Sedative, analgesic, behavioral and clinical effects of intravenous nalbuphine-xylazine combination in camels (Camelus dromedarius).

This study examined the sedative, analgesic, behavioral, and clinical effects of a combination of xylazine (XY) and nalbuphine-xylazine (NA-XY) in camels. A total of five adult camels were used in a prospective randomized cross-over design with a wash out period of two weeks. Camels were allocated randomly to two treatment groups: the XY group (xylazine, 1.1mL/100 kg IV) and the NA-XY group (xylazine, 1.1mL/100 kg IV and nalbuphine, 1 mg/kg IV). The sedative, analgesic, behavioral, and clinical effects of XY and NA-XY combination were evaluated prior to administration (baseline) and at 5, 15, 30, 45, 60, 75, 90, and 120 minutes post-administration. The results showed that the NA-XY combination accelerates the onset of sedation and analgesia and prolongs the durations of both sedation (p < 0.001) and analgesia (p < 0.01). The behavioral parameters showed higher scores with a NA-XY combination than xylazine alone. Although a XY injection resulted in a significant decline in the heart and respiratory rate, the NA-XY combination group revealed a non-significant change in both clinical parameters compared to the baseline. In conclusion, the use of a NA-XY combination in camels improved the sedative and analgesic onset and duration with an improved outcome in the behavioral scores, as well as in both the heart and respiratory rates compared to XY alone.

INTRODUCTION

Injectable anesthetic drugs for deep sedation in camels are commonly used in clinical practice [1]. Alpha-2 adrenergic agonist drugs (xylazine, romifidine, detomidine, and medetomidine) have been used widely for animal restraint, analgesia, muscle relaxation, minor surgery, and diagnostic procedures [2]. The administration of such agents provide an average intra-operative duration of approximately 60 and 40 minutes for sedation and analgesia, respectively [3]. While multiple injections are needed to maintain a certain level of sedation, the higher risk for developing serious complications and cardiopulmonary depression has limited the clinical application of α-2 adrenergic agents. Therefore, an effective and safe drug combination is needed to handle and restrain animals as well as achieve a faster onset and longer duration of sedation and analgesia. In camels, several drug combinations, such as xylazine-ketamine [2], xylazine-propofol-diazepam and xylazine-propofol [4], have been used successfully in clinical settings.

Recently, the analgesic effects of morphine and nalbuphine, a synthetic opiate with mixed agonist-antagonist actions, have been reported [56]. As a novel opioid κ receptor agonist-antagonist, nalbuphine, can agitate the κ receptors to achieve a superior analgesic effect and antagonize the μ receptors to reduce the incidence of adverse reactions [78]. The intravenous administration of nalbuphine in dogs has been reported to affect all behavioral scoring parameters [9]. Previous studies reported the effects of combinations of xylazine and other analgesic drugs on the locomotion and movement of sheep [10] and ponies [11]. The nalbuphine-xylazine combination showed effective analgesic and sedative outcomes with cardiovascular stability in calves [7], dogs [12] and horses [13]. However, the nalbuphine- xylazine combination has not been used in camels. Therefore, this study was designed to evaluate the sedative, analgesic, behavioral, and clinical effects of a nalbuphine-xylazine combination in camels.

MATERIALS AND METHODS

The study procedures were conducted in compliance with the recommendations of the guidelines for the care and use of animals at the college of Veterinary Medicine, Benha University. The protocol was approved by the Ethical Committee for Institutional Animal Use and Care of the College of Veterinary Medicine, Benha University (BUFVTM 01-04-2019).

Animals and study design

A total of five adult dromedary camels (two males and three non-pregnant females) with an average body weight of 385 ± 15.3 kg, aged between 4–5 years were used randomly in a crossover design. All camels were housed in a single pen, fed grass (hay) supplemented with concentrate, and given access to water ad libitum. Prior to enrollment, each camel was subjected to a complete physical examination to ensure that they were clinically healthy. Food and water were withheld for 24 and 12 h before the experiments, respectively. The camels were assigned to receive one of two intravenous anesthesia protocols, with a wash-out period of two weeks. First, xylazine (XY group) (Xylaject 20%, Adwia Co., Egypt) was injected IV at a dose of (1.1mL/100 kg) for each camel. After two weeks, the same camels were injected IV with the nalbuphine-xylazine combination (NA-XY group) at a dose of xylazine (1.1mL/100 kg) (Xylaject 20%, Adwia Co) and nalbuphine (1 mg/kg) (Nalufin, 20 mg-mL, Pharmacia, Egypt). The injections were carried out in the left jugular vein using a 20-gauge catheter (Int. Comp. Med. Nec., Egypt). All injections were performed while the camels were secured and laid down on a sternal recumbency with both the fore and hind limbs tied together. After the injections, they were allowed to stand up immediately. The study was performed outdoors in a quiet environment with natural daylight and an ambient temperature between 22°C–25°C. On all occasions, the person assigned to evaluate the analgesic agents was completely blinded to the anesthetic protocol.

Treatment evaluations

Sedative and analgesic evaluation

The evaluation was done before injecting the drugs at the baseline (0 min) and at 5, 15, 30, 45, 60, 75, 90, and 120 min after injection. The onset and depth of analgesia were recorded each minute after the injection by recording the response of the animal to pinching with a hemostat clamp (closed to the first ratchet). Pinching was applied to both sides of the shoulder, flank area, thigh, perineum, and dorsal fetlock of the hind limb. The forceps were kept in place for 5 sec or until a response appeared. The animal's eyes were covered at the time of pinching to avoid any visually motivated response. A positive pain response was defined as the purposeful avoidance movements of the head, neck, trunk, limbs and tail, attempts to kick and bite, and turning of the head toward the stimulus site. As soon as the camel showed one of the defined responses, the investigator removed the clamp as described elsewhere [14]. The degree of sedation was evaluated by monitoring of the changes in body language as the distance between the ear tips, ataxia, which is defined as a change in locomotion expressed through the abduction of the hind limbs, and distance between the camel head and ground. The time between the appearance and disappearance of sedation and analgesia was considered as the duration of sedation and analgesia, respectively. The depth of sedation and analgesia was graded on a scoring system from 0 to 3, as described elsewhere (Table 1).

Table 1

Description of sedative and analgesic scores

Score	Sedation		Analgesia
	Criteria	Description	Criteria	Description
0	No sedation	Normal movement, head and neck carriage, normal distance between the ears, tongue inside the mouth and no salivation	No analgesia	Strong response to noxious stimulus
1	Mild sedation	Mild change in movement, mild head dropping or deviation to one side, mild increase in the distance between the ears, mild protrusion of the tongue and mild salivation	Mild analgesia	Weak response at all times of noxious stimulus
2	Moderate sedation	Moderate change in movement, moderate head dropping or deviation to one side, moderate increase in the distance between the ears, moderate protrusion of the tongue and moderate salivation	Moderate analgesia	Very weak occasional response
3	Sever sedation	Severe change in movement, severe head dropping or deviated and rest on the back, severe increase in the distance between the ears, severe protrusion of the tongue (whole tongue out of mouth) and severe salivation (excessive salivation)	Severe analgesia	No response to noxious stimulus

Behavioral evaluation

The examiner who was unaware of the injected drugs estimated the behavioral scoring parameters, such as head deviation, tongue protrusion, and salivation on a scale from 0 to 3 (Table 2). The examiner stood a suitable distance from the animals to make a good observation. By scan observation, the examiner observed the head deviation, tongue protrusion, and salivation at the baseline and at each time point post injection.

Table 2

Behavioral scoring parameters

Parameter	Score	Description
Head deviation	0	No deviation
	1	Mild deviation to one side
	2	Moderate deviation to one side
	3	Severe deviation, head rest on the back
Tongue protrusion	0	Tongue inside the mouth, no protrusion
	1	Small part of tongue get out from one side of the mouth
	2	Large part of tongue get out from one side of the mouth
	3	The whole tongue is outside the mouth
Salivation	0	No salivation
	1	Little drops get out from the mouth
	2	Much drops get out from the mouth
	3	Excessive salivation, like strand hanged from the mouth

Heart and respiratory rate evaluation

The clinical parameters, including heart rate and respiratory rate, were also evaluated at the baseline (0 min) and then at 5, 15, 30, 45, 60, 75, 90, and 120 min after injection. The heart rate was assessed by palpation of the middle coccygeal and femoral artery, and is expressed as beats/minute. The respiratory rate was measured by an inspection of the respiratory movements and by auscultation of the trachea and is expressed as breaths/minute.

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics version 23 (IBM Armonk, USA). The normality of the data distribution was evaluated using a Shapiro-Wilk test. Data analysis was performed using an Analysis of variance (ANOVA) and non-parametric Wilcoxon/Kruskal-Wallis Tests where relevant. Significance was set to p < 0.05 and all values are presented as the mean ± standard error of mean.

RESULTS

The results showed that the NA-XY combination had a significant effect on sedative, analgesic, clinical, and behavioral parameters in camels, which are represented by a significant increase in the duration of sedation and analgesia. In addition, it caused a significant change in all recorded behavioral parameters, as well as non-significant change in both the heart and respiratory rates compared to the XY group. An intravenous injection of NA-XY combination resulted in rapid onset of apparent sedative effects (5.40 ± 0.24 and 4.8 ± 0.37) min for the XY group and NA-XY group, respectively. The sedative effects persisted for 44.00 ± 2.44 and 90.00 ± 2.60 min for the XY group and NA-XY group, respectively (Table 3).

Table 3

Onset and duration of sedation and analgesia after IV injection of xylazine and nalbuphine-xylazine combination

Treatment	Sedation		Analgesia
	Onset	Duration	Onset	Duration
XY	5.40 ± 0.24	44.00 ± 2.44b	9 ± 0.58a	28.4 ± 1.68b
NA-XY	4.8 ± 0.37	90.00 ± 2.60a	7.20 ± 0.37b	55.00 ± 7.4a
p value	0.2	< 0.001	< 0.05	< 0.01

Means (± SE) with different superscript letters in the same column are significantly different.

XY, xylazine; NA-XY, nalbuphine-xylazine.

Intravenous injection of NA-XY combination resulted in rapid onset and prolonged analgesic duration than XY alone. The onset of the analgesic effect for the XY group and NA-XY group were 9 ± 0.58 and 7.20 ± 0.37 min, respectively. The analgesic durations for the XY group and NA-XY group were 28.4 ± 1.68 and 55.00 ± 7.4 min, respectively (Table 3).

The post-administration sedative effect was evaluated through a change in body language. The results showed that the NA-XY combination increased the distance between the ear tips significantly throughout all time points post injection. The highest significant difference occurred at T30, where the distance between the ear tips was 20.6 ± 0.81 and 29.2 ± 1.59 cm for both XY and NA-XY groups, respectively (Table 4). The degree of ataxia was also recorded through monitoring the abduction of the hind limbs (distance between the hind limbs), which showed significant changes after injection of NA-XY combination. The maximum distance recorded at T5 post administration as the distance between the hind limbs was 48.8 ± 2.15 and 66.6 ± 1.86 cm for XY group and NA-XY group, respectively (Table 4). The distance between the head and ground was significantly lower in the NA-XY group than the XY group at all times after the injections except at T15, in which the distance was 176.8 ± 0.86 and 185.8 ± 1.68 for the XY group and NA-XY group, respectively. The most obvious head dropping was observed in T30 in the NA-XY group; the distance between the head and ground was 96.2 ± 2.15 cm compared to 188.6 ± 2.4 cm for the XY group at the same time point (Table 4).

Table 4

Body language change of camels before and after IV injection of xylazine and nalbuphine-xylazine combination

Item	Treatment	Pre-treatment	Post-treatment
		T0	T5	T15	T30	T45	T60	T75	T90	T120
Ear tips distance	XY	18.4 ± 1.36	20 ± 1.58	21 ± 1.14	20.6 ± 0.81b	19.25 ± 0.72b	19.33 ± 0.73	19.6 ± 0.74	20 ± 1.37	20.6 ± 1.06
	NA-XY	18.2 ± 0.86	21.4 ± 1.53	22 ± 1.26	29.2 ± 1.59a	23.4 ± 0.92a	20.2 ± 1.59	20.4 ± 1.60	22.6 ± 1.16	21.4 ± 1.13
Abduction of hind limbs	XY	5.6 ± 2.58	48.8 ± 2.15b	24 ± 1.70b	32.6 ± 2.50b	34.2 ± 0.24	32.4 ± 2.50	26.8 ± 1.98b	25 ± 1.58b	25.8 ± 1.60b
	NA-XY	5.4 ± 2.35	66.6 ± 1.86a	37.8 ± 2.61a	38.2 ± 2.41a	36 ± 1.87	35 ± 1.58	39.6 ± 1.63a	36.4 ± 1.86a	36.8 ± 1.52a
Head to ground distance	XY	212.4 ± 2.50	190 ± 1.63	176.8 ± 0.86b	188.6 ± 2.42a	174 ± 2.21a	181.4 ± 2.73a	192.4 ± 2.50a	198.2 ± 2.59a	200.4 ± 2.07a
	NA-XY	212.4 ± 2.50	189 ± 1.70	185.8 ± 1.68a	96.2 ± 2.15b	122.4 ± 2.50b	122 ± 2.54b	118 ± 1.37b	115.8 ± 1.68b	118.2 ± 1.08b

Means (± SE) with different superscript letters in the same column with different treatments are significantly different.

XY, xylazine; NA-XY, nalbuphine-xylazine.

As shown in Table 5, the behavioral scoring parameters were affected at different treatment time points as the head deviation degree was significantly higher in the NA-XY group than the XY group, particularly at T30, T45, and T60 post administration. The degree of tongue protrusion was higher in the XY group than in the NA-XY group at T15 post administration. In contrast, at T60, T75, and T90 post injection, the degree of tongue protrusion was higher in the NA-XY group than the XY group. The degree of salivation was significantly higher in the NA-XY group than the XY group from T30 until T90 post administration.

Table 5

Behavioral scoring parameters of camels before and after IV injection of xylazine and nalbuphine-xylazine combination

Item	Treatment	Pre-treatment	Post-treatment
		T0	T5	T15	T30	T45	T60	T75	T90	T120
Head deviation	XY	0	0	1	1b	1	0b	0	0	0
	NA-XY	0	0	2	3a	2	2a	1	1	0
Tongue protrusion	XY	0	0	2a	1	1	1	0b	0b	0
	NA-XY	0	1	1b	1	1	3a	2a	2a	1
Salivation	XY	0	0	1	0b	1	0b	0b	0b	0
	NA-XY	0	0	1	2a	2	3a	2a	2a	1

Variables with different superscript letters in the same column with different treatments are significantly different.

XY, xylazine; NA-XY, nalbuphine-xylazine.

A clinical assessment of the camels showed that XY administration decreased both the heart and respiratory rates significantly at different time points compared to the baseline. The NA-XY combination showed non-significant changes in both the heart and respiratory rates at different time points compared to the pretreatment baseline (T0), as shown in (Table 6).

Table 6

Clinical evaluation of camels before and after IV injection of xylazine and nalbuphine-xylazine combination

Item	Treatment	Pre-treatment	Post-treatment
		T0	T5	T15	T30	T45	T60	T75	T90	T120
Heart rate	XY	58.8 ± 0.96	52 ± 1.04b	46.8 ± 0.58b	41.6 ± 0.52b	42.8 ± 0.73b	46.4 ± 0.60b	46.6 ± 0.50b	49.8 ± 0.37b	52.4 ± 0.35b
	NA-XY	59.2 ± 0.66	56.6 ± 0.50a	54.6 ± 0.50a	51.6 ± 0.74a	49.2 ± 1.01a	51 ± 0.44a	52 ± 0.31a	54 ± 0.44a	56.8 ± 0.39a
Respiratory rate	XY	30.2 ± 0.86	24.6 ± 0.50b	21.4 ± 0.60b	19.2 ± 0.73b	19.4 ± 0.50b	20 ± 0.70b	20.6 ± 0.60b	22.6 ± 0.92b	23.4 ± 0.25b
	NA-XY	30 ± 1.09	28.2 ± 0.96a	26 ± 0.94a	25.2 ± 0.96a	25.4 ± 0.81a	26.6 ± 0.87a	27.2 ± 0.96a	27.2 ± 0.91a	28.8 ± 0.30a

Means (± SE) with different superscript letters in the same column with different treatments are significantly different.

XY, xylazine; NA-XY, nalbuphine-xylazine.

DISCUSSION

This study indicates that the intravenous administration of a nalbuphine (1 mg/kg) and xylazine (1.1mL/100 kg) combination had great sedative, analgesic, behavioral, and clinical effects on camels. The maximum duration of sedation and analgesia after administering the α-2 agonist is 55 to 65 and 37 to 46 minutes for sedation and analgesia, respectively [141516], which may be insufficient during long lasting operations. Combinations of α-2 agonist and opioids have been reported to decrease the number of side effects and improve the sedation and analgesia durations compared to individual drug injections [1718]. Such combinations of an α-2 agonist and opioids have been used previously in camels as xylazine-tramadol [19] and midazolam-propofol [20]. Nalbuphine is an appealing opioid with a low cost and wide safety margin. The drug appears to be safe for enhancing the neuroleptanalgesic effects with xylazine and possibly other α-2 agonists [12]. The selected dose of nalbuphine in the present study (1 mg/kg) was calculated based on the fact that nalbuphine is considered to be one-fifth as potent as butorphanol when administered parenterally [21]. The safe analgesic dose of butorphanol in camels has been reported to be 0.2 mg/kg IV [22].

Sedation and analgesia

While previous studies have reported the safe and efficient effects of a NA-XY combination in different species [71213], the effects of this combination in camels have not been recorded. Similar to previous studies, the results of the current study showed that a combination of nalbuphine with xylazine resulted in the rapid onset and prolonged duration of sedation and analgesia compared to xylazine alone [23]. Numerous studies in pets and horses have reported improved effects on sedation and analgesia when an α-2 agonist was administered in combination with an opioid due to the synergistic effects of sedatives and opioid drugs [24252627]. Similarly, Coetzee et al. [7] reported that nalbuphine accelerates the onset and extends the duration of both sedation and analgesia in calves. Moreover, the combination of nalbuphine hydrochloride and medetomidine yielded good quality sedation with 50 min of muscular relaxation and acceptable physiological parameters [2829]. The current study recommended the following body language indicators to assess the degree of sedation: distance between the ear tips, abduction of the hind limbs as an ataxia indicator and distance between animal's head and ground as an indicator for muscle relaxation. The increase in the distance between the ear tips and a decreased distance between the head and ground post injection may be attributed to the muscle relaxation and reduction of awareness caused by the sedative effects of the drugs. These results agree with previously published studies [1415].

Ataxia is considered one of the most important indicators of abnormal locomotion. In the current study, the camels were allowed to stand up after drug administration. The distance between the hind limbs was measured as an indicator of ataxia. After xylazine and nalbuphine-xylazine combination injection, the abduction between the hind limbs increased, leading to slow motion or no motion, particularly in the NA-XY group. Coetzee et al. [7] recorded a decrease in the number of steps taken after castration in calves with less pain-associated behaviors due to the sedative effects after nalbuphine administration. The frequency of all recorded adverse behaviors in dogs was less when nalbuphine was combined with xylazine. Nalbuphine-xylazine combination also improved the onset of both sedation and analgesia, decreased the animal movement, and kept the animal calm during the recovery period [12].

Behavioral scoring parameters

The behavioral indicators, such as head deviation, tongue protrusion and salivation, were affected significantly by different treatments. After administration, the behavioral parameters increased, particularly in the NA-XY group due to the CNS excitation produced by the drugs. These results agree with Dhanjal et al. [30] who reported that the use of tramadol as opioids in horses leads to head deviation. Seo et al. [31] also reported that tongue protrusion and salivation increased after administration of a xylazine-tramadol combination.

Heart and respiratory rate

In the XY group, the camels showed a significant decrease in both heart and respiratory rates in all time points post-injection compared to baseline values. Cardiopulmonary suppression is a common concern after an injection of α-2 adrenoceptor agonists because of the central stimulation mediated through the vagus nerve and a decrease in sympathetic tone [32]. Previous studies [3334] in camels reported a significant reduction in heart and respiratory rates after intravenous administration of xylazine. On the other hand, the cardiopulmonary stability was the main feature observed after intravenous administration of NA-XY combination. No significant differences in heart rate and respiratory rate were observed in the NA-XY-treated camels compared to the baseline values. This is consistent with the literature and supports the observation that nalbuphine is an agonist-antagonist opioid analgesic with cardiovascular stability and a smaller capping effect on respiratory depression in humans [35] and calves [7]. The cardiopulmonary protective effect of nalbuphine makes it safe and provides better hemodynamic stability during the early postoperative period compared to morphine in patients with cardiac surgery [36].

In conclusion, nalbuphine is capable of producing an additive-synergistic effect when added to IV xylazine. This effect is evidenced in camels by the rapid onset, greater intensity, and longer duration of sedation and analgesia. An additional discernible feature of this combination is the consequential cardiopulmonary stability. The nalbuphine-xylazine combination also affects the behavioral parameters. The changes in the response of these behavioral parameters indicate a state of deep sedation and analgesia with no agitation and calm recovery.