**1. Introduction**

Animals are frequently used in research, in particular in biomedical studies [1]. The selection of the appropriate animal species and a model for the specific scientific question under investigation are the first steps towards a successful study [2,3]. If the species and model utilized have not been tailored to the study goal, it might prove impossible to answer the research question. Furthermore, when planning an animal study, it is necessary to consider the 3R principles. An animal study should only be considered if no alternative method exists to answer the research question (replace). Further, by careful planning, it should also be ensured that a maximum of relevant information is obtained from as few animals as possible (reduce), and last but not least, the burden of the animals is alleviated as much as possible by adapting standard protocols and procedures to the chosen species and model (refine) [2].

Sheep are commonly used as a preclinical model in orthopedic research [1,4–9]. They are chosen due to their similarities to humans in terms of body weight, rate of bone healing [7], and characteristics of mineralization of long bones [1]. Additionally, the size of several bones can be compared with the one of humans [10]. For example, sheep were employed by Yamamuro et al. to test a prosthesis for the replacement of the lumbar vertebrae [6], and by den Boer et al. to develop a new segmental long bone defect model [7]. In an implant-associated infection study, Boot et al. used a sheep model to investigate different treatment strategies after implantation of a intramedullary implant in the tibia [8]. In all these models and the majority of orthopedic research, surgical intervention is essential to mimic the clinical problem. As in clinical cases, surgical interventions require the implementation of an appropriate analgesic protocol. In research, the analgesic protocol is not only a tool but can also be an important determinant for the study outcome since pain can majorly impact the reproducibility of results [11]. Therefore, an analgesic protocol tailored to the species and model is a necessity not only for animal welfare reasons, but also to ensure the acquisition of high-quality data.

In order to control pain produced by invasive surgeries, analgesic drugs are typically administered pre-, intra- and a postoperatively. Before and during surgery, the sheep is under general anesthesia with a venous access allowing easy administration of analgesic medication intravenously. On the contrary, after surgery, the sheep is awake, and administration of pain medication can be stressful. In addition, pain assessment during the postoperative phase is very challenging and insufficient analgesia could easily be unrecognized [12,13]. An adequate postoperative analgesic protocol should therefore guarantee constant analgesia adapted to the level of pain inflicted with minimal side effects and stress.

Opioids are analgesics for moderate to severe pain, often used to obtain sufficient pain relief due to invasive surgical procedures [14]. Fentanyl is a strong synthetic opioid commonly used in analgesia protocols for humans and animals [15–19]. Due to the short half-life of this drug, systems allowing continuous administration are required [16]. During surgery, fentanyl can be given by continuous rate infusion (CRI) via infusion pumps. Such tools can be heavy and can disturb the freedom of movement of sheep when awake. For this reason, fentanyl, as an injectable medication, is not the method of choice during the postoperative phase. However, fentanyl can also be applied via a transdermal fentanyl patch (TFP), which consists of a polyacrylate adhesive layer allowing absorption of fentanyl through the skin (according to the information provided by Mepha® Pharma AG). With such a TFP providing a continuous and prolonged fentanyl administration, the sheep can be left undisturbed, neither stressful injections nor infusion pumps are needed [20,21]. Therefore, the transdermal application of fentanyl is a suitable method to provide analgesia for a time period of several days [16].

In sheep, the foreleg is a commonly used location for TFP since the application is very simple [20,22]. However, not only the ease of application but also the onset of action as well as the overall achieved fentanyl plasma concentration should be considered. In a study by Ahern et al., the TFP was applied 12 h prior to surgical intervention to compensate the slow uptake and achieve analgesic levels intraoperatively [22]. In dogs, TFP application has been recommended even 24 h before surgery [23]. In horses however, it has been shown that the application of a TFP at the foreleg results in a lower absorption rate and lower fentanyl plasma levels when compared to the application at the groin region or thorax [24]. In another study in horses by Orsini et al., the TFP applied at the thorax showed a fast fentanyl uptake [25]. Considering these findings in horses, it is logical to hypothesize that the thorax might be preferable in sheep as well, because of better skin perfusion, less affected by temperature

variation and lower mobility when compared to the limb. Additionally, the onset of action might be faster, making it unnecessary to apply the TFP many hours before its desired e ffect.

The comparison of di fferent locations for TFP application in sheep has not been described yet.

The aim of this study was to compare the fentanyl uptake resulting from a TFP applied at two di fferent locations, namely the foreleg and the thorax, in healthy adult sheep.

Following TFP application, fentanyl plasma levels were measured at di fferent time points. It was hypothesized that with the TFP applied at the thorax the fentanyl uptake would be faster and a higher peak would be reached than with the TFP applied at the foreleg.

### **2. Materials and Methods**
