Stress and reproductive events detected in North Atlantic right whale blubber using a simplified hormone extraction protocol.

As studies quantifying steroid hormones in marine mammal blubber progress, methodological refinements may improve the utility and consistency of blubber hormone measurements. This study advances blubber extraction methodologies by testing a simplified extraction protocol that reduces time and complexity compared to a protocol widely used in cetacean blubber studies. Using blubber samples archived from remote biopsy (n = 21 live whales) and necropsy collection (n = 7 dead whales) of North Atlantic right whales (NARW; Eubalaena glacialis) of known life history states, we performed analytical and biological validations to assess the feasibility of measuring reproductive (testosterone, progesterone) and glucocorticoid (cortisol) hormones in blubber via enzyme immunoassay following the simplified extraction. Analytical validations (parallelism, accuracy, extraction efficiency, repeatability) showed the simplified extraction produced similar results to the extended protocol, offering a more efficient and consistent technique. In live, apparently healthy whales, blubber testosterone concentrations (mean ± SE) were significantly higher in males (2.02 ± 0.36 ng/g) compared to females (0.81 ± 0.15 ng/g). Blubber progesterone was highest in a confirmed pregnant female (60.3 ng/g), which was 12-fold greater than the mean concentration of non-pregnant females (4.56 ± 0.88 ng/g). Blubber cortisol concentrations in whales that died from anthropogenic causes averaged 5.31 ± 2.28 ng/g, whereas most live, healthy whales had cortisol values below 1 ng/g. Among living whales, a whale actively entangled in fishing gear had the highest blubber cortisol measurement (3.51 ng/g), exhibiting levels similar to whales that died from acute entanglement (2.88 ± 0.42 ng/g). Overall, the highest blubber cortisol concentration (18.0 ng/g) was measured in a dead whale with a severe chronic entanglement, approximately 30-fold greater than mean blubber cortisol of apparently healthy whales (0.58 ± 0.11 ng/g). The methodological approach presented here provides a reference for researchers interested in an alternative, streamlined technique for hormone extraction of cetacean blubber and contributes to the diverse tool set for stress and reproductive assessments of endangered NARWs.

Quantifying hormones in exhaled breath for physiological assessment of large whales at sea.

Exhaled breath analysis is a non-invasive assessment tool that has shown promise in human diagnostics, and could greatly benefit research, management, and conservation of large whales. However, hormone assessment of whale respiratory vapor (blow) has been challenged by variable water content and unknown total volume of collected samples. To advance this technique, we investigated urea (a compound present in narrow range in circulation) as a normalizing factor to correct for blow sample concentration. Normalized progesterone, testosterone, and cortisol concentrations of 100 blow samples from 46 photo-identified North Atlantic right whales (Eubalaena glacialis) were more biologically relevant compared to absolute estimates, varying by sex, age class, or individual. Progesterone was elevated in adult females compared with other cohorts and highest in one independently confirmed pregnant female. For both sexes, testosterone was two-fold higher in reproductively mature whales but studied adult females showed the widest variation. Cortisol was present in relatively low concentrations in blow and demonstrated variation between individual whales, suggesting potential for studies of individual differences in adrenal activity. Incorporation of methodologies that normalize sample concentration are essential for blow hormone analysis of free-swimming whales, and measurement of urea could be used to optimize non-invasive physiological assessment of whales.

Reproductive hormone monitoring of dugongs in captivity: detecting the onset of sexual maturity in a cryptic marine mammal.

Determining the reproductive status of long-term captive animals is essential because the onset of sexual maturity and reproductive activity may necessitate changes in husbandry requirements. This study reports on the first multi-year reproductive hormone monitoring program for captive dugongs of both sexes using feces. Fecal samples were collected from one male (Pig) over 9 years (4-13.2y of age; n=288 samples, 0.8±0.1 samples per week from July 2007 to February 2012) and one female (Wuru) over 7 years (from neonate to 6.9 y; n=171 samples, 0.5±0.1 samples per week from July 2007 to February 2012), and from one solitary female dugong (Gracie) over 10 months (10.5-11.3y of age; n=54 samples, 1.1±0.2 sample per week from September 2008 to June 2009). Using enzyme-immunoassay, fecal progesterone (fP) and estradiol-17ß (fE) concentrations were assayed in the two captive females, and testosterone (fT) concentration in the captive male, and compared these to concentrations in wild dugongs. Female Wuru exhibited increasing fP concentrations at 5+ y, indicating early onset of ovarian cycling typical of non-pregnant adult females. Female Gracie maintained basal fP concentrations consistent with wild immature dugongs, indicating that she had not reached puberty by 11y. Nutritional plane may account for differences in age at sexual maturity in these female dugongs. At age 3-4y, Wuru had fE concentrations 1.4 times greater than maximum concentrations recorded in all wild females, and these concentrations were coincident with a period of rapid weight gain. For the male Pig, increasing fT concentrations at 9y provided early indications of puberty. Pig's tusks erupted by 11y, and sexual maturity (indicated by spermatic semen) was confirmed by 12.8y. Identification of sexual maturation prompted two trials of a male contraceptive treatment using the GnRH agonist, deslorelin (9.4mg administered in 2010 and 15.6mg in 2011). Testosterone production was not significantly suppressed by these dosages, and treatment did not terminate sperm production at week 10-11 post-implantation, even at the larger dose tested. Routine analysis of fecal hormones was helpful for making reproductive management decisions regarding individual captives and in guiding the long-term captive management of this cryptic species.

Diagnosing pregnancy in free-ranging dugongs using fecal progesterone metabolite concentrations and body morphometrics: a population application.

Assessing reproductive status and monitoring reproductive rates is important in the effective management of vulnerable marine mammal species such as the dugong (Dugong dugon). Knowledge of the reproductive physiology of this species is limited, and determining reproductive parameters (e.g., sexual maturation, pregnancy, and reproductive senescence) has been restricted by a lack of non-lethal methods for assessing reproductive status in free-ranging individuals. The aim of this study was to develop a method to identify pregnant individuals in a wild dugong population. Using an enzymeimmunoassay, we quantified concentrations of fecal progesterone metabolites (fP) in 322 dugongs, including confirmed pregnant females (n=10), presumed non-pregnant adult females (n=25), juvenile females (n=24), subadult females (n=41), adult females of unknown pregnancy state (n=63), and males of all sizes (n=159). External body morphometrics of each dugong were measured, and confirmation of pregnancy in adult female dugongs was determined by ultrasonography or observation of subsequent neonates. Concentrations of fP were different between sexes and reproductive size classes (P<0.001), and ∼30-fold higher in confirmed pregnant dugongs (2017-7760 ng/g) compared to presumed non-pregnant females (30-221 ng/g), juvenile females (29-195 ng/g), and males (24-261 ng/g) (P<0.001). Body measures of maximum and anal girths, and teat length were all greater in confirmed pregnant females than presumed non-pregnant females (all P<0.05). We evaluated a Discriminant Function Analysis (DFA) to provide a model for predicting pregnant and non-pregnant dugongs. Cross-validated results showed that the DFA correctly classified 100% of pregnant and non-pregnant females using fP concentrations, body length, fineness ratio (an index of body shape), and teat length (a female reproductive trait). Using the DFA model, we classified the pregnancy status of all female dugongs and identified a total of 30 females as pregnant and 133 females as non-pregnant from the sampled population over the sample period. Pregnant dugongs in the Moreton Bay population are characterized by fecal progesterone metabolite concentrations > 1000 ng/g, body length ≥ 260 cm, maximum girth ≥ 215 cm, anal girth ≥ 126 cm, and teat length ≥ 5 cm long. In summary, analysis of fP concentrations in combination with body morphometrics may be used to diagnose pregnancy in free-ranging dugongs, and provides a new tool to monitor breeding rates of wild sirenian populations.