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Abstract Laboratory mice are typically housed in “shoebox” cages with limited opportunities to engage in natural behaviour. Temporary access to environments with increased space and complexity (playpens) may improve mouse welfare. Previous work by our group has shown that mice are motivated to access and use these environments, but it is unknown how other aspects of welfare are impacted. Female C57BL/6J, BALB/cJ, and DBA/2J mice (n = 21; 7 mice per strain) were housed in mixed-strain trios and given temporary access to a large playpen with their cage mates three times per week. Control mice (n = 21; 7 mice per strain) remained in their home cages. Home cage behaviour (development of stereotypic behaviour over time, aggression following cage-changing) and anxiety tests were used to assess how playpen access impacted welfare. Contrary to our predictions, we found increased time spent performing stereotypies in playpen mice; this difference may be related to negative emotional states, increased motivation to escape the home cage, or active coping strategies. Playpen access resulted in strain-dependent improvements in aggression and some measures of anxiety. Aggression was lower for C57BL/6J mice in the playpen treatment following cage changing than it was for C57BL/6J control mice, while playpen mice, and particularly the C57BL/6J strain, spent more time in the center of the open field test and produced fewer fecal boli during anxiety testing, supporting other research showing that strain differences play an important role in behaviour and stress resiliency.

Intensive silvopastoral systems (ISS) are a sustainable alternative to monoculture systems (MS). The presence of trees and legumes improves animal welfare due to the increased food quality and quantity and the presence of shade while providing a variety of environmental services. As cattle behaviour is greatly affected by environmental conditions, knowledge on the behavioural trade-offs that cattle make to meet their demands while foraging in different grazing systems is important, as this will help us understand the perceived advantages of ISS. This pilot study assessed the behaviour of heifers in an ISS (n=8 heifers) and MS (n=8 heifers) in the Mexican tropics during the dry and rainy seasons, and its relationship with forage availability, mean travelled distance and the temperature humidity index (THI). In both seasons, daily foraging times were longer in the MS than the ISS (P

Silvopastoral systems can be a good alternative for sustainable livestock production because they can provide ecosystem services and improve animal welfare. Most farm animals live in groups and the social organization and interactions between individuals have an impact on their welfare. Therefore, the objective of this study was to describe and compare the social behaviour of cattle (Bos indicus×Bos taurus) in a silvopastoral system based on a high density of leucaena (Leucaena leucocephala) combined with guinea grass (Megathyrsus maximus), star grass (Cynodon nlemfuensis) and some trees; with a monoculture system with C. nlemfuensis, in the region of Merida, Yucatán. Eight heifers in each system were observed from 0730 to 1530 h each day for 12 consecutive days during the dry season and 12 consecutive days during the rainy season. The animals followed a rotation between three paddocks, remaining 4 days in each paddock. The vegetation was characterized in the paddocks of the silvopastoral system to estimate the average percentage of shade provided. To make a comparison between systems, we used a t test with group dispersion, and Mann–Whitney tests with the frequency of affiliative and agonistic behaviours. We assessed differences in linearity and stability of dominance hierarchies using Landau’s index and Dietz R-test, respectively. The distance of cows with respect to the centroid of the group was shorter, and non-agonistic behaviours were 62% more frequent in the intensive silvopastoral system than in the monoculture one. Heifers in the silvopastoral system had a more linear and non-random dominance hierarchy in both seasons (dry season: h’=0.964; rainy season: h’=0.988), than heifers in the monoculture system (dry season: h’=0.571, rainy season: h’=0.536). The dominance hierarchy in the silvopastoral system was more stable between seasons (R-test=0.779) than in the monoculture system (R-test=0.224). Our results provide the first evidence that heifers in the silvopastoral system maintain more stable social hierarchies and express more sociopositive behaviours, suggesting that animal welfare was enhanced.

Laboratory mice are typically housed in "shoebox" cages with limited opportunities to engage in natural behaviour. Temporary access to environments with increased space and complexity (playpens) may improve mouse welfare. Previous work by our group has shown that mice are motivated to access and use these environments, but it is unknown how other aspects of welfare are impacted. Female C57BL/6J, BALB/cJ, and DBA/2J mice (n = 21; 7 mice per strain) were housed in mixed-strain trios and given temporary access to a large playpen with their cage mates three times per week. Control mice (n = 21; 7 mice per strain) remained in their home cages. Home cage behaviour (development of stereotypic behaviour over time, aggression following cage-changing) and anxiety tests were used to assess how playpen access impacted welfare. Contrary to our predictions, we found increased time spent performing stereotypies in playpen mice; this difference may be related to negative emotional states, increased motivation to escape the home cage, or active coping strategies. Playpen access resulted in strain-dependent improvements in aggression and some measures of anxiety. Aggression was lower for C57BL/6J mice in the playpen treatment following cage changing than it was for C57BL/6J control mice, while playpen mice, and particularly the C57BL/6J strain, spent more time in the center of the open field test and produced fewer fecal boli during anxiety testing, supporting other research showing that strain differences play an important role in behaviour and stress resiliency., (© 2024. The Author(s).)

Individual variation in responses to commonly used tests of anxiety and spatial memory is often reported. While this variation is frequently considered to be 'noise', evidence suggests that it is, at least partially, related to consistent individual differences in behavioral responses (i.e., personality). The same tests used to assess anxiety are often used to profile personality traits, but personality differences are rarely considered when testing treatment differences in anxiety. Focusing on the rat literature, we describe fundamental principles involved in anxiety and spatial memory tests and we discuss how personality differences and housing conditions can influence behavioral responses in these tests. We propose that an opportunity exists to increase stress resiliency in environmentally sensitive individuals by providing environmental enrichment. We conclude by discussing different approaches to incorporating personality measures into the design and analysis of future studies; given the potential that variation masks research outcomes, we suggest that a strategy which considers the individual and its housing can contribute to improving research reproducibility., Competing Interests: Conflicts of Interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Evidence indicates that carbon dioxide (CO 2 ) induces negative affective states (including anxiety, fear and distress) in laboratory rodents, but many countries still accept it for euthanasia. Alternative methods (e.g. inhalant anaesthetic) may represent a refinement over CO 2 but are not widely adopted. We conducted an online survey of Canadian and European laboratory animal professionals and researchers ( n  = 592) to assess their attitudes towards the use of CO 2 and alternative methods for rodent euthanasia using quantitative 7-point scale (from 1 (= strongly oppose) to 7 (= strongly favour) and qualitative (open-ended text) responses. CO 2 was identified as the most common method used to kill rodents, and attitudes towards this method were variable and on average ambivalent (mean ± SD score on our 7-point scale was 4.4 ± 1.46). Qualitative analysis revealed four themes relating to participant attitude: (a) the animal's experience during gas exposure; (b) practical considerations for humans; (c) compromise between the animal's experience and practical considerations; and (d) technical description of the procedure or policies. Many participants (51%) felt that there were alternatives available that could be considered an improvement over CO 2 , but perceived barriers to implementing these refinements. Qualitative analysis of these responses revealed five themes: (a) financial constraints; (b) institutional culture; (c) regulatory constraints; (d) research constraints; and (e) safety concerns. In conclusion, concerns regarding the use of CO 2 often focused on the animal's experience, but barriers to alternatives related to operational limitations. New research is now required on to how best to overcome these barriers.

Laboratory mice are commonly euthanised with carbon dioxide (CO 2 ); however, there is ample evidence that this gas is aversive. Previous work suggests that sedation achieved via injection with benzodiazepines prior to CO 2 administration could reduce aversive behaviours during euthanasia. We explored the potential of using a voluntarily ingested sedative (tiletamine-zolazepam, Zoletil ® ) prior to euthanasia. Male and female C57BL/6 mice were allocated into one of the five experimental groups, which differed in the dose of Zoletil: 0, 10, 20, 40, 80 or 100 mg/kg. A dose of 20 mg/kg was found to achieve mild sedation prior to euthanasia; mice which received this dose numerically reared and walked on the cage lid less, and showed ataxia, immobility and recumbency for longer than mice that received a lower dose. During euthanasia, mice that received 20 mg/kg showed fewer aversive responses to CO 2 . Doses of 40 to 100 mg/kg were associated with signs of moderate to severe sedation, but resulted in an incomplete intake of the sedative, which made the interpretation of the aversiveness to CO 2 difficult. Voluntary oral administration of a sedative is an effective, affordable, and easy way to minimize the stress of mice to euthanasia with CO 2 .

Feelings of fear, anxiety, dyspnea and panic when inhaling carbon dioxide (CO2) are variable among humans, in part due to differences in CO2 sensitivity. Rat aversion to CO2 consistently varies between individuals; this variation in aversion may reflect CO2 sensitivity, but other personality traits could also account for individual differences in aversion. The aims of this study were to 1) assess the stability of individual differences in rat aversion to CO2, 2) determine if individual differences in sweet reward motivation are associated with variation in aversion to CO2, and 3) assess whether variation in aversion to CO2 is related to individual differences in motivation to approach gains (promotion focus) or maintain safety (prevention focus). Twelve female Sprague Dawley rats were exposed multiple times at three different ages (3, 9 and 16 months old) to CO2 in approach-avoidance testing to assess motivation to avoid CO2 against motivation to gain sweet rewards. Rats were also tested for motivation to find hidden sweet rewards, and for their motivation to approach rewards or darkness. Tolerance to CO2 increased with repeated exposures and was higher at older ages. Individual differences in aversion to CO2 were highly repeatable but unrelated to motivation for sweet rewards or the strength of promotion and prevention focus. These results indicate that individual differences in aversion to CO2 reflect variation in CO2 sensitivity., Competing Interests: The authors have declared that no competing interests exist.

The aim of this review is to summarize evidence regarding rat emotional experiences during carbon dioxide (CO 2 ) exposure. The studies reviewed show that CO 2 exposure is aversive to rats, and that rats respond to CO 2 exposure with active and passive defense behaviors. Plasma corticosterone and bradycardia increased in rats exposed to CO 2 . As with anxiogenic drugs, responses to CO 2 are counteracted by the administration of anxiolytics, SRIs, and SSRI's. Human studies reviewed indicate that, when inhaling CO 2 , humans experience feelings of anxiety fear and panic, and that administration of benzodiazepines, serotonin precursors, and SSRIs ameliorate these feelings. In vivo and in vitro rat studies reviewed show that brain regions, ion channels, and neurotransmitters involved in negative emotional responses are activated by hypercapnia and acidosis associated with CO 2 exposure. On the basis of the behavioral, physiological, and neurobiological evidence reviewed, we conclude that CO 2 elicits negative emotions in rats.

Carbon dioxide (CO 2 ) is commonly used to kill laboratory rats. Rats find CO 2 aversive and aversion varies between individuals, indicating that rats vary in CO 2 sensitivity. Healthy humans experience feelings of anxiety at concentrations similar to those avoided by rats, and these feelings are diminished by the administration of benzodiazepines. Our aim was to assess the effects of the benzodiazepine midazolam on individual thresholds of rat aversion to CO 2 . Six female Sprague Dawley rats were repeatedly exposed to CO 2 gradual-fill in approach-avoidance testing. The first three exposures were to a control-treatment followed by three exposures to midazolam (0.375 mg/kg). Within each treatment aversion to CO 2 was not affected by exposure number; however, tolerance increased from an average of 10.7% CO 2 avoided during control sessions, to 15.5% CO 2 avoided when treated with midazolam. These results indicate that rats experience anxiety when exposed to CO 2 , and that variation in rat CO 2 sensitivity is driven by individual differences in the onset of these feelings of anxiety. No rat tolerated CO 2 concentrations required to induce loss of consciousness.

Carbon dioxide (CO2) gradual-fill is commonly used to kill laboratory rats, but this use remains controversial due to a lack of agreement between studies. Inconsistencies may arise from differences in behaviors measured (e.g. active versus passive behaviors), in how rats cope with threats, or in rat sensitivity to CO2. The aims of the current study were to 1) describe active and passive responses during CO2 forced exposure, 2) determine if these responses are consistent within individuals and across aversive stimuli, 3) assess individual differences in aversion to CO2 in aversion-avoidance and approach-avoidance tests and 4) determine how responses in aversion tests relate to individual differences in behavior during forced exposure. Twelve Sprague Dawley female rats were exposed twice to three treatments: CO2, oxygen (O2), and fox scent, and were exposed to CO2 twice in each aversion test. The change in behavior from baseline was higher for rearing and locomotion when rats were exposed to CO2 than when exposed to O2 and fox scent. Responses varied among rats but were consistent across multiple tests within rats. For example, rearing was consistent within individuals between two exposures to CO2. Similarly, the strength of aversion was consistent within individuals across multiple exposures to CO2 in aversion-avoidance and approach-avoidance testing. Latency to avoid CO2 in aversion-avoidance tests was negatively correlated with rearing during CO2 forced exposure. Collectively, these results indicate that rat responses to CO2 vary between (but are consistent within) individuals, suggesting that rats vary in CO2 sensitivity. However, even the less responsive rats avoided CO2 concentrations far below those necessary to achieve unconsciousness, indicating that all rats likely experience negative states when euthanized with CO2., Competing Interests: The authors have declared that no competing interests exist.

Intensive silvopastoral systems (ISS) are a sustainable alternative to monoculture systems (MS). The presence of trees and legumes improves animal welfare due to the increased food quality and quantity and the presence of shade while providing a variety of environmental services. As cattle behaviour is greatly affected by environmental conditions, knowledge on the behavioural trade-offs that cattle make to meet their demands while foraging in different grazing systems is important, as this will help us understand the perceived advantages of ISS. This pilot study assessed the behaviour of heifers in an ISS (n=8 heifers) and MS (n=8 heifers) in the Mexican tropics during the dry and rainy seasons, and its relationship with forage availability, mean travelled distance and the temperature humidity index (THI). In both seasons, daily foraging times were longer in the MS than the ISS (P<0.01). The duration of rumination was higher for ISS (P<0.01) and the duration of lying was higher for the dry season (P<0.05). The decrease in foraging times in relation to THI was significantly higher in the ISS than in the MS (mean slope±SE: ISS=-4.64±0.34; MS=-2.34±0.22; t=-14.20, P<0.001). The results suggest that the forage availability and access to shade in the ISS allow cattle to rest longer and increase rumination, whereas cattle in MS spend more time searching for food and foraging at times of the day were the temperatures were higher as a compensatory strategy, which potentially decreases cattle's welfare and production qualities when compared with the ISS. In conclusion, ISS are likely to generate positive behavioural trade-offs that result in better welfare conditions and higher productive potential.

Silvopastoral systems can be a good alternative for sustainable livestock production because they can provide ecosystem services and improve animal welfare. Most farm animals live in groups and the social organization and interactions between individuals have an impact on their welfare. Therefore, the objective of this study was to describe and compare the social behaviour of cattle (Bos indicus×Bos taurus) in a silvopastoral system based on a high density of leucaena (Leucaena leucocephala) combined with guinea grass (Megathyrsus maximus), star grass (Cynodon nlemfuensis) and some trees; with a monoculture system with C. nlemfuensis, in the region of Merida, Yucatán. Eight heifers in each system were observed from 0730 to 1530 h each day for 12 consecutive days during the dry season and 12 consecutive days during the rainy season. The animals followed a rotation between three paddocks, remaining 4 days in each paddock. The vegetation was characterized in the paddocks of the silvopastoral system to estimate the average percentage of shade provided. To make a comparison between systems, we used a t test with group dispersion, and Mann-Whitney tests with the frequency of affiliative and agonistic behaviours. We assessed differences in linearity and stability of dominance hierarchies using Landau's index and Dietz R-test, respectively. The distance of cows with respect to the centroid of the group was shorter, and non-agonistic behaviours were 62% more frequent in the intensive silvopastoral system than in the monoculture one. Heifers in the silvopastoral system had a more linear and non-random dominance hierarchy in both seasons (dry season: h'=0.964; rainy season: h'=0.988), than heifers in the monoculture system (dry season: h'=0.571, rainy season: h'=0.536). The dominance hierarchy in the silvopastoral system was more stable between seasons (R-test=0.779) than in the monoculture system (R-test=0.224). Our results provide the first evidence that heifers in the silvopastoral system maintain more stable social hierarchies and express more sociopositive behaviours, suggesting that animal welfare was enhanced.