doi:10.1111/jfd.12261

Journal of Fish Diseases 2014

Rapid temperature-dependent wound closure following adipose fin clipping of Atlantic salmon Salmo salar L. M Andrews1, M Stormoen1, H Schmidt-Posthaus2, T Wahli2 and P J Midtlyng1 1 Centre of Epidemiology and Biostatistics, Faculty for Veterinary Medicine and Biosciences, Norwegian University of Life Sciences, Oslo, Norway 2 Centre for Fish and Wildlife Health, Institute of Animal Pathology, University of Bern, Bern, Switzerland

Abstract

Introduction

Three groups of Atlantic salmon were kept at a constant temperature of 4, 10 and 14 °C. The adipose fins were removed; six fish/group were sampled at 11 subsequent time points post-clipping. Samples were prepared for histopathological examination to study the course of re-epithelization. A score sheet was developed to assess the regeneration of epidermal and dermal cell types. Wounds were covered by a thin epidermal layer between 4 and 6 h post-clipping at 10 and 14 °C. In contrast, wound closure was completed between 6 and 12 h in fish held at a constant temperature of 4 °C. By 18 h post-clipping, superficial cells, cuboidal cells, prismatic basal cells and mucous cells were discernible in all temperature groups, rapidly progressing towards normal epidermal structure and thickness. Within the observation period, only minor regeneration was found in the dermal layers. A positive correlation between water temperature and healing rates was established for the epidermis. The rapid wound closure rate, epidermal normalization and the absence of inflammatory reaction signs suggest that adipose fin clipping under anaesthesia constitutes a minimally invasive method that may be used to mark large numbers of salmon presmolts without compromising fish welfare.

Atlantic salmon, Salmo salar L., aquaculture has been growing steadily over the past few decades particularly in Norway, Scotland, the Faroe Islands, Canada and Chile. Due to the floating net pen-rearing technology, escapee-ism has become a relatively common occurrence (Zhang et al. 2013). This has triggered concerns especially among salmon anglers and stakeholders of wild salmon populations of the potential negative effects that farm escapees may have on wild stocks (Jonsson & Jonsson 2006; Skaala, Wennevik & Glover 2006). To characterize the perceived hazard, the term ‘genetic pollution’ has been used1, inferring that contributions to the reproduction of river stocks from farmed escapees are negative because they may reduce the genetic variability of the wild salmon populations (Clifford, Mcginnity & Ferguson 1998; Hindar et al. 2006). Besides general measures to reduce the incidence of farm escapes, Norwegian stakeholders requested visible marking of farmed fish to allow for removal via angling and also during management operations to cultivate and enhance natural river stocks (Skaala et al. 2014). This situation prompted the Norwegian Seafood Industry Association (FHL) to call for studies to identify the most effective methods to distinguish between wild and cultured Atlantic salmon2. Genetic testing may be used; however, this technique is not suitable for discrimination in the field

Keywords: animal welfare, epidermis, healing, integument, marking, skin. Correspondence M Andrews, Centre of Epidemiology and Biostatistics, Faculty for Veterinary Medicine and Biosciences, Norwegian University of Life Sciences, Postboks 8146 Dep, Oslo N-0033, Norway (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd

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1 http://en.wikipedia.org/wiki/Genetic_pollution (accessed February 22, 2014). 2 http://fhl.no/dette-er-tiltakene-mot-romming/ (accessed March 09, 2014).

Journal of Fish Diseases 2014

and neither is PIT tagging as a PIT tag reader would be needed to operate (Skaala et al. 2014). The simplest and most easily applicable method for visual marking of farmed salmon would be clipping of the adipose fin that, when performed diligently, fails to regenerate leaving a permanent mark. As the identification of fish with or without this fin needs neither special skills nor technical equipment, fast evaluation of high numbers of fish is possible (Coombs et al. 1990). The common practice of vaccinating all farmed salmon by injection prior to sea transfer would allow for simultaneous fin clipping during anaesthesia, and consequently for recognition and removal of marked fish, that is, escapees that are caught during angling or broodstock capture. The normal histological structure of the fish integument has recently been summarized by Elliott (2011a), while the adipose fin structure was studied by Buckland-Nicks, Gillis and Reimchen (2012). Adipose fin epithelium comprises of an outermost superficial cell layer of evenly flattened cells; a central region comprising cuboidal cells; and an inner layer of prismatic basal cells (Fig. 1). Mucous cells are distributed throughout these layers. A pigment cell layer is located immediately below the basement membrane. The dermis consists of both a dense and a loose connective tissue layer. The dermis of the adipose fin does not contain scale pockets. Little to no adverse effects have been associated with adipose fin clipping (Gunnes & Refstie 1980; Johnsen & Ugedal 1988; Vander Haegen et al. 2005). However, for routine use, its potential to

M Andrews et al. Healing following fin clipping

cause any harmful physiological effects resulting from the presence of open skin lesions particularly at low water temperatures should be investigated. Wahli et al. (2003) conducted one of the few recently published studies of wound healing in salmonids, with emphasis placed on the influence of dietary vitamin C on the wound-healing process of rainbow trout, Oncorhynchus mykiss (Walbaum, 1792). The authors began sampling from day 3 post-wounding and noted that the wound was already covered with an epidermal layer at the first sample point. While the epidermis showed the normal structure in all sampled fish 3 weeks after wounding, the dermal and muscular layers did not regain the normal structure within this time period. Hickey (1982) described initial skin wound closure for salmon larvae (body length 18–27 mm); however, no data are available dealing with the immediate reaction during the first hours following fin clipping of Atlantic salmon presmolts. Furthermore, skin wound healing at different water temperatures has been investigated in various species (Cunha Da Silva et al. 2005) we found no detailed investigation relating to the removal of the adipose fin in salmonids. Injuries of the epidermal barrier may have an effect on osmotic balance that is of relevance to overall fish welfare (Quilhac & Sire 1998; Ashley 2007) and may also be an entry port for pathogens. The purpose of this study was thus to describe the early wound-healing processes following adipose fin clipping, in particular the time course for wound closure and the re-establishment of a normal epithelial barrier, as well as the effect of different water temperatures on this process; in our view, this is important factual data needed to quantify the overall health and welfare effects that this marking technique may have when being employed on an industrial scale or in research.

Materials and methods

Experimental set-up

Figure 1 Overview of an Atlantic salmon adipose fin; the main features discussed in this study are detailed here, including the superficial cell layer (a); cuboidal cells (b); prismatic basal cells (c); mucous cells (d); basement membrane (e); pigment cells (f); and connective tissue (g); 200 9 mag. Ó 2014 John Wiley & Sons Ltd

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The live fish experiment was conducted at the VESO Vikan Research Facility, in mid-Norway. A total of 207 Atlantic salmon smolts (avg. weight 36 g; range 27.7–45.3 g) were randomly divided between three 450-L tanks supplied with municipal freshwater. Subsequently, each tank was adjusted to the intended study temperature, that is, 4, 10 and 14 °C, respectively. Fish were given 2 weeks to

M Andrews et al. Healing following fin clipping

Journal of Fish Diseases 2014

adapt to the temperatures. Each temperature group consisted of 66 fish; an additional six fish were included in the 10 °C group to be used as the immediate post-clipping sample for all three groups. Three unclipped control fish from the same source as the experimental fish were sampled at the start of the experiment. For fin clipping, all fish were netted and transferred into an Aqui-S anaesthetic bath (15 lg L 1 isoeugenol) containing water from the holding tank. The adipose fin was completely removed using small, curved surgical scissors, after which the fish were returned to the holding tank. Each group was processed within a short time period of approximately 30 min. From the 10 °C group, six fish were sampled immediately following fin clipping according to the method described below, serving as 0 h controls for all temperature groups. At 11 subsequent time points (2, 4, 6, 12, 18, 24, 30, 36, 48, 60 and 72 h post-clipping), six fish per temperature group were sampled following a benzocaine (MS222) overdose. Once completely unresponsive, their spinal cord was cut using scissors, and the skin area including the underlying muscle tissue around the base of the adipose fin was carefully excised using a scalpel. Excised samples were immediately placed in 10% neutral buffered formalin and kept at room temperature until processing. Histological analysis A transverse cut through the mid-section of the wound area was prepared for each sample, which were then coded to ensure blind histological processing and evaluation. Fixed samples were paraffin-embedded and routinely processed for

histological examination; sections of 3 lm thickness were cut and stained with haematoxylin and eosin (H&E). One slide from each sample was examined by light microscopy at 2009 magnification. A score sheet (Table 1) adapted from Wahli et al. (2003) was used to evaluate the sections. The score sheet consisted of 11 parameters, including the following seven epidermal parameters: structure, thickness, prismatic basal cells, cuboidal cells, superficial cells, mucous cells and infiltration, and four dermal parameters: structure, cell debris, infiltration and pigment cells. All parameters were scored using a linear scale that ranged from 0 (abnormal) to 30 (normal). The thickness of the epidermis was initially scored with 15 being the normal whereby lower values represented a thinner layer than normal, higher values a thicker than normal layer. The thickness outcomes were transformed to the common range (0 = abnormal/ absence of epidermal cells; 30 = normal thickness). All parameters were scored once for each sample (Table 1) by a single pathologist. Statistical analysis The relationship between time and temperature for each parameter was examined using a multivariable linear regression analysis in Stata SE 11 (StataCorp). Due to the distribution of the epidermal parameters, a time-quadrate term was added to the analysis to achieve a good fit. The dermal parameters did not show a better fit using this method, and the simplest model was thus used. As temperature was included as a categorical variable, a F-test was performed to show the effect of temperature as a whole on the parameter. As

Table 1 Scoring sheet describing the presence and form of the epidermis and dermis cell groups following adipose fin clipping of Atlantic salmon; 0 = absent/abnormal, 30 = present/normal

Epidermis

Dermis

Nr.

Parameter

Description

1

Structure

2 3 4

Thickness Prismatic basal cells Cuboidal cells

5 6 7 8

Superficial cell layer Mucous cells Infiltration Structure

Normal epidermal structure consisting basal cell layer, several layers of round cells with mucous cells distributed throughout and a superficial cell layer Uniform epidermal thickness across the wound area Adjacent to the basement membrane with all cells being cuboidal and columnar Consists of a number of layers located between the basal cell layer and superficial cell layer, all cells are round and cuboidal The outermost cell layer comprising of flattened, elongated cells Mucous cells should be present throughout the epidermis Infiltration granulocytes, lymphocytes or macrophages are present in the wound area Basement membrane, pigment layer, stratum spongiosum (no scales present), stratum compactum and a hypodermal layer present in the area of interest Necrotic cells, oedema or cell debris present in the wound area Infiltration granulocytes, lymphocytes or macrophages are present in the wound area Cell layer immediately below basal membrane

9 10 11

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Cell debris Infiltration Pigment cells

Journal of Fish Diseases 2014

no infiltration was evident, no statistical analysis was performed on these parameters. Post-estimations evaluating independence, normality and homoscedasticity were conducted for each of the models. Results

The structure of the intact adipose fins that were sampled before clipping corresponded to earlier histological descriptions of this organ (Fig. 1). Immediately following adipose fin clipping, the wound area presented a clearly visible cut with signs of oedema being the first immediate reaction (Fig. 2a). At 2 h post-clipping, the primary reaction observed in all three temperature groups was the formation of an oedematous area near the wound mid-point and fibrin exudation covering the excision area (Fig. 2b). Four hours post-clipping, the 4 °C fish exhibited no signs of wound closure, while in contrast, initial re-epithelization of the wound area was observed in both the 10 and 14 °C group. In 33% sampled from 10 °C and 75% of the fishes sampled from 14 °C, the wound was covered with a thin layer of undifferentiated epithelial cells (Fig. 2c). Six hours post-clipping, 66% fishes in the 4 °C group showed faint epithelial coverage of the wound. In fish kept at 10 °C, the epidermal cells had already differentiated into basal epithelial cells, in addition to these cuboidal cells, and superficial flattened cells were discernible in the 14 °C fish (Fig. 2d). Twelve hours post-clipping, near-complete re-epithelization was observed in all the 4 °C fish; however, the epidermis was very stretched and flattened comprising of a few layers of undifferentiated epithelial cells (Fig. 2e). The epidermis had continued to thicken in the 10 and 14 °C fish with all cell types being discernible; however, the overall appearance was uneven with areas of moderate spongiosis (the presence of intercellular oedema within the epidermis) (Fig. 2f). By 18 h post-clipping, superficial cells, cuboidal cells, basal cells and mucous cells were discernible in all temperature groups. All temperature groups were similar to 24 h post-clipping (Fig. 3a) with the epidermis increasing in thickness yet remaining uneven and with spongiotic areas. The numbers of mucous cells increased gradually, and the epidermal structure continued to normalize from Ó 2014 John Wiley & Sons Ltd

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M Andrews et al. Healing following fin clipping

30 h through to 72 h (Fig. 3b). Although regeneration of pigment cells in the dermis below the basal membrane was visible, the re-establishment of a normal pigment cell layer was still incomplete 72 h post-clipping. The time-course development of wound closure as documented by the epidermal structure and thickness scores is summarized in Fig. 4, illustrating the differences between the temperature groups especially during the first 24 h following marking. The statistical analysis of the histology results showed a significant association between time, temperature and all the epidermal parameters in particular structure and thickness. There was a significant difference between the temperatures for all parameters albeit marginally between 4 °C and 10 °C for cuboidal cells and prismatic basal cells. The models for all the epidermal parameters showed a fairly high degree of explanation of the total variation (adjusted R2), ranging from 46% to over 60% (Table 2). Only minor regeneration of the dermis was observed throughout the examination period, and the degree of explanation was only faint. Discussion

The most interesting and also most significant result from the current study is the very swift closure of the wound area by epidermal cells, occurring within 4–12 h depending on the water temperature. This emergency repair of the interrupted epidermal barrier enables control of the electrolyte exchange with the surrounding water and maintenance of osmotic balance that is one of the key features of the fish integument (Elliott 2011b). It strongly illustrates the evolutionary benefits for fish of possessing a live cell epidermis and offers an explanation for the remarkable lack of adverse event reports following the historical widespread use of adipose fin clipping to mark fish in experiments and in field studies (Chart & Bergersen 1988; Vincent-Lang 1993). The results further confirm that water temperature plays an important role in the time course of wound closure of Atlantic salmon. A further important finding is the absence of marked inflammatory reactions and the absence of any features indicative for a pathogen infection, indicating that the fast overgrowth of the wound by epithelial cells prevents the invasion of pathogens. This may have been aided by the fact that

M Andrews et al. Healing following fin clipping

Journal of Fish Diseases 2014

(a)

(b)

(c)

(d)

(e)

(f)

Figure 2 Progress of wound closure following adipose fin clipping of Atlantic salmon; 0 h post-clip, 10 °C (a); 2 h post-clip, 10 °C (b); 4 h post-clip, 14 °C (c); 6 h post-clip, 14 °C (d); 12 h post-clip, 4 °C (e); 12 h post-clip, 14 °C (f). Arrow indicates spongiotic area. 200 9 mag.

throughout the experiment, the fish were held in municipal water rather than in sea water. Wound closure in fish was already addressed by Harabath (1928); however, the details of postwounding epidermal reactions in various finfish species were intensively researched not before the 1970s and early 1980s, using both light (Roberts et al. 1973; Bullock, Marks & Roberts 1978a) Ó 2014 John Wiley & Sons Ltd

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and electron (Phromsuthirak 1977) microscopy. Migration of the surrounding epidermal cells from the wound edge and eventually fusion of the meeting cell layers was found to be the principal mechanism of wound closure. Bullock, Marks and Roberts (1978b) conducted experiments with plaice, Pleuronectes platessa Linnaeus, 1758, showing that wounds were closed within 9 h at 10 °C and

M Andrews et al. Healing following fin clipping

Journal of Fish Diseases 2014

(a)

(b)

Figure 3 Progress of wound closure following adipose fin clipping of Atlantic salmon; 24 h post-clip, 14 °C (a); 60 h post-clip, 14 °C (b). Arrow indicates mucous cells. 200 9 mag.

Thickness, 4 °C

0

0

Score 10 20

Score 10 20

30

30

Structure, 4 °C

2 46

12

22

30 36

48

60

72

246

48

60

72

60

72

60

72

Thickness, 10 °C

0

0

Score 10 20

Score 10 20

30

30

Structure, 10 °C

12 18 24 30 36

2 46

12 18 24 30 36

48

60

72

2 46

48

Thickness, 14 °C

0

0

Score 10 20

Score 10 20

30

30

Structure, 14 °C

12 18 24 30 36

2 46

12 18 24 30 36

48

60

72

2 46

12 18 24 30 36

48

Figure 4 Healing progression in the epidermal structure and thickness of the adipose fin following clipping at 4, 10 and 14 °C. Scoring was conducted using a scale ranging from 0 (absent/abnormal) to 30 (present/normal). Values are provided as median, minimum and maximum values for each sampling point (h) post-clipping.

12 h at 5 °C post-wounding; however, the cells were relatively undifferentiated at both temperatures, and at the lower temperature, they were present in a thinner layer. In a comprehensive study, Hickey (1982) further proved that epidermal migration for wound closure was essentially similar in Atlantic herring, Clupea harengus Ó 2014 John Wiley & Sons Ltd

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Linnaeus, 1758, plaice and salmon larvae. In 24to 27-mm salmon alevins held at 10–11 °C, the coverage of an approximately 0.8 mm wound took between 4 and 8 h, giving an estimated rate of migration between 50 and 120 lm h 1. Experiments in plaice larvae showed that the epidermal migration rate almost doubled between 5 and

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0.4545 0.4527 0.4206 0.3899 0.5530 0.4050 0.1277 0.0334 0.0969 0.6336 0.5939 0.4882 0.4597 0.6284 0.5160 0.1219 0.0306 0.0916 0.001 0.010 0.027 0.027

Rapid temperature-dependent wound closure following adipose fin clipping of Atlantic salmon Salmo salar L.

Three groups of Atlantic salmon were kept at a constant temperature of 4, 10 and 14 °C. The adipose fins were removed; six fish/group were sampled at ...
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