We quantified progesterone in 110 blubber samples from dolphins of known reproductive status in order to test the accuracy of a method to determine pregnancy status in wild cetaceans. The samples were collected from fishery‐bycaught delphinids of three species (Delphinus delphis, Lissodelphis borealis, and Lagenorhynchus obliquidens). We ascertained that blubber progesterone concentrations could clearly distinguish pregnant D. delphis (range 132–415 ng/g, mean 261 ng/g) from non‐pregnant mature and immature ones (range 0.92–48.2 ng/g, mean 15.2 ng/g). We found similar dramatic differences in L. borealis and L. obliquidens. These results were insensitive to various blubber sampling depths and anatomical sampling locations on the body, suggesting relative homogeneity of progesterone levels throughout the blubber. However, no trend was found in blubber progesterone concentration with fetal length, indicating that although blubber progesterone appears to distinguish pregnancy status, it is unlikely to differentiate pregnancy stage. Based on the findings presented here we suggest that this method, when coupled with projectile biopsy procedures, can be used to assess the pregnancy status of free‐ranging cetaceans and thus provide a new tool to determine pregnancy rates of wild populations.

Following the Deepwater Horizon (DWH) oil spill, reproductive success rates in 2 northern Gulf of Mexico (GoM) bottlenose dolphin stocks exposed to oil were evaluated for 4 yr during and after the spill (2010 to 2015) in efforts to assess population-level reproductive health. Pregnancy was determined from either (1) ultrasound examinations of the reproductive tract during capture-release health assessments, or (2) endocrine evaluations of blubber tissue collected from dart biopsies of free-ranging dolphins. Follow-up photo-identification was then used to track the status of pregnant females and any associated neonatal calves for a minimum of 1 yr after the initial pregnancy detection (IPD). For all pregnant females observed following IPD, individuals seen with a calf (reproductive success) and without one (reproductive failure) were recorded. The resulting estimated reproductive success rates for both GoM stocks (19.4%; 7/36) were less than a third of those previously reported in other areas not impacted by the spill (i.e. Sarasota Bay, FL; Indian River Lagoon, FL; and Charleston Harbor, SC) using similar techniques (64.7%; 22/34). We also evaluated the relationships between reproductive success and 13 potential covariates, including stock, ordinal date, progesterone, cortisol, thyroid hormone concentrations, leukocyte count, lung health score, and total body length. Among these, the results only provide strong evidence (Bayes factor > 20) of a relationship between reproductive failure and the total leukocyte count covariate. The high reproductive failure rates measured in both GoM stocks following the DWH oil spill are consistent with mammalian literature that shows a link between petroleum exposure and reproductive abnormalities and failures.

A novel molecular technique was used to measure blubber testosterone (BT) in 114 male short‐beaked common dolphins, Delphinus delphis, collected from incidental fishery bycatch and strandings. When these concentrations were compared across maturity states, the mean (± SEM) BT levels of mature D. delphis (14.3 ± 3.0 ng/g) were significantly higher than those of pubertal (2.5 ± 0.5 ng/g, P= 0.006) and immature animals (2.2 ± 0.3 ng/g, P < 0.0001). BT concentrations in mature males were significantly higher in summer months (53.9 ± 2.0 ng/g) than during the rest of the year (7.9 ± 0.69 ng/g, P < 0.0001), indicating reproductive seasonality. An analysis of BT in different anatomical locations showed that hormone concentrations were not homogenous throughout the body; the levels in the dorsal fin were significantly lower than in most other areas (F= 5.39, P= 0.043). Conversely, we found no significant differences in BT concentration with respect to subepidermal depth (F= 2.09, P= 0.146). Finally, testosterone levels in biopsies from 138 free‐swimming male D. delphis, of unknown maturity state, sampled off California were found to be of concentrations similar to those from the fishery bycatch and stranding samples and revealed an analogous trend with respect to ordinal date.

Bowhead whale Balaena mysticetus progesterone concentrations were measured in different sample matrices (serum, blubber, and urine) to investigate (1) concordance among sample type and (2) variation among life-history class. Samples were collected from subsistencehunted whales (n = 86) taken from 1999 to 2009. In general, irrespective of sample matrix, pregnant females had the highest concentrations by orders of magnitude, followed by mature animals of both sexes, and subadults had the lowest concentrations. Subadult males and females had similar progesterone concentrations in all sample matrices measured. When pregnant animals were included in our analyses, permuted regression models indicated a strong positive relationship between serum and blubber progesterone levels (r 2 = 0.894, p = 0.0002). When pregnant animals were not included, we found no significant relationship between serum and blubber levels (r 2 = 0.025, p = 0.224). These results suggest that progesterone concentrations are mirrored in these sample types over longer periods (i.e. on the order of weeks to months, time frame of reproductive changes) but not shorter periods (i.e. on the order of hours to days, time frame of daily fluctuations). This conclusion is consistent even for progesterone concentrations measured in females that had recently changed pregnancy states (either new mothers or newly pregnant animals), for which blubber progesterone levels seem to lag those in the serum. Finally, urine progesterone had statistically significant positive relationships with serum (r 2 = 0.136, p = 0.0460) and blubber progesterone (r 2 = 0.150, p = 0.0421). Our results suggest that progesterone concentrations first peak in the serum, then in the urine, and finally in the blubber.

No abstract

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 = 0.022ngcortisol deviation/ultissue 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.

Recent studies have validated the use of biopsies as a minimally invasive way to identify pregnant females in several species of wild cetaceans: Balaenaptera acutorostrata , Delphinus delphis , Lissodelphis borealis , and Lagenorhynchus obliquidens . 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 Delphinus capensis (n = 18), Stenella attenuata (n = 8), S . longirostris (n = 6), and Phocoenoides dalli (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 P dalli , 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.

Stress hormones, released into circulation as a consequence of disturbance, are classically assayed from blood samples but may also be detected in a variety of matrices. Blubber and fecal samples can be remotely collected from free‐ranging cetaceans without the confounding hormone elevations associated with chase, capture, and handling required to collect blood samples. The relationship between cortisol concentrations in circulation with that of blubber and feces, however, is unknown. To assess these associations, we elevated cortisol by orally administering hydrocortisone for five days in five bottlenose dolphins. Voluntary blood and fecal samples were collected daily; blubber biopsies were collected on day one, just prior to hydrocortisone administration, and days three and five of hydrocortisone administration. We evaluated subsequent changes in several circulating stress hormones as well as cortisol and glucocorticoid metabolites in blubber and feces, respectively. There was a significant association between cortisol levels in serum and in blubber (F1,12.7 = 14.3, P < 0.01, mR2 = 0.57) despite substantial variability in blubber cortisol levels. Counterintuitively, fecal cortisol metabolite levels were inversely related to serum cortisol. The relationship between serum and blubber cortisol levels suggests blubber samples from remote sampling may be useful to detect stress loads in this species.