Chemical restraint of captive Kinkajous Potos flavus ( Schreber , 1774 ) ( Carnivora : Procyonidae ) using a ketamine , xylazine and midazolam combination and reversal with yohimbine

Detailed information on the anaesthetic and cardiorespiratory effects of drug combinations used for the chemical immobilization of Kinkajous (Potos flavus) is scarce. This study assessed the effects of ketamine (2.5mg/kg), xylazine (1mg/kg) and midazolam (0.5mg/kg) combination in P. flavus. Five clinically healthy adult Kinkajous of both sexes were included. Heart rate, respiratory rate, oxygen saturation, blood pressure and body temperature were recorded at five-minute intervals for 25 minutes. Then, animals received 0.125mg/kg of yohimbine by intramuscular injection. Anaesthetic depth was assessed based on stimulus response and muscle tone. Induction, immobilization, and recovery periods were recorded and qualitatively assessed based on the absence of adverse effects. The durations of the induction, immobilization, and recovery periods were 9.42±1.73, 33.33±2.16, and 31.37±5.82 minutes. All periods showed good quality and adequate anaesthetic depth was achieved. Mean heart and respiratory rates were 99±20 beats/minute and 44±9 breaths/minute. Both parameters decreased over the duration of the anaesthesia but they did not reach levels suggesting either bradycardia or bradypnea. Mean body temperature was 37.1±1.5 0C and it also showed a decreasing trend over the duration of the anaesthesia. Mean oxygen saturation was 92±6% and it showed a mildly increasing trend over the duration of the anesthesia. Mean blood pressure was 129±23 mmHg and mild to moderate hypertension was observed. No mortality occurred and no adverse effects were observed in any of the individuals during the three months following immobilization. The assessed anaesthetic combination effectively immobilized the P. flavus individuals, provided good quality and acceptable duration of both induction and recovery periods. It should, however, not be used in Kinkajous with either known hypertension record or pre-existing target organ disease (e.g., renal failure, retinopathy).


INTRODUCTION
Kinkajous Potos flavus are a medium sized procyonid inhabiting neotropical forests ranging from Mexico to Brazil (Melo et al. 2005;Kays et al. 2008). They are nocturnal, solitary, and arboreal, which may account for the fact that Kinkajous are one of the least known mammals in the Neotropics (Melo et al. 2005;Kays et al. 2008;Sampaio et al. 2011;Monterrubio-Rico et al. 2013). Their diet is mainly composed of flowers and nectar but also includes leaves, fruits, insects, bird eggs, and small vertebrates (Monterrubio-Rico et al. 2013). Currently, it is included in the Least Concern (LC) category of the IUCN Red List of Threatened Species (Kays et al. 2008).
In captivity, procyonids can inflict serious wounds on animal husbandry personnel using their claws and teeth if they are stressed or feel pain during handling (Labate et al. 2001). Hence, physical restraint of procyonids is unsafe, requiring the use of chemical immobilization to perform routine (e.g., physical examination, vaccination) or emergency procedures (e.g., medical treatment) (Labate et al. 2001; Association of Zoos and Aquariums Small Carnivore Taxon Advisory Group 2010). Drug combinations can have synergistic effects that vary across species; consequently, it is necessary to investigate the use of drug combinations in each species (Robert et al. 2012 (Fournier et al. 1998).
The aim of this study was to assess the efficiency of a combination of ketamine, xylazine and midazolam for the immobilization of captive Kinkajous and to describe its anaesthetic and cardiorespiratory effects.

Animals
Five adult Kinkajous of both sexes were immobilized. The animals were kept at El Buen Pastor Zoo (Lima, Peru), were clinically healthy, and the immobilizations were performed in order to allow routine health assessments.

Physical and chemical restraint
The animals were physically restrained by grabbing and holding their tails through the wire mesh surrounding the enclosure. They immediately received a combination of ketamine hydrochloride (2.5mg/kg) (Imalgene®, Merial, France), xylazine hydrochloride (1mg/kg) (Dormi-Xyl®, Agrovet Market, Peru) and midazolam maleate (0.5mg/kg) (Midanex®, AC Farma, Peru) by intramuscular injection using a sole syringe. All animals received anaesthetic drugs for an estimated body weight of 2kg. Once immobilized, animals were weighed and actual administered dosages were calculated.

Record of anaesthetic periods
The following was recorded: first effect time (time from drug administration to the appearance of ataxia), recumbency time (time from drug administration until the animal remained in either sternal or lateral recumbency), induction time (time from drug administration until the animal remained unresponsive to external stimuli), and recovery time (time from administration of the antagonistic drug until the animal remained standing for at least five minutes). Room temperature and humidity were recorded during all anaesthesia procedures.

Record of vital signs
Heart rate, respiratory rate, oxygen saturation, blood pressure, and body temperature were recorded at 5-minute intervals for 25 minutes. Heart rate was assessed by thoracic auscultation using a neonatal stethoscope (Littman, Germany) for 60 seconds. Respiratory rate was assessed by observing thoracic inspiratory movements for 60 seconds. Body temperature was assessed using a digital rectal thermometer. Oxygen saturation was assessed using a pulsoximeter (VS2000V, Ubox, China), placing the probe on the tongue (Heard 2007) (Fig. 1a). Blood pressure was assessed using an oscilloscope (VS2000V, Ubox, China), placing the pressure cuff on the hindlimb (Image 1b) (Heard 2007). R o om ft em p e r a ft u r e a n d h um fi d fi ft y a ft w o r k fi n g fim e r e s p e c fi v e fl y w e r e 2 6 . 8 ± 2 . 3 0 C a n d 6 7 ± 4 % . F fi r s ft e ff e c ft , r e c um b e n c y , fi n d u c fi o n , a n d r e c o v e r y fim e s w e r e 4 . 3 ± 1 . 2 , 6 . 5 ± 0 . 9 , 9 . 4 ± 1 . 7 , a n d 3 1 . 4 ± 5 . 8 m fi n u ft e s .
H e a r ft r a ft e a n . M e a n h e a r ft a n d r e s p fi r a ft o r y r a ft e s w e r e 9 9 ± 2 0 b e a ft s /m fi n u ft e a n d 4 4 ± 1 0 b r e a ft h s /m fi n u ft e a n d m e a n b o d y ft em p e r a ft u r e w a s 3 7 . 1 ± 1 . 5 0 C . M e a n o x y g e n s a ft u r a fi o n a n d b fl o o d p r e s s u r e w e r e 9 2 ± 6% a n d 1 2 9 ± 2 3mm H g . T h e m e a n s
The ketamine dosage used in this study was lower than those used in combinations with medetomidine [KM] in P. flavus (Fournier et al. 1998) and in Procyon lotor (Robert et al. 2012). It was also lower than that used in combination with medetomidine and butorphanol [KMB] in Nasua narica (Georoff et al. 2004), and lower than that used in combination with xylazine [KX] in P. lotor (Deresienski & Rupprecht 1989). It was also lower than the dosages when used as a sole agent [K] in P. lotor (Norment et al. 1994) and in P. flavus (Kollias & Abou-Madi 2007). The xylazine dosage used in this study was lower than those used in combination with ketamine [KX] and with tiletamine-zolazepam [TZX] in P. lotor (Deresienski & Rupprecht 1989;Belant 2004). The midazolam dosage used in this study was within the range recommended for immobilization of procyonids in combination with ketamine (Kollias & Abou-Madi 2007). The use of low dosages for each anaesthetic agent in this study might be explained by the synergistic effects of benzodiazepines and alpha-2 adrenergic agonists, which agrees with the concept that when using balanced anaesthestics the dosages of the individual drugs can be reduced a (Bol et al. 2000;Fahlman et al. 2005;Hendrickx et al. 2008;Painer et al. 2012;Ahmad et al. 2013). It should also be noted that this study was conducted on captive animals; free-ranging animals might require higher dosages as seen in other mammals (Caulkett & Haigh 2007a,b;Citino & Bush 2007;Gunkel & Lafortune 2007;Ølberg 2007).
In this study the mean time to the observation of the first anaesthetic effect was longer than the times reported in N. narica immobilized with KMB (5.8±3.2 minutes) (Georoff et al. 2004) and in P. lotor immobilized using TZX (1.7±0.6 minutes) (Belant 2004). Recumbency time recorded in this study was longer than that observed in N. narica immobilized using KMB (5.8±3.2 minutes) (Georoff et al. 2004). Induction time reported in this study was longer than those recorded in P. lotor immobilized with TZX (4.8±3.8 minutes) (Belant 2004), K (3.2±1.8 minutes) (Norment et al. 1994   immobilized using KM (3±0.9 minutes) (Fournier et al. 1998). In other mammals, higher dosages of anesthetic drugs shortened induction periods (Painer et al. 2012;Wolfe et al. 2014); hence, the lower drug dosages used in our study might have been the reason for induction longer than those found in other studies on procyonids.
Considering that the effects of drug mixtures on induction time vary among species (Robert et al. 2012), the effects of different drugs should always be compared in the same species. Thus, the shorter induction time observed in P. flavus immobilized with KM (Fournier et al. 1998) may be explained by both the higher dosage of ketamine administered in the respective study as well as the use of medetomidine, a drug which provides an earlier onset of sedation than xylazine, probably due to its higher alpha-2 adrenergic receptor specificity (Pawson 2008;Al-Sobayil et al. 2010). The mean recovery time in this study was shorter than those observed in Procyon lotor immobilized using TZ without antagonist (128.5±48.4 minutes) (Belant 2004), and longer than those observed in P. lotor immobilized using KX and antagonized with 0.2mg/kg of yohimbine (23.7±6 minutes) (Deresienski & Rupprecht 1989). Moreover, comparisons of recovery time are difficult as different authors define the time differently; it is defined as "head up time" (from antagonist administration to the first attempt to lift its head) (Robert et al. 2012), or as "sternal time" (from antagonist administration to sternal recumbency) (Georoff et al. 2004), or as "walking time" (from antagonist administration to coordinated walking) (Deresienski & Rupprecht 1989). Differences in duration of immobilizations, species, and specificity of the antagonist drug also influence the length of the recovery time (Deresienski & Rupprecht 1989;Robert et al. 2012;Yu et al. 2014).
An important aspect of the findings was that induction, immobilization, and recovery periods were of good quality, showing no adverse effects. The achieved anaesthetic depth allowed the performance of routine health assessment procedures and provided safety for the involved personnel.
The mean heart rate in this study was higher than those observed in Potos flavus immobilized using KM (Fournier et al. 1998), and also higher than in Procyon lotor immobilized with KM (Robert et al. 2012) and KX (Deresienski & Rupprecht 1989); it was lower than that observed in Nasua narica immobilized using KMB (Georoff et al. 2004), and similar to that recorded in Procyon lotor immobilized with TZX (Belant 2004). The heart rate in the anesthetized animals did not significantly differ over the assessment period in this study. In contrast Fournier et al. (1998) found a significant decrease in heart rate in P. flavus immobilized with KM. In our study, we only observed a mildly decreasing trend in the heart rate, similar to that reported in Procyon lotor, which were immobilized using KM (Robert et al. 2012) and TZX (Belant 2004). The slight increase in heart rate at the beginning of the immobilization period might be attributable to sympathomimetic effects of ketamine on cardiovascular system (Branson 2001;Grimm & Lamont 2007;Pawson & Forsyth 2008), which progressively disappear as the drug is metabolized. Furthermore, considering that heart rates in resting P. flavus range from 76 to 79 beats/minute at room temperatures from 23-29 0 C (Müller & Rost 1983), our results suggest that despite the slight decrease in heart rate the animals did not present bradycardia.
The mean respiratory rate observed in this study was higher than those observed in Potos flavus immobilized with KM (Fournier et al. 1998), higher than in Procyon lotor immobilized using TZX (Belant 2004) and KM (Robert et al. 2012), and higher than in N. narica immobilized with KMB (Georoff et al. 2004). The respiratory rate remained almost stable over the immobilization period in this study only towards the end it showed a mild decrease. Kinkajous, which were immobilized with KM, developed respiratory depression (Fournier et al. 1998); the respiratory rates in our study were higher and suggest that the drug combination we used had no clinically important effects on this parameter.
The body temperatures observed in this study was lower than those observed in Procyon lotor immobilized using TZX (Belant 2004) and KX (Deresienski & Rupprecht 1989) and in those in Nasua narica immobilized with KMB (Georoff et al. 2004); it was similar to the body temperatures that were observed in Procyon lotor immobilized with KM (Robert et al. 2012), and higher than the body temperatures that were observed in Potos flavus immobilized with KM (Fournier et al. 1998). This parameter did not vary significantly over the immobilization period. On the contrary, a significant decrease was observed in Potos flavus immobilized using KM (Fournier et al. 1998). This parameter showed a mild decrease with a difference of 0.4 0 C between the beginning and the end of immobilization period. This finding is similar to what was observed by Belant (2004) in Procyon lotor immobilized using TZX. In our study no animal showed severe hypothermia (i.e. T° <34°C) comparable to that observed in two P. flavus immobilized with KM (Fournier et al. 1998). Room temperature in our study was lower than that recorded by Fournier et al. (1998) in their study, and they do not mention the use of support to maintain the body temperature of the animals, whereas we did take measures to prevent hypothermia in our animals during the study (placing animals on a warm water blanket and under an infrared heat lamp). Considering that the body temperature of Potos flavus ranges from 36±0.6 0 C (during the day) to 38.1±0.4 0 C (during the night) (Ford & Hoffmann 1988), it can be inferred that the KXMid combination we used in our study showed, had only a minimal and clinically insignificant effect on the body temperature. This difference could be explained by the fact that we provided thermal support to animals or because KXMid has less of a hypothermic effect than KM.
The oxygen saturation observed in this study was higher than those recorded in N. narica immobilized using KMB (Georoff et al. 2004) and those in P. flavus immobilized with KM (Fournier et al. 1998). In our study, no animal showed hypoxemia (i.e., SpO 2 <80%), in contrast to observations in Potos flavus, which were immobilized using KM (Fournier et al. 1998). Though no statistically significant variation was observed in this parameter over the immobilization period, it showed an increasing trend. Xylazine produces an initial peripheral vasoconstriction, due to post synaptic α1 and α2 receptor stimulation; later it causes vasodilatation due to both central and peripheral pre synaptic α2 receptors stimulation (Grimm & Lamont 2007;Pawson 2008). This vasoconstrictive effect of xylazine at the beginning of the immobilization, could cause the pulsoximeter to underestimate true oxygen saturation (Hall et al. 2001;Moens & Coppens 2007). Such underestimation would be corrected as the vasoconstrictive effect progressively disappeared (Hall et al. 2001;Longley 2008;Flecknell 2009). The oxygen saturation remained within an acceptable range for anesthetized mammals without oxygen supplementation (i.e. SpO 2 >90%) (Mosley & Gunkel 2007;Sawyer 2007;Flecknell 2009) over most of the immobilization period, suggesting that our anaesthetic combination had no clinically relevant effect on this parameter.
The mean blood pressure observed in this study remained stable over the immobilization period and it is the first study presenting blood pressure for of Kinkajous during anaesthesia. In most mammals, hypotension is defined as a mean blood pressure values lower than 60mmHg and systolic blood pressure values lower than 90mmHg (Mosley & Gunkel 2007;Sawyer 2007) and hypertension is defined as systolic blood pressure values above 150mmHg (Sawyer 2007). According to this scale, most animals immobilized in this study presented mild to moderate hypertension (Citino et al. 2002;Brown et al. 2007), which is a common and undesired side effect of xylazine and ketamine (Mosley & Gunkel 2007;Pawson 2008;Pawson & Forsyth 2008). Unfortunately, there are no blood pressure baseline data for Kinkajous or other procyonids; hence, the actual significance of the observed acute hypertension in our study is unclear. And though no animal died during the anaesthesia or the three months after the immobilization, absence of morbidity could not be unequivocally assumed. In domestic animals, target organ damage (e.g., renal failure, retinopathy, encephalopathy, ventricular hypertrophy) has been described as a possible consequence of transient acute hypertension (Cattet et al. 1999b;Brown et al. 2007). Therefore, we do recommend not to use KXMid in Kinkajous with either known hypertension record or pre-existent disease in any of the target organs.

CONCLUSIONS
The ketamine, xylazine, and midazolam combination assessed in this study provided effective and safe immobilization of Kinkajous Potos flavus.