Sirenian (manatees and dugongs) reproductive endocrinology.

Reproductive hormones are essential to mating systems, behavior, fertility, gestation, parturition, and lactation in mammals and understanding the role of hormones in these processes is essential for species conservation. Sirenia is a unique order of marine mammals that include manatees, dugongs, and the extinct Steller's sea cow. Extant Sirenian species are all listed as vulnerable due to habitat loss, cold stress, boat strike trauma, harmful algal bloom toxicity, entanglements, and illegal hunting. Therefore, successful reproduction is essential to maintaining and increasing Sirenian populations. Understanding Sirenian reproductive behavior, endocrinology, and mating strategies will aid conservation and management efforts to protect and provide the proper conditions for successful reproduction. The objectives of this review were to synthesize the current knowledge regarding reproductive cycles and endocrinology of Sirenians and identify knowledge gaps for future investigation. The current literature on Sirenian reproductive physiology reports reproductive seasonality, sexual maturation, estrous cyclicity and acyclicity, pregnancy, and sex differences. However, there remain significant knowledge gaps on the cyclicity and pulsatile release of gonadotropins, maturation in females, and characterization of pregnancy hormone profiles throughout gestation. To date, there is no explanation for confirmed pattern for ovarian acyclicity, nor understanding of the function of the numerous accessory corpus luteum described in manatees. Research including a greater number of longitudinal and postmortem studies on a wider variety of wild manatee populations are important first steps. Taken together, understanding the reproductive endocrinology of these vulnerable and threatened species is critical for policy and management decisions to better inform protection initiatives.

Reproductive Science Methods for Wild, Fully-Marine Mammals: Current Approaches and Future Applications.

Determining reproductive rates of marine mammal populations can give insight into their persistence and resilience in changing environments. As our marine environments continue to degrade along developed coastal fringes and as mankind's influences extend across even our widest oceans, there is a concern that the reproductive functioning of marine mammals may be affected adversely. Since many marine mammal species and populations are still in the recovery phase post-commercial harvest, and yet others are endangered or threatened by ongoing pressures, further environmental changes may represent direct or indirect threats to their reproductive potential. In this chapter, we review the current methods employed to investigate various aspects of reproductive science in fully-marine mammals, including direct observation of reproductive behavior and output, endocrinology to determine reproductive state, and assisted reproductive technologies to enhance reproductive outcomes. In particular, we focus on the most recent developments and innovations to reproductively sample marine mammals. Two case studies are presented to illustrate the challenges thrown up to researchers studying free-ranging marine mammals, and to highlight diversity in research approach. The North Atlantic right whale is on the brink of extinction through historic overharvest and present-day entanglement and ship strike. Environmental disruption to their migration routes and declining population health has resulted in reduced reproductive rates. In contrast, the main current threats to the reproductive success and survival of the vulnerable dugong are extreme weather events that affect availability of its seagrass diet. Climate disruption with increasingly severe coastal storms and flooding threaten the health of coastal seagrass beds, and consequently reproductive success and survival of this species. It is anticipated that climate change may have diverse and often serious effects on marine mammal reproduction in populations around the globe.

Adrenal responses of large whales: Integrating fecal aldosterone as a complementary biomarker to glucocorticoids.

Until now, physiological stress assessment of large whales has predominantly focused on adrenal glucocorticoid (GC) measures. Elevated GC concentrations in feces (fGC) are known to reflect stressful disturbances, such as fishing gear entanglement and human-generated underwater noise, in North Atlantic right whales (Eubalaena glacialis). However, there can be considerable variation in GC production as a function of sex and life history stage, which may confound the interpretation of fGC levels. Additionally, GC antibodies used in immunoassays can cross-react with other fecal metabolites (i.e., non-target steroids), potentially influencing fGC data. Here, aldosterone concentrations (fALD; aldosterone and related metabolites) were measured in fecal samples from right whales (total n=315 samples), including samples from identified individuals of known life history (n=82 individual whales), to evaluate its utility as a complementary biomarker to fGC for identifying adrenal activation. Concentrations of fALD were positively correlated with fGCs in right whales (r=0.59, P<0.001), suggesting concurrent secretion of these hormones by the adrenal gland. However, fALD levels were less influenced by concentrations of reproductive steroids in feces, minimizing the potential confounder of assay cross-reactivity in samples with highly skewed hormone ratios. Across different life history states for right whales, fALD concentrations showed similar patterns to those reported for fGC, with higher levels in pregnant females (35.9±7.6ng/g) followed by reproductively mature males (9.5±0.9ng/g) (P<0.05), providing further evidence of elevated adrenal activation in these groups of whales. The addition of fALD measurement as a biomarker of adrenal activation may help distinguish between intrinsic and external causes of stress hormone elevations in large whales, as well as other free-living wildlife species, providing a more comprehensive approach for associating adrenal activation with specific natural and anthropogenic stressors.

Methods to examine reproductive biology in free-ranging, fully-marine mammals.

Historical overexploitation of marine mammals, combined with present-day pressures, has resulted in severely depleted populations, with many species listed as threatened or endangered. Understanding breeding patterns of threatened marine mammals is crucial to assessing population viability, potential recovery and conservation actions. However, determining reproductive parameters of wild fully-marine mammals (cetaceans and sirenians) is challenging due to their wide distributions, high mobility, inaccessible habitats, cryptic lifestyles and in many cases, large body size and intractability. Consequently, reproductive biologists employ an innovative suite of methods to collect useful information from these species. This chapter reviews historic, recent and state-of-the-art methods to examine diverse aspects of reproduction in fully-aquatic mammals.

Testosterone and tusks: maturation and seasonal reproductive patterns of live, free-ranging male dugongs (Dugong dugon) in a subtropical population.

Knowledge of male reproductive status and activity in free-ranging animals is vital to understanding reproductive patterns and population dynamics. Until now, almost all information regarding reproductive behavior of the dugong, a cryptic marine mammal, has relied on post-mortem examination. We examined the relationships between body length, tusk eruption (secondary sexual characteristic), seasonality, and group association on fecal testosterone metabolite concentrations in 322 free-ranging dugongs (159 males, 163 females) in subtropical Moreton Bay, Australia. Fecal testosterone concentrations demonstrated biologically meaningful differences in testicular activity between sexes and across reproductive/age classes, and were correlated with circulating concentrations in serum. Male dugongs have a pre-reproductive period that persists until a body length of 240 cm is achieved. Puberty apparently occurs between 240 and 260 cm body length when fecal testosterone levels increase fourfold (>500 ng/g) over juvenile levels, and is associated with tusk eruption. However, social maturity may be delayed until male dugongs are larger than 260 cm with well-developed tusks. In mature males, the lowest (<500 ng/g) fecal testosterone concentrations occur in the austral autumn months with maximal concentrations in September-October, coincident with the onset of a spring mating season. During spring, solitary mature males had fecal testosterone concentrations double those of mature males sampled within groups, potentially suggesting a mating strategy involving roving of reproductively active males. This study demonstrates that single-point physiological data from individuals across a population have value as indicators of reproductive processes. Our approach provides an efficacious non-lethal method for the census of reproductive status and seasonality in live male dugongs.

Comparative geochemical and microbiological characterization of two thermal pools in the Uzon Caldera, Kamchatka, Russia.

Arkashin Schurf (Arkashin) and Zavarzin Spring (Zavarzin), two active thermal pools in the Uzon Caldera, Kamchatka, Russia, were studied for geochemical and microbiological characterization. Arkashin, the smaller of the two pools, had broader temperature and pH ranges, and the sediments had higher concentrations of total As (4,250 mg/kg) relative to Zavarzin (48.9 mg/kg). Glycerol dialkyl glycerol tetraether profiles represented distinct archaeal communities in each pool and agreed well with previous studies of these pools. Although no archaeal 16S rRNA sequences were recovered from Arkashin, sequences recovered from Zavarzin were mostly representatives of the Crenarchaeota and "Korarchaeota," and 13% of the sequences were unclassifiable. The bacterial community in Arkashin was dominated by uncultured "Bacteroidetes," Hydrogenobaculum of the Aquificales and Variovorax of the Betaproteobacteria, and 19% of the sequences remained unclassified. These results were consistent with other studies of As-rich features. The most abundant members of the Zavarzin bacterial community included the Chloroflexi, as well as members of the classes Deltaproteobacteria and Clostridia. In addition, 24% of the sequences were unclassified and at least 5% of those represent new groups among the established Bacterial phyla. Ecological structure in each pool was inferred from taxonomic classifications and bulk stable isotope δ values of C, N, and S. Hydrogenobaculum was responsible for primary production in Arkashin. However, in Zavarzin, the carbon source appeared to be allochthonous to the identified bacterial community members. Additionally, sequences related to organisms expected to participate in N and S cycles were identified from both pools.

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.

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.

Ameliorating transport-related stress in endangered Kemp's ridley sea turtles (Lepidochelys kempii) with a recovery period in saltwater pools.

Sea turtle rehabilitation clinics and aquaria frequently transport stranded sea turtles long distances out of water, e.g. for release at sites with appropriate water temperatures. Endangered Kemp's ridley turtles (Lepidochelys kempii) are known to exhibit an adrenal stress response during such transports. In an opportunistic study of turtles transported by road from Massachusetts to Georgia for release, we tested whether placing turtles in saltwater pools for short periods after transport would help turtles recover from transport-related stress. Eighteen juvenile Kemp's ridley turtles were examined and blood samples collected (1) immediately pre-transport, (2) immediately post-transport and (3) after a 6 h (n = 9) or 24 h (n = 9) post-transport period in unfamiliar pools, after which all turtles were released to the sea. Blood samples were analyzed for corticosterone, glucose, total white blood cell (WBC) count, heterophil/lymphocyte (H/L) ratio, pH, pO2, pCO2, HCO3 (bicarbonate), sodium, potassium, ionized calcium, lactate and hematocrit. Though the majority of turtles remained in good clinical condition, corticosterone, glucose, WBC and H/L elevated significantly during transport, while potassium declined slightly but significantly. After at least 6 h in a saltwater pool, potassium and glucose returned to pre-transport baselines and corticosterone partially recovered toward baseline. Extending the pool time to 24 h did not markedly enhance the physiological recovery of turtles, and two immune measures (WBC, H/L) remained elevated from the effect of transport. Six hours in a saltwater pool appears to facilitate the recovery of Kemp's ridley sea turtles from transport-related stress and may therefore improve their readiness for release.

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.

Get the most out of blow hormones: validation of sampling materials, field storage and extraction techniques for whale respiratory vapour samples.

Studies are progressively showing that vital physiological data may be contained in the respiratory vapour (blow) of cetaceans. Nonetheless, fundamental methodological issues need to be addressed before hormone analysis of blow can become a reliable technique. In this study, we performed controlled experiments in a laboratory setting, using known doses of pure parent hormones, to validate several technical factors that may play a crucial role in hormone analyses. We evaluated the following factors: (i) practical field storage of samples on small boats during daylong trips; (ii) efficiency of hormone extraction methods; and (iii) assay interference of different sampler types (i.e. veil nylon, nitex nylon mesh and polystyrene dish). Sampling materials were dosed with mock blow samples of known mixed hormone concentrations (progesterone, 17ß-estradiol, testosterone, cortisol, aldosterone and triiodothyronine), designed to mimic endocrine profiles characteristic of pregnant females, adult males, an adrenal glucocorticoid response or a zero-hormone control (distilled H2O). Results showed that storage of samples in a cooler on ice preserved hormone integrity for at least 6 h (P = 0.18). All sampling materials and extraction methods yielded the correct relative patterns for all six hormones. However, veil and nitex mesh produced detectable assay interference (mean 0.22 ± 0.04 and 0.18 ± 0.03 ng/ml, respectively), possibly caused by some nylon-based component affecting antibody binding. Polystyrene dishes were the most efficacious sampler for accuracy and precision (P < 0.001), but required an ethanol rinse for improved progesterone recovery (increased 81%; P < 0.001). Awareness of assay interference from exogenous materials is crucial to future studies. This study establishes critical groundwork to help ensure that hormones can be measured accurately in samples obtained from field collections of whale blow.

Assessment of ground transportation stress in juvenile Kemp's ridley sea turtles (Lepidochelys kempii).

Sea turtle rehabilitation centres frequently transport sea turtles for long distances to move animals between centres or to release them at beaches, yet there is little information on the possible effects of transportation-related stress ('transport stress') on sea turtles. To assess whether transport stress is a clinically relevant concern for endangered Kemp's ridley sea turtles (Lepidochelys kempii), we obtained pre-transport and post-transport plasma samples from 26 juvenile Kemp's ridley sea turtles that were transported for 13 h (n = 15 turtles) or 26 h (n = 11 turtles) by truck for release at beaches. To control for effects of handling, food restriction and time of day, the same turtles were also studied on 'control days' 2 weeks prior to transport, i.e. with two samples taken to mimic pre-transport and post-transport timing, but without transportation. Blood samples were analysed for nine clinical health measures (pH, pCO2, pO2, HCO3, sodium, potassium, ionized calcium, lactate and haematocrit) and four 'stress-associated' parameters (corticosterone, glucose, white blood cell count and heterophil-to-lymphocyte ratio). Vital signs (heart rate, respiratory rate and cloacal temperature) were also monitored. Corticosterone and glucose showed pronounced elevations due specifically to transportation; for corticosterone, this elevation was significant only for the longer transport duration, whereas glucose increased significantly after both transport durations. However, clinical health measures and vital signs showed minimal or no changes in response to any sampling event (with or without transport), and all turtles appeared to be in good clinical health after both transport durations. Thus, transportation elicits a mild, but detectable, adrenal stress response that is more pronounced during longer durations of transport; nonetheless, Kemp's ridley sea turtles can tolerate ground transportation of up to 26 h in good health. These results are likely to depend on specific transportation and handling protocols.

Variation in the hindgut microbial communities of the Florida manatee, Trichechus manatus latirostris over winter in Crystal River, Florida.

The Florida manatee, Trichechus manatus latirostris, is a hindgut-fermenting herbivore. In winter, manatees migrate to warm water overwintering sites where they undergo dietary shifts and may suffer from cold-induced stress. Given these seasonally induced changes in diet, the present study aimed to examine variation in the hindgut bacterial communities of wild manatees overwintering at Crystal River, west Florida. Faeces were sampled from 36 manatees of known sex and body size in early winter when manatees were newly arrived and then in mid-winter and late winter when diet had probably changed and environmental stress may have increased. Concentrations of faecal cortisol metabolite, an indicator of a stress response, were measured by enzyme immunoassay. Using 454-pyrosequencing, 2027 bacterial operational taxonomic units were identified in manatee faeces following amplicon pyrosequencing of the 16S rRNA gene V3/V4 region. Classified sequences were assigned to eight previously described bacterial phyla; only 0.36% of sequences could not be classified to phylum level. Five core phyla were identified in all samples. The majority (96.8%) of sequences were classified as Firmicutes (77.3 ± 11.1% of total sequences) or Bacteroidetes (19.5 ± 10.6%). Alpha-diversity measures trended towards higher diversity of hindgut microbiota in manatees in mid-winter compared to early and late winter. Beta-diversity measures, analysed through PERMANOVA, also indicated significant differences in bacterial communities based on the season.

Sex, scarring, and stress: understanding seasonal costs in a cryptic marine mammal.

We investigated variation in adrenal activity in the dugong, a vulnerable cryptic marine mammal species, in a population inhabiting the lower thermal limit of their range. We sampled 319 live wild dugongs and examined faecal glucocorticoid (fGC) concentrations in relationship to sex, reproductive state (immature, sexually mature, or pregnant), and season, as well as associations with body condition, male-induced aggressive conspecific interactions, and environmental temperature. Physiological validations with apparently healthy and unhealthy animals indicated that fGC levels reliably reflect adrenal activation in dugongs. Glucocorticoid levels and body condition varied seasonally, with individuals expressing lowest fGC levels and an improving body condition over summer and autumn. Best body condition was observed in winter, which may have enhanced the ability of the animals to cope with thermal and nutritional challenges and helped to sustain the subsequent costs of reproduction (including mating activity in spring). Immature dugongs, especially those recently estranged from their mothers, may be particularly vulnerable to stress during winter, and also at risk of injury from adult conspecifics. During the spring mating period, mature and sexually active males with large erupted tusks had high fGC and poorer body condition compared with all dugongs, except pregnant females, in all other seasons. This finding was consistent with high levels of body scarring on adults of both sexes, resulting from agonistic behaviour by mature males. Competitive reproductive behaviour, inherent in a promiscuous mating system, may have stressful consequences for male dugongs, especially given that strenuous mating activity follows unfavourable thermal and nutritional conditions. Overall, fGC levels appear to be a good proxy for stressors in dugongs associated with season and/or temperature, intra-specific aggression, and pregnancy, but not lactation and/or maternal care. Understanding baseline fGC levels will allow wildlife managers to identify additional and/or unusual stressors that may threaten wild dugong populations.

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.