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Growing awareness of antimicrobial resistance (AMR) in livestock production has led to calls for the development of alternatives such as antimicrobial peptides (AMPs) that are also able to combat infectious diseases in farm animals. A critical step in the development of AMPs is to understand people's perspectives towards this technology to avoid misalignment with societal expectations. The aim of this study was to investigate university student perspectives of AMP applications in farm animals as alternatives to antibiotic use. We interviewed 20 university students and, using thematic analysis, identified six themes: 1) Initial knowledge, including knowledge of antibiotics and initial impressions of AMPs; 2) Human wellbeing, including the effects of food and animal health on public health, the importance of addressing AMR, and cost for farmers; 3) Animal welfare, including animal health, welfare, and production, and continuation of contentious farm practices; 4) Perceived naturalness of AMPs, including biocompatibility and comparing same and different species transfer of biological material; 5) Unforeseen consequences of AMPs, and the importance of researching unintended consequences of novel technologies; and 6) Public acceptance of AMPs, including trust and lack of awareness. In summary, participants viewed AMPs positively as an alternative to antibiotic use in farm animals to address AMR. However, key concerns centered around unintended harmful effects for food systems, public health, and animal welfare, which may impact public acceptance of AMPs in animal agriculture., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Urloiu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dairy cattle experience a variety of stressors associated with routine farm practices, including injections, pen movements, regrouping, and artificial insemination. The primary objective of this study was to assess ear position (categorized according to 5 positions) and body movement responses of heifers during their first experience of artificial insemination, in comparison with responses during baseline observations before and after this procedure. A secondary objective was to test whether heifers exposed to positive reinforcement training (PRT) responded differently to this procedure. We tested 12 heifers (13 ± 0.7 mo. old); 7 were trained using PRT (as part of another study) and 5 had no experience with PRT. Ear axial and ear forward positions were more frequent during observations before and after artificial insemination relative to during artificial insemination (4.57 ± 0.82 vs. 0.31 ± 0.82 times/period, and 3.45 ± 0.51 vs. 0.50 ± 0.51 times/period, respectively). Backward pinned ear position was observed less frequently before and after versus during artificial insemination (0.12 ± 0.76 vs. 3.76 ± 0.76 times/period). We found no effect of period relative to artificial insemination on any of the 4 body movements assessed (leaning forward, backward, and steps taken with front legs and back legs).We found an effect of PRT on forward ear position (1.00 ± 0.51 times/PRT group vs. 2.95 ± 0.60 times/control group), and found that control heifers leaned forward and took more steps with their front legs than did PRT heifers (0.93 ± 0.57 times/PRT group vs. 3.55 ± 0.67 times/control group and 1.29 ± 0.68 times/PRT group vs. 3.85 ± 0.81 times/control group). We conclude that heifers experiencing artificial insemination for the first time express distinct ear positions that are consistent with responses to a negative emotional state. Further work is required to validate these responses and to determine the extent that they can be used to assess affective responses to this and other procedures., Competing Interests: The authors have read the journal’s policy and have the following competing interests: This study received support from the Dairy Farmers of Canada (Ottawa, ON, Canada), Saputo Inc. (Montreal, QC, Canada), British Columbia Dairy Association (Burnaby, BC Canada), Alberta Milk (Edmonton, AB, Canada), Intervet Canada Corporation (Kirkland, QC, Canada), Boehringer Ingelheim Animal Health (Burlington, ON, Canada), BC Cattle Industry Development Fund (Kamloops, BC, Canada), The Semex Alliance (Guelph, ON, Canada), Lactanet (Sainte-Anne-de-Bellevue, QC, Canada), Dairy Farmers of Manitoba (Winnipeg, MB, Canada), and Sask-Milk (Regina, SK, Canada). There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Heinsius et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dairy cows have a partial preference to access an outdoor deep-bedded pack, but the effects of continuous access to an outdoor area on estrous behaviors has not been studied. Our objective was to investigate if access to an outdoor open deep-bedded pack improves the expression of estrus behaviors. We enrolled 60 lactating Holstein cows directly after calving and followed them each for 12 weeks. Cows were housed in a single freestall pen holding 36 cows at a time, with a dynamic group composition. Half of the cows were randomly assigned to the OUTDOOR treatment; these cows had access to an outdoor open pack via an automated selection gate. INDOOR cows were housed together with OUTDOOR cows but were not allowed outdoor access. All cows were fitted with an automated activity monitor (AAM) 21 ± 3 d before expected calving date. Estrous behaviors (i.e., standing to be mounted and mounting behaviors) were continuously monitored using video, and the intensity of mounting (i.e., the number of standing to be mounted and other mounting behaviors per hour) was calculated per estrus event per cow. Temperature and humidity were monitored by data loggers indoors, and these data were used to calculate the Temperature Humidity Index (THI). Following an alert from the AAM, cows were checked to detect the presence of a dominant preovulatory follicle and an absence of a mature corpus luteum (CL) by rectal ultrasonography following milking, as well as 7 d thereafter to confirm ovulation by the presence of a new CL. A total of 94 estrus events were used in the final analysis. INDOOR cows tended to have a lower mounting intensity with increasing indoor THI, but no such effect was noted for OUTDOOR cows. The total number of cows in estrus positively influenced mounting intensity. OUTDOOR cows had a longer duration of estrus, as measured by the automated monitors, compared with INDOOR cows (12.4 ± 0.7 h versus 9.9 ± 0.8 h). Estrus event number and the total number of cows in estrus were both positively associated with estrus duration. These results indicate that access to an outdoor pack tended to increase mounting behavior for freestall housed dairy cows, especially during periods of elevated THI. We conclude that providing dairy cows access to an outdoor area can help with estrus detection and may thus help improve the reproductive programs on dairy farms., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Smid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Access to brushes allows for natural scratching behaviors in cattle, especially in confined indoor settings. Cattle are motivated to use brushes, but brush use varies with multiple factors including social hierarchy and health. Brush use might serve an indicator of cow health or welfare, but practical application of these measures requires accurate and automated monitoring tools. This study describes a machine learning approach to monitor brush use by dairy cattle. We aimed to capture the daily brush use by integrating data on the rotation of a mechanical brush with data on cow identify derived from either 1) low-frequency radio frequency identification or 2) a computer vision system using fiducial markers. We found that the computer vision system outperformed the RFID system in accuracy, and that the machine learning algorithms enhanced the precision of the brush use estimates. This study presents the first description of a fiducial marker-based computer vision system for monitoring individual cattle behavior in a group setting; this approach could be applied to develop automated measures of other behaviors with the potential to better assess welfare and improve the care for farm animals., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sadrzadeh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Genetic engineering of animals has been proposed to address societal problems, but public acceptance of the use of this technology is unclear. Previous work has shown that the source of information proposing the technology (e.g. companies, universities), the term used to describe the technology (e.g. genome editing, genetic modification), and the genetic engineering application (e.g. different food products) affects technology acceptance. We conducted three mixed-method surveys and used a causal trust-acceptability model to understand social acceptance of genetic engineering (GE) by investigating 1) the source of information proposing the technology, 2) the term used to describe the technology, and 3) the GE application for farm animals proposed. Further, participants expressed their understanding of technology using a range of terms interchangeably, all describing technology used to change an organism's DNA. We used structural equation modelling and confirmed model fit for each survey. In each survey, perceptions of benefit had the greatest effect on acceptance. Following our hypothesized model, social trust had an indirect influence on acceptance through similar effects of perceived benefit and perceived risk. Additional quantitative analysis showed that the source of information and technology term had little to no effect on acceptance. Applications involving animals were perceived as less beneficial than a plant application, and an application for increased cattle muscle growth was perceived as more risky than a plant application. When assessing the acceptability of applications participants considered impacts on plants, animals, and people, trust in actors and technologies, and weighed benefits and drawbacks of GE. Future work should consider how to best measure acceptability of GE for animals, consider contextual factors and consider the use of inductive frameworks., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Koralesky et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

One response to calls for increased openness in animal research is to make protocols publicly accessible, but it is unclear what type of input the public would provide if given this opportunity. In this study we invited public responses to five different research projects, using non-technical summaries intended for lay audiences. Our aim was to assess the potential for this type of public consultation in protocol review, and a secondary aim was to better understand what types of animal research people are willing to accept and why. US participants (n = 1521) were asked (via an online survey) "Do you support the use of these (insert species) for this research", and responded using a seven-point scale (1 = "No", 4 = "Neutral", and 7 = "Yes"). Participants were asked to explain the reasons for their choice; open-ended text responses were subjected to thematic analysis. Most participants (89.7%) provided clear comments, showing the potential of an online forum to elicit feedback. Four themes were prevalent in participant reasoning regarding their support for the proposed research: 1) impact on animals, 2) impact on humans, 3) scientific merit, and 4) availability of alternatives. Participant support for the proposed research varied but on average was close to neutral (mean ± SD: 4.5 ± 2.19) suggesting some ambivalence to this animal use. The protocol describing Parkinson's research (on monkeys) was least supported (3.9 ± 2.17) and the transplant research (on pigs) was most supported (4.9 ± 2.02). These results indicate that public participants are sensitive to specifics of a protocol. We conclude that an online forum can provide meaningful public input on proposed animal research, offering research institutions the opportunity for improved transparency and the chance to reduce the risk that they engage in studies that are out of step with community values., Competing Interests: The authors have declared that no competing interests exist.

Institutions that conduct animal research are often obliged to release some information under various legal or regulatory frameworks. However, within an institution, perspectives on sharing information with the broader public are not well documented. Inside animal facilities, managers exist at the interface between the people who conduct animal research and those charged with providing care for those animals. Their perception of transparency may influence their interpretation of the institutional culture of transparency and may also influence others who use these facilities. The objective of our study was to describe perceptions of transparency among animal research facility managers (all working within the same ethical oversight program), and how these perceptions influenced their experiences. Semi-structured, open-ended interviews were used to describe perceptions and experiences of 12 facility managers relating to animal research transparency. Four themes emerged from the participant interviews: 1) communication strategies, 2) impact on participant, 3) expectations of transparency, and 4) institutional policies. Similarities and differences regarding perceptions of transparency existed among participants, with notable differences between participants working at university versus hospital campuses. These results illustrate differences in perceptions of transparency within one institutional animal care and use program. We conclude that institutions, regulators and the public should not assume a uniform interpretation of a culture of transparency among managers, and that sustained communication efforts are required to support managers and to allow them to develop shared perspectives., Competing Interests: The authors have declared that no competing interests exist.

Rats (Rattus norvegicus) bred for research are typically confined with their litters until weaning, but will spend time away from pups when given the opportunity. We aimed to assess how dam welfare is affected by the ability to escape from their pups. Rat dams (n = 16) were housed in cages either with or without an elevated loft. We measured time dams spent in lofts, time spent nursing, and affective states using elevated plus maze and anticipatory behavior testing. We predicted that 1) dams housed with lofts would use them increasingly as pups aged, 2) dams without a loft would spend more time passively nursing (i.e. initiated by pups rather than the dam) and more total time nursing as pups aged, and 3) dams housed with lofts would show evidence of a more positive affective state. Dams housed with lofts spent more time in the loft with increasing pup age; dams spent on average (mean ± SE) 27 ± 5% of their time in the loft when pups were 1 wk old, increasing to 52 ± 5% of their time at 3 wks. When pups were 3 wks old, dams with lofts spent less time passively nursing (10 ± 2% of total time, compared to 27 ± 4% for dams without a loft) and less time nursing overall (36 ± 4% of time versus 59 ± 2% for dams without a loft). Rats without loft access showed increased anticipatory behavior potentially indicative of negative affective state (24.5±1.8 behaviors per minute in wk 3 compared to 18.8±1.0 in wk 1). These findings indicate that rat dams in laboratories choose to spend time away from their pups when provided the opportunity, particularly later in lactation; an inability to do so is associated with increased passive nursing and negative affect., Competing Interests: The authors have declared that no competing interests exist.

Dairy farm systems have intensified to meet growing demands for animal products, but public opposition to this intensification has also grown due, in part, to concerns about animal welfare. One approach to addressing challenges in agricultural systems has been through the addition of new technologies, including genetic modification. Previous studies have reported some public resistance towards the use of these technologies in agriculture, but this research has assessed public attitudes toward individual practices and technologies and few studies have examined a range of practices on dairy farms. In the present study, we presented participants with four scenarios describing dairy practices (cow-calf separation, the fate of excess dairy calves, pasture access and disbudding). Citizens from Canada and the United States (n = 650) indicated their support (on a 7-point scale) toward five approaches (maintaining standard farm practice, using a naturalistic approach, using a technological approach, or switching to plant-based or yeast-based milk production) aimed at addressing the welfare issues associated with the four dairy practices. Respondents also provided a text-based rationale for their responses and answered a series of demographic questions including age, gender, and diet. Participant diet affected attitudes toward milk alternatives, with vegetarians and vegans showing more support for the plant-based and yeast-based milk production. Regardless of diet, most participants opposed genetic modification technologies and supported more naturalistic practices. Qualitative responses provided insight into participants' values and concerns, and illustrated a variety of perceived benefits and concerns related to the options presented. Common themes included animal welfare, ethics of animal use, and opposition toward technology. We conclude that Canadian and US citizens consider multiple aspects of farm systems when contemplating animal welfare concerns, and tend to favor naturalistic approaches over technological solutions, especially when the latter are based on genetic modification., Competing Interests: The authors have declared that no competing interests exist.

People often express concern for the welfare of farm animals, but research on this topic has relied upon self-report. Facial expressions provide a quantifiable measure of emotional response that may be less susceptible to social desirability bias and other issues associated with self-report. Viewing other humans in pain elicits facial expressions indicative of empathy. Here we provide the first evidence that this measure can also be used to assess human empathetic responses towards farm animals, showing that facial expressions respond reliably when participants view videos of farm animals undergoing painful procedures. Participants (n = 30) were asked to watch publicly sourced video clips of cows and pigs undergoing common management procedures (e.g. disbudding, castration, tail docking) and control videos (e.g. being lightly restrained, standing). Participants provided their subjective rating of the intensity of 5 negative emotions (pain, sadness, anger, fear, disgust) on an 11-point Likert scale. Videos of the participants (watching the animals) were scored for intensity of unpleasantness of the participants' facial expression (also on an 11-point Likert scale) by a trained observer who was blind to treatment. Participants showed more intense facial expressions while viewing painful procedures versus control procedures (mean ± SE Likert; 2.4 ± 0.08 versus 0.6 ± 0.17). Participants who reported more intense negative responses also showed stronger facial expressions (slope ± SE = 0.4 ± 0.04). Both the self-reported and facial measures varied with species and procedure witnessed. These results indicate that facial expressions can be used to assess human-animal empathy., Competing Interests: The authors have declared that no competing interests exist.

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.

Pain induces deficits in appreciation of rewards (i.e. anhedonia) and variation in response to pain may be partly explained by individual differences in general expectations (i.e. optimism). Dairy calves are routinely subjected to painful procedures such as hot-iron disbudding. We tested if female Holstein calves (n = 17) display signs of anhedonia (as evidenced by reduced consumption of a sweet solution) after hot-iron disbudding (performed under general and local anesthesia), and whether individual differences in optimism explain the variation in this response. Individual variation in optimism was measured using responses to two judgment bias tests (performed when calves were 25 d old), and anhedonia was measured by comparing consumption of a sweet solution before and after hot-iron disbudding. We found that intake of the sweet solution declined (by mean ± SD: 48.4 ± 44.3%) on the day after disbudding, and that more pessimistic calves were more affected. Sweet solution consumption did not return to baseline for the duration of the study (i.e. 5 days). Calves reduced their intake of a sweet solution after hot-iron disbudding, consistent with pain-induced anhedonia, and more pessimistic calves showed stronger evidence of anhedonia, suggesting that they were more affected by the procedure. However, our results cannot rule out the possibility that calf responses were driven by anorexia., Competing Interests: The authors have declared that no competing interests exist.

Despite scientific interest in animal empathy, and growing public concern for farm animal welfare, the empathic abilities of farm animals remain under researched. In this study, we investigated empathic responses of young Holstein dairy calves to conspecifics recovering from hot-iron disbudding, a painful procedure common on dairy farms. A combination of social approach and place conditioning was used. First, 'observer' calves witnessed two 'demonstrator' calves recover from either a painful procedure (hot-iron disbudding and sedation) or a sham procedure (sedation alone) in distinct pens. Observer calves spent more time in proximity and paid more attention to calves recovering from the painful procedure compared to sham calves (proximity: 59.6 ± 4.3%; attention: 54.3 ± 1.5%). Observers were then tested for conditioned place aversion (in the absence of demonstrators) at 48h, 72h and 96h after the second demonstration; observers tended to avoid the pen associated with conspecific pain during the second of the three tests, spending 34.8 ± 9.6% of their time in this pen. No strong evidence of pain empathy was found, but our tentative results encourage further research on empathy in animals., Competing Interests: The authors have declared that no competing interests exist.

Genetic modification has been used to create dairy cattle without horns and with increased resistance to disease; applications that could be beneficial for animal welfare, farm profits, and worker safety. Our aim was to assess how different stated purposes were associated with public attitudes toward these two applications using a mixed methods approach. Using an online survey, U.S. participants were randomly assigned to one of ten treatments in a 2 (application: hornless or disease-resistant) x 5 (purposes: improved animal welfare, reduced costs, increased worker safety, all three purposes, or no purpose) factorial design. Each participant was asked to read a short description of the assigned treatment (e.g. hornlessness to improve calf welfare) and then respond to a series of questions designed to assess attitude toward the treatment using 7-point Likert scales (1 = most negative; 7 = most positive). Responses of 957 participants were averaged to creative an attitude construct score. Participants were also asked to explain their response to the treatment. Qualitative analysis of these text responses was used to identify themes associated with the participants' reasoning. Participant attitudes were more favorable to disease resistance than to hornlessness (mean ± SE attitude score: 4.5 ± 0.15 vs. 3.7 ± 0.14). In the 'disease-resistance' group participants had more positive attitudes toward genetic modification when the described purpose was animal welfare versus reduction of costs (contrast = 1.00; 95% CI = 0.12-1.88). Attitudes were less favorable to the 'hornless' application if no purpose was provided versus when the stated purpose was either to improve animal welfare (contrast = 0.95; 95% CI = 0.26-1.64) or when all purposes were provided (contrast = 0.88; 95% CI = 0.19-1.58). Similarly, attitudes were less positive when the stated purpose was to reduce costs versus either improving animal welfare (contrast = 0.86; 95% CI = 0.09-1.64) or when all purposes were provided (contrast = 0.79; 95% CI = 0.02-1.56). Quantitative and qualitative analysis indicated that both the specific application and perceived purpose (particularly when related to animal welfare) can affect public attitudes toward genetic modification., Competing Interests: The authors have declared that no competing interests exist.

Negative social interactions have been extensively studied in dairy cattle, but little is known about the establishment of positive (preferential) relationships. Adult dairy cows are known to spend more time at close proximity to specific social partners, indicating that they establish stronger bonds with these animals, but few studies have explored what happens in socially housed calves. In this study, we explored whether calves that spent their entire life in the same social group established social preferences (i.e. pairs of individuals that interact more) that are stable over time (two 48-h periods, separated by three days), across two types of behavior (standing and lying) and across contexts (change in environment and housing design). When housed in an open pack, calves showed consistent proximity patterns when standing (but not when lying). These preferential relationships persisted even after calves were moved into a new pen fitted with free stalls. At the individual level, calves varied in how selective they were in their social relationships, with some calves spending much more time with specific partners than did others. This degree of selectivity was not associated to Sociability, marginally associated to Fearfulness, but was associated with Pessimism (more pessimistic calves were more selective in their social relationships). In conclusion, calves can form selective relationships that appeared to be consistent over time and across context, and the degree to which calves were selective varied in relation to individual differences in Pessimism., Competing Interests: The authors have declared that no competing interests exist.

Genetic modification of farm animals has not been well accepted by the public. Some modifications have the potential to improve animal welfare. One such example is the use of gene editing (i.e. CRISPR (clustered regularly interspaced short palindromic repeats)) to spread the naturally occurring POLLED gene, as these genetically hornless animals would not need to experience the painful procedures used to remove the horns or horn buds. The aim of the current study was to assess public attitudes regarding the use of GM to produce polled cattle. United States (US) citizens (n = 598), recruited via Amazon Mechanical Turk, were asked "Do you think genetically modifying cows to be hornless would be…", and responded using a 7-point Likert scale (1 = a very bad thing, 4 = neither good nor bad, 7 = a very good thing). Participants were then asked to indicate if they would be willing to consume products from these modified animals. We excluded 164 of the original 598 participants for not completing the survey, failing any of three attention check questions, or providing no or unintelligible qualitative responses. Respondents were then asked to provide a written statement explaining their answers; these reasons were subjected to qualitative analysis. Comparison of Likert scale ratings between two groups was done using the Wilcoxon rank-sum test, and comparisons between more than two groups were done using the Kruskal-Wallis rank test. More people responded that the modification would be good (Likert ≥ 5; 65.7%) than bad (Likert ≤ 3; 23.1%), and that they would be willing to consume products from these animals (Likert ≥ 5; 66.0%) versus not consume these products (Likert ≤ 3; 22.6%). Qualitative analysis of the text responses showed that participant reasoning was based on several themes including animal welfare, uncertainty about the technology, and worker well-being. In conclusion, many participants reported positive attitudes towards GM polled cattle; we suggest that people may be more likely to support GM technologies when these are perceived to benefit the animal., Competing Interests: The authors have declared that no competing interests exist.

A number of studies have shown widespread public concern over housing animals in ways that restrict their ability to move freely. Dairy cows housed in tie stall barns are tethered continuously or for part of the day, but no study has assessed public support for this type of housing system. We report two experiments assessing public perceptions of tie stall housing for dairy cattle using a hypothetical referenda format. In Experiment 1, 65% of participants (n = 430) said they would support a ban on tie stalls. The probability of supporting a ban increased as the duration of time that cows were tethered increased. In Experiment 2, information about possible economic consequences was included. Relatively fewer (55%) participants (n = 372) indicated they would support a ban. Supporters of a ban were willing to pay an average dairy product price premium of 68% to see the ban enacted. Indirect measures of support indicated socially desirable responding was greater in Experiment 2 where the economic impacts of voting behavior were made explicit. In both studies, women and liberals were more likely to support a ban. The majority of participants in Experiment 1 (51%) and Experiment 2 (57%) said they had never heard or read anything about tie stalls before participating in our survey. We conclude that current knowledge of the use of tie stalls is low, but if this situation were to change there may be considerable public concern about the use of this housing method., Competing Interests: The authors have declared that no competing interests exist.

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.

On many dairy farms cows are kept indoors. Providing outdoor access is often considered desirable, but housing can protect animals from aversive climatic conditions. For example, by providing shade and fans, indoor housing can protect cows from heat stress they might otherwise experience on open pasture. This study tested how public attitudes to cattle rearing varied when participants were experimentally assigned to different scenarios using a 2 x 2 factorial design varying pasture versus indoor housing with or without heat stress. Participants (n = 581) were randomly assigned to a single scenario, and attitudes in response to the scenario were measured using a Likert scale (1 = "strongly disagree" to 5 = "strongly agree"). We also asked open-ended questions allowing participants to explain their responses. Participants responded most positively to the scenario that provided both pasture access and protection from heat stress (Likert 4.1±0.08), and least positively to scenario with indoor housing and heat stress (Likert 2.2±0.08). However, when the different animal welfare attributes were in conflict (i.e. naturalness as provided by pasture, and biological functioning/affective state as associated with protection from heat stress), participants placed priority on the latter: they were more supportive of the scenario providing indoor housing that protected cows from heat stress (Likert 3.5±0.08), than they were of a pasture rearing system that exposed cows to heat stress (Likert 2.4±0.08). Open-ended responses indicated that participants viewed the lack of protection from heat stress as a failure in the farmer's duty of care towards the cow. We conclude that participants valued both access to pasture and protection from heat stress for dairy cows, but prioritized protecting animal from heat stress when these features were in conflict., Competing Interests: The authors have declared that no competing interests exist.

The use of animals in research is controversial and often takes place under a veil of secrecy. Lab animal technicians responsible for the care of animals at research institutions are sometimes described as performing 'dirty work' (i.e. professions that are viewed as morally tainted), and may be stigmatized by negative perceptions of their job. This study assessed if transparency affects public perceptions of lab animal technicians and support for animal research. Participants (n = 550) were randomly assigned to one of six scenarios (using a 3x2 design) that described identical research varying only the transparency of the facility (low, high) and the species used (mice, dogs, cows). Participants provided Likert-type and open-ended responses to questions about the personal characteristics (warmth, competence) of a hypothetical lab technician 'Cathy' and their support for the described research. Quantitative analysis showed participants in the low-transparency condition perceived Cathy to be less warm and were less supportive of the research regardless of animal species. Qualitative responses varied greatly, with some participants expressing support for both Cathy and the research. These results suggest that increasing transparency in lab animal institutions could result in a more positive perception of lab animal researchers and the work that they do.

Public concerns regarding the quality of life of farm animals are often focused on specific practices such as separating the cow and calf immediately after birth. The available scientific literature provides some evidence in support of this practice (including reduced acute responses to separation when it does occur), as well as evidence of disadvantages (such as increased risk of uterine disease in cows). The aim of this study is to systematically examine public views around this practice. Specifically, this study analyzes the views of American and German citizens to separation of cow and calf at birth using a quantitative segmentation approach. Although the majority of participants opposed early separation, a small proportion of our sample supported the practice. According to participants' preference for early and later separation and their evaluation of different arguments for both practices, three clusters were identified. US participants were more likely to support early separation compared to German participants. The arguments presented for and against both practices caused different reactions in the three clusters, but did not appear to sway the opinions of most participants. The results show considerable opposition to the practice of early separation in large parts of the sample and suggest that the dairy industry should consider approaches to address this concern.

Citizens' concerns about farm animal welfare are often dismissed on the assumption that they are not well informed about farming practices. We conducted exploratory surveys of interested citizens (n = 50) before and after a self-guided tour of a 500-head dairy farm. 'Before' survey questions explored perceptions, concerns, and values about dairy cattle farming and welfare, in addition to a short knowledge-based quiz on dairy cattle husbandry. An 'after' survey explored the extent to which these constructs shifted after the tour. Before, most participants correctly answered quiz questions about general feeding and housing practices, but scores were low on questions about specific practices such as cow-calf separation. Participants considered several elements as necessary for a 'good' life for dairy cattle: fresh food and water, pasture access, gentle handling, space, shelter, hygiene, fresh air and sunshine, social companions, absence of stress, health, and safety from predators. These elements reflect a diverse conception of animal welfare that incorporates values for physical and mental well-being, natural living, and humane care. The visit had a mixed effect on perceptions of whether dairy cows had a 'good' life, improving perceptions for a quarter of participants, worsening perceptions in a third, with no shift in the remaining participants. The visit appeared to mitigate some concerns (e.g., provision of adequate food and water, gentle humane care) while reinforcing or eliciting others (e.g., lack of pasture access, early cow-calf separation). Moreover, animal welfare-relevant values held by participants (e.g., natural living, care) appeared to play an important role in influencing perceptions of farm practices. These results suggest that education and exposure to livestock farming may resolve certain concerns, but other concerns will likely persist, especially when practices conflict with deeply held values around animal care.

Fish are capable of excellent vision and can be profoundly influenced by the visual properties of their environment. Ambient colours have been found to affect growth, survival, aggression and reproduction, but the effect of background darkness (i.e., the darkness vs. lightness of the background) on preference and aggression has not been evaluated systematically. One-hundred Coho salmon (Oncorhynchus kisutch), a species that is increasing in popularity in aquaculture, were randomly assigned to 10 tanks. Using a Latin-square design, every tank was bisected to allow fish in each tank to choose between all the following colour choices (8 choices in total): black vs. white, light grey, dark grey, and a mixed dark grey/black pattern, as well as industry-standard blue vs. white, light grey, dark grey, and black. Fish showed a strong preference for black backgrounds over all other options (p < 0.01). Across tests, preference strength increased with background darkness (p < 0.0001). Moreover, having darker backgrounds in the environment resulted in less aggressive behaviour throughout the tank (p < 0.0001). These results provide the first evidence that darker tanks are preferred by and decrease aggression in salmonids, which points to the welfare benefits of housing farmed salmon in enclosures containing dark backgrounds.

Laboratory rats are usually kept in relatively small cages, but research has shown that they prefer larger and more complex environments. The physiological, neurological and health effects of standard laboratory housing are well established, but fewer studies have addressed the sustained emotional impact of a standard cage environment. One method of assessing affective states in animals is to look at the animals' anticipatory behaviour between the presentation of a cue signalling the arrival of a reward and the arrival of that reward. The primary aim of this study was to use anticipatory behaviour to assess the affective state experienced by female rats a) reared and housed long-term in a standard laboratory cage versus a semi-naturalistic environment, and b) before and after treatment with an antidepressant or an anxiolytic. A secondary aim was to add to the literature on anticipatory behaviour by describing and comparing the frequency and duration of individual elements of anticipatory behaviour displayed by rats reared in these two systems. In all experiments, total behavioural frequency was higher in standard-housed rats compared to rats from the semi-naturalistic condition, suggesting that standard-housed rats were more sensitive to rewards and experiencing poorer welfare than rats reared in the semi-naturalistic environment. What rats did in anticipation of the reward also differed between housing treatments, with standard-housed rats mostly rearing and rats from the semi-naturalistic condition mostly sitting facing the direction of the upcoming treat. Drug interventions had no effect on the quantity or form of anticipatory behaviour, suggesting that the poorer welfare experienced by standard-housed rats was not analogous to depression or anxiety, or alternatively that the drug interventions were ineffective. This study adds to mounting evidence that standard laboratory housing for rats compromises rat welfare, and provides further scientific support for recommendations that current minimum standards be raised.

Understanding concerns about the welfare of farm animals is important for the development of socially sustainable production practices. This study used an online survey to test how views on group versus stall housing for pregnant sows varied when Canadian and US participants were provided information about these systems, including access to scientific papers, YouTube videos, Google images, and a frequently-asked-questions page (S1 Appendix). Initial responses and changes in responses after accessing the information were analyzed from Likert scores of 242 participants and from their written comments. Participants were less willing to accept the use of gestation stalls after viewing information on sow housing. For example, initially 30.4% of respondents indicated that they supported the use of gestation stalls; this declined to 17.8% after participants were provided additional information. Qualitative analysis of comments showed that supporters of gestation stalls expressed concern about the spread of disease and aggression between animals in less confined systems, whereas supporters of group housing placed more emphasis on the sow's ability to interact socially and perform natural behaviors. These results point to public opposition to the use of gestation stalls, and indicate that the more that the public learns about gestation stalls the less willing they will be to accept their use.

Rodents and primates deprived of early social contact exhibit deficits in learning and behavioural flexibility. They often also exhibit apparent signs of elevated anxiety, although the relationship between these effects has not been studied. To investigate whether dairy calves are similarly affected, we first compared calves housed in standard individual pens (n = 7) to those housed in a dynamic group with access to their mothers (n = 8). All calves learned to approach the correct stimulus in a visual discrimination task. Only one individually housed calf was able to re-learn the task when the stimuli were reversed, compared to all but one calf from the group. A second experiment investigated whether this effect might be explained by anxiety in individually housed animals interfering with their learning, and tested varying degrees of social contact in addition to the complex group: pair housing beginning early (approximately 6 days old) and late (6 weeks old). Again, fewer individually reared calves learned the reversal task (2 of 10 or 20%) compared to early paired and grouped calves (16 of 21 or 76% of calves). Late paired calves had intermediate success. Individually housed calves were slower to touch novel objects, but the magnitude of the fear response did not correlate with reversal performance. We conclude that individually housed calves have learning deficits, but these deficits were not likely associated with increased anxiety.

The objectives of this study were to describe the incidence of postpartum disease and to evaluate the association with serum cholesterol concentrations during the first 3 weeks after calving in grazing dairy cows. The association between non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHBA), calcium and postpartum diseases was also evaluated. A total of 307 Holstein dairy cows from 6 commercial grazing herds in Osorno, Chile, were monitored from calving until 21 days in milk. Cases of retained placenta, clinical hypocalcemia and clinical mastitis were recorded by the farmer using established definitions. Twice weekly, cows were evaluated for metritis by the same veterinarian based on vaginal discharge and body temperature. Postpartum blood samples were collected weekly and analyzed for serum concentrations of cholesterol, NEFA, BHBA and calcium. Cows were considered as having subclinical ketosis if BHBA >1.2 mmol/L, and subclinical hypocalcemia if calcium <2.0 mmol/L in any of the 3 weekly samples. Overall, 56% of the cows studied developed at least one clinical or subclinical disease after calving. Incidence of individual diseases was 8.8% for retained placenta, 4.2% for clinical hypocalcemia, 11.7% for clinical mastitis, 41.1% for metritis, 19.9% for subclinical hypocalcemia and 16.6% for subclinical ketosis. Lower postpartum cholesterol in cows was associated with developing severe metritis or having more than one clinical disease after calving. For every 0.4 mmol/L decrease in serum cholesterol cows were nearly twice as likely to be diagnosed with multiple clinical diseases after calving. Higher BHBA concentrations and lower calcium concentrations during week 1 were associated with severe cases of metritis. Low serum calcium concentration during week 1 was also associated with developing more than one clinical disorder after calving. In conclusion, the incidence of postpartum diseases can be high even in grazing herds and lower serum cholesterol concentrations were associated with occurrence of clinical postpatum disorders.

Negative emotional states in humans are associated with a negative (pessimistic) response bias towards ambiguous cues in judgement tasks. Every mammalian young is eventually weaned; this period of increasing nutritional and social independence from the dam is associated with a pronounced behavioural response, especially when weaning is abrupt as commonly occurs in farm animals. The aim of the current study was to test the effect of separation from the cow on the responses of dairy calves in a judgement task. Thirteen Holstein calves were reared with their dams and trained to discriminate between red and white colours displayed on a computer monitor. These colours predicted reward or punishment outcomes using a go/no-go task. A reward was provided when calves approached the white screen and calves were punished with a timeout when they approached the red screen. Calves were then tested with non-reinforced ambiguous probes (screen colours intermediate to the two training colours). "GO" responses to these probes averaged (± SE) 72±3.6% before separation but declined to 62±3.6% after separation from the dam. This bias was similar to that shown by calves experiencing pain in the hours after hot-iron dehorning. These results provide the first evidence of a pessimistic judgement bias in animals following maternal separation and are indicative of low mood.

Early social housing is known to benefit cognitive development in laboratory animals. Pre-weaned dairy calves are typically separated from their dam immediately after birth and housed alone, but no work to date has addressed the effect of individual housing on cognitive performance of these animals. The aim of this study was to determine the effects of individual versus social housing on two measures of cognitive performance: reversal learning and novel object recognition. Holstein calves were either housed individually in a standard calf pen (n = 8) or kept in pairs using a double pen (n = 10). Calves were tested twice daily in a Y-maze starting at 3 weeks of age. Calves were initially trained to discriminate two colours (black and white) until they reached a learning criterion of 80% correct over three consecutive sessions. Training stimuli were then reversed (i.e. the previously rewarded colour was now unrewarded, and vice-versa). Calves from the two treatments showed similar rates of learning in the initial discrimination task, but the individually housed calves showed poorer performance in the reversal task. At 7 weeks of age, calves were tested for their response to a novel object in eight tests over a two-day period. Pair-housed calves showed declining exploration with repeated testing but individually reared calves did not. The results of these experiments provide the first direct evidence that individual housing impairs cognitive performance in dairy calves.

Zebrafish are becoming one of the most used vertebrates in developmental and biomedical research. Fish are commonly killed at the end of an experiment with an overdose of tricaine methanesulfonate (TMS, also known as MS-222), but to date little research has assessed if exposure to this or other agents qualifies as euthanasia (i.e. a "good death"). Alternative agents include metomidate hydrochloride and clove oil. We use a conditioned place avoidance paradigm to compare aversion to TMS, clove oil, and metomidate hydrochloride. Zebrafish (n = 51) were exposed to the different anaesthetics in the initially preferred side of a light/dark box. After exposure to TMS zebrafish spent less time in their previously preferred side; aversion was less pronounced following exposure to metomidate hydrochloride and clove oil. Nine of 17 fish exposed to TMS chose not to re-enter the previously preferred side, versus 2 of 18 and 3 of 16 refusals for metomidate hydrochloride and clove oil, respectively. We conclude that metomidate hydrochloride and clove oil are less aversive than TMS and that these agents be used as humane alternatives to TMS for killing zebrafish.

Pain is defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage, but emotional states are difficult to directly assess in animals. Researchers have assessed pain using behavioural and physiological measures, but these approaches are limited to understanding the arousal rather than valence of the emotional experience. Cognitive bias tasks show that depressed humans judge ambiguous events negatively and this technique has been applied to assess emotional states in animals. However, limited research has examined how pain states affect cognitive processes in animals. Here we present the first evidence of cognitive bias in response to pain in any non-human species. In two experiments, dairy calves (n = 17) were trained to respond differentially to red and white video screens and then tested with unreinforced ambiguous colours in two or three test sessions before and two sessions after the routine practice of hot-iron disbudding. After disbudding calves were more likely to judge ambiguous colours as negative. This 'pessimistic' bias indicates that post-operative pain following hot-iron disbudding results in a negative change in emotional state.