Invisible Invertebrates: The Welfare of Invertebrates in Public Aquaria.

Awareness of welfare issues within animal collections is increasing as information becomes more accessible for staff and the public. A knowledge gap remains when considering the welfare of invertebrates, particularly when housed in public aquaria. TripAdvisor comments were analyzed for 485 worldwide aquariums. The public focused on anthropogenic features or charismatic organisms within collections. Invertebrate welfare was only presented in 18% of negative welfare comments compared with the 51% of represented vertebrates and 31% of negative general welfare comments. The UK and USA reported a greater number of perceived invertebrate welfare issues. Greater dissemination of information between aquarists and scientists should be encouraged to drive welfare standards and improve husbandry. In addition, incorporating input from invertebrate aquarists while utilizing welfare toolkits are vital for improving overall standards if we are to have greater representation of invertebrate welfare in public aquaria.

Visitors and observers otter-ly influence the behavior and enclosure use of zoo-housed giant otters.

The potential impact of human presence on captive animal behavior has recently been the focus of considerable research interest, especially following 2020 and 2021 periods of enforced closure as a result of COVID-19 opening restrictions. It is important to investigate whether human presence represents an enriching or stressful stimulus to a range of zoo-housed species. In 2020, during an easing of lockdown restrictions, investigations of the "visitor effect" and "observer effect" were carried out, using the giant otter (Pteronura brasiliensis) as a model species. To investigate the impact of both visitor and observer presence, otter behavior and space use was recorded for a pair of on-show and a pair of off-show otters. Observations were conducted using either a human observer, or cameras, allowing the researchers to investigate otter behavior when no one was present at the exhibits. The Electivity Index was used to assess the otters' use of four enclosure zones. Overall, otter behavior was significantly impacted by observer presence, though the impact of an observer differed between individual otters. Visitors had a minimal effect on otter enclosure use, whereas observers had a greater impact, whereby otters used their pools less frequently and houses more frequently when observers were present. However, this change in zone use differed between individuals, with more dominant otters tending to make use of indoor zones more often when observers were present. Zoos should consider the potential impact of human presence on their animals and use both behavior and space use when conducting their investigations.

Guidelines for the Care and Welfare of Cephalopods in Research -A consensus based on an initiative by CephRes, FELASA and the Boyd Group.

This paper is the result of an international initiative and is a first attempt to develop guidelines for the care and welfare of cephalopods (i.e. nautilus, cuttlefish, squid and octopus) following the inclusion of this Class of ∼700 known living invertebrate species in Directive 2010/63/EU. It aims to provide information for investigators, animal care committees, facility managers and animal care staff which will assist in improving both the care given to cephalopods, and the manner in which experimental procedures are carried out. Topics covered include: implications of the Directive for cephalopod research; project application requirements and the authorisation process; the application of the 3Rs principles; the need for harm-benefit assessment and severity classification. Guidelines and species-specific requirements are provided on: i. supply, capture and transport; ii. environmental characteristics and design of facilities (e.g. water quality control, lighting requirements, vibration/noise sensitivity); iii. accommodation and care (including tank design), animal handling, feeding and environmental enrichment; iv. assessment of health and welfare (e.g. monitoring biomarkers, physical and behavioural signs); v. approaches to severity assessment; vi. disease (causes, prevention and treatment); vii. scientific procedures, general anaesthesia and analgesia, methods of humane killing and confirmation of death. Sections covering risk assessment for operators and education and training requirements for carers, researchers and veterinarians are also included. Detailed aspects of care and welfare requirements for the main laboratory species currently used are summarised in Appendices. Knowledge gaps are highlighted to prompt research to enhance the evidence base for future revision of these guidelines.

Cephalopods in neuroscience: regulations, research and the 3Rs.

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of "live cephalopods" became regulated within the European Union by Directive 2010/63/EU on the "Protection of Animals used for Scientific Purposes", giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce "guidelines" and the potential contribution of neuroscience research to cephalopod welfare.

The effects of modulating eNOS activity and coupling in ischemia/reperfusion (I/R).

The in vivo role of endothelial nitric oxide synthase (eNOS) uncoupling mediating oxidative stress in ischemia/reperfusion (I/R) injury has not been well established. In vitro, eNOS coupling refers to the reduction of molecular oxygen to L-arginine oxidation and generation of L-citrulline and nitric oxide NO synthesis in the presence of an essential cofactor, tetrahydrobiopterin (BH(4)). Whereas uncoupled eNOS refers to that the electron transfer becomes uncoupled to L-arginine oxidation and superoxide is generated when the dihydrobiopterin (BH(2)) to BH(4) ratio is increased. Superoxide is subsequently converted to hydrogen peroxide (H(2)O(2)). We tested the hypothesis that promoting eNOS coupling or attenuating uncoupling after I/R would decrease H(2)O(2)/increase NO release in blood and restore postreperfused cardiac function. We combined BH(4) or BH(2) with eNOS activity enhancer, protein kinase C epsilon (PKC ε) activator, or eNOS activity reducer, PKC ε inhibitor, in isolated rat hearts (ex vivo) and femoral arteries/veins (in vivo) subjected to I(20 min)/R(45 min). When given during reperfusion, PKC ε activator combined with BH(4), not BH(2), significantly restored postreperfused cardiac function and decreased leukocyte infiltration (p < 0.01) while increasing NO (p < 0.05) and reducing H(2)O(2) (p < 0.01) release in femoral I/R veins. These results provide indirect evidence suggesting that PKC ε activator combined with BH(4) enhances coupled eNOS activity, whereas it enhanced uncoupled eNOS activity when combined with BH(2). By contrast, the cardioprotective and anti-oxidative effects of the PKC ε inhibitor were unaffected by BH(4) or BH(2) suggesting that inhibition of eNOS uncoupling during reperfusion following sustained ischemia may be an important mechanism.

The role of tetrahydrobiopterin and dihydrobiopterin in ischemia/reperfusion injury when given at reperfusion.

Reduced nitric oxide (NO) bioavailability and increased oxidative stress are major factors mediating ischemia/reperfusion (I/R) injury. Tetrahydrobiopterin (BH(4)) is an essential cofactor of endothelial NO synthase (eNOS) to produce NO, whereas dihydrobiopterin (BH(2)) can shift the eNOS product profile from NO to superoxide, which is further converted to hydrogen peroxide (H(2)O(2)) and cause I/R injury. The effects of BH(4) and BH(2) on oxidative stress and postreperfused cardiac functions were examined in ex vivo myocardial and in vivo femoral I (20 min)/R (45 min) models. In femoral I/R, BH(4) increased NO and decreased H(2)O(2) releases relative to saline control, and these effects correlated with improved postreperfused cardiac function. By contrast, BH(2) decreased NO release relative to the saline control, but increased H(2)O(2) release similar to the saline control, and these effects correlated with compromised postreperfused cardiac function. In conclusion, these results suggest that promoting eNOS coupling to produce NO and decrease H(2)O(2) may be a key mechanism to restore postreperfused organ function during early reperfusion.