Evaluation of the Safety and Gross Pathology of a Sucrose Based Excipient Intended to Deliver Time-Released Spawning Peptides in Warm water Marine Fish
Elizabeth H. Silvy* and Todd D. Sink
Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, Texas
*Corresponding author: Elizabeth H. Silvy,Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, Texas, USA, E-mail: firstname.lastname@example.org
Citation: Silvy E H, and T.D. Sink,(2020). Evaluation of the Safety and Gross Pathology of a Sucrose Based Excipient Intended to Deliver Time-Released Spawning Peptides in Warm water Marine Fish. J Aquat Res Mar Sci 2020: 218-225.
Received Date: 06Jun, 2020; Accepted Date: 21 Jun, 2020; Published Date: 25 Sept, 2020
Currently, the only commercially available slow-release spawning peptides to induce gonad maturation and ovulation in warm water marine finfish are configured as cellulose-based implants. These implants have led to side effects during and post injection. We investigated a novel proprietary time-released spawning peptide to hormonally control spawning in red drum (Sciaenopsocellatus). We assessed the safety and adverse effects of these implants, specifically if the placebo caused a reaction and/or pathology at the site of injection. Red drum (n = 20) were administered 0.1 mL of the excipient by injection into the right dorsal musculature between the first and second dorsal fins and a second 0.1 mL injection of the excipient was administered to the intra-coelomic cavity. Fish were then euthanized 8 days post injection and injection sites were observed for adverse reactions resulting from injections. Survival of all fish was 100% post injection and only 25% of the fish injected showed effects of injections.
Induced spawning, Ovaprim, red drum, Sciaenopsocellatus, spawning implant side effects,
Controlled reproduction in captivity is essential for the continued expansion of aquaculture. As natural fish populations decline, there is an increased effort to develop novel technologies to induced fish spawning [1-3]. In many fish species, induced spawning can be achieved by manipulating water temperature, photoperiod, spawning cycle, or other environmental conditions [4-7]. However, many fish species having economic significance in aquaculture do not reproduce spontaneously in captivity or reproduce under natural conditions that are not easily recreated in a hatchery setting [8, 1, 9]. Hormonal manipulations are the only reliable way to produce fertilized eggs or to promote efficiency in egg or milt production [1, 10]. In addition to breeding desirable fish species, induced spawning can be used to produce hybrids and sterile polyploid fish, produce monosex populations, synchronize reproduction of large numbers of fish for simultaneousspawning, produce larvae outside of the normal spawning season, and maximize survival of larvae in controlled hatchery conditions [1,11,12]).
Hormonal manipulations in fish reproduction have been in use for the past 80 years [13,1,14]. One hormonal manipulation is the injection of crude extracts of the pituitary gland (PE) of mature fish [15,16]. Another is the injection of exogenous luteninizing hormone (LH) that directly affects the gonads . In more recent years, synthetic agonists of gonotropin-releasing hormones (GnRHa) that act at the level of the pituitary to release LH have predominantly been used to induce spawning [6,17], as well as human chorionic gonadotropin (HCG; [18,2]. Purified gonadotropins that stimulate ovaries and testes also are used . Most of the aforementioned hormone injections can be used along with dopamine blockers which enhance potency of LHRHa[1,20,21].
Currently, the only commercially available slow-release spawning peptides to induce gonad maturation and ovulation in warm water marine finfish are configured as cellulose-based implants (e.g., [22-29]). These implants are several millimeters in diameter (up to 3.5 mm), and depending upon dosage and composition, have lengths up to five times their diameter. They are implanted using Ralgro® (Merck & Co. Inc. 2 Giralda Farms, Madison, New Jersey) pellet injectors or Ralgun® implanters (Syndel International Inc., Qualicum Beach, British Columbia, Canada) originally developed for injecting large Ralgro® implants into cattle and other livestock. Implanters create large diameter holes (needle outside diameter of 4.76 mm) in the dermis compared to needles used with aqueous hormones (23-gauge needle outside diameter of 0.64 mm), thus exposing fish to infection by the numerous pathogenic bacteria and fungi present in aquatic environments and eventually causing mortality [30-32].
Despite the large size of implants, relatively small dosages are available in commercial implants. For example, the maximum dosage available for Ovaplant® implants (Syndel International Inc., Qualicum Beach, BC, Canada) is 250 μg of salmon gonadotropin releasing hormone analogue (sGnRHa), and the effective dosage in fish is 50 μgsGnRHa/kg body weight. This means that large broodfish frequently require several implants be administered per fish, creating multiple large holes in the dermis to deliver the appropriate dose of peptide.
In previous studies involving reproductive implants in cobia (Rachycentroncanadum), dermal and musculature lesions and secondary infections have resulted after injection with Ovaplant® implants using the Ralgun® implanter at the Texas A&M University Aquacultural Research and Teaching Facility. Small lesions were observed 2–4 days post-implantation which increased to larger, deep ulcerations 5–10 days post-implantation and the infected broodstock were euthanized 25 days post- implantation after trial criteria for euthanasia was reached and recovery was deemed unlikely to occur by research personnel. Negative side effects of the injection of a similar hormone, Ovaprim®, have been observed [32,33]; these adverse effects included redness and bruising at the injection site and stemmed from degradation of water quality while holding and handling fish and in some cases led to mortality.
Another issue identified from previous studies is a complication related to the fixed dose of each implant [32,34,35,33]. There is no way to ensure fish receive the correct biologically active dosage based on body weight because each implant contains a static dose with large intervals among available implant doses . Small fish can receive an overdose when using even the smallest commercially available implant dosage . Large marine fish can be implanted with multiple implants to get close to the desired biological concentration, but it is difficult to achieve the correct body weight dosage without using multiple implants and implant doses. Hormone implants are extremely costly compared to hormone injections, often costing more than USD$10 per implant, and can only be purchased commercially in implant cartridges containing 24 implants . To purchase and keep on hand multiple implant cartridges, each containingimplants of different doses to achieve dosages close to desired biological concentrations, adds a great deal of expense when considering they only have a one-year shelf-life and only 5–10 broodfish may be implanted in any given year to supply an entire farm. Even then, the applicator can only achieve a dosage close to the desired biological concentration. Due to this, broodfish often receive too much or too little of the hormone when injected with the implant . These observations led other researchers and myself to conclude that a less invasive procedure that can deliver exact biological dosages of peptides based upon fish weight is needed to induce spawning in large marine fish.
Other animal production industries, such as beef and swine production, use liquid- or gel-based excipients to deliver and slowly release peptides over an extended period of time. Examples of these are slow-release bovine somatotropin (bST, Prosilc, Elanco, Greenfield, Indiana) to induceand promote lactation in cows  and LONGRANGE® (eprinomectin 5% w/v extended-release injection, Boehringer Ingelheim Animal Health USA Inc., Duluth, Georgia) to prevent bovine warble fly infections . These excipients could potentially be adapted for use in fish, allowing for customizable peptide doses to be drawn into a syringe and delivered to the fish intramuscularly through needles as small as 23 gauge. These excipients would still allow the same time-release benefit as implants do, in a customized dose for each fish that is less intrusive. In large marine broodstock, this also would result in fewer puncture wounds, one for injection versus multiple for implants, created in the dermis and musculature of the fish. The resulting puncture wound also would be significantly smaller than the 4.76-mm puncture wound created by the Ralgun® implanter used to administer Ovaplant® implants. This would reduce the exposure of the fish to exogenous bacterial and fungal pathogens in the aquatic environment by limiting wound sites and size where pathogens could enter.
One such excipient used in beef and swine production that has potential to be used in fish is a hydrophobic, sucrose-based liquid with a propriety blend of emulsifiers that slowly releases peptides over an extended period. (This excipient is under a non-disclosure agreement). The majority (~60%) of the peptide is released from the excipient within 24 h of injection while the remaining 40% of the peptide is slowly released from 24 to 72 hours post-injection (Dr. Peter McKenzie, Senior VP Product Management, and Dr. Katie Haman, DVM, Director for Spawn Products, Syndel, Ferndale, Washington). This is somewhat analogous to a priming dose and later resolving dose of hormones frequently employed with fish when using aqueous-based peptide injections, but eliminates the need to repeatedly capture, handle, inject, and otherwise stress the fish. Additionally, it delivers a steady flow of peptide for 72 hours post-injection unlike a static resolving dose that delivers the peptide to the fish all at once. This is a novel excipient and it has never been used on any species of fish prior to this clinical trial to make observations of gross pathology of the excipient.
The first step in evaluating the safety and efficacy of an excipient is to determine the safety and conversely any adverse effects when administered to the test animal. In order to establish the effects on the test animal, a “placebo” injection, or simply an injection containing only the excipient and no peptide, must be evaluated in the test animal to determine the effects of only the excipient on the animal. Red drum (Sciaenopsocellatus) have been spawned in captivity since the 1970’s  using either spawning peptides or environmental conditioning, and they are the most readily available species of warm-water marine fish cultured in the United States. Therefore, they are an ideal test subject to evaluate the effects of a sucrose-based excipient in warm-water marine fish.
The primary objective of our clinical trial was to assess the safety and conversely any adverse effects of a sucrose-based excipient when injected into the dorsal musculature and or intra-coelomic cavity (IC) of red drum, specifically investigating if the placebo causes a reaction and/or pathology at the site of injection. Our research hypothesis was that red drum will not show signs of gross pathology and reaction upon injection with placebo replacement of hormones.
Materials and Methods
Red drum (n = 20) were kept in a 4,429-L recirculating tank, at a photoperiod of 14 h of light and 10 h of dark at a water temperature of 24–26°C and a salinity of >10 ppt. The system included a 2m x 0.5m-sump tank filled with two sacks of bioballs/filtration media for water collection, a 2-horsepower pump (Hayward MaxFlo XL 2 HP Dual Speed Pool Pump, One Hayward Industrial Drive Clemmons, North Carolina), a Arias 4000© (Pentair Aquatic Eco-Systems Inc. 2395 Apopka Blvd. Apopka, Florida) bead filter, a 25-micron Water Co.© filter (Water Co.®, Augusta, Georgia), and a Jebao© PU-36 UV clarifier (Jebao®, Dongsheng, Zhongsha, Guangdong,China). One half of the water that exited the UV filter was returned to the sump tank. The other half of the water that exited the UV filter returned to the tank at an angle encouraging uni-directional waterflow in a circular pattern. The tanks were set up with a venturi drain style standpipe, covered in large mesh that let waste water flow into a drainage pipe that deposited waste water into the sump tank for filtration. Aeration was applied using four air stones from an externalblower (Whitewater® Regenerative Blower Pentair Aquatic Eco-Systems Inc. 2395 Apopka Blvd. Apopka, Florida).
Twenty-five milliliters of a proprietary sucrose-based excipient placebo in a sealed syringe vial was supplied by Syndel International Inc. The excipient was stored under refrigeration (2–8°C) until time of the clinical trial to ensure effectiveness and limit potential expiration. Approximately 45 minutes prior to use, the excipient was removed from refrigeration and allowed to warm to room temperature (23°C) to ensure viscosity and ease of drawing the excipient into the syringe.Following removal of the placebo from refrigeration, if liquid was still too viscous to draw into a 21-gauge needle (BD PrecisionGlide™ Needle, 21G x 2 [0.8 mm x 50 mm], Becton, Dickinson and Company, 1 Becton Drive Franklin Lakes, New Jersey), it was warmed by hand for 5 minutes to increase viscosity.
Red drum were removed from the holding tank, anaesthetized using 70 mg/L sodium bicarbonate buffered tricaine methanesulfonate (Western Chemical Incorporated, Ferndale, Washington) and a mixture of Vidalife™ (Syndel International Inc., Qualicum Beach, British Columbia, Canada) and sea water in a holding bath for 2 minutes with oxygen provided from a 6.23-cubic meter compressed-oxygen cylinder at a rate of 2-L/minute using a fine pore, porcelain air stone (69.9 cm x 8.5 cm, Point Four™ Micro Bubble Diffusers, Pentair Aquatic Eco-Systems, Inc., Apopka, Florida) and a regulator (Roscoe Medical, RMI-15H CGA-540 H Regulator, 0-9 LPM, Compass Health Brands, 6753 Engle Road Middleburg Heights, Ohio).
Fish were administered 0.1 mL of the excipient by injection into the right dorsal musculature between the first and second dorsal fins approximately 2.5 cm below where the dorsal fin meets the body musculature. A second 0.1 mL injection of the excipient was administered to the intra-coelomic cavity (IC), approximately 5 cm anterior and to the left side of the urogenital pore. Excipient injections were administered using sterile 3-mL syringes (Becton, Dickinson and Company, Luerlok disposable syringes,1 Becton Drive Franklin Lakes, New Jersey) and 21-gage needles (BD PrecisionGlide™ needle, 21G x 2 [0.8 mm x 50 mm]). A new syringe and needle were used for each fish and injection site to minimize infection risks. After injection fish were released back into the holding tank.
Fish were monitored for mortality and fed their normal diet of Rangen EXTR 400™ (Rangen Inc., Angleton, Texas) 0.32 cm pelleted feed for 8 days post-injection. Eight days (3 days of hormone delivery followed by a 5-day latency period) was determined to be the end point of the clinical trial because had the excipient contained an actual spawning peptide, all viable broodfish receiving the excipient would have spawned within this period. If mortality occurred, the specimen was immediately refrigerated (2–8°C) until a post mortem exam/necropsy could be performed (<12 hours).
At the conclusion of the 8-day clinical trial, all specimens were euthanized by removing them with a net from the holding tank and placing them in a 1,000 L-tank filled with a mixture of seawater and an overdose of isoeugenol (Sigma-Aldrich® PO Box 14508 St. Louis, Missouri) 150 mg/L. Following euthanasia, post-mortem exams/necropsies were conducted. During post-mortem exams, injection sites were first visually observed externally and any abnormalities, lesions, etc. were noted. Next, the scales and dermis covering the injection sites were removed with a scalpel (Disposable safety scalpel 23, Integra LifeSciences, 311 Enterprise Drive, Painsboro, New Jersey) and the fish was inspected between the dermis and outer musculature for signs of pathology including needle punctures, discoloration, hemorrhaging, inflammation, lesions, pustules, nodules, infection, or scar tissue. When any signs of pathology were found, they were recorded and examined microscopically (T720 Amscope, 40X-1,000X Plan Infinity Kohler Laboratory Trinocular Compound Microscope, Irvine, California). The musculature was then sliced layer by layer using a scalpel to investigate for evidence of pathology, as listed above, associated with the injection site or excipient.
After inspection and dissection of the dorsal musculature, the IC injection was then inspected by using a scalpel to open a 6–8 cm incision beginning at the urogenital pore towards the anterior of the fish. The incision was spread open using tweezers and inspected for signs of the excipient or pathology including needle punctures, discoloration, hemorrhaging, inflammation, lesions,pustules, nodules, infection, or scar tissue. The organs around the injection site also were evaluated for reaction to the injection. When any signs of pathology were found, they were recorded and examined microscopically. If gross pathology was observed in a specimen, small pieces of tissue were collected and stored in formalin prior to histopathology. All results of post-mortem exam/necropsies were recorded for later analysis.
Survival of red drum after treatment was 100% ± 0 (SE) and no signs of primary or secondary bacterial infections were noted in or around the injection sites, and all fish appeared healthy. Only 5 (25%) of the 20 fish injected showed effects of injections. Two (10%) of the fish were observed with a small incision in muscle tissue caused by a needle during injection. One (5%) fish had a small nodule of scar tissue in proximity of an injection site and 2 (10%) fish had subdermal inflammations observed at the dorsal injection site.
No lesions or ulcerations were found around the injection sites. Minimal effects of the injections were noted such as scar tissue, puncture wounds, hemorrhaging, discolored livers and globules of the excipient located under the dermis (Figure 1).
Figure 1: In 75% of the fish, the IC injection was completely unremarkable and no adverse effects at the injection site were noted.
In this clinical trial, normal behavior of the fish resumed in approximately one hour and the fish ate vigorously only 3 hours post-injection. For 33% of the IC injections, a small discolored dot on the outer dermis was noted, where the injection was administered (Figure 2).
Figure 2: As observed in this fish, a small discolored dot on the outer dermis where the IC injection was administered was noted on 33% of the study fish.
Observations of the dorsal IM injection site found one fish (5%) had a small incision in interior muscle tissue (Figure 3) probably caused by dorsal injection site (Figure 4).
Figure 3: A small incision in the interior muscle tissue as noted in this fish was caused by the needle during injection.
Figure 4: Note the subdermal inflammation observed at a dorsal injection site of this fish.
There was globular, tacky, gelatinous substance (Figure 5) removed from subdermal pocket (Figure 6).
Figure 5: Globular, tacky, gelatinous substance believed to be part of the injected hormone excipient that was forced out of the muscle tissue at the injection sight of this fish.
Figure 6: A subdermal pocket located at dorsal IM injection site of this fish wherein a tacky, gelatinous substance was observed in this fish.
It was believed to be part of the injected hormone excipient that was forced out of the muscle tissue at the injection sight. It was noted during injection that some of the initial injections were very shallow in the dorsal muscle tissue. It was observed the hydrophobic, sucrose-based liquid excipient with a proprietary blend of emulsifiers, became a sticky, gelatinous globule once injected into the fish. On a fourth fish, an incision was observed in muscle tissue made from needle during injection; however, there was no bleeding, bruising or scar tissue noted. In a fifth fish, a small nodule of scar tissue was observed in muscle tissue in proximity of the injection site. The scar tissue was not immediately discernable as a result of the excipient or injection because of the healed status of the scar tissue in relation to the time of injection to the time of dissection. The cause was not likely due to the injection or excipient. No abnormalities were observed for the other 15 fish injected.
When compared to traditional spawning aids, this sucrose-based excipient was less invasive, easier to tailor dosage, and caused minimal harm and discomfort to the fish. It is important to note that this is the first exploratory clinical trial of this excipient and there is no previous research on the effects of this excipient.
Other spawning aids such as Ovaplant® and Ovaprim® have led to secondary bacterial infections, lesions, and mortality [34,32,]. We also found negative effects of Ovaprim® injection included redness and bruising at the injection site . The time release action of this injection mitigates the prolonged or multiple handlings of this fish which reduces stress and potential mortalities.
Due to the novel nature of this excipient, we reviewed the actual use, protocols, and observations based on the clinical trial. The excipient used in this research was extremely viscous. We allowed the excipient to sit at room temperature (26.7o C) for 25 minutes and it was still difficult to draw into a syringe. We allowed the excipient to remain at room temperature for another 15 minutes before drawing it into the syringe; however, it still required approximately 1 minute to draw a 0.1-mL dose of excipient into the syringe using 23-gauge needles. This was not practical with 40 syringes to fill and would not be acceptable in a commercial setting where hundreds of fish may need to be injected in a source period of time. Therefore, we switched to 21-gauge needles, but the excipient was still somewhat difficult to draw into the syringe. Using 21-gauge needles was more practical and required approximately 15–20 seconds to draw the 0.1-mL dose of excipient into the syringe.
Despite the difficulties of drawing the excipient into the syringe, the excipient was not difficult to inject into the fish. However, the viscosity issue can prove problematic for both large broodfish such as cobia and small broodfish such as ornamentals. For large broodfish where larger volumes or excipient and hormone are required, even a 21-gauge needle may prove impractical to quickly load large volumes and larger needles would be required. Larger needles would of course leave larger holes which could lead to a higher secondary infection rate. For small ornamental fish, the use of a 21-gauge needle or larger could be damaging to the fish.
In all, the viscosity could be an issue, but this excipient would be an improvement over the large and numerous holes in the dermis created by the Ralgun® implanter that are required to put 6–8 implants into a single large broodfish to achieve the correct dosage. However, it could provide a slow-release option for small broodfish where the large cellulose-based implants are impractical and potentially lethal. From this experience with the excipient, it appears to be quite safe and minimally invasive for the fish. As noted, once the excipient was inside the fish, it formed a sticky, gelatinous, globule that did not appear to cause distress or physical damage to the fish.
While this was the first clinical trial using this sucrose-based excipient, it has the potential to replace commonly used hormone implants or injections. It was non-invasive and due to its time-release properties, it limits handling time that may cause undue stress to fish. The positive implications of the results of this clinical trial indicated this excipient could be used as a spawning aid for most species of fish with no or very few adverse effects.