No clear trends in expatriation of non-human primate research from Switzerland between 2004 and 2017

: Animal experimentation is commonly practiced in scientific research worldwide. However, there are no globally accepted standards for regulating the ethical boundaries and accepted practices for animal experimentation. Large differences exist between countries. A report suggested that some researchers, especially from countries with more stringent animal experimentation regulations, may be relocating experimental research to countries with less stringent regulations. We followed a systematic literature review approach to identity publications and determine whether there is an increasing trend in expatriation of non-human primate experimentation by researchers based in Switzerland. We used the Projects People Publications database, which contains projects funded by the Swiss National Science Foundation, to identify researchers conducting experiments using non-human primates. This list of names, together with terms referring to non-human primates were used to search the Web of Science. Publications without an author aﬀiliated to a Swiss institution, no living or only with free non-human primates, and non-original research were excluded. For each publication, we recorded the place of experimentation, funding source, number of animals, species and the statement of ethical approval. We retained 120 publications, involving more than 2,429 non-human primates. Macaca mulatta and Macaca fascicularis were the most common species. We could not confirm an increasing trend in expatriation of non-human primate experimentation outside of Switzerland. Over time, publications appeared to report the ethical approval number more consistently. These results should be interpreted with caution because the sample included only studies that were: 1) published and 2) reported in the Web of Science. Consequently, studies with insignificant results may have been excluded because these studies are rarely published, and studies of poor quality may have been excluded because they are often published in lower quality journals, not indexed by the Web of Science. Zeitschrif-Abstract Animal experimentation is commonly practiced in scientiﬁc research worldwide. However, there are no globally accepted standards for regulating the ethical boundaries and accepted practices for animal experimentation. Large differences exist between countries. A report suggested that some researchers, especially from countries with more stringent animal experimentation regulations, may be relocating experimental research to countries with less stringent regulations. We followed a systematic literature review approach to identity publications and determine whether there is an increasing trend in expatriation of non-human primate experimentation by researchers based in Switzerland. We used the Projects People Publications database, which contains projects funded by the Swiss National Science Foundation, to identify researchers conducting experiments using non-human primates. This list of names, together with terms referring to non-human primates were used to search the Web of Science. Publications without an author afﬁliated to a Swiss institution, no living or only with free non-human primates, and non-original research were excluded. For each publication, we recorded the place of experimentation, funding source, number of animals, species and the statement of ethical approval. We retained 120 publications, involving more than 2,429 non-human primates. Macaca mulatta and Macaca fascicularis were the most common species. We could not conﬁrm an increasing trend in expatriation of non-human primate experimentation outside of Switzerland. Over time, publications appeared to report the ethical approval number more consist-ently. These results should be interpreted with caution because the sample included only studies that were: 1) published and 2) reported in the Web of Science. Conse-quently, studies with insigniﬁcant results may have been excluded because these studies are rarely published, and studies of poor quality may have been excluded because they are often published in lower quality journals, not indexed by the Web of Science. de la science. Par conséquent, les études avec des résultats non signiﬁcatifs peuvent avoir été exclues car ces études sont rarement publiées et les études de mauvaise qualité peuvent avoir été exclues car elles sont souvent publiées dans des revues de moindre qualité, non indexées par le Web of Science.


Abstract
Animal experimentation is commonly practiced in scientific research worldwide. However, there are no globally accepted standards for regulating the ethical boundaries and accepted practices for animal experimentation. Large differences exist between countries. A report suggested that some researchers, especially from countries with more stringent animal experimentation regulations, may be relocating experimental research to countries with less stringent regulations. We followed a systematic literature review approach to identity publications and determine whether there is an increasing trend in expatriation of non-human primate experimentation by researchers based in Switzerland. We used the Projects People Publications database, which contains projects funded by the Swiss National Science Foundation, to identify researchers conducting experiments using non-human primates. This list of names, together with terms referring to non-human primates were used to search the Web of Science. Publications without an author affiliated to a Swiss institution, no living or only with free non-human primates, and non-original research were excluded. For each publication, we recorded the place of experimentation, funding source, number of animals, species and the statement of ethical approval. We retained 120 publications, involving more than 2,429 non-human primates. Macaca mulatta and Macaca fascicularis were the most common species. We could not confirm an increasing trend in expatriation of non-human primate experimentation outside of Switzerland. Over time, publications appeared to report the ethical approval number more consistently. These results should be interpreted with caution because the sample included only studies that were: 1) published and 2) reported in the Web of Science. Consequently, studies with insignificant results may have been excluded because these studies are rarely published, and studies of poor quality may have been excluded because they are often published in lower quality journals, not indexed by the Web of Science. reporting of animal experiments also varies between countries. It ranges from no reporting of any information about animal experimentation conducted within the country to some countries that publish detailed statistics about the number of animals used. 41 In countries with robust legislation, stringent animal welfare standards and strong public concerns about animal experiments, the relocation of animal experiments and researchers outside the country has become a topic of concern. 7, 16,19 Over the years there has been a trend of increasing numbers of international collaborations between scientists from different countries. 24 At least in some of these collaborations, not all experiments are funded by the same agency and they may be implemented in different countries. Collaborations are generally positive and may lead to better harmonization of standards and regulations between countries. 45 But, it is important to recognize that this does not always happen and in some cases animal experiments are relocated to avoid regulations or prohibitions, something that can be referred to as «ethics dumping», a practice which naturally worries funding agencies and the general public. 7,36 The present study was conducted in Switzerland, where the animal welfare legislation relies on the recognition of the dignity of animals -including those enrolled in laboratory experiments -and requires a careful weighing of interests. 42 Animal experiments can only take place if no alternative method is available, after carefully weighting of the harms and benefits and if the husbandry meets the minimal requirements for the concerned species, based on current knowledge about its biology and behavior. 35,36 In Switzerland, the number and species of animals used for experimentation, the purpose of animal experiments and the degree of severity of animal experiments are reported and publicly communicated annually by the Federal Food Safety and Veterinary Office (FSVO). 5 Legal disputes around experiments with non-human primates have occurred and certain licenses to perform these experiments were revoked by local courts or the Federal Supreme court. 39 Along with regulations, public and political pressure against animal experimentation may lead some researchers in Switzerland to shift their non-human primate experiments to other countries with more permissive regulations. 1,8,39 The purpose of our study was to deter-

Introduction
Animal experimentation is a well-established method that is used worldwide in many areas of biomedical research. Ethical issues around animal experimentation have generated intense debate in the scientific community and society, as scientific knowledge and changing societal norms have influenced the perception of animal experimentation. While during the 20 th century there was a sharp increase in the number of animals used in scientific research, there was also an increased recognition of animals as sentient beings and a continued debate on the moral implications of animal experimentation. 4,12 This led to the development of laboratory animal science, the recognition of the importance of animal welfare, the development of the Three Rs Principles (Reduce, Refine, Replace) 44 and to a progressive demand for more transparency, stricter regulations or bans on certain types of experiments. 13 Animal experimentation is still commonly seen as an essential element in some research fields, however there is debate about continuing to use this kind of experimentation in the future. 23,26 Attitudes towards animal experimentation vary greatly between countries, social groups and in respect to the concerned animal species and characteristics of research. 9,13,31 In many European and North American countries, some species of non-human primates are of special concern, especially when it comes to more invasive experiments. 14,46 There are no worldwide animal welfare standards, regulations or laws for the justification, conduct, limitation and reporting of animal experiments. Significant variation exists between countries. 43  Publications were classified as multiple reports from the same experiment if the same animal(s) and experimental protocol(s) were identified in different publications. When this was the case, the report with the earliest date of publication was selected for the study and all others excluded. After exclusion 120 publications remained ( Figure 1).

Data extraction
From each selected publication, we extracted information about the place of experimentation, number and species of animals used, ethical standards reported and funding organization(s). In most publications this information was extracted from the Material and Methods, Notes or Acknowledgement sections. In some cases, it was extracted from other sections or the supplementary materials. Each publication was then classified according to the place of experimentation, the ethical standards reported and the degree of severity (zero or greater).
The place of experimentation was classified into four categories: 1) «Switzerland», if the publication reported experiments conducted or approved only in Switzerland; 2) «Switzerland and Outside», if the publication reported experiments conducted or approved in both Switzerland and another country or countries; 3) «Outside», if the publication reported experiments conducted or approved exclusively outside of Switzerland; 4) «No statement», if there was no information reported about the place of experimentation or approval. In publications where the mine whether non-human primate experimental research by researchers based in Switzerland has been shifted out of the country. We used a systematic literature review approach to identify publications reporting non-human primate experimental research by researchers based in Switzerland and assessed whether there were trends in: 1) location of experimentation, 2) ethical standards reported in these publications and 3) the number of non-human primates used in experiments.

Researcher Selection
To identify researchers based in Switzerland engaged in animal experimentation, we used the Projects People Publications (P3) database of the Swiss National Science Foundation (SNSF). 40 It contained 2487 grants funded by the SNSF for the years between 2001 and 2016. None of the projects using non-human primates had a starting date before 2004.

Literature Search
We followed a systematic literature review approach to identify publications using the Web of Science (WoS) database 47 on the 7 th of March 2018, with the «Advanced Search» function and two search queries performed sequentially. The first query consisted of the project applicants' names that were extracted from the P3 database, in the field tag «AU» (author) combined with the Boolean term «OR». In the second query, the field tag «TS» (topic) included the following species names: «ape$ OR baboon$ OR bonobo$ OR Callithrix jacchus OR chimpanzee$ OR gorilla$ OR guenon$ OR Hominidae OR Macaca fascicularis OR macaque$ OR Maccaca mulatta OR marmoset$ OR monkey$ OR orangutan$ OR Pan$ OR Papio$ OR Pongo$ OR primate$ OR Rhesus macaque$ OR tamarin$ OR vervet$ monkey$ OR vervet$ OR Cercopithecus OR Callimico goeldii OR Simia diana». The two queries were then combined, using the Boolean term «AND», under the «Combine sets» option. The search was restricted to publications in English, classified as «Articles», within the «Timespan» from 2004 to 2017.
The search in the WoS identified 574 publications. One duplicate was removed. Publications then were examined by the first author and excluded for the following reasons: 1) none of the authors was affiliated to a Swiss institution and the publication did not report funding from an institution based in Switzerland (n = 138); 2) publications were classified as literature reviews (n = 21); 3) no living non-human primates were used (n = 137); 4) all the animals involved were wild and in natural reserves or natural habitat (n = 140); and 5) publications were multiple reports from the same experiment (n = 17). Multiple report (n=17) Included (n=120) Classified according to: • Place of experimentation • Ethical approval Recorded: • Number of animals • Species used SAT | ASMV 9 | 2021 Band 163, Heft 9, September 2021, 553-563, © GST | SVS number was reported; 2) «Approval» if the article reported having received approval for the experiment, but an approval number was not reported; 3) «Guidelines Followed» if no approval was reported, but the authors reported having followed guidelines; 4) «No statement» if no statement about approval or guidelines followed was reported. In case two different ethical standards' categories were reported, the publication was classified based on the category with the lowest level of ethical approval, using the following ranking: highest level -reporting of the approval number >> reporting that the experiment was approved >> reporting solely having followed guidelines >> no statement. Publications were also divided in two groups -reporting approval and not reporting approval. The first included all publications classified as «Approval Number» and «Approval» and the second those classified as «Guidelines followed» and «No statement».
Each article was then classified as degree of severity zero or greater, based on the definitions for experimental degree of severity provided by the FSVO. 11 We classified all experiments in manuscripts reporting multiple experiments and chose the highest classification of degree of severity.
Finally, we classified publications into 3 funding categories: 1) publications reporting funding received from Swiss institutions, if all or part of the funding came from at least one Swiss Institution; 2) publications reporting funding exclusively received from non-Swiss institutions; 3) publications not reporting any information about funding sources.

Analysis
Data compilation was done using Microsoft Excel. 27 Data management, bar charts and analysis were performed using the statistical software R V.3.5.2. 34 The non-parametric Mann-Kendall Trend test, performed with the R package «Trend», 33 was used to assess the existence of monotonic trends in the total number of articles and animals and, to consider fluctuations of publications between different years, in the annual percentages of articles classified in the different categories over the years of publication. The threshold of statistical significance was set at 0.05. Time trend lines (loess smoothing) were plotted for each category analyzed.

Number of publications, animals and species used
There were 120 publications with more than 2429 non-human primates, for the years between 2004 and    places of experimentation and approval were different, the place of experimentation was the one considered.
For place of experimentation, the only statistically significant trend found was a decrease in the percentage of publications classified as «No statement» (τ = -0,625, p = 0,003) ( Figure 6 and Table 1). Consistently, for the percentage of animals used in each category of place of experimentation, the only statistically significant trend was a decrease in the percentage of animals from publications classified as «No statement» (τ = -0,512, p = 0,015) ( Table 2).
The only statistically significant trend found was an increase in the percentage of publications reporting an ethical approval number (τ = 0,451, p = 0,037) ( Figure 8). When the percentage of publications was divided in two groups -the total reporting approval («Approval Number» and «Approval») and total not reporting approval («Guidelines followed» and «No statement») -no statistically significant trend was found ( Table 4).

Sources of Funding
Eighty-six publications (71,7%) reported having been totally or partly funded by institutions based in Switzerland. From these, 79 (65,8%) stated funding from the SNSF. Twenty-eight publications (23,3%) reported funding only from institutions based in other countries. Six publications (5,0%) did not report any funding institutions.

Discussion
We found no evidence to support the hypothesis that researchers based in Switzerland have increased the  amount of research conducted in other countries during the study period. When analyzing separately experiments with degree of severity zero and above zero, we found no evidence for significant trends, with exception of a rising trend in the publications with degree of severity zero classified as «Switzerland and Outside» (τ = 0,542, p = 0,013). We also found an increasing trend in the reporting of details relating to the ethical approval and the location of experimentation or approval. Over the study period there was an overall increase in the number of manuscripts published. The temporal trend in the number of animals used followed similar patterns, with an overall increase in the number of animals. We consider these findings to be important for Swiss society and funding agencies, such as the SNSF.
We identified some experiments conducted abroad. This is not unusual, as it has also been reported in other countries 8,15 and it is not surprising in Switzerland, where international scientific collaborations are considered strategic and promoted by the Federal Council of Switzerland. Various funding agencies, including the SNSF, offer grants aimed at establishing international collaborations or for international scientific stays. Experiments conducted outside of Switzerland, or both in Switzerland and other countries, may, at least in part, be a result of these international collaborations. We observed publications with multiple authors based in different countries and funded by different organizations. However, these data were not systematically recorded. The contribution of each author was seldom reported, making it impossible to determine the role played in the study. It is noteworthy that based on the criterion «Protecting animals used in scientific research», within the indicator of «Presence of animal welfare legislation» of the Animal Protection Index, Austria, Denmark, France, Netherlands and Sweden have similar scores to Switzerland. Consequently, shifting experiments to these countries most likely would not be a result of regulatory constraints.
Overall, ninety-five (79,2%) publications reported receiving ethical approval for all the experimental protocols, which is equal to the proportion reported by Yoon et al. 50 We observed an increasing trend in the reporting of specific ethical approval, which may indicate better reporting transparency. Among the 26 publications reporting this number, 23 (88,5%) were published after 2012. Eighteen (90%) of the 20 publications classified as having «No statement» for the place of experimentation were published before 2012. These findings may reflect an increasing endorsement of the ARRIVE Guidelines. Since their publication in 2010, the ARRIVE guidelines emphasize the need for reporting information on ethical approval of animal experiments. 19 However, some authors pointed out that following the ARRI-VE guidelines is still far from universal. 3,18 Within our sample, two (1,7%) publications, from 2006, did not report the number of animals used in the study. In contrast, Carlsson et al. reported in 2004 that 63% did not report the number of animals in the study 6 and Kilkenny et al. reported in 2009 that all publications reported the number of non-human primates used. 20 The discrepancy among these findings may be due to differences in study designs. The first included studies using «some type of primate biological material», while the second included only experiments with live primates. Our study included studies with live primates or where primates were euthanised for the study. Our fin-  ding that Macaca mullata and Macaca fascicularis were the most common species involved in experiments is in agreement with the findings from other studies focused on non-human primate experimental research conducted in Canada, the USA and Sweden. 6,15,23 In nine (7,5%) of the publications in our study, authors reported double affiliation, one being a Swiss institution and the other being an institution outside of Switzerland. These publications did not report receiving funding from a Swiss funding agency. The experimental location in five of these publications was reported to be outside of Switzerland and the other four had no information about the location. It could be that these authors were in transit from an institution outside of Switzerland to a Swiss institution, or vice versa, or had a permanent double affiliation.
The present study has limitations and should thus be interpreted with caution. It is possible that, by relying uniquely on SNSF P3 database to identify researchers working with non-human primates in Switzerland, we introduced some bias towards basic academic research. Even if for this branch of science the SNSF is an important public funding agency, 49 it is not the only one and it is possible that some researchers have never been funded by SNSF for experiments with non-human primates, especially if they work in corporate research and development. Furthermore, for the approval of funding, the SNSF requires the experiments to be performed in places with similar animal welfare conditions to those of Switzerland. This requirement makes it more difficult for researchers to shift experiments to places with lower standards and less regulations. Relying exclusively on a single database (WoS) may have resulted in not identifying some studies published in non-listed journals. In addition, relying uniquely on published experiments may have introduced further bias, as it has been suggested that negative or non-significant results are less likely to be published. 10,25,37 This is of concern, as poorly planned studies without ethical approval may not be published, or may be published in poorly cited journals not included in the WoS. Finally, the time lag between the actual experiment and the publication of the results may have resulted in some of the experiments conducted during the investigated interval not being included.
In several studies it was not easy to find information on the place of experimentation or role played by each researcher. This influenced our classification of publications. In studies where place of experimentation was not reported, we used the country of ethical approval as place of experimentation, if stated. One article reported the country of experimentation at a different location than the country where the approval was issued. It was not possible to identify the reasons why some researchers applied for ethical approval in one country and conducted their research in another. However, we feel that it would be important for researchers to be transparent about this and report their reasons. Journal editors and reviewers could play a role by requesting this information from authors. This also emphasizes the importance of complete and transparent reporting of ethical statements, including the place where experiments are performed and roles and contributions of each author.
It was our opinion that experiments with degree of severity greater than zero may be more likely to be expatriated. For this reason we tested the publications classified as degree of severity zero and greater than zero separately, per place of experimentation, for the presence of a trend. The reason behind an increasing trend for the percentage of articles classified as «Switzerland and Outside» and degree of severity zero could be, at least in part, studies with zoo animals. More than half (52%) of these publications involved animals from zoos, so these studies were expected to be mainly observational and therefore, to have a low degree of severity. This would be in accordance with a report of the increase in studies with zoo animals, namely primates, over the previous years. 17 The lack of other trends could also be due, at least in part, to the small sample size, as there were only a few articles with degree of severity greater than zero per place of experimentation each year, along 14 years.
Unfortunately, it is difficult to have an accurate picture on the real complete trend, due to reasons presented above to three, for the years between 2004 and 2017, but no statistically significant trend in the number of animals involved in experiments with a degree of severity zero. 5 In our study we recorded the total number of animals per year of publication. However, our results cannot be compared with FSVO reported numbers, as in our study there were several publications classified as «Switzerland and Outside» or «No statement» and two of the articles did not report the number of animals involved. In the future, other approaches could consider monitoring these trends and trying to understand what the main reasons behind the decrease were. Changes in legislation and court rulings may have had an important influence on the trends. But we believe that the ultimate causeabandoning certain animal experiments, expatriation of experiments or shifting to another country -cannot be fully confirmed using uniquely the quantitative approach we employed.
Good animal welfare standards, strict regulations and transparency are important, not just from a scientific perspective, but also from a moral and a social perspective. Monitoring changes in animal experimentation helps assuring researchers, funding bodies, legislators and the general public that regulations are respected and effective and can prompt debates about procedures and legislation. Non-human primates represent just a small proportion of the total number of animals used for experimentation. 6 It would be interesting to verify our findings with a more comprehensive systematic literature review of further repositories and grey literature to determine whether the same trends apply to other animal species. It is also important to understand the role and motivations of researchers who are based in Switzerland, but conduct their research outside the country.
SR, JB and FMS conceived and designed the study; SR and FMS acquired and collected the data; SR, JB and FMS analysed and interpreted the data; FM wrote the first draft; all authors revised and approved the final version.