In the last years considerable attention has been paid on the use of seaweeds (SW) as a possible ingredient for aquafeeds. Red, green and brown SW can be taken from their natural habitat and brought to the shore by the action of winds and tides. Otherwise, biomass can be obtained from secondary and tertiary treatment of effluents. Wastewater treatment utilising photosynthetic organisms is an interesting alternative to reduce the ecological impact of domestic, industrial or aquaculture effluents. Generally, high-quality algal biomass is yielded from algal cultivation, representing an excellent source of hydrocolloids, carotenoids, and bioactive substances, which allows different industrial applications. In addition, there is currently an increasing interest for the potential of SW in human and animal nutrition.

1. I N C O R P O R AT I N G
f i s h far m ing t e c h no l og y
March | April 2013
Effect of dietary inclusion of seaweeds on
intestinal proteolytic activity of juvenile sea
bream, Sparus aurata
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The International magazine for the aquaculture feed industry

2. FEATURE
Effect of dietary inclusion
of seaweeds on intestinal
proteolytic activity of juvenile
sea bream, Sparus aurata
by María Isabel Sáez, Tomás Martínez and Javier Alarcón,
Universidad de Almería-CEIA3, Spain
I
n the last years considerable attention (especially under nitrogen-enriched condition) response (Valente et al., 2006). Nonetheless,
has been paid on the use of seaweeds and season. it has been also noted in other publications
(SW) as a possible ingredient for aqua- The high biological value of algal proteins
that high SW inclusion reduces fish growth
feeds. Red, green and brown SW can be makes algae suitable for inclusion both in and feed efficiency. From the literature avail-
taken from their natural habitat and brought animal feeds (especially marine species) and in able it can be deducted that the response of
to the shore by the action of winds and human diets. The high carbohydrate content animals to SW seems to be dose-dependent
tides. Otherwise, biomass can be obtained (30 to 60%) is a very marked characteristic in and species-specific. Moreover, certain sub-
from secondary and tertiary treatment of most SW, comprising mainly soluble carbo- stances with antinutritive activity may be
effluents. Wastewater treatment utilising hydrates, like sugars, and pectins, alginic acid,
present in SW, like lectins, tannins, phytic
photosynthetic organisms is an interest- agar and carrageenan as well. Besides their acid, and protease and amylase inhibitors
ing alternative to reduce (Oliveira et al., 2009). Such
the ecological impact of antinutritional factors might
domestic, industrial or interfere with bioavailability
aquaculture effluents. and/or digestibility of nutri-
Generally, high-quality algal ents.
biomass is yielded from Special emphasis should
algal cultivation, represent- be focused on protease
ing an excellent source of inhibitors. Binding of pro-
hydrocolloids, carotenoids, tease inhibitors to pro-
and bioactive substances, teolytic enzymes causes
which allows different the pancreas to secrete
industrial applications. In larger amounts of digestive
addition, there is currently enzymes to overcome the
an increasing interest negative effects of inhibi-
for the potential of SW tors on the digestion of
in human and animal dietary protein. This fact
nutrition. can lead to decreased
Figure 1: Detail of experimental feeds. UL-25 weight gain, and pancreatic
percent (above) and control (below) hypertrophy in some fish
Seaweed as species. For this reason,
ingredient studies aimed to include
in aquafeeds potential nutritional value, from a techno- SW in aquafeeds must also bring up their
Although nutritional properties of SW are logical point of view, SW can also be used as possible effects on fish digestive physiology.
not as well known as are those of land plant- additives in the feed industry, for instance, as To date, there is scarce literature analysing if
based ingredients, their chemical composition excellent feed agglutinants (improving texture SW inclusion causes negative consequences
may be characterised by low content in lipids, and water stability of pellets), or as attractants on digestive physiology of fish.
moderate in protein, but rich in non-starch (increasing feed intake).
polysaccharides, minerals and vitamins. Lipid Evaluating the effect of seaweeds
contents range from 0.3 to 7.2 percent, The effects of seaweeds on fish on digestive proteases
although algal lipids are rich in PUFA such as Several studies have proved that addition In a recent study, we evaluated the effect
C20:5n3 (eicosapentaenoic acid, EPA) and of small amount of SW in aquafeeds resulted of inclusion of two SW as dietary ingredients
C22:6n3 (docosahexaenoic acid, DHA). The in considerable positive effect on growth on intestinal proteolytic activity of juvenile sea
protein contribution is ranged from 10 to 30 performance and feed utilisation efficiency, bream. Gracilaria cornea (GR) and Ulva rigida
g/100 g dry weight, which may vary greatly carcass quality, physiological activity, intesti- (UL) were chosen in the present study owing
among SW species, environmental conditions nal microbiota, disease resistance, and stress to its fast growth, low-cost production and
38 | InternAtIonAl AquAFeed | March-April 2013

3. FEATURE
FEATURE
Need for novel a partial dietary substitute for fish oil within ies have been recently reviewed (Nasopoulou
sources inhibitors in SW. incubation of digestive proteases with extracts
compound feeds. The same is true of linseed and Zabetakis, 2012).
Dose-response
In order to reduce oil and rapeseed oil, although to a lesser of the experimental diets. The mean inhibition
New, alternative and in a way ‘non-
dependence on fish oil, sig- extent. curves showed orthodox’, from 11 to 48 percent. In general,
ranged sources of lipids need to be
nificant breakthroughs have that UL contained UL-supplemented feeds showed inhibition
Furthermore, the use of palm oil in diets of identified and valorised in order to achieve
occurred over the past few Atlantic salmon and rainbow trout has given to sustainable production of fish GR-supplemented
substances able values higher than the feeds and thus
years in replacing it with plant reduce digestive diets, which did not exceed 16 percent. For
oils. By substituting feeds with proteolytic activity UL diets, it was found that percentage of inhi-
plant oils, it also serves to reduce in sea bream (up B) bition was positively correlated with the SW
costs due to the fact that vegeta- to 77%), whereas a inclusion level, which agrees with the above
ble oils have steadily increasing produc- negligible inhibition mentioned dose-response curve. Inhibition
tion, high availability and better economic by GR was found produced by GR feeds cannot be associated
value. Several studies have been carried out (4%). Obvious dif- to the use of this SW.
to investigate certain vegetable oils as pos- ferences in the
sible sustainable partial substitutes for fish kinetic of inhibition Effect of seaweed on digestive
oils in compounded fish feeds. The most of protease activity proteases of sea bream
common vegetable oils used for fish feed pro- were found for UL. Digestive enzymes were affected by diets,
duction have been soybean, linseed, rapeseed, Equation defining as fish had different enzyme activity level of
Figure 2: Dose-response curves obtained when2: Representative optic micrographs xalkaline proteases sections stained with
Figure different such curve may be 100 of aortic wall
sunflower, palm oil and olive oil. (0 to 300 µg) were incubated with a and eosin from the two experimental groups,after 70 days of feeding
amounts of SW meal haematoxylin where atherosclerotic
used to predict the experimental diets. In general, a decrease
Soybean and amount of proteolytic activity (1 U) in the inhibitory appear as foam cells (↑). (A) Group A (atherogenic diet); (B) Group B
fixed rapeseed oil are considered lesions
assay. Protease inhibition was salmo- (atherogenic diet expected percent- in alkaline protease activity was evidenced
possible alternative lipid sources for expressed as the percentage of enriched with sea bream polar lipids) (adopted from Nasopoulou et
nids, freshwater and marine fish since Such curves are a simple way
reduction in proteolytic activity. they age of reduction when feeds included UL or GR. In particular,
al., 2010). Copyright, “Food Chemistry” Elsevier
to evaluate how hypothetical variations in the inclusion of in protease activ- the proteolytic activities of fish fed Ulva
are rich in PUFAs, especially linoleic (18:2
SW might affect sea bream digestive proteases ity, once protease supplemented-feeds were significantly lower
ω−6) and oleic acid (18:1 ω−9), but devoid growth and feed utilization efficiency compa- enabling the further development of aquacul-
activity in the diges- ture applications. Such promising lipid sources
of n-3 PUFA. However, in some cases, fish rable to fish fed with equivalent levels of fish than those of fish fed on control diet. The
tive tract partial are vegetable oils (VO). The use of VO based
oil substitution by 60 percent rapeseed oil oil. Olive oil could also be used as a and the presence of protease inhibitors in SW may
has been found to decrease European sea substitute for dietary fish oil are European aquafeeds has some strong advantages. Olive
successful integrated culture in fish-farm efflu- amount of feed ingested in known. For be the reason of the progressive decrease
instance, in the case of 40 g sea bream, total in the proteolytic activity in fish fed diet with
ents. Biomass was obtained from the Marine sea bass culture, during growth out phase, pomace (OP) and olive pomace oil (OPO)
bass (Dicentrarchus labrax) growth. Soybean
protease activity released after a meal is around increasing levels of of olive oil produc-
Biotechnology Centre (ULPGC, Spain). SW Atlantic salmon (salmo salar) and rainbow are natural by-products Ulva meal. Supporting
oil appears to be a better plant lipid source
1,300 units. Those fish that consumed 0.5 g of this hypothesis, it has been confirmed that
were cultivated in 750 L semicircular fibreglass trout (Oncorhynchus mykiss) with data showing tion, which contain micro constituents with
regarding gilthead sea bream (Sparus aurata)
a feed containing 15 percent of UL, showed a aqueous extracts of Ulva meal inhibit alkaline
growth while with seawater savings in fishpond similar growth rates to the ones when fish was atheroprotective (substances) activity such as
tanks filled considerable plus the feed
effluents of a pilot aquaculture system (11 m3 fed on 100 percent fish oil diet. All these stud- PAF-inhibitors and phenolic/polyphenolic mol-
costs could be achieved if it could be used as ratio mg UL per unit of activity of 50, which proteases of S. aurata. Moreover, the drop in
with an optimal density of Sparus aurata of 20 determined a
kg m-3, and a water renovation rate of 6–8 reduction nearly
vol day-1). Red and green SW were washed 40 percent in the
with sea water, sun-dried for 48 hours, ground activity of diges-
and sieved through 0.1 mm sieve before being tive proteases.
used as a dietary ingredient. Fortunately, fish
Dry algal biomass was incorporated into have mechanisms
six experimental diets (40% crude protein to compensate
and 12% crude lipid) at increasing levels (5, the effect of die-
VIV Russia 2013
15 and 25%). A feed without SW served tary antinutrients.
as a control diet. Feeds were made at the Zymograms
University of Almeria-CEIA3 facilities (Service obtained after
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stecnicos_spe). Every experimental feed was separation of
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randomly assigned to triplicate group of proteins is a use-
fifteen sea bream juveniles (15.4 g initial body ful tool to know
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Results revealed the presence of protease observed after
March-April 2013 | InternAtIonAl AquAFeed | 23
March-April 2013 | InternAtIonAl AquAFeed | 39

4. FEATURE
Figure 3: Inhibition of
intestinal proteolytic enzymes
by Gracilaria cornea and
Ulva rigida meal. Qualitative
analysis: visualization of
inhibition of active fractions in
zymograms
Figure 5: Total alkaline protease activity measured in extracts of sea bream fed
different experimental diets containing graded levels of SW
compensation mechanism against dietary References
protease inhibitors in juvenile sea bream
Alarcón FJ, Díaz M, Moyano FJ and Abellán E.
has been previously proved by Santigosa et (1998) Characterization and functional properties
al. (2010), who found similar results when of digestive proteases in two sparids; gilthead
fish were fed diets with soybean trypsin sea bream (Sparus aurata) and common dentex
inhibitor. (Dentex dentex). Fish Physiol Biochem. 19: 257-267.
According to the results, it is clear that
Alarcón, FJ, Moyano, FJ and Díaz, M. (1999).
the amount of the pancreatic proteases Effect of inhibitors present in protein sources on
secreted into the intestinal lumen in juvenile digestive proteases of juvenile sea bream (Sparus
S. aurata is affected by the use of SW, par- aurata). Aquatic Living Res. 12: 233-238.
ticularly Ulva. Nevertheless, it is also evident
Oliveira, MN, Ponte-Freitas, AL, Urano-Carvalho, AF,
that these ingredients did not cause qualita-
Taveres-Sampaio, TM, Farias, DF, Alves-Teixera, DI,
tive changes in the composition of alkaline
Figure 4: Inhibition of sea Gouveia, ST, Gomes-Pereira, J and Castro-Catanho
proteases, given that all fish showed the de Sena, MM. (2009) Nutritive and non-nutritive
bream intestinal proteases
after incubation of extracts same pattern of proteolytic enzymes in their attributes of washed-up seaweeds from the coast
with solutions prepared using intestines, and that growth performance of of Ceará, Brazil. Food Chem. 11: 254-259.
experimental diets containing 5, fish was not affected, as deduced from the in
15 and 25 percent of Ulva (UL) Santigosa, E, Sánchez, J, Médale, F, Pérez-Sánchez, J
vivo feeding trial.
and Gracilaria (GR) meal and Gallardo, MA. (2008). Modifications of digestive
enzymes in trout (Onchorynchus mykiss) and sea
Conclusions bream (Sparus aurata) in response to dietary
In vitro protease inhibition assays are a use- fish meal replacement by plant protein sources.
the level of alkaline protease activity was not ful tool to assess the presence of antinutrients Aquaculture 252: 68-74.
accompanied by a decrease of fish growth in SW with potential use in aquafeeds. Based
Santigosa, E, Sáez de Rodigrañez, MA, Rodiles, A,
and feed utilization, since all fish grew equally on the results of this study, SW, especially
García Barroso, F and Alarcón, FJ. (2010). Effect of
(unpublished data). Santigosa et al. (2008) Ulva rigida, have antinutritive factors able to diets containing a purified soybean trypsin inhibitor
reported a similar finding when trout were fed inhibit digestive proteases of S. aurata. Feeding on growth performance, digestive proteases and
on diets including plant proteins. juvenile S. aurata on seaweed-based diets intestinal histology in juvenile sea bream (Sparus
On the other hand, the analysis of decreased the amount of proteolytic activ- aurata L.). Aquaculture Res. 41: e187-e198.
zymograms revealed that the pattern of ity secreted into the intestine. However, the
Valente, LMP, Gouveia, A, Rema, P, Matos, J, Gomes,
intestinal proteases was not modified by inclusion of SW does not alter the pattern
EF and Pinto, IS. (2006) Evaluation of three
inclusion of SW. All sea bream specimens of proteolytic enzymes in sea bream, which seaweeds Gracilaria bursa-pastoris, Ulva rigida
showed the same number and distribu- reveals a compensating mechanism in this and Gracilaria cornea as dietary ingredients in
tion of active fractions as in control group species. Research is being currently conducted European sea bass (Dicentrarchus labrax) juveniles.
(after electrophoretical separation, the pat- to assess the effect of SW on other digestive Aquaculture 252: 85-91.
tern of intestinal proteases in this species enzymes, intestinal microbiota, blood and
is characterized by five groups of active tissue metabolites, and intestine and liver
bands). These results confirmed that the histology after 70 days of feeding SW-based More inforMation:
type of alkaline proteases secreted into the diets. Further research is needed in order María Isabel Sáez Casado
intestinal lumen was not modified by any to known the impact of SW in a long-term Email: msc880@ual.es
of experimental diets. The existence of a feeding assay. ■
40 | InternAtIonAl AquAFeed | March-April 2013

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