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.

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.

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.

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.

Journeys, Journey Conditions, and Welfare Assessment of Broken (Handled) Horses on Arrival at Italian Slaughterhouses.

During horse transportation, the journey conditions are considered a welfare risk. This study aimed to document journeys, journey conditions, and welfare status of handled horses on arrival at two different slaughterhouses in Northern and Southern Italy, to find possible associations between journey conditions and welfare problems. The welfare status of 613 draft-breed and light-breed horses from 32 different journeys was evaluated on arrival at the slaughterhouses with a standardized protocol, using animal-based (ABMs) and environmental-based (EBMs) measures. The drivers' skills and vehicle characteristics were found to be mostly compliant with EC 1/2005. The horses traveled in single bays, 90° to the direction of travel for an average journey duration of 26.5 ± 14 h. On arrival at the slaughterhouses, the horses were unloaded by handlers, via halter and rope. The prevalence of reluctance to unload, injuries, nasal, and lacrimal discharge was 22.2%, 24.6%, 11.6%, and 10%, respectively. Journey duration, unloading duration, vehicle changes, long stops, handlers/drivers' skills, temperature, season, and horse individual characteristics were associated with horses' welfare and health status (all p < 0.05). Our study confirms the hypothesis that appropriate journey conditions are of crucial importance to safeguard the welfare of broken/handled horses transported over long distances for slaughter.

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.

Journeys, Journey Conditions, and Welfare Assessment of Unbroken (Unhandled) Horses on Arrival at a Slaughterhouse in Italy.

Transportation of horses to slaughterhouses can pose a welfare concern, in particular when horses are unbroken/unhandled. This study aimed to describe their journeys and journey conditions, document their welfare status on arrival in a slaughterhouse in Italy, and investigate possible associations between journey conditions and welfare issues. A total of 395 unbroken draft horses in 20 different consignments were assessed with a standardized protocol. The most common departure point (16/20 consignments, 80%) was a French assembly center, but many of these horses had Spanish passports, suggesting they had previously been transported from Spain to France. The average journey duration was 34 ± 14 h, including journey breaks (i.e., short stops inside the vehicle and long resting stops at control posts), while transit time was 24 ± 4 h. The drivers were well experienced, the trucks were well equipped (i.e., forced ventilation, drinkers), and the horses traveled loose in small groups (n ≤ 4 horses) within pens inside the vehicle. On arrival at the slaughterhouse, the horses self-unloaded and showed minimal behavioral and health problems. The prevalence of injuries, diarrhea/abnormal feces, and nasal discharge were 1.52%, 6.58%, and 4.30%, respectively. Cold temperatures, space allowance, and lack of feeding during transport were found to be the main hazards of those problems (all p < 0.05). Our findings confirm that the correct assessment of the fitness for transport, adequate journey conditions, and experienced staff are crucial factors to safeguard the welfare of unbroken horses during long journeys.

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.

Effects of BEMER® physical vascular therapy in horses under training. A randomized, controlled double blind study.

Horses preparation for competition may cause psychological and physical stress. Physical vascular therapy BEMER® is reported to increase vasomotion and microcirculation, supporting body healing. This study aimed at assessing whether BEMER® physical vascular therapy in horses influences recovery rate of hematological and biochemical blood parameters within 1 h after moderate exercise and reduces stress measured by physiological and behavioral indicators. This prospective, randomized, double blinded, placebo-controlled crossover study included twelve warmblood horses (3 mares, 8 geldings, 1 stallion). Additionally to their daily work, horses were subjected to 15 min of exercise on a longe. Horses were randomly divided in two groups: A (n = 6), B (n = 6). Group A underwent first to BEMER® blanket for two weeks, then to Placebo blanket for two weeks. Group B did the opposite. Blood samples, thermographic infrared images, Heart Rate Variability and behavior were analyzed. ANOVA was used to investigate any treatment effect. After two weeks of treatment, although not statistically significant, hematocrit (%) measured immediately post exercise was lower in horses undergoing BEMER® treatment (48.30% ± 3.21) than both No blanket (51.15% ± 3.57) and Placebo blanket (49.58% ± 5.77). While wearing BEMER® blanket and after treatment, horses had a lower LF/HF ratio compared to other groups, although this difference was not statistically significant. These results possibly suggest an effect of BEMER® therapy on vagal activity and relaxation. Substantial progress in recovery after exercise was not confirmed, leading to the need for further investigation on the overall effect of BEMER® therapy.

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.

Use of thermography in pigs: relationship between surface and core temperature.

This study aims to assess the correlation between surface temperature estimated by infrared thermography and core temperature measured with rectal thermometer in weaning and fattening pigs. A total of 108 pigs were used in this study. Thermal images of the eye of each animal were recorded with a thermal imaging camera, rectal temperatures were measured using a calibrated digital thermometer. The average rectal temperature was 38.9 ± 0.4 °C (MIN = 37.9 °C; MAX = 40.1 °C) and the average eye temperature was 36.7 ± 0.1 °C (MIN = 34.8 °C; MAX = 38.8 °C). Our results showed that the mean eye temperature estimated by infrared thermography was significantly correlated (r = .581, P < .01) with rectal temperature. The correlation was significant and strong for weaners (r = .739, P < .01), significant although weak for fatteners (r = .236 P < .05). Thermography could be a valid method to estimate the core temperature of pigs under farm condition.

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.

Influence of Gonadectomy on Canine Behavior.

Due to the lack of unequivocal scientific evidence, gonadectomy's effects on dogs' behavior are still debated. Since veterinarians differ in their opinion, there may be considerable diversity in the advice received by owners. This study aimed to evaluate the effects of gonadectomy on dog behavior across time. Ninety-six dog owners (48 control dogs and 48 experimental dogs) were interviewed twice (T0 and T1, nine months later) to obtain information about their dog's behavior. No change was found in the eating behavior or weight of dogs of both groups. Compared to T0, at T1, experimental dogs were reported to show less mounting behavior, pull on the leash, and roaming behaviors. Marking behavior did not vary across time for both groups of dogs. A tendency to reduce owner-directed aggression was observed at T1 for experimental male dogs, while no change was observed for male controls. The literature reports conflicting information about the effect of gonadectomy on behavior, suggesting that further studies about this topic should be undertaken.

Equine Transport-Related Problem Behaviors and Injuries: A Survey of Italian Horse Industry Members.

An online survey was conducted to determine associations between equine transport management and transport-related injuries and problem behaviors in Italy. The survey was composed of four sections: respondents' demographic information and background, transport management practices, journey details and vehicle design, and transport injuries experienced by the horse in the previous two-year period. Univariable and multivariable logistic regression with a binary outcome variable was performed to explore associations between variables (respondents' and journeys' details and transport practices) and equine transport-related problem behaviors (TRPBs) and injuries. TRPBs were also considered an explanatory variable for injuries. The survey generated 201 responses; only 148 were complete and analyzed. TRPBs were reported by 14.45% of the respondents and the odds of TRPBs was linked to the respondent gender (p = 0.034), the use of tranquilizers prior to transport (p = 0.002), the use of a whip for loading (p = 0.049), the lack of protection equipment (p = 0.050), and shavings (p = 0.025) on the vehicle floor. Horse injuries (11.49%) were reported by more respondents who did not check the brakes of their transport vehicle before traveling (p = 0.043), had vehicles with padding on the chest bar (p = 0.038), and for horses reported to display TRPBs (p = 0.001). Finally, 10 respondents reported they were injured during horse transport (10/140; 7.14%), 50% simultaneously with their horses. The study findings should be interpreted with caution due to small sample size bias and participants' recall bias. Nevertheless, the results are in concordance with the literature, confirming that horse transport is a risk for the horse's and handler's health and well-being. Further studies are needed to identify best management practices to educate equine industry members on how to minimize transport-related problems.

The development of the AWIN welfare assessment protocol for donkeys / O desenvolvimento do protocolo de avaliação de bem-estar AWIN (Animal Welfare Indicators) para jumentos

The donkey population has increased in the last 10 years, with an estimated 50 million donkeys currently worldwide. Donkey welfare, meanwhile, is an increasing global concern that receives close public scrutiny. However, multiple challenges are surrounding how donkey welfare is assessed and recorded. The Animal Welfare Indicators (AWIN) project is the first project, funded by the European Commission, intended to improve donkey welfare by developing a scientifically sound and practical on-farm welfare assessment protocol. The present study describes the procedure for the development of the AWIN welfare assessment protocol for donkeys: 1) selection of promising welfare indicators; 2) research to cover gaps in knowledge; 3) stakeholder consultation; 4) testing the prototype protocol on-farm. The proposed two-level strategy improved on-farm feasibility, while the AWIN donkey app enables the standardized collection of data with prompt results. Although limitations are linked with a relatively small reference population, the AWIN welfare assessment protocol represents the first scientific and standardized approach to evaluate donkey welfare on-farm.(AU)

Na última década, a população de jumentos vem aumentando; estima-se que existam aproximadamente 50 milhões de em todo o mundo. O bem-estar dos jumentos é uma preocupação global crescente, que recebe um escrutínio público próximo. No entanto, existem vários desafios em torno de como o bem-estar do jumento é avaliado e registrado. O projeto Indicadores de Bem-Estar Animal (AWIN) foi o primeiro projeto, financiado pela Comissão Europeia, destinado a melhorar o bem-estar dos jumentos, desenvolvendo um protocolo de avaliação do bem-estar cientificamente válido e prático na fazenda. O presente estudo descreve o procedimento para o desenvolvimento do protocolo de avaliação de bem-estar AWIN para jumentos: 1) seleção de indicadores promissores de bem-estar; 2) pesquisa para cobrir lacunas no conhecimento; 3) consulta às partes interessadas; 4) testando o protocolo do protótipo em fazendas. A estratégia proposta em dois níveis de avaliação melhorou a viabilidade na fazenda, além disso, o aplicativo AWIN donkey permite coletar dados de maneira padronizada e mostrar resultados rapidamente. Embora a limitação esteja ligada a uma população de referência relativamente pequena, o protocolo de avaliação de bem-estar do AWIN representa a primeira abordagem científica e padronizada para avaliar o bem-estar de jumentos em fazendas.(AU)

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.