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Use of a translucent refuge for Xenopus tropicalis with the aim of improving welfare
Laboratory Animals 0(0) 1–4 ! The Author(s) 2017 Reprints and permissions: sagepub.co.uk/ journalsPermissions.nav DOI: 10.1177/0023677217737798 journals.sagepub.com/home/lan
Gavan M Cooke
Abstract Xenopus tropicalis is an increasingly important animal model in a variety of biological research fields. In many countries legislation exists to promote and increase welfare wherever possible, including the ability to view animals during daily husbandry with minimal stress to the animal. X. tropicalis (n ¼ 16) refuge use was investigated; it was found that the animals significantly preferred black opaque overhead cover to openended pipes or closed-off ceramic plants pots in refuge choice experiments. This experiment was repeated by replacing the opaque black overhead cover with red filters. A significant preference for overhead cover was seen for the red translucent cover compared with other available refuges, suggesting that X. tropicalis may adopt translucent refuges due to their visual inabilities. The inability of frogs to see certain wavelengths of light may allow staff to view them whilst simultaneously providing the refuge of choice.
Keywords Xenopus, refuge use, husbandry, welfare Date received: 17 July 2017; accepted: 28 September 2017
Xenopus laevis and Xenopus tropicalis are significant model organisms in biomedical research and related subject areas,1,2 but there are few scientific studies on captive amphibian enrichment.1,3 Major and Wassersug4 found that 46% of Xenopus laboratories provided a refuge; 26% offered cover or shading; and 20% offered sinkable rocks, ceramic plant pots and other refuges capable of partly or entirely covering the animal. Hilken et al.5 have reported that refuge/cover reduced activity and increased growth, and Brown and Nixon6 showed that Xenopus prefer tanks with covers, with a trend towards a greater quantity and quality of eggs produced. Refuges and cover also reduce aggression and cannibalism.7 X. laevis vision is believed to have a peak absorbance sensitivity between 519 nm8 (see Figure 1 in Bridges et al.9) and 522 nm (Figure 1 in Crescitelli10), possessing only one visual pigment,9 indicating they may not be able to perceive colour at longer wavelengths, for example reds and shorter wavelengths in the blue range. Close phylogenetic relatedness means it is plausible that X. tropicalis has similar visual inabilities.
Translucent plastic hides with a red tint, called ‘mouse houses’, have been designed for laboratory mice; mice are thought to perceive translucent red as darkness.11 For this study, 16 laboratory-reared X. tropicalis were sexed (10 females, six males), weighed/measured (mean mass ¼ 20.3 g 1.7; mean length ¼ 107 mm 10.1) and isolated before the experiments began. They were approximately 3 years old, all from the same batch of eggs (strain unknown). They were kept according to standard EU/UK housing regulations (ASPA 1986) and fed a mixture of sinking pellets and blood worms throughout their lives. The first experiment tested preference of refuges typically given to laboratory amphibians.2 A grey opaque tank measuring (l w h) 86.5 cm 40 cm 55 cm was
Anglia Ruskin University, Cambridge, UK Corresponding author: Gavan M Cooke, Anglia Ruskin University, East Road, Cambridge, CM1 1SQ, UK. Email:
[email protected]
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Figure 1. In experiment 1 (a) a significant preference for using the opaque cover over the other refuges was found, but not compared with not using a refuge at all. (Friedman test, p ¼ 0.001; post hoc tests found that there was a significant difference between the black opaque cover and plant pot, p ¼ 0.023); not using any refuge and plant pot (p ¼ 0.02); not using any refuge and pipe (p ¼ 0.012); a strong tendency towards using the black cover more than the pipe (p ¼ 0.055). Experiment 2 (b), ANOVA, p ¼ 0.001. Post hoc testing (Tukey). Frogs spent significantly more time using the red translucent cover than the pipe (p ¼ 0.033), than the plant pot (p ¼ 0.005), but not compared with using no refuge at all (p ¼ 0.995). There was also significantly more time spent using nothing at all than the plant pot (p ¼ 0.01).
filled with water to 38 cm. Three types of typical refuge were tested (terracotta plant pot with 8 cm aperture, 5 cm base, and 8 cm length; a black open-ended PVC pipe measuring 7.5 cm diameter, 9.5 cm length; and a black overhead cover covering one-quarter of the surface area of the tank). One refuge was placed in one of the quadrants in the tank. The other two refuges were placed in two of the remaining three quadrants, and the fourth quadrant was left free as an open area. Each frog was added to the tank and allowed to acclimatise for 5 minutes. A ‘blind’ observer using a stop watch then timed how long the frog used each type of refuge. Time started when a frog stopped within a refuge but not if it simply passed through. Times were collected for each refuge use over 5 minutes, and these times were also compared with the time spent not using any refuge at all (300 seconds minus total time of all refuge use). The positions of the refuges in each quadrant were randomised daily. The second experiment repeated the first experiment with the exception of replacing the black opaque cover with a laminated red filter (Lee FiltersTM 106 ‘primary red’ http://www.leefilters.com/lighting/colour-details. html#106&filter¼cf&sort¼number). All experiments were tested for equality of variances and normality, and statistical analyses were carried out in SPSS v 20. All experiments were conducted after an ethical review carried out by the institution’s Named Animal Care & Welfare Officer and conformed to ARRIVE guidelines.
Significant difference in the amount of time spent in different refuges or non-refuge use was found (Friedman test Chi Square ¼ 10.563 df ¼ 3, p ¼ 0.001) in experiment 1. Post hoc testing (Wilcoxon matched pairs) found significant difference between the black opaque cover and plant pot use (p ¼ 0.023); not using any refuge and plant pot (p ¼ 0.02); not using any refuge and pipe (p ¼ 0.012); and a strong tendency towards black cover use more than the pipe (p ¼ 0.055). Significant difference between the time used in the refuges (translucent red cover, pipe, plant pot) or non-refuge use was found (ANOVA, F6.2883,15, p ¼ 0.001) in experiment 2. Post hoc testing (Tukey) revealed that there was significantly more time spent using the red translucent cover than the pipe (p ¼ 0.033) and the plant pot (p ¼ 0.005), but not compared with using no refuge (p ¼ 0.995). There was also significantly more time spent using no refuge than the plant pot (p ¼ 0.01). There were no significant differences between sexes in either experiment. The results from the first experiment (Figure 1) suggest that overhead cover is preferred as a refuge for X. tropicalis. The surface area of cover offered by the overhead cover far exceeds that of the ceramic plant pot and PVC pipework. The cover gives more protected space for behaviours such as foraging. X. tropicalis utilise translucent red covers in the same way that they utilise black, opaque covers. A non-opaque cover, such as that used in experiment 2, has many advantages; it allows staff to observe
Cooke the frogs without disturbing them and causing stress, and to monitor waste, uneaten food or reproduction. Furthermore, it may allow experimentation or observation of natural behaviours without interference. Given our understanding of Xenopus vision,8–10 it is perhaps plausible that the frogs are unable to see the remaining light after it has passed through the red filter, which therefore provides adequate cover for the frogs. The finding that ‘no cover’ did not differ significantly from using the preferred refuge is possibly due to exploration of the environment, looking for food perhaps; these times include movement between areas, which may have inflated the recorded time for ‘no cover’ use. The use of laboratory animal colour vision to improve welfare has been investigated before in laboratory mice.11 It is to be noted that not all common laboratory mice strains accepted the translucent red refuges,11 and there are numerous laboratory Xenopus strains; some may not react in the same way as the animals studied here. Nevertheless, we believe this pilot study offers future directions for research regarding Xenopus spp. welfare, by taking advantage of an animal’s deficit in colour vision. Declaration of Conflicting Interests The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author received no financial support for the research, authorship, and/or publication of this article.
3 References 1. Chum H, Felt S, Garner J, et al. Biology, behavior, and environmental enrichment for the captive African clawed frog (Xenopus spp). Appl Anim Behav Sci 2013; 143(2–4): 150–156. 2. Green SL. The Laboratory Xenopus sp. Boca Raton: CRC Press, 2009. 3. Archard GA. Refuge use affects daily activity patterns in female Xenopus laevis. Appl Anim Behav Sci 2013; 145(3): 123–128. 4. Major N and Wassersug RJ. Survey of current techniques in the care and maintenance of the African clawed frog (Xenopus laevis). J Am Assoc Lab Anim Sci 1998; 37(5): 57–60. 5. Hilken G, Dimigen J and Iglauer F. Growth of Xenopus laevis under different laboratory rearing conditions. Lab Anim 1995; 29(2): 152–162. 6. Brown MJ and Nixon RM. Enrichment for a captive environment-the Xenopus laevis. Anim Technol Welfare 2004; 3: 87–95. 7. Torreilles SL and Green SL. Refuge cover decreases the incidence of bite wounds in laboratory South African clawed frogs (Xenopus laevis). J Am Assoc Lab Anim Sci 2007; 46(5): 33–36. 8. Dartnall HJA. A study of the visual pigments of the clawed toad. J Physiol 1954; 125(1): 25–42. 9. Bridges CDB, Hollyfield JG, Witkovsky P, et al. The visual pigment and vitamin A of Xenopus laevis embryos, larvae and adults. Exp Eye Res 1977; 24(1): 7–13. 10. Crescitelli F. The visual pigment system of Xenopus laevis: Tadpoles and adults. Vision Res 1973; 13(4): 855–865. 11. Key D and Hewett A. Developing and testing a novel cage insert, the ‘‘Mouse House’’, designed to enrich the lives of laboratory mice without adversely affecting the science. Anim Technol Welfare 2002; 1: 55–64.
´sume ´ Re Le Xenopus tropicalis est un mode `le animal de plus en plus important dans une varie ´te ´ de domaines de recherche biologique. Dans de nombreux pays, la le ´gislation promeut et accroıˆt le bien-e ˆtre dans la mesure du possible, y compris en ayant la possibilite ´ de voir les animaux tous les jours pendant leur e ´levage, en ne leur causant qu’un stress minimal. L’utilisation de refuges destine ´s aux X. tropicalis (n ¼ 16) a e ´te ´e ´tudie ´e. L’e ´tude a montre ´ que ces animaux pre ´fe ´raient nettement des espaces couverts avec de mate ´riaux opaques noirs aux tuyaux a ` bouts ouverts ou aux pots de fleurs en ce ´ramiques lors des expe ´riences portant sur le choix d’un refuge. Cette expe ´rience a e ´te ´ re ´pe ´te ´e en remplac¸ant le mate ´riau noir opaque par des filtres rouges. Une pre ´fe ´rence significative a e ´te ´ constate ´e pour les dispositifs de couverture en mate ´riau translucide rouge, par rapport a ` d’autres refuges disponibles, sugge ´rant que les X. tropicalis peuvent adopter des refuges translucides en raison de leur incapacite ´ visuelle. L’incapacite ´ des grenouilles a ` voir certaines longueurs d’onde de lumie `re peut permettre au personnel de les observer tout en leur fournissant un refuge de choix.
Abstract Xenopus tropicalis ist ein Tiermodell, das in verschiedensten biologischen Forschungsbereichen zunehmend an Bedeutung gewinnt. In vielen La ¨ndern existieren Gesetze zur weitestgehenden Fo ¨rderung und
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Verbesserung des Tierschutzes, wozu auch Mo ¨glichkeiten zur Beobachtung der Tiere bei der ta ¨glichen Haltung za ¨hlen, die Stress fu ¨r die Tiere auf ein Mindestmaß reduzieren. Im Rahmen der Untersuchung der Wahl von Zufluchtsorten von X. tropicalis (n ¼ 16) wurde bei diesbezu ¨ glichen Experimenten festgestellt, dass die Tiere schwarze lichtundurchla ¨ssige obere Schutzabdeckungen gegenu ¨ber offenen Rohren oder geschlossenen Keramiku ¨berto ¨pfen signifikant bevorzugten. Dieses Experiment wurde wiederholt, indem die lichtundurchla ¨ssige schwarze obere Schutzabdeckung durch rote Filter ersetzt wurde. Es wurde eine signifikante Vorliebe fu ¨r die obere rote transluzente Abdeckung im Vergleich zu anderen verfu ¨gbaren Ru ¨ ckzugsorten festgestellt. Dies legt den Schluss nahe, dass X. tropicalis mo ¨glicherweise aufgrund ihres Unvermo ¨gens visueller Wahrnehmung transluzente Ru ¨ckzugsorte aufsuchen. Aufgrund der Unfa ¨higkeit der Fro ¨sche, bestimmte Lichtwellenla ¨ngen zu sehen, ko ¨nnen sie mo ¨glicherweise vom Personal beobachtet werden, wa ¨hrend ihnen zugleich ihre bevorzugten Ru ¨ckzugsorte zur Verfu ¨gung gestellt werden ko ¨nnen.
Resumen El Xenopus tropicalis es un modelo animal cada vez ma ´s importante en una variedad de campos de investigacio ´n biolo ´gica. En muchos paı´ses existe una legislacio ´n para fomentar e incrementar el bienestar en la medida posible, incluida la capacidad de observar a animales durante crı´as diarias con el mı´nimo estre ´s para el animal. Se investigo ´ el uso del refugio en X. tropicalis (n ¼ 16) y se observo ´ que preferı´an significativamente una cobertura opaca negra en tubos abiertos o macetas de cera ´mica cerradas en experimentos con refugio. Este experimento se repitio ´ reemplazando la cobertura negra opaca por filtros rojos. Se observo ´ una preferencia significativa de coberturas translu ´ cidas rojas en comparacio ´n a otros refugios disponibles, lo que sugiere que los X. tropicalis pueden adoptar refugios translu ´ cidos debido a sus limitaciones visuales. La incapacidad de las ranas para ver ciertas longitudes de ondas de luz puede permitir al personal verlas suministra ´ndoles a su vez el refugio de su eleccio ´n.
