Annals of RSCB Vol. XVI, Issue 1

Transcription

1 OBSERVATIONS REGARDING ANESTHESIA AND SOME OF THE HEMATOLOGICAL PARAMETERS IN CARASSIUS GIBELIO, CYPRINUS CARPIO AND ONCORHYNCUS MIKISS C. Peştean, L. Oana, C. Ober, V. Miclăuş, L. Ognean, Cristina Cernea, Florina Iancu UASMV CLUJ-NAPOCA, FACULTY OF VETERINARY MEDICINE Summary Fish display robust neuroendocrine and physiologic stress responses to noxious stimuli. Many anesthetic, sedative, or analgesic drugs used in other vertebrates reduce stress in fish, decrease handling trauma, minimize movement and physiologic changes in response to nociceptive stimuli, and can be used for euthanasia. But extrapolating from limited published anesthetic and sedative data to all fish species is potentially harmful because of marked anatomic, physiologic, and behavioral variations; instead, a stepwise approach to anesthetizing or sedating unfamiliar species or using unproven drugs for familiar species is advisable. The anesthesia with etomidat and clove oil was efficient in all three species; the other anesthetics had variable effects. Fish blood sampling is possible using different techniques, regarding fish size and purpose of the study. The results of hematological profile for the three freshwater species were in range of reference values, with the presence of an anemic syndrome in Cyprinus carpio. Key words: fish, anesthesia, blood sampling, hematology cj10ppc@yahoo.com Introduction Whether or not fish feel pain is an ongoing debate. Although both sides of the argument set the same requirements for pain perception, opinions differ on where the criteria appear phylogenetically and whether or not parallel systems have evolved in different classes and species (Huntingford et al., 2006). Fish display robust neuroendocrine and physiologic stress responses to noxious stimuli (Nicula et al., 2010). They are easily stressed by handling and transport; stress can cause immuno-suppression, physical injury, or even death. In aquaculture, anesthetics are used during transportation to prevent physical injury and reduce metabolism (DO consumption and excretion). They are also used to immobilize fish so they can be handled more easily during harvesting, sampling and spawning procedures (Coyle. et al., 2004). An ideal anesthetic should induce anesthesia rapidly with minimum 234 hyperactivity or stress. It should be easy to administer and should maintain the animal in the chosen state of anesthesia. When the animal is removed from the anesthetic, recovery should be rapid. The anesthetic should be effective at low doses and the toxic dose should greatly exceed the effective dose so that there is a wide margin of safety (Ross, and Ross., 1999). Many factors affect the efficacy of anesthetics in fish. These can be divided into biological and environmental factors. Often, the rate at which anesthetic drugs become effective is related to the gill area to body weight ratio, which can vary considerably among fish species. Aquatic species also have different metabolic rates that affect the rate at which chemicals are absorbed and anesthesia is induced. For example, cold-water species seem to respond to lower concentrations of anesthetic than warm-water species. There are also factors that can affect anesthesia

2 within a particular species. Larger individuals generally require a greater concentration of anesthetic than smaller individuals. In contrast, it has also been reported that the larger, more active fish in a group are anesthetized faster than smaller ones (Harms, Bakal., 1995). The most common method of drug administration is through immersion, a technique analogous to gaseous inhalant anesthesia in terrestrial animals, but the use of injectable anesthetic and sedative agents (primarily intramuscularly, and also intravenously) is increasing. Lowering water temperature will tranquilize or immobilize fish. Lower water temperatures also increase the oxygencarrying capacity of water and reduce the activity and oxygen consumption of fish. Water can be cooled by refrigeration or by adding ice. Gradual cooling is recommended because rapid chilling can produce thermal shock. This technique has been used primarily for transportation. Fish are usually anesthetized by immersing them in an anesthetic bath containing a suitable concentration of drug so that the drug is absorbed through the gills and rapidly enters the blood stream. The simplest procedure is to prepare the required drug concentration in an aerated container and quickly, but gently transfer the fish to the container. The anesthetic bath and recovery tank should use water (at a similar temperature and chemistry) with identical proprieties. Water quality needs to be carefully controlled, especially where large numbers of animals are being handled and baths are being reused. Main concerns involve maintaining proper temperature, adequate dissolved oxygen, low ammonia and a minimum amount of fecal matter (Chandroo et al., 2004). In addition to immersion methods, anesthetics can be delivered orally, intravenously (IV), intracoelomically (ICo), and intramuscularly (IM). With the exception of metomidate, there have been few studies on oral anesthetics (Hansen et al., 2003) and this route likely has limited use because precise dosing is difficult and the rate and degree of absorption are uncertain (Harms 2003). IV injections result in rapid induction and usually a short duration of effect, but have the disadvantage of requiring either manual restraint or prior administration of drugs by another route to allow IV access (Fleming et al. 2003; Hansen et al., 2003). ICo delivery, although effective, has two disadvantages: an increased risk of visceral damage (although this can be minimized by inserting the needle at an acute angle directed anteriorly) and inconsistent induction time, because the drugs must pass through the serosal surface. The most common parenteral route is IM injection by hand syringe, pole syringe, or a darting system (Harms., 1999). When possible, the recommended site for IM administration is the dorsal saddle, an area that surrounds the dorsal fin and extends laterally to just above the lateral line, from the operculum or posterior gill slit caudal to a point approximately onethird the distance between the dorsal fin and the caudal peduncle. IM injections often result in leakage as the needle is withdrawn and the surrounding muscles contract (Peters et al., 2001); drug loss is preventable by holding the fish in hand, placing the needle directly on the dorsal midline between the epaxial muscles, and then laterally inserting it into a muscle. It is important to note that skeletomuscular movement helps blood and lymph circulate, so anesthetic induction time may be delayed in sedentary species. Also, the injection volumes are often large and may contribute to sterile abscess formation in the musculature (Tyler and Hawkins, 1981). Regardless of route, parenteral anesthetics often do not provide adequate sedation or anesthesia and so require supplemental immersion anesthesia. In addition, ventilator support is necessary with parenteral anesthetics, particularly if recovery is prolonged (Harms 1999). There are few published studies evaluating chlorofluorocarbon-based 235

3 anesthetics in fish (Stetter 2001). Their use requires special care as anesthetic depth is difficult to control and the drug s insolubility in water results in localized higher concentrations that may cause overdosage. In addition, volatilization and difficulty in scavenging waste gas are hazardous to personnel. Given the disadvantages associated with the use of isoflurane and similar drugs, a number of researchers and clinicians recommend them only as a last resort for anesthesia (Harms., 1999). Scientific study often requires that fish be sampled for reasons such as hematology, clinical chemistry parameters, bacteriology, parasitological investigations, packed cell volume, etc. The history of applying hematological methods as diagnostic aids in episodes of noninfectious and infectious diseases in confined and free-living populations of fish is quite meager (Blaxhall 1972). The major reason for the lack of utility, as compared with mammalian medicine, is the variability of data. Fish are subjected to many environmental influences which alter the healthy hemogram; i.e., the baseline data for cellular and plasma components. This is not the case in mammals. Thus, when hematological methods are used as aids in fish disease diagnostics, many variables must be taken into account (Miller et al., 1983). Sampling blood from the dorsal aorta can be performed through several places of choice, of which the easiest is through lateral approach just below the spine, especially in large fish. From caudal vein, the sample is taken midline just posterior of the anal fin. The needle is inserted into the musculature perpendicular to the ventral surface of the fish until the spine is reached or blood enters the syringe. If contact with the spine is made, withdraw the needle slightly. The vein is located ventral to the overlying spine. This blood vessel can also be sampled laterally. For salmonids this is the preferred method for blood sampling. 236 Cardiac punctures from the ventral side are sometimes used in fusiform fish or through the operculum in laterally compressed species. Blood is collected from the heart ventricle. The needle is inserted perpendicular to the ventral surface of the fish in the centre of an imaginary line between the anterior most parts of the base of the pectoral fins (Weiss.; Wardrop, 2010). The aim of this study was to evaluate several anesthetics and to assess the hematological profile for three freshwater fish species. Material and methods Thirty Carassius gibelio (~200g), twelve Cyprinus carpio (~2200g) and thirty Oncorhyncus mykiss (~200g) were used; each species was equally divided in six groups. For transportation, induction, maintenance and recovery, we used adequate water containers and oxygen supplementation. Every group (five Carassius gibelio or 2 Cyprinus carpio or five Oncorhyncus mykiss) was exposed separately to a different anesthetic protocol. The fishes from group 1 were administered ketamine 30 mg/kg i.m., group 2 was exposed to halothan (1 ml/3 liters water), group three was exposed to isofluran (2 ml/3 liters water), group four received lidocaine 1mg/kg i.m., group five was anesthetized with etomidat 1 mg/l water, and group six was anesthetized with clove oil 80 mg/l water (fig. 1). To assess anesthetic depth in fish, we have observed reactivity to stimuli, equilibrium (righting reflex), jaw tone and muscle tone. Induction is marked by decreases in caudal fin strokes, swimming, respiratory rate, and reaction to stimuli; the drop in caudal fin stroke activity is usually the first sign, followed by loss of equilibrium and response to stimuli. At surgical anesthesia there is total loss of muscle tone and a further decrease in respiratory rate. A firm squeeze at the base of the tail can be an effective way to

4 determine response to stimuli: if the animal does not respond, general anesthesia has taken effect. For recovery, the fish were placed in anesthetic-free water. Although recovery occurs by different mechanisms, transfer to a new water container is advisable after any route of anesthesia as some of these drugs and their metabolites may be excreted into the water and reabsorbed through the gills and/or skin. Blood sampling was performed only to anesthetized fish, by caudal venous puncture, dorsal aorta puncture or heart puncture. From blood sample, we determined: microhematocrit, hemoglobin (Gowers Sahli method), erythrocyte number, leucocytes number and erytrocytes derivative. Figure 1 (a) Carassius gibelio anesthetized with halothane ; (b) Administration of ketamine to Cyprinus carpio; (c) Oncorhyncus mykiss anesthesia with etomidat Results and discussion Carassius gibelio anesthesia For the group anesthetized with ketamine, we obtained the following results: after 1 minute from ketamine administration, the fin and gill movements were decreased, after 2 minutes we observed the loss of equilibrium and after 3 minutes fish were without response to stimuli. After 12 minutes of profound anesthesia, some gill movements were observed. The complete recovery from anesthesia was recorded at 15 minutes from ketamine administration (fig. 2 a). In group 2 we noticed the loss of equilibrium after 3 minutes from exposure to halothan. Between 7 and 9 minutes the profound level of anesthesia was achieved, with lost of reactivity and depress of gill and fin movements. At 10 minutes after the transfer to a new water container, first signs of recovery (fin movements) were recorded. The complete recovery was noticed after another 5 minutes. The induction of anesthesia with isoflurane, was performed in two steps. First, we administered 1 ml of isoflurane in 237 an aquarium with 3 liters of water. Because after 5 minutes we did not see any change, we administered another 1 ml isoflurane. In 5 minutes the characteristic changes were visible, in 7 minutes a deep level of anesthesia was recorded. The recovery began at 5 minutes and ended at 7 minutes from the transfer to another water container. The anesthesia with lidocaine was of poor quality because after 13 minutes we observed only minor modifications, the reduction of movement frequency. The maximum depth of anesthetic level was recorded after another 2 minutes with only light changes in righting reflex. This level was maintained for 2 minutes, after that some signs of recovery were observed and in another 3 minutes the recovery was complete. The induction of anesthesia with etomidat was very fast, in 45 seconds fishes lost their equilibrium, muscle tone and response to stimuli. The recovery begun and was completed in 6 and 8 minutes respectively, from the transfer to another container.

5 The effects of clove oil, changes of righting reflex and decrease of fin stroke, were noticed after 2 minutes of anesthetic administration. In another 3 minutes fishes lost their equilibrium and response to stimuli. The recovery started after 15 minutes and was completed in only 1 minute from the transfer to a different fresh water container. Cyprinus carpio anesthesia The first signs of anesthesia were observed after 15 minutes from ketamine administration, the depression of righting reflex and gills movement. The anesthetic level was light; the recovery started after 4 minutes and was completed in another 3 minutes. In halothan group, first signs of anesthesia become visible in 12 minutes with light changes in equilibrium and preservation of reactivity and muscle tone. The recovery was appreciated as complete in 7 minutes after the transfer in fresh water container. Induction of anesthesia with isoflurane started in 12 minutes after anesthetic administration, with the decrease of gills and fin strikes. The maximum depth of anesthesia was achieved in another 2 minutes, accompanied by light depression of response to stimuli. The recovery was noticed after 5 minutes from the transfer to another container. The effects of lidocaine were observed at 10 minutes after administration, reduction of movement and some alteration of equilibrium. During the following 5 minutes, the anesthesia was slightly increased, with the decrease of gills activity. All the fish maintained muscle tone and responsiveness to stimuli. The recovery occurred after 12 minutes. In group exposed to etomidat, the lost of reactivity and fin movements decrease begun after 1 minute from administration. The level of anesthesia was profound, without muscle tone and response to stimuli. In 5 minutes after the transfer to fresh water container, we noticed first signs of recovery, completed after another 3 minutes. The anesthesia with clove oil was induced in 3 minutes, starting with minor modification of equilibrium. In only 30 seconds, anesthesia was profound, without any muscle tone or reaction to stimuli. The recovery started in 3 minutes and was finished in 5 minutes after the removal of fish to a different container (fig. 2 b). Oncorhyncus mykiss anesthesia The anesthetic effect of ketamine appears to be light, because after 7 minutes from administration, we observed only a slight decrease of fin movement. We decided to supplement the dosage of ketamine with 60 mg for each fish. In 1 minute the results were visible, with the decrease of gills activity and alteration of equilibrium. All the fish presented agitation, excitability and chaotic swimming as side effects. These effects were attenuated in 3 minutes and the full recovery was recorded in 20 minutes. Fish anesthetized with halothane showed increased excitability with fin extension in 7 minutes after anesthetic administration. After another 2 minutes, this excitement phase continued with rapid and repeated flaring of the opercula and fin extension. The exposure of fish to halothan was terminated after 10 minutes, because no improvement of anesthesia was observed. The side effects stopped immediately after the fish transfer to fresh water container, the recovery was completed in 5 minutes. Subsequent to 20 minutes of careful monitoring of anesthesia induced with isoflurane, we interrupted the exposure of fish to this anesthetic because it was without any effect. Induction of anesthesia with lidocaine started at 20 seconds after anesthetic administration, with dorsoventral swimming and decrease of reaction to stimuli. The recovery was also fast, being complete in 3 minutes. The anesthesia with etomidat was achieved in 30 seconds. The level of anesthesia was constant and profound with the depression of fin and gills movement, 238

6 loss of equilibrium, but without reactivity. The recovery was finished in 10 minutes after the transfer to another container. The effect of clove oil was noticed after 1 minute from fish immersion in water with anesthetic. The anesthesia was profound with seldom fin and gills activity, and without muscle tone. The capacity to swim was restored in 8 minutes from the transfer to a fresh water container (fig. 2 c). Figure 2 (a) Carassius gibelio anesthetized with ketamine; (b) Cyprinus carpio anesthetized with clove oil; (c) Oncorhyncus mykiss anesthetized with etomidat Blood sampling Blood was sampled in all three fish species, following three separate procedures: from dorsal aorta, cardiac puncture and caudal vein (fig. 3). Because the blood sampling was performed on fish with more than 8 cm in length and under general anesthesia, we manage to induce minimal trauma, all the patients survived. Using these three techniques, we were able to collect sufficient volume of blood for hematological assessment. The mean value of blood volume sampled was 1,2 ml for Carassius gibelio, 3,1 ml for Cyprinus carpio and 1 ml for Oncorhyncus mykiss. a Figure 3 Blood sampling: (a) caudal vein; (b) dorsal aorta; (c) cardiac puncture Hematological assesment Hematological changes due to stress have been fairly well characterized in fishes, but other responses are not so well documented. Some progress in the field of fish hematology was made, although much work remains. Generally the hematocrit ranges from 20% to 45% with higher PCV value seen in active species of fish. The hematocrit can increase artifactually from splenic contraction and from RBC swelling that occurs after the sample is collected. Precapture stress or inadequate anesthesia during blood sampling can exacerbate 239 RBC swelling and significantly increase of PCV. Hematocrit values obtained by us are within reference limits. Hemoglobin concentrations are low compared to mammals. As with the PCV, more active fishes have higher hemoglobin values. Active fish with higher oxygen tend to have smaller RBCs and consequently lower MCV. Red blood cell counts are usually lower in fishes than in mammals and vary with the need for oxygen. Leukocyte numbers are variable but

7 generally range from to cells/μl. In Cyprinus carpio at a global analysis, we observed an anemic syndrome with low MCH, possibly due to an iron Specification deficiency. The other mean values, in all three species, were in reference values, but the results must be correlated with lots of factors for a precise interpretation (table 1). Table 1. Hematological assessment Carassius gibelio Cyprinus carpio Oncorhyncus mykiss Reference Mean Reference Mean Reference Mean value value value value value value Hb (g/100 ml) , 5 53, ,1 Hct (%) 9,7-10,6 10, ,8 34, ,63 RBCc( 106 /μl) 2-4,2 2,7 1,10-2,20 1,39 0,77-1,67 1,37 MCV (cm3/erythrocyte) , ,06 MCH (pg) , ,35 14, MCHC (ghb/100 ml) 26 24, ,15 5,6-24,4 17,5 Within each species there have been recorded very different values that can be attributed to the effectiveness of anesthesia. However, mean values reflect the homogeneity of the studied batches. The examination of white cells fractions revealed monocytosis in Cyprinus carpio; this result being a consequence of stress status of the investigated fish. The reference value for carp monocyte percent is 8,7±23% (Martins et al, 2004). Our results had a higher mean value, 33±(9,69%) (fig. 4). Figure 4 Cyprinus carpio monocytes (panoptic stain X 100) Conclusions Of all anesthetics used, the anesthesia with etomidat and clove oil proved to be efficient in all three studied species. We obtain a deep level of anesthesia, suitable for long time and surgical procedures. It should be mentioned the ease with which anesthesia was achieved, with no need for a prior fish restraining. Ketamine induced good anesthesia in Carassius gibelio but light anesthesia in the other two species. Halothan anesthesia had good quality in Carassius gibelio, poor quality in Cyprinus carpio and unsatisfactory in Oncorhyncus mykiss because there was a prolonged excitement phase. To achieve a good level of anesthesia in Carassius gibelio using isoflurane, it was necessary to supplement the initial dosage, but this anesthetic proved to be unsatisfactory for Cyprinus carpio and total inefficient to Oncorhyncus mykiss. The shorter time for induction of anesthesia with lidocaine was recorded in Oncorhyncus mykiss but the quality of anesthesia in all three species was unsatisfactory. The blood sampling was possible using all three techniques, after general anesthesia, but the method and volume of blood sampled should be chosen regarding the fish size and the patient necessity to remain alive. The hematological parameters were in reference values; only in Cyprinus carpio we observed an anemic syndrome with some low value for MCH. 240

8 References Blaxhall, P.C.; The haematological assessment of the health of freshwater fish, J. Fish. Biol Chandroo, K.P.; Duncan, I.J.H.; Moccia, R.D.: Can fish suffer?: perspectives on sentience, pain, fear and stress Applied Animal Behaviour Science, 86: Coyle, S.D.; Mengel, G.J.; Tidwell, J.H.; Webster, C.D.: Evaluation of growth, feed utilization and economics of hybrid tilapia, Oreocromis niloticus x Oreochromis aureus, fed diets containing different protein sources in combination with distillers dried grains with solubles, Aquaculture Research 35: Ghittino, P.: Tecnologia e patologia in acquacoltura, Tipografia Emilio Bono, Torino (Italy) Hansen, M.K.; Nymoen, U.; Horsberg, T.E.; Pharmacokinetic and pharmacodynamic properties of metomidate in turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus), Journal of Veterinary Pharmacology and Therapy 26, , Harms, C.A.: Anesthesia in fish, In: Fowler ME, Miller RE (eds). Zoo and Wild Animal Medicine: Current Therapy 4. Philadelphia: WB Saunders Co, pp , Harms, C.A.: Fish. In: Fowler ME, Miller RE (eds). Zoo and Wild Animal Medicine, 5th ed. Philadelphia: Saunders, pp. 2-20, Harms, C.A.; Bakal, R.S.: Techniques in fish anesthesia, J Sm Exotic Anim. Med. 3: 19-25, Huntingford, F.A.; Adams, C.E.; Braithwaite,V.A.; Kadri, S.; Pottinger, T.G.; Sandoe, P.; Turnbull, J.F.: Current issues in fish welfare, J. Fish Biology 68, , Martins, M.L.; Tavares-Dias, M.; Fujimoto, R.Y.; Onaka, E.M.; Nomura, D.T.: Haematological alterations of Leporinus macrocephalus (Osteichtyes: Anostomidae) naturally infected by Goezia leporini (Nematoda: Anisakidae) in fish pond, Arq. Bras. Med. Vet. Zootec., v.56, n.5, p , 2004, Miller, W.R.; Hendricks, A.C.; and Cairns, J.: Normal ranges for diagnostically important hematological and blood chemistry characteristics of rainbow trout (Salmo gairdneri), Can. J. Fish. Aquat. Sci., 40, 420, Nicula M., Banatean-Dunea I., Gergen I., Harmanescu M., Simiz E., Patruica S., Polen T., Marcu A., Lunca M., Szucs S.: Effect of natural zeolite on reducing tissue bioaccumulation and cadmium antagonism related to some mineral micro- and macronutrients in Prussian carp (Carassius gibelio). AACL Bioflux 3, , Peters, R.C.; Van den Honk, B.; Bretschneider, M.F.; Struik, L.: Saffan : a review and some examples of its use in fishes (pisces : teleostei), Neth. J. Zool., 51 : 1-17, Ross, L.G.; Ross B.: Anaesthetic and Sedative Techniques for Aquatic Animals, 2nd ed. Blackwell Science, U.K, Stetter, M.D.: Fish and amphibian anesthesia, Vet. Clin. N. Am.: Exotic Anim. Prac.t 4:69-82, Tyler, P.; Hawkins, A.D.: Vivisection, anaesthetics and minor surgery, Pages , in A. D. Hawkins, editor. Aquarium systems. Academic Press, Harcourt Brace Jovanovich, London. Weiss, D.; Wardrop, K.J.: Schalm s Veterinary Hematology, John Wiley and Sons,