Clinico-biochemical study on different pre-anesthetic drugs combined with propofol for neutering dogs

Anesthesia in animals has always been challenging as there is no ideal anesthetic available to produce complete sedation, analgesia and muscle relaxation. The  anesthetic  agents  used  in  Pakistan  are  not  safe  in  terms  of  induction, maintenance, duration and recovery (Khan and Merry 2018). These often result in serious anesthetic emergencies and deaths due to failure of anesthesia. Propofol belongs to alkyl phenol family. It is non-barbiturate hypnotic agent that metabolized quickly in the body of animal. The ideal properties of propofol are rapid induction, short duration, not having excitatory side effects on induction and recovery, but side effects of propofol are respiratory depression and apnea. It is the best anesthetic for the liver and kidney disorders patients (Suh et al., 2014). As the analgesic property of propofol is limited due to rapid metabolism and the pharmacokinetics studies showed that propofol has high volume of distribution, rapid clearance when administered by repeated dose (Griffenhagen et al., 2015). Therefore propofol alone is not a good anesthetic and its combination with some pre-anesthetic can provide the ideal anesthesia. In veterinary anesthetics three commonly used pre-anesthetics are- xylazine, diazepam and medetomidine. Each of the preanesthetic has its own advantages and disadvantages. Xylazine is the shortest acting α 2 –adrenergic agonist and has been widely used as preanaesthetic for immobilizing dogs as well as for sedative, muscle relaxant, and analgesic purposes (Sutil et al., 2017). Similarly diazepam is used as preanesthetic in both healthy and cardiovascular compromised dogs however it requires repeated injections and quick endotracheal incubation (Gehrke et al., 2015). Medetomidine hydrochloride is most potent α2-adrenoceptor selective agonist. It reduces the amount of both inhalant and injectable anesthetic agents when used as preanesthetic (Tsukamoto et al., 2015). It has sedative, analgesic and muscle relaxation properties and is widely used as tranquilizing or pre anesthetic medication in veterinary medicine. However, it indices cardiovascular side-effects such as dysrhythmia, hypotension, hypertension and bradycardia (Sinclair, 2003). There is not a single study on the comparative efficacy of these three commonly used preanesthetics. The aim of this prospective study was to identify an ideal preanesthetic on basis of better analgesic effect with least toxicity in body by comparing the efficacy of three above mentioned pre-anesthetics in combination with propofol.

Physical examination of the dogs was carried out before surgical interventions and pre-anesthetic evaluation was performed to state animal fit for anesthetic administration. The neutering was performed on total 18 dogs (mongeral), age ranged (12-24 months) and average body weight (13-21kg) were brought to Pet Centre Lahore. The follow up visit was planned. The dogs were subjected into three groups (n=6) viz A, B and C.
 
Experimental design
 
In group A xylazine (xylaze; 20mg/mL, Farvet, Holland) @ 2.2 mg/kg, group B diazepam@ 0.25 mg/kg and group C medetomidine  (domiter, 1mg/mL) @ 40 µg/kg body weight, were injected intramuscular as preanesthetics. After 3-5 minutes of pre-anesthetic, propofol pofol; 10mg/mL; dongkook pharm, Korea) injection @ 6 mg/kg of body weight was administered intravenous as anesthetic drug in all three groups.
 
Pre-operative consideration
 
All dogs were thoroughly examined twenty four hours before surgery for complete health status, clinical examination and weight. Prophylactically, all the dogs were vaccinated with Rabisin-R vaccine (Mariel Philippines, Inc) and were treated with trichlorophon 98% w/w (Seguvan; of symans pharmaceuticals Pakistan) for the control of ectoparasites. Endoparasites were treated with Albendazole (Zental suspension; of Smith Kline Becham) at the rate 10mg/kg body weight orally. Dogs were withheld for food but not water at least 12 hours before surgery. The dogs were prepared by application of 20 gauge Teflon catheter in right cephalic vein of each animal patient before start of surgery.
 
Preparation of neutering kit
 
All the surgical instruments included drapes, forceps, needle holder; scalpel, scissors, bowls and tray etc. were wrapped in the surgical pack and placed in the autoclave for sterilization before surgery.
 
Preparation of the patient
 
Clipping of the hair and then washing of the site with the soap and water around the area of the testis was performed. The dogs were shifted to the operation theater for the surgery.
 
Parameters evaluated
 
The effect of above designed anesthetic combinations on each group was evaluated on basis of physical findings and haematological parameters.
 
Physical parameters
 
Rectal temperature was recorded at 0, 20, 40, and 60 minutes interval by clinical thermometer.
 
Pulse rate was counted at 0, 20, 40 and 60 minutes from femoral artery.
 
Respiration rate was observed at 0, 20, 40 and 60 minutes by placing the hands in front of nostrils.
 
Sedation parameters
 
The absence or presence of sedation parameters- head down, neck down, gait incoordination, saliva drooling and palpebral reflexes (touching of eyelid) were evaluated after administration of all three pre anesthetics to all groups. The time of onset of sedation was also assessed five minutes before giving anesthetic drug.
 
Analgesic parameters
 
The following parameters were assessed after administration of anesthetic cocktails.

Tail response (+/-) was checked by pricking with needle. Toe pinch reflex/pedal (+/-) was checked by pinching the toe and pedal area with halstated mosquito forcep. The time from awake to anaesthetized state was calculated in minutes after drug administration. The total time of unconsciousness or loss of reflexes was calculated in minutes. Reappearance of consciousness or reflexes till animal was completely alert or awake was estimated in minutes (Table 3).
 
Collection of blood
 
The blood samples were collected from the cephalic vein at two different intervals before and after the surgery. Complete blood count was performed. Serum was separated for liver and kidney function tests.

Surgical procedure of neutering
 
Pre-scrotal approach was used for performing neutering in all dogs. The surgery was performed with all protocols (Slatter. D. 2003).
 
Post-operative consideration
 
After surgical procedure the dogs were kept in the cages of surgery section under complete observation and care. Antibiotic coverage was given with inj. Cephradine (velocef) as 25mg/kg body weight up to 5th day of operation. Analgesics were given for five days inj. Profenid (ketoprofen) @dose of 0.2-1 mg/kg body weight.
 
Statistical analysis
 
The data was analyzed by one way ANOVA followed by multiple comparison tests using Statistical Package for Social Science (SPSS) version 20.0. The proportionate data was analyzed through Z-distribution test. All the parameters were compared and significant value P≤0.05 expressed in Table 1-6.       









 

                  

Rapid onset (9.13 ± 1.41 minutes) and long duration (59.67± 5.50 minutes) of analgesia was observed in group C compared to other groups (onset; 13.661±1.72 A and 20.16 ± 1.75 B minutes) and duration of analgesia (48.56 ± 4.61 A) and (40.66± 5.46 B) minutes (Table 1). There was significant difference in onset and duration (P £0.05). Non significant decrease (P≥0.05) in rectal temperature was noted in all three groups (group A 101.9 ± 0.37 to 100.8 ± 0.58, group B 101.4 ± 0.22 to 100.7 ± 0.41 and group C 101.5 ± 0.38 to 100.3 ± 0.26) during 60 minutes interval. In group C, significant decrease (P≥0.05) in pulse rate (74.9 ± 3.54 to 69.9 ± 1.7), in group A, highly significant decrease (P≥0.05) in pulse rate (75.8 ± 4.5 to 61.2 ± 8.6) and non significant (P≥0.05) decrease in pulse rate in group B (77.8 ± 3.4 to 72.8 ± 4.5) by 60 minutes and returned to normal by 90 minutes (Table 2). The mean respiration rate was decreased significantly (P≥0.05) in all three groups (Table 2). There was non significant increase in value of BUN in group A and C group A (9.04 ± 0.2 to 10.68 ± 0.69) and (14.63 ± 1.45 to 15.23 ± 1.56) respectively. In group B there was significant increase (P≥0.05) in BUN (10.03 ± 0.37 to 13.54 ± 0.75). Serum creatinine was non significantly increased in group A and C, group A (0.71 ± 0.04 to 0.78 ± 0.06, group C (0.82 ± 0.02 to 0.87 ± 0.03) and significant increase (P≥0.05) in group B (0.56 ± 0.03 to 0.73 ± 0.03). There was significant increase (P≥0.05) in ALT values in group B (18.66 ± 2.52 to 20.76 ± 2.31) and there was non-significant increase in group A (24.33 ± 2.52 to 25.94 ± 2.08) and group C (24.66 ± 1.52 to 25.80 ± 1.11) respectively during 24 hours interval.     
 
Recovery from analgesia
 
Dogs of group C showed significantly minimum recovery time (in minutes) 9.13± 1.41 followed by group A 13.66 ±1.72 and group B 20.16 ± 1.75 (Table 1).

Efficacy of xylazine, diazepam and medetomidine HCl with propofol anaesthesia was assessed on the basis of clinical and haematological parameters in dogs.
 
Rectal temperature
 
A non significant reduction (P≥0.05) was observed in rectal temperature of all treatment groups. It has been established that Alpha-2 adrenoceptor inhibitors induce hypothermia and allow better maintenance of body temperature due to peripheral vasoconstriction followed by central redistribution of blood (Quan et al., 1992) which is in agreement with the findings of present study. However this slight difference in the body temperature may be due to variation in the conditions as well as difference in combination. For example medetomidine has also been reported to induce hyperthermia under free ranging conditions in canine species (Gutema et al., 2018).
 
Pulse rate
 
Medetomidine and xylazine treated groups initially showed a decrease in pulse rate. This could be associated with peripheral vasoconstriction followed by vasodilatation. Morever, medetomidine and other Alpha-2 adrenoceptor agonists are also reported to induce bradycardia (Chue and Chue 2017). But bradycardia was more pronounced in xylazine treated group.
 
Onset and duration of analgesia
 
Analgesic onset time period was compared between group A, B and C injected with xylazine, diazepam and medetomidine HCl with propofol. Dogs of group C showed minimum analgesic onset time with maximum analgesia duration that could be an ideal property for any anesthesia (Table 1).
 
lgesia parameters
 
Analgesic potency was evaluated on presence or absence of tail pinch, toe pinch test and palpebral effect at 20 minutes of post drug administration. Maximum dogs of group C were not responsive to pin prick, tail pinch and toe pinch at 20 minutes of post medetomidine-propofol administration. This suggested that medetomidine-propofol produced prolong and potent analgesia in dogs. While in comparison to this the dogs of group A and B showed less potent analgesia. As the previous studies explained that medetomidine alone cannot produce prolonged and rapid analgesia (Baker et al., 2011) therefore this might be due to the synergestic effect of medetomidine along with propofol. Medetomidine has increased specificity for the alpha-2 receptor and increased analgesic potency compared to xylazine (Jena et al., 2014). Medetomidine is preferred over xylazine in dogs (Lemke, 2007). Benzodiazepines do not produce analgesia.
 
Respiratory rate depression
 
It was observed in all three groups due to direct depression of respiratory centre in central nervous system (Table 2). Induction of propofol also depressed the respiratory rate in our study. Our study was in accordance with earliest studies in dogs but decrease was remained in normal acceptable range (Maney et al., 2013 and Singh et al., 2013).
 
Haematological parameters
 
In present study, the values of red blood cells and hemoglobin decreased non-significantly (P>0.05) from the initial base line value in xylazine-propofol, diazepam-propofol and medetomidine-propofol group. It was possibly due to pooling of erythrocytes in to spleen due to decreased in sympathetic stimulation or hemodilution. Our results are in accordance with the (Jena et al., 2014). However, the values of white blood cells non-significantly increased (P>0.05) in xylazine-propofol receiving group as compared to diazepam-propofol and medetomidine propofol group. This variation is may be due to different pharmacokinetics of various pre-anesthetics used in present study by pooling of blood cells in spleen and other reservoir organ.
 
Kidney function tests
 
Increase in BUN and creatinine in all three groups has been recorded and it was due to propofol as blood flow was reduced to kidneys led to retention of nitrogenous substances in the blood (Manat and Kelawala, (2004).
 
Liver function tests
 
We also established liver function test in our study. Liver enzymes AST and ALT were significantly higher in (P≥0.05) in group B during 60 minutes but then returned to normal till 90 minutes post surgery. The liver enzymes were high in all three groups as hypoxia from anaesthetics caused some alteration in cell membrane and leakage of liver enzymes and other enzymes as well (Psatha, 2011). But that rise in liver enzyme lasted for 60 minutes and then returned to normal at 90 minutes interval. The rise in liver enzymes was lowest in medetomidine and lower in xylazine treated group. The results were in accordance with earliest studies (Akbar et al., 2014). The three combinations are safe to use as Propofol, nonbarbiturate, ultrashort acting anesthetic agent is a drug of choice for animals with liver disease. It is eliminated from body by extrahepatic system and its clearance was faster from body than hepatic blood flow. Our three combinations were safe but analgesic and sedation property of medetomidine is superior to other pre anesthetic and our results were supported by (Akbar et al., 2014). 

The present study concluded that medetomidine HCl in combination with propofol produce, rapid onset, and long duration of analgesia, with rapid and smooth recovery and no effects on hematological parameters of dogs.