Laboratory evaluation of different formulations of Stress Coat^® for slime production in goldfish (Carassius auratus) and koi (Cyprinus carpio)

Introduction

The first line of defense for fish against parasites and other invading organisms is the epithelial mucus or “slime” coat. Both external and internal epithelial surfaces of fish are covered with a layer of mucus that provides protection against environmental factors like microorganisms, toxins, pollutants, acidic pH and hydrolytic enzymes (Ræder et al., 2007; Gomez, Sunyer & Salinas, 2013; Guardiola et al., 2013). Slime is produced on a continuous basis that allows for trapping of pathogens that can be washed into the water as mucus. Secretory mucins, produced by goblet cells (Shephard, 1994), are the major constituents of the mucus layer in which several biochemical compounds have been identified, including lysozyme (Fletcher & White, 1973a), antimicrobial peptides (Cole, Weis & Diamond, 1997) and antibodies (Fletcher & White, 1973b). For aquarium hobbyists it is very important that fishes are healthy and stress free to maintain good slime production. It has been reported that stress causes chemical changes in mucus that decreases its effectiveness as a chemical barrier against invading organisms (Francis-Floyd, 2002; Vastos et al., 2010; Ma, Huang & Want, 2013). Stress upsets the normal electrolyte (sodium, potassium, and chloride) balance, resulting in excessive uptake of water by fresh water fish and dehydration in saltwater fish. Handling trauma physically removes mucus from the fish. This results in decreased chemical protection, decreased osmoregulatory function, decreased lubrication (causing the fish to use more energy to swim), and disruption of the physical barrier against invading organisms. Chemical stress (e.g., chemotherapeutant treatment) often damages mucus resulting in loss of osmoregulatory function, loss of lubrication, and damage to the physical barrier created by mucus (Francis-Floyd, 2002).

Several products are available in the ornamental fish industry claiming to maintain a healthy fish slime coat. Stress Coat^®, a patented product of Mars Fishcare Inc., instantly removes chlorine and chloramines, making municipal tap water safe for fish (Goldstein, Patel & Wiley, 1992). The manufacturer claims it can also neutralize heavy metals and replace the natural slime coating that fish need in times of stress due to handling, shipping or tank mate aggression (there is no published evidence for these claims). The product contains aloe vera, “nature’s liquid bandage,” that can help prevent loss of essential electrolytes and protect damaged tissue against pathogens. Aloe vera has been reported to contain pharmacologically active ingredients advocated for the healing of several diseases in humans (Ferreira et al., 2007). One of the active ingredients in Stress Coat^® is polyvinylpyrrolidone (PVP), a compound that has been widely tested and used in human and veterinary medicine as an effective wound healing accelerator and disinfectant when combined with iodine and other compounds (Mayer & Tsapogas, 1993; Burks, 1998; Osborne & Yang, 1999; Kim et al., 2015). Another common name for polyvinylpyrrolidone is povidone. The degree of effectiveness of povidone-iodine and wound healing is up for debate and results have been mixed (Mayer & Tsapogas, 1993; Burks, 1998). Stress Coat^®, according to the manufacturer, may also may help heal torn fins and skin wounds and its use is recommended when setting up aquariums, changing water, or adding fish to a system.

In this study, we evaluated the efficacy of different formulations of Stress Coat^® on slime production in goldfish (Carassius auratus) and koi (Cyprinus carpio). The specific objectives were to: (1) Quantify the amount of slime/mucus produced by goldfish and koi following administration of Stress Coat^®, Compound “X” (PVP formulation only), Compound “Y” (Stress Coat^® without PVP formulation) and saline (sodium chloride) at various time intervals. (2) Study histological changes in slime producing cells of koi exposed to the above compounds.

Materials and Methods

The study was carried out at the North Carolina State University College of Veterinary Medicine with the approval of the NC State Institutional Care and Use Committee Protocol #06-135-0. Effects of Stress Coat^® and its different formulations on slime production were investigated in goldfish and koi. In experiments utilizing goldfish, slime produced was calculated quantitatively by determining the weight of slime from a pre-determined area of skin. With koi, along with the weight of slime produced, 6 mm punch biopsies were obtained to evaluate histological changes, particularly, the number of slime producing cells. The experimental design is outlined below.

Results

Slime weight

Goldfish

Mean weights of slime (mg) from goldfish belonging to different treatment and control groups at various time intervals are presented in Table 1. Goldfish treated with salt had significantly lower mucus weights at 25 h. Goldfish treated with PVP has significantly higher mucus weights at 25 h.

Table 1:

Mean slime weight (mg) from goldfish treated with different compounds at various time intervals.

Values in bold are significantly lower (P ≤ 0.05) then the time 0 values in the same row.

Treatment	Time (hr)
	0	0.25	1	4	25
Salt	11.2	8.6	5.4	7.0	2.0
Stress Coat® w/o PVP	6.2	6.8	8.0	6.4	3.3
Stress Coat®	8.8	8.6	9.2	9.2	4.8
PVP only	7.0	8.0	8.4	7.2	13.8
Control	9.2	7.6	6.0	6.0	7.5

DOI: 10.7717/peerj.3759/table-1

Koi

Mean slime weights (mg) from koi belonging to different experimental groups at three time intervals are presented in Table 2. Koi treated with salt and PVP had significantly lower mucus weights at 1 and 25 h. Control koi had significantly higher mucus at 25 h.

Table 2:

Mean slime weight (mg) from koi treated with different compounds at three time intervals.

Values in bold are significantly different (P ≤ 0.05) then the other values in the same row.

Treatment	Time (hr)
	0	1	25
Salt	12.0	5.0	6.0
Stress Coat® w/o PVP	9.0	7.0	6.0
Stress Coat®	3.0	9.0	8.0
PVP only	10.0	9.0	3.0
Control	4.0	8.0	13.0

DOI: 10.7717/peerj.3759/table-2

Histological changes in koi

Koi from different experimental groups were biopsied at three time intervals. It was found that none of the treatment groups, except the control, showed a marked difference in the slime producing cells (Fig. 1). The control showed a higher number of slime producing cells at 1 and 25 h compared to 0 h. With regards to epidermal thickness (Fig. 2), The Stress Coat^® group showed the highest level of epidermal thickness.

Inflammation and secondary infection

The open biopsy sites in all koi showed clinical signs (hyperemia) of inflammation and secondary bacterial infection (Fig. 3).

Wound healing

Wound healing in biopsied koi was monitored for a period of 2 weeks. Evaluation of wound healing was subjective and based on lesion color, size, and, depth. At the end of 2 weeks, it was determined that the three koi treated with salt and PVP remained healthy and showed a higher degree of healing than other treatment koi and the control group (Fig. 3).

Discussion

Slime weight was significantly lower (P < 0.05) in goldfish that were treated with salt for 25 h. One possibility for this would be the general decrease in stress that salt provides to freshwater fishes and with that stress reduction might come a decreased mucus and inflammatory response. When slime weight for various experimental groups was compared at each time interval, fish treated with PVP for 25 hr showed significantly higher (P < 0.05) mucus weight than fish treated with salt and Stress Coat^® w/o PVP for 25 h (Table 1). The cause for this could be related to stress or inflammation initiated by the PVP (Shephard, 1994; Vastos et al., 2010). Although this finding may lead to speculation that PVP in Stress Coat^® might increase mucus production after 25 h, more detailed studies are needed to confirm the effect since no significant difference (P > 0.05) was observed between PVP-only treated fish and controls. Overall, the findings indicate that the compounds used in the study may not play a significant role in increasing mucus production. However, extensive studies using techniques that evaluate the slime thickness accurately would likely yield conclusive results.

Koi were used primarily to assess the histological changes in slime producing cells. Hence, only one koi was sampled from each experimental group with no replication at three time intervals, which resulted in a small sample size, not worthy of statistical analysis.

Various physiological factors might be associated with the number of slime producing or goblet cells (Shephard, 1994). These include decreased oxygen levels (Vastos et al., 2010), increased nitrate (Vastos et al., 2010), social hierarchy stress (Bonga, 1997), and toxic metals in the water (Bonga, 1997). When epidermal layers (where slime producing cells are located) of koi were compared, koi treated with Stress Coat^® showed higher thickness than other treatments (Fig. 2). However, the koi randomly assigned to this treatment group turned out to be relatively larger than those in the other treatment groups, which might have resulted in this observation.

The control group, with the least amount of observed healing, suggests the compounds tested have some positive effect on wound healing. In addition to relatively poor healing, koi from the control group also developed what appeared to be secondary bacterial infections (presumptive). The observation appears to indicate that PVP in Stress Coat^® contributes positively to wound healing, especially since the fish exposed to Stress Coat^® without PVP showed lesser healing than Stress Coat^® alone. Polyvinylpyrrolidone is known to speed healing in mammals (Osborne & Yang, 1999; Kim et al., 2015), and, the same appears to hold true for cyprinid fishes.

This work adds to our understanding of wound healing and mucus secretion in cyprinid fishes from an applied, clinical angle. Veterinarians and other health care professionals dealing with wounds in fish now have some evidenced based data to rely on when applying Stress Coat^® and/or salt to the water containing goldfish and koi.

Conclusions

The effect of Stress Coat^® on slime production in goldfish was studied. The study also investigated histological changes that might be associated with the slime producing cells and wound healing in koi. It was found that no Stress Coat^® treatments resulted in a significantly higher slime production during any of the time intervals in goldfish. However, slime production in PVP-only exposed goldfish was significantly higher than salt and Stress Coat^® without PVP after 25 h. This gives an indication that PVP present in Stress Coat^® is an important component that results in enhanced mucus production. This observation is further strengthened by the results of wound healing, where fish that were treated with compounds containing PVP showed better wound healing than those that did not receive PVP. Hence, it appears that PVP plays an important role in wound healing and mucus production. In koi, histology results showed no difference in slime producing cells in any of the treatment groups. Further studies with techniques that measure slime more accurately are recommended.

Supplemental Information