From progesterone in biopsies to estimates of pregnancy rates: Large scale reproductive patterns of two sympatric species of common dolphin, Delphinus spp. off California, USA and Baja, Mexico

Blubber progesterone levels were measured in biopsy samples and used to predict the pregnancy status of 507 female common dolphins (204 long-beaked common dolphins, Delphinus capensis, and 303 short-beaked common dolphins, D. delphis). Samples were collected in the coastal waters of the eastern North Pacific between central California, USA and the southern end of Baja California, Mexico. The percentage of females pregnant was similar between the two species: 22.1% (n = 45) of D. capensis and 28.1% (n = 85) of D. delphis. For both species we found strong geographic patterns in pregnancy, suggesting that some areas were more conducive for pregnant females. A sizable drop in percent pregnant from early (38.8%, n = 133) to late (25.3%, n = 91) autumn was found in D. delphis but not in D. capensis. The potential for sample selectivity was examined via biopsies collected either from a large research ship or from a small, rigid-hull inflatable boat (RHIB) launched from the larger ship. An analysis of “Tandem Biopsy Sampling”, replicate biopsy effort on the same schools from each vessel/platform, yielded little evidence that disproportionately more pregnant female common dolphins were biopsied from one platform versus the other. This result plus an analysis of pregnancy status relative to the duration of biopsy operations failed to uncover strong evidence of unaccounted sampling bias with respect to pregnancy state. In total, these results demonstrate the utility of blubber progesterone concentrations to assess pregnancy status in free-ranging cetaceans and they highlight potential factors associated with population-level variation in dolphin pregnancy rates

Validation of blubber progesterone concentrations for pregnancy determination in three dolphin species and a porpoise.

Recent studies have validated the use of biopsies as a minimally invasive way to identify pregnant females in several species of wild cetaceans: Balaenapteraacutorostrata , Delphinusdelphis , Lissodelphisborealis, and Lagenorhynchusobliquidens. These studies found that progesterone (P4) concentrations quantified from blubber attached to biopsy samples is diagnostic of pregnancy. Here we examine a broader group of cetacean species in efforts to investigate how progesterone levels vary between species with respect to pregnancy status. We compared P4 concentrations in blubber collected from fishery bycatch and beach-stranded specimens for 40 females of known reproductive condition from Delphinuscapensis (n = 18), Stenellaattenuata (n = 8), S. longirostris (n = 6), and Phocoenoidesdalli (n = 8). The P4 concentrations were different (t = -7.1, p = 1.79E-08) between pregnant and non-pregnant animals in all species, with the mean blubber P4 concentration for pregnant animals 164 times higher than that of non-pregnant animals. There was no overlap in concentration levels between sexually immature or non-pregnant sexually mature animals and pregnant animals. No significant differences (F = 0.354, p = 0.559) were found between mature non-pregnant and immature D. capensis and Pdalli, suggesting P4 level is not indicative of maturity state in female delphinoids. P4 concentrations in relation to reproductive state were remarkably similar across species. All samples were analyzed with two different enzyme immunoassay kits to gauge assay sensitivity to measure progesterone in small samples, such as biopsies. With the technique now validated for these cetacean species, blubber P4 is a reliable diagnostic of pregnancies across multiple species, and thus expands the utility of this method to study reproduction in free-ranging cetaceans using biopsies

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 of a seasonal analysis of blubber cortisol levels in stranded <i>D</i>. <i>delphis</i>.

<p>The open circles represent cortisol concentrations for each individual. The observed three-month-running averages were calculated for each month (see text) and represented by the solid line. The dotted lines represent the 95% confidence envelope for the total seasonal randomness, i.e., the cortisol measurements were randomized relative to sampling date in 10,000 permuted datasets from which three-month-running averages were calculated. All observed three-month-running average values (represented by the solid line) are contained within the 95% confidence interval of the randomized null distributions indicating there was no evidence of seasonal fluctuations in blubber cortisol value.</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