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Stocking density and trough space allowance can potentially impact sheep welfare during live export voyages. The aim of this study was to assess the welfare implications for sheep housed at five allometric stocking densities, with either unrestricted or restricted trough space allowance. Merino wethers ( n = 720) were housed in 40 pens of 18 heads for 18 days. Two 5-min continuous focal animal observations (n = 3/pen) were conducted on days 3, 5, 11, and 17. Scan sampling of standing and lying behaviours were conducted on the same days at hourly intervals. Live weights and immune cell counts were quantified at the start and end of the experiment, as well as faecal glucocorticoid metabolites (FGCMs), which were also assessed on days 6 and 12. Focal animals housed at higher stocking densities spent less time lying during one of the continuous observation periods, but no important effects on the overall number of animals lying or on the synchronicity of lying were evident. The scan sampling results indicated that the expression of some preferred lying positions was impaired at high stocking densities, and that high stocking densities also resulted in increased agonistic social interactions and displacement events at the start of the trial. There was a slight reduction in day 18 live weights for animals housed at higher stocking densities, but FGCM concentrations and immune cell counts were essentially unaffected. Trough space had no important effects on day 18 live weight, FGCM concentrations, or immune cell counts, and had limited effects on sheep behaviour. The lack of important impacts on biological fitness traits suggests that the behavioural responses observed were sufficient in allowing sheep to cope with their environment. However, we provide evidence that the provision of additional space is beneficial in reducing the time it takes for animals to adapt to their environment and to facilitate the expression of some preferred lying positions. While designed to emulate certain conditions relevant during live export voyages, some factors that may induce stress during this mode of transport were not present such as heat and ocean swell, so the conclusions must be interpreted in the context of the experimental conditions., Competing Interests: Author JM was employed by Jemora Pty Ltd. The study received funding from Meat and Livestock Australia Pty Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mayes, Tait, Cowley, Morton, Doyle, Arslan and Taylor.)

To ensure animal welfare is not compromised, virtual fencing must be predictable and controllable, and this is achieved through associative learning. To assess the influence of predictability and controllability on physiological and behavioral responses to the aversive component of a virtual fence, two methods of training animals were compared. In the first method, positive punishment training involved sheep learning that after an audio stimulus, an electrical stimulus would follow only when they did not respond by stopping or turning at the virtual fence (predictable controllability). In the second method, classical conditioning was used to associate an audio stimulus with an electrical stimulus on all occasions (predictable uncontrollability). Eighty Merino ewes received one of the following treatments: control (no training and no stimuli in testing); positive punishment training with an audio stimulus in testing (PP); classical conditioning training with only an audio stimulus in testing (CC1); and classical conditioning training with an audio stimulus followed by electrical stimulus in testing (CC2). The stimuli were applied manually with an electronic collar. Training occurred on 4 consecutive days with one session per sheep per day. Sheep were then assessed for stress responses to the cues by measuring plasma cortisol, body temperature and behaviors. Predictable controllability (PP) sheep showed no differences in behavioral and physiological responses compared with the control treatment ( P < 0.05). Predictable uncontrollability of receiving the aversive stimulus (CC2) induced a higher cortisol and body temperature response compared to the control but was not different to CC1 and PP treatments. CC2 treatment sheep showed a higher number of turning behaviors ( P < 0.001), and more time spent running ( P < 0.001) than the control and PP treatment groups, indicating that predictability without controllability was stressful. The behavior results also indicate that predicting the event without receiving it (CC1) was less stressful than predicting the event then receiving it (CC2), suggesting that there is a cost to confirmation of uncontrollability. These results demonstrate that a situation of predictability and controllability such as experienced when an animal successfully learns to avoid the aversive component of a virtual fence, induces a comparatively minimal stress response and does not compromise animal welfare., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer DB declared a past co-authorship with one of the authors CL to the handling editor., (Copyright © 2020 Kearton, Marini, Cowley, Belson, Keshavarzi, Mayes and Lee.)