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Vol 13 Issue 4 October 2021

AQUAFEED Advances in processing & formulation An Aquafeed.com publication

ADVANCES IN PROBIOTICS Byproduct-based fishmeal and hydrolysates Mineral recommendations in seabream Intestinal conditioners Published by: Aquafeed.com LLC. Kailua, Hawaii 96734, USA www.aquafeed.com info@aquafeed.com

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AQUAFEED

VOL 13 ISSUE 4 2021

Contents

NOVEL STRAINS OF BACILLUS SUBTILIS IMPROVE SHRIMP AQUACULTURE 28 Three B. subtilis strains can effectively remove the nitrogenous wastes from the culture water and potentially keep the pathogen load under control in commercial shrimp farms.

SHRIMP PROTEIN HYDROLYSATE 16 A new type of protein hydrolysate derived from shrimp via enzymatic hydrolysis is a potential feed ingredient to enhance the palatability of feed and improve the growth and health of aquatic animals.

INTESTINAL CONDITIONER PRONUTRIENTS 47

MICROMINERAL RECOMMENDATIONS FOR GILTHEAD SEABREAM 54

Pronutrients are an effective and natural tool that can be included in tilapia diets to improve culture performance.

A series of studies conducted in juvenile seabream aimed at better understanding the requirements of Mn, Se and Cu in diets with high levels of plant ingredients.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

AQUAFEED

VOL 13 ISSUE 4 2021

Contents

6 Interview: Ardi Budiono, President Director PT Suri Tani Pemuka 10 News Review 15 The revolution of on-site liquid enzyme production in aquafeed 16 Empowering productive and sustainable aquaculture through bioactive marine peptides

21 Byproduct-based fishmeals: Adding to the future of fishmeal production

Novel strains of Bacillus subtilis for improving water quality and controlling pathogens in shrimp aquaculture

33 Hydrolyzed Kluyveromyces fragilis: The all-in-one solution for fish and shrimp

east probiotics to boost fish gut health and improve Y feed efficiency *Cover photo

arine probiotics to positively balance shrimp microbiota M and promote better growth and survival

47 50 54

Effects of intestinal conditioner pronutrients in aquaculture diets

59 62

The gut health revolution

Juvenile gilthead seabream (Sparus aurata) nutrition: Defining micromineral recommendations for more sustainable aquafeeds

A natural algal astaxanthin for aquafeeds and beyond

Innovative tools to improve monitoring of the nutritional supply chain in aquaculture

Columns 26 Steve Dahlblom – Fishmeal outlook 66 Albert Tacon – Aquaculture and aquafeed production in 2019

Calendar of events

To read previous issues in digital format or to order print copies, visit: http://www.aquafeed.com/publications/aquafeed-magazine/

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

New protein ingredients, tailored for aquaculture nutrition. The Andersons is proud to offer ANDVantage 40Y and ANDVantage 50Y highly digestible plant-based feed ingredients that are rich in protein and spent distillers’ yeast. To learn more about how ANDVantage 40Y and ANDVantage 50Y can benefit your aquaculture feed formulations, please call 866-653-1892 or visit us online at www.andersonsgrain.com. The Andersons has been a trusted partner since 1947.

Available in 40% and 50% crude protein options.

Ardi Budiono is President Director of PT Suri Tani Pemuka (STP), Aquaculture Division of JAPFA Group.

INTERVIEW AQ: Please tell us about yourself. What has been your journey in aquafeeds? How did you get to where you are today? AB: I am President Director of STP, Aquaculture Division of JAPFA, one of the leading agri-food companies in Asia. Based in Jakarta, I joined JAPFA over 20 years ago and have led the Aquaculture Division since 2017. Prior to leading the Aquaculture Division, I served in various managerial positions as Head of Feed for JAPFA India and, previously, as Operations Manager in the aquaculture operations. Now I am responsible for the company’s

with Ardi Budiono increasing development and expansion efforts, both domestically and internationally in emerging markets. AQ: STP is known as one of the few fully integrated shrimp operators in Indonesia, and one of the biggest. What is the scope and size of the business? AB: We are proudly the market leader in the aquaculture industry in Indonesia. STP focuses on integrated aquaculture and operates aquafeed mills, shrimp hatcheries and ponds, freshwater fish hatcheries, tilapia grow-out and seafood processing facilities.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Our operations cover customers’ needs and inquiries from all over Indonesia. Upstream, we operate five aquafeed mills in Lampung, Gresik and Banyuwangi in East Java, Medan and Purwakarta in West Java. We also operate shrimp hatcheries in Aceh, Canti in Lampung, Anyer in Banten, Indramayu in West Java, Banyuwangi in East Java, Singaraja and Negara in Bali, Sumbawa and Makassar, while our freshwater fish hatcheries are in Jogjakarta, Purwakarta and West Kalimantan. Midstream, we operate shrimp ponds in Bomo, Karang Tekok and Situbondo in East Java. We also operate tilapia hatcheries and grow-out in Simalungun, Medan. Downstream, we operate three processing, cold storage and value-added facilities for both retail and B2B markets, tilapia processing in Simalungun, Medan, and fish processing in Banyuwangi, East Java. To support the success of our farmer customers, we offer a full array of customer and technical services through our supporting departments such as Shrimp Farm Technician (SFT), Fish Farm Technician (FFT) and Aquaculture Technology Development (ATD). As part of our approach of being a total solution provider for our customers and partners, we perform research and development in various aspects of the value chain through our JAPFA Aquaculture Research Center (JAR). The JAR facilities are located in Banyuwangi for aquaculture research, in Purwakarta for aquaculture technology where we focus on the latest technologies for sustainable aquaculture, and in Cianjur for nutrition research. AQ: Is most of the company's feed for its own farms and hatcheries, or are external feed sales an important part of the business? AB: Most of our feed sales are to our external customers, and only a minor portion is for internal usage. AQ: How does STP support its farmer customers? AB: STP’s vision is to become a total solution provider for our stakeholders in the aquaculture industry. Our mission is to support our customers in achieving performance and profitable business through a farmer-oriented approach, trusted value-added products and top-quality standards. In line with our vision, we provide high-quality feed, high-quality shrimp fry (SPF) and fish fingerlings with traceable quality. Our shrimp farm technical (SFT)

and fish farm technician (FFT) teams are dedicated to supporting our shrimp and fish farmer customers both on-site and off-site with specific support services, such as water quality testing and water dissolve analysis, culture condition analysis, harvest data analysis, feeding program, feeding technique and feed storage training and farm management SOPs, among others. All these functions play a significant role to support the success of both our aquaculture customers as well as our own operations. In addition, STP also has an aquaculture Mobile Diagnostic Lab to help customers access shrimp farming laboratory services. Through this mobile lab, customers can check their shrimps faster on-site. AQ: 2021 marks Japfa’s 50th anniversary. With five aquafeed mills strategically located across Indonesia, do you plan to expand to other markets? AB: Fish consumption in Indonesia is significantly increasing and we believe that aquaculture can become another major pillar for the JAFPA group in addition to poultry, swine and dairy. To support this development, we continue to grow the feed business by expanding the range of feed products as well as increasing the production capacity of existing feed mills or with new locations. We are also expanding our shrimp hatchery business with increased and continued supply to our farmer customers. As the brand is getting stronger, we plan to expand in India and Vietnam through our sister companies. AQ: Japfa participated in the F3 Challenge in 2017. What are the main goals achieved by STP since then to get fish-free feeds? AB: For the past three years, we have been successfully shifting from high to less fishmeal feed. We substantially reduced dietary fishmeal from 15% to 8% without compromising the growth performance and the sales of our feed product have increased tremendously. When it was first introduced in 2018, the sales were only 24% of the total shrimp feed sales volume. Through a strong communication campaign and support by our internal technical team on the importance of using less fishmeal feed for more sustainable and responsible farming, we tripled the sales up to 72% of the total shrimp feed product sales volume by August 2021.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

AQ: Sustainability is key to many feed company’s agendas. What plans does STP have to lower its carbon footprint, or achieve other sustainability goals? AB: We are committed to managing our aquaculture activities sustainably to meet the market demand for sustainable aquaculture products while, at the same time, minimizing the impact on the environment. Every year, we commit to improving our carbon footprint throughout our operations. Our carbon footprint from emissions, waste and water use is audited and reported regularly to the competent authorities. For instance, in our feed mills, our expert nutritionists utilize advanced technology such as Near Infrared Reflectance (NIR) to develop balanced feed formulation and, at the same time, improve production efficiency. We also implement automation in our feed mills to achieve higher efficiency rates. We managed to source 55% of the fish oil supplies locally and continuously strive to reduce imports. In addition, we maximize the use of condensate water for the boilers that can save water use by up to 20% and simultaneously reduce the energy usage for preheating machines. Besides that, following the rece nt disease occurrence in shrimp farms in Indonesia due to improper design of wastewater treatment facilities (WWT) within the farm, we are conducting seminars and meetings with farmers to highlight the importance of a sustainable design of WWT. AQ: The Indonesian government plans to develop a model for sustainable shrimp farming. What are the main challenges to achieve it? AB: The key factors for a successful sustainable shrimp farming area are good quality of shrimp feed, SPF broodstock and shrimp fry or post-larvae (PL), the implementation of biosecurity and a well-executed WWT system in shrimp farming. Besides that, the availability of a good location and qualified manpower are also required. In line with the government program to make Indonesia the first global shrimp producer by 2024, STP believes that if all stakeholders work closely together, Indonesia can be a major shrimp producer globally.

AQ: How about fish? What challenges are there to the production of other major species? AB: In Indonesia, the most widely consumed fish are catfish, pangasius and tilapia. Awareness about fish consumption and demand in Indonesia is increasing due to its high protein content and cheaper price compared to other proteins like beef. The challenge of fish culture relies on good quality disease-free fingerlings. In addition, most fish cultures are located in lakes where water quality could be uncertain. Therefore, to meet the demand of aquaculture with a good quality product, it’s necessary to have high-quality fingerlings, high-quality fish feed and ample space to operate. However, in Indonesia, finding the perfect location for fish farming can be difficult due to the limitation of free land. Looking at this challenge, STP has started to apply biofloc technology for our customers. Through this technology, we are able to maintain water and culture quality more sustainably. In addition, our freshwater fish hatcheries apply a Marker Assisted Selection (MAS) to ensure our fish fingerlings are pathogen-free. AQ: Japfa recently organized Japfa Feeds the Future Challenge. What do you think will be the next innovations that will boost aquaculture farming? AB: We believe that digitalization will be very important in aquaculture, in particular Technology 4.0. For instance, we see a lot of farmers using automatic feeders. To support digitalization in aquaculture farming, we also provide an automatic feeder using IoT for Kampung Perikanan Digital (Digital Fishing Village), a collaboration program with e-Fishery, Telkomsel and West Java government in Cirebon, West Java. In STP, to monitor farms, we use android-based systems such as IoT, digitalization and big data analytics through TKT Mobile – an android-based mobile app for tracking shrimp farm production that can be accessed by our Shrimp Farm Technician (SFT) and QC Sharp, Quality Control Shrimp Hatchery Accurate Rapid Program, a web-based mobile app that enables us to monitor the growth and development of shrimps in an efficient yet precise way to support sustainable farming. In the future, we are also moving towards the use of satellites for farm site monitoring.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

AQUACULTURE

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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

De Heus breaks ground on new animal feed plant in Ivory Coast

The company started the construction of a greenfield compound feed factory in Abidjan, in the new industrial zone of PK in Attingue’, Ivory Coast, with an initial capacity of 100,000 tons per year. The factory will produce high-quality complete feed for fish, poultry and cattle, which will expand the current portfolio of Koudijs concentrates and specialty feeds. Production is planned to commence during the second half of 2022.

BioMar to expand capacity in Ecuador BioMar unveiled a comprehensive investment plan for its business unit in Ecuador. It includes new land, a transformation of the current factory layout, an upgrade of existing pelletizing equipment, and four new lines for extruded feed. The two-phased installation will bring a total of four new lines with a combined capacity of 200,000 tons at BioMar’s facilities in Guayaquil. The investments are part of the preparation for the introduction of new advanced shrimp feed concepts, which are planned for launch in the market from 2022.

Aquafeed Technology Center opens in Norway The state-of-the-art center, located at Nofima’s facilities in Bergen, aims to work towards a more environmentally friendly fish feed. Its infrastructure and expertise offers are within bioprocessing, process technology, extrusion and feed technology, and analytical platforms. The center’s new equipment allows studying the impact the processing of new raw materials has on the ingredients and the final product.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Aller Aqua’s carbon footprint labeling verified for its fish feeds Aller Aqua achieved the verification of CO2-labelling of fish feed at its European factories, being the world’s first fish feed company verified. “It is a huge milestone for us to be the first company to be able to label our entire product portfolio by

the recognized PEFCR (Product Environment Footprint Category Rules) principles Feed for foodproducing animals. This means that we can offer customers a full carbon footprint declaration on each type of feed,” the company said.

Grobest Group, Universal Aquaculture to develop world’s first functional performance shrimp feed for RAS The companies partnered to collaborate on the development of the world’s first next-generation Functional Performance Shrimp Feed for UniAqua’s proprietary Hybrid Biological Recirculating

System™. In initial trials, Grobest functional performance feeds have outperformed competitive products by yielding healthier, tastier shrimp and significantly better water quality.

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Lowers mortality in shrimps and increases resistance against the White Spot Syndrome Virus (WSSV)

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Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Nutreco, Unga Group joint venture to expand Skretting’s footprint in East Africa Nutreco formed two joint ventures with the Kenyabased, largest feed miller in East Africa, Unga Group Plc, to meet the growing demand for high-quality protein within the East African region. The yet-tobe-named joint ventures will focus on fish feed development in East Africa and complete animal feed development in Uganda. The Kenyan entity will produce fish feeds intended for East African markets at Unga’s existing extruded fish feed plant in Nairobi and market them under the Skretting & Fugo brands.

New feeds in the market

INVE Aquaculture unveiled breakthrough rotifer substitution feed line and protocol that reduces live rotifer diets by at least 50% in marine fish hatcheries. Skretting introduced a new hatchery diet, GEMMA Neo, inspired by nature that reduces

the dependence on traditional feed ingredients, while also providing ideal nutrient composition, unprecedented production system performance and weaning flexibility. BioMar launched a new feed for meagre (Argyrosomus regius). EFICO 2152 is a specialized solution that allows meagre farmers to lower production costs, increase efficiency, and thereby, control the economic performance of their farm under challenging market conditions. Aquasoja developed a nutritional solution for red porgy, PAGRUS, designed to meet the nutritional requirements of red porgy, and that contains natural and synthetic pigments to obtain the desired skin color.

Cambodia opens first aquafeed mill Agrimaster, part of Medivet Group, recently opened Cambodia’s first aquafeed mill. The facility started supplying an additional 600 tons per month of highquality fish feed to the local growing aquaculture sector. The state-of-the-art aquafeed mill introduced Famsun’s single-screw extrusion system to help Agrimaster become competitive in aquafeed production. Famsun also constructed Haida Group’s first shrimp feed line in the Americas. The new facility, located in Guayas Duran, Ecuador, has five shrimp feed production

lines with a total hourly output of 13 tons and a total annual output of 120,000 tons of shrimp feed.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

New US R&D center to strengthen insect farming

Grobest appoints new CEO

A newly awarded $2.2 million grant from the National Science Foundation has established the Center for Environmental Sustainability through Insect Farming. The Texas A&M College of Agriculture and Life Sciences has been assigned as the lead site for the center, which will be a collaborative effort with Mississippi State University (MSU) and Indiana University-Purdue University, Indianapolis (IUPUI). Joining the universities will be 34 U.S. and global industrial partners including Mars Inc., Tyson Foods and insect farming pioneers such as Aspire Food Groups, Protix and Beta Hatch Inc. Researchers will examine optimization in the production and development of food as well as feed products for poultry, swine, aquaculture and pets.

BÜCHI introduces NIR sensor for essential process control

Grobest appointed Hua Du as the company’s new CEO following a decision by current CEO, Samson Li to step back from his role. Du joins Grobest after 11 years at Solvay, where he held the position of president of Global Business Units and was a member of Executive Committee, and 13 years at the Rohm and Haas Company.

Cargill, Freshwater Institute partnership to improve feeds for RAS salmon farming

BÜCHI Labortechnik AG introduced NIR-Online X-Sential™, a new process sensor that takes advantage of more than 15 years of in-the-field experience with process NIR technology and applications. The X-Sential™ was developed and designed as a cost-effective, fit-for-purpose process sensor.

The Conservation Fund’s Freshwater Institute and Cargill announced a multi-year agreement to develop, evaluate and enhance feeds for the growing land-based aquaculture industry. This partnership builds on recent efforts between the two groups that validated the effectiveness of Cargill’s new diet for Atlantic salmon grown in land-based RAS.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

PRODUCT FOCUS

The revolution of on-site liquid enzyme production in aquafeed Heat processing of feed, including pelleting and extrusion, has been a practice in the feed industry for decades. To overcome the degradation of heatsensitive additives, adding enzymes as liquids via post pelleting systems (PPLA) avoids the loss of enzyme activity. These liquid enzymes are produced in water, adding stabilizing substances and delivered in IBCs to the feed mill. However, using liquid enzymes poses challenges. In particular, in summer conditions and in tropical regions where aquaculture production is diverse, the stability of these liquid enzymes may be compromised, in particular the requirement for temperature-controlled transport and storage.

Instant water-soluble enzyme powders The basic thought of this concept is quite simple: Why not produce liquid enzymes in the feed mill itself? This was the question that led to the development of the Huvematic Concept by Huvepharma. This concept has two elements: the development of water-soluble enzyme powders (WSP) and the development of a tool to automatically produce liquid enzymes on-demand at the feed mill. Huvepharma’s know-how on the production and processing of enzymes has led to the development of a range of instant water-soluble enzymes powders (WSP) like Hostazym® X WSP (NSPase), OptiPhos® WSP. These products are 50-150 times more concentrated than the usually available enzyme powders and are soluble, even in cold and hard water, within 30 seconds. A tool called the Huvematic® automatically produces liquid enzymes from the WSP powders. Batches of 5 to 15 liters have been developed in collaboration with external partners. This equipment accurately weighs

the required quantities of WSP enzymes and water and mixes them together so that the liquid enzyme is produced at the correct required concentration. The Huvematic® can be fully integrated into factory production, along with automatization and its reporting system, to completely comply with the regulatory quality control and traceability standards. One Huvematic® has enough production capacity to serve multiple PPLA lines within the same feed mill.

Worldwide implementation This recent innovation by Huvepharma is new in most areas of the world. However, it has already been a common practice for several years in the U.S. where it has been intensively used for land animal species. To aquaculture applications, Huvematic can help improve use of the increasing demand of enzymes used in functional feeds. This success has encouraged Huvepharma to launch the concept of the WSP enzymes globally for the aquaculture industry. Major benefits have been communicated by customers. Nutritionists value freshly prepared enzymes and have no worry about activity-loss of the liquid enzyme due to storage time and conditions. Also, the ability to produce any desired enzyme concentration is considered advantageous and allows flexibility in dosing. There is also a health and safety implication in that the plant manager/production leader appreciates that no more IBC’s need to be stored, replaced, and manipulated, saving storage space and labor costs.

More information: Daniel Arana Global Product Manager Aquaculture Huvepharma NV E: daniel.arana@huvepharma.com

Empowering productive and sustainable aquaculture through bioactive marine peptides Zhou Hu-Ping, Rahul Mathew, Siok Thing Tan, Koen Meynen, Rajalekshmi M., Kemin AquaScience™

Protein utilization in aquafeed Fully exploited fish stocks stress the aquafeed market in terms of fishmeal supply. To increase sustainability, aquaculture tends to switch from fishmeal to plantbased protein sources. Replacement of fishmeal with new alternatives is complicated and involves many considerations in terms of nutritional profiles, feed palatability, digestibility and the compensation of essential micronutrients that come along with fishmeal. Efforts have been made in replacing fishmeal, which is the main focus in the aquaculture industry now, and the molecules that can functionally compensate for the loss of nutrients, palatability, digestibility and micronutrients when replacing fishmeal with alternatives are crucial in determining replacement success. Functional protein hydrolysate in aquaculture Low molecular weight peptides of protein hydrolysates have been studied for their positive effect in diets for many aquaculture species. They have been reported to increase feed intake, feed utilization and somatic growth (Refstie et al., 2004; Aksnes et al., 2006; Zheng et al., 2011, 2013; Khosravi et al., 2015), as well as promote the immune system (Kotzamanis et al., 2007; Ovissipour et al., 2014; Khosravi et al., 2015, among others) and enhance the harmonious development of the skeleton and digestive systems in ﬁsh larvae (Cahu et al., 1999; Gisbert et al., 2012; Delcroix et al., 2014; Johannsdottir et al., 2014). In aquaculture, there is increasing interest to replace fishmeal with functional protein hydrolysate from animal byproducts modified through chemical, enzymatic or microbial hydrolysis of proteins to

generate peptides that have both nutritional and physiological functions in animals (Al-Souti et al., 2019). The quality of these functional protein hydrolysates highly depends on the abundance and diversity of different short peptide chains which are subjected to the source of raw materials and methods used for hydrolysis. Presently, there are several origins of animal hydrolysate utilized as protein sources in aquaculture, including shrimp, tuna and squid. The bioactive peptides and corresponding benefits from these origins in animals can vary due to the differences in resulting peptides. Although the complete replacement of fishmeal with animal-derived hydrolysate remains controversial, these hydrolysates still draw a lot of interest from the market to supply the demands of fishmeal alternatives in aquaculture and supplement the key molecules that are similar in fishmeal in the feed formulation adjusted with the protein alternatives. On the other hand, researchers have progressed to seeking bioactivities from these functional hydrolysates such as growth promotion, immunostimulation and palatability enhancement.

New generation shrimp protein hydrolysate Proprevia™ N 100 LQ, a new type of protein hydrolysate derived from shrimp via enzymatic hydrolysis, is a potential feed ingredient to enhance the palatability of feed and improve the growth and health of aquatic animals due to its enriched profile of free amino acids and peptides with different molecular weights that can stimulate the chemo-sensory mechanism of animals. Compared to typical fish hydrolysate, Proprevia™ N 100 LQ contributes 68% higher essential amino acids

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 1. (A) Essential amino acid profile (% total product weight)* Source: KID SPRE-20-18690. (B) Palatant amino acid profile (% total product weight)* Source: KID SPRE-20-18690.

and four times higher lysine content (Fig. 1A). Lysine, the first limiting amino acid, is known to produce maximum feeding response and feed ingestion of shrimp (Chutima, 2014). What’s more, alanine, arginine, glutamic acid, glycine, histidine, proline, tyrosine, taurine are known to be chemo-attractants for both fish and shrimp (Chutima, 2014, Yacoob I, 2003, David TI, 2021). Comparably, enzymatic hydrolysis is more gentle than other methods such as acid and chemical hydrolysis and higher consistency in hydrolysate quality is achieved. The molecular mass distribution of Proprevia™ N 100 LQ indicates that over 85% of total peptides fall below 1kDa (Fig. 2), which are the peptides with a low molecular weight that have higher digestibility and can be easily absorbed by the animals. Several studies also reported that low molecular weight fractions (<3kDa) possess higher antioxidant capacity than fractions of higher molecular weights.

Effects on palatability and feed intake One of the key benefits of fishmeal application is its palatability contribution to feed. This chemosensory characteristic of the feed could indirectly affect the ingestion or rejection of the feed to fish and thus the performance of animals. Studies were conducted in fish and shrimp to evaluate the Proprevia™ N 100 LQ in palatability, feed intake, and growth performance.

Asian seabass trial In an Asian seabass trial, the impact on feed palatability with different inclusion rates coated on the top of the feed was determined. Juvenile Asian seabass were randomly allocated into nine glass tanks of 100L with six fish in one tank. A total of three diets were tested which included the basal diet and two basal diets topcoated respectively with 1% and 3% Proprevia™ N 100 LQ. Fish were hand-fed with a fixed number of diets two times a day and the total number of consumed pellets were recorded. To minimize the reliance of fish towards given diets and accurately determine the preference discrepancies of fish towards assigned diets, the diets given every meal to each tank were swapped randomly. In addition, the formulation of the basal diet was readjusted to partially remove ingredients that could largely contribute to feed palatability to remove interference for the experiment. Results showed that the total feed intake for diets coated with 1% and 3% was enhanced by 3% and 15.7%, respectively, compared to the control diet within an 8-day experiment period. Moreover, it was found that fish fed on the feed coated with the shrimp protein hydrolysate at a faster rate (0.67 and 0.88 pellet/ sec/tank corresponding to 1% and 3%, respectively), compared to the control diet (0.43 pellet/sec/tank). The fast feeding time by aquatic animals can ensure the high nutrient availability from the feed by avoiding the nutrient leaching when feed immerses in water. The water quality can be also improved since less feed wastage is generated.

Figure 2. Low molecular weight peptides distribution of Proprevia™ N 100 LQ. Source: KID SPRE-20-18690.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 3. Growth performance of shrimp in normal and low-fishmeal diet. Source: KID SD-21-24007.

Figure 4. DPPH radical scavenging activity of Proprevia N 100 LQ and BHT. Each point is the average of three measurements. Source: KID SD-21-23938.

Figure 5. ABTS radical scavenging activity of Proprevia N100 LQ and ascorbic acid. Each point is the average of three measurements. Source: KID SD-21-23938.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Shrimp trial Similar studies were conducted in Pacific white shrimp (Litopenaeus vannamei). Feed intake was compared between feed sample top-coated with 2% Proprevia™ N 100 LQ and uncoated feed. The coated and uncoated feed were placed at different ends of 70L glass tanks. The experiment was done in triplicate tanks, each tank having six shrimps. Prior to placing the feed, shrimps were confined to the middle of the tank using nylon nets and were released 30 seconds after placing feed samples at the two ends. The choice of the shrimps between the two feeds was measured in terms of feed intake. The leftover feed after one hour of feeding was collected and dried to calculate the feed intake. It was observed that the intake of feed coated with Proprevia™ N 100 LQ was significantly (p<0.05) higher than that of the uncoated feed. The effect of shrimp protein hydrolysate in partially replacing fishmeal in vannamei diets was evaluated in an 8-week growth trial conducted in net-caged earthen ponds with a salinity range of 6-10 ppt. Shrimp juveniles having an initial body weight of 0.68 grams were fed either a 20% fishmeal-containing diet or a 15% fishmeal diet containing 2% Proprevia™ N 100 LQ, 3 times a day at 3-5% of body weight. At the end of the feeding period, both the groups had similar body weight gain, FCR, specific growth rate (SGR) (Fig. 3). The survival rates in the control and Proprevia™ N 100 LQ-supplemented groups were 87.19% and 88.75%, respectively.

Effects on immune function and antioxidant activity The health and immunity-related parameters were also measured (Table 1). No significant difference (p>0.05) in the measured parameters was seen between the reduced fishmeal group supplemented

Table 1. Immunity indicator of shrimp in normal and low-fishmeal diet. Source: KID SD-21-24007.

Control diet (20% fishmeal)

Treatment diet (15% fishmeal + 2% PropreviaTM N100 LQ

Haemocyte count (x105cells/ml) 8.5

7.8

Phenoloxidase activity (unit/mg protein)

148.7

149.2

Super oxide dismutase activity (unit/ml)

22.6

23.5

No statistically significant difference was observed between the two groups, in any of the parameters (p>0.05).

with 2% Proprevia™ N 100 LQ and the control group with higher fishmeal. Apart from the palatability and effects on growth, protein hydrolysates possess functional benefits like antioxidant properties. The interactions, properties and sequences between amino acids play a big role in exhibiting biological function. For example, cysteine and tryptophan are reported to make a significant contribution due to their structural properties to the antioxidant activities if they are present in the same peptide (Zou et al., 2016). The unique enzymatic hydrolysis process results in the superiority of Proprevia™ N 100 LQ in exhibiting functional benefits for aquatic animals. One example is resistance against oxidative stress, which could be rendered by environmental and

nutritional factors in aquatic animals. Oxidative stress results from the imbalance between the production of Reactive Oxygen Species (ROS) and the inherent ROS buffering system such as glutathione, superoxide dismutase and catalase which serve as a defense mechanism in aerobic organisms to resist the damage caused by ROS. It is also reported that the oxidative stress encountered by animals has been correlated to an increased likelihood of diseases occurrence (Blier, 2014). The antioxidant property of Proprevia™ N 100 LQ has been evaluated by three different in vitro assays – DPPH radical scavenging, ABTS radical scavenging and linoleic acid oxidation assay. Its antioxidant activity was compared to that of BHT and ascorbic acid in DPPH and ABTS assays, respectively. The comparison

Figure 6. Linoleic acid oxidation test. 50mM linoleic acid in 99.5% ethanol was incubated in the dark at 40°C. The degree of linoleic acid oxidation was measured at regular intervals using the ferric thiocyanate method. The presence of lipid peroxides increases absorption. Source: KID SD-21-23938.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

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was made between the concentration of the test substance needed to obtain a radical scavenging activity of 50%. It was found that Proprevia™ N 100 LQ has a radical scavenging activity equivalent to 1% and 3% of the activity of BHT and ascorbic acid, respectively (Fig. 4, 5). In the third essay, it was found that the oxidation of linoleic acid decreased with an increased concentration of Proprevia™ N 100 LQ (Fig. 6). With the recommended inclusion level of Proprevia™ N 100 LQ in feed ranging from 1 to 5%, the added radical scavenging potential is equivalent with BHT concentrations sufficient to protect feed against oxidation. However, since the activity of an antioxidant in the feed not only depends on the radical scavenging activity but also on the physical distribution in the feed, the actual impact on the stability of extruded feed will have to be verified in a storage study. For these peptides to exert their function in vivo, after oral uptake, they must be able to reach the site of activity. Active uptake of peptides in the intestine is effectuated by peptide transporters that shuttle di- and tripeptides. Therefore, only the free amino acids and di- and tripeptides present in the hydrolysate have the potential to act as in vivo antioxidants. The typical molecular weight profile of Proprevia™ N 100 LQ shows that it is rich in very small peptides.

Conclusion The introduction of new generation shrimp protein hydrolysate brings more possibilities to aquafeed formulation, through superior benefits on nutritional profiles, palatability, digestibility and immunity. Application of Proprevia™ N 100 LQ enables more flexible feed formulation and offsets the potential deviations from various sourced raw materials to ensure the performance of aquatic animals. Aquafeed millers are able to create a more competitive product, and farmers will benefit from consistent and profitable yield. With the application of Proprevia™ N 100 LQ, better sustainability is adopted by lower dependence on wild-caught marine resources and optimized carbon footprint. In addition, less nutrient waste is generated and nutrient leaching in water is minimized by reducing the duration needed for animals feeding and feed immersion in water by increasing the palatability of feed and feed intake of animals, thereby ensuring the easier practice of pond environment management. References available on request.

More information: Zhou Hu-Ping Marketing Manager Kemin AquaScience™ E: Ronnie.zhou@kemin.com

www.hydronix.com enquiries@hydronix.com

Hydronix-Aquafeed advert-July 2021 58.4x228.6mm.indd 1

22/06/2021 20:19

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Byproduct-based fishmeals: Adding to the future of fishmeal production Brett D. Glencross, Enrico Bachis, IFFO, The Marine Ingredients Organisation Changing the face of the marine ingredients industry There was once a time when nearly all the world’s fishmeal and fish oil supplies were obtained from forage fisheries, those that have a very limited direct food market and are using high-abundance low-trophic level fish with annual recruitment cycles. Still to this day, some of these fisheries, like the Peruvian anchoveta and the North Atlantic capelin fisheries, are among the largest single-species fisheries on earth. Despite the introduction of strict fishery quotas and third-party certification systems to substantiate sustainability credentials of these fisheries, it is widely recognized that we are generally at the limits of the global supply of forage fish. With a finite supply of forage fish, there has been an increasing focus on the use of byproducts to provide

the raw material biomass for fishmeal and fish oil production. Arguably, today this represents one of the best bio-circularity stories around in terms of capturing the full value of animal production/ capture in our food chain. Increasingly, contributing to the story in providing additional biomass input are the byproducts from the aquaculture industry itself. Over the past twenty years, the volume of byproduct fishmeals has grown from 1.2 million tons (about 19% of total fishmeal production) to the present day where it now represents more than 1.4 million tons (about 29% of total fishmeal production) (Fig. 1). In 2020 it is estimated that 29% of all fishmeals and 48% of all fish oils are from byproducts (Fig. 2). Combined, that means almost one-third of all fishmeal and fish oil are now being sourced from byproducts in 2020.

Figure 1. Global fishmeal production by raw material input origin 1996 – 2030 estimates.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 2. Global fishmeal and fish oil production by raw material input origin 2020 estimates.

Figure 3. Production of global byproduct meals by category in 2020.

Figure 4. Production of global byproduct oils by category in 2020.

Gaining momentum as aquaculture grows Of the global production of fishmeals and oils from byproducts, there is a bit of a dichotomy. More than 69% of byproduct fishmeal production comes from wild-fishery byproducts and only just over 30% from aquaculture byproducts (Fig. 3). For fish oil, the opposite is the case, with almost 60% of fish oil byproduct production coming from aquaculture and just over 40% from wild-fishery byproducts (Fig. 4). It is estimated that globally there is an additional 11.7 million tons of byproduct produced in processing plants that are currently not collected for the production of fishmeal and fish oil. At a typical processing yield of 4:1, this would realize another 3 million tons of fishmeal and fish oil from existing resources. While the majority of byproduct fishmeals still come from biomass obtained from wild-fisheries sourcing fish for direct human consumption (DHC), the supply of biomass from aquaculture is rapidly catching up. Presently three major aquaculture sectors contribute most of the aquaculture byproduct fishmeal, though undoubtedly there are more that we are yet to account for (Fig. 3). Salmon aquaculture byproducts are the largest contributor, followed by pangasius byproducts and then tilapia. Notably, we have not included byproducts from shrimp aquaculture in these estimates, although we know a substantial volume of this product exists. Among fish oils, the main aquaculture contributors are both salmon and pangasius aquaculture sectors, with both contributing about 45% of the byproduct oil from

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 5. In vivo (Atlantic salmon) protein digestibility of a selection of byproduct fishmeals, two whole fish fishmeals and several plant protein ingredients. Data IFFO (2021). Notable is the consistency in digestibility values of both the byproduct and forage-fishery fishmeals.

aquaculture (Fig. 4).

Not so variable and still high-quality One of the criticisms that byproduct fishmeals and fish oils attract is that they are seen as second-rate compared to other resources. While this may have been the case in the past, recent evidence does not support this with a growing volume of work showing they can

be produced to high-quality specifications. Much of this comes from adopting modern processing technologies using low-temperature drying and maintenance of coldchain management systems for the raw material, to ensure biogenic amine production is kept down prior to processing. Recent work undertaken by IFFO comparing the quality of various byproduct fishmeals against several other fishmeal and grain product

Figure 6. Mean feed intake by Atlantic salmon of a standard commercial reference diet formulation (BASAL) and diets where 30% of the diet has been made of the test ingredient. Included as test ingredients are a selection of byproduct fishmeals, two whole fish fishmeals and several plant protein ingredients. Data IFFO (2021). Notable is that all fishmeals result in a numerical improvement in feed intake, while each of the plant ingredients has the opposite effect.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

24 Table 1. Example of the composition (g/kg as is) of different byproduct fishmeals compared to whole fish fishmeals and common plant protein ingredients.

Name

Moisture

Protein

Lipid Ash

Energy

ARG

CYS

HIS

ILE LEU LYS MET PHE THR VAL

Blue Whiting BP

675

107

159

20.1

Herring BP

100

660

137

163

20.9

Tuna BP

559

116

224

17.7

Atlantic Mackerel BP

672

115

133

20.4

Anchoveta

670

109 139

20.1

30 50 53 20 27 25 37

Capelin

667

157 116

21.9

27 54 53 22 27 30 36

Soy Pro Conc (SPC)

588

19.2

Soy Pro Isolate (SPI)

843

21.5

Corn Gluten

102

557

20.9

100

ingredients showed that both their digestibility and palatability criteria can be equally as good as that of those other resources (Fig. 5, 6). Analysis of the composition of these resources does often show that they are lower in protein (Table 1) and omega-3 (Fig. 7) than their wild-fish counterparts but given that the “best” parts of the fish have already gone for DHC, then this is perhaps not surprising. Irrespective, there are still resources with characteristics equal to that of many whole-fish fishmeals and fish oils and in

most cases, they are still superior to that provided by plant protein and oil ingredients.

The future for byproducts Looking to the future, we see positive signs. There is improving management of global fisheries throughout the world. Cuts to total allowable catches (TACs) in the early 2000’s are now reaping benefits in terms of stabilizing fishery biomasses in many of the large forage fisheries around the world. Combined with

Figure 7. Fatty acid profiles of various oils, including salmon and pangasius byproduct, whole fish oils from anchoveta and capelin and rapeseed and soybean vegetable oils.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 8. Projections of future fishery and aquaculture production yields with byproduct biomass potentials overlaid (dark blue = forage fishery, light-blue = byproduct from fishery, green = byproduct from aquaculture). Analysis suggest that global fishmeal and oil production could more than double over the next thirty years on the back of improved fishery byproduct management and continuing aquaculture growth.

the increased use of third-party sustainability certification systems, like MarinTrust and MSC, market forces are being used to drive responsible resource use across the sector. However, despite these positive signs, it is unlikely that we will see total forage fishery catch exceed much more than 16 million tons a year into the future. This means that fishmeal and fish oil yields from that resource are constrained to about 4 million tons a year (assuming a meal yield of 22% and oil yield of 3%). These same improvements are extending beyond the forage fisheries into other large DHC fisheries. This of course also means that with improvements in fishery sustainability for DHC we are also improving our biomass base for the production of byproduct fishmeals and oils. Based on FAO estimates for fishery yields in 2050 of 80 million tons, we suggest that of the 64 million tons that is predicted to be caught for DHC, that we should be able to repurpose up to 33% of that biomass as byproducts (20 million tons) producing another 5 million tons of fishmeal and oil. We are presently processing almost 5 million tons of biomass (~1.25 million tons of fishmeal and oil), yet there are still many more processors around the world where we could repurpose more of this valuable resource.

Aquaculture is another dimension to add to this story. Over the past twenty years, we have seen unprecedented growth in this food production sector and predictions are for this to continue. Based on FAO estimates for aquaculture production in 2050, we could see production reach 140 million tons (Fig. 8). Even if we take a conservative view of only being able to repurpose up to 15% of that biomass as byproducts (~20 million tons) by 2050, then that would contribute another 5 million tons of fishmeal and oil. Presently in 2020 we already repurpose more than 3 million tons of aquaculture byproducts and there is still much more that could be done to improve the supply and use of these products. But one thing is for certain, so long as we keep fishing and growing fish in aquaculture for human food, then we will always have fishmeal and fish oil available as strategic feed resources. More information: Brett D. Glencross Technical Director IFFO, The Marine Ingredients Organisation E: bglencross@iffo.com

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

COLUMN

Fishmeal outlook Steve Dahlblom Steve Dahlblom is the global marine director for Scoular which handles more than 200,000 MT of various types and qualities of fishmeal. The company recently released a new brand, Encompass, for its global fishmeal products and services and is opening fishmeal facilities in Myanmar and Warrenton, USA, in the coming year. E: sdahlblom@scoular.com.

Importance of fishmeal in aquafeeds Fishmeal is an important ingredient in aquafeeds for many reasons. It is nutrient-rich and provides a highly digestible source of amino acids and important omega3 fatty acids, in addition to acting as a strong attractant in feed. As ingredient innovation across the globe rapidly continues, fishmeal remains one of the most nutritionally balanced ingredients for aquafeed, piglet and pet diets. In aquaculture diets, fishmeal inclusion ranges from 0% to 80% depending on the marine species and where it is being raised. U.S. catfish diets use little to no fishmeal, while eel feed in Asia requires high inclusion levels. A typical salmon diet uses 10% to 15% inclusion, while shrimp and seabass are 15% to 30%. All feed diets must contain protein, and the protein’s nutritional value sets one ingredient apart from another. To promote growth, essential amino acids must be supplied in the feed. Plant-based concentrates and meals may measure up in protein content but cannot compete with fishmeal's robust source of essential amino acids such as lysine, methionine and cystine. Fishmeal substitutes like vegetable concentrates and proteins also contain less-digestible nitrogen, which

leads to excess ammonia in the feces that can negatively affect water quality in shrimp ponds, tilapia ponds, trout streams and Recirculating Aquaculture Systems (RAS).  The fatty acid profile is another reason why fishmeal is a beneficial ingredient. The majority of fish oil is extracted when the raw material is processed into fishmeal, leaving between 6% to 10% fat by weight in the meal. This percentage provides valuable polyunsaturated fatty acids, specifically the essential omega-3’s (EPA and DHA). Most oils of vegetable origin contain high concentrations of Omega-6 fatty acids but lack the Omega-3 fatty acids.  Feed producers face a difficult decision when trying to reduce fishmeal inclusion in the diet. While fishmeal can be a relatively expensive ingredient, producers are challenged to reduce inclusion rates any further without the quality of the feed deteriorating.

Main challenges of fishmeal production  Fishmeal and fish oil production share many challenges with traditional agriculture, such as seasonality and inclement weather. However,

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

fishmeal and fish oil production are more complex.  Even with the best acoustic equipment and perfect weather, finding and catching the targeted species is never guaranteed.  One of the fishmeal industry’s most pressing considerations is how to best manage fisheries sustainably to ensure the longevity and long-term success of the biomass. The industry, in partnership with third parties, works hard to responsibly manage fisheries around the world, including measuring and monitoring biomasses, spawning, juveniles, setting quotas (i.e. catching limits) and enforcing fishing bans. While 51% of the global fishmeal market is certified sustainable through third-party certifications under the Marin Trust and MSC standards, it is not always economical or feasible for many small-scale producers in lower volume producing countries to get these certifications. Many these producers are acting as good stewards while following similar benchmarks of the third party certifications. It is important to understand their fishing operations and support them for doing the right thing and not ignore the source completely. Another challenge is catching and properly handling the fish both at sea and on land to maximize its nutritional value and quality. Fishing operations must keep the catch as fresh as possible until processing. If nets or vessels are too full, fish can be damaged and cause enzymes to break down amino acids, lowering protein levels, quality and value. Processing temperatures also directly impact product quality and value. For example, fish must be processed using a low temperature and without direct flame to achieve the highest level of digestibility. Factors such as weather, food source, location of the fish, size and fat of the fish, and volume of the catchable supply all impact fishmeal and oil quality. From day to day and season to season, fishmeal is hard to predict, making delivery of consistent supply and quantity challenging. Inevitably, feed millers want and depend on consistent product quality, performance and availability. Over the past year, the world container market has turned supply management on its head. Longer lead times have created a greater need for reserve stocks on hand for buyers. The current freight environment appears it will be long-lasting and underestimating the risk associated with that would be a mistake. Working with high-integrity trade partners who stand by their

contracts matters more than ever in a high-volatility freight environment.   Supply chain management, along with seasonality, weather issues, sustainability, inconsistency and quality, are among the challenges Scoular continues to navigate in our decades-old fishmeal business, now branded as Encompass. Our large multi-national team and strategically located fishmeal facilities in Myanmar, Mexico, Indonesia and across the United States, along with fish oil tanks in Dutch Harbor, Alaska, help suppliers and customers efficiently manage their evercomplicated pipelines.

Future perspectives  Demand for all proteins is growing rapidly due to increasing global populations with higher disposable incomes, and we must find creative ways to feed 10 billion people by 2050. To help meet higher demand, aquaculture is the fastestgrowing sector in agriculture. Fishmeal production, which is critical for the aquaculture industry, remains fairly flat year-over-year, and the demand is outpacing the supply. Ocean-caught fishmeal has decreased and increases from fishmeal made from trimmings and offals from the filet and human consumption processing does not meet the increased demand. Therefore, sustainability is crucial to the fishmeal industry’s future. As previously stated, 51% of the fishmeal produced globally is certified for sustainability, of which approximately 30% is produced from trimmings up-cycled into sustainable fishmeal. Sustainable ingredients along with sustainable aquaculture practices will become necessary, and companies without a strong sustainability strategy will struggle to exist. At Scoular, for example, fishmeal is a key ingredient in our portfolio, and in 2020, we launched a sustainability strategy with marine sourcing as a key component. Additionally, we believe a formula for success is to partner with suppliers and customers who share the same vision and values. This drives success for each party as well as for the overall industry. In closing, many challenges and disruptions exist in the fishmeal business, and there will always be market volatility. However, one constant will be that fishmeal is a highly valuable ingredient in global feed rations.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Novel strains of Bacillus subtilis for improving water quality and controlling pathogens in shrimp aquaculture Karthik Masagounder, Michelle Dargatz, Evonik Operations GmbH, Germany, Sarah He, Evonik (China) Co., Ltd., China

Whiteleg shrimp production has doubled in the past decade, growing from 2.6 million metric tons (mMT) to 4.97 mMT between 2010 and 2018. It had the largest share (53%) of total crustacean production (FAO, 2020) over this period too. This significant increase in production is partly because of the intensification of shrimp farming, which is gradually shifting from semi-intensive to intensive and superintensive farming. As farming practices intensify, stocking density has gone up from 50 shrimp/m2 to 200-400 shrimp/m2. Unfortunately, the rapid growth and intensification of shrimp farming create tremendous pressure on the water quality and nutrient load of the surrounding environment. Clean water is an essential resource for the sustainable growth of shrimp farming. With stocking density continuing to increase, there is an escalating challenge to maintain the ideal water quality parameters needed for healthy shrimp. Shrimp feed contains 35-45% crude protein, of which only about 35% is retained in the body. The rest is largely catabolized, contributing to nitrogen waste. One way to improve feed protein utilization is to formulate feed following the ideal protein concept. This means meeting the exact amino acid requirements of shrimp with no deficiency and no, or little, excess. In practical diets – formulated using ingredients such as fishmeal, soybean meal and wheat products – except for the first limiting amino acid, methionine (Met), the other amino acids are provided in excess

and are inevitably catabolized, contributing to nitrogen excretion. With the use of supplemental amino acids such as AQUAVI® Met-Met (DL-Methionyl DL-Methionine), Biolys® (Lysine Sulfate) and ThreAMINO® (L-Threonine) in diet formulation, we can reduce the inclusion of protein-rich ingredients, and minimize the excess levels of dietary protein or amino acid levels – still meet the requirements for all the limiting amino acids. This will increase the protein utilization to 40-45% (Nunes et al., 2019; Masagounder et al., 2021). However, while this approach can reduce the nitrogen load, there is still a lot going into the culture water which needs to be removed. Ammonia is the principal nitrogen waste product excreted by shrimp. Ammonia from the hemolymph (blood) of shrimp is largely excreted into the water across the gills via diffusion process in the form of un-ionized ammonia. Assuming 35-45% protein retention, this means, for 10 g shrimp stocked at 100/m2 in a 1 hectare (ha) pond (water depth 1 m) and fed at 3% body weight per day with a feed containing 35% crude protein, about 11,000-13,000 g ammonia per ha or 1.1-1.3 parts per million (ppm) NH3 is added into the pond every day. In other words, for every 100 kg feed, about 0.4 ppm of NH3 is added into the pond water. If this is not removed, this will quickly increase the ammonia concentration in water. As the concentration of ammonia in the water increases, the diffusion gradient

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

of NH3 from hemolymph into the water decreases – leading to accumulation of NH3 in the blood causing serious physiological stress and illness to the shrimp. The exact LC50 of total ammonia nitrogen (TAN) on shrimp depends on various parameters such as salinity, pH, species and age and varies, 30-110 mg/L over 48 h. Although ammonia is the principal nitrogenous waste from shrimp, we also find nitrite accumulation in the culture water over time. Nitrite is an intermediate product in the nitrification or de-nitrification cycle. In the nitrification process, ammonia is oxidized by highly aerobic, chemoautotrophic bacteria. During denitrification, heterotrophic bacteria use nitrate in the absence of oxygen (anaerobic process) and nitrate is reduced into nitrite and eventually into a gaseous form of nitrogen. Both ammonia (mainly un-ionized form) and nitrite are toxic to shrimp. As a consequence, the toxicity of NH3-N and NO2-N negatively impacts excretion, respiration, osmoregulation, immunity, antioxidant defense, molting, growth, feed utilization and eventually survival of the animal (Zhao et al., 2020).

A multi-strain Bacillus subtilis-based product for water application Probiotics are increasingly used in aquaculture to control disease, improve water quality, and enhance the health status of fish and shrimp. Bacillus-based probiotics with species including B. subtilis (Liu et al., 2010; Maia et al., 2016), B. licheniformis (Zhang et al., 2011), B. amyloliquefaciens (Xie et al., 2013) and B. pumilus (Nimrat et al., 2012) are commonly used in shrimp aquaculture for both water and feed application. Gram-positive spore-forming bacteria from the species Bacillus subtilis preferably use glutamine as a nitrogen source but in absence of such, it can incorporate inorganic nitrogen sources, such as ammonia into organic biomolecules. B. subtilis has the genetic features to take up ammonia, nitrite, or nitrate from the environment, reduce nitrate and nitrite to ammonia and feed this into glutamine synthesis. The efficiency of this assimilation of inorganic nitrogen varies among strains as the activities of the required enzymes are usually under strict control. By applying efficient ammonia and nitrite assimilating B. subtilis strains to polluted water, reduction of toxic ammonia and nitrite can be archived by simple growth and biomass formation of the added bacteria. With this background, Evonik developed a new product called AQUAVI® Pro-Pond (Pro-Pond).

Figure 1. Ammonia and nitrite removal of the three specific B. subtilis strains included in Pro-Pond under 0, 10 (DSM 3351 and 3352), 15 (CCTC M 2020356) and 30 ppt salinity after 48 hours.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Pro-Pond is composed of three B. subtilis strains that were selected based on their ammonia and nitrite removal capabilities under a wide range of salinities. To ensure product stability, Pro-Pond contains the strains in the form of endospores with a total concentration of 1.5×1010 CFU/g. Ammonia and nitrite removal capacities under salinities ranging from 0 to 30 ppt for the single product strains are depicted in Figure 1. The efficacy of the product containing the combined spores of the three strains was tested under high ammonia concentrations after 24 and 48 hours and is illustrated in Figure 2.

Figure 2. Ammonia and nitrite removal of Pro-Pond after 24 and 48 hours.

Figure 3. Trend of total ammonia level over the whole culture period in the Pro-Pond treated ponds (purple, 0.25 ppm) versus commercial control ponds treated with multiple products (C1, C2, C3). Solid dots indicate the days when the pond was treated with its respective product. Bigger solid grey dots in C3 indicate the days when two different products were applied on the same day. Purple arrows in the top panel indicate drop in NH3 level following the Pro-Pond treatment.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Evaluation in a semi-intensive shrimp farm in China To evaluate the efficacy of AQUAVI® Pro-Pond on the nitrogen removal efficacy, a commercial pond trial was conducted in collaboration with Prof. Dr. Shijun Chen, South China Agricultural University at the Guangdong province in China between November 2020 and March 2021. Three earthen ponds at a stocking density of ~80 shrimp/m2 were allotted to the Pro-Pond treatment. The average culture period of the three ponds lasted for 108 days (97, 112, and 115 days for the three ponds). Three other earthen ponds (average area 3,557 m2 or 5.3 mu) were assigned to commercial control and the average culture period lasted 102 days. The three ponds (average area 2,668 m2 or 4 mu) allotted for Pro-Pond group were treated with Pro-Pond at 11.3 times per pond on an average (34 times in total for the three ponds) at a dose of 0.25 ppm or 2.5 kg per ha during each application over the whole culture period (108 days). On the other hand, the three commercial control ponds were treated with four different commercial products at 10.7 times per pond on an average (32 times in total for the three ponds) over the 102 day culture period. These four commercial products were based on probiotics (Bacillus, nitrifying bacteria, Enterococcus faecalis), yeast and/or enzyme mix. Product selection, dose and application frequency were decided by the farmer based on his experience and the recommendation from the respective product supplier. Ammonia nitrogen and nitrogen were recorded daily for each pond until harvest. Given the differences in product application in the commercial control ponds, the average of Pro-Pond treated ponds were compared with individual commercial control pond on the ammonia level over the culture period (Fig. 3). Pro-Pond was able to keep the ammonia and nitrite levels down (Fig. 3, 4). When the ammonia level increased, the product was applied and we could see that in the following one to two days, the NH3 level went down. Overall, Pro-Pond was marginally better than the commercial treatment on controlling the NH3-N level (0.45 mg/l versus 0.53 mg/l; p-value = 0.10, t-test). There were no differences between the two groups on the nitrite level. Although multiple products were applied in the control ponds by the farmer, Bacillus subtilis alone was sufficient to control nitrogen

Figure 4. Overall concentration of NH3-N and NO2-N in the Control and Pro-Pond groups. NH3-N level in the Pro-Pond treated ponds was marginally lower (-15%) than that in the control ponds which were treated with four different products (p-value = 0.10, t-test).

Figure 5. Final yield of shrimp (kg/mu) for the commercial control and Pro-Pond treated groups.

waste in the commercial ponds. At the end of culture, the average harvest yield of shrimp was 505 kg/mu for the control group and 535 kg/mu for the B. subtilis group (6% higher). Results demonstrate that Pro-Pond can be used to control nitrogen waste and improve shrimp production.

Potential on controlling common shrimp and fish pathogens A co-incubation of the Pro-Pond product strains and relevant aquaculture pathogens via antagonism assay was applied to determine the inhibitory potential. The inhibitory zone/halo formed in bacterial lawn of the pathogen after 24 hours was determined and classified in: a negative sign (-) means no inhibition, a positive sign (+) means mild inhibition, double positive sign (++)

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Table 1. In vitro inhibition profiles of Pro-Pond strains towards different pathogens relevant for aquaculture.

DSM 33351

DSM 33352

CCTCC M 2020356

Vibrio parahaemolyticus +

Vibrio penaecida

- + -

Vibrio haarveyi

- + +

Vibrio anguilarum - - +

Vibrio alginolyticus ++ + +

Aeromonas hydrophila ++ ++ Not tested

Streptococcus agalactiae +++

+++ Not tested

means medium inhibition, and finally triple positive sign (+++) means strong inhibition. The combination of strains yields a broad pathogen inhibition profile is illustrated in Table 1. Overall, our study results demonstrate that AQUAVI Pro-Pond® developed with three unique strains of B. subtilis bacteria can be used for water application to effectively remove the nitrogenous wastes (NH3N, NO2-N and NO3-N) from the culture water and potentially keep the pathogen load under control in commercial shrimp farms.

More information: Karthik Masagounder, Head of Aqua Research Evonik Operations E: animal-nutrition@evonik.com Michelle Dargatz Scientist Evonik Operations

Sarah He TSM and R&D Manager, Aqua Evonik (China) Co. Ltd.

References are available on request.

CLEAN FEED. CLEAN WATER. Wenger Extrusion Solutions for RAS Feed Production Wenger innovative extrusion solutions deliver clean, durable, nutritional feeds specifically designed for the most efficient RAS operations. Feeds produced on Wenger systems maintain their integrity better and longer, for clean and clear water. So you feed the fish, not the filter. Learn more about the Wenger RAS advantage. Email us at aquafeed@wenger.com today. PHONE: 785.284.2133 | EMAIL: AQUAFEED@WENGER.COM | WENGER.COM USA

BELGIUM

TAIWAN

BRASIL

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

CHINA

Hydrolyzed Kluyveromyces fragilis: The all-in-one solution for fish and shrimp Valentin Eckart, Biochem

Aquafeeds must meet high standards to meet the challenges of the modern aquaculture industry. Rising raw material prices are forcing feed manufacturers to use cheaper alternative protein sources to maintain the high nutritional value of their feeds without unduly driving up costs for farmers. These new feed formulations often have off-flavors that must be covered up with attractants. Speaking of farmers, emerging diseases, the effects of climate change and high stocking densities have raised awareness of the need for proper stress management in fish and shrimp. In short, modern aquafeed must be palatable, nutrient-rich, health-promoting and affordable in increasingly challenging times.

Fish prefer Kluyveromyces fragilis To achieve this, Biochem has developed the product TechnoYeast, a hydrolyzed yeast based on Kluyveromyces fragilis (also known as K. marxianus). In its unhydrolyzed form, K. fragilis has already been shown to modulate the gut microbiota and counteract soy meal-induced enteritis in Atlantic salmon at 200 g/kg intake (Grammes et al., 2013). Other studies have found that K. fragilis can replace up to 40% of LT fishmeal in Atlantic salmon diets without affecting growth. No comparable results were found for the most commonly used yeast species Saccharomyces cerevisiae (Øverland et al., 2013). Accordingly, K. fragilis appears to possess some beneficial properties that could be even more effective after splitting into their functional components by hydrolysis.

Figure 1. The average consumption of shrimp feed from the control diet (CON1), the 20 g/kg squid meal diet (SM20), and the 20 g/kg TechnoYeast diet (TY20) after 30 minutes. Due to the higher feed consumption of SM20 (+11%) and TY20 (+13%), the two feeds had a stronger attraction effect than CON1 (n=40).

Figure 2. White blood cell (WBC) density in blood. Fish fed increasing levels of TechnoYeast (TY 0.5%, TY 1.0%, TY 2.0%) had lower WBC counts – an indicator for reduced pathogenic pressure. Different letters accompanying bars denote significant differences (P < 0.001; n=3).

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Table 1. Data on fish weight, weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR) and survival. Values are the means. Different letters in a row indicate significant differences (P < 0.001) (± standard deviation, n=30).

CON2

TY 0.5%

TY 1.0%

TY 2.0%

Initial weight (g)

17.2 ± 0.5

17.8 ± 0.7

17.1 ± 1.2

12.9a ± 2.5

Final weight (g)

81.4a ± 2.0

91.2b ± 4.6

94.6b ± 6.5

102.9c ± 5.3

WG (g)

64.2a ± 1.5

73.5b ± 5.0

77.5b ± 7.0

90.0c ± 7.3

SGR (%BW/day)

1.73a ± 0.01

1.82a ± 0.08

1.90a ± 0.12

2.31b ± 0.24

FCR

0.79a ± 0.01

0.73a ± 0.04

0.69a ± 0.05

0.61a ± 0.05

Survival (%)

99.98a 99.33a 99.67a 99.98a

Increase feed intake of plant-based diets Nowadays, attractants are very common in state-ofthe-art feed formulations with reduced fishmeal content and high content of plant material. For example, it was recently reported that ingredients such as krill meal and fishmeal hydrolyzate in soybeanbased diets accelerate and increase feed intake of shrimp (Soares et al., 2021). To test whether these effects were also observed when hydrolyzed K. fragilis was used, three isonitrogenous shrimp diets were formulated - one with no attractants (CON1), one with 20 g/kg squid meal (SM20), and one with 20 g/kg TechnoYeast (TY20). Five grams of each of these diets were randomly placed in the center of the marked feeding areas in a tank containing 100 Pacific white shrimp (size 8-10 g). After 5, 15, and 30 min, the number of shrimp within

the marked area and next to the feeds was counted to determine shrimp feeding preference. Then, the remaining feed was removed, dried, weighed and compensated for leaching loss to calculate feed consumption. The experiment was repeated 40 times at different feeding sites. There was a clear preference for the two feeds with attractants compared to the control. Finally, measurement of the consumed feed confirmed that hydrolyzed K. fragilis provided at least the same attractiveness as other commercial shrimp attractants such as squid meal (Fig. 1).

Pushing feed efficiency to the limits The COVID-19 pandemic has shown how fragile the world market can be. There have been shortages of feed raw materials and a drop in the price of harvested

Figure 3. Composition of white blood cells (WBC). A WBC composition with increased neutrophils may be a sign of acute inflammation (min. sig. level compared to CON2: lymphocytes P < 0.05; neutrophiles P < 0.001; No significance for monocytes & eosinophiles; n=3).

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 4. Liver enzyme analysis of lactate dehydrogenase (LDH), aspartate transaminase (AST) and alanine transaminase (ALT). Elevated values are an indicator of bad liver condition and tissue damage. Different letters accompanying bars denote significant differences (P < 0.01; n=3).

fish and shrimp. As aquafeed is still the biggest cost factor for farmers, high feed efficiency is important to maintain competitiveness. In this context, TechnoYeast was tested in a dose-response trial with rainbow trout (Oncorhynchus mykiss). Over a 90-day period, 4 groups of 200 fish (initial weight 16.3 g) were fed a commercial trout diet (CON2) with different doses of TechnoYeast - 0.5% (TY 0.5%), 1.0% (TY 1.0%) and 2.0% (TY 2.0%) with 3 replicates each.

Results showed that fish performance was significantly better with increasing doses of TechnoYeast (Table 1). The average FCR was reduced from 0.79 to 0.61, and the final weight was improved by up to 26%. Hydrolyzed K. fragilis is rich in free amino acids, dipeptides, tripeptides, and nucleotides, but also contains decent amounts of prebiotic components such as mannanoligosaccharides (MOS) and β-1,3-1,6-glucans. According to the above-mentioned modulation of the

Figure 5. Consumed feed from standard feeding regime during thermal challenge. Feeding was started on the second day at 14°C water temperature. TechnoYeast treated groups (TY 0.5%, TY 1.0%, TY 2.0%) continued feeding at high rates even at temperatures above 20°C. The negative control (not shown) was normally feeding at a constant temperature of 10°C (n=3).

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 6. Survival of fish during a 7-day thermal challenge. The temperature was increased by about 2.5°C every day. Treatment groups, which were fed TechnoYeast at 1% and 2% inclusion (TY 1.0% & TY 2.0%) had about 10-20% lower mortality at different temperature levels compared to the control group (CON2). There was no mortality observed in the negative control group at constant 10°C water temperature (n=3).

gut microbiota and the demonstrated attractant effect, this combination of functional components must be the reason for the improved performance and feed efficiency shown here.

Reduce stress for better growth There are many types of stress in the life of an aquacultured fish, such as handling stress, suboptimal temperature, low oxygen, pathogens, or high stocking densities. Blood analyses are often used to get an idea of the current immune status of animals, such as by the number and composition of white blood cells (WBC) as part of the innate immune system. Peters and Schwarzer (1985) described the relationship between elevated levels of WBC with a dominant abundance of neutrophils in the blood of rainbow trout and exposure to various types of stress. Blood analysis of rainbow trout fed TechnoYeast showed a significant reduction in the total number of WBCs and a significantly lower abundance of neutrophils compared to untreated fish (Fig. 2, 3). Despite the same production environment, these fish appeared to be less stressed. These observations were confirmed by analysis of liver enzymes such as lactate dehydrogenase (LDH), aspartate transaminase (AST) and alanine transaminase (ALT) (Fig. 4). LDH, AST, and

ALT are commonly used as indicators for assessing liver health in relation to intoxication and tissue damage (Bury et al., 2003, Parveen et al., 2017). Good to know, but what is the benefit to the fish farmer? If a fish has to spend less effort on strengthening its immune system, it can use the saved energy for growth - as shown in the previous section.

Break the limits: Improve stress tolerance Alternatively, an energy reserve can be used as a buffer to cope with stressful situations. For example, instead of producing WBC, the organism may produce relevant enzymes, such as superoxide dismutase (SOD), to compensate for stress-induced production of radical oxygen species (ROS) (Slaninova et al., 2009). According to this hypothesis, fish fed hydrolyzed K. fragilis should have higher stress tolerance. To see if this was true, trout from the dose-response experiment were exposed to various challenges after the 90-day feeding period. To make a long story short: TechnoYeast-treated fish showed higher survival and vitality under all five stresses - high and low water pH, low oxygen, thermal stress and bacterial stress (Aeromonas hydrophila). The thermal stress results were particularly interesting because warm water is a seasonal threat to many trout farms. Due to the

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

relatively long observation period of one week with increasing temperatures (10 to 28°C in 7 days), the feeding regime was continued. Although trout prefer temperatures well below 20°C, fish fed hydrolyzed K. fragilis continued to consume feed at high rates above 20°C, whereas fish in the control group radically reduced their feed intake (Fig. 5). In addition, the survival rate of TechnoYeast-treated fish was 10% to 20% higher than that of the control group at each temperature level (Fig. 6). Thus, the original hypothesis was confirmed.

Conclusion The initial statement “modern aquafeed must be palatable, nutrient-rich, health-promoting and affordable in increasingly challenging times” applies perfectly to a functional feed material based on hydrolyzed K. fragilis, like TechnoYeast. The last aspect regarding the cost can be answered considering the wide range of benefits derived from the use of this product. The addition of 2% hydrolyzed K. fragilis is a source of highly digestible proteins that accelerates the production cycle, increases feed efficiency and feed intake even in stressful situations, and reduces the risk of excessive losses due to various types of stress. Acknowledgements: Many thanks to the researchers from the Iranian Fisheries Research Organization (IFRO), Iran, and CreveTec, Belgium, who conducted these trials. References available on request.

More information: Valentin Eckart Product Manager Aqua Applications Biochem E: veckart@biochem.net

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Yeast probiotics to boost fish gut health and improve feed efficiency Otavio Serino Castro, Nadège Richard, Phileo by Lesaffre

Gut health results from interactions of host-specific intrinsic characteristics with several factors such as environment microbiome, nutritional status, feed quality, biotic and abiotic stress. Disturbances can be hard to predict and are observed as an acute occurrence or generated after long exposure to a single or a combination of stress factors. Aquatic animals under farming conditions are constantly being exposed to stressors, with the risk to have gut health disrupted by dysbiosis caused by pathogenic bacteria, parasites, mucosa inflammatory process and impaired gut function induced by antinutritional factors, stress due to low water quality, pollutants, etc. In aquaculture, yeast postbiotics rich in bioactive compounds such as mannooligosaccharides, beta-glucans and nucleotides were subject of intense research in the past few decades, with a well-documented mode of action and benefits, especially targeting to improve innate immunity and increase disease resistance. On the other hand, feed supplementation with live yeast as a probiotic and its benefits on gut health has received less attention

SBM14

SBM14 + Actisaf

SBM28

SBM28 + Actisaf

Figure 1. Nile tilapia final body weight after 48 days of feeding. Means with different letter differ significantly (p < 0.05). Source: Ran et al., 2015.

and remains an area with great potential to be furthered explored.

Yeast as a probiotic Saccharomyces yeast is commonly found in marine and freshwater aquaculture environments in water, soil, plants, animals, or microorganisms. They can play different roles supporting aquatic environmental balance such as reducing ammonia, removal of toxic

Figure 2. mRNA gene expression in Nile tilapia hindgut. (A) Heat shock protein 70 (Hsp70) expression; (B) Interleukin 1 beta (il1β) expression; (C) Transforming growth factor-β expression (tgfβ). Means with different letter differ significantly (p < 0.05). Source: Ran et al., 2015.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 3. Nile tilapia (A) survival rate; (B) specific growth rate; (C) apparent protein digestibility and (D) feed conversion rate after 63 days of feeding 40% crude protein diets with and without Actisaf supplementation in different densities. Means with different letter differ significantly (p < 0.05). Source: Lara-Flores et al., 2003.

elements such as heavy metals, reduction of phenolic compounds and integrating the food chain serving as food to invertebrates and zooplankton. The yeast Saccharomyces cerevisiae is a singlecell eucaryotic fungi widely used as a probiotic supplement to improve gut health and microbiota balance in both human and livestock. Potential interest for farmed aquatic animals relies on the ability of yeast to produce and secrete enzymes, nutrients and bioactive compounds that benefit the host and the beneficial bacteria in the gut. Its also relies on its ability to neutralize harmful toxins and produce antimicrobial substances to promote in the gut mechanisms of competitive exclusion with pathogens and stimulate innate immunity and enzymes of the antioxidant system.

Benefits in Nile tilapia Ran et al. (2015) evaluated the effect of the supplementation of a commercial yeast probiotic Actisaf® Sc 47 (Saccharomyces cerevisiae, Lesaffre

proprietary strain NCYC Sc 47/ CNCM I-4407) on tilapia fingerings with 0.7 g of body weight fed for 48 days on diet SBM14 (with inclusion of 14% of soybean meal (SBM), 28% rapeseed meal (RSM) and 10% of fishmeal (FM)) and diet SBM28 (with 28% of SBM, 20% RSM and 5% of FM). Both diets were supplemented with 1 g/kg of Actisaf® Sc 47 top coated in the feed (diets SBM14 + Actisaf and SBM28 + Actisaf). Results indicated significant differences generated by the different formulas, where diet SBM28 outperformed diet SBM14. Yeast probiotic supplementation significantly improved microvilli length and density in the midgut, weight gain and final body weight in both diets (Fig. 1). Overall FCR was improved from 1.56 to 1.44 with yeast supplementation. Additionally, yeast probiotics promoted a significant increase in the abundance of the genus Lactococcus sp. in the allochthonous microbiota, and downregulated significantly the expression of stress related and inflammatory cytokines

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 4. In vivo apparent digestibility coefficients for dietary (A) lipids and (B) crude fiber. Means with different letter differ significantly (p < 0.05). Source: Manuscript in preparation.

in the gut mucosa (hsp70: involved in protein folding, indicator of ER stress; il1β: proinflammatory cytokine; tgfβ: anti-inflammatory (Fig. 2)). In a second trial, Ran et al. (2016) evaluated the impact of Actisaf® Sc 47 feed supplementation during 56 days on Nile tilapia fingerlings (9.8 g body weight) performance in high density (436 fish/m3) compared to a control condition (218 fish/m3). The higher density significantly reduced zootechnical performance and impacted negatively the gut morphology of the nonsupplemented group. Yeast probiotic supplementation generated positive improvements such as significant improvement of intestinal microvilli length, reduced hsp70 gene expression in the intestine compared to the high-density group, increased trypsin activity compared to both groups and reduced activity of gut alkaline phosphatase 24 h post-challenge by Aeromonas

hydrophila. These results help to further elucidate the significant improvements on zootechnical performance and protein digestibility previously observed by Lara-Flores et al. (2003), who observed significant improvements supplementing Actisaf® Sc 47 (1 g/kg) on Nile tilapia fingerlings (0.15 g) reared at different densities (500 and 1,000 fish/m3) for 63 days (Fig. 3).

Benefits in rainbow trout Sheikhzadeh et al. (2015) observed benefits supplementing Actisaf® Sc 47 (1g/kg) on the rainbow trout Oncorhynchus mykiss (12 g body weight) feeds for 60 days. Results demonstrated significant improvement of final body weight and improved innate immune parameters starting after 30 days of feeding. Serum activity of lysozyme and alternative complement system were improved, and total immunoglobulin

Table 1. Microvilli height (µm) in the middle and posterior intestine. Means with different letter differ significantly (p < 0.05). Source: Manuscript in preparation.

Treatment

Middle intestine

Posterior intestine

26% CP

630.4 ± 80.6

684.0 ± 91.4

26% CP + 0.5g Actisaf®

649.3 ± 93.0ab

681.7 ± 136.6

26% CP + 1g Actisaf®

819.5 ± 204.6

678.6 ± 251.0

28% CP

496.5 ± 198.5

474.7 ± 103.3

28% CP + 0.5g Actisaf®

777.1 ± 73.7b

597.7 ± 56.2

28% CP + 1g Actisaf®

807.9 ± 174.9b

614.8 ± 187.9

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

count increased compared to the control group. Fish were subsequently challenged with pathogenic Yersinia ruckeri (BCCM/LMG 3279) via intraperitoneal injection. The yeast probiotic supplementation delayed mortality evolution in the first day’s post-challenge, reaching at day 7 mortality of 30% compared to more than 50% in the control group. Farzad & Kuhn (2020) evaluated the inclusion of Actisaf® Sc 47 supplementation (3 g/kg) in juvenile rainbow trout (52.4 g) diets for 56 days with or without association with yeast postbiotics. Authors reported a positive trend to decrease feed conversion ratio by 20% and improvement on the activity of antioxidant enzymes such as superoxide dismutase.

Benefits in pangasius A recent trial conducted in Can Tho University (manuscript in preparation) evaluated Actisaf® Sc 47 supplementation (0.5 and 1 g/kg) on striped catfish Pangasianodon hypophthalmus (36.4g) diets with 26% and 28% of crude protein for 56 days. No significant differences were observed in survival. An overall trend to achieve higher growth, final body weight, and improved feed conversion rate were observed in animals fed on 28% CP diet. The best FCR and protein efficiency ratio was achieved in the group fed on the 28% CP diet and supplemented with 0.5 g/kg of Actisaf® Sc 47. No clear effect on protein and dry matter in vivo apparent digestibility was observed. Actisaf significantly improved fiber digestibility at 1 g/kg inclusion in both diets, and lipid apparent digestibility on 28% CP diets (Fig. 4). Microvilli height of supplemented groups was also significantly higher compared to respective control groups. The best gut structure was achieved

by supplementing both diets with 1 g/kg, or 0.5 g/kg in the 28% CP diet (Table 1).

Conclusions Yeast probiotic Actisaf® Sc 47 is a sustainable and effective tool to improve gut health management and performance in aquaculture. Its unique characteristics can bring valuable functionalities to the feed, with potential to complement and creating synergies with other solutions. Further studies to better elucidate benefits and its impact on fish welfare and sustainability key indicators would be of high interest to the industry. Ongoing advances in production processes and yeast probiotic physical characteristics are expected to facilitate industrial-scale adoption of yeast probiotics in aquafeeds. References available on request.

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

Nadège Richard Research & Development Manager Aquaculture Phileo by Lesaffre, France

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Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Marine probiotics to positively balance shrimp microbiota and promote better growth and survival Carine Le Ker, Emilie Giudicelli, Fanny Giudicelli, Marine Akwa

The microbiota is known to interact with its host and contribute to a number of key host processes including nutrition, development, immunity and behavior. It is therefore involved in the regulation of shrimp health and disease. Its composition changes with different factors: the stage of development, the feed quality, environmental conditions and the presence of pathogens. Microbiota of farm-raised shrimp is less diverse than in wild shrimp due to lower microbial diversity in pond farms and industrial feed lacking live microorganisms. An alternative to the imbalance of farmed shrimp’s microbiota is the introduction of beneficial bacteria to restore bacterial diversity. Not all bacteria can be successfully introduced in shrimp microbiota or its water environment. Shrimp production is commonly

performed in salted water, so probiotics (i.e. live microorganisms) used in shrimp production must be active in a salinity range from 2-35‰. Most of the probiotics used on the market are of terrestrial origin and, therefore, not fully adapted to the aquatic living environment or the host itself. Stressed terrestrial bacteria slow down or stop their metabolism and reproduction, and consequently stop playing their probiotic function. An illustration is the use of a terrestrial Pediococcus isolated from natural-pasture Gramineae in salmon feed supplementation. The terrestrial probiotic did not persist in salmon microbiota following the transfer from freshwater to seawater. However, benefits have been observed over time (Jaramillo-Torres et al., 2019).

Figure 1. Experimental design of trials from nursery to pre-grow out.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Table 1. Growth parameters at the end of the experiment in control and treated shrimp with marine probiotics consortium encapsulated in algae.

T test, p< 0,05

Marine probiotics encapsulated in algae Probiotics have to resist the salted environment and be able to enter shrimp or fish mucus and compete for space and nutrients in the animal’s gut. For that purpose, the use of “host-associated probiotics” from fish and shrimp has gained attention (Van Doan et al., 2020). Our team strategy was to focus on sessile organisms with holobiont free of aquaculture pathogens genus (more specifically Vibrio). When facing a predator or a pathogen, sessile organisms cannot run and have developed an arsenal of bioactive molecules to fight. Many of them are produced thanks to the symbiotic bacteria they harbor. From our screening and characterization of marine bacteria studies, we developed two formulas dedicated

to fish and shrimp, named AKWABIOTIC, consisting in marine Bacillus probiotics consortium encapsulated in algae providing synergic and beneficial effects to shrimp and fish health and growth. The marine probiotics consortium encapsulated in algae was evaluated in triplicate on shrimp from nursery (3,500 PLs/tank) to pre-grow out (350 PLs/tank) in two experimental stations in China and Peru (Fig. 1).

Growth performance and pathogen challenges Marine probiotics encapsulated in algae significantly increased the final body weight by 11%, weight gain rate by 27%, specific growth rate by 6% and decreased the feed coefficient rate by 15% after the 80 days of feed supplementation (Table 1). Under normal controlled conditions, marine probiotic solutions increase the

Figure 2. Survival with (B) and without (A) salinity stress of control and treated of PL30 shrimp with marine consortium probiotics encapsulated in algae (n=125 shrimps/tank).

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 3. Shrimp survival during the challenge against Vibrio parahaemolyticus (carrying the phage responsible for the Early Mortality Syndrome), at PL30 (A) and after an 80-day experiment (B), treated or not with the marine probiotics consortium encapsulated in algae (n=125 shrimps/tank). Vibrio colonies counted from shrimp’s hepatopancreas on TCBS (C).

survival rate by 6% (Fig. 2). During salinity stress, where salinity was reduced to 1‰, the probiotic solution also improved the shrimp resistance showed 15% more survivors. In challenges against several aquaculture pathogens, marine probiotics at least doubled the number of survivors and shift half-life by 30% (data not published). The challenge with Vibrio parahaemolyticus (carrying the phage responsible for the acute hepatopancreatic necrosis disease) led to an increase of survival by 120% at PL30 (end of nursery) and by 125% at the end of the experiment after the 80-days probiotics supplementation, with mortalities, in control group, being 83% and 72%, respectively (Fig. 3). These high survival rates were associated with a low level of Vibrio in the gut when marine probiotics were administered (Fig. 3).

Figure 4. Relative abundance of hepatopancreas microbiota at phylum level in control or treated shrimp with marine probiotics consortium encapsulated in algae (n=10 HP).

Microbiota profiles After the 80-day trial, both hepatopancreas and intestines were isolated and analyzed to build microbiota profiles (only results from hepatopancreas are presented). At the phylum level, most of the

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 5. Relative abundance of hepatopancreas microbiota at genus level in control and treated shrimp with marine probiotics consortium encapsulated in algae (n=10 HP).

bacteria population in the control group belonged to Proteobacteria phylum, up to 95% of relative abundance (Fig. 4). The dominance of this phylum is standard with marine animals’ microbiota (Yukgehnaish et al., 2020). The use of marine probiotics increased the richness and diversity of bacterial flora. The Shannon index for probiotics treated shrimps was 2,913 points higher than the control. An enrichment of phyla containing

beneficial bacteria with probiotics activities like Firmicutes and Actinobacteria (producing antibacterials), and Bacterioides (digesting vegetal carbohydrates) was observed in hepatopancreas microbiota of treated shrimp with marine probiotics. At genus level, Vibrio spp. dominate, with up to 65% (Fig. 5) in the control group. The use of marine probiotics decreased the content of Vibrio by 63%. Looking at the specific pathogen species, probiotics

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Table 2. Relative abundance of probiotics and Vibrio in hepatopancreas microbiota of control and treated shrimps with marine probiotics encapsulated in algae.

solution decreased by a factor of 3 the relative abundance of Vibrio harveyi and V. parahaemolyticus (Table 2). This decrease was associated with an increase of probiotics genus abundance, here Bacillus, and perfectly illustrates the mechanism of exclusive competition between probiotics and pathogens in the animal gut (Knipe et al., 2021). In this study, Bacillus probiotics colonized microbiota at a relative abundance of 4%. It is important to remember that both introduced probiotics and beneficial commensal bacteria don’t need to be abundant to play a crucial function and act positively on animal physiology (Wang et al., 2020). Probiotic purpose is not to replace microbiota. Above all, probiotics modulate microbiota composition in favor of beneficial bacteria presence, including probiotic ones. At genus level, we also observed the emergence of beneficial bacteria in shrimp treated with marine probiotics encapsulated in algae. Bacterioides are known to be the major producer of B12 vitamin and powerful vegetal fiber digesters (Nathan & Eduardo, 2017). They also are associated with resistance to low salinity in shrimps’ microbiota (Landsman et al., 2019). Dietzia and Bacillus genera produce antibacterial, antifungal, anti-parasite and antiviral substances (Elshaghabee et al., 2017; Modolon et al., 2020). On the other hand, Nautella, an indicator of bad health, disappeared from microbiota when shrimp were treated with marine probiotics (Zheng et al., 2017).

Conclusion The evaluated AKWABIOTIC shrimp formula made of a marine Bacillus probiotics consortium encapsulated in algae allowed the controlled release of the right bacteria at the right place for a colonization and action of adapted marine Bacillus in the shrimp gut. Marine probiotics and algae showed antagonist effects against Vibrio populations. In addition, marine probiotics and algae positively modulated shrimp microbiota to provide a balanced good/bad bacteria ratio. Induced microbiota modulations improve shrimp health and growth and marine probiotics also improve survival under normal and both biotic- and abiotic-challenged conditions. The preventative use of marine probiotics solutions strengthens the bacterial shield which is the microbiota and decreases the need for antibiotics or other chemical inputs during shrimp production. When used after an event, for example, environmental stress, the appearance of pathogens, the use of antibiotics or disinfectants, the marine probiotic solution allows to correct the caused dysbiosis. Finally, the synergy between algae and marine probiotics significantly improves growth and feed efficiency for better productivity. References available on request.

More information: Carine Le Ker R&D Manager Marine Akwa E: carine@marineakwa.com

Fanny Giudicelli Sales Manager Marine Akwa E: fanny@marineakwa.com

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Effect of intestinal conditioner pronutrients in aquaculture diets Ekaitz Maguregui Matellanes, Biovet

Pronutrients: What are they? The term pronutrient was first described by Dr. Gordon Rosen in the 1950s as a micro ingredient included in the diet in relatively small quantities with the aim of improving animal physiology, the intrinsic nutritional value of the feed and prevent the presence of pathogenic microorganisms. Further studies carried out by Biovet S.A., together with universities and scientific research centers all over the world, have described pronutrients as complex molecules of botanical origin derived from shikimic acid, capable of stimulating or regulating animal physiology without causing a pharmacological effect, and whose administration strengthens and maintains animal health. The mechanism of action of pronutrients is based on stimulating certain genes in the DNA of target cells related to specific cellular functions. Such gene stimulation is related to a greater production of tissuespecific proteins, which vary according to the target cell of each pronutrient. Pronutrients are classified into different groups depending on their function and target cell (Table 1), and intestinal conditioners are the ones that improve gut health. Effect of intestinal conditioner pronutrients in aquaculture diets Intestinal conditioner pronutrients are active molecules present in different botanical extracts capable of improving the physiological state of the intestinal mucosa. These pronutrients act directly on enterocytes, accelerating their rate of renewal and improving the structural integrity of intestinal villi. At the same time, intestinal conditioner pronutrients strengthen the tight junctions between enterocytes, which prevents

microorganisms and different toxins from reaching the organism through the paracellular pathway. The supplementation of feeds with intestinal conditioner pronutrients is especially interesting in fingerlings, during the first phases of feeding with dry feeds since these active molecules ensure the complete development of the intestinal mucosa, improving the absorption of the nutrients necessary for their growth. A trial was conducted at the Universidad Científica del Sur (UCSUR) in Peru to evaluate the effect of Table 1. Pronutrients classification depending on their activity and target cell.

Types of pronutrients

Target cell

Intestinal conditioners

Enterocytes

Intestinal optimizers

Cells of the gut local immune system

Immunostimulants

Immune cells

Liver conditioners

Hepatocytes

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Table 2. Control and experimental diet composition.

Ingredients (%)

Control diet

Experimental diet

Fishmeal

22.8

Corn flour

25.2

24.26

Wheat flour

15.21

15.1

Soybean meal

35.25

35.3

DL-Methionine

0.5

Dicalcium phosphate

0.02

Vitamin C

0.2

Common salt

0.5

Premix

0.1

0.2

0.02

Mound inhibitor

BHT

Functional ingredient

Total (%)

100

Table 3. Performance results in the control group fed on the standard diet and the experimental group supplemented with intestinal conditioner pronutrients.

Performance

Control

Intestinal conditioner pronutrients

Initial weight (g)

0.967 ± 0.010

0.961 ± 0.004

Final weight (g)

28.303 ± 2.420

31.363 ± 1.719

Weight gain (g)

27.336 ± 2.422

30.402 ± 1.717

Specific growth rate (%weight/day)

4.819 ± 0.131

4.977 ± 0.078

Total intake/individual (g)

33.185 ± 1.330

34.135 ± 1.793

Feed conversion rate (FCR)

1.220 ± 0.107

1.123 ± 0.026

Survival rate (%) 100 100

intestinal conditioner pronutrients, Alquernat Nebsui from Biovet S.A., on growth in Nile tilapia fingerlings (Oreochromis niloticus).

Experimental design A hundred and twenty tilapia fingerlings were used in eight RAS experimental units of 40 liters each. Two experimental treatments (Table 2) were used in the trial with 4 replicates per treatment: A control group fed on a standard diet without intestinal conditioner pronutrients and an experimental group fed on a standard diet with intestinal conditioner pronutrients. Both diets were isoproteic and isocaloric, with 36% of CP and 4,200 Kcal/kg. Animals were fed

ad libitum, 4 times per day (8:00 am, 11:00 am, 14:00 am and 17:00 am). Water temperature was 28.01 ± 1.34°C, water flow was 0.6 l/min (10% daily renewal) and 12L/12D photoperiod. Nile tilapia fingerlings were raised for 70 days. The following productive parameters were measured: initial and final weight (g), weight gain (g), specific growth rate (% weight/day), total intake per animal (g), feed conversion rate (FCR) and survival rate (%).

Results The productive performance results of both groups are shared in Table 3. The initial average weight in the control group was 0.967 g and 0.961 g in the

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 1. Average growth (g) in the control group with the standard diet and the experimental group supplemented with intestinal conditioner pronutrients.

experimental group. The final weight was higher in the group supplemented with intestinal conditioner pronutrients (31.363 g) than in the control group (28.303 g; Fig. 1). The specific growth rate (SGR) was lower in the group with the standard diet, 4.819% weight/day, compared to the experimental diet, 4.977% weight/day. A slight increase was observed in the total intake per animal in the group with pronutrients (34.135 g/ animal), in comparison with the control group (33.185 g/animal). No differences were observed between both batches regarding the FCR. The survival rate was 100% both in the control and in the experimental groups.

Conclusion The intensive production of tilapia demands strategies based on the use of additives that allow maintaining gut health since the efficiency of diet utilization and the performance results depend on it. Ensuring the development and function of the digestive system is essential to achieve good production parameters and ensure the profitability of farms. Intestinal conditioner pronutrients are an effective and natural tool that can be included in tilapia diets to improve the SGR by 3.6% while maintaining the same

conversion rate thanks to their positive effect on gut health, which is related to an improvement of feed digestion and nutrient absorption. In addition, pronutrients are active molecules that do not affect aquatic ecosystems. Since they are completely natural, they do not leave residues in meat or water and lack a withdrawal period.

More information: Ekaitz Maguregui Matellanes Technical Area Manager Biovet E: ekaitz@biovet-alquermes.com

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

The gut health revolution Marta Arredondo Luque, Novation 2002

Ensuring efficient and sustainable aquatic production is not always an easy task. Aquaculture encompasses a high diversity of species with very diverse feeding habits and cycles which hinders the use of a unique solution. Aquaculture production has been growing exponentially for years and the World Bank estimates that by 2030, 62% of fish produced worldwide will come from aquaculture. To meet the future demand, aquaculture must find a solution to challenges, such as fishmeal (FM) and fish oil (FO) substitution, reduction of chemical use and antibiotic resistance and improve immune response against pathogenic challenges.

Fishmeal and fish oil substitution Aquatic production is extremely dependant on FM and FO supply, pressuring wild stocks and making unsustainable aquaculture grow. Numerous plant have been tested as protein and oil sources for feeds. However, these protein sources have antinutritional factors and can have detrimental effects on growth performance and welfare as well as increasing feed and nutritional waste. Soybean meal (SBM) is a high protein source and is currently a commonly used protein source in aquafeed including carnivorous species. However, high inclusions of SBM have detrimental effects on gut health causing enteritis, high vulnerability against pathogenic challenges (bacteria, parasites, or toxins), and changes in the absorptive cells provoking inflammation and villi shortening. Improve resistance against pathogenic challenges Over-replacement of FM can also lead to inferior performance and poor disease resistance, negatively affecting the oxidant-stressed status and induced SBM inflammatory response. Several studies have demonstrated that high inclusions of SBM disturb oxidative homeostasis in fish. The imbalance between

reactive oxygen species (ROS) generation and elimination can cause oxidative stress. Sensitivity to novel protein raw materials in feed has also been observed in several aquatic species, resulting in strong immune responses. Induced SBM enteritis in Atlantic salmon, carp and turbot has been long known. Cytokines play a key role in the regulation of the immune response. Intestinal inflammatory response with the combination of proteins and antimicrobial peptides are crucial for the control of pathogens. Previous studies confirm that inflammation response might be deteriorated via down-regulation of anti-inflammatory cytokines (IL-1β and TNFα) and up-regulating of pro-inflammatory cytokines (TGF-β and IL-10).

Reduction of chemical use and antibiotic resistance The intensification of aquaculture in the past forced the industry to look for solutions to increase survival even when welfare was not ideal. The constant bacterial and parasitic outbreaks were difficult to control, and the use of antibiotics and chemical products was the only solution at the time. In the past, and currently in some countries, there is excessive use of antibiotics to prevent and/or control infectious diseases caused by bacterial pathogens and for growth-promoting purposes. Public awareness of the potential harm to public health due to antibiotic residue bioaccumulation and antibiotic resistance led to their ban on animal feed formulation. All these challenges led to the search for feed additives for a more sustainable aquaculture. The benefits of using butyric acid in aquaculture Aquafeeds use additives to supplement the nutrients to counteract the lack of some essential nutrients. However, additives look into a more functional approach. These so-called “functional nutrients” affect

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Feed Conversion Ratio (FCR) FCR

0,85 a

Control

FCR

0,85 ab

Competitor 0.5%

0,80 b

FCR

Butirex C4 0.5%

Final Biomass (g) 93,57b g Control

96,34 abg Competitor 0.5%

100,06a g Butirex C4 0.5%

Yield (Kg/m³) Kg/m³

1,17 b

Control

Kg/m³

1,20ab

Competitor 0.5%

Kg/m³

1,25 a

Butirex C4 0.5%

Figure 1. Performance rates of tilapia with and without the use of Butirex-C4.

metabolism and physiology, directly and indirectly, helping mitigate the detrimental effects due to the intensification of aquaculture. Short-chain organic acids (C1-C7) and their salts or mixtures, the so-called “acidifiers”, are natural metabolic products of organisms. The supplementation of feed with organic acids in terrestrial livestock such as poultry and swine have consistently reported improved performance, such as increased feed intake, growth, feed utilization efficiency and improved health and immunity. While various feed additives have been studied to substitute the use of antibiotics as growth promoters, organic acids are increasing in popularity due to their strong antimicrobial and prophylactic activity against several pathogenic bacteria. This positions them as a suitable alternative. Acidifiers have been widely used for terrestrial animals giving outstanding effects. Although only ten-year research accompanies the use of acidifiers in aquaculture, results from research and fieldwork in fish and shrimp have shown acidifiers can help the

aquaculture industry in various ways. Depending on the species, culture conditions and health management practices, effectiveness and applicability can vary. Butyric acid (C4) is formed by microbial fermentation of non-digestible carbohydrates in the gastrointestinal tract of animals. Butyric acid and its salts, such as sodium and calcium butyrate, have drawn attention in animal nutrition, initially in terrestrial nutrition and now in aquaculture formulation, due to its numerous favorable effects both on intestinal and peripheral tissues. Based on results obtained using butyric acid in terrestrial animal nutrition, studies performed in aquatic species have shown improved performance, growth, feed efficiency, antioxidant capacity and immune response.

Butyric acid, the ally to FM replacement Butyric acid has been a good ally to improve fish intestinal health when feeding low-FM aquafeeds. Its inclusion has helped counteract the impaired growth and performance.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Recent results conducted with rainbow trout supplemented with Butirex-C4, a commercial protected sodium butyrate, support previous studies evaluating butyric acid as a booster of intestinal health when reducing the use of FM. This is attributable to improved digestibility and optimization of nutrient uptake (Fig. 1).

Why does the inclusion of butyric acid in diets improve performance? In aquatic species, the first line of defense against infectious diseases is the innate immune system, which is divided into physical barriers, cellular and humoral components. The fish intestine not only takes a role in digestion and absorption of nutrients but also has a crucial role in the immune response. The gut barrier not only act as a physical barrier allowing the uptake of nutrients but also prevents pathogens from entering the blood flow (Fig. 2). The epithelial barrier, formed by epithelial cells chained together by tight junction proteins, plays a key role in maintaining the integrity and function of the physical barrier. Previous studies have shown that optimal butyrate supplementation up-regulated tight junction proteins such as OCLD, ZO and claudin. Some studies indicate the potential of butyrate improving fish gut structure integrity, such as a recent trial in rainbow trout supplemented with Butirex-C4 showing improved tight junction proteins gene expression (Fig. 3).

Tight junction reinforcement

Zonula Occludens-1

0,97a

1,16a

4,10c

2,13b

3,26a

2,86a

5,48b

9,45c

65,55a

70,33a

109,55b

105,22b

CONTROL

BUTIREX C4 0.15%

BUTIREX C4 0.25%

BUTIREX C4 0.50%

Figure 3. Relative tight junction protein gene expression results.

Figure 2. Illustration of bacterial infection in the gut barrier.

Intestinal enterocytes have surface-enlarging microvilli that are responsible for nutrient absorption. The absence of healthy villi and microvilli translates into a reduced nutrient absorption surface area in fish intestines. The replacement FM with plant-based diets in aquafeed also revealed villi shortening, making aquafeed less efficient and fish feed not converted effectively. The trial conducted in rainbow trout also revealed improved villi growth and development in Butirex-C4 supplemented diets (Fig. 4). This promotes nutrient absorption area boosting growth and performance due to effective nutrient utilization. More efficient barrier functions, improved antioxidant status and inflammation control can also help counteract the deleterious effects of plantbased diets. Diets containing low FM content and supplemented with Butirex-C4 in L. vannamei resulted in an increased total haemocyte count, phenol oxidase, catalase and antioxidant activity. Shrimp gut is an organ that plays a vital role in immune response, studies might indicate a connection between the improvement in gut physiology and gut histology. Controlled inflammatory response can also be key to improved resistance to pathogenic or chemical challenges. The intestinal immune-related genes that initiate an inflammation effect can also be counteracted using functional additives. Trials conducted with protected Butirex-C4 have demonstrated an efficient down-regulation of proinflammatory cytokines and up-regulation of anti-

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Villi growth and development a

a 377.5 a 33.0a 386.2 32.16

443.0c 45.16b

410.2 b 41.16 a

Villi length Villi thickness Muscular thickness

160.7 a

160.8 a

169.7 ab

178.0 b

CONTROL

BUTIREX C4 0.15%

BUTIREX C4 0.25%

BUTIREX C4 0.50%

Figure 4. Villi length and development with and without the use of Butirex-C4.

inflammatory cytokines. This translates to fish that are not wasting energy due to inflammation caused by antinutritional factors of plant proteins and can save energy to be able to fight against pathogenic or chemical challenges.

Conclusion In summary, the use of organic acids like butyric acid has a global effect on fish welfare. Butyric

acid modulates immune components, cytokines and improves intestinal integrity, which translates to a boost in performance. All this makes butyric acid a good candidate to improve fish and shrimp resistance to disease and pathogenic challenges. Studies conducted to date can give us a hint that protected butyric acid is an effective intestinal booster to reach soon our common objective of sustainable aquaculture. References available on request.

More information: Marta Arredondo Luque Aquaculture Product Manager Novation 2002 E: marredondo@novation2002.com

Unique protected sodium butyrate for aquafeed C

CMY

The gut health revolution www.novation2002.com

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Juvenile gilthead seabream (Sparus aurata) nutrition: Defining micromineral recommendations for more sustainable aquafeeds David Dominguez, Daniel Montero, Marisol Izquierdo, Aquaculture Research Group (IU-ECOAQUA), University of Las Palmas de Gran Canaria, Ramón Fontanillas, Skretting Aquaculture Research Centre Introduction Although, it is possible today to replace, at least to a large extent, fishmeal and fish oil in the formulation of alternative aquaculture feeds for several species, little is known on the long-term effects of changes in diet formulations on cultured fish species. Additionally, there is still a need for further research for an optimized formulation of aquaculture feed rations for the different farmed species over their life cycle with reduced fishmeal and fish oil percentages.

In this context, simultaneous supplementation with micro-nutrients, such as minerals, must be considered in more detail. Analysis of available knowledge on the quantitative requirements of fish (NRC, 2011) for each of the minerals shows a considerable lack of precise information for some of the major species of interest to European aquaculture, including gilthead seabream (Sparus aurata). Minerals are essential inorganic elements for all life processes in animals, including fish. They intervene in

Table 1. Ingredient composition and analyzed Se contents of the experimental diets supplemented with increasing levels of sodium selenite.

Ingredient (g kg-1)

Basal diet

Soya concentrate, CJ SELECTA S.A. (Brazil)

230.0

Wheat gluten, Cargill B.V. (The Netherlands)

216.6

Corn gluten, Cargill B.V. (The Netherlands)

150.0

Wheat, Lantmannen Ek. For. Lantbruk Handel, Malmö (Sweden)

116.9

Fish meal, Norsildmel AS. (Norway)

100.0

Fish oil South American, Copeinca S.A. (Peru)

60.0

Faba beans, Cafetra, B.V. (The Netherlands)

50.0

Rapeseed oil, Thywissen GmbH (Germany)

30.0

Palm oil, Cargill B.V. (The Netherlands)

16.4

Linseed oil, Cargill B.V. (The Netherlands)

8.2

Micronutrient premix†, Trouw Nutrition (The Netherlands)

21.9

† Micronutrient premix includes: methionine (10.6 g kg -1), lysine (28.5 g kg-1), phosphate (6.7 g kg-1), vitamin premix (1.8 g kg-1) and mineral premix excluding the target mineral for each trial (1.1 g kg -1).

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 1. Liver gene expression of MnSOD analyses of gilthead seabream fed increasing levels of dietary Mn for 42 days. Different letters indicate significant differences (p<0.05, means ± SD, n = 3).

the formation of the skeleton, regulation of the acidbase equilibrium and maintenance of the colloidal systems. Many minerals are also components of enzymes, metalloproteins and hormones. Marine fish can obtain certain minerals from the water, while others must be included in the diet (NRC, 2011). Some of these minerals used to be included in enough quantities through fish-derived ingredients, however, the increased substitution of these by plant ingredients has radically altered the dietary mineral balance. In previous studies (Dominguez et al., 2020a), a complete mineral premix with several nutrients was

supplied in six different concentrations in seabream practical diets. The diets were designed with high levels of inclusion of plant ingredients. A great variety of specific biomarkers for each mineral were used in order to provide a good approximation of the optimal dietary levels for the different minerals and, in particular, the requirements for inorganic zinc (Zn). However, in this study, the optimal levels for other minerals such as manganese (Mn), selenium (Se) or copper (Cu) weren’t clarified. Therefore, within the PERFORMFISH project, a series of studies with various levels of individual minerals

Figure 2. Relationship between dietary selenium level and final weight for gilthead seabream fed increasing levels of dietary selenium for 42 days as described by a broken-line model. According to the plot, the optimal dietary level of selenium is 0.91 mg kg -1 (p<0.05, means ± SD, n = 3).

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 3. Liver gene relative expression of gilthead seabream fed increasing levels of dietary selenium for 42 days. * indicates significant differences (p<0.05, means ± SD, n = 3).

were conducted in juvenile seabream fed practical diets aimed at better understanding the requirements of Mn, Se and Cu. More specifically, the objectives of this project include: • Determine the optimal level of Mn supplementation for juvenile seabream fed diets with high levels of plant ingredients. • Define the effect of supplementation with Se in practical diets for juvenile seabream that contain high levels of plant ingredients. • Establish safe levels of Cu supplementation in diets for juvenile seabream with high levels of plant ingredients.

Material and methods Three independent trials were conducted using a common plant-based diet (FM 10% and FO 6%, Table 1) supplemented with either manganese sulphate, sodium selenite or copper sulphate in five levels. The five diets per trial were supplemented to contain 19, 27, 30, 41 and 66 mg Mn kg-1; 0.45, 0.68, 0.86, 1.00 or 1.70 mg Se

kg-1 diet; or 5.5, 7.4, 9.3, 11.0 and 32.0 mg Cu kg-1 diet respectively. For each trial 450 seabream fingerlings weighing 12.6 ± 1.5 g (mean ± S.D.) were distributed into 15 tanks and fed until apparent satiation thrice daily over 42 days. Growth and productive parameters were monitored and samples for biochemical, mineral, histological and gene expression analyses were taken at the end of the trial.

Results Manganese Mn supplementation did not alter growth, feed utilization, whole-body chemical composition or liver morphology, indicating that the Mn content present in the basal diet (19 mg Mn kg-1) already covered the requirement of juvenile gilthead seabream fed practical plant-based diets, most probably due to the higher concentrations of Mn in plant ingredients than animal sources. This level remains lower than the maximum tolerable levels established by EFSA (2013) for fish (100 mg Mn kg-1). Nevertheless, fish fed higher Mn dietary

Table 2. Growth performance and feed utilization in gilthead seabream fed increasing contents of Cu for 42 days.

Dietary Cu (mg/kg)

Cu5.5

Cu7.4

Cu9.3

Cu11.0

Cu32.0

FW (g)

36.2 ± 1.4b

36.0 ± 1.1b

35.5 ± 1.5b

31.5 ± 0.7a

34.9 ± 0.3ab

FCR (g)

1.04 ± 0.02ab

1.01 ± 0.02a

1.05 ± 0.05ab

1.15 ± 0.02b

1.06 ± 0.03ab

FE (g)

0.96 ± 0.02ab

0.99 ± 0.02b

0.95 ± 0.05ab

0.87 ± 0.02a

0.94 ± 0.03ab

FW: Final weight. FCR: Feed Conversion Ratio. FE: Feed efficiency.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 4. Hepatic histological analyses of gilthead seabream fed increasing levels of dietary Cu for 42 days. Histological observations were analysed by pair evaluators in a 0–3 scale, where 0 was absence of observation and 3 presence in most of the liver. Figure A represents liver steatosis, Figure B liver peripheral nucleus, Figure C liver broken cell margin and Figure D liver sinusoid dilatation. Different letters indicate significant differences, p<0.05, n=3.

levels showed a reduction in markers of oxidative risk suggesting a higher requirement to improve antioxidant protection (Fig. 1) (Dominguez et al., 2020b). Selenium Results of this study suggest that the optimum dietary levels of total Se are around 0.94 mg Se kg-1 to promote the growth of gilthead seabream juveniles (Fig. 2) when fed diets with 10% FM. Moreover, and despite the current maximum limit for total Se in animal feeds has been set at 0.5 mg kg-1 feed (EC 1831/2003 and amendments), feed levels of 0.85-1.00 mg Se kg-1 did not produce a negative effect on growth, catalase expression, or liver morphology, and were therefore

safe. On the contrary, dietary levels of 1.70 mg Se kg-1 were toxic as they caused growth reduction, increased catalase expression (Fig. 3) and hydropic degeneration in the liver, thus should be avoided in gilthead seabream formulated feeds containing low levels of marine ingredients (Dominguez et al., 2019a). Copper Basal dietary Cu content of 5.5 mg kg-1, was enough to cover Cu requirements for growth in gilthead seabream. Further supplementation up to 9.3 mg Cu kg-1 did not cause negative effects on any of the parameters studied, suggesting that no Cu supplementation is required in gilthead seabream fed practical diets based

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 5. Visual representation of the recommended levels for Mn, Se and Cu for gilthead seabream as described by Dominguez et al. (2020a) within the ARRAINA project and the current results obtained from the PERFORMFISH project (Dominguez et al., 2019a,b, 2020b , compared to Atlantic salmon (Salmo salar) within NRC (2011). Excess levels were described within the PERFORMFISH project (Dominguez et al., 2019a,b).

that, though the maximum dietary Cu content allowed by EFSA (2014) is 25 mg kg-1, even lower levels can already cause toxicity (Dominguez et al., 2019b).

Final remarks Minerals are essential nutrients that can also be potential pollutants. Their supplementation in aquafeeds must therefore be thoroughly considered. In this series of trials, we successfully demonstrated that feeds for gilthead seabream juveniles with high inclusions of plant ingredients already contain sufficient amounts of Mn and Cu, and thus no further supplementation is required. On the other hand, Se supplementation must be considered only when the levels in the feed are low, and always take into account the narrow margin existing between optimum and toxic levels (Fig. 5). References available on request.

on plant protein sources as long as these provide at least 5.5 mg Cu kg-1. On the other hand, dietary contents of 11-32 mg Cu kg-1 negatively affected gilthead seabream performance by reducing fish growth (Table 2), increasing oxidative risk and inducing hepatic damage and cholestasis (Fig. 4), thus demonstrating

More information: David Dominguez Postdoc Aquaculture Research Group (IU-ECOAQUA),University of Las Palmas de Gran Canaria E: david.dominguez@ulpgc.es

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

A natural algal astaxanthin for aquafeeds and beyond Adelheid Kuehnle, Robert Schurr, Kuehnle AgroSystems

Photo credit: Gordon Wallace of Kuehnle AgroSystems.

Aquafeeds need a lower cost option for natural astaxanthin, a pigmented antioxidant included in diets to benefit animal well-being and provide wildcaught coloration to farmed fish and crustaceans. A chemically synthesized form of astaxanthin - Carophyll® Pink, by Hoffman La Roche/DSM - has dominated red seafood since its debut in 1985 because it supplies adequate coloration but is cheaper than current natural astaxanthin alternatives. This contradicts a growing consumer demand for healthy eating and using sustainably derived naturally sourced feed ingredients.

Natural algal astaxanthin Kuehnle AgroSystems (KAS), a microalgae products discovery and development company located in Honolulu, Hawaii, has patented low-cost technologies for the fermentation of Haematococcus pluvialis. KAS’s fermentation technology offers the potential for the factory scale manufacturing of natural algal astaxanthin from Haematococcus at a cost that rivals synthetic astaxanthin.

Historically, Haematococcus algae was the first industrial source of natural astaxanthin, when Cyanotech commenced cultivation in 1988 using outdoor raceways in Hawaii. Haematococcus naturally accumulates the highest cellular amount of astaxanthin of any organism, at about 5%, an order of magnitude higher than krill oil at 0.12% or wild-type Phaffia yeast with about 0.03%. As such, it requires no genetic engineering or irradiation mutation. Cyanotech’s original intent was to replace synthetic astaxanthin used in aquaculture. Their cracked cell product, NatuRose®, demonstrated excellent performance across relevant species. It is approved in the USA and Canada for salmonids (FDA 21 CFR 73.185; CFIA Reg. No. 990535); in Japan for all animal feeds; in New Zealand for organic feeds; and in the EU comprises dried algae (Regulation (EU) 2017/1017 of 15 June 2017 amending Regulation (EU) No 68/2013, Annex Part C 7.1.2). However, after gaining both FDA approval and market traction for its extracted algal astaxanthin as a human nutritional supplement, Cyanotech discontinued marketing to aquafeeds in late 2007.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Table 1. Some barriers to low-cost and sustainable natural algal astaxanthin (AX) using legacy production eliminated by KAS's novel fermentation technology.

Legacy Barrier

KAS's Fermentation Solution

Cell encystment to accumulate AX, AX accumulation in vegetative requiring cell milling macrozooids, which require no milling

Outcome Bioavailability in salmonid and shrimp feeds and stability in storage demonstrated using uncracked cells

Slow growth using only photosynthesis

Fast growth with fed organic acid and software controlled precision nutrition

Growth rate well over one magnitude faster

Slow carotenogenesis in maturation ponds or PBRs

Rapid AX accumulation in macroozoids in the absence of light

Up to 7 times faster

Inconsistent production, losses from predation or inclement weather

Aseptic conditions and containment maintained during fermentation production

Consistent production cycles, no crop failure

Discontinuous process for vegetative growth and encystment

Elimination of light dependence for carotenogenesis, software controlled precision nutrition

Carotenogenesis is precisely timed and can occur in the same vessel

High cell titers with fed organic acid High water usage to eliminate inefficiencies of cell shading

Removing barriers to market entry KAS hypothesized that if it was technically feasible to grow Haematococcus by fermentation, it would offer significant cost reduction advantages over current algae production methods. The company’s strategy was to remove key production barriers to yield a costcompetitive natural algal ingredient (Table 1) as an appealing “green” alternative while also promoting resource conservation. KAS developed and patented a new algal fermentation method using salt-free organic acids as carbon feedstock in combination with sufficient aeration for oxygenation to dramatically boost growth rates under nutrient replete conditions. This method primes cells to rapidly accumulate pigments within hours and without encystment or light, once nitrogen or other nutrients become depleted (U.S. patent number 11,034,968). Results of cultivating Haematococcus pluvialis using fermentation The result is Naturekrome™, a dried whole-cell ingredient with chemistry identical to NatuRose® (Table 2) but at a significantly lower cost of production. Unlike synthetic, yeast or bacterial astaxanthin, algal astaxanthin is esterified like that of krill, a natural component of the food chain, but in a much more concentrated form. Algal astaxanthin is recognized to

Cell titers well over one magnitude higher; over 24 times higher rate of AX productivity per unit water

provide excellent flesh/skin coloration in some animals, notably shrimp, while also delivering vital benefits to animal health for value beyond color (Guerin, 2019; Lorenz, 1998; Lorenz, 1999; Stachowiak & Szulc, 2021). Esterified astaxanthin is thought to have increased oxidative stability, which benefits applicability and also pigmentation efficiency after cooking (Ju et al., 2011; Lorenz, 2001; Seabra & Pedrosa, 2010). Functionally, esterification may enhance antioxidant properties with implications for enhancing the immunological response, notably preventing lipid peroxidation in several animal models and activation of energy metabolism, i.e., vigor (Aoi et al., 2018; Seabra & Pedrosa, 2010). A recent study documents anti-aging effects by reversing damage caused by oxidative stress using KAS’s AstaFusion® heterotrophic Haematococcus extract with a human skin model (Yamawaki et al., 2021). The fermentation product accumulates at least 2.5% dry weight astaxanthin in complete darkness, even higher in light, plus other carotenoids such as lutein and beta-carotene that contribute to flesh and skin color. The production strains are still capable of oxygenic photosynthesis and can switch rapidly from predominantly heterotrophic to mixotrophic and to autotrophic modes as required. In the wholecell mass, astaxanthin and the other carotenoids are associated with healthy oils that comprise a slightly

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Table 2. General compositions with typical astaxanthin isomer and ester chemistry for Haematococcus pluvialis whole cell biomass grown in the field (NatuRose®) or fermentor (Naturekrome™).

Property

NatuRose®

Naturekrome™

Predominant carotenoid

85% astaxanthin of total carotenoids

86% astaxanthin of total carotenoids

Crude protein

20-30%

25-35%

Carbohydrates

30-40%

20-35%

7-25%

28-33%

C16:0 palmitic, C18:1 oleic, C18:2 linoleic, C18:3 linolenic

5-15%

5-7%

6-9%

3-5%

Crude fat

Predominant fatty acids

Ash

Moisture

Particle size

5-25 microns

10-15 microns

Astaxanthin esterification

70% monoester, 25% diester, 5% free

74% monoester, 23% diester, 3% free

Predominant enantiomer

3S,3'S

higher proportion in Naturekrome™ and a decreased carbohydrate value due to the absence of cyst walls (Table 2). The profile of fatty acids from heterotrophicallygrown Haematococcus is almost identical to that of photosynthetically grown Haematococcus. Polyunsaturated fatty acids comprise about 50% of the fatty acids, with the essential linoleic (C18:2n6) and alpha-linolenic (C18:3n3) as main components with their recognized metabolic and physiological functions (Lin et al., 2019). Initial industry tests indicate promising feed performance for coloration, equivalent to synthetic astaxanthin, with no milling required. KAS’s dried algae works both pre- and post-pelleting. Beyond feed applications, the uncracked biomass has proven to be well-suited for supercritical CO2 or solvent extractions to produce an oleoresin for restoring photodamaged skin (Yamawaki et al., 2021) and for nutraceutical applications.

Concluding remarks Multinational companies are keen on boosting their portfolio of sustainable feed ingredients as part of their long-term strategy in both animal and human nutrition. They play a vital role in influencing the adoption of bio-based products in the market (Gaffey et al., 2021). KAS’s game-changing fermentation platform for algae

ingredients, starting with whole-cell Haematococcus, removes key barriers to deliver sustainable and costeffective natural esterified astaxanthin from a trusted source. Significantly improved production efficiency reduces the cost of production and coupled with the lower natural resource usage of this production method, delivers to the aquaculture market, for the first time, cost-effective sustainably sourced natural algal astaxanthin. References available on request.

More information: Adelheid Kuehnle Co-Founder & CTO Kuehnle AgroSystems E: Heidi.kuehnle@kuehnleagro.com

Robert Schurr Senior Scientist Kuehnle AgroSystems

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Innovative tools to improve monitoring of the nutritional supply chain in aquaculture Dominique P Bureau, University of Guelph, Jamie M. Hooft , Stephen J. Gunther, Wittaya Aqua International

The aquaculture industry is a highly dynamic and rapidly evolving sector that is continually faced with the challenge of improving its efficiency and sustainability. Much of global aquaculture production relies on the supply of nutritious feeds supporting high growth, health and welfare of animals. Considerations also need to be made in order to ensure that the feeds and feeding enable the production of high-quality, highly marketable products in an environmentally sustainable and profitable manner (Fig. 1).

With feed cost representing 50% to 70% of the total production cost, processes focused on optimizing nutrition have a significant impact on the overall economic viability of aquaculture operations. Aquaculture production relies on a “nutritional supply chain”, which includes the production and procurement of feed ingredients, feed formulation and manufacturing and the feeding of animals. Tracking the nutritional supply chain in aquaculture, from ingredients, to feed, to fish is regarded as increasingly important for a variety of reasons.

Figure 1. The nutritional supply chain in aquaculture.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 2. A digital ecosystem linking farms and feeds.

Variations in feed quality can have profound impacts on the performance of animals, quality of the final products, amount of waste outputs and associated potential environmental impacts and profitability of aquaculture operations. Variations in feed quality may be attributable to variations in the quality of the feed ingredients used in the feed formulations or due to issues during formulation, manufacturing and handling of the finished feeds. Feed manufacturers and aquaculture producers also need to be able to ensure traceability of their products and be able to trace back problems to their source.

Tools for feed manufacturers Feed manufacturers invest considerable efforts in analyzing raw materials and finished feeds as part of their Quality Assurance and Quality Control (QA/QC) program. However, QA/QC programs typically generate an inordinate amount of data which is most often underutilized for a variety of reasons. Most notably because of reliance on a tedious and idiosyncratic collection of data (record sheets, spreadsheets, etc.) and a limited capacity to collate, analyze and use the data in a meaningful fashion.

Sustained interactions with feed industry stakeholders worldwide suggest that many are relying on relatively static, potentially outdated, information about the nutritive value of their feed ingredients. Nutrition and QA/QC teams often spend several hours or even days tracking information about the quality of feed ingredients in preparation for monthly or quarterly management meetings. Large feed manufacturers can afford tailor-made ERP systems that can enable tracking of this kind of information. However, for many medium and small-size manufacturers, such systems are overly expensive and complicated. Innovative platforms addressing this challenge are now available to feed manufacturers of any size. These include BESTMIX® Quality Control from Adifo (adifo.com), KAllix from A-Systems (a-systems.fr) and the Raw Material Map (RMM) which is part of the AquaOp Feed platform (wittaya-aqua.ca). These solutions can help streamline QA/QC data collection and handling. They allow feed manufacturers to save time, avoid errors, update feed ingredient composition databases used in formulation software on a more regular basis, improve the monitoring and benchmark of the quality of raw materials sourced from different

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

suppliers and thus support decisions making by the nutrition and QA/QC teams. While there is no doubt that the analyses carried out as part of QA/QC programs are of crucial importance to ensuring stable quality, the ultimate measure of quality is in the performances of the animals fed these feeds. Many of the measurements carried out as part of QA/QC programs are surrogates for true measures of nutritive value of ingredients and feeds. For example, crude protein is not an indication of the true protein content of an ingredient. Nutrient analyses, such as amino acid analysis, do not provide any information about the digestibility and bio-availability of the nutrients. There would be great value for feed manufacturers in effectively being able to link animal performance with data on feed formulation and composition. Feed manufacturers cannot afford to test every batch of raw materials and finished feeds through animal growth trials. Consequently, they are highly dependent on monitoring the performance of their feeds under commercial conditions at their clients’ operations. However, obtaining accurate and detailed information about animal performance from clients can be difficult due to challenges associated with sharing of information. In addition, performances are affected by a multitude of factors and it is difficult to attribute effects to feeds or other factors.

Tools for aquaculture operations While virtually all aquaculture operations are actively involved in the collection of animal performance data, they are also relying on ineffective collection of data using idiosyncratic formats and have a very limited capacity to effectively use the data collected. Farm data is often collected on paper, on whiteboards or in a myriad of spreadsheets. Collected data are often incomplete, contain errors or bias and are often rough estimates or simply “made up” (e.g. estimates of live weight based on feed served and an expected FCR). The diversity of data compilation formats makes it difficult and tedious to integrate and analyze data. Good management of aquaculture operations begins with effective capture, vetting, compilation, and analysis of information. Accordingly, there exists a significant opportunity for aquaculture operations to better utilize the data generated.

While large aquaculture operations can rely on complex and expensive software, including FishTalk (www.akvagroup.com) and Mercatus (www.scaleAQ.com) to track production data, more affordable and user-friendly aquaculture farm management software have been developed for aquaculture operations of any size. Software, such as AquaManager (www.aqua-manager.com), Aquanetix (aquanetix.co.uk), Jala (jala.tech) and AquaOp Farm (wittaya-aqua.ca) to name a few, are increasingly popular among small- and medium-sized farms. AquaOp Farm is a cloud-based farm management platform that helps farms manage their operations more effectively. AquaOp Farm enables efficient uploading, tracking and safe-keeping of production data. A cloud-based system provides secure warehousing and rapid access to data from anywhere with confidentiality. The platform is easy to use, comprehensive, and adaptable to a very wide variety of aquaculture species, from salmon and trout to tilapia and shrimp. It is able to accommodate the complexity of the production process on farms, from lot splitting to merging to partial harvest. Data such as water quality, sample weights and feed amount can be easily entered and monitored. AquaOp Farm generates a series of reports including forecasts for growth, feed requirements, waste outputs, oxygen requirements, etc. These reports can help aquaculture producers improve their feeding practices, reduce wastes and improve FCR and help farms become more efficient, profitable and sustainable.

Approaches and tools to link field performance and feed composition Adoption by feed manufacturers and aquaculture operations of systems enabling the efficient capture, compilation, integration and vetting of data would be an important step towards establishing an appropriate linkage between feed composition information and commercial field performance. Integrating information from the entire nutrition supply chain could help better characterize the effects of various factors (e.g. ingredient type, source and quality, nutritional specifications, physical characteristics of the feeds, etc.) on different parameters (growth, survival, FCR, etc.) under field conditions and potentially enable fine-tuning of

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

feed formulation to improve sustainability and profitability of the sector. AquaOp Farm and AquaOp Feed are uniquely integrated to provide a feedback loop between commercial field performance and feed ingredient composition and nutritional characteristics of the feed (Fig. 2). These two platforms are powered by a series of proprietary, cutting-edge hybrid knowledge and data-driven mathematical models. The platforms are designed to foster confidential, seamless and noninterfering cross-cooperation amongst stakeholders (aquaculture producers, feed manufacturers, hatcheries/breeders, processing plants, etc.). Moreover, the platforms can also be used as objective reporting tools to third parties such as regulatory agencies, certification bodies and financial institutions.

The use of platforms such as AquaOp Feed and AquaOp Farm can be a highly effective solution to improving production efficiency and capacity and ultimately, environmental and economic sustainability.

More information: Stephen Gunther Director of Business Development Wittaya Aqua E: stephen.gunther@wittaya-aqua.ca

LACTIC ACID BACTERIA FOR AQUACULTURE

Believe in what you see

We can see it inside, you will see it from the outside! BACTOCELL activates and associates with the gut mucosa, which is the key to a true probiotic effect. If you need to see more, years of research and field applications have provided compiling evidence of BACTOCELL’s modes of action and benefits at cellular, animal and farm level. Discover the world of BACTOCELL, the pioneering probiotic in aquaculture. Not all products are available in all markets nor all claims allowed in all regions.

LALLEMAND ANIMAL NUTRITION SPECIFIC FOR YOUR SUCCESS www.lallemandanimalnutrition.com

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

COLUMN

Aquaculture and aquafeed production in 2019 Albert G. Tacon, Ph.D. Dr. Albert Tacon is a Technical Editor at Aquafeed.com and an independent aquaculture feed consultant. E: agjtacon@aquahana.com

Updated FAO aquaculture data for 2019 from FAO Aquaculture, Capture and Global production databases. Table 1. Top feed fish and crustaceans in 2019 (FAO FishStatJ release 4.01.1 May 2021). *Excludes 13.25 Mt of silver carp, bighead carp, catla and rohu or 23.5% total farmed fish production in 2019 (FAO, 2021). EFAO estimate, not country value.

Table 2. Top fish and crustaceans fed commercial feeds in 2019.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 1. Chinese fed carp production from 2015 to 2019 (FAO, 2021) and estimated commercial feed usage from 2000 to 2025.

Figure 2. Catfish production from 2015 to 2019 (FAO, 2021) and estimated commercial feed usage from 2000 to 2025.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 3. Tilapia production from 2015 to 2019 (FAO, 2021) and estimated commercial feed usage from 2000 to 2025.

Figure 4. Shrimp production from 2015 to 2019 (FAO, 2021) and estimated commercial feed usage from 2000 to 2025.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Figure 5. Marine fish production from 2015 to 2019 (FAO, 2021) and estimated commercial feed usage from 2000 to 2025.

Figure 6. Salmon production from 2015 to 2019 (FAO, 2021) and estimated commercial feed usage from 2000 to 2025.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

Industry Events

Send your meeting details to: editor@aquafeed.com

2021 NOVEMBER 3 – 4:

2021 Equipment Manufacturers Conference, USA

www.afia.org

DECEMBER 2 – 4: 11 – 14: 12 – 17:

Taiwan International Fisheries & Seafood Show, Taiwan Aquaculture Africa, Egypt International Symposium of Fish Nutrition and Feeding, South Korea

www.taiwanfishery.com www.was.org www.isfnf2020busan.com

Extrusion Scale-Up & Process Transfer, Americas, Online

www.fie.com.au

Fish International, Germany Aquafarm, Italy Aquaculture 2022, USA

www.fishinternational.de www.aquafarm.show www.was.org

2022 JANUARY 13 – 15:

FEBRUARY 13 – 15: 16 – 17: 28 – March 4:

MARCH 7 – 9: Engineering, Management & Operation of Extrusion Plants, South Africa 8 – 10: 2022 Purchasing and Ingredient Suppliers Conference, USA 10 – 11: Food & Feed Drying Technology, South Africa 24 – 25: 3rd International Webinar on Aquaculture and Marine Biology, Online 28 – April 1: XVI International Symposium on Aquaculture Nutrition, Online

www.fie.com.au www.afia.org www.fie.com.au www.conferencemind.com aema.mx

MAY 3 – 5: 24 – 27: 31 – June 2:

Aquaculture UK World Aquaculture 2021, Mexico VICTAM & VIV Europe, The Netherlands

www.aquacultureuk.com www.was.org victaminternational.com

JUNE 5 – 9: 8 – 10: 13 – 15:

XX International Symposium on Fish Nutrition and Feeding, Italy www.isfnf2022.org Aquaculture Symposium Guatemala export.co.gt Applied Food & Feed Extrusion, Thailand www.fie.com.au

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

AQUAFEED VOL 13 ISSUE 3 2021

CONTACT US Editorial: editor@aquafeed.com Editor: Lucía Barreiro Executive Editor/Publisher: Suzi Dominy Technical Editors: Peter Hutchinson, Albert Tacon, Ph.D Assistant Editor: Marissa Yanaga

We are grateful to the following companies for sponsoring this issue of the magazine. Their support allows us to make our publications available without charge. We thank them for partnering with us to support the development of our industry through education and information.

Conferences and webinars: info@aquafeed.com

Andritz........................................................................................................ 2

Advertising Enquiries/request media pack: sales@aquafeed.com

Adisseo..................................................................................................... 9

The Andersons.................................................................................... 5

Technical Feed Consulting: Senior Technical Consultant: Warren Dominy, Ph.D consulting@aquafeed.com Accounts & All Other Enquiries: info@aquafeed.com

Provita....................................................................................................... 11 Phileo by Lesaffre......................................................................13 Hydronix Limited UK................................................................20 Wenger................................................................................................... 32 Liptosa...................................................................................................... 37

ARTICLE SUBMISSION You are encouraged to submit scientifically/technically-oriented feature articles describing new products, practical nutrition or processing research, applications, feeding trial results, case studies, feed management, storage, handling and delivery. GUIDELINES l 1,000 words plus tables, charts etc. l Send text as a Word document. l Also attach tables and graphs in original Excel document, if possible. l Please include photos whenever possible (high resolution – at least 300dpi – JPEF preferred). l References should be included. If extensive, please omit and state "References available by request". l Author's name, job, title, company, email, photo (+ contact for reader follow-up if different from Author). l Keep it descriptive and technical with minimal use of product name: please no "sales puff". NEWS ITEMS New items are also welcome for publication in our newsletters and magazines. Please include a photo whenever possible (at least 300 dpi – JPEG preferred). Submission does not guarantee acceptance and the editors reserve the right to edit for content, style, clarity and grammar.

SUBSCRIBE Digital editions are free to industry subscribers. You may also purchase print copies. Subscribe at Aquafeed.com to receive your own digital copy of our publications.

Extru-Tech............................................................................................. 41 Novation................................................................................................ 53 Lallemand............................................................................................ 65 Aquafarm..........................................................................................72 Taiwan International.............................................................73 World Aquaculture Society........................................... 74

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Aquafeed: Advances in Processing & Formulation Vol 13 Issue 4 2021

16-17 FEBRUARY 2022 FIERA DI PORDENONE INTERNATIONAL CONFERENCE & TRADE SHOW ON AQUACULTURE, ALGACULTURE AND FISHING INDUSTRY

5th

edition

news 2022

3rd

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Find business opportunities at Taiwan’s No.1 Online x Offline international fishery trade show. Virtual Booth is Now Available

Ocean Fishery & Fishing Equipment Smart Aquaculture & Fish Seeding Technology Seafood & Value-added Process Fishing Tackle & Water Equipment

Taiwan External Trade Development Council Exhibition Section VI, Exhibition Department Tel: +886-2-2725-5200 #2781 Ms. Vicki Chiu / #2783 Mr. Albert Hsu Email: taiwanfishery@taitra.org.tw

My Exhibition Co. Ltd TEL: +886-2-2587-5802 ＃204 Mr. Tim Wu Email: tim_wu@myexhibition.com.tw

N DA EW TE S

World Aquaculture Singapore 2022

Nov. 29 - Dec. 2, 2022

Singapore EXPO Convention & Exhibition Centre and MAX Atria

May 24-27, 2022

The Annual International Conference & Exposition of World Aquaculture Society

Mérida, Mexico

Asian Paciﬁc Aquaculture 2020 – Annual Meeting of Asian Paciﬁc Chapter, WAS

Centro Internacional de Congresos de Yucatán, CIC

Hosted by Singapore Food Agency

Annual global meeting of the World Aquaculture Society

Conference Sponsors Temasek Polytechnic, Nanyang Technological University National University of Singapore, James Cook University Republic Polytechnic 3rd International Symposium on Perch and Bass WAS Premier Sponsors

WA2020 Partner @WASAPC

@WASingapore WASAPC

Associate Sponsors Aquaculture Engineering Society International Association of Aquaculture Economics & Management WorldFish

WAS Premier Sponsors

Aquaculture 2022 Come one, Come all, for Aquaculture Large and Small

Sustainable Aquaculture – Feeding Africa AQUACULTURE AFRICA 2021 Alexandria Egypt • December 11-14, 2021 The 1st Annual International Conference & Exposition of the African Chapter of the World Aquaculture Society (AFRAQ2021) Egypt is the biggest aquaculture producer in the continent. Both local and international aquaculture delegates will converge for the event at the beautiful City of Alexandria, the Pride of the Mediterranean Sea.

February 28 - March 4, 2022

Town and Country Resort & Conference Center San Diego, California

Hosted by

Conference Management Exhibits & Sponsors WAS - African Chapter worldaqua@was.org Mario Stael Blessing Mapfumo Chapter Founding Gold Sponsor Conference Sponsor and www.was.org mario@marevent.com africanchapter@was.org Egyptian Aquaculture Society (EgAS) AFRAQ 2021 Gold Sponsor

Silver Sponsor

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Aquacultural Engineering Society Aquaculture Association of Canada Aquaculture Feed Industry Association Catﬁsh Farmers of America Global Aquaculture Alliance

For More Information Contact:

International Association of Aquaculture Economics and Management Latin America & Caribbean Chapter WA US Trout Farmers Association World Aquatic Veterinary Medical Association Zebraﬁsh Husbandry Association