Anaesthetic, clinical, morphometric, haematological, and serum chemistry evaluations of an Andean Cat Leopardus

: The Andean Cat Leopardus jacobita , one of the most rare and endangered feline species in the world, is distributed from central Peru to central Argentina. The aim of this study was to evaluate the health and morphometry of a subadult male Andean Cat that was rescued from wildlife trade in Bolivia and held captive for 165 days before being released back into its natural habitat. Physical immobilizations followed by anaesthesia using ketamine hydrochloride (KH) and xylazine hydrochloride (XH) were performed to obtain clinical, morphometric, haematological and serum chemical parameters. Physical immobilizations were efficient using capture nets. The combination of KH + XH had an average initial sedation effect within 12min with a range of 10–16 min after intramuscular application. Anaesthetic average plane lasted 41.7min with a range of 40–45 min and was extended to 64.5min (63–66 min range) with an addition of KH. The individual was underweight on arrival and gradually reached an ideal condition and was overweight before its release. Morphometry parameters showed that it grew during the captive period. It was released back into the wild when it was considered healthy. This is the first report of a protocol of physical and chemical immobilization, physiological values, and biometric variation of an Andean Cat under captive conditions.


INTRODUCTION
The Andean Cat Leopardus jacobita is one of the rarest feline species in the world and the most threatened in America (Andean Cat Alliance 2011). The species is classified as Endangered and is threatened by loss and degradation of habitat, opportunistic or palliative hunting and extremely low genetic diversity (Cossíos et al. 2012;Villalba et al. 2016). In Bolivia's Red List Species Book, it is listed as Critically Endangered, as it is threatened by hunting, fragmentation and alteration of habitat and by declining prey populations ).
The species is distributed from central Peru to central Argentina (Sorli et al. 2006;Cossíos et al. 2007;Novaro et al. 2010). In Bolivia, its presence was confirmed in the high Andean region of the departments of Potosí in southwestern Bolivia, of Oruro and Cochabamba in the central Andes of Bolivia and of La Paz in western Bolivia (Villalba et al. 2012;Huaranca et al. 2013).
To our knowledge, there is no published information on haematology, serum chemistry and clinical values of the Andean Cat. According to phylogenetic studies, the closest related species to the Andean Cat is the Pampas Cat Leopardus colocola (Johnson et al. 2006), for which only isolated data on chemical immobilization, clinical parameters and haematology in one freeranging individual are available (Beltrán-Saavedra et al. 2009). To date, morphometric information on Andean Cats is scarce, because most of the data correspond to measurements of museum skins, and just a few live individuals were measured (Pine et al. 1979;Yensen & Seymour 2000;García-Perea 2002;Noss et al. 2010;Tellaeche et al. 2018).
The aim of this study was to evaluate an anaesthetic protocol and determine clinical, morphometric, haematological and serum chemistry parameters, and their variations in an Andean Cat rescued from wildlife trade. The animal was kept in captivity at the Vesty Pakos Zoo in La Paz, Bolivia, prior to being relocated to its natural habitat.

STUDY AREA
The Vesty Pakos Zoo in the Municipal Autonomous Government of La Paz is a wildlife custody centre legally established and recognized by the National Environmental Authority, through administrative Resolution VMABCC # 34/15. It is located in La Paz city (-16.572°S & -68.083°W) at an elevation of 3,265m, with an extension of 201,522.15m 2 . On 15 March 2016, a subadult male Andean Cat was handed over by the La Paz Department Authority to the Vesty Pakos Zoo, which maintained the feline in temporary quarantine for 165 days. The animal was visually isolated from humans, and environmental enrichment was applied until it was considered healthy for release. This study was carried out on the basis of five evaluations, the details and dates of each are presented in Table 2.

MATERIAL AND METHODS
On arrival of the individual, Ixodidae ticks were removed from the edges of the ears, and through the coproparasitological enrichment technique faeces analyses were performed, finding eggs of nematodes of the order Strongylida (parasitic load 2 eggs/g faeces).
The Andean Cat was dewormed with a broad-spectrum anthelmintic (Oralmec Gold, ivermectin 1mg/g, pyrantel pamoate 80mg/g, praziquantel 35mg/g, Biomont S.A. Peru; ivermectin 0.2 mg/kg, pyrantel pamoate 16mg/ kg, praziquantel 7mg/kg oral route). Subsequent coproparasitological faeces analyses remained negative for endoparasites. Every day, the animal was observed using a remote camera (GoPro Hero 4, GoPro Inc., USA) and a digital camera (Nikon D5200, 300 mm lens, Nikon Corp. Japan). Various postures adopted by the animal were photographed to evaluate its attitude, bilateral symmetry, appearance, conformation, body condition, tail movements, motor activity, breathing, among others, that indicate possible abnormalities compatible with pathological signs. Inspections of fluids, urine or secretions were carried out during cleaning and environmental enrichment events.
During the captive period, the Andean Cat was fed every day for the first four weeks with a diet composed of live Guinea Pigs Cavia porcellus (595g average; diet proportion: 95.1%) and mice (30.7g average; diet proportion 4.9%), providing the mice once a week. Thereafter, to simulate natural conditions of feeding and because of its low activity inside the enclosure, the cat was fed every other day with a diet of red meat (600g average; diet proportion: 25.5%), heart (550g average; diet proportion: 23.4%) and live Guinea Pigs (800g average; diet proportion: 51.1%); this last item was provided once a week.

Physical and chemical immobilization
The Andean Cat was captured on two occasions with a 106.7 x 38.1 x 50.8 cm folding cage trap (Tomahawk Live Trap Co., USA) following free-ranging capture J TT methodologies (Beltrán-Saavedra et al. 2009). On the following three occasions, the individual was captured with a capture net and immediately covered with cloth to minimize stress.
Chemical immobilization was performed with a combination of dissociative anaesthesia ketamine hydrochloride (KH) (Ketamine 10%, 100mg/ml, Alfasan International B.V., The Netherlands; 10mg/kg intramuscular [i/m] route) and the muscle sedative-relaxant xylazine hydrochloride (XH) (Xilazine 2%, 20mg/ml, Alfasan International B.V., The Netherlands; 1-2 mg/kg i/m). This combination was administered with a hand syringe in the posterior extremity musculature, with the animal covered with cloth until the pharmacological effect was achieved. The veterinary staff remained silent during this time. If drug supplementation was necessary, only KH was used. We did not use any antagonist, as the individual was kept in all occasions in a Kennel cage of 55.9 x 35.6 x 35.6 cm covered with cloth during the recovery phase. After it woke up and was able to stand, its pupillary response to light and motor coordination was evaluated before it was released into the enclosure and after the last evaluation, into the wild.
Morphometric parameters were obtained according to previous publications (Emmons 1999;García-Perea 2002;Yensen & Seymour 2000;Noss et al. 2010;Tellaeche et al. 2018) and our own elaborated guide (Table 1; Figure  1). On the first four evaluations, measurements and body weight were recorded, and on the fifth evaluation only body weight was obtained as the individual was on the way to being released.
The individual was weighed with a digital scale (Inmobiliaria y Constructora TOR S.A. de C.V., San Nicolas MR 66480 México) to the nearest 0,001kg. Body measurements were obtained using a digital vernier caliper (Truper S.A. de C.V., Jilotepec MX 54240 México) to the nearest 0.1mm; and a measure tape and a ruler to the nearest 0.1cm. The increase in body size was calculated from the difference of the last measurement made for each parameter in relation to the first one; and considering that the tail is one of the most relevant characteristics for the identification of the species (Cossíos et al. 2007), the proportion of tail length in relation to body length was calculated.
Following García-Perea (2002) criteria regarding age, the parameters of head and body length (HBL) and tail length (TL), were also used to verify the initial determination of the individual's age as a subadult, which was based on its size and overall appearance.
Biometric data were compared to all available bibliographic data on the species (Table 1).

Blood collection, haematological and serum chemistry evaluations
Before haematological and serum chemistry evaluations, the individual had a 12-hour fasting period. On the first three chemical immobilizations, blood samples of the cephalic vein were collected, keeping 4ml in tubes with ethylenediaminetetraacetic acid (EDTA) for haematology studies, and 3ml in tubes without additives for serum chemistry. Haematological and serum chemistry parameters were determined in a commercial laboratory (Laboratorio Clínico Científico, La Paz, Bolivia) two hours after blood sampling.

Protocol of physical and chemical immobilization
The first two physical immobilizations with folding cage traps allowed the capture of the Andean Cat individual. On the third occasion, it avoided entering the cage, so that a capture net was used, allowing to reduce the time between the capture and the application of the anaesthetic drug.
The combination of KH + XH had an average effect within 12min (range of 10-16 min), after intramuscular (i/m) application to the start of sedation; the average duration of the anaesthetic plane was 41.7 min (range of 40-45 min); and when HK had to be added, it lasted on average 64.5min (range of 63-66 min) ( Table 2; Images 1 and 2).

Clinical and morphometric evaluations
Clinical evaluations showed that the individual arrived with underweight (first weight= 4.100kg). Between the evaluation II and IV, it was judged to have an ideal body weight, reaching 5.946kg at the evaluation V, when it was considered overweight. The Andean Cat increased 1.846kg during the captive period before it was released back into the wild (Table 2).   *Additional doses of ketamine were used after ~40-45 min to extend the duration of sedation, when more handling had to be done with the individual. The data recorded for body temperature (˚C), heart rate (min) and respiratory rate (min) are presented in the Table 3.
Morphometric parameters obtained from the individual in the first four evaluations are presented in Table 4, establishing changes in some parameters; these are compared with those reported by other authors. In the measurement of HBL and TL, the tail represented 57.4% of the total body length upon arrival and gradually increased to 58.1%.
The measurements of HBL and TL taken in the evaluations I to IV were within the range of measurements taken for subadults (Table 4). Therefore, when the Andean Cat was released into the wild, it was still considered to have been in this age category.

Haematological and serum chemical evaluations
Haematological and serum chemistry parameters obtained from the first three immobilizations are reported in Table 5.

DISCUSSION
The present work reports for the first time a protocol of chemical immobilization, haematological and serum chemistry parameters of a healthy Andean Cat individual; and contributes to the morphometry of the species.

Protocol of physical and chemical immobilization
In this study, the individual "learned" not to enter the cage trap, and the use of capture nets proved to be a more appropriate method of physical immobilization in a closed environment.
Chemical immobilization of other feline species such as Bobcat Lynx rufus, Guigna Leopardus guigna, Ocelot L. pardalis, and Pampas Cat suggest the use of ketamine and xylazine on doses ranging from 7.65-14.7 mg/kg KH and 0.74-1.4 mg/kg XH (Beltrán & Tewes 1995;Acosta et al. 2007;Tellaeche et al. 2020). For this individual Andean Cat under captive conditions, however, we had J TT a good and safe 40 minutes sedation period using a dose of 8.68mg/kg KH and 1.74mg/kg XH.

Clinical and morphometric evaluations
The Andean Cat was considered underweight on the evaluation I of its body condition, which may be related to its natural condition of being a wild subadult individual (García-Perea 2002), and a presumable fourday fasting period prior to its arrival at the Vesty Pakos Zoo. Subsequently, its body condition was judged ideal and within ranges of 4.0-5.8 kg recorded for the species by García-Perea (2002), Villalba et al. (2004), and Tellaeche et al. (2018). At the evaluation V, it was judged overweight, which was probably the effect of a low energy expenditure, and also by medical and nutritional care given during captive conditions.
Although hypothermia and hyperthermia are considered common adverse effects of chemical immobilizations of wild felids (e.g., Tellaeche et al. 2020), the Andean Cat showed none, probably because it was not too stressed prior to anaesthetic injection and because it was kept warm using hot-water bottles wrapped in cloth during procedures.
During sedation, heart and respiratory rates higher than the averages were initially recorded (129.7/min y 36.2/min respectively), possibly due to capture stress, which stabilized during the course of the sedations. Similar records were observed in a free-ranging wild Pampas Cat (Beltrán-Saavedra et al. 2009).
The cat's tail length ratio to the body length (58.1%) was slightly lower (60-75%) than those obtained of freeranging adult Andean Cats (Yensen & Seymour 2000;García-Perea 2002), and from skins (66-75%) of this species (Cossíos et al. 2007). Its body measurements, however, were similar or within the ranges reported for the species (Yensen & Seymour 2000;García-Perea 2002;Tellaeche et al. 2018). We report measurements of testis length of an Andean Cat for the first time. For further morphological measurements, we suggest to evaluate both left and right size of canines and testicles to identify the existence of possible asymmetries.

Haematological and serum chemical evaluations
In the evaluation I, the haematological values of red blood cells, mean cell haemoglobin concentration (MCHC), eosinophils, basophils and lymphocytes obtained were all within the reference ranges of Domestic Cats, free-ranging and captive Ocelots (International Species Information System 2002;Aiello & Moses 2016;Widmer et al. 2016), and similar to those reported in a free-ranging Pampas Cat (Beltran-Saavedra et al. 2009 Widmer et al. 2016). These differences, however, were not associated with pathological signs and abnormal haematological morphologies such as reticulocytosis, anisocytosis or polychromasia, which are compatible with anaemia or haemolysis (Aguiló 2001;Cowell 2004).
Also, in the evaluations I to III, the platelet counts (180, 190, and 260 10 9 /L) were lower than the reference ranges of Domestic Cats, free-ranging and captive Ocelots (International Species Information System 2002; Aiello & Moses 2016; Widmer et al. 2016). Ectoparasite ticks on the ears and a low parasite load of one type of nematode that were detected at the individual's arrival could be the cause for initial lower platelet count, which increased after removal of ticks and the deworming treatment. The low platelet count, however, was not associated with pathological signs such as bleeding, haemorrhagic diathesis or petechiae described by Nuñez-Ochoa (2007).
All the values of serum chemistry obtained in the evaluations I to III were within the reference ranges of Domestic Cats and free-ranging and captive Ocelots (International Species Information System 2002;Aiello & Moses 2016;Widmer et al. 2016), with little variation of creatinine, globulin and total protein values in freeranging Ocelots. In addition, total protein was similar to the values reported in a free-ranging Pampas Cat (Beltrán-Saavedra et al. 2009).
The only potassium value obtained in the evaluation I was a little lower than the reference ranges of Domestic Cats, whereas blood urea nitrogen and aspartate aminotransferase values were higher than the same reference ranges (Aiello & Moses 2016). There were, however, no clinical abnormalities, and these serum values are among those reported for free-ranging and captive Ocelots (International Species Information System 2002;Widmer et al. 2016).
The value of albumin/globulin proportion obtained in the evaluation I was higher than the ranges reported for free-ranging Ocelots (Widmer et al. 2016). The parameters of erythrosedimentation were not comparable, since previous authors did not report these data.
Using capture nets was more suitable than cage traps for the containment of this individual. The procedures using KH+XH with an intended dose of 10:2mg/kg proved to be safe and efficient for 40 minutes of procedure for this subadult male Andean Cat under captive conditions. Clinical parameters indicated that the individual remained in good body condition during captivity, showed a constant increase in size and weight, and was considered healthy following haematological and serum chemistry evaluations. This study contributed to the knowledge of physiological and morphological parameters of the Andean Cat, and together with other health and biological parameters allowed to determine the timing of release of this individual into its natural habitat. We strongly recommend to conduct more research on the Andean Cat to obtain additional data relevant for both in situ and ex situ conservation and management of this little-known cat.