1. Investigación

Platelet functionality plays a crucial role in marine mammals. Alterations in platelet function can result from stress, pathologies, or exposure to xenobiotics, among others. The early detection of platelet function abnormalities is essential in these species to prevent advanced pathology and mitigate potential risks. Our main objective was to establish a range of physiological values of platelet function in bottlenose dolphins (Tursiops truncatus), beluga whales (Delphinapterus leucas), sea lions (Otaria flavescens) and walruses (Odobenus rosmarus). Intraplatelet Ca2+ mobilization using adenosine diphosphate (ADP) as a platelet agonist was used as a platelet function biomarker, adapting the methodology previously described by us in dolphins (Felipo-Benavent et al., 2022) to the rest of the species. The assay was also adapted to a seal (Phoca vitulina). Numerical indicators of intraplatelet Ca2+ mobilization kinetics were established, and statistical analyses were performed to compare the effects of species, sex, age, aquarium and species. Significant differences were observed between species, being the platelets of the sea lions the more reactive to the agonist. This work demonstrates the usefulness of this assay in the diagnosis or monitoring of animals with hemostatic diseases, showing two clinical cases in which intraplatelet calcium mobilization values were altered in marine mammals suffering haemorrhages. This assay may also serve as a means to monitor environmental changes and their potential impact on the health of marine mammal populations.

To provide new insight into the pathophysiological mechanisms underlying gas emboli (GE) in bycaught loggerhead sea turtles (Caretta caretta), we investigated the vasoactive characteristics of the pulmonary and systemic arteries, and the lung parenchyma (LP). Tissues were opportunistically excised from recently dead animals for in vitro studies of vasoactive responses to four different neurotransmitters: acetylcholine (ACh; parasympathetic), serotonin (5HT), adrenaline (Adr; sympathetic) and histamine. The significant amount of smooth muscle in the LP contracted in response to ACh, Adr and histamine. The intrapulmonary and systemic arteries contracted under both parasympathetic and sympathetic stimulation and when exposed to 5HT. However, proximal extrapulmonary arterial (PEPA) sections contracted in response to ACh and 5HT, whereas Adr caused relaxation. In sea turtles, the relaxation in the pulmonary artery was particularly pronounced at the level of the pulmonary artery sphincter (PASp), where the vessel wall was highly muscular. For comparison, we also studied tissue response in freshwater sliders turtles (Trachemys scripta elegans). Both PEPA and LP from freshwater sliders contracted in response to 5HT, ACh and also Adr. We propose that in sea turtles, the dive response (parasympathetic tone) constricts the PEPA, LP and PASp, causing a pulmonary shunt and limiting gas uptake at depth, which reduces the risk of GE during long and deep dives. Elevated sympathetic tone caused by forced submersion during entanglement with fishing gear increases the pulmonary blood flow causing an increase in N2 uptake, potentially leading to the formation of blood and tissue GE at the surface. These findings provide potential physiological and anatomical explanations on how these animals have evolved a cardiac shunt pattern that regulates gas exchange during deep and prolonged diving.

During a 12 month period, a group of 14 medusa-stage jellies of the genus Chrysaora, including Pacific sea nettle (Chrysaora fuscescens, n = 11) and Japanese sea nettle (Chrysaora pacifica, n = 3), that were maintained in a public aquarium developed progressive ulcerative umbrellar lesions. In 6 cases (42.9%), ulceration was deep, transmural, and perforated through the mesoglea and subumbrella. In 6 cases (42.9%), ciliated protozoa histomorphologically consistent with scuticociliates were observed in the mesoglea and gastrovascular cavity. In 2 cases (14.3%), commensal dinoflagellates (zooxanthellae) were in the mesoglea and in the cytoplasm of the scuticociliates. During this period, water quality parameters including temperature [°C], pH, oxidation-reduction potential (ORP) [mV], salinity [psu], dissolved oxygen [%], ammonia (NH3), and nitrite (NO2) levels were monitored daily or weekly. The main water quality abnormalities were increased NO2 and pH levels above recommended reference ranges for C. fuscescens and elevated temperature above recommended reference ranges for C. pacifica tank. After correction of water quality parameters, apparent improvement of jellies was observed. In this case, environmental factors were considered the most likely predisposing factors for the development of ulcerative lesions, and ciliated protozoa were considered secondary rather than primary pathogens.

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 signaltransduction 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.

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 17 -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.