Influence of glyceryl guaiacolate ether on anesthetics in tilapia compared to benzocaine and eugenol

Objective. The study aimed to investigate the effectiveness of glyceryl guaiacolate ether (GGE) and compare the times of induction, recovery, hematological changes, total protein and glycaemia among anesthetics in Nile tilapia, Oreochromis niloticus. Materials and methods. A total of 60 tilapia distributed in 3 aquariums (N=20) were used, which formed the group benzocaine (100 mg/L), eugenol (50 mg/L) and guaiacol glyceryl ether (9.000 mg/L). After the induction of anesthesia fish blood samples were collected to determine the complete hemogram and glycemia. Then the animals were placed in aquariums with running water for assessing the anesthesia recovery. Results. It was verified that GGE showed longer induction and recovery times as well a significant increase (p<0.05) of glycemia, when compared with the other groups (p<0.05). The concentration of total protein did not differ between groups (p>0.05). An increase in the number of monocytes in the group treated with benzocaine (p <0.05) was observed in the analysis of the hematological parameters with no difference between groups for other variables. Conclusions. Eugenol and benzocaine allow rapid induction and recovery in Nile tilapia, without evidence of stress during handling and GGE showed high induction and recovery times, being inadequate for anesthetic use in Nile tilapia.


INTRODUCTION
The expansion and intensification of fish farming creates handling and health problems, because an increase in density leads to a loss in water quality, adverse handling conditions and others that result in stress with a decrease in defense mechanisms (1).
Within this context, the use of anesthetics in fish is widely used to reduce the adverse effects in handling as a way to ensure the integrity of animals, as well as reduce mortality and facilitate manipulation (2).The anesthetics mostly used in fish include the tricaine methanesulfonate (MS222), benzocaine, quinaldine, 2-phenoxyethanol, clove oil and its derivatives (methyleugenol, eugenol and isoeugenol) (2).
In Brazil there are no laws that regulate the use of anesthetics in fish, so the recommendations of the Food and Drugs Administration (FDA) have been adapted.In this way, alternatives are being sought for having safer and more effective anesthetics for use in fish farming.The effect of different anesthesia on the physiological parameters of fish is varied in relation to the different anesthetics and species used.
Benzocaine acts by blocking sodium channels and reduces the action potential of cell membranes (3).Depending on the intended degree of depression in the fish, the addition of a solution of benzocaine with ethanol or acetone in water at a dose of 25 to 100 mg/L is indicated.For this reason, fish under anesthesia with benzocaine and intended for feeding must be subjected to quarantine (4).
Clove oil is a phenolic compound resulting from the distillation of leaves and flowers of the clove trees in India (Syzgium aromaticum), with eugenol being its active ingredient, ranging from 70% to 95% (5).It is listed as a safe,
Protocolo experimental.Los peces fueron divididos en tres grupos (n=20) en un delineamiento enteramente al azar.Todos los peces fueron sometidos a anestesia por el método de inmersión, effective, affordable anesthetic with a wide safety margin for fish, as well as being non-toxic for the operator in the doses mentioned.Another advantage of this compound is that metabolism and excretion are faster, allowing consumption without waiting for a quarantine period (6).The Food and Agriculture Organization (FAO) indicates as acceptable a daily intake of 2.5 mg eugenol/ kg in humans.In fish anesthetized with 450 mg/L of clove oil, it was demonstrated that after 48 hours prior to abate there was a retention of just 0.32 mg of eugenol/kg, a value that is within the acceptable levels for the FAO (7).
Guaiacol glyceryl ether (GGE) or guaifenesin is a white powder, with a bitter taste, little soluble in water.It is a muscle relaxant of central action that blocks polysynaptic reflexes and depresses the transmission of nerve impulses in spinal cord neurons, widely used in horses (8).Although this compound is associated with muscle relaxation through sedation and a minimal analgesic effect, its effects on fish are yet to be evaluated.
The objective of this study was to assess the time of induction and recovery as well as determine glycemia, total protein concentration and hematological variables in Nile tilapias, Oreochromis niloticus, subjected to the anesthetic effects of benzocaine, eugenol, and guaiacol glyceryl ether in order to assess the latter for use in fish manipulation in routine procedures.

Animals and conditioning. Sixty Nile tilapias,
Oreochromis niloticus, were used with an average weight of 95.0±1.5 g distributed in 3 aquariums with a capacity of 350 L (n=20), supplied with water from a well, a water flow of 1 L/min and artificial aeration.Water quality was monitored daily and fish fed twice a day (9:00 and 17:00 h) with commercial fish concentrated with 20% of crude protein, with a biomass offer of 3%.
Experimental protocol.Fish were divided into three groups (n=20) in an entirely random outline.All the fish were subjected to anesthesia through the immersion method, each prepared with different concentrations.Benzocaine (BEN) was diluted in ethanol (0.1 g/ml) and in water for cada uno preparada en concentraciones diferentes.La benzocaína (BEN) fue diluida en alcohol etílico (0.1 g/ml) y esta en agua para concentración final de 100 mg/L (9).El eugenol (EUG) y gliceril éter guayacolato (EGG) se diluyeron directamente en el agua a una concentración de 50 mg/L (10) y 9.000 mg/L (dosis establecida en las pruebas anteriores), respectivamente.Para el ensayo, cada pez fue capturado con nasa al azar y colocado en recipientes plásticos con volumen útil de 50 L, donde los peces fueron mantenidos hasta el estado IV (Tabla 1). a final concentration of 100 mg/L (9).Eugenol (EUG) and guaiacolate glyceryl ether (GGE) were diluted directly in water at a concentration of 50 mg/L (10) and 9.000 mg/L (dose established in previous tests), respectively.For the assay, each fish was captured with a creel at random and placed in plastic containers with a volume of 50 L, where fish were maintained until state IV (Table 1).

Hematological parameters.
Approximately 2 mL of blood were collected from each fish after the induction, by means of the caudal venipuncture.These samples served to determine the glycemia (Accu-Check ® Active-Roche), hematocrit percentage (12), total plasma protein concentration by the biuret method (545 nm wavelength), hemoglobin rate and erythrocytes count in a Neubauer chamber (13).Once these data were determined, the hematimetric indexes, composed of the mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), were calculated.Total leukocytes were counted through the Neubauer chamber following the procedures described by Martins et al (14) and stains according to Tavares-Dias & Moraes (15).
Exposure times to anesthetics.To evaluate the anesthetic recovery, the fish were placed in aquariums with 90 L of tap water, with a flow of 1 L/min.During this period were observed the variables described by Grush et al (16) where: i) reemergence of opercular movements, ii) partial return of balance and swimming ability, iii) total recovery of balance, iv) swimming ability and reaction to external stimuli still unsteady, and v) total balance recovery and normal swimming ability.The times required for induction and anesthetic recovery were evaluated with a digital stopwatch.During the exposure period the animals were evaluated by direct observation for signs of intoxication, taking into account the alterations of behavior and mortality.
Statistics.The results were subjected to analysis of variance and comparison of means by the Tukey test with five percent significance (p<0.05)(17).

RESULTS
The quality of water remained within the comfort values of the fish (DO=5.1 mg/L: T 0 =26.47°C;pH=7.66 and electrical conductivity=117.96μS/cm) (18).The three substances used as anesthetics in Nile tilapia were able to annul the reactions to handling and material collection, including fish out of the  (11).

State Description
Behavioral response in fish 0 Normal Reaction to external stimuli, normal opercular movements, normal muscular reaction.

I Mild sedation
Reaction to external stimuli, slow swimming movements, small opercular movements, normal balance.

II Deep sedation
Total loss to external stimuli, even to strong pressure, reduced opercular movements.

III Narcosis
Partial loss of muscle tone, erratic swimming, increased opercular movements, reaction to tactile stimuli and vibration.

IV Deep anesthesia
Total loss of muscle tone, balance, slow but constant opercular movements.

V Surgical anesthesia
Total absence of reaction, slow and irregular opercular movements, decreased heart rate, and complete loss of all reflexes.

VI Spinal collapse
Decreased heart rate and total loss of reflexes.
water.However, there were differences in the induction and recovery and glycemia determined after anesthetic induction.
With respect to induction, animals in the GGE group showed times (p<0.05)higher than those observed in the groups BEN and EUG (Figure 1).The mean induction times for each anesthetic were on average 1.6±0.33minutes for group EUG; 2.58±0.51minutes for group BEN and 10.88±2.09minutes for group GGE; demonstrating that the preselected dose of each compound was efficient for anesthetic induction and manipulation, including venipuncture, with no reaction in the animals.
The same can be observed with regard to recovery times in the GGE group with an average time of 37.54±30.49minutes for the recovery of fish, which was higher (p<0.05)than the BEN and EUG groups, with times of 3.88±0.89and 2.38±1.25 minutes, respectively (Figure 2).
In the group anesthetized with GGE the glycemia rate was higher (p<0.05)(Figure 3) than the BEN and EUG groups.In the analysis of hematological parameters (Table 2), the substances tested did not differ (p>0.05) in relation to the total plasma protein concentration.Meanwhile, it was observed that the GGE significantly decreased (p<0.05) the values of circulating monocytes in relation to other anesthetics (Table 2).

DISCUSSION
The times observed for induction and recovery in the BEN group were lower than those found by Delbon and Paiva (19), who used the same dose and reported times of 1.5 and 4.7 minutes, respectively.This difference can be attributed to the difference in the concentration of dissolved oxygen (DO) in this test compared to the averages of 2.3 mg/L DO observed by said authors.In addition, the weight of the fish used in this study was different since they were adults (100±12 g) compared to those used by Delbon e Paiva (19) which were fingerlings, there being a correlation of weight with the reduction of the induction and recovery time, also verified in carps, Cyprinus carpio (20).
The induction time found for animals exposed to eugenol were lower than those observed in the literature for juvenile Nile tilapia (19), a difference that can be attributed to the metabolic rate, weight and age (21).A higher induction time was also observed in juvenile pampo (Trachinotus marginatus), anesthetized with 50 mg/L of eugenol (22), probably due to physiological and metabolic differences between the species used.The recovery time for EUG coincides with those found for tilapia (19).
The induction and deep anesthesia must have a short latency time (60 to 180 seconds) and recovery should not exceed 300 seconds (23).
In this test, tilapia specimens were exposure to benzocaine and eugenol at a concentration of 100 mg/L and 50 mg/L respectively.These values confirm the results of studies conducted within the same species (22).
Fish anesthetized with GGE showed higher induction and recovery times, possibly because of the pharmacokinetics of the compound (24).Fish anesthetized with GGE did not lose the balance and the induction was confirmed by manipulation without reaction.This behavior can result from the action mechanism of the compound, which is not a central depressant but a muscle myorelaxant (8), which would enable the maintenance of the balance of the fish.No reference was found in the literature consulted on the use of guaiacolate glyceryl ether in fish.
The level of stress can be estimated from the increase of serum glucose levels, as glycemia is one of the secondary physiological responses to stress, to meet the increased energy demand (25) The hematologic variables of fish have an ecological, physiological and pathological interest.These analyses help to understand of the relationship between blood characteristics and the environment (28).The results of the hemogram and total protein concentration in animals subjected to anesthesia with all the compounds tested, did not show significant differences, corroborating the values found in tilapia anesthetized with benzocaine and eugenol in different concentrations (29).
In tambaquis (Colossoma macropomum) anesthetized with eugenol, no alterations in blood variables were verified, except for the increase in hematocrits.The authors attributed this fact to the higher hydration of red blood cells due to the stressful stimulus to which animals were exposed (30), a fact not observed in this test.
A possible explanation for the increase in the number of monocytes observed in group BEN can be attributed to the fact that benzocaine has irritative properties such as p-aminobenzoic acid, leaving the fish agitated during the start of the test and as a result the mobilization of the marginal pool of monocytes and the increase of the same in blood circulation.This behavioral alteration in the beginning of the induction process was also observed by Delbon (29).
The results of this study suggest that the anesthetics did not cause hematological alterations.Benzocaine (100 mg/L) and eugenol (50 mg/L) enabled a rapid induction and recovery in Nile tilapia without evidence of stress during handling.The guaiacolate glyceryl ether in the concentration tested (900 mg/L), despite having induced the anesthesia of the fish, showed a high induction and recovery time and was inappropriate for use in O. niloticus.

Table 1 .
Stages of anesthesia according to Ross y Ross

Table 2 .
Average values (standard error) and analysis of variance of hematological variables of Oreochromis niloticus under BEN, EUG and GGE anesthetics.