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The objective was to evaluate industrial and research slaughter methods for Atlantic salmon (Salmo salar), gilt-head seabream (Sparus auratus) and eel (Anguilla anguilla) with respect to welfare and quality. As a general term of reference, an optimal slaughter method should render fish unconscious until death without avoidable excitement, pain or suffering prior to killing. For Atlantic salmon, commercial slaughter methods (carbon dioxide stunning followed by gill cutting, and gill cutting alone) are not in conformity with the general term of reference, as the fish are not rendered unconscious immediately and possibly experience stress. Evaluation of automated percussive stunning remained unconclusive. More research should enable us to ascertain whether loss of consciousness is instantaneous. Electrical stunning can be humane if applied properly. However, because flesh of electrostunned fish was characterized by occasional bloodspots, optimization of the electrical parameters is required. Prototypes for percussive and electrical stunning of salmon have been recently developed. This implies that humane slaughter of salmon is feasible for industry. For gilt-head seabream, neither aphyxia in air nor transfer of the fish to an ice slurry were considered to be humane: the methods did not induce immediate brain dysfunction and vigorous attempts to escape occurred. Percussive and electrical stunning can be in conformity with the general term of reference. However, conditions for stunning whole batches of seabream have not been established. Quality of the fish slaughtered by percussive stunning was similar to that obtained by the industrial method, i.e. immersion in an ice slurry. Further work is required to establish optimal stunning conditions and to develop prototypes. For eel, desliming in a salt-bath followed by evisceration, electrical stunning performed under the conditions prescribed by the German legislation, and live chilling and freezing were not considered to be humane. In contrast, it was established that a 10±20 kg batch of eels in fresh water could be rendered unconscious immediately and until death by applying electricity in combination with nitrogen gas. The conditions used were 0.64 A dm À2 for 1 s, followed by 0.17 A dm À2 combined with nitrogen flushing for 5 min. A preliminary assessment of flesh quality suggests that it may be improved by application of the latter method, compared with the salt bath. The results clearly indicated that humane slaughter of eels is possible in practice.

A method for the evaluation of brain function in fish has been developed which is based on assessments of self-initiated behaviours, responses to stimulation, and reflexes. These assessments were validated in several freshwater and marine species and applied to evaluate the brain function of fish while they were anaesthetised and while they were being killed by a variety of methods. The results of these investigations were compared with published neurophysiological observations and it is concluded that some of the tests can be used to identify, with reasonable confidence, the state of awareness, and thus the capacity to experience suffering of several species of fish.

We investigated how adult growth in Atlantic salmon (Salmo salar L.) was affected by changing embryonic temperature from fertilization until the completion of eye pigmentation. Fertilized eggs from several hundred families were divided between four temperature treatments (2, 5, 8 or 108C) and subsequently reared in identical conditions in replicated tanks. Fish exposed to 2 and 58C treatments were significantly smaller at smoltification than groups at higher temperatures, but showed substantial compensatory catch-up growth. Remarkably, temperature during this short window of embryogenesis dictated adult myogenic phenotype three years later with significant treatment effects on the muscle fibre final number (FFN), maximum diameter, nuclear density and size distribution. FFN was highest for the 58C treatment and was reduced at higher and lower treatment temperatures. Our results require direct temperature effects on embryonic tissues, such as the stem cell-containing external cell layer, in order to produce persistent effects on juvenile and adult growth.

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Influenza A viruses (IAVs) can jump species barriers and occasionally cause epidemics, epizootics, pandemics, and panzootics. Characterizing the infection dynamics at the target tissues of natural hosts is central to understanding the mechanisms that control host range, tropism, and virulence. Canine influenza virus (CIV; H3N8) originated after the transfer of an equine influenza virus (EIV) into dogs. Thus, comparing CIV and EIV isolates provides an opportunity to study the determinants of influenza virus emergence. Here we characterize the replication of canine, equine, and human IAVs in the trachea of the dog, a species to which humans are heavily exposed. We define a phenotype of infection for CIV, which is characterized by high levels of virus replication and extensive tissue damage. CIV was compared to evolutionarily distinct EIVs, and the early EIV isolates showed an impaired ability to infect dog tracheas, while EIVs that circulated near the time of CIV emergence exhibited a CIV-like infection phenotype. Inoculating dog tracheas with various human IAVs (hIAVs) showed that they infected the tracheal epithelium with various efficiencies depending on the virus tested. Finally, we show that reassortant viruses carrying gene segments of CIV and hIAV are viable and that addition of the hemagglutinin (HA) and neuraminidase (NA) of CIV to the 2009 human pandemic virus results in a virus that replicates at high levels and causes significant lesions. This provides important insights into the role of evolution on viral emergence and on the role of HA and NA as determinants of pathogenicity. IMPORTANCEInfluenza A viruses (IAVs) have entered new host species in recent history, sometimes with devastating consequences. Canine influenza virus (CIV) H3N8 originated from a direct transfer of an equine influenza virus (EIV) in the early 2000s. We studied the infection patterns of IAVs that circulate in dogs or to which dogs are commonly exposed and showed that CIV emergence was likely caused by an adaptive driver, as evolutionarily distinct EIVs display distinct infection phenotypes. We also showed that many human viruses can infect dog tracheas and that reassortment with CIV results in viable viruses. Finally, we showed that the hemagglutinin and neuraminidase of CIV act as virulence factors. Our findings have significant implications because they show that dogs might act as "mixing vessels" in which novel viruses with pandemic potential could emerge and also provide experimental evidence supporting the role of viral evolution in influenza virus emergence.