Plasma concentrations of itraconazole following a single oral dose in juvenile California sea lions (Zalophus californianus).

The objective of this study was to establish a single-dose pharmacokinetic profile for orally administered itraconazole in California sea lions (Zalophus californianus). Twenty healthy rehabilitated juvenile California sea lions were included in this study. Itraconazole capsules were administered orally with food at a target dose of 5-10 mg/kg. Blood samples were collected from each animal at 0 hr and at two of the following timepoints: 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 hr. Quantitative analysis of itraconazole in plasma samples was performed by high-performance liquid chromatography. An average maximum concentration of 0.22 µg/ml ± 0.11 was detected 4 hr after administration. The average concentration fell to 0.12 µg/ml ± 0.11 by 6 hr and 0.02 µg/ml ± 0.02 at 12 hr. At no point did concentrations reach 0.5 µg/ml, the concentration commonly accepted for therapeutic efficacy. While this formulation was well tolerated by the sea lions, oral absorption was poor and highly variable among individuals. These data indicate that a single oral dose of itraconazole given as a capsule at 5-10 mg/kg, under the conditions used in this study, does not achieve therapeutic plasma concentrations in California sea lions.

EVALUATION OF SERIAL BLOOD LACTATE AND THE USE OF A POINT-OF-CARE LACTATE METER IN LIVE-STRANDED PINNIPEDS.

Live-stranded pinnipeds often present to rehabilitation centers systemically debilitated with dehydration and poor perfusion. In many terrestrial mammals, blood lactate elevation has been correlated with global tissue hypoxia and decreased circulating blood volume. Serial blood lactate measurements in companion animals and humans have been used to guide fluid resuscitation therapy and evaluate prognosis. The primary objective of this study was to evaluate the prognostic value of serial blood lactate levels in live-stranded pinnipeds in a rehabilitation setting. The secondary objectives were to evaluate the use of a point-of-care lactate meter, and potassium oxalate-sodium fluoride (gray-top) tubes for storing samples for lactate analysis in pinniped patients. Fifty-five live-stranded pinnipeds (30 northern elephant seals [Mirounga angustirostris], 21 Pacific harbor seals [Phoca vitulina richardsi], and four California sea lions [Zalophus californianus]) that presented to a rehabilitation center were manually restrained for blood collection and lactate measurement using a point-of-care analyzer (Lactate Plus™) prior to fluid or other medical therapy. Lactate measurements were repeated 72 hr post admission and prior to euthanasia or release. Plasma samples from gray-top tubes were frozen for 30-100 days at -80°C, then thawed and evaluated using both the LactatePlus and bench-top (ABL 815) analyzers. The point-of-care analyzer was successful in measuring lactate in pinnipeds. Gray-top tubes were effective at preserving lactate levels in frozen plasma samples for up to 100 days. Released animals had significantly greater lactate clearance at 72 hr (P = 0.039) than animals that died or were euthanized. Therefore, lactate clearance, determined by serial blood lactate measurements, may be useful for evaluating prognosis in live-stranded pinnipeds. Initial lactate and lactate values prior to euthanasia or release were not significantly associated with outcome. Given these findings, clinicians should interpret isolated lactate values in pinnipeds with caution.

REGIONAL AND AGE-RELATED VARIATIONS IN HAPTOGLOBIN CONCENTRATIONS IN STELLER SEA LIONS ( EUMETOPIAS JUBATUS) FROM ALASKA, USA.

Varying concentrations of the highly conserved acute phase response protein, haptoglobin, can indicate changes to the health and disease status of mammals, including the Steller sea lion (SSL; Eumetopias jubatus). To better understand factors relating to acute phase response in SSLs, circulating haptoglobin concentrations (Hp) were quantified in plasma collected from 1,272 individuals sampled near rookeries and haulouts off the coast of Alaska, US. We compared Hp in SSLs between sexes and among different age classes (young pups, young-of-the-year, yearlings, subadults, and adults) sampled within distinct regions in Alaska (Aleutian Islands, Gulf of Alaska, Southeast Alaska). Regional and age-related differences were observed, particularly in younger SSLs. No sex-related differences were detected. We identified weakly significant relationships between Hp and hematology measurements including white blood cell counts and hematocrit in young pups from the Aleutian Islands and Southeast Alaska. No relationship between Hp and body condition was found. Lastly, a nonlinear relationship of plasma Hp and whole blood total mercury concentrations (THg) was observed in SSLs from the endangered western distinct population segment in Alaska. These results demonstrated that regional variation in Hp, especially in younger SSLs, may reflect regional differences in health and circulating THg.

REFERENCE RANGES AND AGE-RELATED AND DIVING EXERCISE EFFECTS ON HEMATOLOGY AND SERUM CHEMISTRY OF FEMALE STELLER SEA LIONS ( EUMETOPIAS JUBATUS).

Decreased health may have lowered the birth and survival rates of Steller sea lions ( Eumetopias jubatus) in the Gulf of Alaska and Aleutian Islands over the past 30 yr. Reference ranges for clinical hematology and serum chemistry parameters needed to assess the health of wild sea lion populations are limited. Here, blood parameters were serially measured in 12 captive female Steller sea lions ranging in age from 3 wk to 16 yr to establish baseline values and investigate age-related changes. Whether diving activity affects hematology parameters in animals swimming in the ocean compared with animals in a traditional aquarium setting was also examined. Almost all blood parameters measured exhibited significant changes with age. Many of the age-related changes reflected developmental life history changes, including a change in diet during weaning, an improvement of diving capacity, and the maturity of the immune system. Mean corpuscular hemoglobin and mean corpuscular volume were also higher in the ocean diving group compared with the aquarium group, likely reflecting responses to increased exercise regimes. These data provide ranges of hematology and serum chemistry values needed to evaluate and compare the health and nutritional status of captive and wild Steller sea lions.

Serum estradiol and progesterone profiles during estrus, pseudopregnancy, and active gestation in Steller sea lions.

While the proximate driver behind the decline of the Western stock of Steller sea lions (Eumetopias jubatus, >80% since 1970s) is likely multifactorial, the population reduction may have been powered by a decrease in fecundity. A harvest of Steller sea lions in the 1970s and 80s revealed a 30% reduction in the proportion of pregnant females from early (October-November) to late gestation (April-May). Identification and quantification of these reproductive failures are difficult when we lack species-specific data on endocrinology associated with discrete stages of the reproductive cycle (i.e., estrus, implantation, and gestation). We tracked changes in serum estradiol and progesterone in three adult female Steller sea lions from 2011 to 2015. In all years and most females, a discrete increase in estradiol was observed during the breeding season (June-August), indicative of estrus. Estradiol concentrations from October to May in a pregnant female compared to her corresponding values when non-pregnant did not consistently differ through gestation. An elevation in progesterone was observed in all females and all years beginning approximately in June and lasting through November. This likely results from progesterone production by the corpus luteum in both pregnant and pseudopregnant females. Serum progesterone shows promise as a diagnostic tool to identify pregnancy during months 3-5 (December-February) of the 8-month active gestation following embryonic implantation. This study provides ranges of key hormones during estrus, embryonic diapause/pseudopregnancy, and gestation in pregnant and non-pregnant females for studying reproduction in Steller sea lions.

Atypical Red Blood Cells Are Prevalent in California Sea Lion Pups Born during Anomalous Sea Surface Temperature Events.

To date, there is limited knowledge of the effects that abnormal sea surface temperature (SST) can have on the physiology of neonate pinnipeds. However, maternal nutritional deficiencies driven by alimentary restrictions would expectedly impact pinniped development and fitness, as an adequate supply of nutrients is essential for growth and proper functioning of all body systems, including red blood cell synthesis and clearance. Here, we investigated red blood cell morphology of California sea lion (CSL) pups from the San Benito Archipelago born during the 2014 and 2015 anomalously high SST events recorded in the northeastern Pacific Ocean. We examined whether atypical erythrocyte morphologies were more common in 2015, when the high SST event was more pronounced, and whether the stable isotope signature of pup fur, as an indicator of maternal feeding strategies, accounted for the number of atypical cells. Various atypical erythrocyte morphologies were more prevalent and more abundant than reference values. Evidence of iron deficiency was found in both years, and only pups born in 2014 showed evidence of active erythropoiesis. Microcytes and reticulocytes were more common in pups with higher isotopic δ13C and lower δ15N values, suggesting a probable relationship between maternal feeding strategies and the effect of climatic anomalies on red blood cell physiology of their pups. As developing pinnipeds require increased oxygen storage capacity for diving and foraging, the presence of atypical erythrocytes could be relevant to CSL pup fitness if the underlying cause is not reverted. This study is a first step to explore the effects that climatic alterations in the marine environment can have on the blood physiology of developing individuals.

Flipper stroke rate and venous oxygen levels in free-ranging California sea lions.

The depletion rate of the blood oxygen store, development of hypoxemia and dive capacity are dependent on the distribution and rate of blood oxygen delivery to tissues while diving. Although blood oxygen extraction by working muscle would increase the blood oxygen depletion rate in a swimming animal, there is little information on the relationship between muscle workload and blood oxygen depletion during dives. Therefore, we examined flipper stroke rate, a proxy of muscle workload, and posterior vena cava oxygen profiles in four adult female California sea lions (Zalophus californianus) during foraging trips at sea. Flipper stroke rate analysis revealed that sea lions minimized muscle metabolism with a stroke-glide strategy when diving, and exhibited prolonged glides during the descent of deeper dives (>100 m). During the descent phase of these deep dives, 55±21% of descent was spent gliding, with the longest glides lasting over 160 s and covering a vertical distance of 340 m. Animals also consistently glided to the surface from 15 to 25 m depth during these deeper dives. Venous hemoglobin saturation (SO2 ) profiles were highly variable throughout dives, with values occasionally increasing during shallow dives. The relationship between SO2 and flipper stroke rate was weak during deeper dives, while this relationship was stronger during shallow dives. We conclude that (1) the depletion of oxygen in the posterior vena cava in deep-diving sea lions is not dependent on stroke effort, and (2) stroke-glide patterns during dives contribute to a reduction of muscle metabolic rate.

A comparison of blood nitric oxide metabolites and hemoglobin functional properties among diving mammals.

The ability of marine mammals to hunt prey at depth is known to rely on enhanced oxygen stores and on selective distribution of blood flow, but the molecular mechanisms regulating blood flow and oxygen transport remain unresolved. To investigate the molecular mechanisms that may be important in regulating blood flow, we measured concentration of nitrite and S-nitrosothiols (SNO), two metabolites of the vasodilator nitric oxide (NO), in the blood of 5 species of marine mammals differing in their dive duration: bottlenose dolphin, South American sea lion, harbor seal, walrus and beluga whale. We also examined oxygen affinity, sensitivity to 2,3-diphosphoglycerate (DPG) and nitrite reductase activity of the hemoglobin (Hb) to search for possible adaptive variations in these functional properties. We found levels of plasma and red blood cells nitrite similar to those reported for terrestrial mammals, but unusually high concentrations of red blood cell SNO in bottlenose dolphin, walrus and beluga whale, suggesting enhanced SNO-dependent signaling in these species. Purified Hbs showed similar functional properties in terms of oxygen affinity and sensitivity to DPG, indicating that reported large variations in blood oxygen affinity among diving mammals likely derive from phenotypic variations in red blood cell DPG levels. The nitrite reductase activities of the Hbs were overall slightly higher than that of human Hb, with the Hb of beluga whale, capable of longest dives, having the highest activity. Taken together, these results underscore adaptive variations in circulatory NO metabolism in diving mammals but not in the oxygenation properties of the Hb.

Reference Intervals of Mineral Elements in Plasma of Anesthetized Free-Ranging Adult Females of South American Sea Lion, Otaria flavescens.

Levels of zinc (Zn), copper (Cu), potassium (K), calcium (Ca), magnesium (Mg), and phosphorus (P) in plasma of Otaria flavescens females (n = 29) were evaluated. Reference intervals were established for each element, being the first report for this species.

Seals and sea lions are what they eat, plus what? Determination of trophic discrimination factors for seven pinniped species.

RATIONALE: Mixing models are a common method for quantifying the contribution of prey sources to the diet of an individual using stable isotope analysis; however, these models rely upon a known trophic discrimination factor (hereafter, TDF) that results from fractionation between prey and animal tissues. Quantifying TDFs in captive animals is ideal, because diet is controlled and the proportional contributions and isotopic values of all prey items are known. METHODS: To calculate TDFs for the Hawaiian monk seal, northern elephant seal, bearded seal, ringed seal, spotted seal, harbor seal, and California sea lion, we obtained whiskers, serum, plasma, red blood cells, and prey items from nine captive individuals. We obtained δ(13) C and δ(15) N values using continuous-flow isotope-ratio mass spectrometry. The average δ(13) C and δ(15) N values from bulk and lipid-corrected prey from the diet were subtracted from the δ(13) C and δ(15) N values of each blood and whisker sample to calculate tissue-specific TDFs for each individual (∆(13) C or ∆(15) N). RESULTS: The ∆(13) C values ranged from +1.7 to +3.2‰ (bulk prey) and from +0.8 to +1.9‰ (lipid-corrected prey) for the various blood components, and from +3.9 to +4.6‰ (bulk prey) or +2.6 to +3.9‰ (lipid-corrected prey) for whiskers. The ∆(15) N values ranged from +2.2 to +4.3‰ for blood components and from +2.6 to +4.0‰ for whiskers. The TDFs tended to group by tissue, with whiskers having greater ∆(13) C values than blood components. In contrast, the ∆(15) N values were greater in serum and plasma than in red blood cells and whiskers. CONCLUSIONS: By providing the first TDF values for five seal species (family Phocidae) and one otariid species (family Otariidae), our study facilitates more accurate mixing models for these species. These values are particularly important for critically endangered Hawaiian monk seals and the three Arctic seal species (bearded, ringed, and spotted) that are faced with a rapidly changing environment.

ASSOCIATION BETWEEN POSITIVE CANINE HEARTWORM (DIROFILARIA IMMITIS) ANTIGEN RESULTS AND PRESENCE OF ACANTHOCHEILONEMA ODENDHALI MICROFILARIA IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

This study establishes a relationship between positive canine heartworm (Dirofilaria immitis) test results frequently observed in California sea lions (Zalophus californianus) and infection with the filarid nematode Acanthocheilonema odendhali. Four commercially available canine heartworm antigen tests were evaluated for cross-reaction with A. odendhali in California sea lions. Sera were tested from fifteen California sea lions with A. odendhali-associated microfilaremia, confirmed by blood smear, and with no evidence of D. immitis infection at necropsy. Ninety-five percent of tests were falsely positive for D. immitis. This study also determined that the prevalence of A. odendhali infection in stranded California sea lions from central California is approximately 23% by comparing the number of findings of mircofilaremia to the total number of California sea lions sampled at The Marine Mammal Center between 2005 and 2011, inclusive. Acanthocheilonema odenhali microfilaremia in California sea lions is likely to cross-react with canine heartworm antigen tests, and clinicians should interpret results with caution.

Dive, food, and exercise effects on blood microparticles in Steller sea lions (Eumetopias jubatus): exploring a biomarker for decompression sickness.

Recent studies of stranded marine mammals indicate that exposure to underwater military sonar may induce pathophysiological responses consistent with decompression sickness (DCS). However, DCS has been difficult to diagnose in marine mammals. We investigated whether blood microparticles (MPs, measured as number/µl plasma), which increase in response to decompression stress in terrestrial mammals, are a suitable biomarker for DCS in marine mammals. We obtained blood samples from trained Steller sea lions (Eumetopias jubatus, 4 adult females) wearing time-depth recorders that dove to predetermined depths (either 5 or 50 meters). We hypothesized that MPs would be positively related to decompression stress (depth and duration underwater). We also tested the effect of feeding and exercise in isolation on MPs using the same blood sampling protocol. We found that feeding and exercise had no effect on blood MP levels, but that diving caused MPs to increase. However, blood MP levels did not correlate with diving depth, relative time underwater, and presumed decompression stress, possibly indicating acclimation following repeated exposure to depth.

Long-lasting concentrations of cefovecin after subcutaneous and intramuscular administration to Patagonian sea lions (Otaria flavescens).

Cefovecin is a third-generation cephalosporin developed as an aqueous solution for use by the subcutaneous route in dogs and cats. This study evaluated the duration of cefovecin plasma concentrations after single intramuscular (IM) or subcutaneous (SC) injection at different doses in 10 Patagonian sea lions (Otaria flavescens). Blood samples were collected serially from the day of the injection up to 60-90 days post-injection. Plasma drug concentrations were determined by high performance liquid chromatography-UV detection and pharmacokinetic parameters were calculated by non-compartmental analysis. No reactions or side effects associated with the drug were observed in any of the studied animals. Both routes showed very similar pharmacokinetic behaviour. Elimination half-life (11.3-21.6 days, SC; 13.1-15.9 days, IM) and mean residence time (17.6-36.8 days SC; 16.5-25.4 days IM) were, in all cases and doses, considerably longer than those previously reported for any other species. Based on these findings, and preliminary data on specific pathogen sensitivity, cefovecin was found to be a very promising antimicrobial for Patagonian sea lions, in particular those that are difficult to access or that are under certain rehabilitation conditions.

Health Status of Galápagos Sea Lions (Zalophus wollebaeki) on San Cristóbal Island Rookeries Determined by Hematology, Biochemistry, Blood Gases, and Physical Examination.

The Galápagos sea lion, Zalophus wollebaeki, is an endemic and endangered species subject to population decline associated with environmental variability, such as El Niño events, constant feeding stress, and exposure to diseases through contact with introduced species. Reference blood parameter intervals have been published for some pinniped species, but baseline biochemical and blood gas values are lacking from Z. wollebaeki. We analyzed blood samples from 30 juvenile Galápagos sea lions (19 females, 11 males) captured in two rookeries on San Cristóbal Island. A portable blood analyzer (iSTAT) was used to obtain near-immediate field results for pH, partial pressure of O2, partial pressure of CO2, bicarbonate (HCO3(-)), hematocrit (Hct), hemoglobin, Na, K, ionized Ca, and glucose, and blood lactate was measured using a portable Lactate Plus(TM) analyzer. Average heart rate, biochemistry, and hematology parameters were comparable with healthy individuals of other pinniped species. Hemoglobin was significantly correlated with body condition of juvenile Galápagos sea lions. When compared with available blood values of clinically healthy California sea lions, Galápagos sea lions had higher total protein and Hct and lower Ca and K levels. Our results provide baseline data that may be useful in comparisons among populations and in detecting changes in health status among Galápagos sea lions.

PHARMACOKINETICS OF TRAMADOL HYDROCHLORIDE AND ITS METABOLITE O-DESMETHYLTRAMADOL FOLLOWING A SINGLE, ORALLY ADMINISTERED DOSE IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

Tramadol is a synthetic, centrally acting, opiate-like analgesic that is structurally related to codeine and morphine. The objective of this study was to determine the pharmacokinetics of tramadol hydrochloride and its major active metabolite O-desmethyltramadol (M1) in the California sea lion (Zalophus californianus). A single dose of tramadol was administered orally in fish at 2 mg/kg to a total of 15 wild California sea lions admitted for rehabilitation. Twenty-four total blood samples were collected post drug administration at 10, 20, 30, and 45 min and at 1, 3, 5, 6, 8, 12, and 24 hr. Blood plasma was separated and stored at -80°C until analysis with high-performance liquid chromatography was performed to determine levels of tramadol and M1, the major active metabolite. The results indicate that the plasma levels of parent tramadol are low or negligible during the first 30-45 min and then reach the predicted mean maximum plasma concentration of 358 ng/ml at 1.52 hr. The M1 metabolite was not detectable in 21 of 24 plasma samples, below the level of quantification of 5 ng/ml in one sample, and detectable at 11 and 17 ng/ml in two of the samples. This study suggests that a 2 mg/kg dose would need to be administered every 6-8 hr to maintain concentrations of tramadol above the minimum human analgesic level for mild to moderate pain. Based on dosing simulations, a dose of 4 mg/kg q8 hr or q12 hr, on average, may represent an adequate compromise, but further studies are needed using a larger sample size. Pharmacodynamic studies are warranted to determine if tramadol provides analgesic effects in this species. The potential for tramadol toxicosis at any dose also has not been determined in this species.

PHARMACOKINETICS OF SINGLE-DOSE ORALLY ADMINISTERED CIPROFLOXACIN IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

Ciprofloxacin is commonly selected for clinical use due to its broad-spectrum efficacy and is a frequently administered antibiotic at The Marine Mammal Center, a marine mammal rehabilitation facility. Ciprofloxacin is used for treatment of California sea lions ( Zalophus californianus ) suffering from a variety of bacterial infections at doses extrapolated from other mammalian species. However, as oral absorption is variable both within and across species, a more accurate determination of appropriate dosage is needed to ensure effective treatment and avoid emergence of drug-resistant bacterial strains. A pharmacokinetic study was performed to assess plasma concentrations of ciprofloxacin in California sea lions after a single oral dose. Twenty healthy California sea lions received a single 10-mg/kg oral dose of ciprofloxacin administered in a herring fish. Blood was then collected at two of the following times from each individual: 0.5, 0.75, 1, 2, 4, 8, 10, 12, 18, and 24 hr postingestion. Plasma ciprofloxacin concentration was assessed via high-performance liquid chromatography. A population pharmacokinetics model demonstrated that an oral ciprofloxacin dose of 10 mg/kg achieved an area under the concentration vs. time curve of 6.01 µg hr/ml. Absorption was rapid, with ciprofloxacin detectable in plasma 0.54 hr after drug administration; absorption half-life was 0.09 hr. A maximum plasma concentration of 1.21 µg/ml was observed at 1.01 hr, with an elimination half-life of 3.09 hr. Ciprofloxacin administered orally at 10 mg/kg produced therapeutic antibacterial exposure for only some of the most susceptible bacterial organisms commonly isolated from California sea lions.

Health assessment of free-ranging endangered Australian sea lion (Neophoca cinerea) pups: effect of haematophagous parasites on haematological parameters.

Evaluation of the health status of free-ranging populations is important for understanding the impact of disease on individuals and on population demography and viability. In this study, haematological reference intervals were developed for free-ranging endangered Australian sea lion (Neophoca cinerea) pups within the context of endemic hookworm (Uncinaria sanguinis) infection and the effects of pathogen, host, and environment factors on the variability of haematological parameters were investigated. Uncinaria sanguinis was identified as an important agent of disease, with infection causing regenerative anaemia, hypoproteinaemia, and a predominantly lymphocytic-eosinophilic systemic inflammatory response. Conversely, the effects of sucking lice (Antarctophthirus microchir) were less apparent and infestation in pups appears unlikely to cause clinical impact. Overall, the effects of U. sanguinis, A. microchir, host factors (standard length, body condition, pup sex, moult status, and presence of lesions), and environment factors (capture-type and year of sampling) accounted for 26-65% of the total variance observed in haematological parameters. Importantly, this study demonstrated that anaemia in neonatal Australian sea lion pups is not solely a benign physiological response to host-environment changes, but largely reflects a significant pathological process. This impact of hookworm infection on pup health has potential implications for the development of foraging and diving behaviour, which would subsequently influence the independent survival of juveniles following weaning. The haematological reference intervals developed in this study can facilitate long-term health surveillance, which is critical for the early recognition of changes in disease impact and to inform conservation management.

A nutrigenomic approach to detect nutritional stress from gene expression in blood samples drawn from Steller sea lions.

Gene expression profiles are increasingly being used as biomarkers to detect the physiological responses of a number of species to disease, nutrition, and other stressors. However, little attention has been given to using gene expression to assess the stressors and physiological status of marine mammals. We sought to develop and validate a nutrigenomic approach to quantify nutritional stress in Steller sea lions (Eumetopias jubatus). We subjected 4 female Steller sea lions to 3 feeding regimes over 70-day trials (unrestricted food intake, acute nutritional stress, and chronic nutritional stress), and drew blood samples from each animal at the end of each feeding regime. We then extracted the RNA of white blood cells and measured the response of 8 genes known to react to diet restriction in terrestrial mammals. Overall, we found that the genomic response of Steller sea lions experiencing nutritional stress was consistent with how terrestrial mammals respond to dietary restrictions. Our nutritionally stressed sea lions down-regulated some cellular processes involved in immune response and oxidative stress, and up-regulated pro-inflammatory responses and metabolic processes. Nutrigenomics appears to be a promising means to monitor nutritional status and contribute to mitigation measures needed to assist in the recovery of Steller sea lions and other at-risk species of marine mammals.

Steller sea lions (Eumetopias jubatus) have greater blood volumes, higher diving metabolic rates and a longer aerobic dive limit when nutritionally stressed.

Marine mammal foraging behaviour inherently depends on diving ability. Declining populations of Steller sea lions may be facing nutritional stress that could affect their diving ability through changes in body composition or metabolism. Our objective was to determine whether nutritional stress (restricted food intake resulting in a 10% decrease in body mass) altered the calculated aerobic dive limit (cADL) of four captive sea lions diving in the open ocean, and how this related to changes in observed dive behaviour. We measured diving metabolic rate (DMR), blood O2 stores, body composition and dive behaviour prior to and while under nutritional restriction. We found that nutritionally stressed sea lions increased the duration of their single long dives, and the proportion of time they spent at the surface during a cycle of four dives. Nutritionally stressed sea lions lost both lipid and lean mass, resulting in potentially lower muscle O2 stores. However, total body O2 stores increased due to rises in blood O2 stores associated with having higher blood volumes. Nutritionally stressed sea lions also had higher mass-specific metabolic rates. The greater rise in O2 stores relative to the increase in mass-specific DMR resulted in the sea lions having a longer cADL when nutritionally stressed. We conclude that there was no negative effect of nutritional stress on the diving ability of sea lions. However, nutritional stress did lower foraging efficiency and require more foraging time to meet energy requirements due to increases in diving metabolic rates and surface recovery times.

Proteomic Analysis of Plasma from California Sea Lions (Zalophus californianus) Reveals Apolipoprotein E as a Candidate Biomarker of Chronic Domoic Acid Toxicosis.

Domoic acid toxicosis (DAT) in California sea lions (Zalophus californianus) is caused by exposure to the marine biotoxin domoic acid and has been linked to massive stranding events and mortality. Diagnosis is based on clinical signs in addition to the presence of domoic acid in body fluids. Chronic DAT further is characterized by reoccurring seizures progressing to status epilepticus. Diagnosis of chronic DAT is often slow and problematic, and minimally invasive tests for DAT have been the focus of numerous recent biomarker studies. The goal of this study was to retrospectively profile plasma proteins in a population of sea lions with chronic DAT and those without DAT using two dimensional gel electrophoresis to discover whether individual, multiple, or combinations of protein and clinical data could be utilized to identify sea lions with DAT. Using a training set of 32 sea lion sera, 20 proteins and their isoforms were identified that were significantly different between the two groups (p<0.05). Interestingly, 11 apolipoprotein E (ApoE) charge forms were decreased in DAT samples, indicating that ApoE charge form distributions may be important in the progression of DAT. In order to develop a classifier of chronic DAT, an independent blinded test set of 20 sea lions, seven with chronic DAT, was used to validate models utilizing ApoE charge forms and eosinophil counts. The resulting support vector machine had high sensitivity (85.7% with 92.3% negative predictive value) and high specificity (92.3% with 85.7% positive predictive value). These results suggest that ApoE and eosinophil counts along with machine learning can perform as a robust and accurate tool to diagnose chronic DAT. Although this analysis is specifically focused on blood biomarkers and routine clinical data, the results demonstrate promise for future studies combining additional variables in multidimensional space to create robust classifiers.