The effect of social isolation, gender and familiarity with the experimental procedure on tests of porcine nociceptive thresholds.

OBJECTIVE: To investigate the effects of habituation and isolation on mechanical nociceptive thresholds in pigs at the pelvic limbs and at the tail. STUDY DESIGN: Prospective randomized multifactorial study. ANIMALS: Thirty-two healthy castrated male (experiment 1), and 12 castrated male and 12 female (experiment 2) Danish Landrace×Yorkshire pigs, weighing 63.5±0.8 kg and 55.4±0.6 kg (the mean±SD, experiment 1 and 2, respectively). METHODS: Mechanical nociceptive thresholds were quantified with a von Frey anesthesiometer applied to two distinct anatomical regions (tail and pelvic limbs). Pigs receiving the mechanical challenge in the pelvic limbs were tested inside a cage, whereas pigs exposed to stimuli at the tail region were tested in an open arena. For both experiments, the effect of familiarity to the procedure was evaluated by comparing thresholds of nociception in habituated versus naïve pigs. The presence of a companion animal was also evaluated in pigs receiving stimuli at the pelvic limbs. RESULTS: Pigs tested inside the cage were affected by the habituation to the procedure as indicated by the increase in willingness and time spent by the animals in the test cage. This effect was reflected in the lower mechanical nociceptive thresholds (medians with 25-75 percentiles) recorded for familiar pigs compared with naïve animals [495 g (302-675) versus 745 g (479-1000), respectively; p=0.026]. Mechanical nociceptive thresholds measured at the tail of the pigs in the open arena were not affected by the familiarity of the animals with the experimental procedure. CONCLUSIONS AND CLINICAL RELEVANCE: The current results reiterate the value of habituation in research involving animal behaviour. Further characterization of the methodology is needed to allow its application in the evaluation of clinical conditions in pigs.

Lower isoflurane concentration affects spatial learning and neurodegeneration in adult mice compared with higher concentrations.

BACKGROUND: Volatile anesthetics such as isoflurane are widely used in clinical and research contexts. Concerns have been raised that the effects of these drugs on the central nervous system may result in long-term impairment after surgery or general anesthesia. Hence, this study aimed to detect how different isoflurane concentrations influence spatial learning and cell death in adult mice. METHODS: Fifty-two C57BL/6 mice were randomly divided in four groups. Mice in three groups were exposed to different concentrations of isoflurane (1, 1.5, and 2%) for 1 h; the control group was not exposed to anesthesia. Five mice per group were killed 3 h after anesthesia to perform histopathologic and immunohistochemical analyses (hematoxylin-eosin staining; caspase-3 activation). Eight mice per group were used for behavioral tests (open field, T-maze spontaneous alternation, and water maze) on subsequent days. RESULTS: There were no differences between groups in the T-maze spontaneous alternation test or in the open field (no confounding effects of stress or locomotion). The group anesthetized with 1% isoflurane performed worse in the water maze task on day 1 (550.4 ±162.78 cm) compared with the control group (400.1 ± 112.88 cm), 1.5% isoflurane (351.9 ± 150.67 cm), and 2% isoflurane (364.5 ± 113.70 cm; P ≤ 0.05) and on day 3 (305.0 ± 81.75 cm) compared with control group (175.13 ± 77.00 cm) and 2% isoflurane (204.11 ± 85.75 cm; P ≤ 0.038). In the pyramidal cell layer of the region cornu ammonis 1 of the hippocampus, 1% isoflurane showed a tendency to cause more neurodegeneration (apoptosis) (61.4 ± 26.40, profiles/mm) than the group with 2% of isoflurane (20.6 ± 17.77, profiles/mm; P = 0.051). CONCLUSION: Low isoflurane concentration (1%) caused spatial learning impairment and more neurodegeneration compared with higher isoflurane concentrations. Results for mice receiving the latter concentrations were similar to those of control mice.

Nociceptive Threshold of Calves and Goat Kids Undergoing Injection of Clove Oil or Isoeugenol for Disbudding.

In this preliminary study, we compared changes in mechanical nociceptive thresholds (MNT) of calves and goat kids injected with clove oil or isoeugenol under the horn bud as a potential, more welfare-friendly alternative to hot-iron disbudding. Twenty male calves and goat kids were randomly allocated to clove oil (n = 10 per species) or isoeugenol (n = 10 per species) injection under the horn buds. MNT was measured via a pressure algometer in calves and kids at several locations around the horn buds at several time points before and up to 24 h after injection. In kids, von Frey filaments were used additionally at the same time points. In calves, linear mixed models revealed an effect on MNT of time point (p = 0.010) and side (p = 0.007), but not of injection (p = 0.298), nor of the interaction 'injection*time point', MNT waslowest 9 h post-injection. In goats, there was an effect of injection depending on time point (interaction injection*time point, p = 0.03) with MNT being lowest 24 h post-injection for clove oil, while MNT was similar to pre-injection in isoeugenol. In both species, variation in the individual response post-injection was very high. Our results suggest that clove oil and isoeugenol induced hypersensitivity, which was higher for clove oil, in goat kids, but they also suggest a transient anaesthetic effect in some animals and locations.

Transcriptomics Analysis of Porcine Caudal Dorsal Root Ganglia in Tail Amputated Pigs Shows Long-Term Effects on Many Pain-Associated Genes.

Tail amputation by tail docking or as an extreme consequence of tail biting in commercial pig production potentially has serious implications for animal welfare. Tail amputation causes peripheral nerve injury that might be associated with lasting chronic pain. The aim of this study was to investigate the short- and long-term effects of tail amputation in pigs on caudal DRG gene expression at different stages of development, particularly in relation to genes associated with nociception and pain. Microarrays were used to analyse whole DRG transcriptomes from tail amputated and sham-treated pigs 1, 8, and 16 weeks following tail treatment at either 3 or 63 days of age (8 pigs/treatment/age/time after treatment; n = 96). Tail amputation induced marked changes in gene expression (up and down) compared to sham-treated intact controls for all treatment ages and time points after tail treatment. Sustained changes in gene expression in tail amputated pigs were still evident 4 months after tail injury. Gene correlation network analysis revealed two co-expression clusters associated with amputation: Cluster A (759 down-regulated) and Cluster B (273 up-regulated) genes. Gene ontology (GO) enrichment analysis identified 124 genes in Cluster A and 61 genes in Cluster B associated with both "inflammatory pain" and "neuropathic pain." In Cluster A, gene family members of ion channels e.g., voltage-gated potassium channels (VGPC) and receptors e.g., GABA receptors, were significantly down-regulated compared to shams, both of which are linked to increased peripheral nerve excitability after axotomy. Up-regulated gene families in Cluster B were linked to transcriptional regulation, inflammation, tissue remodeling, and regulatory neuropeptide activity. These findings, demonstrate that tail amputation causes sustained transcriptomic expression changes in caudal DRG cells involved in inflammatory and neuropathic pain pathways.

Determination of stable reference genes for RT-qPCR expression data in mechanistic pain studies on pig dorsal root ganglia and spinal cord.

RNA expression levels for genes of interest must be normalised with appropriate reference or "housekeeping" genes that are stably expressed across samples and treatments. This study determined the most stable reference genes from a panel of 6 porcine candidate genes: beta actin (ACTB), beta-2-microglobulin (B2M), eukaryotic elongation factor 1 gamma-like protein (eEF-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), Ubiquitin C (UBC) in sacral dorsal root ganglia and spinal cord samples collected from 16 tail docked pigs (2/3rds of tail amputated) 1, 4, 8 and 16weeks after tail injury (4 pigs/time point). Total RNA from pooled samples was measured by SYBRgreen real-time quantitative PCR. Cycle threshold values were analysed using geNorm, BestKeeper and NormFinder PCR analysis software. Average expression stability and pairwise variation values were calculated for each candidate reference gene. GeNorm analysis identified the most stable genes for normalisation of gene expression data to be GAPDH>eEF-1>UBC>B2M>ACTB>SDHA for dorsal root ganglia and ACTB>SDHA>UBC>B2M>GAPDH>eEF-1 for spinal cord samples. Expression stability estimates were verified by BestKeeper and NormFinder analysis. Expression stability varied between genes within and between tissues. Validation of most stably expressed reference genes was performed by normalisation of calcitonin gene related polypeptide beta (CALCB). The results show similar patterns of CALCB expression when the best reference genes selected by all three programs were used. GAPDH, eEF-1 and UBC are suitable reference genes for porcine dorsal root ganglia samples, whereas ACTB, SDHA and UBC are more appropriate for spinal cord samples.

Characterization of short- and long-term mechanical sensitisation following surgical tail amputation in pigs.

Commercial pigs are frequently exposed to tail mutilations in the form of preventive husbandry procedures (tail docking) or as a result of abnormal behaviour (tail biting). Although tissue and nerve injuries are well-described causes of pain hypersensitivity in humans and in rodent animal models, there is no information on the changes in local pain sensitivity induced by tail injuries in pigs. To determine the temporal profile of sensitisation, pigs were exposed to surgical tail resections and mechanical nociceptive thresholds (MNT) were measured in the acute (one week post-operatively) and in the long-term (either eight or sixteen weeks post-surgery) phase of recovery. The influence of the degree of amputation on MNTs was also evaluated by comparing three different tail-resection treatments (intact, 'short tail', 'long tail'). A significant reduction in MNTs one week following surgery suggests the occurrence of acute sensitisation. Long-term hypersensitivity was also observed in tail-resected pigs at either two or four months following surgery. Tail amputation in pigs appears to evoke acute and sustained changes in peripheral mechanical sensitivity, which resemble features of neuropathic pain reported in humans and other species and provides new information on implications for the welfare of animals subjected to this type of injury.

Docking piglet tails: How much does it hurt and for how long?

Tail docking in pigs has the potential for evoking short- as well as long-term physiological and behavioural changes indicative of pain. Nonetheless, the existing scientific literature has thus far provided somewhat inconsistent data on the intensity and the duration of pain based on varying assessment methodologies and different post-procedural observation times. In this report we describe three response stages (immediate, short- and long-term) through the application of vocalisation, behavioural and nociceptive assessments in order to identify changes indicative of potential pain experienced by the piglets. Furthermore, we evaluated the following procedural differences: (1) cautery vs. non-cautery docking; (2) length of tail removal. Sound parameters showed a significantly greater call energy and intensity exhibited by docked vs. sham-docked piglets (P<0.05). Observations of general activity of the animals in a test situation failed to detect a difference among treatments (P>0.05) up to 48h post-tail docking. Similarly, no difference in mechanical nociceptive thresholds indicative of long term pain was observed at 17weeks following neonatal tail docking (P>0.05). The present results highlight the potential for the use of measures of vocalisation to detect peri-procedural changes possibly associated with evoked pain. Nonetheless, activity and nociceptive measures failed to identify post-docking anomalies, suggesting that alternative methodologies need to be implemented to clarify whether tail docking is associated with short- and long-term changes attributable to pain experienced by the piglets.

A Review of Pain Assessment in Pigs.

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

Application of a handheld Pressure Application Measurement device for the characterisation of mechanical nociceptive thresholds in intact pig tails.

The assessment of nociceptive thresholds is employed in animals and humans to evaluate changes in sensitivity potentially arising from tissue damage. Its application on the intact pig tail might represent a suitable method to assess changes in nociceptive thresholds arising from tail injury, such as tail docking or tail biting. The Pressure Application Measurement (PAM) device is used here for the first time on the tail of pigs to determine the reliability of the methods and to provide novel data on mechanical nociceptive thresholds (MNT) associated with four different age groups (9, 17, 24 and 32weeks) and with proximity of the target region to the body of the animal. We recorded an overall acceptable level of intra-individual reliability, with mean values of CV ranging between 30.1 and 32.6%. Across all age groups, the first single measurement of MNT recorded at region 1 (proximal) was significantly higher (P<0.05) than the following two. This was not observed at tail regions 2 and 3 (more distal). Age had a significant effect (P<0.05) on the mean thresholds of nociception with increasing age corresponding to higher thresholds. Furthermore, a significant effect of proximity of tail region to the body was observed (P<0.05), with MNT being higher in the proximal tail region in pigs of 9, 17 and 24weeks of age. There was also a significant positive correlation (P<0.05) between mechanical nociceptive thresholds and age/body size of the animals. To the best of our knowledge, no other investigation of tail nociceptive thresholds has been performed with the PAM device or alternative methods to obtain mechanical nociceptive thresholds in intact tails of pigs of different age/body size. The reliability of the data obtained with the PAM device support its use in the measurement of mechanical nociceptive threshold in pig tails. This methodological approach is possibly suitable for assessing changes in tail stump MNTs after tail injury caused by tail docking and biting.

The Assessment of Facial Expressions in Piglets Undergoing Tail Docking and Castration: Toward the Development of the Piglet Grimace Scale.

Many piglets are exposed to potentially painful husbandry procedures within the first week of life, including tail docking and castration, without the provision of either anesthesia or analgesia. The assessment methods used to evaluate pain experienced by piglets are often affected by low specificity and practical limitations, prompting the investigation of alternative methodologies. The assessment of changes in facial expression following a painful event has been successfully applied to several species. The objective of this pilot study was to evaluate the utility of a Grimace Scale applied to neonatal pigs to evaluate pain evoked by tail docking and castration. Eight female piglets, Sus scrofa domesticus (Landrace/Large White X synthetic sire line) underwent tail docking and 15 male piglets (75% Large White and 25% Belgian Landrace) were exposed to the castration procedure. Clear images of the faces of the piglets were collected immediately pre- and post-procedure. The images were used by experienced observers to identify facial action units (FAUs) which changed in individuals over this period, and a scoring scale was depicted in a training manual. A set of randomly selected images were then combined in a scorebook, which was evaluated after training by 30 scorers, blind to the treatment. The scale for most FAU was used with a high level of consistency across all observers. Tail docking induced a significant change (P < 0.05) in free moving piglets only in the "orbital tightening" FAU, whereas no change in any unit was observed in castrated piglets, which were restrained at the time of assessment. In this initial stage of development, orbital tightening seems to have the potential to be applied to investigate painful conditions in neonatal pigs. Nonetheless, more studies are needed to assess its full effectiveness and to evaluate the influence of possible confounds (e.g., handling stress) on the observed changes in FAUs.

Capsaicin-induced neurogenic inflammation in pig skin: a behavioural study.

Topical capsaicin is a well-established model of experimental hyperalgesia. Its application to the study of animals has been limited to few species. The effect of topical capsaicin on hyperalgesia in porcine skin was evaluated as part of a study of inflammatory pain in the pig. Two experiments were carried out on pigs of 27 ± 5 kg (n = 8) and 57 ± 3 kg (n = 16). Thermal and mechanical noxious stimuli were provided (CO2 laser and Pressure Application Measurement device) to assess avoidance behaviours. Capsaicin induced significant thermal hyperalgesia in the smaller pigs (P < 0.05), while no mechanical hyperalgesia was observed in either animal group. The present model of topical capsaicin application may be useful to investigate the mechanisms of primary hyperalgesia in this species, although some experimental conditions, such as the administration route and cutaneous morphology, need to be evaluated.