Identification of intestinal and fecal microbial biomarkers using a porcine social stress model.

Understanding the relationships between social stress and the gastrointestinal microbiota, and how they influence host health and performance is expected to have many scientific and commercial implementations in different species, including identification and improvement of challenges to animal welfare and health. In particular, the study of the stress impact on the gastrointestinal microbiota of pigs may be of interest as a model for human health. A porcine stress model based on repeated regrouping and reduced space allowance during the last 4 weeks of the finishing period was developed to identify stress-induced changes in the gut microbiome composition. The application of the porcine stress model resulted in a significant increase in salivary cortisol concentration over the course of the trial and decreased growth performance and appetite. The applied social stress resulted in 32 bacteria being either enriched (13) or depleted (19) in the intestine and feces. Fecal samples showed a greater number of microbial genera influenced by stress than caecum or colon samples. Our trial revealed that the opportunistic pathogens Treponema and Clostridium were enriched in colonic and fecal samples from stressed pigs. Additionally, genera such as Streptococcus, Parabacteroides, Desulfovibrio, Terrisporobacter, Marvinbryantia, and Romboutsia were found to be enriched in response to social stress. In contrast, the genera Prevotella, Faecalibacterium, Butyricicoccus, Dialister, Alloprevotella, Megasphaera, and Mitsuokella were depleted. These depleted bacteria are of great interest because they synthesize metabolites [e.g., short-chain fatty acids (SCFA), in particular, butyrate] showing beneficial health benefits due to inhibitory effects on pathogenic bacteria in different animal species. Of particular interest are Dialister and Faecalibacterium, as their depletion was identified in a human study to be associated with inferior quality of life and depression. We also revealed that some pigs were more susceptible to pathogens as indicated by large enrichments of opportunistic pathogens of Clostridium, Treponema, Streptococcus and Campylobacter. Generally, our results provide further evidence for the microbiota-gut-brain axis as indicated by an increase in cortisol concentration due to social stress regulated by the hypothalamic-pituitary-adrenal axis, and a change in microbiota composition, particularly of bacteria known to be associated with pathogenicity and mental health diseases.

Impact of Maternal High Stocking Density during the Dry Period on Dairy Calf Health, Behaviour, and Welfare.

This study aimed to investigate the effect of maternal stocking density during late pregnancy (approximately 60 ± 4 days before calving) on offspring performance during the pre-weaning period. Forty-five dairy calves were born to cows that went through either industry minimum standards (H: n = 24, high stocking density) or more extensive space allowances (L: n = 21, low stocking density) during the dry period. Body weight and average daily gain during the pre-weaning period (day 1-49) were measured. Observations were made of: (i) activity levels (day 2-6); ii) the level of training required to use an automatic feeder, and behavioural reactions to the group environment (d7); (iii) feeding and social behaviour in the group pen (day 7-21); and (iv) responses to weaning (day 40-49) and disbudding (day 28+). Compared to L calves, H calves made more frequent social contacts with pen mates in the group pen (p = 0.003) and decreased their lying time around weaning (p = 0.045). Among the healthy calves, L calves displayed more severe behavioural reactions to the disbudding procedure (p < 0.001), a significant increase in salivary cortisol concentrations (p = 0.013), and more frequent pain-related behaviour (p = 0.036). This study indicated associations between maternal stocking density during late pregnancy and some welfare-relevant offspring outcomes during the pre-weaning period; these effects were found to be modulated by offspring health status.

Periparturient Behavior and Physiology: Further Insight Into the Farrowing Process for Primiparous and Multiparous Sows.

Giving birth is a critical time for many species and is often the most painful event ever experienced by females. In domestic species, like the pig, pain associated with parturition represents a potential welfare concern, and the consequences of pain can cause economic losses (e.g., by indirectly contributing to piglet mortality as pain could slow post-farrowing recovery, reduce food and water intake, reducing milk let-down). This study investigated pain assessment and its management in primiparous (gilts) and multiparous (sows) breeding pigs, including the provision of a non-steroidal anti-inflammatory drug (NSAID) post-parturition. Individuals were randomly allocated to receive the NSAID ketoprofen (3 mg/kg bodyweight) (n = 11 gilts, 16 sows) or the equivalent volume of saline (n = 13 gilts, 16 sows) by intramuscular injection 1.5 h after the birth of the last piglet. Data collected included putative behavioral indicators of pain (back leg forward, tremble, back arch), salivary cortisol concentrations pre-farrowing and up to 7 days post-injection. In addition, post-partum biomarkers of inflammation, including the acute phase protein C-reactive protein (CRP) and 3 porcine cytokines [interleukin-1 ß (IL1 ß), interleukin-6 (IL6), and tumor necrosis factor α (TNF α)] were measured in plasma collected 6 h following the injection. Behaviors were analyzed using generalized linear mixed models, and physiological variables with linear mixed models. No difference in putative pain behaviors, salivary cortisol, CRP, or cytokines were found between individuals treated with ketoprofen or those administered the saline control. However, there were some differences between gilts and sows, as sows exhibited more putative pain behavior than gilts, had higher salivary cortisol on the day of farrowing and had higher plasma TNF α. Conversely, gilts had higher salivary cortisol than sows on day 3 post-farrowing and had higher CRP. This indicates that, like human females, multiparous sows experience more pain from uterine activity following birth than primiparas. This study provides useful information for developing management practices relating to post-farrowing care for breeding pigs.

Survey of dry cow management on UK commercial dairy farms.

Dry period management of the dairy cow focuses on maximising milk production during the subsequent lactation but may include procedures that negatively affect dry cow health and welfare. A survey of dairy farmers in the UK was conducted to investigate dry cow management procedures. The questionnaire was completed by 148 farmers. Most farms (84 per cent) kept dry cows in dynamic social groups. The median length of the dry period was 56 days, and 83 per cent of farms stopped milking abruptly, regardless of milk production level at dry-off. Twenty-seven per cent of cows from respondent farms produced more than 20 kg of milk per day at dry-off. The majority of farms (78 per cent) used antibiotic dry cow intramammary tubes at dry-off, in combination with internal or external teat sealants. Procedures that were commonly practised and potentially stressful for dry cows included abrupt cessation of milking of high yielding cows and frequent changes in diet and social environment.

A Review of Pain Assessment in Pigs.

There is a moral obligation to minimize pain in pigs used for human benefit. In livestock production, pigs experience pain caused by management procedures, e.g., castration and tail docking, injuries from fighting or poor housing conditions, "management diseases" like mastitis or streptococcal meningitis, and at parturition. Pigs used in biomedical research undergo procedures that are regarded as painful in humans, but do not receive similar levels of analgesia, and pet pigs also experience potentially painful conditions. In all contexts, accurate pain assessment is a prerequisite in (a) the estimation of the welfare consequences of noxious interventions and (b) the development of more effective pain mitigation strategies. This narrative review identifies the sources of pain in pigs, discusses the various assessment measures currently available, and proposes directions for future investigation.

The identification of potential behavioural indicators of pain in periparturient sows.

Periparturient pain is a welfare concern and could contribute to piglet losses. This has led to studies investigating post-farrowing analgesia. A clear reduction in pain has not been demonstrated, partly due to a lack of pain indicators. This study quantified behaviours as potential pain indicators (PPIn) in sows: i) before, during and after farrowing, and ii) 2min before and after piglet births. Twenty-five sows were observed during and after, and ten pre-farrowing. Behaviour recorded included: 1) back leg forward (back leg pulled forward and/or in); 2) tremble (movement as if shivering); 3) back arch (leg(s) stretch forming an arched back); 4) paw (leg scraped in pawing motion); and 5) tail flick (tail moved rapidly up and down). Behaviours were analysed using generalized linear models and Spearman's rank correlations. All PPIn were rare or absent pre-farrowing, highest during farrowing, and back leg forward, tremble and back arch were greater in the early post-farrowing period. Several significant positive correlations between PPIn during and post-farrowing were found. Back arch, tail flick and paw were higher before than after a piglet birth, and were more frequent earlier in the birth order. Back leg forward and tremble did not differ before and after births, and tremble increased with birth order. These behaviours, which were absent or rare pre-farrowing, present during farrowing and were lower afterwards, and showed consistent individual variation, may be quantitatively associated with pain. Spontaneous behaviours could be used to test the efficacy of analgesics or identify sows that may benefit.

Prenatal stress produces anxiety prone female offspring and impaired maternal behaviour in the domestic pig.

Numerous studies have shown that prenatal stress (PNS) can have profound effects on postnatal well-being. Here, the domestic pig (Sus scrofa) was used to investigate PNS effects owing to the direct relevance for farm animal welfare and the developing status of the pig as a large animal model in translational research. Pregnant primiparous sows were exposed, in mid-gestation, to either a social stressor (mixing with unfamiliar conspecifics) or were kept in stable social groups. The ratio of levels of mRNAs for corticotropin releasing hormone (CRH) receptors 1 and 2 in the amygdala, measured for the first time in the pig, was substantially increased in 10-week-old female, but not male, PNS progeny indicating a neurobiological propensity for anxiety-related behaviour. Mature female offspring were observed at parturition in either a behaviourally restrictive crate or open pen. Such PNS sows showed abnormal maternal behaviour in either environment, following the birth of their first piglet. They spent more time lying ventrally, more time standing and showed a higher frequency of posture changes. They were also more reactive towards their piglets, and spent longer visually attending to their piglets compared to controls. Associated with this abnormal maternal care, piglet mortality was increased in the open pen environment, where protection for piglets is reduced. Overall, these data indicate that PNS females have their brain development shifted towards a pro-anxiety phenotype and that PNS can be causally related to subsequent impaired maternal behaviour in adult female offspring.

Qualitative Behavioural Assessment of emotionality in pigs.

Scientific assessment of affective states in animals is challenging but vital for animal welfare studies. One possible approach is Qualitative Behavioural Assessment (QBA), a 'whole animal' methodology which integrates information from multiple behavioural signals and styles of behavioural expression (body language) directly in terms of an animal's emotional expression. If QBA provides a valid measure of animals' emotional state it should distinguish between groups where emotional states have been manipulated. To test this hypothesis, QBA was applied to video-recordings of pigs, following treatment with either saline or the neuroleptic drug Azaperone, in either an open field or elevated plus-maze test. QBA analysis of these recordings was provided by 12 observers, blind to treatment, using a Free Choice Profiling (FCP) methodology. Generalised Procrustes Analysis was used to calculate a consensus profile, consisting of the main dimensions of expression. Dimension one was positively associated with terms such as 'Confident' and 'Curious' and negatively with 'Unsure' and 'Nervous'. Dimension two ranged from 'Agitated'/'Angry' to 'Calm'/'Relaxed'. In both tests, Azaperone pre-treatment was associated with a more positive emotionality (higher scores on dimension one reflecting a more confident/curious behavioural demeanour) than control pigs. No effect of drug treatment on dimension two was found. Relationships between qualitative descriptions of behaviour and quantitative behavioural measures, taken from the same recordings, were found. Overall, this work supports the use of QBA for the assessment of emotionality in animals.

The impact of prenatal stress on basal nociception and evoked responses to tail-docking and inflammatory challenge in juvenile pigs.

The consequences of tail-docking (at 2-4 days) and prenatal stress (maternal social stress during the 2nd third of pregnancy) on baseline nociceptive thresholds and responses to acute inflammatory challenge were investigated in juvenile pigs in two studies. Nociceptive thresholds were assessed on the tail root and on the hind foot using noxious mechanical and cold stimulation before and after acute inflammatory challenge by intradermal injection of 30 µg capsaicin (study 1) or 3% carrageenan (study 2) into the tail root. Four groups of 8 (study 1, n=14-16 pigs/treatment) or 5 (study 2, n=6 pigs/treatment/sex) week-old pigs were exposed to the main factors: maternal stress and treatment (docked vs. intact tails). In study 1, tail docking did not significantly alter thresholds to noxious mechanical stimulation, whilst prenatally stressed pigs had significantly higher baseline thresholds to noxious mechanical stimulation on the tail root and on the hind foot than unstressed pigs, whether tail-docked or intact. Capsaicin injection induced localised mechanical allodynia around the tail root in all treatment groups, but had no effect on noxious plantar mechanical responses; however prenatally stressed offspring exhibited significantly attenuated response thresholds to capsaicin compared to controls. In study 2 tail docking did not alter thresholds to either mechanical or noxious cold stimulation. Baseline response durations to noxious cold stimulation of the tail root were significantly shorter in both sexes of prenatally stressed pigs, whilst male but not female prenatally stressed pigs exhibited significantly higher baseline thresholds to mechanical stimulation than controls, although results in female pigs tended towards significance. Carrageenan injection into the tail root induced localised mechanical and cold allodynia in all treatment groups, effects that were attenuated in prenatally stressed pigs. Collectively, these findings indicate that prenatal stress can induce long-term alterations in nociceptive responses, manifest as a reduced sensitivity to noxious mechanical and cold stimulation and evoked inflammatory allodynia. Neonatal tail-docking does not lead to long-term alterations in nociception in pigs.

Emotionality in growing pigs: is the open field a valid test?

The ability to assess emotionality is important within animal welfare research. Yet, for farm animals, few tests of emotionality have been well validated. Here we investigated the construct validity of behavioural measures of pig emotionality in an open-field test by manipulating the experiences of pigs in three ways. In Experiment One (pharmacological manipulation), pigs pre-treated with Azaperone, a drug used to reduce stress in commercial pigs, were more active, spent more time exploring and vocalised less than control pigs. In Experiment Two (social manipulation), pigs that experienced the open-field arena with a familiar companion were also more exploratory, spent less time behaviourally idle, and were less vocal than controls although to a lesser degree than in Experiment One. In Experiment Three (novelty manipulation), pigs experiencing the open field for a second time were less active, explored less and vocalised less than they had done in the first exposure to the arena. A principal component analysis was conducted on data from all three trials. The first two components could be interpreted as relating to the form (cautious to exploratory) and magnitude (low to high arousal) of the emotional response to open-field testing. Based on these dimensions, in Experiment One, Azaperone pigs appeared to be less fearful than saline-treated controls. However, in Experiment Two, exposure to the arena with a conspecific did not affect the first two dimensions but did affect a third behavioural dimension, relating to oro-nasal exploration of the arena floor. In Experiment Three, repeat exposure altered the form but not the magnitude of emotional response: pigs were less exploratory in the second test. In conclusion, behavioural measures taken from pigs in an open-field test are sensitive to manipulations of their prior experience in a manner that suggests they reflect underlying emotionality. Behavioural measures taken during open-field exposure can be useful for making assessments of both pig emotionality and of their welfare.

Development of a mechanical stimulator and force measurement system for the assessment of nociceptive thresholds in pigs.

A mechanical stimulator and force measurement system was developed to quantify withdrawal thresholds to noxious mechanical stimulation of the foot in young pigs. The device and associated PC software have design and control features not previously used in other mechanical stimulators. The device, capable of delivering stimulus rates between 2 and 17 mm/s, maximum force 27 N, was validated in a cross-over study on 8 juvenile pigs (6-8 weeks of age) to check the repeatability and reliability of force threshold measurement and assess its ability to measure changes in force threshold following an inflammatory challenge. Threshold force measurements were obtained over several time periods before and after the pigs received a 0.25 ml subcutaneous injection of 3% carrageenan in 0.01 M phosphate buffered saline (PBS) or PBS in the hind foot. Consistent withdrawal thresholds were measured in injected (ipsilateral) and contralateral feet, 24 h and 30 min prior to injection (mean 8.4; 95% CI 7.1-9.7 N). Carrageenan injection, but not PBS injection, induced a significant decrease in withdrawal thresholds 90 min after injection (4.6+/-0.9 N) which remained reduced for 6h after injection. The testing system provided reliable and reproducible measurements of foot withdrawal thresholds to noxious mechanical force in young pigs (weight range 32-39 kg), and was capable of detecting and monitoring changes in threshold sensitivity following the induction of acute local inflammation in the foot. The system is suitable for studying nociceptive mechanisms in pigs.

Pre-natal stress amplifies the immediate behavioural responses to acute pain in piglets.

Pre-natal stress (PNS) or undernutrition can have numerous effects on an individual's biology throughout their lifetime. Some of these effects may be adaptive by allowing individuals to tailor their phenotype to environmental conditions. Here we investigated, in the domestic pig Sus scrofa, whether one possible consequence of a predicted adverse environment could be altered pain perception. The behavioural response of piglets to the surgical amputation ('docking') of their tail or a sham procedure was measured for 1 min in piglets born to mothers who either experienced mid-gestation social stress or were left undisturbed throughout pregnancy. A behavioural pain score was found to predict the docked status of piglets with high discriminant accuracy. Piglets exposed to PNS had a significantly higher pain score than controls, and for each litter of tail-docked piglets, the average pain score was correlated with mid-gestation maternal cortisol levels. The data presented here provide evidence that the experience of stress in utero can result in a heightened acute response to injury in early life. Speculatively, this may represent an adaptive alteration occurring as a consequence of a pre-natal 'early warning' of environmental adversity.

Lameness prevalence and risk factors in organic and non-organic dairy herds in the United Kingdom.

This study aimed to compare the prevalence of lameness on organic and non-organic dairy farms in the United Kingdom (UK) and to assess which cow and farm factors influenced lameness levels. Forty organic and 40 non-organic dairy farms across the UK were repeatedly visited over a 2.5 year period. On each visit all milking cows were locomotion scored, and information about farm housing, management and husbandry practices was recorded on-farm. Over the whole study, the mean herd lameness prevalence was 16.2%, 16.3% and 19.3% in the autumn, winter and spring observation periods, respectively. Lameness prevalence was lower (P=0.012) on organic farms compared to non-organic farms. Numerous specific factors were found to significantly influence the prevalence of lameness. This study provided evidence that organic management reduced herd lameness. It supported previous research which suggested that lameness is a serious problem on many farms in the UK and further emphasised the multi-factorial aetiology of lameness problems.

The responses of growing pigs to a chronic-intermittent stress treatment.

Many of the stressor treatments used in animal models of depression have parallels in the normal experiences of domestic pigs. The experiment described here aimed to assess whether a chronic-intermittent stress regime caused behavioural or physiological changes, indicative of depression, in domestic pigs. Ten juvenile male pigs were exposed to a social and environmental stress regime. Over the stressor period, weight gain was significantly lower in test pigs than in control pigs. Stress treatment had a significant effect on salivary cortisol levels, with test pigs having a higher salivary cortisol concentration than control pigs after the stress treatment but not before. Test pigs showed less ventral lying than control pigs in the post-stress observation. A detrended fluctuation analysis (DFA) of postural behavioural organisation showed that test pigs had a more structured pattern of activity than controls in the post-stress observation and a tendency towards a more structured pattern in the pre-stress observation. There were no major behavioural differences between the two groups during three repeated open field tests. The results suggest that the stressor treatment did create a mild chronic stress, as indicated by the hypercortisolaemia and lower weight gain in the test pigs. However, no unambiguous behavioural indicators of depression were seen. The behavioural analysis did show that fractal techniques, such as DFA, could be applied to pig behaviour and that they can reveal extra novel information about the structure of an individual's behavioural organisation and how it changes in response to complex environmental stressors.

Visual fixation of a landing perch by chickens.

Chickens were video recorded while making jumps or flights toward a landing perch, to test hypotheses about visual fixation behaviour. In the first experiment, varying the height above the landing perch of the food container providing the incentive to jump had no effect on head orientation, indicating that the birds were not fixating this object. In the second experiment, hens jumped over six combinations of perch height and distance, and a linear relationship was found at take-off between head orientation and the angular distance of the perch from the horizontal at the eye. This relationship is consistent with fixation of the perch by a linear combination of head and eye rotations, with the head component contributing 73% of the total response. The image of the perch is fixated 20 masculine below that of the bill tip, outside any region of the chicken retina specialised for high acuity vision. Fixation of the perch before jumping must therefore have some function other than inspection with high acuity, such as providing a constraint that enables precise visual control of trajectory and landing manoeuvres.