A double ovulation protocol for Xenopus laevis produces doubled fertilisation yield and moderately transiently elevated corticosterone levels without loss of egg quality.

The African claw-toed frog, Xenopus laevis, is a well-established laboratory model for the biology of vertebrate oogenesis, fertilisation, and development at embryonic, larval, and metamorphic stages. For ovulation, X. laevis females are usually injected with chorionic gonadotropin, whereupon they lay typically hundreds to thousands of eggs in a day. After being rested for a minimum of three months, animals are re-used. The literature suggests that adult females can lay much larger numbers of eggs in a short period. Here, we compared the standard "single ovulation" protocol with a "double ovulation" protocol, in which females were ovulated, then re-ovulated after seven days and then rested for three months before re-use. We quantified egg number, fertilisation rate (development to cleavage stage), and corticosterone secretion rate as a measure of stress response for the two protocol groups over seven 3-month cycles. We found no differences in egg number-per-ovulation or egg quality between the groups and no long-term changes in any measures over the 21-month trial period. Corticosterone secretion was elevated by ovulation, similarly for the single ovulation as for the first ovulation in the double-ovulation protocol, but more highly for the second ovulation (to a level comparable to that seen following shipment) in the latter. However, both groups exhibited the same baseline secretion rates by the time of the subsequent cycle. Double ovulation is thus transiently more stressful/demanding than single ovulation but within the levels routinely experienced by laboratory X. laevis. Noting that "stress hormone" corticosterone/cortisol secretion is linked to physiological processes, such as ovulation, that are not necessarily harmful to the individual, we suggest that the benefits of a doubling in egg yield-per-cycle per animal without loss of egg quality or signs of acute or long-term harm may outweigh the relatively modest and transient corticosterone elevation we observed. The double ovulation protocol therefore represents a potential new standard practice for promoting the "3Rs" (animal use reduction, refinement and replacement) mission for Xenopus research.

Social roles influence cortisol levels in captive Livingstone's fruit bats (Pteropus livingstonii).

A critical component of conserving and housing species ex situ is an explicit scientific understanding of the physiological underpinnings of their welfare. Cortisol has been repeatedly linked to stress, and therefore used as an indicator of welfare for many species. In order to measure cortisol in the Livingstone's fruit bat (Pteropus livingstonii; a critically endangered keystone species) without disturbing the captive population, we have developed and validated a non-invasive, novel hormone extraction procedure and faecal glucocorticoid assay. A total of 92 faecal samples, 73 from the P. livingstonii breeding colony at Jersey Zoo, Channel Islands and 19 samples from P. livingstonii housed at Bristol Zoological Gardens, UK, have been collected and analyzed. Mixed-effect modelling of the influence of physiological state variables on cortisol concentration revealed that lactating females had higher cortisol levels than non-lactating females, indicating that our assay is measuring biologically relevant hormone concentrations. Males and older bats also had higher cortisol than non-lactating females and younger individuals. Further analysis applied social network methodology to compare the cortisol levels of bats with different social roles. We found that individuals that linked social groups possessed higher than average cortisol levels and conversely, individuals with high-quality, positive relationships had lower cortisol levels. These results demonstrate, for the first time in a bat species, social mediation of stress hormones. Lastly, the frequency of vocalisation was found to positively correlate with cortisol concentration in males, suggesting that this behaviour may be used by animal management as a visual indicator of a bat's hormonal status. Hence, this research has provided unique insights and empirical scientific knowledge regarding the relationship between the physiology and social behaviour of P. livingstonii, therefore allowing for recommendations to be made to optimise bat welfare at the individual level.

Effects of transportation, transport medium and re-housing on Xenopus laevis (Daudin).

Understanding the immediate and longer-term effects of transportation and re-housing in a laboratory species is crucial in order to refine the transfer process, enable the optimal introduction of new animals to a novel environment and to provide a sufficient acclimatisation period before usage. Whilst consideration of animal welfare in most model vertebrate species has received attention, little quantitative evidence exists for the optimal care of the common laboratory amphibian Xenopus laevis. Techniques for the non-invasive welfare assessment of amphibians are also limited and here a non-invasive physiological assay was developed to investigate the impacts of transportation, transport medium and re-housing on X. laevis. First the impacts of transportation and transport medium (water, damp sponge or damp sphagnum moss) were investigated. Transportation caused an increase in water-borne corticosterone regardless of transport medium. Frogs transported in damp sphagnum moss also had a greater decrease in body mass in comparison to frogs not transported, suggesting that this is the least suitable transport medium for X. laevis. Next the prolonged impacts of transportation and re-housing were investigated. Frogs were transported between research facilities with different housing protocols. Samples were collected prior to and immediately following transportation, as well as 1 day, 7 days and 35 days after re-housing. Water-borne corticosterone increased following transportation and remained high for at least 7 days, decreasing to baseline levels by 35 days. Body mass decreased following transportation and remained lower than baseline levels across the entire 35 day observation period. These findings suggest the process of transportation and re-housing is stressful in this species. Together these findings have important relevance for both improving animal welfare and ensuring optimal and efficient scientific research.