Assessment of a non-invasive approach to pregnancy diagnosis in gray whales through drone-based photogrammetry and faecal hormone analysis.

Knowledge of baleen whales' reproductive physiology is limited and requires long-term individual-based studies and innovative tools. We used 6 years of individual-level data on the Pacific Coast Feeding Group gray whales to evaluate the utility of faecal progesterone immunoassays and drone-based photogrammetry for pregnancy diagnosis. We explored the variability in faecal progesterone metabolites and body morphology relative to observed reproductive status and estimated the pregnancy probability for mature females of unknown reproductive status using normal mixture models. Individual females had higher faecal progesterone concentrations when pregnant than when presumed non-pregnant. Yet, at the population level, high overlap and variability in progesterone metabolite concentrations occurred between pregnant and non-pregnant groups, limiting this metric for accurate pregnancy diagnosis in gray whales. Alternatively, body width at 50% of the total body length (W50) correctly discriminated pregnant from non-pregnant females at individual and population levels, with high accuracy. Application of the model using W50 metric to mature females of unknown pregnancy status identified eight additional pregnancies with high confidence. Our findings highlight the utility of drone-based photogrammetry to non-invasively diagnose pregnancy in this group of gray whales, and the potential for improved data on reproductive rates for population management of baleen whales generally.

Evaluating Dermal Bone as a Novel Source of Endocrine Information in Ninespine and Threespine Stickleback Fish.

Monitoring the physiology of small aquatic and marine teleost fish presents challenges. Blood samples, often the first choice for endocrinologists, can be difficult or even impossible to obtain and alternative matrices currently used for hormone analyses do not occur in fishes (e.g., hair, feathers etc.) or are not easily collected from small aquatic organisms (e.g., urine and feces). Some teleosts, however, have enlarged bony dermal elements that possibly accumulate and store steroid hormones in physiological relevant concentrations. Both threespine stickleback (Gasterosteus aculeatus) and ninespine stickleback (Pungitius pungitius) have a series of external, lateral bony plates, dorsal spines, and a pair of pelvic spines attached to the pelvic girdle. We investigated if cortisol, the primary circulating glucocorticoid in teleosts, could be extracted from stickleback dermal bone and quantified using a commercially available enzyme immunoassay (EIA). We successfully validated a cortisol EIA for dermal bone extracts, determined that cortisol was detectable in both species, and found that dermal bone cortisol levels significantly correlated with cortisol levels in whole body homogenate. Ninespine stickleback had significantly higher dermal bone cortisol concentrations than threespine stickleback and female threespine stickleback tended to have over twice the mean dermal bone cortisol concentration than males. Because both stickleback species are widely used for ecotoxicological studies, using dermal bone as a source of endocrine information, while leaving the body for contaminant, genomic, histological, and stable isotope analyses, could be a powerful and parsimonious tool. Further investigation and physiological validations are necessary to fully understand the utility of this new sample matrix.

Spanish (translation provided by Luis C. Beltran)El monitoreo de la fisiología de pequeños peces teleósteos acuáticos y marinos presenta desafíos. Las muestras de sangre, a menudo la primera opción para los endocrinólogos, pueden ser difíciles o incluso imposibles de obtener y las matrices alternativas que se utilizan actualmente para los análisis hormonales no son aplicables en peces (p. ej., pelo, plumas, etc.) o no se obtienen fácilmente de pequeños organismos acuáticos (p. ej., orina y heces). Sin embargo, algunos teleósteos tienen elementos dérmicos óseos agrandados que posiblemente acumulan y almacenan hormonas esteroides en concentraciones fisiológicas relevantes. Tanto el espinoso de tres espinas (Gasterosteus aculeatus) como el espinoso de nueve espinas (Pungitius pungitius) tienen una serie de placas óseas laterales externas, espinas dorsales y un par de espinas pélvicas unidas a la cintura pélvica. Nosotros investigamos si el cortisol, el principal glucocorticoide circulante en los teleósteos, podría extraerse del hueso dérmico del espinoso y cuantificarse mediante un inmunoensayo enzimático (EIA, por sus siglas en inglés) disponible comercialmente. Validamos con éxito un EIA de cortisol para extractos óseos dérmicos, determinamos que el cortisol era detectable en ambas especies, y descubrimos que los niveles de cortisol óseo dérmico se correlacionaron significativamente con los niveles de cortisol en el cuerpo entero homogeneizado. El espinoso de nueve espinas tenía concentraciones de cortisol óseo dérmico significativamente más altas que el espinoso de tres espinas y el espinoso de tres espinas hembra tendía a tener más del doble de la concentración media de cortisol óseo dérmico que los machos. Debido a que ambas especies de espinosos se usan ampliamente para estudios ecotoxicológicos, el uso del hueso dérmico como fuente de información endocrina, mientras se deja el cuerpo para análisis de contaminantes, genómicos, histológicos e isótopos estables, podría ser una herramienta poderosa y parsimoniosa. Se necesitan más investigaciones y validaciones fisiológicas para comprender completamente la utilidad de esta nueva matriz de muestra.

Portuguese (translation provided by Jonathan M. Branco)Monitorar a fisiologia de pequenos peixes teleósteos aquáticos e marinhos apresenta desafios. Amostras de sangue, muitas vezes a primeira escolha para endocrinologistas, podem ser difíceis ou mesmo impossíveis de obter e matrizes alternativas atualmente usadas para análises hormonais não ocorrem em peixes (e.g., pelos, penas etc.) ou não são facilmente coletadas em pequenos organismos aquáticos (e.g., urina e fezes). Alguns teleósteos, no entanto, possuem elementos dérmicos ósseos ampliados que possivelmente acumulam e armazenam hormônios esteroides em concentrações fisiológicas relevantes. Ambos Gasterosteus aculeatus e Pungitius pungitius apresentam uma série de Placas ósseas externas laterais, espinhos dorsais e um par de espinhos pélvicos ligados à cintura pélvica. Investigamos se o cortisol, o principal glicocorticoide circulante em teleósteos, poderia ser extraído do osso dérmico de G. aculeatus e P. pungitius e quantificado usando um imunoensaio enzimático (EIA) comercialmente disponível. Validamos com sucesso um EIA de cortisol para extratos de ósseos dérmicos, determinamos que o cortisol era detectável em ambas as espécies e descobrimos o cortisol ósseo dérmico em níveis significativamente correlacionados com os níveis de cortisol em todo o corpo homogeneizado. P. pungitius apresentou concentrações de cortisol ósseo dérmico significativamente mais altas do que G. aculeatus e fêmeas de G. aculeatus tenderam a ter mais que o dobro da concentração média de cortisol ósseo dérmico de machos. Como ambas as espécies são amplamente utilizadas para estudos ecotoxicológicos, usando o osso dérmico como fonte de informação endócrina, deixando o corpo para análises de contaminantes, genômicas, histológicas e de isótopos estáveis, pode ser uma ferramenta poderosa e parcimoniosa. Mais investigações e validações fisiológicas são necessários para entender completamente a utilidade desta nova matriz de amostra.

The influence of androgens on hibernation phenology of free-living male arctic ground squirrels.

Free-living ground squirrel species are sexually dimorphic in hibernation phenology. The underlying causes of these differences are not yet known. Androgens, testosterone (T) in particular, inhibit hibernation. To determine the influence of endogenous androgens on annual timing of hibernation we first measured circulating levels of T and dehydroepiandrosterone (DHEA), an adrenal androgen implicated in non-mating season aggression in other species, in free-living male arctic ground squirrels (Urocitellus parryii, AGS). We also manipulated endogenous androgen levels by surgical castration, and consequently compared body temperature records from intact (n=24) and castrated (n=9) males to elucidate the influence of endogenous androgens on annual body temperature cycles. The highest T levels (0.53±0.10ng/mL) were in reproductively mature male AGS in spring; whereas, both immature males in spring and all males in late summer had T levels an order of magnitude lower (0.07±0.01 and 0.06±0.00ng/mL, respectively). DHEA levels were higher in males during the late summer compared to reproductively mature males in spring (120.6±18.9 and 35.9±2.3pg/mL, respectively). Eliminating gonadal androgens via castration resulted in males delaying euthermy by extending heterothermy significantly in spring (Apr 22 ±2.9) than reproductive males (Mar 28 ±3.9) but did not change the timing of hibernation onset (castrate: Oct 12 ±1.0 vs. intact: Oct 3 ±3.1). We conclude that while androgens play a significant role in spring hibernation phenology of males, their role in fall hibernation onset is unclear.

Thermogenic capacity at subzero temperatures: how low can a hibernator go?

Abstract Hibernation in mammals is a physiological and behavioral adaptation to survive intervals of low resource availability through profound decreases in metabolic rate (MR), core body temperature (Tb), and activity. Most small mammalian hibernators thermoconform, with Tb approximating ambient temperature (Ta); arctic species are an exception, since they must actively defend what can be large thermal gradients between Tb and Ta. Here we compare the thermogenic capacity of the arctic ground squirrel (Urocitellus parryii) to that of the golden-mantled ground squirrel (Callospermophilus lateralis), a temperate-zone montane hibernator. We allowed animals to reenter torpor at sequentially lower Ta's and found that arctic ground squirrels maintained steady state torpor at Ta's as low as -26°C, through a 36-fold increase in torpid MR (TMR), compared to their minimum TMR, exhibited at a Ta of 0°C. Golden-mantled ground squirrels are able to maintain steady state torpor at Ta's at least as low as -8°C, through a 13.5-fold increase in MR, compared to their minimum TMR at a Ta of 2°C. In a second experiment, torpid animals were exposed to continuously decreasing Ta's (0.25°C/30 min); individuals of both species increased their metabolism while remaining torpid at low Ta's (as low as -30°C for arctic ground squirrels and -10°C for golden-mantled ground squirrels). Although the capacity to hibernate at subfreezing Ta's is not unique to arctic ground squirrels, their large body size, greater torpid metabolic scope, and previously ascribed capacity to supercool allow them to occupy much colder hibernacula for prolonged seasons of hibernation.

Effects of ambient temperature on metabolic rate, respiratory quotient, and torpor in an arctic hibernator.

Arctic ground squirrels (Spermophilus parryii) overwinter in hibernaculum conditions that are substantially below freezing. During torpor, captive arctic ground squirrels displayed ambient temperature (T(a))-dependent patterns of core body temperature (T(b)), metabolic rate (TMR), and metabolic fuel use, as determined by respiratory quotient (RQ). At T(a) 0 to -16 degrees C, T(b) remained relatively constant, and TMR rose proportionally with the expanding gradient between T(b) and T(a), increasing >15-fold from a minimum of 0.0115 +/- 0.0012 ml O(2). g(-1). h(-1). At T(a) 0-20 degrees C, T(b) increased with T(a); however, TMR did not change significantly from T(b) 0 to 12 degrees C, indicating temperature-independent inhibition of metabolic rate. The overall change in TMR from T(b) 4 to 20 degrees equates to a Q(10) of 2.4, but within this range of T(b), Q(10) changed from 1.0 to 14.1. During steady-state torpor at T(a) 4 and 8 degrees C, RQ averaged 0.70 +/- 0.013, indicating exclusive lipid catabolism. At T(a) -16 and 20 degrees C, RQ increased significantly to >0.85, consistent with recruitment of nonlipid fuels. RQ was negatively correlated with maximum torpor bout length. For T(a) values <0 degrees C, this relationship supports the hypothesis that availability of nonlipid metabolic fuels limits torpor duration in hibernating mammals; for T(a) values >0 degrees C, hypotheses linked to body temperature are supported. Because anterior body temperatures differ from core, overall, the duration torpor can be extended in hibernating mammals may be dependent on brain temperature.

Calories, fat and cancer.

The experiments reported are part of our effort to dissociate the tumor-enhancing effects of dietary fat and high caloric intake. Rats either were fed ad libitum diets containing 4% corn oil or their calories were restricted by 40% and their diets contained 13.1% corn oil. Incidence of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors was 80% in rats fed ad libitum and 20% in those fed the calorie-restricted diets. Incidence of 1,2-dimethylhydrazine (DMH)-induced colon tumors was 100% in rats fed ad libitum and 53% in those whose caloric intake was restricted by 40%. The tumor yield (tumors per tumor-bearing rat) was significantly lower in rats on caloric restriction. In another series, rats were fed diets containing 5, 15 or 20% corn oil ad libitum or were fed calorie-restricted (by 25%) diets which provided 20 or 26.6% corn oil (therefore, the same absolute amount of fat was consumed in each of the pair-fed groups). Tumor incidence and tumor yield in the two calorie-restricted groups were similar to those seen in the rats fed 5% fat ad libitum; tumor burden (total g of tumor) was 45-65% lower in the calorie-restricted rats. The data suggest that caloric intake is a more stringent determinant of tumor growth than fat intake.

Caffeine and ephedrine stimulated thermogenesis in LA-corpulent rats.

Resting metabolic rate (RMR), as well as caffeine (CAF) and ephedrine (EPH) stimulated thermogenesis (VO2) were measured in young adult corpulent (corp) LA/N-cp (LA-corpulent) rats. RMR of lean was greater than corp. Administration of EPH, CAF and EPH + CAF resulted in 32, 48 and 50% increases in VO2, respectively, in both lean and corp rats. The time to attain maximal VO2 was similar for both drugs in both phenotypes, but the duration of maximal VO2 averaged 50, 26 and 42% longer in corp than lean for EPH, CAF and EPH + CAF, respectively. Acute weight loss following these treatments was greater for corp than lean, and corresponded with the duration of elevated VO2. These results are consistent with a normally functioning end-organ sympathomimetic receptor system in the corp phenotype of the LA/N-cp rat, and suggest that obesity in this model may be caused by factors other than defective brown fat thermogenesis at the end organ level.