Clinico-physiological response of dexmedetomidine and fentanyl citrate as premedicants to zolazepam-tiletamine-isoflurane anaesthesia in canine

Introduction

Perfect anaesthesia is essential for successful surgical intervention, ensuring adequate hypnosis, analgesia and muscle relaxation. General anaesthesia is a reversible drug-induced depression of the central nervous system with loss of sensory, motor and autonomic functions (Tranquilli and Grimm, 2015) [75] . Balanced anaesthesia, achieved through the use of multiple drug combinations, enhances anaesthetic efficacy while minimizing individual drug side effects (Campagna et al., 2003; Kumar et al., 2012) [7,43] . In veterinary practice, anaesthesia is induced using injectable agents and commonly maintained with inhalation anaesthetics like isoflurane due to better control and rapid recovery (McKenzie, 2008; Bustamante et al., 2018) [6,55] . Premedicants such as atropine, dexmedetomidine and fentanyl play a vital role in stabilizing physiological responses and improving analgesia (Lerche, 2015; Bloor et al., 1992; Keating et al., 2013) [4,32,51] . The tiletamine-zolazepam combination provides effective dissociative anaesthesia with muscle relaxation, while isoflurane ensures safe maintenance with minimal cardiopulmonary depression (Plumb, 2018; Matthews et al., 2003) [54,61] . However, limited studies in India have evaluated this combination; therefore, the present study was undertaken to assess the efficacy and suitability of tiletamine-zolazepam-isoflurane anaesthesia with different preanaesthetics in canine surgical patients.

Materials and Methods

The present study was conducted at the Department of Veterinary Surgery and Radiology and Teaching Veterinary Clinical Complex (TVCC), College of Veterinary Science and Animal Husbandry, Anjora, Durg (C.G.). A total of 18 mongrel dogs of either sex, weighing between 15-30 kg body weights, were selected for the study. All animals were dewormed prior to experimentation and randomly divided into three groups (Group I, II and III), each comprising six animals. The animals were fasted for 12 hours, with water withheld for 6

Hours before administration of anaesthesia. A pilot study was conducted to standardize the drug dosages, and the minimum effective doses were selected for the experiment. The animals were subjected to three different anaesthetic protocols. In Group I, atropine sulphate (0.04 mg/kg IM) was used as premedication, followed by induction with zolazepamtiletamine (3 mg/kg IV) and maintenance with 1.5-2% isoflurane in 100% oxygen. In Group II, atropine sulphate (0.04 mg/kg IM) was combined with dexmedetomidine (10 µg/kg IV), while in Group III, atropine sulphate (0.04 mg/kg IM) was combined with fentanyl citrate (4 µg/kg IV), with induction and maintenance protocols similar to Group I. Drug dosages were calculated based on body weight. Baseline values of all parameters were recorded prior to administration of premedicants. Following premedication, animals were allowed for onset of sedation. Intravenous access was established, and normal saline was administered at a rate of 5 ml/kg/h. Anaesthesia was induced 10 minutes after premedication using zolazepam-tiletamine administered intravenously until loss of pedal reflex. Endotracheal intubation was performed using appropriatesized tubes, and anaesthesia was maintained with isoflurane (1.5-2%) in 100% oxygen for a duration of 90 minutes using a closed rebreathing system. After completion of the procedure, isoflurane was discontinued and oxygen was continued for an additional 5 minutes. Clinical parameters including onset and quality of sedation, induction time, quality of induction, reflex responses (jaw relaxation, palpebral and pedal reflex), and recovery characteristics such as extubation time, sternal recumbency time, standing time, complete recovery time and duration of anaesthesia were recorded using standardized scoring systems (Amarpal et al., 1996; Carter et al., 2013; Tamura et al., 2014) [2,9,73] .

Physiological parameters including rectal temperature (°F), respiration rate (breaths/min) and heart rate (beats/min) were recorded at predetermined intervals. Haemodynamic parameters including systolic, diastolic and mean arterial blood pressure were recorded using a non-invasive monitor, while capillary refill time was assessed manually. Mean blood pressure was calculated using the formula: MBP = DBP + 1/3 (SBP -DBP).

Results

3.1Assessment of anaesthesia 3.1.1Onset of Sedation

The onset of anaesthesia in all the three groups has been shown in Table 1 and represented in Fig. 1. The mean±S. E. onset of sedation was recorded as 0, 5.50±0.42 and 8.83±0.47 minutes in Group I, II and III, respectively, with significant differences between the groups (mathbb p<0.05). No sedation was observed in Group I. Group II exhibited rapid and marked sedation with lateral recumbency, whereas Group III showed delayed and mild sedation. Overall, dexmedetomidine produced a faster and more pronounced sedative effect compared to fentanyl citrate.

Sedation score

The Sedation score in all the three groups has been shown in Table 2 and represented in Fig. 2. Intervals in all the three groups are depicted in Table 3 and Fig. 3. The Mean ± S.E. values of palpebral reflex scores were 0.00±0.00 at 0 min and after atropine administration in all the groups, indicating intact reflexes. In Group I, scores increased to 0.67±0.21 at 10 min post-induction and reached 3.00±0.00 by 20 min, remaining at this level up to 90 min, showing complete abolition of reflex. In Group II, scores increased significantly to 1.17±0.17 after sedation and reached 3.00±0.00 as early as 10 min post-induction, which persisted up to 90 min. In Group III, scores increased to 0.50±0.22 after sedation and 2.67±0.21 at 10 min postinduction, with complete abolition (3.00±0.00) from 20 min onwards. Within-group comparison revealed a significant (mathbf p<0.05) increase in scores from baseline in all groups. Inter-group comparison indicated a significantly earlier abolition of palpebral reflex in Group II followed by Group III and Group I.

Relaxation of jaw

The Mean±SE values of Relaxation of jaw score at various Intervals in all the three groups are depicted in Table 4 and Fig. 4. In Group I, scores increased to 2.33±0.21 at 10 min post-induction and reached 3.00±0.00 by 20 min, remaining at this level up to 60 min, followed by a slight decrease at later intervals. In Group II, scores increased significantly to 1.33±0.21 after sedation and reached 3.00±0.00 as early as 10 min postinduction, which persisted up to 90 min. In Group III, scores increased to 0.67±0.21 after sedation and 2.50±0.22 at 10 min post-induction, with complete relaxation (3.00±0.00) from 20 min onwards, followed by a gradual decline at later intervals. Within-group comparison revealed a significant (mathbf p<0.05) increase in scores from baseline in all groups. Inter-group comparison indicated a significantly earlier and more sustained relaxation of jaw in Group II followed by Group III and Group I. Various intervals in all the three groups are depicted in Table 6 and Fig. 6. 0-Strong reaction to pinching of the inter-digital skin of the foot, 1-Weak responses to pinching of the inter-digital skin of foot, 2-Occasional response to pinching of the interdigital skin of the foot, 3-No response to pinching of the inter-digital skin of the foot. The Mean±S.E. values of analgesia scores were 0.00±: 0.00 at 0 min and after atropine administration in all the groups, indicating absence of analgesia. In Group I, scores increased to 1.33±0.21 at 10 min post-induction and reached 3.00±0.00 by 20 min, remaining at this level up to 90 min, indicating complete analgesia. In Group II, scores increased significantly to 1.50±0.22 after sedation and reached 3.00±0.00 as early as 10 min post-induction, which persisted up to 90 min. In Group III, scores increased to 0.67±0.21 after sedation and 2.67±0.21 at 10 min post-induction, with complete analgesia (3.00±0.00) from 20 min onwards. Within-group comparison revealed a significant (mathbf p<0.05) increase in scores from baseline in all groups. Inter-group comparison indicated a significantly earlier onset and deeper analgesia in Group II followed by Group III and Group I.

Induction of Anaesthesia

The mean±SE values of Induction of Anaesthesia (seconds) in all the three groups has been summarized in Table 7 and represented in Fig. 7. In the present study induction of anaesthesia was recorded from the time of injection of Zolazepam-Tiletamine until pedal reflex was completely lost. After administration of Zolazepam-Tiletamine, the induction of anaesthesia was rapid, smooth and free from any untoward reactions like struggling and paddling in all the three groups. The mean anaesthetic induction time was 87.3±1.22, 36.3±2.94 and 67.3±2.18 sec. in groups I, II and III respectively. The induction time in group II was significantly (p˂0.05) rapid as compared to group I and group III.

Quality of Anaesthetic induction

The mean±SE values of Quality of anaesthetic induction in different experimental groups have been depicted in Table 8. The mean±SE scores of quality of anaesthetic induction was observed to be in 2.00±0.00, 4.00±0.00 and 2.50±0.22 in Group I, Group II and Group III respectively. Whereas induction was very smooth in Group II, quite smooth in Group III whereas moderately smooth induction was found in Group I. The quality of anaesthetic induction in Group II varied significantly (p<0.05) as compared to groups I and III

Quality of Anaesthetic recovery

The mean±SE values of Quality of Anaesthetic Recovery in different experimental groups have been depicted in Table 9. The mean±SE values of Quality of anaesthetic recovery was observed to be in 2.30±0.21, 3.50±0.22 and 2.33±0.21 in Group I, Group II and Group III respectively. The quality of anaesthetic recovery in Group II varied significantly (p<0.05) compared to groups I and III.

Inhalant anaesthesia recovery Time (min)

The mean±SE values of Inhalant anaesthesia recovery time In different experimental groups have been depicted in Table 10 and Fig. 8.

Table 10: Mean ± S.E. of Inhalant anaesthesia recovery time (min) in different groups in different treatment groups in canine

Group

Inhalant anaesthesia recovery Time (min) 1 0.72±0.20 a 2 14.17 ± 0.47 c 3 6.70 ± 0.78 b Values bearing different superscript vary significantly (P˂0.05) between groups The mean Inhalant anaesthesia recovery time in group II was significantly (p<0.05) longer (14.17±0.47 mins) than in group I (0.72±0.20 mins) and group III (6.70±0.78 mins). A significant difference (p<0.05) was observed between all the three groups.

Dose of Induction and Maintenance agent

Induction dose of Tiletamine-Zolazepam undertaken in the present study i.e., @ 3mg/kg b.wt. Was found to be sufficient for good induction and intubation amongst animals of all the three groups. However, evaluating the MAC% of Isoflurane somehow varied amongst the groups. In group I animals, it was found that three out of six animals required Isoflurane concentration 2.5-3% after 45 minutes of anaesthesia for excellent maintenance. In group II, all the animals were efficiently maintained within 1.5 -2% MAC%. In group III animals, two out of six animals required Isoflurane concentration 2.1-2.5% for efficient maintenance of anaesthesia.

Extubation time (min) The mean±SE values of extubation time (min) in different

Experimental groups have been depicted in Table 11 and Fig. 9. The mean extubation time in group II was significantly (p˂0.05) longer (8.82±0.30 mins) than in group I (6.21±0.47 mins) and group III (7.60±0.33 mins). A significant difference (p˂0.05) was observed between all the three groups. 12 and represented in Fig. 10.

The mathbf Me an±mathbf SE of time taken by animals to regain sternal recumbency after last supply of isoflurane was calculated. The mathbf Mean±mathbf SE of Total amount of isoflurane consumed was calculated in each group. There was significant (p˂0.05) difference in total amount of isoflurane consumed by group Ⅰ compared to group and group. ⅠⅡ Ⅱ 22 and represented in Fig. 13. In Group I, rectal temperature showed a non-significant decrease from baseline up to 30 min, followed by a significant (p<0.05) decrease from 45 min to 90 min, reaching 98.46±0.29°F. In Group II, rectal temperature exhibited a non-significant decrease up to 10 min after sedation, followed by a significant (p<0.05) decline from 10 min post-induction onwards, reaching the lowest value of 95.48±0.76°F at 90 min. In Group III, a non-significant decrease was observed up to 10 min after sedation, followed by a significant (p<0.05) decrease from 10 min postinduction to 90 min, reaching 97.18±0.09°F. Inter-group comparison revealed a significant (p<0.05) difference in Groups II and III compared to Group I from 45 min to 90 min, with Group II showing the maximum decline followed by Group III. In Group I, heart rate showed a significant (p<0.05) increase following atropine administration and continued to increase significantly up to 30 min post-induction, reaching 122.67±2.62 beats/min. Thereafter, a gradual decrease was observed from 45 min to 90 min, returning near baseline values. In Group II, heart rate increased significantly (p<0.05) after atropine administration, followed by a significant decrease after dexmedetomidine administration. Subsequently, heart rate increased significantly post-induction up to 30 min and then showed a gradual decline up to 90 min, approaching baseline values. In Group III, heart rate increased significantly (p<0.05) after atropine administration, followed by a non-significant decrease after fentanyl citrate administration. Post-induction, heart rate increased significantly up to 30 min and thereafter decreased gradually up to 90 min, returning towards baseline. Inter-group comparison revealed a significant (p<0.05) difference in Group II at multiple intervals (10 min after sedation up to 75 min postinduction) when compared to Groups I and III, with comparatively lower heart rate values observed in Group II. The mathbf Mean±mathbf S. E. values of respiration rate (breaths/min) showed a declining trend following sedation and induction in all the groups. In Group I, respiration rate decreased significantly (p<0.05) from 30.17±1.56 at baseline to 21.56±0.44at90 min. In Group II, a significant (p<0.05) decrease was observed from 33.33±4.52 to 21.00±0.73 after sedation, further declining up to 16.53±0.43 and remaining below baseline up to 90 min. In Group III, respiration rate decreased significantly (mathbf p ≥ts0.05) from 27.33±2.72 to 24.33±0.28 after sedation and continued to decline up to 20.67±1.31 at 90 min.Withingroup comparison revealed significant (mathbf p<0.05) reduction in respiration rate over time in all groups. Intergroup comparison indicated significantly lower respiration rates in Group II at early intervals followed by Group III and Group I. In animals of Group I, systolic blood pressure showed a non-significant increase from baseline (0 min) to after premedication (139.67±5.52 to 140.17±3.51). It then decreased nonsignificantly (p>0.05) at 60 minutes (128.33±8.50) before rising non-significantly again to (135.17±8.28) at 90 minutes when compared to the baseline values. In animals of Group II, systolic blood pressure increased significantly ≤ft (p<0.05right) from baseline (0 min) to 10 min. after sedation (132.67±2.36to151.00±2.48). Following ZT induction, it decreased significantly (mathbf p<0.05) over the next 20 minutes and non-significantly The mathbf Mean±mathbf S. mathbf E. Values of diastolic blood pressure (mmHg) showed mild variations across time intervals in all the groups. In Group I, values showed a non-significant change from baseline (95.50±5.37) and gradually decreased to 92.83±3.66 at 90 min. In Group II, diastolic pressure increased significantly (mathbf p<0.05) to 102.00±3.50 after sedation, followed by a nonsignificant decline up to 86.00±1.57 at 90 min. In Group III, values increased significantly (p<0.05) to 95.17±3.98 after sedation and then decreased non-significantly to 80.33±2.04 at 90 min. Within-group comparison revealed significant (mathbf p<0.05) changes mainly after sedation in Groups II and III, while inter-group comparison indicated significant differences (mathbf p<0.05) in Group II at certain intervals compared to Groups I and III. The Mean±S.E. values of mean blood pressure (mmHg) showed an initial increase following atropine and sedation, followed by a gradual decline during maintenance in all the groups. In Group I, mean blood pressure increased up to 105.33±1.31 and then decreased significantly (mathbf p<0.05) from 93.67±1.48 at 10 min post-induction to 87.50±1.65 at 90 min. In Group II, values increased significantly (mathbf p<0.05) to 119.50±1.26 after sedation, followed by a gradual decline up to 98.33±0.80 at 90 min. In Group III, a non-significant increase was observed up to 112.33±1.20 after sedation, followed by a gradual decline with slight rise at 90 min. Withingroup comparison revealed significant (mathbf p<0.05) changes over time, while intergroup comparison indicated significantly higher mean blood pressure values in Group II at most intervals compared to Groups I and III.

Discussion 4.1 Assessment of anaesthesia 4.1.1 Onset of sedation

In the present study, animals of Group II receiving dexmedetomidine exhibited an earlier and more profound onset of sedation compared to other groups. Atropine alone did not produce any sedative effect, whereas fentanyl citrate induced mild to moderate sedation within approximately 7-8 minutes. Dexmedetomidine produced marked sedation characterized by ataxia, muscle relaxation, recumbency, head lowering and eyelid closure, thereby facilitating easier handling of animals. These findings are consistent with previous reports describing similar sedative effects of α2adrenergic agonists in dogs (Kuusela et al., 2001, 2003) [46] . The onset of sedation observed in this study aligns with earlier findings of 5-15 minutes following dexmedetomidine administration (Granholm et al., 2007) [16] , while Ahmad et al. (2013) [1] reported a mean onset time of 4.5±0.96 minutes. The higher sedative potency of dexmedetomidine compared to medetomidine has also been well documented (Kuusela et al., 2000) [45] . In contrast, fentanyl citrate produced only mild sedation, supporting previous observations that opioid analgesics induce moderate central nervous system depression in dogs (Branson et al., 2001) [5] , with onset of action reported within 4-7 minutes (Hall et al., 2001a) [19] .

Sedation score

In the present study, animals of Group I receiving atropine sulphate did not exhibit any sedation, whereas those in Group II administered dexmedetomidine showed profound sedation. In contrast, Group III animals receiving fentanyl citrate exhibited only mild sedation. These findings are in agreement with Dent et al. (2019) [11] , who reported a sedation score of 3±0.8 for dexmedetomidine irrespective of the route of administration. Similar observations were reported by Singh et al. (2013a), who documented a sedation score of 3±0.09 with dexmedetomidine alone or in combination with fentanyl citrate. Kuusela et al. (2001) [46] also reported deep sedation at higher doses of medetomidine/dexmedetomidine, while lower doses produced only mild sedative effects. In the present study, fentanyl citrate induced mild sedation (score 0-1), which is consistent with Hamad et al. (2022) [20] . Additionally, moderate salivation, nasal secretions and panting were observed in some animals, with onset of panting around 7 minutes post-administration, corroborating the findings of Hughes and Nolan (1999) [25] .

Record of various reflexes 4.2.1 Palpebral reflex

In the present study, intact and strong palpebral reflex was observed in Groups I and III following premedication with atropine sulphate and atropine-fentanyl, respectively, whereas Group II (atropine-dexmedetomidine) showed a weak and sluggish reflex. Following induction with zolazepam-tiletamine, the palpebral reflex became markedly reduced but was not completely abolished, while complete abolition was observed during maintenance with isoflurane in all groups. These findings are consistent with Lin et al. (1993) [53] and Ko et al. (1998) [37] , who reported variable palpebral reflex responses following TZ administration. Dexmedetomidine has been reported to cause a moderate to marked reduction in palpebral reflex, with eyelids relaxed but reflex remaining sluggish (Sabbe et al., 1994; Kuusela et al., 2001) [46,65] . Greater suppression of the reflex with drug combinations compared to dexmedetomidine alone has also been documented (Ahmad et al., 2013) [1] , indicating synergistic effects. Similar observations of mild depression during sedation and complete abolition after induction have been reported by Khattri et al. (2013) [33] and Carroll et al. (1998) [8] .

Relaxation of Jaw

In the present study, animals of Groups I and III did not allow opening of the jaw following premedication with atropine sulphate and atropine-fentanyl, respectively, whereas Group II (atropine-dexmedetomidine) showed mild resistance with quick closure of the jaw. Following induction and maintenance, excellent jaw relaxation was observed in all groups. Tiletamine-zolazepam administered alone is known to provide inadequate muscle relaxation and may cause muscle rigidity and spontaneous movements (Dugassa et al., 2018; Gomez et al., 2013) [13] ; however, such effects were not evident in the present study, likely due to the muscle relaxant effect of isoflurane masking these responses. Previous studies have also reported inadequate muscle relaxation with TZ alone (Lin et al., 1993) [53] , whereas improved relaxation has been observed with the use of α2-agonists (Sinclair, 2003) [69] . The findings of good to excellent muscle relaxation in the present study are in agreement with earlier reports using similar drug combinations (Jadon et al., 1995; Ksenija et al., 2012; Santosh et al., 2013; Khattri et al., 2013) [27,33,39,67] , suggesting a synergistic effect of the anaesthetic agents.

Pedal reflex

In the present study, animals of Groups I and III exhibited intact and strong pedal reflex following premedication with atropine sulphate and atropine-fentanyl, respectively, whereas Group II (atropine-dexmedetomidine) showed a weak and sluggish pedal reflex. Complete abolition of pedal reflex was achieved after induction with zolazepamtiletamine and during maintenance with isoflurane in all groups. However, the reflex returned earlier in groups receiving TZ alone, indicating comparatively shorter duration of analgesia. These findings are in agreement with Wilson et al. (1993) [77] , who reported inadequate analgesia with TZ alone, which can be enhanced by α2-agonists due to synergistic action at central and spinal receptors. Dexmedetomidine provides analgesia through spinal and supraspinal mechanisms (Sinclair, 2003; Murrell and Hellebrekers, 2005) [56,69] , resulting in prolonged suppression of pedal reflex. Similar prolonged abolition of pedal reflex with drug combinations has been reported by Chen et al. (2005) [10] and Ahmad et al. (2013) [1] . In contrast, fentanyl citrate produced only partial analgesia, as evidenced by slow withdrawal response, which may be attributed to its opioidmediated analgesic effect (Wagner, 2002) [76] .

Analgesia

In the present study, Group II animals receiving dexmedetomidine-tiletamine-zolazepam exhibited superior quality and longer duration of analgesia compared to Groups I and III. This prolonged analgesic effect may be attributed to dexmedetomidine, which acts primarily at the spinal level by inhibiting nociceptive transmission in the dorsal horn (Kuusela et al., 2000; Talukdar and Hikas, 2009) [45] . Similar findings of enhanced and prolonged analgesia with α2-agonist combinations have been reported by Chen et al. (2005) [10] and Ko et al. (2007) [33] . Previous studies have also demonstrated moderate to prolonged analgesia with tiletamine-zolazepam combinations (Won et al., 2010; Ksenija et al., 2012) [39,78] , with further enhancement when combined with other agents (Hampton et al., 2019; Ratnu et al., 2021; Koli et al., 2021) [21,38,63] .In contrast, fentanyl citrate in Group III produced only mild analgesia (score 0-1), possibly due to the lower dose used, although its analgesic effect persisted for approximately 45 minutes, in agreement with Hall et al. (2001) [19] . While fentanyl is a potent μ-opioid agonist, peak analgesic effects may vary depending on dose and timing (Stoelting and Hillier, 1999; Gutstein et al., 2001; Hamad et al., 2022) [17,20,71] . Overall, dexmedetomidine and fentanyl contributed to improved analgesia during induction and maintenance with tiletamine-zolazepam and isoflurane.

Induction of Anaesthesia

In the present study, Group II exhibited a significantly ≤ft (p<0.05right) shorter induction time compared to Groups I and III, which may be attributed to the sedative effects of dexmedetomidine facilitating rapid induction. Tiletaminezolazepam administered intravenously is known to induce anaesthesia within 60-90 seconds (Kastner, 2007; Lin, 1991) [30, 52] , with induction times decreasing at higher doses (11.2±3.2 to 16.0±4.8 seconds) (Hellyer et al., 1989) [24] . Similar rapid induction has been reported with dexmedetomidine-based protocols, including 57.33±0.99 seconds (Arunkumar et al., 2017) [3] and 33.10±2.59 seconds (Koli et al., 2021) [38] , supporting the present findings. These results are further corroborated by studies of Rafee et al. (2015a), Hafez et al. (2017) [18] , Karasu et al. (2018) [29] and Ratnu et al. (2021) [63] , indicating that α2agonists significantly enhance the efficiency of anaesthetic induction.

Quality of Anaesthetic induction

In Group II, induction quality was excellent, with most animals allowing smooth endotracheal intubation on the first attempt. This may be attributed to effective preanaesthetic sedation with atropine and dexmedetomidine, resulting in reduced salivation and adequate muscle relaxation. In contrast, Groups I and III showed comparatively poorer induction quality, with persistence of pharyngeal reflexes and requirement of multiple intubation attempts, likely due to insufficient analgesia leading to coughing, excitement and movement during induction. These findings are in agreement with Hafez et al. (2017) [18] and Hampton et al. (2019) [21] , who reported similar variations in induction quality and intubation ease under comparable protocols. Post-induction apnoea and transient respiratory depression were observed following rapid intravenous administration of the TZ combination, which resolved spontaneously within a few minutes. Similar observations have been reported by Savvas et al. (2005) [68] and Hampton et al. (2019).

Quality of Anaesthetic recovery

Recovery quality was smooth in the majority of animals, with Group II showing comparatively better recovery than Groups I and III. In Groups I and III, transient emergence delirium was observed, characterized by hyperactive motor activity such as paddling, flailing, vocalization and excitation during the immediate post-anaesthetic period. This rough recovery may be attributed to the longer duration of action of tiletamine compared to zolazepam, leading to residual dissociative effects such as muscle rigidity, sympathetic stimulation and delirium (Landry and Maza, 2020) [49] . The smoother recovery observed in Group II could be due to the sedative and calming effects of dexmedetomidine. Isoflurane may further improve recovery quality by attenuating the excitatory effects of tiletamine during prolonged anaesthesia. These findings are in agreement with Pablo and Bailey (1999) [58] and Hampton et al. (2019a).

Inhalant anaesthesia recovery time

Inhalant anaesthesia recovery time, assessed by reappearance of pedal reflex, was shortest in Group I (TZisoflurane alone) and longest in the dexmedetomidine premedicated group, followed by the fentanyl premedicated group. The prolonged recovery in the dexmedetomidine group may be attributed to its potent sedative and CNS depressant effects, which delay return of reflexes. These findings are consistent with previous reports by Nam et al. (2013) [57] and Hampton et al. (2019) [21] . Ko et al. (1998) [37] also noted that recovery time decreases when lower doses of TZ are combined with sedatives, whereas Santosh et al. (2012) [66] reported prolonged recovery with increasing doses of dexmedetomidine.

Extubation time (min)

In the present study, all dogs were extubated within 10 minutes after discontinuation of anaesthesia. Extubation time was comparatively longer in the dexmedetomidine premedicated group (Group II) than in the fentanyl group (Group III), likely due to the prolonged sedative effects of dexmedetomidine. These findings are in agreement with Di Franco et al. (2024) [12] and Jarosinski et al. (2021) [28] . Group III animals showed shorter extubation time compared to Group I, which may be attributed to the additive effects of opioids in reducing anaesthetic requirements and facilitating faster recovery (Hunt et al., 2014) [26] . Prolonged extubation with higher doses of dexmedetomidine has also been reported by Kuusela et al. (2001) [46] . Variations in extubation time across studies may be due to differences in anaesthetic protocols and agents used, as also observed by Gomez et al. (2006) and Hampton et al. (2019) [21] .

Sternal recumbency time (min)

Preanaesthetic administration of dexmedetomidine significantly prolonged sternal recumbency time (SRT) in Group II compared to Groups I and III. This may be attributed to the enhanced sedative and synergistic effects of dexmedetomidine with tiletamine-zolazepam, leading to delayed recovery and redistribution of drugs. These findings are in agreement with Kwon et al. (2003) [48] and Hellyer et al. (1989) [24] , who reported prolonged SRT with α₂-agonist combinations and higher doses of TZ, respectively. Santosh et al. (2012) [66] also observed increased SRT with higher doses of dexmedetomidine. In contrast, shorter SRT reported by Pereira et al. (2019) [60] may be due to lower drug dosages. Overall, dexmedetomidine significantly prolongs SRT due to its potent sedative effects.

Standing time (min)

In the present study, animals in Groups I and III required considerable effort to attain standing, often showing excitement, paddling and flailing, with multiple attempts needed to achieve sternal recumbency and standing. These findings are in agreement with Hampton et al. (2019a). In contrast, Group II animals exhibited delayed but smoother standing without such excitatory signs, likely due to the sedative effects of dexmedetomidine. However, standing time was prolonged in this group, consistent with reports by Singh et al. (2013a) and Santosh et al. (2013) [67] , indicating that α₂-agonists such as dexmedetomidine increase recovery and standing time.

Complete recovery time (min)

Complete recovery time (CRT) was significantly lower when tiletamine-zolazepam (TZ) was administered alone. However, TZ at doses of 5-10 mg/kg IV may produce inadequate anaesthesia with prolonged and sometimes rough recovery (Gomez et al., 2013). CRT was comparatively higher in animals premedicated with dexmedetomidine than with fentanyl citrate, likely due to the prolonged sedative and tranquilizing effects of mathtt α 2 -ag on is ts. These findings are consistent with Kwon et al. (2003) [48] , who reported increased recovery times with xylazine and medetomidine combinations compared to TZ alone. Recovery was smoother in mathtt α 2 -ag on is t groups due to their sustained tranquilizing effects, which aligns with the present observations using dexmedetomidine. In contrast, tiletamine-zolazepam alone may result in less smooth recovery, possibly due to the longer half-life of tiletamine relative to zolazepam (Chen et al., 2005) [10] .

Duration of anaesthesia (min)

Duration of anaesthesia was comparatively shorter when tiletamine-zolazepam (TZ) was used alone or in combination with fentanyl citrate, whereas it was highest in the dexmedetomidine-TZ-isoflurane group. The shorter duration with TZ alone may be due to its limited anaesthetic effect at standard doses, although increasing the dose can prolong surgical anaesthesia (Hellyer et al., 1989) [24] . The prolonged duration observed with dexmedetomidine may be attributed to its potent sedative, analgesic and muscle relaxant effects, enhancing the depth and duration of anaesthesia. Similar findings have been reported by Kwon et al. (2003) [48] , who observed longer anaesthetic duration with α₂-agonist combinations, and by Koli et al. (2021) [38] , Pablo and Bailey (1999) [58] , and Hampton et al. (2019) [21] , supporting the prolongation of anaesthesia with such combinations.

Amount of Isoflurane consumed

In the present study, the total isoflurane consumption was highest in Group I, likely due to the absence of preanaesthetic medication and inadequate depth of anaesthesia achieved with Zoletil alone, resulting in a higher MAC requirement. In contrast, Group III showed comparatively lower isoflurane consumption, which may be attributed to the analgesic effect of fentanyl citrate, known to reduce MAC (Hellyer et al., 2001) [23] . Group II exhibited the least isoflurane requirement, possibly due to the combined sedative, analgesic and muscle relaxant effects of dexmedetomidine, leading to a pronounced MAC-sparing effect. These findings are in agreement with previous reports indicating reduced isoflurane requirement following administration of α₂-agonists such as dexmedetomidine and medetomidine (Kuusela et al., 2001; Kastner et al., 2006; Singh et al., 2013a) [46,70] .

Physiological Parameters 4.11.1 Rectal Temperature (ºF)

Rectal temperature (RT) decreased non-significantly following administration of atropine and other preanaesthetics in all groups, and showed a significant decline after induction with zolazepam-tiletamine and maintenance with isoflurane, remaining below baseline throughout the 90-minute observation period. In Group I, RT decreased non-significantly up to 30 minutes postinduction, followed by a significant decline until 90 minutes. The reduction in RT may be attributed to the combined effects of anaesthetic agents causing CNS depression, muscle relaxation and decreased metabolic rate. Dexmedetomidine-induced hypothermia is associated with reduced muscular activity and central depression (Sinclair, 2003; Kumar et al., 2018) [42,69] . Fentanyl citrate produced a mild, non-significant decrease in temperature, likely due to reduced basal metabolic rate and increased heat loss through panting (Hamad et al., 2022; Haskins, 2013) [20,22] . Additionally, anaesthesia impairs thermoregulation, further contributing to hypothermia (Ko and Krimins, 2014) [36] . Similar observations have been reported by Lemke (2007) [50] .

Heart rate(beats/min)

Heart rate (HR) increased significantly in all groups following atropine administration due to its vagolytic action blocking parasympathetic influence on the heart (Ko et al., 2001) [35] . Subsequent administration of dexmedetomidine and fentanyl citrate resulted in a significant and nonsignificant decrease in HR in Groups II and III, respectively. Induction with Zoletil (tiletamine-zolazepam) caused a significant increase in HR up to 30 minutes, followed by a gradual decline from 45 to 90 minutes during maintenance with isoflurane anaesthesia. The tachycardia observed with Zoletil may be attributed to its sympatheticstimulating effect along with the vagolytic action of atropine, as reported by Dugassa et al. (2018) [13] , Hellyer et al. (1989) [24] and Tarraga et al. (2000) [74] . Isoflurane further contributed to increased HR, possibly as a compensatory response to decreased peripheral vascular resistance and negative inotropic effects (Eger, 1984; Hellebrekers, 1986). Dexmedetomidine-induced bradycardia may be due to α₂-adrenoceptor-mediated vasoconstriction, reflex vagal stimulation and reduced cardiac output (Lemke, 2007; Murrell and Hellebrekers, 1986; Nam et al., 2013) [50,57] . Fentanyl citrate produced a mild, non-significant decrease in HR, likely due to suppression of sympathetic tone and enhanced vagal activity acting on the sinoatrial node (Loeb et al., 1984), although some studies have reported minimal or no effect (Arndt et al., 1984; Grimm et al., 2005). Overall, HR changes observed in the present study reflect the combined pharmacodynamic effects of atropine, Zoletil, dexmedetomidine, fentanyl and isoflurane.

Respiration rate (breaths/min)

Respiration rate (RR) showed a non-significant increase in all groups following atropine administration, followed by a significant decrease in Groups II and III after administration of dexmedetomidine and fentanyl citrate, respectively. Subsequently, RR further decreased in all groups after zolazepam-tiletamine administration and during maintenance with isoflurane anaesthesia. The initial increase may be attributed to the stimulatory effect of atropine on the cardiopulmonary system (Robaj et al., 2014) [64] , whereas the subsequent decrease is likely due to central nervous system depression caused by α2-agonists and opioids (Kuusela et al., 2001; Ahmad et al., 2013; Nishimura et al., 2018) [1,46] . Similar reductions in RR with dexmedetomidine-based protocols have been reported by Santosh et al. (2013) [67] and Ratnu et al. (2021) [63] . Fentanyl-induced respiratory depression may be due to its action on pontine and medullary respiratory centres (Bailey et al., 1990), with reports of apnoea and reduced respiratory activity during fentanyl-isoflurane anaesthesia (Keating et al., 2013; Bufalari et al., 2007) [32] . The overall decrease in RR observed in the present study may be attributed to the combined depressant effects of tiletamine-zolazepam and isoflurane (Hellyer et al., 1989; Lin et al., 1989) [24] . 4.12. Haemodynamic Parameters 4.12.1 Systolic blood pressure (mmHg) Systolic blood pressure (SBP) showed a non-significant increase in all groups following atropine administration, with a significant (p<0.05) rise in Group II and a nonsignificant increase in Group III at 10 minutes post-sedation. Thereafter, SBP decreased non-significantly following administration of zolazepam-tiletamine and remained stable during maintenance with isoflurane. The initial increase in SBP may be attributed to atropine and peripheral α2adrenoceptor-mediated vasoconstriction induced by dexmedetomidine (Vainio et al., 1989; Bisht et al., 2018a, 2018b), while the subsequent decline may be due to reduced peripheral vascular resistance following tiletaminezolazepam administration (Hellyer et al., 1989) [24] . Similar biphasic changes in SBP with α2-agonists have been reported by Gomez [20] . The subsequent decrease in SBP during maintenance may be attributed to the combined effects of tiletamine-zolazepam and isoflurane, which has also been reported in previous studies (Keating et al., 2013; Liehmann et al., 2006) [32] . 4.12.2 Diastolic blood pressure (mmHg) Diastolic blood pressure (DBP) showed a significant (p<0.05) increase in Groups II and III following atropine administration up to 10 minutes post-sedation, followed by a non-significant decrease after administration of tiletaminezolazepam and during maintenance with isoflurane. The initial rise in DBP may be attributed to peripheral α2adrenoceptor-mediated vasoconstriction induced by dexmedetomidine, whereas the subsequent decline reflects reduced sympathetic tone and decreased peripheral vascular resistance (Zornow et al., 1989; Sinclair, 2003) [69] . Similar biphasic responses have been reported by Rafee et al. (2015) [62] . The decrease in DBP following tiletamine-zolazepam administration is consistent with Hellyer et al. (1989) [24] , who attributed it to reduced vascular resistance. In Group III, fentanyl-induced increase in DBP may be due to increased systemic vascular resistance (Grimm et al., 2005), with a transient rise followed by gradual decline as reported by Hamad et al. (2022) [20] . However, contrasting findings of decreased or non-significant changes in DBP with fentanyl combinations have also been documented (Keating et al., 2013; Gardocki and Yelnosky, 1964; Hughes et al., 1999) [25,32] .

Mean blood pressure (mmHg)

Mean blood pressure (MBP) showed a significant (p<0.05) increase in Group II and a nonsignificant increase in Group III following atropine administration up to 10 minutes postsedation. Thereafter, MBP decreased significantly ≤ft (p<0.05right) in Group II and non-significantly ≤ft (p>0.05right) in Group III following administration of tiletamine-zolazepam and during maintenance with isoflurane. The initial rise in MBP may be attributed to the effects of atropine and peripheral α 2 • -adrenoceptor stimulation by dexmedetomidine (Vainio et al., 1989; Bisht et al., 2018a, 2018b), whereas the subsequent decline reflects reduced peripheral vascular resistance and the biphasic response of α2-agonists, characterized by transient hypertension followed by prolonged hypotension (Hellyer et al., 1989; Ruffolo et al., 1993; Tibirica et al., 1991) [24] . Isoflurane further contributed to the decrease in MBP by reducing peripheral vascular resistance (Hikasa et al., 2000). In Group III, the non-significant changes in MBP may be associated with the variable cardiovascular effects of fentanyl, including transient increases in vascular resistance followed by mild hypotension. Similar reductions in MBP during isoflurane-opioid anaesthesia have been reported by Liehmann et al. (2006) and Grimm et al. (2005), although variable responses with fentanyl have also been documented (Hamad et al., 2022) [20] .

Capillary Refill Time (seconds)

Capillary refill time (CRT) did not show any significant change (p<0.05) throughout the observation period in any of the groups. CRT, an indicator of peripheral perfusion, normally remains below 2 seconds and prolongs in cases of circulatory compromise (Kumar et al., 2013) [41] . In the present study, consistent CRT values and normal mucosal coloration indicated adequate peripheral circulation, suggesting that the anaesthetic protocols did not adversely affect haemodynamic stability. Similar findings have been reported by Kumar et al. (2013) in dogs.

Conclusion

Dexmedetomidine as a premedicant with zolazepamtiletamine-isoflurane provided superior sedation, analgesia, smoother induction and reduced isoflurane requirement, though with prolonged recovery. Fentanyl citrate showed moderate effects, while atropine alone was inadequate. All protocols were physiologically safe; however, dexmedetomidine-based anaesthesia was found to be the most effective for balanced anaesthesia in canines.

Acknowledgement

The authors express their sincere gratitude to the Dean, College of Veterinary Science and Animal Husbandry, Anjora, Durg, for providing the necessary facilities to carry out this study. They are also thankful to the Professor and Head, Department of Veterinary Surgery and Radiology, for their valuable guidance and support. Appreciation is extended to the staff of the Teaching Veterinary Clinical Complex (TVCC) and to the dog owners for their cooperation throughout the study.

Conflict of Interest

The authors affirm that there is no conflict of interest related to the publication of this research.

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