A Standardized Protocol for Measuring Bioelectrical Impedance in Green Turtles (Chelonia mydas).

AbstractBioelectrical impedance analysis (BIA) is gaining popularity in wildlife studies as a portable technology for immediate and nondestructive predictions of body composition components, such as fat-free and fat masses. Successful application of BIA for field-based research requires the identification and control of potential sources of error, as well as the creation of and adherence to a standardized protocol for measurement. The aim of our study was to determine sources of error and to provide a standardization protocol to improve measurement precision of BIA on juvenile green turtles (Chelonia mydas; n=35). We assessed the effects of altered environmental temperature (20°C-30°C), postprandial state (2-72 h), and time out of the water (2 h) on five impedance parameters (resistance at infinite frequency [Rinf], resistance at zero frequency [R0], resistance at 50 kHz [R50], phase angle at 50 kHz [PhA50], and intracellular resistance [Ri]) using a bioimpedance spectroscopy device. Technical reproducibility of measurements and interanimal variability were also assessed. We found an inverse exponential relationship between change in environmental temperature and impedance parameters Rinf, R0, and R50. Postprandial state significantly increased Rinf and Ri 72 h after feeding. BIA measurements were reproducible within individual juvenile green turtles at temperatures from 20°C to 30°C. Significant variation in impedance values was found between animals at all temperatures, sampling times, and postprandial states, but the relative differences (%) were small in magnitude. Our study suggests that measurement precision is improved by measuring animals at consistent environmental temperatures close to their preferred thermal range. We propose a standardized protocol of measurement conditions to facilitate laboratory and field use of BIA for body composition assessment studies in turtles.

Field-based adipose tissue quantification in sea turtles using bioelectrical impedance spectroscopy validated with CT scans and deep learning.

Loss of adipose tissue in vertebrate wildlife species is indicative of decreased nutritional and health status and is linked to environmental stress and diseases. Body condition indices (BCI) are commonly used in ecological studies to estimate adipose tissue mass across wildlife populations. However, these indices have poor predictive power, which poses the need for quantitative methods for improved population assessments. Here, we calibrate bioelectrical impedance spectroscopy (BIS) as an alternative approach for assessing the nutritional status of vertebrate wildlife in ecological studies. BIS is a portable technology that can estimate body composition from measurements of body impedance and is widely used in humans. BIS is a predictive technique that requires calibration using a reference body composition method. Using sea turtles as model organisms, we propose a calibration protocol using computed tomography (CT) scans, with the prediction equation being: adipose tissue mass (kg) = body mass - (-0.03 [intercept] - 0.29 * length2/resistance at 50 kHz + 1.07 * body mass - 0.11 * time after capture). CT imaging allows for the quantification of body fat. However, processing the images manually is prohibitive due to the extensive time requirement. Using a form of artificial intelligence (AI), we trained a computer model to identify and quantify nonadipose tissue from the CT images, and adipose tissue was determined by the difference in body mass. This process enabled estimating adipose tissue mass from bioelectrical impedance measurements. The predictive performance of the model was built on 2/3 samples and tested against 1/3 samples. Prediction of adipose tissue percentage had greater accuracy when including impedance parameters (mean bias = 0.11%-0.61%) as predictor variables, compared with using body mass alone (mean bias = 6.35%). Our standardized BIS protocol improves on conventional body composition assessment methods (e.g., BCI) by quantifying adipose tissue mass. The protocol can be applied to other species for the validation of BIS and to provide robust information on the nutritional and health status of wildlife, which, in turn, can be used to inform conservation decisions at the management level.

Importance of health assessments for conservation in noncaptive wildlife.

Wildlife health assessments help identify populations at risk of starvation, disease, and decline from anthropogenic impacts on natural habitats. We conducted an overview of available health assessment studies in noncaptive vertebrates and devised a framework to strategically integrate health assessments in population monitoring. Using a systematic approach, we performed a thorough assessment of studies examining multiple health parameters of noncaptive vertebrate species from 1982 to 2020 (n = 261 studies). We quantified trends in study design and diagnostic methods across taxa with generalized linear models, bibliometric analyses, and visual representations of study location versus biodiversity hotspots. Only 35% of studies involved international or cross-border collaboration. Countries with both high and threatened biodiversity were greatly underrepresented. Species that were not listed as threatened on the International Union for Conservation of Nature Red List represented 49% of assessed species, a trend likely associated with the regional focus of most studies. We strongly suggest following wildlife health assessment protocols when planning a study and using statistically adequate sample sizes for studies establishing reference ranges. Across all taxa blood analysis (89%), body composition assessments (81%), physical examination (72%), and fecal analyses (24% of studies) were the most common methods. A conceptual framework to improve design and standardize wildlife health assessments includes guidelines on the experimental design, data acquisition and analysis, and species conservation planning and management implications. Integrating a physiological and ecological understanding of species resilience toward threatening processes will enable informed decision making regarding the conservation of threatened species.

Importancia de los exámenes diagnósticos para la conservación de fauna silvestre Resumen Los exámenes diagnósticos de fauna silvestre ayudan a identificar poblaciones en riesgo por desnutrición, enfermedades infecciosas y disminución poblacional, causadas por impactos antropogénicos. Revisamos los estudios disponibles que llevaron a cabo exámenes diagnósticos en fauna silvestre y diseñamos un marco de trabajo para integrar dichos exámenes en monitoreos poblacionales. Empleando un enfoque sistemático, evaluamos aquellos estudios que examinaban múltiples indicadores de salud en vertebrados no cautivos entre 1982 y 2020 (n = 261 estudios). Cuantificamos las tendencias estadísticas, clasificadas por taxones, del diseño del estudio y de los métodos diagnósticos usando modelos lineales generalizados, análisis bibliométricos y representaciones visuales del lugar de estudio versus los hotspots (puntos calientes) de biodiversidad. Sólo el 35% de los estudios incluían colaboraciones internacionales o transfronterizas, y los países ricos en biodiversidad y especies amenazadas estaban gravemente subrepresentados. Las especies no clasificadas como amenazadas en la Lista Roja de la Unión Internacional para la Conservación de la Naturaleza representaban el 49% de las especies examinadas; una tendencia posiblemente asociada al enfoque regional de la mayoría de los estudios. Recomendamos encarecidamente seguir protocolos diagnósticos y manuales de técnicas del estudio de la fauna silvestre, además de usar tamaños muestrales estadísticamente adecuados al establecer rangos de referencia. Los métodos diagnósticos más comunes para todos los taxones fueronanálisis sanguíneos (89%), evaluaciones de composición corporal (81%), exámenes físicos (72%) y análisis fecales (24% de los estudios). Presentamos un marco conceptual para mejorar y estandarizar los exámenes diagnósticos en estudios de fauna silvestre; dicho marco incluye guías para el diseño experimental, para la obtención y el análisis de datos, y para elaborar planes de acción para especies amenazadas. La combinación de conocimientos fisiológicos y ecológicos, relacionados con la resiliencia biológica de especies amenazadas, facilitará una toma de decisiones eficiente para el manejo y para la conservación de la biodiversidad.

Haematological and biochemical reference intervals for wild green turtles (Chelonia mydas): a Bayesian approach for small sample sizes.

Animal health is directly linked to population viability, which may be impacted by anthropogenic disturbances and diseases. Reference intervals (RIs) for haematology and blood biochemistry are essential tools for the assessment of animal health. However, establishing and interpreting robust RIs for threatened species is often challenged by small sample sizes. Bayesian predictive modelling is well suited to sample size limitations, accounting for individual variation and interactions between influencing variables. We aimed to derive baseline RIs for green turtles (Chelonia mydas) across two foraging aggregations in North Queensland, Australia, using Bayesian generalized linear mixed-effects models (n = 97). The predicted RIs were contained within previously published values and had narrower credible intervals. Most analytes did not vary significantly with foraging ground (76%, 22/29), body mass (86%, 25/29) or curved carapace length (83%, 24/29). Length and body mass effects were found for eosinophils, heterophil:lymphocyte ratio, alkaline phosphatase, aspartate transaminase and urea. Significant differences between foraging grounds were found for albumin, cholesterol, potassium, total protein, triglycerides, uric acid and calcium:phosphorus ratio. We provide derived RIs for foraging green turtles, which will be helpful in future population health assessments and conservation efforts. Future RI studies on threatened species would benefit from adapting established veterinary and biomedical standards.

Geographical variation in the standard physiology of brushtail possums (Trichosurus): implications for conservation translocations.

Identifying spatial patterns in the variation of physiological traits that occur within and between species is a fundamental goal of comparative physiology. There has been a focus on identifying and explaining this variation at broad taxonomic scales, but more recently attention has shifted to examining patterns of intra-specific physiological variation. Here we examine geographic variation in the physiology of brushtail possums (Trichosurus), widely distributed Australian marsupials, and discuss how pertinent intra-specific variation may be to conservation physiology. We found significant geographical patterns in metabolism, body temperature, evaporative water loss and relative water economy. These patterns suggest that possums from warmer, drier habitats have more frugal energy and water use and increased capacity for heat loss at high ambient temperatures. Our results are consistent with environmental correlates for broad-scale macro-physiological studies, and most intra-generic and intra-specific studies of marsupials and other mammals. Most translocations of brushtail possums occur into Australia's arid zone, where the distribution and abundance of possums and other native mammals have declined since European settlement, leading to reintroduction programmes aiming to re-establish functional mammal communities. We suggest that the sub-species T. vulpecula hypoleucus from Western Australia would be the most physiologically appropriate for translocation to these arid habitats, having physiological traits most favourable for the extreme Ta, low and variable water availability and low productivity that characterize arid environments. Our findings demonstrate that geographically widespread populations can differ physiologically, and as a consequence some populations are more suitable for translocation to particular habitats than others. Consideration of these differences will likely improve the success and welfare outcomes of translocation, reintroduction and management programmes.

Calibration of the HemoCue point-of-care analyser for determining haemoglobin concentration in a lizard and a fish.

Haemoglobin concentration ([Hb]) is measured for a wide variety of animal studies. The use of point-of-care devices, such as the HemoCue, is becoming increasingly common because of their portability, relative ease of use and low cost. In this study, we aimed to determine whether the [Hb] of blue-tongued skink (Tiliqua nigrolutea) blood can be determined accurately using the HemoCue and whether the HemoCue overestimates the [Hb] of reptile blood in a similar manner to fish blood. Additionally, we aimed to test whether ploidy affected [Hb] determined by the HemoCue using blood from diploid and triploid Atlantic salmon (Salmo salar). The HemoCue Hb 201(+) systematically overestimated [Hb] in both blue-tongued skinks and Atlantic salmon, and there was no difference between calibration equations determined for diploid or triploid salmon. The overestimation was systematic in both species and, as such, [Hb] determined by the HemoCue can be corrected using appropriate calibration equations.

Pregnancy limits lung function during exercise and depresses metabolic rate in the skink Tiliqua nigrolutea.

High gestational loads have been associated with a range of ecological costs, such as decreased locomotor ability; however, the physiological mechanisms that underpin these changes are poorly understood. In this study, breathing patterns, metabolic rates, lung volume and lung diffusing capacity were measured at rest and during exercise in the pregnant skink Tiliqua nigrolutea. Breathing patterns were largely unaffected by gestation; however, decreases in metabolic rate (rate of oxygen consumption) in the late stages of pregnancy induced a relative hyperventilation. The reductions in metabolic rate during late pregnancy prevent the calculation of the maintenance cost of pregnancy based on post-partum and neonatal metabolic rates. Despite the high relative litter mass of 38.9±5.3%, lung diffusing capacity was maintained during all stages of pregnancy, suggesting that alterations in diffusion at the alveolar capillary membrane were not responsible for the relative hyperventilation. Lung volume was increased during pregnancy compared with non-pregnant females, but lung volume was significantly lower during pregnancy compared with post-partum lung volume. Pregnant females were unable to produce the same metabolic and ventilatory changes induced by exercise in non-pregnant females. This lack of ability to respond to increased respiratory drive during exercise may underpin the locomotor impairment measured during gestation in previous studies.

The use of spirometry to evaluate pulmonary function in olive ridley sea turtles (Lepidochelys olivacea) with positive buoyancy disorders.

This study utilized computed spirometry to compare the pulmonary function of two stranded olive ridley sea turtles (Lepidochelys olivacea) presenting with a positive buoyancy disorder with two healthy captive olive ridley sea turtles held in a large public aquarium. Pulmonary function test (PFT) measurements demonstrated that the metabolic cost of breathing was much greater for animals admitted with positive buoyancy than for the normal sea turtles. Positively buoyant turtles had higher tidal volumes and significantly lower breathing-frequency patterns with significantly higher expiration rates, typical of gasp-type breathing. The resulting higher energetic cost of breathing in the diseased turtles may have a significant impact on their long-term survival. The findings represent a method for clinical respiratory function analysis for an individual animal to assist with diagnosis, therapy, and prognosis. This is the first study, to our knowledge, to evaluate objectively sea turtles presenting with positive buoyancy and respiratory disease using pulmonary function tests.

Gestation increases the energetic cost of breathing in the lizard Tiliqua rugosa.

High gestational loads result in fetuses that occupy a large proportion of the body cavity and may compress maternal organs. Compression of the lungs results in alterations in breathing patterns during gestation, which may affect the energetic cost of breathing. In this study, the energetic cost of breathing during gestation was determined in the viviparous skink Tiliqua rugosa. Radiographic imaging showed progressive lung compression during gestation and a 30% reduction in the lung inflation index (rib number at which the caudal margin of the lung was imaged divided by total rib number). Pneumotachography and open flow respirometry were used to measure breathing patterns and metabolic rates. Gestation induced a twofold increase in minute ventilation via increases in breathing frequency, but no change in inspired tidal volume. The rates of O(2) consumption and CO(2) production did not change significantly during gestation. Together, these results suggest that a relative hyperventilation occurs during gestation in T. rugosa, which in turn suggests that diffusion and/or perfusion limitations may exist at the lung during gestation. The energetic cost of breathing was estimated as a percentage of resting metabolic rate using hypercapnia to stimulate ventilation at different stages of pregnancy. The energetic cost of breathing in non-pregnant lizards was 19.96±3.85% of resting metabolic rate and increased threefold to 62.80±10.11% during late gestation. This significant increase in the energetic cost of breathing may have significant consequences for energy budgets during gestation.

The accessory role of the diaphragmaticus muscle in lung ventilation in the estuarine crocodile Crocodylus porosus.

Crocodilians use a combination of three muscular mechanisms to effect lung ventilation: the intercostal muscles producing thoracic movement, the abdominal muscles producing pelvic rotation and gastralial translation, and the diaphragmaticus muscle producing visceral displacement. Earlier studies suggested that the diaphragmaticus is a primary muscle of inspiration in crocodilians, but direct measurements of the diaphragmatic contribution to lung ventilation and gas exchange have not been made to date. In this study, ventilation, metabolic rate and arterial blood gases were measured from juvenile estuarine crocodiles under three conditions: (i) while resting at 30°C and 20°C; (ii) while breathing hypercapnic gases; and (iii) during immediate recovery from treadmill exercise. The relative contribution of the diaphragmaticus was then determined by obtaining measurements before and after transection of the muscle. The diaphragmaticus was found to make only a limited contribution to lung ventilation while crocodiles were resting at 30°C and 20°C, and during increased respiratory drive induced by hypercapnic gas. However, the diaphragmaticus muscle was found to play a significant role in facilitating a higher rate of inspiratory airflow in response to exercise. Transection of the diaphragmaticus decreased the exercise-induced increase in the rate of inspiration (with no compensatory increases in the duration of inspiration), thus compromising the exercise-induced increases in tidal volume and minute ventilation. These results suggest that, in C. porosus, costal ventilation alone is able to support metabolic demands at rest, and the diaphragmaticus is largely an accessory muscle used at times of elevated metabolic demand.

Breathing with big babies: ventilation and oxygen consumption during pregnancy in the lizard Tiliqua rugosa.

We determined the effects of high gestational loads on ventilation and the rate of oxygen consumption (VO2) in the scincid lizard Tiliqua rugosa. Tiliqua rugosa is a large viviparous lizard that gives birth to one to four young after 6-7 mo gestation. Pregnant females gave birth to large young, weighing 89.5+/-5.9 g, which represents 21.6%+/-2.6% of maternal body mass. As the embryos developed, they occupied an increasingly large proportion of the body cavity, decreasing food consumption and compressing the gastrointestinal tract. Computerized axial tomography scans demonstrated that the lungs were compressed and/or regionally collapsed by the developing embryos, potentially compromising ventilation. Both minute ventilation (VE) and tidal volume decreased as gestation progressed, but no compensatory changes in breathing frequency or in the duration of the nonventilatory period were observed. The total rate of VO2, consisting of contributions from both maternal and fetal tissues, did not change during gestation, suggesting that maternal VO2 decreases as fetal VO2 increases. Pregnant females demonstrated a decreased ventilatory response to increased respiratory drive (triggered via inhalation of hypoxic hypercapnic gas), which may be associated with the increased energetic cost of ventilating a compressed lung or the desensitization of chemoreceptors during gestation. The decreased ability of the respiratory system to respond to increased respiratory drive may have important consequences for locomotor performance and predator avoidance in pregnant lizards.

Terrestrial locomotion does not constrain venous return in the American alligator, Alligator mississippiensis.

The effects of treadmill exercise on components of the cardiovascular (heart rate, mean arterial blood pressure, central venous pressure, venous return) and respiratory (minute ventilation, tidal volume, breathing frequency, rate of oxygen consumption, rate of carbon dioxide production) systems and on intra-abdominal pressure were measured in the American alligator, Alligator mississippiensis, at 30 degrees C. Alligators show speed-dependent increases in tidal volume and minute ventilation, demonstrating that the inhibition of ventilation during locomotion that is present in some varanid and iguanid lizards was not present in alligators. Exercise significantly increases intra-abdominal pressure; however, concomitant elevations in central venous pressure acted to increase the transmural pressure of the post caval vein and thus increased venous return. Therefore, despite elevated intra-abdominal pressure, venous return was not limited during exercise in alligators, as was the case in Varanus exanthematicus and Iguana iguana. Respiratory cycle variations in intra-abdominal pressure, central venous pressure and venous return indicate that, at high tidal volumes, inspiration causes a net reduction in venous return during active ventilation and thus may act to limit venous return during exercise. These results suggest that, while tonically elevated intra-abdominal pressure induced by exercise does not inhibit venous return, phasic fluctuations during each breath cycle may contribute to venous flow limitation during exercise.

Elevated intra-abdominal pressure limits venous return during exercise in Varanus exanthematicus.

The effects of treadmill exercise on components of the cardiovascular (venous return, heart rate, arterial blood pressure) and respiratory systems (minute ventilation, tidal volume, breathing frequency, oxygen consumption, carbon dioxide production) and intra-abdominal pressure were investigated in the Savannah monitor lizard, Varanus exanthematicus B., at 35 degrees C. Compared with resting conditions, treadmill exercise significantly increased lung ventilation, gular pumping, intra-abdominal pressure, mean arterial blood pressure and venous return (blood flow in the post caval vein). However, venous return declines at high levels of activity, and mean arterial pressure and venous return did not attain peak values until the recovery period, immediately following activity. Elevating intra-abdominal pressure in resting lizards (via saline infusion) resulted in significant reductions in venous return when the transmural pressure of the post caval vein became negative (i.e. when intra-abdominal pressure exceeded central venous pressure). Together these results suggest that increments in intra-abdominal pressure compress the large abdominal veins and inhibit venous return. During locomotion, the physical compression of the large abdominal veins may represent a significant limitation to cardiac output and maximal oxygen consumption in lizards.

Regenerating lizard tails: a new model for investigating lymphangiogenesis.

Impaired lymphatic drainage in human limbs causes the debilitating swelling termed lymphoedema. In mammals, known growth factors involved in the control of lymphangiogenesis (growth of new lymph vessels) are vascular endothelial growth factors-C and -D (VEGF-C/D). Here we characterize a model of lymphangiogenesis in which the tail of lizards is regenerated without becoming oedematous. Three weeks after the tail is shed (autotomy), there are a small number of large diameter lymphatic vessels in the regenerated tail. Thereafter, the number increases and the diameter decreases. A functional lymphatic network, as determined by lymphoscintigraphy, is established 6 wk after autotomy. The new network differs morphologically and functionally from that in original tails. This lymphatic regeneration is associated with an up-regulation of a reptilian homologue of the VEGF-C/D protein family (rVEGF-C/D), as determined by Western blot analysis using a human reactive VEGF-C polyclonal antibody. Regenerating lizard tails are potentially useful models for studying the molecular basis of lymphangiogenesis with a view to developing possible treatments for human lymphoedema.