GALERA RC612 (1).pdf

Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido:09/01/2025 Aceptado:26/02/2025 Publicado: 20/04/2025 hps://doi.org/10.52973/rcfcv-e35612 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 9 Eﬀects of Xylazin on sedave, analgesic and some liver enzymes in rats Efectos de la Xilazina sobre los efectos sedantes, analgésicos y algunas enzimas hepácas en ratas Nazlı Halitoğullari 1 , Ferit Yildiz 2 , Musa GenÇcelep 3 ¹Van Metropolitan Municipality, Department of Business Administraon and Aﬃliates, 65100 Van, Türkiye. ²Provincial Directorate of Agriculture and Forestry, 49100 Muş, Türkiye. ³Van Yuzuncu Yil University, Faculty of Veterinary, Department of Surgery, TR-65100 Van, Türkiye. *Corresponding author: ferit49@gmail.com ABSTRACT This study was conducted to determine the sedaon level of rats aﬅer using two diﬀerent doses of Xylazine, to measure the damage that may occur in the liver, and to determine the change in pain response due to sedaon. The use of analgesic substances is one of preferred methods in pain control studies. Analgesic eﬀects are also present in drugs used for sedave purposes. Xylazine is used for pain relief, calming (sedave), sleep inducing (hypnoc), and striated muscle relaxaon. It is also used for premedicaon purposes before using diﬀerent anesthec drugs. 24 rats used in the study were randomly selected and divided into three groups. Only physiological saline was given to the rats in the control group (KO). The second group was given Xylazine (K10) 10 mg/kg intraperitoneally (ip), and the third group was given Xylazine (K15) 15 mg/kg/ip. Sedaon assessment was performed according to the behavior of the rats to which sedave drugs were applied to the smuli. Sedaon was classiﬁed as mild, moderate and severe. The degree of pain-relieving properes of Xylazine in rats was measured with a pain test. The pain test was performed with a hotplate test and a hotplate apparatus. Aﬅer the pain tests, the rats were sacriﬁced aﬅer taking blood as needed. AST, ALT and corcosteroid values were measured in the blood serum. In this study, it was determined that Xylazine had pain-relieving properes and did not change liver enzyme values when used at a dose of 15 mg/kg in rats, and it was concluded that it could be preferred for safe sedaon in cases where short-term complete immobility was needed. Key words: Pain; rat; sedaon degree; Xylazine RESUMEN Este estudio se realizó para determinar el nivel de sedación de ratas después de usar dos dosis diferentes de Xilacina, y medir el daño que puede ocurrir en el hígado y para determinar el cambio en la respuesta al dolor debido a la sedación. El uso de sustancias analgésicas es uno de los métodos preferidos en los estudios de control del dolor. Los efectos analgésicos también están presentes en los medicamentos ulizados con ﬁnes sedantes. La Xilacina se uliza para aliviar el dolor, calmar (sedante), inducir el sueño (hipnóco) y relajar el músculo estriado. También se uliza con ﬁnes de premedicación antes de usar diferentes medicamentos anestésicos. Se seleccionaron al azar 24 ratas ulizadas en el estudio y se dividieron en tres grupos. Solo se administró solución salina ﬁsiológica a las ratas del grupo de control (KO). El segundo grupo recibió xilacina (K10) 10 mg/kg por vía intraperitoneal (ip), y el tercer grupo recibió xilacina (K15) 15 mg/kg/ip. La evaluación de la sedación se realizó de acuerdo con el comportamiento de las ratas a las que se les aplicaron fármacos sedantes ante los esmulos. La sedación se clasiﬁcó como leve, moderada y severa. El grado de propiedades analgésicas de la xilacina en ratas se midió con una prueba de dolor. La prueba de dolor se realizó con una prueba de placa calefactora y un aparato de placa calefactora. Después de las pruebas de dolor, las ratas fueron sacriﬁcadas después de extraerles sangre según fuera necesario. Se midieron los valores de AST, ALT y corcosteroides en el suero sanguíneo. En este estudio, se determinó que la xilacina tuvo propiedades analgésicas y no modiﬁcó los valores de las enzimas hepácas cuando se usó una dosis de 15 mg/kg en ratas, y se concluyó que podría ser la opción preferida para una sedación segura en casos en los que se necesitara inmovilidad completa a corto plazo. Palabras clave: Dolor; rata; grado de sedación; Xilacina

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Sedaon is a common procedure used to reduce the response of an animal that needs to remain calm to external smuli, to facilitate small applicaons and to increase safety [1]. It is the depression of the central nervous system with drowsiness due to the applicaon of sedave drugs. It is the calming of the animal by pung the central nervous system under mild pressure. The paent is relaxed and calm despite being awake [2]. Such drugs are used in paents to reduce fear and anxiety, to minimize the total dose of anesthec to be administered, to provide easier inducon, to provide easier and more comfortable recovery from anesthesia, to provide pain relief, to reduce bronchial and saliva secreon, and to eliminate the vasovagal reﬂex [3]. The sedave and analgesic eﬀects depend on the acvaon of the supraspinal and spinal regions of alpha2-adrenoceptors [4 , 5]. The primary purpose of using alpha2-adrenoceptor agonists is to cause sedaon in animals. In addion to these eﬀects, they also cause analgesia due to their central and spinal eﬀects [6]. In mammals, alpha2-adrenoceptors are found in many ssues and systems such as the gastrointesnal tract, uterus, and platelets, and their smulaon causes diﬀerent eﬀects. Sedaon and analgesia occur as a result of smulaon of alpha 2-adrenoceptors located in the central nervous system [6]. Their eﬀects on the respiratory system vary according to animal species. In dogs (Canis familiaris), cats (Felis catus) and horses (Equus caballus), doses that provide advanced sedaon may reduce the respiratory rate and, to a lesser extent, decrease the Paral Arterial Carbon Dioxide Pressure (PaCO 2 ) value. In ruminants, tachypnea may occur, respiraon is provided with eﬀort and the PaCO 2 level decreases [6]. Requirements for ideal sedaon First of all, the animals must be hungry. A calm environment/ room is required before and aﬅer the injecon for the maximum eﬀect of sedave drugs. There should be no animal sounds of the same species/breed and diﬀerent genders. Especially for cats and caged poultry, there should be no sounds of animals of the dog type. For pets, there should be no general noise or buzzing in the environment, as well as the smell of previous animals, and if possible, there should be dim lighng [2 , 6]. Pain The most valid deﬁnion of the concept of pain today was made by the Internaonal Associaon for the Study of Pain (IASP). According to this organizaon, pain; It is an unpleasant sensory and emoonal experience that accompanies exisng or potenal ssue damage or can be deﬁned by this damage [7 ,8]. Animal pain studies deﬁnitely require animal material. The more suitable this material is for the study, the more accurate the results are. The assessment of pain in animals is quite problemac. An animal that cannot express itself verbally will show itself by giving similar reacons in similar events. Its response is usually simple reﬂexes, somemes in the form of vocalizaon or escape. What is important in such experiments is to learn when the animal perceives pain. Methods using mechanical, thermal and chemical smuli have been developed to understand the presence of pain [9 , 10]. Xylazine Xylazine is a non-narcoc compound used in veterinary medicine as a sedave, analgesic, and muscle relaxant, and is oﬅen referred to as “anestesia de caballo” or “horse anesthesia”. It is not approved for human use as an anhypertensive because of its extreme depression of the central nervous system, profound hypotension, but is introduced for veterinary use as a sedave, emec, analgesic, and muscle relaxant [11]. It is a premedicant drug used before anesthesia in the vast majority of animal species. It is applied by iv. or im. in horses (Equus caballus), cale (Bos taurus), sheep (Ovis aries), goats (Capra hircus), dogs (Canis familiaris), cats (Felis catus), pigs (Sus scrofa domescus) and poultry (Gallus gallus domescus). It produces varying degrees of muscle relaxaon and analgesia depending on the animal species [6 , 12]. Xylazine is oﬅen used together with ketamine as an anesthec agent, especially in experimental studies on rabbits (Leporidae) and rats (Raus) [11]. Aﬅer Xylazine applicaon, cardiac output may decrease by 1/3 or even 1/2 and blood pressure may decrease by 1/4 to 1/3. These eﬀects occur in almost all species. Aﬅer the applicaon of the recommended clinical dose of Xylazine, respiratory rate decreases, while arterial pH, PaCO 2 and PaO 2 levels do not change in cats and dogs, while there is a minimal decrease in horses [6]. Xylazine and sedaon degrees Some researchers classiﬁed the stages aﬅer the onset of sedaon in sheep with Xylazine into 3 as mild, moderate and severe [13]. Mild sedaon occurs when Xylazine is administered to sheep at 0.1 mg/kg/iv [14]. In cale, mild, moderate and severe sedaon has also been classiﬁed [2 , 6]. In a study invesgang the clinical eﬀects of Xylazine premedicaon on water buﬀalo calves (Bubalus bubalis) castrated under general anesthesia, 10 animals were administered 0.1 mg/kg/im and 0.15 mg/kg/im Xylazine, anesthesia was induced with Ketamine (2 mg/kg/iv) and maintained with Isoﬂurane-Oxygen-air. Safe and consistent sedaon was achieved in both groups, which were evaluated every 5 min [15]. Topal [6] reported that administering 1 mg/kg/ iv Xylazine to horses caused mild sedaon. In a study in which 1.1 mg/kg/iv. xylazine was administered to seven clinically healthy male adult miniature donkeys (Equus asinus) and the degree of sedaon was controlled, it was determined that mild or moderate sedaon was observed in the donkeys between 5 and 60 min aﬅer treatment and that no sedaon was observed aﬅer 90 min, and that all animals recovered from sedaon without complicaons within 2 h [16]. In another study conducted in Sri Lanka on 15 young Asian elephants (Elephas maximus), the ﬁrst signs of sedaon occurred within 5-18 min aﬅer injecon of Xylazine (50-110 mg per elephant; 0.09-0.15 mg/kg/im), and all elephants were sedated in standing posion. Twelve elephants remained standing during the sedaon period, while 3 elephants were reported to have recumbent [17]. Pain classiﬁcaon according to origin According to the region where it originates, pain is classiﬁed as visceral, somac, sympathec and peripheral pain. Visceral pain can be widespread, diﬃcult to localize and reﬂected. It is also generally called pain originang from internal organs. Somac pain is a type of pain that originates from somac nerves, starts suddenly, is sharp and well localized. Vascular pain that occurs with the acvaon of the sympathec nervous system is called sympathec pain. The source of peripheral pain can be muscle, tendon or peripheral nerves [18]. 2 of 9

Xylazine sedaon and pain sensaon in rats / Halitoğullari et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Pain classiﬁcaon according to mechanisms a-Nocicepve pain: Free nerve endings that respond to thermal, mechanical and chemical smuli that cause ssue damage are called “nociceptors”. The enre series of events that create the pain felt with ssue damage is called “nocicepon” [19]. The processes that occur in physiopathology occur as a result of the smulaon of pain receptors called nociceptors. Nociceptors are found in all ssues and organs, as well as in the nervous system [20]. Nocicepve analgesia is perceived in four physiological stages [21 , 22]. In the periphery; transducon and transmission, in the medulla spinalis; modulaon, in the transmission to the central nervous system; percepon plays a role [23]. b-Neuropathic (non-nocicepve) pain: The Internaonal Associaon for the Study of Pain (IASP) reports neuropathic analgesia as a pain caused by damage or dysfuncon of any part of the nervous system [24]. The formaon of neuropathic pain occurs as a result of compression, cut, ischemia, inﬁltraon or damage of neurons. The source of neuropathic pain occurring in any part of the body may be the peripheral nervous system, central nervous system or autonomic nervous system. Accordingly, it is classiﬁed as centrally or peripherally sourced neuropathic pain [25]. Evaluaon of neuropathic pain models in animals Von frey test Von frey ﬁlaments are of various thicknesses (0.25-2 g) and are performed to detect tacle allodynia. In order to detect allodynia, measurements should ﬁrst be made on a normal subject. In the von frey test, diﬀerent ﬁlaments are pressed against the subject’s skin in the mid-plantar areas for measurement. A total of ﬁve presses are made at a frequency of twice per second. If the animal withdraws its paw, this ﬁlament is recorded as the threshold value in the records. Over me, a decrease in the threshold will be observed in applicaons made to the extremity. In order to talk about tacle allodynia, it must withdraw its paw signiﬁcantly with the lowest smulus [9]. The subject is placed in a closed test box and the device is placed so that it coincides with the subject. With the automac version, the values of the mechanical pain threshold are observed with the Dynamic Plantar Anesthesiometer. With the help of a mirror, a force that can increase up to 50 g is applied to the plantar surface of the animal’s hind paw and as the animal withdraws its paw, the value on the device’s screen is accepted as the nocicepve threshold value. This value is automacally recorded by the device [9]. Hotplate test The hotplate test is performed by placing a mouse (Mus musculus) or rat (Raus norvegicus) in a gap with an open-ended cylinder placed on a metal surface. Although ﬁrst described by Woolfe and MacDonald in 1944. It usually consists of a surface (copper or aluminum) heated to 50-56 degrees. In order for the animal to remain on the heated surface in certain areas, it must be used in glass cylinders that are large enough to not restrict its movements. The me elapsed from the moment it is placed on the surface unl the animal withdraws its hind leg is observed. It is one of the most preferred methods in the sources. On the other hand, the observaon criteria for the animal feeling the sensaon of pain vary. There are problems in understanding which of the behaviors it shows is a pain indicator. Aﬅer a constant temperature is achieved, the reacon me of a mouse or rat placed in a cavity is used to evaluate two behaviors. The behavior shown may be pulling the animal’s foot, or it may be licking, kicking, shaking, dancing or jumping. When examining the animal’s licking behavior, the licking of the hind legs is taken into consideraon more. Because it can lick its front legs at normal mes. The test is terminated when the animal jumps because it tends to try to escape when it touches the ground. The me aﬅer the test is terminated is recorded. The average duraon of the reacons a mouse can give varies between 5-20 s. In order not to cause ssue damage, the test should not be applied for more than 30 s [9 , 10]. This study was conducted to determine whether Xylazine has analgesic properes aﬅer injecon of a sedave dose into rats, to reveal the damage it may cause to the liver, and to clinically grade sedaon in rats. MATERIALS AND METHODS Twenty four female, adult and healthy Wistar albino rats were used in the study. Rats were housed in an environment with an average temperature of 20-24 o C and 50-60% humidity, with 12 h of light and 12 h of darkness. Standard pellet rat feed and drinking water were given ad libitum for feeding the rats. Rats were randomly divided into three groups, with 8 rats in each group. Hotplate device (May AHP 0603 Analgesic Hot Plate, Commat, 2018, Turkey) (FIG. 1) and Modiﬁed hotplate apparatus (10 cm handle, 15 mm diameter and 20 mm height brass cylinder) were used for the tests. The apparatus was applied for tesng when it reached 56 o C (Sous vide cooker, Proﬁ Cook PC-SV-1126, 2019, France) (FIG. 2A-B). Control group (KO group n=8): Each animal in this group was injected with 0.10 mL of Physiological Saline (NaCl 0.9%) intraperitoneally. Xylazine 10 mg group (K10 group, n=8): In order to create sedaon, 10 mg/kg dose of Xylazine was injected intraperitoneally to the animals in this group. Xylazine 15 mg group (K15 group, n=8): In order to create sedaon, 15 mg/kg dose of Xylazine was injected intraperitoneally to the animals in this group. 3 of 9

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 1. Hotplate device used in the study FIGURE 2. Hotplate apparatus (A) and temperature stabilizer (B) Sedaon assessment Aﬅer administering physiological saline in the KO group and sedave drugs in the K10 and K15 groups, observaons were made at the 1st, 2nd, 3rd, 4th, 5th, 10th, 15th, 20th, 30th and 45th min to grade sedaon. To evaluate sedaon, rats were placed in transparent 15-liter containers immediately aﬅer injecon and monitored without being disturbed. When they became immobile during observaon, they were smulated audibly and mechanically by hing two metal pieces together (a 6-8 cm metal plate was hit 2-3 mes). Their reacons to aempts to catch, hold, and turn were checked. The disappearance of the Righng Reﬂex (RR) (coming to lateral or dorsal recumbency) and the me for RR return (the animal coming to sternal recumbency aﬅer lying lateral or dorsal) were checked. Aﬅer the sedave agent was applied, the movements and behaviors of the animals were monitored and sedaon was graded according to the criteria below, using the sedaon classiﬁcaons of many researchers. The presence and evaluaon of sedaon were done separately for each rat [13 , 26 , 27]. No sedaon (Degree 0): The animal is acve, mobile, and has the acvaon to respond to all kinds of environmental smuli. It makes an escape movement during the manual capture process with the help of a towel or cloth. It can bite and scratch when caught. RR is present. Mild Sedaon (Degree 1): Its mobility has decreased but it is sll acve. It may be sleepy, it may wait in lateral recumbency. It may stand or walk slowly. It may lean/hit the edges of the container or cage wires it is in. Its reacons have weakened aﬅer the manual capture process. RR has not disappeared. Moderate Sedaon (Degree 2): It is very sleepy, there is dorsal or lateral recumbency. However, it may walk slowly or make diﬀerent foot/head movements during the manual capture process with the help of a towel or cloth. RR is sll present. Severe Sedaon (Degree 3): It is immobile and unresponsive to environmental smuli in lateral, abdominal or dorsal recumbency. It remains in the same posion when its posion is changed. There is no response during and aﬅer the capture/ holding process. In the lying posion, movements such as tail, leg and paral tremors in the head can be observed. RR is always negave. Conducng a temperature test to evaluate the analgesic eﬀect To determine whether Xylazine has pain-relieving properes in rats, two materials with the same temperature were used. In the pre-sedaon period, rats that were not sedated were placed on a hot surface in the sternal (abdominal) posion in the hotplate device and tested. Aﬅer sedaon began, a pain test was performed using the hotplate apparatus. When a rat is placed on a hot surface in the hotplate device heated to 56 o C, it will be able to change its body posion, perform a ﬂexor reﬂex or lick its foot due to the pain/ache it experiences. However, when it is placed on a hot surface in the same device in the sternal posion during the sedaon period, it will not be possible for it to change its body posion or perform a ﬂexor reﬂex even if it feels the pain or ache. Therefore, since misleading results may occur, it was found appropriate to develop a hotplate apparatus and check the presence of ﬂexor or extensor reﬂexes against heat at the same temperature in the lateral lying posion under sedaon. 4 of 9

Xylazine sedaon and pain sensaon in rats / Halitoğullari et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico In order to keep a rat that has not been sedated in the lateral posion before sedaon, it is necessary to hold it ghtly from the nape and tail base to reduce its movements. In addion, the rat will make an intense eﬀort to get out of this posion. It was thought that these procedures would aﬀect the state/me of response to the hot apparatus applied to the sole of the foot and that incorrect results would occur. Considering these two situaons, the pain test was performed with a hotplate device before sedaon and with a hotplate apparatus with the same temperature during sedaon. Acclimaon to the device before sedaon In order for the rats to get used to the hotplate device environment before sedaon, each rat was kept on the device unl it came out of the glass globe or for 3 min while the device was closed and then taken from the device and placed in their cages. Performance of the pain test with the hotplate device before sedaon (BS) Aﬅer the device was turned on before the sedave drug was applied and reached a constant temperature of 56°C, each of the 24 rats was placed on the hot surface and their behaviors were evaluated in terms of determining the reacon me of the rat. Each rat was monitored for behavioral changes by two observers. Aﬅer the rat was placed on the hot ﬂoor, it was evaluated whether it quickly pulled/liﬅed its legs away from the hot ﬂoor (ﬂexor reﬂex), licked the sole surfaces of its feet or jumped. Liﬅing and licking the forelimbs from the ﬂoor is a behavior that can occur at normal mes. However, it was also taken into account that the me of liﬅing and licking the feet from the ﬂoor may be diﬀerent due to the hot ﬂoor. Performing the pain test with the hotplate apparatus during sedaon (DS). One liter of normal water was placed in a suitable container and started to be heated with a circulatory water heater (FIG. 2B). When the water temperature reached 56 °C, this process was automacally terminated by the device and the water temperature was kept constant due to its feature. The modiﬁed hotplate apparatus was kept inside from the me the water started to heat up. Ten min was waited for the mild sedaon that occurred aﬅer the sedave agent was administered. At the end of this period, the pain test was performed considering that the rat would not pass to the moderate/severe level of sedaon. Pain reﬂex test was performed at the 10th min in rats with moderate sedaon and at the 10th min aﬅer severe sedaon. When the test was to be performed under sedaon, the rat was ﬁrst placed in the right and then leﬅ lateral recumbency posion and the upper forefoot and hindfoot soles were touched by holding the handle of a modiﬁed hotplate apparatus at 56 °C (FIGS. 6-7). The me between the rat’s withdrawal of its foot (ﬂexor reﬂex, withdraw reﬂex) aﬅer the foot sole was touched was recorded. Serum aspartate transaminase (AST), Alanine aminotransferase (ALT) and corsol parameters were measured (DAS Elisa Reader, 2019, Halomedicals Systems Limited, Italy) to determine the liver damage status. Variance analysis (ANOVA), Duncan mulple comparison and Paired Samples T test for dependent samples were applied in the stascal evaluaon of the data (SPSS 22). RESULTS AND DISCUSSION Clinical results in the KO group No changes were detected in the behavior of the rats in the control group during the 45 min aﬅer the physiological saline injecon. Clinical results in the K10 group As seen in TABLE I, it was observed that 7 out of 8 rats entered sedaon at the 5th min, 6 of them entered the moderate sedaon stage at the 10th min and remained in this state unl the me of awakening, and 2 of them entered the severe sedaon stage very quickly from mild sedaon (TABLE I). TABLE I. Sedaon levels determined in the K10 group K10 Group 1. min 2. min 3. min 4. min 5. min 10. min 15. min 20. min 30. min 45. min K10-1 0 0 0 0 1 1 2 2 1 — K10-2 0 0 0 0 1 1 3 3 2 — K10-3 0 0 0 0 1 3 3 3 2 — K10-4 0 0 0 0 2 2 2 2 1 — K10-5 0 0 0 0 1 2 2 2 1 — K10-6 0 0 0 1 1 2 2 2 1 — K10-7 0 0 0 0 0 2 2 2 1 — K10-8 0 0 0 1 1 2 2 2 1 — 0: no sedaon, 1: mild sedaon, 2: moderate sedaon, 3: severe sedaon, min: minute, —: no evaluaon was made. At the 30th min, it was observed that the current sedaon state in all rats had eased and at the 45th min, it was thought that there was no need to evaluate sedaon and the monitoring process was terminated. Blood samples were taken from all rats and the rats were sacriﬁced. The blood samples were centrifuged and the serum was separated. Clinical results in the K15 group As seen in TABLE II, all rats were sedated at the 5th min, 7 of them were in moderate sedaon and 1 was in severe sedaon at the 10th min. Sedaon connued regularly and without any problems unl the 20th min except for 1. A total of 3 rats were in severe sedaon (TABLE II) (FIGS. 3, 4 and 5). 5 of 9

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE II. Sedaon levels and sedaon duraons in the K15 group. K15 Group 1. min 2. min 3. min 4. min 5. min 10. min 15. min 20. min 30. min 45. min K15-1 0 0 0 0 1 2 2 3 3 2 K15-2 0 0 0 0 1 2 3 3 2 — K15-3 0 0 0 1 2 2 2 2 1 — K15-4 0 0 0 1 1 2 2 2 1 — K15-5 0 0 0 0 1 3 2 2 1 — K15-6 0 0 0 0 1 2 2 2 1 — K15-7 0 0 0 1 1 2 2 2 1 — K15-8 0 0 1 1 1 2 2 1 — — 0: no sedaon, 1: mild sedaon, 2: moderate sedaon, 3: severe sedaon, min: minute, —: no evaluaon. In the 30th min control, it was observed that sedaon had eased in 7 of the rats. In the 45th min control, it was observed that the degree of sedaon had eased in the last one, and the monitoring process was terminated. Blood samples were taken from all rats and the rats were sacriﬁced. The blood samples were centrifuged and their serums were separated. Pain test results It was determined that the rats placed in the hotplate device for the purpose of accustoming them ﬁrst sniﬀed the device, calmly walked around inside it, and then tried to get out. It was observed that some of them sat on their haunches and calmly licked their front feet and some their hind feet. Aﬅer being placed in the heated device to perform the pain test before sedaon, the sniﬃng and walking process was performed again. However, it was determined that seconds later, they quickly pulled their feet oﬀ the ground (ﬂexor reﬂex), then pulled their other foot, quickly licked it, then pulled their other foot and quickly licked it, and quickly got out of the container. When these behaviors were determined, the rat was taken oﬀ the device. It was determined that the foot pulling and licking behavior was much faster than normal behaviors. A pain test was performed with the hotplate device before applying physiological saline to the rats in the KO group. However, since no sedaon occurred as a result of the physiological saline applicaon, pain tesng could not be performed with the hotplate apparatus. When the hotplate apparatus at 56 °C was applied to the feet of the rats under sedaon (K10, K15), only ﬂexor reﬂexes occurred in the subjects. The mean reﬂex mes in the KO, K10 and K15 groups are presented in TABLE III. TABLE III. The mean pain test reﬂex mes of all groups. Groups BS (second) DS (second) P KO 11.62 — K10 12.37 13.50 0,051 K15 13.12 17.87 0,017* —: test could not be performed. * Since p<0.05, there is a stascally signiﬁcant diﬀerence between BS and DS in the K15 group. BS: Mean duraon of foot withdrawal, licking and jumping behaviors on the hotplate device before sedaon. DS: Mean duraon of foot wit- hdrawal behavior aﬅer contact with the hotplate device during sedaon In TABLE III, the pain sensivity and response mes of rats in the K0, K10 and K15 groups before and during sedaon were measured and their average mes were compared. Accordingly, it was observed that the reﬂex mes were longer in the K10 and K15 groups both before and during sedaon than in the KO group. In addion, the fact that the response mes of the K15 group were signiﬁcantly longer than in the K10 group before and during sedaon showed that beer sedaon was provided. Biochemical results Serum AST, ALT and corsol values of all groups are presented in TABLE IV. 6 of 9

Xylazine sedaon and pain sensaon in rats / Halitoğullari et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE IV. Mean values of biochemical parameters Groups KO K10 K15 Mean P AST 53.5000 52.1250 72.8750 59.5000 0.089 ALT 27.2500 25.3750 30.3750 27.6667 0.546 Corsol 1.6138 b 2.2025 a 2.2763 a 2.0308 0.001 * * Since p<0.05, there is a signiﬁcant diﬀerence between the groups (ANOVA – analysis of variance). Duncan mulple comparison test was used. There is a stascally signiﬁcant diﬀerence between those with diﬀerent leers (a, b, c) In TABLE IV, serums of all rats were taken and liver enzymes (AST, ALT) and corsol values were measured and the average values of these biochemical parameters were compared. Accordingly, while no diﬀerence was observed in terms of AST and ALT enzymes in the K0, K10 and K15 groups, it was observed that the corsol value was lower in the K0 group and was close to each other in the K10 and K15 groups and there was no signiﬁcant diﬀerence. Xylazine was ﬁrst reported in animals in the late 1960s and is currently approved for use in veterinary medicine as a nonopioid tranquilizer. Xylazine is frequently used in conjuncon with ketamine as an anesthec in experimental studies in dogs, cats, horses, rabbits, and rats [11]. Xylazine has been implicated in signiﬁcant morbidity and mortality in the United States and worldwide in recent years due to its muldrug use and its diﬀerent mechanism of acon compared to illicit opioids such as heroin and fentanyl. Therefore, it is extremely important to be aware of the threat that these powerful drugs pose when used illicitly [28]. Karasu and Gençcelep [13] classiﬁed the stages aﬅer the start of sedaon in sheep with Xylazine into 3 as mild, moderate and severe. Carvalho [14] stated that mild sedaon occurs when Xylazine 0.1 mg/kg/iv is administered to sheep. Mild, moderate and severe sedaon classiﬁcaons have also been made in cale [2 , 6]. Mild sedaon occurs when Xylazine 1 mg/kg/iv is administered to horses [6]. It has been reported that mild, moderate and severe sedaon occurs in cats and dogs using Xylazine and other chemical agents [12 , 29 , 30]. In a study evaluang the eﬀecveness of Xylazine-Ketamine anesthesia on reﬂexes and vital signs during and aﬅer tendon surgery in rabbits; Xylazine (5 mg/kg/im) and Ketamine (35 mg/ kg/im) injecons were administered to rabbits, and reﬂexes, ear pinch reﬂex and pedal reﬂex were measured before and aﬅer the anesthesia injecon. It was stated that when Xylazine- Ketamine was administered together, the return of reﬂexes was delayed compared to the group administered only Ketamine, the duraon of surgical anesthesia was longer, and adequate anesthesia was provided for the rabbits as evidenced by good cardiovascular and other clinical indices [31]. In the study, it was determined that three diﬀerent degrees of sedaon emerged in both groups by looking at the movement/behavioral changes of rats that were administered Xylazine at two diﬀerent doses. It was also observed that the quality of sedaon was beer in the K15 group than in the K10 group. Karasu and Gençcelep [13] determined the lateral recumbency of the animal as the limit in the clinical disncon between moderate and severe sedaon in their study on sheep. In this study, the loss of the righng reﬂex in rats was evaluated as the most important disnguishing point of severe sedaon. Xylazine is frequently applied to horses and ruminants for sedaon and analgesia. The sleepiness aﬅer Xylazine applicaon may connue for 1-2 h, but the analgesic eﬀect is as short as 15-30 min. Painful intervenons should be performed within 10-15 min of the sedave eﬀect. It is known that Xylazine is more eﬀecve than opioids in eliminang pain in horses and ponies [6]. Xylazine is an alpha-2 adrenergic receptor smulant that was ﬁrst used in veterinary medicine for analgesic and sedave purposes [32]. It is reported that Xylazine has analgesic properes according to animal species. Gençcelep and Karasu [27] stated that in the sedaon they performed with Xylazine in sheep, it did not have analgesic properes in mild and moderate sedaon, but mild analgesic properes in severe sedaon. In the study, the fact that the response mes aﬅer contact with the hot plate apparatus in the K10 and K15 groups were longer than in the KO group was evaluated as the presence of the analgesic property of Xylazine in rats. However, the fact that the serum corsol level had diﬀerent values compared to the control group was interpreted as the analgesic property of Xylazine being very low. Corsol is a major glucocorcoid that is necessary for life and is found aﬅer physiological stress. Corsol levels remain high for a long me aﬅer trauma and systemic stress [18]. Some researchers reported that Xylazine reduces serum corsol levels, but corsol levels increase under the inﬂuence of diﬀerent stresses [33]. In a study conducted on ten healthy female Merino meat sheep; sedaon with Xylazine was applied to sheep lying on their backs, assuming that hoof trimming creates a short but intense stress situaon, and there was no increase in respiratory rate and rectal temperature, but a decrease in heart rate, and a decrease in escape, defense and general stress behavior (reducon in head and leg movements, sing on the knees and licking) were observed. Serum corsol concentraons were also measured at 2.28 mes lower levels compared to control sheep [34]. In the study, the fact that the response mes were longer in the K10 and K15 groups aﬅer the contact with the hot plate apparatus compared to the KO group was evaluated as the presence of the analgesic property of Xylazine in rats. The increase in serum corsol levels in the K10 and K15 groups compared to the KO group was thought to be due to the pain stress resulng from the contact of the hot plate apparatus with the animals’ feet.AST and ALT biochemical values are used to evaluate the damage status in the liver in terms of laboratory. In the liver, muscle damages and metabolic disorders, the levels of Aspartate amino transferase (AST) and Alanine amino transferase (ALT) enzymes in the blood are important in terms of indicators of damage [35 , 36 , 37]. Enzymes produced in hepatocytes by liver cells are the ﬁrst structures that determine the damage in the liver. Under normal condions, liver cells store these enzymes. However, when an injury or damage occurs in the liver cells, these enzymes mix with the blood and can be detected with blood tests. The enzymes that are speciﬁc to the liver and are most commonly used to reveal liver damage are amino transferases (AST and ALT). AST and ALT tests are among the tests used in the diagnosis of hepatocellular damage [38]. As a result of this study, the fact that no change was detected in serum AST and ALT values was interpreted as Xylazine did not cause any damage to the liver. 7 of 9

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico In a study on castraon of water buﬀalo calves, it was emphasized that among the two groups premedicated with 0.1 mg/kg/im and 0.15 mg/kg/im Xylazine, a faster and more sasfactory sedaon occurred in the group administered 0.15 mg/kg/im Xylazine [15]. CONCLUSION In this study, especially in rats, which are frequently used as experimental animals for many diseases, treatments, medicaons, surgical intervenons and other experimental purposes; It was determined that Xylazine could be administered at doses of 10 and 15 mg/kg/ip as a sedave, analgesic and muscle relaxant, and could be used easily and safely as it did not change AST and ALT values and therefore did not cause liver damage. 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