Research paperEffects of transportation, transport medium and re-housing on Xenopus laevis (Daudin)
Introduction
High-quality animal welfare is ethically important and ensures physiologically and psychologically healthy animals that can fulfil their original purposes of captivity, e.g. reliable experimental results (Poole, 1997) or the quality of meat (Schwartzkopf-Genswein et al., 2012). Transportation of captive animals, for research, agriculture or conservation purposes, is a significant stressor in a range of species, e.g. mammals (Minka and Ayo, 2007); birds (Leche et al., 2013); fish (Boerrigter et al., 2015); reptiles (Mancera et al., 2014); and amphibians (Narayan and Hero, 2011). Refinement of transportation and re-housing methods can reduce potential negative impacts (Conour et al., 2006) and may lead to shorter acclimatisation periods before animals recover for use (Obernier and Baldwin, 2006). It is therefore crucial to understand the immediate and longer-term effects of animal transportation and re-housing (Grandin, 1997).
Despite their numbers in captivity (for purposes of conservation, education, research or as pets) the welfare of amphibians has generally received less attention than that of other classes of tetrapods, in part due to challenges in interpreting amphibian behaviour (Alworth and Harvey, 2007) and quantifying the hypothalamic-pituitary-adrenal axis (HPA) response. Xenopus laevis (Daudin) is a common model organism in scientific research with over 6000 procedures performed in the UK in 2013 alone (Home Office, 2014). There is, however, little quantitative evidence concerning the optimal care of this species in captivity, particularly in comparison to other model organisms (Reed, 2005). For example, three media are suggested for X. laevis transportation: water, damp sphagnum moss, and damp sponge (Green, 2010, Leadley Brown, 1970, Reed, 2005, Swallow et al., 2005), yet whilst these media are likely to provide different experiences in terms of support, vibration, tactility and novelty, there is no evidence as to which might be most suitable for X. laevis welfare and all three methods are currently used (survey results from 77 respondents: water (68.8%), moss (40.3%) and sponge (20.8%), multiple answers permitted; Holmes et al., 2015). Understanding the impacts of transportation and re-housing on X. laevis is consequently vital to allow laboratories to better prepare for new arrivals and leave sufficient acclimatisation periods to ensure robust scientific data.
Specific stressors associated with transferring animals between facilities include: the initial capture, handling and confinement; vibration, disruption and conditions during transportation; and the introduction to novel housing, individuals and care protocols on arrival (reviewed in Conour et al., 2006, Santurtun and Phillips, 2015, Swallow et al., 2005). In amphibians physiological and/or behavioural changes indicative of stress have been observed following capture and handling (e.g. Narayan et al., 2012, Ricciardella et al., 2010, Woodley and Lacy, 2010), exposure to water vibrations (Davis and Maerz, 2011), and in response to a novel social environment (Cikanek et al., 2014). The amphibian hypothalamic-pituitary-interrenal axis (HPI, analogous to the mammalian hypothalamic-pituitary adrenal axis; Rollins-Smith, 2001), releases corticosterone when activated and increases in corticosterone have been observed following both infection and physical stressors in amphibians (Gabor et al., 2013b, Yao et al., 2004). In X. laevis increases in corticosterone or corticotropin-releasing factor peptide content have been observed following tank agitation (Boorse and Denver, 2004, Glennemeier and Denver, 2002, Yao et al., 2004), as well as after brief handling and exposure to a novel environment (Archard and Goldsmith, 2010). These studies, however, do not represent the full spectrum of disturbance resulting from transportation and re-housing and there was no prolonged monitoring after cessation of the stressors.
Amphibian hormones, such as corticosterone, have traditionally been quantified using invasive measures such as cardiac puncture (e.g. Crespi et al., 2015, Mosconi et al., 2006) or whole body homogenisation (e.g. Thurmond et al., 1986). These methods present ethical problems and may mask experimental results by inducing additional stress (Palme, 2012). Sampling of urine, saliva or faeces can reduce invasiveness; however these practices may still involve prolonged handling or cloacal penetration (reviewed in Narayan, 2013) and remain problematic for fully aquatic species. Research into fish welfare has led to the extraction of cortisol from the surrounding water, a much less intrusive method (Scott and Ellis, 2007, Scott et al., 2008, Scott et al., 2001), and this technique has had some application in amphibians (Gabor et al., 2013a, Gabor et al., 2013b, Gabor et al., 2015, Gabor et al., 2016). It has been suggested that trends in glucocorticoids should be supported with other physiological measures (Breuner et al., 2013, Dawkins, 2006, Otovic and Hutchinson, 2015), and reductions in body mass resulting from transportation have been noted in terrestrial animals (reviewed in Schwartzkopf-Genswein et al., 2012). The use of changes in body mass as an indicator of welfare has received less attention in amphibians, however in Ocoee salamanders (Desmognathus ocoee) chronic handling stress produced a decrease in body mass (Bliley and Woodley, 2012).
Here a water-borne corticosterone assay was developed and validated for X. laevis and combined with measures of body mass in order to investigate the effects of transportation, transport medium and re-housing in this species. To investigate the impacts of transportation and transport medium X. laevis were transported in water, sponge or sphagnum moss and the changes in water-borne corticosterone and body mass compared against frogs that were similarly sampled but not transported. Whilst body condition index scores have often been used in comparisons with hormone data, body mass was selected here to avoid the disturbance that obtaining snout-vent length in this fully aquatic species requires. This was from both a welfare perspective and to ensure sampling method didn’t impact on hormone results. It was expected that transportation might result in an increase in corticosterone and a decrease in body mass but no prediction was made regarding the impact of transport medium. In the second experiment X. laevis were transported between facilities and re-housed in a novel physical, social and feeding environment as might be experienced by frogs during stock animal purchasing. Water-borne corticosterone and body mass were monitored across 5 weeks to investigate the longer-term impacts of transportation and re-housing.
Section snippets
Subjects and housing
Subjects were wild-type Xenopus laevis, purchased from the European Xenopus Resource Centre (EXRC, University of Portsmouth) and housed at the University of Chester. Housing parameters were selected from Reed, 2005, Green, 2010 and A. Jafkins at EXRC (personal communication). X. laevis were housed in same-sex groups of five individuals per glass tank (584 mm × 431 mm × 305 mm, Clearseal), in dechlorinated mains water at a depth of 140 mm with a temperature range 20–23 °C (air temperature
Effect of transportation and transport medium
There was a significant effect of treatment group (no transport, water, sponge, moss transportation; F(6,38) = 3.44, p = 0.008; Fig. 2), which when analysed further showed treatment group differences for both corticosterone change (F(3,19) = 6.05, p = 0.005) and body mass change (F(3,19) = 4.29, p = 0.02). Post-hoc pairwise comparisons (Gabriel’s) revealed that all three transport groups showed an increase in corticosterone compared to the non-transport group (water p = 0.04, sponge p = 0.01,
Discussion
Understanding the immediate and longer-term impacts of transportation and re-housing in a laboratory species is crucial in order to refine the transportation process, enable the optimal introduction of new animals to a novel environment and to provide a sufficient acclimatisation period before usage (Obernier and Baldwin, 2006). In line with transportation studies in mammals, birds and fish (e.g. Boerrigter et al., 2015, Leche et al., 2013, Minka and Ayo, 2007, Schwartzkopf-Genswein et al., 2012
Acknowledgments
This study was funded by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs, NC/K000497/1). NC3Rs had no further role in study design, the collection, analysis and interpretation of data, the writing of the manuscript or the decision to submit the paper for publication. We are grateful to members of the Department of Biological Sciences at the University of Chester and to Alan Jafkins and Matthew Guille at the European Xenopus Resource Centre,
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