**4. Discussion**

The goal of this survey among sheep users was to gather information on the current situation, with emphasis on animal selection criteria and issues encountered in practice if sheep are used for biomedical research. This survey has revealed that, currently, there is a strong need for improvement regarding sheep selection for biomedical research. The major issue identified was the animals' health status, which was, unfortunately, often insufficiently known when the animals were enrolled in experiments. With about 60% of the participants reporting on having encountered health issues unrelated to the experimental protocol, this seems to be a common problem. This may have a grea<sup>t</sup> impact on the experimental results, on the reproducibility of the findings and engender unnecessary ethical and scientific costs.

In this survey, only half of the participants followed a health monitoring program in sheep, whereas such programs are considered to be state of the art in rodent facilities. Furthermore, if there are sheep monitoring program in place, they differ substantially from institution to institution and are focused on only a few pathogens. It has to pointed out that the EU Directive 2010/63 states Annex III (requirements for establishments and for the care and accommodation of animals) in Section 3.1 that "establishments shall have a strategy in place to ensure that a health status of the animals is maintained that safeguards animal welfare and meets scientific requirements. This strategy shall include regular health monitoring, a microbiological surveillance program and plans for dealing with health breakdowns and shall define health parameters and procedures for the introduction of new animals" [2]. Beside the legal requirement, in the authors' opinion, health and welfare monitoring is refinement. The definition of "refinement" in animal experimentation has evolved significantly in recent decades. Russel and Burch, the creators of the 3Rs concept, described as refinement "simply to reduce to an absolute minimum the amount of stress imposed on those animals that are still used" [5]. They further characterized refinement as "an art or an ability to improvise", mentioning that "the greatest experimenters have been artists in this sense". Several decades later, Buchanan-Smith et al. [6] proposed a "harmonised progressive definition" of refinement, "in line with changes in animal ethics and animal welfare science" that included "health and welfare monitoring". Unfortunately, in the 21st century, despite guidelines and recommendations from experts [3,7–9], there are still no reports of the implementation of this harmonized definition in terms of health monitoring for sheep used for research purposes. The recently published recommendations of best practices for the health managemen<sup>t</sup> of ruminants and pigs used for scientific and educational purposes of the FELASAmight be a first step in establishing best health and welfare managemen<sup>t</sup> practices (i.e., refinement) at institutions [10].

Considering that the animals' availability ranked second among the criteria for selecting a sheep provider, it could be useful in the future to have specialized sheep breeders for biomedical research that ensure a good level of control and knowledge of the animals' health status.
