Validating a Non-Invasive Method for Assessing Cortisol Concentrations in Scraped Epidermal Skin from Common Bottlenose Dolphins and Belugas.

Society is showing a growing concern about the welfare of cetaceans in captivity as well as cetaceans in the wild threatened by anthropogenic disturbances. The study of the physiological stress response is increasingly being used to address cetacean conservation and welfare issues. Within it, a newly described technique of extracting cortisol from epidermal desquamation may serve as a non-invasive, more integrated measure of a cetacean's stress response and welfare. However, confounding factors are common when measuring glucocorticoid hormones. In this study, we validated a steroid hormone extraction protocol and the use of a commercial enzyme immunoassay (EIA) test to measure cortisol concentrations in common bottlenose dolphin (Tursiops truncatus) and beluga (Delphinapterus leucas) epidermal samples. Moreover, we examined the effect of sample mass and body location on cortisol concentrations. Validation tests (i.e., assay specificity, accuracy, precision, and sensitivity) suggested that the method was suitable for the quantification of cortisol concentrations. Cortisol was extracted from small samples (0.01 g), but the amount of cortisol detected and the variability between duplicate extractions increased as the sample mass decreased. In common bottlenose dolphins, epidermal skin cortisol concentrations did not vary significantly across body locations while there was a significant effect of the individual. Overall, we present a contribution towards advancing and standardizing epidermis hormone assessments in cetaceans.

Dolphin-WET-Development of a Welfare Evaluation Tool for Bottlenose Dolphins (Tursiops truncatus) under Human Care.

Ensuring high standards of animal welfare is not only an ethical duty for zoos and aquariums, but it is also essential to achieve their conservation, education, and research goals. While for some species, animal welfare assessment frameworks are already in place, little has been done for marine animals under human care. Responding to this demand, the welfare committee of the European Association for Aquatic Mammals (EAAM) set up a group of experts on welfare science, cetacean biology, and zoo animal medicine across Europe. Their objective was to develop a comprehensive tool to evaluate the welfare of bottlenose dolphins (Tursiops truncatus), named Dolphin-WET. The tool encompasses 49 indicators that were either validated through peer review or management-based expertise. The first of its kind, the Dolphin-WET is a species-specific welfare assessment tool that provides a holistic approach to evaluating dolphin welfare. Inspired by Mellor's Five Domains Model and the Welfare Quality®, its hierarchical structure allows for detailed assessments from overall welfare down to specific indicators. Through combining 37 animal-based and 12 resource-based indicators that are evaluated based on a two- or three-level scoring, the protocol offers a detailed evaluation of individual dolphins. This approach allows for regular internal monitoring and targeted welfare management, enabling caretakers to address specific welfare concerns effectively.

Age-associated changes in intestinal health biomarkers in dogs.

The gut microbiome is critical for maintaining host health. In healthy humans, the aging process is one of the main factors modulating the changes in the intestinal microbiota. However, little is known about the relationship between gut health, microbiota, and the aging process in dogs. The present study aims to explore the differences in the intestinal microbiota and intestinal health based on fecal biomarkers in a population of dogs of different ages. The study involved 106 dogs of different breeds aged between 0.2 and 15 years categorized as senior (>7 years; n = 40), adult (2-7 years; n = 50), and junior (< 2 years; n = 16). Fecal samples were collected during the same period at the same facilities. The analysis included the following gut health indicators: 16S rRNA gene sequencing to investigate the differences in the fecal microbiota; qPCR to determine the dysbiosis index; fecal short-chain fatty acid concentrations; fecal calprotectin; and immunoglobulin A. Beta diversity analysis revealed a significant difference with a small effect size (p = 0.003; R = 0.087) among age categories based on the unweighted UniFrac metric, but no significance was observed based on the weighted UniFrac metric or Bray-Curtis distances. There were no significant differences in the alpha diversity measures or the fecal dysbiosis index among age categories. Senior dogs had significantly higher relative abundance proportions in phyla Bacteroidota and Pseudomonadota and the genus Faecalibacterium, but not on qPCR analysis. At the family level, Ruminococcaceae, Uncl. Clostridiales.1, Veillonellaceae, Prevotellaceae, Succinivibrionaceae, and Bacteroidaceae abundances were higher in the senior category than in the adult and/or junior categories. Relative proportions, but not concentrations of fecal acetate, were higher in the senior category, while butyrate, isovaleric acid, and valeric acid were lower. The valeric acid concentration was significantly lower in the senior category than in the adult category. Calprotectin and immunoglobulin A levels did not differ significantly across groups. In conclusion, this study observed multiple minor changes in the fecal microbiota composition and the relative amount of short-chain fatty acids in dogs among different age groups, but studies in larger populations representative of all ages are warranted to refine the present results.

A Protocol to Assess the Welfare of Patagonian Huemul (Hippocamelus bisulcus) in Conservation Centers.

Animal-welfare-assessment protocols are important for identifying welfare problems in conservation programs. This study aimed to develop a baseline welfare protocol for the assessment of the welfare of Patagonian huemuls (Hippocamelus bisulcus) in conservation centers. This protocol is based on the Welfare Quality® (WQ) framework for cattle and was developed with the consideration of the available research on the behaviors, ecology, conservation, and captive management of this species, as well as welfare-assessment protocols for other ungulate species. As a result, the protocol was specifically developed for Patagonian huemuls and included four principles, 12 criteria, and 23 animal- and resource-based indicators. The twelve criteria of the WQ protocol were reduced to nine, and three new criteria were added because they were both feasible and essential for welfare assessment in captive Patagonian huemuls. This protocol is mainly intended to identify welfare problems in endangered species in the context of conservation centers (reproduction, rescue, rehabilitation, or treatment centers). Thus, the aggregation of different measures to obtain a global score was not proposed. However, a scoring system that assigns a value on a 0-2 scale (0 = no welfare concern; 1 = welfare concern; 2 = urgent welfare concern) was proposed for each category. Although further research is still needed to fully validate the protocol, this is the first development of a protocol to assess Patagonian huemul welfare, and it can be used as a basis for the development of welfare-assessment protocols for other deer species in captivity.

Zoo Animal Welfare Assessment: Where Do We Stand?

Zoological institutions, such as zoos and aquariums, have made animal welfare a top priority, as it is not only a moral obligation but also crucial for fulfilling their roles in education and conservation. There is a need for science-based tools to assess and monitor animal welfare in these settings. However, assessing the welfare of zoo animals is challenging due to the diversity of species and lack of knowledge on their specific needs. This review aims to discuss the advantages and disadvantages of existing methodologies for assessing zoo animal welfare through: (1) A critical analysis of the main approaches to zoo animal welfare assessment; (2) A description of the most relevant animal-based welfare indicators for zoo animals with a particular focus on behavioural and physiological indicators; and (3) An identification of areas that require further research.

Evaluation of Two Practical Tools to Assess Cognitive Impairment in Aged Dogs.

Cognitive dysfunction syndrome is the most common cause of cognitive decline in aged dogs. Early diagnosis is crucial because the sooner treatment is implemented, the greater the chance of slowing the progression of the disease. Assessment tools to assess cognitive decline may differ depending on the environment in which the dogs live. The aims of this study were threefold, first, to describe two feasible methods to evaluate cognitive impairment in aged dogs living in different environments: (i) a Canine Cognitive Assessment Scale (CCAS) for dogs living in a home environment and (ii) a practical cognitive test (PCT) potentially useful for dogs not living in a home environment (NHE); second, to assess the effect of age on the outcome of both tools and, finally, to compare the results of the CCAS with those of the PCT. Both methods were found to be practical to perform. Age was found to significantly predict the score obtained by the CCAS (p = 0.0011) and the outcome of the PCT (p = 0.009). However, the reversal phase from the PCT did not significantly predict the outcomes of the CCAS (p = 0.97). Taken together, these findings suggest that the CCAS is a practical method to evaluate age related cognitive changes in owned dogs. The fact that the PCT has not been proven to be related with the CCAS calls into question the use of the PCT as a sensitive tool to assess cognitive impairment. Further studies in this field are suggested.

Systematic review of animal-based indicators to measure thermal, social, and immune-related stress in pigs.

The intense nature of pig production has increased the animals' exposure to stressful conditions, which may be detrimental to their welfare and productivity. Some of the most common sources of stress in pigs are extreme thermal conditions (thermal stress), density and mixing during housing (social stress), or exposure to pathogens and other microorganisms that may challenge their immune system (immune-related stress). The stress response can be monitored based on the animals' coping mechanisms, as a result of specific environmental, social, and health conditions. These animal-based indicators may support decision making to maintain animal welfare and productivity. The present study aimed to systematically review animal-based indicators of social, thermal, and immune-related stresses in farmed pigs, and the methods used to monitor them. Peer-reviewed scientific literature related to pig production was collected using three online search engines: ScienceDirect, Scopus, and PubMed. The manuscripts selected were grouped based on the indicators measured during the study. According to our results, body temperature measured with a rectal thermometer was the most commonly utilized method for the evaluation of thermal stress in pigs (87.62%), as described in 144 studies. Of the 197 studies that evaluated social stress, aggressive behavior was the most frequently-used indicator (81.81%). Of the 535 publications examined regarding immune-related stress, cytokine concentration in blood samples was the most widely used indicator (80.1%). Information about the methods used to measure animal-based indicators is discussed in terms of validity, reliability, and feasibility. Additionally, the introduction and wide spreading of alternative, less invasive methods with which to measure animal-based indicators, such as cortisol in saliva, skin temperature and respiratory rate via infrared thermography, and various animal welfare threats via vocalization analysis are highlighted. The information reviewed was used to discuss the feasible and most reliable methods with which to monitor the impact of relevant stressors commonly presented by intense production systems on the welfare of farmed pigs.

Welfare and Performance of Post-Weaning Sows and Piglets Previously Housed in Farrowing Pens with Temporary Crating on a Spanish Commercial Farm: A Pilot Study.

The study investigated the effect of farrowing environment on the weaning adaptability of sows and piglets. One farrowing crate (FC) and two farrowing pens with temporary crating (TC: SWAP and JLF15) were compared. Sixty-four sows and 663 piglets were followed until 5 days post-weaning. At weaning (D24), sows and piglets were moved to group pens and nursery pens, respectively. Sows and piglets' behaviors were observed on D24, D25, and D26. On D23, D25, and D26, piglets' skin lesions were counted, and sows and piglets' saliva samples were collected for stress biomarkers (cortisol and chromogranin A, CgA). Piglets were weighed on D23 and D29. All the piglets' skin lesions increased on D25 and decreased on D26 (p < 0.05). Compared to D23, cortisol of JLF15 and CgA of FC piglets increased, whereas those of SWAP piglets remained similar after weaning (p < 0.05). Post-weaning performance in piglets was similar across farrowing systems. SWAP sows vocalized more than FC and JLF15 on D24 and D25 (p < 0.001). Results suggested that SWAP piglets showed a lower weaning stress response. Frequent post-weaning vocalization in SWAP sows might be linked with a negative effect of the abrupt separation from the piglets.

Alteration in Activity Patterns of Cows as a Result of Pain Due to Health Conditions.

The main conditions and diseases considered painful in dairy cows are mastitis, lameness, calving (including dystocia and caesarean section) and metritis. The cattle literature reports that deviation from normal daily activity patterns (both increased and/or reduced daily lying time) can be indicative of painful conditions and diseases in cows. This narrative review discusses on how pain due to several health conditions in dairy cows modifies its activity pattern and explores if non-steroidal anti-inflammatory drugs (NSAIDs) are capable of restoring it. Divergent outcomes may differ depending upon the painful cause, the severity and the moment, and consequently its interpretation should be properly explained. For instance, cows with clinical mastitis reduced their time lying and increased the number of lying bouts and stepping due to pain caused by the swollen udder when cows are lying. However, lame cows show longer lying times, with a lower number of lying bouts and longer and more variable lying bouts duration, as compared to non-lame cows. When the relationship between painful disorders and daily activity patterns is studied, factors such as parity, bedding type and severity of disease are important factors to take into consideration. The potential benefits of the NSAIDs treatment in painful health disorders depend upon the type of drug administered, its dosage and administration mode, and the time of administration relative to the painful health disorder. This narrative review can be used as a tool to properly interpret and grade pain in cows through behavioural activity patterns and proposes directions for future investigations.

Changes in salivary biomarkers of oxidative status in calves at weaning and grouping.

BACKGROUND: Saliva is being increasingly used as a sample for measuring biomarkers in several species and shows a high potential of use to detect and monitor stress. The weaning and grouping in dairy calves are a particularly stressful time. Therefore, the objectives of this study were to evaluate a panel of antioxidant and oxidant biomarkers in the saliva of calves on the day of weaning (W0), 2 days after weaning or milk withdrawal (W + 2), and 4 days after grouping (G + 4). In addition, to verify if cortisol and oxytocin concentrations are related to the biomarkers measured. RESULTS: Salivary cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of saliva (FRAS), Trolox equivalent antioxidant capacity (TEAC), advanced oxidation protein products (AOPP), and ferrous oxidation-xylenol orange (FOX) were significantly higher (P < 0.02) 4 days after grouping than the day of weaning and 2 days after. The increases were 50 and 54% for CUPRAC, 93 and 116% for FRAS, 117 and 135% for TEAC, 22 and 49% for AOPP and 10 and 5% for FOX in comparison with weaning and 2 days after, respectively. In addition, oxytocin and cortisol showed significant negative and positive correlations (P < 0.05) respectively with the biomarkers of oxidative status. CONCLUSIONS: Our results showed that calves after grouping show increases in antioxidants and oxidants concentrations, indicating that a balance between these molecules has been tried to maintain during this stressful situation. The dynamic changes of biomarkers of oxidative status should be explored and characterised in other stressful conditions.

Caretaker Score Reliability for Personality Assessment of Bottlenose Dolphin (Tursiops truncatus).

The evaluation of zoo animals' personalities can likely lead to a range of benefits, including improving breeding success, creating stable social groups, and designing and developing environmental enrichment programmes. The goal of this study was to use caretakers scores to evaluate personality in bottlenose dolphins and to assess the reliability of scores within each rater and among raters from each centre. To this end, 24 caretakers from 3 countries (Spain, France, and Argentina), including a total of 5 dolphinariums and 6 groups of dolphins, used a questionnaire based on the Five-Factor Model of Personality to score bottlenose dolphins on a number of personality traits in three different contexts. Each caretaker evaluated the animals under their care twice, ensuring that raters did not share thoughts nor impressions with other raters. Our findings showed a good degree of agreement between each rater's scores and a fair degree of agreement among scores of raters from the same centre. We also identified which raters and centres had significant mean score differences and detected that 4 out of 24 raters from two different centres showed such differences systematically. The evaluation of raters' reliability and the identification of particular inconsistent raters and centres is critical to make more appropriate and realistic management decisions that, in turn, directly impact animals' welfare.

Effect of Finishing Diet and Lairage Time on Steers Welfare in Uruguay.

The objective of this experiment was to evaluate the effect of two different pasture-based finishing strategies and lairage time on steers welfare in Uruguayan conditions. Sixty Hereford (H) and Braford (B) steers were assigned to two different diets for finishing purposes: (D1) native pasture plus corn grain (1% of live weight) (H n = 15, B n = 15) and (D2) high-quality pasture (H n = 15, B n = 15). The average daily gain was registered every 14 days, and temperaments were individually assessed one week before slaughter by three individual tests: crush score, flight time and exit speed, building a multicriterial temperament index (TIndex). Animals were slaughtered the same day in two groups (50% from D1 and 50% from D2 in each group) after traveling for 3.5 h and staying 15 (long lairage) and 3 h (short lairage) in the lairage pens, respectively. The behaviors were observed during lairage, and physiological indicators were used to assess stress at the farm after transport, after lairage and at slaughter. Bruises incidence and final pH were registered at the abattoir as a means of assessing the overall animal welfare. Calmer animals had higher average daily gains with no differences either between diets or between breeds. Calmer animals also had a lower stress response during all preslaughter stages, regardless of the time in lairage. Transport did not imply psychological stress (cortisol) for any slaughter group, but physical stress was evident after transport in both groups through NEFA and CPK increases. Bruise incidences did not differ between lairage groups. The short lairage group did not have enough time to cope with the environment before slaughter, with the consequent deleterious effects on the carcass pH. Animals from the long lairage group had a higher metabolic response shown through NEFA values, but they had enough time to rest and recover overnight, reaching final pH values lower than 5.8, considered the upper limit of the normal range. According to this experiment, with pasture-based animals without fasting on the farm and after 3.5 h of transportation, a resting period of 15 h in lairage should be better than a 3-h one.

A Systematic Review on Validated Precision Livestock Farming Technologies for Pig Production and Its Potential to Assess Animal Welfare.

Several precision livestock farming (PLF) technologies, conceived for optimizing farming processes, are developed to detect the physical and behavioral changes of animals continuously and in real-time. The aim of this review was to explore the capacity of existing PLF technologies to contribute to the assessment of pig welfare. In a web search for commercially available PLF for pigs, 83 technologies were identified. A literature search was conducted, following systematic review guidelines (PRISMA), to identify studies on the validation of sensor technologies for assessing animal-based welfare indicators. Two validation levels were defined: internal (evaluation during system building within the same population that were used for system building) and external (evaluation on a different population than during system building). From 2,463 articles found, 111 were selected, which validated some PLF that could be applied to the assessment of animal-based welfare indicators of pigs (7% classified as external, and 93% as internal validation). From our list of commercially available PLF technologies, only 5% had been externally validated. The more often validated technologies were vision-based solutions (n = 45), followed by load-cells (n = 28; feeders and drinkers, force plates and scales), accelerometers (n = 14) and microphones (n = 14), thermal cameras (n = 10), photoelectric sensors (n = 5), radio-frequency identification (RFID) for tracking (n = 2), infrared thermometers (n = 1), and pyrometer (n = 1). Externally validated technologies were photoelectric sensors (n = 2), thermal cameras (n = 2), microphone (n = 1), load-cells (n = 1), RFID (n = 1), and pyrometer (n = 1). Measured traits included activity and posture-related behavior, feeding and drinking, other behavior, physical condition, and health. In conclusion, existing PLF technologies are potential tools for on-farm animal welfare assessment in pig production. However, validation studies are lacking for an important percentage of market available tools, and in particular research and development need to focus on identifying the feature candidates of the measures (e.g., deviations from diurnal pattern, threshold levels) that are valid signals of either negative or positive animal welfare. An important gap identified are the lack of technologies to assess affective states (both positive and negative states).

Intestinal Parasites and Fecal Cortisol Metabolites in Multi-Unowned-Cat Environments: The Impact of Housing Conditions.

Housing conditions were assessed in different unowned multi-cat management models in order to evaluate their impact on the occurrence of intestinal parasites and fecal cortisol metabolite (FCM) levels. Fresh stool fecal samples were collected from rescue shelters, catteries and feline colonies for coprological analyses in order to detect intestinal parasite patency and fecal cortisol metabolites. A questionnaire provided information about the facilities, management and housing conditions of cats, including information about dog exposure, enclosure size, environment enrichment and changes to group composition. Overall, intestinal parasite infection was detected in 58.2% of fecal samples collected. The occurrence of intestinal parasites detected in free-roaming cats was 82.2%, mainly due to helminth infection. The parasite infection rate was 57.3% in rescue shelters and 34.6% in catteries. In confined cats, protozoa infection was more likely detected in rescue shelters than in catteries (RR = 2.02 (1.30-3.14), p = 0.0012). Although the FCM values were very variable between cats, the enclosure size and parasite infection were correlated with the average FCM. A small enclosure size was correlated with high fecal cortisol metabolites (p = 0.016). Protozoa-positive samples showed higher FCM levels than negative samples (p = 0.0150). High dog exposure was statistically associated with protozoa infection (p = 0.0006). The results indicated that improving housing, especially in terms of floor space and avoiding dog exposure, reduces stress and can thus be applied to make control strategies in multi-unowned-cat environments more efficient, especially when cats are confined.

Effect of Different Finishing Strategies and Steer Temperament on Animal Welfare and Instrumental Meat Tenderness.

The aim of this experiment was to evaluate the effect of different fattening systems from pasture to concentrate and temperament on animal welfare (AW) and meat quality (MQ). Eighty-four Hereford steers were randomly assigned to the following groups: T1, pasture (4% of animal live weight: LW); T2, pasture (3% LW) plus concentrate (0.6% LW); T3, pasture (3% LW) plus concentrate (1.2% LW); T4, an ad libitum concentrate treatment. Temperament was assessed by three individual tests: crush score, flight time, and exit speed, building a multicriterial temperament index (TIndex). The flight zone was also registered for each treatment. AW was assessed through the integration of indicators of productivity, physiology, and behavior, as well as by monitoring the health status within each treatment. Shear force was registered for MQ. Differences in average daily gain were due to the different energetic composition of the diets (T4 > T3 > T2 > T1) and were not attributable to animal welfare problems. Animals from T4 had the higher average daily gain (ADG) but welfare was negatively affected, being evident through physiological indicators, the restriction or deprivation of relevant behaviors, diet-related diseases, and mortality. T1, T2, and T3 did not appear to compromise animal welfare. However, strict preventive measures and monitoring should be taken during the habituation process and when using any new diet that includes concentrate, because of possible dietary diseases. Shear force values were lower in T1. None of the animals in our experiment were excitable or aggressive, but there was a positive response to handling in all treatments. In addition, regardless of diet, calmer animals had higher average daily gain and lower shear force values; thus, temperament appears to have a significant influence on productivity and meat quality.

Oxytocin in bovine saliva: validation of two assays and changes in parturition and at weaning.

BACKGROUND: The possible use of oxytocin in saliva as an indicator of positive emotions in bovine species has been poorly investigated. In the present study, two new assays (one using a monoclonal antibody and the other using a polyclonal antibody) for the measurement of oxytocin in bovine saliva were developed and validated. Also, the changes in oxytocin in saliva were explored in two different situations. One was around parturition, and for this purpose, saliva samples from 13 cows were collected at three different times: 7 days before the parturition, the day of parturition and 7 days after the parturition. The second situation was weaning and grouping of calves, and for this purpose, saliva from 25 calves was collected at three different times: 1 day before weaning, 2 days after weaning or milk withdrawal and 4 days after grouping calves. RESULTS: In cows, oxytocin concentrations showed an increase on the day of parturition with both assays, while in calves, oxytocin concentrations showed a decrease 4 days after the grouping. CONCLUSIONS: The assays validated in this report could be used for the measurement of oxytocin in bovine saliva and detect changes in this analyte that can occur in different physiological or productive situations such as parturition and weaning.

Oral Meloxicam Administration in Sows at Farrowing and Its Effects on Piglet Immunity Transfer and Growth.

Many factors can lead to an inadequate development of piglets during their first days of life, including poor maternal behavior, which can be due to pain caused by farrowing, and reduced colostrum ingestion. This study investigates the action of meloxicam administered orally at farrowing on piglet weight gain and immunity transfer. Thirty-five multiparous sows were divided into two groups and treated with 0.4 mg/kg of oral meloxicam (oral meloxicam group; n = 18) or with a mock administration (control group; n = 17). A total of 382 piglets were individually weighed on the farrowing day (day 0), as well as on days +9 and +20. Immunoglobulin G (IgG) and A (IgA) concentrations in piglet serum and in sow's saliva, colostrum and milk were measured. Additionally, Interleukin-2 (IL-2), Interleukin-4 (IL-4) and Interferon gamma (IFN-⋎) in serum of piglets and in sow's milk or colostrum were studied. All samples were obtained on days +1, +9, and +20. Piglets from sows in the oral meloxicam group tended to grow faster from day +9 to day +20 than did piglets from control sows (p = 0.059), and this difference was also observed in piglets with low body weight (BW) at birth (p = 0.056). The oral meloxicam group sows tended to increase the colostrum levels of IgA and IgG, as compared with control sows on day +1 (p = 0.068 and p = 0.072, respectively). IgA levels in piglet serum from the oral meloxicam group were significantly higher than in the control group on day +1 and +9 (p = 0.019 and p = 0.011 respectively). Furthermore, IL-2 and IL-4 levels in the serum of piglets from sows in the oral meloxicam group tended to be higher than that in the control group on day +9 (p = 0.078 and 0.056, respectively). The administration of meloxicam orally at the beginning of farrowing in multiparous sows increased immunoglobin and cytokine concentrations in colostrum, improving both humoral and cellular immune response of piglets. Pre-weaning growth of piglets born with a low BW improved in the meloxicam-treated group.

Sex Differences in Group Composition and Habitat Use of Iberian Free-Range Pigs.

The aim of the present work was to study group size, group composition and habitat use of Iberian pigs along the year when reared outdoor. This consists of a regimen in which animals are reared free range from 2 months of age until at least 14 months of age. In a first stage, animals are supplemented with concentrates, and in a second, called montanera, pigs eat just natural resources in areas with no more than two pigs per hectare. In these systems, males are castrated to avoid boar taint and females spayed to avoid the attraction and mounting by wild boars. The study was carried out in five different farms allocated in the south-west of Spain during 2 consecutive years, from March 2012 to February 2014, under the montanera regimen, and with a total of 995 animals observed (498 males and 497 females). The data were analyzed with SAS by means of general models and proc mixed. Mean group size along the year was of 17 ± 12.9 individuals, but this was significantly lower (P < 0.05) during the montanera (12 ± 0.8) and at midday (13 ± 0.8). Groups were bigger (P < 0.05) when they were more than 50 m from a tree (23 ± 1.8), or <10 m from the shelter (25 ± 1.5), the feeding area (31 ± 3.1) and the water-bath area (25 ± 1.5). Nine percent of the groups were solitary animals, being higher (P = 0.0286) during the montanera (11%) than the rest of the year (8%) and being formed in 68% by males. Males were less involved in mixed groups than were females (75% vs. 91%), especially in spring, where the largest (P < 0.0001) male groups were found. Female groups were less frequent and smaller (P < 0.0001) than were male and mixed groups. In conclusion, although males were castrated at a very young age, they showed a different behavior than females, forming in bachelor groups during the spring and being less involved in mixed groups and with more solitary animals. During the montanera, when animals were feeding on acorns and other natural resources, groups were smaller and closer to the trees, solitary males reaching a maximum percent.