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Friday, February 13, 2015

13/02/2015: Microalgae as an ingredient in aquafeeds

Inclusion of microalgae in diets for gilthead sea bream (Sparus aurata L.) juveniles and the effect on intestinal functionality

by Antonio Vizcaíno, María Isabel Sáez, Tomás Martínez
and Javier Alarcón, LifeBioencapsulation SL Almería, Spain

First published in International Aquafeed, January - February 2015

Fishmeal is currently the main source of protein utilised in carnivorous fish feeding but overexploitation of fisheries combined with growing demand has caused its price to rise continuously. Consequently, future expansion of aquaculture production based on the use of fishmeal as a major ingredient for aquafeeds will be unsustainable. protein sources such as soybean meal, rapeseed meal, corn gluten meal or wheat gluten are commonly used as ingredients in commercial aquafeeds. However such protein ingredients often contain anti-nutritional factors that can have negative effects on the digestive tract of fish (Santigosa et al., 2008; Merrifield et al., 2009).

Microalgae as an ingredient in aquafeeds
In this regard, and owing to their chemical composition, microalgae appear as a promising alternative for enhancing the nutritive value of conventional feeds and for use, at least partially, as a substitute for fishmeal (Lupatch, 2009).

In general microalgae contain a protein level ranging from 30-55 percent DM; about 13-33 percent DM total lipids, with considerable amounts of highly unsaturated fatty acids (HUFA) which are indispensable in fish nutrition; and high vitamin content. They are also rich in pigments such as chlorophylls, carotenoids and phycobiliproteins (Vizcaino et al., 2014).

Microalgae therefore stand out as a promising protein source for aquaculture and thus might well reduce the ecological impacts associated with current fishmeal-based intensive fish farming if sufficient quantities of algal biomass become available at a suitable price (Shields and Lupatch, 2012).

Uses of Scenedesmus almeriensis microalgae in fish
In a recent study we evaluated the effect of inclusion of Scenedesmus almeriensis microalgae as a dietary ingredient on intestinal proteolytic activity of juvenile sea bream. Scenedesmus is a native microalga and it was chosen in the present study owing to its high protein content, fatty acid profile with substantial amounts of linolenic acid, 18:3(n-3), and remarkable productivity.
Biomass was obtained from the facilities at the Estación Experimental Las Palmerillas–Fundación Cajamar (Almería, Spain). The Scenedesmus alga was cultivated in a large-scale tubular photobioreactor. The algal biomass was harvested using a RINA continuous centrifuge (Riera Nadeu SA, Spain) then frozen, freeze-dried and finally milled to obtain a homogenised powder (<100 µm) that was stored in the dark at −20 °C until use.

Dry algal biomass was incorporated into four experimental diets (40 percent crude protein and 10 percent crude lipid) at increasing levels (12, 20, 25 and 39 percent). A feed without Scenedesmus served as a control diet. Feeds were made at the University of Almeria-CEIA3 facilities (Service of Experimental Diets;

Every experimental feed was randomly assigned to triplicate groups of fifteen sea bream juveniles (8.0 g initial body weight). Fish were fed by hand twice per day (9:00 and 13:00) at a rate of 3 percent of their body weight over 45 days. At the end of the trial, fish were killed according to the requirements of the Directive 2010/63/UE, and the digestive tract was removed and processed to obtain enzymatic extracts. Digestive enzymes’ activity levels were differentiated in two groups: total alkaline protease, trypsin, chymotrypsin and α-amylase activity levels were used as indicators of digestive capacity, while leucine aminopeptidase and alkaline phosphatase activity levels were used as indicators of intestinal absorptive capacity.
In order to visualise the active proteases present in intestinal extracts of fish fed on different experimental feeds, substrate-SDS-PAGE electrophoresis gels were carried out (Alarcón et al., 1998). In addition, the presence of protease inhibitors in SA was tested according to Alarcón et al. (1999).

Possible impacts of the different diets on the ultrastructure of the intestinal mucosa were studied by transmission electron microscopy (TEM), giving information about the length and diameter of the microvilli. The intestines of three fish from each tank were collected for examination and TEM samples were prepared according to Vizcaino et al. (2014).

Effect of SA on digestive enzyme activities of sea bream
The ability of fish to use the ingested nutrients depends on the presence of an adequate set of digestive enzymes. Hence, knowledge on nutrient hydrolysis in the digestive tract is an important factor in optimising fish feeds (Deguara et al., 2003). Intestinal enzymes are correlated with the nutritional status of fish, and their activities have been used as indicators of the digestive capacity of farmed fish (Alarcón et al., 1998). In fact fish are capable of modulating their digestive enzyme pattern in response to the feed source, quality and concentration of dietary nutrients (Santigosa et al., 2008).

The use of Scenedesmus meal to replace fishmeal protein did not decrease the enzyme activities secreted into the intestinal lumen at any of the inclusion levels tested, albeit increased activities attributable to algae inclusion have been observed. For trypsin activity, which plays a decisive role in dietary protein hydrolysis and zymogen activation, fish fed on SC12 showed significantly higher activity than those animals fed on a microalgae-free diet. In addition, alkaline phosphatase and L-aminopeptidase activities increased significantly in the SC20 group compared to the Control and SC39 groups (Fig 1). Nevertheless, total alkaline protease, chymotrypsin and α-amylase activities remained unaffected by Scenedesmus inclusion.
Zymograms carried out on intestinal extracts obtained after electrophoretic separation of proteins are shown in Fig 2. The profile of the intestinal proteases seems not to be influenced by the inclusion of Scenedesmus biomass, given that all the animals showed the same number and distribution of active fractions as control-fed fish, characterised by five groups of active bands.
Another important aspect to consider when using ingredients alternative to fishmeal is the presence of anti-nutritive factors that might interfere with nutrient digestion and absorption (Alarcón et al., 1999). Among the wide range of such factors, protease inhibitors are well known as substances that can affect dietary protein utilisation. In this study, neither Scenedesmus meal (unlikely other protein sources) nor experimental feeds contained substances able to inhibit the digestive proteases of gilthead sea bream juveniles, given that inhibition never reached more than 5 percent (data not shown).

Checking effects by histological study of the intestine
In addition to digestive enzyme activities, the structure and morphology of the intestinal mucosa play a key role in nutrient absorption. TEM analysis of anterior and posterior intestine (Fig. 3a and 3b, respectively) revealed that the inclusion of microalgae reduced microvilli length (ML), except for the SC20 group (with higher and similar ML values compared to those of the CT group in the anterior and posterior intestine, respectively) and the SC39 group (with similar and higher ML values compared to those of control fish in the anterior and posterior intestine, respectively), and that microvilli diameter in the anterior intestine was greater in Scenedesmus-fed fish. Thus, the overall effects of both results were increased absorptive surface and improved contribution of the intestinal mucosa as a physical barrier.

The inclusion of S. almeriensis increased the level of intestinal enzyme activities as well as the intestinal absorptive surface. Therefore, the combination of these effects, together with the lack of anti-nutritional factors, confirms that Scenedesmus biomass can be used as a dietary ingredient for juvenile sea bream diets, and furthermore, that an inclusion level of 20 percent is recommended according to the positive effects observed on gut functionality at such a ratio.

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