Contribution of gut content to the nutritional value of Brachionus plicatilis used as

prey in larviculture
S. Romero-Romero, M. Yfera
Instituto de Ciencias Marinas de Andaluca (ICMAN-CSIC), Apartado Ocial, 11510 Puerto Real, Cdiz, Spain
a b s t r a c t a r t i c l e i n f o
Article history:
Received 21 May 2012
Received in revised form 31 July 2012
Accepted 3 August 2012
Available online 15 August 2012
Keywords:
Brachionus plicatilis
Enrichment
Microalgae
Gut lling pattern
Gut evacuation pattern
Energy content
With the aimof assessing the potential value of gut content in the rotifer Brachionus plicatilis during the standard
procedures of enrichment and posterior residence in the larval tanks, we have examined by image analysis the
changes in gut volume during the lling and subsequent evacuation process when feed is no more available. The
gut lling pattern has been examined at different microalgal concentrations of Nannochloropsis gaditana ranging
between 0.4 and 20.810
6
cell ml
1
. The rotifer gut was completely lled in 120 min and the gut volume
became signicantly higher in rotifers fed at the highest microalgae concentrations tested. The gut volume
accounted for up to 15% of the body volume. When harvested with a non-submerged lter and resuspended in
cleanseawater, the rotifers evacuatedthe gut quickly, losing 60% of its content inthe rst 5 min. Contrarily, when
the rotifers were rinsed carefully using a submerged lter while maintaining the water volume, the gut was
evacuated progressively and needed 1 h to lose 60% of the content. Moreover, we have examined the changes in
dry mass and energy. After 2 h of gut evacuation the rotifers lost 20% of the initial dry weight, and 38% after 24 h
of starvation. In terms of energy the rotifers lost 43% of the caloric content after 24 h of starvation. The ndings of
this study conrm the importance of performing appropriate feeding protocols to supply living prey of high
nutritional quality in larviculture.
2012 Elsevier B.V. All rights reserved.
1. Introduction
Different species and lineages of the Brachionus plicatilis cryptic
species complex are widely used as live prey to feed marine sh larvae
during the rst weeks. The advantages and constraints of their use as
food in aquaculture are well known (Conceio et al., 2010; Lubzens and
Zmora, 2003; Lubzens et al., 1989). One of the main concerns is the
nutritional quality when the rotifers are produced in mass culture.
Rotifers are produced under different methods such us batch, semi-
continuous and continuous cultures, as well as under high-density
superintensive, water recirculation and automatic-controlled systems
(Alver et al., 2010; Bentley et al., 2008; Dhert et al., 2001; Kostopoulou et
al., 2012; Lubzens et al., 1989; Yoshimura et al., 1997; Yfera, 2001). The
culture system may affect the body biochemical composition. In some
cases, the food is almost exhausted at harvesting and the animals may
present pre-starvation symptoms affecting the body dry mass and
biochemical composition with the consequent poor nutritional quality
(Kotani et al., 2009; Makridis and Olsen, 1999; Szyper, 1989). Likewise,
the use of non-expensive food sources such as baker yeast, some
commercial products, and some microalgae species usually yield rotifers
with nutritional deciencies of the essential fatty acids (Ben-Amotz et
al., 1987; Rainuzzo et al., 1989, 1994; Watanabe et al., 1983), vitamins
and minerals (Gimnez et al., 2007; Hamre et al., 2008). The potentially
inappropriate nutritional quality of rotifers as food for sh larvae has
been solved by re-feeding them with some microalgae species, oil
emulsions, commercial microparticulated products and tailored boost-
ers (Demir and Diken, 2011; Fernndez-Reiriz et al., 1993; Olsen et al,
1989; Palmtag et al., 2006; Rodrguez et al., 1996) before being supplied
to the rearing tanks.
This post-harvesting enrichment process has a double purpose.
One is the incorporation of desired specic nutrients to the rotifer
tissues, process that requires between 12 and 24 h of enrichment
(Kotani et al., 2010; Rainuzzo et al., 1994). The other is to use the
rotifer body, and most specically the gut, as a living capsule for
transferring a given feed containing a specic compound to the sh
larval gut (bioencapsulation). In this second aspect, the nal quality
of the ingested food depends widely on the larval feeding protocol.
Gut transit time in rotifers is relatively fast and consequently the time
the rotifers spend in the rearing tank before being eaten will affect the
nal quality as a prey (Olsen et al., 1989; Yamamoto et al., 2009).
Furthermore, the survival and normal development of larvae depends
not only on the adequate nutrients but also on the ingestion of the
sufcient calories. Therefore, the nutritional value of rotifers depends
on their biochemical composition as well as on their dry mass and
caloric content (Lubzens et al., 1989).
The objective of this study is to assess the nutritional quality in
terms of mass and caloric content of rotifers during the common
practices of feeding larvae in the hatcheries. With this aim, we have
Aquaculture 364365 (2012) 124129
Corresponding author: Tel.: +34 956 632612; fax: +34 956 934701.
E-mail address: manuel.yufera@icman.csic.es (M. Yfera).
0044-8486/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aquaculture.2012.08.011
Contents lists available at SciVerse ScienceDirect
Aquaculture
j our nal homepage: www. el sevi er . com/ l ocat e/ aqua- onl i ne
rstly determined the gut lling pattern of rotifers fed with different
microalgal cell concentrations as well as the evacuation pattern in
clean and green seawater, and secondly we have compared the dry
weight and the carbon, nitrogen and energy content of overfed and
starved rotifers.
2. Material and methods
B. plicatilis sensu stricto, strain S-1 (Dooms et al., 2007; Yfera,
1982) was cultured in 1 L asks at a temperature of 20 C and a salinity
of 33 ppt with gentle aeration. The microalgae Nannochloropsis gaditana
was provided as food in all experiments.
2.1. Gut lling pattern
To determine the gut lling pattern the rotifers were rst maintained
under starvation conditions during 24 h to empty the guts. The rotifers
were fed with algae at six different concentrations, 0.410
6
, 1.210
6
,
2.610
6
, 5.210
6
, 10.410
6
and 20.810
6
cell ml
1
. The density of
the rotifers (3040 individual ml
1
) was low enough to avoid that the
ingestion affected the microalgal cell concentration during the experi-
mental time. Rotifers were sampled in the six treatments at different
times for a period of 240 min and xed in formalin (4%v/v). Then, the
body and gut volume were measured as described in Morphometric
analyses section, and to avoid the variability associated with body size
the gut volume was presented as percentage of the total female body
volume. The following asymptotic function was tted to the set of data
points showing the increase of gut volume:
V a 1e
bT

:
Excepting for the treatment with 0.410
6
cell ml
1
, in which the
best t was obtained with a sigmoid equation:
V a= 1 e
cT =b

where V is the gut volume (% body volume), T is time (min), a is the
asymptote, and b and c are regression parameters.
2.2. Gut evacuation pattern
To determine the evacuation pattern, rotifers were rst overfed
with microalgae at high concentration (20.810
6
cell ml
1
) for
180 min. Then, microalgal cells were removed by ltration through a
71 m mesh sieve using three different procedures. In the rst
procedure (non-submerged ltration), the rotifers (rotifers+culture
medium) were poured over the lter and the collected rotifers were
rinsed and transferred to a newask with clean seawater. The second
procedure (non-submerged ltration+microalgae) was the same as
the rst one but after rinsing, the rotifers were transferred to a ask
containing seawater with 0.410
6
cell ml
1
of N. gaditana in order to
simulate the conditions of green water rearing techniques. In the third
procedure (submerged ltration), the rotifers were poured over a
sieve submerged in a recipient with water to maintain the water
volume during the washing process. Then, clean seawater was added
to recipient to remove the microalgal cells.
Rotifers were sampled at different times for a period of 180 min and
xed in formalin. The rst sample was taken just before the ltration
(considered as time 0).The declining of gut volume was presented as
percentage of the total body volume. The following exponential equation
was tted to the experimental points:
V ae
bT
ce
dT
where V is the gut volume (% body volume), T is time (min) and a, b, c
and d are the regression parameters.
2.3. Morphometric analyses
Determination of body and gut volume was carried out by image
analysis. Between 6 and 10 rotifers of each sample were photographed
under a microscope at 250 magnication. The pictures were analysed
using the free software UTHSCSA ImageTool (University of Texas Health
Science Center, San Antonio, TX, http://ddsdx.uthscsa.edu/). Body width
and length and the gut area were measured in each individual (Fig. 1).
For the gut measurement, the criterion was to consider just the green-
coloured area of the image. This area could be composed of either 1 or 2
spots, corresponding to the so-called stomach and intestine cavities
(Kleinow et al., 1991). Rotifers after starvation may exhibit a nominal
gut lumen volume of 0 m
3
, this is due to the absence of microalgae, i.e.
there is no coloured part in the image, as shown in Fig. 1A.
Rotifers body volume was calculated according to an ellipsoid of
revolution. For eachindividual, the widthandlength(m) were obtained
measuring the lorica without spines (Yfera, 1982). Assuming the cross
section in this Brachionus strain is circular; depth and width are equal,
and the volume was calculated as follows:
Rotifer volume m
3

4=3ab
2
where a and b are one-half of the major and minor axes (length and
width), respectively.
The gut cavities were considered to have a spherical shape and the
volume was calculated fromthe area. For the calculation of the volume,
the following formula was used:
Gut volume m
3

4=3 A
1
A
1
=

4=3 A
2
A
2
=

where A
1
and A
2
correspond to the area (m
2
) of the two gut cavities
measured in the pictures.
2.4. Dry mass and energy content
Dry weight was determined in rotifers with guts full of microalgae
(fed 120 min with 20.810
6
cell ml
1
), after 120 min of evacuation in
cleanwater without microalgae, and after 24 hof starvation. Dry weight
was determined by drying triplicate samples of 9001000 individuals at
60 C. The samples were obtained by concentrating the rotifers with a
submerged lter. After rinsing with distilled water to remove saltwater
and microalgae the lter was takenout of the recipient withwater anda
sample was placed in pre-weighed glass covers. The egg/female ratio
was quantied due to its positive correlation to dry weight (Yfera and
Pascual, 1989). Carbon and nitrogen content were analysed in rotifers
with their guts completely full and after 24 h of starvation. Three 1 mg
subsamples per treatment were analysed using an elemental CNHS
analyser (Thermoquest, mod. Flash 1112), using sulphanilamide as
standard. Energy content was estimated using the factor of 43 J per mg
of carbon (Yfera et al., 1997).
2.5. Statistical analysis
In order to identify signicant differences of rotifer gut fullness
among the six treatments, the maximumexperimental meanreachedin
a specic time of each treatment were compared. The maximum per-
centages of gut volume were contrasted using one-way ANOVA fol-
lowed by a post-hoc Tukey's test to identify which groups were
signicantly different.
3. Results
The body volume of the rotifers ranged between 0.73 and
4.2110
6
m
3
(average: 2.050.6810
6
m
3
) (Fig. 2). The maxi-
mum gut volume average obtained from rotifers once the asymptote
125 S. Romero-Romero, M. Yfera / Aquaculture 364365 (2012) 124129
was attained was 0.230.0610
6
m
3
, although gut volumes up to
0.3510
6
m
3
were observed.
The fullness of the gut during the enrichment experiment followed a
saturation curve (r>0.963; Pb0.006 in all cases), achieving its
asymptote after approximately 2 h (Fig. 3; Table 1). However, for the
lowest cell concentration (0.410
6
cell ml
1
) the best t was obtained
with a sigmoid curve (r=0.998; Pb0.0001) (Fig. 3; Table 1).
The gut completely full of microalgae occupied between 13.5 and
15.0% of the body volume (Table 2). In all cases the maximum gut
volume occurred between 120 and 180 min after the microalgae was
supplied, and then declined slightly at 240 min. A signicant dif-
ference in the maximum gut volume was observed between the
three highest microalgae concentrations tested (5.210
6
, 10.410
6
and 20.810
6
cell ml
1
) and concentrations of 0.410
6
and
1.210
6
cell ml
1
(Table 2).
Once the microalgae had been removed from the culture me-
dium, the gut volume decreased quickly. In the case of the rst
ltering procedure (rinsing directly over a non-submerged lter), a
sharp drop was noticed during the rst few minutes from 13.515.0
to 6.07.0% of the body volume. Afterwards, the gut maintained this
volume during the following 2 h after ltration, but it became
completely empty after 24 h. In the case of adding a microalgae
concentration of 0.410
6
cell ml
1
to the rotifers once they have
been ltrated and resuspended in water, no signicant differences in
the evacuation pattern were found. Contrarily, when the ltration
was carried out using the third procedure (using a submerged lter),
the gut volume decreased at a lower rate (Fig. 4).
Dry weight, carbon and nitrogen content, and energy content are
presented in Table 3. Dry weight was obtained in a population with
egg/female ratio of 0.39 that declined to 0.17 egg/female after 24 h of
starvation. The rotifers experienced a dry weight decreased of 128 ng
(19.71% of body weight) after 2 h of starvation, and of 254 ng (38.22%
of body weight) after 24 h of starvation. Both carbon and nitrogen
content decreased during 24 h of starvation but this drop was more
moderate for nitrogen and consequently C/N ratio decreased from
4.96 to 4.72. The energy content of the rotifer biomass declined from
19.78 to 18.39 J mg
1
DW representing a loss of 42.65% in each in-
dividual (Table 3).
4. Discussion
Body size distributionof a populationof Brachionus spp is affectedby
different environmental factors such as feeding conditions and temper-
ature, but it is mainly dened by the lineage and strain (Kostopoulou
et al., 2009; Snell and Carrillo 1984; Yfera 1982; Yfera, 2001). The
morphometric measurements and the extrapolated body volume of the
B. plicatilis strain S-1 found in the present study were quite similar to
previous measurements carried out in this strain (Yfera 1982; Yfera et
al., 1993).
Inaddition to the effect of the body size whichis related to the age of
individuals (Kostopoulou et al., 2009), the gut volume varied widely
depending on its microalgae content, that is, on the time passed since
the start of the enrichment and on the time spent without microalgae in
the water after the enrichment process.
The lling process occurred relatively fast. In our B. plicatilis strain
and at a temperature of 20 C, the gut was almost full in nearly an hour
Fig. 1. Examples of Brachionus plicatilis used for the experiments. The gut content is outlined in yellow. A) After 24 h of starvation. B) After 15 min feeding on 20.810
6
cell ml
1
of
microalgae. C) After 30 min feeding on 20.810
6
cell ml
1
of microalgae.
Fig. 2. Frequency histogram of the body volume (m
3
) of all the rotifers used in this
study.
Fig. 3. Gut lling pattern in Brachionus plicatilis fed on six different microalgae
concentrations. All values are means, n=610.
126 S. Romero-Romero, M. Yfera / Aquaculture 364365 (2012) 124129
from the start of the enrichment, reaching the 80% of the total gut
volume, and in 2 h the rotifers exhibited a full gut (Fig. 3). Koiso and
Hino (1999) also found an important size increase in the digestive
organs of B. plicatilis when fed on microalgae during 15 min at a
temperature of 26 C. In our study when the gut volume was full it
reaches values up to 230 pL (15% of the rotifer body volume), which is a
notable part of the prey unit ingested by the sh larvae. Kleinow et al.
(1991) estimated the gut volume of other B. plicatilis strain (200 m
lorica length) in 60120 pL. Baer et al. (2008) working with the lineage
Brachionus Cayman (168 m lorica length) fed on latex beads found
much lower values (18 pL; 1.18% of body volume). Besides the different
methodology and environmental conditions, this difference can be due
to a lower ingestion of the inert beads compared to microalgae as well
as to an underestimation of the ingested volume because the space
between the rigid beads was not taken into account as stated by the
authors.
Interestingly, the gut volume after the enrichment depended on the
microalgal concentration and follows a saturation pattern (Table 2). The
maximum gut fullness was achieved above a concentration threshold
between 2.610
6
and 5.210
6
cell ml
1
. There was no signicant
variation in the gut fullness for the three highest microalgae concentra-
tions tested. Alternatively, concentrations lower than 5.210
6
cell ml
1
resulted in lower gut fullness, although at 2.610
6
cell ml
1
the dif-
ference was not signicant, and therefore the rotifers would not con-
tribute to the larval diet with all their potential caloric content. In fact,
these results reect to some extent the ingestion behaviour observed for
this species. The ingestion rate of B. plicatilis like other zooplankters
depends on the algal concentration and also follows a saturation pattern
(Hotos, 2003; Montagnes et al., 2001; Yfera, 2007; Yfera and Pascual,
1985).
The protocol followed before rotifers are provided to the larvae
deeply inuenced their quality as live feed. It has been reported that the
concentration and harvest of rotifers should be performed under water;
otherwise the animals could be damaged (Dhert, 1996). We found as
well that the careless ltration of rotifers using non-submerged lters
generates a sudden decrease of 60% of the gut volume in the rst 5 min.
However when the microalgae removal was carried out in a gently way,
with submerged lters, the gut volume decrease occurred progressively
and needed 1 h to lose 60% of the content (Fig. 4).
Quite similar gut evacuation curves were obtained when the rotifers
were resuspended in clean water and in water with 0.410
6
cell ml
1
of Nannochloropsis. The use of the green water has been proven to have
a positive correlation with growth and survival of the sh larvae when
addedalong with the rotifers to the tanks (Reitanet al., 1993). However,
the low concentration used in this study did not contribute in a
signicant way to gut volume at least during the rst 2 h. In fact, the gut
volume after 15 min of evacuation is the same as the maximum
obtained when we fed the rotifers with 0.410
6
cell ml
1
.
We found that the time necessary for an optimal short-term en-
richment ranged between 120 and 180 min. A longer enrichment pe-
riod, as shown in Fig. 3, led to a decrease in gut volume probably due to
a rebound effect. Baer et al. (2008) also found the same phenomenon,
that may be the consequence of the start of evacuation after a given gut
residence time.
The dry weighof a single individual inthis strainof B. plicatilis ranged
between 200 and 900 ng depending on temperature and fecundity
status (Yfera et al., 1997). Average values in mass culture and with
0.150.60 egg/female ranged between 400 and 700 ng. We found that
the gut content after the post-harvesting enrichment may account for
the 38% of the mass of a single female. Besides, we found that 20% of the
rotifer weight is lost in 2 h in the rearing tanks or even in only few
minutes when the rotifers are harvested and transferred to the tanks
without enough care. Furthermore, a prolonged starvation, as canbe the
case of uneaten rotifers spending the night in the rearing tanks, may
lead to a loss of 43% of the total calories ingested by sh larvae when
they eat one rotifer (Table 3).
A single individual of this strain ingests around 350 cell min
1
of
N. gaditana when fed at 2010
6
cell min
1
of N. gaditana (Yfera,
2007). Considering 627 ng as the dry weight of an individual at 20 C
and 0.40 egg/female (Yfera et al., 1997) and 3.3 pg the dry weight of
a single cell of N. gaditana (Yfera and Pascual, 1985), each rotifer
would ingest the 22.11% its own body weight (137 ng) in the 2 h
required to ll completely the gut. These calculations are close to the
results found in this study (Table 3).
Table 1
Regression parameters of the curves describing the lling and evacuation patterns.
Filling
Algae concentration (cell ml
1
) a b c R
0.410
6
6.1057 13.0356 64.3676 0.9976
1.210
6
6.9127 0.0467 0.9634
2.610
6
9.2225 0.0478 0.9710
5.210
6
13.7317 0.0358 0.9756
10.410
6
12.8481 0.0472 0.9733
20.810
6
12.8627 0.0523 0.9722
Evacuation
Filtering procedure a b c d R
Non-submerged 6.9995 0.2220 5.4759 1.444710
11
0.9356
Non-submerged+microalgae 7.5058 3.1430 6.1711 0.0017 0.9083
Submerged in clean water 3.6301 0.6073 11.0027 0.0098 0.9864
Table 2
Maximum volume (percentage of body volume) occupied by the gut of B. plicatilis at different cell concentrations. Letters indicate signicant differences within treatments. Values
expressed as meanS.D. Tukey's test (F
(5,35)
, Pb0,0001).
Cell conc. (cell.ml
1
) 0.410
6
1.210
6
2.610
6
5.210
6
10.410
6
20.810
6
Maximum volume (%) 6.441.59
a
7.254.42
a
11.553.70
ab
14.973.48
c
13.581.67
bc
14.282.87
bc
127 S. Romero-Romero, M. Yfera / Aquaculture 364365 (2012) 124129
This study conrms the signicant contribution of the gut content to
the nutritional quality of the rotifers offered as food for sh and
crustacean larvae. Results also conrm that harvesting and enriching
procedures have a notable impact on gut content. This experiment has
been done with the microalgae N. gaditana which is commonly used in
larviculture. Some changes in the demographic parameters and in the
ingestion and evacuation pattern may occur when fed on other fresh
microalgae species/strains (Sayegh et al., 2007; Yfera et al., 1983) or
commercial diets andenriching formula (Dhert et al., 2001; Kotani et al.,
2009; Naz, 2008) also usual in the rotifers culture and enrichment.
Likewise, some variations may occur in other Brachionus strains with
different body size (Hotos, 2003; Yfera and Pascual, 1985) or with
different environmental conditions (Ferreira et al., 2009; Lebedeva and
Orlenko, 1995; Montagnes et al., 2001). However these variations would
probably not change substantially the results obtained in this study.
Acknowledgements
This research was supported by the National Research Programof
Spain (project RIDIGEST, AGL2011-23722). We thank Dr. Tim Attack
(Viking Fish Farms Ltd, Ardtoe Marine Laboratory, Scotland) who
asked us how long the rotifers maintain the gut full of algae in the
larval rearing tanks? origin of the present study. We also thank two
anonymous reviewers whose suggestions greatly improved the man-
uscript. Sonia Romero-Romero was supported with an undergraduate
scholarship from CSIC (JAE Intro-2009-00763).
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Table 3
Dry weight (DW), carbon content (%C), nitrogen content (%N), C:N ratio per female
and energy content (J.mg
1
DW and mJ ind
1
) as well as percentage of dry mass and
energy lost under starvation in rotifers with the gut full of microalgae, after 2 h of
starvation and after 24 h of starvation. Egg:female ratio is also included for comparison.
Values are expressed as meanS.D.
Gut full Starved for 2 h Starved for 24 h
DW (ng) 663.3109.5 532.515.2 409.782.5
Egg/female 0.39 0.43 0.17
%C 46.010.13 42.780.96
%N 9.270.04 9.070.17
C/N 4.960.02 4.720.08
J mg
1
DW 19.780.06 18.390.42
mJ ind
1
13.121.18 7.520.48
Lost DW (%) 19.71 38.22
Lost energy (%) 42.65
Fig. 4. Gut evacuation pattern in Brachionus plicatilis in the three methods tested (see
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seawater. Green line: rotifers harvested in non-submerged lter and resuspended in
sea water with 0.410
6
cell ml
1
of microalgae. Red line: rotifers harvested in
submerged lter. MeanS.D.
128 S. Romero-Romero, M. Yfera / Aquaculture 364365 (2012) 124129
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