Heavy Pigs Reared for Italian Dry-Cured Products: Does Immunocastration Influence the Fatty Acid Profile of Loins and Backfat?

The Italian pig sector requires heavy pigs (raised for at least nine months and slaughtered at >160 kg). In order to avoid boar taint and lower the impact on welfare, immunocastration provides an alternative to surgical castration. This study investigated the effects of immunocastration compared to surgical castration on the chemical composition and fatty acid profile of loins (longissimus dorsi muscle) and adipose tissue in Italian heavy pigs raised for dry-cured ham. Twenty-four male pigs were subjected to surgical castration (n = 12) or immunocastration (n = 12). Carcass parameters were monitored at slaughter, and samples of longissimus dorsi muscle and subcutaneous fat were analysed. This study showed no significant differences in carcass characteristics and proximate composition of fresh meat. However, variations were observed in the fatty acid profiles of meat and adipose tissue between groups. Notably, saturated fatty acids, particularly stearic acid (18:0), were higher in the intramuscular fat (IMF) of the immunocastrated pigs compared to the surgically castrated pigs. Conversely, monounsaturated fatty acids, predominantly oleic acid (18:1n-9), were higher in the IMF from the surgically castrated pigs compared to the immunocastrated pigs. While immunocastration may offer benefits in terms of animal growth and carcass composition, it could lead to unfavourable lipid changes in fresh loin meat for Italian heavy pigs.

Management of Donkeys in Assisted Interventions: A Snapshot.

People working in the field of Animal-Assisted Interventions (AAIs) often state that they perceive animal welfare as a matter of paramount importance; nevertheless, most scientific literature focuses on the effectiveness of interventions from the user's perspective. Before focusing on the animals' management and welfare during their interactions with users, it is important to ensure animal welfare during their "ordinary lives". This article reports information and considerations about the management of donkeys involved in AAIs in Northern Italy. Six facilities with several years of experience in Donkey-Assisted Interventions were visited for the purpose of an initial data collection regarding the management of donkeys involved in AAIs. Some knowledge gaps regarding the nutritional needs of the donkey and its preventive medicine have been identified; this study also highlighted a need for efforts to create a more stimulating and enriched environment for animals involved in AAIs. Some possible areas for improvement in the management of donkeys involved in AAIs have been highlighted. Further studies are necessary to provide a more comprehensive picture of the welfare of donkeys involved in this context.

Non-invasive estimation of in vivo optical properties and hemodynamic parameters of domestic animals: a preliminary study on horses, dogs, and sheep.

Biosensors applied in veterinary medicine serve as a noninvasive method to determine the health status of animals and, indirectly, their level of welfare. Near infrared spectroscopy (NIRS) has been suggested as a technology with this application. This study presents preliminary in vivo time domain NIRS measurements of optical properties (absorption coefficient, reduced scattering coefficient, and differential pathlength factor) and hemodynamic parameters (concentration of oxygenated hemoglobin, deoxygenated hemoglobin, total hemoglobin, and tissue oxygen saturation) of tissue domestic animals, specifically of skeletal muscle (4 dogs and 6 horses) and head (4 dogs and 19 sheep). The results suggest that TD NIRS in vivo measurements on domestic animals are feasible, and reveal significant variations in the optical and hemodynamic properties among tissue types and species. In horses the different optical and hemodynamic properties of the measured muscles can be attributed to the presence of a thicker adipose layer over the muscle in the Longissimus Dorsi and in the Gluteus Superficialis as compared to the Triceps Brachii. In dogs the absorption coefficient is higher in the head (temporalis musculature) than in skeletal muscles. The smaller absorption coefficient for the head of the sheep as compared to the head of dogs may suggest that in sheep we are indeed reaching the brain cortex while in dog light penetration can be hindered by the strongly absorbing muscle covering the cranium.

The Feasibility of Animal-Based Indicators of Consciousness and Unconsciousness for Stunning in Sheep: A Systematic Review.

BACKGROUND: According to EU legislation, animal-based indicators (ABMs) are used to evaluate the efficacy of stunning methods to ensure that animals do not regain consciousness. EFSA has provided a list of ABMs for electrical and mechanical stunning in sheep; however, there is still a lack of information on their feasibility. We aimed to identify and evaluate the feasibility constraints of ABMs commonly applied in slaughterhouses to assess proper stunning in sheep. METHOD: For this systematic review, we searched the Scopus and Web of Science databases from 2000 to 8 August 2022, including full peer-reviewed papers written in English on the welfare of sheep at the stunning and restraint phases. We excluded studies using a gas stunning method or without prior stunning, as well as manuscripts in which indicators were applied after sticking. RESULTS: Of 1289 records identified, only 8 papers were eligible for the critical evaluation of physical aspects that affect the feasibility of ABMs. These aspects were defined as a given definition of the feasibility of ABMs, and information was summarized and critically evaluated. The results highlighted a lack of information on the feasibility of ABMs which should be considered in the various conditions of commercial slaughterhouses.

Does housing system affect horse welfare? The AWIN welfare assessment protocol applied to horses kept in an outdoor group-housing system: The 'parcours'.

Outdoor group housing is generally reported as being beneficial to the welfare of horses compared to single boxes, being considered to show greater similarities with the living conditions of feral horses, allowing full expression of behaviours such as grazing, social interactions and free movement. However, concerns persist regarding the ability to maintain a good nutritional state and the possibility of acquiring injury. No data reporting a comprehensive assessment of welfare for horses in outdoor group-housing systems are currently available. The present study aimed at applying a scientifically valid welfare assessment protocol to group-housed outdoor horses in 'parcours', a particular management system used in the south of France. 'Parcours' are semi-natural areas, grazed by domestic herbivores located in lowland, mountain, or marsh. One hundred and seventy-one horses older than a year pertaining to six farms and kept on 'parcours' were evaluated by a trained veterinarian using a modified version of the second level Animal Welfare Indicators (AWIN) welfare assessment protocol for horses. No major welfare issues were detected. Horses in 'parcours' displayed few abnormal behaviours, they could move freely for most of the day and interact with conspecifics, maintaining a healthy state of nutrition and a good relationship with humans. The main welfare concerns were related to the presence of superficial integument alterations such as alopecia, difficulty in reaching quality controlled water sources and a lack of shelter. As the number of facilities involved in this study is relatively limited, further harmonised data collection should aim to enlarge the sample size and allow comparison with different outdoor group-housing conditions.

Events with Different Emotional Valence Affect the Eye's Lacrimal Caruncle Temperature Changes in Sheep.

Infrared thermography (IRT) has been recently applied to measure lacrimal caruncle temperature non-invasively since this region is related to the sympathetic response, and it seems a promising technique that is able to infer negative emotions in sheep (e.g., fear). However, the scientific literature so far is limited in understanding whether a caruncle's temperature changes also in response to positive emotional states in sheep. Through classical conditioning, we aimed to assess how a positive or a negative event affects the physiological (lacrimal caruncle temperature measured with IRT and cortisol levels) and behavioral responses of sheep (ear position). Fourteen ewes from the same flock were randomly assigned to two treatment groups: positive (n = 7) and negative (n = 7). Each group was then trained through classical conditioning to associate a neutral auditory (ring bell) stimulus to an oncoming event: for the positive group, the presence of a food reward (maize grains), while for the negative one, the opening of an umbrella. After three weeks of training, before (at rest) and after (post-treatment), lacrimal caruncle temperature was non-invasively measured via IRT, and saliva samples were gently collected to measure cortisol levels. During treatment, sheep behavior was videorecorded and then analyzed using a focal animal sampling technique. At rest, the eye's lacrimal caruncle temperature was similar in both groups, while post-treatment, a significant increase was shown only in the negative group (t-test; p = 0.017). In the anticipation phase, sheep in the positive group kept their ears forward longer compared to those in the negative one (Mann-Whitney; p < 0.014), 8.3 ± 2.1 s and 5.2 ± 4.2 s, respectively. The behavioral response observed reflects a learnt association between a neutral stimulus and events with different emotional valence. Cortisol concentration slightly increased in both groups post-treatment. Our results confirm that IRT is a non-invasive technique that can be useful when applied to assess how positive and negative events may affect the physiological response in sheep.

Application of QBA to Assess the Emotional State of Horses during the Loading Phase of Transport.

To identify feasible indicators to evaluate animals' emotional states as a parameter to assess animal welfare, the present study aimed at investigating the accuracy of free choice profiling (FCP) and fixed list (FL) approach of Qualitative Behaviour Assessment (QBA) in horses during the loading phase of transport. A total of 13 stakeholders were trained to score 2 different sets of videos of mixed breed horses loaded for road transport, using both FCP and FL, in 2 sessions. Generalized Procustes Analysis (GPA) consensus profile explained a higher percentage of variation (80.8%) than the mean of 1000 randomized profiles (41.2 ± 1.6%; p = 0.001) for the FCP method, showing an excellent inter-observer agreement. GPA identified two main factors, explaining 65.1% and 3.7% of the total variation. Factor 1 ranging from 'anxious/ to 'calm/relaxed', described the valence of the horses' emotional states. Factor 2, ranging from 'bright' to 'assessing/withdrawn', described the arousal. As for FL, Principal Component Analysis (PCA) first and second components (PC1 and PC2, respectively), explaining on average 59.8% and 12.6% of the data variability, had significant agreement between observers. PC1 ranges from relaxed/confident to anxious/frightened, while PC2 from alert/inquisitive to calm. Our study highlighted the need for the use of descriptors specifically selected, throughout a prior FCP process for the situation we want to evaluate to get a good QBA accuracy level.

Unhandled horses classified with broken/unbroken test (BUT) exhibit longer avoidance, flight reactions, and displacement behaviors when approached by humans.

Horses with a low level of tameness are at higher risk for transport-related disease and injury; hence, European regulations for the protection of animals during transport (EC 1/2005) are stricter for unhandled (unbroken) horses. However, the regulation does not provide adequate tools for unhandled horse identification. The Broken/Unbroken Test (BUT) was developed and validated to easily identify whether a horse is broken (handled) or not. As a further validation step, the aim of this study was to assess whether there is any correspondence between the BUT classification and the behavioral response of the horse. A total of 100 healthy Italian Heavy Draft horses were video recorded when assessed with the BUT. In total, 90 videos (48 handled and 42 unhandled horses) matched the inclusion criteria and were assessed. The behavior of each horse was evaluated by three observers blinded as to the horses' experience with a focal animal continuous recording method. Behaviors were classified in four categories: stress, avoidance, displacement, and aggression. A Mann-Whitney test was used to identify differences in behavioral patterns between horses classified as handled or unhandled with the BUT. Unhandled horses showed not only a significantly longer time to be approached by the handler but also more avoidance and flight reactions (p < 0.001). Unhandled horses showed significantly longer displacement behaviors, such as sniffing (p < 0.001). These findings further validate the BUT classification and confirm that horses classified as unhandled are more prone to show avoidance and flight reactions when approached by humans. For this reason, the adoption of the BUT could be helpful to minimize humans' horse-related injuries and, if applied regularly before loading, to contribute to safeguard the welfare of horses during transport.

Does Immunocastration Affect Behaviour and Body Lesions in Heavy Pigs?

Immunocastration has been pointed out as an alternative to surgical castration; though, most of the scientific studies were performed in light pig production. This study aimed to evaluate the effect of immunocastration on animal welfare in heavy pig production through the evaluation of behaviour and body lesions. A total of 188 commercial-hybrid pigs were randomly allocated into two treatment groups: surgical castration (SC) and immunocastration with Improvac® (IC). Data on behaviour, body lesions, and salivary testosterone levels were collected the day before each vaccination at 15, 22, 32, and 36 weeks of age. IC and SC pigs were slaughtered at 40 and 41 weeks of age, respectively; productive and carcass traits data were also collected. Considering productive performance, our results confirmed that IC pigs grew faster and presented a higher weight at slaughter. A critical period for pig welfare was observed before 32 weeks: testosterone concentration and body lesion score were significantly higher in IC pigs compared to SC pigs; active behaviours were significantly more frequent in IC at 15 weeks. Immunocastration may represent a suitable alternative to surgical castration with profitable productive performances, whereas the impairment of welfare during the period before the effective vaccination should be further investigated as a potential critical aspect in heavy pig production.

The Impact of Noise Anxiety on Behavior and Welfare of Horses from UK and US Owner's Perspective.

Noise anxiety is an over-reaction to loud noises commonly detected among pets and can greatly impact on their welfare and on their management. When exposed to noisy events, horses can show intense escape attempts, which may cause severe accidents for the horse and the rider/handler. The aim of the present study was to investigate, through a web survey, UK and US owners' perception of noise anxiety severity in their horses, their management strategies and perceived efficacy. The questionnaire was shared via social networking and advertised as "What is your horse afraid of?". Over a total of 1836 questionnaires filled out; 409 owners reported that their horse has shown unusual behavior during a noise event. A two-step cluster analysis identified two groups: very anxious (VA) and slightly anxious (SA). VA horses were reported to have higher frequency of anxiety behaviors; higher frequency of signs of noise reactivity; and their anxiety did not improve with time. The most used management strategies consisted in providing hay throughout the night, turning in/out their horse or moving it to a paddock. A binomial logistic regression identified that horses that have reported injuries during noise events were more likely to be clustered as VA (OR = 0.24, 95% CI: 0.08-0.76); while providing hay throughout the night was more likely to be very effective management strategy in SA horses (OR = 0.41, 95% CI: 0.16-1.01). Our results confirmed that noise anxiety is a growing behavioral problem that can lead to important welfare concerns for horses. New management strategies, including the use of medicinal products, should be considered to reduce behavioral and physiological signs and help horses to cope with noisy events.

Development and Validation of a Test for the Classification of Horses as Broken or Unbroken.

Regulation EC 1/2005 has stricter rules for transportation of unbroken (untamed) vs. broken (tamed) horses, but does not provide adequate tools for their identification. This study aimed to develop and validate such a tool. A behavioural test (Broken/Unbroken Test (BUT)) based on approaching, haltering, and leading was applied to 100 horses. Physiological and additional behavioural data were also collected, and the horses' status (broken/unbroken) was assessed by the expert who administered the BUT. Each horse's behaviour during the BUT was scored by four trained observers blinded to the horse's history. The BUT score showed excellent inter-observer, intra-observer, and test-retest reliability (all intraclass correlation coefficients (ICCs) > 0.75). It was also negatively associated with respiratory rate, avoidance distance, and time needed to approach, halter, and lead the horse (p < 0.05 for all). The optimal BUT score cut-off for discrimination between broken and unbroken horses (gold standard: expert judgment) showed 97.8% sensitivity and 97.3% specificity. There was almost perfect agreement between BUT-based and expert classification of horses (ICC = 0.940). These findings confirm the BUT's construct and criterion validity. The BUT could provide officials with a feasible, reliable, and valid tool to identify a horse's broken/unbroken status and, consequently, direct stakeholders towards correct transport procedures.

Towards an improved pain assessment in castrated horses using facial expressions (HGS) and circulating miRNAs.

BACKGROUND: Pain in horses is an emergent welfare concern, and its assessment represents a challenge for equine clinicians. This study aimed at improving pain assessment in horses through a convergent validation of existing tools: we investigated whether an effective analgesic treatment influences the horse grimace scale (HGS) and the concentration of specific circulating microRNAs (miRNAs). METHODS: Eleven stallions underwent routine surgical castration under general anaesthesia. They were divided into two analgesic treatment groups: castration with the administration of preoperative flunixin and castration with preoperative flunixin plus a local injection of mepivacaine into the spermatic cords. HGS and levels of seven circulating miRNAs were evaluated pre-, 8 and 20 hours post-procedure. RESULTS: Compared to pre-castration, HGS, miR-126-5p, miR-145 and miR-let7e increased significantly in horses receiving flunixin at 8 hours post-castration (Friedman test, p < 0.05). Both behavioural and molecular changes occurred in horses receiving flunixin only, confirming that the addition of local mepivacaine is an effective analgesic treatment. CONCLUSIONS: Combining the use of HGS and circulating miRNAs, particularly miR-145, could be meaningful to monitor acute pain conditions in horses. Our results further validate the HGS as a method to assess acute pain in horses and point out miR-145 as a promising biomarker to identify pain.

A Systematic Review on Commercially Available and Validated Sensor Technologies for Welfare Assessment of Dairy Cattle.

In order to base welfare assessment of dairy cattle on real-time measurement, integration of valid and reliable precision livestock farming (PLF) technologies is needed. The aim of this study was to provide a systematic overview of externally validated and commercially available PLF technologies, which could be used for sensor-based welfare assessment in dairy cattle. Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a systematic literature review was conducted to identify externally validated sensor technologies. Out of 1,111 publications initially extracted from databases, only 42 studies describing 30 tools (including prototypes) met requirements for external validation. Moreover, through market search, 129 different retailed technologies with application for animal-based welfare assessment were identified. In total, only 18 currently retailed sensors have been externally validated (14%). The highest validation rate was found for systems based on accelerometers (30% of tools available on the market have validation records), while the lower rates were obtained for cameras (10%), load cells (8%), miscellaneous milk sensors (8%), and boluses (7%). Validated traits concerned animal activity, feeding and drinking behavior, physical condition, and health of animals. The majority of tools were validated on adult cows. Non-active behavior (lying and standing) and rumination were the most often validated for the high performance. Regarding active behavior (e.g., walking), lower performance of tools was reported. Also, tools used for physical condition (e.g., body condition scoring) and health evaluation (e.g., mastitis detection) were classified in lower performance group. The precision and accuracy of feeding and drinking assessment varied depending on measured trait and used sensor. Regarding relevance for animal-based welfare assessment, several validated technologies had application for good health (e.g., milk quality sensors) and good feeding (e.g., load cells, accelerometers). Accelerometers-based systems have also practical relevance to assess good housing. However, currently available PLF technologies have low potential to assess appropriate behavior of dairy cows. To increase actors' trust toward the PLF technology and prompt sensor-based welfare assessment, validation studies, especially in commercial herds, are needed. Future research should concentrate on developing and validating PLF technologies dedicated to the assessment of appropriate behavior and tools dedicated to monitoring the health and welfare in calves and heifers.

Habituation to Transport Helps Reducing Stress-Related Behavior in Donkeys During Loading.

Adopting proper animal management strategies, including training, might reduce to a substantial extent the adverse effects of transport-related stress in animals. The aim of this study was to evaluate the effect of habituation to transport on stress-related behaviors and physiological indicators during loading and unloading in donkeys. Fourteen donkeys were recruited and divided in two treatment groups: Habituation (H; M = 5, F = 2) and Control (C; M = 5, F = 2). H donkeys were gradually habituated to be transported, traveling together with their mothers and other adult donkeys well-accustomed to transport, while C donkeys had never been transported before. Loading and unloading phases were video recorded and behavior was analyzed. Saliva samples for cortisol concentration determination were collected at rest and after unloading. Latency time to load was significantly shorter for H donkeys than C donkeys (Mann-Whitney; p = 0.004). C donkeys also showed significantly more stress-related behaviors (Mann-Whitney; p = 0.026) and required a higher but not statistically significant number of human interventions to load. Cortisol concentration increased in both groups, but no differences were found between them (Mann-Whitney; p > 0.05). These results suggest that habituation to transport could mitigate stress during loading procedures in donkeys reducing loading time, frequency of stress-related behaviors and diminishing the need of human intervention.

Analysis of Hindgut Microbiome of Sheep and Effect of Different Husbandry Conditions.

The microbiome is now seen as an important resource to understand animal health and welfare in many species. However, there are few studies aiming at identifying the association between fecal microbiome composition and husbandry conditions in sheep. A wide range of stressors associated with management and housing of animals increases the hypothalamic-pituitary axis activity, with growing evidence that the microbiome composition can be modified. Therefore, the purpose of the present study was to describe the core microbiome in sheep, characterized using 16S rRNA gene sequencing, and to explore whether exposure to stressful husbandry conditions changed sheep hindgut microbiome composition. Sheep (n = 10) were divided in two groups: isolated group (individually separated for 3 h/day) and control group (housed in the home pen for the entire trial period). Sheep core microbiome was dominated by Firmicutes (43.6%), Bacteroidetes (30.38%), Proteobacteria (10.14%), and Verrucomicrobia (7.55%). Comparative results revealed few operational taxonomic units (OTUs) with significantly different relative abundance between groups. Chao1, abundance-based coverage estimator (ACE), and Fisher's alpha indices did not show differences between groups. OTU-based Bray-Curtis distances between groups were not significant (p-value = 0.07). In conclusion, these results describing the core microbiome of sheep do not suggest a strong effect of stressful husbandry conditions on microbial composition.

Reliability of fNIRS for noninvasive monitoring of brain function and emotion in sheep.

The aim of this work was to critically assess if functional near infrared spectroscopy (fNIRS) can be profitably used as a tool for noninvasive recording of brain functions and emotions in sheep. We considered an experimental design including advances in instrumentation (customized wireless multi-distance fNIRS system), more accurate physical modelling (two-layer model for photon diffusion and 3D Monte Carlo simulations), support from neuroanatomical tools (positioning of the fNIRS probe by MRI and DTI data of the very same animals), and rigorous protocols (motor task, startling test) for testing the behavioral response of freely moving sheep. Almost no hemodynamic response was found in the extra-cerebral region in both the motor task and the startling test. In the motor task, as expected we found a canonical hemodynamic response in the cerebral region when sheep were walking. In the startling test, the measured hemodynamic response in the cerebral region was mainly from movement. Overall, these results indicate that with the current setup and probe positioning we are primarily measuring the motor area of the sheep brain, and not probing the too deeply located cortical areas related to processing of emotions.

May Salivary Chromogranin A Act as a Physiological Index of Stress in Transported Donkeys? A Pilot Study.

Road transport is known to be a stressful animal husbandry procedure as it induces the activation of two main physiological stress-related pathways: the hypothalamic-pituitary-adrenal cortex axis and the sympathetic-adrenal medulla axis. This preliminary study aimed to investigate whether salivary chromogranin A (CgA) concentration, known as a biomarker of the sympathetic activity system during psychological stress, may represent a novel physiological index of transportation-induced stress in donkeys. Nineteen Romagnolo donkeys, raised in groups on paddocks, were subject to two transportations, following the farm's routine procedures, for a mean duration of 64 min each on two consecutive days. Salivary samples were gently collected 15 min before and 15 min after each transportation. Salivary CgA was measured by a commercially available enzyme-linked immunosorbent assay test. Results showed that CgA salivary levels significantly decreased after both transportations. The physiological mechanisms underlying this result may be related to catestatin activity, a bioactive product of the proteolytic cleavage of CgA, that acts as an inhibitor of catecholamine release. This hypothesis requires further investigation, particularly considering the limited number of subjects involved in this preliminary study. The identification of a reliable and non-invasive stress-marker would represent a useful tool for improving farm animals' welfare in transport conditions.

Is the Piglet Grimace Scale (PGS) a Useful Welfare Indicator to Assess Pain after Cryptorchidectomy in Growing Pigs?

Pig cryptorchidism is a congenital anomaly, which requires surgical intervention. Pain assessment in pig farming has some limitations and consumers are increasingly concerned about suffering linked to on-farm procedures. The PGS seems to be a promising tool for pain assessment in piglets, although it has not been investigated whether this tool is applicable to growing pigs. This study was designed to determine whether the PGS could be useful to assess pain in growing pigs undergoing on-farm cryptorchidectomy. Ten mixed-breed cryptorchid pigs were pre-medicated with azaperone and ketamine. Anesthesia was induced and maintained using IV sodium based. Pigs were filmed pre- and 6 h post-surgery to evaluate their behavior (scan sampling every minute). Besides, 36 pictures of the face expressions (18 pre- and 18 6 h post-surgery) were scored with the PGS by three treatment-blind observers. The pre-surgery pig's activity ratio was 59%, while it was 2% 6 h post-surgery. While the PGS inter-observer reliability was excellent (Interclass Correlation Coefficient value of 0.87), the PGS score increased significantly in pigs 6 h post-surgery (Paired sample t-test, p = 0.02). The PGS proved to be a potentially effective method to assess pain associated with cryptorchidectomy. However, further validation studies are required to validate this tool for other potentially painful procedures.

Text Mining Analysis to Evaluate Stakeholders' Perception Regarding Welfare of Equines, Small Ruminants, and Turkeys.

Welfare of animals significantly depends on how stakeholders perceive their needs and behave in a way to favor production systems that promote better welfare outcomes. This study aimed at investigating stakeholders' perception of the welfare of equines, small ruminants, and turkeys using text mining analysis. A survey composed by open-ended questions referring to different aspects of animal welfare was carried out. Text mining analysis was performed. A total of 270 surveys were filled out (horses = 122, sheep = 81, goats = 36, turkeys = 18, donkeys = 13). The respondents (41% veterinarians) came from 32 different countries. To describe welfare requirements, the words "feeding" and "water" were the most frequently used in all the species, meaning that respondents considered the welfare principle "good feeding" as the most relevant. The word "environment" was considered particularly important for turkeys, as well as the word "dry", never mentioned for other species. Horses stakeholders also considered "exercise" and "proper training" important. Goat stakeholders' concerns are often expressed by the word "space", probably because goats are often intensively managed in industrialized countries. Although the sample was too small to be representative, text mining analysis seems to be a promising method to investigate stakeholders' perception of animal welfare, as it emphasizes their real perception, without the constraints deriving by close-ended questions.

Can grimace scales estimate the pain status in horses and mice? A statistical approach to identify a classifier.

Pain recognition is fundamental for safeguarding animal welfare. Facial expressions have been investigated in several species and grimace scales have been developed as pain assessment tool in many species including horses (HGS) and mice (MGS). This study is intended to progress the validation of grimace scales, by proposing a statistical approach to identify a classifier that can estimate the pain status of the animal based on Facial Action Units (FAUs) included in HGS and MGS. To achieve this aim, through a validity study, the relation between FAUs included in HGS and MGS and the real pain condition was investigated. A specific statistical approach (Cumulative Link Mixed Model, Inter-rater reliability, Multiple Correspondence Analysis, Linear Discriminant Analysis and Support Vector Machines) was applied to two datasets. Our results confirm the reliability of both scales and show that individual FAU scores of HGS and MGS are related to the pain state of the animal. Finally, we identified the optimal weights of the FAU scores that can be used to best classify animals in pain with an accuracy greater than 70%. For the first time, this study describes a statistical approach to develop a classifier, based on HGS and MGS, for estimating the pain status of animals. The classifier proposed is the starting point to develop a computer-based image analysis for the automatic recognition of pain in horses and mice.