Plasma cortisol is the most commonly used indicator of stress in fish but, as the blood sampling procedure itself can be a source of stress, it would be helpful to measure cortisol using less invasive matrices. It is also necessary to find alternative matrices as stress indicators in dead fish in which blood sampling is impossible. In the present study, we investigated transport stress in three aquaculture species, European sea bass (Dicentrarchus labrax L.), common carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum), by cortisol determination (radioimmunoassay) in plasma and other matrices (skin mucus, gut content, lateral muscle and caudal fin). Cortisol significantly increased after transport in all species and matrices, except in the sea bass gut content, where it remained unchanged. The three species responded to transport stress by producing different cortisol levels. In conclusion, the significant correlation found between plasma cortisol and most of the other matrices opens up the possibility of using them to evaluate stress in fish: mucus sampling is a less invasive method than blood sampling, and in addition to muscle and fin sampling, it can be used in postmortem fish.

At weaning (33 days of age), 246 hybrid rabbits (782 6 53 g live weight) were divided into six experimental groups and fed ad libitum six iso-ADF diets formulated according to a bifactorial arrangement with two protein levels (152 and 162 g/kg) and three soluble fibre-to-starch ratios (0.2, 0.6 and 1.5), the latter obtained by replacing starch (from 209 to 91 g/kg) with soluble fibre (from 48 to 136 g/kg). The trial lasted for 42 days until slaughter. The rabbits that were fed the diet with the highest protein level and the lowest soluble fibre-to-starch ratio showed the highest mortality rate (17.1% v. 1.7% on average; P , 0.001) and sanitary risk (mortality 1 morbidity: 20.0% v. 8.1%; P 5 0.04) compared with the rabbits fed the other diets. With increasing dietary crude protein level, the digestibility of dry matter (DM; 0.615 to 0.626) and gross energy (0.620 to 0.630) as well as aNDF (without sodium sulphite; 0.298 to 0.323) and hemicelluloses (0.417 to 0.461) significantly (0.001 , P , 0.10) improved. Moreover, total volatile fatty acids (VFAs) in the caecal content increased (59.0 to 68.4 mmol/l; P 5 0.01) and ileum crypt depth tended to reduce (P 5 0.07). Neither growth performance nor slaughter results were affected by the protein level. When increasing soluble fibre-to-starch ratio, the digestibility of DM and gross energy did not change, whereas the digestibility of aNDF (0.264 to 0.352), ADF (0.167 to 0.267) and hemicelluloses (0.400 to 0.470) linearly increased (P , 0.001). At caecum, N-ammonia tended to decrease linearly (P 5 0.08), total VFA concentration (56.0 to 67.3 mmol/l) and acetate proportion (80.4 to 83.3 mmol/100 mmol VFA) linearly increased (P , 0.01), whereas butyrate and valerate proportions decreased (0.01 , P , 0.05). Growth performance was similar among groups, whereas at slaughter the proportion of the gastrointestinal tract linearly increased (177 to 184 g/kg; P , 0.01) without effect on dressing percentage, however. As soluble fibre-to-starch ratio increased, meat pH linearly decreased and lightness (L*), redness (a*) and yellowness (b*) colour indexes increased (0.01 , P , 0.05).

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During growth (27 to 75 days of age), a total of 384 rabbits were kept in 72 individual cages, 48 bicellular cages (2 rabbits/cage) and 24 collective cages (9 rabbits/cage). To evaluate the effects of the housing system on the fear level and behavioural patterns of rabbits at the two ages (39 to 45 days and 66 to 73 days), a tonic immobility test and an open-field test were conducted and their behaviour was video recorded. In the tonic immobility test, the number of attempts to induce immobility (1.38) was lower, and the duration of immobility (47.8 s) was higher (0.05 , P , 0.01) in the rabbits housed in individual cages than in those kept in bicellular (1.72 attempts and 25.0 s of immobility) and collective cages (1.99 attempts and 25.0 s of immobility). During the open-field test, the rabbits from individual and bicellular cages showed higher latency (38.8 and 40.3 v. 27.0 s), a lower number of total (73.3 and 81.7 v. 91.9) and central displacements (3.6 and 2.8 v. 5.4) and a shorter running time (11.8 and 13.6 s v. 17.7 s) and the time biting the pen (5.5 and 9.1 s v. 28.2 s) compared with the rabbits kept in collective cages (0.05 , P , 0.001). During the 24-h video recording, the rabbits in individual and bicellular cages spent less time allogrooming (0.34% and 0.19% v. 1.44%), moving (0.74% and 0.60% v. 1.32%) and running (0.08% and 0.03% v. 0.21%) than the rabbits in the collective cages (0.01 , P , 0.001). The lowest numbers of alerts and hops were observed in the rabbits kept in bicellular cages. With increasing age, a lower number of rabbits were sensitive to the immobility test and more rabbits entered the pen spontaneously during the open-field test (P , 0.001). In conclusion, the rabbits in individual cages exhibited the highest fear level and incomplete behavioural patterns; the rabbits housed in collective cages showed the lowest fear levels and had the possibility of expressing a wider range of behaviour; and the rabbits in bicellular cages exhibited an inconsistent pattern of fear in the tonic immobility and open-field tests. Probably, these rabbits were in a less stressful condition compared with animals in individual cages because social contacts were allowed, even if freedom of movement was more limited.Keywords: housing system, fear level, behaviour, growing rabbits ImplicationsPeople are concerned about the welfare status of rabbits reared for meat production in small cages. This paper demonstrates that housing rabbits in individual or bicellular cages threatens their welfare by increasing their fear level and by limiting their possibility of expressing normal behaviour. In collective cages (9 rabbits/cage), social contacts and movements are permitted, and both fear response and behavioural patterns improve. The tonic immobility and the open-field tests can be used to compare the fear level of rabbits in different housing systems.

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During growth (from 27 to 75 days of age), 384 rabbits were kept in different types of wire-net cages: 72 individual cages (72 rabbits; 10 animals/m 2 ), 48 bicellular cages (96 rabbits; 2 rabbits/cage; 18 animals/m 2 ) and 24 collective cages (216 rabbits; 9 rabbits/cage; 18 animals/m 2 ). The rabbits housed in individual cages showed higher daily weight gain both during the fattening period (from 52 to 75 days of age) and during the whole period of growth (43.0 v. 41.8 and 41.5 g/day; P , 0.05), and they had a higher final live weight at 75 days of age (2678 v. 2619 and 2602 g; P , 0.05) compared with the rabbits in the bicellular and collective cages, respectively. Rabbits in individual cages ingested more feed (133 v. 127 and 126 g/day; P , 0.01), but the feed conversion did not differ significantly among rabbits housed in the three types of cages. At slaughter, the carcass traits and meat quality were weakly affected by the housing system. The transport losses were higher in rabbits kept in individual and bicellular cages compared with those reared in collective cages (3.1% and 2.9% v. 2.2%; P , 0.01). In rabbits kept in individual cages, the hind leg muscle to bone ratio was higher (6.35 v. 6.19 and 5.91; P , 0.05) compared with the bicellular and collective cages, respectively. The pH and colour of the longissimus lumborum did not change with the housing system, while the b* index of the biceps femoris was lower (3.04 and 3.32 v. 4.26; P , 0.001) in the rabbits kept in individual and bicellular cages, respectively, than in those kept in collective cages. In conclusion, the rabbits housed in individual cages showed higher daily growth than rabbits kept in bicellular or collective cages, but they had a similar feed conversion and carcass quality. Differently, neither in vivo performance nor slaughter results differed among the rabbits kept in bicellular cages or in collective cages. The meat colour may be affected by the housing system, but to an extent that is hardly perceivable by the final consumer.