Buffalo (Bubalus bubalis), often referred to as the ‘Black gold’ of India, plays a pivotal role in the rural economy and holds immense potential for poverty alleviation and promotion of rural entrepreneurship. Water buffalo is a valuable livestock species with excellent zootechnical attributes for both milk and meat production (Guerri et al., 2021). In veterinary practice, the selection of an appropriate anaesthetic protocol is crucial for ensuring safe and effective surgical interventions in this species.
       
Tiletamine-zolazepam (Zoletil®/Telazol®) is a non-opioid, non-barbiturate injectable anaesthetic combination consisting of a dissociative anaesthetic agent, tiletamine and   a benzodiazepine tranquilizer, zolazepam (Dewangan et al., 2025). Tiletamine alone is associated with poor muscle relaxation and may cause excitement during recovery, whereas zolazepam possesses anticonvulsant and muscle relaxant properties. Tiletamine-Zolazepam is a potent dissociative anaesthetic combination consisting of two distinct pharmacological agents, tiletamine and zolazepam, which are formulated together in a precise fixed ratio of 1:1. Clinical studies and veterinary practice have demonstrated that the specific analgesic effect provided by tiletamine is significantly greater and more profound than that of ketamine when used under similar conditions (Sulekha et al., 2023).

Ketamine combined with xylazine, diazepam and   butorphanol is a safe anaesthetic protocol for buffalo calves, ensuring smooth induction and recovery without adverse cardiopulmonary effects. However, the xylazine-ketamine combination provides a significantly longer duration of surgical anaesthesia and a smoother recovery than the other combinations (Singh et al., 2026).
       
The combination of these agents results in improved muscle relaxation, enhanced analgesia, prolonged duration of action and smoother recovery, while minimizing the risk of convulsions (Lin and Walz, 2014).
       
Glycopyrrolate is commonly used as a preanaesthetic agent to reduce salivary and respiratory secretions and to decrease gastrointestinal motility. Dexmedetomidine, an a2‚ -adrenoceptor agonist, is widely used for its sedative, analgesic and anaesthetic-sparing effects. Butorphanol, an opioid agonist-antagonist, provides effective analgesia along with antitussive action and is associated with minimal respiratory, gastrointestinal and cardiovascular depression (Svozil et al., 2007).
       
Despite the widespread use of these agents, there is limited information regarding their combined use with tiletamine-zolazepam in buffalo calves, particularly under Indian conditions. Therefore, the present study was undertaken to evaluate the haemato-biochemical and oxidative stress responses associated with these anaesthetic protocols.
       
Plasma cortisol concentration is one of the most widely used biomarkers for assessing stress responses associated with surgical interventions and anaesthesia (Singh et al., 2005). In addition, evaluation of oxidative stress indices such as malondialdehyde (MDA), a marker of lipid peroxidation and reduced glutathione (GSH), an important endogenous antioxidant, provides valuable insight into anaesthesia-induced oxidative stress. These parameters are useful for early postoperative monitoring and for minimizing surgical and anaesthetic complications (Mahalingam et al., 2014).
       
In ruminants, most surgical procedures are commonly performed under local or regional analgesia. However, general anaesthesia becomes essential in complex and invasive procedures such as diaphragmatic herniorrhaphy, thoracopericardiotomy, repair of ruptured suspensory ligaments, orthopaedic surgeries, keratoplasty and ventral hernia repair (Riazuddin et al., 2004). General anaesthesia ensures complete unconsciousness, adequate analgesia, optimal muscle relaxation and suppression of reflex activity, thereby facilitating safe and effective surgical manipulation. The pharmacological combination of Guaifenesin and ketamine produces a range of excellent to good muscle relaxation effects in the subjects, demonstrating a highly effective and reliable level of neuromuscular blockade and physical ease (Nirmale et al., 2025).
       
Although tiletamine-zolazepam has been extensively studied in dogs, cats and wild animals, there is a paucity of literature regarding its use in buffalo calves, particularly in combination with preanaesthetic agents such as dexmedetomidine or butorphanol under Indian conditions. Furthermore, limited information is available on balanced anaesthetic protocols in buffaloes with respect to haemato-biochemical and oxidative stress responses. Therefore, the present study was undertaken to evaluate the haemato-biochemical and oxidative stress alterations and to assess the suitability of tiletamine-zolazepam alone and in combination with dexmedetomidine or butorphanol in buffalo calves.

The present study was conducted in the Department of Veterinary Surgery and Radiology, College of Veterinary Science and Animal Husband ry, Anjora, Durg (Chhattisgarh). A total of 18 clinically healthy, non-descript male buffalo calves, aged between 6 months and 1 year and weighing 80-100 kg, were selected for the study. The animals were rand omly divided into three groups (A, B and C), comprising six animals in each group. An acclimatization period of 7 days was allowed prior to the commencement of the experiment.
       
All animals were dewormed with fenbendazole @ 5 mg/kg body weight orally 15 days prior to the start of the trial. The animals were maintained under uniform feeding and managemental conditions throughout the experimental period. Prior to administration of anaesthesia, all animals were fasted for 24 hours with free access to water withheld for 12 hours.
       
Glycopyrrolate @ 0.01 mg/kg body weight was administered intramuscularly 10 minutes prior to induction of anaesthesia in all groups. In Group A, animals received tiletamine-zolazepam @ 2.5 mg/kg body weight administered intravenously as a slow bolus injection. In Group B, dexmedetomidine @ 2 µg/kg body weight was administered intravenously, followed 10 minutes later by tiletamine-zolazepam @ 2.5 mg/kg body weight administered intravenously. Similarly, animals in Group C received butorphanol @ 0.075 mg/kg body weight intravenously, followed after 10 minutes by tiletamine-zolazepam @ 2.5 mg/kg body weight administered intravenously as a slow bolus.
       
A pilot study was conducted on six buffalo calves to stand ardize the effective dose of tiletamine-zolazepam using different dose rates (2.0, 2.5 and 3.0 mg/kg body weight). The dose of 2.5 mg/kg body weight was selected based on optimal analgesia, sedation and muscle relaxation. The animals used in the pilot study were excluded from the main experiment.
       
The study was carried out over a period of two years (2024-2025). Prior approval for conducting the experiment was obtained from the Institutional Animal Ethics Committee (CPCSEA) (IAEC Approval No.: VSR-PhD-1/2025).
 
Sample collection and laboratory analysis
 
Approximately 5 ml of blood was collected aseptically from the external jugular vein at different time intervals. Out of this, 1 ml of blood was transferred into EDTA-containing vials for haematological analysis, 2 ml into clot activator tubes for serum biochemical analysis and the remaining 2 ml into vials containing acid citrate dextrose (ACD) solution (1.5 ml per 10 ml of blood) for the estimation of oxidative stress parameters.
       
Blood samples were collected at baseline (0 min; prior to premedication) and at 15, 30, 60 and 120 minutes following induction of anaesthesia. Haematological parameters were analysed using an automated veterinary haematology analyser (Merilyzer CelQuant Vet).
       
For biochemical analysis, blood samples collected in clot activator tubes were allowed to clot at room temperature for 5 minutes and subsequently centrifuged at 3000 rpm for 15 minutes. The separated serum was carefully aspirated using a micropipette and transferred into properly labelled Eppendorf tubes, which were stored at 2-4°C until further analysis. Serum biochemical parameters were analysed using a semi-automated biochemical analyser (Erba Mannheim).
       
Oxidative stress parameters were assessed by estimating serum cortisol, reduced glutathione (GSH) and malondialdehyde (MDA). Serum cortisol levels were determined by radioimmunoassay (RIA) using a stand ard cortisol estimation kit. Reduced glutathione (GSH) levels were estimated according to the method described by Prins and Loos (1969), while lipid peroxidation, expressed as malondialdehyde (MDA), was determined as per the method of Placer et al., (1966). All analyses were carried out on the same day of sample collection to avoid analytical variation.
 
Statistical analysis
 
The data obtained in the present study were analysed using Statistical Package for the Social Sciences (SPSS) software (Version 25.0). One-way analysis of variance (ANOVA) was employed to evaluate the differences among groups at different time intervals. Wherever significant differences were observed, the means were further compared using Duncan’s multiple range test. The results are presented as mean±stand ard error (Mean±S.E.). Differences were considered statistically significant at P<0.05.

Haematological parameters
 
The mean±S.E. values of various haematological parameters are presented in Table 1. Haemoglobin (Hb) concentration and packed cell volume (PCV) exhibited a non-significant decrease in Group A, whereas a significant (P<0.05) reduction was observed in Groups B and C (Fig 1). The decline in Hb and PCV following tiletamine-zolazepam anaesthesia may be attributed to anaesthesia-induced stress, splenic sequestration of erythrocytes and fluid redistribution between intravascular and extravascular compartments to maintain cardiac output (Kumar et al., 2014; Singh, 2021). These findings are in agreement with earlier reports in cattle, calves and   sloth bears subjected to similar anaesthetic protocols (Ukkali, 2022; Sindak et al., 2003; Anitha et al., 2019).




       
A gradual, non-significant decrease in total erythrocyte count (TEC) was recorded in all groups. This reduction may be associated with anaesthesia-induced vasodilation and subsequent plasma volume expansion, leading to haemodilution and vascular pooling. Comparable observations have been reported in goats under diazepam-ketamine-propofol and tiletamine-zolazepam-based anaesthesia (Ragab et al., 2022; Sahu, 2024).
       
Total leukocyte count (TLC) showed a non-significant decrease across all groups, which could be attributed to sequestration of circulating leukocytes in the spleen and other reservoirs due to reduced sympathetic tone during anaesthesia (Ragab et al., 2022; Singh, 2021).
       
Differential leukocyte count revealed a non-significant increase in neutrophil percentage accompanied by a corresponding decrease in lymphocyte count. These changes may be related to stress-induced adrenocortical stimulation leading to neutrophilia and lymphocytopenia (Jayakrishnan, 2023). Monocytes, eosinophils and basophils exhibited non-significant fluctuations, which might be due to the immunomodulatory effects of anaesthetic agents and adrenal activation (Anitha et al., 2019). These observations are consistent with previous studies conducted in cattle and buffalo calves under comparable anaesthetic regimens (Gill, 2013; Ukkali, 2022; Jayakrishnan, 2023).
 
Biochemical parameters
 
The mean±S.E. values of various biochemical parameters are presented in Table 2. A significant (P < 0.05) increase in serum glucose levels was observed in animals of all three groups at different time intervals (Fig  2). The hyperglycaemic response following tiletamine-zolazepam administration may be attributed to increased circulating catecholamines after premedication, along with the effect of anaesthetic agents on subcortical centres regulating adrenocorticotropic hormone (ACTH). Additionally, decreased peripheral glucose utilization, impaired insulin activity and stress-induced glucocorticoid release may contribute to elevated blood glucose levels (Ragab et al., 2022).




       
Total serum protein values exhibited a non-significant decrease across all groups at various time intervals. This reduction may be due to haemodilution resulting from increased plasma volume following fluid shifts, as well as redistribution of fluids from the extravascular to intravascular compartments, leading to dilution of plasma proteins (Fararh et al., 2011). Similar findings have been reported in buffalo calves (Kumar et al., 2014).
       
Serum urea nitrogen (SUN) and creatinine levels showed a non-significant increase in Groups A and C, whereas a significant (P<0.05) increase was recorded in Group B (Fig 3 and 4). The transient elevation of these renal biomarkers may be attributed to a temporary reduction in renal blood flow and glomerular filtration rate caused by anaesthetic-induced haemodynamic alterations, resulting in the retention of nitrogenous waste products (Okwudili et al., 2014). Comparable observations have been reported by Abou-Ghanema et al. (2014) in buffalo calves and by Malik (2011) in buffaloes.




       
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities showed non-significant increases at different time intervals in all groups. The mild elevation of these hepatic enzymes may be associated with transient alterations in hepatocellular membrane permeability due to haemodynamic changes induced by anaesthetic agents or as an immediate response to altered cardiac function (Anitha et al., 2019). Similar findings have been documented by Singh et al., (2006) and Kumar et al., (2014) in buffalo calves. The transient nature of these changes may also reflect hepatic metabolism of the administered drugs.
       
Gamma-glutamyl transferase (GGT) values showed a non-significant increase in all groups at various time intervals. The slight elevation in GGT activity may be indicative of mild hepatic stress induced by anaesthetic agents. However, the absence of significant changes suggests minimal adverse effects on hepatic and biliary function. Similar findings have been reported in buffaloes and buffalo calves (Sharma et al., 2020; Vijay Pal et al., 2016).
 
Oxidative stress parameters
 
The mean±S.E. values of oxidative stress parameters are presented in Table 3. A significant (P<0.05) increase in serum cortisol levels was observed at different time intervals in all three groups (Fig 5). The elevation in cortisol concentration may be attributed to increased secretion or a reduced metabolic clearance rate (MCR) of the hormone under anaesthesia-induced stress conditions. Similar findings have been reported by Choudhury et al. (2022) in goats following tiletamine–zolazepam anaesthesia, while Malik (2011) also documented increased plasma cortisol levels in water buffaloes administered butorphanol-medetomidine. However, Sekhar et al., (2020) reported that dexmedetomidine is a superior adjuvant to clonidine for epidural analgesia in cattle, offering better cardiorespiratory and biochemical stability. The combination of ropivacaine and dexmedetomidine provides enhanced analgesia with minimal physiological impact, making it recommended for use in cattle.

A significant (P<0.05) decrease in reduced glutathione (GSH) levels was observed at various time intervals in all groups (Fig 6). The decline in GSH may be associated with increased oxidative stress resulting from anaesthetic-induced mitochondrial dysfunction and enhanced generation of reactive oxygen species (ROS), leading to increased utilization of endogenous antioxidant reserves. Peak depletion of GSH may coincide with transient hypoxaemia, acidosis and enhanced lipid peroxidation (Mahalingam et al., 2014). Comparable observations have been reported in cattle and sheep under similar anaesthetic conditions (Gnanasekar and Vijayalakshmi, 2016; Ceylan et al., 2007).







Lipid peroxidation, assessed in terms of malondialdehyde (MDA) levels, showed a significant (P<0.05) increase in all groups (Fig 7). The elevated MDA levels indicate enhanced oxidative damage to cellular membranes, which may be attributed to hepatic metabolism of tiletamine-zolazepam, as both agents undergo extensive biotransformation in the liver (Fig 8). Similar findings have been reported in sheep (Ceylan et al., 2007), as well as in studies involving combinations with xylazine in sheep (Aydilek, 2007) and tramadol in miniature pigs (Jiang et al., 2014).

The present study evaluated tiletamine-zolazepam alone and in combination with dexmedetomidine or butorphanol in buffalo calves under Indian conditions. Haemoglobin and packed cell volume showed significant reductions in Groups B and C, whereas total erythrocyte count, total leukocyte count and differential leukocyte counts exhibited non-significant alterations. Serum glucose and cortisol levels increased significantly following induction, while other biochemical parameters showed only mild and transient changes. Oxidative stress indices revealed increased cortisol and malondialdehyde levels along with decreased reduced glutathione levels.
       
Importantly, no mortality or major complications were observed during the anaesthetic trials. All three anaesthetic protocols provided effective, safe and reliable anaesthesia, with haemato-biochemical and oxidative stress parameters remaining within physiological limits and returning to baseline values during recovery. These findings indicate that tiletamine-zolazepam, alone or in combination with dexmedetomidine or butorphanol, can be safely employed for both minor and major surgical procedures in buffalo calves.

The present study was supported by Dau Shri Vasudev Chand rakar Kamdhenu Vishwavidyalaya (DSVCKV) Durg, Chhattisgarh.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Institute of Animal Ethical Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish or preparation of the manuscript.