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 Aquaculture 556 (2022) 738288

Contents lists available at ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Evaluation of Nannochloropsis gaditana raw and hydrolysed biomass at low

inclusion level as dietary functional additive for gilthead seabream (Sparus
aurata) juveniles
María Isabel Sáez a, Alba Galafat a, Antonio Jesús Vizcaíno a, Elena Chaves-Pozo b,
María Dolores Ayala c, Marta Arizcun b, Francisco Javier Alarcón a, María Dolores Suárez a,
Tomás Francisco Martínez a, *
a
Departamento de Biología y Geología, Escuela Superior de Ingeniería, CEIMAR, Universidad de Almería, 04120 Almería, Spain
b
Centro Oceanográfico de Murcia, Instituto Español de Oceanografía (IEO – CSIC), Carretera de la Azohía s/n, Puerto de Mazarrón 30860, Murcia, Spain
c
Departamento de Anatomía y Anatomía Patológica Comparada, Facultad de Veterinaria, Universidad de Murcia, 30100 Murcia. Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Abundant research is being carried out in the last years aimed at exploring microalgal biomass as nutrient source
Bioactive compounds for different species of aquacultured fish. Some microalgae species, such as Nannochloropsis gaditana, have thick
Cellulase cell walls rich in cellulose, which might well reduce the bioavailability of intracellular active compounds. Among
Functional additive
the alternatives aimed at overcoming this limitation, cellulase enzyme hydrolysis is proposed as a convenient and
Lipid oxidation
Microalgae hydrolysis
practical solution. In this regard, an in vitro assay was carried out, in which N. gaditana biomass was treated with
cellulase (5% w/w basis) and the release of soluble compounds (reducing sugars, free amino acids and total
phenolics) into the reaction medium was measured and compared to untreated raw biomass. The results
confirmed increased yields of those compounds as a result of the enzyme pre-treatment. A 90-d feeding trial was
also carried out in order to assess in vivo the influence of the inclusion of N. gaditana in feeds on juvenile gilthead
seabream (Sparus aurata) growth, digestive physiology and body composition. Microalgal biomass was added at
two inclusion levels (25 and 50 g kg− 1 dry weight) in four experimental feeds, either crude or enzymatically pre-
treated. Animals (15.1 g initial body weight) were randomly assigned to five dietary treatments (two inclusion
levels, 2.5 and 5%, and two microalgae formats, raw and enzymatically hydrolysed, plus a microalgae-free
control), and distributed triplicate tanks per dietary treatment. Fish were withdrawn after 45 and 90 days,
and proximate composition, muscle fatty acid and amino acid profiles, muscle and liver lipid oxidation,
instrumental skin colour, digestive enzyme activities, as well as structural and ultrastructural changes in the
intestinal mucosa were determined. No differences attributable to the dietary treatments were found with regard
to fish growth or proximate composition at the end of the feeding trial. On the contrary, the inclusion of
microalgal biomass, irrespectively of the cellulase pre-treatment, caused beneficial effects on some physiological
parameters (namely digestive mucosa structure and functionality, oxidative status of muscle lipids, and instru­
mental colour). The only clear improvement found in fish attributable to the cellulase pre-treatment of the
microalgal biomass was related to the prevention of muscle lipid oxidation. Overall, the results suggest that
N. gaditana used as additive (at inclusion level below 5%) in feeds might represent a valuable nutritional strategy
for S. aurata juveniles, even if growth was not affected.

1. Introduction aquaculture feed manufacturing (Yarnold et al., 2019).

However, they are also drawing the attention of nutritionists as a
Interest in microalgae has emerged strongly in recent years, as they valuable source of bioactive compounds, many of which remain to be
present a valuable and still relatively unexploited potential to reduce identified. Different studies have pointed out that these substances can
dependence on unsustainable ingredients, namely fishmeal, in exert positive effects on several aspects of fish physiology, even if added

* Corresponding author.
E-mail address: tomas@ual.es (T.F. Martínez).

https://doi.org/10.1016/j.aquaculture.2022.738288
Received 23 December 2021; Received in revised form 30 March 2022; Accepted 22 April 2022
Available online 28 April 2022
0044-8486/© 2022 Elsevier B.V. All rights reserved.
M.I. Sáez et al.
at low inclusion level (e.g., less than 10%) in feeds (Becker, 2003; Kiron,
2012). Given their current production costs, the interest in microalgae is
turning from large-scale use as main ingredient towards their use as
functional additives at low inclusion levels in feeds. The search for
bioactive compounds aimed at improving not only fish growth, but also
the general condition of the animals is nowadays a thriving field in
aquaculture research. This concept is based upon numerous reports
indicating that many microalgae species are valuable sources of essen­
tial n-3 long chain polyunsaturated fatty acids (n3-PUFAs), vitamins,
minerals, pigments, and polyphenols, among others (Sansone et al.,
2020; Teimouri et al., 2013; Tibaldi et al., 2015; Shah et al., 2018). In
this context, Nannochloropsis gaditana, by virtue of its richness in eico­
sapentanoic acid (EPA, C20:5n-3), pigments and other natural antioxi­
dants and other bioactive compounds (Kilian et al., 2011; Tibbetts et al.,
2017; Cerón-García et al., 2018), together with their availability at in­
dustrial or semi-industrial scale (Heredia et al., 2021; Kavitha et al.,
2021), is a promising candidate as a commercial additive in aquafeeds.
However, it has been reported in the literature that the theoretical
nutritional potential of microalgae might not always be reflected
straight on the animals, neither in terms of fish growth nor on physio­
logical condition (Cerezuela et al., 2012; Cardinaletti et al., 2018). This
could be explained by the existence of a cell wall that limits the
bioavailability of inner microalgal compounds (Wu et al., 2017; Yong
et al., 2020). Specifically, the existence of a cellulose-rich cell wall in
certain genus, such as Nannochloropsis, together with the lack of diges­
tive cellulase activity in fish, might well limit their further practical
utilization. In fact, cellulose accounts for 75% of cell walls dry matter in
N. gaditana (Scholz et al., 2014).
When it comes to improving bioavailability prior to their inclusion in
aquafeeds, several strategies have been proposed in the literature aimed
at weakening or disrupting microalgae cell walls, all of them with ad­
vantages and disadvantages (Gomes et al., 2020; Rojo et al., 2021;
Pagels et al., 2021). Studies have reported that both enzymatic (Agboola
et al., 2019; Batista et al., 2020; Galafat et al., 2020) and physical
(Timira et al., 2021; Rojo et al., 2021) disruption strategies (such as bead
milling, ultrasonication and high-pressure homogenization, Lee et al.,
2010; Günerken et al., 2015) are able to increase the yield of antioxidant
compounds (Almendinger et al., 2021), as well as the nutrient avail­
ability and digestibility of algae by fish (Teuling et al., 2019). However,
species-specific factors must be taken into account when selecting the
most appropriate method to disrupt microalgal cell walls (Batista et al.,
2020).
Even if successful at laboratory scale, however, methods of physical
disruption may not end up in scalable, and economically feasible pro­
cedures applicable to the feed processing industry, taking into account
that additional costs should be added to the production prices of
microalgal biomass (Batista et al., 2020). Consequently, there is still
considerable scope for developing simple, economical, and cost-
effective cell wall disruption protocols. The use of hydrolytic enzymes
capable of weakening cell walls prior to its incorporation into feeds
could overcome many of the existing limitations. Fibrolytic enzymes,
not least cellulases, have a wide range of industrial applications, and
hence they are available at affordable prices, and generally speaking,
any bioprocess including enzymes could be certainly scalable at indus­
trial level.
In this context, we hypothesize that the use of cellulases capable of
weakening N. gaditana cell walls may represent a valuable strategy to
improve the bioavailability of nutrients and bioactive compounds of the
biomass when added into gilthead seabream (Sparus aurata) experi­
mental diets. To this end, either crude or enzymatically hydrolysed
N. gaditana was added at low inclusion levels (2.5 and 5% w/w) to diets
for gilthead seabream juvenile. This is, the microalgal biomass was
assessed as a potential functional additive rather than as a main ingre­
dient. A 90-d feeding trial was carried out, and the occurrence of po­
tential effects of the microalgae on fish growth, muscle composition,
lipid oxidative status, skin pigmentation, and digestive structure and
2
Aquaculture 556 (2022) 738288
functionality were assessed.
2. Materials and methods
2.1. Microalgae biomass and enzyme hydrolysis
Nannochloropsis gaditana was cultured in tubular photobioreactors at
the pilot plant (EU-H2020 SABANA facilities) of the Universidad de
Almería (Spain) as reported by Menegol et al. (2019). The culture pH
was maintained at 8 by the on-demand addition of CO2. The culture was
harvested daily by centrifugation (at a dilution rate of 0.3 d− 1), then the
concentrated biomass was freeze-dried. Raw microalgae biomass
(approx. 15% dry matter) was freeze-dried and stored at − 20 ◦ C until
further use. The proximal composition of N. gaditana meal yielded
44.5% crude protein, 33.3% carbohydrates, 4.5% ash, and 17.7% crude
lipid on dry matter basis.
Enzymatic hydrolysis was carried out by mixing the microalgal
biomass, at a final concentration of 150 g dry weight L− 1 in 50 mM
sodium citrate buffer solution (pH 5.5), and incubated at 45 ◦ C under
continuous agitation for 5 h. Commercial cellulase (from Aspergillus
oryzae, Sigma-Aldrich, Madrid, Spain) was added at an enzyme-to-
microalgae ratio of 0.05 (50 g cellulase kg− 1 dry microalgae). The
estimation of the microalgae hydrolysis was carried out by monitoring
the amount of reducing sugars (expressed as g of glucose equivalents
100 g− 1 biomass, according to Miller, 1959) and total amino acids
(expressed as g L-leucine 100 g− 1 protein, according to Church et al.,
1983) released into the reaction vessel at different sampling times (0, 15,
30, 60, 90, 120, 180, 240, and 300 min). Total polyphenols (expressed as
mg gallic acid equivalents 100 g− 1 biomass, according to Singh et al.,
2002) were also measured in reaction vessels at the beginning and at the
end of the in vitro hydrolysis. A control assay was also carried out under
the same experimental conditions, without the addition of cellulase
enzyme.
Following the hydrolysis, the mixture was immediately used for
manufacturing aquafeeds.
2.2. Experimental diets
Four isonitrogenous and isolipidic experimental diets containing
Nannochloropsis gaditana biomass were elaborated at the CEIA3-Uni­
versidad de Almería facilities (Servicio de Piensos Experimentales, htt
p://www.ual.es/stecnicos_spe). Two inclusion levels (25 and 50 g
kg− 1 w/w), and two microalgae formats (raw and enzymatically
hydrolysed) were considered. Therefore, diets were designed as R25 and
R50 for raw microalgae lots, and H25 and H50 for enzymatically-
hydrolysed biomass. A microalgae-free diet was used as control (CT).
The formulation and proximal composition of the experimental diets is
shown in Table 1. In addition, fatty acid and amino acid profiles of each
diet are presented in Table 2 and Fig. 1, respectively. Feed ingredients
were finely ground and mixed in a vertical helix mixer (Sammic 13 M-
11, 5-L capacity, Sammic SA, Azpeitia, Spain) for 20 min. Then the
microalgae (crude or hydrolysed) were added at the specified inclusion
level, and water content was adjusted to provide 400 mL per kg of the
ingredient mixture, in order to obtain homogenous dough. The dough
was passed through a single screw laboratory extruder (Miltenz 51SP, JS
Conwell Ltd., New Zealand), provided with matrixes so as to obtain 2
and 3 mm pellets, according to the size of fish. The feeds were dried with
forced-air circulation (Airfrio, Almería, Spain) at 30 ◦ C for 24 h, and
then stored at − 20 ◦ C until use.
2.3. Fish maintenance and experimental design
The feeding trial was carried out at the aquaculture facilities (REGA:
ES300261040017) of Centro Oceanográfico de Murcia (Mazarrón,
south-eastern Spain), Instituto Español de Oceanografía (IEO-CSIC).
Gilthead seabream juveniles (15.06 ± 1.40 g average initial body
M.I. Sáez et al. Aquaculture 556 (2022) 738288

Table 1 keeping seawater renewal rate (37‰ salinity) at 500 L h− 1 and ammonia
Ingredients and composition of the experimental diets. and nitrite values (<0.1 mg L− 1) suitable for gilthead seabream culture.
Diets Animals were kept under natural photoperiod (ranging from 10 to 13
daylight hours per day) and the water temperature was kept at 18 ±
Ingredients CT R25 R50 H25 H50
(% dry matter) 0.5 ◦ C. Light intensity ranged from 100 to 150 lx. Tanks were equipped
with aerators to maintain an adequate level of oxygenation (above 6 mg
Fish meal LT941 15.0 15.0 15.0 15.0 15.0
Raw N. gaditana2 – 2.5 5.0 – –
L− 1).
Hydrolysed N. gaditana – – – 2.5 5.0 After 45 and 90 days of the feeding trial, twenty fish per tank (60
Squid meal3 2.0 2.0 2.0 2.0 2.0 animals per dietary treatment) were withdrawn at each sampling time
CPSP904 1.0 1.0 1.0 1.0 1.0 and killed by anaesthetic overdose (200 mg L− 1 clove oil; isoeugenol)
Krill meal5 2.0 2.0 2.0 2.0 2.0
followed by spine severing. Body weight was recorded, and growing
Gluten meal6 15.0 15.0 15.0 15.0 15.0
Soybean protein parameters were calculated as follows: feed conversion rate (FCR): (total
40.0 38.8 37.3 38.8 37.3
concentrate7 feed being consumed/weight gain) and specific growth rate (SGR) (% d
Fish oil8 11.4 11.0 10.5 11.0 10.5 − 1): 100 × {(ln final weight – ln initial weight)/days}. Immediately
Soybean lecithin9 1.0 1.0 1.0 1.0 1.0 after slaughtering, instrumental colour parameters were determined on
Wheat meal10 5.4 4.5 4.0 4.5 4.0
Choline chloride11 0.5 0.5 0.5 0.5 0.5
the right side of the anterior dorsal skin of fish. Then, sampled fish were
Betain12 0.5 0.5 0.5 0.5 0.5 dissected, and the digestive tract and dorsal muscle were removed. The
Lysine13 1.5 1.5 1.5 1.5 1.5 liver and portions of muscle (5 g per fish) were stored at − 80 ◦ C for lipid
Methionine14 0.6 0.6 0.6 0.6 0.6 oxidation determinations (TBARS). The rest of individual muscle sam­
Vitamin and mineral
2.0 2.0 2.0 2.0 2.0 ples were freeze-dried and stored at − 20 ◦ C for further analysis of
premix15
Vitamin C16 0.1 0.1 0.1 0.1 0.1 proximate composition and fatty acids. Small segments (around 5 mm
Guar gum17 2.0 2.0 2.0 2.0 2.0 long) of the anterior intestine from five fish per tank were collected for
Crude protein
45.2 ± 46.1 ± 46.4 ± 45.4 ± 45.9 ± examination by optical, transmission (TEM) and scanning (SEM) elec­
0.2 1.1 0.1 0.5 1.0 tron microscopy. For digestive enzyme activity determinations, in­
15.2 ± 15.9 ± 15.5 ± 15.8 ± 15.8 ±
Crude lipid
0.1 0.3 0.1 0.7 0.1
testines from 15 fish per tank were randomly pooled (5 fish per pool, 3
11.8 ± 11.9 ± 12.2 ± 12.0 ± 11.7 ± pools per tank and sampling time) to obtain three enzymatic extracts
Ash
0.3 0.1 0.0 0.1 0.3 from each experimental tank (a total of 9 pooled extracts per dietary
Moisture
5.4 ± 5.6 ± 5.7 ± 5.3 ± 5.6 ± treatment and sampling time).
0.2 0.4 0.1 0.2 0.1

CT: control diet. R25 and R50: diets including 25 and 50 g kg− 1 raw microalgal 2.4. Proximate composition, fatty acid and amino acid analysis
biomass, respectively. H25 and H50: diets including 25 and 50 g kg− 1 hydro­
lysed microalgae, respectively. Moisture (drying at 105 ◦ C in an oven, J.P. Selecta S.A., Barcelona,
1
69.4% crude protein, 12.3% crude lipid (Norsildemel, Bergen, Norway); Spain), crude protein (Kjeldahl Nx6.25, Foss KT200 Kjeltecdistiller,
2
Nannochloropsis gaditana (44.5% crude protein, 33.3% carbohydrates, 4.5%
FOSS, Denmark), and ash (mufla oven 1100 ◦ C, J.P. Selecta S.A., Bar­
ash, and 17.7% crude lipid);
3, 4,5 celona, Spain) were determined in feeds and muscle samples according
purchased from Bacarel (UK). CPSP90 is enzymatically pre-digested fish­
meal.
to AOAC (2000) procedures.
6
78% crude protein (Lorca Nutrición Animal SA, Murcia, Spain). Lipids were extracted from samples following Folch (1957) meth­
7
Soybean protein hydrolysate, 65% crude protein, 8% crude lipid (DSM, odology using chloroform/methanol (2:1 v/v) as solvent, and crude lipid
France). content was calculated gravimetrically. Fatty acid (FA) profiles of
8
AF117DHA (Afamsa, Spain). N. gaditana, feeds and muscle samples were determined by gas chro­
9
P700IP (Lecico, DE). matography (Hewlett Packard, 4890 Series II, Hewlett Packard Com­
10
Local provider (Almería, Spain). pany, Avondale, PA) following the method described in Rodríguez-Ruiz
11,12, 13,14
Lorca Nutrición Animal SA (Murcia, Spain). et al. (1998), using a modification of the direct transesterification
15
Lifebioencapsulation SL (Almería, Spain). Vitamins (mg kg− 1): vitamin A method described by Lepage and Roy (1984) that requires no prior
(retinyl acetate), 2000,000 UI; vitamin D3 (DL-cholecalciferol), 200,000 UI;
separation of the lipid fraction. Briefly, 150 mg of sample were mixed
vitamin E (Lutavit E50), 10,000 mg; vitamin K3 (menadione sodium bisulphite),
with 1 mL of n-hexane, and an internal standard solution (pentadecanoic
2500 mg; vitamin B1(thiamine hydrochloride), 3000 mg; vitamin B2 (ribo­
flavin), 3000 mg; calcium pantothenate, 10,000 mg; nicotinic acid, 20,000 mg; acid, C15:0) was added to each tube. Then, 1 mL of methylation mixture
vitamin B6 (pyridoxine hydrochloride), 2000 mg; vitamin B9 (folic acid), 1500 (methanol and acetyl chloride 20:1 v/v) was added to each tube and
mg; vitamin B12 (cyanocobalamin), 10 mg vitamin H (biotin), 300 mg; inositol, placed in a thermoblock (100 ◦ C, 30 min) with the purpose of obtaining
50,000 mg; betaine (Betafin S1), 50,000 mg. Minerals (mg kg− 1): Co (cobalt the corresponding fatty acid methyl esters (FAMEs). Once methylated, 1
carbonate), 65 mg; Cu (cupric sulphate), 900 mg; Fe (iron sulphate), 600 mg; I mL distilled water was added to each tube in order to remove the hexane
(potassium iodide), 50 mg; Mn (manganese oxide), 960 mg; Se (sodium sele­ phase, and tubes were centrifuged (3500 g, 5 min). The hexane phase
nite), 1 mg; Zn (zinc sulphate) 750 mg; Ca (calcium carbonate), 18.6%; that accumulated the FAMEs was removed from the tubes, and then
(186,000 mg); KCl, 2.41%; (24,100 mg); NaCl, 4.0% (40,000 mg). inserted in vials for fatty acid identification and quantification by gas
16
TECNOVIT, Spain.
17
chromatography.
EPSA, Spain.
Amino acid profiles of N. gaditana and feeds were determined by ion
exchange chromatography with post column derivatization with
weight) were selected and randomly distributed in 15 tanks (triplicate ninhydrin (Biochrom 30+ amino acid analyser, Biochrom LTD Cam­
tanks per dietary treatment) of 500 L capacity to reach an initial average bridge, UK) after hydrolysis of the samples (20 mg mL− 1 HCl 6 M,
biomass of 1200 g m− 3 (40 fish tank− 1). 110 ◦ C, 24 h, under N2 atmosphere), using norleucine as standard.
Fish were fed with CT diet (microalgae-free) during a 15-d acclima­
tion period prior to the beginning of the feeding trial. Afterwards, the 2.5. Digestive enzyme activities
experimental diets were offered thrice per day (9:00, 14:00 and 18:00)
at 2% of the biomass, until triplicating initial body weight. The amount For intestinal extracts, samples were homogenized in distilled water
of feed ingested was recorded daily in each tank. (0.5 g mL− 1) at 4 ◦ C. Supernatants were obtained after centrifugation
The 90-d feeding trial was carried out in an open flow circuit, (16,000 g, 12 min, 4 ◦ C) and stored in aliquots at − 20 ◦ C until further

3
M.I. Sáez et al. Aquaculture 556 (2022) 738288

Table 2
Fatty acid profile of N. gaditana meal and experimental diets (% of total fatty acids).
Diets

Fatty acids N. gaditana CT R25 R50 H25 H50 P1

14:0 5.60 ± 0.01 3.11 ± 0.00 3.09 ± 0.03 3.13 ± 0.02 3.15 ± 0.04 3.17 ± 0.037 n.s.
16:0 22.4 ± 0.02 21.60 ± 0.10 21.51 ± 0.04 21.49 ± 0.18 22.03 ± 0.35 21.70 ± 0.07 n.s.
18:0 21.30 ± 0.02 5.95 ± 0.05 b 5.77 ± 0.01 ab 5.59 ± 0.09 a 5.89 ± 0.07 b 5.67 ± 0.03 a 0.007
16:1n7 4.21 ± 0.02 a 4.59 ± 0.03 b 5.06 ± 0.04 b 4.63 ± 0.03 c 5.06 ± 0.06 c < 0.001
18:1n7 2.45 ± 0.01 b 2.42 ± 0.00 ab 2.35 ± 0.02a 2.45 ± 0.02 b 2.36 ± 0.03 a 0.008
18:1n9 15.17 ± 0.12 b 14.91 ± 0.04 b 14.42 ± 0.14 a 14.95 ± 0.11 b 14.54 ± 0.09 a 0.004
20:1n9 4.0 ± 0.01 1.40 ± 0.02 1.42 ± 0.01 1.62 ± 0.36 1.42 ± 0.01 1.38 ± 0.06 n.s.
18:2n6 11.64 ± 0.11 b 11.54 ± 0.10 b 11.34 ± 0.04 ab 11.51 ± 0.13 ab 11.12 ± 0.10 a 0.020
18:3n3 3.7 ± 0.01 1.61 ± 0.07 1.50 ± 0.02 1.51 ± 0.11 1.44 ± 0.08 1.41 ± 0.06 n.s.
16:2n4 0.74 ± 0.02 0.56 ± 0.22 0.69 ± 0.00 0.69 ± 0.00 0.56 ± 0.19 n.s.
16:3n4 0.94 ± 0.01 0.83 ± 0.17 0.93 ± 0.01 0.97 ± 0.00 0.82 ± 0.18 n.s.
18:4n3 0.46 ± 0.02 a 0.41 ± 0.02 a 0.66 ± 0.06 b 0.71 ± 0.01 b 0.67 ± 0.00 b < 0.001
20:4n6 0.31 ± 0.01 0.27 ± 0.00 0.27 ± 0.00 0.31 ± 0.01 0.28 ± 0.04 n.s.
20:4n3 9.5 ± 0.02 1.41 ± 0.01 a 1.57 ± 0.00 b 1.68 ± 0.04 c 1.50 ± 0.01 b 1.80 ± 0.01 c < 0.001
20:5n3 (EPA) 33.4 ± 0.05 6.10 ± 0.02 a 6.52 ± 0.05 b 7.10 ± 0.08 c 6.47 ± 0.06 b 7.15 ± 0.02 c < 0.001
22:5n3 1.23 ± 0.04 1.26 ± 0.03 1.28 ± 0.06 1.26 ± 0.05 1.25 ± 0.08 n.s.
22:6n3 (DHA) 17.14 ± 0.27 b 16.31 ± 0.21 a 15.60 ± 0.18 a 16.10 ± 0.12 a 15.77 ± 0.11 a 0.003
Others 4.54 ± 0.56 5.45 ± 0.78 5.23 ± 0.50 4.53 ± 0.94 5.30 ± 0.82 n.s.
∑
SFA 30.65 ± 0.15 30.38 ± 0.05 30.21 ± 0.29 31.07 ± 0.47 30.53 ± 0.07 n.s.
∑
MUFA 23.23 ± 0.13 23.34 ± 0.02 23.45 ± 0.16 23.45 ± 0.15 23.34 ± 0.12 n.s.
∑
PUFA 38,14 ± 0.22 39.91 ± 0.29 39.44 ± 0.37 39.29 ± 0.13 39,45 ± 0.25 n.s.
∑
n-3 27.95 ± 0.15 27.56 ± 0.33 27.83 ± 0.31 27.48 ± 0.20 28.05 ± 0.27 n.s.
∑
n-6 11.95 ± 0.12 b 11.83 ± 0.10 ab 11.60 ± 0.04 ab 11.818 ± 0.14 ab 11.40 ± 0.14 a 0.025
n3/n6 2.34 ± 0.01 a 2.33 ± 0.01 a 2.40 ± 0.02 b 2.33 ± 0.01 a 2.46 ± 0.01 c < 0.001
EPA/DHA 0.36 ± 0.01 a 0.40 ± 0.00 b 0.46 ± 0.00 c 0.40 ± 0.00 b 0.45 ± 0.00 c < 0.001
1
The statistical comparison was carried out among the experimental diets, excluding N. gaditana meal. Therefore, P-values illustrate the statistical significance of
differences among CT, R25, R50, H25 and H50 diets. CT: control diet. R25 and R50: diets including 25 and 50 g kg− 1 raw microalgal biomass, respectively. H25 and
H50: diets including 25 and 50 g kg− 1 hydrolysed microalgae, respectively Different lower-case superscripts indicate significant differences among diets within each
row (P < 0.05). Values (n = 3) are mean ± standard deviation. n.s.: not significant.

Fig. 1. Amino acid profile of N. gaditana meal and the experimental diets. A: essential amino acids; B: non-essential amino acids. Results (n = 3) are expressed as % of
total amino acids.

use. Total soluble protein was determined according to Bradford (1976)
using bovine serum albumin as standard.
Total alkaline protease activity in intestinal extracts was measured
spectrophotometrically following the procedure described by Alarcón
et al. (1998), using 5 g L− 1 casein in 50 mM Tris-HCl (pH 9.0) as sub­
strate. One unit of total protease activity was defined as the amount of
enzyme that released 1 μg of tyrosine per min in the reaction mixture,
considering an extinction coefficient of 0.008 μg− 1 mL− 1 cm− 1 for
tyrosine, measured at 280 nm wavelength. Trypsin and chymotrypsin
activities were assayed using 0.5 mM BAPNA (N-α-benzoyl-DL-arginine-
4-nitroanilide) as substrate according to Erlanger et al. (1961), and 0.2
mM SAPNA (N-succinyl-(Ala)2-Pro-Phe-p-nitroanilide) according to Del
Mar et al. (1979), respectively, in 50 mM Tris-HCl, 10 mM CaCl2 buffer,
pH 8.5. Leucine aminopeptidase activity was determined by using 2 mM

4
M.I. Sáez et al.
L-leucine-p-nitroanilide in 100 mM Tris-HCl buffer, pH 8.8, as substrate
(Pfleiderer, 1970), and alkaline phosphatase was assayed using 450 mM
p-nitrophenyl phosphate in 1 M diethanolamine, 1 mM MgCl2 buffer, pH
9.5 (Bergmeyer, 1974) as substrate. For trypsin, chymotrypsin, and
leucine aminopeptidase activities, one enzyme activity unit (U) wasde­
fined as the amount of enzyme releasing 1 μmol of p-nitroanilide (pNA)
per minute, considering as extinction coefficient 8800 M cm− 1,
measured spectrophotometrically at 405 nm. For alkaline phosphatase,
one activity unit was defined as the amount of enzyme that released 1 μg
of nitrophenyl per min considering an extinction coefficient of 17,800 M
cm− 1 for p-nitrophenol, measured also at 405 nm. All assays were per­
formed in triplicate, and specific enzymatic activity was expressed as
units (U) g tissue− 1.
2.6. Histology of the intestinal mucosa
Intestine samples were fixed in phosphate-buffered formalin (4% v/
v, pH 7.2) for 24 h, then dehydrated and embedded in paraffin according
to standard histological techniques, as described in Vizcaíno et al.
(2018). Samples were cut in transversal sections (5 μm), and the slides
were stained with haematoxylin-eosin (H&E). The preparations were
examined under light microscope (Olympus ix51, Olympus, Barcelona,
Spain) equipped with a digital camera (CC12, Olympus Soft Imaging
Solutions GmbH, Muenster, Germany). Images were analysed with
specific software (Image J, National Institutes of Health, USA). The
length of mucosal folds and total enterocyte height were determined in
intestinal samples (10 independent measurements performed at 4
different optical areas of each section from 5 fish per tank; 2 sections per
fish).
2.7. Ultrastructure of the intestinal mucosa
Intestine samples for scanning electron microscopy (SEM) were
washed with 1% S-carboxymethyl-L-cysteine (Sigma Chem.) for 20 s,
with the aim of removing the epithelial mucus, prior to fixation. Then,
specimens were fixed in phosphate-buffered formaldehyde (4% v/v, pH
7.2) for 24 h; next excess glutaraldehyde was removed by washing
samples in 0.1 mol L− 1cacodylate buffer, pH 7.2, and then dehydrated
with a series of increasing concentrations of ethanol (50% to 100% v/v).
Samples were critical point dried in absolute ethanol as intermediate
fluid, and CO2 as transition fluid (CDP 030 Critical point dryer, Leica
Microsystems, Madrid, Spain). After drying, specimens were mounted
on aluminium stubs, immobilized with graphite (PELCO® Colloidal
Graphite, Ted Pella INC., Ca, USA), and then gold sputter coated (SCD
005 Sputter Coater, Leica Microsystems). Finally, all samples were
screened with a scanning electron microscope (HITACHI S-3500, Hitachi
High-Technologies Corporation, Japan).
Samples for transmission electron microscopy (TEM) were fixed (4 h,
4 ◦ C) in 25 g L− 1 glutaraldehyde, 40 g L− 1 formaldehyde in phosphate
buffer saline (PBS), pH 7.5. Next, intestine sections were washed with
PBS for 20 min and then, a post-fixation step with 20 g L− 1 osmium
tetroxide was carried out. Samples were dehydrated by consecutive
immersion in increasing concentrations of ethanol, embedded for two
hours, in 1:1 mixture of Epon resin and 100% (v/v) ethanol under
continuous shaking, and then, included in pure Epon resin, and let
polymerize at 60 ◦ C. Finally, ultrathin cuts were obtained from resin
blocks, and placed on a 700 Å copper mesh and stained with uranyl
acetate and lead citrate. The mesh observation was performed with a
Zeiss 10C TEM at 100 Kv (Carl Zeiss, Barcelona, Spain).
SEM and TEM visualization fields were recorded and digital images
were analysed using UTHSCSA ImageTool software (University of Texas
Health Science Center, San Antonio, TX). Microvilli length (ML) and
microvilli diameter (MD) were determined in TEM micrographs ac­
cording to (Vizcaíno et al., 2014). SEM images were used to obtain
measurements of enterocyte apical area (EAA). Finally, data obtained
from TEM and SEM images were used to estimate the total absorption
5
Aquaculture 556 (2022) 738288
surface per enterocyte (TAS) according to Vizcaíno et al. (2014).
2.8. Lipid oxidation
Lipid oxidation was estimated by thiobarbituric acid-reactive sub­
stances (TBARS) analysis in muscle and liver according to the method of
Buege and Aust (1978). Briefly, samples (2 g each) were homogenized in
4 mL 50 mM NaH2PO4, 0.1% (v/v) Triton X-100 solution. The mixture
was centrifuged (10,000 g, 20 min, 4 ◦ C) and supernatants were mixed in
a ratio 1:5 (v/v) with 2-thiobarbituric acid (TBA) reagent (0.375% w/v
TBA, 15% w/v TCA, 0.01% w/v 2,6-dibutyl hydroxytoluene (BHT) and
0.25 N HCl). The mixture was heated for 15 min and then centrifuged
(3600 g, 10 min, 4 ◦ C), and the absorbance of supernatants was
measured at 535 nm. The amount of TBARS was expressed as mg of
malonyl dialdehyde (MDA) per kg of muscle after comparing with a
MDA standard.
2.9. Instrumental colour determination
For all fish samples colour was measured on skin dorsal portion by
L*, a*, and b* system (CIE, 1986), using a Minolta Chroma meter CR400
device (Minolta, Osaka, Japan). The parameter lightness (L*, on a 100-
point scale from black to white), redness (a*, assesses the position be­
tween red, positive values, and green, negative values), and yellowness
(b*, assesses the position between yellow, positive values, and blue,
negative values) were recorded.
2.10. Statistics
The effect of the categorical variables “pre-treatment” and “doses”,
as well as their interactions, were determined for each numeric
parameter studied by fitting a generalized lineal multifactorial statistical
model (GLM analysis) that relates measured parameters to predictive
factors, using specific software (SPSS 25, IBM Corporation Inc.). Least
square means were tested for differences using Fisher’s least significant
difference (LSD) procedure. Unless otherwise is specified, a significance
level of 95% was considered to indicate statistical differences (P < 0.05).
When measurements were expressed as a percentage (e.g., fatty acids
profile), arcsine transformation of their square root was carried out in
order to normalize data prior to the statistical analysis.
3. Results
3.1. Microalgae hydrolysis
The concentration of reducing sugars in the reaction vessels
increased throughout the in vitro assay owing to the addition of the
commercial cellulase enzyme (Fig. 2A), being differences significant (P
< 0.001) at each sampling time throughout the hydrolysis. Results
indicate that enzyme-treated (5% cellulase) microalgal biomass yielded
final values in the region of 8 g glucose equivalents (GE) per 100 g
microalgae dry mass. This value was about 4-fold the amount of
reducing sugars released from untreated raw algae (control), which
accounted for stable values about 2–2.5 g GE throughout the complete
assay (300 min).
Analogously, the total amount of amino acids released (Fig. 2B)
during the assay indicated that cellulase hydrolysis increased signifi­
cantly (P < 0.001) their concentration in the reaction vessels, compared
to raw biomass. Under the assay conditions, final concentration of free
amino acids in enzyme-treated batches reached 12 g 100 g protein− 1,
compared to 6–7 g free amino acids 100 g protein− 1 measured in con­
trols (Fig. 3).
The quantification of total polyphenols at the beginning and at the
end of the enzymatic assay revealed a 70% increase of these substances
when the algal biomass was enzymatically treated (Fig. 3). The final
concentration of total phenolics in supernatants was significantly higher
M.I. Sáez et al. Aquaculture 556 (2022) 738288

Fig. 2. Time-course of the concentration in reducing sugars (A, expressed as D-glucose equivalents, GE, 100 g dry biomass-1) and total free amino acids (B, expressed
as g L-leucine, L-Leu, 100 g protein− 1) measured from raw and cellulose-hydrolysed biomass of N. gaditana during the in vitro assay. Within each sampling time,
asterisks indicate significant differences between values (P < 0.05). Values (n = 6) are mean ± standard deviation.

Table 3
Fish biometric parameters and muscle proximate composition at the end of the
feeding trial (90 d).
Diets

CT R25 R50 H25 H50 P

Final BW 49.10 ± 51.30 ± 49.50 ± 49.90 ± 50.2 ± n.

(g) 5.69 4.75 5.69 5.69 5.69 s.
1.07 ± 1.04 ± 1.01 ± 1.01 ± 1.01 ± n.
FCR 0.09 0.09 0.19 0.09 0.19 s
SGR (% 1.47 ± 1.49 ± 1.54 ± 1.54 ± 1.52 ± n.
d− 1) 0.09 0.09 0.09 0.09 0.09 s
Crude
protein 17.68 ± 18.36 ± 18.29 ± 17.99 ± 17.86 ± n.
(% DM) 0.28 0.34 0.33 0.48 0.27 s.
Crude lipid 7.33 ± 7.05 ± 7.19 ± 7.21 ± 7.18 ± n.
(% DM) 0.87 0.77 0.75 0.81 0.74 s.
Ash (% 1.81 ± 1.76 ± 1.75 ± 1.75 ± 1.77 ± n.
DM) 0.06 0.04 0.05 0.06 0.11 s.
Moisture 72.59 ± 72.61 ± 71.91 ± 71.9 ± 72.18 ± n.
(%) 0.64 0.66 0.79 0.73 0.49 s.

Fig. 3. Total phenolics released from raw and cellulase-hydrolysed N. gaditana
biomass at the beginning and at the end of the in vitro hydrolysis. Results are
represented as mg gallic acid equivalents (GAE) 100 g microalgal dry bio­
mass− 1. Within each sampling time, asterisks indicate significant differences
between values (P < 0.05). Data (n = 6) are mean ± standard deviation.
(P < 0.01) in cellulase-treated N. gaditana (reaching 70 mg gallic acid
equivalents (GAE) 100 g dry microalgae− 1), than that measured in
controls (40 mg GAE 100 g− 1). In absence of cellulase, no significant
differences were observed in total phenolics measured in the reaction
vessel after the 5-h (300 min) incubation period.
3.2. Fish biometric parameters, muscle proximate composition and fatty
acid profile
Experimental diets were well accepted by the fish, and feed intake
was similar in all groups. Mortality was below 1%. During the experi­
mental period, no differences were observed regarding growth param­
eters (final BW, FCR and SGR) among experimental lots at the end of the
feeding trial (Table 3). Throughout this period, final body weight
(approx. 50 g) at least triplicated initial values (approx. 15 g). No
BW: final body weight. FCR: feed conversion rate. SGR: specific growth rate. CT:
control diet. R25 and R50: diets including 25 and 50 g kg− 1 raw microalgal
biomass, respectively. H25 and H50: diets including 25 and 50 g kg− 1 hydro­
lysed microalgae, respectively. Different lower-case superscripts indicate sig­
nificant differences among diets within each row (P < 0.05). Values are mean ±
standard deviation. For proximate composition n = 15. For biometric parame­
ters n = 90. n.s.: not significant.
significant differences (P > 0.05) were observed for any of the param­
eters of muscle proximate composition. Although not significantly,
microalgae-enriched diets tended to decrease slightly total muscle lipid
content compared to CT diet, no matter the microalgae concentration or
treatment considered.
Overall results on muscle fatty acid profile indicated that the inclu­
sion of raw or hydrolysed microalgae yielded significant changes in FA
profile (Table 4), especially with regard to MUFAs and PUFAs, which
displayed opposing tendencies. Thus, microalgae-enriched diets reduced
total MUFAs compared to CT, being this decrease more evident in diets
including raw biomass (R25 vs. H25, and R50 vs. H50; P < 0.05).
Regarding individual MUFAs, oleic acid (18:1n9) was the predominant
FA, and its tendency paralleled that of total MUFA values. On the other

6
M.I. Sáez et al. Aquaculture 556 (2022) 738288

Table 4 compared to controls (Table 5), with the exception of leucine amino­
Effects of the dietary inclusion of N. gaditana on fatty acid profile of gilthead peptidase (LAP) activity. Also considering all the activities as a whole,
seabream muscle after a 90-d feeding trial (% of total fatty acids). significant differences were attributable to sampling time, as values
Diets measured at the end of the assay (90 d) where significantly higher (P <
Fatty CT R25 R50 H25 H50 P
0.05) than those measured at day 45, irrespectively of the dietary
acids treatment, with some exceptions for LAP activity again.
After 45 days of feeding, significant differences (P < 0.05) were
2.00 ± 1.51 ± 1.47 ± 1.78 ± 1.86 ±
14:0
0.01d 0.00a 0.02a 0.04b 0.02c
0.020 observed in trypsin activity attributable to both biomass pre-treatment
16.54 ± 16.57 ± 16.83 ± 16.68 ± 16.96 ± and inclusion level, whereas only microalgae hydrolysis influenced
16:0 0.015
0.14a 0.15a 0.15ab 0.12a 0.26b total alkaline protease and alkaline phosphatase activities (P < 0.05). No
4.70 ± 5.51 ± 5.52 ± 5.10 ± 5.10 ± changes due to these factors were observed for leucine aminopeptidase.
18:0 0.022
0.04a 0.00c 0.04c 0.05b 0.08b
4.69 ± 3.58 ± 3.82 ± 4.63 ± 4.40 ±
At the end of the feeding trial, the influence of both variables was sig­
16:1n7 0.033 nificant on total alkaline protease, trypsin and chymotrypsin and ac­
0.02c 0.02a 0.05b 0.05c 0.02c
2.69 ± 2.53 ± 2.53 ± 2.69 ± 2.44 ± tivities, but only a hydrolysis-dependent effect was observed for and
18:1n7 0.036
0.02b 0.12ab 0.01b 0.11b 0.09a leucine aminopeptidase activity.
19.64 ± 14.76 ± 15.76 ± 17.68 ± 18.35 ±
18:1n9 0.024
0.18e 0.31a 0.14b 0.07c 0.09d
1.49 ± 1.32 ± 1.28 ± 1.36 ± 1.48 ± 3.4. Intestinal mucosa histology
20:1n9 0.006
0.05b 0.14ab 0.09a 0.09ab 0.05b

18:2n6
8.37 ± 8.06 ± 8.14 ± 8.50 ± 8.52 ±
0.014 The histological characteristics of intestinal sections obtained from
0.08b 0.03a 0.08a 0.16b 0.07b fish receiving the experimental dietary treatments at the end of the
0.97 ± 0.86 ± 0.86 ± 1.03 ± 1.05 ±
18:3n3
0.01b 0.02a 0.02a 0.02b 0.08b
0,014 feeding trial are shown in Fig. 4, and results of the measurements carried
0.59 ± 0.49 ± 0.51 ± 0.55 ± 0.56 ± out on haematoxylin-eosin stained sections are summarized in Table 6.
16:4n3 0.019
0.01c 0.02a 0.06a 0.01b 0.01b Neither evidence of lipid droplet accumulation in the intestinal epithe­
0.60 ± 0.43 ± 0.44 ± 0.55 ± 0.54 ± lium nor inflammatory changes in the lamina propria were observed.
18:4n3 0.019
0.01c 0.01a 0.02a 0.05bc 0.01b
Consequently, no apparent damage attributable to any of the dietary
1.72 ± 2.20 ± 2.37 ± 1.78 ± 1.88 ±
20:4n6
0.02a 0.01c 0.03d 0.06ab 0.11b
0.033 treatments was found. Enterocytes presented aligned nucleus, homog­
0.52 ± 0.53 ± 0.50 ± 0.51 ± 0.53 ± enous supranuclear vacuolization and adequate cell shape (columnar
20:4n3 n.s.
0.00 0.01 0.01 0.02 0.01 and high). Intercellular spaces were not visible between enterocytes, and
20:5n3, 4.76 ± 5.63 ± 5.72 ± 5.23 ± 5.25 ± goblet cells were evenly dispersed throughout the epithelium.
0,014
EPA 0.03a 0.04c 0.09c 0.11b 0.04b
2.22 ± 2.12 ± 2.12 ± 2.14 ± 2.19 ±
Image analysis of the preparations indicated that no significant dif­
22:5n3 0.031 ferences in fold length or enterocyte height were found among the di­
0.02c 0.01a 0.09a 0.05ab 0.03ab
22:6n3, 22.84 ± 23.75 ± 23.96 ± 23.42 ± 23.56 ± etary treatments. However, differences attributable to the inclusion
0.001
DHA 0.08a 0.07c 0.16c 0.10b 0.26bc level were observed, as the animals receiving R50 and H50 diets
5.17 ± 9.52 ± 7.76 ± 5.61 ± 4.70 ± <
Other FA showing a significantly thinner lamina propria than the rest of the
0.38a 0.51d 0.36c 0.25 ab 0.40b 0.001
∑ 23.24 ± 23.58 ± 23.67 ± 23.70 ± 23.92 ± experimental batches, irrespectively of the enzyme pre-treatment.
SFA n.s
0.18 0.16 0.16 0.23 0.37
∑
MUFA
28.50 ± 22.19 ± 23.39 ± 26.35 ± 26.67 ±
0.032 3.5. Ultrastructure of the intestinal mucosa
0.26d 0.31a 0.06b 0.17c 0.16c
∑ 41.82 ± 43.62 ± 44.10 ± 43.16 ± 43.53 ± <
PUFA
0.17a 0.02c 0.12d 0.16b 0.17bc 0.001
TEM and SEM observations indicated that none of the experimental
∑ 31.73 ± 33.33 ± 33.59 ± 32.87 ± 33.13 ± < diets caused any perceptible damage on the enterocyte brush border
n-3
0.19a 0.03b 0.12bc 0.03c 0.20b 0.001 ultrastructure (Fig. 5), since specimens from animals from all the
∑ 10.09 ± 10.28 ± 10.50 ± 10.29 ± 10.40 ± experimental groups presented a well-defined and organized intestinal
n-6 0.026
0.08a 0.09b 0.10b 0.13b 0.09b
brush border membrane. Moreover, no intercellular spaces were visible
3.15 ± 3.24 ± 3.20 ± 3.19 ± 3.19 ± <
n3/n6
0.02a 0.01b 0.01b 0.02b 0.03ab 0.001 in the apical zone of the epithelium. Image analysis (Table 7) showed
EPA/ 0.21 ± 0.24 ± 0.24 ± 0.22 ± 0.22 ± < that only 5% inclusion level caused changes in all the parameters
DHA 0.00a 0.00c 0.01c 0.01 b 0.00b 0.001 studied. R50 treatment yielded higher microvilli diameter (MD) than the
CT: control diet. R25 and R50: diets including 25 and 50 g kg− 1 raw microalgal rest of treatments, whereas only H50 caused significant increase in
biomass, respectively. H25 and H50: diets including 25 and 50 g kg− 1 hydro­ microvilli length (ML). Both R50 and H50 treatments increased signifi­
lysed microalgae, respectively. Values with different lowercase superscript cantly enterocyte apical area (EAA). The factor that accounted for most
within each row indicate significant differences in muscle lipids attributed to of the changes observed in total enterocyte absorption surface (TAS) was
dietary treatments (p < 0.05). SFA: saturated fatty acids; MUFA: mono­ the biomass pre-treatment, given that both H25 and H50 batches
unsaturated fatty acids; PUFA: polyunsaturated fatty acids; EPA: eicosapentae­ showed significantly higher values for this parameter compared to CT
noic acid; DHA: docosahexaenoic acid. Values (n = 15) are expressed as average and R25.
± standard deviation. n.s.: not significant.
3.6. Muscle and liver lipid oxidation (TBARS)
hand, increased total PUFAs in muscle was observed in fish fed on
microalgae-containing diets compared to CT lot. As indicated for total The overall tendency for TBARS values (Table 8) indicates that CT
MUFAs, although with opposite trend, the hydrolyzed biomass was batch yielded the highest values for this parameter, irrespectively of the
responsible for significantly higher values (P < 0.05) of muscle PUFAs tissue, sampling time, and dietary treatment considered (P < 0.001).
than raw biomass within each inclusion level. It is worth mentioning Nevertheless, not all factors were responsible for significant differences
that both EPA and DHA paralleled such increase. in all cases.
With regard to muscle, R50, H25 and H50 treatments decreased
3.3. Digestive enzyme activities significantly lipid oxidation compared to CT batch at both sampling
times (45 and 90 d). Within each dietary treatment, only H50 showed
In general, the supplementation with N. gaditana increased signifi­ differences attributable to sampling time.
cantly (P < 0.05) the enzyme activities measured in intestinal extracts The effects of the microalgae-enriched diets were even more evident

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M.I. Sáez et al. Aquaculture 556 (2022) 738288

Table 5
Enzyme activities (U g tissue− 1) measured in intestinal extracts of Sparus aurata juveniles fed with the experimental diets.
Diets

Sampling time Enzyme activity CT R25 R50 H25 H50 P

Total alkaline protease 491.55 ± 50.47 a,I 567.54 ± 23.68 b,I 589.56 ± 24.92 b,I 553.14 ± 37.69 b,I 560.90 ± 28.50 b,I <0.001
45 d Trypsin (x10− 3) 16.60 ± 2.30 a,I 33.02 ± 1.76 c,I 35.77 ± 2.22 d,I 28.71 ± 1.58 b,I 28.90 ± 1.26 b,I <0.001
Chymotrypsin 2.49 ± 0.17 a,I 2.45 ± 0.13 a,I 3.05 ± 0.32 b,I 2.34 ± 0.20 a,I 2.32 ± 0.13 a,I <0.001
LAP (x10− 3) 0.43 ± 0.04 I 0.41 ± 0.04 I 0.41 ± 0.04 0.44 ± 0.04 0.42 ± 0.04 n.s.
Alkaline phosphatase 11.0 ± 0.36 a,II 10.44 ± 0.51 a,I 10.78 ± 0.40 a,I 13.25 ± 0.40 b,I 13.38 ± 0.27 b,I <0.001
Total alkaline protease 676.46 ± 38.27 a,II 909.67 ± 57.50 b,II 1015.58 ± 22.37 c,II 849.97 ± 49.89 b,II 862.73 ± 29.72 b,II <0.001
90 d Trypsin (x10− 3) 22.85 ± 2.03 a,II 46.57 ± 1.75 c,II 53.31 ± 2.57 d,II 36.78 ± 2.44 b,II 34.46 ± 2.30 b,II <0.001
Chymotrypsin 2.10 ± 0.10 a,II 3.21 ± 0.15 c,II 3.79 ± 0.11 d,II 2.81 ± 0.27 b,II 2.80 ± 0.11 b,II <0.001
LAP (x10− 3) 0.37 ± 0.04 a,I 0.52 ± 0.05 c,II 0.41 ± 0.05 ab 0.51 ± 0.04 c 0.43 ± 0.07 b <0.001
Alkaline phosphatase 14.60 ± 0.81 a,II 15.22 ± 0.97 a,II 16.59 ± 0.49 ab,II 17.25 ± 0.92 b,II 15.09 ± 0.78 a,II <0.001

CT: control diet. R25 and R50: diets including 25 and 50 g kg− 1 raw microalgal biomass, respectively. H25 and H50: diets including 25 and 50 g kg− 1 hydrolysed
microalgae, respectively. LAP: Leucine aminopeptidase. Values (n = 9) are mean ± standard deviation. Values in the same row with different lowercase superscript
indicate significant differences owing to dietary treatments (P < 0.05). Values of each enzyme activity with different superscript in Roman numerals indicate sig­
nificant differences due to sampling time (P < 0.05). n.s.: not significant.

Fig. 4. Light microscopy details of intestine sections of S. aurata juveniles fed on the experimental diets for 90 days. H&E stain, magnification x100 (upper images)
and ×400 (lower images). CT: control diet. R25 and R50: diets including 25 and 50 g kg− 1 raw microalgal biomass, respectively. H25 and H50: diets including 25 and
50 g kg− 1hydrolysed microalgae, respectively.

Table 6
Measurements in histological preparations of the intestinal mucosa of S. aurata juveniles fed with the experimental diets during 90 days.
Parameters Diets

CT R25 R50 H25 H50 P

Fold length (μm) 940.82 ± 216.24 1173.12 ± 414.47 1193.48 ± 269.95 1053.49 ± 205.41 1013.00 ± 239.95 n.s.
Enterocyte height (μm) 46.94 ± 7.47 57.21 ± 8.26 46.95 ± 3.95 45.61 ± 4.94 50.01 ± 8.33 n.s.
Lamina propria (μm) 13.76 ± 3.62b 13.06 ± 2.50b 6.35 ± 1.40a 11.23 ± 1.95b 7.63 ± 2.39a < 0.001

Values in the same row with different lowercase indicate significant differences (P < 0.05) owing to dietary treatments. CT: control diet. R25 and R50: diets including
25 and 50 g kg− 1 raw microalgal biomass, respectively. H25 and H50: diets including 25 and 50 g kg− 1 hydrolysed microalgae, respectively. Values are expressed as
mean ± standard deviation. n.s.: not significant.

in liver, given that all treatments, irrespectively of biomass hydrolysis or
inclusion level, yielded lower TBARS values than CT group. In this tis­
sue, significant differences attributable to biomass hydrolysis (lower
values for H25 and H50 in comparison with R25 and R50, respectively)
and inclusion level (lower values for R25 and H25 compared to R50 and
H50, respectively) were also observed. Same as found in muscle, also in
liver only H50 showed differences attributable to sampling time.
N. gaditana were significantly lower thanthose of CT group, indicating a
more greenish colorationat both 45 and 90 d. Nevertheless, no differ­
ences attributable to biomass pre-treatment (R25 vs. H25; R50 vs. H50)
or inclusion level (2.5% vs. 5.0%) were observed (P > 0.05). Similarly,
all treatments including algae biomass tended to increase b* parameter
(more yellowish pigmentation of the skin) compared to CT batch,
although differences were significant only for R50 treatment at day 45,
and for R25, R50 and H25 specimens at day 90.

3.7. Instrumental colour determinations

4. Discussion

After 45 days of the feeding trial, skin L* values were similar in all
Given that cellulose accounts for the most abundant structural car­
lots (Table 9). The same lack of significant differences was found at the
bohydrate in N. gaditana, the breakage of cell walls by hydrolysis with
end of the experimental period for this parameter, although L* values
cellulase enzymes emerges as a promising alternative aimed at
were influenced for the factor sampling time (P < 0.05). Skin a*
improving nutrient bioavailability and digestibility. The following may
parameter presented negative values in all specimens, irrespectively of
be cited as advantages of this procedure: i) owing to the wide variety of
the sampling time. Figures for fish fed with any diet supplemented with

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M.I. Sáez et al. Aquaculture 556 (2022) 738288

Fig. 5. Transmission (upper images, A) and scanning (lower images, B) electron microscopy micrographs from the anterior intestinal region of juvenile gilthead
seabream at the end of the feeding trial (90 days). CT: control diet. R25 and R50: diets including 25 and 50 g kg− 1 raw microalgal biomass, respectively. H25 and
H50: diets including 25 and 50 g kg− 1 hydrolysed microalgae, respectively.

Table 7 Table 8
Microvilli morphological parameters obtained from transmission electron mi­ Estimation of lipid oxidation (TBARS) in muscle and liver of juvenile fish fed on
croscopy ultramicrographs of the anterior intestine of S. aurata juveniles fed the different experimental diets.
with the experimental diets during 90 days. Diets
Parameters Diets
Time CT R25 R50 H25 H50 P
CT R25 R50 H25 H50 P
2.17 1.42
1.68 ± 1.31 ± 1.04 ± <
2.07 ± 2.16 ± 2.59 ± 2.28 ± 2.73 ± 45 d ± ±
ML (μm) 0.008 0.14bc 0.14ab 0.13a,I 0.001
0.59a 0.15a 0.20ab 0.13ab 0.14b 0.37c 0.12b
Muscle
0.11 ± 0.10 ± 0.13 ± 0.10 ± 0.11 ± 2.01 1.15
MD (μm) 0.026 1.71 ± 1.26 ± 0.92 ± <
0.01a 0.01a 0.01b 0.01a 0.01a 90 d ± ±
0.05b 0.20a 0.09a,II 0.001
19.36 20.75 26.14 20.45 25.92 0.39b 0.23a
EAA (μm2) <0.001
± 3.57a ± 4.20a ± 4.03b ± 4.02a ± 4.03b 4.62 3.19
3.86 ± 2.77 ± 2.52 ± <
587.63 620.47 692.94 772.71 917.96 45 d ± ±
0.39c 0.19a 0.18a,I 0.001
TAS (μm ) 2
± ± ± ± ± 0.006 0.43d 0.17b
Liver
88.60a 55.46a 81.93ab 38.01b 56.62b 4.64 3.03
3.73 ± 2.67 ± 2.15 ± <
90 d ± ±
ML: microvilli length; MD: microvilli diameter; EAA: enterocyte apical area; 0.26d 0.20b 0.07a,II 0.001
0.19e 0.09c
TAS: total enterocyte absorption surface. CT: control diet. R25 and R50: diets
including 25 and 50 g kg− 1 raw microalgal biomass, respectively. H25 and H50: CT: control diet. R25 and R50: diets including 25 and 50 g kg− 1 raw microalgal
diets including 25 and 50 g kg− 1 hydrolysed microalgae, respectively. Values in biomass, respectively. H25 and H50: diets including 25 and 50 g kg− 1 hydro­
the same row with different lowercase superscripts indicate significant differ­ lysed microalgae, respectively. TBARS stands for thiobarbituric acid reactive
ences (P < 0.05) owing to the dietary treatment. Values (n = 15) are expressed as substances, expressed as mg malonyldialdehyde (MDA) kg tissue− 1. Values in
mean ± standard deviation. the same row with different lowercase superscripts indicate significant differ­
ences (P < 0.05) owing to dietary treatments. Within each tissue, values with
different superscript in Roman numerals indicate significant differences due to
industrial applications, cellulases are reasonably inexpensive; ii) no sampling time (P < 0.05). Values (n = 15) are mean ± standard deviation.
complex equipment is needed for hydrolysis, as incubators widely uti­
lized in the food and feed industry can be used; iii) negative impacts on
line with previous studies on this microalgae genus carried out on
thermolabile compounds are not expected, as mild temperatures are
several species of commercial fish (Qiao et al., 2019; Sørensen et al.,
involved in the hydrolysis process; and iv) given the specificity of the
2017; Walker and Berlinsky, 2011). Nevertheless, other reports pointed
catalytic action on cellulose, the remaining released compounds would
to improved fish growth owing to microalgae inclusion in diets, but
not be hydrolysed by the enzymatic pre-treatment.
higher inclusion levels were evaluated (up to 10% inclusion in diets for
Under this perspective, this study evaluatedin a 90-d feeding trial the
Nile tilapia, Abdel-Tawwab and Ahmad, 2009; from 10 to 39% for
effects of a cellulase pre-treatment on Nannochloropsis gaditana biomass
S. aurata, in Vizcaíno et al., 2014, 2016, 2018). It is likely that the low
prior to its incorporation into feeds for gilthead seabream juveniles. It
inclusion levels considered have accounted for this lack of effect on
was expected that nutrient bioavailability would increase as a result of
growth.
the enzyme pre-treatment, and according to the results of the enzyme
With regard to fish muscle proximal composition, overall, no dif­
hydrolysis (Figs. 2 and 3), the increased release of reducing sugars, free
ferences attributable to the experimental diets were observed, in
amino acids and polyphenols taking place in the reaction vessels seem to
agreement with previous studies on N. gaditana enriched diets (Qiao
confirm this hypothesis.
et al., 2019; Vizcaíno et al., 2018; Sales et al., 2021). Only slight, but not
Even if the enzyme treatment increased in vitro bioavailability,
significant differences in lipid and protein contents were measured
however, no impact on fish growth was observed for any of the exper­
among the experimental groups (Table 3). The study by Galafat et al.
imental batches throughout the 90-d feeding trial. These results are in

9
M.I. Sáez et al. Aquaculture 556 (2022) 738288

Table 9 the contrary, control diet showed the highest DHA figures, but yielded
Instrumental colour determinations on the skin surface of juvenile fish fed with the lowest content in fish muscle. In this regard, previous studies have
the different experimental diets. also reported certain selective retention of this structural FA owing to
Diets the addition of both macro and microalgae (Hussein et al., 2013; Viz­
Time Colour CT R25 R50 H25 H50 P
caíno et al., 2014, 2016; Kousoulaki et al., 2016; Sáez et al., 2020). In
parameters short, the results suggested that N. gaditana, even at the low inclusion
levels studied, was responsible for some selective retention of n-3-PUFA
73.74 74.98 73.36 72.40 72.13
L* ± 4.22 ± 3.24 ± 3.44 ± 5.19 ± 5.33 n.s. in muscle (mostly owing to DHA, the most abundant n3-PUFA), whilst
I I I I I the opposite effect was observed with regard to MUFAs (Table 4). Such
- 1.00 - 1.88 - 1.85 - 1.89 - 1.80 decrease in total MUFAs is in agreement with the evidenced lower oleic
45 d a* ± ± ± ± ± 0.047 acid content, which is the main MUFA in muscle of gilthead seabream.
0.39a 0.44b 0.40b 0.39b 0.32b
5.25 7.79 8.54 7.09 7.88
Nevertheless, disparate results have been reported in the literature
b* ± ± ± ± ± 0.033 regarding the effects of dietary microalgae on S. aurata lipid metabolism
1.40a 1.81ab 1.04b 1.09ab 1.36ab (Perera et al., 2020), and further studies aimed at fully understanding
89.75 87.91 87.91 88.07 87.37 the intrinsic mechanisms underlying the results observed are needed.
L* ± 1.41 ± 3.24 ± 3.25 ± 2.06 ± 1.45 n.s.
II II II II II On the other hand, microalgae are acknowledged as valuable source
- 1.48 - 2.01 - 2.16 - 1.89 - 1.94 of pigments and phenolic compounds with antioxidant capacity
90 d a* ± ± ± ± ± 0.021 (Koyande et al., 2019; Almendinger et al., 2021; Sáez et al., 2021), many
0.16a 0.38b 0.11b 0.12b 0.08b of which remain unidentified (Sansone et al., 2020).
5.53 8.16 8.28 8.02 7.25 Due to the interest of the pharmaceutical industry in pigments, these
<
b*
substances have received more attention than phenolics, but some au­
± ± ± ± ±
0.001
1.13a 0.46b 0.46b 0.92b 1.07ab
thors suggested that both groups of substances might contribute simi­
Values in the same row with different lowercase superscripts indicate significant larly to the antioxidant activity of microalgae (Almendinger et al.,
differences (P < 0.05) owing to dietary treatments. CT: control diet. R25 and
2021). Nevertheless, the relative contribution of phenolics and pigments
R50: diets including 25 and 50 g kg− 1 raw microalgal biomass, respectively. H25
to the antioxidant capacity of most microalgae species remains to be
and H50: diets including 25 and 50 g kg− 1 hydrolysed microalgae, respectively.
Values (n = 30) are mean ± standard deviation. Parameters L*, a* and b* as
ascertained (Goiris et al., 2012). N. gaditana contains chlorophylls,
defined in M&M section. n.s.: not significant. β-carotene, violaxanthin y vaucheriaxanthin, as well as trace amounts of
astaxanthin (Cerón-García et al., 2018), which might explain our results
pointing to higher antioxidant response in muscle and liver of fish
(2020) reported a significant increase inmuscle protein in juvenile
supplemented with the microalgal biomass. Teimouri et al. (2016, 2019)
gilthead seabream fed enzymatically hydrolysed Arthrospira platensis
also described this effectas a result of the inclusion of microalgae in
added at 2% inclusion level, as well as a significant decrease in total
feeds, and more specifically, Qiao et al. (2019) reported lower TBARS
lipids when added at 4% inclusion level. Reduced muscle lipid storage
values both in liver and serum in Scophtalmus maximus juveniles fed on
has also been reported not only for microalgae species (Hussein et al.,
5% N. gaditana diets. A recent study (Sales et al., 2021) has shown that
2013; El-Sheekh et al., 2014; Vizcaíno et al., 2014, 2016), but also for
purified extracts of the unsaponifiable fraction of N. gaditana, rich in
macroalgae (Ortiz et al., 2006; Yildirin et al., 2009; Sáez et al., 2020).
carotenoids, included in feeds to partially replace fish oil yielded potent
These findings suggest the existence of bioactive compounds in algae
antioxidant effects in muscle of S. aurata juveniles.
capable of influencing protein and lipid metabolism, although the na­
The cellulase pre-treatment of the microalgal biomass was respon­
ture of such substances or the underlying mechanisms involved in such
sible for a trend towards increased in vivo antioxidant effects on muscle
effects have not yet been fully ascertained. Recent evidence in this re­
lipids (Table 8), compared to untreated N. gaditana. Such increase
gard was provided by Perera et al. (2020), although microalgae-
attributable to enzyme hydrolysis reached statistical significance in the
containing commercial products rather than pure microalgae biomass
case of liver lipids. These results suggest that increased release and
were considered in the study.
further bioavailability of some inner bioactive compounds contained in
Whilst no quantitative differences in muscle lipid content were
the cells might have occur, as was the case for total phenolics (Fig. 3).
observed, however, qualitative differences were found in this analytic
Galafat et al. (2020) also found lower TBARS values in muscle of
component. It is known that fish muscle lipids reflect dietary FA profiles
S. aurata juveniles fed with Arthrospira sp. protease hydrolysates
(Grigorakis et al., 2002; Grigorakis, 2007; Yildiz, 2007), and this might
included at low inclusion level (2 and 4%) in diets. In agreement, and
explain the significant increase of EPA muscle content of fish fed with all
with regard to phenolics, N. gaditana contains certain amount of these
the algae-containing diets (Table 4), compared to control batch.
substances in raw biomass, in line with previous studies (Kherraf et al.,
N. gaditana is rich in EPA (33% of total FA), and consequently, all
2017; Haoujar et al., 2019), which might explain the potent antioxidant
microalgae-supplemented diets, either raw or hydrolysed (Table 4),
effects found on fish lipids in our study. Noticeably, as mentioned, total
were enriched in this specific FA in a dose-dependent manner, but not
phenolics measured in the reaction vessels increased as a result of the
influenced by the enzymatic pre-treatment of the biomass (no significant
cellulase treatment (Fig. 3).
differences, between R25 and H25, or between R50 and H50, Table 2).
Physical treatments, even if valuable when it comes to increasing the
Although all the microalgae-enriched diets yielded muscle EPA
yield of microalgae main compounds (i.e. protein and lipid fractions),
contents higher than those found in CT batch (Table 4), this effect wasn’t
might jeopardize the chemical integrity of thermolabile minor com­
dose-dependent. Interestingly, and contrary to what was expected, the
pounds (Schafberg et al., 2020), and consequently, impair their func­
enzymatic treatment of the biomass yielded lower EPA in muscle
tional activity.
compared to the raw microalgae. A possible explanation would be that
Given the susceptibility of pigments, especially carotenoids, to
EPA released from cells could be more susceptible to structural damage
different factors (temperature, oxygen, light, acidic pH, etc., Schieber
than that remaining within the microalgae cells. In other words, intact
and Weber, 2016), and even if the extraction procedures increase the
cell walls might have acted as a sort of “natural microcapsule” for EPA.
releasing of inner compounds, it should also be born in mind that
All the experimental batches fed with the supplemented diets yielded
microalgal biomass, as part of the ingredient mixture, will be extruded
significantly higher DHA muscle content than control fish. This fact
during the elaboration of the experimental diets, a process involving
can’t be explained by differences in this FA in the experimental diets
high pressure and temperature. Consequently, doubts could arise related
(Table 2), given that no DHA was measured in N. gaditana biomass. On
to the integrity and the subsequent in vivo bioavailability of some of the

10
M.I. Sáez et al.
compounds released in vitro. Previous research suggests that the
resulting balance of disrupting strategies is favourable to the enrichment
of aquafeeds (Schafberg et al., 2020), and our results coincide with that
idea. But given the diversity and complex nature of the antioxidant
substances involved, this specific issue deserves further research. Espe­
cially the balance between phenolics and carotenoids in a given
microalgae species is likely that could determine the persistence of the
antioxidant effects in feeds after processing procedures.
Although instrumental colour measurements at early stages of the
productive cycle have no interest in practical terms of fish quality
assessment, they can still provide valuable information about pigment
deposition and antioxidant effects in growing fish tissues. The favour­
able influence of microalgae on fish colour parameters found in our
study (increased a* and b*, Table 9) has been documented previously
(Teimouri et al., 2013; Cardinaletti et al., 2018; Galafat et al., 2020;
Kousoulaki et al., 2020; Sales et al., 2021). Although tendencies
observed for skin pigmentation suggest that raw microalgae intensified
the effects compared to hydrolysed biomass (Table 9), however, no
significant differences attributable to the enzymatic treatment were
found. This might well be explained again by the fact that some pig­
ments contained in the hydrolysed biomass might have been damaged to
a higher extent than those from raw biomass due to feed processing.
The activity of digestive enzymes is acknowledged not only as a
marker of their digestive and absorptive capacity (Alarcón et al., 1998),
but also as a reliable indicator of the nutritional status of aquacultured
fish. More specifically, the activity of some brush border membrane
enzymes, such as leucine aminopeptidase (LAP) and alkaline phospha­
tase, reveals the integrity and the absorptive capability of the intestinal
mucosa (Silva et al., 2010). Disparate results have been reported on the
effects of raw microalgae on digestive enzyme activities, and thus, Qiao
et al. (2019) found increased trypsin activity in juvenile turbot supple­
mented with N. gaditana biomass at 7.5% inclusion level after a 10-week
feeding trial. On the contrary, Jorge et al. (2019) observed no effects on
total alkaline protease, trypsin, α-amylase and lipase activities in
response to dietary N. gaditana supplementation, although the low in­
clusion levels considered (0.5, 1, and 1.5%) together with the short
duration of the feeding trial (37 d) might well have accounted for such
lack of effects. Few studies are available assessing the physiological
consequences of microalgae enzyme hydrolysates on such activities.
Galafat et al. (2020) described higher trypsin and LAP activities as a
result of protease hydrolysates of Arthrospira sp. at low inclusion level (2
and 4%) in S. aurata juveniles. More recently, Galafat et al. (2022) re­
ported increased trypsin, chymotrypsin and leucine aminopeptidase
activities in gilthead seabream fry as a result of supplementing starting
diets with Arthrospira platensis at 5 and 10% compared to control fish. In
addition, within each inclusion level, animals fed with diets that
included the hydrolysed biomass yielded consistently higher digestive
enzyme activities than those receiving the crude biomass. The results
obtained in this study indicate that N. gaditana supplementation, even at
the low inclusion levels tested, overall increased the enzyme activities
assayed compared to control fish, irrespectively of sampling time (45 or
90 d), or biomass pre-treatment, with the exception of leucine amino­
peptidase activity at day 45 (Table 5). It is also worth mentioning that
the favourable effects of the experimental diets on intestinal digestive
activities observed in this work concur, roughly speaking, with the ul­
trastructural determinations (Table 7) carried out on the intestinal
mucosa at the end of the feeding trial, especially at the highest con­
centration assayed (5%).
Overall, concerning the enzyme pre-treatment considered in this
work, no decisive effects were observed in terms of fish growth, muscle
composition, or digestive functionality, but the remarkable influence of
this treatment on the oxidative status of fishlipids could result in
beneficial effects on other parameters linked to the health status of
aquacultured fish, a fact that deserves further investigation as well. It
should also be born in mind that any feeding trial under controlled
conditions and short duration has evident limitations in terms of further
11
Aquaculture 556 (2022) 738288
applicability on-farm, bearing in mind the numerous additional factors
involved in the operation of long-term production cycles in commercial
fish farms.
5. Conclusions
Although N. gaditana biomass at low inclusion level in feeds had no
impact on growth and muscle proximal composition, however, it is
worth mentioning that the lack of detrimental effects, together with
some beneficial effects on other physiological parameters (digestive
structure and functionality, oxidative status of muscle and liver lipids,
and skin colour), overall indicate that might represent a valuable ad­
ditive in long-term S. aurata production cycles.
The results obtained evidenced the effectiveness of the cellulase pre-
treatment when it comes to in vitro releasing of intracellular compounds
from N. gaditana cells, which could improve not only extraction yields in
industrial applications, but also increase the bioavailability of certain
metabolites with potentially bioactive and functional effects. However,
no conclusive evidence was found regarding the impact of this strategy
on most of the physiological parameters tested, with the exception of the
enhanced effects on lipid oxidation.
It would be interesting to carry out further research aimed at
assessing the possible influence of N. gaditana hydrolysates on other
valuable physiological aspects, such as their influence on the intestinal
microbiome, the intermediary metabolism (not least lipid metabolism)
and the immune response of gilthead seabream, especially at early
stages of the production cycle.
Authors’ contributions
Sáez, M.I., Alarcón, F.J. and Martínez T.F. conceived and designed
the experiments. Alarcón, F.J. and Galafat, A. prepared the aquafeeds.
Galafat, A., Sáez, M.I., Vizcaíno,A.J. and Martínez, T.F. performed fish
sampling. Arizcun, M., Chaves-Pozo, E.and Ayala, M.D. participated in
sampling, fish care and maintenance, and in biometric and proximal
analysis. Suárez, M.D. performed and interpreted fatty acid analysis.
Galafat, A., Sáez, M.I., Martínez, T.F., Suárez, M.D., Arizcun M., and
Chaves-Pozo E. performed analytical analysis and discussed the data.
Sáez, M.I., Alarcón, F.J. and Martínez, T.F. drafted the manuscript. T.F.
Martinez and M. Arizcun obtained the necessary funds for conducting
the research. All authors critically revised and approved the manuscript.
Funding information
This research was funded by Spanish MCIU-FEDER (grant #
RTI2018-096625-B-C33 and grant # RTI2018–096625-B-C31), SABANA
project (the European Union’s Horizon 2020 Research and Innovation
program, grant # 727874), AquaTech4Feed (grant # PCI2020-112204)
granted by MCIN/AEI/10.13039/501100011033, the EU “NextGener­
ationEU”/PRTR within the ERA-NET BioBlue COFUND). Servicio de
piensos experimentales was granted by EQC2018-004984-P and
EQC2019-006380-P.
Statement of informed consent, human/animal rights
The authors state that no conflicts, informed consent, human or
animal rights are applicable. All studies involving fish were conducted in
accordance with the requirements of the Directive 2010/63/EU, and the
Spanish legislation (Real Decreto 53/2013, as amended by RD218/
2021), regarding the ethical rules applicable in research involving lab­
oratory animals. Thereby, all the procedures were authorized by the
Bioethics and Animal Welfare Committee of the Instituto Español de
Oceanografía (REGA code ES300261040017) with the approval of the
Ministry of Water, Agriculture and Environment of the Autonomous
Community Region of Murcia (Spain; A13200101).
M.I. Sáez et al.
Declaration of Competing Interest
The authors declare that they have no conflict ofinterest.
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 Russian colonists in 1892 and
containing forty-one families. This
village and the neighbouring one of
Poltavski, founded in 1896 and where
there are thirty-five families, are the
most southern settlements within the
Russian Empire. The inhabitants exist
almost entirely by the exportation of
inconsiderable quantities of wheat, hay
and straw to Kushkinski Post for the
purposes of the garrison.
Kushkinski Post station, 306.4
sagenes above sea-level, is 293 versts
from Merv. It possesses a fine buffet.
The military post, situated near the
frontier in the broad valley of the Kushk
river, is bounded by the undulating
slopes of the Bend Chengurek chain,
an off-shoot of the Paropamisus. With
the completion of the Murghab railway,
hindu traders at pendjeh Kushkinski Post immediately attained
special importance and, in 1900, it was declared a fortress of the
fourth rank. The hoisting of the Imperial standard over the walls was
carried out in the presence of the late Minister of War, General
Kuropatkin. In the early days, before the lines of the fortress had
been planned, Kushkinski Post comprised a number of detached
works within which the various arms were quartered. At that time,
too, the officers’ accommodation, consisting of one-storey buildings
roughly constructed out of mud, was in the railway settlement where,
pending the completion of the main works, long narrow sheds for the
use of the troops had been erected. Now improvement has followed
upon preliminary chaos and the men are comfortably housed in cool
barracks upon the upper slopes of the adjacent heights. The officers
are disposed with equal care and convenience elsewhere. Public
buildings likewise have improved upon their original sites. The
military hospital, the post and telegraph bureau and the Custom
House have taken up locations upon high ground, their positions
crowned, if not protected by forts upon the crest of these very useful
eminences. Kushkinski Post, therefore, may be said to be a thriving
settlement where, if the hours are wearisome and the days charged
with ennui, there is always the prospect of a “dust up.”
Attempts have been made from time to time, by officers stationed
at Kushkinski Post, to become familiar with the officers in command
of the Afghan posts across the frontier. More often these attempts at
friendliness have been rebuffed, the Afghan soldiery neither
accepting advances from the Russians nor making any overtures
themselves. Strained relations exist, as a rule, between military
posts on either side of any frontier, although, in regard to the Russo-
Afghan frontier, there was an occasion when friendly conditions
prevailed between the Russians and the Afghans.[10] At that time the
staff of the frontier regiment on guard along the Afghan side of the
border had accepted an invitation to the mess at the Russian post.
They arrived in due course—appearing in all the full-dress grandeur
of second-hand railway uniforms! The officer commanding the
detachment exhibited on the collar of his tunic the mystic words
“Ticket Collector”; his subordinate, a subaltern, was content with the
less exalted label of “Guard.” Out of courtesy to their guests the
Russians suppressed their merriment, receiving nevertheless the
impression that a portion of the subsidy, granted by the Government
of India to the Amir of Afghanistan, was taken out in the castoff
uniforms of British public companies. The facts were that the Amir,
through his Agent in India, had acquired a large parcel of discarded
clothing at one of the annual sales of condemned stores in Northern
India.
This exchange of courtesies on the frontier illustrates only the
pleasant side of service in this region. More serious incidents occur.
Occasionally in the heat of the chase, when parties of Russian
officers have crossed the frontier in pursuit of their quarry they have
been fired upon by the Afghan patrols or ridden down by Afghan
sowars. Sporting trips around Kushkinski Post or in the valleys of the
Murghab are infrequent among the Russians, although wild boar
abound in the thick patches of reeds which hem in the banks of the
rivers; the tufts of grass, the hardy scrub and the patches of bush
also afford excellent cover for partridges and pheasants. The
scarcity of good water at any distance from the railway is the great
drawback to such excursions, since the transport of water is both
costly and cumbersome. In cantonments goat-skins of the precious
fluid are brought for sale by water-sellers who come round, earning a
precarious livelihood by their industry.
This custom, which prevails throughout the East, was once turned
to account by an Afghan who was afterwards discovered to be an
Hazara sapper from the Kabul garrison. Disguised as a water-seller
he spent three weeks at Kushkinski Post, conducting an exhaustive
inspection of the works and coming every night and morning to the
fort with his supplies of water. Chance, which in Asia plays no less a
part in the affairs of man than in Europe, threw across his path a
native who had visited Kabul some weeks before with letters from
the Governor-General of Turkestan. The Afghan had been deputed
by the Amir to attend to the Turkestani. He had met and escorted
him to the capital and back again to the western boundary. As the
Russian had entered Afghanistan from the Kushkinski Post, along
the Hari Rud valley, he was conducted from the capital to the frontier
by the route he had first followed. At the frontier he had dismissed
his Afghan attendant, who promptly proceeded to disguise himself as
a water-carrier and to obtain admission to the station. Here he
busied himself daily until, meeting of a sudden his late charge,
recognition upon the part of the Russian subject was immediate and
the spy was arrested in the act of escaping from the precincts of the
fort. Suspicion as to the man’s identity became assured when a
packet of notes was found, wrapped in a rubber sheath, at the
bottom of the goat-skin water-bag.
Until the advent of the railway the colony at Kushkinski Post apart
from the garrison, comprised a few Armenian and Persian traders.
With the prolongation of the line from Merv the civilian population
began to increase rapidly. There is, of course, no hotel in the station;
although the officers of the garrison have established a small military
club wherein they mess together and where, when the bi-weekly
trains bring the supply of ice, there is usually an animated gathering
of desolated humanity. At the present time there are in Kushkinski
Post 123 buildings, of which some thirty odd belong to private
persons. Apart from the garrison the civil population numbers fifty
people.

native water-sellers

Kushkinski Post station consists of a handsome, spacious

structure in the white stone which is brought from quarries in the
basin of the Kushk. The railway buildings include a depôt with
workshops, eight bungalows for the heads of the staff and special
quarters for the employés. There are also large barracks for the 6th
Company of the 1st Trans-Caspian Railway Battalion, who are not
included in the field state of the post. All buildings are lighted by
electricity and the workshops are furnished with electric motors,
while the water is drawn from springs on Gumesli mountain.
Kushk region is malarial in consequence of the marshy nature of
the surrounding country. For some years past measures have been
undertaken with a view to draining the swamps and regulating the
running of the streams. By these means it has been hoped to render
more healthy the general environment of the station, including the
fortress works, Kushkinski village and the district lying between the
Afghan frontier post of Kara Teppe and the Russian Alexeieffski and
Poltavski villages.
The specific disease which makes duty in the Murghab and Kushk
valleys peculiarly obnoxious is a low fever of an endemic nature. Its
pathological history is still undetermined and, although investigations
have been made into its character and numerous experiments
essayed, the malady is usually fatal. In general, the patient is
stricken suddenly when the liver would appear immediately to be
affected, the skin becoming yellow and the sufferer lapsing into
unconsciousness within a few hours of the attack. Systematic study
of the disease has enabled the medical authorities to trace it
indirectly to the soil from which, just as in Africa and any of the
countries lying within the fever belt, germs are released whenever it
is disturbed. In this way the most infectious points in the Kushk and
Murghab valleys are those lying within the cultivated areas, more
especially around those places where digging operations are of
frequent occurrence. As the order of life becomes more settled and
the necessity for any interference with the soil disappears, it is
anticipated that the extreme virulence of the disease may diminish.
At one time the soldiers of the Railway Battalions were so
susceptible to its ravages that its course assumed the appearance of
an epidemic.
No commercial importance belongs to Kushkinski Post and it is
solely the strategic considerations which attach to it that give it so
much value. In the hands of Russia and commanding the trade
routes into Afghanistan, as well as the road to Herat, Kushkinski
Post well might play a leading part in the settlement of questions still
outstanding between Russia and Great Britain in respect of
Afghanistan. Whether the existence of the post will promote the
development of trade relations, which are now restricted by the
Amir’s Government and directed by the Afghan frontier authorities
through Khorassan, remains to be seen. Nothing can underestimate
its significance. The post, together with the whole of this branch line,
is a deliberate military measure against Afghanistan, the boundaries
of which kingdom can almost be seen from the ramparts of the forts
which crown the crest of the hills.

khorassan dervish

Eighteen versts to the south of the fortress, at Chahil Dukteran,

there is the post of the Russian Frontier Guard and the present
terminus of the Murghab Valley railway. Beyond may be noted the
solitary figures of the Russian sentinels keeping their beat along the
extensive line of their position; while southward and serving at the
moment for a caravan route lies the road to Herat. As an interesting
link in the chain of evidence which points to the future use of this
road in another way, there is the existence of a large store of light
railway plant prepared for the purposes of extending it into
Afghanistan itself, whenever the troops of Russia may require to be
carried forward to the walls of Herat through the passes of the
Paropamisus, a little less than 80 miles.
To Englishmen another, perhaps less direct and more ficticious,
interest attaches to this railway. A glance at the map of the Eastern
hemisphere will show that the shortest practicable line of
communication between London and India lies through Russia and
across Central Asia. The direction would be viâ Calais, Berlin,
Warsaw, Rostov-on-Don, Petrovski, Baku, Krasnovodsk, Merv,
Kushkinski, Girishk and Kandahar. The whole of this distance has
now been covered by railway, with the exception of the span of 195
miles across the Caspian Sea, between Baku and Krasnovodsk and
the gap of 500 miles which still separates Kushkinski Post from New
Chaman. If these sections were bridged the journey from London to
India might be very considerably shortened, assuming that the
present rate of speed—32 miles an hour on the European and 25 on
the Asiatic lines—were maintained. The net saving in time, if the
railway were completed, would be seven days; while the horrors of
the Red Sea and the monsoon would be but bad dreams to the
Anglo-Indian traveller. The country between Kushkinski Post and
New Chaman presents no obstacle to the engineer; the
Paropamisus range could be crossed by the Ardewan or the
Chashma Sabz pass, neither of which is more than 3400 feet above
sea-level or 1000 feet higher than that of the tableland on either side.
From this point Herat, the garden city of Afghanistan and the key of
India, is distant only 30 miles; thence the line would be carried by
way of Sabzawar, Farah, Girishk and Kandahar to New Chaman.
However if further railway construction in this region is to take
place, it will be in connection with the development of plans which
concern the requirements of potential strategy rather than the
humours of experimental fantasies. For some time past there have
been abundant signs that Russia is proposing to find compensation
in the Middle East for the downfall of her prestige in Further Asia.
Certainly there is a field for her energies lying fallow in Central Asia.
The precise quarter where the furrows are waiting to be ploughed is
between the Central Asian railway and the frontiers of Northern
Persia and Northern Afghanistan. It is to-day evident that sooner or
later Russia will improve her communications in this direction by
adding to the Orenburg-Tashkent system its natural complement—an
extension to Termes on the Oxus, where there is a Russian
fortress—or by imparting to her position on the Perso-Afghan border
that little requisite attention which it merits—a railway to Meshed in
Khorassan. Long since is it that these schemes entered the domain
of practical politics, the Russian military position on the Middle Oxus
requiring an alternative line of communications to that offered by the
Amu Daria, which, when frozen in winter with the post-roads across
the mountains blocked by snow, wraps in dangerous isolation the
Russian garrisons at Termes, Kelif and elsewhere along this section
of the frontier. Preliminary surveys for a railway were conducted in
1902, when the routes selected followed from Samarkand the Shar-i-
Sabz, Huzar, Shirabad caravan highway to Termes; and, from Farab
to Termes, the trade route along the Oxus through Burdalik and Kelif.
Further extensions in this direction would provide railway
communication between Huzar and Karki by a bridge across the
river, by which Huzar would become as important a railway junction
as it is a caravan and trading centre. Still more in the future is the
strong probability that Karki will be joined with the Afghan frontier at
Imam Nasar, by following the caravan route from the river, or with
Pendjeh across the fringe of the Kara Kum.
 the murghab valley railway

Equally determined has been the intention to open up railway

communication with the north, north-eastern frontier of Persia, the
original surveys taking place simultaneously with the parties working
towards the Oxus. For purposes of the Persian railway two routes
were also inspected in this quarter, the Askhabad-Meshed line
receiving the earliest attention and warmest support. This scheme,
after passing through the defiles between Firuza, the summer resort
of Askhabad society, and Badjira, entered Persian territory at
Kettechinar; running up the Deregez valley and leaving the Atrek
waters near their source at Kuchan, it then broke into the Keshef
Rud valley, striking the caravan road to Meshed between Durbadam
and Imam Kuli. Great initial outlay was made in connection with this
railway. Its course had been pegged out under the supervision of M.
Stroieff, dragoman of the Russian Consulate at Meshed, with the
help of the Ikram-ul-Mul late Karguzar of Kuchan, to whom 12,000
roubles were presented. Further, it was arranged to open a branch of
the Imperial Russian Bank at Meshed to assist the financing of the
work, the staff comprising an official from St. Petersburg as
manager-in-chief, an assistant manager from Teheran, with Ali Askar
Khan, the interpreter of the State Bank at Askhabad. The outbreak of
hostilities in Manchuria imposed a temporary check upon the labours
of the construction parties, the reflection thus obtained giving rise to
the advantage of dropping a branch line from Tejend station on the
Central Asian railway viâ Sarakhs, Daulatabad, Pul-i-Khatun to
between Zulfikar and Kala Kafir, wherever some future extension of
the Askhabad-Meshed line, following the Keshef Rud to its meeting
with the Hari Rud on the actual Perso-Afghan frontier, may
terminate. The Tejend Rud is the name given to the lower waters of
the Hari Rud which, flowing by Herat, receives midway in its course
the Keshef Rud and thence runs close to Sarakhs, presenting to any
line running along the Had Rud valley an alternative approach to the
Afghan city.
That Herat and Meshed are the objectives of Russian railway
policy is obvious from a pamphlet issued in 1902 by the
Topographical Bureau in St. Petersburg and entitled Railways Across
Persia. In its pages a railway was projected from Kara Kliss, a
station midway between Tiflis and Erivan, viâ Tabriz, Teheran,
Shahrud to Meshed. The mileage, cost, the number of sidings and
names of stations were all laid down. The principal stations in the
first section—Kara Kliss to Tabriz—were Erivan and Julfa. At this
moment the span from Kara Kliss to Julfa a distance of 135 miles is
completed, the first hundred miles—Kara Kliss to Erivan—being
open to traffic and the remaining thirty-five miles—Erivan to Julfa—in
working order. From Julfa a carriageway, constructed under Russian
auspices and in all essentials a Russian military road, runs to Tabriz,
so that Russian schemes for broad gauge railways to Herat and
Meshed are at least removed from their incipient obscurity.
 native school

[10] “All the Russias.” H. V. Norman.

 CHAPTER VI

THE MURGHAB VALLEY

The river Murghab, which, with the Kashan
and the Kushk streams, waters the Merv
oasis and then disappears in the sands of
the Kara Kum desert, rises in the mass of
mountains connecting the eastern
extremities of the Safed Koh and Tir Band-i
Turkestan ranges. It flows in a westerly
direction through the great valley separating
these mountain chains and, after receiving
the waters of numerous tributaries, turns
towards the north-west to pass the Afghan
fortress of Bala Murghab and the post of
Karawal Khana. At this latter point it receives
the waters of the Kaisar affluent. Continuing
the russian cossack on
the afghan border in a north-westerly direction it flows past
Maruchak, lying on the right bank, where a
short distance below it is joined by the Kashan stream. Pendjeh and
Ak Tepe are both situated upon the left bank. At Ak Tepe the Kushk
river, which rises in the Paropamisus range, unites with it and from
this point the Murghab runs in a due northerly direction past Yulatan
to Merv, thence running dry in the desert.
Within Russian territory the Murghab river irrigates exclusively the
Merv district, and its length within the Trans-Caspian province is
about 400 versts. If its numerous bends were taken into account the
length of the stream would be 850 versts. The Kushk river waters
Russian territory for a distance of 100 versts, from the Russo-Afghan
frontier to its confluence with the Murghab; the Kashan for 60 versts.
The width of the Murghab at the Kaushut-Khan-Band, 28 versts
above the town of Merv, is about 23 sagenes; but at Merv itself it
narrows to 12 sagenes. Its mean depth is 7 feet. The rise of the
water begins in the middle of March and the fall finishes three
months later. Between June and the middle of October the level of
the river is determined by the rain-fall and snow in the neighbouring
mountains. About June, when the river has fallen, the population
experiences the want of the water which is necessary for the autumn
crop of cotton. In years of drought, when the dearth of water is felt
much earlier—during the period of the ripening of the crops, in fact—
the population are obliged to abandon the greater portion of their
harvest.
The country through which these rivers flow is, in the main, a
mixture of desert waste and cultivated strip, with rising uplands
carpeted in spring by bright flowers and hidden in winter by heavy
snows. Roads meander along the valleys, sometimes by means of
rocks and boulders crossing and recrossing the stream many times
in short stretches or, at others, wandering far away from the
waterside to traverse the broken spurs of hills. Where signs of
cultivation exist, there are indications that the population has
regained confidence in the Russian domination of the district. Fields
and irrigation canals have been cleaned and restored; the sparkle of
the little rills is reflected in the brilliant sunshine.
 tamarisk scrub in the river valley

From the broad uplands of the watershed, from where to the river
bed below there is in general a tedious scramble across a confusion
of stones and brushwood, the tumbled masses of the rounded
slopes are seen to sink into long undulating sweeps. Where the
Kushk and Murghab valleys become entangled, a line of sand cliffs
disappears in one direction into the haze of the Kara Kum and
merges in another with the Karabyl plateau. In the distance the river,
spreading itself over a labyrinth of canals, passes through a rapid
succession of changing scenes, until, in the broad arid wastes of the
Kara Kum, its waters are finally and completely lost. South-west of
Bala Murghab the valley narrows to the dimensions and rugged
outlines of a defile. Through this the river rolls, tumbling with
thunderous clamour, towards Pendjeh oasis, where it acquires a
breadth of 1 to 3 miles. At Pul-i-Khisti, identical with Tash Kepri and a
little above the Russian settlement of Takhta Bazaar, the stream is
joined by the waters of the Kushk rivulet, when it is not consumed in
irrigation. From this point the united rivers flow onward to the oases
of Yulatan and Merv, passing through a broad flat valley, 2 miles in
width, bounded on either side by sandstone heights. In this stage the
river is slow running, deep and difficult to cross, and possessing but
few fords. Its average breadth varies between 40 and 70 yards and
the most prominent feature is its extreme sinuosity. Beyond Bala
Murghab the river valley is contained on the left bank by an
undulating chain of low hills, high rocky gorges enclosing the right. At
this point the sides are steep, with a possible height of 24 feet and a
surface growth of shrubs and willows. A narrow, level strip, tufted
with scattered grasses, lies between the water’s edge and the hills
on the left bank. The river itself flows in a single channel, clinging
rather closely to the left of the valley. It possesses a mean breadth of
70 yards and a maximum current of 5 miles. The depth of the ford is
between 3 to 4 feet.
The valleys which debouch upon the river are quite spacious and
contain small plots of cultivated ground, with here and there a
village. Unfortunately, while the banks of the river are fertile the
valleys themselves are exceedingly unhealthy—a low fever,
pathologically identical in the two districts of Murghab and Kushk,
permeating them. Although the great majority of the inhabitants avow
themselves immune from the disease, they are averse to settling in
the valleys. A feature of the river is the abruptness with which the
broad open spaces are changed to narrow gorges of no remarkable
height. This trait in the character of an otherwise respectable inland
river compresses so great a volume of water into so small a channel
that its passage is attended with risk. It is not until the spreading
expanses of the Pendjeh, Yulatan and Merv oases have exhausted it
that the stream is crossed with convenience. At Bala Murghab,
where the current is very strong and the depth uncertain, deep holes
in the bottom and masses of protruding rocks, added to the hidden
dangers from quicksands, make the task of fording an intricate
proceeding. There are two fords at this point, and a similar number
are in use at Maruchak, Karawal Khana and Pendjeh, while the
Russians have restored many stone bridges which formerly existed
in the Kushk valley near the junction of the Murghab and Kushk
rivers, at Maruchak and Bala Murghab. The liability of the two rivers
to sudden floods renders all fords uncertain and insecure,
particularly in the lower stretches between Pendjeh and Merv. More
often than not necessity dictates the prudence of stripping to the
skin, when the native, a prayer to Allah on his lips and his
possessions strapped in a bundle on his head, flounders through the
water to arrive damp, disconsolate and very scared on the opposite
side. Nevertheless, the best fords are found usually where the
stream flows swiftly through a narrow bed. At such a crossing there
is a firm bottom and foothold is readily secured.
Many contrivances are used to cross the rivers of High Asia.
Where the current is sluggish an inflated goatskin is employed. This
system is in vogue on the Oxus and, in lesser degree, on the
Helmund, where rafts of bushes are preferred. Along the Murghab
the indifferent nature of the fords and the swiftness of the current in
the narrow channels of the river make the use of a boat, drawn along
a hawser, more suited to the needs of the occasion. Fords on the
Murghab are not so frequent as on the Oxus.
The Kushk Valley extends in Russian and Afghan territory some
14 miles. It possesses an average breadth of three-quarters of a
mile. Its hills, low and rounded, are a conglomerate of clay and red
sand, but bare of trees and with their faces dotted with mud-cabins.
An extensive system of irrigation is fed by the river and there is much
cultivation on the tops and sides of the hills. The produce of the
fields is only sufficient for the immediate wants of the native settlers,
although the Russians hope, now a garrison has been established at
Kushkinski Post, that the demands of the troops will spur the
villagers to greater agricultural activity. In Afghan territory the valley
is the habitat of the Jamshidis, who, quiet and tractable, reveal few
wants and even fewer interests. Excessive irrigation has done so
much to spread the fever that the population throughout the valley
has been dwindling gradually. There are now less than 4000 families
in the entire valley, years of peace and prosperity seeming to
accentuate the restlessness which underlies the nature of all
nomadic people. A weekly bazaar is held at Kushkinski Post; similar
gatherings taking place at Afghan Kushk, Bala Murghab, Maruchak
and in the Pendjeh oasis at Takhta Bazaar. Salt, rice, soap, carpets
and horses are all brought to the markets, while the Russians
encourage the native merchants under their protection to display
stocks of Russian sugar, matches and cotton prints. Silks from
Meshed and Bokhara are also in evidence, but nothing of any
English or Indian origin. French, American and German products are
barred no less rigorously, although German matches and French
sugar occasionally escape the specific ostracism which applies to
British manufactures.
In the Kushk valley the fertility of the land is dependent upon the
flooding of the river by the spring rains. As a consequence an ever-
present feeling of irritation exists in the lower parts of the Kushk
valley against the Afghan villagers, who control the head waters of
the river and divert it to their own fields. This difficulty prevails along
the entire line of the frontier in this region, the demarcation of the
boundary between the two races leaving the heads of the canals in
Afghan territory. There are many exceptions to the misfortune, and,
so far as possible, the division is arranged in a spirit of mutual
ownership, although the natives, on the Russian side of the frontier,
have no claim to compensation if there should be an insufficient
quantity. With a river like the Kushk, which possesses an irregular
volume, the difficulty is much greater than in the case of the
Murghab or even the Hari Rud. Water means to these primitive
peoples life and existence; and, as cultivation is only rendered
possible by most assiduous irrigation, the task of conserving the
supply involves incessant labour. Although agricultural activity
prevails principally in the Murghab and Kushk district there are a few
cultivated places in the Kashan valley. It would be useless to make
any comparison between the former valleys and the Kashan. The
Kashan valley contains a small strip, level, well watered and about
half a mile in width, through which percolates a narrow stream. In
spite of its culturable soil the Kashan district is not frequently
inhabited, as in the extreme hot weather the Kashan river is
exhausted by the claims made upon it for purposes of irrigation;
below Robat-i-Kashan, except during the spring floods, there is no
trace of water. A similar condition of affairs characterises its
companion stream the Kushk; at the point of union with the Murghab
it is frequently reduced to a mere trickle. None the less during the
spring rains each of these rivers is liable to sudden floods. Prior to
the advent of the railway at Tanur Sangi there were but few
settlements in the valley. There was one at Karawal Khana on the
right bank of the Murghab and 12 miles south of Maruchak, while the
next of any consequence was at Bala Murghab, upon the same bank
and more than 20 miles away from Maruchak. At the time when the
Anglo-Russian Commission was adjusting the line of the Russo-
Afghan frontier in this region, the absence of habitation and human
settlement of any kind was most marked. Time has brought a
change.
Tanur Sangi is now one of the termini of the Murghab valley
railway. Barracks for the troops who are occupying the post have
been built on the heights of the valley, the dense vegetation has
been burnt off and a system of drainage applied to the neighbouring
swamps. For the moment the Maruchak district, extending equally
within Russian and Afghan territory, is pregnant with prospects and
the advent of the Russians there has been followed by an influx of
native settlers. Upon the Afghan side of the river there are similar
indications, by reason of the arrival of the levies who garrison the
Afghan forts at Bala Murghab, Maruchak, Kala Nao and elsewhere.
 bokharan traders at pendjeh

The river is the dividing-point between Russian and Afghan

possessions at Maruchak for 15 miles. Still it is interesting to note
that the natural frontier between Maruchak and Pendjeh is at the
northern end of the Maruchak valley, where the hills, closing in upon
the river on both sides, separate the Maruchak acres from those of
the Pendjeh oasis. Formerly, too, the Murghab flowed down the
northern end of the Maruchak valley, washing the western face. It
has now changed its course and, sweeping from west to east,
washes the eastern aspect. This deviation had an important bearing
upon the findings of the Anglo-Russian Commission. Under their
correct and literal interpretation of the protocol the Russians were
debarred from exercising any claim over the waters of canals
employed for irrigation, provided their heads were in Afghan territory.
By the change in the direction of the Murghab the head of the waters
supplying the Pendjeh oasis, which proceed from the Band-i-Nadir
canal on the left bank of the Murghab, was placed within Afghan
territory. A modification of the situation was urged; finally the
boundary was made to pass from Zulfikar on the Hari Rud to the
Kushk and from the head of the canal in the Kashan valley to the
head of the Band-i-Nadir on the Murghab, due west of Maruchak
instead of to a point north of it. This re-adjustment permitted control
of the head waters of the Band-i-Nadir to revert to Russia.

the russo-afghan boundary

The Afghan fortress of Maruchak has experienced a varying

fortune, the vicissitudes of which once brought it to ruin and caused
its defences to be abandoned. Since then the advance of the
Russians has thrown it into prominence again. Its walls have been
restored, although it can never serve any other purpose than that of
a frontier post of observation. The fortress is in the shape of a
square of which the outer walls, measuring some 600 yards, rise 20
feet from the side of a moat. The main entrance faces the river on
the west. Other entrances of less importance are placed at the north-
east and south-east angles. In the centre, rising from a circular