Introduction

Skin is a barrier that protects the fish's body from the ambient ecosystem (Naiel et al 2022b). Keeping the skin's integrity is crucial to ensure the immune system's capacity of aquatic organisms (Grada et al 2018). It is recorded that wound induction in farmed fish species and disease outbreaks are highly associated with each other and are considered a constraint for production (Bruno et al 2013).

Despite the similarity of the essential function of the skin in most animals, the organization and composition of fish differ from other vertebrates (Naiel et al 2023). Fish can serve as an excellent model for studying wound healing due to the distinct similarity between the wound healing process in fish and other mammalian systems (Roohani et al 2013; Sveen et al 2020). The ambient water temperature degree and other environmental factors, such as habitat and diversity in fish species, can reveal specific differences in wound healing in fish (Naiel et al 2021). Moreover, skin injury could disrupt the balance of fish barrier function, consequently threatening aquatic animal welfare (Abdi et al 2023). Besides, full-thickness skin wounds must be healed as soon as possible to avoid contamination of the wounded areas and blood loss (Shaw and Martin 2009).

Similar to mammals, fish skin has a dermis, where wound healing varies depending on the wounded portion of the skin, and the epidermis of superficial wounds in fish may require a shorter time to heal than the deeper wounds (Rakers et al 2010; Roohani et al 2013; Richardson et al 2016; Sveen et al 2020). In the initial stages of wound healing, keratinocytes migrate from the intermediate layer of the epidermis to cover a surface area of wounds, which is followed by inflammation, where neutrophils and macrophages are relocated to the wounded area, as well as, activating growth factors signals to provoke cell proliferation and the formation of granulation tissue to replace the injured area (Gomez and Primm 2021).

The surface skin bacterial load is intimately associated with general health status and entire physiological disorders (Naiel et al 2022a). The relationship between total skin bacterial load and the various cell types responsible for wound healing modulates the immune response and promotes barrier repair activities (Jiang et al 2020). In addition, vast types of bacterial strains can migrate to the wounded area after injury, which plays a crucial role in wound healing (Di Domizio et al 2020). Those bacterium start to move to the dermis to recruit neutrophils and increase the expression of growth factors at the site of a wound (Nazeer et al 2023). Certain products, minerals, elements, chemical compounds, other medicinal plants, and blood-derived products play an essential role in the wound healing process in vertebrates due to their role in improving the immune status and eliciting various physiological functions (Mei et al 2020). Specifically, numerous essential trace elements, such as selenium, zinc, and copper, have shown remarkable efficacy in wound healing due to their immune-promoting features across the body (Lukáč et al 2009; Jensen et al 2015). For example, platelet-rich plasma and fibrin demonstrated higher wound healing efficiency of the abdominal wall and skin defects in canine and equine models, respectively (Abouelnasr et al 2017; Hamed et al 2019).

Zinc (Zn) is an essential trace element for fish welfare and general health status (Gharib et al 2022). Also, zinc is vital for growth, stimulates the immune system, and improves antioxidant activities and fast-moving the wound healing process (Roohani et al 2013; Song et al 2017). Furthermore, zinc-dependent proteins are linked to a variety of functions inside the cell, including the activation of DNA repair pathways, the regulation of gene transcription, the modulation of metabolic processes, and the stimulation of the redox state (Zhang et al 2010; Pawlak et al 2012; Cho et al 2016).

It is well known that wound healing is one of the most complicated processes in vertebrates, comprising several phases, including hemostasis/inflammation, proliferation, and remodeling (Sveen et al 2018). Thus, a lack of balance in any of these phases can result in different types of damage (Okur et al 2019). During wound healing, zinc can be implicated in various phases, such as wound hemostasis, inflammation, re-epithelization and granulation tissue formation, extracellular matrix remodeling, and its role in membrane repair (Li et al 2015, 2017). Until now, more investigation is required to determine zinc's preventive ability and mode of action during wound healing process in fish.

Therefore, the current study was conducted to investigate the beneficial role of different concentrations of immersion bath of zinc sulfate on the wound healing process in common carp (Cyprinus carpio) at other time points within two weeks of the healing process through analyzing gene expression of growth factors associated with wound healing, antioxidant defence system, and histological re-epithelization scoring.

Materials and methods

Fish welfare and used chemicals

The experimental fish was brought from a private hatchery located at Dakahlia governorate, Egypt. Fish were randomly allocated into glass aquaria (with dimensions: 45 × 30 × 60 cm) pre-filled with 60 L of dechlorinated clean water. The aquaria were supplied with continuous aeration and internal filters. Senso Direct 150 (Lovibond, Germany) checked water physicochemical parameters daily. The fish diet was formulated based on a diet conducted by El‐Adl et al. (2018). Fish were fed twice daily (3% of their biweekly biomass). At the beginning of the feeding trial period, the fish were acclimated for 14 days. Aquarium wastes were removed every day using the siphon technique, and the rearing water and extra chemicals were restored afterwards. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Z0251) was bought from Sigma-Aldrich (Germany). The 96 h LC50 values used in this experiment were chosen based on a study by El‐Adl et al. (2018), which employed probit analysis, where the 96 h LC50 of ZnSO4·7H2O was 45.74 mg/L and referred to an equivalent of 10.5 mg/L of Zn.

Experimental design

A total of 240 healthy common carp fish (52.3 ± 0.9 g weight and 30.5 ± 2.5 cm total length) were randomly divided into four equal groups in triplicate (20 each). During the feeding trial period, all experimental fish were receiving commercial diets. As described by El‐Adl et al. (2018) and Przybylska-Diaz et al (2013), all experimental fish groups were imperiled to mechanically generated wounds (with a diameter of 6 mm) employing a biopsy punch needle (Gamhoria Co., Egypt) between lateral line and dorsal fin at the end of dorsal fin. After wound induction, the treatment groups were subjected to varied tested Zinc sulfate immersion bath dosages once daily. The first group was assigned as control (without zinc sulfate immersion bath), while the second group through the fourth group subjected into zinc sulfate immersion bath of 2.09, 1.05, and 0.53 mg/L corresponding to 1/5, 1/10, and 1/20 of 96 h of Zn, and named Zn/5, Zn/10, and Zn/20, respectively. Then, ten fish specimens were collected from every group after three different time intervals as follows: 6 h, two days, three days, and two weeks. Before collecting samples, the fish were euthanized with buffered 400 mg/L Ms222 (E10521, Sigma-Aldrich, Germany) solution (Matthews and Varga 2012). Then, fish musculature specimens were collected from wounds and were divided into three parts. The first part was stored directly at − 80 °C for gene expression analysis. The second part was kept at 10% neutral buffered formalin for histopathological and histochemical examination. In contrast, the third portion was stored at phosphate buffer saline (PBS) pH 7.6 to determine antioxidant and oxidative stress markers.

Expression analysis of genes associated with wound healing

For gene expression analysis, 50 mg of musculature tissues was homogenized on GENEzol reagent (Geneaid, Taiwan) in a 1.5-ml microcentrifuge tube, and RNA extraction was conducted using TriRNA pure kit (Geneaid, Taiwan) following the instructional manual. RNA concentration was measured and checked for purity using a Quawell nano spectrophotometer (USA). RNA was also checked for integrity with Agarose gel electrophoresis (1.5%). A total of 2 µg of RNA was converted to cDNA using a High-capacity cDNA kit. Gene expression analysis was conducted using primer pairs (El‐Adl et al. 2018) of vascular endothelial growth factor-alpha (VEGF-A) (XM_019097650), transforming growth factor beta-1 (TGF‐β1) (AF136947), collagen alpha 1 (COL1α1) (HM363526), fibroblast growth factor-7 (FGF-7) (KP293853) and metallothionein (Mt) (AF249875). Relative expression of those genes was normalized against housekeeping gene β-actin (M24113) on the Step One TM system (Thermo Fisher Scientific). PCR cycling condition was started with an initial denaturation step of 95 °C/9 min and followed by 35 cycles of denaturation step at 95 °C/40 s, annealing step at 59 °C/30 s, and extension step 72 °C/30 s, the PCR was terminated with a final extension at 72 °C/5 min. The cycle threshold (Ct) value of all quantified genes was normalized against both control and housekeeping genes according to the method of Livak and Schmittgen (2001).

Determination of antioxidant and oxidative stress markers

Hundred milligrams of musculature from the wounded area was homogenized in 900 L PBS pH 7.6 in a 1.5-ml microcentrifuge tube and centrifuged at 3000×g per 15 min at 4 °C. Supernatant was collected and used for the determination of superoxide dismutase (SOD) activity, catalase (CAT) activity, and reduced glutathione levels (GSH), as mentioned in the research work of El‐Adl et al. (2018). All commercial kits required to determine antioxidants were purchased from Bio-diagnostic. Egypt. The levels of tissue total protein were evaluated by the method of Lowry et al (1951).

Histological evaluation

The collected samples were fixed in 10% neutral buffered formalin. After 48 h, the samples were subjected to dehydration in ascending concentrations of ethyl alcohol series, clearing in a xylene series, immersion, then embedding in liquid paraffin wax and sectioned (5 μm thick) using a rotatory microtome. According to Bancroft and Gamble (2008) procedure, the sections were stained with hematoxylin and eosin stain(H&E) for general histopathology or with Mallory trichrome for collagen density (Bancroft 2008). Histopathological sections were examined under a light microscope. Wound healing was assessed according to the histological score described by Abramov et al (2007). The criterion evaluated for wound healing in histopathological sections consisted of re-epithelialization, granulation tissue maturation, collagen deposition, and inflammatory cell infiltration. Each criterion score was individually assessed in three sections of each wound in 5 high-power fields (× 400) for each section. The calculated values were expressed as means ± standard error (SE).

Statistical analysis

The normality test was employed using Shapiro–Wilk’s test, where the homogeneity test (Levene’s test) was done to check the homogeneity of variance at a probability lower than 0.05. The two-way ANOVA test was used to investigate the factors of dose and time on the different parameters in the current study. Tukey’s test was used as a post hoc test for determining the difference between means at p < 0.05 using SPSS IBM v.23 (Arbuckle 2011). Data were expressed as mean ± SEM.

Results

Monitoring the wound healing on fish skin

Healing of the mechanically induced wound was different in each group in a positive relation corresponding to Zn sulfate concentration. After three days of therapy, the wound edges in the Zn/5 group were completely healed. In contrast, wound closure was found in both the Zn/10- and Zn/5-treated groups after 14 days compared to the control or Zn/20 groups (Fig. 1), whereas, after 14 days of bath therapy, the Zn/20-treated group showed partial wound healing.

Fig. 1
figure 1

A picture illustrates the wounds created by a biopsy bunch that induced a 6-mm incisional wound in partly scaled common carp in all examined groups (Control, Zn/20, Zn/10, and Zn/5) from the start of the experiment (Day/0) and after 6 h, 2 days, 3 days and 14 days after that. (Control) shows wound closure times in fish from the control group at previously mentioned time points after wound induction. The Zn/20, Zn/10, and Zn/5 represent a fish from zinc sulfate-treated groups with 1/20, ZS 1/10 and ZS 1/5, respectively, after 6 h, 2 days, 3 days or 14 days of wound induction

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Expression analysis of genes associated with wound healing

The expression of COL1α1 displayed the maximum expression level in Zn/5 that was significantly elevated compared to other experimental groups after 6 h, 2 and 3 days of wound induction (p < 0.05) with a non-significant change between both Zn/5 and Zn/10 after 14 days of wound induction (p > 0.05). The expression of COL1α1 in Zn/10 showed the same magnitude as Zn/20 after 2 and 3 days of wound induction. When compared to the control group, the Zn/20 fish group demonstrated significant increases in COL1α1 expression only two days after wound induction (p > 0.05) (Fig. 2A).

Fig. 2
figure 2

The relative expression level of the COL1α1 (A), FGF‐7 (B), TGF‐β1 (C), VEGF‐A (D), and Met (E) of wound tissue after 6 h, 2 days, 3 days, and 14 days from wound initiation. All estimated genes were regulated against the housekeeping gene (β‐actin) employing 2−ΔΔct calculating method. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, n = 10

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Furthermore, the Zn/5 fish group had the most significant FGF-7 expression at all estimated periods, followed by the Zn/10 group, which enhanced FGF-7 expression relative to both the Zn/20 and the control groups after 6 h, 2 days, and 14 days of post-wound induction. After 14 days of wound induction, the expression of FGF-7 showed a stepwise increase, with the highest expression in the Zn/5 group and the lowest expression in the control group, as shown in Fig. 2B.

The magnitude of expression of TGF‐β1 was the highest in the Zn/5 group at all time points while TGF‐β1 was significantly downregulated in the Zn/10 group after 2 and 14 days of wound induction. The expression of TGF‐β1 revealed a non-significant change between both Zn/20 and the control group at all time points. Moreover, the expression level of TGF‐β1 was the same among Zn/10, Zn/20, and the control group at two days and 14 days of wound induction (Fig. 2C).

The angiogenesis marker (VEGF-A) showed the highest expression in the Zn/5 group between all studied groups at different time points. In contrast, the Zn/10 and Zn/20 demonstrated that the same VEGF-A expression magnitude at every point was approximately the same level as the control group (Fig. 2D).

In addition, the expression of Mt was the highest in the Zn/5 group, while the expression of Mt between Zn/10 and the other groups (control group and Zn/20) was the same except after 2 days of wound induction (Fig. 2E).

Redox status biomarkers associated with wound healing

Activities of SOD, CAT, and GSH are represented in Fig. 3. The activity of SOD was increased significantly in the Zn/5 group when compared with the other studied groups at different time points (6 h, 2, and 3 days) of wound induction, where the activity of SOD showed the same level with other ZnSO4-treated groups after 14 days of wound induction. Throughout the trial, SOD activity in the Zn/10 and Zn/20 groups remained similar. The highest concentration of Zn improved the activity of CAT by nearly threefold after 6 h, 2, and 3 days of wound induction when compared with a control group. Furthermore, the concentration of GSH was significantly elevated in the Zn/5-treated group compared with the other studied groups at 6 h, 2, and 3 days of wound induction. In contrast, 14 days after wound induction, the level of GSH concentration remains the same in all ZnSO4-treated groups.

Fig. 3
figure 3

redox status biomarkers related to wound healing treatments employing several levels of ZnSO4 in common carp. SOD, superoxide-dismutase, CAT, catalase and GSH, glutathione peroxidase

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Histopathological and inflammatory scoring

The microscopic examination from all groups at 6 h (Fig. 4:1a, 1b, 1c, and 1d) revealed that after the wound incision, the wound surface had already been covered by some migratory front, but the epidermal organization and re-epithelization score were still incomplete. Also, some improvement in the re-epithelization score was remarkably detected (p < 0.05) in the Zn/5 fish group (the mean score was 1 ± 0.148) compared to the other treated and non-treated groups (Fig. 4: A). The melanocytes or the pigment cells were also detected beneath the basement membranes (sub-epidermal) in the Zn/5 group. Two days after wound induction (Fig. 4:2a, 2b, 2c, and 2d), the score of the epithelization significantly increased after ZnSO4 treatment compared to the control group. Still, the highest score was detected in the Zn/5 group (Fig. 4: A), as the epidermis was wholly anchored to the underlying dermis. As the average skin area, some club cells were detected in the epidermal layer. However, the thickness of the epidermis was still thinner than the normal region (Fig. 4:2d). After 3 days following wound incision (Fig. 4:3a, 3b, 3c, and 3d), a complete re-epithelization score was significantly increased (p < 0.05) in Zn/5 group in compared to the other groups (Fig. 4: A). Finally, at 14 days of post-wound incision, the re-epithelization score was also ultimately recorded in the Zn/10-treated group, and the club, mucus, and pigment cells were intensity observed at the normal skin area (Fig. 4:4 b, 4c, 4d, and A) compared to the non-treated one.

Fig. 4
figure 4

photomicrograph of the wounded site stained with H&E showing epidermal layer (EP), immature granulation tissue (GT), inflammatory cells (black arrowhead), collagen fibers (black arrow), wide intercellular spaces (green arrowhead), disrupted basement membrane(black circle), club cells(black-tailed arrow), melanocyte (black vertical arrow), stratum spongiosum (SP), stratum compactum (SC), mucous cells (green tailed arrow), the epidermis of wounded skin (black double armed arrow) and normal skin (blue double armed arrow). A Represents the mean re-epithelization scores, B represents the mean score of granulation tissue maturation, and c illustrates the mean inflammatory cells score. Data were expressed as mean ± SME. Bars carrying different superscripts are significantly different (p < 0.05)

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The inflammatory cell infiltration was detected mainly in the dermis of all groups at 6 h (Fig. 4:1a, 1b, 1c, and 1d). After that, at any time, the ZNSO4 immersion bath treatment suppressed the inflammatory cell infiltration (Fig. 4C).

Granulation tissue maturation scores and collagen density %

After ZNSO4 immersion bath treatment, especially in the Zn/5 group, some improvement of granulation tissues and collagen fibers arrangement was represented at 6 h compared to the control (Fig. 5:1a, 1b, 1c and 1d). After that, on the 2,3, and 14 days of post-wound induction, the granulation tissue scores (Fig. 4:2–4a, 2–4 b, 2–4c, 2–4d and B) and collagen density % (Fig. 5:2–4a, 2–4b, 2–4 c, 2–4d and A) exhibited markedly increases (p < 0.05) compared to the non-treated group. Generally, ZnSO4 immersion bath treatment accelerated the granulation tissue maturation as the collagen fibers had organized parallel to the epidermal layers (in two layers; stratum spongiosum and compactum) and appeared thicker and denser.

Fig. 5
figure 5

photomicrograph of the wounded site stained with Mallory trichrome showing immature granulation tissue (GT), dispersed collagen fibers (black arrow), wide inter-fiber spaces (vertical arrow), clumped collagen fibers (black-tailed arrow), condensed and parallel arranged collagen fibers (green tailed arrow). A Representing the mean % of collagen fiber density. Data were expressed as mean ± SME. Bars carrying different superscripts are significantly different (p < 0.05)

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Discussion

The immunity of fish mucosal epithelium is distinguished from other vertebrates through the absence of keratinized epithelium, which accelerates fish metabolic activity (Press and Evensen 1999; Zhang et al 2010). This rapid metabolic activity is associated with migrating epithelial cells building up wound gaps without requiring initial inflammatory responses for wound re-epithelization (Richardson et al 2013). Zinc is conventionally applied as a cofactor of several metabolic processes and metalloenzymes that play an essential role in the repair processes of cell membranes, immune function, and cellular proliferation (Lin et al 2018). Specifically, cell receptors interact with the extracellular matrix and other intercellular components to modulate signal transduction, cellular proliferation, inflammation, and apoptosis (Castillo-Briceño et al 2010).

The expression of COL1α1 is associated with tissue structural configuration in skin and muscles (Herr and Farndale 2009). Applying zinc compounds and other biomaterials can sustain the release of Zn+2 ions in the environment, contributing to upregulating specific osteogenic genes, such as Col type 1, in zebra fish (Gistelinck et al 2016). Moreover, the upregulation of COL1α1 gene expression in a Zn dose-dependent manner indicated the rise of collagen biosynthesis in the incidence wound (Todorovic 2002; McDowell 2003). However, the sudden decline following 14 days of immersion bath treatment in all examined groups can be attributed to the depletion of collagen biosynthesis due to wound closure. The obtained results found to be similar Fukuda et al (2015) finding that the upregulation level of COL1α1 gene expression reached its maximum level post 7 days of treatment period and started to decline significantly following 10 days post-wound induction.

Similarly, the expression of FGF-7 showed a continuous increase in expression by increasing the zinc concentration in immersion bath water as well as the exposure period. It is well known that the FGF-7, the keratinocyte growth factor (KGF), has a significant role in re-epithelization during the first hours of wound induction via the proliferation of keratinocyte migration (Lafosse et al 2016). Zinc can stimulate keratinocyte migration to the wound injury site, promoting its proliferation by modulating integrin function (Tenaud et al 2000). Moreover, different salts of zinc, such as zinc sulfate and zinc histidine, are incorporated with an intense proliferation of keratinocytes in natural human keratinocytes (Deters et al 2003). In Atlantic salmon, a diet supplemented with Zn significantly improved the wound healing process, with a marked increase in mucus cells during 2 weeks (Jensen et al 2015). In zebrafish, FGFs are incorporated in different developmental pathways (Itoh and Konishi 2007), while FGF-7 acts specifically on epithelial cells in adult mice (Miki et al 1991) with a paracrine effect on wound sites. The level of FGF-7 expression showed the highest level of expression after two days and four days after wound induction in skin and muscle, respectively, in male mice (Koike et al 2020).

The expression level of TGF‐β1 showed a stepwise increase by increasing the concentration of Zn in immersion water bath. TGF‐β1 is incriminated in several phases of the wound healing process, including several proliferative, regulatory, and inductive processes (Bitzer et al. 1998). In the proliferative phase, TGF‐β1 stimulated the process of angiogenesis (Sporn et al 1986) as well as controlling the extracellular matrix components in the maturation phase (Shah et al 1994) and could improve tissue repair function via stimulation immune response (Wahl 1992). Zn is a crucial TGF‐β1/SMAD pathway cofactor that promotes granulation tissue production (Maywald et al 2017b). Interestingly, the excessive production of TGF‐β1 can cause maladaptation of repair condition (Boo and Dagnino 2013), which clarified the decrease in expression of TGF‐β1 from the second day of wound induction till the end of the experiment under the control of Mitsugumin 53, which controls cell membrane repair process (Cai et al 2009).

Zinc, as a main biologically trace element, is characterized by its role in maintaining cellular physiology (Petry et al 2016; Maywald et al 2017a), as well as its incorporation of transporters protein named SLC39A, which has a crucial role in vascular development and the process of angiogenesis (Jin et al 2015). Furthermore, applying zinc chloride stimulated the production of VEGF-A and B in chondrocytes and improved the chondrogenesis process in the ATDC5 cell line (Hozain et al 2021). The stimulatory effect of zinc on FGF-7 and its incorporation with keratinocyte migration would stimulate the process of establishment of new blood vessel production (Saghiri et al 2015).

Metallothionein (Mt) is considered a low molecular weight protein rich in cysteine residues, which has a specific role in the metabolism of zinc (Hamer 1986). In the current trial, the expression of Mt in the wounding area reached its highest after 24 h of wound induction. The same result was demonstrated after 12 h in human epidermal keratinocyte cell lines during the acute phase of host damage (Salesa et al 2021). Mt expression gradually decreased after 18 h due to zinc accumulation in the wounded area (Iwata et al 1999). The critical role of metallothionein in wound healing is through the incorporation of Mt in the biosynthesis of large amounts of enzymes required for cell proliferation and tissue remodeling (Lansdown 2002).

In the early stages of wounds, reactive oxygen species increase on the site of the wound and cause imbalances between oxidant/antioxidant systems that would increase tissue damage and delay wound healing in unhealthy wounds or immuno-compromised individuals (Torres et al 2013). GSH is categorized as a critical antioxidant in several biochemical pathways that require the elimination of free radicals, where any disturbance in both oxidized and reduced forms of glutathione can result in a change in redox status in the wound site and can affect the expression of genes that is responsible for healing process (Chen et al 2017; Wang et al 2017). Meanwhile, SOD is considered an effective antioxidant enzyme that has a role in the disproportion of superoxide radicals which eventually breaks apart in adverse conditions and aggravates wound healing (Radhakumary et al 2011; Zhuang et al 2015). Meanwhile, catalase is found to be reduced in skin wound areas of rats suffering from diabetes mellitus, and catalase activity is reduced in old groups of rats compared to young-aged rats (Rasik and Shukla 2000). The hyperproliferation in would epithelium in wound edges shows the highest expression of catalase and glutathione peroxidase in all layers of the wound epidermis (Steiling et al 1999). Generally, waterborne zinc immersion bath treatment in an experimental study in Carassius auratus achieves a maximum increase in SOD, CAT, and glutathione peroxidase activity (Wang et al 2020). The level of reduced glutathione is also increases at different pH values (Qiao et al 2014). In erythrocytes of Oreochromis niloticus, ½ LC50 of Zn (5 mg/L) can conduct a significant increase in activity of both GSH and CAT after seven days of treatment that is decreased after 14 days in GSH, while CAT activity remain constant (Fırat and Kargın 2010).

As always, the increase in CAT activity would be strongly associated with the significant production of H2O2 at the wound site (Basha and Rani 2003), which is considered a stimulator of the wound healing process by stimulating wound granulation (Rai et al 2021). In an experiment on sublethal toxicity of zinc chloride on Clarias gariepinus, 1/10 LC50 of ZnCl2 (1.5 mg Zn/L) caused an initial increase of SOD and CAT activity and GSH levels within seven days of intoxication, but all measured parameters dropped after 14 days (Saliu and Bawa-Allah 2012).

The epidermis plays a critical role in protecting against external environmental hazards, so a rapid wound closure by an epidermal cover is crucial to avoid irreversible damage (Roubal and Bullock 1988). The intercellular spaces are reduced until the wound is completely closed, and then the epidermis thickens and differentiates (Pastar et al 2014). In the carp fish, the primary function of the epidermis was mainly attributed to the secretions of the mucous and club cells (Dash et al 2018). The epithelial cells in the common carp were protected by mucus gel covering secreted from the epidermal mucous (Wainwright and Lauder 2017). In addition, extracellular proteins, such as antimicrobial peptides, immunoglobulins, and enzymes, are active in the mucus gel, protecting the underlying epithelia (Dash et al 2018; Reverter et al 2018). In the current study, the regeneration of the mucous cells was enhanced by ZnSO4 application, especially in the Zn/5 group, compared to the control group, as some of the mucous cells were detected in the Zn/10 group on 3 days and completely regenerated in Zn/5 at the same time, but not observed in the non-treated group till the end of the trial period.

For a long time, it was suggested that the club cells are mainly restricted to the alarm function (Chia et al 2019). Still, recent studies exhibited that the club cells are part of the fish's innate immune system as the alarm substances' content has an immune function against parasites, pathogens, and external environmental hazards (Wisenden and Stacey 2005; Wisenden and Chivers 2006). In addition, some fish species' club cells contain keratin and chondroitin, which could help in damaged tissue repair (Iger and Abraham 1990; Ralphs and Benjamin 1992; Zaccone et al 1999). Applying ZnSO4 has also accelerated the club cells' regeneration rapidly compared to the control group, as some club cells were detected early after two days of ZnSO4 immersion bath therapy in the Zn/5 group. Conversely, in the control group, nearly most of the club cells still degenerated till the end of the trial period.

The stratum compactum in the dermal layer is formed of dense collagen fibers separated by rows of fibroblast cells (Le Guellec et al 2004). The arrangement of the collagen fibers in the stratum compactum allows muscular contraction, so it was early thought that the skin acts as an external tendon (Summers and Long Jr 2005). The collagen density in the stratum compactum enhanced the wound strength and the puncture resistance of the skin (Szewciw and Barthelat 2017). In the current study, the granulation tissue maturation and the collagen density % were markedly detected after ZnSO4 application compared to the control group.

Although the inflammatory response is necessary to clear the wound from the damaged tissue and enhance regeneration (Richardson et al 2013; Negm et al 2021), a robust inflammatory reaction might cause prolonged edema, delay the healing process, or impair the skin constituents (Kasuya and Tokura 2014). The current study detected distinct signs of inflammation in the dermis and the underlying muscles in the control group. Contrarily, applying ZnSO4 was accompanied by a rapid and significant decrease in the inflammatory cell’s infiltration.

Conclusions

In conclusion, this study showed that ZnSO4 immersion bath at 1/5 of the calculated LC50 effectively improved the healing of the incisional wound in mirror common carp via upregulation of the growth factor genes and improvement of the antioxidant status of fish. Thus, it has been suggested as a potential method for improving accidental wound healing and skin re-epithelization in the influenced fish species throughout the transportation and handling process.