Health reference intervals and values for common bottlenose dolphins (Tursiops truncatus), Indo-Pacific bottlenose dolphins (Tursiops aduncus), Pacific white-sided dolphins (Lagenorhynchus obliquidens), and beluga whales (Delphinapterus leucas).

This study reports comprehensive clinical pathology data for hematology, serum, and plasma biochemistry reference intervals for 174 apparently healthy common bottlenose dolphins (Tursiops truncatus) and reference values for 27 Indo-Pacific bottlenose dolphins (Tursiops aduncus), 13 beluga whales (Delphinapterus leucas), and 6 Pacific white-sided dolphins (Lagenorhynchus obliquidens) in zoos and aquariums accredited by the Alliance for Marine Mammal Parks and Aquariums and the Association of Zoos & Aquariums. Blood samples were collected as part of a larger study titled "Towards understanding the welfare of cetaceans in zoos and aquariums" (colloquially called the Cetacean Welfare Study). Two blood samples were collected following a standardized protocol, and two veterinarian examinations were conducted approximately six months apart between July to November 2018 and January to April 2019. Least square means, standard deviations, and 95% confidence intervals were calculated for hematology, serum, and plasma biochemical variables. Comparisons by age, gender, and month revealed statistically significant differences (p < 0.01) for several variables. Reference intervals and values were generated for samples tested at two laboratories for up to 56 hematologic, serum, and plasma biochemical variables. To apply these data, ZooPhysioTrak, an iOS mobile software application, was developed to provide a new resource for cetacean management. ZooPhysioTrak provides species-specific reference intervals and values based on user inputs of individual demographic and sample information. These data provide a baseline from which to compare hematological, serum, and plasma biochemical values in cetaceans in zoos and aquariums.

Hematologic profile of Amazon river dolphins Inia geoffrensis and its variation during acute capture stress.

Hematological values are of primary importance when investigating the health and physiological status of populations as they reflect the biological equilibrium of aquatic ecosystems. The objectives of this study are to produce baseline values for hematological parameters of the Amazon River dolphin (Inia geoffrensis), as well as to investigate significant variations according to sex, age, reproductive status and stress level. One-hundred-and-ten dolphins from Mamirauá Sustainable Development Reserve (3°3'S, 64°51'W), Central Amazon, Brazil, were live captured and sampled in November 2004 and 2005. Further, the means, standard deviations, minimum and maximum values and reference values (90% CI) were calculated. Correlations were performed to assess the relationships among blood values and cardiac rate (CR), respiratory frequency (RF), handling time and level of stress. No significant differences were found between sexes. Also, no differences occurred among pregnant and non-pregnant females, pregnant females and adult males or non-pregnant females and adult males. Calves had a higher white blood cell (WBC) count, and the neutrophil and lymphocyte absolute counts were significantly higher in calves than adults. The level of stress determined by empirical observation positively correlated with the WBC, neutrophil, lymphocyte and monocyte absolute counts and CR and RF. It was found that less stressed animals tend to present lower platelet counts and lower CR. The handling time of the dolphins was positively correlated with hematocrit (Hct), red blood cells (RBC) and Hb level. The hematological and physiological parameters varied according to time of handling and proved to be a good bioindicator of acute stress in Amazon River dolphins. The data provided here can complement long-term monitoring and identify the early warning indicators of health problems at the population level.

Substituted diphenylamine antioxidants and benzotriazole UV stabilizers in blood plasma of fish, turtles, birds and dolphins from North America.

Substituted diphenylamine antioxidants (SDPAs) and benzotriazole UV stabilizers (BZT-UVs) are additives used in industrial and commercial applications to prevent degradation by oxidation and are contaminants of emerging environmental concern. Little is known about the fate of these contaminants in wildlife, particularly in reptiles, birds and marine mammals. Nine SDPAs and six BZT-UVs were measured in blood plasma of seven fish species, snapping turtles (Chelydra serpentina), double-crested cormorants (Phalacrocorax auritus), and bottlenose dolphins (Tursiops truncatus) from various locations in North America. Plasma SDPAs were more frequently (90-100%) detected and with higher concentrations (median: 25-270 pg g-1, wet weight (ww)) in organisms from urban areas than rural locations (median:  double-crested cormorants > bottlenose dolphins. Of the three quantifiable BZT-UVs, 2-(2H-benzotriazol-2-yl)-4,6-di-tert-pentylphenol (UV328) showed higher detection frequency in most species of fish, bird and turtle (range of 0-67%), indicating the widespread distribution of UV328 in the aquatic environment of lower Great Lakes region.

BLOOD CHEMISTRY AND HEMATOLOGY VALUES IN HEALTHY AND REHABILITATED ROUGH-TOOTHED DOLPHINS ( STENO BREDANENSIS).

Rehabilitation efforts for live stranded marine mammals are guided by diagnostic measures of blood chemistry and hematology parameters obtained from each individual undergoing treatment. Despite the widespread use of blood parameters, reference values are not available in the literature from healthy rough-toothed dolphins ( Steno bredanensis) with which to infer the health status of an animal. We examined serum or plasma chemistry and hematology data from 17 rough-toothed dolphins either housed at Dolphin Quest French Polynesia or during their rehabilitation at the Dolphin and Whale Hospital in Sarasota, Florida, US between 1994 and 2005. Blood parameters were compared among healthy animals, rehabilitation animals that were eventually released, and rehabilitation animals that died. This study indicated significant differences in many blood parameters for the poorly known rough-toothed dolphin that are likely to vary between healthy and sick animals. These included aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, bicarbonate, and globulins, which were greater in sick dolphins, and alkaline phosphatase and total protein which were greater in healthy individuals. Total white blood cell counts were lower in healthy animals as were the absolute numbers of neutrophils, monocytes, and eosinophils. Analysis of first blood sample levels for glucose, sodium, and erythrocyte sedimentation rate may have value for triage and prognostic evaluation.

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.

Characterization of alpha-fetoprotein levels in three dolphin species: development of sensitive immunoassays for analysis of the pregnancy-associated variations.

A single radial immunodiffusion (SRID) assay and a chemiluminescent immunoassay (CLIA) were initially developed for alpha-fetoprotein (AFP) of the striped dolphin. Utilizing these developed assays, we investigated pregnancy-associated changes in the levels of AFP in the sera of fetuses and pregnant females of three dolphin species; samples were either collected from captive individuals or obtained as fishery by-products. The concentrations of AFP in the fetal serum ranged from 419.0 to 2026.3 µg/ml in the striped dolphin, 12.6 to 1218.7 µg/ml (for an AFP equivalent; eqAFP) in the common bottlenose dolphin and 770.6 to 3129.1 µg eqAFP/ml in the Risso's dolphin. AFP levels decreased with increased fetal size in fetuses over 20 cm in length. The concentrations of AFP in sera of pregnant females ranged from 7.18 to 8068.7 ng/ml in the striped dolphin, 6.6 to 1241.1 ng eqAFP/ml in the common bottlenose dolphin and 3.4 to 2868.7 ng eqAFP/ml in the Risso's dolphin. The levels in most pregnant females were equal to or lower than those found in males and nonpregnant individuals, although a few pregnant females exhibited extremely high levels (in the range of hundreds to thousands of nanograms per milliliter). Such high levels of AFP were not observed during pseudopregnancy. To our knowledge, this is the first report on basal profiles for serum AFP levels in small odontocetes. The profiles indicated that AFP may play a significant role during embryonic development, although maternal levels do not appear to be a diagnostic biomarker for monitoring pregnancy.

Bubbles in live-stranded dolphins.

Bubbles in supersaturated tissues and blood occur in beaked whales stranded near sonar exercises, and post-mortem in dolphins bycaught at depth and then hauled to the surface. To evaluate live dolphins for bubbles, liver, kidneys, eyes and blubber-muscle interface of live-stranded and capture-release dolphins were scanned with B-mode ultrasound. Gas was identified in kidneys of 21 of 22 live-stranded dolphins and in the hepatic portal vasculature of 2 of 22. Nine then died or were euthanized and bubble presence corroborated by computer tomography and necropsy, 13 were released of which all but two did not re-strand. Bubbles were not detected in 20 live wild dolphins examined during health assessments in shallow water. Off-gassing of supersaturated blood and tissues was the most probable origin for the gas bubbles. In contrast to marine mammals repeatedly diving in the wild, stranded animals are unable to recompress by diving, and thus may retain bubbles. Since the majority of beached dolphins released did not re-strand it also suggests that minor bubble formation is tolerated and will not lead to clinically significant decompression sickness.

Effect of breath-hold on blood gas analysis in captive Pacific white-sided dolphins (Lagenorhynchus obliquidens).

The effect of a breath-hold on blood gas was evaluated in captive Pacific white-sided dolphins (Lagenorhynchus obliquidens). Serial blood collections were performed from a vessel on the ventral surface of the flukes during breath-hold. In total, 178 blood samples were taken from three dolphins for five trials in each animal. During a breath-hold, partial pressure of oxygen (Po2) decreased from 152.5 to 21.8 mmHg and partial pressure of carbon dioxide (Po2) conversely increased from 31.8 to 83.6 mmHg. The range of pH was 7.54 to 7.25, suggesting drastic change from alkalemia to acidemia. These wide ranges of blood gas imply a considerable change of oxygen affinity caused by the Bohr effect during breath-hold, which enable effective uptake and distribution of oxygen to metabolizing tissues.

Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene.

BACKGROUND: Iron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit). Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron. METHODS: Sequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR) fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas). The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA. RESULTS: Dolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99-100%, (99-->98) ; L: 98-100%). The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species. CONCLUSION: These results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed.

Natriuretic peptides in cetaceans: identification, molecular characterization and changes in plasma concentration after landing.

Dolphins are aquatic animals free from gravity, and this may have imposed significant changes in their cardiovascular status and its hormonal regulation compared with terrestrial animals. This study molecularly characterized two major cardiovascular hormones, atrial and B-type natriuretic peptides (ANP and BNP) and measured their changes in dolphin plasma concentrations in relation to the cardiovascular status of the animal. We initially identified ANP and BNP in three species of dolphins (Lagenorhynchus obliquidens, Phocoenoides dalli and Tursiops truncatus). ANP precursors are highly conserved in most mammals, but dolphin BNP precursors were more variable. In molecular phylogenetic analyses, dolphin ANP and BNP precursors grouped with those of artiodactyls, particularly to the camel peptides. The chromatographic characterization of tissue and plasma molecular forms using specific radioimmunoassays showed that the predominant ANP and BNP in the atrium are prohormone and mature peptide, respectively, whereas mature ANP and BNP are circulating in the dolphin blood. A mass spectrometric analysis showed that atrial BNP consists of 26 amino acids, rather than the 32-amino-acid form detected in other mammals. Finally, changes in plasma ANP and BNP concentrations were examined in captive bottlenose dolphins (Tursiops truncatus) after their pool was drained. Plasma ANP and BNP concentrations did not change after landing, unlike terrestrial mammals. Plasma angiotensin II and cortisol concentrations did not change either, showing minor stress after landing. Since landed dolphins show a different cardiovascular status on land than terrestrial mammals, plasma ANP and BNP concentrations seem to reflect the cardiovascular status characteristic of dolphins.

Animal behaviour: continuous activity in cetaceans after birth.

All mammals previously studied take maximal rest or sleep after birth, with the amount gradually decreasing as they grow to adulthood, and adult fruitflies and rats die if they are forcibly deprived of sleep. It has therefore been assumed that sleep is necessary for development and serves a vital function in adults. But we show here that, unlike terrestrial mammals, killer-whale and bottlenose-dolphin neonates and their mothers show little or no typical sleep behaviour for the first postpartum month, avoiding obstacles and remaining mobile for 24 hours a day. We find that neonates and their mothers gradually increase the amount of time they spend resting to normal adult levels over a period of several months, but never exceed these levels. Our findings indicate either that sleep behaviour may not have the developmental and life-sustaining functions attributed to it, or that alternative mechanisms may have evolved in cetaceans.

Evaluation of the polymorphonuclear cell functions of bottlenose dolphins.

The functions of polymorphonuclear cells (PMN) are the important non-specific defense mechanisms in the immune system. Especially marine mammals are protected by these mechanisms from the aquatic environment with a large variety of microorganisms. Therefore, we examined the PMN functions of bottlenose dolphins in order to obtain the normal ranges and to standardize the techniques. PMNs were isolated by using lymphocyte isolate solution whose density was 1.077; superoxide production was assessed by nitroblue tetrazolium reduction test (NBT) and phagocytosis was tested by using polystyrene latex beads. We showed that the optimal incubation time was 30 min in NBT assay and 12 hr in phagocytosis assay for dolphin PMNs.

Diurnal and annual changes in serum cortisol concentrations in Indo-Pacific bottlenose dolphins Tursiops aduncus and killer whales Orcinus orca.

Until present, fundamental studies on cortisol secretory patterns have not been conducted in cetaceans. The objectives of this study were: (1) to examine diurnal changes in serum cortisol concentrations in Indo-Pacific bottlenose dolphins Tursiops aduncus and killer whales Orcinus orca, (2) to investigate annual cortisol changes in killer whales, and (3) to investigate the relationship between cortisol and sex steroids (testosterone and progesterone) concentrations in killer whales. Diurnal changes in serum cortisol concentrations were investigated at various intervals in the two species. In Indo-Pacific bottlenose dolphins, serum cortisol levels exhibited the same episodic fluctuations for 24 h as did diurnal terrestrial mammals: cortisol levels were lower at 18:00 h and higher in the early morning. In killer whales, cortisol concentrations continued to decrease until 18:00 h, after which they fluctuated, and then increased in the next morning. Annual changes in cortisol levels were investigated by collecting blood samples every two weeks from two male killer whales and a pregnant female one twice per day (during 09:00-10:00 and 16:00-17:00 h) throughout a one-year period. Regarding sera collected during 09:00-10:00 h from the female, cortisol concentrations showed cyclic changes having about 4-month intervals. In males, cortisol showed higher concentrations in winter and lower concentrations during the summer season. There was a negative correlation between cortisol and progesterone levels in the female and a negative correlation was also observed between cortisol and testosterone in male no. 2. In the female and male no. 1, cortisol levels during 09:00-10:00 h were significantly higher than those during 16:00-17:00 h, and their data are considered to support observations regarding diurnal changes in cortisol levels in the two cetacean species.

Seasonal changes of blood composition in captive bottlenose dolphins.

To determine how blood values in bottlenose dolphins changed during the year, 504 blood samples were taken from 9 dolphins from 1991 to 1999 and clinical blood examinations were undertaken monthly including 3 hematological and 19 serum chemistry tests. In creatinine, significant seasonal changes were found among three groups of adult males, adult females and juveniles, and the average values in summer were 15-38% higher than those in winter. In two out of three groups the average total cholesterol value were highest in winter, and the lowest of all groups were in summer. In two other groups the peaks of average FFA value were recorded in summer, and the lows were in winter.

Evaluation of mitogen-induced responses in marine mammal and human lymphocytes by in-vitro exposure of butyltins and non-ortho coplanar PCBs.

The effects of exposure to butyltin compounds (BTs: tributyltin; TBT, dibutyltin; DBT and monobutyltin; MBT) and non-ortho coplanar PCBs (IUPAC 77, 126 and 169) on marine mammals and human lymphocyte were evaluated. Peripheral blood mononuclear cells (PBMCs) isolated from Dall's porpoises (Phocoenoides dalli), bottlenose dolphins (Tursiops truncatus), a California sealion (Zalophus californianus), a larga seal (Phocoa largha) and humans (Homo sapiens) were exposed at varying concentrations of BTs and coplanar PCBs. Concanavalin A (Con A)-stimulated mitogenesis found significantly suppressed (P<0.01) when the cells were exposed at 300 nM (89 ng/ml) of TBT and 330 nM of DBT (77 ng/ml), while MBT showed little cytotoxicity at treatment levels of up to 3,600 nM (620 ng/ml). BTs concentrations in the liver of Dall's porpoises from Japanese coastal waters ranged between 81-450 ng/g for TBT and 200-1,100 ng/g (wet wt.) for DBTs, which is greater than the cytotoxic levels registered in this study. In contrast, non-ortho coplanar PCBs did not suppress cell proliferation at concentrations of up to 30 nM (10 ng/ml). The residue levels of coplanar PCBs in the blubber of Dall's porpoises were 0.12-1.3 ng/g, which were one order of lower than those levels that do cell proliferation. When cells were exposed to a mixture of TBT/DBTand coplanar PCBs, the proliferation was significantly reduced to 33 nM DBT plus 34 nM CB-77 and 33 nM DBT plus 28 nM CB-169 mixtures, respectively. The investigations relating the contaminant-induced immunosuppression in marine mammals have been focused on persistent organochlorines such as PCBs. pesticides and dioxin compounds. However, this study suggested the possibility of BTs could also pose a serious threat to the immune functions in free-ranging marine mammals and humans.

Measurement of serum immunoglobulin concentration in killer whales and sea otters by radial immunodiffusion.

Killer whales and sea otters maintained in captivity are the subjects of routine health monitoring programs, and interest in immunologic studies in sea otters has been rising recently in response to potential impacts from infectious disease and environmental pollution on the threatened southern sea otter population. Development of species-specific reagents for immunologic studies in these two marine mammals is currently in its infancy. In this study, killer whale and sea otter immunoglobulin-specific polyclonal antibodies were generated, and used to develop tests for serum Ig concentration in the killer whale (Orcinus orca) and the southern (Enhydra lutris nereis) and northern sea otter (Enhydra lutris lutris). Killer whale serum IgG was purified using caprylic acid/ammonium sulfate precipitation. Sea otter plasma IgG was purified using protein-A-agarose. Polyclonal anti-Ig antisera were produced in rabbits, and specificity confirmed by immunoelectrophoresis. Radial immunodiffusion was used to measure Ig concentration in serum or plasma samples derived from 21 captive killer whales, 18 wild and 4 captive southern sea otters and 15 wild and 4 captive northern sea otters grouped by age. Mean killer whale serum Ig concentration (+/-95% confidence interval) ranged from 15.04 +/- 3.97 g/l for animals aged 0-5 years to 26.65 +/- 9.8 g/l for animals aged >10 years. Mean sea otter serum Ig concentration (+/-95% confidence interval) ranged from 28.39 +/- 11.00 g/l for southern sub-adults to 32.76 +/- 11.58 g/l for southern adults. No significant difference in serum Ig concentration was found between southern and northern sea otters. Serum Ig concentrations in two northern sea otter pups were low compared to those of adult sea otters. The two serum Ig quantitation assays produced were highly specific and reproducible and will be useful additions to the limited number of tests available for immune function in these marine mammal species.

Molecular cloning and identification of bottle-nosed dolphin p40(phox), p47(phox) and p67(phox).

The bottle-nosed dolphin NADPH oxidase cytosolic components, p40(phox), p47(phox) and p67(phox) cDNA's were cloned from mitogen stimulated peripheral white blood cell mRNA utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that dolphin p40(phox), p47(phox) and p67(phox) clones contained open reading frames encoding predicted polypeptides of 339, 391 and 526 amino acids, respectively. Analysis of the p47(phox) and p67(phox) amino acid sequences showed two potential Src homology three domains and p40(phox) one. Comparison of the deduced amino acids showed that dolphin p40(phox) sequence shared 88.8% similarity with the human p40(phox), that dolphin p47(phox) sequence shared 87.7% similarity with the bovine p47(phox), and that dolphin p67(phox) shared 88.1% similarity with the bovine p67(phox). Western blot analysis using anti-human p40(phox), p47(phox) and p67(phox) antibodies demonstrated that dolphin neutrophil possesses p40(phox), p47(phox) and p67(phox) with similar molecular masses and structures, to each counterpart in human neutrophils, except for the p67(phox) COOH-terminus. These results suggest that dolphin NADPH oxidase cytosolic components have functional activities equivalent to those of human.

Serum lipid peroxide and alpha-tocopherol concentrations and superoxide dismutase activity in captive bottle-nosed dolphins.

OBJECTIVE: To evaluate serum lipid peroxide (LPO) and alpha-tocopherol concentrations and superoxide dismutase (SOD) activity in captive bottle-nosed dolphins and to evaluate effects of storage on production of LPO in various marine fish. ANIMALS: 16 bottle-nosed dolphins. PROCEDURE: 8 dolphins (group A) were fed chub mackerel and herring (high fat) and arabesque greenling and banded blue-sprat (low fat); the other 8 dolphins (group B) were fed chub mackerel and Pacific saury (high fat) and shishamo smelt and Japanese horse mackerel (low fat). Each group had been on these respective diets for 3 years. Serum LPO and alpha-tocopherol concentrations, serum SOD activity, and superoxide production by neutrophils were measured. All types of marine fish were frozen at -20 C for 6 months, and concentrations of LPO were measured at various time points. RESULTS: Serum LPO concentrations in group-A dolphins were significantly higher than those in group B. Serum alpha-tocopherol concentrations and SOD activity in group A were significantly lower than those in group B. A significant negative correlation was found between serum LPO and alpha-tocopherol concentrations in all 16 dolphins. The LPO concentrations in mackerel and herring fed to group-A dolphins were higher than those of other fish. Concentrations of LPO in herring stored for 3 and 6 months at -20 C were higher than those in herring before freezing and in herring stored for 1 month. CONCLUSIONS AND CLINICAL RELEVANCE: Serum LPO and alpha-tocopherol concentrations in captive bottle-nosed dolphins may be strongly influenced by high amounts of polyunsaturated fatty acid and LPO found in marine fatty fishes. High concentrations of serum LPO, as found in group-A dolphins, were associated with decreased antioxidative states. Monitoring of serum LPO and alpha-tocopherol concentrations and serum SOD activity may be useful for the management of captive marine mammals.

Oxygen radical generation and expression of NADPH oxidase genes in bottlenose dolphin (Tursiops truncatus) neutrophils.

Oxygen radical generation by stimulation with phorbol myristate acetate (PMA) was evaluated in bottlenose dolphin neutrophils. A Cypridina luciferin analog-dependent chemiluminescent assay demonstrated that dolphin neutrophils generate superoxide by the addition of PMA, and that its superoxide-forming activity is completely suppressed by diphenylene iodonium, a specific inhibitor of NADPH oxidase. These results indicate that dolphin neutrophils possess NADPH oxidase activity. Furthermore, the NADPH oxidase activity (hydrogen peroxide production) in dolphin neutrophils, as well as in human neutrophils, was greater at 37 degrees C than at a lower temperature. RT-PCR with specific primers revealed that dolphin neutrophils expressed the mRNAs of the major NADPH oxidase components, which included membrane-associated flavocytochrome b (gp91(phox) and p22(phox)) and cytosolic factors (p40(phox), p47(phox), and p67(phox)), implying the existence of these protein homologues in dolphin neutrophils.