Dolphin-WET-Development of a Welfare Evaluation Tool for Bottlenose Dolphins (Tursiops truncatus) under Human Care.

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.

Pharmacokinetics of the analgesic and anti-inflammatory drug meloxicam after administration of multiple doses to nursehound sharks (Scyliorhinus stellaris).

OBJECTIVE: To determine the pharmacokinetics of meloxicam in the nursehound shark (Scyliorhinus stellaris) during multiple dose administration. STUDY DESIGN: Prospective experimental trial. ANIMALS: A total of eight clinically healthy adult nursehounds (four males, four females). METHODS: Meloxicam was administered intramuscularly at a dose of 1.5 mg kg-1 once daily for 7 days. Blood samples were collected from the caudal vein for pharmacokinetic analysis at 2.5 hours and 24 hours after drug administration. After a 4 week washout period, meloxicam was administered orally at the same dose at 12 hour intervals for three repeated doses. Blood samples were collected at 1, 2, 4, 6, 8, 12, 24, 36 and 48 hours after the first administration. Sharks were visually monitored during each study and 4 weeks afterwards for side effects or signs of toxicity. Time required to achieve steady state was assessed by visual inspection and statistical comparison of peak and trough concentrations using a Friedman test; comparison between sexes was performed using a Mann-Whitney U test and p-value was set at 0.05. RESULTS: No animal died or showed clinical signs of toxicity during the study. Meloxicam administered orally did not produce detectable concentrations in plasma. After intramuscular administration, steady state was achieved after five doses, and mean trough and peak plasma concentrations at steady state were 1.76 ± 0.21 µg mL-1 and 3.02 ± 0.23 µg mL-1, respectively. Mean peak concentration accumulation ratio was 2.50 ± 0.22. CONCLUSIONS AND CLINICAL RELEVANCE: This study shows that intramuscular posology produces plasma concentrations considered therapeutic for other species. However, meloxicam was not detected in plasma after oral administration. These results suggest that meloxicam administered intramuscularly may be a useful non-steroid anti-inflammatory drug in nursehound sharks. Further pharmacodynamic studies are needed to fully evaluate its clinical use in this species.

New insights into the pathogenesis and transmission of Brucella pinnipedialis: systemic infection in two bottlenose dolphins (Tursiops truncatus).

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.

Tension pneumothorax in small odontocetes.

Pneumothorax, the accumulation of air in the pleural cavity, occurs when air enters the pleural space by the pleuro-cutaneous, pleuro-pulmonary, or pleuro-oesophageal-mediastinal route. Tension pneumothorax is an infrequent and severe form of pneumothorax where a positive pressure in the pleural space is built up during at least part of the respiratory cycle, with compression of both lungs and mediastinal vessels, and, if unilateral, with midline deviation towards the unaffected hemithorax. We describe 9 cases of tension pneumothorax in 3 species of small cetaceans (striped dolphin Stenella coeruleoalba, common dolphin Delphinus delphis, and common bottlenose dolphin Tursiops truncatus) from the western Mediterranean coast of Spain, and one case from a dolphinarium. Computed tomography (CT) imaging performed in 2 carcasses before necropsy showed lung compression, midline deviation, and pressure on the diaphragm, which was caudally displaced. Tension pneumothorax was recognized at necropsy by the presence of pressurized air in one of the hemithoraces. Seven of the pneumothorax cases were spontaneous (2 primary and 5 secondary to previous lung pathology). In the other 2 dolphins, the pneumothorax was traumatic, due to oesophageal-pleural perforation or rib fractures. We hypothesize that pneumothorax in dolphins is predominantly tensional because of their specific anatomical and physiological adaptations to marine life and the obligate exposure to extreme pressure changes as diving mammals.

First description of a lesion in the upper digestive mucosa associated with a novel gammaherpesvirus in a striped dolphin (Stenella coeruleoalba) stranded in the Western Mediterranean Sea.

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).

New insights into risk variables associated with gas embolism in loggerhead sea turtles (Caretta caretta) caught in trawls and gillnets.

Tissue and blood gas embolism (GE) associated with fisheries bycatch are likely a widespread, yet underestimated, cause of sea turtle mortality. Here, we evaluated risk factors associated with tissue and blood GE in loggerhead turtles caught incidentally by trawl and gillnet fisheries on the Valencian coastline of Spain. Of 413 turtles (303 caught by trawl, 110 by gillnet fisheries), 54% (n = 222) exhibited GE. For sea turtles caught in trawls, the probability and severity of GE increased with trawl depth and turtle body mass. In addition, trawl depth and the GE score together explained the probability of mortality (P[mortality]) following recompression therapy. Specifically, a turtle with a GE score of 3 caught in a trawl deployed at 110 m had a P[mortality] of ~50%. For turtles caught in gillnets, no risk variables were significantly correlated with either the P[GE] or GE score. However, gillnet depth or GE score, separately, explained P[mortality], and a turtle caught at 45 m or with a GE score between 3 and 4 had a P[mortality] of 50%. Differences in the fishery characteristics precluded direct comparison of GE risk and mortality between these gear types. Although P[mortality] is expected to be significantly higher in untreated turtles released at sea, our findings can improve estimates of sea turtle mortality associated with trawls and gillnets, and help guide associate conservation efforts.

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets of bottlenose dolphins (Tursiops truncatus).

Marine mammals may suffer alterations in platelet function and hemostasia due to multiple pathologies, environmental conditions (including stress) or exposure to different contaminants that induce platelet activation. Detecting early alterations in platelet function in these animals could be an especially relevant diagnostic tool in these species because they typically do not show signs of weakness or disease until the pathology is in advanced state, in order to avoid attracting predators in natural conditions. The study of early markers of platelet activation is relevant for the detection, monitoring and therapy of inflammation and hemostasis disorders. Flow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular Ca2+ , using sensitive fluorescent indicators that can be loaded into intact cells. In order to study intraplatelet Ca2+ mobilization in marine mammals, we have adapted a kinetic assay of human platelet activation to study platelet activation in whole-blood samples of bottlenose dolphins (Tursiops truncatus) using the Ca2+ -sensitive dye Fluo-4AM and a clone of the platelet-specific antibody CD41-PE that recognizes dolphin platelets. This no-wash, no-lyse protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signal-transduction responses to platelet agonists under near-physiological conditions. The adaptation of this technique to marine mammals represents a methodological advance for basic and clinical veterinary applications but also for general environmental studies on these species.

Fungal Diseases in Elasmobranchs and Their Possible Treatment with a Special Mention to Azole Antifungal Agents.

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.

Hepatitis E Virus Infections in Free-Ranging and Captive Cetaceans, Spain, 2011-2022.

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.

HEMATOLOGY AND PLASMA BIOCHEMISTRY REFERENCE VALUES OF JUVENILE UNDULATE RAYS (RAJA UNDULATA) UNDER HUMAN CARE.

Despite the paucity of published literature on elasmobranch hematology and biochemistry, great interspecific diversity has been observed. Blood samples from 43 undulate rays (Raja undulata) (23 males, 20 females) hatched and raised at Oceanogràfic Aquarium, were analyzed for hematology and plasma biochemistry. Animals were divided into two age groups: 1 yr old (28 skates) and 2 yr old (15 skates). All individuals were clinically healthy on physical examination. Weight, total length, standard length, and disc width were recorded. No statistically significant differences were observed between male and female juvenile skates for the evaluated morphometric, hematologic, and plasma biochemical values. Once reference intervals (RI) were determined, blood samples from seven healthy adult skates housed at the same aquarium were collected for comparison. Statistically significant differences were observed in cholesterol, triglycerides, alkaline phosphatase, blood urea nitrogen, chloride, and sodium between juvenile and adult skates. This is the first article describing hematological and plasma biochemical RI for this species, increasing the clinical knowledge on elasmobranch blood analytics. These data will serve as a valuable diagnostic and research tool for professionals working with undulate rays and closer relatives in aquariums and in the field. Further studies using larger elasmobranch sample sizes are needed to determine reliable species-specific baseline health values and to evaluate the effect of intrinsic and extrinsic parameters on blood analytics more accurately.

Absence of atrial smooth muscle in the heart of the loggerhead sea turtle (Caretta caretta): a re-evaluation of its role in diving physiology.

Contraction of atrial smooth muscle in the hearts of semi-aquatic emydid turtles regulates ventricular filling, and it has been proposed that it could regulate stroke volume during characteristic rapid transitions in cardiac output associated with diving. For this hypothesis to be supported, atrial smooth muscle should be widely distributed in diving Testudines. To further understand the putative function and evolutionary significance of endocardial smooth muscle in Testudines, we studied the hearts of loggerhead sea turtles, Caretta caretta (n=7), using immunohistochemistry and histology. Surprisingly, we found no evidence of prominent atrial smooth muscle in C. caretta. However, smooth muscle was readily identified in the sinus venosus. Our results suggest that atrial smooth muscle does not contribute to the diving capabilities of C. caretta, indicating that the possible roles of smooth muscle in emydid turtle hearts require a re-evaluation. In sea turtles, the sinus venosus may instead contribute to regulate cardiac filling.

Increasing the data on elasmobranch plasma protein electrophoresis: electrophoretogram reference values determination in the undulate skate (Raja Undulata) and the nursehound shark (Scyliorhinus stellaris) maintained under human care.

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.

Hematology and Plasma Chemistry Reference Values in Nursehound Shark (Scyliorhinus Stellaris) Maintained Under Human Care.

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.

PHARMACOKINETICS OF MELOXICAM AFTER A SINGLE 1.5 MG/KG INTRAMUSCULAR ADMINISTRATION TO NURSEHOUND SHARKS (SCYLIORHINUS STELLARIS) AND ITS EFFECTS ON HEMATOLOGY AND PLASMA BIOCHEMISTRY.

A single-dose meloxicam pharmacokinetic (PK) study was performed with eight clinically healthy nursehound sharks (Scyliorhinus stellaris) maintained under human care. Meloxicam was administered IM at a dosage of 1.5 mg/kg to six animals; two animals were administered elasmobranch physiological saline solution (EPSS) IM as a negative control group. Blood samples were obtained prior to and at 12 predetermined times during the first 36 h after administration. Effects on hematology and plasma biochemistry were compared prior to and 24 h after administration. No animal died or showed clinical signs during the study. A significant increase in creatinine kinase and aspartate aminotransferase was found in both EPSS and meloxicam groups and could be considered a direct consequence of sampling and handling required for the PK study. Observed mean time to maximum plasma concentration ± SEM was 2.58 ± 0.47 h and observed mean maximum plasma concentration ± SEM was 806 ± 66 ng/ml; mean terminal half-life ± SEM was 15.97 ± 1.20 h; mean residency time ± SEM was 23.40 ± 2.25 h. Area under the plasma concentration-versus-time curve extrapolated to infinity ± SEM was 15.52 ± 1.70 h·µg/ml. This study suggests that meloxicam 1.5 mg/kg IM in nursehound sharks is likely to result in clinically relevant plasma levels for periods of 24 h without producing significant alterations in blood analytics, although further PK studies with meloxicam IV in sharks are needed. Future PK and pharmacodynamic studies with different drugs and doses are needed in elasmobranchs to establish safe and effective treatment protocols.

Antimicrobial Resistant Salmonella in Chelonians: Assessing Its Potential Risk in Zoological Institutions in Spain.

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.

Pharmacokinetics of the Anti-Inflammatory Drug Meloxicam after Single 1.5 mg/kg Intramuscular Administration to Undulate Skates (Raja undulata).

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.

Pharmacokinetic Studies in Elasmobranchs: Meloxicam Administered at 0.5 mg/kg Using Intravenous, Intramuscular, and Oral Routes to Nusehound Sharks (Scyliorhinus stellaris).

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.

Systematic Determination of Herpesvirus in Free-Ranging Cetaceans Stranded in the Western Mediterranean: Tissue Tropism and Associated Lesions.

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.

Comparative Study of Semen Parameters and Hormone Profile in Small-Spotted Catshark (Scyliorhinus canicula): Aquarium-Housed vs. Wild-Captured.

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 17ß-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.

OPTIMIZING A PLATELET-RICH PLASMA CONCENTRATION PROTOCOL FOR SOUTH AMERICAN SEA LIONS (OTARIA FLAVESCENS).

Accelerated healing in wild or captive South American sea lions (Otaria flavescens) is a key tool to help minimize infection and complications associated with open wounds, dental disease, and ocular pathology. Platelet-rich plasma (PRP) is an autogenous source for growth factors based on platelet concentration, which can be obtained by centrifuging whole blood collected in sodium citrate anticoagulant. Currently, there are well-defined PRP concentration protocols for humans and most domestic companion animal species. However, there is no clear centrifugation protocol for obtaining PRP in most marine mammal species. This study aimed to optimize the platelet concentration protocol based on whole blood centrifugation using speeds ranging from 500 to 5,000 rpm and times ranging from 3 to 6 min. Blood was drawn from seven adult South American sea lions, placed into 1-ml sodium citrate tubes, and centrifuged following 12 different centrifugation protocols. PRP was designated as the lower third fraction of the centrifuged plasma. Platelet counts were performed using flow cytometry and statistical analysis was carried out to establish a well-defined protocol for efficient PRP production. Transmission electron microscopy (TEM) analysis was performed to evaluate possible platelet degranulation during the different centrifugation protocols and measure platelet areas. Maximum concentration of platelets in PRP was 4.73-fold higher than the number of platelets in equal volume of whole blood, and significant differences in the concentrations obtained were found between the 12 centrifugation protocols evaluated using different speed and time combinations. The best one-step centrifugation protocol resulted from using 900-rpm speed for 3 min. The highest-fold increase was achieved using a two-step centrifugation protocol, which combined the most efficient one-step centrifugation protocol (900 rpm, 3 min) with a second centrifugation using 2,000-rpm speed for 6 min. TEM analysis confirmed that platelets were complete and maintained integrity after the proposed protocol.