Source: KAMPACHI FARMS, LLC submitted to NRP
FORMULATED SARDINES: A HIGH-MOISTURE, SUSTAINABLE DIET FOR FASTIDIOUS FEEDING HIGH-VALUE MARINE FISH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1011072
Grant No.
2016-33610-25860
Cumulative Award Amt.
$599,082.00
Proposal No.
2016-04520
Multistate No.
(N/A)
Project Start Date
Sep 1, 2016
Project End Date
Aug 31, 2019
Grant Year
2016
Program Code
[8.7]- Aquaculture
Recipient Organization
KAMPACHI FARMS, LLC
1 KEAHOLE POINT RD
KAILUA KONA,HI 967402637
Performing Department
(N/A)
Non Technical Summary
The current dependence of grouper and tuna aquaculture on "wet fish" diets poses challenges for the long term viability and scalability of such aquaculture in the U.S. and internationally. There is a recognized need for alternative feed formulations (those including agricultural oils and proteins, such as soybean meal, soy protein concentrate, wheat and corn gluten, canola, poultry and other animal processing by-products, microalgae, etc.), but these top-end predators are often highly averse to eating conventional hard, extruded pellets; and, in aquaculture, are often fed a diet of "trash fish" bycatch or whole fresh/frozen forage fisheries products such as sardines or pilchards. Continued reliance on these limited, price-volatile, wild-caught "wet fish" resources present a critical constraint to the sustainable expansion of high-end marine aquaculture and its ability to produce healthy, efficiently grown, desirable fish for a growing and increasingly prosperous global population. Kampachi Farms has sought to resolve this issue by adapting the USDA-developed "Formulated Sardine" - a fibrous, high-moisture, soft extruded strip of formulated feed which more closely mimics the texture of natural fish - to high-end marine fish such as groupers and tunas. By switching to a formulated diet, sustainable, scalable agricultural oils and proteins can be incorporated into the diets of these high-value fish, rendering their expanded commercial production both more economically attractive to investors, and more environmentally sound.Phase I research thoroughly demonstrated the feasibility of this concept by conducting palatability and grow-out trials to determine if Formulated Sardines would be accepted by high-value marine carnivores and generate acceptable growth rates attractive to commercial culture. Several experimental formulations of Formulated Sardines were developed and manufactured by USDA ARS fish nutritionist Dr. Frederic Barrows, including the early deployment of a 100% fishmeal-free blend, which became the focus of grow-out trials during this study. All experimental diets were formulated to be nutritionally suitable for the two species tested: the relatively sedentary Giant Grouper (Epinephelus lanceolatus) and the more active kampachi (Seriola rivoliana). Growth, feed conversion ratio (FCR) and feed waste metrics were compared between populations of both species fed wet fish diets, pelleted diets and Formulated Sardines. The results of these trials were promising, with Formulated Sardines accepted by both grouper and yellowtail at comparable rates to their standard feeds, producing no significant differences in growth performance or FCR. The fact that these results were attained with a 0% fishmeal formulation, months to years ahead of when that development was expected, only underscores the Formulated Sardine feed format's potential for commercial utility in the culture of high-value marine finfish such as grouper and tuna. The apparent palatability of these diets in Grouper and Yellowtail studies also suggests several additional areas across the hatchery production cycles of these species where Formulated Sardine-type diets could provide significant advantages over traditional feeds.Kampachi Farms therefore proposes, through this Phase II research, to bring this novel aquafeed format closer to commercial viability by expanding into trials with Pacific Bluefin and Yellowfin Tunas, assessing its efficacy in specialty formulated broodstock diets, and determining the effectiveness of a derivative soft "crumble" product in accelerating weaning of highly prey-selective, difficult-to-rear marine finfish larvae onto formulated feeds.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30737191010100%
Knowledge Area
307 - Animal Management Systems;

Subject Of Investigation
3719 - Other cultured finfish;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
The current dependence of grouper and tuna aquaculture on "wet fish" diets poses challenges for the long term viability and scalability of such aquaculture in the U.S. and internationally. There is a recognized need for alternative feed formulations (those including agricultural oils and proteins, such as soybean meal, soy protein concentrate, wheat and corn gluten, canola, poultry and other animal processing by-products, microalgae, etc.), but these top-end predators are often highly averse to eating conventional hard, extruded pellets; and, in aquaculture, are often fed a diet of "trash fish" bycatch or whole fresh/frozen forage fisheries products such as sardines or pilchards. Continued reliance on these limited, price-volatile, wild-caught "wet fish" resources present a critical constraint to the sustainable expansion of high-end marine aquaculture and its ability to produce healthy, efficiently grown, desirable fish for a growing and increasingly prosperous global population. Kampachi Farms has sought to resolve this issue by adapting the USDA-developed "Formulated Sardine" - a fibrous, high-moisture, soft extruded strip of formulated feed which more closely mimics the texture of natural fish - to high-end marine fish such as groupers and tunas. By switching to a formulated diet, sustainable, scalable agricultural oils and proteins can be incorporated into the diets of these high-value fish, rendering their expanded commercial production both more economically attractive to investors, and more environmentally sound.Phase I research thoroughly demonstrated the feasibility of this concept by conducting palatability and grow-out trials to determine if Formulated Sardines would be accepted by high-value marine carnivores and generate acceptable growth rates attractive to commercial culture. Several experimental formulations of Formulated Sardines were developed and manufactured by USDA ARS fish nutritionist Dr. Frederic Barrows, including the early deployment of a 100% fishmeal-free blend, which became the focus of grow-out trials during this study. All experimental diets were formulated to be nutritionally suitable for the two species tested: the relatively sedentary Giant Grouper (Epinephelus lanceolatus) and the more active kampachi (Seriola rivoliana). Growth, feed conversion ratio (FCR) and feed waste metrics were compared between populations of both species fed wet fish diets, pelleted diets and Formulated Sardines. The results of these trials were promising, with Formulated Sardines accepted by both grouper and yellowtail at comparable rates to their standard feeds, producing no significant differences in growth performance or FCR. The fact that these results were attained with a 0% fishmeal formulation, months to years ahead of when that development was expected, only underscores the Formulated Sardine feed format's potential for commercial utility in the culture of high-value marine finfish such as grouper and tuna. The apparent palatability of these diets in Grouper and Yellowtail studies also suggests several additional areas across the hatchery production cycles of these species where Formulated Sardine-type diets could provide significant advantages over traditional feeds.Kampachi Farms therefore proposes, through this Phase II research, to bring this novel aquafeed format closer to commercial viability by expanding into trials with Pacific Bluefin and Yellowfin Tunas, assessing its efficacy in specialty formulated broodstock diets, and determining the effectiveness of a derivative soft "crumble" product in accelerating weaning of highly prey-selective, difficult-to-rear marine finfish larvae onto formulated feeds.
Project Methods
7.1 Feed formulation and packagingAll feed formulation and manufacturing work will be conducted by Dr. Frederic Barrows and his team at the USDA-ARS Feed and Nutrition Lab at the Fish Technology Center in Bozeman, MT.Stability trialA small sample batch of Formulated Sardines including a chemical mold inhibitor (propionic acid), will be manufactured and packaged into three retort pouches, and then autoclaved to sterilize and stabilize the product. These packages, along with an un-stabilized quantity of frozen Formulated Sardines (the original Phase I formula) will then be shipped to Kampachi Farms, where a short shelf stability trial will assess this approach's success. Once in Hawaii, the pouches will be stored in refrigeration (<40F), an air-conditioned storage trailer(~75F), and under ambient atmospheric conditions (~85F) for a period of two weeks. After this storage trial, the pouches will be opened and assessed for product degredation and rancidity (via peroxide analyses). The frozen untreated diet will be defrosted, and a short acceptance trial using four tanks of juvenile Kampachi will be conducted to ensure that the treated feeds are accepted at the same rate as the untreated diet. The opened pouches will then be clipped closed and placed in a refrigerator for one week. The pouches will be checked for mold growth daily7.2 Tuna acceptance and feeding efficiency trials To determine the suitability of the Formulated Sardine for use in Yellowfin (Thunnus albacares) and Pacific Bluefin (Thunnus orentalis) tuna culture, Kampachi Farms will collaborate with Dr. Barbara Block and staff of the Tuna Research and Conservation Center at Stanford University's Hopkins Marine Station in Monterrey, CA to conduct palatability trials similar to those accomplished in Phase I for grouper and kampachi. Dr. Block and her team maintain a 7500-sq-ft facility at TRCC which has been designed to house up to twenty captive Yellowfin and Pacific Bluefin tunas in separate 109,000L tanks. Tank-acclimated tuna will be implanted with archival body temperature monitoring tag (Lotek and Amitrix technology). The project team will record and analyze the thermal signatures of each animal during feeding to obtain estimates of the energy delivered by tuna-optimized Formulated Sardines over the course of a 3-4 week palatability/acceptance trial. Total feed consumption and wastage will also be measured and recorded. These metrics will be compared with archival values from fish fed the standard wet fish and squid diet, which are well-described in previous studies. If consumption and energy delivery rates are acceptable, the research will move on to more precise specific dynamic action analyses. Individual tuna will be hand-fed a known quantity of Formulated Sardines while researchers watch from an over-tank gantry and record the exact amount ingested. A settling period of around two hours will be allowed after which the fish be captured in a sling and transferred to a Loligo Systems flume tank respirometer which will measure metabolic oxygen consumption over the course of the post-prandial period (up to 24 hours). Each individual in the captive populations will be measured, and this data compared to a large body of archival metabolic data from previous studies using wet fish diets.7.3 Broodstock conditioning trials12 wild mature Seriola rivoliana broodstock (six males and six females) will be captured using the standard hook-and-line protocol, and transferred into oxygenated totes for delivery to Kampachi Farms' research facility. The fish will be stocked into two 68,000L HDPE tanks (three males and three females per tank) and put through a three-week hyposalinity quarantine. After the broodstock fish have started eating regularly, one of the tanks will be transitioned onto the Formulated Sardine feed, while the other remains on the traditional wet fish diet. This regimen will be maintained for the remainder of the project, to allow the fish to become acclimated to their captivity, to reach spawning condition, and to establish a reproductive history over the course of approximately 12-18 months in captivity. After the fish begin spawning (usually 3-6 months after capture), Kampachi Farms researchers will measure, record, and compare spawn frequency, spawn quality (% viability), fecundity, and hatch rate for both broodstock groups. If, by the end of one year of conditioning, the broodstock population being fed the Formulated Sardine diet is producing spawns at least of a quantity and quality consistent with the control group, then specialty marine finfish broodstock feeds may be selected as an area of concentration for Formulated Sardine commercialization.7.4 Larval weaning trialsGiant Grouper broodstock will be maintained as per routine until four days prior to the full moon in July 2017, when the grouper will be anaesthetized and injected with Western Chemical OvaRH® (a synthetic salmon GnRH analogue), with a resolving dose of Chorulon® HCG being administered on the day of the full moon. Eggs from the resulting spawn will be hatched and run through Kampachi Farms' standard larval rearing procedure, until about 15 days post-hatch, when the larvae are consuming only live Artemia brine shrimp. At this point, the larvae will be split evenly into six one-ton (1000L) tanks. Three of these tanks will start receiving Otohime B1, an industry-standard powdered microdiet, and three will start receiving a high-moisture micro-crumble manufactured at the Bozeman FTC. As the weaning process progresses, and the size of the artificial diets administered increases, twice-daily larval samples will be taken from each tank and analyzed under a dissecting microscope to determine gut content composition over time. Under Kampachi Farms' larval rearing SOP's, gut content analysis is what drives the weaning schedule; as the fish grow and become acclimated to artifical feeds, the ratio of Artemia to artifical feeds found in the gut declines, and the ration of Artemia administered to the tank is reduced. If the soft-textured high-moisture crumbles are indeed more effective than traditional dry microdiets, we would expect to see the weaning process completed more rapidly in that treatment. In addition to this assessment, researchers will measure and record larval growth rate and survival over the course of the trial. Resulting fingerlings will be randomized and transferred into six 4000L HDPE trial tanks for a 2-month post-larval growout trial. Three of these tanks will be administered larger Formulated Sardine crumbles (1.5-4mm dia) and three will be administered a minced wet fish diet. Growth rate, feed conversion ratio, and survival will be recorded.

Progress 09/01/16 to 08/31/19

Outputs
Target Audience:As an SBIR, the results are not widely disseminated to the public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Opportunity for professional development was given to one researcher at Kampachi Farms, who executed the project and worked alongside one of the leading fish feed formulators in the country during the project. How have the results been disseminated to communities of interest?As an SBIR the results are not widely disseminated. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The initial dietary formulation of the experimental EcoSardine, evaluated in Phase I, has been modified several times over the course of the second phase of this project to address palatability concerns, shelf stability, production costs, and species and life-stage specific nutritional requirements. The initial results from feed trials with adult Pacific bluefin tuna, Thunnus orientalis, and juvenile Coral Trout, Plectropomous leopardus, indicated that dietary modification was necessary in order to improve the diet's palatability and acceptance. Additional formulations were required for the development of a marine growout and larval micro-diet for larval Seriola rivoliana. These formulations, designed with Dr. Rick Barrows, were informed by the established nutrition requirements for these species and the nutrition work conducted at Kampachi Farms, Hopkins Marine Station, and Oceanic Institute. Kampachi Farms was successful in developing formulated diets for adult Pacific yellowfin tuna (Thunnus albacares), larval and juvenile kampachi (Seriola rivoliana), and juvenile coral trout (Plectropomous leopardus) using a high moisture aquafeed extrusion process pioneered by the USDA lab in Bozeman, MT.

Publications


    Progress 09/01/17 to 08/31/18

    Outputs
    Target Audience:Kampachi Farms is pursuing a collaborative business agreement with feed company, Zeigler Brothers Inc., who is obtaining an exclusive use license of the moist feed production technology from the USDA-ARS. This collaboration will provide Kampachi Farms with a producer for experimental diets, expertise on formulation, and access to existing marine fish markets. Discussions with Zeigler have been ongoing over the course of the Phase II period, and they are receptive to the value of our research in the species and life stage-specific formulations for marine fish. We are working to reach an agreement for sales and production of the feeds being developed through this project on the path to commercialization. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Work Forecast: Over the course of the next year, Kampachi Farms will focus its work towards commercialization in the following categories: A continuation of dietary formula/ process development and a demonstration of the diet's value in various commercial settings: Marine larval weaning moist microdiets Grouper/Coral trout grow-out moist pellet Seriola grow-out moist pellet Tuna grow-out moist bar For each of the above categories, our intention is to design and test formulas in order to inform their development and build proof of value for each product. Evaluating the physical properties of these products to determine the viable applications; specifically: Leaching properties Apparent digestibility in Seriola Spp. Palatability

    Impacts
    What was accomplished under these goals? Description of work carried out: The original dietary formulation of the experimental EcoSardine, evaluated in Phase I, has been modified to address species and life-stage specific nutritional requirements and palatability concerns. Necessity for dietary modification was made evident by the initial acceptance results from adult Pacific Bluefin Tuna, Thunnus orientalis, and juvenile Coral Trout, Plectropomus leopardus feed trials. Kampachi Farms has investigated multiple strategies for extending the shelf-life of the moist feed and has demonstrated success in limiting microbial spoilage during transport and storage. ??Research Objectives: 1. Through collaborative efforts with USDA-ARS researchers, develop shelf-stable formulation and packaging for high-moisture feeds. 2. Using observations from palatability/acceptance and feed efficiency trials, develop effective moist feeds and demonstrate their value as grow-out diets for Pacific Bluefin Tuna, Seriola spp. Evidence from the trial results will be used to encourage commercial collaboration and farm-scale evaluations of the product. 3. Through a comparative larval rearing study, determine whether a small-particle EcoSardine effectively accelerates the weaning process in hatchery-reared Seriola spp. larvae. 4. Determine the effect of dietary moisture on protein utilization and digestibility in Seriola rivoliana. Research Work Carried Out: Shelf Stability Trial The high nutrient density and moisture content in the moist feed provide an ideal substrate for microbial growth, which presents an issue for sanitary transportation and storage of the feed. To address the relatively poor shelf-stability at room or refrigerated temperatures, several strategies for increasing the shelf-life of the feed were pursued: reduction of water activity using the humectant glycerin, and addition of an anti-microbial, sorbic acid (potassium sorbate). Nutritionally isonutritional diets were produced at the U.S.D.A.-A.R.S. facility in Bozeman with the addition of 0.1% potassium sorbate, 8% glycerin, or with no anti-microbial agents. All feed was tested for microbial growth by sampling and agar plate inoculation (plating methods adapted from those used in Sanders, 2012). Glycerin and Potassium Sorbate were both successful in limiting the amount of microbial growth in the frozen and refrigerated samples of feed. However, neither inhibited mold growth entirely. Following 7 days of storage at 4°C, there was a significant increase in the number of bacterial colonies found in the control diet, and no significant microbial growth in the two experimental treatments (Figure 1., Appendix Figure 2.). The two mechanisms for preservation (Potassium Sorbate, Glycerin) used in this study are disparate, and could potentially provide a beneficial compounding effect when used in conjunction with one another. Identifying compounds and inclusion levels that successfully inhibit microbial growth is an important step towards developing a shelf-stable product to reduce transportation and storage costs. Currently, the most effective method of feed preservation appears to be desiccation immediately following production. Using a flow-through, pulse bed dryer, the feed can be dried to a moisture of <10% and then rehydrated with freshwater immediately prior to feeding, which allows for long term unrefrigerated storage and significantly reduced transportation cost. Figure 1. Relative fungal and bacterial presence on moist fish feed when frozen, and when thawed at 4? for 7 days. Pacific Bluefin Tuna Acceptance/ Energy Utilization Under the supervision of Dr. Barbara Block, the Tuna Research and Conservation Center Lab at Hopkins Marine Station (HMS) in Monterey, CA examined feed acceptance (EcoSardine), and post-prandial heat increment and heart rate in captively held Pacific Bluefin Tuna. This study was a supplement to previous work conducted at HMS where bluefin tuna were provided the formulated sardine as a sole ration rather than co-fed with natural prey items, as was practiced in the previous study. Prior to the beginning of the trial, Pacific Bluefin Tuna were implanted with acoustic and archival tags to measure heart rate and internal temperature. There was an extended acclimation period, during which, the tuna were allowed to recover from surgery and weaned from cut fish to the EcoSardine moist feed. After a period of several weeks, a portion of the captive tuna were observed and filmed readily accepting the formulated diet (Figure 2; Appendix, Figure 3). Several fish did not consume the feed over a period of 1.5 months during the feeding study, indicating a texture or palatability issue with the feed. Video footage of each feeding event was recorded to determine the proportion of the tank that accepted food. Although feed consumption was observed in many of the fish, and validated by the internal temperature loggers (post prandial specific dynamic action) from several fish, there was a persistent issue with the overall palatability of the diet for Pacific Bluefin Tuna. This trial was ended after the 1.5 month feeding period, so that the fish which had not yet consumed the formulated diet could have an opportunity to feed on wet fish. The formulation has been revisited and modified to improve the diet's texture and palatability (Appendix, Table 3.). Additionally, Kampachi Farms is collaborating with a company, FinnTan, which specializes in the design of species-specific finfish attractants for feed and tackle. FinnTan has designed an attractant to promote EcoSardine consumption in PBT. If the newest formulations are successfully accepted by the PBT, there will be a very strong proof of value for this feed as a more sustainable, scalable grow-out diet for such high-value species. It may also generate sufficient interest for a commercial site-test using this feed, which represents an important opportunity on the path to commercialization of the high-moisture feed. Figure 2. Graph of thermal data recorded from acoustically instrumented Pacific Bluefin Tuna during the period of acclimation to the formulated sardine feed. Peaks in internal temperature signify successful feeding events and consequent post prandial specific dynamic action. Eco-Sardine: Kampachi Nutrition To compare the success of a formulated, moist feed to commercially available diets for the grow-out of Seriola spp, hatchery-reared Seriola rivoliana were stocked into triplicate treatment tanks to be fed one of the following diets: Commercially available, extruded marine grow-out feed (EWOS Yellowtail) Cut, previously frozen anchovies Formulated, high moisture extruded diet (Appendix, Table 2) We are currently conducting a nutrition trial to compare the growth rate and FCR of the three diets. This trial will continue for an additional 8-12 weeks from the time of reporting. If the moist diet performs comparably to, or outperforms other treatments, we will have successfully demonstrated proof of value of the product to our potential commercial partners at Zeigler, and will have foundational research for initial sales and marketing efforts. Marine Larval Weaning Diets In collaboration with the USDA, we have been developing a microparticle diet using this feed production technology for use as a larval weaning diet. A formula has been developed that caters to the general nutritional requirements of larval marine fish. This formula is also comprised of feed ingredients that are allowed for importation into Mexico, which may enable us to evaluate this diet in the Cabo Kampachi commercial marine hatchery in La Paz, MX. If successful, the commercial operation in La Paz may adopt this feed into our routine larval production cycles, which would potentially establish a steady demand for the microdiet.

    Publications


      Progress 09/01/16 to 08/31/17

      Outputs
      Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?A greater quantity of the high-moisture tuna formulation will be milled for a follow-on feeding study with both yellowfin and bluefin tuna; 200kg of the preferred diet will be milled and shipped to TRCC for a trial to determine the amount of usable energy the fish extract from the diet. In addition, Dr. Barrows will perform proximate analyses on the diet, which will provide the fractions of major components to aid in Dr. Block's analysis of the usable energy in the diet. In mid-Sept, students and staff from Dr. Block's lab, along with twoKampachi Farms staff will convene for a bluefin tuna collection charter off the coast of San Diego. Tuna from this trip will be outfitted with Dr. Block's temperature data logger technology and used in all subsequent feeding studies. More wild kampachi broodstock will be sourced for the feed conditioning study and subsequent larval weaning experimentation.

      Impacts
      What was accomplished under these goals? The scope of the project was officially modified (on 5/10/17) to focus on the near-term commercial viability of the high-moisture feed technology as it applies to kampachi and tuna culture. Grouper culture is indeed important, and in need of formulated diets, but does not hold such immediate commercial traction. Dr. Barbara Block has tested two formulations of high-moisture feed on both yellowfin and bluefin tuna at TRCC, San Diego. Two isonutritional high-moisture diets were milled by Dr. Rick Barrows based on a dietary composition suggested by Dr. Block, the only difference being: one was treated for shelf-stability, and one required freezing. Both yellowfin and bluefin tuna have accepted both of the feeds. All of the TRCC tuna are equipped with heat sensing equipment, which passively log internal temperature data from the tunas. After feedings, the bluefin tuna showed a marked increase in internal body temperature, confirming their ingestion and caloric uptake from the diet. The yellowfin tuna transitioned onto the high-moisture feeds very readily. They do not conserve heat in their viscera as bluefin tuna do, and thus, must be put into a flume to measure their caloric uptake, which is planned for follow-on experimentation. Wild-sourced Seriola rivoliana broodstock are currently being housed at the Kampachi Farms facility in Kona. They will be used in a high-moisture feed acceptance study, and their offspring will furnish larval weaning trials on high-moisture crumble.

      Publications