Influences of type of anaesthesia on cortisol, β-endorphin and heart rate in pigs H Gerard, Pl Sensky, Dm Broom, S Perremans, R Geers

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H Gerard, Pl Sensky, Dm Broom, S Perremans, R Geers. Influences of type of anaesthesia on cortisol, β-endorphin and heart rate in pigs. Veterinary Research, BioMed Central, 1996, 27 (3), pp.219-226. ￿hal-00902416￿

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Influences of type of anaesthesia on cortisol, β-endorphin and heart rate in pigs

H Gerard1 PL Sensky DM Broom S Perremans R Geers1

1 Laboratorium Agrarische Bouwkunde, Katholieke Universiteit Leuven, Kard Mercierlaan 92, B-3001 Heverlee, Belgium; 2 Department of Clinical and Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, UK

(Received 5 April 1995; accepted 16 January 1996)

Summary ― Two experiments were carried out using a total of 20 growing pigs of approximately 20 kg in order to investigate the effect of different types of anaesthesia (metomidate versus ketamine) on plasma levels of cortisol and (3-endorphin on the heart rate. The animals were housed individually within a zone of thermal neutrality. Feed and water were available ad libitum. After becoming accus- tomed to human contact (being approached and touched), a catheter was implanted in the jugular vein, exteriorized in the neck region. This operation was performed under halothane anaesthesia. After recovery, hourly blood samples were taken over a 27 hour period, and cortisol and beta-endor- phin concentrations were measured in the sample plasma. After this reference period, half of the ani- mals were anaesthetized with metomidate, the other half with ketamine, and blood samples were taken at regular intervals. During a second experiment the heart rate was monitored after being anaes- thetized as described before. An influence of the type of anaesthesia was observed on the plasma con- centration of cortisol, but no effect was observed with respect to the (3-endorphin concentration. A fivefold increase of cortisol concentration was observed after injection with ketamine, while a twofold increase was observed after injection with metomidate. This finding suggests a suppressive effect of metomidate on the plasma cortisol levels. A fivefold increase was also observed for the (3-endorphin concentration after injection with either ketamine or metomidate. The increase in the heart rate was related to the awakening activity, which was more difficult after the ketamine injection. pig / anaesthesia / cortisol / [3-endorphin / heart rate

* Correspondence and reprints Résumé ― Influence du type d’anesthésie sur le taux de cortisol, de fi-endorphines et sur le rythme cardiaque chez le porc. Au cours de deux expérimentations l’effet de l’anesthésie (métomi- date ou kétamine) a été recherché sur les taux plasmatiques de cortisol et de {3-endorphine, ainsi que sur le rythme cardiaque. Vingt porcelets d’environ 20 kg de poids vif ont été placés dans des loges individuelles. Les conditions thermiques étaient optimales ; la nourriture et l’eau étaient dis- tribuées ad libitum. Quand les porcelets ont été habitués au contact humain, un cathéter a été implanté dans la veine jugulaire sous anesthésie à l’halothane. Après la guérison, chaque heure, un échantil- lon de sang a été prélevé pendant une période de 27 heures pour déterminer les valeurs de référence. Puis la moitié des porcelets a été anesthésiée par une injection de métomidate ou de kétamine, suivie par la collecte des échantillons de sang. Dans le plasma les taux de cortisol et de j3-endorphine ont été déterminés. Au cours d’une autre épreuve, le rythme cardiaque a été mesuré avant, pendant et après l’anesthésie. Le type d’anesthésie a eu une influence sur le taux de corüsol et le rythme cardiaque. Le taux de cortisol a augmenté cinq fois après une injection de kétamine, de même que le taux de {3-endor- phine après une injection de kétamine ou métomidate. Le taux de corüsol a augmenté seulement deux fois après une injection de métomidate. Ce résultat suggère un effet suppressif du métamidate sur le taux de cortisol plasmatique. L’augmentation du rythme cardiaque était liée à l’activité d’éveil, plus laborieuse en cas d’injection de kétamine. porc / anesthésie / cortisol / {3-endorphine / rythme cardiaque

INTRODUCTION sol levels normally starts about 10 min after injection. At the level of the adrenal cortex, The release of ACTH and [3-endorphin from however, etomidate blocks the stimulating the anterior pituitary gland during stress effect of ACTH, thus inhibiting the increase reactions is controlled by the corticotrophin in cortisol levels (Preziosi and Vacca, 1988). hormone et releasing (CRH) (Bagdy al, Several drugs are used for pig anaes- Vale et 1990a; al, 1981 Adrenocorticotropic thesia. Azaperone, a butyrophenone deriva- hormone (ACTH) induces the production tive, acts as a neuroleptic drug. It is partic- and release of glucocorticoids such as cor- ularly useful in pigs as a premedicant for tisol from the adrenal cortex. Plasma (3- anaesthesia or for reducing distress (Green, endorphin also originates from the interme- 1979). Metomidate and etomidate are both diary lobe of the pituitary gland (Bagdy et hypnotic agents. They have marked mus- al, 1990b; Murburg et al, 1993). The type cle relaxant properties in pigs, but produce of stress may determine however, the level no analgesia. In combination with azaperone of production of p-endorphin from the ante- however, they produce a state that resem- rior or intermediary lobe of the pituitary gland bles general anaesthesia (Callear and Van (Kjaer et al, 1995). Diurnal rhythmicity has Gestel, 1973; Green, 1979). Etomidate is been reported for plasma levels of p-endor- not often used in pigs, mainly because it phin in rats (Bagdy et al, 1991) and humans has to be injected intravenously (Holzchuh (Iranmesh et al, 1989; Veldhuis et al, 1990), and Cremonesi, 1991 ). Ketamine hydrochlo- and for plasma cortisol levels in pigs (Becker ride produces loss of consciousness and et al, 1985; Bottoms et al, 1972; Dalin et al, peripheric analgesia, but no muscle relax- 1993; Favre and Moatti, 1977). In humans, ation. Therefore, azaperone is used in com- the stimulation of the hypothalamus-pitu- bination with ketamine to complete analge- itary-adrenal axis by ketamine and other sia and to reduce muscle tone (Green, anaesthetics is well known (Monk et al, 1979). It is therefore necessary to consider 1992; Oyama et al, 1970; Preziosi and one’s research objectives in order to select Vacca, 1988). An increase in plasma corti- an appropriate type of anaesthesia. The aim of our research was to find out the anaesthetic was injected intravenously at 9 am and 3 Blood taken whether metomidate has the same sup- pm. samples were by the i/v catheter at 60 min and 1 min prior to the pressing effect as etomidate on plasma administration of anaesthetic and 5, 30, 60 and cortisol levels and Ketamine rhythmicity. 120 or 180 min after its administration. Samples was used as a reference method because were handled as previously described. The same it increases cortisol plasma level. Plasma procedure was repeated two days later with an (3-endorphin, the release of which is con- intramuscular injection of azaperone and ketamine comitant with ACTH (Vale et al, 1981 and and an intraperitoneal injection of metomidate which is less affected by storage condi- (table I). tions after blood sampling than ACTH (ICN Plasma hormone levels were determined with available kits. Because the Biochemicals, 1984), was evaluated as a commercially assays needed to be carried out two months after sam- second parameter. Heart rate, which is ple collection, p-endorphin was analysed instead affected anaesthetic was also by agents, of ACTH. [3-Endorphin is released concomitantly measured. with ACTH, but is less affected by duration of storage of the sample at -20 °C when no preser- vatives are added (ICN-Biochemicals, 1984). MATERIALS AND METHODS Plasma cortisol was evaluated by enzyme immunoassay (Biogenesis Ltd, Bournemouth, UK). This heterogeneous enzyme immunoassay method is based on the principle of competitive 1 Experiment binding. Cortisol is released from serum binding proteins in a two-step process. First, the dilution of the serum in the weakens its Eight Large White x Landrace piglets (four male, protein assay the four female) weighing between 18 and 20 kg were binding capacity. Secondly, higher affinity more for the cor- housed individually on straw. Food and water antibody competes effectively tisol than the weaker serum were provided ad libitum. Lights were on contin- binding proteins. uously in order to allow blood sampling. After The total amount of cortisol present is free to becoming accustomed to human approach and compete with an enzyme-labelled cortisol for a limited number of available sites on an contact, a catheter was inserted in the jugular binding for cortisol. The bound and free vein and exteriorized in the neck region. The oper- antibody specific ation was performed under halothane anaesthe- enzyme-labelled cortisol fractions are separated. The is the sia. Patency of the catheters was maintained by bound enzyme quantified by enzy- daily flushing with 50 units/mL heparinized saline. matic conversion of a specific substrate to a When the pigs had recovered, three days after coloured product. The colour intensity is inversely the operation (Dalin et al, 1993), hourly blood related to the amount of cortisol in the sample. sampling started at noon and continued for 27 h. Intra- and inter-assay variability were respec- The first three samples allowed the animals to tively 4.9 ± 1.2% and 6.4 ± 1.7%. The mean become used to frequent sampling and were not recovery value was 99 ± 2%, and the cross-reac- included in the results. About 10 mL of blood was tivity was 100% for cortisol, 14.8% for 11-deoxy- collected in cooled EDTA tubes and immediately cortisol, 11.5% for corticosterone and lower than centrifuged. Plasma was stored at -20 °C until 2% for other related compounds. Total plasma (3- further analysis. Two days later, the animals were endorphin content was evaluated by Radio randomly divided into two groups (four pigs each). immuno assay (RIA) (Peninsula Laboratories, One group was injected intravenously with aza- St Helens, UK). This kit is designed to measure perone (Stresnil, 40 mg/mL, Janssen Pharma- specifically (3-endorphin (rat) levels, and its ceutica, Beerse, Belgium) in combination with related peptides by a competitive RIA. Intra- and ketamine (Vetalar, 100 mg/mL, Parke Davis, inter-assay variability were respectively 7.6 ± Gwent, UK), and the other group with azaperon in 3.3% and 9.4 ± 4.1 %. The mean recovery value combination with metomidate (Hypnodyl, was 88 ± 4%. Cross-reactivity for porcine p- 50 mg/mL, Janssen Pharmaceutica, Beerse, Bel- endorphin was 100%. Figure 1 shows the simi- gium). The experimental protocol and doses are larity of the dilution curves for three samples of shown in table I. On the first experimental day pig plasma. Experiment 2 able. Because the pigs themselves were the experimental units and randomly allocated to the fixed main effects, statistical analysis was based Twelve female 20 of an pigs (= kg) homozygous on a randomized lay-out. Treatment effects on halothane negative line were used (Seghers mean values were contrasted by using analysis of Hybrid, Buggenhout, Belgium). Six were anaes- variance within a generalized linear model, includ- thetized with an intramuscular injection of azap- ing the time factor as repeated measures were erone (2 mg/kg) and an intraperitoneal injection of made in the same animal (SAS Guide, 1988). metomidate (10 mg/kg). The others were injected Statistical differences between the times of day with azaperone and ketamine (10 mg/kg IM) were expressed as two-tailed probability values (Imalgene, Rh6ne M6rieux, France). After anaes- within a Students t-test procedure. thesia, an ambulatory heart rate monitoring device, using five electrodes positioned on the chest, was fixed on the back of the pig (ViII6 et al, RESULTS 1993). Data acquisition covered the whole mea- suring period. The pigs were housed individually within their zone of thermal neutrality and were fed ad libitum. Influence of type of anaesthesia on plasma cortisol and /3-endorphin (Experiment 1) Statistics The two days prior to the anaesthetics injec- The individual pigs were chosen at random from tion were considered as reference days. different litters within the population of pigs avail- Plasma cortisol levels were high (up to 80 ng/mL) from 00.00-12.00 h, and low min after injection is shown in figure 4. About (down to 30 ng/mL) between 13.00-24.00 h 2 h after the injections, the concentrations (P < 0.001) (fig 2). The measurements of returned to basal levels. This retum took a lit- R-endorphin were too close to the detection tle bit longer after metomidate anaesthesia. limits of the radioimmunoassay (< 100 The method of drug injection (IV or IM/IP) pg/mL), and could not be used for statistical had no significant effect. analysis. A fivefold increase in cortisol lev- els was observed within 30 min after the Influence of of anaesthesia ketamine injection in the morning, and a type on heart rate threefold rise in the afternoon (P < 0.005) (Experiment 2) (fig 3). About 2 h after the injection, the plasma values returned to normal levels. Figure 5 presents the hourly mean heart Injection with metomidate produced only a rate (beats/min) as a function of time after twofold increase in basal levels (P< 0.05), injection with metomidate or ketamine. Heart but the return to basal level took about 6 h rate is strongly related to body activity dur- (fig 3). A statistically significant (P < 0.005) ing awakening. As early as 30 min after the fivefold increase in plasma p-endorphin 60 ketamine injection the pigs demonstrated high muscular activity and screamed. This afternoon and evening, was the same as was not the case after the metomidate injec- that reported by Becker et al (1985) and tion, where awakening began about 2 h after Dalin et al (1993). This variation in cortisol injection. The awakening process was less levels must be taken into account when cor- difficult for metomidate, as indicated by the tisol is used as an indicator of welfare or of lower heart rate (P < 0.05). response to treatment (Broom and John- son, 1993). Since a normal circadian rhythm was observed, there is no evidence that the DISCUSSION blood sampling as such affected plasma hormone levels, nor were they affected by The diurnal rhythm of cortisol levels the injection technique (with or without a observed in this study, with higher levels catheter). After injection with ketamine, a during the night and morning than in the fivefold increase was observed in the plasma cortisol levels. This result is in agree- ing of the heart rate after metomidate anaes- ment with Oyama et al (1970). Preziosi and thesia (fig 5). Heart rate variability, however, Vacca (1988) reported that the injection of can also be explained by differences in the etomidate in rats and humans induced awakening process. In human studies, hal- ACTH release which was not accompanied lucinations and psychomotoric activities by an increase in cortisol levels. This was have been described after ketamine anaes- explained by the strong adrenal suppres- thesia (Green, 1979), which may also be sion capability of etomidate, with 11-[3- related to the increase in heart rate. A low- hydroxylase as the most sensitive target ering of the heart rate may be beneficial enzyme. Our results suggested a similar when working with pigs that are suscepti- but less severe effect of metomidate on the ble to malignant hyperthermia. pig adrenal cortex. We could not measure ACTH, however, and thus we have no direct evidence for (a) lower stimulation of pitu- ACKNOWLEDGMENTS itary adrenocortical activity, or (b) an impaired response of the adrenal cortex to was EC-DG VI ACTH. The increases in cortisol and Funding provided by (AIR pro- (3- gramme). B Parrott from the AFRC Babraham were after the endorphin comparable Institute (UK) gave advice on the blood sampling ketamine injection . This provided some evi- method. The Laboratory of Animal Physiology dence for a pituitary-adrenal dissociation (KU Leuven) supported the assay work. Further after metomidate, since a-endorphin release technical support was provided by J Van Bael was at least as great as with ketamine, but and W Duchateau. R Geers is a Research Direc- tor of the NFWO the cortisol response was much lower. (Belgium). Further research should show that there is no difference between the effects of eto- REFERENCES midate and ketamine on ACTH release, since p-endorphin release was stimulated by both metomidate and by ketamine. The Bagdy G, Calogero AE, Szemeredi K, Chrousos GP, stimulation of (3-endorphin production (May Gold PW (1990a) Effects of cortisol treatment on brain and adrenal corticotropin-releasing hormone et and the of cortisol al, 1991) suppression (CRH) content and other parameters regulated by (Korte et al, 1992) might explain the lower- CRH. 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