Validation of indicators used to assess unconsciousness in veal calves at slaughter.

European legislation states that after stunning regular checks should be performed to guarantee animals are unconscious between the end of the stunning process and death. When animals are killed without prior stunning these checks should be performed before the animal is released from restraint. The validity of certain indicators used to assess unconsciousness under different stunning and slaughter conditions is under debate. The aim of this study was to validate the absence of threat-, withdrawal-, corneal- and eyelid reflex as indicators to assess unconsciousness in calves subjected to different stunning and slaughter methods. Calves (201±22 kg) were randomly assigned to one of the following four treatments: (1) Captive bolt stunning followed by neck cut in an inverted position (n=25); (2) Non-stunned slaughter in an upright position (n=7); (3) Non-stunned slaughter in an inverted position (180° rotation) (n=25); (4) Non-stunned slaughter in an upright position followed by captive bolt stunning 40 s after the neck cut (n=25). Each calf was equipped with non-invasive electroencephalogram (EEG) electrodes before the slaughter procedure. All reflexes were verified once before the slaughter procedure. At the beginning of the procedure (T=0 s) calves were stunned (treatment 1) or neck cut in an upright position (treatment 2, 4) or inverted position (treatment 3). Calves of treatment 4 were captive bolt stunned 34±8 s after the neck cut. Reflexes were assessed every 20 s from T=15 s for all treatments until all reflex tests resulted in a negative response three times in a row and a flat line EEG was observed. In addition, reflexes were assessed 5 s after captive bolt stunning in calves of treatments 1 and 4. Visual assessment of changes in the amplitude and frequency of EEG traces was used to determine loss of consciousness. Timing of loss of consciousness was related to timing of loss of reflexes. After captive bolt stunning, absence of threat-, withdrawal-, corneal- and eyelid reflex indicated unconsciousness as determined by EEG recordings. After non-stunned slaughter, both threat- and withdrawal reflex were on average lost before calves were unconscious based on EEG recordings. The eyelid- and corneal reflex were on average lost after calves had lost consciousness based on EEG recordings and appeared to be distinctly conservative indicators of unconsciousness in non-stunned slaughtered calves since they were observed until 76±50 and 85±45 s (mean±SD), respectively, after EEG-based loss of consciousness.

Validation of behavioural indicators used to assess unconsciousness in sheep.

The validity of behavioural indicators to assess unconsciousness under different slaughter conditions is under (inter)national debate. The aim of this study was to validate eyelid-, withdrawal-, threat reflex and rhythmic breathing as indicators to assess unconsciousness in sheep. Sheep were monitored during repeated propofol anaesthesia (n=12) and during non-stunned slaughter (n=22). Changes in the EEG and behavioural indices of consciousness/unconsciousness were assessed and compared in sheep. Threat reflex and rhythmic breathing correlated with EEG activity during propofol anaesthesia whilst absence of non-rhythmic breathing or threat reflex indicated unconsciousness. None of the behavioural indicators correlated with EEG activity during non-stunned slaughter. Absence of regular breathing and eyelid reflex was observed 00:27±00:12 min and 00:59±00:17 min (mean±SD) respectively after animals were considered unconscious, indicating that absence of regular breathing and eyelid reflex are distinctly conservative indicators of unconsciousness during non-stunned slaughter in sheep.

Indicators used in livestock to assess unconsciousness after stunning: a review.

Assessing unconsciousness is important to safeguard animal welfare shortly after stunning at the slaughter plant. Indicators that can be visually evaluated are most often used when assessing unconsciousness, as they can be easily applied in slaughter plants. These indicators include reflexes originating from the brain stem (e.g. eye reflexes) or from the spinal cord (e.g. pedal reflex) and behavioural indicators such as loss of posture, vocalisations and rhythmic breathing. When physically stunning an animal, for example, captive bolt, most important indicators looked at are posture, righting reflex, rhythmic breathing and the corneal or palpebral reflex that should all be absent if the animal is unconscious. Spinal reflexes are difficult as a measure of unconsciousness with this type of stunning, as they may occur more vigorous. For stunning methods that do not physically destroy the brain, for example, electrical and gas stunning, most important indicators looked at are posture, righting reflex, natural blinking response, rhythmic breathing, vocalisations and focused eye movement that should all be absent if the animal is unconscious. Brain stem reflexes such as the cornea reflex are difficult as measures of unconsciousness in electrically stunned animals, as they may reflect residual brain stem activity and not necessarily consciousness. Under commercial conditions, none of the indicators mentioned above should be used as a single indicator to determine unconsciousness after stunning. Multiple indicators should be used to determine unconsciousness and sufficient time should be left for the animal to die following exsanguination before starting invasive dressing procedures such as scalding or skinning. The recording and subsequent assessment of brain activity, as presented in an electroencephalogram (EEG), is considered the most objective way to assess unconsciousness compared with reflexes and behavioural indicators, but is only applied in experimental set-ups. Studies performed in an experimental set-up have often looked at either the EEG or reflexes and behavioural indicators and there is a scarcity of studies that correlate these different readout parameters. It is recommended to study these correlations in more detail to investigate the validity of reflexes and behavioural indicators and to accurately determine the point in time at which the animal loses consciousness.

Pain behaviour after castration of piglets; effect of pain relief with lidocaine and/or meloxicam.

Behavioural responses and the effect of lidocaine and meloxicam on behaviour of piglets after castration were studied. A total of 144 piglets of 2 to 5 days of age were allocated to one of six treatments: castration (CAST), castration with lidocaine (LIDO), castration with meloxicam (MELO), castration with lidocaine and meloxicam (L + M), handling (SHAM) and no handling (NONE). Behaviour was observed for 5 days after the procedure, growth until weaning was recorded and characteristics of the castration wound noted. MELO piglets showed significantly (P < 0.05) more no pain-related behaviour than CAST and LIDO at the afternoon after castration, and were not significantly different from SHAM and NONE. LIDO piglets showed an increase (P < 0.001) in tail wagging, lasting for 3 days. This increase was not seen in L + M piglets. The occurrence of several behaviours changed with age, independent of treatment. A treatment effect on growth was not found. Wound healing was rapid in all treatments, but thickening of the heal was observed in several piglets, suggesting perturbation in the cicatrization process. Our study showed a pain-relieving effect of meloxicam after castration. Local anaesthesia resulted in piglets performing more tail wagging during the first few days after castration, which was prevented by administering meloxicam in combination with local anaesthesia.

Effects of a local anaesthetic and NSAID in castration of piglets, on the acute pain responses, growth and mortality.

The present study addresses the questions whether on-farm use of local anaesthesia with lidocaine leads to a reduction in pain responses during castration, and whether the non-steroidal anti-inflammatory drug meloxicam improves technical performance after castration of piglets. Five treatments were included in the study: (1) castration without anaesthesia or analgesia (CAST), (2) castration after local anaesthesia with lidocaine (LIDO), (3) castration after administration of meloxicam (MELO), (4) castration after lidocaine and meloxicam (L + M) and (5) sham castration (SHAM). To reduce litter influences, each treatment was present in each of the 32 litters (n = 32 per treatment). During castration, vocalizations were recorded continuously. Blood samples were collected 15 min before and 20 min after castration for determination of plasma levels of total cortisol, glucose, lactate and creatine kinase (CK). Mortality was registered and piglets were weighed several times to calculate growth. Several aspects of vocalizations during castration showed consistent and significantly different levels in CAST compared with LIDO, L + M and SHAM. CAST piglets squealed longer, louder and higher. Vocalizations of MELO piglets most resembled those of CAST. An increase in cortisol was seen in all treatments. However, in SHAM piglets this increase was significantly lower than in the other treatments. LIDO piglets showed a significantly smaller increase in plasma cortisol levels compared with CAST and MELO. L + M piglets differed significantly only from the SHAM group. Lactate levels differed significantly between LIDO and MELO, the level in LIDO being decreased after castration. In the other treatments an increase was measured. No treatment effects were found in plasma glucose and CK levels, nor in growth and mortality of the piglets. In conclusion, on the basis of vocalizations and plasma cortisol, local anaesthesia with lidocaine reduces pain responses in piglets during castration. A positive effect of meloxicam on technical performance was not found.

Effects of fentanyl on isoflurane minimum alveolar concentration and cardiovascular function in mechanically ventilated goats.

The effects of fentanyl on the minimum alveolar concentration (MAC) of isoflurane and cardiovascular function in mechanically ventilated goats were evaluated using six healthy goats (three does and three wethers). Following induction of general anaesthesia with isoflurane delivered via a mask, endotracheal intubation was performed and anaesthesia was maintained with isoflurane. The baseline MAC of isoflurane (that is, the lowest alveolar concentration required to prevent gross purposeful movement) in response to clamping a claw with a vulsellum forceps was determined. Immediately after baseline isoflurane MAC determination, the goats received, on separate occasions, one of three fentanyl treatments, administered intravenously: a bolus of 0.005 mg/kg followed by constant rate infusion (CRI) of 0.005 mg/kg/hour (treatment LFENT), a bolus of 0.015 mg/kg followed by CRI of 0.015 mg/kg/hour (treatment MFENT) or a bolus of 0.03 mg/kg followed by CRI of 0.03 mg/kg/hour (treatment HFENT). Isoflurane MAC was redetermined during the fentanyl CRI treatments. Cardiopulmonary parameters were monitored. A four-week washout period was allowed between treatments. The observed baseline isoflurane MAC was 1.32 (1.29 to 1.36) per cent. Isoflurane MAC decreased to 0.98 (0.92 to 1.01) per cent, 0.75 (0.69 to 0.79) per cent and 0.58 (0.51 to 0.65) per cent following LFENT, MFENT and HFENT respectively. Cardiovascular function was not adversely affected. The quality of recovery from general anaesthesia was good, although exaggerated tail-wagging was observed in some goats following MFENT and HFENT.

Lumbosacral spinal cord somatosensory evoked potentials for quantification of nociception in horses.

REASONS FOR PERFORMING STUDY: There is a need for objective evaluation and quantification of the efficacy of analgesic drugs and analgesic techniques in horses. OBJECTIVES: To determine whether lumbosacral spinal cord somatosensory evoked potentials (SSEP) can be a useful and reliable tool to assess nociception in equines. METHODS: SSEPs and electromyograms (EMG) from the epaxial muscles were recorded simultaneously, following electrical stimulation applied to the distal hindlimb in lightly anaesthetised Shetland ponies (n=7). In order to validate the model, the effect of increasing stimulus intensity was documented and the conduction velocities (CV) of the stimulated nerves were calculated. The effect of epidurally applied methadone (0.4 mg/kg bwt) in a randomised, crossover design was investigated. RESULTS: Two distinct complexes (N1P1 and N2P2) were identified in the SSEP waveform. Based on their latency and conduction velocity and the depressant effect of epidurally applied methadone, the SSEP N2P2 was ascribed to nociceptive Adelta-afferent stimulation. The SSEP N1P1 originated from non-nociceptive Abeta-afferent stimulation and was not influenced by epidurally applied methadone. CONCLUSIONS AND POTENTIAL RELEVANCE: The nociceptive Adelta component of the SSEP, the N2P2 complex, is presented as a valid and quantitative parameter of spinal nociceptive processing in the horse. Validation of the equine SSEP model enables the analgesic effects of new analgesics/analgesic techniques to be quantified and analgesia protocols for caudal epidural analgesia in equidae improved.

Sedative and cardiopulmonary effects of acepromazine, midazolam, butorphanol, acepromazine-butorphanol and midazolam-butorphanol on propofol anaesthesia in goats.

UNLABELLED: The sedative, propofol-sparing and cardiopulmonary effects of acepromazine, midazolam, butorphanol and combinations of butorphanol with acepromazine or midazolam in goats were evaluated. Six healthy Boer - Indigenous African crossbreed goats were by randomised cross-over designated to 6 groups: Group SAL that received saline, Group ACE that received acepromazine, Group MID that received midazolam, Group BUT that received butorphanol, Group ACEBUT that received acepromazine and butorphanol and Group MIDBUT that received midazolam and butorphanol as premedication agents intramuscularly on different occasions at least 3 weeks apart. The degree of sedation was assessed 20 minutes after administration of the premedication agents. Thirty minutes after premedication, the dose of propofol required for induction of anaesthesia adequate to allow placement of an endotracheal tube was determined. Cardiovascular, respiratory and arterial blood-gas parameters were assessed up to 30 minutes after induction of general anaesthesia. Acepromazine and midazolam produced significant sedation when administered alone, but premedication regimens incorporating butorphanol produced inconsistent results. The dose of propofol required for induction of anaesthesia was significantly reduced in goats that received midazolam alone, or midazolam combined with either acepromazine or butorphanol. The quality of induction of anaesthesia was good in all groups, including the control group. Cardiovascular, respiratory and blood-gas parameters were within normal limits in all groups and not significantly different between or within all groups. IN CONCLUSION: sedation with midazolam alone, or midazolam combined with either acepromazine or butorphanol significantly reduces the induction dose of propofol with minimal cardiopulmonary effects in goats.

Differences in hepatic cytochrome P450 activity correlate with the strain-specific biotransformation of medetomidine in AX/JU and IIIVO/JU inbred rabbits.

Medetomidine is an alpha(2)-adrenoceptor agonist with sedative and analgesic properties. Previously we demonstrated significant differences in the response to medetomidine between two inbred rabbit strains, denoted IIIVO/JU and AX/JU. The aim of the present study was twofold: first, to compare the hepatic CYP450 enzyme activities between these rabbit strains [n = 13(male male,7 female female)/strain]. To this end, liver microsomes were incubated with known fluorescent substrates for the major drug-metabolizing CYP450 isoforms. A comparison of the obtained results indicated significant gender differences as well as differences between the two rabbit inbred strains. Secondly, the biotransformation rate of medetomidine in liver microsomes of both rabbit strains was determined using liquid chromatography coupled to tandem mass spectrometry. The rate of hydroxymedetomidine and medetomidine carboxylic acid formation was found to be significantly higher in the AX/JU strain. Specific CYP2D and CYP2E inhibitors could decrease the formation of both metabolites. Significant correlations were found between the rate of biotransformation of medetomidine and the activities of CYP2D and CYP2E, as well as between CYP450 enzyme activities and the anaesthetic response to medetomidine.

Application of a modified form of the Glasgow pain scale in a veterinary teaching centre in the Netherlands.

The Glasgow Composite Measure Pain Scale was developed to measure acute pain in dogs in a hospital setting. In this investigation a modified version of the scale was applied in a centre with a different surgical case load and analgesic protocols, and where English is not the first language, to test its validity in a different clinical environment. The modified scale was used to score pain in 60 dogs during the 24 hours after surgery. Their levels of sedation and a clinical impression of their pain were scored at the same time. Three questions were considered; first, how the modified pain score was related to the pain assessed subjectively, secondly, how it related to variables such as the surgical procedure and the dog's health and thirdly, how it changed over time. The mean modified pain scores for the dogs rated subjectively as having no, mild, moderate or severe pain were significantly different, indicating that the modified scale distinguished between pain of different severities. The changes in the dogs' scores also followed the expected changes in their level of pain with time, providing empirical evidence that the scale measures pain.

The effects of buprenorphine on behaviour in the ACI and BN rat inbred strains.

Buprenorphine is a partial mu, kappa agonist that has been shown to influence spontaneous behaviour in animals. Previously, we have demonstrated significant differences in the analgesic response to buprenorphine between the August Copenhagen Irish (ACI)/SegHsd and the Brown Norway (BN)/RijHsd inbred rat strains. The purpose of this study was to determine whether these strains also differed in their behavioural response to buprenorphine in order to provide an additional parameter for the genetic analysis and localization of genes involved in this response. Male and female rats of both strains were used (n = 6/strain/sex) for this study. Each rat was subjected, respectively, to three treatment regimens at 15:00 h: (A) unchallenged; (B) intravenous saline; (C) intravenous buprenorphine (0.05 mg/kg) according to a crossover design. The relative duration (s/h) of locomotion, grooming, drinking and eating behaviour was subsequently determined from 15:30 to 07:00 h using the automatic registration system, Laboratory Animal Behaviour Registration and Analysis System(trade mark). Significant strain differences were observed in unchallenged behaviour between the ACI and the BN rats. ACI rats, but not BN rats, responded to buprenorphine treatment with decreased levels of locomotion, drinking and eating behaviour. The same treatment resulted in an increased grooming behaviour in both strains. Slight but significant sex differences were observed for locomotion and eating in the analysis of variance procedure, but did not reach the level of statistical significance in the multiple comparison procedure. The results of this study emphasize the possibility that strain-specific effects must be taken into account when using behavioural parameters for the assessment of the analgesic effects of buprenorphine in rats.

Differences in response to anaesthetics and analgesics between inbred rat strains.

Differences in response to analgesic and anaesthetic drugs can partly be attributed to variations in the genetic background of experimental animals. This study was carried out to determine differences in the response of inbred rat strains to a selection of analgesics and drugs used in anaesthetic protocols. A cross between the most contrasting strains can then be phenotyped in future studies in order to localize quantitative trait loci (QTLs) involved in analgesic/anaesthetic drug sensitivity. Eight inbred strains (n = 6 rats/strain) were selected for the study: the pigmented ACI, BN and COP strains and the albino F344, LEW, SHR, WAG and WKY strains. Each rat was injected intravenously with two analgesics (buprenorphine 0.05 mg/kg and nalbuphine 1 mg/kg) and three drugs used in anaesthetic protocols (propofol 25 mg/kg, medetomidine 50 microg/kg and ketamine 10 mg/kg), respectively, using a crossover design. Analgesic responses were assessed using an analgesiometric procedure. The sleep time of the rat and, where applicable, the interval between injection and loss of righting reflex were used to determine the anaesthetic response. Six out of eight strains responded significantly different from each other to the analgesic effect of buprenorphine with the ACI strain as hyper-responder. The tail withdrawal latency at 55 degrees C of the F344 and WKY rats using buprenorphine was not significantly different from baseline tail withdrawal latencies. In this study, all strains were non-responsive to the analgesic effects of nalbuphine. The response to all three drugs used in anaesthetic protocols differed significantly among the strains. The F344 and BN strains were relatively resistant to the sedative effects of medetomidine. Use of ketamine was abandoned in the ACI and BN strains when the first two animals of both strains died soon after induction. With all three drugs the sleep time of albino rats was significantly longer compared with that of the pigmented ones. We conclude that the results from this study can be used in future studies where QTLs for the sensitivity to anaesthetic/analgesic drugs are localized.

Pharmacodynamic effects and pharmacokinetic profile of a long-term continuous rate infusion of racemic ketamine in healthy conscious horses.

Ketamine (KET) possesses analgesic and anti-inflammatory activity at sub-anesthetic doses, suggesting a benefit of long-term KET treatment in horses suffering from pain, inflammatory tissue injury and/or endotoxemia. However, data describing the pharmacodynamic effects and safety of constant rate infusion (CRI) of KET and its pharmacokinetic profile in nonpremedicated horses are missing. Therefore, we administered to six healthy horses a CRI of 1.5 mg/kg/h KET over 320 min following initial drug loading. Cardiopulmonary parameters, arterial blood gases, glucose, lactate, cortisol, insulin, nonesterified fatty acids, and muscle enzyme levels were measured, as were plasma concentrations of KET and its metabolites using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Levels of sedation and muscle tension were scored. Respiration and heart rate significantly increased during the early infusion phase. Glucose and cortisol significantly varied both during and after infusion. During CRI all horses scored 0 on sedation. All but one horse scored 0 on muscle tension, with one mare scoring 1. All other parameters remained within or close to physiological limits without significant changes from pre-CRI values. The mean plasma concentration of KET during the 1.5 mg/kg/h KET CRI was 235 ng/mL. The decline of its plasma concentration-time curve of both KET and norketamine (NKET) following the CRI was described by a two-compartmental model. The metabolic cascade of KET was NKET, hydroxynorketamine (HNK), and 5,6-dehydronorketamine (DHNK). The KET median elimination half-lives (t1/2alpha and t1/2beta) were 2.3 and 67.4 min, respectively. The area under the KET plasma concentration-time curve (AUC), elimination was 76.0 microg.min/mL. Volumes of C1 and C2 were 0.24 and 0.79 L/kg, respectively. It was concluded that a KET CRI of 1.5 mg/kg/h can safely be administered to healthy conscious horses for at least 6 h, although a slight modification of the initial infusion rate regimen may be indicated. Furthermore, in the horse KET undergoes very rapid biotransformation to NKET and HNK and DHNK were the major terminal metabolites.

Clinical investigation of remifentanil and propofol for the total intravenous anaesthesia of dogs.

Fifteen adult dogs underwent elective ovariectomy. They were premedicated with 0.5 mg/kg methadone and 0.05 mg/kg(-1) atropine administered intramuscularly, and anaesthesia was induced with propofol and maintained with intravenous infusions of remifentanil at 0.6 microg/kg/minute and propofol; the mean (sd) rate of infusion of propofol throughout the period of anaesthesia was 0.33 (0.03) mg/kg/minute. The dogs were ventilated continuously with oxygen while they were anaesthetised. Their haemodynamic parameters were clinically acceptable during the period of anaesthesia. Two dogs received additional atropine to correct bradycardias of less than 60 bpm and several dogs received additional boluses of remifentanil or propofol to maintain an adequate depth of anaesthesia, as determined by a clinical assessment. The mean (range) time to the return of spontaneous respiration after stopping the remifentanil infusion was 11.1 (6.0 to 17.0) minutes, and the mean (range) time to the dogs standing was 38.0 (20.0 to 80.0) minutes. The quality of recovery was good in 12 of the dogs, two showed mild excitation in the immediate postoperative period and the other dog required additional analgesia with methadone.

Development of a rat model to assess the efficacy of the somatosensory-evoked potential as indicator of analgesia.

Drug-induced changes in somatosensory-evoked potentials (SEPs) are considered to reflect an altered nociceptive state. Therefore, the SEP is proposed to be a parameter of analgesic efficacy. However, at present, SEPs have not been studied in relation to animal pain. The present study aims to develop a rat model in which this relationship can be studied based on Pavlovian fear conditioning. Therefore, rats, implanted with epidural electro-encephalogram recording electrodes, were randomly assigned to either a paired or random-control group and subjected to an aversive-to-appetitive transfer paradigm. During the aversive phase, the SEP-stimulation paradigm (5 mA square wave pulses, n = 72, of 2 ms duration each, with a stimulus frequency of 0.5 Hz; total duration 144 s) was used as the unconditioned stimulus (US), while a tone (40 s, 1500 Hz, 85 dB sound pressure level) was used as the conditioned stimulus (CS). During the appetitive phase, the CS was presented paired to the presentation of a sugar pellet. When compared to the random-control group, the paired group showed significantly more freezing behavior and significantly less reward-directed behavior in response to the CS in the appetitive phase. In addition, SEPs were not significantly affected by fear conditioning. Based on these results, we conclude that the SEP-stimulation paradigm can be successfully employed as a US in fear conditioning. In future studies, fear conditioning can be carried out under different levels of an analgesic regimen to allow the changes in SEP parameters to be compared to changes in fear-induced behavior making this model potentially useful to validate SEP parameters as indicators of analgesia.