The use of social network analysis to describe the effect of immune activation on group dynamics in pigs.

The immune system can influence social motivation with potentially dire consequences for group-housed production animals, such as pigs. The aim of this study was to test the effect of a controlled immune activation in group-housed pigs, through an injection with lipopolysaccharide (LPS) and an intervention with ketoprofen on centrality parameters at the individual level. In addition, we wanted to test the effect of time relative to the injection on general network parameters in order to get a better understanding of changes in social network structures at the group level. 52 female pigs (11-12 weeks) were allocated to four treatments, comprising two injections: ketoprofen-LPS (KL), ketoprofen-saline (KS), saline-LPS (SL) and saline-saline (SS). Social behaviour with a focus on damaging behaviour was observed continuously in 10 × 15 min bouts between 0800 am and 1700 pm 1 day before (baseline) and two subsequent days after injection. Activity was scan-sampled every 5 min for 6 h after the last injection in the pen. Saliva samples were taken for cortisol analysis at baseline and at 4, 24, 48, 72 h after the injections. A controlled immune activation affected centrality parameters for ear manipulation networks at the individual level. Lipopolysaccharide-injected pigs had a lower in-degree centrality, thus, received less interactions, 2 days after the challenge. Treatment effects on tail manipulation and fighting networks were not observed at the individual level. For networks of manipulation of other body parts, in-degree centrality was positively correlated with cortisol response at 4 h and lying behaviour in the first 6 h after the challenge in LPS-injected pigs. Thus, the stronger the pigs reacted to the LPS, the more interactions they received in the subsequent days. The time in relation to injection affected general network parameters for ear manipulation and fighting networks at the group level. For ear manipulation networks, in-degree centralisation was higher on the days following injection, thus, certain individuals in the pen received more interactions than the rest of the group compared to baseline. For fighting networks, betweenness decreased on the first day after injection compared to baseline, indicating that network connectivity increased after the challenge. Networks of tail manipulation and manipulation of other body parts did not change on the days after injection at the group level. Social network analysis is a method that can potentially provide important insights into the effects of sickness on social behaviour in group-housed pigs.

Development and validation of a multiplex fluorescent microsphere immunoassay assay for detection of porcine cytokines.

Cytokines are cell signalling proteins that mediate a number of different physiological responses. The accurate measurement of cytokine profiles is important for a variety of diagnostic and prognostic scenarios in relation to animal health and welfare. Simultaneous quantification of cytokine profiles in a single sample is now possible using fluorescent microsphere immunoassays (FMIA). We describe the development and validation of a novel multiplex assay using the Bio-Plex® 200 system to quantify cytokines in five different porcine tissues (brain, placenta, synovial tissue and fluid, plasma). The cytokine profiles are both tissue, and research hypothesis, -dependent but include Interleukin-1beta (IL-1ß), Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10) and Tumor necrosis factor (TNF-α). This methods paper is reported in two parts: the development of a FMIA for porcine tissues and validation of pre-treatment for optimal cytokine recovery in porcine brain, placenta, synovial tissue and plasma. Validation steps are critical in ensuring an assay is suitable for novel sample types. This technique advances traditional ELISAs by: •FMIA provides insight into the profiles of multiple porcine cytokines in certain situations (e.g. disease, parturition).•Use of the Bio-Plex® 200 system to investigate novel sample types, including brain, placenta and synovial tissue.•Multiplexing utilises a fraction of the sample volume compared with multiple ELISAs.

Histopathological Characterization of Tail Injury and Traumatic Neuroma Development after Tail Docking in Piglets.

Tail docking of neonatal pigs is widely used as a measure to reduce the incidence of tail biting, a complex management problem in the pig industry. Concerns exist over the long-term consequences of tail docking for possible tail stump pain sensitivity due to the development of traumatic neuromas in injured peripheral nerves. Tail stumps were obtained post mortem from four female pigs at each of 1, 4, 8 and 16 weeks following tail amputation (approximately two-thirds removed) by a gas-heated docking iron on post natal day 3. Tissues were processed routinely for histopathological examination. Non-neural inflammatory and reparative epidermal and dermal changes associated with tissue thickening and healing were observed 1 to 4 months after docking. Mild neutrophilic inflammation was present in some cases, although this and other degenerative and non-neural reparative changes are not likely to have caused pain. Traumatic neuroma and neuromatous tissue development was not observed 1 week after tail docking, but was evident 1 month after tail docking. Over time there was marked nerve sheath and axonal proliferation leading to the formation of neuromata, which were either localized and circumscribed or comprised of multiple axons dispersed within granulation tissue. Four months after tail resection, neuroma formation was still incomplete, with possible implications for sensitivity of the tail stump.

Physiological responses to low atmospheric pressure stunning and the implications for welfare.

In low atmospheric pressure stunning (LAPS), poultry are rendered unconscious before slaughter by gradually reducing oxygen tension in the atmosphere to achieve a progressive anoxia. The effects of LAPS are not instantaneous, so there are legitimate welfare concerns around the experience of birds before loss of consciousness. Using self-contained telemetry logging units, high-quality continuous electroencephalogram (EEG) and electrocardiogram (EKG) recordings were obtained from 28 broiler chickens during exposure to LAPS in a commercial poultry processing plant. Application of LAPS was associated with changes in the EEG pattern in the form of increases in total power, decreases in mean frequency, and in particular, increases in slow-wave (delta) activity, indicating a gradual loss of consciousness. Increased delta wave activity was seen within 10 s of LAPS onset and consistently thereafter, peaking at 30 s into LAPS at which point the EEG signal shared characteristics with that of birds in a surgical plane of anesthesia. During LAPS, heart rate consistently decreased, with more pronounced bradycardia and arrhythmia observed after 30 s. No heart rate increases were observed in the period when the birds were potentially conscious. After an initial quiescent period, brief body movements (presumed to be ataxia/loss of posture) were seen on average at 39 s into the LAPS process. Later (after 120 s on average), artifacts related to clonic (wing flapping) and tonic (muscle spasms) convulsions were observed in the EKG recordings. Based on EEG analysis and body movement responses, a conservative estimate of time to loss of consciousness is approximately 40 s. The lack of behavioral responses indicating aversion or escape and absence of heart rate elevation in the conscious period strongly suggest that birds do not find LAPS induction distressing. Collectively, the results suggest that LAPS is a humane approach that has the potential to improve the welfare of poultry at slaughter by gradually inducing unconsciousness without distress, eliminating live shackling and ensuring every bird is adequately stunned before exansguination.

Behavioural and physiological measures following treadmill exercise as potential indicators to evaluate fatigue in sheep.

The welfare consequences of long-distance transportation of animals remain a controversial topic. Animals that stand for most of the long journey (especially if additional muscular activity is required to deal with postural instability) are at risk of developing fatigue. Previous observational studies of behaviour and physiology suggested either that sheep do not become markedly fatigued by long journeys or that previous methods did not adequately identify fatigue. A range of behavioural and physiological measures were made on eight pairs of sheep during and after treadmill exercise. Within each pair of sheep, a treatment sheep was walked on a treadmill at 0.5 m/s for up to 5 h or until the sheep voluntarily stopped exercising or showed other signs of reduced performance, and a control sheep was exercised for two 10-min periods on either side of the exercise period for the treatment sheep. With the exception of one sheep that only walked for 4.5 h, all treatment sheep walked for 5 h without apparent difficulty. After exercise, the plasma cortisol concentration of treatment sheep was significantly greater than that of control sheep. However, there were no significant treatment effects on plasma creatine kinase activity or blood lactate concentration. After 5 h of exercise, there was a proportionate decrease in the median frequency of the electromyogram recorded over the m. semitendinosus, and this was significantly different from control sheep. There was no evidence that treatment sheep lay down sooner or for longer after treadmill exercise than controls. In sheep tested in a maze to examine whether there was increased motivation to rest after exercise, there was no significant difference between the times taken by treatment and control sheep to obtain a food reward. Qualitative behavioural assessment of the sheep by a panel of observers identified two main dimensions of sheep demeanour, but among descriptors elicited from observers only one person used a term associated with fatigue. No significant difference was found between the scores of treatment and control sheep on these two demeanour dimensions. Thus, there was little evidence that prolonged gentle walking exercise fatigues sheep. Further development of methods to both repeatedly induce and to identify fatigue in sheep is required.

Quantifying genetic (co)variation and effects of genetic selection on tibial bone morphology and quality in 37 lines of broiler, layer and traditional chickens.

1. Tibial bone morphology and quality was assessed at 6, 8 and 10 weeks of age in 12 broiler, 12 layer and 13 traditional lines of chickens. 2. High intraclass correlations (0.65-0.96) were estimated for tibial weight, length, width, strength, stiffness, stress radiodensity, plateau angle but not for cortical width and tibial torsion at 6 and 10 weeks of age and for weight, length, width, strength radiodensity and plateau angle at 8 weeks of age. Lower intraclass correlations (0.4-0.8) were estimated within category (broiler, layer and traditional) for weight, length, width at 6, 8 and 10 weeks, and for stiffness, stress and radiodensity at 6 and 10 weeks, and were lower (<0.33) at 8 weeks of age. Intraclass correlations for tibial torsion angles were low (< or =0.4) at all ages. 3. Tibia from layers and traditional lines were similar suggesting that intensive genetic selection for high rates of egg laying has not changed bone size, shape or quality. Tibia from broilers, as expected, were heavier and larger and the differences were greatest at 6 weeks of age suggesting that the broilers were earlier maturing than layers and traditional lines. Broiler tibiae were more radiodense, stronger and stiffer and had lower stress values than bones from layers and traditional lines. Plateau angles were higher in broilers and torsion angles had higher external rotation compared with layers and traditional lines.

Quantifying the effects of genetic selection and genetic variation for body size, carcass composition, and meat quality in the domestic fowl (Gallus domesticus).

A multistrain experiment was conducted to quantify the extent of genetic differences in carcass and muscle yields, muscle quality, support organs, and taste panel assessments of cooked breast muscle of 296 birds from 37 lines of commercial broiler, layer, and traditional chickens. The birds were reared as broilers and 4 males from each line were slaughtered at 6 and 10 wk of age. The extent of genetic variation was measured as the intraclass correlation. The intraclass correlation for live weight; carcass yields; breast, drum, and wing portions; and associated muscle yields were high, whereas those for the thigh portion and yield were low. Broilers had more breast and thigh muscle but similar drum muscle as a proportion of carcass weight compared with layer and traditional lines. Genetic variation for muscle quality (plasma creatine kinase activity) was high; that for muscle color (L, a, and b) and hemorrhage score were moderate in size and were greater at 10 than at 6 wk of age. Broiler lines had greater creatine kinase activity indicative of greater muscle pathology; breast muscle was lighter, less red and yellow in color, and had a greater hemorrhage score than muscle from layer and traditional lines, which were similar. Intraclass correlations for taste panel scores were low and generally not significant except for texture, chicken flavor intensity, flavor liking, and overall liking at 6 wk of age. Significantly greater scores from broiler compared with layer and traditional lines for texture, chicken flavor intensity, and overall liking were observed. At 10 wk of age, chicken flavor intensity did not differ between broiler or layer birds but was significantly greater in both groups than traditional birds. Genetic variation for relative weight of abdominal fat, spleen, and heart was moderately high and greater at 10 than at 6 wk of age. Broiler carcasses had a relatively high proportion of abdominal fat and smaller spleen and heart weights.

A comparison of breast muscle characteristics in three broiler great-grandparent lines.

Genetic selection of broiler chickens has led to a gross overdevelopment of the broiler breast muscle pectoralis major. This may have resulted in increased myopathy and detrimental effects on meat quality. The present study examined 3 commercial great-grandparent lines (lines A, B, and C). Lines A and B are female lines, and line C is a male line. The mean BW of line C (2.7 kg) was significantly greater than those of lines A and B (both 2.3 kg). However, the mean breast yield of both lines B and C (8.9 and 8.7%, respectively) was significantly greater than that of line A (6.9%). Line B therefore matched the meat yield of line A while maintaining a high reproductive capacity. The mean breast fillet weight of line A (169 g) was significantly lower than lines B (207 g) and C (235 g). No differences were observed between lines in either mean fiber size or amount of connective tissue. Therefore, additional fibers must provide the additional weight in the breast fillet of lines B and C, compared with A. Plasma creatine kinase activity, a commonly used marker of muscle damage, was significantly higher in line A (1368 IU/L) than in lines B (995 IU/L) and C (982 IU/L). However, qualitative evaluations of muscle pathology revealed no differences among lines. Selection for increased embryonic muscle fiber number, rather than for increased radial fiber growth, could improve growth potential and may also alleviate muscle damage.

Skeletal muscle fibre growth and growth associated myopathy in the domestic chicken (Gallus domesticus).

1. Genetic selection of broilers may have pushed muscle fibres to their maximum functional size constraints. Broiler (B), female great-grandparent (GGP) and layer (L) lines were weighed, blood sampled and killed from 5 to 25 weeks of age. 2. At 25 weeks, Pectoralis major (Pm) fibre size reached by the B (65.9 microm) and GGP (59.8 microm) were 1.5 times greater than the L (38.1 microm). In the B and GGP lines, fibre growth of the Pm markedly exceeded that of the Biceps femoris (Bf) muscle. However, in the L line, fibre growth of the Pm and Bf muscle was comparable. Connective tissue content was generally higher in the Bf than in the Pm of all lines. 3. Centralised nuclei were observed predominantly in Pm, and may regulate fibre size. Both large muscle fibres and inadequate capillary supply may induce metabolic stress in B and GGP lines due to the large diffusion distances for oxygen, metabolites and waste products. 4. Enzyme markers of muscle damage (creatine kinase (CK), lactate dehydrogenase (LDH) and aspartate aminotransaminase (AST)) and histopathological analysis of Pm and Bf indicated greater myopathy in B and GGP vs L. 5. Regenerative processes were associated with oestrogen secretion. Reduced CK and LDH preceded egg yolk precursor production and increased calcium uptake for eggshell synthesis in all three lines. Oestrogen may stimulate muscle fibre regeneration and recovery as a myo-protective adaptation to potentially detrimental changes in calcium economy during egg production.

Thermoregulatory capacity and muscle membrane integrity are compromised in broilers compared with layers at the same age or body weight.

1. The effects of acute heat stress (2 h at 32 degrees C and 75% RH) on body temperature and indices of respiratory thermoregulation and skeletal muscle function were examined in two divergently selected male grandparent lines of broiler and layer-type chickens at two ages (35 and 63 d), or at a similar body weight (approximately 2.2 kg). 2. The two chicken lines exhibited markedly different baseline blood acid-base and skeletal muscle characteristics. At the same age or live weight, birds from the broiler line had significantly higher venous blood carbon dioxide tensions associated with lower blood pH. Plasma creatine kinase (CK) activities reflecting muscle membrane damage were also greatly elevated in the broiler line. 3. Exposure to acute heat stress caused an increase in deep body temperature, panting-induced acid-base disturbances and elevated plasma CK activity in both lines of chicken, an effect that increased with age. The extent of disturbances in acid-base regulation and heat-stress-induced myopathy were more pronounced in the broiler than the layer line at the same age or similar live weights. 4. It is suggested that genetic selection for high muscle growth in broiler lines has compromised their capacity to respond to an acute thermal challenge, leading to detrimental consequences for muscle function. This reduction in heat tolerance may have important implications for bird welfare and subsequent meat quality.

The role of sodium ions in the pathogenesis of skeletal muscle damage in broiler chickens.

The effect of sodium ions (Na+) on calcium (Ca2+)-mediated muscle damage in broiler chickens was investigated using an in vitro muscle preparation. Muscle Ca2+ accumulation was determined by 45Ca2+ uptake. Muscle damage was assessed by measurement of the efflux of the intracellular enzyme creatine kinase (CK) into the incubation medium. Loading muscle cells with Na+ by means of the sodium ionophore monensin led to concentration-dependent (25 to 200 microM) increases in 45Ca2+ uptakes and corresponding and proportional CK losses. The greatest responses occurred at 100 microM ionophore or greater, reflected in a 49% increase (P < 0.05) in 45Ca2+ uptake and an associated 140%-fold increase (P < 0.001) in CK efflux. Inhibition of muscle Na+/K+-ATPase activity with ouabain (2 mM) induced a 56% increase in 45Ca2+ uptake and a 60%-fold increase (P < 0.001) in total CK loss. The combined use of ionophore and ouabain resulted in 90 and 130%-fold elevations in 45Ca2+ uptake and CK loss, respectively. In monensin-treated muscles, inhibition of external Ca2+ influx from the incubation medium by chelation with 1,2 bis(2-aminophenoxy)ethane-N,N,N',N' tetracetic acid (5 mM) markedly reduced 45Ca2+ uptake (38%: P < 0.05) but increased CK release by 85% (P < 0.001). The results demonstrate that initial elevations in muscle Na+ can facilitate increases in muscle Ca2+ and lead to alterations in muscle cell membrane integrity and CK loss. The Na+-induced increases in myocellular Ca2+ may be mediated via direct extracellular Ca2+ entry or redistribution from internal Ca2+ stores. It is proposed that in order to reduce or prevent myopathies in poultry, exposure to conditions that may lead to elevations in muscle Na+ (e.g., increased muscle activity and stress or accidental ionophore toxicosis) should be avoided. The findings of this study have implications for management strategies of bird welfare, muscle pathology, and product quality.

Myopathy in broiler chickens: a role for Ca(2+)-activated phospholipase A2?

The role of Ca(2+)-dependent phospholipase A2 (PLA2) in the mechanism of skeletal muscle damage in broiler chickens was examined in vitro using a novel, synthetic, PLA2-specific inhibitor Ro31-499/001 (Ro31). Muscle damage was assessed by measurement of creatine kinase (CK) efflux from isolated muscles into the incubation medium. Treatment with the specific Ca(2+)-ionophore 4-Br-A23187 (5 microM) caused a 72% elevation (P<0.05) in muscle 45Ca2+ accumulation, which was associated with a marked increase (P<0.001) in muscle CK efflux (7.6-fold). Incubation with Ro31 (50 microM) reduced (P<0.001) CK efflux from muscles treated with ionophore (45%) but was without effect on 45Ca accumulation. Treatment with the Na+ ionophore monensin (100 microM) induced 55% (P< 0.05) elevation in 45Ca2+ accumulation with a concomitant 2.5-fold increase (P<0.001) in CK loss. Muscles incubated with monensin in the presence of Ro31 exhibited a 49% reduction (P<0.001) in CK leakage but showed no change in 45Ca2+ uptake. The results indicate that increasing external Ca2+ entry, directly or indirectly, and elevation of intracellular Ca2+, significantly alters sarcolemmal integrity resulting in increased CK efflux from broiler skeletal muscle. This process is, at least in part, dependent upon activation of PLA2 activity and thus inhibitable by Ro31. It is further proposed that muscle damage in poultry induced by a range of stresses, and insults may also be mediated by a Ro31 sensitive, PLA2-dependent component. The findings have implications for strategies to reduce or prevent myopathies in poultry affecting bird welfare and product quality.

Acute heat stress-induced alterations in blood acid-base status and skeletal muscle membrane integrity in broiler chickens at two ages: implications for meat quality.

The effects of acute heat stress (AHS) on indices of respiratory thermoregulation and skeletal muscle damage (myopathy) were examined in broiler chickens at two ages (35 and 63 d of age); the relationships of these responses with changes in meat quality were assessed. Exposure to AHS significantly increased deep-body temperatures, panting-induced acid/base disturbances, and plasma creatine kinase (CK) activities, reflecting heat stress-induced myopathy (HSIM). The extent of the hyperthermia and disturbances in acid/base status and myopathy was significantly (P < 0.05) higher in the older birds. Consistent with AHS-induced alterations in thermoregulatory indices and muscle membrane integrity were changes in breast muscle glycolytic metabolism as indicated by lower muscle pH immediately postslaughter (pHi), increased water loss, and increased incidence of breast muscle hemorrhages. Values of pHi were lower and hemorrhage scores greater in the AHS birds at 63 d; drip losses were significantly higher in the 35-d-old birds. Exposure to AHS did not affect breast meat eating quality, although overall reductions in flavor attributes were observed in the older birds. We concluded that exposure to AHS induced disturbances in blood acid/base status and had a detrimental effect upon skeletal muscle membrane integrity. Muscle from broilers exhibited an increased sensitivity to AHS exposure with age. Alterations in antemortem blood acid/base status and muscle membrane integrity induced by AHS were associated (though not necessarily causally) with adverse effects upon breast meat quality. It is recommended that preslaughter exposure of broiler chickens to AHS should be avoided in order to reduce alterations in muscle metabolism and membrane integrity and undesirable meat characteristics.

Skeletal muscle damage following halothane anaesthesia in the domestic fowl: plasma biochemical responses.

The effects of a short period (10 minutes) of halothane anaesthesia upon skeletal muscle have been examined in broiler chickens. Integrity of the muscle membrane was assessed by measurement of plasma activities of intracellular enzymes. Creatine kinase activity was greatly increased during the first 12 hours post-anaesthesia and remained elevated for at least 48 hours. Lactate dehydrogenase activity exhibited a similar pattern which was less pronounced. Halothane anaesthesia induced transient hypercapnic acidosis and hypokalemia and increased corticosterone secretion but there was no significant effect upon deep body temperature. It is proposed that halothane may act directly upon skeletal muscle, perhaps influencing intracellular calcium homeostasis, to alter membrane permeability and increase enzyme efflux reflecting a degree of post-anaesthetic muscle damage. The transient changes in electrolyte and acid-base balance may contribute to these effects. Precautions should be taken to minimise the incidence or extent of halothane induced myopathy in birds particularly in rapidly growing broiler chickens where susceptibility may be increased.

Interaction of age, strain, sex and food restriction on plasma creatine kinase activity in turkeys.

1. Plasma creatine kinase activity was determined at 4, 8, 12, 16, 20, 24, 28, 38 and 48 weeks of age in male and female traditional turkeys fed ad libitum, in male and female turkeys of a sire-line fed ad libitum or restricted to 0.5 during rearing and subsequently to 0.8 of sex-specific ad libitum-fed body weight, and in sire-line males fed ad libitum to 18 weeks and 0.8 of ad libitum body weight thereafter. 2. Plasma creatine kinase activity was low in traditional turkeys and increased rapidly after 12 weeks of age in males and females of the sire-line of turkeys. 3. Food restriction decreased the activity of plasma creatine kinase. 4. There was no difference in plasma creatine kinase activity between the sexes during rearing. After the onset of lay, the activity in plasma from females decreased at 38 weeks of age and rose dramatically in restricted females at 48 weeks when the birds had ceased laying. 5. The changes in plasma creatine kinase activity in females were associated with concomitant changes in ovarian activity as reflected in altered plasma triglyceride concentrations.

Creatine kinase isoenzyme profiles in the plasma of the domestic fowl (Gallus domesticus): effects of acute heat stress.

Creatine kinase isoenzyme activities in extracts of plasma, skeletal muscle, heart and brain tissue of domestic fowls were separated by anion exchange chromatography and tissue specific distributions of the isoenzyme designated MM-CK, BB-CK1 and BB-CK2 were demonstrated. The muscle isoenzyme (MM-CK) was the predominant form in plasma (99 per cent) and its activity increased in response to an episode of acute heat stress.