Aquafeed Vol 14 Issue 3 2022

Vol 14 Issue 3 July 2022

AQUAFEED Advances in processing & formulation An Aquafeed.com publication

FISH GUT HEALTH Phytogenics Moisture management Functional protein hydrolysates Published by: Aquafeed Media, S.L.U. www.aquafeed.com info@aquafeed.com

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AQUAFEED

VOL 14 ISSUE 3 2022

Contents

A FUNCTIONAL PROTEIN HYDROLYSATE FOR SHRIMP 34 An overview of functional hydrolysate’s performance for higher growth rate, higher resistance to infectious challenges and resulting survival rates, among others.

FEED ADDITIVE PROTECTS AGAINST INTESTINAL ENDOPARASITE 15

DRYING AND COOLING PROCESSES FOR PELLETED SHRIMP FEED 38

The beneficial effects of a functional feed additive on gut health and fish performance complemented by the protective impact against an endoparasite.

The latest drying and cooling technology in pelleted shrimp feed allows manufacturers total moisture control and saves drying energy.

OREGANO ESSENTIAL OIL 57 A natural oregano essential oil contains multiple compounds which work in synergy to offer several well-documented properties and functions in aquaculture species.

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AQUAFEED CONTACT US Editorial: editor@aquafeed.com Editor/Publisher: Lucía Barreiro Consulting Editor: Suzi Dominy Technical Editors: Peter Hutchinson, Albert Tacon, Ph.D Assistant Editor: Marissa Yanaga Conferences and webinars: info@aquafeed.com Advertising enquiries/request media pack: sales@aquafeed.com Accounts & all other enquiries: info@aquafeed.com

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Adisseo...................................................................................... 2 Evonik........................................................................................... 5 Phileo by Lesaffre....................................................................... 9 Aquafeed.com......................................................................13 Wenger......................................................................................18 Huvepharma............................................................................26 Sparos......................................................................................29 Oxiris ..........................................................................................33 Extru-Tech..................................................................................42 Phodé........................................................................................45 Hatchery Feed & Management...........................................52 PlusVet Animal........................................................................56 WAS ..........................................................................................64

VOL 14 ISSUE 3 2022

Contents 6

Interview with Hugo Carrillo

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News Review

15 Functional feed additive for gut integrity and protection against the intestinal myxosporean endoparasite Enteromyxum leei

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*Cover story

23 Botanical compounds for fish gut health:

Case of soybean meal-induced enteritis in Atlantic salmon

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lack soldier fly improves the health status of rainbow trout B increasing their high gut microbial richness

34 A functional protein hydrolysate to support better shrimp performance

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nderstanding drying and cooling processes for pelleted U shrimp feed

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I nstant insights into aquafeed and ingredients with portable on-site analyzers

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Managing moisture in aquafeed production

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A pplications of phytogenics in shrimp farming

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N atural oregano essential oil sustainably supports gut health for optimal performance

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A ntioxidant technology helps optimize feed costs and performance

Columns 30 Ronald W. Hardy – History of fish nutrition. Part II (1957-2000) 46 Louis D’Abramo & Thomas R. Zeigler - Development of aquafeeds.

Reflections and future perspectives II: The transformational years (1980-2019)

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E nhancing gut health in tilapia with yeast-based probiotic for better feed efficiency and growth

Calendar of events

To read previous issues in digital format or to order print copies, visit: www.aquafeed.com

Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

From field to app. To you.

AMINODat® 6.1 – the most extensive web-based database for animal nutritionists

The most comprehensive animal nutrition database in the world supports you in mastering the challenge of reducing costs while meeting livestock needs. It covers over 1,000 raw materials and more than 278,000 samples from all over the world, based on more than 13,000,000 analytical results. Find out what AMINODat® 6.1 can do for you. animal-nutrition@evonik.com www.aminodat.com

d Update test with la crops!

Hugo Carrillo is CEO of Vitapro.

INTERVIEW with Hugo Carrillo AQ: How did you get to where you are today?

HC: Each step in my life has prepared me to lead Vitapro.

Throughout my career, I have gained considerable experience in both B2B and B2C business lines in many Latin American countries. This has allowed me to develop a solid vision focused on customers, an essential key to guiding the industry through two transcendental and necessary changes to reach our current position.

The first change happened during my first few years at Vitapro. I found myself in a business and sector with extraordinary growth forecast, with clients who progressively needed greater support and personalized service, in an increasingly growing competitive environment. This led us to the first moment of change in which, along with a team of experts with extraordinary talent, we challenged ourselves to give a new meaning to our value proposal, joining our two greatest strengths under a holistic approach: high-quality nutritional solutions and technical advisory services, in a proposal that put our clients at the center of the decision-making.

It seemed that this enhancement of our value proposition that had led us to success, until that point, would be enough. But very soon, we saw that our commitment to the industry’s future would drive us to be the bridge between our clients and digital transformation. Taking this route has led us to serving them better and being more responsible to the world. We made a change in our value proposal through the creation of Vitapro Ventures (Digital and Technology Innovation Hub) and the GTE (Specialized Aquaculture Technical Management).

We acquired skills that allowed us to develop different tools and digital solutions that we have been offering to our clients and that in some way, have complemented and strengthened the service that we had already been providing, evolving our approach into a fully comprehensive one. So today, we can say that we have shifted from developing nutritional solutions to providing comprehensive ones under a more holistic perspective of services that cover all the needs of our customers in order to achieve more efficient results in a more sustainable manner.

So, we could say that I have come this far thanks to valuable previous experience which gave me the right vision to lead the transformation of a company with enormous capabilities, but above all with an incredible team that knows very well the needs of our clients in Latin America.

AQ: For more than 30 years, Vitapro has been developing specialized feed solutions in Latin America through its Nicovita and Salmofood brands. What have been the main market challenges throughout these years?

HC: The salmon and shrimp industries are going through totally different stages; the salmon industry is mature, while shrimp is undergoing a transformation process that salmon has already experienced, and is leading Ecuador to become the largest producer of shrimp in the world. Within this context, the main transversal challenges we face in the industry are related to the contribution that companies, like ours,

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can provide to reduce the conversion factor, as well as enhance efficiency, productivity, and sustainability of the industry.

AQ: Salmofood is the salmon brand. Where is the company now in terms of size, markets served and aquafeed volume?

HC: To explain Salmofood's current position, it is necessary to go back to when Salmofood became part of Vitapro because its strategy changed from that moment on. In the past, it was seen as a local company that served small and medium-sized clients with a not-so-robust proposal. Since Salmofood joined Vitapro, it has become a challenging player in the market, and highly competitive in the industry. Thus, we have focused our efforts on delivering a unique proposal for each client based on three pillars: smart nutrition, personalized support, and world-class nutritional solutions. As a consequence, we have solid growth and we are targeting to reach a volume of more than 170,000 tons this year, a significant increase in the past ten years of the company.

in addition to consolidating the production plant and storage capacity acquired in 2021, we also announced in March that we will invest more than $80 million to build a new production plant in Ecuador.

The shrimp industry in Ecuador has maintained a significant growth that is enabling the country to consolidate itself as the most important producer of white shrimp in the world. This new investment is a tangible reflection of our commitment to being part of the exponential growth of the sector in the coming years.

With this investment, we aim to expand our production and storage capacity of animal feed for the country by approximately 45%, by building a new plant with cuttingedge technology, more efficient production systems and processes, under high-quality standards aligned with the Nicovita brand seal of trust.

AQ: Salmofood offers tailor-made feeds with specific supplements adapted to each farm's challenges, through its Care Blocks. What are the main solutions offered and how have they been accepted by farmers?

HC: Care Blocks are a result of Vitapro's global emphasis on R&D through our Aquaculture Experimental Centers (CEA) in Latin America. Its implementation is complementary, meaning that, when using a high-energy diet it is possible to complement it with Care Blocks, according to the specific needs of each producer with the aim for the best productive performance adjusted to their reality.

This innovation has been well accepted by our clients, especially those that complement the use of Care Blocks with our PatagonIA digital tool – an analytical and predictive model that projects conversion and growth factors using artificial intelligence.

AQ: Nicovita offers GenIA, a digital platform to improve production management. What are the main services it offers and how has it been implemented by farmers?

HC: Digital and technological innovation is an important pillar to consolidate our value proposal as a company that provides comprehensive aquaculture solutions to our customers. For this reason, through our digital and technology innovation hub, Vitapro Ventures, we created GenIA by Nicovita, the Nicovita Technological Ecosystem. It combines digitization, advanced analytics, Internet of Things (IoT), and the knowledge of specialized technical advisors to maximize productivity and efficiency in pools. GenIA allows us to generate patterns and predictions that develop personalized recommendations for each farm, thus promoting data-based decisionmaking on shrimp feeding and, consequently increasing shrimp farmers’ productivity, offering an unprecedented value proposal. A very important aspect of the development and implementation process of GenIA is that it has been developed hand-in-hand with our clients in Latin America.

AQ: Nicovita, the shrimp feed brand, is investing $80 million in a new shrimp feed facility in Ecuador. What is Nicovita's goal in terms of volume produced and what are the main technology improvements the new facility features?

HC: From the beginning, we have maintained a steady track record of continuous investments that have accompanied the growth strategy of our brands and supported the evolution of the industry. This year,

AQ: Through a series of acquisitions and expansions, Vitapro has a strong network of research facilities. What are the current R&D projects the company is working on?

HC: The constant development in innovation and capabilities is definitely a priority for Vitapro and its brands in both of its categories (fish and shrimp). Adding the Experimental Aquaculture Centers that we have implemented in recent years in Chile and Mexico to the previously installed capacity and our pilot plant, we

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can affirm that Vitapro has the most complete and important research and innovation network for shrimp at a global level.

Our most representative projects are related to five fronts: nutrition, health, technology, supplies, and management, seeking: to deepen our understanding of the nutritional requirements in the different shrimp life stages and farming systems, as part of our continuous improvement process and aiming to optimize formulas; challenge our understanding of diseases to create food solutions for preventive treatments; design and determine the best practices that we can recommend to our clients, through specialized technical advice; create new production management technologies and Artificial Intelligence models, such as GenIA; and promote the search for alternative raw materials, validating both their availability and digestibility for use in shrimp formulations.

AQ: Vitapro has a technical advisory team that provides personalized service to customers. What are the main services the company offers?

HC: Within the transformations that Vitapro has undergone, the experience in the technical consulting area is one of the most important. Our firm decision to place the client and their needs at the core of our operation has led us to design solutions tailored to their reality. This also results in technical advice that has taken a quantitative leap, going from a focus on delivering a technical consultancy by specialty, to a comprehensive technical consultancy nurtured by several specialists. We currently approach clients using agile methodologies, building “squads” with a multidisciplinary team that solves the problem and provides a solution from multiple perspectives.

AQ: Vitapro recently partnered with the Aquaculture Stewardship Council for responsible practices in the Latin American shrimp farming industry. What are the main challenges in the region?

HC: As a feed supplier, we are part of an industry that plays a fundamental role in the present and the future of food security. The role of our industry also leads us to face environmental challenges and generate commitments to sustainable development, and the conservation and preservation of marine resources.

In the case of the alliance with ASC, both ASC and Vitapro share a common purpose of transforming aquaculture, as well as a common vision of advancing

environmentally and socially responsible aquaculture and improving standards across the industry.

Since sustainability is at the core of Vitapro's operation, the company seeks to promote the strict adoption of ASC standards in all territories where the Nicovita brand is present. In this context, the main challenges of the industry focus on raising its standard in terms of sustainability, promoting best practices that allow the increase of global competitiveness, and working together with suppliers of marine ingredients to endorse compliance with sustainable standards in raw materials.

AQ: How about sustainability? What are Vitapro's main sustainability commitments?

HC: Sustainability is part of our DNA and is embedded in all our initiatives. Following this line, Vitapro has a sustainability strategy based on three pillars: promoting a healthy nutrition and wellness, enhancing value chain sustainable development, and as an active agent, encouraging environmental care.

The transformation of the aquaculture industry remains the biggest challenge, and to achieve this, innovation and digital transformation are necessary as drivers. Along these lines, Vitapro´s commitment to the industry becomes tangible through the impulse of innovative development of tools that help to transform shrimp farmers' businesses, offering them a variety of solutions and practical improvements that maximize their performance; a digital transformation in the technical advice provided; the application of new technologies such as advanced analytics to enhance decision-making; research for a portfolio development of products focused on nutritional profiles and the reduction of the FCA (Food Conversion Factor).

Additionally, Vitapro works hand-in-hand with its suppliers to encourage its value chain to adopt the best sustainability practices. In this sense, in 2021 through the Accelerator program for Sustainable Aquaculture Ingredients, the company designed a program to promote among producers of marine ingredients in Latin America the adoption of the certification that guarantees responsible and sustainable production, and thus, ensure the suitability and high quality of the ingredients used in our production processes.

This comprehensive approach allows us to generate value, maximize clients' results, care about the raw materials' origin and the marine ecosystem, and promote local growth and well-being.

Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

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NEWS REVIEW Highlights of recent news from Aquafeed.com Sign up at Aquafeed.com for our free weekly newsletter for up-to-the-minute industry news

Guangdong Haid Group opens aquafeed mill in Vietnam

Green Plains signs aquafeed partnership with Riverence Green Plains partnered with the Riverence Group, the largest trout producer in the Americas, to form a joint venture to expand aquafeed production in Idaho. The venture will produce trout and salmon feeds for the Riverence Group, utilizing wholesome, sustainable ingredients including the 60%+ fermented protein product.

Sheng Long Bio-Tech International, a subsidiary of the Chinese Guangdong Haid Group, opened a new aquafeed facility in Hòa Phúc Industrial Zone, Longhu County, Vinh Long Province, in southern Vietnam. With the commissioning of the new feed mill, the company has 15 shrimp feed and 7 extruded fish feed production lines for the international market, with a total annual production capacity of nearly 650,000 tons.

Calysseo starts production at Skretting partners with its first industrial-scale facility Andfjord Salmon for world's first salmon feed with Calanus in China Andfjord Salmon, Skretting Norway and Zooca® (Calanus AS) have signed a letter of intent that facilitates the provision of a specifically designed salmon feed for Andfjord Salmon and long-term cooperation to increase the utilization of Calanus finmarchicus in salmon feed. Andfjord Salmon is the first salmon farmer in the world that utilizes a commercially developed feed that contains Calanus.

The joint venture between Adisseo and Calysta is commissioning and starting up activities at the world’s first industrial-scale FeedKind® facility in Chongqing, China. The facility will produce 20,000 tonnes of FeedKind® Aqua protein annually. Initially, production will be available for use in China.

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Aquafeed companies acquire shrimp feeding and management technology BioMar acquired AQ1 Systems, the world’s leading producer of acoustic feeding technology for the shrimp farming industry, aiming to create new synergies between farming technologies and feed solutions. Nutreco acquired Eruvaka which develops cloud-based aquaculture pond management solutions, including real-time monitoring and smart feeders.

Tietjen develops hammer mill for fine grinding

ADM acquires feed mill in Southern Mindanao, Philippines The company acquired a feed mill in Polomolok, South Cotabato, from South Sunrays Milling Corporation. The mill, along with existing ADM feed production facilities in Cebu and Bulacan, will support customer growth in the region with a wide range of innovative pet food, complete feed, aquaculture, and premix solutions.

Tietjen presented the large chamber mill FD, the latest of its hammer mills optimized for fine grinding, at VICTAM International. It achieves a very high throughput of over 15 t/h and reaches a high fineness at the same time.

In combination with a metering screw and the new airgravity separator AGS AD, the efficiency of the grinding system is increased even further.

Andritz introduces micro feed system and new sensor The company recently launched two innovations for the aquafeed industry. The Micro Feed System is a dedicated extruder upgrade system designed for aquafeeds that are less than 2 mm in size. The system can increase capacity by up to 40% when compared to running micro feed at reduced speeds on standard extrusion lines, and represents a breakthrough in the industry, the company said.

Metris Vibe is a wireless vibration and temperature sensor that can monitor the health status of mechanical equipment regardless of the manufacturer. The sensor is wireless, battery-

powered and measures vibration in three directions.

It can be applied to existing equipment at feed mills.

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Marel acquires Wenger Manufacturing Marel successfully closed the USD 540 million acquisition of Wenger Manufacturing LLC. The Wenger platform will form a new business segment that will constitute Marel’s fourth pillar alongside Poultry, Meat and Fish, and will be focused on the sizeable and attractive growth markets of pet food, plant-based proteins and aquafeed.

Cargill acquires Delacon to create a leading plant-based phytogenic feed additives business

Van Aarsen unveils highcapacity conditioning and pelleting line Van Aarsen introduced at VICTAM International its CU1200 Titan, a highcapacity conditioning and pelleting line.

This high-capacity process line uses European-made quality machines, is easy to use, easy to maintain and has a compact design. It has the capacity to process up to 70 tons per hour. It also offers low operational costs per ton of feed.

Geelen Counterflow delivers first electric dryer for Cargill-Ewos in Norway

Cargill has signed a binding agreement to acquire Delacon, the globally leading expert in plant-based phytogenic additives. The companies will build sustainable customer solutions focused on animal health, feed efficiency, and performance.

DSM expands intelligent sustainability service to salmon farming Sustell™ is the only environmental lifecycle assessment system with 3rd party, independent ISO certification by DNV and ISO assurance certified to ISO 14044/44. Salmon producer and cooperation partner Bakkafrost, based in the Faroe Islands, was the development partner for the Sustell™ salmon module and is one of the first to utilize Sustell™ to model and reduce the environmental footprint of their salmon farming operations.

The company has installed its first electric counterflow dryer at the Cargill-Ewos aquafeed plant in Bergneset, Norway. Energy consumption for drying is reduced by 75% (15000 MWh per year) and CO2 emissions from drying (previously 3,000 tons per year) are eliminated because gas burners have been replaced by electric heat pumps running on renewable electricity.

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Optimizing efficiency and lowering aquafeed costs using the ideal protein concept Ewen McLean, Kelly B. Alfrey, Delbert M. Gatlin III, T. Gibson Gaylord, Frederick T. Barrows A new analysis of the essential amino acids (EAA) of ten major farm-raised fish and shrimp species can help the industry drive down the cost of aquaculture feeds and make them more efficient. To our knowledge, this analysis is the first to examine such a wide variety of species' EAA using the same laboratory.

The analysis of EAA was motivated by the ideal protein concept (IPC) that advocates optimizing the blend of essential amino acids in feeds to precisely meet the target animal’s requirements for growth and maintenance. An animal’s EAA profile is considered to reflect an optimal blend for dietary EAAs. For over 50 years, swine and chicken feeds have been formulated with the IPC philosophy. The goal is a more efficient feed, in terms of cost, decreased waste and pollution, including nitrogen excretion.

Fishmeal has long been included in aquafeeds due to its favorable amino acid profile which closely resembles that of its consumers. However, with scarcity in wild-caught fish driving fishmeal prices higher, the IPC has gained momentum in aquafeed formulation over the last several years with the arrival of new and innovative protein sources, such as single-cell proteins and insect meals that offer a greatly expanded ingredient pantry for research and development.

By focusing on matching animal and feed EAA profiles, feed formulators can create more efficient formulations using less expensive ingredients.

Understanding EAA requirements is critical to formulating cost-efficient feeds. And yet, of the over 230 species of aquatic animals currently being aquacultured, the complete EAA requirements are known for only a handful of them.

When an animal’s nutritional requirements are unknown, the EAA analysis provides a good starting point to optimize that animal’s feed. The IPC relies on knowing the quantitative requirements of a reference EAA. Because Lysine (Lys) is one of the first limiting amino acids and its analysis is uncomplicated, it is often the first analyzed. All other requirements for EAA can then be expressed as a percentage of Lys.

In our recent study published in Aquaculture and Fisheries, we evaluated EAA muscle profiles of commercially important species, such as largemouth bass (Micropterus salmoides), channel catfish (Ictalurus punctatus), grass carp (Ctenopharyngodon Idella), Nile tilapia (Oreochromis niloticus), red drum (Sciaenops ocellatus), pompano (Trachinotus carolinus), longfin yellowtail (Seriola rivoliana), Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), yellowfin tuna (Thunnus albacares) and Pacific whiteleg shrimp (Litopenaeus vannamei), which represent nine orders.

The analyses were conducted by the same lab to eliminate variation due to lab technique so that differences in EAA could be detected across species, facilitating the efficient formulation of feed that mirrors the EAA muscle requirements.

In the fish group, little difference in EAA ratios (the A/E ratio) among the 10 species was detected and Lys represented the dominant muscle EAA measured, ranging between 7.06 and 9.58 g/100 g protein.

However, distinct differences were seen between the EAA of whiteleg shrimp and fish. For the shrimp, arginine (Arg) was the principal EAA followed by Lys. Good correlations have been found between wholebody and muscle EAA profiles. Further requirements have been estimated using the A/E ratio.

The A/E ratio does not provide the quantitative requirement for EAAs but the relative balance among the ten EAAs. Preparing feeds with this exact balance provides the means of creating low-protein feeds that return identical performance, in terms of growth rates and feed conversion ratio, when compared to an unbalanced, high-protein feed. IPC also reduces feed costs since proteins are one of the most expensive nutrients in feed, and feed is one of the most expensive variable costs in aquaculture production. Therefore, the more efficient the feed, the more profitable and more sustainable aquaculture can become. This study offers a good starting point to better formulate aquafeeds to minimize costs and maximize efficiency.

The study was supported by the F3 – Future of Fish Feed’s Feed Innovation Network.

Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

GUT HEALTH

Functional feed additive for gut integrity and protection against the intestinal myxosporean endoparasite Enteromyxum leei Panos G. Kalatzis, Maria Mercè Isern-Subich, Waldo G. Nuez-Ortín, Adisseo The importance of gut in fish health and performance A healthy alimentary canal is a fundamental element for the optimal growth and performance of all cultured fish.

Apart from nutrient absorption and digestion, which are its main functions, the gut plays also a major role in sustaining fish health (Kristen et al., 2012; Tarnecki et al., 2017). However, there are several, both non-infectious and infectious factors, that may cause imbalance among the major components of the gut: microbiota, epithelium and intraepithelial leukocytes. The amount of scientific research on gut microbiota has been growing exponentially over the past few years (Egerton et al., 2018; Bozzi et al., 2021), however, the complexity of gut microbiome and the potential interventions that may lead to better fish health and performance has

only scratched the surface (Montalban et al., 2015).

The clinical picture of such an imbalanced gut is called enteritis and occurs in the form of generalized intestinal inflammation, triggered by the activation of the intraepithelial immune cells such as macrophages.

The inflammatory response of the intestine is the defensive mechanism of the organism due to an infection or an injury. Although it is rather beneficial in the short term, if prolonged, it can lead to chronic inflammation which undermines both nutrient absorption and immunity functions of the gut. The combination of nutritional factors, such as the use of plant-based ingredients in the feed and gut pathogens, for instance the myxosporean parasite Enteromyxum leei, can further cause severe disruption of the gut integrity, undermining the growth and performance of

Figure 1. (A) Transepothelial resistance of anterior intestine with and without SANACORE GM® supplementation (0.5%). Tissue resistance in the intestine of healthy seabream juveniles of 100g is expected to be >150 Ω cm 2, therefore a threshold has been set with dotted line. (B) A statistically significant difference of 30% higher tissue resistance is reported under SANACORE GM® supplementation (0.5%). Statistical significance is stated by *.

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Figure 2. (A) Internal:External perimeter ratio. On the left section (SANACORE GM®) the ratio is higher by 7% compared to the right (CONTROL) and the intestine is presented intact and well-developed with many well-shaped villi. (B) External:Internal diameter ratio. On the left section (SANACORE GM®) the ratio is higher by 11% compared to the right (CONTROL) because the development of the villi is more consistent, leading to a smaller lumen space.

the fish (Sitjà-Bobadilla et al., 2019; Piazzon et al., 2022).

It should also be noted that a well-functioning gut is a prerequisite for the avoidance of the subclinical picture of the fish, which may not show direct consequences at once but will do so as soon as any abrupt or steep changes eg. suboptimal temperature, high stocking densities, opportunistic pathogens, vaccination or transfer stress, etc. appear in the environment.

Therefore, functional feed additives which are able to enforce and protect gut health may claim today a prominent role in the formula.

The objective of the present article is to assess and present the impact that the functional feed additive under the commercial name SANACORE GM® can render on the host’s gut. The documented beneficial effects on gut integrity, villi formation, antiinflammatory response and consequent protective activity against the myxosporean parasite E. leei draw the picture of a healthier gut which not only generates better performance indices but also makes it harder for pathogens to infiltrate and damage the tissue.

SANACORE GM® promotes gut integrity and protects against E. leei infestations SANACORE GM® by Adisseo is a broad-spectrum, health-promoting additive based on components that

deliver gut integrity and anti-inflammatory action. The positive impact of SANACORE GM® on gut integrity and health promotion has been extensively documented in the literature and reported by EU aquaculture projects such as ParaFishControl.

The functionality of the intestine for both digestion and health performance requires gut integrity to be intact. The transepithelial electrical resistance (TER) of the intestine, as an indicator of gut integrity, has been measured in gilthead seabream, Sparus aurata, fed a 10% fishmeal diet supplemented with SANACORE GM®.

TER was 30% significantly higher under supplementation and in relation to the control group (Fig. 1). The tighter the junctions among the cells, the more difficult for the electrical current to pass, hence, a tangible improvement of gut integrity is capitalized to better nutrient absorption, improved growth performance, and more robust fish health.

Morphometrical studies on the seabream’s gut, corroborate the previous result by focusing on the length and surface of intestinal villi. The villi from the hindgut, which is the most sensitive to inflammation part of the gut, indicate that internal:external gut perimeter and external:internal gut diameter fractions are higher by 7% and 11%, respectively, under SANACORE GM® supplementation (Fig. 2). The biological

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Figure 3. Resilience to inflammation following a cytomix stimulus. The ELISA results showed that in all 4 different concentrations (0.1x, 0.2x, 0.4x and 1x) and both time points (2h and 16h), SANACORE GM® was the most efficient additive in maintaining the inflammatory status below threshold.

meaning of these numbers is translated into more consistently developed villi, less inflamed villi, larger lumen space, bigger mucosal surface and consequently improved gut integrity.

The inflammatory process is mediated by the secretion of proinflammatory cytokines including interleukins (IL).

IL-8 has been particularly studied and extensively used in research as a biomarker for the inflammatory cascade acting as a neutrophil leukocyte chemoattractant or activator (Kim et al., 2019; Laing et al., 2002; Laing et al., 2004). The in vitro model using the caco-2 cell line (van de Walle et al., 2010), which originates from epithelial cells mimicking a functional intestine barrier, was used in order to assess the inflammatory response to SANACORE GM® and two other commercial feed additives. The cells were exposed to a mixture of proinflammatory cytokines (cytomix) and it was shown that supplementation with SANACORE GM® was the most efficient in maintaining the IL-8 levels below the anti-inflammatory effect threshold of 90%, whereas the other tested additives generated either no effect (90-110%) or proinflammatory effect (>110%) for most concentrations and time points (Fig. 3).

Supporting gut integrity and intestinal health is directly reflected in the successful application of SANACORE GM® as a preventive strategy against pathogenic

infestations. E. leei is a myxosporean endoparasite that colonizes the gut and occasionally can also be found in the bile. Enteromyxosis in gilthead seabream is a more chronic, rather than acute, condition since instead of causing immediate mortality, it gradually causes anorexia, weight loss, muscle reduction and eventually death. However, when illustrated in more sensitive fish, such as the sharpsnout seabream (Diplodus puntazzo) and the red seabream (Pagrus major) mortality can be induced much faster (Palenzuela et al., 2020; Henry et al., 2020). The route of infection for the parasite passes through the already loose junctions among the epithelial cells and further exacerbates the clinical picture of enteritis. The disruption of gut integrity compromises the host’s health while rendering it vulnerable to secondary infections as well. E. leei is a case in point for the Mediterranean aquaculture, particularly for gilthead and sharpsnout seabream (Palenzuela et al., 2020; Henry et al., 2020). In the former case, SANACORE GM® could mitigate the SGR decrease caused by the infection by up to 15% versus the untreated infected control.

A functional feed could also lower the prevalence of the infestation by 30% whereas the mean parasitic abundance per fish was significantly lower compared to untreated control (Palenzuela et al., 2020). In the case of sharpsnout seabream, SANACORE GM® had an

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immune-promoting effect on the antibacterial, antiprotease and ceruloplasmin activity compared to the untreated positive control (Henry et al., 2020).

Efforts for sustainable aquaculture and high prices that accompany the ingredients of marine origin have led the industry to constantly increase the inclusion of plantbased components and encourage the development of alternative nutrient sources in aquaculture diets. A recent study has proved that reinforcing a zero fishmeal (FM) diet with SANACORE GM® to support fish in dealing with E. leei infection is as effective as a 20% FM in avoiding the increased intestinal susceptibility to the parasite (Piazzon et al., 2022).

Conclusion The economic losses accompanied by hampered productivity along with the increasing research for plant-origin ingredients coupled with the stressors and pathogens to which culture fish are exposed,

highlight the importance and necessity of functional feed additives in the feed formula. Under these circumstances, the beneficial effects SANACORE GM® on gut health and fish performance complemented by the protective impact against E. leei, enforce its role as a core functional feed additive for the Mediterranean aquaculture.

References available on request.

More information: Panos G. Kalatzis Regional Manager Aquaculture Europe Adisseo E: panos.kalatzis@adisseo.com

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Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

MICRO

GUT HEALTH

Enhancing gut health in tilapia with yeast-based probiotic for better feed efficiency and growth Otavio Castro, Nadège Richard, Alban Caratis, Phileo by Lesaffre Suboptimal water quality conditions, pathogens, feed composition and quality, or pollutants are all factors that can lead to dysbiosis and gut inflammation in fish, ultimately reducing growth, feed efficiency and overall health status. The use of probiotics in aquaculture has taken a prominent place in preventively addressing these challenges while simultaneously providing an alternative to the systematic use of chemical therapeutics.

Leveraging more than 160 years of expertise in the fermentation of yeasts, Phileo® by Lesaffre developed ActiSaf®, a yeast-based probiotic that improves gut health and thus, feed efficiency and growth performance of farmed tilapia.

What gut health is and why it is important The gut has the dual role of secreting digestive enzymes and triggering some immune functions in fish. A healthy gut with a balanced microbiota means fish can process

Group

Midgut microvilli length (µm2)

Diet 1 Diet 1 + ActiSaf® Diet 2 Diet 2 + ActiSaf®

0.93 ± 0.02a 1.02 ± 0.02b 1.05 ± 0.03b 1.17 ± 0.02c

Figure 1. ActiSaf® supplementation improves midgut microvilli length and reduces inflammatory expression in Nile tilapia fingerlings.

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Figure 2. ActiSaf® supplementation improves survival, growth performance, protein efficiency ratio, and apparent protein digestibility in Nile tilapia reared under low-density (LD) and high-density (HD) conditions.

feed more efficiently, have a better immune system and are less susceptible to the pathogens present in their environment. On the other hand, without a stable microbiota, they tend to be more susceptible to stress and are more likely to develop some conditions.

The stability of the gut microbiome, as in any living organism, is a fragile balance that can be disturbed by the conditions and the variety of stress factors that fish encounter throughout production.

For instance, the increasing price of commodities

and the pressure to reduce feed formula costs have necessitated the utilization of more low-value vegetable ingredients in aquafeeds. These are also known sources of anti-nutritional factors, such as mycotoxins, enzymatic activity inhibitors and nonstarch polysaccharides, that can cause chronic gut inflammation and change the gut microbiome.

The gut health status of tilapia can also be disrupted by pathogen-related dysbiosis. This process happens when harmful toxins are produced by host-colonizing

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Figure 3. Feed conversion performances of tilapia fed with Actisaf® supplementation. Source: Schwarz et al. (2016).

pathogens which subsequently damage the mucosa, resulting in an inflammatory response that affects intestinal morphology. This diminishes the fish’s nutrient absorption capacity and growth, thus making them more vulnerable to infections. Also, infections and other disease outbreaks in fish farming have a long history of being tackled using antibiotics. In addition to the sustainability issues that are tied to the use of antibiotics, such as resistance development and food safety concerns, their administration can significantly reduce intestinal bacterial diversity and suppress the fish’s immune response (He et al., 2011; Zhu et al., 2018). Gut health is therefore strongly connected to farm productivity and may lead to increased production costs.

Gut health management practices in aquaculture The use of probiotics in aquaculture has grown along with the necessity of managing fish gut microbiome during production in order to optimize performances at harvest while minimizing the use of therapeutics. Probiotics are living micro-organism additives that are widely used in aquaculture to enhance fish production as they help improve fish microbiota and thus contribute to immune response enhancement and disease prevention (Islam, Rohani, and Shahjahan, 2021).

Although probiotics were initially used for controlling pathogens' pressure in the water, their use in aquaculture has extended to improving fish growth and feed conversion through feed application. In this context, the supplementation of live yeast-based probiotics in aquafeeds is gaining interest and their benefit on gut health has already been assessed in different trials (Ran, Huang, Hu et al., 2016; Ran et al., 2015), with some promising results reported in tilapia.

Maintaining gut health for higher production performances with ActiSaf® ActiSaf® is a yeast probiotic that is produced by Phileo® from a selected proprietary strain of Saccharomyces cerevisiae that is exclusively dedicated to the animal production industry.

Ran et al. (2015, 2016) showed that ActiSaf® supplementation in the diet of Nile tilapia fingerlings (at the optimized ratio of 1 kg/ton of feed) increased midgut microvilli length, thus supporting higher nutrient absorptive capacity and final body weight. ActiSaf® also reduced the expression of inflammatory and stressrelated cytokines such as IL1β and TGFβ in the gut mucosa (Fig. 1).

ActiSaf® also proved to be helpful in both high and low fish density conditions, increasing microvilli length, improving trypsin activity in the intestine (a

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Table 1. Strategies for the application of Actisaf® for the improvement of gut health and feed efficiency in tilapia, per production stage.

Gut health & feed efficiency support ActiSaf®

Fry 0.1-2g

Fingerling 2-20g

Growth I 20-50g

Growth II 150-400g

Finishing 400g-harvest

C

C

C

C

C

1 kg/ton

1 kg/ton

1-2 kg/ton

1-2 kg/ton

1-2 kg/ton

C = Continuous application. P = pulsed application with minimum of 14-21 days of supplementation before challenge peak.

digestive enzyme), and reducing hsp70 expression (thus decreasing crowding-induced stress) and alkaline phosphatase activity (Ran, Huang, Liu et al., 2016). Furthermore, ActiSaf® showed a beneficial impact on tilapia microbiota by enriching beneficial bacteria Lactococcus spp. in allochthonous microbiota (Ran et al., 2015). Additionally, Lara-Flores et al.

(2003) established the outperformance of yeast probiotics over bacterial probiotics (Streptococcus faecium and Lactobacillus acidophilus) in the improvement of feed efficiency and growth performance characteristics in tilapia. Results from this study also demonstrated that Actisaf® can improve tilapia survival and specific growth rate independently of the density (high-density HD, and low-density LD), and support feed digestion under HD and more stressful conditions (Fig. 2).

By improving gut microbiota and nutrient absorption capacity, ActiSaf® significantly reduces FCR and generates significant savings on feed costs (Fig. 3).

Consequently, the growth performance and overall health status of tilapia are also improved.

Conclusion The shift of aquaculture practices towards more intensive systems and the necessity to optimize feed costs are two major root causes of gut issues in tilapia farming. In some areas, these issues are likely to grow in complexity due to the continued development of production, the demand for affordable products, the emergence of antimicrobial-resistant pathogens and climate change. Therefore, the use of probiotics for maintaining gut health in aquaculture will be increasingly required. As demonstrated earlier, ActiSaf® offers producers the possibility to prevent problems related to gut inflammation and dysbiosis, thus optimizing production performances at harvest while maintaining low feed costs.

Based on the results obtained in the lab and commercial trials reported here, we provide a variety of ActiSaf® recommendations ranging from 1 to 2 kg per tonne of feed depending on the extent of the gut issues and your own production objectives (Table 1).

For additional information, visit Aquasaf tilapia program.

References available on request.

More information: Otavio Serino Castro Global Species Manager Aquaculture Phileo by Lesaffre E: o.castro@phileo.lesaffre.com

Nadège Richard Research & Development Manager Aquaculture Phileo by Lesaffre E: n.richard@phileo.lesaffre.com

Alban Caratis Global Program Manager Aquaculture Phileo by Lesaffre E: a.caratis@phileo.lesaffre.com

Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

GUT HEALTH

Botanical compounds for fish gut health: Case of soybean meal-induced enteritis in Atlantic salmon Siméon Fagnon, Phytosynthese Gut health is defined by complex homeostasis between gut immunity and microbiota. Considering gut health is key for fish health and performance, its management has been the focus of much recent research for feed strategies. At the same time, fish farming environment could be the source of many stresses which challenge gut heath.

In carnivorous species, the replacement of fish ingredients is needed for sustainable production.

Soybean meal is currently one of the prominent alternatives for fishmeal replacement in aquafeed.

Although its nutritious value has been demonstrated as an excellent protein and almost balanced amino acid complex, the high-level inclusion of this ingredient in aquafeed remains challenging (Wu et al., 2021). Soybean meal contains some antinutritional factors especially saponins which cause negative performances and degradation of intestinal health in some carnivorous fish species (Miao et al., 2018). In salmonids, the main effects of feed containing high antinutrients are gut inflammation induction, loss of important digestive functions and diarrhea, as well as reduced nutrient digestibility and subsequently growth.

Botanicals for intestinal health Many strategies have been developed to overcome this issue. Natural botanical compounds have recently been identified as a potential option. Plants produce uncountable secondary metabolites, some of which have been used for millenniums by humans and wild animals to improve environmental resilience. Over recent years, botanical compounds were evaluated

in the aquaculture sector for anti-inflammation, immunomodulation, antioxidant, microbiota balance and growth improvement (Fagnon et al., 2020).

Regarding these well-documented properties in intestinal tissues, natural botanical compounds might contribute to alleviating the inflammatory reactions caused by standard soybean meal and subsequent consequences on overall health.

Phytosynthese is an independent French company specialized in sourcing and analytical chemistry of plants for 25 years. Its feed supplements provide high and constant concentration of botanical secondary metabolites. Recently developed by the company, Phyto AquaMeric is a combination of different flavonoids and terpenoids from botanicals and Brazilian green propolis. The formula was specifically designed

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Table 1. Diet composition.

Ingredients Soybean meal Wheat gluten Faba bean dehulled Soy protein concentrate Pea Protein Conc. 50 Lucatin pink, 10% Fish oil NA Fish meal NA Rapeseed oil Wheat Mineral and Vitamines MCP Ytrium premix DL methionine Ca-carbonate

Soy-based feed 25 20.74 5 12 5 0.01 9.3 5 8.6 6 0.5 2 0.1 0.3 0.45

to reduce intestinal inflammation and ensure overall health when faced with high-level inclusion of soybean in carnivorous fish.

Materials and methods The trial was conducted at Nofima AS’ research station at Sunndalsøra (Norway). Post salmon of average weight of 234 grams were fed 10% in excess based on weighed daily rations and anticipated growth over 56 days.

Thirty-five fish were stocked per tank, in 6 cylindrical, 1000 L, fiberglass tanks supplied with saltwater and

Figure 1. Specific growth rate (SGR) for the reference feed (0%) (CTR) and 0.05% Phyto AquaMeric (PAM0.05).

maintained at 7.8°C. Each diet was fed to fish in three replicate tanks per feed, using automatic belt feeders.

Fish were fed with the following diets: a reference feed (CTR) as described in Table 1 and a supplemented reference diet with 0.05% of Phyto AquaMeric. The diets were formulated to fulfill the requirements of fish and contained 44% protein and 21% lipid.

At the end of the feeding period, six fish were randomly taken from each tank and euthanized. The weight of individual fish was recorded. Blood and plasma were collected from the caudal vein. The intact intestinal tract of the fish was removed, and divided into 3 regions: proximal, mid and distal intestines. Tissue samples were collected from all three segments and gene expression analyses were performed on distal intestine. Samples were stored for histology and gene expression according to Sahlmann et al. (2013).

Growth performance On the triplicate, no significant effect on specific growth rate (SGR) was observed in the group supplemented with Phyto AquaMeric (p>0.05). However, Phyto AquaMeric group showed the highest value of SGR (+9.5%) compared to the reference diet. The same tendency was found for the thermal growth coefficient (TGC).

Histology An histology improvement was found in the group supplemented with Phyto AquaMeric compared to the reference diet. In the proximal submucosa, the percentage of normal fish in the Phyto AquaMeric group was higher than the control with the same benefit for pyloric steatosis (Fig. 2). Phyto AquaMeric protected more individuals from inflammatory changes.

In mid intestine submucosa, a significant reduction of inflammatory changes was also observed in Phyto AquaMeric group with no fish showing severe steatosis.

No visual differences were observed in the distal intestine related to the high feed challenge.

Gene expression In the distal intestine, gene expression revealed a positive effect of PhytoAquaMeric (Fig. 3). For example, interleukin (IL) 17α showed a down-regulation expression in Phyto AquaMeric group. The opposite was found in many inflammation regulatory markers

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Figure 2. Number of mid intestine tissue sections that were scored “normal”, “moderate”, “marked” or “severe” for (A) enterocyte steatosis or hyper-vacuolization, and (B) increases in the width and inflammatory cell infiltration in the submucosa.

Figure 3. Relative expression levels in distal intestine of pro-inflammatory cytokine IL17α; anti-inflammatory markers IL4; immunomodulation markers (mmp13 & pcna); barrier function genes; plotted as mean tank levels (n=3) +/-SEM.

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such as iIL4 and TGF-β. Other immune-related genes that revealed an up-regulation expression were extra-cellular matrix modulator collagenase (mmp13) and proliferating cell nuclear antigen (pcna).

This observation might indicate a change in epithelial tissue remodeling or reorganization as an effect of the dietary supplement. Moreover, gene coding for tight-junction proteins like E-Cadherin (Ecad) or Zonula occludens 1 (ZO1) were up-regulated. These results indicate Phyto AquaMeric might contribute to restoring the integrity of intestinal barriers through the regulation of such genes.

Conclusion In a highly challenging trial, Phyto AquaMeric improved growth performances. Results revealed alleviation of enteritis at proximal and mid intestines. This effect of Phyto AquaMeric on the first segments of intestines could be explained by immunomodulation activity expressed by the gene expression in the distal part of the intestine. Selected botanical compounds could contribute to reducing the consequences of intestinal inflammation in fish. The reproducibility of these results should be evaluated on a larger scale.

Botanical ingredient quality was shown essential to answering aquafeed industry needs.

References available on request.

More information: Siméon Fagnon Innovation Product Manager Phytosynthese E: simeon.fagnon@phytosynbthese.fr

Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

GUT HEALTH

Black soldier fly improves the health status of rainbow trout increasing their high gut microbial richness Alex Diana, Andrew Richardson, Innovafeed As it has been widely demonstrated in human health, the microbiota affects many vital functions. It contributes to the regulation of the immune system, digestion of food, production of vitamins, and much more. Recent research demonstrates the impact of insect ingredients on fish species' microbiome.

The stomach is often called the second brain of the human body. It represents 100 million nerve cells that form the enteric nervous system (ENS) and gut microbes may stimulate immune cells, which then signal to the brain, meaning your gut’s health can have a very realworld impact on your well-being. Indeed, by modulating

gut microbiome composition through proper nutrition and probiotics, we also help decrease anxiety and depression in humans. This is because Lactobacilli and inflammation are also recognized to affect the brain pathway and when an imbalance occurs, mood disorders develop.

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Figure 1. Relative abundance within the two diets.

Aquaculture growth comes with important challenges that nutritionists, farmers, and formulators have to face daily, of which maintaining survival rate and strengthening immune development are the main priorities. These problems have been addressed primarily by advancements in automated feeding machines, reduced reliance on fishmeal-based feeds, and even disease control through commercial adoption of vaccine development along with biological control of parasite infection using cleaner fish.

Gut communities in aquaculture species In aquatic animals, gut microbial communities shift with host development and living environments.

Understanding the mechanism by which the environment impacts the gut microbial communities of aquatic animals is crucial for assessing and managing aquatic ecosystem health. Initially, fish embryos develop in a relatively constant bacteria-free environment (i.e., within the egg), although some environmental microbes quickly colonize the egg surface from the surrounding water after spawning.

After hatching, environmental microbes colonize the gut of larvae through the ingestion of water. After the first feeding, new microbial communities are introduced into the gut with the diet, increasing microbial diversity. The gut microbiota further shifts with host development and with changes in diet. At an early stage, the gut microbiota is influenced mainly by the introduction of environmental microbes with water and diet. However, as the immune system and nutrition metabolism develop, gut microbes are selected and enriched gradually and can be altered with nutritional elements.

For this reason, the research project aimed to explore the impact of novel ingredients on these gut communities using metabarcoding analysis of feed- and gut-associated microbial communities. The feeding trial was set at a recirculation aquaculture system (RAS) with a growth phase of 87 days. A control diet containing only fish and plant proteins (CTRL) and an insect diet containing 10% of Hermetia illucens meal (Hi10) were tested in rainbow trout (Oncorhynchus mykiss) were used to source comprehensive samples of the gut.

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Results In agreement with the majority of studies on the partial substitution of fishmeal with insect meal, the experimental diet Hi10 had an important effect in modulating the intestinal microbial communities of trout. In particular, the Hi10 diet increased significantly gut microbial richness and numerically the diversity (Shannon diversity index p=0.05) as compared to control fish (Fig. 1).

In general, high gut microbial richness and diversity are considered desired features because they are usually associated with the health status of the host.

Dietary inclusion of insect meal influenced the microbial intestinal profiles of trout both qualitatively and quantitatively and allowed a dominance of Firmicutes.

Indeed, multivariate analysis of bacterial communities revealed a significant relationship between diet and microbiota associated with fish intestines.

The analysis of microbiota showed a significant increase in Bacillales in the intestine of trout fed with Hi meal. In this case, we can exclude the influence of feed’s microbiota on trout’s gut microbiota since the relative abundance of Bacilli was very similar between the two feeds. A possible explanation could be that chitin, contained in insect meal, may have acted as a substrate to increase the proliferation of gut chitinolytic bacteria, which include many Bacillus species.

Several studies in fish have shown that dietary Bacillus subtilis administration enhances immune responses

and disease resistance in salmonids. Moreover, other genera of bacilli have been reported to produce high levels of lipase and therefore can help to break down fats from the feed diet, and therefore they can be better absorbed in the intestine.

Conclusion Our results indicate that dietary inclusion of Hi meal at 10% modified trout gut microbiota by increasing its richness and therefore improving the health status of the fish and its microbiome. This is an important step when guaranteeing the animal welfare of the fish in a comprehensive approach with other good practices.

More information: Alex Diana Product Manager Aquaculture Innovafeed E: alex.diana@innovafeed.com

Andrew Richardson Product and Account Manager Innovafeed

Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

COLUMN

History of fish nutrition Part II (1957-2000) Ronald W. Hardy, Distinguished Professor Emeritus, Aquaculture Research Institute, University of Idaho

During the middle of the 20th century, a confluence of factors initiated a new era in fish nutrition research that led to the identification of essential nutrient requirements for a number of important farmed fish species. In just a few decades, the field of fish nutrition advanced more than it had in the previous century.

New research approaches yielded reliable nutritional requirement information that made it possible to formulate feeds to match the nutrient requirements of selected fish species. As a result, a number of fish feed manufacturing companies became global providers of high-quality fish feeds that fueled the emergence of industrial fish and shrimp farming across the globe.

Halver’s foundational studies The first factor was the development of a vitaminfree, semi-purified experimental diet that supported reasonable fish growth. Researchers first used this diet to determine the vitamin requirements of juvenile salmon and later to determine the requirements of salmon and other farmed fish and shrimp for vitamins, amino acids, minerals and, eventually, essential fatty acids. The approach in these studies was similar to that used in nutritional studies of livestock, poultry and lab animals; one vitamin (or other nutrient) was deleted from an otherwise nutritionally complete diet and added back to experimental diets at incremental levels. The dietary level above which no further growth response (or other dependent variable such as tissue level, enzyme activity or other health metric) was measured was considered the nutritional requirement.

The semi-purified diet that changed the course of fish nutrition research was developed at the University

of Washington in Seattle. A graduate student in the Department of Biochemistry was assigned by the department chair to help a professor in the College of Fisheries investigate the persistent problem of anemia in chinook salmon fingerlings fed standard hatchery diets. The graduate student, John Halver, was an WWII army veteran whose experiences in post-war Germany with malnutrition inspired him to study human nutrition. His assignment to investigate potential solutions to salmon anemia was supposed to be a minor detour in his studies. Vitamin B12 had just been synthesized and the Department of Biochemistry had secured a few milligrams that Halver injected along with folic acid into anemic juvenile salmon. The fish recovered in 10 days.

Halver’s career goal changed; he switched to fish nutrition and undertook the challenge of developing a vitamin-free, semi-purified diet for fish as his Ph.D. research project. After a few attempts, Halver developed a semi-purified diet formulation that worked (Halver & Coates, 1957). He then used this diet to show that several B vitamins were essential in the diets of juvenile chinook salmon (Halver, 1957). Over time, the formulation of this diet changed as new information about the nutritional requirements of fish became available, but the general concept of the diet remained the same.

A second factor was the decision by the US Fish & Wildlife Service to build a second fish nutrition research laboratory in the western USA to complement the Cortland Hatchery in New York. Halver became the first director of this laboratory, located in Washington State along the Columbia River. The research focused

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on the quantitative vitamin and later amino acid requirements of chinook salmon, coho salmon and rainbow trout fingerlings (Halver, 1972) (NRC, 1973).

Halver’s training was in nutritional biochemistry, and he employed rigorous experimental approaches at the new laboratory that were subsequently used by researchers in the USA, Europe and Japan in the 1960s and 1970s to identify dietary requirements of salmonids, channel catfish, carp and other farmed fish. In the USA, catfish farmers were shifting to intensive production that depended on the use of nutritionally complete feeds. Research conducted by university and government researchers led to the development of high-quality and economical catfish feeds, resulting in a tremendous growth of the US catfish industry in the 1970s and 1980s.

An excellent historical review of catfish nutrition research was recently published (Robinson & Li, 2019). By the 1970s, most of the quantitative nutrient requirements of juvenile salmon and catfish were known (NRC, 1973).

Fish nutrition research around the world Aquaculture has a long history in Japan and in the second half of the 20th century, researchers in Japan made substantial contributions to fish nutrition, although many of their studies were published in Japanese with English abstracts. This limited recognition of their work by fish nutritionists in western countries. Examples of early fish nutrition research by Japanese scientists included amino acid composition of hatchery diets (Ogino, 1957), digestibility (Nose & Toyama, 1966), nutritional requirements of Penaeus japonicus, Japanese prawn (Kanazawa et al., 1971), vitamin requirements of trout (Kitamura et al., 1965; 1967), and dietary fatty acid requirements (Toyomizu et al., 1963; Toyomizu et al., 1963; Kayama, 1964; Higashi et al., 1966; Kaneko et al., 1966. By the late 1970s, Japanese researchers had estimated the dietary requirements for amino acids, fatty acids, vitamins and minerals for trout, carp, shrimp and other farmed species. Tokyo University of Fisheries and Kagoshima University under the leadership of T. Watanabe and S. Kanazawa established strong research programs that attracted and trained many international students who returned to their home countries to lead fish nutrition programs.

Fish nutrition research also took hold in European countries in the 1970s, particularly in the UK, France, Germany, Italy, Norway, Greece, Poland and Hungary.

In the UK, the Unit for Research into Fish Nutrition was established as a part of the National Institute for Research into Dairying at the University of Reading by C. Cowey, who subsequently joined the Institute of Marine Biochemistry in Aberdeen, Scotland, and led a team of researchers who approached fish nutrition from a biochemical perspective. Cowey organized the first International Symposium on Fish Nutrition and Feeding in Aberdeen, Scotland in 1985. This symposium became a biannual event rotating from Europe, the Americas and Asia. The most recent symposium was held in June 2022 in Sorrento, Italy. Germany hosted the first World Symposium on Finfish Nutrition and Fishfeed Technology in Hamburg, Germany in 1979 under the umbrella of the European Inland Fisheries Advisory Committee (EIFAC) of the FAO (Halver & Tiews, 1979). EIFAC also organized a dedicated workshop on Methodology for Determination of Nutrient Requirements in Fish in 1993 in Eichenhau, Germany.

Other government and academic centers in Europe added fish nutrition to their research portfolios. The Institute of Aquaculture, University of Stirling, Scotland, became a center of expertise in lipid nutrition and metabolism. In Norway, the Institute of Nutrition in Bergen added fish nutrition to its programs on animal and human nutrition in the late 1970s. Basic and applied fish nutrition research continues to this day at the Institute of Marine Research in Bergen.

In the 1970s, with the rapid development of Atlantic salmon farming in Norway, another major group dedicated to fish nutrition, Akvaforsk, now NOFIMA, was created to conduct research and development in fish nutrition and feeding. In France, Luquet established the first laboratory dedicated to fish nutrition as part of animal nutrition group in the National Institute for Agricultural Research (INRA) in Jouy en Josas. This laboratory then moved to the southwest of France in Saint Pee sur Nivelle in 1976, where the group became a leading laboratory conducting research on fish nutrition and metabolism. Currently, in almost all European countries, research teams as part of universities or public research institutes deal with dedicated work on various aspects of nutrition of different species of farmed fish at such locations as the Hellenic Centre for

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Marine Research, Athens, Greece, the University of Las Palmas, Spain, the University of Porto, Portugal, and the University of Udine in Italy, just to mention a few.

China accounts for over 62% of global farmed fish and crustacea production but it was a relative latecomer to the study of fish nutrition. Throughout China's more than 3000-year history of aquaculture, the production relied on natural food in ponds to support fish growth.

In the early 1950's, China was the first to succeed in the artificial propagation of four major fishes (black carp, grass carp, silver carp and bighead carp). Artificially propagated fingerings replaced wild sources to stock ponds and production depended on natural food, often enhanced by adding agricultural by-products to ponds to increase natural food production. Research on quantitative nutrient requirements of fish did not start until after 1980. In fact, in the mid-1980s, not a single feed company in China exclusively produced fish feeds.

However, the situation changed quickly and within a few decades, China had over 12,000 fish feed factories.

Availability of high-quality fish feeds in China enabled rapid intensification of freshwater finfish production through intensification and rapid expansion of marine finfish aquaculture production, including intensive production of many other species with pelleted feeds, including turtles and frogs.

First aquafeeds In the early days of Pacific salmon hatchery operations, feeds were made on-site using frozen, adult salmon carcasses as a feed component. However, feeds containing salmon carcasses were discovered to be a vector for fish tuberculosis, a disease caused by Mycobacterium chelonei (Wood & Ordahl, 1958).

This disease was a significant problem for the Pacific salmon hatchery programs because the main clinical manifestation of chronic infection was incomplete gonadal development in maturing fish. Hatchery programs were sustained by collecting and fertilizing eggs from salmon that returned to hatcheries and fish tuberculosis prevented hatcheries from meeting production goals due to a lack of viable eggs (Wood & Ordahl, 1958). Research showed the pathogen was not affected by freezing salmon carcasses, but heat treatment (pasteurization) killed it. Subsequently, pasteurized fish carcasses and byproducts from fish processing were used in an open-formula, semi-moist

salmon feed called the Oregon Moist Pellet (OMP) that became the standard feed used in hatchery programs (Hublou, 1963). The OMP was produced in central locations by commercial companies, frozen and shipped to hatcheries. Hatchery-made feeds were dropped. The adoption of the OMP eliminated fish tuberculosis, improved hatchery water quality and supported consistent and predictable juvenile salmon growth at hatcheries.

The OMP had one major drawback; it was a highmoisture feed (~28%) and therefore had to remain frozen until it was fed. It was also relatively expensive.

Research by the US Fish & Wildlife Service at the Cortland Hatchery and the Abernathy Fish Technology Center in Washington State led to develop open formulas for dry, compression-pelleted trout and salmon feeds (Phillips et al., 1964; Fowler & Burrows, 1971). The open-formula feeds were produced by commercial feed companies who bid to supply state, federal and tribal salmon hatcheries, and commercial trout farmers. The formulations were later changed by feed companies and became proprietary, closedformula feeds. The most important advantage of compressed pellets compared to moist feeds was that pellets did not require frozen storage. Another advantage was that feed formulations for compressed could include a greater number of common feed ingredients than moist feeds because moist feeds required a precise combination of feed ingredients with binding properties to hold pellets together.

Compressed pellets have several disadvantages as aquaculture feeds. First, they are dense and rapidly sink in water. Second, the water stability of compressed pellets is limited. Third, they can fracture if not handled carefully during shipping and storage, creating dust (fines) that are essentially wasted feed. Finally, their high density limits the amount of fat or oil that can be applied by top-dressed pellets to a maximum of 18-20% total lipid.

Extrusion technology Using cooking-extrusion technology to produce fish feeds overcame the disadvantages of compressed pellets and propelled the aquaculture industry to much higher and efficient fish production.

The first major application of extrusion pelleting in aquaculture was by the US catfish industry. The

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density of extruded pellets can be controlled by the feed formulation and conditions of pelleting to produce floating, slowly sinking or sinking pellets.

Catfish are primarily farmed in large ponds and using floating feeds allowed farmers to see feeding activity and adjust feeding levels accordingly. However, when extrusion was first used to produce trout feeds by a major USA feed company, the results were disastrous. Feeding extruded pellets to rainbow trout caused high mortality from fatty liver disease. Trout farmers blamed the extrusion process and did not use extruded feeds for decades, even after extruded feeds were widely and successfully used in Europe by trout and salmon farmers.

Fatty liver disease in trout was, of course, not caused by the extrusion process per se but rather by using feed formulations designed for compressed pellets.

These formulations specified relatively high starch levels. Cooking-extrusion processing greatly increased the degree of starch gelatinization, thereby increasing starch digestibility and raising blood glucose levels.

Trout have limited ability to metabolize glucose, so excess glucose was metabolized into fat and stored in the liver, causing fatty liver disease. When starch levels were reduced in high-lipid, high-energy, extruded feed formulations, the level of digestible starch was also reduced to levels that salmon and trout could tolerate.

The adoption of extruded feeds by salmon and trout farmers improved feed efficiency, reduced environmental pollution from lost feed and reduced production costs. Control of feed density allowed feed producers to control feed buoyancy and increase lipid levels of feeds beyond 30%. The adoption of highenergy feeds allowed feed protein levels to be reduced, leading to lower feed conversion ratios, around 1.0 to 1.2 for commercial salmon farms. All of this improved the economics of salmon and trout aquaculture, making salmon the second-most valuable farmed fish species in the world, behind shrimp.

The period between 1958 and 2000 looks, in retrospect, to have been a golden age for fish nutrition research. New information on the nutritional requirements for a wide range of fish and shrimp species, combined with improvements in feed manufacturing processes, provided the foundation for tremendous increases in the amount and efficiency of global aquaculture production of fed aquaculture species. However, the rapid growth of aquaculture production created new challenges for the industry, notably bringing sustainability issues to the forefront of fish nutrition research. This will be the subject of the final installment of this series of columns.

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Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

MARINE INGREDIENTS

A functional protein hydrolysate to support better shrimp performance Mikael Herault, Paul Seguin, Pablo Antonio Zevallos Valero, Symrise Aqua Feed

Sustainable innovations are driving the development of the aquaculture industry and especially that of aquatic feed formulation. The 30-year-old trend to reduce fishmeal (FM) inclusion in formulas has led manufacturers to find new alternatives to marine raw materials. Co-products resulting from seafood and food processing are valuable sources of raw materials as long as freshness and supply chain consistency are guaranteed. Tuna, tilapia, squid, shrimp, whitefish as well as chicken bases are among the most common sources. If the meal process is today dominating the

valorization of such seafood co-products, the hydrolysis process can be considered a better alternative to generate new functionalities to finished products i.e. functional hydrolysates, like higher palatability, digestibility and bioactivities.

ActiTuna, a functional protein hydrolysate Functional hydrolysates are made by enzymatic hydrolysis of protein raw materials. The very specific equipment used by Symrise Aqua Feed, combined with the high level of control applied during the hydrolysis

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35

process ensures the production of standardized products with consistent performance from batch-tobatch over time contrary to many other processes and their resulting products. Such product performance is intimately linked to its peptide profile where quantification of peptide sizes and diversity are essential for predicting the responses of animals fed feed with functional hydrolysates. Any change in the peptide profile could lead to deviation in product performance, either in terms of palatability, digestibility or bioactivity.

ActiTuna is a product manufactured by Symrise Aqua Feed in Thailand and Ecuador, through a fully controlled hydrolysis (process) of tuna by-products, produced by the tuna processing industry. Symrise Aqua Feed transforms these raw materials into a functional product releasing a high proportion of bioactive peptides and other low molecular weight nitrogen compounds, so important in aquaculture species such as whiteleg shrimp. Such listed functional compounds will help stimulate shrimp feeding behavior, improve gut health and support animal immunity to help it better resist biotic and abiotic challenges.

ActiTuna can bring multiple benefits to aquafeed manufacturers and farmers: enhancement and standardization of feed performance, replacement of raw materials showing unpredictable quality, dietary balance of low molecular weight nitrogen compounds (nucleotides and peptides), in particular in low fishmeal feed formula, development of specific feed segments, better pond productivity and the guarantee of traceability.

Critical challenges met by shrimp farming industry The recent Rabobank shrimp industry survey (2020) confirmed that in 2020, the two first challenges met by the shrimp industry were feed costs and shrimp diseases. Growth rate, through feed intake and shrimp welfare, can also be compromised by environmental conditions when temperature or salinity changes are brutal, or when water turbidity is changing. Limited growth rates, and feed intake, will impair shrimp yield directly (harvested biomass) but also indirectly by impairing shrimp health status and resistance to disease outbreaks, or by resulting in longer growth cycles under unfavorable sanitary conditions.

With more than ten years of experience, Symrise Aqua Feed has performed hundreds of trials, in its own testing facilities (Aqualis) and with partners, for developing and analyzing many types of functional hydrolysates, assessing their performance in different species and under different testing conditions. The following data will give an overview of ActiTuna performance for different targeted benefits: higher growth rate through higher feed intake or assimilation, higher resistance to infectious challenges and resulting survival rates among others.

Bioactivity performance of ActiTuna protein hydrolysate - in vitro Among the many different bioactivities found in marine peptides, antimicrobial ones can find very interesting applications in shrimp. Dietary antimicrobial peptides might improve shrimp resistance to opportunistic

Figure 1. In vitro anti-microbial activity of ActiTuna. Each green bar represents an ActiTuna distinct peptide fraction while the black curve represents ActiTuna peptide size profile measured by HPLC.

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Table 2. Symrise Aqua Feed meta-study of trials implemented over ten years in whiteleg shrimp using ActiTuna as a performance enhancer ingredient. FCR: Feed Conversion Ratio, WGR: Weekly Growth Rate.

disease thanks to the enhancement of non-specific health, as well as the modulation of microbiota, reducing the relative load of pathogen bacteria, like Vibrio strains, in the bacteria population.

To demonstrate the antimicrobial performance of ActiTuna, we ran in vitro trials after a separation of the hydrolysate into 30 different peptide fractions based on their molecular weight sizes (HPLC). Each hydrolysate/ peptide fraction was tested on four different culture solutions of bacterial strains, responsible for major aquaculture diseases. After six hours, the absorbance was read to determine whether this peptide fraction had resulted in bacteria growth or inhibition. The results presented in Figure 1 demonstrate the presence of antimicrobials peptides in ActiTuna, effective against distinct pathogens.

More than 90% of the protein of ActiTuna is composed of peptides smaller than 1000Da. Besides antimicrobial activities, low molecular weight peptides and free amino acids are also involved in feed attractivity and palatability, as well as in feed digestibility.

Meta-analysis on ActiTuna used in shrimp diets During the last ten years, Symrise Aqua Feed has implemented 22 trials with ActiTuna included in shrimp feed: 14 of them were dedicated to the replacement of over-exploited marine resources (fishmeal or squid meals) while eight other trials were focusing on “a mere” ActiTuna supplementation (3% on average, from 6% in larval feed to 2% in grower feed) on existing commercial formulations, preferably by its inclusion in feed matrixes.

Results are consolidated in Table 2 with a colored logo illustrating the trend of each trial outcome and bold figures showing average performance values for each scope of use: fishmeal/squid meal substitution with ActiTuna combined to plant-based raw materials or ActiTuna supplementation (on top of the other raw materials of feed formula). In addition to the topic of research, we have distinguished as experimental factors: the experimental conditions and type of rearing facilities, and the type of process implemented to prepare experimental diets. To normalize performance

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Table 3. Expressed p-value of a two-way ANOVA implemented on 3 factors and 4 performance variables.

KPI: key performance indicators; FCR: feed conversion ratio; WGR: weekly growth rate.

Observed p-value for KPIs

Topic of study

Experimental Conditions

Yield (kg/unit of space) 0,001 FCR 0,003 Survival rates (%) 0,96 Growth rate (WGR: g/week) 0,07

indicators obtained under different rearing or dietary conditions, we’ve decided to base our analysis on the relative increase/reduction of performance observed between the ActiTuna diets and their control ones, expressed as a percentage.

In both scopes, the global trend confirms that using ActiTuna leads to improved shrimp performance, with a reduction of the average FCR and an increase in growth, survival and yield parameters. Nevertheless, it appears obvious that observed performance gains are much higher when the scope of the research was supplementation vs. the replacement of over-exploited marine resources, as for the last one, the objective is to reach, at least, the same performance as the control diet containing more fishmeal and/or squid meal.

For instance, on average, yield differences obtained between ActiTuna diets, and their controls, were 14% in the case of ActiTuna supplementation vs. 5% in the case of fish/squid meal substitution studies. A two-way ANOVA implemented on these factors and zootechnical variables confirmed that only the scope of research (factor: Topic) had had a significant impact on observed differences between these in vivo assays (Table 3) without questioning the positive outcome of marine meals replacement with plant proteins and ActiTuna functional hydrolysate. This may sound intuitive, but we experienced, and demonstrated in a consistent way, that it was possible to maintain shrimp farming productivity and even increase it, without necessarily relying on over-exploited marine resources, as long as the shrimp's dietary nutritional requirements were met.

Another key learning of this meta-study, and the related ANOVA analysis, is that in both scopes of research, fishmeal replacement or supplementation, the differences observed for survival rates were similar i.e. not significantly different. This indicates that ActiTuna potency as a health-enhancing ingredient was not nuanced by the significant replacement of overexploited marine resources.

Diet process

0,65 0,14 0,74 0,13 0,66 0,58 0,28 0,80

Finally, it is also noted that diet process, or experimental facility types, didn’t statistically influence the trends observed between ActiTuna diets and their respective controls.

A cost-effective performance enhancer ingredient in shrimp feed formulation This review illustrated the process of evaluation of novel ingredients as implemented by Symrise Aqua Feed to meet shrimp industry needs in terms of costeffective solutions enhancing shrimp performance, and productivity. It was demonstrated that these performance were tightly related to better shrimp survival rates, which were directly increasing shrimp harvested biomass while lowering feed production costs. In our studies, we have correlated the positive trend observed in terms of shrimp health performance under field conditions with lab in vitro and in vivo assays (results available upon request) demonstrating ActiTuna stimulation of shrimp immune defenses or antimicrobial potency against aquaculture major bacterial pathogens.

All experimenters know that in vivo trials, especially with shrimp, can be subjected to unpredictable and/ or unknown biases, resulting in unexpected results.

Only meta-study analysis, following several repetitions under different environmental conditions, can provide consistent and reliable conclusions about the efficiency of novel dietary solutions.

More information: Mikaël Herault Performance Measurement Manager Symrise Aqua Feed E: mikael.herault@symrise.com

Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

TECHNOLOGY

Understanding drying and cooling processes for pelleted shrimp feed Albert Wang, IDAH Co., Ltd.

Development of shrimp feed A few decades ago, a Japanese scientist Dr. Motosaku Fujinaga (also known as Dr. Hudinaga), became a pioneer in the technology of shrimp spawning and larval rearing. This technology development is an important milestone that marks the start of in-land shrimp cultivation. In the 1970s, commercial shrimp feed was sold to the public. These pellets are fast sinking and water-stable (Chamberlain, 2010).

Pelleted feed production in the early days Early day formulation of shrimp feed incorporated a high percentage of an aquatic animal protein source, such as fishmeals or squid meal, which was readily digestible for the shrimp digestive tract (higher FCR) (Yun et al., 2017).

The early pelleting process only incorporated a small amount of moisture and no significant drying process was needed.

The limitation and rising price of the animal-based feed source promoted the usage of a reasonable amount of plant-based protein, such as soybean meal as a protein source substitute (Hasan, 2001; Suárez et al., 2009). The incorporation of plant-based meals induced the need for a better ingredient cooking process because the cooking process can denaturate these plant proteins and promote starch cooking.

The first generation of shrimp feed pelleting system The first-generation (G1) of shrimp feed production machine, popular from the 1980s until the 2010s, consisted of a pellet mill, a post-conditioner, and a cooler. This machine setup can prolong the mash cooking while taking out a small amount of moisture. The pre-conditioner system with direct steam spraying incorporated into the pelleting system in the 1980s helped increase the moisture content of the mash by around 2-3% (wb). The higher moisture

Figure 1. The third-generation (G3) shrimp feed pelleting system development offers total moisture control and saves drying energy.

(IDAH, Taiwan)

improvement can increase the feed stability in water.

The following post-conditioning and cooling process will remove approximately 1-2% moisture from the feed, resulting in the finished product with a moisture content of 10-11%, a shelf-stable pelleted feed moisture content during storage.

Incorporation of the carousel dryer to shrimp feed pelleting lines Even though the G1 technology is still a popular option in many Asian countries, there is a growing demand to increase the incorporation of sustainable plant-based protein while keeping the water stability (Bae et al., 2020). One way to improve the water stability of plantbased protein is by adding moisture to the mash. The benefits of increasing the moisture content during mash cooking are the increase of starch cooking and

Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

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the lubricating effect of the mash when passing the pelleting hole.

Carousel dryer was first incorporated into the shrimp pelleting line back in 2013 in a large Indian shrimp feed manufacturing company. This started the secondgeneration (G2) pelleting system. This dryer can fulfill all conditions needed to dry the shrimp feed properly and produce high-quality shrimp feed. Nowadays, the G2 system has become popular and utilized in many different pelleting lines in Asia.

The incorporation of the carousel dryer after the postconditioning process in the production line opens up the possibility of increasing the mash moisture before the pelleting process, due to the ability of the dryer to take out 4-5% of moisture content from the feed. The significant improvement of G2, when compared with the G1 in shrimp feed drying, are: • Optimum drying moisture control. The carousel dryer can dry material with ±0.5% moisture variance, much less than the G1 (± 1%). Having adequate moisture control means the water content in pellets can be precisely controlled and indirectly reduce the loss of raw materials.

• The dryer optimally utilizes the drying air, thus minimizing energy use.

• The G2 technology can reduce steam usage for the post-conditioning system by up to 15%.

Latest drying and cooling technology for pelleted shrimp feed So what is the latest technology in drying and cooling shrimp feed? We considered all the requirements and the experience in the field and have developed a Generation 3 (G3) pelleting system that incorporates the dryer and cooler (TK-series) in one machine (Fig. 1).

IDAH sold the TK-series machine to China and India back in 2021 (Fig. 2). The two benefits of this G3 system when compared to G2 system are: • Total moisture control: easy and fast. The TK-series can produce feed with ±0.5% moisture variance in both dryer and cooler. This combination solved the high moisture variance problem that usually occurs when applying the conventional box cooler. This TK-series machine lessens the point to control in the production flow.

• Saving drying energy. In a carousel dryer, the counterflow drying airflow will support efficient heat transfer

Figure 2. The ±0.5% moisture variance in the carousel dryer-cooler lowers the amount of production flow control point in the shrimp feed pelleting production line. (IDAH, Taiwan)

and give a low exhaust air temperature. The system utilizes the air exhaust from the cooler as makeup air for the dryer. The dust-free makeup air discharged from the cyclone (or jet filter) still contains some amount of heat and can lower the energy use (1530%) and reduce the amount of air emission (30-50%) and decrease odor problems.

Conclusion In the past 40 years, there have been breakthroughs in shrimp feed production technology. Along with the changes in the formulation from animal-based to become plant-based, we also utilized new technologies to enable the production of high-quality shrimp feed. The main changes were, first, the moisture level increase during the pre-pelleting process, which improved the cooking process and water stability; second, the post-conditioning system prolonged the cooking of the pellets; and lastly, the incorporation of carousel dryers, which gave total moisture control (+/0.5%), energy-saving, and lower dust/odor exhaust that benefited feed producers.

References available on request.

More information: Albert Wang, M.Sc. Marketing Manager (IDAH Co., Ltd.) E: albert.wang@idah.com

Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

FEED QUALITY

Instant insights into aquafeed and ingredients with portable on-site analyzers Chris Larkin, NeoSpectra by Si-Ware

Aquaculture production is projected to increase globally by 8-15% over the next decade. Meeting the increased demand for seafood will require aquafeeds to meet nutritional and environmental sustainability standards.

But new pressures imposed on aquafeed manufacturers pose a challenge to meeting increased demand. Factors like unstable global markets for core commodity ingredients, new aquafeed ingredient sources, and market and end-use changes threaten to obstruct industry production.

Like other livestock feed producers, aquaculture feed manufacturers are challenged to create recipes for a

multitude of aquaculture feeds utilizing many different ingredients, often sourced globally, to balance the palatability and proper nutritional profile at the lowest price point.

Formulating feed recipes on a least-cost basis is not a simple task. On the quality side of the equation, aquaculture feed ingredients are natural products with inherent variability in composition based on growing environments, variety, processing, and storage conditions. Wheat bran can vary from 14-17% protein, as an example, while fishmeals can range from 60-75% protein depending on type and season.

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The cost side of the equation is equally complex. From production and supply chain factors in the last two years to the Russian aggression in Ukraine in 2022, the global commodity market has seen record prices in 2022 for many aquaculture feed ingredients. Wheat, for example, hit record prices above USD 1200/bu in March and May of 2022 before declining to around USD 800/ bu, still an 8-year high. Soybeans have shown typical trends, reaching prices of over USD 1700/bu in June of 2022, almost double the pre-pandemic price in the USD 900/bu range.

But it’s not all bad news. Necessity and conflict often elicit a reaction from innovators. Accurate and frequent analysis of feed ingredients can provide the insight needed to optimize the production of nutritionally complete aquafeed at reduced costs.

NIR to optimize production costs of aquafeeds Many aquafeed manufacturers are already familiar with the benefits of using NIR instrumentation technology to test ingredients for nutritional composition. This technology is commonly used in laboratories via benchtop instruments, but these instruments tend to be expensive and require skilled operators.

In terms of cost, NIR analysis is less expensive and faster than wet chemistry methods in general, with similar or better precision. For example, the cost of routine proximate analysis is only about one-third of the cost of wet chemistry, and the analysis is typically completed in less than a minute.

The benefits of frequent analysis can be seen throughout the aquafeed value chain. Incoming raw ingredients can be inspected to ensure they meet contract claims, providing evidence for claims where the quality is below specification and valuable data to take advantage of when quality is higher.

Aquafeed producers can use this information to adjust formulations based on commodity or ingredient prices and still maintain proper nutritional profiles for the various final compound feeds.

Final compound aquafeed can be monitored at production to ensure labeled specifications and at intake at fish farms to validate each batch of incoming feed. Industry leaders are creating calibrations for new ingredients, such as insect proteins, so feed manufacturers can properly incorporate novel new materials into formulations. However, the actual value of frequent NIR testing can only be realized by

Table 1. Calibration statistics from a soy meal calibration created on a current benchtop FT-NIR and a NeoSpectra by Si-Ware Scanner analyzer.

Reprinted with permission from Klevtech Consulting.

Leading Benchtop FT-NIR

NeoSpectra Scanner Portable FT-NIR

Protein

N SECV R2 Range

138 0.58 0.91 39.7 - 49.8

137 0.54 0.91 39.7 – 49.6

Fat

N SECV R2 Range

135 0.298 0.93 0.47 – 8.1%

145 0.32 0.95 0.4 – 8.7%

Fiber

N SECV R2 Range

138 0.3 0.75 2.49 – 7.67%

143 0.29 0.78 2.49 – 7.67%

Moisture

N SECV R2 Range

95 0.19 0.85 10.2 – 11.7%

95 0.19 0.83 9.5 - 11.5%

N = number of samples in the calibration set SECV = standard error of cross-validation in the calibration set R2 = coefficient of determination for the cross-validation in the calibration set Range = minimum and maximum reference values in the calibration set

Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

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easy, on-site testing throughout the product chain. As technology has advanced, NIR has left the confines of the laboratory and become portable and accessible to feed manufacturers in their facilities. NIR is about putting more information into the hands of the feed manufacturer and livestock farms.

Portable NIR With the cost of bench-top instrumentation ranging from 40 to 100K dollars and a concurrent requirement for a clean and stable environment and well-trained operators, many feed manufacturers are looking for reliable instrumentation that is portable, rugged, and adaptable to a wide range of sample types, feeding operations, and ways of handling feed. Additional requirements for the next-generation solutions are that they must also operate at every point in the manufacturing process (raw material through final product) at an affordable price point and still provide the benefits of improved efficiency and lowering costs.

Portable NIR devices have been on the market for some time, but many have not delivered the performance, reliability, and ease of use required for intuitive on-site analysis.

With the earliest examples of handheld NIR technology, the biggest issue was the poor transferability of calibrations from robust benchtop instruments to the portable platform. With nextgeneration spectrometers, such as those implementing MEMS technology, it is possible to eliminate these issues and transfer calibrations reliably. FT-based MEMs

spectrometers also deliver accuracy and performance comparable to much more expensive benchtop units, even when faced with challenging conditions.

One recent study examined the performance of a portable MEMS-based NIR analyzer from NeoSpectra

by Si-Ware with an industry-standard benchtop FT-NIR analyzer for soy meal analysis. Table 1 shows that the portable handheld NeoSpectra scanner performs as well as a benchtop instrument for protein, oil, fiber, and moisture analysis.

As the data above show, new portable FT-NIR analyzers can provide the insight required by aquafeed manufacturers to standardize consistent feed quality while minimizing raw material costs in an end-to-end quality program from ingredient to aquaculture farm.

Cloud-based Intuitive mobile apps bring smart-phone ease of use to the operator, and cloud-based software aggregates and summarizes reported data for powerful insights into the operation.

The next-generation portable NIR instruments are ushering in a new era of aquafeed analysis with affordable on-site analysis in easy-to-use handheld devices.

More information: Chris Larkin Senior Applications Engineer NeoSpectra by Si-Ware E: Chris.larkin@si-ware.com

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Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

at 785-284-2153 or visit us online at extru-techinc.com P.O. Box 8 100 Airport Road Sabetha, KS 66534, USA Phone: 785-284-2153 Fax: 785-284-3143 extru-techinc@extru-techinc.com www.extru-techinc.com

1/28/21 8:32 AM

FEED QUALITY

Managing moisture in aquafeed production John Williamson, Devenish

Extruding aquafeed requires high moisture addition during processing. Moisture is added at various stages of production: in the mixer, in the conditioner and the extruder. The moisture content of the raw materials during production is pushed to levels up to 25% to ensure good cooking, particle adhesion and starch gelatinization. Aquafeed raw materials need to absorb this high moisture in a very short period of time.

This is a challenge as raw materials used in producing aquafeed are generally hydrophobic and do not easily absorb moisture.

SmartMoisture, a new patented solution from Devenish, will accelerate moisture acceptance in feed particles during the various processes of extruding aquafeed. It is a novel patented technology that contains a combination of propionic acid esterified to glycerol, buffered organic acids, and surfactants.

The rate at which molds and bacteria grow after feed processing depends on factors such as pH, temperature, oxygen availability, and most importantly, water activity of the feed. The decision on what the final moisture content will be is based on risk. Lower moisture content in the final feed is targeted considering the volume of moisture used in the extrusion process and the hydrophobic nature of aquafeed raw materials to avoid free water or high-water activity. SmartMoisture technology is very effective in reducing water activity in aquafeed thereby reducing risk and allowing manufacturers to produce feed with higher moisture.

Stabilizing propionic acid SmartMoisture contains unique glycerol esters of propionic acid and surfactants. Glycerol esters of propionic acid, are covalent bonds that stabilize

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Figure 1. Stability of propionic acid from SmartMoisture.

propionic acid, allowing for a higher concentration and longer retention in aquafeed. The bond increases the retention time of propionic acid in feed ensuring longerlasting microbial inhibition (Fig. 1).

In addition, glycerol can hold up to 1,000 times its own weight in water. It works in synergy with surfactants.

Surfactants reduce the water’s surface tension, improving moisture distribution. At the same time, the glycerol acts as a humectant drawing the moisture inside the feed particles and locking it inside. The moisture is now locked into feed particles and water activity is reduced. The propionic acid attached to the glycerol is absorbed by the feed particles.

Aquafeed trial A trial was conducted by a commercial aquafeed producer who exports aquaculture globally. Due to challenges associated with shipping products to tropical regions, the company reduces its feed moisture to Table 1. Water activity (aW) of aquafeeds treated and not treated with SmartMoisture.

Figure 2. Higher SmartMoisture inclusion rates lead to higher aquafeed moisture.

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below 6% in order to keep water activity below their rejection value of 0.6 aW. The trial assessed three treatments: • Control: No SmartMoisture with moisture level reduced to 5.7% .

• Treatment 1: 0.5g/kg SmartMoisture with moisture level reduced to 8.5%.

• Treatment 2: 0.5g/kg SmartMoisture with moisture level reduced to 9.7%.

SmartMoisture was added to the mixer with all the usual liquid additions. Water activity was measured for all three treatments.

The humectant properties of SmartMoisture at the inclusion of 500g per ton stabilized the water activity when the aquafeed was left at a higher moisture content and even at a 4% moisture increase over the control, SmartMoisture achieved water activity below 0.55 aW (Table 1).

More tests were done and SmartMoisture inclusion was increased to 1 kg pushing moisture to 10.5 (Fig. 2).

SmartMoisture at higher inclusion allowed for lower water activity at higher moisture content and gave this

customer confidence to ship their aquafeed with higher moisture content.

The benefits of using SmartMoisture in aquafeed SmartMoisture’s novel technology allows aquafeed manufacturers to: • Manage moisture levels.

• Keep water activity low and stable.

• Improve steam absorption.

• Protect feed against microbial and mold contamination.

• Improve starch gelatinization.

• Save energy by reducing drying and cooling.

• Increase yield.

More information: Janus Fouche SmartMoisture Product Manager Devenish E: janus.fouche@devenish.com

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Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

COLUMN

Development of aquafeeds: Reflections and future perspectives II. The transformational years: 1980-2019 Louis R. D’Abramo, Professor Emeritus, Department of Wildlife, Fisheries and Aquaculture, Mississippi State University Thomas R. Zeigler, Senior Technical Advisor, Past President and Chairman, Zeigler Bros., Inc.

During the foundational years of 1940 -1979, basic knowledge of aquatic animal nutrition provoked the early development of feed manufacturing techniques.

This period was followed by an arbitrarily defined period of 40 years (1980-2019) when information about specific nutritional requirements began to be systematically generated. The accumulation of this information ultimately led to efforts of synthesis whereby noteworthy publications were produced to serve as sources of knowledge and guidance. For the manufacturing of aquafeeds, a notable transition was realized. The choice of feed ingredients in formulations was moving from a domain based on an indiscriminate and rather arbitrary choice to selection guided by scientifically-based knowledge that specifically targeted the nutrition and physiology of a species.

The aquafeed manufacturing sector resourcefully introduced technological improvements designed to achieve the efficient provision of nutrients to yield good survival and a reduction in the amount of feed consumed per increase in body weight (feed conversion ratio, FCR). These goals translated into an operational reduction in feed costs and the detrimental effects of feed waste on water quality. The aquafeed marketplace further compelled the industry to develop improved technologies of manufacture to address distinct needs.

As a result, the global aquaculture enterprise entered a period that was truly transformational with the revolutionary development and use of aquafeeds.

Synthesis of information about nutrient requirements of fish and crustacean species Many efforts to synthesize the growing knowledge of nutrition of aquaculture species began to occur resulting in published books, manuals, and handbooks (Fig. 1).

A chronology of some of the most notable publications that appeared from 1981 through 2011 follows.

In 1987, the Food and Agricultural Organization (FAO) published three “field documents” that were inspired by the utilitarian need to produce training manuals to promote an understanding of the nutrition and feeding of farmed fish and shrimp. Three publications that carried the same title, The Nutrition and Feeding of Farmed Fish and Shrimp with different subtitles, the essential nutrients (Field Document 2), nutrient sources and composition (Field Document 5) and feeding methods (Field Document 7), were prepared

by Dr. Albert G. J. Tacon. The manuals were derived from Tacon’s notes that he used to instruct trainees enrolled in the Fourth Senior Aquaculturists Training Course in Brazil during a period of approximately two weeks in 1986.

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Figure 1. Some notable texts were published from 1981 to 2011 during the transformational years. They provided nutrient requirements and feeding information that contributed to advances in aquafeed development.

Preceding these publications was a 1981 publication produced by the National Research Council (NRC) of the National Academy of Sciences (NAS) titled Nutrient Requirements of Cold Water Fishes. In 1988, the book Nutrition and Feeding of Fish was published. The author, Tom Lovell of Auburn University, focused on channel catfish nutrition and feeding, including a chapter on feed formulation and processing. A group of researchers contributed additional chapters that addressed practical feeding of tilapia, penaeid shrimp, eels, salmon, trout, and crawfish. In 1993, the NRC followed with a publication titled Nutrient Requirements of Fish and Shrimp. This publication was updated by a select group of international aquatic animal nutritionists and published by NRC in 2011. The pioneering publication, Fish Nutrition (1st edition), edited by Dr. John Halver, became available in 1979 and was followed by 2nd and 3rd editions edited by Halver and Dr. Ron Hardy, and published in 1989 and 2002, respectively. In 1997, the first publication that offered a comprehensive synthesis of crustacean nutrition, Crustacean Nutrition, edited by Drs. Lou D’Abramo, Douglas Conklin, and Dean Akiyama, was published by the World Aquaculture Society as part of its book series.

The fate of proposed standardized reference diets for nutrient requirement research As more information was being generated, a noteworthy effort, designed to promote a wellorganized generation of knowledge of crustacean nutrition, was collaboratively initiated by a 1985 publication by Dr. John Castell and colleagues in Canada and the United States. In the genre of Halver’s early work using a specific test diet to determine vitamin requirements of salmonids, they proposed a rather revolutionary approach designed to improve the value of crustacean nutrition studies. It was founded on the desire to reduce confusion arising from the lack of control of a very important experimental variable, the ingredient composition of the diet, used in the performance of nutritional experiments. The goal was to establish a universally available, standardized reference diet that would ideally be used by a majority of researchers in their nutritional requirement studies.

Accordingly, the knowledge of nutrient requirements of crustacean species that was being increasingly generated could take on a greater significance and application among research laboratories.

The ingredient compositions of two proposed “standard reference diets” were presented and the results of comparative testing with different crustacean

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species were reported. However, concerns arose about the ability to reliably provide the standard reference diet(s) to meet the research-based demand, and the possible inability to control inconsistencies in the nutrient composition of some ingredients and therefore different batches. Additionally, the lack of sufficient standardization of other experimental variables combined with differences in the feeding habits and habitat of a wide array of crustacean species introduced potentially confounding rather than elucidating information about nutrient requirements among species.

Clarification of the protein requirement of aquatic animal species The interest in the protein content of aquafeeds was fueled by its integral role in the growth and its proportionally higher contribution to the overall ingredient cost. Good relationships between the ratio of an essential amino acid relative to total essential amino acids (A/E ratio) of muscle or whole-body protein and those of the dietary requirements of the ten essential amino acids were identified. The application of this relationship proved to be an effective way to estimate dietary requirements of essential amino acids without the need to conduct time-consuming, doseresponse experiments for each essential amino acid.

This method was reviewed by Toshio Akiyama, Ichiro Oohara and Takeshi Yamamoto in a 1997 publication and they alluded to a specificity of essential amino acid requirements among fish species or families.

The publication, Dietary protein requirements of fishes: a reassessment, authored by Stephen Bowen in 1987, provided insight into the protein requirements of aquatic vs. terrestrial animals. His analysis of protein requirements found in published research results revealed how the median values of absolute protein requirements, defined as protein intake per gram of body weight consumed per day, were, for the most part, similar among aquatic and terrestrial species.

In contrast, the relative dietary protein requirements, expressed as a percent of dietary protein, were found to be higher for aquatic species. This unstandardized difference is actually a consequence of reduced consumption of food by poikilotherms (fish, crustaceans and other invertebrate organisms) to meet the need for dietary energy in comparison to homeotherms

(terrestrial vertebrates) that have higher energy needs. Consequently, the amount of feed consumed per increase in body weight (feed conversion ratio), an index of farm performance, was lower for fish and crustaceans. Other superior performance outcomes associated with aquatic species, such as edible protein yield and protein retention, were not addressed. However, this paradigm of comparative protein/energy requirements of aquatic versus terrestrial production species was foundational to the understanding of preferences in qualitative and quantitative dietary energy sources among different species of fish and crustaceans.

The rising consciousness of the roles of dietary protein and energy ultimately led to the recognition of the importance of dietary protein: energy ratios in the formulation of aquafeeds. Ratios were uniquely associated with physiological differences specific to life-history stages. Additionally, efficient utilization of dietary energy provided as lipid or carbohydrate was also species-dependent, principally associated with the feeding habits of the species. As a result, choice of feed ingredients used in the formulation of feeds was guided by the relative ability to use carbohydrates, lipids, or a combination as these sources of energy. This effort was aligned with the goal of minimizing the use of protein derived from comparatively high-cost ingredients as a source of energy, a focus termed “protein sparing”.

Specific requirements of lipid in addition to an energy source Lipid serves as a source of not only energy but also essential fatty acids, polyunsaturated fatty acids (PUFA) as well as long-chain polyunsaturated fatty acids (LC-PUFA). Choice of sources of dietary lipid would be guided by the provision and availability of these nutrients that are essential for growth and other physiological processes. For larval and juvenile forms of crustaceans, unique requirements for lecithin, often composed of an array of phospholipids have been identified. Those phospholipids with the greatest activity were determined to be phosphatidylcholine and phosphatidylinositol. For crustaceans, a dietary requirement for cholesterol has been consistently reported. Plant-derived sterols (phytosterols) have not conferred the same growth performance when substituted for cholesterol on a 1:1 basis. However,

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there is some evidence of the sparing of the cholesterol requirement using a comparatively small proportional amount of phytosterols.

Addressing particle size and feed stability in aquafeed manufacturing With the increased knowledge of nutrient requirements, the objective of effective provision of nutrients via stability and availability of manufactured aquafeed became an important area of investigation. The resulting improvements contributed to meeting overarching goals of environmental and economic sustainability of aquafeed.

Stability encompasses a variety of characteristics that include reduced rate of loss of activity of nutrients, maintenance of physical integrity when handled, ability to retain size and shape, and reduction of loss due to leaching. Availability refers to the size and enhancement of encounters for consumption. The increase in the number of farmed species of fish and crustaceans and respective life history stages introduced many challenges within the aquafeed industry. Most manufacturing goals were driven by the need to produce feed to meet size and feeding behavior requirements whereby efficient consumption and delivery of essential nutrients were achieved.

For example, animals produced in hatcheries and nurseries increased the demand for smaller feed particles for consumption. This specific need was effectively addressed by improvements in the ability to grind ingredients to finer particle sizes so that a homogenous mixture was maintained in the formulations. Grinding of feed ingredients to finer particles also helped enhance water stability and improved the digestibility of feeds.

Water stable feeds were especially critical to the success of the ever-increasing shrimp aquaculture enterprise globally. Maintenance of the physical integrity of the feed was important because shrimp were protracted feeders that commonly used their mouthparts to break up feed into smaller particles prior to consumption. The increased surface area and duration of exposure to water exacerbated the leaching of water-soluble nutrients. Specific dietary ingredients were added to formulations to confer stability in conjunction with the feed manufacture process and evaluated. Urea-formaldehyde was identified as a highly

successful binder, but the use of this and other effective chemical compounds was short-lived, eventually being prohibited in the European Union and by the Food and Drug Association (FDA) within the United States. In response to these policies, focus turned to the use of specific carbohydrates that would convey the desired level of feed stability.

A demand for larger feed size, from 1.27 cm to 2.54 cm, or even 3 to 4 cm in diameter, to satisfy needs for the farming of larger marine species also introduced challenges specifically related to manufacturing by extrusion. The larger pellets needed to be thoroughly dried to avoid deleterious mold growth and loss of nutrient activity within the core. However, utmost caution needed to be exercised because short-term drying at too extreme a temperature would risk denaturation of dietary protein and reduction of levels of activity of nutrients located on or close to the surface of the pellet. With the desire to increase the efficiency of production and economic sustainability, the salmon cage farming industry sought slow-sink feeds to maximize the time of encounter and consumption.

Through combined manufacturing modifications in extruders and forced hot air dryers and changes in proportional amounts of carbohydrate ingredients, successful operational protocols were established for the production of a slow sink, floating, and large pellet feeds.

Maximizing availability of dietary nutrients The objective of maximizing the availability of essential nutrients in aquafeeds has evolved and improved over time and is essentially founded on four strategies, use of highly digestible ingredients, supplementation to existing levels, protection/preservation of heatsensitive, highly degradable types, and provision of specific additives. Knowledge of the ability of particular aquaculture species to digest particular feedstuffs is essential in reducing waste and efficiently providing required dietary nutrients. The increased digestibility of dietary carbohydrates in a gelatinized form, a byproduct of feed manufacturing by extrusion, increased the amount of available dietary energy. New grinding equipment, designed to reduce the particle size of feedstuffs, contributed to increased digestion and was instrumental in the manufacture of small feed particles of homogeneous composition for juvenile

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and larval stages. Variable speed and temperature dryers associated with the manufacturing of extruded feeds were introduced with the objective of effectively reducing nutrient loss.

Use of feed additives to maximize performance, health and associated economic benefits The positive benefits of the inclusion of specific additives in formulated aquafeeds are well established.

New or improved additives in aquafeeds will first be based on the additional cost for effective inclusion followed by that cost passed on to the consumer relative to the performance realized. As a feed additive, dietary ascorbic acid in crystalline form is highly watersoluble and subject to degradation during relatively short durations (~ 30 days) of feed storage. Fish that consumed feed that contained reduced ascorbic acid activity due to prolonged storage were afflicted with a vitamin C deficiency called lordosis which is an inward curvature of the spine. Required dietary levels of vitamin C, provided in crystalline form, for crustaceans were reported to be 10-20x those of fish. The presumed higher dietary requirements were misleading and actually reflective of a significant loss due to leaching into the water due to a protracted duration of feeding and exposure of smaller particles (higher surface area) derived from the consumptive behavior of crustaceans.

A process developed by Seib and Liao to produce ascorbic tripolyphosphate, a compound composed of a mixture of mono, di, and tri phosphorylated esters of ascorbic acid, served to launch a major breakthrough for the aquafeed industry. Adding this compound to aquafeed extended the durations of feed storage by 90 to 120 days with a gradual loss of activity as influenced by temperature, moisture and sunlight. Products providing these ester forms of ascorbic acid are also comparatively less water-soluble. Dietary additives of ascorbic acid in the form of phosphorylated esters (Stay-C) are preferably used in aquafeeds to meet the vitamin C requirement and contain approximately 35% active ascorbic acid.

Plant-derived ingredients, commonly found in aquafeed formulations as grains and oil seeds, contain a compound called phytate which is able to effectively sequester minerals and thereby make them metabolically unavailable. To remove the “antinutritional” effects of phytate, an enzyme additive,

phytase, has been included as an aquafeed additive.

The presence of active phytase allows important dietary elements (minerals) that would otherwise be lost to the environment to become physiologically available for absorption. Like all enzymes, phytase is a protein that is heat sensitive and subject to denaturation during the process of manufacturing by extrusion unless precautionary measures are taken.

During the last decade of the 20th century, the physiological role of both probiotics and prebiotics and the advantage of inclusion in aquafeeds to promote disease resistance and growth through their influential role in the composition of the gut microbiome were revealed. This recognition has paralleled what occurred in the 1970s in the poultry and swine production industries. However, one of the major challenges confronting the application of probiotics and prebiotics as additives in aquafeeds is the myriad of aquatic species that are currently farmed globally. Species specificity would seem to be a prime determinant of success and accordingly, the concept of “one size fits all” for application in the aquaculture industry does not appear to be likely. The benefits of particular probiotics would most probably be influenced by such factors as the trophic level of species, phylogenetic family, and production system. Additionally, caution needs to be exercised based on the acknowledgment of the possible confounding interactive effects of both quantity and quality of these additives within commercially produced aquafeeds. Specific feed manufacturing precautions are needed to preserve desired activity. For the aquafeed industry to meet the anticipated demand of the marketplace, these additives must be characterized by predictability and consistency. Dietary supplementation of nucleotides has also been shown to enhance immunocompetence and stress tolerance of fish.

Loss and waste of dietary nutrients due to heat sensitivity are protected by either of two approaches, encapsulation or top coating. Encapsulation produces a barrier that prevents steam and high temperature of the extrusion process from diminishing or eliminating the level of activity of a nutrient.

Top coating was an innovation introduced by the aquafeed equipment industry. Quantities of lipid, alone or containing a heat-sensitive ingredient, are introduced into a special mixer wherein a vacuum is created. The lipid is then sprayed onto the recently

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Figure 2. The four directives collectively contribute to the production of economically and environmentally sustainable aquafeeds as part of a dynamic and ongoing process of development.

produced extruded feed while it is still warm, and is drawn into the feed when released at atmospheric pressure. Incorporation of this technique into the manufacturing process also serves to attain dietary lipid levels that are not possible under normal conditions of extruded feed manufacture. Feeds containing levels of lipid of up to 35% are possible. Additional lipid provides a greater source of energy than that available from equivalent levels of carbohydrate. These higher levels of dietary lipid can also be effective in sparing the use of dietary protein as a source of energy that will correspondingly reduce the excretion of nitrogen into the culture environment.

The feed manufacturing industry has also introduced vitamin and mineral premixes and crystalline sources of first limiting essential amino acids, commonly methionine, lysine, and threonine for fish. Arginine is often a limiting amino acid in crustacean feed formulations and commonly will require supplementation. Taurine, a product of the metabolism of sulfur-containing amino acids, does not contain the characteristic carboxyl group of amino acids and has been characterized as a conditionally required metabolic nutrient. It is added in powder form to feeds formulated for marine and freshwater fish and shrimp species to compensate for deficiencies when plantbased sources of dietary protein proportionally exceed

those that are animal-based. If needed, the level of supplementation appears to be influenced by species, life history stage, and process of feed manufacture. For shrimp feeds, the efficacy of crystalline amino acids and taurine as additives to meet dietary requirements is problematic based on solubility in water. Protracted periods of leaching into the aquatic medium occur due to characteristic delays in consumption of feed that is broken into smaller sizes. A possible option may be the inclusion of chelates of 2-hydroxy-4-methylthiobutyrate which are significantly less soluble in water, However, cost considerations vs. performance level must be considered. Reduction of activities of certain vitamins and mineral availability occurs due to manufacturing conditions and leaching. Accordingly, the dietary levels of these required nutrients that are added compensate for these losses.

Sustainable ingredient substitutes in feeds For decades, marine-derived meals and oils have been the mainstay ingredients in aquafeeds produced for fish and crustacean aquaculture. It has become clear that these particular ingredients themselves are not critical, but rather the essential nutrients they contain.

Thus, an ongoing objective was launched to produce cost-effective feeds using highly available plant-derived alternatives to these ingredients, such as soybean meal,

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processed soybean meal, high protein corn gluten meal and corm distillers dried grains. These feedstuffs are commonly consistent in nutrient composition and not subject to vagaries in availability; however, as singular substitutes, they have inherent nutrient deficiencies.

Mixtures of these sources appear to be a plausible solution. Other possible substitutes that potentially have merit are single-cell protein sources and insect meal as demonstrated by success in laboratory-based research.

These potential feed ingredients also have the added advantage of consistency in a composition achieved through highly controlled production parameters.

The process of the manufacturing of aquafeed has embraced the circular bioeconomy approach by using feed ingredients that are recycling products. In addition, byproducts (waste) of the rendering of terrestrial animal production offer highly acceptable and efficient substitutes with the added feature of their reduced carbon footprint. The search for oil sources that can potentially serve as effective substitutes for marinederived oils as sources of essential PUFAs and LC-PUFAs in sustainable aquafeeds remains an ongoing challenge.

Conclusion At this point in our historical journey, we offer what we believe to be overarching directives that have evolved in concert with transformational changes fueled by

advances in knowledge of aquatic animal nutrition and the ensuing market demands for manufacture of high performance aquafeed (Fig. 2). As established, these “directives”, are reflective of the quintessential goal of producing environmentally and economically sustainable aquafeed. Future research endeavors that yield more knowledge about the nutritional physiology of farmed aquatic species and will be complemented by dynamic innovation in feed formulation and methodology of the manufacturing of aquafeed. The greatest challenge resides in the variety of fed species being cultured globally. Collectively, some of the contributions based on each of the four overarching directives will take on a species-specific quality.

Aquafeeds will move to a level of being characteristically “designed” to meet the needs of different species or arrays of species and their respective production systems. In the next issue of Aquafeed Magazine, the third and final part of the history of the development of aquafeeds will be presented as Development of Aquafeeds: Reflections and Future Perspectives III. The Sustainable Future. We will take on the challenge of addressing the fate of the most current trends. We will engage in speculating novel strategies that may prove to be rewarding in meeting the need to produce sustainable aquafeeds as an essential component of sustainable aquaculture enterprise globally.

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PHYTOGENICS

Applications of phytogenics in shrimp farming Ruby Yu, Teresa M. Agulles Teixidó, PlusVet Animal Health

For the past two decades, the farming industry is moving towards a reduction in the use of antibiotics to prevent the emergence of antibiotic-resistant strains in humans. Feeds without antibiotic growth promoters (AGPs) are a reality in many countries, while others are looking into ways to restrict them. Different types of feed additives can be used to replace antimicrobial growth promoters, always combined with other types of interventions such as changes in feed formulation and improvements in biosafety and management practices.

Since prehistoric times, humans have used plants to cure their own diseases and those of their animals.

In the past 150 years, the use of medicinal plants has been shown to have a scientific basis: plants contain an abundance of chemicals, called “secondary compounds” or “phytochemicals”, that enable them to perform metabolic and ecological functions, such as resisting attacks by microorganisms and insects, and have been proved to exert useful health-promoting effects in human beings and animals.

In the world of animal nutrition, plant derivatives are called “phytogenics” or “botanicals”. The group includes plant extracts, essential oils, purified natural compounds such as saponins, flavonoids, and tannins, and synthetic compounds that are identical to natural substances.

Mechanisms of action of phytogenics Since antibiotic growth promoters were banned in Europe in 2006, phytogenics have emerged as an effective alternative and their use is becoming widespread. When they began to be used on commercial farms, their mechanism of action was poorly understood, which made it difficult for nutritionists to include phytogenics in their feeds.

However, in recent years, research work has multiplied,

and it has uncovered that the mechanism of action of plant derivatives as AGPs replacers is complex and multifunctional. Such complexity represents an advantage over other types of feed additives.

Antimicrobial activity Many phytogenics are microbiocidal against Grampositive and Gram-negative bacteria, yeasts and fungi.

As the microbiocidal activity of phytogenics arouses interest as substitutes for antibiotics, a multitude of research works has been carried out trying to determine their mechanism of action.

It has been determined that their antibacterial activity takes place through different mechanisms: • Alteration of quorum sensing. When the number of bacteria within a population is large enough, each individual secretes and receives small signal molecules that allow the whole colony to act as a group. This communication system is called quorum sensing.

Thanks to quorum sensing, bacterial colonies can form biofilms, become more virulent and secrete toxins, multiply faster and cross the intestinal barrier to enter the bloodstream. Since quorum sensing is responsible for pathogen virulence, finding active principles that can inhibit this communication system is a promising strategy to control pathogenic bacteria.

Many phytogenics have been proven to disrupt bacterial quorum sensing at concentrations much lower than those needed to kill bacteria.

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• Direct effect on the bacterial cell. Toxicity to bacteria can occur in different ways, but the best known is the disruption of the bacterial wall. Thanks to the lipophilic nature of many phytochemicals, they bind to the bacterial wall and increase its permeability, which ultimately results in the death of the microorganism. Gram-positive bacteria are more sensitive to phytogenics, as their plasma membrane is covered by a cell wall layer consisting of peptidoglycans, which are lipophilic. On the other hand, the outermost layer of Gram-negatives, called the outer membrane, is made of hydrophilic lipopolysaccharides, making it more difficult for phytogenics and other hydrophobic antimicrobials to penetrate. When formulating products based on plant-derived compounds, it is possible to weaken the outer membrane of the Gram-negative bacteria by adding permeabilizes, substances that act by disintegrating the outer membrane.

A little-known aspect of the use of plant derivatives is that, when antibiotic therapy is necessary, phytogenics and antibiotics often act in synergy, increasing the effectiveness of the antibiotic.

Antiparasitic action Several phytogenics have been confirmed as antiparasitic agents. Certain essential oils kill ciliated protozoa and flagellates by disrupting their cellular structure. In the case of nematodes, trematodes and leeches, phytochemicals damage their cuticle and their gastrointestinal tract. The mechanism of action of Myxozoans and Monogeneans is unknown.

Antioxidant and anti-inflammatory activity In healthy animals, protection against the harmful effects of free radicals is achieved through maintaining a delicate equilibrium between the production of free radicals, that are needed for certain physiological processes, and their destruction through the antioxidant defense system. This equilibrium is called “the redox balance”.

Whenever the redox balance is disrupted, either due to an overproduction of free radicals or because of a malfunction of the antioxidant defense system, free radicals trigger a harmful chain reaction, called oxidative stress, that damages all kinds of biological molecules and starts inflammatory pathways.

A growing body of evidence in vitro and in vivo shows that some phytogenics are powerful antioxidants and can prevent and ameliorate the dysfunctions caused by oxidative stress.

On the other hand, inflammation is a complex biological reaction to a harmful event, such as the presence of oxidative stress, pathogens, toxins, or damaged cells. Although inflammation is a beneficial process aimed at the repair of the damaged tissue, when it persists, it may lead to disease and productivity losses. Intestinal inflammation is associated with poor nutrient absorption, the disruption of the gut barrier function and the diversion of nutrients and energy that would have been destined for growth toward the inflammatory reaction.

Some compounds derived from plants have anti-inflammatory effects, either thanks to their antioxidant activity or by blocking metabolic processes related to inflammation.

Stimulation of the immune system Immunostimulants are compounds that stimulate the non-specific defense mechanisms in shrimp. When there is immune stimulation, the number of hemocytes circulating in the hemolymph increases.

Research has demonstrated that some plant-derived compounds increase the count of hemocytes by more than 60% in healthy animals and by more than 30% during diseases. Cellular immune responses and the secretion of immune-related enzymes, such as phagocytic activity, superoxide anion production, phenoloxidase, acid phosphatase, alkaline phosphatase and lysozyme activity are also increased.

Use of phytogenics in shrimp farming Most of the studies on the application of phytogenics have been conducted with poultry and pigs.

However, since fish and shrimp farming industries are constantly under threat due to the outbreak of infectious diseases, there is increasing interest in natural products, not only to replace antibiotics and promote productivity but also to prevent diseases.

The detailed mechanisms of action of phytogenics on fish and shrimp have been little studied compared to other species.

The following are the main applications of phytogenics in shrimp farming, according to our field experience.

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Productivity improvement Application of plant-derived compounds results in better productive parameters, such as: • Yield of the crop increased by 15-35%.

• 16 - 4 4% more survivability in healthy animals, and 35-78% more survivability during certain diseases.

• 5-18% better final weight.

• 3-10% better feed conversion ratio.

Successful start at the hatchery Phytogenics in water-soluble forms, added to water tanks at the hatchery, help reduce the load of pathogenic bacteria, promote growth and enhance the survival rate.

Figure 1. Culture in Vibrio parahaemolyticus agar, at 35°C for 24 hours. Inoculum of 5*104 CFU/plate. According to results, 0.5 g/L PhytoAqua slightly inhibited the growth of Vibrio parahaemolyticus, and 1 g/L PhytoAqua or higher more completely inhibited its growth.

Improvement of sensory quality and shelf life The administration of certain phytogenics improves the sensory quality of shrimp meat by increasing the percentage of taste-related amino acids. On the other hand, antioxidant phytochemicals delay lipid oxidation and prolong shrimp’s shelf life.

Balance of gut flora In recent years, it has been discovered that the digestive flora is involved in processes as diverse as feed digestion, immune response and inflammatory reactions.

Phytogenics are able to modulate the composition of bacterial communities, positively impacting the functioning of the digestive system and the overall health of the animal.

Prevention of diseases Outbreaks of infectious diseases are continuously endangering shrimp farming around the world. Much of the losses are induced by infections of bacterial origin and among the different types of pathogenic bacteria, the genus Vibrio stands out. Vibrio sp.

Figure 2. Culture in Vibrio parahaemolyticus agar, at 35°C for 24 hours. Inoculum of 3.7*105 CFU/plate. According to results, 0.5 g/L GrowthPlus significantly inhibited the growth of Vibrio parahaemolyticus, and 1 g/L GrowthPlus or higher more completely inhibited its growth.

Figure 3. Culture in Vibrio parahaemolyticus agar, at 35°C for 24 hours. Inoculum of 3.7*105 CFU/plate. According to results, 0.5 g/L AroMar slightly inhibited the growth of Vibrio parahaemolyticus, and 1 g/L AroMar or higher more completely inhibited its growth.

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are Gram-negative bacteria, which are found in seawater environments all around the world, and cause lesions, immune suppression, discoloration and death. Overuse of antibiotics to control vibriosis has led to drug resistance.

Vibrio parahaemolyticus is the causative agent of hepatopancreas necrosis disease (AHPND). AHPND can cause up to 100% mortality within 20-30 days and is an emerging disease that has severely damaged the global shrimp industry. Figures 1-3 show a trial testing three products in vitro to prove their antibacterial effect against Vibrio parahaemolyticus, which lays the basis for their application in aquaculture. All three products contain phytogenics and permeabilizing agents to increase their effectiveness against Gram-negatives.

Field trials that have not yet been published show that these products are effective in reducing the total number of Vibrio bacteria found in the hepatopancreas and intestine of white shrimp.

Conclusion Plant-based additives have been used in aquaculture for relatively few years, and are still unknown to many

farmers and nutritionists, but they are proving to have great potential, either to prevent disease, such as to replace antibiotics and promote growth. It is necessary to continue working, both in the investigation of their mechanisms of action and in the demonstration of their effects in the field, to achieve more scientific and targeted application.

More information: Ruby Yu Research and Development PlusVet Animal Health E: yuhaixia@plusvet.cn

Teresa M. Agulles Teixidó Director of Operations PlusVet Animal Health E: operations@plusvet.eu

Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

PHYTOGENICS

Natural oregano essential oil sustainably supports gut health for optimal performance Lawrence Brown, Anpario plc

In aquaculture production, supporting optimum gut health can have a beneficial impact on health and performance. Ensuring a healthy gut, in terms of both the gut structure and microbial population, can be fundamental in maximizing producer profitability which is of great importance, particularly with rising costs of raw materials and feed manufacturing.

A well-balanced and diverse gut microbiome is known as a state of eubiosis. It is well recognized that eubiosis has a profound impact on metabolic and immune homeostasis, ultimately having a direct effect on fish and shrimp health and wellbeing. Optimal gut health is associated with the proliferation of beneficial bacteria, as well as assisting in natural digestive processes and endogenous enzyme activity. This helps support the absorption of nutrients, leading to improved feed conversion and better growth performance.

With proven efficacy across a multitude of species, phytogenic feed additives can provide a multitude of benefits in supporting aquaculture health and performance. The Orego-Stim range from Anpario is developed from a unique source of 100% natural oregano essential oil (OEO) and contains multiple compounds which work in synergy to offer several welldocumented properties and functions. This includes antimicrobial, anti-inflammatory, immunomodulatory and antioxidant functions. Such properties mean that fish and shrimp health and performance can be maintained while supporting antibiotic-free production systems.

While there are many phytogenic products available on the market for aquaculture producers, with the aim of supporting profitability, aqua health and performance and a reduction in antibiotic use, it is

important that a natural source of the plant oil is used for optimum efficiency. 2009 research conducted on catfish has shown that natural sources of OEO, such as that found in Anpario’s Orego-Stim range, are more efficient than synthetic oils. Zheng et al. (2009) reported a significantly greater body weight gain, a significantly improved feed conversion ratio and numerically greater

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Figure 1. Effect of Orego-Stim Plus (Anpario plc) supplementation on shrimp performance and incidence of infection.

survivability in catfish supplemented with Orego-Stim compared to synthetic sources of thymol, carvacrol or a combination of the two.

Shrimp trials Oxytetracycline (70%) (OTC-70) is an antibiotic commonly used in shrimp farming for the control of Necrotising Hepatopancreatitis (NHP) and other bacterial diseases. A commercial study was recently undertaken at a shrimp farm in Ecuador. This farm routinely used 7 kg/tonne of OTC-70 for one week to assist in the control of NHP. The study aimed to determine if Orego-Stim Plus could maintain shrimp performance and a low incidence of bacterial challenge in the absence of OTC-70. The pond contained 952,000 shrimp with a starting weight of 8.3 g. Shrimp were fed a standard diet supplemented with 6

A

kg/tonne of Orego-Stim Plus for one week, instead of the 7 kg/tonne of OTC-70.

Shrimp diets supplemented with Orego-Stim Plus maintained shrimp health and performance. Shrimp demonstrated above-average growth compared to previous OTC-70 treatment, decreased mortality, and a reduction in NHP, White Spot Syndrome Virus (WSSV) and Vibrio (Fig. 1). This particular farm estimated a cost saving of approximately USD 500 over the course of the one-week period when using Orego-Stim Plus compared to the cost of treating shrimp with OTC70. Supplementation of this phytogenic containing 100% natural OEO not only offers producers the potential to reduce production costs but can also help reduce industry antibiotic usage, safeguarding their future efficacy.

B

C

Figure 2. (A) Average increase in total biomass (kg) over the study period. (B) Average feed conversion ratio (FCR) for the study period.

(C) Average mortality (%) over the study period.

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Tilapia trials In addition to proven efficacy in shrimp production, trials have demonstrated success in tilapia production, particularly in the absence of antibiotics. Florfenicol is a broad-spectrum antibiotic, commonly used to treat bacterial infection in aquaculture species.

A commercial trial conducted in Mexico over a 28-day period showed that 100% natural OEO supplementation provides a cost-effective solution to maintain tilapia production in the absence of florfenicol. Tilapia were subjected to one of two dietary treatments, either a medicated diet with florfenicol (20 mg/kg biomass from 1-7 days) or a standard diet supplemented with OEO (Orego-Stim Plus at 5 kg/t from 1-14 days).

Compared to tilapia treated with florfenicol, supplementation of the natural, sustainable OEO additive resulted in a 50% higher total biomass increase (Fig. 2A) and a 23% improvement in feed conversion ratio (Fig. 2B). In addition, mortality figures were 5.1% lower than tilapia treated with florfenicol (Fig. 2C), reducing the incidence of mortality by 48%. These benefits resulted in a return on investment of 30:1 for this production system.

Conclusions The Food and Agriculture Organization of the United Nations reported that fish from aquaculture for human consumption was projected to rise to 59% by 2030, an increase of approximately 19 million tonnes from 2022 to 2030. It is, therefore, more important than ever to provide aquaculture producers with natural, sustainable solutions to help meet the demands of the growing population, while supporting antibiotic-free production in the fight against antimicrobial resistance. The properties of 100% natural oregano essential oil make it one such solution, helping support aquaculture gut health to optimize health, performance and producer profitability in the absence of antibiotics.

References available on request.

More information: Dr Lawrence Brown Senior Technical and Account Manager for Aqua Anpario plc E: lawrence.brown@anpario.com

Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022

PHYTOGENICS

Antioxidant technology helps optimize feed costs and performance Dr. Stephanie Ladirat, NUQO© Every day, animals face various kinds of stress: changes in diet or raw material quality, hot temperature, diseases, handling and manipulations, etc. Stress triggers the production of free reactive radicals in cells that oxidize lipids or proteins. These free radicals are normally regulated but under stress conditions, free radicals’ level can increase dramatically and results in significant damage to cell structures and, to some extent, affect the performance and/or health of the animal, including fish and shrimp species.

A strategic approach of natural antioxidants for aquaculture In order to avoid negative effects from oxidative stress, cells contain naturally present antioxidant mechanisms.

In an ideal situation, a balance between oxidation and antioxidant mechanisms is established safeguarding cell integrity and function. Antioxidant mechanisms mitigate cell oxidative stress by neutralizing reactive radicals.

In highly productive animals or during stressful periods, additional components with antioxidant capacity are often provided on top of the naturally present antioxidant mechanisms in the body to support the health of animals. Many ingredients have shown interesting antioxidant effects. More precisely, several groups of molecules have shown an effect to help balance oxidation and antioxidant mechanisms in the organism. Among these groups, different types of natural ingredients containing polyphenols have shown antioxidant effects.

Polyphenols are natural compounds present in plants with numerous biological activities. Several studies have explored the properties of polyphenols in antiinflammation and oxidation, the mechanisms involved in signaling pathways activated upon oxidative stress,

as well as the possible roles of polyphenols in specific disorders. Polyphenols are present in herbs, spices, stems, flowers, etc. and even seaweeds. They are the secondary metabolites of plants involved in the defensive system.

It is difficult to determine the best source of natural antioxidants. Environmental factors, such as the type of the soil, exposure to light, rainfall, culture methods, and fruit yield per tree, have a strong impact on final concentration and activity. In vitro methods are a good preliminary approach to evaluating antioxidants, nevertheless, in vivo trials are necessary to validate solutions. The scientific approach to determine the

Figure 1. Antioxidant activity and variability among sources.

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ideal solution can therefore be split into two steps: the evaluation of individual ingredients and their variability; and the formulation of optimal ingredients.

To illustrate the variability of ingredients, a recent work (Fig. 1) measured the antioxidant activity of phlorotannins from various seaweeds and various geographic origins. Phlorotannins are a type of polyphenols found in brown algae such as kelps and that can only be found in seaweeds. This study showed that between seaweeds or even for the same species, variability can be very high. The same applies to other plants. This is why formulators need to carefully select the source and the quality of antioxidants to guarantee consistent quality and efficacy in feed.

Regarding formulation or the selection of solutions, considering the numbers of natural antioxidants, this can be a very time-consuming activity, but not to say a total waste of time. More importantly, several studies have highlighted the potential synergies between different sources of polyphenols (Liu, 2004). When combining polyphenols from four different sources, the dose-response curve of antioxidant activity was shifted to the left and the median effective dose (EC50) of the combination was five times lower than the EC50 of each single source, suggesting synergistic effects when combining sources. As a matter of fact, combining various sources of polyphenols looks like the most strategic and most resilient approach.

Strengthening the antioxidant pool for more resilience Countless studies have shown the potential of natural antioxidants to strengthen or complement the activity of other antioxidants, such as vitamin E, in feed. This effect is well documented, for many commercial solutions. The interest for such application is driven by technical and economic considerations and is of high interest in case of a price crisis.

Recent trials made with an exclusive blend of natural antioxidants, including polyphenols from fruits, vegetables and seaweeds, showed how these solutions could be used to optimize formulations and maintain the performance of animals. There is now sufficient evidence globally to invest in such a strategy, even though this depends of course on the level of supplementation and the formulation of feed, as well as the economic environment and potential savings.

Figure 2. Impact of stress on farm animals and possible applications of antioxidants.

Optimizing feed costs and performance While farm animals are expected to perform at the top of their genetic potential, oxidative stress tends to occur during normal conditions as well.

Supplementation of natural antioxidants supports the animal to overcome all kinds of environmental stressors during its highly productive life. It can also be used to optimize formulation and vitamin supplementation.

Recently, a trial was set up to measure the effects of partially replacing vitamin E (control) with a solution combining polyphenols from selected plants and seaweeds/algae origin. In this test, shrimp received a commercial diet with a level of vitamin E equivalent to 600 IU or alternative treatments where part of Vitamin E was substituted with a combination of polyphenols from plants and seaweeds. Results showed that replacing >50% of the vitamin E with the new technology did not impair the performance of shrimp. The alternative treatments (combinations with vitamin E and natural antioxidants) even numerically decreased FCR and improved survival rate at 45 days.

Regarding the impact on economics, the combination triggered substantial savings for the producer for equal performance. In addition, the producer got the benefits and income of the additional yield per pond observed (NUQO©, 2021).

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Optimizing formulation and securing performance In conclusion, natural antioxidants based on a rigorous process of selection and formulation, can be considered a flexible tool for nutritionists, to optimize feed formulations over time, and represent as well a good tool to mitigate the negative effects of various stress and challenges. The new generation of natural antioxidants, combining high quality and consistent sources of phytogenics and phycogenics (ie, metabolites from plants and seaweeds) now offer new perspectives to nutritionists to support the performance of shrimp and fish farms, reduce health challenges, maintain performance and/or reduce feed costs.

The results of this field trial showed that partially replacing vitamin E with this new technology, combining polyphenols from plants and seaweeds, did not impair growth performance after 42 days. Including this technology in the diets thus allows nutritionists to optimize feed formulation to reduce cost or manage risks during crises or price increases of ingredients like vitamin E.

More information: Dr. Stephanie Ladirat Technology and R&D Director NUQO© E: info@nuqo.eu

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Aquafeed: Advances in Processing & Formulation Vol 14 Issue 3 2022