Skin swabbing is a refined technique to collect DNA from model fish species

Abstract

Model fish species such as sticklebacks and zebrafish are frequently used in studies that require DNA to be collected from live animals. This is typically achieved by fin clipping, a procedure that is simple and reliable to perform but that can harm fish. An alternative procedure to sample DNA involves swabbing the skin to collect mucus and epithelial cells. Although swabbing appears to be less invasive than fin clipping, it still requires fish to be netted, held in air and handled-procedures that can cause stress. In this study we combine behavioural and physiological analyses to investigate changes in gene expression, behaviour and welfare after fin clipping and swabbing. Swabbing led to a smaller change in cortisol release and behaviour on the first day of analysis compared to fin clipping. It also led to less variability in data suggesting that fewer animals need to be measured after using this technique. However, swabbing triggered some longer term changes in zebrafish behaviour suggesting a delayed response to sample collection. Skin swabbing does not require the use of anaesthetics and triggers fewer changes in behaviour and physiology than fin clipping. It is therefore a more refined technique for DNA collection with the potential to improve fish health and welfare.

Figure 1

Early indicators of stress activation. (a, b) Changes to number of opercular beats per minute and (c, d) cortisol excretion in stickleback (a, c) and zebrafish (b, d). Manipulation groups: 1 undisturbed; 2 netted under water; 3 netted air; 4 swabbed; 5 MS-222; 6 MS-222 and swabbed; 7 MS-222 and fin clipped. Letters on graphs not shared in common between groups indicate significant differences.

Figure 2

Long-term behavioural alterations in stickleback. (a) Distance swum in novel tank diving test, day 1. (b) Distance swum in novel tank diving test, day 2. (c) Distance swum in novel tank diving test, day 7. (d) Time in bottom of novel tank, day 1. (e) Time in bottom of novel tank, day 2. (f) Time in bottom of novel tank, day 7. (g) Distance swum in open field test, day 1. (h) Distance swum in open field test, day 2. (i) Distance swum in open field test, day 7. (j) Time in centre of open field tank, day 1. (k) Time in centre of open field tank, day 2. (l) Time in centre of open field tank, day 7. (m) Time in white half of black/white tank, day 1. (n) Time in white half of black/white tank, day 2. (o) Time in white half of black/white tank, day 7. Manipulation groups: 1 undisturbed; 2 netted under water; 3 netted air; 4 swabbed; 5 MS-222; 6 MS-222 and swabbed; 7 MS-222 and fin clipped. The Y axis is log-transformed in (j)–(o) to better depict data. All statistical analyses were performed on raw (untransformed data).

Figure 3

Long-term behavioural alterations in zebrafish. (a) Distance swum in novel tank diving test, day 1. (b) Distance swum in novel tank diving test, day 2. (c) Distance swum in novel tank diving test, day 7. (d) Time in bottom of novel tank, day 1. (e) Time in bottom of novel tank, day 2. (f) Time in bottom of novel tank, day 7. (g) Distance swum in open field test, day 1. (h) Distance swum in open field test, day 2. (i) Distance swum in open field test, day 7. (j) Time in centre of open field tank, day 1. (k) Time in centre of open field tank, day 2. (l) Time in centre of open field tank, day 7. (m) Time in white half of black/white tank, day 1. (n) Time in white half of black/white tank, day 2. (o) Time in white half of black/white tank, day 7. Manipulation groups: 1 undisturbed; 2 netted under water; 3 netted air; 4 swabbed; 5 MS-222; 6 MS-222 and swabbed; 7 MS-222 and fin clipped. The Y axis is log-transformed in (j)–(o) to better depict data. All statistical analyses were performed on raw (untransformed data).

Figure 4

Expression of stress marker genes in stickleback. qPCR data showing expression of (a) brain-derived neurotrophic factor (bdnf) day 1. (b) brain-derived neurotrophic factor, day 2. (c) brain-derived neurotrophic factor, day 7. (d) corticotropin releasing hormone a (crha) day 1. (e) corticotropin releasing hormone a, day 2. (f) corticotropin releasing hormone a, day 7. (g) corticotropin releasing hormone b (crhb) day 1. (h) corticotropin releasing hormone b, day 2. (i) corticotropin releasing hormone b, day 7. (j) galanin (galn) day 1. (k) galanin day 2. (l) galanin day 7. (m) neuropeptide y (npy) day 1. (n) neuropeptide y day 2. (o) neuropeptide y day 7. Manipulation groups: 1 undisturbed; 2 netted under water; 3 netted air; 4 swabbed; 5 MS-222; 6 MS-222 and swabbed; 7 MS-222 and fin clipped.

Figure 5

Expression of stress marker genes in zebrafish. qPCR data showing expression of (a) brain-derived neurotrophic factor (bdnf) day 1. (b) brain-derived neurotrophic factor, day 2. (c) brain-derived neurotrophic factor, day 7. (d) corticotropin releasing hormone a (crha) day 1. (e) corticotropin releasing hormone a, day 2. (f) corticotropin releasing hormone a, day 7. (g) corticotropin releasing hormone b (crhb) day 1. (h) corticotropin releasing hormone b, day 2. (i) corticotropin releasing hormone b, day 7. (j) galanin (galn) day 1. (k) galanin day 2. (l) galanin day 7. (m) neuropeptide y (npy) day 1. (n) neuropeptide y day 2. (o) neuropeptide y day 7. Manipulation groups: 1 undisturbed; 2 netted under water; 3 netted air; 4 swabbed; 5 MS-222; 6 MS-222 and swabbed; 7 MS-222 and fin clipped.

Figure 6

Health and condition indicators in stickleback and zebrafish. Growth and health indicators 28 days after swabbing or fin clipping. (a) Stickleback body length. (b) Stickleback body weight. (c) Stickleback hepatosomatic index. (d) Stickleback splenosomatic index. (e) Stickleback gonadosomatic index. (f) Stickleback nephrosomatic index. (g) Zebrafish body length. (h) Zebrafish body weight. (i) Zebrafish hepatosomatic index. (j) Zebrafish splenosomatic index. (k) Zebrafish gonadosomatic index. Manipulation groups: 1 undisturbed; 2 netted under water; 3 netted air; 4 swabbed; 5 MS-222; 6 MS-222 and swabbed; 7 MS-222 and fin clipped.

Figure 7

Summary of changes to stress axis activation, behaviour, gene expression and condition indicators following fin clipping or skin swabbing. Red shading indicates skin swabbing without anaesthetic (group 4) or fin clipping (group 7) for comparison with un-manipulated control animals (group 1). Arrow indicate increases (↑) or decreases (↓) of readouts, where a—indicates no change.