Jeremy Kientz, Nthn Huysmn, Michel E. Brnes,＊

aSouth Dakota Department of Game, Fish and Parks, 4130 Adventure Trail, Rapid City, SD, 57702, USA

bSouth Dakota Department of Game, Fish and Parks, McNenny State Fish Hatchery, 19619 Trout Loop, Spearfish, SD, 57783, USA

Keywords:

ABSTRACT

1.Introduction

Acoustic transmitters are commonly used in fisheries management studies to help understand survival, movement, and other dynamics of fisheries populations (Guy, Blankenship, & Nielsen, 1996). They are typically inserted in the fish body cavity through an incision, followed by one or more sutures for wound closure (Jepsen, Koed, Thorstad, &Baras, 2002). However, suturing increases handling time and is best performed by trained or experienced surgeons (Wagner & Cooke, 2005).

Cyanoacrylate adhesives are an alternative to sutures for wound closure in fish (Nemetz & MacMillan, 1988). These solvent-free, low--toxicity, fast-acting adhesives are esters of cyanoacrylic acid and are used widely in industry, human medicine, and personal households(Klenovich & Vetrova, 2007). However, cyanoacrylate use and its impacts on wound closure and healing in fish remains largely inconclusive,highlighting the need for additional research. In a study comparing cyanoacrylates to sutures in black crappies (

Pomoxis nigromaculatus

)Petering and Johnson (1991) reported that cyanoacrylate adhesives decreased the time required for surgery but increased the time needed for wound healing. However, Kaseloo, Weatherly, Lotimer, and Farina(1992) noted that rainbow trout (

Oncorhynchus mykiss

) wounds reopened less than 24 h after surgery using cyanoacrylate. Furthermore,Baras and Jeandrain (1998) reported that cyanoacrylate decreased tagging incision inflammation and increased the survival of European yellow eels

Anguilla

receiving adhesives instead of sutures for wound closure. A novel component of their research was the application of a cut fragment of eel fin over the cyanoacrylate as a biological bandage which significantly improved wound healing times (Baras & Jeandrain, 1998).Fin clipping is a standard fish marking technique (McFarlane, Wydoski,& Prince, 1990), and removal of a single fin does not affect growth or survival (Gjerde & Refstie, 1988; Mears & Hatch, 1976; Stauffer &Hansen 1969).Although cyanoacrylate adhesives likely reduce surgery time, their effect on tag retention is uncertain. Higher tag loss with the use of cyanoacrylate adhesives in comparison to sutures has been reported in black crappies (Petering & Johnson, 1991) and brown trout (

Salmo trutta

) (Jepsen, Larsen, & Aarestrup, 2017). In a review of tagging techniques, Wargo Rub, Jepsen, Liedtke, Moser, and Scott Weber (2014)recommended against the use of adhesives for wound closure in fish due to concerns related to wound healing and tag retention. In contrast,Raoult, Brown, and Wiliamson (2012) had 88% passive integrated transponder tag retention for juvenile mulloway (

Argyosomus japonicus

)whose incisions were closed with cyanoacrylate adhesive.

Transmitter retention and fish health are the primary objectives of the tagging procedure (Jepsen et al., 2002). Because sutures delay healing and adhesives have been associated with high tag loss, other wound closure techniques need to be more fully evaluated. In salmonids,aside from the investigation of cyanoacrylate use with rainbow trout by Kaseloo et al. (1992) and brown trout by Jepsen et al. (2017), no other wound closure adhesive work has been done. Furthermore, there is also a paucity of literature on the use of fin tissue as a biological bandage.

Due to inconsistencies in the literature regarding the use of cyanoacrylate for wound closure, wound healing, and associated tag retention,and the lack of information on the benefits and use of fin tissues as biological bandages, further experimentation is needed. Thus, the objectives of this study were to evaluate the effects of cyanoacrylate and suturing wound closure techniques on wound healing, handling time,tag retention, and growth of rainbow trout tagged with acoustic transmitters. Rainbow trout were used because of previous cayanoacrylate research (Kaseloo et al. (1992), and because they are considered a model fish species for experimentation (Thorgaard, Bailey, Williams, Buhler,Kaattari, Ristow, Hansen, Winton, Bartholomew, Nagler, & Walsh,2002).

2.Material and methods

2.1.Experimental design

Experimentation occurred at McNenny State Fish Hatchery, rural Spearfish, South Dakota, USA. Well water (11̊ C; total hardness as CaCO, 360 mg/L; alkalinity as CaCO210 mg/L; pH, 7.6; total dissolved solids, 390 mg/L) was used throughout the study. All fish used in this study were hatched from eyed-eggs and reared as a part of normal hatchery production. Sixty-three Erwin strain rainbow trout [mean (SE)initial length and weight =364 (4) mm and 646 (81) g respectively]were assigned to one of seven treatments, with nine fish in each treatment. The seven treatments consisted of a control group where fish were only handled, two suture groups (tagged, non-tagged), and four adhesive groups (non-tagged, tagged with adipose fin applied as a biological bandage, tagged with anal fin applied, and non-tagged with a waterproof bandage applied). All 63 fish were divided into subsets of 21 fish (three fish per treatment), with each subset placed into one of three hatchery raceways (4.7 m long x 2.4 m wide x 0.5 m deep).

2.2.Surgery

Fish surgeries were performed by a single trained and experienced surgeon (Deters et al., 2010; Jepsen et al., 2002; Mulcahy, 2003). Prior to surgery, fish were anesthetized using tricaine methanosulfate(MS-222; Argent Chemical Laboratories, Ferndale, Washington, USA), at 0.092 g/L to stage 4 anesthesia and maintained using the same concentration throughout the procedure (Hikasa, Takase, Ogasawara, &Ogasawara, 1986). Procedures were completed on all fish within a treatment prior to the next treatment. All surgical instruments and inert acoustic tags were disinfected using iodine prior to surgery. After anesthesia, fish were measured to the nearest mm (total length),weighed to the nearest g, and tagged with a unique alphanumeric visible implant alpha (VIA) tag (1.2 mm ×2.7 mm; Northwest Marine Technology, Inc., Seattle, Washington, USA) inserted into the postorbital tissue following methods described by Davis, Barnes, Kientz, and Galinat (2014). Control fish only experienced anesthesia and handling.Trout in the experimental treatments were placed ventral side up in a slotted foam pad and a 10 mm incision was made 3 mm from the midventral line, anterior to either of the pelvic fins. In tagged groups, an inert acoustic transmitter (9 ×24 mm, 3.6 g weight in air; VEMCO,Bedford, Nova Scotia, Canada) was inserted into the peritoneal cavity.Initial tag burdens on the fish (0.95–1.02%) were well below the recommended 2% tag-to-body ratio (Jepsen, Schreck, Clements, & Thorstad, 2005). Incisions were then closed using two simple uninterrupted sutures (Oasis Nylon Monofilament sutures 4–0, Glendora, California,USA) or one of the three adhesive methods. In all adhesive groups a thin layer of Vetbond adhesive (3M Animal Care, St. Paul, Minnesota, USA)was applied to the incision. In the adhesive treatments, adhesive was applied to the incision, and, when using a covering, the covering (adipose fin, anal fin, waterproof bandage) was put on top of the adhesive,and then another thin layer of adhesive was applied on top of the covering. Fins used for coverings were applied to the same fish from which they were removed. Fins were removed at the base of the fin using disinfected surgical scissors. Handling time was defined from first exposure to anesthesia until surgery completion and fish placement back into fresh water. Fish were fed floating feed (4.5 mm floating Classic Trout, Skretting North America, Tooele, Utah, USA) once per day at a level beyond satiation starting on the first day post-surgery.

2.3.Healing metrics

At 14-day (two-week) intervals for the 98-day (14-week) duration of the study, each fish was inspected for tag loss, measured (total length to the nearest mm) and weighed to the nearest g. Their wound area was also photographed. Each photo was viewed and scored by four individuals using an adaptation of the Paukert, Chavala, Heikes, and Brown (2001) method, with scores recorded for wound gape (no gape =0; gape

<

1.0 mm =1; gape

>

1.0 mm =2) and wound redness (none =0;localized to incision and sutures =1; extended beyond incision and sutures =2) (Table 1). An average of the four scores for each photograph was then used for subsequent analysis. The 14-week study duration was dictated by the space available at the hatchery for experimentation. The following formulas were used:

Tag retention (%) =100 x (initial number of fish tagged/final number retaining tags)

Gain =final weight - initial weight

Specific Growth Rate (SGR) =100 x [(Ln end weight – Ln start weight)/number of days]

2.4.Data analysis

Data were analyzed using the SPSS (24.0) statistical analysis program (IBM, Chicago, Illinois, USA). Two-way analysis of variance(ANOVA) was initially used to test for a raceway effect among treatment groups, but because no significant raceway effects were observed, all fish were combined for further analysis using one-way ANOVA. Wound redness severity and wound gape severity data were initially analyzedusing repeated measures ANOVA, because these variables were sampled in successive events using the same fish. However, a significant interaction between the treatments and week sampled was observed,necessitating subsequent one-way ANOVA and Tukey’s post-hoc means testing. ANOVA was used with SGR, gain, and tag retention, and if significant differences were detected, Tukey’s post-hoc means comparison procedures were conducted. Significance was pre-determined at

p <

0.05.

Table 1 Scoring criteria for wound gape severity and wound redness severity used to determine surgical wound healing for rainbow trout (N =63) following dummy acoustic transmitter insertion surgery and utilizing three different wound closure methods.

3.Results

3.1.Survival

No mortalities occurred during this study. Thus, no significant differences in survival were observed among the treatments.

3.2.Surgery time

Both suture treatments (with or without tags) had significantly longer handling times (F=36.863;

p <

0.001) than any of the adhesive treatments or control (Table 2). Using sutures for wound closure took over 6 min, which was 50% longer than any of the adhesive treatments.

3.3.Tag retention

In the treatments where dummy tags were inserted, retention was similar in the suture and adhesive anal fin treatments at 77.8% and 88.9% respectively. However, retention in the adhesive adipose fin treatment was significantly lower (F=11.467;

p <

0.001) at only 11.1%; eight of the nine tagged fish in this treatment lost their tags. Tag loss for all treatments occurred by expulsion through the side of the abdominal wall, and abdominal bulging was first observed at 42 days post-surgery (Fig.1).

3.4.SGR and gain

Initial lengths, initial weights, final lengths, final weights, and SGR were not significantly different among treatments (Table 3). However,compared to the control, weight gain was significantly less (F=2.958;

p

=0.014) in the adhesive adipose fin and adhesive bandage treatments.

3.5.Wound redness

At the end of the study, wound redness severity was significantly higher (F=2.958;

p

=0.029) in the tagged suture treatment compared to the control (Table 4). There were no significant differences among any of the other treatments.

Table 2 Mean (±SE) individual fish growth handling time (s) and tag retention for sixtythree rainbow trout subjected to one of seven post-surgery wound closure treatments (N =9 fish per treatment) with and without insertion of a dummy acoustic transmitter. Means in a column followed by different letters are significantly different based on Tukey’s post-hoc multiple comparisons (p <0.05).

3.6.Wound gape

Wound gape severity was significantly higher (F=5.526;

p <

0.001) in the adhesive adipose fin treatment than in the control, adhesive only (no fin or bandage), suture, and tagged suture treatments(Fig.2). However, there was no significant difference in wound gape severity among the adhesive adipose fin, adhesive anal fin, and adhesive bandage treatments. Wound gape never completely closed in the adhesive adipose group. In comparison, wounds were completely closed between four- and six-weeks post-surgery in both the tagged suture and tagged adhesive anal fin treatments.

4.Discussion

With any tagging method, fish survival and tag retention are primary concerns (Panther, Brown, Gaulke, Deters, Woodley, & Eppard, 2011).While all the fish in this study survived the entire trial, tag retention among treatments varied. The combination of cyanoacrylate adhesive and anal fin provided similar tag retention as sutures while adhesive and adipose fin resulted in poor tag retention. This difference in retention between the two fin bandages could be due to the structure of the fins. In particular, the adipose fin is fleshy and non-rayed ( Buckland-Nicks,Gillis, & Reimchen, 2011) in comparison to the more rigid structure of the rayed anal fin (Lauder, 2015). The bony structure of the anal fin may allow it to better adhere to the cyanoacrylate adhesive, thereby preventing tag loss. Similarly, the lack of structure in the waterproof bandage may also explain its relatively poor performance. Removing a bony fin for incorporation into a wound covering should not be problematic for the fish, given that clipping a single fin has not been shown to affect fish survival or growth (Gjerde & Refstie, 1988; Mears & Hatch,1976; Stauffer & Hansen 1969).

The extremely poor tag retention observed in the adhesive-only treatment in this study supports the recommendation by Wargo Rub et al. (2014) against the use of adhesives because of issues with wound healing and subsequent tag retention. Similarly, Kaseloo et al. (1992)noted that rainbow trout wounds closed with cyanoacrylate adhesives reopened less than 24 h post-surgery. Increased wound healing times and high tag loss with cyanoacrylates was also reported in brown trout(Jepsen et al., 2017) and black crappie (Petering & Johnson, 1991).

Tag expulsion in other studies has been noted to occur through the abdominal cavity, incision site, or through the intestinal wall (Chisholm& Hubert, 1985; Jepsen et al., 2002; Knights & Lasse, 1996; Welch,Batten, & Ward, 2007). Signs of tag expulsion were first observed around week six of the study when the fish began to exhibit abdominal bulging. Tag expulsions occurred only through the abdominal wall or adjacent to the surgical site, similar to that reported by Urbaniak,Barnes, and Davis (2016). No tags were lost due to suture failures(Meyers, Thuemler, & Kornely, 1992). The adhesive with adipose fin fish exhibited reduced wound closure at the 6-week mark compared to other treatments, suggesting that even though the expulsion did not occur through the wound, the poor healing of the wound may have played a role in the fish actively expelling the transmitter.

Tag burden to the fish is an important consideration when conducting transmitter surgeries. Although numerous studies have sought to extend the recommended maximum 2% tag-to-body ratio (Jepsen et al., 2005) with varying degrees of success (Brown, Cooke, Anderson,& McKinley, 1999; Brown, Geist, Deters, & Grassell, 2006; Chittenden et al., 2009; Klinard, Halfyard, Fisk, Stewart, & Johnson, 2018; Rechisky& Welch, 2009; Smircich & Kelly, 2014), the starting ratios of approximately 0.95–1.02% used in this study were well below this threshold. In addition, the colder water temperature required for salmonids used in this study should not have affected the tag retention results, although much warmer water has been shown to increase infection and mortality risks (Bunnell & Isely, 1999; Knights & Lasse, 1996).

Fig.1.Photographs of rainbow trout representing various stages of healing and illustrating tag loss following surgical procedures where fish received different postsurgical closure methods with and without insertion of a dummy acoustic transmitter.

Table 3 Mean (±SE) individual fish lengths (mm), weights (g), gain (g), and specific growth rate (SGR)＊ for sixty-three rainbow trout subjected to one of seven postsurgery wound closure treatments (N =9 fish per treatment). Means in a column followed by different letters are significantly different based on Tukey’s post-hoc multiple comparisons (p <0.05).

The increased wound redness observed in the tagged and sutured fish may have been due to the types of sutures used. Different suturing materials can cause inflammatory responses in different species of fish(Mulcahy, 2003). However, the monofilament in this study is the most commonly selected suturing material due to its reduction in causing inflammatory responses compared to other suture types (Wagner &Cooke, 2005). Although the fish epidermis can heal quickly (Fontenot &Neiffer, 2004; Noga, 2000), the lack of wound closure in the cyanoacrylate adhesive with an adipose fin treatment fish is very undesirable.This poor healing, combined with the poor tag retention observed using this treatment, emphasizes the unsuitability of this surgical wound-closure technique.

The significantly longer handling times associated with suturing in this study are not unusual. Due to the complexity of the procedure, suturing techniques typically result in increased handling time when compared to cyanoacrylate and stapling techniques (Baras & Jeandrain,1998; Murray, 2002; Petering & Johnson, 1991; Swanberg, Schmetterling, & McEvoy, 1999). Even though the adhesive with adipose fin had similar surgery times compared to the adhesive with anal fin, the fish had to be flipped in the v-trough during surgery to access the fin and the surgical location. This could result in a greater potential for surgicalerror and increased handling.

Table 4 Bi-weekly mean (±SE) wound redness scores from sixty-three rainbow trout subjected to one of seven post-surgery wound closure treatments (N =9 fish per treatment) with and without insertion of a dummy acoustic transmitter as evaluated from four separate reviewers. Means in a column followed by different letters are significantly different from each other based on Tukey’s post hoc multiple comparisons (N =9; p <0.05)..

The use of sutures requires experienced surgeons to minimize damage to the fish (Jepsen et al., 2002; Mulcahy, 2003) and positively influence tag retention (Deters et al., 2010). Cyanoacrylate adhesives eliminates the need for suture training. The positive results of this study on tag retention, wound healing, and handling time using an adhesive with a bony fin cover provides a viable alternative to traditional suturing methods during acoustic tag surgeries on rainbow trout.

Fig.2.Bi-weekly mean wound gape severity scores from rainbow trout (N =63) subjected to one of seven post-surgery wound closure treatments with and without dummy acoustic transmitter insertion into the peritoneal cavity.

Although samples sizes were relatively small at only nine fish per treatment in this study, the results were very consistent and uniform and provide a solid basis for future experiments. These results may be species-specific to rainbow trout. Different surgical techniques may be required for different fish species (Jepsen et al., 2002). However, as a model organism (Thorgaard, Bailey, Williams, Buhler, Kaattari, Ristow,Hansen, Winton, Bartholomew, Nagler, & Walsh, 2002), it is likely that the results should be applicable at least to other salmonid fishes, at least at fish sizes comparable to those used in this study. It is unknown how these novel surgical techniques would perform in smaller or larger fish;however, future studies could apply this methodology for tagging juvenile trout as well as compare the effects of unskilled versus experienced surgeons.

5.Conclusions

The results from this study indicate that cyanoacrylate adhesive used in conjunction with anal fin tissue may be an acceptable post-tagging surgical treatment for rainbow trout and is worthy of future study.Wound healing and tag retention were comparable or better than using traditional sutures, and the tagging surgeries could be completed much more quickly using an adhesive in conjunction with an anal fin for wound closure. Because this adhesive and fin method requires only minimal training, it could be widely and easily used, particularly if it produces acceptable results with all sizes of trout. In brief, the use of an adhesive in conjunction with an anal fin for wound closure provides an opportunity to improve fish tagging results and efficiencies.

CRediT authorship contribution statement

Jeremy Kientz:

Conceptualization, Methodology, Investigation,Writing - original draft, Visualization.

Nathan Huysman:

Investigation,Writing - original draft, Writing - review & editing.

Michael E. Barnes:

Conceptualization, Methodology, Formal analysis, Writing - review &editing, Visualization, Supervision, Project administration.

Acknowledgements

We thank Patrick Nero, Eric Krebs, and Kelsen Young for their assistance with data collection throughout the study and to the anonymous reviewers for their comments and suggestions to improve the manuscript.