The mean P4 values obtained from Diagnostic Systems Laboratory (DSL) and Enzo Life Sciences/Assay Designs (AD) EIA kits according to reproductive class.

<p>Note that the y-axis is presented on a log₁₀ scale to show a wide range of P4 values; whiskers indicate the standard error of the non-log values.</p

The mean P4 values for this study are combined with those from Kellar et al [13] to illustrate species-specific variability.

<p>Note that the y-axis is presented on a log₁₀ scale to show a wide range of P4 values; whiskers indicate the standard error of the non-log values (D cap – <i>Delphinus</i><i>capensis</i>, D del - <i>Delphinus</i><i>delphis</i>, L bor - <i>Lissodelphis</i><i>borealis</i>, L obl - <i>Lagenorhynchus</i><i>obliquidens</i>, P dal - <i>Phocoenoides</i><i>dalli</i>, S att - <i>Stenella</i><i>attenuata</i>, S lon - <i>Stenella</i><i>longirostris</i>).</p

Non-pregnant individuals are plotted with their corresponding P4 concentrations.

<p>Animal length is standardized for the plot as the ratio of total body length to the species-specific average length at sexual maturity.</p

The P4 concentration in blood serum of pregnant and non-pregnant animals.

<p>The data were taken from previous studies on <i>Phocoenoides</i><i>dalli</i> (P dal [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069709#B2" target="_blank">2</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069709#B3" target="_blank">3</a>]), <i>Stenella</i><i>coeruleoalba</i> (S coe [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069709#B1" target="_blank">1</a>]), and <i>Globicephala</i><i>macrorhynchus</i> (G mac [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069709#B1" target="_blank">1</a>]).</p

Blubber Cortisol: A Potential Tool for Assessing Stress Response in Free-Ranging Dolphins without Effects due to Sampling

<div><p>When paired with dart biopsying, quantifying cortisol in blubber tissue may provide an index of relative stress levels (i.e., activation of the hypothalamus-pituitary-adrenal axis) in free-ranging cetacean populations while minimizing the effects of the act of sampling. To validate this approach, cortisol was extracted from blubber samples collected from beach-stranded and bycaught short-beaked common dolphins using a modified blubber steroid isolation technique and measured via commercially available enzyme immunoassays. The measurements exhibited appropriate quality characteristics when analyzed via a bootstraped stepwise parallelism analysis (observed/expected = 1.03, 95%CI: 99.6 – 1.08) and showed no evidence of matrix interference with increasing sample size across typical biopsy tissue masses (75–150mg; r² = 0.012, p = 0.78, slope = 0.022ng_{cortisol deviation}/ul_{tissue extract added}). The relationships between blubber cortisol and eight potential cofactors namely, 1) fatality type (e.g., stranded or bycaught), 2) specimen condition (state of decomposition), 3) total body length, 4) sex, 5) sexual maturity state, 6) pregnancy status, 7) lactation state, and 8) adrenal mass, were assessed using a Bayesian generalized linear model averaging technique. Fatality type was the only factor correlated with blubber cortisol, and the magnitude of the effect size was substantial: beach-stranded individuals had on average 6.1-fold higher cortisol levels than those of bycaught individuals. Because of the difference in conditions surrounding these two fatality types, we interpret this relationship as evidence that blubber cortisol is indicative of stress response. We found no evidence of seasonal variation or a relationship between cortisol and the remaining cofactors.</p></div

Comprehensive Screening Links Halogenated Organic Compounds with Testosterone Levels in Male <i>Delphinus delphis</i> from the Southern California Bight

While environmental pollutants have been associated with changes in endocrine health in cetaceans, efforts to link contaminant exposure with hormones have largely been limited to a list of known, targeted contaminants, overlooking minimally characterized or unknown compounds of emerging concern. To address this gap, we analyzed a suite of potential endocrine disrupting halogenated organic compounds (HOCs) in blubber from 16 male short-beaked common dolphins (<i>Delphinus delphis</i>) with known maturity status collected from fishery bycatch in the Southern California Bight. We employed a suspect screening mass spectrometry-based method to investigate a wide range of HOCs that were previously observed in cetaceans from the same region. Potential endocrine effects were assessed through the measurement of blubber testosterone. We detected 167 HOCs, including 81 with known anthropogenic sources, 49 of unknown origin, and 37 with known natural sources. The sum of 11 anthropogenic and 4 unknown HOC classes were negatively correlated with blubber testosterone. Evidence suggests that elevated anthropogenic HOC load contributes to impaired testosterone production in mature male <i>D. delphis</i>. The application of this integrative analytical approach to cetacean contaminant analysis allows for inference of the biological consequences of accumulation of HOCs and prioritization of compounds for future environmental toxicology research

Results from matrix interference assessment.

<p>A standard solution (final concentration 240pg/ml) was spiked with either phosphate-buffered saline or a set of serial dilutions of a pooled sample (“uL of sample added”) composed of blubber cortisol extracts from four individuals to make a final equivalent volume of 240ul. The concentration of cortisol contributed from the pooled sample (neat = ± 398.5pg/ml) was subtracted from each sample-spiked measurement so its contribution would be factored out of the assessment. Little to no evidence of matrix interference was observed (r² = 0.012, p = 0.78).</p

Blubber cortisol concentrations for bycaught (n = 40) and beach stranded (n = 23) <i>D</i>. <i>delphis</i>.

<p>Horizontal box lines represent the lower quartile, median, and upper quartile values. Whiskers lines indicate range of concentrations. Points of inflection represent upper and lower bounds to the 95% confidence interval. Stranded individuals (24.3 ng/g) had on average 6.1 times more blubber cortisol than bycaught animals (3.99 ng/g).</p

Mean cortisol concentrations in the blubber of immature and mature male and female <b><i>Delphinus delphis</i></b> (bycatch specimens) of various reproductive states.

<p>Mean cortisol concentrations in the blubber of immature and mature male and female <b><i>Delphinus delphis</i></b> (bycatch specimens) of various reproductive states.</p

Results from linearity assessment of cortisol enzyme immunoassay (EIA) with blubber tissue extracts.

<p>Serial dilutions of extracts (open circles) show parallelism with the standards of the cortisol EIA (dark squares) (observed/expected = 1.01, 95%CI: 99.3–1.06); an indication that the assay is measuring the same antigens in the blubber as in the standards and therefore suitable for use with the short-beaked common dolphin blubber tissue extracts. Four individuals were represented in the pooled blubber extracts; 1 male and 3 female.</p