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Stress is an important factor in animal welfare. Hair or wool cortisol concentrations are considered to be potential long-term indicators of stress experienced by an animal. Using Swifter sheep, we investigated whether ewe parity and litter size affect the wool cortisol concentrations in ewes and their offspring. We hypothesized that multiparous ewes and their offspring would have higher wool cortisol concentrations than primiparous ewes and their offspring, that ewes with larger litters and their offspring would have lower wool cortisol concentrations than ewes with smaller litters and their offspring, that male lambs would have higher wool cortisol concentrations than female lambs, and that the wool cortisol concentrations in the wool of ewes and their lambs would be correlated. Lamb wool grows in utero during the third trimester of pregnancy. In ewes, the shave–reshave method was used so that wool samples from ewes also covered approximately the last trimester of pregnancy. Our study confirmed that litter size affected ewe wool cortisol concentrations: ewes that gave birth to larger litters (i.e., 3 or 4 lambs) had higher wool cortisol concentrations than ewes that gave birth to smaller litters (i.e., 1 or 2 lambs). There was no evidence that the wool cortisol concentrations of the ewes and their lambs were correlated. Neither litter size nor parity of the ewe affected wool cortisol in the lambs. Our study confirms that wool cortisol can be reliably measured in ewes and their newborn lambs, and suggests that it may be useful as a retrospective indicator of stress during the last trimester of pregnancy.

Translating theoretical concepts of animal welfare into quantitative assessment protocols is an ongoing challenge. Glucocorticoids (GCs) are frequently used as physiological measure in welfare assessment. The interpretation of levels of GCs and especially their relation to welfare, however, is not as straightforward, questioning the informative power of GCs. The aim of this systematic mapping review was therefore to provide an overview of the relevant literature to identify global patterns in studies using GCs as proxy for the assessment of welfare of vertebrate species. Following a systematic protocol and a-priory inclusion criteria, 509 studies with 517 experiments were selected for data extraction. The outcome of the experiments was categorized based on whether the intervention significantly affected levels of GCs, and whether these effects were accompanied by changes in behavior,morphology and physiology. Additional information, such as animal species, type of intervention, experimental set up and sample type used for GC determination was extracted, as well. Given the broad scope and large variation in included experiments,meta-analyses were not performed, but outcomes are presented to encourage further, in-depth analyses of the data set. The interventions did not consistently lead to changes in GCs with respect to the original authors hypothesis. Changes in GCs were not consistently paralleled by changes in additional assessment parameter on behavior, morphology and physiology. The minority of experiment quantified GCs in less invasive sample matrices compared to blood. Interventions showed a large variability, and species such as fish were underrepresented, especially in the assessment of behavior. The inconclusive effects on GCs and additional assessment parameter urges for further validation of techniques and welfare proxies. Several conceptual and technical challenges need to be met to create standardized and robust welfare assessment protocols and to determine the role of GCs herein.

Animal welfare is a multifaceted issue that can be approached from different viewpoints, depending on human interests, ethical assumptions, and culture. To properly assess, safeguard and promote animal welfare, concepts are needed to serve as guidelines in any context the animal is kept in. Several different welfare concepts have been developed during the last half decade. The Five Freedoms concept has provided the basis for developing animal welfare assessment to date, and the Five Domains concept has guided those responsible for safeguarding animal welfare, while the Quality of Life concept focuses on how the individual perceives its own welfare state. This study proposes a modified and extended version of an earlier animal welfare concept - the Dynamic Animal Welfare Concept (DAWCon). Based on the adaptability of the animal, and taking the importance of positive emotional states and the dynamic nature of animal welfare into account, an individual animal is likely in a positive welfare state when it is mentally and physically capable and possesses the ability and opportunity to react adequately to sporadic or lasting appetitive and adverse internal and external stimuli, events, and conditions. Adequate reactions are elements of an animal’s normal behavior. They allow the animal to cope with and adapt to the demands of the (prevailing) environmental circumstances, enabling it to reach a state that it perceives as positive, i.e., that evokes positive emotions. This paper describes the role of internal as well as external factors in influencing welfare, each of which exerts their effects in a sporadic or lasting manner. Behavior is highlighted as a crucial read-out parameter. As most animals under human care are selected for certain traits that may affect their behavioral repertoire it is crucial to have thorough ethograms, i.e., a catalogue of specific behaviors of the species/strain/breed under study. DAWCon highlights aspects that need to be addressed when assessing welfare and may stimulate future research questions.

Background Lifestyle influences endocrine, metabolic and cardiovascular homeostasis. This study investigated the impact of diet and oral anti-diabetic medication on cardio-metabolic health in human-sized diabetic pigs. Methods After a growing pre-phase from ~30 to ~69 kg during which domestic pigs were fed either a low fat, low sucrose diet (group A) or a fast food-type diet elevated in lard (15%) and sucrose (40%) (group B), the pigs were subdivided in 5 groups (n = 7–8 pigs per group). Group 1, normal pigs from group A on a low fat, low sugar (L) pig diet and group 2, normal pigs from group B on a high lard (25%), sucrose-fructose (40%), cholesterol (1%) fast food-type (F) diet. Diabetes (D) was induced in group B pigs by streptozotocin and group 3 received the F diet (DF), group 4 received the F diet with Anti-diabetic medication metformin (2 g.day-1)-pioglitazone (40 mg.day-1) (DFA) and group 5 switched to a Plant-Fish oil (25%), Slowly digestible starch (40%) diet (DPFS). The F and PFS diets were identical for fat, carbohydrate and protein content but only differed in fat and carbohydrate composition. The 5 pig groups were followed up for 7 weeks until reaching ~120 kg. Results In normal pigs, the F diet predisposed to several abnormalities related to metabolic syndrome. Diabetes amplified the inflammatory and cardiometabolic abnormalities of the F diet, but both oral FA medication and the PFS diet partially corrected these abnormalities (mean±SEM) as follows: Fasting plasma TNF-ɑ (pg.ml-1) and NEFA (mmol.l-1) concentrations were high (pDF (193±55 and 0.79±0.16), intermediate in DFA (136±40 and 0.57±012) and low in DPFS pigs (107±31 and 0.48±0.19). Meal intolerance (response over fasting) for glucose and triglycerides (area under the curve, mmol.h-1) and for lactate (3-h postprandial, mmol.l-1) was high (pDF (489±131, 8.6±4.8 and 2.2±0.6), intermediate in DFA (276±145, 1.4±1.1 and 1.6±0.4) and low in DPFS (184±62, 0.7±1.8 and 0.1±0.1). Insulin-mediated glucose disposal (mg.kg-1.min-1) showed a numerical trend (p = NS): low in DF (6.9±2.2), intermediate in DFA (8.2±1.3) and high in DPFS pigs (10.4±2.7). Liver weight (g.kg-1 body weight) and liver triglyceride concentration (g.kg-1 liver) were high (pDF (23.8±2.0 and 69±14), intermediate in DFA (21.1±2.0 and 49±15) and low in DPFS pigs (16.4±0.7 and 13±2.0). Aorta fatty streaks were high (pDF (16.4±5.7%), intermediate in DFA (7.4±4.5%) and low in DPFS pigs (0.05±0.02%). Conclusion This translational study using pigs with induced type 2 diabetes provides evidence that a change in nutritional life style from fast food to a plant-fish oil, slowly digestible starch diet can be more effective than sole anti-diabetic medication.

Many protocols have been developed to assess farm animal welfare. However, the validity of these protocols is still subject to debate. The present study aimed to compare nine welfare assessment protocols, namely: (1) Welfare Quality© (WQ), (2) a modified version of Welfare Quality (WQ Mod), which has a better discriminative power, (3) WelzijnsWijzer (Welfare Indicator, WW), (4) a new Welfare Monitor (WM), (5) Continue Welzijns Monitor (Continuous Welfare Monitor, CWM), (6) KoeKompas (Cow Compass, KK), (7) Cow Comfort Scoring System (CCSS), (8) Stall Standing Index (SSI) and (9) a Welfare Index (WI Tuyttens). In addition, a simple welfare estimation by veterinarians (Estimate vets, EV) was added. Rank correlation coefficients were calculated between each of the welfare assessment protocol scores and mean hair cortisol concentrations from 10 cows at 58 dairy farms spread over the Netherlands. Because it has been suggested that the hair cortisol level is related to stress, experienced over a long period of time, we expected a negative correlation between cortisol and the result of the welfare protocol scores. Only the simple welfare estimation by veterinarians (EV) (ρ = −0.28) had a poor, but significant, negative correlation with hair cortisol. This correlations, however, failed to reach significance after correction of p-values for multiple correlations. Most of the results of the different welfare assessment protocols had a poor, fair or strong positive correlation with each other, supporting the notion that they measure something similar. Additional analyses revealed that the modified Welfare Quality protocol parameters housing (ρ = −0.30), the new Welfare Monitor (WM) parameter health (ρ = −0.33), and milk yield (ρ = −0.33) showed negative correlations with cortisol. We conclude that because only five out of all the parameter scores from the welfare assessment protocols showed a negative, albeit weak, correlation with cortisol, hair cortisol levels may not provide a long term indicator for stress in dairy cattle, or alternatively, that the protocols might not yield valid indices for cow welfare.

In this study, we investigated whether alterations in plasticity markers such as brain-derived neurotrophic factor (BDNF), p75 neurotrophin receptor (p75(NTR)) and tyrosine receptor kinase B (TrkB) are underlying iron deficiency (ID)-induced cognitive impairments in iron depleted piglets. Newborn piglets were either fed an iron depleted diet (21 mg Fe/kg) or an iron-sufficient diet (88 mg Fe/kg) for four weeks. Subsequently, eight weeks after iron repletion (190-240 mg Fe/kg) we found a significant decrease in mature BDNF (14 kDa) and proBDNF (18 kDa and 24 kDa) protein levels in the ventral hippocampus, whereas we found increases in the dorsal hippocampus. The phosphorylation of CAMP response element binding protein (CREB) follows the mature BDNF protein level pattern. No effects were found on BDNF and CREB protein levels in the prefrontal cortex. The protein levels of the high affinity BDNF receptor, TrkB, was significantly decreased in both dorsal and ventral hippocampus of ID piglets, whereas it was increased in the prefrontal cortex. Together, our data suggest a disrupted hippocampal plasticity upon postnatal ID.

Simple Summary ‘Crowding’, keeping too many birds per m2, is one of the largest welfare concerns in the poultry industry. It is therefore worrisome that there is a gap in research investigating the effects of high stocking densities during the rearing phase of laying hens. This study evaluated anxious behavior and corticosterone levels, a hormone involved in the stress response, during the first 10 weeks of laying hen chicks housed under three different crowding conditions: undercrowding, conventional crowding, and overcrowding. We found that overcrowded chicks displayed more anxious behavior compared to undercrowded chicks. Corticosterone levels were elevated in both extreme groups in week 3, but dropped to values of the conventional crowding group at week 10. We conclude that current conventional stocking densities do not seem to impair the welfare state of the laying hen chick, and that a three-fold in- or decrease of density influences stress and anxiety, but within the adaptive capacity of the chick. Important side-notes to this conclusion are that an increase of stocking density did result in a slower rate of adaptation, and that we currently do not know if there are long-term consequences of different crowding densities reaching into the laying period. Abstract The recent increases in stocking density, in extreme cases resulting in ‘crowding’, have a major impact on poultry welfare. In contrast to available research on adult laying hens, there is a gap in the literature studying the rearing phase. The present study investigated the effects of stocking density during the rearing period on the welfare of the laying hen chick. The chicks were housed under one of three crowding conditions, increasing with age: undercrowding (500-1000-1429 cm2 per chick), conventional crowding (167-333-500 cm2 per chick), or overcrowding (56-111-167 cm2 per chick). The parameters evaluated encompassed behavioral and physiological factors related to anxiety and stress. We found that during the first 6 weeks, overcrowded chicks displayed more anxious behavior than undercrowded chicks, and both extreme densities induced higher corticosterone levels compared to chicks housed under conventional crowding. At 10 weeks of age, plasma corticosterone had dropped to the level of conventional crowding group in both groups, whereas feather corticosterone remained high only in the overcrowded group. We conclude that current conventional stocking densities do not seem to impair the welfare state of the laying hen chick, and that a three-fold increase or decrease of density influences corticosterone levels and anxious behavior, but within the adaptive capacity of the chick. Important side notes to this conclusion are that an increase of stocking density did result in a slower rate of adaptation, and that there could be long-term consequences of both the different stocking densities and/or increased costs of adaptation.

Meloxicam, a preferential COX-2 inhibitor, is a commonly used nSAID in pigs. Besides having potential side effects on the gastrointestinal tract, this type of drug might potentially affect osteogenesis and chondrogenesis, processes relevant to growing pigs. Therefore, the effects of long-term meloxicam treatment on growing pigs were studied. Twelve piglets (n=6 receiving daily meloxicam 0.4 mg/kg orally from 48 until 110 days of age; n=6 receiving only applesauce (vehicle control)) were subjected to visual and objective gait analysis by pressure plate measurements at several time points. Following euthanasia a complete postmortem examination was performed and samples of the talus and distal tibia, including the distal physis, were collected. Trabecular bone microarchitecture was analysed by microCT scanning, bone stiffness by compression testing and growth plate morphology using light microscopy. Animals were not lame and gait patterns did not differ between the groups. Pathological examination revealed no lesions compatible with known side effects of nSAIDs. Trabecular bone microarchitecture and growth plate morphology did not differ between the two groups. The findings of this in vivo study reduce concerns regarding the long-term use of meloxicam in young, growing piglets.

Studies of the cognitive abilities of pigs are increasing in number, due to their relevance for the fields of animal welfare and biomedical research. While both female and male pigs have been used in cognitive tasks, possible sex differences in performance have not yet received extensive attention. This is of interest, as sexual dimorphism in cognitive abilities has been documented for a variety of species. The aim of this study was to assess the effects of sex on pigs’ performance in two cognitive tasks. Spatial learning and memory of ten female and ten male pigs was compared in a spatial holeboard task. Working and reference memory, as well as measures of motivation and exploration were assessed. Both females and males acquired the task and no differences were found between sexes for any measures of spatial memory. However, female pigs performed more successfully during reversal trials (shorter latency to first reward, higher number of rewards found), indicating greater response flexibility. This difference between sexes was transient, with males eventually reaching the same level of performance as the females. Judgment bias, a cognitive measure of affective state, was subsequently assessed using an active choice judgment bias task. Pigs were trained to respond differently to a negative and a positive stimulus, signaling either a small or a large reward. During judgment bias testing, pigs were presented with ambiguous cues and their trained ‘positive’ and ‘negative’ responses were recorded as optimistic or pessimistic choices, respectively. Both females and males displayed a slightly optimistic judgment bias. Optimistic choosing decreased with repeated testing for both groups. It is likely the pigs learned about the unrewarded outcome of ambiguous cues, rendering them no longer ambiguous. Further improvement of the judgment bias task as a cognitive measure of affective state is deemed necessary. Overall, our results indicate that sex is not a confounding factor when measuring baseline performance of pigs in the spatial holeboard or judgment bias task. Sex effects were only found when subjecting the pigs to a reversal task, warranting further study of sex differences in response flexibility. Such a difference would have implications for pig welfare, as it suggests that males are slower to cope with changes in their environment.

Simple summary Intensive farming systems are confronted with a number of animal welfare issues such as injuries from horns in cattle and feather pecking in poultry. To solve these problems, mutilating procedures, such as dehorning in cattle and goats and beak trimming in laying hens, are applied routinely. These and other procedures such as early maternal separation, overcrowding, and barren housing conditions impair animal welfare. Scientific underpinning of the efficacy of these interventions and management practices is poor. We advocate that all stakeholders, in particular animal scientists and veterinarians, take the lead in evaluating common, putative mutilating and welfare reducing procedures and management practices to develop better, scientifically supported alternatives, focused on adaptation of the environment to the animals, to ensure uncompromised animal welfare. Abstract A number of mutilating procedures, such as dehorning in cattle and goats and beak trimming in laying hens, are common in farm animal husbandry systems in an attempt to prevent or solve problems, such as injuries from horns or feather pecking. These procedures and other practices, such as early maternal separation, overcrowding, and barren housing conditions, raise concerns about animal welfare. Efforts to ensure or improve animal welfare involve adapting the animal to its environment, i.e., by selective breeding (e.g., by selecting “robust” animals) adapting the environment to the animal (e.g., by developing social housing systems in which aggressive encounters are reduced to a minimum), or both. We propose adapting the environment to the animals by improving management practices and housing conditions, and by abandoning mutilating procedures. This approach requires the active involvement of all stakeholders: veterinarians and animal scientists, the industrial farming sector, the food processing and supply chain, and consumers of animal-derived products. Although scientific evidence about the welfare effects of current practices in farming such as mutilating procedures, management practices, and housing conditions is steadily growing, the gain in knowledge needs a boost through more scientific research. Considering the huge number of animals whose welfare is affected, all possible effort must be made to improve their welfare as quickly as possible in order to ban welfare-compromising procedures and practices as soon as possible.

Judgment bias tasks (JBTs) are considered as a family of promising tools in the assessment of emotional states of animals. JBTs provide a cognitive measure of optimism and/or pessimism by recording behavioral responses to ambiguous stimuli. For instance, a negative emotional state is expected to produce a negative or pessimistic judgment of an ambiguous stimulus, whereas a positive emotional state produces a positive or optimistic judgment of the same ambiguous stimulus. Measuring an animal’s emotional state or mood is relevant in both animal welfare research and biomedical research. This is reflected in the increasing use of JBTs in both research areas. We discuss the different implementations of JBTs with animals, with a focus on their potential as an accurate measure of emotional state. JBTs have been successfully applied to a very broad range of species, using many different types of testing equipment and experimental protocols. However, further validation of this test is deemed necessary. For example, the often extensive training period required for successful judgment bias testing remains a possible factor confounding results. Also, the issue of ambiguous stimuli losing their ambiguity with repeated testing requires additional attention. Possible improvements are suggested to further develop the JBTs in both animal welfare and biomedical research.

Lameness in pigs decreases animal welfare and economic profit for the farmer. An important reason for impaired welfare in lame animals is pain due to lameness. No direct measurement of pain is possible in animals, and methods to indirectly detect and quantify the amount of pain an animal is experiencing are urgently needed. In this study, two methods to assess pain associated with lameness in pigs were evaluated to determine if they were sensitive enough to detect a lameness reduction as an effect of an experimental analgesic medication. Asymmetry associated with lameness was objectively quantified using pressure mat kinetic parameters: peak vertical force (PVF), load rate (LR), vertical impulse (VI) and peak vertical pressure (PVP). Locomotor activity was assessed in an open field test. A dose of 0.04 mg/kg buprenorphine, a strong analgesic, was used to treat 10 lame pigs, while eight other lame pigs, treated with physiological saline solution, served as controls. Buprenorphine decreased lameness-associated asymmetry for pressure mat LR (P = 0.002), VI (P = 0.003) and PVP (P = 0.001) and increased activity of the lame pigs in the open field (P = 0.023), while saline-treated animals did not show any changes in asymmetry and became less active in the open field (P

Low birth weight (LBW) is common in humans and has been found to cause lasting cognitive and developmental deficits later in life. It is thought that the primary cause is intra-uterine growth restriction (IUGR) due to a shortage of oxygen and supply of nutrients to the fetus. Pigs appear to be a good model animal to investigate long-term cognitive effects of LBW, as LBW is common in commercially farmed breeds of pigs. Moreover, pigs are developmentally similar to humans and can be trained to perform complex tasks. In this study, we trained ten very low birth weight (vLBW) piglets and their ten normal birth weight (NBW) siblings in a spatial cognitive holeboard task in order to investigate long-term cognitive effects of LBW. In this task, four out of sixteen holes contain a hidden food reward, which allows measuring working memory (WM) (short-term memory) and reference memory (RM) (long-term memory) in parallel. Piglets were trained for 46–54 trials during the acquisition phase, followed by a 20-trial reversal phase in which a different set of four holes was baited. Both groups acquired the task and improved their performance over time. A mixed model repeated measures ANOVA revealed that vLBW piglets showed better RM performance than NBW piglets in both the acquisition and reversal phase. Additionally, WM scores in the vLBW were less disrupted than in the NBW animals when switched to the reversal phase. These findings are contrary to findings in humans. Moreover, vLBW pigs had lower hair cortisol concentrations (HCCs) than NBW pigs in flank hair at 12 weeks of age. These results could indicate that restricted intra-uterine growth causes compensatory mechanisms to arise in early development that result in beneficial effects for vLBW piglets, increasing their low survival chances in early-life competition.

Low-birth-weight (LBW) children are born with several risk factors for disease, morbidity and neonatal mortality, even if carried to term. Placental insufficiency leading to hypoxemia and reduced nutritional supply is the main cause for LBW. Brain damage and poor neurological outcome can be the consequence. LBW after being carried to term gives better chances for survival, but these children are still at risk for poor health and the development of cognitive impairments. Preventive therapies are not yet available. We studied the risk/efficacy of chronic prenatal treatment with the anti-oxidative drug allopurinol, as putative preventive treatment in piglets. LBW piglets served as a natural model for LBW. A cognitive holeboard test was applied to study the learning and memory abilities of these allopurinol treated piglets after weaning. Preliminary analysis of the plasma concentrations in sows and their piglets suggested that a daily dose of 15 mg.kg(-1) resulted in effective plasma concentration of allopurinol in piglets. No adverse effects of chronic allopurinol treatment were found on farrowing, birth weight, open field behavior, learning abilities, relative brain, hippocampus and spleen weights. LBW piglets showed increased anxiety levels in an open field test, but cognitive performance was not affected by allopurinol treatment. LBW animals treated with allopurinol showed the largest postnatal compensatory body weight gain. In contrast to a previous study, no differences in learning abilities were found between LBW and normal-birth-weight piglets. This discrepancy might be attributable to experimental differences. Our results indicate that chronic prenatal allopurinol treatment during the third trimester of pregnancy is safe, as no adverse side effects were observed. Compensatory weight gain of treated piglets is a positive indication for the chronic prenatal use of allopurinol in these animals. Further studies are needed to assess the possible preventive effects of allopurinol on brain functions in LBW piglets.

BACKGROUND: Feather pecking and cannibalism are major concerns in poultry farming, both in terms of animal welfare and farm economics. Genetic selection and introduction of (aspects of) maternal care have been suggested as potential interventions to reduce feather pecking in laying hens. Altered brain development has been proposed to reflect welfare states in animals, and can provide more insight into the underlying processes involved in feather pecking. Both vasotocin (the avian homologue of vasopressin) and dopaminergic neural circuitry have roles in control of social behaviors as well as in the stress response, and may be linked to feather pecking. Thus, the hypothalamus of adult laying hens selected for low early mortality (LML), which show low feather pecking, was examined and compared with a control line of adult laying hens selected for production characteristics only (CL). The effect of foster hen rearing on the two genetic lines and their hypothalamic morphology was also investigated. RESULTS: We demonstrated an increase in the number of neurons positive for the rate-limiting enzyme in dopamine production, tyrosine hydroxylase, in the periventricular area of the hypothalamus in the LML hens compared to CL hens. Hen-reared chicks showed more vasotocin -positive neurons in the medial pre-optic area compared to the hens raised without a hen. No correlations were found between behavior in an open field at 5-6 weeks of age, and the histology of the same hens at adulthood. CONCLUSION: The hypothalamic dopaminergic and vasotinergic systems are altered in hens following genetic selection or maternal care, indicating a potential role for these systems in feather pecking. Keywords: Hypothalamus, Vasotocin, Vasopressin, Tyrosine hydroxylase, Dopamine, Welfare, Laying hen