Pancreatic PYY but not PPY expression is responsive to short-term nutritional state and the pancreas constitutes the major site of PYY mRNA expression in chickens.

PP-fold peptides such as peptide YY (PYY) and pancreatic polypeptide (PPY) are known to play key roles in vertebrate energy homeostasis. Until recently, no gene sequence was available for avian PYY and therefore a gap in knowledge of regulation of its expression exists in avian species. Here we further evidence the mRNA sequence for chicken PYY and show that the pancreas is the major site of its mRNA expression, with a secondary peak of expression around the distal jejunum, in contrast to mammals where the large intestine is the major site of PYY expression. We also demonstrate that pancreatic PYY expression is responsive to short-term and long-term nutritional state, increasing within hours of feeding, in contrast to intestinal PYY which does not fluctuate to the same extent, and pancreatic PPY which appears to be primarily determined by long-term energy state. Both pancreatic PYY and PPY expression were found to exhibit ontogeny, being evenly distributed throughout the pancreas in young (2wk) chicks but having a decreasing splenic to duodenal gradient by adolescence (12wk).

The Effects of Commercially-Relevant Disturbances on Sleep Behaviour in Laying Hens.

Ensuring the welfare of commercially kept animals is a legal and ethical responsibility. Sleep behaviour can be sensitive to environmental perturbations and may be useful in assessing welfare state. The objective of this study was to use behavioural and electrophysiological (EEG) measures to observe the effects of 24 h stressors followed by periods of no stressors on laying hen sleep behaviour, and to investigate the use of sleep behaviour as a means of welfare assessment in commercial poultry. Ten laying hens surgically implanted with EEG devices to record their brain activity over four batches were used. Hens were subjected to undisturbed, disturbed and recovery periods for 24 h. Disturbed periods consisted of either feed deprivation, increased ambient temperature (28 °C) or simulated footpad pain via injection of Freund's adjuvant into the footpad. Sleep state was scored using behaviour data from infrared cameras and EEG data. Over all periods, hens engaged in both SWS (average 60%) and REM sleep (average 12%) during the lights-off period. Feed deprivation and footpad pain had little to no effect on sleep states, while increased ambient temperature significantly reduced REM sleep (to near elimination, p < 0.001) and SWS (p = 0.017). During the lights-on period, footpad pain increased the proportion of time spent resting (p = 0.008) and in SWS (p < 0.001), with feed deprivation or increased ambient temperature (p > 0.05) having no effect. Increasing ambient temperatures are likely to affect sleep and welfare in commercially-kept laying hens in the face of global climate change.

The Effects of Mild Disturbances on Sleep Behaviour in Laying Hens.

The positive welfare of commercial animals presents many benefits, making the accurate assessment of welfare important. Assessments frequently use behaviour to determine welfare state; however, nighttime behaviours are often ignored. Sleep behaviour may offer new insights into welfare assessments. This study aimed to establish a baseline for sleep behaviour in laying hens and to then apply mild short-term disturbances and observe the subsequent effects. Twelve laying hens were divided into four batches and were surgically implanted with electroencephalogram (EEG) devices to record their brain activity. The batches were subjected to undisturbed, disturbed and recovery types of nights. Disturbed nights consisted of systematic sequences of disturbance application (wind, 90 dB noise or 20 lux light) applied one at a time for 5 min every 30 min from 21:00 to 03:00 (lights off period: 19:00-05:00). Sleep state was scored using EEG data and behaviour data from infrared cameras. Over all the types of night hens engaged in both SWS (58%) and REM sleep (18%) during lights off. When applied, the disturbances were effective at altering the amounts of wakefulness and SWS (Time × Type of Night, p < 0.001, p = 0.017, respectively), whereas REM sleep was unaltered (p = 0.540). There was no evidence of carry-over effects over the following day or night. Laying hens may be resilient to short-term sleep disruption by compensating for this in the same night, suggesting that these disturbances do not impact their long-term welfare (i.e., over days). Sleep behaviour potentially offers a unique means of assessing an aspect of animal welfare that, to date, has been poorly studied.

Do Hens Use Enrichments Provided in Free-Range Systems?

Hens in free-range systems are given enrichments to increase foraging and limit injurious pecking, but the efficacy of enrichment types requires investigation. We studied hen behaviour and feather cover in eight commercial free-range flocks each given access to four enrichments within the shed. Sheds were split into quarters, in which two enrichments (jute ropes (R) + other) were installed. Other enrichments were: lucerne hay bales (B), pecking blocks (PB), pelleted feed (PF), or further R (control). Hens were observed at three ages, at three times per age (−1, 0, ≥1 h relative to PF application), in 1 m diameter circle locations around ropes (ControlR), Enrich (B, PB, PF, R), and Away from each enrichment. Feather scores were recorded at all ages/times, at the Away location only. Significantly more birds were in Enrich locations where PB, B, and PF were available, and least near R, ControlR, and Away locations (p < 0.001). Proportions of birds interacting with enrichments were significantly higher for PB, B, and PF than R (p < 0.001), but enrichments did not generally affect proportions of birds foraging in the litter, apart from a significant decrease (p < 0.001) in PF birds foraging in the Enrich location because they were directing behaviour at PF instead. Feather scores worsened with age (p < 0.001) but were not consistently affected by enrichment. Enrichment replacement rates varied between farms. Enrichments costs were highest for PB and cheapest for R. Enrichments except R were used by hens, but with no obvious effect on feather cover. A balance has to be struck between enrichment benefits to hens and economics, but evidence suggested that hens did not benefit from R.

The effects of feed restriction, time of day, and time since feeding on behavioral and physiological indicators of hunger in broiler breeder hens.

Broiler breeder chickens are commercially feed restricted to slow their growth and improve their health and production, however, there is research demonstrating that this leads to chronic hunger resulting in poor welfare. A challenge in these studies is to account for possible daily rhythms or the effects of time since last meal on measures relating hunger. To address this, we used 3 feed treatments: AL (ad libitum fed), Ram (restricted, fed in the morning), and Rpm (restricted, fed in the afternoon) to control for diurnal effects. We then conducted foraging motivation tests and collected home pen behavior and physiological samples at 4 times relative to feeding throughout a 24-h period. The feed treatment had the largest influence on the data, with AL birds weighing more, having lower concentrations of plasma NEFA, and mRNA expression of AGRP and NPY alongside higher expression of POMC in the basal hypothalamus than Ram or Rpm birds (P < 0.001). R birds were more successful at and had a shorter latency to complete the motivation test, and did more walking and less feeding than AL birds in the home pen (P < 0.01). There was little effect of time since last meal on many measures (P > 0.05) but AGRP expression was highest in the basal hypothalamus shortly after a meal (P < 0.05), blood plasma NEFA was higher in R birds just before feeding (P < 0.001) and glucose was higher in Ram birds just after feeding (P < 0.001), and the latency to complete the motivation test was shortest before the next meal (P < 0.05). Time of day effects were mainly found in the difference in activity levels in the home pen when during lights on and lights off periods. In conclusion, many behavioral and physiological hunger measures were not significantly influenced by time of day or time since the last meal. For the measures that do change, future studies should be designed so that sampling is balanced in such a way as to minimize bias due to these effects.

Effects of Different Scratch Mat Designs on Hen Behaviour and Eggs Laid in Enriched Cages.

Laying hens in the UK and EU must be provided with litter for pecking and scratching. In enriched cages, this is commonly provided by dispensing layer's feed onto a scratch mat. Mats vary in design and size, which might affect hen behaviour and egg quality, since eggs are sometimes laid at the mats. We investigated if four different scratch mats (BD, K, V, Z) provided to hens in enriched cages resulted in differences in behaviour on the mats and external egg quality. Twenty-four 60-bird cages (6 cages/bank × 4 banks) with 2 mats/cage at one tier of a commercial enriched cage unit were used. Mats were allocated to cages in a balanced design prior to the flock arriving. Hens and eggs were studied at 30, 50 and 79 weeks of age, with three behaviour observations (before, during or after scratch feed application). The data were analysed by GLMMs or LMMs. The vast proportions of birds on the mats were standing (0.720) or sitting (0.250). Bird proportions on the mats were low overall and declined from 0.028 (30 weeks) and 0.030 (50 weeks) to 0.020 (79 weeks) (p < 0.001). The greatest proportion of hens were observed on Z (p < 0.001), which had the largest area, but relative to the available area least birds were on Z and most were on K (p < 0.001). Foraging was not affected by bird age or mat type but was greater at the second observation (p < 0.001). Most eggs were laid in the nest box and were clean. Clean eggs declined, and dirty eggs increased, significantly with age, particularly at the scratch mat (p < 0.001). Dirty eggs were not affected by mat design. Cracked eggs were highest at 79 weeks of age, particularly with BD mats (p < 0.001). Overall, scratch mat designs had minimal effects on behaviour (but few hens were seen there) and egg quality.

Spectral entropy of early-life distress calls as an iceberg indicator of chicken welfare.

Chicks (Gallus gallus domesticus) make a repetitive, high energy 'distress' call when stressed. Distress calls are a catch-all response to a range of environmental stressors, and elicit food calling and brooding from hens. Pharmacological and behavioural laboratory studies link expression of this call with negative affective state. As such, there is an a priori expectation that distress calls on farms indicate not only physical, but emotional welfare. Using whole-house recordings on 12 commercial broiler flocks (n = 25 090-26 510/flock), we show that early life (day 1-4 of placement) distress call rate can be simply and linearly estimated using a single acoustic parameter: spectral entropy. After filtering to remove low-frequency machinery noise, spectral entropy per minute of recording had a correlation of -0.88 with a manual distress call count. In videos collected on days 1-3, age-specific behavioural correlates of distress calling were identified: calling was prevalent (spectral entropy low) when foraging/drinking were high on day 1, but when chicks exhibited thermoregulatory behaviours or were behaviourally asynchronous thereafter. Crucially, spectral entropy was predictive of important commercial and welfare-relevant measures: low median daily spectral entropy predicted low weight gain and high mortality, not only into the next day, but towards the end of production. Further research is required to identify what triggers, and thus could alleviate, distress calling in broiler chicks. However, within the field of precision livestock farming, this work shows the potential for simple descriptors of the overall acoustic environment to be a novel, tractable and real-time 'iceberg indicator' of current and future welfare.

Cell Proliferation in the Adult Chicken Hippocampus Correlates With Individual Differences in Time Spent in Outdoor Areas and Tonic Immobility.

Access to outdoor areas is provided as a means of enhancing welfare in commercial systems for laying hens (Gallus gallus domesticus), but substantial individual differences exist in their proportional use. Baseline cell proliferation levels of Adult Hippocampal Neurogenesis (AHN) have been associated with individual differences in reactive vs. proactive coping style, and in both mammals and birds, AHN is upregulated by positive experiences including environmental enrichment and exercise. We thus sought to explore whether individual differences in use of outdoor areas and in tonic immobility responses (indicative of fearfulness) were associated with hippocampal cell proliferation and neuronal differentiation. Radio frequency identification technology was used to track the ranging behavior of 440 individual focal hens within a commercially-relevant system over a 72-days period, after which tonic immobility durations were measured. Following hippocampal tissue collection from 58 focal hens, proliferation and neuronal differentiation were measured through quantitative PCR for proliferating cell nuclear antigen (PCNA) and doublecortin mRNA, respectively. Individual differences in tonic immobility duration positively correlated with PCNA expression over the whole hippocampal formation, while greater time spent in outdoor areas (the grassy range and stone yard) was associated with higher proliferation in the rostral subregion. Basal proliferation in the chicken hippocampal formation may thus relate to reactivity, while levels in the rostral region may be stimulated by ranging experience. Doublecortin expression in the caudal hippocampus negatively co-varied with time on the grassy range, but was not associated with tonic immobility duration. This suggests that ranging outside may be associated with stress. Within laying hen flocks, individual differences in hippocampal plasticity thus relate to coping style and use of external areas.

Welfare assessment of novel on-farm killing methods for poultry.

There is a need for novel mechanical devices for dispatching poultry on farm following the introduction of EU Regulation (EC) no. 1099/2009 On the Protection of Animals at the Time of Killing. We examined three novel mechanical killing devices: Modified Armadillo, Modified Rabbit Zinger, a novel mechanical cervical dislocation device; and traditional manual cervical dislocation. The four killing methods were tested on 230 chickens across four batches. We measured behavioural, electroencephalogram and post-mortem outcomes in anesthetized laying hens and broilers at two life stages (juveniles and adults/slaughter age). Graeco Latin-Square designs systematically randomized killing treatment, bird type, age and kill order. All birds were lightly anaesthetized immediately prior to the killing treatment with inhalation of Sevoflurane. The novel mechanical cervical dislocation method had the highest kill success rate (single application attempt only, with no signs of recovery) of a mechanical method (96%). The Modified Armadillo was the least reliable with 49% kill success. Spectral analysis of electroencephalogram signals at 2 s intervals for successfully killed birds only revealed progressive decreases in median frequency alongside increases in total power. Later, total power decreased as the birds exhibited isoelectric electroencephalogram signal. Latencies to pre-defined spectral ranges associated with unconsciousness showed that birds subjected to manual and novel mechanical cervical dislocation achieved these states sooner than birds subjected to the modified Armadillo. Nevertheless all methods exhibited short latencies (<4 s). The Modified Rabbit Zinger had the shortest duration of reflex persistence for nictitating membrane, pupillary and rhythmic breathing post method application. Of the methods tested, the novel mechanical cervical dislocation device is the most promising mechanical method for killing poultry on-farm based on a range of behavioural, electroencephalogram and anatomical parameters. This device has the potential to fulfil the current need for a mechanical alternative to manual cervical dislocation.

On Farm Evaluation of a Novel Mechanical Cervical Dislocation Device for Poultry.

Urgent development of alternative on-farm killing methods for poultry is required following the number restrictions placed on the use of traditional manual cervical dislocation by European Legislation (EU 1099/2009). Alternatives must be proven to be humane and, crucially, practical in commercial settings with multiple users. We assessed the performance and reliability of a novel mechanical cervical dislocation device (NMCD) compared to the traditional manual cervical dislocation (MCD) method. NMCD was based on a novel device consisting of a thin supportive glove and two moveable metal finger inserts designed to aid the twisting motion of cervical dislocation. We employed a 2 × 2 factorial design, with a total of eight stockworkers from broiler and layer units (four per farm) each killing 70 birds per method. A successful kill performance was defined as immediate absence of rhythmic breathing and nictitating membrane reflex; a detectable gap in the vertebrae and only one kill attempt (i.e., one stretch and twist motion). The mean stockworker kill performance was significantly higher for MCD (98.4 ± 0.5%) compared to NMCD (81.6 ± 1.8%). However, the MCD technique normally used by the stockworkers (based previous in-house training received) affected the performance of NMCD and was confounded by unit type (broilers), with the majority of broiler stockworkers trained in a non-standard technique, making adaption to the NMCD more difficult. The consistency of trauma induced by the killing methods (based on several post-mortem parameters) was higher with NMCD demonstrated by "gold standard" trauma achieved in 30.2% of birds, compared to 11.4% for MCD (e.g., dislocation higher up the cervical region of the spine i.e., between vertebrae C0-C1, ≥1 carotid arteries severed), suggesting it has the potential to improve welfare at killing. However, the results also suggest that the NMCD method requires further refinement and training optimization in order for it to be acceptable as an alternative across poultry industry, irrespective of previous MCD technique and training.

Welfare Risks of Repeated Application of On-Farm Killing Methods for Poultry.

Council Regulation (EC) no. 1099/2009 on the protection of animals at the time of killing restricts the use of manual cervical dislocation in poultry on farms in the European Union (EU) to birds weighing up to 3 kg and 70 birds per person per day. However, few studies have examined whether repeated application of manual cervical dislocation has welfare implications and whether these are dependent on individual operator skill or susceptibility to fatigue. We investigated the effects of repeated application (100 birds at a fixed killing rate of 1 bird per 2 min) and multiple operators on two methods of killing of broilers, laying hens, and turkeys in commercial settings. We compared the efficacy and welfare impact of repeated application of cervical dislocation and a percussive killer (Cash Poultry Killer, CPK), using 12 male stockworkers on three farms (one farm per bird type). Both methods achieved over 96% kill success at the first attempt. The killing methods were equally effective for each bird type and there was no evidence of reduced performance with time and/or bird number. Both methods of killing caused a rapid loss of reflexes, indicating loss of brain function. There was more variation in reflex durations and post-mortem damage in birds killed by cervical dislocation than that found using CPK. High neck dislocation was associated with improved kill success and more rapid loss of reflexes. The CPK caused damage to multiple brain areas with little variation. Overall, the CPK was associated with faster abolition of reflexes, with fewer birds exhibiting them at all, suggestive of better welfare outcomes. However, technical difficulties with the CPK highlighted the advantages of cervical dislocation, which can be performed immediately with no equipment. At the killing rates tested, we did not find evidence to justify the current EU limit on the number of birds that one operator can kill on-farm by manual cervical dislocation.

Erratum: Martin, J.E., et al. Welfare Risks of Repeated Application of On-Farm Killing Methods for Poultry. Animals 2018, 8, 39.

The authors wish to make the following correction to their paper[...].

Can Non-Beak Treated Hens be Kept in Commercial Furnished Cages? Exploring the Effects of Strain and Extra Environmental Enrichment on Behaviour, Feather Cover, and Mortality.

Commercial laying hens are prone to injurious pecking (IP), a common multifactorial problem. A 2 × 2 × 2 factorial design assessed the effects of breed (Lohmann Brown Classic (L) or Hyline Brown (H)), beak treatment (infra-red treated (T) or not (NT)), and environment (extra enrichment (EE) or no extra enrichment (NE)) on mortality, behaviour, feather cover, and beak shape. Hens were allocated to treatments at 16 weeks of age and data were collected every four weeks from age 19 to 71 weeks. Data were analysed in Genstat using mixed models. L hens had higher all and IP-related mortality than H hens (p < 0.003), whilst NT hens had higher mortality than T hens but only due to culling of whole cages (p < 0.001). Feather cover for L hens deteriorated more quickly with age at most body sites than H hens (age × breed × body site p < 0.001). For NT hens, feather cover was worse at most body sites (beak treatment × body site p < 0.001), and worsened more quickly with age (age × beak treatment p = 0.014) than T hens. L and NE hens performed more bird-to-bird pecking than H and EE hens, respectively (breed p = 0.015, enrichment p = 0.032). More damage to mats and ropes was caused by L and NT hens than by H and T hens, respectively (age × breed p < 0.005, beak treatment p < 0.001). Though H hens had fewer mortalities and better feather cover, breed effects may have been influenced by farm management practices, as they may have been better suited to H than L hens. Though EE hens performed less bird-to-bird pecking, the enrichments were less effective at reducing feather cover damage and mortality than expected.

Behaviour of food restricted broilers during rearing and lay--effects of an alternative feeding method.

Broiler breeders are subjected to quantitative food restriction in order to control their growth, and this restriction is particularly severe during rearing. While such restriction improves some welfare problems associated with ad libitum feeding, it causes others: birds routinely show abnormal oral behaviours and have elevated plasma corticosterone concentration (PCC) and changes in white blood cell counts (WBCs). The aim of this study was to examine if feeding birds qualitatively restricted diets ad libitum during rearing could reduce signs of impaired welfare, as judged by behaviour and blood indices of stress, while also meeting commercially desired growth rates and uniformity. Furthermore, we examined what carry-over effects such a feeding method had on birds in the laying phase when all birds were fed on a conventional quantitative restriction regime. During rearing (1-20 weeks of age), pens of birds were either fed limited amounts of standard basal diets (Control, i.e. quantitative restriction), or ad libitum diets consisting of standard basal diets with gradually increasing levels of calcium propionate (CaP) and a constant level of oat hulls (OH), designated CaP+OH (i.e. qualitative restriction). Results showed that, during rearing, weights and weight uniformity were similar for the two groups. During feeding motivation tests, Control birds always consumed more food than CaP+OH birds. This suggests that Control birds were more highly motivated to feed than CaP+OH birds, although care has to be taken in interpreting these results. Treatment did not affect PCCs or WBCs, but there was a general decline in PCCs with bird age. All reported behaviours differed significantly between treatment groups during rearing, but disappeared during lay when all birds were fed a similar amount of food. Control birds spent up to 50% of time in object pecking during rearing periods, but this behaviour was virtually non-existent in birds in the qualitative feeding regime. Overall, the results indicate that broiler breeders can be successfully limited in growth rates by qualitative food restriction and this results in significant changes of behaviour that suggest improvements to bird welfare.

Avian reflex and electroencephalogram responses in different states of consciousness.

Defining states of clinical consciousness in animals is important in veterinary anaesthesia and in studies of euthanasia and welfare assessment at slaughter. The aim of this study was to validate readily observable reflex responses in relation to different conscious states, as confirmed by EEG analysis, in two species of birds under laboratory conditions (35-week-old layer hens (n=12) and 11-week-old turkeys (n=10)). We evaluated clinical reflexes and characterised electroencephalograph (EEG) activity (as a measure of brain function) using spectral analyses in four different clinical states of consciousness: conscious (fully awake), semi-conscious (sedated), unconscious-optimal (general anaesthesia), unconscious-sub optimal (deep hypnotic state), as well as assessment immediately following euthanasia. Jaw or neck muscle tone was the most reliable reflex measure distinguishing between conscious and unconscious states. Pupillary reflex was consistently observed until respiratory arrest. Nictitating membrane reflex persisted for a short time (<1 min) after respiratory arrest and brain death (isoelectric EEG). The results confirm that the nictitating membrane reflex is a conservative measure of death in poultry. Using spectral analyses of the EEG waveforms it was possible to readily distinguish between the different states of clinical consciousness. In all cases, when birds progressed from a conscious to unconscious state; total spectral power (PTOT) significantly increased, whereas median (F50) and spectral edge (F95) frequencies significantly decreased. This study demonstrates that EEG analysis can differentiate between clinical states (and loss of brain function at death) in birds and provides a unique integration of reflex responses and EEG activity.

Thermal nociception as a measure of non-steroidal anti-inflammatory drug effectiveness in broiler chickens with articular pain.

Pain associated with poultry lameness is poorly understood. The anti-nociceptive properties of two non-steroidal anti-inflammatory drugs (NSAIDs) were evaluated using threshold testing in combination with an acute inflammatory arthropathy model. Broilers were tested in six groups (n=8 per group). Each group underwent a treatment (saline, meloxicam (3 or 5mg/kg) or carprofen (15 or 25mg/kg)) and a procedure (Induced (arthropathy-induction) or sham (sham-handling)) prior to testing. Induced groups had Freund's complete adjuvant injected intra-articularly into the left intertarsal joint (hock). A ramped thermal stimulus (1°C/s) was applied to the skin of the left metatarsal. Data were analysed using random-intercept multi-level models. Saline-induced birds had a significantly higher skin temperature (± SD) than saline-sham birds (37.6 ± 0.8°C vs. 36.5 ± 0.5°C; Z=-3.47, P<0.001), consistent with an inflammatory response. Saline was associated with significantly lower thermal thresholds (TT) than analgesic treatment (meloxicam: Z=2.72, P=0.007; carprofen: Z=2.58, P=0.010) in induced birds. Saline-induced birds also had significantly lower TT than saline-sham birds (Z=-2.17, P=0.030). This study found direct evidence of an association between inflammatory arthropathies and thermal hyperalgesia, and showed that NSAID treatment maintained baseline thermal sensitivity (via anti-nociception). Quantification of nociceptive responsiveness in a predictable broiler pain model identified thermal anti-hyperalgesic properties of two NSAIDs, which suggested that therapeutically effective treatment was provided at the doses administered. Such validation of analgesic strategies will increase the understanding of pain associated with specific natural broiler lameness types.

Preen gland function in layer fowls: factors affecting morphology and feather lipid levels.

The effects of age and external factors on preen gland morphology and feather lipid concentrations in layer fowl (Gallus domesticus) have not previously been described. This work examines how floor substrate, whether a bird is feather pecked or not, and age influence both gross and histological morphology. Furthermore, the effects of age, beak trimming and floor substrate on feather lipid concentration (mg lipid per g feathers) are investigated. Neither absolute nor relative preen gland weight were reliably affected by floor substrate or feather pecked status. However, these parameters were strongly affected by changes in bird age and related body weight changes. The microstructures of the gland also increased consistently with age. Feather lipid concentration generally increased with age. Feather lipid was strongly influenced by the presence or absence of a dustbathing substrate. Of all the parameters tested here, the age and growth of the bird had the greatest effects on preen gland development. Feather lipid concentration is also influenced by age, and by the presence or absence of a dustbathing substrate.