Pain management in zebrafish : Report from a FELASA Working Group.

Empirical evidence suggests fishes meet the criteria for experiencing pain beyond a reasonable doubt and zebrafish are being increasingly used in studies of pain and nociception. Zebrafish are adopted across a wide range of experimental fields and their use is growing particularly in biomedical studies. Many laboratory procedures in zebrafish involve tissue damage and this may give rise to pain. Therefore, this FELASA Working Group reviewed the evidence for pain in zebrafish, the indicators used to assess pain and the impact of a range of drugs with pain-relieving properties. We report that there are several behavioural indicators that can be used to determine pain, including reduced activity, space use and distance travelled. Pain-relieving drugs prevent these responses, and we highlight the dose and administration route. To minimise or avoid pain, several refinements are suggested for common laboratory procedures. Finally, practical suggestions are made for the management and alleviation of pain in laboratory zebrafish, including recommendations for analgesia. Pain management is an important refinement in experimental animal use and so our report has the potential to improve zebrafish welfare during and after invasive procedures in laboratories across the globe

Data from: Towards the validation of endogenous steroid testing in wildlife hair

1. Hair is emerging as a popular tool to examine steroid hormone levels in wild mammals. The reliability of this approach, however, depends on an understanding of steroid hormone incorporation into hair as well as appropriate validations. 2. We reviewed studies that have examined steroid hormones in wildlife hair with the goal of summarizing the analytical, physiological, and biological evidence that this approach is meaningful. Accordingly, we differentiated among validations aimed at evaluating the reliability of the analytical method versus those designed to assess whether hormone levels in hair reflect physiologically meaningful processes in the target species. 3. Our literature survey revealed that endogenous steroids have been examined in hair from 42 species of non-human animals across 7 mammalian classes. Although the majority (85%) of 72 studies reported analytical validations of the method, physiological validations have only been reported for five species. Moreover, results of physiological validations were inconsistent among studies, highlighting the need for further research designed carefully to differentiate among the multiple purported models of steroid incorporation into hair in species with different types of hair and hair growth patterns. 4. To complement our review of published studies, we present new data supporting a positive relationship between levels of the steroid, cortisol, in hair and blood across eight mammalian species. In addition, we present novel results from a laboratory-based study showing variable hair growth in genetically identical laboratory mice that were kept under controlled conditions. 5. Synthesis and Applications: Collectively, this synthesis reveals substantial progress towards the validation of endocrine assays in hair from a variety of wildlife species. Further validations of other steroids, combined with appropriate physiological validations, would expand the potential applications of hair testing in wildlife research. As a key example, physiological data can provide mechanistic insight into species’ responses to change and may therefore contribute to conservation planning

Identification of a novel RNA virus lethal to tilapia

Tilapines are important for the sustainability of ecological systems and serve as the second most important group of farmed fish worldwide. Significant mortality of wild and cultured tilapia has been observed recently in Israel. The etiological agent of this disease, a novel RNA virus, is described here, and procedures allowing its isolation and detection are revealed. The virus, denominated tilapia lake virus (TiLV), was propagated in primary tilapia brain cells or in an E-11 cell line, and it induced a cytopathic effect at 5 to 10 days postinfection. Electron microscopy revealed enveloped icosahedral particles of 55 to 75 nm. Low-passage TiLV, injected intraperitoneally in tilapia, induced a disease resembling the natural disease, which typically presents with lethargy, ocular alterations, and skin erosions, with > 80% mortality. Histological changes included congestion of the internal organs (kidneys and brain) with foci of gliosis and perivascular cuffing of lymphocytes in the brain cortex; ocular inflammation included endophthalmitis and cataractous changes of the lens. The cohabitation of healthy and diseased fish demonstrated that the disease is contagious and that mortalities (80 to 100%) occur within a few days. Fish surviving the initial mortality were immune to further TiLV infections, suggesting the mounting of a protective immune response. Screening cDNA libraries identified a TiLV-specific sequence, allowing the design of a PCR-based diagnostic test. This test enables the specific identification of TiLV in tilapines and should help control the spread of this virus worldwide

Retinal structural changes well correlated with the ERG findings.

<p>(A) Representative retinal sections stained with hematoxylin and eosin demonstrate marked decrease of retinal thickness in KO eyes by the age of 6 months with further thinning by 10 months. Original magnification x20; Scale bar = 100μm. (B) Throughout the experiment, total retinal thickness was reduced in KO eyes as compared with WT littermates. (C) Thickness of the outer nuclear layer (ONL, containing the photoreceptor nuclei) did not differ between WT and KO mice at the age of 1 month, but by the age of 6 and 10 months, the ONL was significantly thinner in animals lacking SIRT6 activity. Results are presented as mean ± SEM; n = 5–7 for WT, n = 4–5 for KO mice. * <i>P</i> < 0.05.</p

Effect of SIRT6 deficiency on body weight and adiposity.

<p>(A) Average body weight of WT and KO male (left panel) and female (right panel) mice. In both genders, the weight of SIRT6 KO mice was significantly lower as compared with age-matched WT animals. (B) KO mice showed a trend towards lower percentage of total body fat in comparison to their WT littermates. The percentage of total body fat was measured at 6–8 months of age. Results are presented as mean ± SEM. n = 3–14 per group.</p

Genotypes of progeny from crosses of F1 SIRT6+/− mice.

<p>Genotypes of progeny from crosses of F1 SIRT6+/− mice.</p

Characterization of physiological defects in adult SIRT6^-/- mice

<div><p>The NAD+-dependent SIRT6 deacetylase was shown to be a major regulator of lifespan and healthspan. Mice deficient for SIRT6 develop a premature aging phenotype and metabolic defects, and die before four weeks of age. Thus, the effect of SIRT6 deficiency in adult mice is unknown. Here we show that SIRT6^-/- mice in mixed 129/SvJ/BALB/c background reach adulthood, allowing examination of SIRT6-related metabolic and developmental phenotypes in adult mice. In this mixed background, at 200 days of age, more than 80% of the female knock-out mice were alive whereas only 10% of male knock-out mice survived. In comparison to their wild-type littermates, SIRT6 deficient mice have reduced body weight, increased glucose uptake and exhibit an age-dependent progressive impairment of retinal function accompanied by thinning of retinal layers. Together, these results demonstrate a role for SIRT6 in metabolism and age-related ocular changes in adult mice and suggest a gender specific regulation of lifespan by SIRT6.</p></div

Increased glucose uptake in SIRT6 deficient mice.

<p>Glucose tolerance test (GTT) in WT and KO male (A) and female (B) mice. Area under curve (AUC) is shown on the right for each GTT. Both male and female KO mice show increased glucose uptake in comparison to WT mice. (C) Insulin tolerance test in male (left panel) and female (right panel) mice showed no significant difference between genotypes. (D) Insulin blood levels after glucose stimulation as determined by ELISA. All assays were done at 3–4 months old. Results are presented as mean ± SEM. n = 5–14 per group. * <i>P</i> < 0.05, ** <i>P</i> < 0.01</p

Gender specific effect of SIRT6 deficiency on mouse survival.

<p>Kaplan—Meier survival curves of WT, HET and KO male (left panel) and female (right panel) mice in 129/SvJ/BALB/c background. 90% of KO male mice died before 200 days of age and more than 75% of KO female mice survived over 300 days of age. Males, n = 23–39 per group; Females, n = 14–40 per group.</p