Alternatives to Carbon Dioxide in Two Phases for the Improvement of Broiler Chickens' Welfare during Stunning.

This study evaluated the exposure to gas mixtures of carbon dioxide (CO2) associated with nitrogen (N2) as alternatives to CO2 in two phases to improve the welfare of broiler chickens at slaughter. Broilers were exposed to one of three treatments: 40C90C (1st phase: <40% CO2 for 2 min; 2nd phase: >90% CO2 and <2% O2 for 2 min, n = 92), 40C60N (40% CO2, 60% N2, and <2% O2 for 4 min, n = 79), or 20C80N (20% CO2, 80% N2, and <2% O2 for 4 min, n = 72). Brain activity (EEG) was assessed to determine the onset of loss of consciousness (LOC) and death. Behavioural assessment allowed for characterisation of an aversive response to the treatments and confirmed loss of posture (LOP) and motionlessness as behavioural proxies of LOC and brain death in 40C60N and 20N80C. However, the lack of quality of the EEG traces obtained in 40C90C did not allow us to determine the onset of LOC and brain death for this treatment. The onset of LOC in 40C60N was found at 19 s [14-30 s] and in 20C80N at 21 s [16-37 s], whereas a LOP was seen at 53 s [26-156 s] in 40C90C. Birds showed brain death in 40C60N at 64 s [43-108 s] and in 20C80N at 70 s [45-88 s]), while they became motionless in 40C90C at 177 s [89-212 s]. The 40C90C birds not only experienced more events of aversive behaviours related to mucosal irritation, dyspnoea, and breathlessness during induction to unconsciousness but were at risk of remaining conscious when the CO2 concentration was increased in the 2nd phase (known to cause severe pain). From an animal welfare point of view, 40C60N proved to be the least aversive of the three treatments tested, followed by 20C80N and 40C90C.

Changes in cortisol and cortisone in hair of pigs reared under heat stress conditions.

Heat stress accounts for millions of dollars in losses for swine producers worldwide. The aim of the present study was to determine and evaluate cortisol and cortisone in hair as indicators of thermal stress in growing pigs reared under high environmental temperatures. The study was carried out in two independent batches of commercial crosses of Lean Duroc and Pietrain in trials 1 and 2, respectively, during the growing period (from 40 to 100 kg; 81 days in trial 1 and 77 days in trial 2) in the same commercial farm in Spain during the summers of 2020 and 2021. In both cases, four rooms were used. In Trial 1, Room 1 had cooling and 11 pigs per pen; Room 2 had no cooling and 13 pigs per pen; Room 3 had no cooling and 11 pigs per pen, and Room 4 had cooling and 13 pigs per pen. In Trial 2, Rooms 2 and 3 had cooling and rooms 1 and 4 had no cooling, and all of them had 13 pigs per pen. Mean THI value was higher (p < 0.0001) in rooms without cooling systems (75.0 trial 1; 74.9 trial 2) than with them (71.3 trial 1; 71.7 trial 2). A total of four pens per room (16 in total) was selected for analysis of hair corticoids and all pigs inside were sampled at the end of the study. Fifty percent of the pigs were males (castrated and intact in trial 1 and 2, respectively) and 50% females. In total, 44, 52, 44, and 52 pigs, respectively, were sampled in four rooms from the first trial and 52 for each of four rooms in Trial 2. Cortisol concentrations in hair did not show any significant change in relation to cooling-non-cooling in any trial. However, hair cortisone concentration was 172.3 pg./mg and 105.8 pg./mg less (p < 0.001) in pigs housed with cooling systems compared to those without them in Trial 1 and 2, respectively. In addition, the cortisone/cortisol ratio, which is an estimator of the activity of 11ß-hydroxysteroid dehydrogenase (11ß-HSD) type 2, was also greater in rooms without cooling than in rooms with cooling in both trials (p < 0.0001 and p = 0.0105 for Trials 1 and 2, respectively). In relation to the sex effect, the results showed greater levels in females than in castrated males both in cortisone and the cortisol/cortisone ratio while cortisol hair levels were greater in intact males than in females. Therefore, the use of cortisone and the estimation of 11ß-HSD type 2 activity in hair is recommended to evaluate the chronic stress produced by high environmental conditions in pigs instead of using hair cortisol concentrations alone.

Reducing Stocking Densities and Using Cooling Systems for More Adapted Pigs to High Temperatures When Reared in Intensive Conditions.

This study is aimed at evaluating the effect of reducing stocking density and using cooling systems to mitigate the negative effects of high temperatures in growing pigs (females and castrated males) reared in intensive conditions (from 25 to 100 kg) during summer (June to October 2020). The experimental design was a 2 × 2 factorial where pigs were provided with an evaporative cooling system and/or raised at regular or at lower stocking densities (i.e., 0.68 to 0.80 m2/animal). Treatments were distributed in four different rooms containing sex-balanced pens with either castrated males or females. Temperature and humidity were recorded throughout the experiment, and the temperature-humidity index was calculated. Heat stress (HS) on pigs was measured through changes in animals' performance, animal-based indicators (dirtiness and activity budget) and physiological indicators (neutrophil/lymphocyte ratio and hair cortisol). The use of cooling, lowering stocking density and the combination of both strategies had positive effects on pigs' final body weight (+5 kg, +3 kg, +9 kg, respectively; p < 0.001). The prevalence of dirtiness was similar at the stocking densities tested, and no clear effect of the cooling system was found. Both mitigation strategies lowered the physiological indicators of stress, although only hair cortisone can be considered an indicator of HS. In conclusion, both mitigation strategies are effective in improving pig welfare and performance, especially when both are combined. The severity of the stocking density effect may depend on the severity of the temperature.

Relevant Indicators of Consciousness after Waterbath Stunning in Turkeys and Stunning Efficiency in Commercial Conditions.

The prevalence and inter-observer repeatability of the four most valid and feasible indicators of consciousness after waterbath stunning in turkeys were evaluated before bleeding (i.e., tonic convulsion, breathing, spontaneous blinking and vocalization) and during bleeding (i.e., fluttering, breathing, spontaneous swallowing and head shaking). Furthermore, correlations between them were calculated to further understand the association between such indicators. This study compared the assessments of four observers on 7877 turkeys from 28 flocks slaughtered in eight different abattoirs. Repeatability between observers was assessed by combining the crude percentage of agreement and the Fleiss' kappa. Before bleeding, tonic seizure was observed to be not reliable under commercial conditions and spontaneous blinking and vocalization had poor repeatability. During bleeding, spontaneous swallowing was not observed and head shaking had poor repeatability. Hence, the most relevant indicators of consciousness before bleeding is breathing while during bleeding is breathing and wing flapping. Moreover, the stunning efficiency of the key electrical parameters used in the different slaughterhouses was assessed. Therefore, a refined list of indicators of the state of consciousness after waterbath stunning is proposed to be used in commercial turkey slaughterhouses to ensure consistency of controls. On the other hand, the range of the prevalence of turkeys with indicators of consciousness within a batch found before bleeding [0-16%] and after bleeding [0-18%] highlights the importance of controls.

Inter-Observer Repeatability of Indicators of Consciousness after Waterbath Stunning in Broiler Chickens.

This study evaluated the prevalence and tested the inter-observer repeatability of the most valid and feasible animal-based indicators of the state of consciousness after waterbath stunning in broilers before bleeding (tonic seizure, breathing, spontaneous blinking and vocalization) and during bleeding (wing flapping, breathing, spontaneous swallowing and head shaking). In addition, correlations among them were computed to better understand their relationship and offer insights into the reliability of such indicators. This was aimed at proposing a refined list of indicators that could be used in commercial slaughterhouses to ensure consistent assessments. This study compared the assessments of three observers of 5241 broilers from 19 batches in six different slaughterhouses. Inter-observer repeatability was assessed through the combination of the crude percentage of agreement and the Fleiss' kappa coefficient and interpretation. Before bleeding, the results led us to recommend assessing breathing over spontaneous blinking and vocalizations and to neglect tonic seizure in commercial conditions. During bleeding, the recommended indicators are breathing, wing flapping and head shaking while spontaneous swallowing can be neglected.

Intramuscular vs. Intradermic Needle-Free Vaccination in Piglets: Relevance for Animal Welfare Based on an Aversion Learning Test and Vocalizations.

The aim of the present study was to compare intramuscular injection with a needle and intradermic needle-free vaccinations against porcine reproductive and respiratory syndrome (PRRS) in piglets at 28 days old by studying behavioral and physiological reactions. A total of 72 piglets divided into 2 sex-balanced batches were assessed. Within each batch, the piglets were divided into three treatments, which were Hipradermic (0.2 ml of UNISTRAIN® PRRS vaccine administered with an intradermic needle-free device), Intramuscular (IM, 2.0 ml of vaccine), and Control (not vaccinated). Before the vaccination, the piglets were trained to cross a 4-m-long raceway to perform an aversion learning test. The day of vaccination, the time taken to cross the raceway was registered for each piglet at different times: prior to the vaccination and 10 min, 2, 24, 48, and 72 h after the vaccination, to measure variations in these times as signs of aversion to the vaccination process. Vocalizations, as potential signs of pain, were recorded as well at the end of this raceway to analyze their frequency (Hz), duration, and level of pressure (dB) at the moment of vaccination. Salivary cortisol, as a sign of the HPA-axis activity, was assessed 10 min after the vaccination. In addition, activity budgets, local reaction to the vaccine, and serological titer were also considered in the study. Ten minutes after the vaccination, the IM piglets took longer (p < 0.001) to cross the raceway than did the Hipradermic and Control piglets. Vocalizations were significantly different between the three treatments: the Control piglets produced vocalizations with the lowest frequency (p < 0.001) and level of pressure (p < 0.001), and IM with the highest, with Hipradermic in a significant intermediate position (p < 0.001). Accordingly, the day of the vaccination, IM and Hipradermic animals were lying on the side of the vaccine administration a greater proportion of time than were the Control piglets (10, 11, and 6%, respectively; p = 0.027). Salivary cortisol was not significantly different between treatments. The serum titer of antibodies against the PRRS was higher (p < 0.001) in both vaccinated treatments in comparison to the Control piglets. It is concluded that the Hipradermic needle-free vaccination may result in a less aversive experience in piglets than did intramuscular vaccination.

Milk yield, milk composition, and milk metabolomics of dairy goats intramammary-challenged with lipopolysaccharide under heat stress conditions.

Heat stress and mastitis are major economic issues in dairy production. The objective was to test whether goat's mammary gland immune response to E. coli lipopolysaccharide (LPS) could be conditioned by heat stress (HS). Changes in milk composition and milk metabolomics were evaluated after the administration of LPS in mammary glands of dairy goats under thermal-neutral (TN; n = 4; 15 to 20 °C; 40 to 45% humidity) or HS (n = 4; 35 °C day, 28 °C night; 40% humidity) conditions. Milk metabolomics were evaluated using 1H nuclear magnetic resonance spectroscopy, and multivariate analyses were carried out. Heat stress reduced feed intake and milk yield by 28 and 21%, respectively. Mammary treatment with LPS resulted in febrile response that was detectable in TN goats, but was masked by elevated body temperature due to heat load in HS goats. Additionally, LPS increased milk protein and decreased milk lactose, with more marked changes in HS goats. The recruitment of somatic cells in milk after LPS treatment was delayed by HS. Milk metabolomics revealed that citrate increased by HS, whereas choline, phosphocholine, N-acetylcarbohydrates, lactate, and ß-hydroxybutyrate could be considered as putative markers of inflammation with different pattern according to the ambient temperature (i.e. TN vs. HS). In conclusion, changes in milk somatic cells and milk metabolomics indicated that heat stress affected the mammary immune response to simulated infection, which could make dairy animals more vulnerable to mastitis.

Heat stress modifies the lactational performances and the urinary metabolomic profile related to gastrointestinal microbiota of dairy goats.

The aim of the study is to identify the candidate biomarkers of heat stress (HS) in the urine of lactating dairy goats through the application of proton Nuclear Magnetic Resonance (1H NMR)-based metabolomic analysis. Dairy does (n = 16) in mid-lactation were submitted to thermal neutral (TN; indoors; 15 to 20°C; 40 to 45% humidity) or HS (climatic chamber; 37°C day, 30°C night; 40% humidity) conditions according to a crossover design (2 periods of 21 days). Thermophysiological traits and lactational performances were recorded and milk composition analyzed during each period. Urine samples were collected at day 15 of each period for 1H NMR spectroscopy analysis. Principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) assessment with cross validation were used to identify the goat urinary metabolome from the Human Metabolome Data Base. HS increased rectal temperature (1.2°C), respiratory rate (3.5-fold) and water intake (74%), but decreased feed intake (35%) and body weight (5%) of the lactating does. No differences were detected in milk yield, but HS decreased the milk contents of fat (9%), protein (16%) and lactose (5%). Metabolomics allowed separating TN and HS urinary clusters by PLS-DA. Most discriminating metabolites were hippurate and other phenylalanine (Phe) derivative compounds, which increased in HS vs. TN does. The greater excretion of these gut-derived toxic compounds indicated that HS induced a harmful gastrointestinal microbiota overgrowth, which should have sequestered aromatic amino acids for their metabolism and decreased the synthesis of neurotransmitters and thyroid hormones, with a negative impact on milk yield and composition. In conclusion, HS markedly changed the thermophysiological traits and lactational performances of dairy goats, which were translated into their urinary metabolomic profile through the presence of gut-derived toxic compounds. Hippurate and other Phe-derivative compounds are suggested as urinary biomarkers to detect heat-stressed dairy animals in practice.

Effects of chronic heat stress on lactational performance and the transcriptomic profile of blood cells in lactating dairy goats.

High temperature is a major stress that negatively affects welfare, health, and productivity of dairy animals. Heat-stressed animals are more prone to disease, suggesting that their immunity is hindered. Although productive and physiologic responses of dairy animals to heat stress are well known, there is still limited information on the response at the transcriptome level. Our objective was to evaluate the changes in performance and blood transcriptomics of dairy goats under heat stress. Eight multiparous Murciano-Granadina dairy goats in mid-lactation were assigned to 1 of 2 climatic treatments for 35 d. Treatments and temperature-humidity index (THI) were: (1) thermal neutral (TN: n = 4; 15-20 °C, 40-45%, THI = 59-65), and (2) heat stress (HS: n = 4; 12 h at 37 °C-40%, THI = 86; 12 h at 30 °C-40%, THI = 77). Rectal temperature, respiratory rate, feed intake and milk yield were recorded daily. Additionally, milk composition was evaluated weekly. Blood samples were collected at d 35 and RNA was extracted for microarray analyses (Affymetrix GeneChip Bovine Genome Array). Differences in rectal temperature and respiratory rate between HS and TN goats were maximal during the first 3 d of the experiment, reduced thereafter, but remained significant throughout the 35-d experimental period. Heat stress reduced feed intake, milk yield, milk protein and milk fat contents by 29, 8, 12, and 13%, respectively. Microarray analysis of blood revealed that 55 genes were up-regulated, whereas 88 were down-regulated by HS. Bioinformatics analysis using the Dynamic Impact Approach revealed that 31 biological pathways were impacted by HS. Pathways associated with leukocyte transendothelial migration, cell adhesion, hematopoietic cell lineage, calcium signaling, and PPAR signaling were negatively impacted by HS, whereas nucleotide metabolism was activated. In conclusion, heat stress not only negatively affected milk production in dairy goats, but also resulted in alterations in the functionality of immune cells, which would make the immune system of heat-stressed goats less capable of fending-off diseases.