Biological Variation and Reference Change Value of Routine Hematology Measurands in a Population of Managed Bottlenose Dolphins (Tursiops truncatus).

Hematological analyses are particularly useful in assessing a dolphin's health status. However, the creation of appropriate reference intervals for this species is difficult due to the low number of reference individuals. The implementation of individual reference intervals (iRIs) allows researchers to overcome this limitation and, moreover, also consider the within-individual variability. The aims of this study were (1) to evaluate the biological variations in some hematological measurands, including erythrocytes (RBC), hematocrit (Hct), mean cellular volume and hemoglobin content (MCV and MCHC, respectively), RBC distribution width (RDW), leukocytes (WBC), and platelets (PLT); and (2) to calculate the index of individuality (IoI) and reference change value (RCV), which enable the production of iRIs, in healthy managed bottlenose dolphins. Seven dolphins were included, and the results of six hematological exams were analyzed for each animal. Analytical imprecision (CVa), within-dolphin variation (CVi), and between-dolphins variations (CVg) were calculated, and the IoI and RCV were derived for each measurand. All the hematological measurands had intermediate IoI except WBC, for which Iol was low. The calculated RCV ranged from 10.33% (MCV) to 186.51% (WBC). The results reveal that the majority of hematological measurands have an intermediate level of individuality in dolphins, and thus the application of iRIs is appropriate. The calculated RCV can also be applied to other managed dolphins and could be useful in interpreting serial CBC exams.

Comparative Study of the Gastric Mucosa of Risso's Dolphin (Grampus griseus) and Bottlenose Dolphin (Tursiops truncatus): A Key to Manage the Diet in Captive Conditions.

To histologically evaluate the gastric compartments of Risso's (Grampus griseus) and bottlenose dolphins (Tursiops truncatus) and provide suggestions for the diet of Risso's dolphins in captivity, we examined 12 stomachs from both species. While slight differences in keratinization were observed in the forestomach, significant differences came to light in the second stomach's mucosa. At this level, in Risso's dolphin, the principal cells are markedly reduced in size and located externally to the parietal cells, not interspersed between them, compared to bottlenose dolphins; differences were also observed in the structure and concentration of the parietal and principal cells of the gastric body glands (p < 0.0001). The quantitative results of G- and D-cell counts in the gastric mucosa show a clear difference, with a higher concentration of G cells in the mucosa of Risso's dolphin (t = 7.334; p < 0.0001) and a higher level of D cells in bottlenose dolphin mucosa (t = 3.123; p = 0.0049). These results suggest that parietal cells undergo greater stimulation by gastrin produced by G cells, with greater acid secretion in G. griseus. Further studies are needed to understand whether an inappropriate diet could lead to severe clinical signs due to gastric acidity in Risso's dolphin.

Trace and rare earth element bioaccumulation in the spotted dogfish (Scyliorhinus stellaris).

Metals (trace elements and rare earth elements, REEs) were analysed by inductively coupled plasma-mass spectrometry in blood, the liver, the kidney and muscle of ex situ spotted dogfish (Scyliorhinus stellaris). The controlled environment in which these elasmobranchs were hosted allowed to assess a baseline level of metals in the different organs since exposure via water and food can be easily monitored. The highest arsenic, chromium, copper, and iron values were found in the liver, cobalt in the kidney, and cadmium and rubidium in muscle. The highest total trace elements content was found in the trend liver (75 mg kg-1) > blood (33 mg kg-1) > muscle (31 mg kg-1) > kidney (10 mg kg-1), while the ΣREEs was the liver (30 µg kg-1) > muscle (15 µg kg-1) > kidney (13 µg kg-1) > blood (4.1 µg kg-1). Between REEs, the most represented element was scandium. Significant differences in the concentration of metals among organs were observed for almost all elements. Nonessential elements were generally lower and essential elements higher in the examined specimens compared to wild elasmobranchs, suggesting a close relationship between a balanced diet and animal welfare.

Milk composition, fatty acids profile and fat globule size of bottlenose dolphin (Tursiops truncatus, Montagu 1821) milk at early lactation.

Research data on milk composition in cetaceans are scattered and fragmentary. This paper analyses the gross and mineral composition, the fatty acids profile and the fat globule size of bottlenose dolphin (Tursiops truncatus) colostrum and milk at early lactation. The milk samplings were carried out on three lactating female of bottlenose dolphins at the 1st, 4-5 and 20- 42 day post partum. High percentages of dry matter (51.88%), fat (26.08%) and protein (13.83%) were found in the colostrum on the first day, while there was a tendency for these components to decrease in the milk. The average diameter the milk fat globule (7.07 µm) of this species was assessed for the first time. The milk was rich in unsaturated fatty acids, which were more than twice compared to the saturated fatty acids (unsaturated / saturated ratio = 2.6). The main fatty acids of dolphin milk were C16: 0, C16:1 n-7, C18: 1n-9, C20: 1 n-11 C20: 5 and C22:6n-3. Several FA tended to increase from colostrum to milk: C12: 0 doubled, C20: 1 n-7 and C22:1 n-11 tripled, C20: 1 n-9 quintupled in later milk samples. Differently, other FA tended to decrease: C18: 3 n-3 was approximately 4 times lower and C24: 0 and C24: 1 halved in milk compared to colostrum. In conclusion, this paper contributes to the knowledge of the nutritional value of milk from bottlenose dolphins maintained under human care. Higher content of nutritional components such as fat and protein was found in the first day colostrum compared to milk. Moreover, the findings of this study highlight abundance of unsaturated fatty acids and large sized milk fat globules in dolphin milk.

Physiological Parameters Monitored on Bottlenose Dolphin Neonates (Tursiops truncatus, Montagu 1821) over the First 30 Days of Life.

Bottlenose dolphins (Tursiops truncatus) have been bred under human care for more than 60 years. Calves up to 30 days of life have presented the highest mortality rate, although comparable data for free-ranging neonates within this age group is not available. Husbandry measures to increase survivability have therefore been constantly improved. This work shows the results of a structured veterinary program that established the procedures to collect relevant physiological parameters on 13 calves during their first 30 days of life. Standardized observation protocols facilitated statistical analysis of the respiratory pattern, nursing, morphometric measurements and bloodwork. These allow early detection of health issues. Healthy neonates had longer apnea duration, despite the shape and size of the facility. The nursing pattern showed that successful calves started nursing 3 to 18 h postpartum. Although with different individual patterns, a steady increase in suckling time occurs during the first 24 h of life. The healthy neonates grew 0.428 ± 0.102 kg per day and the complete blood count profile, glucose, iron, blood urea nitrogen (BUN), total protein, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (GGT), creatinine and electrolytes values considered normal for healthy calves are provided. Furthermore, cholesterol, triglycerides, α-amylase, lipase, magnesium and cortisol are reported for the first time for such young calves. A list of indications for prompt intervention is included.

Differential Bioaccumulation of Trace Elements and Rare Earth Elements in the Muscle, Kidneys, and Liver of the Invasive Indo-Pacific Lionfish (Pterois spp.) from Cuba.

The Indo-Pacific lionfish is a saltwater fish that inhabits the Red Sea waters and the Indian and Pacific oceans; it is an invasive species in the western Atlantic and was recently introduced into the local diet in the USA, Central and South America, and the Caribbean with the aim of controlling the invasion of this species. Due to its predatory nature, it tends to bioaccumulate metals and other contaminants via the marine food web and could thus constitute a suitable species for monitoring aquatic ecosystems. The presence and distribution of 23 trace elements and 16 rare earth elements (REEs) were investigated by inductively coupled plasma-mass spectrometry (ICP-MS) in the muscle, liver, and kidneys of lionfish from Cuba. Significant differences in metal concentrations were found in the different fish organs. The liver and kidneys registered the highest concentrations for most trace elements and for ΣREE, thus demonstrating that they are effective bioindicators of possible pollution on the environment in which fish live, and assuming great importance in the choice of early biomonitoring. Trace element concentrations in the muscle are instead of crucial interest for consumer safety. The limits set by EU regulations and Cuban guidelines for Cd and Pb in fish muscle were never exceeded, suggesting that lionfish from Cuba could therefore represent a good source of minerals and proteins for the local population. Graphical abstract.

Isolation and phenotyping of potential stem cells from the umbilical cord of the bottlenose dolphin(Tursiops truncatus).

We have successfully isolated cells with stem-like properties from bottlenose dolphin (Tursiops truncatus) umbilical cord. Our results show that this cetacean species has embryonic fetal and adult stem cells as do humans and other studied mammals. This accomplishment allows to eventually investigate whether dolphins, due to their unique adaptations to aquatic environments, have special stem cell lineages or distinctive mechanisms of cell programming. Further characterization of their potency to differentiate into multiple cell lineages would fulfill numerous applicative purposes. We characterized, developed and refined a new protocol for obtaining potential stem cells from umbilical cord tissues of the bottlenose dolphin. Tissue samples were taken from umbilical cords of successful deliveries immediately after placenta ejection and collection from the water. Umbilical cord samples (2-3 cm3) were excised and subjected to enzymatic digestion and mechanical dissociation. Viable cells from specimens resident in the Oceanografic Valencia were cultured and subsequently isolated and tested for pluripotent characteristics (cell morphology, phenotype and expression of surface markers). Cell viability was confirmed also after freezing/thawing. The established protocol is suitable for collection/isolation/culture of dolphin potential mesenchymal stem cells from dolphin umbilical cord, which can be deposited in cell banks for future research needs.

Death Associated to Methicillin Resistant Staphylococcus aureus ST8 Infection in Two Dolphins Maintained Under Human Care, Italy.

The present study describes the isolation of Methicillin-resistant Staphylococcus aureus (MRSA) from respiratory tract of 2 dolphins of different origin, a stranded juvenile Risso's dolphin (Grampus griseus) and a captive born common bottlenose dolphin (Tursiops truncatus) calf, which died in the same institution at 1-month distance from the other. A complete microbiological and genetic investigation confirmed the presence of MRSA clone-complex 8, sequence type (ST) 8, spa-type t008 in both individuals. This strain differs from the one previously reported in walruses and dolphins and has never been described in dolphins before, but it is randomly isolated from Italian human patients. Vertical transmission of the infection may also occurs in other species and considering the description and location of the pathological lesions, this seems to be the most likely route of transmission implied in the young bottlenose dolphin. Staphylococcus aureus is known as an opportunistic agent, usually secondary to other pathogens, but its multiple antibiotic resistance and its zoonotic implications suggest a thorough and strict application of animal management hygiene protocols.

The bottlenose dolphin (Tursiops truncatus) faecal microbiota.

Cetaceans have evolved from herbivorous terrestrial artiodactyls closely related to ruminants and hippopotamuses. Delphinidae, a family included in this order, represent an extreme and successful re-adaptation of mammalian physiology to the marine habitat and piscivorous diet. The anatomical aspects of Delphinidae success are well understood, whereas some physiological aspects of their environmental fitness are less defined, such as the gut microbiota composition and its adaptation to their dietary niche. Here, we explored the faecal microbiota structure of nine adult bottlenose dolphins (Tursiops truncatus) and one breast-fed calf living in a controlled environment. According to our findings, dolphins possess a unique microbiota profile within the Mammalia class, highly resembling that of carnivorous marine fishes. The breast-fed calf showed a distinctive compositional structure of the gut microbial ecosystem, which partially overlaps with the mother's milk microbiota. Taken together, our data indicate that in dolphins the adaptation to the marine niche and piscivorous diet involved the convergence of their gut microbiota structure with that of marine fishes, overcoming the gut microbiota phylogenetic inertia previously described in terrestrial mammalians.

Hiatal hernia in a harbor seal (Phoca vitulina) pup.

A 2-wk-old stranded harbor seal (Phoca vitulina) rescued by Mystic Aquarium showed signs of the presence of a hiatal hernia during rehabilitation. Contrast radiographs of esophagus and stomach revealed an intrathoracic radiodensity that contains filling defects typical of stomach, consistent with gastric rugal folds. Mural thickening was observed at the level of the cardia consistent with a diagnosis of a hiatal hernia. Although clinical improvement was noted with medical therapy and tube feeding, surgical correction of the hiatal hernia was considered necessary for full resolution. However, owing to the animal's low body weight, the corrective hernia surgery was postponed until the body condition improved. The seal needed to be surgically treated for a corneal ulcer, and while anesthetized with isoflurane, the seal became dyspneic and developed cardiac arrhythmias; ultimately cardiac arrest ensued. Resuscitation was unsuccessfully attempted and the seal was euthanized. Necropsy confirmed the radiographic diagnosis and further characterized a paraesophageal hiatal hernia.

Ovarian interstitial cell tumor in a South American sea lion (Otaria flavescens).

A case of an ovarian tumor is reported in an 8-yr-old South American sea lion (Otaria flavescens) kept in a marine park in Malta (35.57 degrees N, 14.25 degrees E). The neoplasm was a solid mass of dense sheets and nests of round to polyhedral, irregularly shaped cells with abundant, finely vacuolated cytoplasm. The nuclei were uniformly small and round to oval. The supporting stroma contained thecal cells. The tumor cells were positive for positive inhibin and vimentin and focally positive for cytokeratin by immunohistochemistry. The neoplasm was diagnosed as an ovarian sex cord-stromal tumor, specifically an interstitial cell tumor.

Squamous cell carcinoma with presence of poxvirus-like inclusions in the foot of a pink-backed pelican (Pelecanus rufescens).

Squamous cell carcinoma (SCC) or avian keratoacanthoma is a neoplastic skin lesion of unknown aetiology that has been well described in birds. Some studies have reported that poxviruses may contribute to the onset of SCC. Here we describe a case of SCC on the underside of a pelican's foot. Histologically, the tumour consisted of irregular cords of pleomorphic epithelial cells that invaded the adjacent tissues. Additionally, keratinized epithelial cells and moderate numbers of keratin pearls were observed. Intracytoplasmic inclusions, a characteristic of this virus, were observed in some of these cells, and viral particles were characterized by electron microscopy. Although the aetiology of the carcinoma in this case may have been secondary to chronic focal trauma, the possibility of a latent or chronic form of fowlpox should be considered in the pathogenesis of the lesion.