LIVER ULTRASONOGRAPHY IN DOLPHINS: USE OF ULTRASONOGRAPHY TO ESTABLISH A TECHNIQUE FOR HEPATOBILIARY IMAGING AND TO EVALUATE METABOLIC DISEASE-ASSOCIATED LIVER CHANGES IN BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS).

The objective of this study was to establish a comprehensive technique for ultrasound examination of the dolphin hepatobiliary system and apply this technique to 30 dolphins to determine what, if any, sonographic changes are associated with blood-based indicators of metabolic syndrome (insulin greater than 14 µIU/ml or glucose greater than 112 mg/dl) and iron overload (transferrin saturation greater than 65%). A prospective study of individuals in a cross-sectional population with and without elevated postprandial insulin levels was performed. Twenty-nine bottlenose dolphins ( Tursiops truncatus ) in a managed collection were included in the final data analysis. An in-water ultrasound technique was developed that included detailed analysis of the liver and pancreas. Dolphins with hyperinsulinemia concentrations had larger livers compared with dolphins with nonelevated concentrations. Using stepwise, multivariate regression including blood-based indicators of metabolic syndrome in dolphins, glucose was the best predictor of and had a positive linear association with liver size (P = 0.007, R2 = 0.24). Bottlenose dolphins are susceptible to metabolic syndrome and associated complications that affect the liver, including fatty liver disease and iron overload. This study facilitated the establishment of a technique for a rapid, diagnostic, and noninvasive ultrasonographic evaluation of the dolphin liver. In addition, the study identified ultrasound-detectable hepatic changes associated primarily with elevated glucose concentration in dolphins. Future investigations will strive to detail the pathophysiological mechanisms for these changes.

Novel Atlantic bottlenose dolphin parainfluenza virus TtPIV-1 clusters with bovine PIV-3 genotype B strains.

Parainfluenza virus 3 (PIV-3) is a common viral infection not only in humans, but also in many other species. Serological evidence suggests that nearly 100 % of children in the United States have been infected with PIV-3 by 5 years of age. Similarly, in cattle, PIV-3 is commonly associated with bovine respiratory disease complex. A novel dolphin PIV-3 (TtPIV-1) was described by Nollens et al. in 2008 from a dolphin that was diagnosed with an unknown respiratory illness. At that time, TtPIV-1 was found to be most similar to, but distinct from, bovine PIV-3 (BPIV-3). In the present study, similar viral growth kinetics and pro-inflammatory cytokine (IL-1ß, IL-6, and CXCL8) production were seen between BPIV-3 and TtPIV-1 in BEAS-2B, MDBK, and Vero cell lines. Initial nomenclature of TtPIV-1 was based on partial sequence of the fusion and RNA polymerase genes. Based on the similarities we saw with the in vitro work, it was important to examine the TtPIV-1 genome in more detail. Full genome sequencing and subsequent phylogenetic analysis revealed that all six viral genes of TtPIV-1 clustered within the recently described BPIV-3 genotype B strains, and it is proposed that TtPIV-1 be re-classified with BPIV-3 genotype B strains.

Fetal omphalocele in a common bottlenose dolphin (Tursiops truncatus).

A routine pregnancy ultrasound examination of a 30-yr-old, multiparous, common bottlenose dolphin, Tursiops truncatus, detected an approximately 16-wk (gestational age) fetus with an omphalocele, an abdominal wall defect at the base of the umbilical cord. Throughout the pregnancy, ultrasound allowed for identification of the omphalocele contents, which included a portion of the liver and intestinal loops. The maximum diameter of the omphalocele was 11.4 cm at an estimated 51-wk gestation. Color Doppler was utilized to study the blood flow within the omphalocele as well as diagnose an associated anomaly of the umbilical cord, which contained three vessels instead of four. Gross necropsy and histopathology confirmed the ultrasound diagnoses. This is the first report of an omphalocele in a T. truncatus fetus, and the first report of a fetal and umbilical cord anomaly diagnosed with ultrasound in a cetacean.

Serologic response in bottlenose dolphins Tursiops truncatus infected with Brucella sp. using a dolphin-specific indirect ELISA.

Marine-origin Brucella infections and serologic evidence of exposure have been documented in multiple cetacean species. A dolphin-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to screen bottlenose dolphin sera for anti-Brucella antibodies. A total of 131 serum samples collected over a 2 to 18 yr period from 6 bottlenose dolphins Tursiops truncatus with confirmed Brucella infections were analyzed for the presence and magnitude of antibody titers against marine-origin Brucella to compare individual antibody responses to various disease manifestations. Additionally, an epidemiologic serologic survey of a managed population of 64 bottlenose dolphins was performed to evaluate for the presence of antibodies and to determine whether there were any clinical pathology predictors for exposure or infection. The serologic results revealed that the dolphins with Brucella-associated abortions were seronegative for 7 to 18 yr until after the abortion and maintained positive titers for several years, with 2 of 3 animals returning to seronegative status. In contrast, the dolphins with Brucella-associated pulmonary or bone lesions maintained persistent positive titers for 2 to 18 yr. The population serosurvey revealed no significant differences in antibody levels among males and females, and dolphins between the ages of 17 and 25 yr were 6.8 times more likely to be Brucella antibody positive compared to those that were younger or older. Seropositive dolphins did not have significant inflammation compared to seronegative dolphins but were more likely to have higher levels of aspartate aminotransferase and gamma-glutamyl transpeptidase. Among 16 dolphins that tested seropositive, 13 (81.3%) had previously been seropositive for at least 3 to 5 yr.

Broader and safer clinically-relevant activities of pentadecanoic acid compared to omega-3: Evaluation of an emerging essential fatty acid across twelve primary human cell-based disease systems.

A growing body of evidence supports that pentadecanoic acid (C15:0), an odd-chain saturated fat found in butter, is an essential fatty acid that is necessary in the diet to support long-term metabolic and heart health. Here, dose dependent and clinically relevant cell-based activities of pure C15:0 (FA15TM) were compared to eicosapentaenoic acid (EPA), a leading omega-3 fatty acid, as well as to an additional 4,500 compounds. These studies included 148 clinically relevant biomarkers measured across 12 primary human cell systems, mimicking various disease states, that were treated with C15:0 at four different concentrations (1.9 to 50 µM) and compared to non-treated control systems. C15:0 was non-cytotoxic at all concentrations and had dose dependent, broad anti-inflammatory and antiproliferative activities involving 36 biomarkers across 10 systems. In contrast, EPA was cytotoxic to four cell systems at 50 µM. While 12 clinically relevant activities were shared between C15:0 and EPA at 17 µM, C15:0 had an additional 28 clinically relevant activities, especially anti-inflammatory, that were not present in EPA. Further, at 1.9 and 5.6 µM, C15:0 had cell-based properties similar to bupropion (Pearson's scores of 0.78), a compound commonly used to treat depression and other mood disorders. At 5.6 µM, C15:0 mimicked two antimicrobials, climabazole and clarithromycin (Pearson's scores of 0.76 and 0.75, respectively), and at 50 µM, C15:0 activities matched that of two common anti-cancer therapeutics, gemcitabine and paclitaxel (Pearson's scores of 0.77 and 0.74, respectively). In summary, C15:0 had dose-dependent and clinically relevant activities across numerous human cell-based systems that were broader and safer than EPA, and C15:0 activities paralleled common therapeutics for mood disorders, microbial infections, and cancer. These studies further support the emerging role of C15:0 as an essential fatty acid.

Evaluation of population health among bottlenose dolphins (Tursiops truncatus) at the United States Navy Marine Mammal Program.

OBJECTIVE: To evaluate health indicators for a population of bottlenose dolphins in the US Navy Marine Mammal Program (MMP) by use of data acquired from 1988 through 2007. DESIGN: Retrospective cohort study. ANIMALS: 167 bottlenose dolphins. PROCEDURES: The following indicators were used to evaluate the health of dolphins during the 20-year period: 5-year age structure, median survival age, annual survival rates, mortality rates, and neonatal and calf survival and mortality rates. Limitations of these population measurements as health indicators for dolphins were assessed. RESULTS: Crude mortality rates of dolphins for 1988 through 1992, 1993 through 1997, 1998 through 2002, and 2003 through 2007 were 3.1%, 4.7%, 3.6%, and 2.4%, respectively; during these same 4 study periods, median survival ages were 14.3, 14.4, 17.7, and 26.1 years, respectively, and mean survival rates were 0.98, 0.97, 0.97, and 0.99, respectively. From 1988 through 1997, 1998 through 2002, and 2003 through 2007, neonatal mortality rates were 4 of 16, 5 of 20, and 2 of 14 neonates, respectively. During these 3 study periods, mean annual survival rates for calves < 3 years old (excluding neonates that died at < 30 days old) were 0.97, 0.92, and 0.99, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Although there were limitations to the measurement of some health indicators, use of multiple methods indicated that the health of dolphins in the MMP population was comparable to, if not better than, that published for other dolphin populations. The MMP population of dolphins may provide useful reference values of health indicators for use in assessment of other managed dolphin populations.

Use of phlebotomy treatment in Atlantic bottlenose dolphins with iron overload.

CASE DESCRIPTION: 3 adult (24- to 43-year-old) Atlantic bottlenose dolphins (Tursiops truncatus) with chronic episodic malaise and inappetence associated with high serum aminotransferase (alanine aminotransferase and aspartate aminotransferase) activities, high serum iron concentration, and serum transferrin saturation > 80% were evaluated. CLINICAL FINDINGS: Results of histologic examination of liver biopsy specimens revealed hemosiderosis in all 3 dolphins. Except for chronic lymphocytosis in 1 dolphin, results of extensive diagnostic testing revealed no other abnormalities. For each dolphin, a diagnosis of iron overload of unknown origin was made. TREATMENT AND OUTCOME: Phlebotomy treatment was implemented to reduce body stores of iron. Each phlebotomy procedure removed 7% to 17% (1 to 3 L) of estimated blood volume. Treatment consisted of an induction phase of weekly phlebotomy procedures for 22 to 30 weeks, which was complete when serum iron concentration and aminotransferase activities were within reference ranges and serum transferrin saturation was < or = 20% or Hct was < or = 30%. Total amount of iron removed from each dolphin was 53 to 111 mg/kg (24.1 to 50.5 mg/lb) of body weight. One dolphin required maintenance procedures at 8- to 12-week intervals when high serum iron concentration was detected. CLINICAL RELEVANCE: Although the cause of the iron overload and high serum aminotransferase activities remained unknown, phlebotomy treatment successfully resolved the clinicopathologic abnormalities, supporting a role of iron overload in the hepatopathy of the 3 dolphins.

Development and testing of species-specific ELISA assays to measure IFN-γ and TNF-α in bottlenose dolphins (Tursiops truncatus).

Monitoring the immune status of cetaceans is important for a variety of health conditions. Assays to quantify cytokines, especially pro-inflammatory cytokines, could be employed, in addition to currently available diagnostic assays, to screen for alterations in the health status of an animal. Though a number of immunological assays are readily available for humans and mice, specific assays for many veterinary species, including cetaceans such as bottlenose dolphins (Tursiops truncatus), are more limited. Herein, we describe the development of IFN-gamma (IFN-γ) and TNF-alpha (TNF-α) enzyme-linked immunosorbent assays (ELISAs) specific to bottlenose dolphins. Utilizing these assays, we monitored the immune status of bottlenose dolphins from a managed population over a period of eleven months. The ELISA assays developed for bottlenose dolphins were used to measure IFN-γ and TNF-α in serum or in culture supernatants from peripheral blood mononuclear cells (PBMCs) stimulated with varying concentrations of mitogens concanavalin A (ConA) or phytohemagglutinin (PHA). Induction of TNF-α in PBMC cultures was consistently highest with 1 µg/mL ConA, while 1 µg/mL PHA induced the highest secretion of IFN-γ. Serum levels of TNF-α and IFN-γ remained relatively constant for each animal over the time period examined. CBC and plasma chemistry variables measured concurrently in the bottlenose dolphins were then examined as independent predictors of cytokine levels. We found these clinical variables were more likely to predict linear changes in serum IFN-γ and TNF-α levels compared to concentrations of these cytokines in mitogen-stimulated PBMC culture supernatants. Cytokine assays developed will be of substantial benefit in monitoring bottlenose dolphin health as an adjunct to currently available diagnostic tests.

Big brains and blood glucose: common ground for diabetes mellitus in humans and healthy dolphins.

Healthy Atlantic bottlenose dolphins (Tursiops truncatus) have a sustained postprandial hyperglycemia, producing a prolonged glucose tolerance curve and a transient, diabetes mellitus-like state during 6 to 72 h of fasting. To further assess dolphins as comparative models for diabetes in humans, we hypothesized that a suite of hematological and clinical biochemistry changes during the fasting state may mimic those reported in humans with diabetes. We conducted a retrospective analysis of covariance to compare fasting and nonfasting hematologic and serum biochemical data, including 1161 routine blood samples from 52 healthy bottlenose dolphins (age, 1 to 49 y; male and female) collected during 1998 through 2005. Most changes found in dolphins during the fasting state--including significantly increased glucose, platelets, gamma-glutamyl transpeptidase, and alkaline phosphatase; significantly decreased serum uric acid; and shifts toward a metabolic acidodic state (significantly increased blood CO2)--have been previously associated with diabetes mellitus in humans. Therefore, healthy bottlenose dolphins may be the first complete and natural comparative animal model for diabetes mellitus in humans. Similarities between dolphins and humans, including metabolic changes associated with high-protein, low-carbohydrate diets; large brain-to-mass ratios; high central nervous system demands for glucose; and similarly unique blood glucose-carrying capacities should be further assessed to better understand the potential evolutionary paths of diabetes mellitus in these 2 species.

Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR.

Both veterinarians caring for dolphins in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin (Tursiops truncatus) health. Quantitative PCR (qPCR) can be used to assess cytokine transcription patterns of peripheral blood mononuclear cells (PBMC). This can supplement currently available blood tests with information on immune status. Full realization of this potential requires establishment of normal ranges of cytokine gene transcription levels in bottlenose dolphins. We surveyed four dolphins over the span of seven months by serial bleeds. PBMC were stimulated with phytohaemagglutinin (1, 5, and 10 µg/mL) and concanavalin A (1 µg/mL) for 48 H in vitro. RNA from these cultures was probed by qPCR using Tursiops truncatus-specific primers (IL-1α, IL-1ß, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-18, IFN-γ and TNF-α). Two blood samples from an additional bottlenose dolphin diagnosed with acute pulmonary disease add further perspective to the data. We observed that mitogen choice made a significant difference in the magnitude of gene transcription observed. On the other hand, most cytokines tested exhibited limited intra-animal variation. However, IL-6 and IL-12p40 differed between older and younger dolphins. Furthermore, the magnitude of mitogenic response clusters the tested cytokines into three groups. The data provide a reference for the selection of target cytokine mRNAs and their expected range of mitogen-stimulated cytokine gene transcription for future studies.

Identification of monoclonal antibodies cross-reactive with bottlenose dolphin orthologues of the major histocompatibility complex and leukocyte differentiation molecules.

The slow progress in understanding immunotoxic effects of environmental contaminants and their influence on disease susceptibility in whales is largely due to the limited information available on the immune systems and immune function of species included in the Cetancodontamorpha clade. Studies in species in the other major clades included in the Artiodactylamorpha, Ruminantiamorpha, Suinamorpha, and Camelidamorpha have revealed the immune systems are similar, but not identical. The present study was undertaken to expand the available monoclonal antibody reagents needed to gain insight into the composition, function, and evolution of the immune system in Cetancodontamorpha, using the dolphin (Tursiops truncatus) as a model cetacean species. Screening of a set of mAbs that recognize highly conserved epitopes expressed on the major histocompatibility complex (MHC) and leukocyte differentiation molecules (LDMs) in cattle by flow cytometry revealed some of the mAbs recognize epitopes conserved on dolphin orthologues of MHC class I, MHC class II, CD11a, CD14, CD16, CD18, CD163 and CD172a. Comparison of the amino acid sequences of dolphin and bovine orthologues revealed limited changes in sequence have occurred during speciation, suggesting an approach for developing cross-reactive mAbs for use in cetacean research.

Evaluation of annual survival and mortality rates and longevity of bottlenose dolphins (Tursiops truncatus) at the United States Navy Marine Mammal Program from 2004 through 2013.

Objective-To evaluate annual survival and mortality rates and the longevity of a managed population of bottlenose dolphins (Tursiops truncatus). Design-Retrospective cohort study. Animals-103 bottlenose dolphins at the US Navy Marine Mammal Program (MMP). Procedures-Population age structures, annual survival and crude mortality rates, and median age at death for dolphins > 30 days old were determined from 2004 through 2013. Results-During 2004 through 2013, the annual survival rates for MMP dolphins ranged from 0.98 to 1.0, and the annual crude mortality rates ranged from 0% to 5%, with a mean of 2.7%. The median age at death was 30.1 years from 2004 through 2008 and increased to 32 years from 2009 through 2013. The maximum age for a dolphin in the study was 52 years. Conclusions and Clinical Relevance-Results indicated that the annual mortality rates were low and survival rates were high for dolphins in the MMP from 2004 through 2013 and that the median age at death for MMP dolphins during that time was over 10 years greater than that reported in free-ranging dolphins. These findings were likely attributable to the continually improving care and husbandry of managed dolphin populations.

Increased Dietary Intake of Saturated Fatty Acid Heptadecanoic Acid (C17:0) Associated with Decreasing Ferritin and Alleviated Metabolic Syndrome in Dolphins.

Similar to humans, bottlenose dolphins (Tursiops truncatus) can develop metabolic syndrome and associated high ferritin. While fish and fish-based fatty acids may protect against metabolic syndrome in humans, findings have been inconsistent. To assess potential protective factors against metabolic syndrome related to fish diets, fatty acids were compared between two dolphin populations with higher (n = 30, Group A) and lower (n = 19, Group B) mean insulin (11 ± 12 and 2 ± 5 µIU/ml, respectively; P < 0.0001) and their dietary fish. In addition to higher insulin, triglycerides, and ferritin, Group A had lower percent serum heptadecanoic acid (C17:0) compared to Group B (0.3 ± 0.1 and 1.3 ± 0.4%, respectively; P < 0.0001). Using multivariate stepwise regression, higher percent serum C17:0, a saturated fat found in dairy fat, rye, and some fish, was an independent predictor of lower insulin in dolphins. Capelin, a common dietary fish for Group A, had no detectable C17:0, while pinfish and mullet, common in Group B's diet, had C17:0 (41 and 67 mg/100g, respectively). When a modified diet adding 25% pinfish and/or mullet was fed to six Group A dolphins over 24 weeks (increasing the average daily dietary C17:0 intake from 400 to 1700 mg), C17:0 serum levels increased, high ferritin decreased, and blood-based metabolic syndrome indices normalized toward reference levels. These effects were not found in four reference dolphins. Further, higher total serum C17:0 was an independent and linear predictor of lower ferritin in dolphins in Group B dolphins. Among off the shelf dairy products tested, butter had the highest C17:0 (423mg/100g); nonfat dairy products had no detectable C17:0. We hypothesize that humans' movement away from diets with potentially beneficial saturated fatty acid C17:0, including whole fat dairy products, could be a contributor to widespread low C17:0 levels, higher ferritin, and metabolic syndrome.

Development and application of specific cytokine assays in tissue samples from a bottlenose dolphin with hyperinsulinemia.

Chronic inflammation has been associated with insulin resistance and type 2 diabetes (T2D) in humans. Postmortem hepatic and splenic tissue from a 46-year-old geriatric male bottlenose dolphin (Tursiops truncatus) with insulin resistance (chronic hyperinsulinemia with hyperglycemia), chronic inflammation (white blood cell count greater than 12,000 cells/µL), and mild fatty liver disease was evaluated for elevated pro-inflammatory mediators. Cytokine mRNA expression in postmortem hepatic and splenic tissue, as determined by real-time PCR, included an array of cytokines: TGF-ß, TNF-α, IFN-γ, IL-2, IL-4, IL-10, IL-12p40, IL-13, and IL-18. Values from this dolphin were compared to a younger reference dolphin with no known chronic metabolic perturbations or inflammation. Levels of TGF-ß, TNF-α, and IL-4 were higher in the case dolphin's liver compared to that of the reference dolphin. In the case dolphin's spleen, IL-10 and IFN-γ mRNA was upregulated while IL-4 was less than the reference dolphin. IL-18 and IL-13 were upregulated in both tissues. Fluorescent immunohistochemistry (IHC) utilized the following antibodies: anti-porcine IL-6, anti-bovine IFN-γ, IL-4, and IL-10, anti-human TGF-ß, anti-ovine IL-1ß, and anti-dolphin IL-8. Fluorescent IHC in spleen from the case dolphin revealed staining of IL-4, IL-6, IL-8, and TGF-ß throughout the tissue. IL-10 and IFN-γ were seen to predominate in areas surrounding the follicles of splenic tissue. This is the first characterization of cytokine levels in dolphin hepatic and splenic tissue. While there are limitations to a case study, this report of inflammatory biomarkers in tissues of a dolphin with insulin resistance and fatty liver disease are similar to those observed in human patients.

Characterization of California sea lion polyomavirus 1: expansion of the known host range of the Polyomaviridae to Carnivora.

The genome of a novel polyomavirus first identified in a proliferative tongue lesion of a California sea lion (Zalophus californianus) is reported. This is only the third described polyomavirus of laurasiatherian mammals, is the first of the three associated with a lesion, and is the first known polyomavirus of a host in the order Carnivora. Predicted large T, small t, VP1, VP2, and VP3 genes were identified based on homology to proteins of known polyomaviruses, and a putative agnoprotein was identified based upon its location in the genome. Phylogenetic analysis of the predicted late region proteins found that the laurasiatherian polyomaviruses, together with Squirrel monkey polyomavirus and Murine pneumotropic virus, form a monophyletic clade. Phylogenetic analysis of the early region was more ambiguous. The noncoding control region of California sea lion polyomavirus 1 is unusual in that only two apparent large T binding sites are present; this is less than any other known polyomavirus. The VP1 of this virus has an unusually long carboxy-terminal region. A quantitative polymerase chain reaction was developed and utilized on various samples from 79 additional animals from either managed or wild stranded California sea lion populations, and California sea lion polyomavirus 1 infection was found in 24% of stranded animals. Sequence of additional samples identified four sites of variation in the t antigens, three of which resulted in predicted coding changes.

Characterization of San Miguel sea lion virus populations using pyrosequencing-based methods.

San Miguel Sea Lion Virus (SMSV) is a small RNA virus in the genus Vesivirus with an unusually broad host range. Three populations of SMSV were examined by PCR amplification of the capsid precursor and putative helicase genes, followed by pyrosequencing. The populations were nasal swabs from two SMSV infected California sea lions (Zalophus californianus) from two different years, and a virus isolate from the earlier swab that was passaged in cell culture five times. In the capsid precursor, extensive deletions were prevalent in the passaged virus but uncommon in the clinical samples. A greater prevalence of point mutations was seen in the capsid precursor gene than in the putative helicase gene. In culture, the minority sequence in the capsid precursor at nucleotide position 5826 rapidly shifted after five passages to become the majority sequence. Levels of diversity at individual sites showed much more similarity between the two clinical samples than between the earlier clinical sample and the passaged culture from the same sample. SMSV appears to behave as a quasispecies. Assessment of original patient samples is preferable for understanding clinical SMSV populations.

Hypocitraturia in common bottlenose dolphins (Tursiops truncatus): assessing a potential risk factor for urate nephrolithiasis.

Numerous cases of urate nephrolithiasis in managed collections of common bottlenose dolphins (Tursiops truncatus) have been reported, but nephrolithiasis is believed to be uncommon in wild dolphins. Risk factors for urate nephrolithiasis in humans include low urinary pH and hypocitraturia. Urine samples from 94 dolphins were collected during April 2006 through June 2009 from 4 wild populations (n = 62) and 4 managed collections (n = 32). In addition, urine uric acid and pH were tested in a subset of these animals. Our null hypothesis was that wild and managed collection dolphins would have no significant differences in urinary creatinine, citrate, and uric acid concentrations and pH. Among urine samples from all 94 dolphins, the urinary levels (mean +/- SEM) for creatinine, citrate, uric acid, and pH were 139 +/- 7.6 mg/dL, 100 +/- 20 mg citrate/g creatinine, 305 +/- 32 mg uric acid/g creatinine, and 6.2 +/- 0.05, respectively. Of the 4 urinary variables, only citrate concentration varied significantly between the 2 primary study groups; compared with wild dolphins, managed collection dolphins were more likely to have undetectable levels of citrate in the urine (21.0% and 81.3%, respectively). Mean urinary citrate concentrations for managed collection and wild dolphin populations were 2 and 150 mg citrate/g creatinine, respectively. We conclude that some managed collections of dolphins, like humans, may be predisposed to urate nephrolithiasis due to the presence of hypocitraturia. Subsequent investigations can include associations between metabolic syndrome, hypocitraturia, and urate nephrolithiasis in humans and dolphins; and the impact of varying levels of seawater ingestion on citrate excretion.