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Multiple techniques have been used for estimating age in bottlenose dolphins (Tursiops truncatus). The longest established technique is via counts of growth later groups (GLGs) in the longitudinal section of an extracted mandibular tooth. Previous studies to validate GLGs have primarily used younger dolphins (less than 16yrs old) due to the limited number of known age older animals. This study assessed the accuracy of age estimates for bottlenose dolphins using both GLG analysis and a newer technique, pectoral flipper radiography (PFR) for dolphins ranging from 0 to 58yrs, with a majority of samples (70%) over 16yrs. GLGs were assessed by two expert independent readers on tooth sections from 52 different dolphins (85% over 16yrs, maximum age 58yrs), and assessments of PFRs were performed by two experienced veterinarians on 37 dolphins (54% over 16yrs, maximum age 54yrs). Results demonstrated both techniques became less accurate in older dolphins, particularly those > 30yrs, with errors as large as 37yrs for GLG estimates. The root mean squared error (RMSE) for age estimation using GLGs for dolphins ≤15 years of age was 1.46yrs compared to 1.58yrs using PFRs demonstrating the accuracy of both techniques in this age bracket. For dolphins >30yrs, RMSE increased to 17yrs in GLG aging and 8.25yrs in PFR. Challenges in obtaining accurate age estimates in GLGs were primarily due to sectioning and staining difficulties in visualizing the newest GLG layers, in the very old animals. Complications in reading the layers included obtaining a good section, the presence of accessory layers, GLG compression, and tooth curvature or wear removing the neonatal line influencing aging biases. In pectoral flipper aging, the primary challenge was obtaining a true dorsoventral radiograph in a live dolphin during a health assessment, as well as the subjective assessment of scoring and reliance on degenerative changes accurately estimate age in geriatric dolphins. While access to the radiography equipment can be a limiting factor, the improved accuracy for age estimation in adult dolphins, the less invasive nature, and reduced processing time to results make pectoral radiography a preferred alternative technique for estimating age in live dolphins.

Abstract Incidental capture, or ‘bycatch’ in fishing gear is a major global threat to sea turtle populations. A recent study showed that underwater entrapment in fishing gear followed by rapid decompression may cause gas bubble formation within the blood stream (embolism) and tissues leading to organ injury, impairment, and even mortality in some bycaught individuals. We analyzed data from 128 capture events using logistic and ordinal regression to examine risk factors associated with gas embolism in sea turtles captured in trawls and gillnets. Likelihood of fatal decompression increases with increasing depth of gear deployment. A direct relationship was found between depth, risk and severity of embolism, which has not been previously demonstrated in any breath-hold diving species. For the trawl fishery in this study, an average trawl depth of 65 m was estimated to result in 50% mortality in by-caught turtles throughout the year. This finding is critical for a more accurate estimation of sea turtle mortality rates resulting from different fisheries and for devising efforts to avoid or minimize the harmful effects of capture.

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), considered a zoonotic agent of wildlife origin, can infect various animal species, including wildlife in free-range and captive environments. Detecting susceptible species and potential reservoirs is crucial for preventing the transmission, spread, genetic evolution, and further emergence of viral variants that are major threats to global health. This study aimed to detect exposure or acute infection by SARS-CoV-2 in 420 animals from 40 different wildlife species, including terrestrial and aquatic mammals, from different regions of Spain during the 2020–2023 coronavirus disease 19 (COVID-19) pandemic. In total, 8/137 animals were positive for SARS-CoV-2 antibodies against the receptor binding domain and/or viral nucleoprotein according to independent ELISAs. However, only one ELISA-positive sample of a captive bottlenose dolphin (Tursiops truncatus) tested positive for SARS-CoV-2 neutralizing antibodies with a low titre (SNT50 38.15) according to a virus neutralization test. Cetaceans are expected to have a high risk of infection with SARS-CoV-2 according to early predictive studies due to the similarity of their angiotensin converting enzyme 2 cell receptor to that of humans. Moreover, of 283 animals analysed for SARS-CoV-2 RNA using RT-qPCR, none tested positive. Our results reinforce the importance of considering cetaceans at risk for SARS-CoV-2 infection and support taking preventive biosecurity measures when interacting with them, especially in the presence of individuals with suspected or confirmed COVID-19. Although most animals in this study tested negative for acute infection or viral exposure, ongoing surveillance of wildlife species and potentially susceptible animals is important to prevent future spillover events and detect potential novel reservoirs.

In chondrichthyans, despite males in ex situ conservation programs exhibiting good sperm quality, offspring production is confined to specific species. This study hypothesizes that the aquarium's environmental footprint may contribute to this loss of reproductive capacity. Consequently, the research initially focused on comparing the blood metabolomic profiles of the Small-Spotted Catshark (Scyliorhinus canicula) in both an aquarium environment at Oceanogràfic of Valencia and in wild populations along the Valencia Region coast (Spain). Furthermore, the study compared the blood metabolomic profiles with those of seminal plasma. This comparison aimed to identify potential variations in seminal plasma composition and its relationship with in vitro seminal characteristics. Samples were collected from S. canicula present in the aquarium (n = 7) and wild captured individuals (n = 12). Despite conventional semen quality assessments showing no significant disparities in semen volume and sperm concentration, considerable differences were observed in sperm motility and viability rates. Specifically, aquarium specimens showed slightly reduced metrics. The untargeted metabolic study in blood revealed significant differences between aquarium and wild individuals, with 158 semi-polar and 233 non-polar differential metabolites. A parallel analysis of seminal plasma similarly revealed substantial differences, with 2 semi-polar and 74 non-polar differential metabolites. Additionally, the analysis showed 104 semi-polar and 188 non-polar metabolites shared between the blood and seminal plasma of both animal groups. A further targeted analysis of 28 metabolites in blood identified significantly lower concentrations of phenylalanine, tyrosine, lysine, taurine, arginine, N-acetyl-L-glutamate, and creatinine in aquarium sharks. In seminal plasma samples, aquarium animals exhibited elevated levels of 7-Dehydrodesmosterol but reduced levels of phenylalanine, pantothenic acid (Vit B5), and spermine. In conclusion, these findings highlight alterations in both the blood and seminal plasma metabolomic profiles between aquarium-housed and wild animals, demonstrating the impact of the aquarium's environmental footprint, which could partly explain the reduced reproductive success of sharks in captivity.

IntroductionFungal diseases are frequently associated with elevated mortality rates in elasmobranchs. Currently, there is a notable absence of scientifically validated therapeutic medications that can ensure both effectiveness and safety when administered to this group of animals. The empirical prescription of azole antifungal agents, particularly voriconazole, has been posited as a potentially efficacious treatment approach for addressing most common mycoses in sharks and rays. However, there are still no published pharmacokinetic studies supporting its use in elasmobranchs and there is a lack of scientific base for its utilization in elasmobranchs.MethodsFor this study, voriconazole was administered intravenously (IV) and intramuscularly (IM), at a single dose of 4 mg/kg to six adult undulate skates (Raja undulata). A washout period of 8 weeks was left between each route of administration. Blood samples were collected both before and at ten predetermined intervals after each dosing (0.25, 0.5, 1, 1.5, 2, 4, 8, 12, 24, and 36 h after drug administration). Plasma concentrations were quantified using a validated high-performance liquid chromatography method, and pharmacokinetic (PK) data was analyzed through non-compartmental methods.ResultsThe mean extrapolated concentration at 0 h (C0) after IV administration was 27.19 ± 7.15 μg/mL and the mean peak plasma concentrations (Cmax) ± SEM after IM administration resulted 2.98 ± 0.28 μg/mL at a mean time to maximum concentration (T max) of 1.33 ± 0.17 h. Terminal half-lives were calculated and resulted 11.18 ± 1.32 h for IV injections and 9.59 ± 1.38 h for IM injections. The area under the curve extrapolated to infinity was determined as 58.14 ± 2.79 h·μg/ml following IV injections and 37.60 ± 6.67 h·μg/ml following IM injections. The IM-administered voriconazole exhibited a mean absolute bioavailability of 64.67 ± 11.47%.DiscussionThese discoveries provide backing for the possible application of voriconazole through the intramuscular route in undulate skates and support using lower dosage regimens compared to those required for oral administration, emphasizing the importance of conducting further pharmacokinetic studies with antifungals in elasmobranchs.

In human medicine, various pathologies, including decompression sickness, thrombocytopenia, and rheumatoid arthritis, have been linked to changes in cellular microparticles (MP) formation, particularly platelet microparticles (PMP). Similar disorders in marine mammals might be attributed to anthropogenic threats or illnesses, potentially impacting blood PMP levels. Thus, detecting platelet phosphatidylserine (PS) exposure and PMP formation could serve as a crucial diagnostic and monitoring approach for these conditions in marine mammals. Our group has developed a methodology to assess real-time PS exposure and PMP formation specifically tailored for marine mammals. This method, pioneered in species such as bottlenose dolphins, beluga whales, walruses, and California sea lions, represents a novel approach with significant implications for both clinical assessment and further research into platelet function in these animals. The adapted methodology for evaluating PS exposure and PMP formation in marine mammals has yielded promising results. By applying this approach, we have observed significant correlations between alterations in PMP levels and specific pathologies or environmental factors. These findings underscore the potential of platelet function assessment as a diagnostic and monitoring tool in marine mammal health. The successful adaptation and application of this methodology in marine mammals highlight its utility for understanding and managing health concerns in these animals.

The study of the immune function in marine mammals is essential to understand their physiology and can help to improve their welfare in the aquariums. Dedicating efforts to studying marine mammal physiology, pathophysiology, and implementing new diagnostic and therapeutic tools promote progress towards preventive medicine in aquariums by facilitating early detection and treatment of diseases. However, biological and clinical research on marine mammals is currently very limited due to difficult access to these species and their biological samples. With this objective, our group has adapted to marine mammals a commercially available assay routinely used to evaluate the phagocytic capacity of monocytes and granulocytes in human whole blood samples. We adapted IngoflowEx kit to bottlenose dolphins (Tursiops truncatus), beluga whales (Delphinapterus leucas), walruses (Odobenus rosmarus), Patagonian sea lions (Otaria flavescens), and harbor (Phoca vitulina). In this paper, we report the modifications carried out on the original protocol for their correct functioning in marine mammals. We obtained physiological values of phagocytic capacity in each species after repeated sampling for 4 years in various individuals of each species. Specific results revealed that the % phagocytic cells that ingested E.coli in bottlenose dolphins were 59.6 ± 1.27, in walruses 62.6 ± 2.17, in sea lions 57.5 ± 4.3, and in beluga whales 61.7 ± 1.4. In the case of the % phagocytic cells producing respiratory burst in bottlenose dolphins were 34.2 ± 3.6, in walruses 36.3 ± 4.3, in sea lions 40.8 ± 10.2, and in beluga whales 26.3 ± 3.7. These preliminary results can be used as a reference to detect alterations in phagocytic capacity either by immunosuppression or by exacerbation of the response in infectious inflammatory processes. Clinical applicability of the assay was verified in two clinical cases in which Ingoflow was useful to detect immune alterations in two diseased individuals, before and after the onset of clinical signs.

Abstract Background A wide variety of lesions have been associated with herpesvirus in cetaceans. However, descriptions of herpesvirus infections in the digestive system of cetaceans are scarce. Case report A young female striped dolphin stranded in the Valencian Community (Spain) on the 6th August 2021. The animal showed external macroscopic lesions suggestive of an aggressive interaction with bottlenose dolphins (rake marks in the epidermis). Internally, the main findings included congestion of the central nervous system and multiple, well-defined, whitish, irregularly shaped, proliferative lesions on the oropharyngeal and laryngopharyngeal mucosa. Histopathology revealed lymphoplasmacytic and histiocytic meningoencephalitis, consistent with neuro brucellosis. The oropharyngeal and laryngopharyngeal plaques were comprised histologically of focally extensive epithelial hyperplasia. As part of the health surveillance program tissue samples were tested for cetacean morbillivirus using a real-time reverse transcription-PCR, for Brucella spp. using a real-time PCR, and for herpesvirus using a conventional nested PCR. All samples were negative for cetacean morbillivirus; molecular positivity for Brucella spp. was obtained in pharyngeal tonsils and cerebrospinal fluid; herpesvirus was detected in a proliferative lesion in the upper digestive mucosa. Phylogenetic analysis showed that the herpesvirus sequence was included in the Gammaherpesvirinae subfamily. This novel sequence showed the greatest identity with other Herpesvirus sequences detected in skin, pharyngeal and genital lesions in five different species. Conclusions To the best of the authors’ knowledge, this is the first report of a proliferative lesion in the upper digestive mucosa associated with gammaherpesvirus posititvity in a striped dolphin (Stenella coeruleoalba).

Society is showing a growing concern about the welfare of cetaceans in captivity as well as cetaceans in the wild threatened by anthropogenic disturbances. The study of the physiological stress response is increasingly being used to address cetacean conservation and welfare issues. Within it, a newly described technique of extracting cortisol from epidermal desquamation may serve as a non-invasive, more integrated measure of a cetacean’s stress response and welfare. However, confounding factors are common when measuring glucocorticoid hormones. In this study, we validated a steroid hormone extraction protocol and the use of a commercial enzyme immunoassay (EIA) test to measure cortisol concentrations in common bottlenose dolphin (Tursiops truncatus) and beluga (Delphinapterus leucas) epidermal samples. Moreover, we examined the effect of sample mass and body location on cortisol concentrations. Validation tests (i.e., assay specificity, accuracy, precision, and sensitivity) suggested that the method was suitable for the quantification of cortisol concentrations. Cortisol was extracted from small samples (0.01 g), but the amount of cortisol detected and the variability between duplicate extractions increased as the sample mass decreased. In common bottlenose dolphins, epidermal skin cortisol concentrations did not vary significantly across body locations while there was a significant effect of the individual. Overall, we present a contribution towards advancing and standardizing epidermis hormone assessments in cetaceans.

In the ex situ conservation of chondrichthyan species, successful reproduction in aquaria is essential. However, these species often exhibit reduced reproductive success under human care. A key aspect is that conventional sperm analyses do not provide insights into the functional competence of sperm. However, proteomics analysis enables a better understanding of male physiology, gaining relevance as a powerful tool for discovering protein biomarkers related to fertility. The present work aims to build the first proteome database for shark semen and to investigate the proteomic profiles of seminal plasma and spermatozoa from small-spotted catsharks (Scyliorhinus canicula) related to the underlying adaptations to both natural and aquarium environments, thereby identifying the reproductive impact in aquarium specimens. A total of 305 seminal plasma and 535 spermatozoa proteins were identified. Among these, 89 proteins (29.2% of the seminal plasma set) were common to both spermatozoa and seminal plasma. In the seminal plasma, only adenosylhomocysteinase protein showed differential abundance (DAP) between wild and aquarium animals. With respect to the spermatozoa proteins, a total of 107 DAPs were found between groups. Gene Ontology enrichment analysis highlighted the primary functional roles of these DAPs involved in oxidoreductase activity. Additionally, KEGG analysis indicated that these DAPs were primarily associated with metabolic pathways and carbon metabolism. In conclusion, we have successfully generated an initial proteome database for S. canicula seminal plasma and spermatozoa. Furthermore, we have identified protein variations, predominantly within spermatozoa, between aquarium and wild populations of S. canicula. These findings provide a foundation for future biomarker discovery in shark reproduction studies. However, additional research is required to determine whether these protein variations correlate with reproductive declines in captive sharks.

ABSTRACT The emergence of Brucella infections in marine mammals is a growing concern. The present study reports two cases of systemic Brucella pinnipedialis infection detected in bottlenose dolphins (Tursiops truncatus) pair stranded together in the Cantabrian coast of Spain. Both animals showed systemic lesions associated with the Brucella infection, more severe in the younger dolphin, considered the likely offspring of the other individual. Real-time PCR, bacterial culture, and whole-genome sequencing were used to detect and characterize the Brucella strains involved in both dolphins. The phylogenetic analysis performed on the Brucella genomes retrieved revealed that the species involved was B. pinnipedialis (ST25). Both animals resulted seropositive in a commercial multispecies blocking ELISA but tested negative in the standard Rose Bengal test. To the best of our knowledge, this is the first report of a systemic infection resulting in various lesions associated with Brucella pinnipedialis (ST25) in two bottlenose dolphins. It is also the initial isolation of Brucella in the milk of a non-pregnant or non-aborting female cetacean likely stranded with its offspring. These findings provide new insights into the epidemiology and clinical impact of B. pinnipedialis infection in cetaceans and underscore the importance of continued diagnostic surveillance to gain better understanding of brucellosis effects and transmission in marine mammal populations. IMPORTANCE Brucella spp. are zoonotic pathogens that can affect both terrestrial and marine mammals. Brucella ceti has been identified in various cetacean species, but only one sequence type (ST27) has been reported in humans. However, it is important to conduct surveillance studies to better understand the impact of marine Brucella species on marine mammals, a typically understudied host group. Here, we describe a systemic infection by two related strains of Brucella pinnipedialis (ST25) in a couple of live-stranded bottlenose dolphins, with more severe lesions in the younger animal. Furthermore, B. pinnipedialis was first detected in milk from a female cetacean that stranded with its offspring. Our study reveals novel insights into the epidemiology and pathological consequences of B. pinnipedialis infections in cetaceans, emphasizing the crucial importance of ongoing surveillance and accurate diagnosis to understand the impact of this pathogen on marine mammal populations.

Epidemiologic surveillance of hepatitis E virus in over 300 free-ranging and captive cetaceans in waters off Spain revealed extensive exposure to this pathogen. We suggest the persistent and widespread presence of hepatitis E in the marine environment off the coast of Spain may be driven by terrestrial sources of contamination.

Age parameters in cetaceans allow examining conservation and studying individuals with growth affection. The age and sexual maturity of 51 stranded Stenella coeruleoalba striped dolphins from the Eastern Tropical Pacific (ETP) of Costa Rica, most suffering brucellosis (95.6%), were assessed. In order to ascertain the dolphins’ ages, we measured the length and growth of dentin-layer group counts (GLGs) and assessed flipper bone radiography without (FBSA) and with a formula (FBF). Sexual maturity was determined through gonadal histology and sexual hormone serum levels. Compared with a model based on S. coeruleoalba ages estimations in other latitudes, the striped dolphin studied displayed deficient growth parameters, with considerable variability in length, teeth, and flippers bone development. Close to 43% (n = 15) of GLGs’ measurements were below the body length average ranges for the predicted age, suggesting developmental abnormalities. Likewise, 34.4% and 31.2% of the dolphins assessed by FBSA and FBF were also below the body length based on age prediction curves, also indicating developmental abnormalities. This information is supported by the poor correlation between GLGs, FBSA, and FBF. Inconsistencies between sexually mature males and females related to GLGs, FBSA, and FBF were evident. Although the different oceanic settings of the ETP, such as contamination, food access, diseases, and other parameters, may influence size variation, our data also suggest that long-lasting debilitating brucellosis may account for detrimental growth in the ETP striped dolphins. Our study highlights the possible deleterious consequences of chronic infectious diseases in the cetacean populations already confronting distressful conditions.

Abstract Background This study determined plasma protein electrophoresis (PPE) reference intervals in two elasmobranch species: the undulate skate (Raja undulata) and the nursehound shark (Scyliorhinus stellaris), using a reference population of 48 undulate skates (27 males, 21 females) and 62 nursehounds (32 males, 30 females), considered to be clinically healthy. Plasma samples were analyzed using capillary zone electrophoresis (CZE). Results The undulate skate electrophoretogram resembled those previously reported in other batoids and could be divided into seven consistent fractions. No statistically significant differences were detected between sexes and developmental stages. The nursehound electrophoretogram was similar to that previously described in other shark species and could be divided into eight consistent fractions. Fraction 5% was significantly higher in juvenile nursehounds when compared to adults, while fraction 6 concentration and percentage were significantly higher in adults. Fraction 4% was higher in males than in females. Albumin band was not detected, and pre-albumin was negligible in both studied species. Alpha-globulins were predominant in the undulate skate, while beta-globulins were predominant in nursehounds. Statistically significant differences were found in all electrophoretogram fraction percentages and concentrations between the two species. Conclusion To the authors knowledge, this is the first study reporting PPE values in undulate skates and nursehounds, and the first study using CZE in elasmobranch plasma. These findings can serve as a primary reference for health monitoring in both species and will add to the limited data available on PPE in elasmobranchs.

Ensuring high standards of animal welfare is not only an ethical duty for zoos and aquariums, but it is also essential to achieve their conservation, education, and research goals. While for some species, animal welfare assessment frameworks are already in place, little has been done for marine animals under human care. Responding to this demand, the welfare committee of the European Association for Aquatic Mammals (EAAM) set up a group of experts on welfare science, cetacean biology, and zoo animal medicine across Europe. Their objective was to develop a comprehensive tool to evaluate the welfare of bottlenose dolphins (Tursiops truncatus), named Dolphin-WET. The tool encompasses 49 indicators that were either validated through peer review or management-based expertise. The first of its kind, the Dolphin-WET is a species-specific welfare assessment tool that provides a holistic approach to evaluating dolphin welfare. Inspired by Mellor’s Five Domains Model and the Welfare Quality®, its hierarchical structure allows for detailed assessments from overall welfare down to specific indicators. Through combining 37 animal-based and 12 resource-based indicators that are evaluated based on a two- or three-level scoring, the protocol offers a detailed evaluation of individual dolphins. This approach allows for regular internal monitoring and targeted welfare management, enabling caretakers to address specific welfare concerns effectively.

The microbiome is a crucial influencer in animal development, immune function and health, and it has complex and dynamic interactions with the environment, but little is known about the microbial signatures of inner body fluids. Recent evidence suggests that inner body fluids could be an indicator of the environmental interactions that fish experience. In the present study, we provide a comparative analysis of the microbial profile found in small-spotted catsharks’ blood plasma and seminal plasma and how microbial signatures vary between aquarium and wild animals. In the blood plasma microbiome, the habitat did not affect the α- and β-diversity, while in seminal plasma, both α- and β-diversity differed between both habitats. Proteobacteria are the main bacteria dominated independently the inner body fluid and habitats. No core microbiome was identified at the genus level, with only Pseudomonas and Cloacibacterium present in both inner body fluids and habitats. Of the 14 genera identified in blood plasma, only four were shared between habitats (making up 45.17% and 51.03% of relative abundance for wild and aquarium, respectively). Similarly, of the 100 genera identified in seminal plasma, only 41 were shared between habitats (84.79% and 64.83%, respectively). Moreover, in the seminal plasma, using ANCOM approaches, Serratia, Salinisphaera and Cutibacterium were found significantly enriched in aquarium animals. None potentially pathogenic bacteria were identified in the blood samples, while Coxiella, Prevotella, Coprococcus, Haemophilus and Phocoenobacter were potentially pathogenic bacteria identified in the seminal plasma samples. In summary, this study provides evidence of a circulating blood and seminal plasma microbiome in healthy small-spotted catsharks. Furthermore, dynamic changes were observed in the microbiome of these inner body fluids, which differed between the aquarium and wild habitats.

Introduction: Elasmobranchs currently constitute an important part of the animal collection of many aquariums worldwide. Their maintenance under human care has allowed us to describe and identify new pathogens and diseases affecting them, as well as to determine different treatments for these diseases. Great advances in elasmobranch husbandry have been developed. Methods: A search was performed on scientific databases as PubMed and other specialized sources (IAAAM archive). Results: Little information on pharmacotherapeutics is available in this taxonomic group, and treatments lack a scientific base and instead are frequently dependent on empirical knowledge. Pharmacokinetic studies are the first step to determining therapeutic protocols that are safe and effective. The available bibliography shows that a majority of the mycoses recorded in cartilaginous fish are severe, aggravated by the fact that the antifungal treatments administered, following the guidelines used for teleost species, are ineffective in elasmobranchs. Azoles appear to be a promising group of antifungals for use in treating systemic mycoses in sharks and rays. Conclusions: Based on the findings of this review, it is essential to investigate the pharmacokinetics of the different antifungals in these species in order to provide therapeutic options for fungal infections in cartilaginous fish.

Studies determining baseline hematological reference intervals (RI) in elasmobranchs are very limited. In this study, blood samples were collected from 94 clinically healthy Nursehound Shark (Scyliorhinus stellaris) maintained under human care. Median (RI) in major leukocyte types were similar to other Carcharhinid sharks as lymphocytes were the predominant leukocyte with 38.0 (28.2–53.5)%, followed by coarse eosinophilic granulocytes with 20.0 (12.2–31.7)%, fine eosinophilic granulocytes with 6.0 (1.2–12.8) %, monocytes with 2.0 (0.0–6.0)%, and neutrophils with 2.0 (0.0–6.0)%. Nursehound Shark produced granulated thrombocytes, which were classified as granulocytes and represented 28.5 (12.4–39.7)% of all leukocytes. Median (RI) manual red blood cell and white blood cell counts were 177.50 (132.50–210.00) x 109 cells/l and 8.26 (5.24–14.23) x 109 cells/l, respectively. Median (RI) plasma chemistry values showed alkaline phosphatase 7.7 (4.2–13.0) U/l, aspartate aminotransferase 7.6 (3.3–17.1) U/l, blood urea nitrogen 281.6 (261.2–305.0) mmol/l, calcium 3.97 (3.59–4.47) mmol/l, total cholesterol 2.04 (1.02–3.91) mmol/l, chloride 233.0 (215.2–259.0) mmol/l, iron 3.79 (1.74–6.93) μmol/l, glucose 0.87 (0.47–1.44 mmol/l), potassium 3.8 (2.9–4.6) mmol/l, sodium 243.0 (227.7–271.0) mmol/l, phosphorus 1.58 (1.13–2.10) mmol/l, total protein 24.0 (20.0–35.0) g/l, and triglycerides 0.97 (0.49–3.35) mmol/l. Creatine kinase, gamma glutamyl transferase, and lactate dehydrogenase levels were below the instrument reading range.

Abstract Background Herpesvirus infections in cetaceans have always been attributed to the Alphaherpesvirinae and Gammaherpesvirinae subfamilies. To date, gammaherpesviruses have not been reported in the central nervous system of odontocetes. Case presentation A mass stranding of 14 striped dolphins (Stenella coeruleoalba) occurred in Cantabria (Spain) on 18th May 2019. Tissue samples were collected and tested for herpesvirus using nested polymerase chain reaction (PCR), and for cetacean morbillivirus using reverse transcription-PCR. Cetacean morbillivirus was not detected in any of the animals, while gammaherpesvirus was detected in nine male and one female dolphins. Three of these males were coinfected by alphaherpesviruses. Alphaherpesvirus sequences were detected in the cerebrum, spinal cord and tracheobronchial lymph node, while gammaherpesvirus sequences were detected in the cerebrum, cerebellum, spinal cord, pharyngeal tonsils, mesenteric lymph node, tracheobronchial lymph node, lung, skin and penile mucosa. Macroscopic and histopathological post-mortem examinations did not unveil the potential cause of the mass stranding event or any evidence of severe infectious disease in the dolphins. The only observed lesions that may be associated with herpesvirus were three cases of balanitis and one penile papilloma. Conclusions To the authors’ knowledge, this is the first report of gammaherpesvirus infection in the central nervous system of odontocete cetaceans. This raises new questions for future studies about how gammaherpesviruses reach the central nervous system and how infection manifests clinically.

Infectious and inflammatory diseases are the most frequently diagnosed pathologies in elasmobranchs maintained under human care. Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used in veterinary medicine for their anti-inflammatory, analgesic, and antipyretic properties. Meloxicam is a commonly prescribed NSAID in elasmobranchs, but there are still no published pharmacokinetic (PK) studies supporting its use in this group of animals. In this study, meloxicam was administered at a single dose of 0.5 mg/kg to eight healthy adult nursehound sharks (Scyliorhinus stellaris) intravenously (IV), intramuscularly (IM), and orally (PO), with a minimum 4-week washout period between administrations. Blood samples were obtained both beforehand and at predetermined times after each administration. Plasma concentrations were measured using a validated high performance liquid chromatography method, and PK data was obtained using a non-compartmental analysis. Meloxicam administered orally did not produce detectable concentrations in blood plasma, while mean peak plasma concentration was 0.38 ± 0.08 μg/ml after IM administration. The mean terminal half-life was 10.71 ± 2.77 h and 11.27 ± 3.96 h for IV and IM injections, respectively. The area under the curve extrapolated to infinity was 11.37 ± 2.29 h·μg/ml after IV injections and 5.98 ± 0.90 h·μg/ml after IM injections. Meloxicam administered IM had a mean absolute bioavailability of 56.22 ± 13.29%. These numbers support meloxicam as a promising drug to be used IM in nursehounds, questions the efficacy of its single PO use in elasmobranchs, elucidate the need for higher dosage regimes, and evidence the need for further PK studies in sharks and rays.

Sea turtles, like other air-breathing diving vertebrates, commonly experience significant gas embolism (GE) when incidentally caught at depth in fishing gear and brought to the surface. To better understand why sea turtles develop GE, we built a mathematical model to estimate partial pressures of N2 (PN2), O2 (PO2), and CO2 (PCO2) in the major body-compartments of diving loggerheads (Caretta caretta), leatherbacks (Dermochelys coriacea), and green turtles (Chelonia mydas). This model was adapted from a published model for estimating gas dynamics in marine mammals and penguins. To parameterize the sea turtle model, we used values gleaned from previously published literature and 22 necropsies. Next, we applied this model to data collected from free-roaming individuals of the three study species. Finally, we varied body-condition and cardiac output within the model to see how these factors affected the risk of GE. Our model suggests that cardiac output likely plays a significant role in the modulation of GE, especially in the deeper diving leatherback turtles. This baseline model also indicates that even during routine diving behavior, sea turtles are at high risk of GE. This likely means that turtles have additional behavioral, anatomical, and/or physiologic adaptions that serve to reduce the probability of GE but were not incorporated in this model. Identifying these adaptations and incorporating them into future iterations of this model will further reveal the factors driving GE in sea turtles.

Proteins play an important role in many reproductive functions such as sperm maturation, sperm transit in the female genital tract or sperm-oocyte interaction. However, in general, little information concerning reproductive features is available in the case of aquatic animals. The present study aims to characterize the proteome of both spermatozoa and seminal plasma of bottlenose dolphins (Tursiops truncatus) as a model organism for cetaceans. Ejaculate samples were obtained from two trained dolphins housed in an aquarium. Spermatozoa and seminal plasma were analyzed by means of proteomic analyses using an LC-MS/MS, and a list with the gene symbols corresponding to each protein was submitted to the DAVID database. Of the 419 proteins identified in spermatozoa and 303 in seminal plasma, 111 proteins were shared by both. Furthermore, 70 proteins were identified as involved in reproductive processes, 39 in spermatozoa, and 31 in seminal plasma. The five most abundant proteins were also identified in these samples: AKAP3, ODF2, TUBB, GSTM3, ROPN1 for spermatozoa and CST11, LTF, ALB, HSP90B1, PIGR for seminal plasma. In conclusion, this study provides the first characterization of the proteome in cetacean sperm and seminal plasma, opening the way to future research into new biomarkers, the analysis of conservation capacity or possible additional applications in the field of assisted reproductive technologies.

Salmonella is mostly noted as a food-borne pathogen, but contact with chelonians has also been reported as a source of infection. Moreover, high levels of antimicrobial resistance (AMR) have been reported in Salmonella isolated from wild and captive reptiles. The aim of this study was to assess the occurrence of Salmonella AMR carriage by chelonians admitted to two zoological institutions in Spain, characterizing the isolates to assess the Salmonella AMR epidemiology in wildlife. To this end, 152 chelonians from nine species were sampled upon their arrival at the zoological nuclei. Salmonella identification was based on ISO 6579-1:2017 (Annex D), isolates were serotyped and their AMR analysed according to the EU Decision 2013/652. Moreover, the genetic relationship of the isolates was assessed by pulsed-field gel electrophoresis (PFGE). Results showed 19% (29/152) of the chelonians positive to Salmonella, all of them tortoises. For all isolates, 69% (20/29) were resistant and 34% (10/29) multidrug-resistant (MDR) strains. PFGE clustered isolates according to the serovar, confirming a low genetic diversity. In conclusion, this study shows a high presence of MDR Salmonella strains in tortoises at their entry into zoological nuclei. This condition highlights the need to establish Salmonella detection protocols for the entry of animals into these centres.

The therapy database currently used in elasmobranchs is still mostly based on empirical data, and there are few efficacy and safety studies supporting clinical practice. In this study, meloxicam pharmacokinetics (PK) were evaluated after a single 1.5 mg/kg IM administration to a group of seven clinically healthy adult undulate skates (Raja undulata Lacepède, 1802). Blood samples were collected before administration and at 15, 30, 60 and 90 min and 2, 4, 8, 12, 24 and 48 h after the IM injection. The meloxicam concentrations in plasma were determined using high-performance liquid chromatography, and PK parameters were calculated using a non-compartmental model approach. The mean ± SEM values of the main PK values were 1.84 ± 0.31 μg/mL for peak plasma concentration, 1.5 ± 0.24 h for time to maximum plasma concentration, 11.43 ± 2.04 h·µg/mL for area under the plasma concentration vs. time curve, 3.55 ± 0.65 h for elimination half-life, and 5.37 ± 0.94 h for mean residency time. No adverse reactions were detected. The relatively high plasma concentration and short time to maximum plasma concentration suggest that meloxicam could turn into an efficient analgesic and anti-inflammatory candidate drug to be used in skates. Further efficacy, pharmacodynamic, and multiple-dose studies with meloxicam are needed in elasmobranchs.

The recent finding of gas embolism (GE) and decompression sickness (DCS) in loggerhead sea turtles (Caretta caretta) in the Mediterranean Sea challenged the conventional understanding of marine vertebrate diving physiology. Additionally, it brought to light a previously unknown source of mortality associated with fisheries bycatch for this vulnerable species. In this paper, we use ultrasonography to describe GE in a leatherback sea turtle (Dermochelys coriacea), a green sea turtle (Chelonia mydas), and an olive ridley sea turtle (Lepidochelys olivacea) from accidental capture in a gillnet, bottom trawl, and pair-bottom trawl, respectively. This is the first description of this condition in these three species worldwide. These cases of GE suggest that this may be a threat faced by all sea turtle species globally.

Abstract Background In the last 20 years, Cetacean Morbillivirus (CeMV) has been responsible for many die-offs in marine mammals worldwide, as clearly exemplified by the three dolphin morbillivirus (DMV) epizootics of 1990–1992, 2006–2008 and 2011 that affected Mediterranean striped dolphins (Stenella coeruleoalba). Systemic infection caused by DMV in the Mediterranean has been reported only during these outbreaks. Results We report the infection of five striped dolphins (Stenella coeruleoalba) stranded on the Spanish Mediterranean coast of Valencia after the last DMV outbreak that ended in 2011. Animal 1 stranded in late 2011 and Animal 2 in 2012. Systemic infection affecting all tissues was found based on histopathology and positive immunohistochemical and polymerase chain reaction positive results. Animal 3 stranded in 2014; molecular and immunohistochemical detection was positive only in the central nervous system. Animals 4 and 5 stranded in 2015, and DMV antigen was found in several tissues. Partial sequences of the DMV phosphoprotein (P), nucleoprotein (N), and hemagglutinin (H) genes were identical for Animals 2, 3, 4, and 5, and were remarkably different from those in Animal 1. The P sequence from Animal 1 was identical to that of the DMV strain that caused the epizootic of 2011 in the Spanish Mediterranean. The corresponding sequence from Animals 2–5 was identical to that from a striped dolphin stranded in 2011 on the Canary Islands and to six dolphins stranded in northeastern Atlantic of the Iberian Peninsula. Conclusions These results suggest the existence of an endemic infection cycle among striped dolphins in the Mediterranean that may lead to occasional systemic disease presentations outside epizootic periods. This cycle involves multiple pathogenic viral strains, one of which may have originated in the Atlantic Ocean.

Several chondrichthyan species are threatened, and we must increase our knowledge of their reproductive biology in order to establish assisted reproductive protocols for ex situ or in situ endangered species. The small-spotted catshark (Scyliorhinus canicula) is one of the most abundant shark species of the Mediterranean coast and is easy to maintain in aquaria; therefore, it is considered an ideal reproductive model. This study aimed to compare S. canicula male reproductive function in aquarium-housed (n = 7) and wild-captured animals, recently dead (n = 17). Aquarium-housed animals had lower semen volume (p = 0.005) and total sperm number (p = 0.006) than wild-captured animals, but similar sperm concentrations. In terms of sperm parameters, aquarium-housed sharks showed higher total sperm motility (p = 0.004), but no differences were observed regarding sperm viability, mitochondrial membrane potential, or membrane integrity. A morphometric study pointed to a significantly longer head (p = 0.005) and acrosome (p = 0.001) in wild-captured animals. The results of the spermatozoa morphological study of S. canicula were consistent with previous results obtained in other chondrichthyan species. With regard to sex hormones, testosterone levels were significantly lower in aquarium-housed animals (p ≤ 0.001), while similar levels of 17β-estradiol and progesterone were found. In short, the present study provides evidence of good in vitro semen quality in S. canicula housed in an aquarium, underlining their excellent potential for application in reproductive technologies for this and other chondrichthyan species.

The monitoring of herpesvirus infection provides useful information when assessing marine mammals’ health. This paper shows the prevalence of herpesvirus infection (80.85%) in 47 cetaceans stranded on the coast of the Valencian Community, Spain. Of the 966 tissues evaluated, 121 tested positive when employing nested-PCR (12.53%). The largest proportion of herpesvirus-positive tissue samples was in the reproductive system, nervous system, and tegument. Herpesvirus was more prevalent in females, juveniles, and calves. More than half the DNA PCR positive tissues contained herpesvirus RNA, indicating the presence of actively replicating virus. This RNA was most frequently found in neonates. Fourteen unique sequences were identified. Most amplified sequences belonged to the Gammaherpesvirinae subfamily, but a greater variation was found in Alphaherpesvirinae sequences. This is the first report of systematic herpesvirus DNA and RNA determination in free-ranging cetaceans. Nine (19.14%) were infected with cetacean morbillivirus and all of them (100%) were coinfected with herpesvirus. Lesions similar to those caused by herpesvirus in other species were observed, mainly in the skin, upper digestive tract, genitalia, and central nervous system. Other lesions were also attributable to concomitant etiologies or were nonspecific. It is necessary to investigate the possible role of herpesvirus infection in those cases.

Accurate age estimation in wildlife conservation is an important diagnostic tool in the interpretation of biological data, necropsy examination, reproductive status and population demographics. The most frequently utilized methods to age bottlenose dolphins (Tursiops truncatus) include tooth extraction; counting dental growth layer groups and dental radiography. These methods are inaccurate in dolphins > 13 years old, due to overlapping of the growth layer groups in dolphins and worn teeth. Establishing a non-invasive method of accurately aging bottlenose dolphins across the entire age range is important to long term conservation efforts to understand health status, lifespan, reproduction and survivability. A database of 126 radiographs from 94 dolphins of known chronological age was utilized to establish the stages of skeletal ossification over time. A numerical score from -1 to 8 was assigned to 16 anatomic locations on the pectoral radiograph, to create a formula to estimate age. The most informative areas to evaluate morphologically were the metaphyseal regions of the radius and ulna, and the proximal and distal epiphysis of metacarpals II and III. Third order polynomial regression calculated separate age predictor formulas for male and female dolphins, with females reaching sexual maturity earlier than males. Completion of epiphyseal closure of the long bones correlated with average sexual maturity. Managed care dolphin ages could be properly estimated with decreasing precision from within 3 months in animals < 5 years old, to within 5 years in animals > 30 years old. This diagnostic tool could also be applied to diagnose atypical ossification patterns consistent with nutritional, developmental or growth abnormalities, and identifying subclinical health issues. In conclusion, knowledge of the lifespan and the onset of sexual maturity for each species will allow this model to be applied to other cetaceans, facilitating age estimation via pectoral radiography in future research.

Species of Balaenophilus are the only harpacticoid copepods that exhibit a widespread, obligate association with vertebrates, i.e., B. unisetus with whales and B. manatorum with marine turtles and manatees. In the western Mediterranean, juveniles of the loggerhead sea turtle, Caretta caretta are the only available hosts for B. manatorum, which has been found occurring at high prevalence (>80%) on them. A key question is how these epibionts are transmitted from host to host. We investigated this issue based on experiments with live specimens of B. manatorum that were cultured with turtle skin. Specimens were obtained from head-started hatchlings of C. caretta from the western Mediterranean. Hatched nauplii crawled only on rough substrates and lacked the ability to swim. Only copepodites IV and V, and adults, were able to perform directional swimming. Legs 2, 3 and 4 played a major role in swimming and were only well-developed in these stages. Nauplii reared in wells with turtle skin readily fed on this item. Late copepodites and adults also fed on turtle skin but did not consume other potential food items such as fish skin, baleen plates or planktonic algae. Evidences suggest that the transmission of B. manatorum should rely on hosts' bodily contacts and/or swimming of late developmental stages between spatially close hosts. The possibility of long-ranged dispersal is unlikely for two reasons. First, all developmental stages seem to depend on turtle skin as a food resource. Second, the average clutch size of ovigerous females was small (< 70 eggs) for free-living phases to successfully contact turtles that occur at very low densities (< 0.6 turtles·km-2) in the western Mediterranean. The high prevalence of B. manatorum in loggerhead turtles in this area raises the question whether these turtles have contacts, or tend to closely aggregate, more than is currently believed.

Dolphins exhibit an extraordinary capacity to heal deep soft tissue injuries. Nevertheless, accelerated wound healing in wild or captive dolphins would minimize infection and other side effects associated with open wounds in marine animals. Here, we propose the use of a biological-based therapy for wound healing in dolphins by the application of platelet-rich plasma (PRP). Blood samples were collected from 9 different dolphins and a specific and simple protocol which concentrates platelets greater than two times that of whole blood was developed. As opposed to a commonly employed human protocol for PRP preparation, a single centrifugation for 3 minutes at 900 rpm resulted in the best condition for the concentration of dolphin platelets. By FACS analysis, dolphin platelets showed reactivity to platelet cell-surface marker CD41. Analysis by electron microscopy revealed that dolphin platelets were larger in size than human platelets. These findings may explain the need to reduce the duration and speed of centrifugation of whole blood from dolphins to obtain a 2-fold increase and maintain proper morphology of the platelets. For the first time, levels of several growth factors from activated dolphin platelets were quantified. Compared to humans, concentrations of PDGF-BB were not different, while TGFβ and VEGF-A were significantly lower in dolphins. Additionally, adipose tissue was obtained from cadaveric dolphins found along the Spanish Mediterranean coast, and adipose-derived mesenchymal stem cells (ASCs) were successfully isolated, amplified, and characterized. When dolphin ASCs were treated with 2.5 or 5% dolphin PRP they exhibited significant increased proliferation and improved phagocytotic activity, indicating that in culture, PRP may improve the regenerative capacity of ASCs. Taken together, we show an effective and well-defined protocol for efficient PRP isolation. This protocol alone or in combination with ASCs, may constitute the basis of a biological treatment for wound-healing and tissue regeneration in dolphins.