons.

Lizards in the family Chamaeleonidae have been described as wiping a viscous substance from a pouch (the temporal pouch) at the angle of the jaw on branches and then capturing flies that land near the area where the wiping occurs. We confirmed the presence of this pouch in Jackson's chameleons. Histological work suggested that the material contained within is a result of decomposition of food and sloughed skin that has been trapped in the pouch rather than a glandular secretion. Gas chromatography-mass spectrometry indicated the presence of compounds that are both volatile and odiferous and similar to insect pheromones. Choice tests with houseflies revealed attraction to the temporal pouch material. Some authors have speculated that the temporal pouch material serves a function in territory marking and/or predator deterrence. While it may play these roles, our results suggest that it also plays a role in chemical luring of prey.

Effects of Functional Electrical Stimulation on Denervated Laryngeal Muscle in a Large Animal Model.

Bilateral vocal fold paralysis (BVCP) is a life-threatening condition that follows injury to the Recurrent Laryngeal nerve (RLn) and denervation of the intrinsic laryngeal musculature. Functional electrical stimulation (FES) enables restoration and control of a wide variety of motor functions impaired by lower motor neuron lesions. Here we evaluate the effects of FES on the sole arytenoid abductor, the posterior cricoarytenoid (PCA) muscle in a large animal model of RLn injury. Ten horses were instrumented with two quadripolar intramuscular electrodes in the left PCA muscle. Following a 12-week denervation period, the PCA was stimulated using a once-daily training session for 8 weeks in seven animals. Three animals were used as unstimulated controls. Denervation produced a significant increase in rheobase (P < 0.001). Electrical stimulation produced a 30% increase in fiber diameter in comparison with the unstimulated control group (33.9 ± 2.6 µm FES+, 23.6 ± 4.2 µm FES-, P = 0.04). A trend toward a decrease in the proportion of type 1 (slow) fibers and an increase in type 2a (fast) fibers was also observed. Despite these changes, improvement in PCA function at rest was not observed. These data suggest that electrical stimulation using a relatively conservative set of stimulation parameters can reverse the muscle fiber atrophy produced by complete denervation while avoiding a shift to a slow (type 1) fiber type.

Frequency dependence of power and its implications for contractile function of muscle fibers from the digital flexors of horses.

The digital flexors of horses must produce high force to support the body weight during running, and a need for these muscles to generate power is likely limited during locomotion over level ground. Measurements of power output from horse muscle fibers close to physiological temperatures, and when cyclic strain is imposed, will help to better understand the in vivo performance of the muscles as power absorbers and generators. Skinned fibers from the deep (DDF) and superficial (SDF) digital flexors, and the soleus (SOL) underwent sinusoidal oscillations in length over a range of frequencies (0.5-16 Hz) and strain amplitudes (0.01-0.06) under maximum activation (pCa 5) at 30°C. Results were analyzed using both workloop and Nyquist plot analyses to determine the ability of the fibers to absorb or generate power and the frequency dependence of those abilities. Power absorption was dominant at most cycling frequencies and strain amplitudes in fibers from all three muscles. However, small amounts of power were generated (0.002-0.05 Wkg(-1)) at 0.01 strain by all three muscles at relatively slow cycling frequencies: DDF (4-7 Hz), SDF (4-5 Hz) and SOL (0.5-1 Hz). Nyquist analysis, reflecting the influence of cross-bridge kinetics on power generation, corroborated these results. The similar capacity for power generation by DDF and SDF versus lower for SOL, and the faster frequency at which this power was realized in DDF and SDF fibers, are largely explained by the fast myosin heavy chain isoform content in each muscle. Contractile function of DDF and SDF as power absorbers and generators, respectively, during locomotion may therefore be more dependent on their fiber architectural arrangement than on the physiological properties of their muscle fibers.

Echolocation call production during aerial and terrestrial locomotion by New Zealand's enigmatic lesser short-tailed bat, Mystacina tuberculata.

Linkage of echolocation call production with contraction of flight muscles has been suggested to reduce the energetic cost of flight with echolocation, such that the overall cost is approximately equal to that of flight alone. However, the pattern of call production with limb movement in terrestrially agile bats has never been investigated. We used synchronised high-speed video and audio recordings to determine patterns of association between echolocation call production and limb motion by Mystacina tuberculata Gray 1843 as individuals walked and flew, respectively. Results showed that there was no apparent linkage between call production and limb motion when bats walked. When in flight, two calls were produced per wingbeat, late in the downstroke and early in the upstroke. When bats walked, calls were produced at a higher rate, but at a slightly lower intensity, compared with bats in flight. These results suggest that M. tuberculata do not attempt to reduce the cost of terrestrial locomotion and call production through biomechanical linkage. They also suggest that the pattern of linkage seen when bats are in flight is not universal and that energetic savings cannot necessarily be explained by contraction of muscles associated with the downstroke alone.

Role of the hypoglossal nerve in equine nasopharyngeal stability.

The equine upper airway is highly adapted to provide the extremely high oxygen demand associated with strenuous aerobic exercise in this species. The tongue musculature, innervated by the hypoglossal nerve, plays an important role in airway stability in humans who also have a highly adapted upper airway to allow speech. The role of the hypoglossal nerve in stabilizing the equine upper airway has not been established. Isolated tongues from eight mature horses were dissected to determine the distal anatomy and branching of the equine hypoglossal nerve. Using this information, a peripheral nerve location technique was used to perform bilateral block of the common trunk of the hypoglossal nerve in 10 horses. Each horse was subjected to two trials with bilateral hypoglossal nerve block and two control trials (unblocked). Upper airway stability at exercise was determined using videoendoscopy and measurement of tracheal and pharyngeal pressure. Three main nerve branches were identified, medial and lateral branches and a discrete branch that innervated the geniohyoid muscle alone. Bilateral hypoglossal block induced nasopharyngeal instability in 10/19 trials, and none of the control trials (0/18) resulted in instability (P<0.001). Mean treadmill speed (+/-SD) at the onset of instability was 10.8+/-2.5 m/s. Following its onset, nasopharyngeal instability persisted until the end of the treadmill test. This instability, induced by hypoglossal nerve block, produced an expiratory obstruction similar to that seen in a naturally occurring equine disease (dorsal displacement of the soft palate, DDSP) with reduced inspiratory and expiratory pharyngeal pressure and increased expiratory tracheal pressure. These data suggest that stability of the equine upper airway at exercise may be mediated through the hypoglossal nerve. Naturally occurring DDSP in the horse shares a number of anatomic similarities with obstructive sleep apnea. Study of species with extreme respiratory adaptation, such as the horse, may provide insight into respiratory functioning in humans.

Growth and development of two species of bats in a shared maternity roost.

Skeletogenesis was studied in two species of bats, Myotis austroriparius (southeastern brown bat) and Tadarida brasiliensis (Brazilian free-tailed bat), occupying a maternity roost in central Florida. These bats often use distinct maternity roost environments, so this provided an opportunity to examine differential patterns of long bone growth while fetuses and newborn developed under similar environmental conditions. Some differences in the timing of onset of osteogenesis were revealed in the bats, indicating that some elements of the hindlimb develop relatively more rapidly in T. brasiliensis than in M. austroriparius. Some variance was also noted, with similarity to other species previously studied by others, in the exact timing and elongation of both long bones, as well as carpal and tarsal bones. In contrast to many elements of the long appendicular skeleton of developing Mus musculus, the bats all exhibit relatively precocial patterns of osteogenesis during which cartilaginous precursors are replaced by bone tissue. The relative advanced timing of osteogenesis in select hindlimb bones of T. brasiliensis may account for its relatively low neonatal mortality compared to M. austroriparius newborn in the same roost.

Forelimb versus hindlimb skeletal development in the big brown bat, Eptesicus fuscus: functional divergence is reflected in chondrocytic performance in Autopodial growth plates.

The morphology of the chiropteran forelimb demonstrates musculoskeletal specializations for powered flight essentially unique among mammals, including extreme elongation of the distal skeletal elements. Recent studies have focused primarily on the relative timing and levels of gene expression during early stages of endochondral ossification in the chiropteran embryo for clues to the molecular basis of the evolutionary origins of flight in these species. The goal of the current study was to examine how elongation of skeletal elements of the forelimb autopod is achieved through a combination of cellular proliferation, cellular enlargement and matrix synthesis during a period of rapid postnatal growth in Eptesicus fuscus. Quantitative analyses were done of multiple performance parameters of growth plate chondrocytes during all phases of the differentiation cascade. Fourteen autopodial growth plates from the forelimb and hindlimb of one individual, as well as the proximal tibial growth plate, were collected and analyzed. Significant differences were seen in all performance parameters examined. Particularly striking were the differences between growth plates of the manus and pes in the size of the pool of chondrocytes in all cellular zones and rates of turnover of terminal cells. The magnitude of hypertrophy of chondrocytes in growth plates of the manus in E. fuscus far exceeded what has been reported previously in any species, even in rapidly elongating rodent long bones. Volume changes approaching x70 and height changes of 50-60 mum/cell (paralleling the direction of growth) occurred after proliferation in the most rapidly growing growth plates. The data demonstrate that final differences in lengths of homologous skeletal elements in the autopod of the forelimb and hindlimb of this species result not just from an initiating factor early in development, but from continued quantitative differences in chondrocytic performance during postnatal bone elongation as measured by multiple kinetic-based parameters.

Postnatal bone elongation of the manus versus pes: analysis of the chondrocytic differentiation cascade in Mus musculus and Eptesicus fuscus.

Bones elongate postnatally by endochondral ossification as cells of the cartilaginous growth plate undergo a differentiation cascade of proliferation, cellular hypertrophy and matrix synthesis. Interspecific comparisons of homologous bones elongating at different rates has been a useful approach for studying the dynamics of this process. The purpose of this study was to measure quantitative stereological parameters of growth plates of the third digit of the manus and pes of the laboratory mouse, and make comparisons to chondrocytic performance parameters in the homologous bones of the big brown bat, Eptesicus fuscus, where extremely rapid postnatal elongation of bones of the manus is associated with skeletal modifications for powered flight. Measurements were made across all zones of forelimb and hindlimb autopod growth plates by dividing each growth plate into strata of equal height (from thirteen 200-mum-high strata in the metacarpus to five 40-mum-high strata in phalangeal bones of the pes). Results indicate that all chondrocytic performance parameters known to quantitatively contribute to the elongation potential of a growth plate change together. A significant finding was that in growth plates of the chiropteran manus, final hypertrophic cell size and shape were achieved early in the zone of hypertrophy, indicating that interstitial expansion of the growth plate resulting from the incremental chondrocytic height increase in the direction of elongation was completed soon after the transition from the cessation of proliferation to the initiation of hypertrophy. This is unlike what has been reported in most mammalian growth plates previously analyzed, but is the situation in the proximal tibial growth plate of rapidly growing frogs and precocial birds. This suggests that a similar adaptation for stabilization of a rapidly elongating bone has evolved independently in three widely separated groups that have in common rapid growth in limbs to be used for early active, powered locomotion.

In vitro model for testing novel implants for equine laryngoplasty.

OBJECTIVE: To develop an in vitro laryngeal model to mimic airflow and pressures experienced by horses at maximal exercise with which to test laryngoplasty techniques. STUDY DESIGN: Randomized complete block. SAMPLE POPULATION: Cadaveric equine larynges (n=10). METHODS: Equine larynges were collected at necropsy and a bilateral prosthetic laryngoplasty suture was placed with #5 Fiberwire suture to achieve bilateral maximal arytenoid abduction. Each larynx was positioned in a flow chamber and subjected to static flow and dynamic flow cycling at 2 Hz. Tracheal pressure and flow, and pressure within the flow chamber were recorded at a sampling frequency of 500 Hz. Data obtained were compared with the published physiologic values for horses exercising at maximal exercise. RESULTS: Under static flow conditions, the testing system produced inspiratory tracheal pressures (mean+/-SEM) of -33.0+/-0.98 mm Hg at a flow of 54.48+/-1.8 L/s. Pressure in the flow chamber was -8.1+/-2.2 mm Hg producing a translaryngeal impedance of 0.56+/-0.15 mm Hg/L/s. Under dynamic conditions, cycling flow and pressure were reproduced at a frequency of 2 Hz, the peak inspiratory (mean+/-SEM) pharyngeal and tracheal pressures across all larynges were -8.85+/-2.5 and -35.54+/-1.6 mm Hg, respectively. Peak inspiratory flow was 51.65+/-2.3 L/s and impedance was 0.57+/-0.06 mm Hg/L/s. CONCLUSIONS: The model produced inspiratory pressures similar to those in horses at maximal exercise when airflows experienced at exercise were used. CLINICAL RELEVANCE: This model will allow testing of multiple novel techniques and may facilitate development of improved techniques for prosthetic laryngoplasty.

Horse soleus muscle: postural sensor or vestigial structure?

The soleus muscle of horses is rather diminutive with respect to the overall size of adjacent synergist muscles in the hind limb of the horse. Whether or not such a muscle might be vestigial or may be providing some essential function has not been determined. We have studied the horse's soleus muscle using histochemical (ATPase), immunocytochemical (myosin isoform identification), and SDS-PAGE analysis to demonstrate that it is largely composed of 100% type I, presumed slow-twitch fibers. Only one soleus muscle studied (out of 13 adult horses) contained any type II muscle fibers. Given this consistent high percentage of slow-oxidative fibers, we hypothesized that the soleus muscle could have a significant role in proprioceptive function, essentially functioning as a proprioceptive organ instead of a significant force-generating muscle during locomotion. We tested this by examining three whole soleus muscles and assessing their muscle spindle content, which proved to have a spindle index of about 12. This value provided equivocal support for the hypothesis since it did not approach values reported for other mammalian proprioceptive muscles that were approximately 40-50 spindles per gram of muscle mass. Other parameters, such as motoneuron number and muscle unit size, may be useful in understanding these data.

Terrestrial locomotion of the New Zealand short-tailed bat Mystacina tuberculata and the common vampire bat Desmodus rotundus.

Bats (Chiroptera) are generally awkward crawlers, but the common vampire bat (Desmodus rotundus) and the New Zealand short-tailed bat (Mystacina tuberculata) have independently evolved the ability to manoeuvre well on the ground. In this study we describe the kinematics of locomotion in both species, and the kinetics of locomotion in M. tuberculata. We sought to determine whether these bats move terrestrially the way other quadrupeds do, or whether they possess altogether different patterns of movement on the ground than are observed in quadrupeds that do not fly. Using high-speed video analyses of bats moving on a treadmill, we observed that both species possess symmetrical lateral-sequence gaits similar to the kinematically defined walks of a broad range of tetrapods. At high speeds, D. rotundus use an asymmetrical bounding gait that appears to converge on the bounding gaits of small terrestrial mammals, but with the roles of the forelimbs and hindlimbs reversed. This gait was not performed by M. tuberculata. Many animals that possess a single kinematic gait shift with increasing speed from a kinetic walk (where kinetic and potential energy of the centre of mass oscillate out of phase from each other) to a kinetic run (where they oscillate in phase). To determine whether the single kinematic gait of M. tuberculata meets the kinetic definition of a walk, a run, or a gait that functions as a walk at low speed and a run at high speed, we used force plates and high-speed video recordings to characterize the energetics of the centre of mass in that species. Although oscillations in kinetic and potential energy were of similar magnitudes, M. tuberculata did not use pendulum-like exchanges of energy between them to the extent that many other quadrupedal animals do, and did not transition from a kinetic walk to kinetic run with increasing speed. The gait of M. tuberculata is kinematically a walk, but kinetically run-like at all speeds.

Muscular design in the equine interosseus muscle.

We studied the forelimb interosseus muscle in horses, Equus caballus, to determine the muscular properties inherent in its function. Some authors have speculated that the equine interosseus contains muscle fibers at birth only to undergo loss of these fibers through postnatal ontogeny. We describe the muscle fibers in eight interosseus specimens from adult horses. These fibers were studied histochemically using myosin ATPase studies and immunocytochemically using several antibodies directed against type I and type II myosin heavy chain antibodies. We determined that 95% of the fibers were type I, presumed slow-twitch fibers. All fibers exhibited normal morphological appearance in terms of fiber diameter and cross-sectional area, suggesting that the muscles are undergoing normal cycles of recruitment. SDS-PAGE studies of myosin heavy chain isoforms were consistent with these observations of primarily slow-twitch muscle. Fibers were determined to be approximately 800 microm long when studied using nitric acid digestion protocols. Short fiber length combined with high pinnation angles suggest that the interosseus muscle is able to generate large amounts of force but can produce little work (measured as pulling the distal tendon proximally). While the equine interosseus muscle has undergone a general reduction of muscle content during its evolution, it remains composed of a significant muscular component that likely contributes to forelimb stability and elastic storage of energy during locomotion.

Testing the hindlimb-strength hypothesis: non-aerial locomotion by Chiroptera is not constrained by the dimensions of the femur or tibia.

In the evolution of flight bats appear to have suffered a trade-off; they have become poor crawlers relative to terrestrial mammals. Capable walking does occur in a few disparate taxa, including the vampire bats, but the vast majority of bats are able only to shuffle awkwardly along the ground, and the morphological bases of differences in crawling ability are not currently understood. One widely cited hypothesis suggests that the femora of most bats are too weak to withstand the compressive forces that occur during terrestrial locomotion, and that the vampire bats can walk because they possess more robust hindlimb skeletons. We tested a prediction of the hindlimb-strength hypothesis: that during locomotion, the forces produced by the hindlimbs of vampire bats should be larger than those produced by the legs of poorly crawling bats. Using force plates we compared the hindlimb forces produced by two species of vampire bats that walk well, Desmodus rotundus (N=8) and Diaemus youngi (N=2), to the hindlimb forces produced during over-ground shuffling by a similarly sized bat that is a poor walker (Pteronotus parnellii; N=6). Peak hindlimb forces produced by P. parnellii were larger (ANOVA; P<0.05; N=65) and more variable (93.5+/-36.6% body weight, mean +/- s.d.) than those of D. rotundus (69.3+/-8.1%) or D. youngi (75.0+/-6.2%). Interestingly, the vertical components of peak force were equivalent among species (P>0.6), indicating similar roles for support of body weight by the hindlimbs in the three species. We also used a simple engineering model of bending stress to evaluate the support capabilities of the hindlimb skeleton from the dimensions of 113 museum specimens in 50 species. We found that the hindlimb bones of vampires are not built to withstand larger forces than those of species that crawl poorly. Our results show that the legs of poorly crawling bats should be able to withstand the forces produced during coordinated crawling of the type used by the agile vampires, and this indicates that some mechanism other than hindlimb bone thickness, such as myology of the pectoral girdle, limits the ability of most bats to crawl.

Biomechanics: independent evolution of running in vampire bats.

Most tetrapods have retained terrestrial locomotion since it evolved in the Palaeozoic era, but bats have become so specialized for flight that they have almost lost the ability to manoeuvre on land at all. Vampire bats, which sneak up on their prey along the ground, are an important exception. Here we show that common vampire bats can also run by using a unique bounding gait, in which the forelimbs instead of the hindlimbs are recruited for force production as the wings are much more powerful than the legs. This ability to run seems to have evolved independently within the bat lineage.