Deadly diving? Physiological and behavioural management of decompression stress in diving mammals.

Decompression sickness (DCS; 'the bends') is a disease associated with gas uptake at pressure. The basic pathology and cause are relatively well known to human divers. Breath-hold diving marine mammals were thought to be relatively immune to DCS owing to multiple anatomical, physiological and behavioural adaptations that reduce nitrogen gas (N(2)) loading during dives. However, recent observations have shown that gas bubbles may form and tissue injury may occur in marine mammals under certain circumstances. Gas kinetic models based on measured time-depth profiles further suggest the potential occurrence of high blood and tissue N(2) tensions. We review evidence for gas-bubble incidence in marine mammal tissues and discuss the theory behind gas loading and bubble formation. We suggest that diving mammals vary their physiological responses according to multiple stressors, and that the perspective on marine mammal diving physiology should change from simply minimizing N(2) loading to management of the N(2) load. This suggests several avenues for further study, ranging from the effects of gas bubbles at molecular, cellular and organ function levels, to comparative studies relating the presence/absence of gas bubbles to diving behaviour. Technological advances in imaging and remote instrumentation are likely to advance this field in coming years.

Evidence of injury caused by gas bubbles in a live marine mammal: barotrauma in a California sea lion Zalophus californianus.

A yearling male California sea lion Zalophus californianus with hypermetric ataxia and bilateral negative menace reflexes was brought to The Marine Mammal Center, Sausalito, California, U.S.A., in late 2009 for medical assessment and treatment. The clinical signs were due to multiple gas bubbles within the cerebellum. These lesions were intraparenchymal, multifocal to coalescing, spherical to ovoid, and varied from 0.5 to 2.4 cm diameter. The gas composed 21.3% of the total cerebellum volume. Three rib fractures were also noted during diagnostic evaluation and were presumed to be associated with the gas bubbles in the brain. The progression of clinical signs and lesion appearance were monitored with magnetic resonance imaging, cognitive function testing and computed tomography. Gas filled voids in the cerebellum were filled with fluid on follow up images. Clinical signs resolved and the sea lion was released with a satellite tag attached. Post release the animal travelled approximately 75 km north and 80 km south of the release site and the tag recorded dives of over 150 m depth. The animal re-stranded 25 d following release and died of a subacute bronchopneumonia and pleuritis. This is the first instance of clinical injury due to gas bubble formation described in a living pinniped and the first sea lion with quantifiable cerebellar damage to take part in spatial learning and memory testing.

Simultaneous measurement of phagocytosis and respiratory burst of leukocytes in whole blood from bottlenose dolphins (Tursiops truncatus) utilizing flow cytometry.

Phagocytic and respiratory burst activity was simultaneously measured by flow cytometry in polymorphonuclear leukocytes (PMN) and monocytes in whole blood from bottlenose dolphins (Tursiops truncatus). Blood was collected from 16 adult dolphins, 12 males (6-34 years of age) and 4 females (11-30 years) and subsequently incubated with a bacteria-to-leukocyte ratio of 25:1 and 10 µl of 500 µM 2',7'-dichlorofluorescein diacetate for 70 min at 37°C. PMN (44.5 ± 3.2%) and monocytes (33.5 ± 3.0%) were positive for propidium iodide-labeled Staphylococcus aureus, indicating phagocytosis. Respiratory burst activity after 70 min as measured by the mean fluorescence intensity (MFI) was 68.0 ± 14.4 in PMN and 47.0 ± 10.3 in monocytes. There were no significant differences in MFI or percentage of phagocytizing PMN (p > 0.094) or monocytes (p > 0.275) after storage at 4°C for 24h when compared to activity measured in fresh blood. Nor was there an effect of storage on respiratory burst activity (MFI or percentage) in PMN (p > 0.420) or monocytes (p > 0.301). This assay may be particularly useful to assess the ability of dolphins to effectively combat bacterial pathogen challenges with minimal amounts of blood.

Seasonality, estrous cycle characterization, estrus synchronization, semen cryopreservation, and artificial insemination in the Pacific white-sided dolphin (Lagenorhynchus obliquidens).

The reproductive physiology of the Pacific white-sided dolphin, Lagenorhynchus obliquidens, was characterized to facilitate the development of artificial insemination (AI) using cryopreserved spermatozoa. Specific objectives were to: 1) describe reproductive seasonality of the Pacific white sided dolphins; 2) describe urinary LH and ovarian steroid metabolites during the estrous cycle; 3) correlate LH and ovarian steroidal metabolite patterns to ultrasound-monitored follicular growth and ovulation; and 4) assess the efficacy of synchronizing estrus, sperm collection/cryopreservation, and intrauterine insemination. Ovulations (64%, n=37) and conceptions (83%, n=18) occurred from August to October. Peak mean serum testosterone (24 ng/ml), cross-sectional testicular area (41.6 cm(2)), and sperm concentration (144.3 x 10(7) sperm/ml) occurred in July, August, and September respectively. Spermatozoa were only found in ejaculates from July to October. Estrous cycles (n=22) were 31 d long and were comprised of a 10 d follicular and 21 d luteal phase. Ovulation occurred 31.2 h after the onset of the LH surge and 19.3 h after the LH peak. Follicular diameter and circumference within 12 h of ovulation were 1.52 and 4.66 cm respectively. Estrus synchronization attempts with altrenogest resulted in 17 (22%) ovulatory cycles with ovulation occurring 21 d post-altrenogest. Ten AI attempts using cryopreserved semen resulted in five pregnancies (50%). The mean gestation length was 356 days (range 348-367). These data provide new information on the Pacific white-sided dolphin's reproductive physiology and collectively enabled the first application of AI in this species.

Neuronal migration defect: a case of subcortical heterotopia in a California sea lion.

A 2 and a half-year-old male California sea lion (Zalophus californianus) presented with a history of intermittent generalized seizures. Magnetic resonance imaging revealed a large focal mass occupying the right cerebral hemisphere with moderate dilatation of the contralateral lateral ventricle. At necropsy, the right cerebral hemispheric white matter was expanded by numerous irregularly shaped, pale pink nodules up to 10 mm in diameter. The overlying cortex was characterized by increased numbers of small, poorly developed gyri with shallow, often indistinct, sulci (polymicrogyria). Microscopically, nodules were composed of neurons, oligodendroglia, microglia, and supporting neuropil and were well delineated from the surrounding white matter. The gross, histological, and immunohistochemical features of this lesion are consistent with a neuronal migration defect resulting in unilateral subcortical heterotopia.

Toxoplasma gondii, Neospora caninum, Sarcocystis neurona, and Sarcocystis canis-like infections in marine mammals.

Toxoplasma gondii, Neospora caninum, Sarcocystis neurona, and S. canis are related protozoans that can cause mortality in many species of domestic and wild animals. Recently, T. gondii and S. neurona were recognized to cause encephalitis in marine mammals. As yet, there is no report of natural exposure of N. caninum in marine mammals. In the present study, antibodies to T. gondii and N. caninum were assayed in sera of several species of marine mammals. For T. gondii, sera were diluted 1:25, 1:50, and 1:500 and assayed in the T. gondii modified agglutination test (MAT). Antibodies (MAT > or =1:25) to T. gondii were found in 89 of 115 (77%) dead, and 18 of 30 (60%) apparently healthy sea otters (Enhydra lutris), 51 of 311 (16%) Pacific harbor seals (Phoca vitulina), 19 of 45 (42%) sea lions (Eumetopias jubatus) [corrected] 5 of 32 (16%) ringed seals (Phoca hispida), 4 of 8 (50%) bearded seals (Erignathus barbatus), 1 of 9 (11.1%) spotted seals (Phoca largha), 138 of 141 (98%) Atlantic bottlenose dolphins (Tursiops truncatus), and 3 of 53 (6%) walruses (Odobenus rosmarus). For N. caninum, sera were diluted 1:40, 1:80, 1:160, and 1:320 and examined with the Neospora agglutination test (NAT) using mouse-derived tachyzoites. NAT antibodies were found in 3 of 53 (6%) walruses, 28 of 145 (19%) sea otters, 11 of 311 (3.5%) harbor seals, 1 of 27 (3.7%) sea lions, 4 of 32 (12.5%) ringed seals, 1 of 8 (12.5%) bearded seals, and 43 of 47 (91%) bottlenose dolphins. To our knowledge, this is the first report of N. caninum antibodies in any marine mammal, and the first report of T. gondii antibodies in walruses and in ringed, bearded, spotted, and ribbon seals. Current information on T. gondii-like and Sarcocystis-like infections in marine mammals is reviewed. New cases of clinical S. canis and T. gondii infections are also reported in sea lions, and T. gondii infection in an Antillean manatee (Trichechus manatus manatus).

Epizootic vesicular disease in captive California sea lions.

An epizootic of vesicular disease occurred in a group of semi-domesticated California sea lions (Zalophus californianus) during the months of April and May 1997. Ten castrated mature male sea lions, ages 12 to 19 yr, were housed in three adjacent open-ocean net enclosures in San Diego Bay (California, USA). Four animals (40%) developed oral and extremity vesicles, anorexia, and were reluctant to perform learned behaviors. One animal developed vesicles but maintained a normal appetite and behavior. The remaining animals showed no clinical signs of infection. Virus (designated FADDL 7005) was isolated from four of the five animals that developed vesicles. Serum antibody titers to FADDL 7005, a previously untyped calicivirus, were demonstrated in animals that showed any combination of clinical signs and in two animals that did not show any clinical signs. No virus was isolated from five fecal samples collected from four of the group animals. Clinical signs lasted 4 to 20 days in affected animals. All affected animals recovered from infection. An experimental swine was inoculated with FADDL 7005 and developed vesicular disease, which was transmitted to another experimental swine upon contact. It is proposed that FADDL 7005 is a new San Miguel sea lion virus.

Effects of in vitro hemolysis on serum biochemistry values of the bottlenose dolphin (Tursiops truncatus).

The effects of in vitro hemolysis on 23 biochemical analytes were assessed in sera from 14 clinically healthy Atlantic bottlenose dolphins (Tursiops truncatus). Each serum sample was divided into three portions for analysis: 1) nonhemolyzed control; 2) moderate hemolysis, simulated by adding hemolyzed serum to a final concentration of approximately 150 mg/dl Hb; and 3) severe hemolysis, simulated by adding hemolyzed serum to a final concentration of approximately 500 mg/dl Hb. Moderate hemolysis resulted in statistically significant increases in the mean values of iron, lactate dehydrogenase, potassium, and uric acid and a decrease in creatinine (P < 0.001). Severe hemolysis resulted in statistically significant changes in the mean values of the above analytes in addition to the following increases: alanine aminotransferase, calcium, and serum globulins (P < 0.001) and albumin and total protein (P < 0.01). Total bilirubin and gamma glutamyl transferase levels were lower in the severely hemolyzed sample (P < 0.001). Differences in mean values for alkaline phosphatase between nonhemolyzed and hemolyzed serum were not significant but did show a downward trend in the hemolyzed sera. The presence and severity of hemolysis must be considered in the interpretation of the serum chemistry values.