Identification of novel biomarkers of acute phase response in chickens challenged with Escherichia coli lipopolysaccharide endotoxin.

BACKGROUND: The chicken's inflammatory response is an essential part of the bird's response to infection. A single dose of Escherichia coli (E. coli) lipopolysaccharide (LPS) endotoxin can activate the acute phase response (APR) and lead to the production of acute phase proteins (APPs). In this study, the responses of established chicken APPs, Serum amyloid A (SAA) and Alpha-1-acid-glycoprotein (AGP), were compared to two novel APPs, Hemopexin (Hpx) and Extracellular fatty acid binding protein (Ex-FABP), in 15-day old broilers over a time course of 48 h post E.coli LPS challenge. We aimed to investigate and validate their role as biomarkers of an APR. Novel plant extracts, Citrus (CTS) and cucumber (CMB), were used as dietary supplements to investigate their ability to reduce the inflammatory response initiated by the endotoxin. RESULTS: A significant increase of established (SAA, AGP) and novel (Ex-FABP, Hpx) APPs was detected post E.coli LPS challenge. Extracellular fatty acid binding protein (Ex-FABP) showed a similar early response to SAA post LPS challenge by increasing ~ 20-fold at 12 h post challenge (P < 0.001). Hemopexin (Hpx) showed a later response by increasing ∼5-fold at 24 h post challenge (P < 0.001) with a similar trend to AGP. No differences in APP responses were identified between diets (CTS and CMB) using any of the established or novel biomarkers. CONCLUSIONS: Hpx and Ex-FABP were confirmed as potential biomarkers of APR in broilers when using an E. coli LPS model along with SAA and AGP. However, no clear advantage for using either of dietary supplements to modulate the APR was identified at the dosage used.

Measuring the impact of dietary supplementation with citrus or cucumber extract on chicken gut microbiota using 16s rRNA gene sequencing.

This study investigated the effects of dietary supplements, citrus (CTS) and cucumber (CMB), on the jejunum and cecum microbiota of 14- and 28-days old broiler chickens to evaluate their impact on the gut health and assess their role as alternatives to antibiotic growth promoters (ABGPs). 16SrRNA gene sequencing revealed the overall bacterial microbiota composition was significantly affected by the gut site (p?

Effect of an immune challenge and two feed supplements on broiler chicken individual breast muscle protein synthesis rate.

Optimization of broiler chicken breast muscle protein accretion is key for the efficient production of poultry meat, whose demand is steadily increasing. In a context where antimicrobial growth promoters use is being restricted, it is important to find alternatives as well as to characterize the effect of immunological stress on broiler chicken's growth. Despite its importance, research on broiler chicken muscle protein dynamics has mostly been limited to the study of mixed protein turnover. The present study aims to characterize the effect of a bacterial challenge and the feed supplementation of citrus and cucumber extracts on broiler chicken individual breast muscle proteins fractional synthesis rates (FSR) using a recently developed dynamic proteomics pipeline. Twenty-one day-old broiler chickens were administered a single 2H2O dose before being culled at different timepoints. A total of 60 breast muscle protein extracts from five experimental groups (Unchallenged, Challenged, Control Diet, Diet 1 and Diet 2) were analysed using a DDA proteomics approach. Proteomics data was filtered in order to reliably calculate multiple proteins FSR making use of a newly developed bioinformatics pipeline. Broiler breast muscle proteins FSR uniformly decreased following a bacterial challenge, this change was judged significant for 15 individual proteins, the two major functional clusters identified as well as for mixed breast muscle protein. Citrus or cucumber extract feed supplementation did not show any effect on the breast muscle protein FSR of immunologically challenged broilers. The present study has identified potential predictive markers of breast muscle growth and provided new information on broiler chicken breast muscle protein synthesis which could be essential for improving the efficiency of broiler chicken meat production. SIGNIFICANCE: The present study constitutes the first dynamic proteomics study conducted in a farm animal species which has characterized FSR in a large number of proteins, establishing a precedent for biomarker discovery and assessment of health and growth status. Moreover, it has been evidenced that the decrease in broiler chicken breast muscle protein following an immune challenge is a coordinated event which seems to be the main cause of the decreased growth observed in these animals.

Body surface temperature of rats reveals both magnitude and sex differences in the acute stress response.

Understanding how biological markers of stress relate to stressor magnitude is much needed and can be used in welfare assessment. Changes in body surface temperature can be measured using infrared thermography (IRT) as a marker of a physiological response to acute stress. While an avian study has shown that changes in body surface temperature can reflect the intensity of acute stress, little is known about surface temperature responses to stressors of different magnitudes and its sex-specificity in mammals, and how they correlate with hormonal and behavioural responses. We used IRT to collect continuous surface temperature measurements of tail and eye of adult male and female rats (Rattus norvegicus), for 30 minutes after exposure to one of three stressors (small cage, encircling handling or rodent restraint cone) for one minute, and cross-validated the thermal response with plasma corticosterone (CORT) and behavioural assessment. To obtain individual baseline temperatures and thermal responses to stress, rats were imaged in a test arena (to which they were habituated) for 30 seconds before and 30 minutes after being exposed to the stressor. In response to the three stressors, tail temperature initially decreased and then recovered to, or overshot the baseline temperature. Tail temperature dynamics differed between stressors; being restrained in the small cage was associated with the smallest drop in temperature, in male rats, and the fastest thermal recovery, in both sexes. Increases in eye temperature only distinguished between stressors early in the response and only in females. The post stressor increase in eye temperature was greater in the right eye of males and the left eye of females. In both sexes encircling may have been associated with the fastest increase in CORT. These results were in line with observed behavioural changes, with greater movement in rats exposed to the small cage and higher immobility after encircling. The female tail and eye temperature, as well as the CORT concentrations did not return to pre-stressor levels in the observation period, in conjunction with the greater occurrence of escape-related behaviours in female rats. These results suggest that female rats are more vulnerable to acute restraint stress compared to male rats and emphasise the importance of using both sexes in future investigations of stressor magnitude. This study demonstrates that acute stress induced changes in mammalian surface temperature measured with IRT relate to the magnitude of restraint stress, indicate sex differences and correlate with hormonal and behavioural responses. Thus, IRT has the potential to become a non-invasive method of continuous welfare assessment in unrestrained mammals.

The perspectives of UK personnel towards current killing practices for laboratory rodents.

Rodents are the predominant species used for scientific research and must be humanely killed upon completion of the work. In the UK this is regulated by Schedule 1 of the Animals Scientific Procedures Act 1986, which lists permitted methodologies considered capable of humane killing, including overdose of an anaesthetic, exposure to carbon dioxide (CO2) gas, dislocation of the neck and concussion of the brain by striking the cranium. Although all are permitted, operator motivations behind method selection and individual operator preference remain unknown. The views of 219 laboratory animal personnel on institutional availability and use of Schedule 1 killing methods for laboratory rodents were obtained. Only 10% of participants reported that all four methods were available at their institution with 57.5% of respondents preferring cervical dislocation. For CO2, only 18.6% of participants reported using the recommended flow rate, while 45.5% did not know the flow rate employed. We highlight the urgent requirement for the development of quality-controlled training programmes, to improve knowledge and confidence in the selection and application of killing methods. We advocate for continuous review of killing practices to ensure best practice is reflected in legislation and achieve optimal protection of the welfare of laboratory rodents during killing.

A novel dynamic proteomics approach for the measurement of broiler chicken protein fractional synthesis rate.

RATIONALE: The study of protein synthesis in farm animals is uncommon despite its potential to increase knowledge about metabolism and discover new biomarkers of health and growth status. The present study describes a novel dynamic proteomics approach for the measurement of protein fractional synthesis rate (FSR) in broiler chickens. METHODS: Chickens received a 10 g/kg oral dose of 2 H2 O at day 21 of their life. Body water 2 H abundance was measured in plasma samples using a portable Fourier transform infrared spectrometer. Free and protein-bound amino acids (AAs) were isolated and had their 2 H enrichment measured by gas chromatography with mass spectrometry (GC/MS). Peptide 2 H enrichment was measured by proteomics analysis of plasma and muscle samples. Albumin, fibrinogen and muscle protein FSR were calculated from GC/MS and proteomics data. RESULTS: Ala appeared to be more enriched at the site of protein synthesis than in the AA free pools. Glu was found to be the AA closest to isotopic equilibrium between the different AA pools. Glu was used as an anchor to calculate n(AA) values necessary for chicken protein FSR calculation in dynamic proteomics studies. FSR values calculated using proteomics data and GC/MS data showed good agreement as evidenced by a Bland-Altman residual plot. CONCLUSIONS: A new dynamic proteomics approach for the measurement of broiler chicken individual protein FSR based on the administration of a single 2 H2 O oral bolus has been developed and validated. The proposed approach could facilitate new immunological and nutritional studies on free-living animals.

Non-invasive assessment of positive affective state using infra-red thermography in rats.

With recent increased focus on positive welfare in animal welfare science, there is demand for objective positive welfare indicators. It is unclear whether changes in body surface temperature can be used to non-invasively identify and quantify positive states in mammals. We recorded continuous measurements of tail surface temperature using infra-red thermography (IRT) and concurrent behavioural observations in male and female Wistar rats (Rattus norvegicus). If tail surface temperature can differentiate between positive and negative experiences, we expect a qualitatively different response compared to negative experiences. Three groups of rats were presented with increasing magnitudes of food rewards (neutral/none, one and three rewards). The rats were placed in an arena to which they were habituated and filmed for 30 s before and 30 min after exposure to different rewards. Tail temperature initially decreased from the pre-reward baseline and subsequently returned towards baseline temperature. The overall pattern of the change was the same as for rats subjected to negative stimuli in previous studies. Nevertheless, dynamic changes in tail temperature, specifically the rate of recovery and the behavioural response (exploration), differed between neutral and rewarded rats but failed to distinguish reward magnitude. Sex differences were found in both thermal and behavioural responses, unrelated to reward magnitudes. Female rats exhibited a greater initial response with a slower recovery than male rats, emphasising the value of using of both sexes in animal welfare research. This study improves our understanding of the effects of positive emotions induced by food reward on peripheral body temperature and behaviour.

Characterizing candidate decompression rates for hypobaric hypoxic stunning of pigs. Part 1: Reflexive behavior and physiological responses.

Alternatives to carbon dioxide (CO2) stunning for the commercial slaughter of pigs are urgently needed because there is robust evidence that exposing pigs to hypercapnic environments is associated with pain, fear, and distress. Hypobaric hypoxia (via gradual decompression, also known as Low Atmospheric Pressure Stunning or LAPS) has been validated in poultry as a humane option, but its potential to improve the welfare of pigs at slaughter is unknown. We investigated the potential of hypobaric hypoxia to reliably elicit a non-recovery state in anesthetized weaner-grower pigs within a commercially viable timeframe. We determined the effect of candidate decompression rates (40, 60, 80, 100 ms-1, at two cycle durations 480 s and 720 s) on a range of physiological and reflexive behavioral indicators of hypoxia and death. We found that the decompression rates tested caused a 100% death rate. As expected, the decompression rate had overarching effects on behavioral and physiological markers of hypoxia and death, with faster decompression rates resulting in shorter latencies to cardiac arrest and cessation of breathing. We observed a higher proportion of pigs displaying repeated and prolonged whole-body movements (likely indicative of convulsive activity) at higher frequencies when we applied the slowest decompression rate (40 ms-1) compared to all other rates. Since these responses may impact the carcass and meat quality, the slower rate of decompression (40 ms-1) should be excluded as a candidate decompression rate. Furthermore, given the marginal effects of decompression rate on physiological indicators of death and reflexive behavioral parameters, we also recommend that the fastest rate tested (100 ms-1) is excluded in further study on conscious pigs (to prevent conscious animals from being exposed to unnecessary faster decompression rates which may compromise animal welfare). This work represents a necessary proof of principle step and confirms the potential of gradual decompression for stunning purposes in pigs. Importantly, however, the data presented provide no information on the welfare outcomes associated with decompression in conscious pigs. Subsequent work should focus on the comprehensive welfare assessment of intermediate decompression rates to determine the potential of hypobaric hypoxia to provide a humane stunning method for pigs.

Characterizing candidate decompression rates for hypobaric hypoxic stunning of pigs. Part 2: Pathological consequences.

Pigs are commonly stunned pre-slaughter by exposure to carbon dioxide (CO2), but this approach is associated with significant welfare concerns. Hypobaric hypoxia, achieved with gradual decompression (also known as Low Atmospheric Pressure Stunning or LAPS) may be an alternative, allowing the retention of welfare friendly handling approaches and group stunning. Although validated in poultry, the feasibility and welfare consequences of gradual decompression for pigs are unknown. Here, we characterize pathological changes in 60 pigs resulting from exposure to a range of candidate decompression curves (ranging from 40 to 100 ms-1 ascent equivalent, with two cycle durations 480 and 720 s). To protect welfare, we worked on unconscious, terminally anesthetized pigs which were subject to detailed post-mortem examinations by a specialized porcine veterinary pathologist. All pigs were killed as a result of exposure to decompression, irrespective of cycle rate or length. Pigs showed no external injuries during ante-mortem inspections. Exposing pigs to decompression and the unavoidable subsequent recompression resulted in generalized congestion of the carcass, organs and body cavities including the ears, oral cavity, conjunctivae and sclera, mucosa of other external orifices (anus and vulva), nasal planum, nasal cavities including nasal conchae, frontal sinuses, cranium, meninges, brain, larynx, trachea, lungs, heart, parietal pleura of the thoracic cavity, peritoneum of the abdominal cavity, stomach, small intestine, caecum, colon, liver, spleen and kidneys and representative joint cavities in the limbs (stifles and elbows). Various severities of hemorrhage were observed in the conjunctivae and sclera, mucosa of other external orifices (anus and vulva), nasal cavities including nasal conchae, frontal sinuses, cranium, meninges, brain, larynx, tracheal lumen, lungs, parietal pleura of the thoracic cavity, liver, spleen and kidneys and representative joint cavities in the limbs (stifles and elbows). In general, faster decompression rates produced higher scores, but in the conjunctivae, sclera and kidneys, faster decompression rates were associated with marginally lower congestion scores. There was considerable individual variation in pathological scores across all body regions. The congestion and hemorrhage observed could translate into welfare harms in conscious pigs undergoing this type of stunning, depending when in the cycle the damage is occurring, but no welfare related conclusions can be drawn from the responses of unconscious pigs. Since recompression is always required, its effects cannot be separated from decompression, however cessation of cardiac activity several minutes before recompression should have eliminated any haemodynamic effects relating to cardiac function and blood pressure. This study represents the first systematic attempt to identify candidate rate profiles to underpin future explorations of decompression as a stunning method for pigs. These pathological findings also inform discussions about the likely carcass quality implications of this novel stunning method.

A review of methods used to kill laboratory rodents: issues and opportunities.

Rodents are the most widely used species for scientific purposes. A critical pre-requisite of their use, based on utilitarian ethical reasoning, is the provision of a humane death when necessary for scientific or welfare grounds. Focussing on the welfare challenges presented by current methods, we critically evaluate the literature, consider emerging methodologies that may have potential for refinement and highlight knowledge gaps for future research. The evidence supports the conclusion that scientists and laboratory personnel should seek to avoid killing laboratory rodents by exposing them to carbon dioxide (CO2), unless exploiting its high-throughput advantage. We suggest that stakeholders and policymakers should advocate for the removal of CO2 from existing guidelines, instead making its use conditionally acceptable with justification for additional rationale for its application. With regards to physical methods such as cervical dislocation, decapitation and concussion, major welfare concerns are based on potential inaccuracy in application and their susceptibility to high failure rates. There is a need for independent quality-controlled training programmes to facilitate optimal success rates and the development of specialist tools to improve outcomes and reliability. Furthermore, we highlight questions surrounding the inconsistent inclusion criteria and acceptability of physical methods in international regulation and/or guidance, demonstrating a lack of cohesion across countries and lack of a comprehensive 'gold standard' methodology. We encourage better review of new data and championing of open access scientific resources to advocate for best practice and enable significant changes to policy and legislation to improve the welfare of laboratory rodents at killing.

Determining Candidate Hypobaric Hypoxia Profiles for Humane Killing of Laboratory Mice.

Millions of mice are used annually in scientific research and must be humanely killed. Despite significant welfare concerns, carbon dioxide exposure remains the most common killing method, primarily because there is no practical and humane alternative. We explored whether hypobaric hypoxia via gradual decompression could induce a non-recovery state in anesthetized male C57BL/6 and Balb/c laboratory mice. We aimed to determine if this was possible in a feasible timescale with minimal pathological consequences, as a proof-of-principle step. Systematic evaluation of two decompression rates (75, 150 ms-1) and three profile shapes (accelerated, linear, gradual) in a factorial design revealed that hypobaric hypoxia effectively induced a non-recovery state in anesthetized laboratory mice, irrespective of decompression rate and shape. Mice took longer to reach a non-recovery state with the 75 ms-1 decompression rate (75 ms-1: 257 ± 8.96 vs. 150 ms-1: 214 ± 7.26 s), with longer latencies in gradual and linear shaped profiles. Accelerated shaped profiles were least susceptible to meaningful refinement via rate. The only pathological changes of concern were moderate middle ear congestion and hemorrhage. These findings suggest that hypobaric hypoxia has potential, and subsequent work will evaluate the welfare consequences of gradual decompression in conscious mice, to identify decompression profiles that minimize welfare harms associated with ear barotrauma.

Administration of ketoprofen affects post-partum lying behaviours of Holstein dairy cows regardless of whether parturition is assisted.

BACKGROUND: Assistance at parturition in cattle is common. Although analgesia is commonly provided, its value in terms of welfare improvement is not known. Few previous studies include both parturition assistance and analgesic treatment status as well as appropriate control groups in a factorial design. METHODS: Seventy-two Holstein cattle (37 assisted and 35 unassisted) were randomly allocated to receive a single dose of the NSAID ketoprofen or a saline placebo within 3 h of parturition. Detailed behavioural observations allowed a time budget to be constructed for each cow for 48 h post-partum. RESULTS: Cows experiencing assisted parturition spent more time in lateral recumbency (overall and with the head rested) (p < 0.05) - an infrequently adopted posture suggestive of illness or pain. Cows treated with ketoprofen spent less time in lateral recumbency (p < 0.05) (overall and with the head rested); this was independent of assistance status, and lateral recumbency was not affected by the interaction between assistance and treatment status. Additionally, cows treated with ketoprofen spent more time with the head rested when in sternal recumbency (p = 0.009) - a behaviour associated with comfortable resting. CONCLUSION: These differences in lying postures exhibited by cows receiving ketoprofen analgesia suggest that, regardless of whether parturition is assisted, a single dose of ketoprofen in the immediate post-partum period has the potential to improve cow comfort in the first 48 h post-partum.

Use of a Tri-Axial Accelerometer Can Reliably Detect Play Behaviour in Newborn Calves.

Traditionally, the welfare assessment of farm animals has focused on health and production outcomes. Positive welfare is, however, not merely the absence of negative welfare and is an important part of a life worth living. Play behaviour is widely considered to be an indicator of positive emotions because it is a "luxury" behaviour. Direct visual observation is considered the most accurate method of behavioural analysis, but it is time consuming and laborious. There is increasing interest in the use of remote monitoring technology to quantify behaviour. We compared the data output ("motion index" (MI)) from a commercially available tri-axial accelerometer fitted to newborn dairy calves to video footage of the same calves, with a focus on play behaviour. The motion index values over 48 h were positively correlated with both the duration of play behaviour and the number of play bouts. The motion index threshold in each sample interval with the optimal sensitivity and specificity for the identification of play behaviour was MI ≥ 2.5 at a 1 min resolution (sensitivity (Se) = 98.0%; specificity (Sp) = 92.9%) and MI ≥ 24.5 at a 15 min resolution (Se = 98.0%; Sp = 89.9%), but these values consistently overestimated the overall proportion of sample intervals in which play was observed. The MI that best reflected the results obtained from visual one-zero sampling was MI ≥ 23 for 1 min intervals and MI ≥ 62 for 15 min intervals-this may therefore be the basis of a more conservative approach to the identification of play behaviour from accelerometer-generated data. Our results indicate that accelerometer-generated data can usefully indicate the amount of play behaviour shown by newborn calves for up to 48 h, providing an efficient method for identifying this important parameter in future work.

"The Cone of Shame": Welfare Implications of Elizabethan Collar Use on Dogs and Cats as Reported by their Owners.

Elizabethan collars are used in companion animals primarily to prevent self-trauma and associated negative welfare states in animals. However, they have been anecdotally associated with negative impacts on animal health and welfare including distress, abraded/ulcerated skin and misadventure. This study aimed to characterise the welfare impacts of Elizabethan collar use on companion dogs and cats, as reported by owners. Owners of pets who wore an Elizabethan collar during the past 12 months were surveyed about the impacts that the use of Elizabethan collars had on animal activities, in particular sleep, eating, drinking, exercise, interactions with other animals, as well as overall quality of life (QOL). The majority of 434 respondents (77.4%) reported a worse QOL score when their companion animal was wearing the collar, significantly so when the Elizabethan collar irritated their pet or impacted on their ability to drink or play. While other factors are likely to impact animal welfare during veterinary treatment that necessitates the use of Elizabethan collars, this study suggests that Elizabethan collars themselves might have negative welfare impacts in a range of domains including nutrition, environment, health, behaviour and mental state. We recommend that animal owners are informed about potential negative impacts of Elizabethan collars and harm minimisation strategies. Where possible, alternative methods of preventing self-trauma should be explored.

Eye region surface temperature dynamics during acute stress relate to baseline glucocorticoids independently of environmental conditions.

Reactions to acute stressors are critical for survival. Yet, the challenges of assessing underlying physiological processes in the field limit our understanding of how variation in the acute stress response relates to fitness in free-living animals. Glucocorticoid secretion during acute stress can be measured from blood plasma concentrations, but each blood sample can only provide information for one point in time. Also, the number of samples that can be extracted from an individual in the field is usually limited to avoid compromising welfare. This restricts capacity for repeated assessment, and therefore temporal resolution of findings within- and between-acute stress responses - both of which are important for determining links between acute stress and fitness. Acute stress induces additional body surface temperature changes that can be measured non-invasively, and at high frequencies using thermal imaging, offering opportunities to overcome these limitations. But, this method's usefulness in the field depends on the extent that environmental conditions affect the body surface temperature response, which remains poorly understood. We assessed the relative importance of individual physiology (baseline glucocorticoid concentrations) and environmental conditions (air temperature and relative humidity) in determining the eye region surface temperature (Teye) response to acute stress, in wild blue tits (Cyanistes caeruleus) during trapping, handling and blood sampling. When controlling for between-individual baseline variation, Teye initially dropped rapidly below, and then recovered above baseline, before declining more slowly until the end of the test, 160 s after trap closure. One measure of the amplitude of this response - the size of the initial drop in Teye - was dependent on environmental conditions, but not baseline corticosterone. Whereas, two properties defining response dynamics - the timing of the initial drop, and the slope of the subsequent recovery - were related to baseline corticosterone concentrations, independently of environmental conditions. This suggests inferring the acute stress response using thermal imaging of Teye will be practical under fluctuating environmental conditions in the field.

The Views of the UK Public Towards Routine Neutering of Dogs and Cats.

Despite being routinely recommended by veterinarians, neutering of dogs and cats has both positive and negative impacts on animal welfare and is ethically problematic. We examined attitudes of a sample of the UK public towards routine neutering of dogs and cats using a questionnaire. Respondents indicated their level of agreement with statements describing welfare and ethical reasons 'for' and 'against' the neutering of male and female dogs and cats. We conducted a general linear model (GLM) analysis to investigate the effects of demographic factors on agreement scores. Respondents (n = 451) expressed views both supporting and opposing neutering. The predominant view (>80%) supported neutering, justified primarily by prevention of unwanted offspring and reproductive diseases. Around 10% of the respondents disagreed and felt that neutering should only be done for medical reasons. Men were less likely than women to support neutering (p < 0.001). Those with meat reduction diets were more likely to be against neutering (p < 0.05) and cat owners supported neutering more than non-cat owners (p < 0.05). Although the data reflected a wide range of ethical views, our findings show that the UK public generally supports the routine neutering of dogs and cats. This insight has implications for future policy-making and compliance with veterinary advice.

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.

Quantitative proteomics using tandem mass tags in relation to the acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin.

The inflammatory response in chickens (Gallus Gallus domesticus) is an integral part of the bird's response to infection. Detailing proteomic changes occurring during infection would be beneficial to the poultry industry, offering opportunities for comparative pathophysiological analysis. The objective of this study was to quantify the changes in the plasma proteome in chickens challenged with lipopolysaccharide (LPS), a bacterial endotoxin known to stimulate the host innate immune system. Plasma from chicken (N = 6) challenged with Escherichia coli (LPS) (2 mg/kg body weight) was collected pre (0 h) and at 12, 24, 48, and 72 h post-injection along with plasma from a control group (N = 6) challenged with sterile saline. Samples were analysed by a quantitative Tandem Mass Tags approach using a Q-Exactive-Plus mass-spectrometer. Identification and relative quantification were performed using Proteome Discoverer, and data were analysed using R. Gene Ontology terms were analysed by Cytoscape based on the Gallus gallus database. Finally, 87 significantly regulated proteins were found, including serum-amyloid-A, ovotransferrin and alpha-1-acid-glycoprotein, showing a significant effect of time post-injection in the LPS-treated group. Different pathways related with protein activation cascade and heterotopic cell-cell adhesion were affected by LPS-challenge. LPS-challenged chickens demonstrate significant changes to the plasma proteome with both increases and decreases of individual proteins within 12 h of challenge. SIGNIFICANCE: The injection of chicken with bacterial lipopolysaccharide followed by sequential plasma and clinical analysis of the bird, is a long established and a widely used model for inflammation and infection studies. This study, utilising and combining proteomic and immunoassay analysis with bioinformatic analysis, revealed that several biological pathways are modulated during this early period of inflammation. In addition, proteins with biomarker potential were identified and successfully validated. This experimental model also demonstrated potential for pathophysiological mechanism investigation and as an inflammatory model for biomedical research. There is, despite plasma being an easily accessible biological matrix which is representative of the health status of the bird, scarce data on the chicken plasma proteome. This research makes a positive contribution to the current field, generating significant data for continuing comparative analysis.

Postpartum ketoprofen treatment does not alter stress biomarkers in cows and calves experiencing assisted and unassisted parturition: a randomised controlled trial.

Dystocia is considered painful and stressful for both the dam and the calf, although systematic evidence of this is limited. Few studies have investigated biochemical markers of stress and pain postpartum and whether any adverse effects are ameliorated by administration of analgesia. In this study, cow-calf pairs experiencing both mild to moderate farmer assistance and no assistance at parturition were randomly assigned to either treatment or placebo group in a two-by-two design (animals subject to veterinary intervention were excluded). The treatments were the NSAID ketoprofen or saline, administered within three hours of parturition. Blood samples taken in the immediate postpartum period, and at 24 hours, 48 hours and 7 days after parturition, were analysed for plasma concentrations of creatine kinase and cortisol (cows and calves) and plasma L-lactate and total protein concentration (calves). Stress biomarkers were highest in the immediate postpartum period and declined over time (P<0.05). Cow plasma cortisol was higher in animals experiencing assisted parturition in the immediate postpartum period (P=0.023); by 24 hours no difference was evident. Intervention with NSAID analgesia did not result in beneficial changes in stress biomarkers. Based on biomarkers alone, this suggests limited benefits of NSAID treatment in unassisted or mild to moderately assisted parturition.

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[...].