Clinico-physiological assessment of dexmedetomidine and butorphanol as preanaesthetics to tiletamine-zolazepam anaesthesia in buffalo calves (Bubalus bubalis)

Introduction

Water buffalo (Bubalus bubalis) is a valuable species with excellent zootechnical characteristics for both milk and meat production (Guerri et al., 2021) [14] . Anaesthesia is achieved through agents that reduce neural activity at local, regional or central levels. In surgical practice, balanced anaesthesia is crucial, as it provides the required combination of hypnosis, analgesia and adequate muscle relaxation. General anaesthesia is regarded mandatory for any surgical intervention as it provides complete unconsciousness, better insensitivity to pain, good muscle relaxation, freedom from reflex responses and loss of motor ability (Thurmon et al., 1996) [38] . Though most surgical procedures in ruminants are carried out under local or regional analgesia, general anaesthesia may be preferred in certain complicated procedures like diaphragmatic herniorrhaphy, thoraco-pericardiotomy, repair of ruptured suspensory ligaments, orthopaedic surgery, keratoplasty and repair of ventral hernia etc. (Riazuddin et al., 2004) [28] . Preanesthetic medication makes the patient easier to handle, decreases secretions and minimizes the total dose of anaesthetics to produce the desired level of anaesthesia (Arya et al., 2021) [3] . Induction of anaesthesia of intractable animals safer, greatly reduces the incidence and the intensity of any adverse effects, provides pre-emptive analgesia and smooth recovery (Kaufman et al., 2005) [17] . Tiletamine-zolazepam is commonly employed for anaesthetic induction because it produces a fast onset of action along with effective muscle relaxation. Dexmedetomidine, a potent α2-adrenergic agonist, induces sedation analgesia and muscle relaxation with minimal respiratory depression (Ragab et al., 2022) [27] . Butorphanol, a mixed κ-agonist and µ-antagonist opioid, provides effective analgesia and mild sedation (Maidanskaia et al., 2023) [25] .

Comparative evaluation of these agents in buffalo calves as preanaesthetics to tiletamine-zolazepam is limited. Thus, the present study was designed to evaluate and compare the clinico-physiological effects produced by dexmedetomidine or butorphanol when used in combination with tiletaminezolazepam anaesthesia.

Materials & Methods Selection of Animals and Experimental Design

The study was carried out in the Department of Veterinary Surgery and Radiology, College of Veterinary Science and Animal Husbandry, Anjora, Durg (C.G.). Eighteen healthy, non-descript male buffalo calves weighing 80-100 kg and aged 8 months to 1 year were randomly assigned to three groups (A, B, and C), each comprising six animals. Prior to the experiment, all calves were dewormed orally with fenbendazole (Panacur ® 150 VET) @ 5 mg/kg body weight. Feed was withheld for 24 hours and water for 12 hours before anaesthetic administration. Standard management and feeding practices were maintained throughout the study. A pilot trial was undertaken to determine the minimum effective drug doses for the experiment. Glycopyrrolate (Pyrolate ® ) @ 0.01 mg/kg intramuscularly was administered to all animals 10 min before the trial. In Group A, anaesthesia was induced with tiletaminezolazepam (Zoletil ® 50) @ 2.5 mg/kg body weight via slow intravenous injection. Group B animals received dexmedetomidine (Dextomid * ) @ 2 µg/kg intravenously, followed 10 min later by tiletamine-zolazepam at the same dose. In Group C, butorphanol (Butodol ® -1) @ 0.075 mg/kg body weight was administered intravenously before implementing the designated anaesthetic protocol. Ethical approval for the experimental procedures was obtained from the Institutional Animal Ethics Committee (IAEC Approval No.-VSR/PhD-1/2025).

Parameters Studied Clinical Parameters

The assessment of anaesthesia was done on the basis of the following clinical parameters were recorded on before sedation (0), 5 mins. after sedation, on induction and 5, 15, 30, 45, 60 and 120 min interval after anaesthesia. The following clinical parameters were assessed viz. onset of sedation, induction of anaesthesia, record of various reflexes and behavioural responses like sedation, analgesia, jaw relaxation, palpebral reflex, pedal reflex, salivation, muscle relaxation of limb, muscle relaxation of abdomen, relaxation of anal sphincter, ataxia, regurgitation, position of eye ball, duration of anaesthesia, quality of anaesthesia, return to head rightening reflex, sternal recumbency time, standing time and complete recovery time. All the aforementioned clinical parameters, along with their corresponding numeric scoring system, are presented in Table 1, as adopted and modified by Singh et al., (2013) [34] and Guerri et al., (2021) [14] .

Table 1: Numeric score system used for recording of various reflexes and responses (as adopted and modified by Singh et al., 2013 [34] and Guerri et al., 2021) [14] .

No. Parameters

Physiological Parameters

The physiological parameters were observed at 0 min before anaesthesia, 10 min after premedication and post induction at 10, 20, 40, 60, 120 min post anaesthesia. Physiological parameters included rectal temperature, heart rate and respiration rate.

Statistical Analysis: Statistical analysis of the data was done using SPSS 25v statistics software program and ANOVA technique according to the method described by Snedecor and Cochran (1994) [36] . Statistically significant difference was considered at 5 per cent level.

Results

 Clinical Parameters  Assessment of Anaesthesia

Onset of sedation

The onset of sedation (Fig 1) in group B was significantly (P<0.05) longer (4.36±0.21 min) than group A (0 min) and C (0 min).

Induction time/Induction of anaesthesia

The induction time (Fig 1) was non-significant among all the three groups A, B and C which was recorded as 0.38±0.04, 0.34±0.03 and 0.39±0.05 min respectively.

Quality of anaesthesia

Quality of anaesthesia (Fig 9) was excellent (scored 4) after administration with tiletamine-zolazepam in all the three groups and characterized by rapid induction, excellent muscle relaxation and analgesia (as adopted and modified by Bodh et al., 2013) [4] . Respiration rate: The respiration rate was noted a significant (P<0.05) decreased up to 20 min in group A. In group B, there was a significant (P<0.05) decrease in the respiration rate after premedication with dexmedetomidine which further significantly (P<0.05) decreased up to 40 min after induction. The animals of group C, showed a nonsignificant decrease in respiration rate after premedication with butorphanol which further significantly (P<0.05) decreased up to 20 min after induction. This trend is evident from the Table 2 and illustrated in Fig 12.

Complication

All animals in Groups A and B exhibited smooth, uncomplicated recoveries following tiletamine-zolazepam anaesthesia, with no post-anaesthetic complications observed, whereas animals in Group C displayed varying degrees of excitement during recovery.

Discussion

Sedation began earlier in group B than in the other groups, likely due to the rapid onset of dexmedetomidine associated with its lipophilicity (Dewangan et al., 2025) [10] . Gurukar (2016) [15] similarly recorded a sedation onset of 3.17±0.36 min following intravenous dexmedetomidine at 2.5 µg/kg body wight in cattle. Cagnardi et al. (2017) [6] also noted that dexmedetomidine produced dependable and prolonged sedation in calves. Animals premedicated with dexmedetomidine exhibited a shorter induction time compared with the other groups, probably due to its synergistic effects, which provided adequate sedation before tiletamine-zolazepam administration. This enhanced response may be attributed to dexmedetomidine's α2receptor agonism in the locus coeruleus, which promotes sedation, hypnosis and analgesia (Sinclair, 2003) [31] . In the present study, rapid induction (<60 seconds) was observed across all three groups with no significant intergroup variation, consistent with the findings of Ukkali (2022) [40] , who reported an induction time of 35.84±0.57 seconds in cattle receiving tiletamine-zolazepam. Tiletamine-zolazepam produced a rapid onset of sedation in all groups, which aligns with its known pharmacokinetic profile in large ruminants, where tiletamine's NMDAreceptor antagonism and zolazepam's benzodiazepine-like action contribute to rapid central nervous system depression (Akaraphutiporn et al., 2024) [2] . Analgesia following induction was excellent across treatments, likely due to tiletamine's potent interaction with the PCP site of the NMDA receptor and its non-competitive antagonistic effect, which can interrupt sensory transmission and sustain

Time interval (min)

analgesia beyond the period of anaesthetic action (Lin et al., 1993) [24] . Previous studies also support the superior analgesic efficacy of tiletamine-zolazepam compared with other anaesthetic combinations: Singh (2021) [33] reported better analgesia and a longer duration of effect than ketamine or propofol with midazolam premedication in calves, while Ukkali (2022) [40] found it superior to ketamine-midazolam in cattle. Sindak (2003) [32] recorded excellent analgesia with xylazine-tiletamine-zolazepam relative to xylazine-ketamine in calves. Additional evidence from buffaloes and small ruminants indicates similarly strong analgesic and muscle-relaxant properties (Bodh et al., 2015; Sahu, 2024) [5,29] . Lin and Walz (2014) [21] reported that combination of tiletamine and zolazepam produce better muscle relaxation, analgesia and duration of action than ketamine alone. Similarly, Carroll et al. (1997) [6] documented that tiletamine-zolazepam @ 5.5 mg alone induced sufficient analgesia for ovariectomy operation in goats. In group B, jaw relaxation and overall muscle relaxation persisted for up to 30 minutes after TZ administration, a longer duration than in groups A and C, likely due to the additional α2-agonist, which enhances muscle relaxation through inhibition of α2-adrenoceptors at the spinal interneuron level (Sinclair, 2003) [31] . Following induction with TZ, all groups demonstrated marked muscle and anal sphincter relaxation, attributable to the combined relaxant effects of zolazepam and the α2-agonist dexmedetomidine, whereas tiletamine itself does not significantly affect muscle tone or cranial and spinal reflexes (Dewangan et al., 2025) [10] . Similar findings were reported by Sulekha et al. (2024) [37] , who attributed strong muscle relaxation to zolazepam's inhibition of internuncial neurons in the spinal cord (Hall et al., 2001) [16] . Saini et al. (2017) [30] noted that dexmedetomidine provided profound analgesia and muscle relaxation and when combined with TZ, prolonged jaw relaxation, supporting its suitability as a premedicant. Palpebral reflex was very weak in group A and C while abolished completely in group B. This effect is likely due to the combined action of zolazepam, a benzodiazepine, and tiletamine, a dissociative anaesthetic, which together exert complementary modulation at the interneuronal junction by influencing both benzodiazepine and NMDA receptors (Ukkali, 2022) [40] Similarly, Singh et al. (2013) [34] observed complete abolition of palpebral reflex after administration of α2-agonist in buffaloes.

Following induction with TZ anaesthesia, pedal reflex was completely absent in all three groups, indicating achievement of a surgical plane of anaesthesia. This surgical depth was maintained for a longer period in group B, persisting for up to 30 min. These findings were in concurrence with Pawde et al. (2000) [26] in buffalo calves and Ragab et al. (2022) [27] in goats after ketamine, propofol or ketamine and ketamine-propofol anaesthesia respectively up to 60 min. The extended duration of anaesthesia observed in group B appears to result from the additive effects of dexmedetomidine combined with tiletamine-zolazepam (Sahu, 2024) [29] . Similar findings have been documented in goats, where tiletamine-zolazepam-xylazine produced a longer anaesthetic period (100.83±43.37 min) than ketamine-xylazine (95.17±12.32 min) (Gicana et al., 2021) [13] . In the present study, animals in group B also demonstrated significantly (P<0.05) longer times for return of head-righting reflex, sternal recumbency, standing time and complete recovery compared with groups C and A. The deeper sedation and prolonged analgesia produced by dexmedetomidine when used with TZ likely contributed to slower metabolic activity and delayed redistribution and metabolism of the drugs. Conversely, the shorter recovery periods in groups A and C suggest more rapid metabolic clearance of TZ in the absence of dexmedetomidine. The α2-agonist may have further influenced recovery in group B by enhancing sedation or prolonging zolazepam elimination (Dewangan et al., 2025) [10] . Comparable prolongation of standing time after TZ anaesthesia was reported by Ukkali (2022) [40] , while Singh (2021) [33] noted extended sternal recumbency, standing time and total recovery duration in calves given tiletamine-zolazepam compared with ketaminemidazolam. Animals in groups A and C of the present study regained standing more quickly. The inclusion of dexmedetomidine in group B likely accounted for the lengthened recovery, consistent with its known capacity to deepen and extend anaesthesia. Animals premedicated with butorphanol displayed brief agitation such as restlessness, excessive sniffing, and exaggerated tail movement along with a shorter recovery phase. This pattern aligns with earlier reports that opioids can induce central nervous system stimulation in ruminants (Maidanskaia et al., 2023; Dzikiti et al., 2016; Delgado et al., 2021) [25,9,12] . Overall, recovery from dexmedetomidine-TZ and TZ alone was smooth and uncomplicated, whereas butorphanol-TZ anaesthesia produced transient behavioural abnormalities. Statistical comparison among the groups confirmed a significant (P<0.05) difference in recovery times. Administration of TZ produced excellent anaesthetic quality across all three groups, marked by rapid induction, strong muscle relaxation and effective analgesia. These effects are consistent with the rapid CNS uptake of TZ following intravenous administration, followed by swift redistribution and efficient metabolic clearance (Ko et al., 2007) [18] . Complete abolition of reflexes in all groups after induction confirmed attainment of a surgical plane of anaesthesia. Among the treatment protocols, dexmedetomidine premedication resulted in the best overall anaesthetic quality, with group B maintaining optimal anaesthesia for up to 30 minutes, whereas groups C and A exhibited excellent anaesthesia for approximately 15 minutes. Although TZ alone provided good muscle relaxation, its duration was shorter, likely reflecting the central musclerelaxant action of zolazepam, as seen in group A. The enhanced and prolonged muscle relaxation observed in the dexmedetomidine group suggests that the α2-agonist augmented TZ-induced sedation and muscle relaxation, findings that are in line with observations reported by Singh et al. (2024) [35] . The decline in rectal temperature across groups can be attributed to reduced basal metabolic rate, decreased muscular activity and depression of the hypothalamic thermoregulatory centre (Kumar et al., 2014) [19] . The changes were consistent with indicating hypothermia with α2-agonists due to CNS depression and reduced skeletal muscle tone. Dexmedetomidine-induced hypothermia has been similarly attributed to α2-adrenergic stimulation, lowered metabolic rate and muscle relaxation (Tranquilli, 2015) [39] . The initial tachycardia observed in Groups A and C aligns with the known sympathomimetic properties of tiletamine and the excitatory, κ-receptor-mediated CNS stimulation seen with butorphanol. Tiletamine enhances cardiac output through increased sympathetic tone and catecholamine release, explaining the marked elevation in heart rate (Abalos et al., 2016) [1] . Similarly, butorphanol is recognized to induce behavioural excitement and sympathetic activation, contributing to persistent tachycardia during the early anaesthetic period (Maidanskaia et al., 2023) [25] . Conversely, dexmedetomidine induced significant bradycardia in Group B due to its potent α2-adrenergic agonist action, decreasing norepinephrine release and increasing vagal tone (Pawde et al., 2000) [26] . Following tiletamine-zolazepam induction, however, a compensatory increase in heart rate occurred, reflecting the opposing sympathomimetic action of tiletamine (Lin et al., 1989) [22] . The respiratory depression following tiletamine-zolazepam was consistent with its pharmacological actions. Zolazepam potentiates GABAergic inhibition, reducing respiratory centre responsiveness, while tiletamine's NMDA antagonism suppresses medullary respiratory neurons, contributing to apneustic and irregular breathing (Chang et al., 2024) [8] . The marked decrease in respiration rate in dexmedetomidine-treated animals aligns with α₂-agonistinduced central depression of respiratory centres and diminished chemoreceptor sensitivity (Cagnardi et al., 2017) [6] . In contrast, butorphanol initially increased respiration rate due to κ-opioid receptor-mediated CNS excitation and sympathetic activation (Maidanskaia et al., 2023) [25] , before the depressant influence of tiletamine-zolazepam emerged. Overall, all the physiological alterations in all the groups were transient, values returning towards baseline by 120 minutes. All values were within acceptable physiological limits, indicating a wide margin of safety of these drug combinations.

Conclusion

Intravenous administration of tiletamine-zolazepam produced a short and safe anaesthetic period with smooth recovery in buffalo calves. Premedication with dexmedetomidine before induction provided superior anaesthetic quality, as evidenced by rapid and smooth induction, profound analgesia, excellent muscle relaxation and prolonged anaesthetic duration without observable complications. Butorphanol premedication provided adequate analgesia and moderate muscle relaxation with a comparatively shorter anaesthetic duration of anaesthesia, although transient excitatory behaviour was observed during sedation and recovery in some animals. Among the anaesthetic protocols evaluated, the glycopyrrolatedexmedetomidine-tiletamine-zolazepam combination was found to be the most balanced and effective regimen, compared to glycopyrrolate-butorphanol-tiletaminezolazepam and glycopyrrolate-tiletamine-zolazepam alone, in terms of overall anaesthetic quality, duration and recovery characteristics. All the changes in physiological parameters remained within normal limits and were transient in nature, returning to baseline values by 120 min, indicating that all the three protocols were clinically safe and resulted in uneventful recovery without any complications in buffalo calves.

Acknowledgement

AcknowledgementsBeing part of Ph.D. research work of the first author, funded by Dau Shree Vasudev Chandrakar Kamdhenu Vishwavidyalaya Durg, the authors are grateful to the University for all the support rendered. The authors also express their sincere gratitude to the College of Veterinary Science and Animal Husbandry, Anjora Durg, for providing laboratory facilities and support.

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