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 are 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.

Outbreak of multidrug-resistant Salmonella infections in people linked to pig ear pet treats, United States, 2015-2019: results of a multistate investigation.

Background: International distribution of contaminated foods can be a source of Salmonella infections in people and can contribute to the spread of antimicrobial-resistant bacteria across countries. We report an investigation led by the United States Centers for Disease Control and Prevention, the Food and Drug Administration (FDA), and state governmental officials into a multistate outbreak of salmonellosis linked to pig ear pet treats. Methods: Pig ear treats and companion dogs were tested for Salmonella by state officials and the FDA. Products were traced back to the country of origin when possible. Cases were defined as outbreak illnesses in people associated with one of seven Salmonella serotypes genetically related to samples from pig ear pet treats, with isolation dates from June 2015 to September 2019. Whole genome sequencing (WGS) of isolates was used to predict antimicrobial resistance. Findings: The outbreak included 154 human cases in 34 states. Of these, 107 of 122 (88%) patients reported dog contact, and 65 of 97 (67%) reported contact with pig ear pet treats. Salmonella was isolated from 137 pig ear treats, including some imported from Argentina, Brazil, and Colombia, and from four dogs. WGS predicted 77% (105/137) of human and 43% (58/135) of pig ear treat isolates were resistant to ≥3 antimicrobial classes. Interpretation: This was the first documented United States multistate outbreak of Salmonella infections linked to pig ear pet treats. This multidrug-resistant outbreak highlights the interconnectedness of human health and companion animal ownership and the need for zoonotic pathogen surveillance to prevent human illness resulting from internationally transported pet food products. Funding: Animal Feed Regulatory Program Standards award. Animal and product testing conducted by FDA Vet-LIRN was funded by Vet-LIRN infrastructure grants (PAR-22-063).

Whole-Genome Sequencing of Shiga Toxin-Producing Escherichia coli for Characterization and Outbreak Investigation.

Shiga toxin-producing Escherichia coli (STEC) causes high frequencies of foodborne infections worldwide and has been linked to numerous outbreaks each year. Pulsed-field gel electrophoresis (PFGE) has been the gold standard for surveillance until the recent transition to whole-genome sequencing (WGS). To further understand the genetic diversity and relatedness of outbreak isolates, a retrospective analysis of 510 clinical STEC isolates was conducted. Among the 34 STEC serogroups represented, most (59.6%) belonged to the predominant six non-O157 serogroups. Core genome single nucleotide polymorphism (SNP) analysis differentiated clusters of isolates with similar PFGE patterns and multilocus sequence types (STs). One serogroup O26 outbreak strain and another non-typeable (NT) strain, for instance, were identical by PFGE and clustered together by MLST; however, both were distantly related in the SNP analysis. In contrast, six outbreak-associated serogroup O5 strains clustered with five ST-175 serogroup O5 isolates, which were not part of the same outbreak as determined by PFGE. The use of high-quality SNP analyses enhanced the discrimination of these O5 outbreak strains into a single cluster. In all, this study demonstrates how public health laboratories can more rapidly use WGS and phylogenetics to identify related strains during outbreak investigations while simultaneously uncovering important genetic attributes that can inform treatment practices.

Population structure and genetic diversity of non-O157 Shiga toxin-producing Escherichia coli (STEC) clinical isolates from Michigan.

Non-O157 STEC are increasingly linked to foodborne infections, yet little is known about the diversity and molecular epidemiology across locations. Herein, we used whole genome sequencing to examine genetic variation in 894 isolates collected from Michigan patients between 2001 and 2018. In all, 67 serotypes representing 69 multilocus sequence types were identified. Serotype diversity increased from an average of four (2001-2006) to 17 (2008-2018) serotypes per year. The top six serogroups reported nationally caused > 60% of infections in 16 of the 18 years; serogroups O111 and O45 were associated with hospitalization as were age ≥ 65 years, diarrhea with blood and female sex. Phylogenetic analyses of seven multilocus sequence typing (MLST) loci identified three clades as well as evidence of parallel evolution and recombination. Most (95.5%) isolates belonged to one clade, which could be further differentiated into seven subclades comprising isolates with varying virulence gene profiles and serotypes. No association was observed between specific clades and the epidemiological data, suggesting that serogroup- and serotype-specific associations are more important predictors of disease outcomes than lineages defined by MLST. Molecular epidemiological studies of non-O157 STEC are important to enhance understanding of circulating strain distributions and traits, genetic variation, and factors that may impact disease risk and severity.

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.

Preoperative Intravenous Versus Oral Acetaminophen in Outpatient Surgery: A Double-Blinded, Randomized Control Trial.

PURPOSE: Preoperative acetaminophen is recognized as an effective part of the multimodal approach to perioperative pain management. The present study, conducted between April 12, 2018 and February 14, 2019, examined whether there are differences in patient-reported pain, postoperative opioid consumption, negative opioid effects, length of postanesthesia care unit stay, and patient satisfaction with pain control between patients who receive intravenous (IV) acetaminophen and patients who receive oral acetaminophen. DESIGN: This double-blinded, randomized controlled trial was conducted among 120 patients undergoing outpatient surgery. METHODS: Patients were randomized to receive preoperatively either intravenous (IV) acetaminophen (and oral placebo) or oral acetaminophen (and IV placebo). Results were analyzed using SPSS statistical software; statistical analyses consisted of Mann-Whitney U test, independent samples t test, and χ2 test. In all analyses, a P value less than .05 was considered significant. FINDINGS: There were no significant differences in any outcome measures based on the route of acetaminophen administration. CONCLUSIONS: The findings of the present study support the practice of administering oral acetaminophen, as opposed to IV acetaminophen, preoperatively as part of the multimodal approach to manage postoperative pain in patients able to tolerate preoperative oral medications.

Expanding Tiny Earth to genomics: a bioinformatics approach for an undergraduate class to characterize antagonistic strains.

The evolution of multidrug resistant pathogens and the diminishing supply of effective antibiotics are global crisis. Tiny Earth (TE) is undergraduate curriculum that encourage students to pursue science careers by engagement in authentic drug discovery research. Through the TE program, students identify environmental strains that inhibit other bacteria. Although these isolates may produce antibiotics based on the antagonistic phenotype, understanding the activity in regard to genome content remains elusive. Previously, we developed a transposon mutagenesis module for use with TE to identify genes involved in antibiotic production. Here, we extend this approach to a second semester undergraduate course to understand the origin of antagonism and genome diversity. Using a bioinformatics strategy, we identified gene clusters involved in activity, and with annotated genomes in hand, students were able to characterize strain diversity. Genomes were analyzed using different computational tools, including average nucleotide identity for species identification and whole genome comparisons. Because the focus of TE involves the evolution of drug resistance, predicted products in strains were identified and verified using a drug susceptibility assay. An application of this curriculum by TE members would assist in efforts with antibiotic discovery.

Loci Encoding Compounds Potentially Active against Drug-Resistant Pathogens amidst a Decreasing Pool of Novel Antibiotics.

Since the discovery of penicillin, microbes have been a source of antibiotics that inhibit the growth of pathogens. However, with the evolution of multidrug-resistant (MDR) strains, it remains unclear if there is an abundant or limited supply of natural products to be discovered that are effective against MDR isolates. To identify strains that are antagonistic to pathogens, we examined a set of 471 globally derived environmental Pseudomonas strains (env-Ps) for activity against a panel of 65 pathogens including Achromobacter spp., Burkholderia spp., Pseudomonas aeruginosa, and Stenotrophomonas spp. isolated from the lungs of cystic fibrosis (CF) patients. From more than 30,000 competitive interactions, 1,530 individual inhibitory events were observed. While strains from water habitats were not proportionate in antagonistic activity, MDR CF-derived pathogens (CF-Ps) were less susceptible to inhibition by env-Ps, suggesting that fewer natural products are effective against MDR strains. These results advocate for a directed strategy to identify unique drugs. To facilitate discovery of antibiotics against the most resistant pathogens, we developed a workflow in which phylogenetic and antagonistic data were merged to identify strains that inhibit MDR CF-Ps and subjected those env-Ps to transposon mutagenesis. Six different biosynthetic gene clusters (BGCs) were identified from four strains whose products inhibited pathogens including carbapenem-resistant P. aeruginosa BGCs were rare in databases, suggesting the production of novel antibiotics. This strategy can be utilized to facilitate the discovery of needed antibiotics that are potentially active against the most drug-resistant pathogens.IMPORTANCE Carbapenem-resistant P. aeruginosa is difficult to treat and has been deemed by the World Health Organization as a priority one pathogen for which antibiotics are most urgently needed. Although metagenomics and bioinformatic studies suggest that natural bacteria remain a source of novel compounds, the identification of genes and their products specific to activity against MDR pathogens remains problematic. Here, we examine water-derived pseudomonads and identify gene clusters whose compounds inhibit CF-derived MDR pathogens, including carbapenem-resistant P. aeruginosa.

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.

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.

The effectiveness of ultrasound guided hydrodistension and physiotherapy in the treatment of frozen shoulder/adhesive capsulitis in primary care: a single centre service evaluation.

BACKGROUND: Evidence for optimal non-operative treatment of frozen shoulder is lacking. The present study aimed to evaluate a treatment strategy for stage II to III frozen shoulder provided by the current primary care musculoskeletal service. METHODS: General practioner referrals of shoulder pain to the musculoskeletal service diagnosed with stage II to III frozen shoulder and who opted for a treatment strategy of hydrodistension and guided physiotherapy exercise programme over a 12-month period were evaluated for 6 months. Thirty-three patients were diagnosed with stage II to III frozen shoulder by specialist physiotherapists and opted for the treatment strategy. Outcome measures included Shoulder Pain Disability Index (SPADI) and Shortened Disabilities of the Arm, Shoulder and Hand (QuickDASH), pain score and range of movement. Data were collected at baseline, as well as at 6 weeks, 12 weeks and 6 months. RESULTS: All patients significantly improved in shoulder symptoms on the SPADI and QuickDASH scores (p < 0.001). Pain scores and range of shoulder movement flexion, abduction, external rotation showed significant improvement at all time points (p < 0.001). CONCLUSIONS: This service evaluation demonstrates that management of frozen shoulder stage II to III, as conducted by physiotherapists in a primary care setting utilizing hydrodistension and a guided exercise programme, represents an effective non-operative treatment strategy.

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.

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.

Congenital Plantar Angiofibroma-Like Hamartoma.

A 2-year-old girl presented with a congenital, slow-growing, exophytic nodule on the plantar surface of the foot. Histopathology revealed a hamartoma consisting of dermal angiofibroma features with overlying epidermal verruciform hyperplasia.

Growth hormone deficiency after mild combat-related traumatic brain injury.

OBJECTIVE: Traumatic brain injury (TBI) has been recognized as a cause of growth hormone deficiency (GHD) in civilians. However, comparable data are sparse in veterans who incurred TBI during combat. Our objective was to determine the prevalence of GHD in veterans with a history of combat-related TBI, and its association with cognitive and psychosocial dysfunction. DESIGN: Single center prospective study. PATIENTS: Twenty male veterans with mild TBI incurred during combat 8-72 months prior to enrollment. MEASUREMENTS: GHD was defined by a GH peak <3 µg/L during glucagon stimulation test. Differences in neuropsychological, emotional, and quality of life of the GHD Veterans were described using Cohen's d. Large effect sizes were considered meaningful. RESULTS: Mean age was 33.7 years (SD 7.8) and all subjects had normal thyroid hormone and cortisol levels. Five (25%) exhibited a subnormal response to glucagon. Sixteen participants (80%) provided sufficient effort for valid neuropsychological assessment (12 GH-sufficient, 4 GHD). There were large effect size differences in self-monitoring during memory testing (d = 1.46) and inhibitory control (d = 0.92), with worse performances in the GHD group. While fatigue and post-traumatic stress disorder were comparable, the GHD group reported more depression (d = 0.80) and lower quality of life (d = 0.64). CONCLUSIONS: Our study found a 25% prevalence of GHD in veterans with mild TBI as shown by glucagon stimulation. The neuropsychological findings raise the possibility that GHD has adverse effects on executive abilities and mood. Further studies are needed to determine whether GH replacement is an effective treatment in these patients.

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.

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.