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.

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.

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.