Evaluation of Haemato-biochemical Effects of Tiletamine-zolazepam Alone and in Combination with Xylazine or Xylazine-ketamine in Atropinized Dogs

General anaesthesia is a state of unconsciousness in which there is a loss of protective reflexes after administration of single or combination of anaesthetic agents. An ideal anaesthetics does have characteristics like sedation, amnesia, analgesia and muscle relaxation. A sole agent cannot provide all these characteristics, hence a combination of drugs is usually preferred. This is known as balanced anaesthesia (Billah et al., 2017).
       
Xylazine is an alpha-2 adrenergic agonist with properties like sedation, analgesia and muscle relaxation. The most common effect of xylazine is bradycardia with reduction in cardiac output up to 30 per cent (Landry and Maza, 2020). Ketamine is a non-competitive antagonist at NMDA receptor and by binding to the phencyclidine binding site, they prevent the binding of excitatory neurotransmitter, glutamate. However ketamine have poor muscle relaxation property and visceral analgesia so to compensate for this sedatives like xylazine can be co-administered (Dugassa and Fromsa, 2018).
       
Tiletamine-Zolazepam is an anaesthetic combination of two drugs tiletamine and zolazepam present in the ratio of 1:1. Analgesic effect tiletamine is greater than ketamine. Zolazepam belongs to benzodiazepine class. Zolazepam intensifies the effect of tiletamine on the central nervous system and reduces the hyperactivity of the skeletal muscle (Lee et al., 2018).
       
In present study three different anaesthetic combinations using anaesthetic agents tiletamine- zolazepam, xylazine-tiletamine-zolazepam and xylazine-ketamine-tiletamine-zolazepam are compared for their anaesthetic efficacy and for their effects on haemato-biochemical parameters as previously not much details are available for the same in canines. The aim of this study is to evaluate the effect of above said anaesthetic combination on haemato-biochemical parameters.

Animals
 
The study was conducted in 18 dogs irrespective of age, breed, sex and body weight presented to the Teaching Veterinary Clinical Complex, Pantnagar for various surgical corrections during January to July 2022. Approval for conducting the study on the clinical cases requiring anaesthesia for various surgical procedures was obtained vide letter no. IAEC/CVASc/VSR/474 dated 25/10/2021 from the Institutional Animal Ethics Committee of College of Veterinary and Animal Sciences, GBPUAT, Pantnagar. Animals were kept off feed for 12 hours and water was withheld for 6 hours prior to the surgery. These animals were randomly divided into three groups viz., A, B and C.
 
Anaesthetic protocols
 
Atropine sulphate (0.025 mg/kg SC) was administered in all the groups, xylazine (0.5 mg/kg IM) in group B and xylazine (0.5 mg/kg), ketamine (2.5 mg/kg IV) combining in group C. A gap of 10 minutes was kept between administration of each drug. In all the three groups induction was done by administering tiletamine-zolazepam (till effect IV). A single shot of tiletamine-zolazepam was given without maintenance dose. Induction dose is the amount of the tiletamine-zolazepam (zoletil) required in respective groups for induction of anaesthesia adequate to allow placement of an endotracheal tube. It was calculated in mg/kg after completion of each trial.
 
Monitoring of anaesthesia
 
Monitoring of anaesthesia was done using veterinary patient monitor (model no. MMED 8000-CV Beijing Choice Electronic Technology Co. Ltd., Beijing, China.
 
Clinico-physiological parameters
 
Clinical parameters evaluated for the study are induction dose (in mg/kg body weight), induction time (in seconds), duration of anaesthesia (minutes), jaw relaxation score and pedal reflex score. The values of these parameters are tabulated in Table 2. Evaluation of jaw relaxation score and pedal reflex score was done as per the method described by Zhang et al., (2021) and Nam et al., (2013) respectively. Scoring parameters of jaw relaxation and pedal reflex is represented in Table 1. Physiological parameters like heart rate, respiration rate, rectal temperature and haemoglobin oxygen saturation (SpO₂) were monitored by veterinary patient monitor. Jaw relaxation score, Pedal reflex score and physiological parameters were evaluated at 0, 10, 15, 25, 35, 45, 60, 75, 90 and 105 minutes.
 

 

 
Haematological and biochemical parameters
 
Haematological parameters estimated includes haemoglobin (g/dl), total erythrocyte count (106/cu mm), packed cell volume (%), differential leucocyte count (%) and total leucocyte count (10³/cu mm). Biochemical parameters includes total protein (g/dl), plasma glucose (mg/dl), blood urea nitrogen (mg/dl), serum creatinine (mg/dl), total bilirubin (mg/dl), alanine aminotransferase (IU/litre) and aspartate aminotransferase (IU/litre). These parameters were estimated at baseline 0 minute, 30 minutes, 1 hour, 6 hour and 24 hours post induction.
 
Statistical analysis
 
Data was expressed as Mean±Standard error (SE). To compare the Mean values at different time intervals with their base values in each group paired “t” test was used (Snedecor and Cochran, 1994). Analysis of variance (ANOVA) was used to compare the mean values between different groups. All data were analysed using SPSS v 15.0 statistics software program.

Clinical parameters
 
Induction of anaesthesia was fast and smooth in all the groups. The Mean±SE values of various clinico-physiological parameters like heart rate, respiratory rate, rectal temperature, SpO₂, jaw relaxation score and pedal reflex score are tabulated in Table 3. Induction dose in group A, B and C was 6.6, 5.5 and 4 mg/kg BW respectively. Significant difference (p<0.05) was observed between the groups subjected to different pre-anaesthetic protocols. Reduction in induction dose of tiletamine-zolazepam in subsequent groups could be due to addition of premedicants like xylazine in group B and xylazine-ketamine in group C. In a similar study by Landry and Maza (2020) combination of xylazine (0.88 mg/kg), ketamine (3.52 mg/kg) and tiletamine-zolazepam (4.4 mg/kg) was used in a single syringe. Induction time of 38.0±2.0, 31.83±2.17 and 25.0±7.55 seconds was reported in group A, B and C respectively. Administration of xylazine in group B and xylazine-ketamine in group C significantly lowered the dose of induction and induction time. Dugassa and Fromsa (2018) have reported that tiletamine-zolazepam takes 45-90 seconds for induction after intravenous administration and due to higher lipid solubility along with hepatic metabolism of tiletamine-zolazepam is responsible for longer recovery time. Duration of anaesthesia in minutes was found to be in an increasing order and being highest in group C, intermediate in group B and lowest in A. This increase was significant (p<0.05). Koli et al., (2021) have stated that combination of xylazine and tiletamine-zolazepam produces longer duration of anaesthesia in dogs as compared to tiletamine-zolazepam given alone. Maximum value of jaw relaxation score in group A and B was 3 and in group C it was 2.6. Score of 2.6 indicates minute resistance to opening of jaw and score of 3 indicates no resistance to opening of jaw and excellent muscle relaxation. In all the groups very good muscle relaxation was due to inhibition of internuncial neurons at spinal cord by zolazepam (Hall et al., 2001). In all the three groups zero value of pedal reflex score was reported at baseline which means normal pedal reflex and maximum value reported was 3 within 5 minutes of induction. A score of 3 indicates absence of pedal reflex which showed animals were in a good analgesic state. Xylazine acts by inhibiting the interneural transmission in CNS which depresses CNS resulting in muscle relaxation and analgesia (Williams et al., 2002). Dissociative anaesthetics bind to opioid receptors and also binds to monoaminergic receptors contribute to anti-nociception (Dugassa and Fromsa, 2018).
 

 
Physiological parameters
 
Heart rate values showed significant (p<0.05) increase in first 25 minutes in all three groups followed by a non-significant drop. Again, a second rise in heart rate (A: 126.60±8.80, B: 131.30±15.70 and C: 136.00±3.46) was reported in all the groups at 75 minutes. Both tiletamine and ketamine increases the sympathetic tone and decreases vagal tone which leads to increased heart rate (Landry and Maza, 2020). Kim et al., (2007) have stated that positive inotropic effect of tiletamine counter balances the bradycardic effect of xylazine. Respiration rate (A:17.00±1.73, B:19.00±3.0 and C:20.30±6.08) was significantly (p<0.05) lowered in three groups post induction at 25 minutes. However, this drop was transient and values again increased. Similar results were obtained by Lee et al., (2018) in dogs using tiletamine- zolazepam where respiratory frequency decreased after 5 minutes of administration. Findings similar to group B were reported by Salve et al., (2020) and Flores et al., (2009) which could be due to depression of respiratory centre located in medulla oblongata by anaesthetic agents. According to Gomez-Villamandos (2013) tiletamine-zolazepam leads to mild to moderate respiratory depression. Rectal temperature was reported to be highest at 0 minute and it kept on decreasing throughout th2e anaesthetic period and in all the groups lowest values were reported at 105 minutes. Findings similar to group A and B have been reported by Flores et al., (2009). Significant reduction in rectal temperature could be due muscle relaxation, reduction in metabolic rate and depression of thermoregulatory centre by anaesthetic agents (Munif et al., 2021 and Lu et al., 2014). Haemoglobin oxygen saturation values reported drop at 25 minutes in all three groups followed by an increasing trend in the values throughout the anaesthetic period. Group A showed non-significant changes. Similar results have been reported by Flores et al., (2009) where immediate non-significant drop in oxygen saturation values post induction was observed followed by an increase throughout the anaesthetic period. This could be due to depression of respiratory centre by anaesthetic agents.
 
Haemato-biochemical parameters
 
Haematological parameters in all the three groups followed similar trend. The Mean±SE values of various haematological parameters are present in Table 4. The Mean±SE values of Haemoglobin (A:13.33±2.64, B:10.86± 0.88, C:11.90±0.81), total erythrocyte count (A:5.90±1.20, B:5.90±0.20, C:4.74±1.32), packed cell volume (A:38.66±7.0, B:40.80± 10.40, C:40.90±10.01), total leucocyte count (A:12.50±6.30, B:11.56±5.20,C:10.74±0.96), lymphocytes (A:30.00±3.46, B:26.30±8.00,C:22.00±0.50), monocytes (A:1.66±1.00, B:1.00±1.73,C:1.60±1.00), eosinophils (A:0.33±1.00, B: 0.33±1.00, C: 0.33±1.00) and basophils (A:0.10±0.02, B: 0.15±0.01, C:0.10±0.02) showed transient decrease in their values at 60 minutes. Their values started increasing thereafter reaching almost the baseline value by 12 hours. All these variations present were within the normal physiologic values. According to Hampton et al., (2019) reduction in haemoglobin was seen in dogs while inducing with tiletamine-zolazepam and it was attributed to haemodilution with no fluid loss via conduction or evaporation. Hafez et al., (2017) have observed a drop in the values of haemoglobin, PCV, TEC and TLC values with tiletamine-zolazepam anaesthesia. The spleen enlarges during general anaesthesia which results in sequestration of red blood cells and leads to reduced haematocrit and haemoglobin values (Hawkey, 1985). Abdul et al., (2015) have attributed significant reduction in granulocytes due to stress involved in the procedure. In a study by Yohannes et al., (2018) reduced value of eosinophils have been reported due to pooling of circulating blood cells in the spleen and other reservoirs post ketamine-xylazine anaesthesia. Neutrophils showed an increase at 60 minutes followed by reduction at 6 hours and reached baseline value by 24 hours. Neutrophilia could be attributed to the stress caused by anaesthetic drugs, surgical trauma and activation of the adrenal cortex (Rubio et al., 2022). All these changes in the haematological parameters were within the normal physiologic range.
 

       
Biochemical parameters were within normal physiologic range throughout the study period in all the three groups and reached near to baseline value by 24 hours. The Mean±SE values of various biochemical parameters are tabulated in Table 5. The Mean±SE value of total protein showed a decrease at 60 minutes whereas plasma glucose, Blood urea nitrogen (BUN), serum creatinine, total bilirubin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) showed an increase till 60 minutes followed by a reverse trend thereafter approaching the baseline value by 24 hours. Hampton et al., (2019) presented similar report where drop in total protein values were observed post induction with tiletamine- zolazepam. The decrease in total proteins could be due to hemodilution or secondary elevation of globulins (Dewangan et al., 2016). Surgical and anaesthetic stress leads to the altered endocrine secretion of the insulin antagonisms like growth hormone (Rubio et al., 2022). Increase in BUN levels might be due to temporary inhibitory effect of drugs on renal blood flow resulting in consequent reduction in glomerular blood flow (Dewangan et al., 2016). After induction with tiletamine-zolazepam anaesthesia with decreasing creatinine value in dogs (Koli et al., 2021). Kamal et al., (2019) observed increased bilirubin concentration at 20 and 40 minutes post induction under xylazine-ketamine anaesthesia in dogs.
 

       
Hafez et al., (2017) in a study have reported similar changes in ALT and AST and contrary results have been reported by Koli et al., (2021) with regards to BUN and serum creatinine. General anaesthetics reduces the blood flow to the liver which can cause harm and alter the permeability of hepatocytes resulting in leakage of liver enzymes and their elevated levels (Vikers et al., 1984; Pandey and Sharma, 1994; Tiwari et al., 1999).

Tiletamine-zolazepam resulted in fast and smooth induction, however premedication with xylazine in group B and xylazine-ketamine in group C significantly reduced its required dose for induction. Transient changes were seen in haematological and biochemical parameters which were within the normal physiologic range. So based on the above study it can be concluded that the various combinations of tiletamine-zolazepam with pre-medicants like xylazine and xylazine-ketamine was found to be safe with regard to their effect on haemato-biochemical parameters.

The author would like to thank the Department of Veterinary Surgery and Radiology, Pantnagar for their support and assistance during the research period. This research did not receive any specific grant from any funding agencies.

Authors declare no conflict of interest.