Aquaculture 420421 (2014) 2431

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Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Dietary medium chain fatty acids from coconut oil have little effects
on postprandial plasma metabolite proles in rainbow
trout (Oncorhynchus mykiss)
L. Luo a, M. Xue b,, C. Vachot c, I. Geurden c, S. Kaushik c
a
Beijing Fisheries Research Institute, Beijing 100068, China
b
Feed Research Institute, The Chinese Academy of Agricultural Sciences, China
c
UR 1067, Nutrition, Metabolism and Aquaculture Unit, INRA, Ple d'Hydrobiologie, 64310 St-Pe-sur-Nivelle, France

a r t i c l e i n f o a b s t r a c t

Article history: This study examined the effect of dietary medium-chain triglycerides supplied by coconut oil on postprandial
Received 24 July 2013 plasma metabolite proles in rainbow trout. The sh (initial body weight 71.3 0.3 g, 17 C) were fed one of
Received in revised form 18 October 2013 four practical diets containing either 5% sh oil (FO low-fat, FL), 15% sh oil (FO high-fat, FH), 5% coconut oil
Accepted 20 October 2013
(CO low-fat, CL) or 15% coconut oil (CO high-fat, CH) for 3 weeks. At the end of the trial, the sh were weighed
Available online 30 October 2013
and plasma sampled to determine glucose, non-esteried fatty acids (NEFA), triglyceride (TG), cholesterol, high
Keywords:
density lipoprotein-cholesterol (HDL-cholesterol), and myeloperoxidase (MPO) at 3, 6, 9, 12, 15 and 24 h after
Rainbow trout (Oncorhynchus mykiss) the last meal. Plasma total ketone bodies (KB) were determined at 6, 12 and 24 h after meal. Blood nitroblue
Coconut oil tetrazolium (NBT) tests were also performed in samples withdrawn at 24 h after meal.
Fish oil Plasma glucose was higher in sh fed the low fat level diet than those fed high fat level, and peaked at postpran-
Growth dial 912 h. Fish fed CH showed higher plasma TG than CL at 3 h after meal, and there was no signicant differ-
Postprandial plasma metabolite ence in plasma TG at the other time points. The peak of TG appeared 12 h after the meal. No clear pattern was
found for cholesterol and HDL-cholesterol (HDL-C) in any of the groups. However, sh fed diet FH had the highest
postprandial plasma HDL-cholesterol level and HDL-C/cholesterol ratio. The peak of NEFA was observed at
1215 h after meal and plasma NEFA of sh fed CH was the highest. Plasma total KB decreased with postprandial
time, and sh of FH groups had higher KB than that of CL group at 6 h. Besides, NBT in sh fed FH was signicantly
higher than that of CH, but there were no differences in MPO between groups. In summary, time-course changes
in plasma proles related to dietary fat level were as expected whereas those related to dietary fat source were
relatively small.
2013 Elsevier B.V. All rights reserved.

1. Introduction
The trend in aquaculture diets, especially in salmon and trout
farming, during the last decades has been to increase the energy content
in the diets by increasing the dietary fat content to improve feed or
protein utilization efciency and to reduce nitrogen losses. Fish oil
(FO) is the most extensively used fat source in salmon feeds because
of its relatively rich n3 long-chain polyunsaturated fatty acid
(LC-PUFA) levels to ensure optimal growth and reproduction (Sargent
et al., 1995, 1999). However, marine resources in general and FO in
particular might be in shortage for future feed production due to an
increasing aquaculture production and at best stable or declining
catches for FO production (Tacon and Metian, 2008). In search for alter-
natives to FO, several studies undertaken with salmonids have shown
Corresponding author at: National Aquafeed Safety Assessment Station, Feed
Research Institute, The Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Tel./fax: +86 10 82109753.
E-mail address: xuemin@caas.cn (M. Xue).
that substitution of FO with vegetable oil may not affect the growth
and survival of sh (Bell et al., 2003, 2004; Greene and Selivonchick,
1990; Richard et al., 2006; Torstensen et al., 2004; Turchini et al., 2009).
Medium chain fatty acids (MCFA) are dened by a fatty acid chain
length from 6- to 12-carbon atoms (Marten et al., 2006). Coconut oil
(CO) is a natural plant source, rich in MCFA with lauric acid (C12:0)
representing 4050% of total fatty acids. CO is rich in saturated fat
content with a high melting point making it heat stable and resistant
to peroxidation, allowing its long-term storage (Bruce, 2005). It might
be supposed that in warm blooded mammals, the utilization of CO
will be easier than in sh reared at low temperatures. However, CO has
been proven to be an efcient fat source in compound diets for rst-
feeding larvae of African catsh (Heterobranchus longilis) (Legendre
et al., 1995) and of common carp (Cyprinus carpio L.) (Fontagne et al.,
2000). Similarly, no negative effects were reported following dietary
inclusion of CO on growth of Ayu (Plecoglossus altielis) (Nakagawa
and Kimura, 1993), red drum (Sciaenops ocellatus) (Craig and Gatlin,
1995), milksh (Chanos chanos) (Alava, 1998) and even, the cold
water species, rainbow trout (Figueiredo-Silva et al., 2012b), or on

0044-8486/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aquaculture.2013.10.024
 L. Luo et al. / Aquaculture 420421 (2014) 2431 25

reproductive performances in rainbow trout (Ballestrazzi et al., 2006). Table 1

On the contrary, Hsieh et al. (2007) found that CO inclusion led to Composition (%) of the four experimental diets fed to rainbow trout.

lower growth than with FO in hybrid tilapia (Oreochromis niloticus Experimental diets
Oreochromis aureus). Also for polka-dot grouper (Cromileptes altivelis),
FL FH CL CH
fed diets containing high levels of CO (15%) displayed signicantly
Ingredients
reduced growth compared to those fed FO (Williams et al., 2006).
Fish meal 38 38 38 38
Although growing similarly well with FO or CO containing diets, there Wheat gluten 20 20 20 20
were signicant effects on the respiratory and cardiovascular physiology Whole wheat 12 12 12 12
in the European eel (Anguilla anguilla L.) and Adriatic sturgeon Gelatinized starch, maize 21 11 21 11
(Acipenser naccarii) (McKenzie, 2001). Coconut oil 5 15
Fish oil 5 15
The role of MCFA as energy-yielding substrates has been largely Vitamin mixturea 1 1 1 1
investigated in terrestrial animals (Aurousseau et al., 1989; Bozzolo Mineral mixtureb 1 1 1 1
et al., 1993). In mammals, MCFA have been reported to directly enter Binder (Sodium alginate) 2 2 2 2
the portal vein, bypassing the intestinal re-acylation and chylomicron Proximate composition (% dry matter)
packaging process, resulting in a more rapid absorption and plasma Dry matter (% diet) 91.6 91.8 91.7 90.9
appearance. Moreover, MCFA do not need carnitine palmitoyl- Crude protein 48.0 47.5 48.0 47.8
transferase-1 (CPT-1) mediated transport to enter mitochondria Crude fat 10.8 21.3 11.1 21.1
Ash 6.1 6.1 6.3 6.1
which may accelerate their oxidation. As a result, only small amounts
a
of MCFA are recovered in peripheral tissues (Johnson et al., 1990; Vitamin mixture (IU or mg/kg diet): DL-a-tocopherol acetate, 60 IU; sodium menadione
Lhuillery et al., 1988). Also in sh, dietary inclusion of MCFA (mainly bisulfate, 5 mg; retinyl acetate, 15,000 IU; DL-cholecalciferol, 3000 IU; thiamin, 15 mg;
riboavin, 30 mg; pyridoxine, 15 mg; B12, 0 05 mg; nicotinic acid, 175 mg; folic acid,
8:0 and 10:0) resulted in decreased fat deposition in the muscle, liver 500 mg; inositol, 1000 mg; biotin, 2.5 mg; calcium panthotenate, 50 mg; choline chloride,
and in intra-peritoneal fat with similar or improved growth rates 2000 mg (Unit de Prparation des Aliments Exprimentaux, Jouy-en-Josas, INRA, France).
(Nakagawa and Kimura, 1993). Negative effects on the efciency of b
Mineral mixture (g or mg/kg diet): calcium carbonate (40% Ca), 2.15 g; magnesium
energy or lipid retention were also seen in Atlantic salmon (Salmo oxide (60%Mg), 1.24 g; ferric citrate, 0.2 g; potassium iodide (75% I), 0.4 mg; zinc sulfate
(36% Zn), 0.4 g; copper sulfate (25% Cu), 0.3 g; manganese sulfate (33%Mn), 0.3 g; dibasic
salar) fed puried MCFA (Nordrum et al., 2003) and in polka-dot
calcium phosphate (20% Ca, 18% P), 5 g; cobalt sulfate, 2 mg; sodium selenite (30% Se),
grouper fed CO, suggesting high levels of C12--oxidation (Williams 3 mg; KCl, 0.9 g; NaCl, 0.4 g (Unit de Prparation des Aliments Exprimentaux, Jouy-en-
et al., 2006). By comparison, Figueiredo-Silva et al. (2012b) revealed Josas, INRA, France).
high C12:0 retention from CO, with C12 representing over 20% of total
body fatty acids compared to less than 1% in trout fed FO. Other studies (sixty sh per tank). Each of the four experimental diets was fed by
in mammals (Allan et al., 2001; Mohamed et al., 2002; Nagao and hand (twice per day) to visual satiation to four replicate groups of sh
Yanagita, 2010) documented the different effects of CO compared to for 3 weeks. The sh in each tank were weighed at the start and end
LCFA oils on plasma parameters related with the metabolic syndrome of the trial in order to calculate the initial and nal daily body weight.
(glycemia, cholesterol, lipoprotein levels, ketone bodies, etc.). In sh, Weight gain (%) = 100% (nal body weight initial body weight) /
little is known on the effect of feeding CO on these plasma parameters. initial body weight; daily food intake (FI, %BW per d) = 100% dry
The dietary model used in this study was to feed rainbow trout high-
fat (21%) or low-fat diets (11%) containing either a highly saturated fat
(CO) or a highly unsaturated fat (FO) as the only fat source. The aim of Table 2
Fatty acid composition of the diets (mg/g of diet).
this study was to compare the effects of the source and level of these
dietary fats on postprandial plasma parameters known to be affected Diets FL FH CL CH
by dietary fat source i.e. glucose, triglycerides, NEFA, cholesterol, HDL, 10:0 ND 0.20 2.32 6.48
LDL, ketone bodies and on parameters related with health status (NBT 12:0 0.20 1.41 20.65 62.40
and MPO). 14:0 5.82 11.66 11.27 28.45
16:0 16.65 32.17 13.59 22.96
18:0 3.07 6.64 2.74 5.49
2. Material and methods 20:0 0.20 0.40 0.11 0.20
Saturates 25.95 52.48 51.09 126.57
2.1. Experimental diets 16:1 4.70 9.85 2.42 2.35
18:1 15.02 31.97 11.38 19.62
20:1 6.13 10.86 4.11 4.32
Four shmeal based diets (Table 1) were formulated to be 22:1 7.15 9.05 6.53 6.67
isonitrogenous, but to contain two levels of crude fat supplied either MUFA 33.00 61.73 24.54 33.16
by sh oil (FO) or coconut oil (CO). Diet FH (sh oil high fat) contained 18:2n6 5.82 7.64 6.64 9.62
15% FO, whereas diet FL (sh oil low fat) contained 5% FO and 10% extra 18: 3n6 0.10 0.20 0.11 ND
20: 2n6 0.20 0.40 0.21 0.20
gelatinized starch. Diet CH (coconut oil high fat) contained 15% CO,
20: 3n6 0.10 0.20 ND ND
whereas diet CL (coconut oil low fat) contained 5% CO and 10% extra 20: 4n6 0.61 1.41 0.32 0.39
gelatinized starch. The four diets were manufactured using a twin- n6 6.85 9.85 7.27 10.40
screw extruder (Clextral, France) at the experimental feed unit (Donzacq, 18:3n3 1.02 1.61 0.84 0.98
France) of the French National Institute of Agronomy Research (INRA, 18:4n3 1.63 3.22 1.05 0.98
20:3n3 0.10 0.20 0.11 ND
France). The analyzed composition is provided in Table 1. Table 2 20:4n3 0.72 1.61 0.42 0.39
gives the details of the fatty acid prole of the diets. 20:5n3 8.28 18.90 4.21 4.32
22:5n3 1.84 4.42 0.84 0.39
2.2. Fish and experimental conditions 22:6n3 12.57 26.34 7.69 3.73
n3 26.16 57.51 15.17 10.99
PUFA 36.68 74.40 23.81 22.76
The experiment was carried out in a ow-through sh rearing SFA:PUFA 0.7 0.7 2.2 5.6
system (INRA Donzacq facilities, France) using natural spring water of FA mg/g diet 102.17 201.07 105.34 196.23
constant temperature (17 C) and under natural photoperiod condi- ND: not detected; FA, fatty acid.
tions (October). Rainbow trout juveniles (71.3 0.3 g initial body Diets: FL: sh oil, low-fat; FH: sh oil, high-fat, CL: coconut oil, low-fat; CH: coconut oil,
weight) were randomly distributed among sixteen 150-liter tanks high-fat.
26 L. Luo et al. / Aquaculture 420421 (2014) 2431

feed intake / ((initial tank biomass + nal tank biomass) / 2 days of 3. Results
trial duration); feed efciency (FE, %) = 100% weight increase / dry
feed intake. 3.1. Growth performance

The survival rate of the sh during the 3-week feeding trial was 100%
2.3. Sampling procedures, circulating and postprandial plasma lipids analysis
in all dietary treatments. Final sh body weight and relative weight gain
were the highest in the FH group (P b 0.05) and were signicantly
At the end of the feeding trial which lasted 3 weeks, nine individuals
higher than in sh fed diet CH with no difference between FL and CL
from each dietary treatment were taken randomly at 3, 6, 9, 12, 15 and
groups. Feed intake values were also improved in FO sh compared to
24 h after the meal. For each dietary treatment and at each sampling
CO sh as shown in Table 3. Feed efciency had no signicant difference
time, we sampled sh from one of the four replicate tanks. Blood
among all treatments (P N 0.05).
samples were withdrawn from the caudal part of the sedated sh
(2-phenoxy ethanol, 0.15 ml/l water) using anticoagulant syringes
3.2. Postprandial plasma lipids
with 2% NaF and 4% oxalic acid. Plasma was separated from whole
blood by centrifugation (1500 g, 5 min) and was frozen at 80 C
Both dietary fat level and sampling time points had highly signicant
until subsequent analyses. The TG, glucose, cholesterol, and HDL-
effects on plasma glucose (P b 0.01). Plasma glucose level was higher in
cholesterol of the plasma were analyzed using enzymatic procedures,
sh fed low fat diets compared to sh fed high fat diets. Following the
commercialized by Biomerieux (Craponne, France). NEFA and total
meal, plasma glucose peaks of high fat groups (FH and CH) were
ketone bodies of the plasma were analyzed by enzymatic procedures
found at 12 h. Among the low-fat groups, postprandial peak glycemia
of Wako Chemicals GmbH (Neuss, Germany). LDL = CHO-TG/5-HDL
was observed at 9 h for FO groups and 15 h for CO groups (Fig. 1(a)).
(based on the HDL test protocol).
There was a signicant interaction between the fat source and time
The NBT assay was carried out following the protocol of Anderson
point (three-way ANOVA, F5, 166 = 2.71, P b 0.05) (Table 4). The
and Siwicki (1995). Briey, 0.1 ml of heparinized blood was taken in
glucose level in FO fed groups (FL and FH) increased from 0.96 g/l at
an Eppendorf to which 0.1 ml of 0.2% NBT (Sigma, USA) solution was
3 h to 1.42 g/l at 24 h, whereas the plasma glucose levels in sh fed
added. The mixture was incubated for 30 min at 25 C. From the
CO (CL and CH) increased from 0.96 g/l at 3 h to 1.43 g/l at 15 h, then
resultant suspension, 50 l was taken, added to 1.0 ml N, N-dimethyl
decreased to 1.02 g/l at 24 h.
formamide (Sigma, USA) in a glass tube and centrifuged at 3000 g for
Plasma TG was signicantly affected by time point (P b 0.01)
5 min. The optical density (OD) of the supernatant was measured at
(Table 4). The peak of TG appeared at 12 h after the meal (Fig. 1(b)).
540 nm in the microplate reader (Bio-Tek, USA).
Fish fed CH showed higher plasma TG than that of CL group at 3 h
Total MPO content present in plasma was measured according to
after meal (one-way ANOVA, P b 0.05), and there was no signicant
Quade and Roth (1997) with slight modication. About 10 l of serum
difference between treatments in plasma TG at other time points.
was diluted with 90 l of Hank's balanced salt solution (HBSS) without
Plasma NEFA levels were signicantly affected by fat source, fat level
Ca2+ or Mg2+ in 96-well plates. Then 35 l of 20 mM 3, 3, 5, 5-
and postprandial interval point. Fish fed CO showed signicantly higher
tetramethylbenzidine hydrochloride (TMB) system (Sigma, USA) was
plasma NEFA levels than those of the FO groups (P b 0.05). High-fat
added. The color change reaction was stopped after 2 min by adding
groups had signicantly higher plasma NEFA than that of low-fat
35 l of 0.5 M sulfuric acid (H2SO4). The OD was read at 450 nm in a
groups (P b 0.01). The peak of NEFA was observed at 1215 h after
microplate reader (Bio-Tek, USA).
meal (Fig. 1(c)). Fat source fat level interaction (three-way ANOVA,
F1, 167 = 5.09, P b 0.05) (Table 4) showed that plasma NEFA of sh
2.4. Statistical analyses fed CH was the highest at all time points.
Plasma cholesterol levels were signicantly affected by fat source
Statistical analyses were performed using STATISTICA 8.0 (StatSoft and time points after meal with an interaction between fat level and
Inc., Tulsa, OK, USA). The mean and error term was computed from time (F5, 168 = 2.99, P b 0.05) (Table 4). Trout fed the FO diets had
the values of nine sh from a single tank in the sampling period. The signicantly higher plasma cholesterol levels than CO groups (P b 0.01).
growth performance was subjected to a two-way ANOVA with different A small peak occurred at 6 h, but no clear plateau was found for choles-
fat sources and fat levels as factors. The effects of time point and diet terol in any of the groups (Fig. 1(d)). For low-fat diet groups, a signi-
with different fat sources and fat levels on plasma parameters were cant peak of plasma cholesterol level was observed at 6 h, reaching a
analyzed by a three-way ANOVA, followed by one-way ANOVA in case plateau phase from 9 h to 24 h. For FH diet groups, cholesterol level
of a signicant interaction. NBT activity of treatments was analyzed by remained stable during 9 h after meal, but dropped at 12 h after meal,
a one-way ANOVA test. Signicant differences between means were and then continued to rise to the level of 3 h at 24 h after meal. For
evaluated by the Duncan's test. The signicance level was set at CH diet groups, cholesterol level was stable during 15 h after meal,
P b 0.05. and then the level increased and peaked at 24 h after meal.

Table 3
Body weight and feed intake of rainbow trout fed the feed with different fat sources (S) and fat level (L) for 3 weeks (means S.E.M, n = 3).

Diets P

Fish oil Coconut oil

FL FH CL CH S L SL

IBW (g) 71.19 0.69 71.24 0.69 71.29 0.58 71.38 0.37 ns ns ns
FBW (g) 114.10 0.34a 125.38 1.16b 115.00 1.89a 118.57 0.86a ns ** *
Weight gain (%) 60.30 1.52a 76.01 0.91c 61.32 2.05a 66.83 0.37b * * *
FI (%BW per d) 2.03 0.01ab 2.18 0.03b 2.01 0.03ab 1.88 0.11a * ns *
FE (%) 1.09 0.02 1.20 0.01 1.11 0.03 1.14 0.06 ns ns ns

Mean values with unlike letters were signicantly different (P b 0.05).

P-value for fat source (S), fat level (L) and interaction between the main effects of the two tested factors (S v. L) are as follows: ns: no signicant difference; * P b 0.05; **P b 0.01 (two-way
ANOVA).
Diets: FL: sh oil, low-fat; FH: sh oil, high-fat, CL: coconut oil, low-fat; CH: coconut oil, high-fat.
 L. Luo et al. / Aquaculture 420421 (2014) 2431 27

FO groups had signicantly higher plasma HDL-cholesterol levels
than CO groups (P b 0.01). High-fat diet groups had signicantly higher
plasma HDL levels than low-fat diet groups. There was a plateau from 9 h
to 12 h in FO-fed groups, while no such plateau was found for HDL in CO-
fed groups because of the highest HDL level recorded at 3 h after the
meal (P b 0.01) (Fig. 1(e)). Interaction between fat source fat
level showed a signicant effect: FH N CH N FL N CL (F1, 167 = 4.97;
P b 0.05). Interaction between fat source time point (F5, 167 = 4.31;
P b 0.01) showed that there was a signicant peak at 12 h in FO-fed
groups, and at 9 h in CO-fed groups. The three-way ANOVA performed
on plasma HDL-cholesterol indicated signicant effects of fat source
fat level time interaction (F5, 167 = 3.82; P b 0.01) (Table 4). The
HDL in sh fed the same fat source showed the same curve change at
different levels following the meal. And the sh groups fed the same
fat level reached the same HDL concentrations at 24 h.
The plasma HDL:total cholesterol ratio (%) was higher in sh fed the
high compared to low fat level (P b 0.01) (Table 4). Following the meal,
HDL/cholesterol levels rst showed a slight decrease (at 6 h), where
after they increased up to a peak at 912 h (Fig. 1(f)). Interaction
between fat level time point (F5, 167 = 3.97; P b 0.01)showed that
there was a signicant peak at 12 h in FO-fed groups, but there was
no signicant peak at the same time point in CO-fed groups. The
three-way ANOVA performed on plasma HDL/cholesterol indicated
signicant effects of the interaction of fat source, fat level and time

(a) (b)
2.0 TG (g/l)
FL glucose (g/l) 7.5
FL
FH
1.8 FH
CL
CL CH
1.6
CH
5.5
1.4

1.2
3.5
1.0

0.8

0.6 1.5
0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24
Time following the meal (h) Time following the meal (h)

(c) NEFA (meq/l)

(d)
FL
0.18 FL Cholesterol (g/l)
FH
FH 4.5
0.16 CL
CL
CH
0.14 CH 4.0

0.12 3.5
0.10
3.0
0.08
2.5
0.06

0.04 2.0
0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24
Time following the meal (h) Time following the meal (h)

(e) (f)
HDL cholesterol (g/l) HDL Chol, % tot chol
35
0.8 FL FL

FH
FH
0.7 30
CL
CL
25 CH
0.6 CH

0.5 20

0.4 15

0.3 10

0.2 5
0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24
Time following the meal (h) Time following the meal (h)

Fig. 1. Changes in circulating plasma lipids in juvenile rainbow trout during the 24 h following the administration of four isonitrogenous meal FL (), FH(), CL(--) and CH (--).
Values are means with their standard errors of the mean of nine sh from one tank per treatment at per sampling point. Statistical differences in postprandial plasma lipid proles related
to the dietary treatment and time point are given in the Results section and Table 4.
28 L. Luo et al. / Aquaculture 420421 (2014) 2431

(g) (h)
LDL (g/l)
3.0 ketone bodies (umol/l)
FL
FH 35
FL
2.5 CL
30 FH
CH
CL
2.0
25 CH

1.5
20

1.0 15

0.5 10
0 3 6 9 12 15 18 21 24 3 6 9 12 15 18 21 24
Time following the meal (h) Time following the meal (h)

(i)
MPO (OD)
1.0 FL
FH
0.9
CL
CH
0.8

0.7

0.6

0.5

0.4

0.3
0 3 6 9 12 15 18 21 24
Time following the meal (h)

Fig. 1 (continued).

(F5, 167 = 3.19; P b 0.01) (Table 4). HDL as a percent of total cholesterol Based on the three-way ANOVA performances (Table 4), sh fed FL
in all the treatment groups tended to a xed value at 24 h. had the highest MPO activity compared with the CH and FH diets
FO groups had signicantly higher plasma LDL than CO groups (F1, 167 = 4.06; P b 0.05). Low fat groups had signicantly higher
(P b 0.05). Dietary fat level had no signicant effect on plasma LDL MPO than high fat groups at 24 h after meal (P b 0.05, Fig. 1(i)).
(P N 0.05). The time points had an extremely signicant inuence on The results of NBT test is reported in Fig. 2. The production of
plasma LDL (P b 0.01). There was a clear valley for LDL in all groups at oxygen radicals by phagocytic cells, measured by the NBT assay, was
12 h after meal (P b 0.01) (Fig. 1(g)). The interaction terms were not signicantly higher in FH-fed sh groups than that of in CH-fed sh
signicant (P N 0.05). groups (P b 0.05).
There were just three sampling time points for ketone bodies (6H,
12 h, and 24 h). Total KB of sh signicantly decreased with postpran-
dial time (P b 0.01) (Fig. 1(h)). Three-way ANOVA performed on
plasma KB indicated a signicant interaction of fat source and fat level
(F1, 79 = 4.13; P b 0.05) (Table 4). Fish of FH groups showed signi-
cantly higher KB than that of other group (P b 0.05).

Table 4
Summary of the statistical analyses (3-way ANOVA) on circulating plasma nutrients and
metabolites in juvenile rainbow trout during different sampling points (Time, T) taken
over the 24 h following the administration of one of the four isonitrogenous diets
differing in fat source (S) and fat level (L) (means of 9 sh from one tank per treatment).

Source Level Time SL ST LT SLT Pooled

(S) (L) (T) S.E.M

Glucose ns ** ** ns * ns ns 0.11
TG ns ns ** ns ns ns ns 0.72
NEFA * ** ** * ns ns ns 0.01
Cholesterol ** ns * ns ns * ns 0.26
HDL-Chol ** ** ** * ** ns ** 0.04
HDL/Chol ns ** ** ns ns ** ** 1.99
LDL * ns ** ns ns ns ns 0.22
Ketone bodies ns ns ** * ns ns ns 1.36
Fig. 2. Mean nitroblue tetrazolium (NBT) activities in rainbow trout sampled 24 h after
MPO ns ns ns * ns ns ns 0.08
of the meal. Bars represent mean S.E.M of 9 numbers of sh in each treatment. Bars
ns: no signicant difference; ** highly signicant (P b 0.01); * signicant (P b 0.05). bearing different superscript (a, b) are signicantly different (P b 0.05).
 L. Luo et al. / Aquaculture 420421 (2014) 2431
4. Discussion
The postprandial rise in plasma TG of rainbow trout was apparent after
6 h and reached a maximum after 12 h. This is later than that found in
common carp where peak levels appeared 8 h after feeding (Geurden
et al., 2008), probably related to the higher temperature in the study
with carp, known to accelerate lipid absorption in sh (Wallaert and
Babin, 1994). Intestinal synthesis and re-acylation pathways of TG in tele-
ost do not seem to differ substantially from those in mammals (Caballero
et al., 2006; Oxley et al., 2005). The high level of plasma TG in the CH group
at the time of absorption indicates the efcient intestinal uptake and also
the efcient re-acylation of the CO fatty acids. Indeed, at the digestive
level, the medium-chain fatty acid 12:0 from CO seems to be released at
high rates and efciently absorbed by salmonids, such as the Arctic charr
(Salvelinus alpinus) (Olsen et al., 1998).
Plasma NEFA levels are the most metabolically dynamic fraction of
lipids in vertebrate blood and reect the importance of lipids in energy
metabolism because NEFA transport in the blood helps to sustain fatty
acid oxidation in tissues (Henderson and Tocher, 1987). Fatty acids
are selectively mobilized from adipose tissue into serum. During fasting,
fatty acids are released into serum as NEFA (Raclot et al., 1995). So it is
reasonable that the peaks of plasma NEFA come after those of TG. Our
results conrmed this theory. The peaks of plasma NEFA (1215 h
after meal) appeared almost 3 h later than those of TG (912 h). On
the other hand, preferential mobilization of fatty acids from adipose
reserves is also known to depend on carbon chain length, degree of
unsaturation, and positional isomerism (Raclot and Groscolas, 1993;
Raclot et al., 1995). Saturated fatty acids respective fractional turnover
rate is stable at the whole-organism level and accurately reect the
turnover rate of total NEFA. In contrast, long-chain PUFA exhibits a
different kinetic behavior (Hagenfeldt, 1975). Thus, the more saturated
fatty acids are, the lower the turnover rate of total NEFA will be. In the
present trial, trout fed high CO diets had the highest level of plasma
NEFA. One of the reasons might be that more than 24 fold of saturated
fatty acids in CH diet resulted in lower turnover rate of total NEFA, and
more NEFA remained in the plasma of sh fed CH diet.
Studies in mammalian vertebrates (Carlson and Kottke, 1990)
including humans (Fisher et al., 1983; Katan et al., 1994; Mattson and
Grundy, 1985) have shown that total plasma cholesterol concentrations
rise when saturated fat (especially CO) replaces monounsaturated or
polyunsaturated fat. In contrast, plasma cholesterol levels in the
present study were lower in trout fed diets containing CO compared
to those fed FO. This conrms previous studies with trout (Richard
et al., 2006) and other sh (Turchini et al., 2009) who report that total
replacement of FO by vegetable oils leads to a decrease in plasma cho-
lesterol. The decrease is to be attributed, at least partially, to the de-
creased content of cholesterol in vegetable compared to that in FO
(Turchini et al., 2009). Thus the cholesterol-lowering effect of FO ob-
served in mammals is not generally recorded in sh. Panserat et al.
(2008) also found that cholesterol biosynthetic pathway was reduced
in the liver of trout fed a vegetable oil based diet in comparison to that
in trout fed a FO based diet. It is well known that 7080% cholesterol
is synthesized by liver and 10% by gut, with plasma cholesterol
reecting mostly that from biosynthesis of acetyl-CoA in the liver
(Wen and Wang, 2002). Further studies are warranted to fully elucidate
the relative importance of dietary cholesterol and that from endogenous
biosynthesis in sh.
High-density lipoproteins (HDL), known to be involved in the trans-
port of cholesterol from plasma to liver, act as cleaners of body tissue
cholesterols in mammals. Their plasma level is known to correlate
negatively with development of cardiovascular diseases (Miller and
Miller, 1975). Rat displayed higher HDL-cholesterol in serum when
fed menhaden (sh) or olive oil than when fed CO (Mohamed et al.,
2002). Also in this study, sh fed FH diets had the highest plasma HDL-
cholesterol. The ratio of HDL to total cholesterol (HDL/Chol) followed
a similar trend.
29
Cholesterol-rich LDL plays a role in the transport of cholesterol from
liver to body tissues. High levels of LDL-cholesterol may overburden
blood vessel and tissues (Wen and Wang, 2002). In this study, dietary
FO had a hypercholesterolemic effect signicantly raising not only
HDL- but also LDL-cholesterol concentrations. This nding differs from
reports in humans showing that saturated compared to polyunsaturated
fatty acids increase serum total and LDL-cholesterol (Katan et al., 1994),
whereas no signicant differences in LDL-cholesterol levels was found
in pigs fed diets containing FO, milkfat, CO or olive oil (Allan et al.,
2001). A possible explanation for the high plasma LDL levels may be
related with the effect of acyl-coenzyme A: cholesterol acyltransferase
(ACAT), a key hepatic enzyme involved in the esterication of free
cholesterol to cholesteryl ester with a preference for unsaturated rather
than saturated fatty acids as the substrate for esterication (Levy, 1995).
As such, the FO-diets, rich in unsaturated fats may have increased ACAT
activity with more cholesteryl ester being transported into LDL particles,
increasing plasma LDL-cholesterol levels. It is also worth stating that LDL
concentrations were calculated here as the difference between total and
HDL-cholesterol. Whereas total cholesterol was signicantly higher in
FO-fed than in CO-fed sh, the percentage of HDL-cholesterol to total
cholesterol remained mostly below 25%. The high total cholesterol in
FO-fed sh hence resulted in high LDL concentrations with a signicant
positive correlation relationship between total and LDL-cholesterol
concentrations (r = 0.772, P b 0.0001).
The ingestion of MCFA in both animals and humans can result in
increased ketogenesis, related to their more rapid oxidation compared
to LCFA as MCFA are taken up by more tissues and have easier access
to mitochondria for subsequent -oxidation (Leonhardt and Langhans,
2004; Nanton et al., 2003; Smith et al., 2005). Acetyl CoA is one of the
main products of -oxidation. Ketone bodies, including acetoacetate
(AA) and D--hydroxybutyrate (-HB), are high-energy fuels produced
primarily from acetyl CoA by liver mitochondria and then exported to
peripheral tissues for oxidation in mammals (Laffel, 1999). In the afore-
mentioned work of Craig and Gatlin (1995), high plasma levels of the
ketone body, -hydroxybutyric acid, were found in red drum when
tricaprylin, containing only 8-carbon fatty acids, was included at dietary
inclusion rates of 4.6%6.0%. They attributed the ketone body produc-
tion in the sh to an overwhelming of the TCA cycle brought about by
accelerated -oxidation of the tricaprylin fatty acids. In the present
study, however, trout fed the CO diet did not have higher plasma ketone
bodies than trout fed the FO diets. This may be due to the high retention
of the absorbed C12 in body lipids in rainbow trout, implying that
the absorbed CO MCFA are not preferentially directed towards
-oxidation in rainbow trout (Figueiredo-Silva et al., 2012b).
In the present study, dietary fat source did not signicantly affect
postprandial glycemia. But a signicant interaction effect between fat
source and time points was found, with glucose levels in FO-fed trout
being lower than in CO-fed trout at several time points (3 h, 9 h, 12 h
and 15 h) after the meal. Regarding changes in dietary fat level, high
dietary fat has been reported to negatively affect plasma glucose clear-
ance (Figueiredo-Silva et al., 2012a; Panserat et al., 2002) in rainbow
trout, known to have a poor capacity to utilize glucose as a fuel with
an insulin resistance-like metabolic behavior (Hemre et al., 2002;
Moon, 2001; Polakof et al., 2011). Higher glycemia in rainbow trout
fed high vs low-fat diets was also seen at the end of a 15-week feeding
trial using the same diets as here (Figueiredo-Silva et al., 2012b). As
opposed, high fat diets in the present study did not increase plasma
glucose levels. This is probably due to the short feeding duration
(3 wks), as suggested from a recent study with trout which showed
that the interfering effect of dietary fat on glucose utilization is not
immediate (Figueiredo-Silva et al., 2012a). Instead, plasma glucose
levels in the present trout were higher when fed the low fat diets, in
line with the higher levels of carbohydrates in these diets as opposed
to the high fat diets. In rainbow trout increased digestible carbohydrate
intakes are well known to result in higher plasma glucose concentra-
tions (Bergot, 1979; Palmer and Ryman, 1972).
30 L. Luo et al. / Aquaculture 420421 (2014) 2431
The NBT reduction product obtained after reaction with superoxides
is considered as an indicator of the health status or the immune system
in sh (Anderson et al., 1992). The signicantly higher level of NBT
activity in trout fed diet FH indicates their better immune status
compared with that observed in CH-fed groups. The effect of dietary
fatty acid composition on health and immune function of sh has
been examined in a few studies. It has been suggested that diets high
in n6 PUFAs enhance the immune response because of the high levels
of proinammatory AA-derived eicosanoids, and diets high in n3
PUFAs may be immunosuppressive because of the high levels of EPA-
derived anti-inammatory eicosanoids (Lall, 2001). However, this
predicted effect is rarely clear as illustrated by the discrepancies in
results between studies. Positive effects of high n3 fatty acid intake
on immune response have been reported in some studies (Ashton
et al., 1994; Sheldon and Blazer, 1991). Balfry et al. (2006) observed
that Atlantic salmon fed diet with FO containing higher n3 fatty
acid levels exhibited higher postvaccination levels of peripheral blood
leukocyte respiratory burst activity and higher serum antibody titers
against Listonella anguillarum, compared to sh fed diets with canola
oil and poultry fat. Sheldon and Blazer (1991) found that high n3
PUFA (menhaden oil) may enhance immune response (enhanced
bactericidal activity of macrophages) of channel catsh compared to
diets with soybean oil or beef tallow, independent of water tempera-
ture. On the other hand, Fracalossi and Lovell (1994) found that channel
catsh fed 7% menhaden oil had a higher mortality rate when exposed
to Edwardsiella ictaluri than sh fed a similar diet containing beef tallow.
High level of n3 fatty acids decreased antibody titers and survival in
Atlantic salmon (Erdal et al., 1991). It appears that the balance of n3
and n6 fatty acids is the most important with regard to increasing
the immune competence of sh (Sargent et al., 2002). Balfry et al.
(2006) also suggested that the ratios of n6 to n3 fatty acids in the
dietary lipids may play an important role in balancing the composition
of the membrane PUFAs, and subsequently in their ability to produce
an array of eicosanoids that do not compromise the immune response.
Their data suggest a relationship between a low dietary n6/n3
ratio and an optimized immune system. Higher antibody and NBT
activity levels in sh fed FO diet were associated with lower n
6/n3 fatty acids in the dietary and tissue lipids. In similar studies
with Atlantic salmon, Thompson et al. (1996) found that low dietary
n6/n3 ratios resulted in increased B lymphocyte responses and im-
proved sh survival following a disease challenge. In the present trial,
n6/n3 ratio of FH diet was 0.17, it was far lower than 0.94 of CH
diet. So the lower n6/n3 ratio resulted in an optimized immune
system.
Similarly, MPO is a major enzyme having antimicrobial activity.
It utilizes hydrogen peroxide during respiratory burst to produce
hypochlorous acid (Dalmo et al., 1997). Reduced activity may indicate
the presence of contaminants or stress (Anderson and Siwicki, 1995). It
appears that FL-fed sh showed well-developed immune status with a
very high MPO activity. On the contrary, an increase in the fat level (CH
and FH groups) reduced MPO activity. However, this result was based
on the average of values from all six time points. As with NBT, MPO values
were not signicantly different among all treatment at 24 h.
In summary, feeding rainbow trout with FO or CO at different
inclusion levels altered proles in postprandial plasma metabolites
and parameters related with innate immune status. The plasma lipid
proles related to time-course changes and dietary fat level were as
expected whereas those related to dietary fat source were relatively
small.
Acknowledgments
Financial support was provided by the Beijing Municipal Bureau
of Agriculture, China, project No. SCGWZJ 20131103-1/-2; the
Beijing Municipal Science &Technology Commission, project No.
Z12110500282114; the National Natural Science Foundation of China,
project No. 31072220; No. 31101907; 31372539; and the Special Fund
for Agro-Scientic Research in the Public Interest (201203015;
201003020).
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