44 SEPTEMBER 2023 • WORLD AQUACULTURE • WWW.WAS.ORG enzyme supplementation in aquafeeds, which can ultimately result in more sustainable production. Role of Enzymes in the Concept of Designer Fish All vertebrates, including fish, lack Δ12 and Δ15 (ω3) desaturases and so cannot form 18:2n-6 (linoleic acid, LA) and 18:3n-3 (linolenic acid, LNA). Therefore, linoleic acid and linolenic acid are essential fatty acids in the diets of vertebrates. Upon further desaturation and elongation, physiologically essential polyunsaturated fatty acids can then be formed. Marine fish species generally require substantial dietary levels of highly unsaturated fatty acids (HUFA). In contrast, freshwater species possess the ability to synthesize three important HUFAs: EPA (20:5 n-3) and DHA (22:6 n-3) from linolenic acid and ARA (20:4 n-6) from linoleic acid. This relates to the presence of desaturase and elongase enzymes responsible for two important steps in the HUFA biosynthesis pathway, desaturation and elongation. In aquaculture nutrition, DHA, EPA and ARA are widely studied in order to determine the optimal dietary requirement levels of broodstock, juveniles and larvae of cultured species. HUFAs such as EPA, ARA and DHA, play an important role in growth, development and reproduction-related processes in multicellular organisms (Innis 1991, Heird and Lapillonne 2005). DHA, EPA and ARA are broadly responsible for a generalized role in gene regulation, cellular signaling, and cellular membrane integrity, maintenance and energy metabolism (Jump 2002). More specifically, these HUFAs also serve as precursors for eicosanoids, a paracrine hormone group responsible for development, immunity and reproduction-like physiological processes. As a result, fish consumption has been anticipated to be the most realistic way to address the issue of a correct n-6:n-3 ratio in the diets of humans (Racine and Deckelbaum 2007). Fatty acid desaturases (Fads) help in desaturation during the synthesis of ARA and EPA from LA and LNA respectively (Sprecher 1981). Similarly, a desaturase is required for the production of DHA from EPA (D’Andrea et al. 2002). Most of the freshwater fish species synthesize HUFA from shorter carbon chain precursors but this capacity is impaired in marine species due to subsequent loss of HUFA biosynthesis capacity (Tocher 2010). Fish is the most important source of n- 3 HUFA for humanity but wild harvests around the globe are not increasing, so increased sources of fish will depend on aquaculture (Tidwell and Allan 2002). More and more fish are being used in fish oil production for fish feed and human consumption and the only sustainable alternative to fish oils are currently plant oils that abundantly contain C18 PUFA, but are devoid of the n-3 HUFA found in fish oils (Sargent et al. 2002). Accordingly, tissue fatty acid compositions in fish fed with vegetable oils have high levels of C18 PUFA and decreased levels of C20/22 HUFA, compromising their nutritional value (Regost et al. 2003). As a result, an important research focus depends on understanding and optimizing the molecular mechanism of HUFA biosynthesis and its regulation in fish to facilitate the efficient and effective use of plant-based oils in aquaculture. Significant progress has been made to describe the functional characterization of fatty acid desaturases involved in HUFA synthesis recently. The inclusion of these enzymes in aquafeeds for species in which they are lacking may improve fatty acid profiles and nutritional value. Successful Interventions Using Exogenous Enzymes in Aquafeeds There are several studies reported in which exogenous enzymes were supplemented successfully for the inactivation of harmful antimetabolites in plant ingredients for fish feeds. One such report is on phytase supplemented diets in Channel catfish in which feed intake, food conversion efficiency and growth were improved while the phosphorus load in fecal matter was reduced (Jackson et al. 1996). Similarly, trout fed with phytase-supplemented soybean-based diets showed a 22 percent growth improvement over control fish and phosphorus availability was increased from 46 percent to over 70 percent (Forster et al. 1999). In another study phosphorus availability and protein digestibility were improved in Atlantic salmon when fed with microbial phytase-supplemented diets (Carter and Hauler 2000). The importance of supplementation was also reported in a feeding trial conducted with tilapia Oreochromis niloticus fingerlings in Brazil, in which feed was supplemented with the commercial phytase enzyme “Natuphas” at 0, 500, 1500 and 3000 units per kilogram of feed. In this study fish fingerlings fed with 500 units of Natuphas showed an improved food conversion ratio (1.8 to 1) and higher weight gain. In another tilapia study, the supplementation of exogenous enzymes and a probiotic was conducted to evaluate the combined effect on intestinal morphology, growth and microbiome composition. Total enterocyte absorptive surface, microvilli diameter and Goblet cell number were higher in the group supplemented with enzyme and probiotic than in those fed a control diet. This combination of enzymes and probiotic improved tilapia growth and intestinal morphology without deleterious effects on the intestinal microbial composition (Adeoye et al. 2016). Another study evaluated the effects of high soybean meal / low fish meal supplementation-based diets in white seabream (Diplodus sargus) juveniles with Natugrain® TS, a non-starch carbohydratase (NSPase) enzyme complex (Magalhaes et al. 2016). It was reported that the fish fed a diet almost devoid of FM performed well. Additionally, supplementation of the enzyme NSPase to highly plant-based diets improved feeding and nutrient utilization efficiency while reducing fecal waste production. Plant-based ingredients contain significant quantities of carbohydrates, and most fishes have a limited ability to utilize dietary carbohydrates as energy sources for growth. Among the carbohydrates, cellulose is not at all digested by most fish. Thus, in one study the enzyme cellulase was supplemented along with duckweed (Zhou et al. 2013), which promoted a better growth rate and increased levels of various digestive enzyme activities in grass carp. The intestinal microbiota of fish was altered in terms of bacterial species as well as in overall density. Another study investigated the effects of dietary cellulase supplementation for improving the nutritive value of Chlorella in juvenile crucian carp Carassius auratus (Shi et al. 2016). The results showed improvements in growth performance, digestive activities and nutrient digestibility at a dietary cellulase supplementation levels of 1.0–1.5 g kg−1. Conclusions Enzymes can play a great role in formulation of eco-friendly aquafeeds. The utilization of exogenous enzymes has the potential of replacing fishmeal partially or completely. This, in turn, can help to reduce the demand for fishmeal from the aquaculture sector in upcoming years.