Data on different factors such as fish species, initial body weight, final body weight, dura-tion of feeding trial, diet type, P concentradura-tion in basal diet, number of dietary P lev
Trang 11,2 2 1 1
Nutrition, Metabolism and Aquaculture (NuMeA), UR 1067, Pole d’Hydrobiologie INRA, Saint-Pee-sur-Nivelle, France;
2
Aquaculture and Fisheries Group, Wageningen Institute of Animal Sciences (WIAS), Wageningen University,Wageningen, The Netherlands
A meta-analysis of available data on dose response to
die-tary phosphorus (P) in fish from over 70 feeding trials
reported in 64 published studies covering over 40 species of
fish was performed Broken-line regression was used to
model the data sets The meta-analysis showed that
esti-mated minimal dietary P level varies with the response
cri-terion and that estimates should preferably be expressed in
terms of available P than in terms of total P Estimates
based on whole-body P concentration (4.7 g available P
Use of ingredients rich in P or of diets with high basal P
content or high levels of water P concentration can affect
the estimations Among the different response criteria
tested, WG was found to be the most reliable and
whole-body P concentration to be the most stringent criterion to
estimate P requirement of a given fish species Expressing
available P requirement as g P per unit DM or digestible
energy (DE) in the diet was equally effective, but
more precise
regression, response criteria, weight gain, whole-body P
Received 7 August 2012; accepted 27 December 2012
Correspondence: S J Kaushik, INRA, UR 1067, Nutrition, Metabolism
and Aquaculture (NuMeA), Pole d’Hydrobiologie INRA, F-64310
Saint-Pee-sur-Nivelle, France E-mail: kaushik@st-pee.inra.fr
Available data on mineral and trace element requirements
of fish and crustaceans have been updated and rized recently by the National Research Council (NRC2011) Of all minerals considered essential for fish,requirement for phosphorus (P) is the most extensivelystudied Although there is a significant amount of dataavailable in the literature on response to dietary P and Prequirement based on a wide range of response variables,direct comparison of results is often difficult due to inter-study differences Meta-analysis of data from the availableliterature is a relevant solution for interstudy comparisonsand for summarizing data (Sauvant et al 2008) Meta-analytic approach has been used to analyse data on die-tary requirements for selected amino acids in animals (Si-mongiovanni et al 2011) as well as in fish (Kaushik &Seiliez 2010), to investigate nutrient balance in growinganimals (Schulin-Zeuthen et al 2007) and to study theeffects of fish meal and fish oil replacement in fish diets(Drakeford & Pascoe 2008; Sales 2009; Sales & Glencross2011; Hua & Bureau 2012) Careful selection and stan-dardization of data and the use of appropriate mathemati-cal model for data analysis are essential to make themeta-analysis to be biologically relevant Till date, morethan 70 studies on P requirement or P utilization havebeen reported for over 40 different species of fish About90% of the studies were undertaken in the past two dec-ades (1991–2011) and over 60% in the past decade alone
importance of P in growth and skeletal development butalso the environmental implications of P discharge intowater P requirement of fish has been estimated using awide range of response criteria: (i) production traits such
.
Aquaculture Nutrition
Trang 2as weight gain (WG), growth rate or feed efficiency, (ii)
levels of ash/P in whole body, plasma/serum, vertebrae,
scale or skin, (iii) mineral balance indices such as P gain,
retention and, (iv) to a limited extent, expression of genes
involved in the absorption of P from the gastrointestinal
tract The major and widely used response criteria are in
the order of WG, whole-body P, vertebral P, plasma P
and whole-body P balance Urinary P excretion has also
been occasionally used, especially for large fishes The
present work aims at reanalysing available data on P
requirement of fish for estimating the minimal dietary P
level required based on different response criteria and to
identify the most stringent criterion and mode of
expres-sion through meta-analysis These were performed by
delineating the interstudy differences through
standardiza-tion of dependent and independent variables following
selection, segregation and grouping of data
Data from published literature on P requirement or P
P levels were collected Data on different factors such as
fish species, initial body weight, final body weight,
dura-tion of feeding trial, diet type, P concentradura-tion in basal
diet, number of dietary P levels tested and corresponding
dietary P concentrations, P availability, feed efficiency,
major protein source, source of supplemental P, time of
sampling after the last meal, type of rearing system,
water temperature, salinity, P concentration of the
rear-ing water, response variables tested, mathematical model
used, if any, response criterion based on which optimal
dietary P level was estimated and the corresponding
estimate values were registered A database was created
with the foresaid information and the corresponding
data from a total of over 70 feeding trials reported in
In each study, data from treatment groups, in which
dietary P concentration alone was the only quantitative
variable, were selected and grouped for inclusion in the
meta-analysis
Every study reported data on one or more of the different
response criteria employed The widely used four criteria
(WG, whole-body P, vertebral P and plasma P) were
cho-sen for a comparative analysis across studies Initially,meta-analysis was performed on the complete data setincluding all fish species (All) Further to assess whether
P requirement differs between fish species, the total dataset was split into two, a data set of only rainbow trout(Rbt) and a data set with all species excluding rainbow
sub-data sets for the four response criteria in relation toboth dietary total P and available P concentration
Detailed description of the different data sets is reported
in Table 1
response unit of expression prior to meta-analysis Forexample, growth or WG data were normalized by takingthe maximum body WG in a given study as 100 and cal-culating the WG of the other groups in the study as a rel-ative percentage to the maximum WG (Simongiovanni
of the growth data, which would as such be difficult due
to differences in fish size, duration of trial and magnitude
of somatic WG in absolute terms Dietary P concentrationexpressed as fed or on a dry matter (DM) basis in the
and available P basis, wherever possible Response dataexpressed in different units in different studies for a givencriteria such as whole-body P, vertebral P and plasma P
respectively
1=3
¼ weight of fish (g) whole body P concentration (%)
.
Trang 3Table 1 Summary of minimal dietary P level (g kg DM, g MJ DE and g kg BW day ) as estimated through the meta-analysis
et al (2007), Choi et al (2005), Coloso et al (2003), Davis & Robinson (1987), Dougall et al (1996), Elangovan & Shim (1998), Fontagne
et al (2009), Furuya et al (2008), Ketola & Richmond (1994), Ketola (1975), Kim et al (1998), Kousoulaki et al (2010), Liang et al (2012), Lovell (1978), Luo et al (2009, 2010), Mai et al (2006), Nwanna et al (2009, 2010), Ogino & Takeda (1976, 1978), Oliva Teles & Pi- mentel Rodrigues (2004), Paul et al (2004), Phromkunthong & Udom (2008), Qiu-shan et al (2009), Robinson et al (1987), Rodehutscord (1996),Rodehutscord et al (2000), Pimentel Rodrigues & Oliva Teles (2001), Roy & Lall (2004), Roy et al (2002), Satoh et al (2003), Schae- fer et al (1995), Shao et al (2008), Shim & Ho (1989), Skonberg et al (1997), Sugiura et al (2000a,b, 2007), Sukumaran et al (2009), Vi- elma & Lall (1998), Vielma et al (2002), Wang et al (2005), Wen et al (2008), Xie et al (2011), Xu et al (2011), Yang et al (2005), You
et al (1987), Li et al (2008), Yuan et al (2011), Zhang et al (2006), Zhao et al (2008a,b), Zheng et al (2007), Zhou et al (2004).
(2010), Nwanna et al (2010), Oliva Teles & Pimentel Rodrigues (2004), Phromkunthong & Udom (2008), Robinson et al (1987), cord (1996),Rodehutscord et al (2000), Pimentel Rodrigues & Oliva Teles (2001), Roy & Lall (2003), Skonberg et al (1997), Sugiura et al (2007), Vielma & Lall (1998), Vielma et al (2002), Wang et al (2005), Xu et al (2011), Yang et al (2005).
Nwan-na et al (2010), Phromkunthong & Udom (2008), Rodehutscord (1996), Sugiura et al (2007), Vielma & Lall (1998), Vielma et al (2002).
(1987), Dougall et al (1996, 1996), Elangovan & Shim (1998), Ketola & Richmond (1994), Ketola (1975), Kim et al (1998), Kousoulaki
et al (2010), Liang et al (2012), Luo et al (2010), Nwanna et al (2009, 2010), Ogino & Takeda (1976, 1978), Oliva Teles & Pimentel drigues (2004), Paul et al (2004), Phromkunthong & Udom (2008), Robinson et al (1987), Rodehutscord (1996), Rodehutscord et al (2000), Pimentel Rodrigues & Oliva Teles (2001), Roy & Lall (2003, 2004), Schaefer et al (1995), Shao et al (2008), Shim & Ho (1989),
Trang 4Ro-Average Metabolic weight gainðg kg BW0:8day1Þ
rela-tion to dietary available P level was used to assess whether
this relationship (growth versus available P) was dependent
on rearing system (flow-through versus recirculation system)
This was carried out for the ‘Rbt’ sub-data set, which
con-tained two of the experiments carried out in recirculation
system (Rodehutscord 1996; Rodehutscord et al 2000)
Meta-analysis was performed on this data set including and
excluding these studies Moreover, a comparison of growth
data (calculated as TGC) was made between two studies in
rainbow trout (Ketola & Richmond 1994; Rodehutscord
1996) of relatively similar initial body weights (35 and 50 g)
but differing in the concentration of P in the rearing water
rendered by the nature of the rearing system (P
concentra-tion in water not determined, flow-through system, and
large differences between studies in the basal diet
composi-tion In order to study whether the relationship betweenresponse criteria and dietary P level was affected by thebasal diet concentration of available P, the total data setfor the response variable WG was split into two sub-datasets: one with studies having a concentration of available P
Meta-analyses were performed with the two subsets
required for growth can be expressed in different ways
be argued if requirements are not related to dietary ent concentration (e.g DE content) In order to assesswhether this unit of expression (per unit of DM versus perunit of DE) improved the prediction of the responsecriteria, a dataset was framed comprising data from studieswherein WG per unit DM as well as DE could be
values of the regression
Two models, namely simple linear broken-line regression(Robbins et al 1979) and Mercer’s four-parameter nutrientsaturation kinetic model (Mercer 1982), were tested tomodel the different data sets with a fixed-effect approach
Preliminary analysis showed that the linear broken-lineregression model had a better fit and was preferred over
Skonberg et al (1997), Sukumaran et al (2009), Vielma & Lall (1998), Wang et al (2005), Xie et al (2011), Xu et al (2011), Yang et al.
(2005), Yuan et al (2011), Zhao et al (2008b), Zheng et al (2007), Luo et al (2009).
et al (1998), Kousoulaki et al (2010), Liang et al (2012), Nwanna et al (2010), Phromkunthong & Udom (2008), Rodehutscord (1996),
Rodehutscord et al (2000), Schaefer et al (1995), Shao et al (2008), Sukumaran et al (2009), Vielma & Lall (1998), Xie et al (2011),
Yuan et al (2011).
Rich-mond (1994), Kousoulaki et al (2010), Liang et al (2012), Luo et al (2009, 2010), Mai et al (2006), Nwanna et al (2009), Ogino &
Take-da (1976, 1978), Oliva Teles & Pimentel Rodrigues (2004), Phromkunthong & Udom (2008), Rodehutscord (1996), Rodehutscord et al.
(2000), Pimentel Rodrigues & Oliva Teles (2001), Schaefer et al (1995), Shao et al (2008), Skonberg et al (1997), Sukumaran et al.
(2009), Xie et al (2011), Xu et al (2011), Yang et al (2005), Yuan et al (2011), Zhang et al (2006).
9
Brown et al (1992), Coloso et al (2003), Fontagne et al (2009), Ketola & Richmond (1994), Kim et al (1998), Kousoulaki et al (2010),
Liang et al (2012), Mai et al (2006), Phromkunthong & Udom (2008), Rodehutscord (1996), Rodehutscord et al (2000), Schaefer et al.
(1995), Shao et al (2008), Sukumaran et al (2009), Vielma et al (2002), Xie et al (2011), Yuan et al (2011), Zhang et al (2006).
et al (2009), Nwanna et al (2009), Ogino & Takeda (1976), Phromkunthong & Udom (2008), Robinson et al (1987), Roy & Lall (2003),
Schaefer et al (1995), Vielma & Lall (1998), Wang et al (2005), Yang et al (2005), Yuan et al (2011), Zheng et al (2007).
(1995), Vielma & Lall (1998), Yuan et al (2011), Zhang et al (2006).
Xie et al (2011).
Lall (1998), Vielma et al (2002), Xie et al (2011).
.
Trang 5Mercer’s nutrient kinetic model The description of the
model used is as follows:
basis
Of all the 64 studies (Table 1) that report a quantitative
die-tary P requirement or a dose–response relationship to graded
dietary P levels for a given fish species, only 40% (25 studies)
provide data on the dietary available P levels in the diets
used Of all the studies that report dietary P requirement in
fish (Table 2), the majority (59%) have used primarily
somatic growth as the response criterion Other response
variables such as vertebral ash or phosphorus content
(39.7%), whole-body phosphorus content (24.4%), P
reten-tion (10.3%), scale P (10.3%), non-faecal P excrereten-tion
(6.4%), plasma P (7.7%) and gene expression of P
transport-ers (1.5%) have also been used to estimate the dietary P
requirement of fish
Background information on the studies used in this
meta-analysis of data on P requirement is presented in Table 2
Estimation of the minimal dietary P levels based on
differ-ent response criteria on differdiffer-ent data sets, using the
bro-ken-line regression, is shown in Table 1 Within the
different data sets (complete and sub-data sets), the minimal
dietary P level was lower for available P compared with
analysis was higher for available P compared to total P
Meta-analysis of the complete data set on all species (All)
showed that minimal dietary P in terms of dietary
P per unit of DE in the diet, the requirement was estimated
Meta-analysis of the different sub-data sets based on
WG showed that minimal dietary P levels for attainingmaximal growth were lower in trout compared to the non-trout fish species, having non-overlapping confidence inter-vals (Table 1) The estimated minimal dietary P concentra-tion to attain maximum WG (Fig 1a; expressed per unit
excluding rainbow trout However, the minimal dietary level estimate for rainbow trout showed no difference withother species when studies conducted in recirculatory sys-tems were excluded from the data set Minimal dietary Pconcentration for maximal WG expressed in relation to DE
sub-data set on all species except rainbow trout (Fig 1b).When P requirement was estimated from the relationship
in the requirement for rainbow trout versus all species
Table 1) This was indicated by the overlapping confidenceinterval of the break points, being 0.07 g available P intake
spe-cies excluding rainbow trout (Fig 1c)
In comparison with WG, using whole-body P tion as the response criterion led to higher estimates for theminimal dietary P levels for all data sets (Table 1) Esti-mated minimal dietary P level for attaining maximal
complete data set (All) For the sub-data sets on ‘Rbt’ and
Trang 9Due to a limited number of studies on rainbow trout usingvertebral P content as the response criterion, minimal die-tary P levels based on this criterion were estimated only onthe complete data set (All) The estimated minimal dietary
P levels expressed as total P were higher when vertebral Pcontent was used as the response criterion compared towhole-body P content, but when expressed as available P,the estimated minimal dietary P levels were almost similarbetween vertebral and whole-body P concentration as theresponse criteria (Table 1) The estimated minimal dietary
DM) for all species combined (Fig 3)
Due to the low number of studies providing data onplasma P concentration, the estimated minimal dietary Plevels had a relatively large 95% confidence interval andoverlapped with most other response criteria Moreover, nobreak point could be observed on the sub-data set on allspecies excluding rainbow trout (Fig 4) Based on plasma Pconcentration, the minimal dietary P level was estimated as
rain-bow trout (Fig 4)
Besides interspecies differences, variations in experimentalconditions were also encountered among studies on Prequirements of fish In order to rule out species differ-ences, the impact of rearing system was assessed withinthe rainbow trout sub-data set In this data set, of theseven studies, two were carried out in recirculation sys-tems, while five in flow-through systems Using WG asthe response criterion, the estimated minimal dietary avail-able P level was found to be high when the two studies inrecirculation systems were excluded from the sub-data set.The minimal dietary available P level for maximal WG
–5.5) for rainbow trout reared in flow-through systems(Table 1) Moreover, as an example for the differencebetween flow-through and recirculation systems in the esti-mated minimal dietary available P level, Fig 5 gives acomparison of the relationship between dietary available Pand growth (calculated as thermal growth coefficient) of
Trang 10two rainbow trout studies with a similar initial bodyweight but differing in rearing system (Ketola & Rich-mond 1994; Rodehutscord 1996).
Broken-line analysis of the data resulted in a requirement
flow-through system, while with the recirculation systemhaving high P concentration in water, it was estimated to
95% confidence limits
a1 b1 Xbp b2
Rbt 57.0 12.3 2.70
~ 0.011
All - Rbt 37.3 11.7 4.71
Rbt 45.79 258.1 0.1592
~ 2.220e-018
All - Rbt 34.44 230.3 0.2461
Rbt 7.19 132 0.0651
~ 0.011
All - Rbt 2.60 105 0.0718
~ 0.011
(a)
(b)
(c)
Figure 1 Minimal dietary available P levels estimated through
meta-analysis, based on weight gain for ‘Rbt’ (filled circles, solid
line) and ‘All-Rbt’ sub-data sets (open circles, dotted line)
Rbt (Rainbow trout) All -Rbt (All except Rbt)
95% confidence limit
a1 b1 Xbp
Rbt 1.001 0.6554 5.852
All -Rbt 2.5 0.38 6.4
live weight) for ‘Rbt’ (filled circles, solid line) and ‘All-Rbt’ data sets (open circles, dotted line).
0 5 10 15
All species data 95% confidence limit
a1 b1 Xbp b2
All species 4.909 0.8671 5.156
~ 2.359e-018
vertebral P content (% of dry weight) estimated through analysis for ‘All’ data set (open circles, dotted line).
meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta- meta-.
Trang 11When constructing the data set (and various sub-data sets),
we noticed that there was a large variability in dietary P
content, both in range of dietary P levels and the lowest
level of P content (basal diet P concentration) tested Using
WG as the response criterion, minimal dietary available P
using all species (25 studies; Table 1) Of the 25 studies, 12
studies used diets made of non-refined (practical)
ingredi-ents for quantifying P requiremingredi-ents and 14 studies (56%)
had an available P concentration in the basal diet above
studies The mean basal dietary P concentration of the 14
DM) Analysis of data from these studies showed that
the minimal dietary available P level (i.e break point) was
the other studies having an available P content in the basal
con-fidence limits of the estimated minimal dietary P levels did
not overlap between these two sub-data sets
It is usual practice to express data on P requirement of fish
and/or minimal dietary P levels in terms of total P in the
diet (as fed or as on dry matter basis), despite the large
variability in the availability of dietary P to fish Minimal
dietary P level expressed in terms of available P gives morereliable quantification of the requirement than total P, asindicated by the wider confidence intervals (Table 1) Theavailability of P from different ingredients and inorganicforms of mineral supplements used in fish diets is known tovary (Kaushik 2001; Lall 2003), depending upon the fishspecies and type of inorganic source (Ogino et al 1979;Satoh et al 1997), feed processing conditions (Satoh et al.2002; Cheng & Hardy 2003), dietary phosphorus concen-tration (Riche & Brown 1996; Sugiura et al 2000b) Hence,
it is considered essential to measure the availability of Pfrom the diets in studies aiming at determining a quantita-tive P requirement of fish
Almost all P requirement studies in fish report data on
WG, most often used as a response criterion to estimate the
P requirement However, the use of WG as the response terion to determine minimal dietary levels of the micronutri-ents such as vitamins, minerals and trace elements for fish isquestionable (NRC 2011) Hardy et al (1993) reported thatgrowth was not affected in rainbow trout when fed a P-defi-cient diet, until the body P reserves depleted to below 80%
cri-of the initial body P levels This implies that the estimatedminimal dietary P level using WG as the response can belower compared with the estimate with whole-body P con-centration as the response criterion, depending on the dura-tion of the trial In the current study, the estimated minimaldietary available P level for achieving maximum WG was
Rbt 0.4227 1.274 3.006
through meta-analysis based on plasma P concentration (mmol
Figure 5 Possible impact of rearing system and high water P centration on the available P requirement estimate in rainbow trout Growth data (TGC) from Ketola & Richmond (1994), initial body weight 35 g, water P concentration not reported, flow-
and from Rodehutscord (1996), initial body weight 53 g, water P
Trang 12approximately 25–30% lower than for the requirement
esti-mate using whole-body P concentration as the response
cri-terion from data sets on all species and all species excluding
rainbow trout (Tables 1 & 2), which, in the case of rainbow
trout alone, was even 50% lower This might be due to an
effect of rearing system in the rainbow trout sub-data set
where two of the seven studies were undertaken in
recircu-lating aquaculture systems (RAS) with levels of up to 5 mg
When these two RAS studies were excluded (Rbt-RAS),
minimal dietary P level for maximum WG as the response
compared to the estimate based on whole-body P
concen-tration Also, the difference between minimal dietary P
lev-els for ‘Rbt’ and ‘All-Rbt’ sub-data sets was reduced to
abil-ity of fish to absorb phosphorus from water is still debated
Although few studies (Coffin et al 1949; Winpenny et al
1998) have denied this, few others have confirmed (Mullins
1950; Al-Kholy et al 1970) the uptake of P by fish from
water through radioisotope studies Moreover, some studies
looking into a P budget in fish have suspected the
possibil-ity of P uptake by fish from water when fed P-deficient diets
(Cowey 1995; Pimentel Rodrigues & Oliva Teles (2001);
Dias et al 2005) In line with the suggestion of P uptake
from water, reanalysis of WG data (TGC) from Ketola &
Richmond (1994) on rainbow trout (35 g initial body
regression (Fig 5) showed that WG plateaued at a dietary
analy-sis of the data on weight gain (TGC) from Rodehutscord(1996) showed that the plateau was reached at 2.2 g avail-
response study undertaken in an RAS (Rodehutscord et al
2000) wherein the data on P concentration in the rearingwater were not presented, plateaued at 1.7 g available P
by rainbow trout and whereby the estimate of minimal tary P level is affected This, however, requires clear confir-mation through more specifically designed studies
die-Comparing the minimal dietary P levels as estimatedthrough meta-analysis (Table 1) and mean of the values of
P requirements reported in different studies using differentresponse criteria (Table 2) reveals that the values frommeta-analysis are lower (25–43% for WG; 13–25% forwhole-body P; and 14% for vertebral P) than the valuesfrom Table 2 The large difference in WG can be possiblydue to methodological as well as physiological and environ-mental factors Methodologically, with WG, as the maxi-mum response was restricted to a value of 100 and
the plateau to be lower, forcing a lower breakpoint valuethan it would be if actual WG data were used as such(Table 1; Fig 1a,b) and difference in models used in differ-ent studies (Table 2) So, an attempt made to observe therelationship between dietary P and WG in absolute terms
Basal P > 3
48.13 7.047 6.400
~ 0.0110
Basal P < 3
40.12 13.69 3.710
~ 0.0110
Figure 6 Effect of high available P concentration in the basal diet
on the estimate of minimal dietary P level for maximal weight
circles, dotted line) in the basal diet.
Spearman r 95% confidence interval
P value (two-tailed)
P value summary
0.9787 0.9718 to 0.9840
< 0.0001
****
Slope 0.003652 ± 2.671e-005
with varying body weight (g) from multispecies data.
.
Trang 13confidence limits when expressed in absolute terms indicate
that the differences might not solely be due to the
method-ological approach but may have also been influenced by
physiological and environmental factors like fish size,
growth rate, feeding method, water temperature and
tro-phic level of the species Similar data analysis focusing on
the effect of these variables on the minimal dietary P level
would improve precision of our estimation of P
require-ment of fish
Another widely used approach to determine P
require-ment of fish is the use of whole-body P as the response
var-iable When whole body P is used as the criterion, the
minimal dietary P levels are estimated to be 20–30% higher
than those estimated with WG The possible reasons
under-lying this have been discussed earlier However, P retention
be more reliable response criteria for estimating the P
requirement of fish, provided the fish are not in a status of
P deficiency (Sugiura et al 2000a) and the duration of the
trial is long enough to nullify any effects due to the initial
P status of the fish Available data are limited to verify
whether there is a relationship between P retention and P
requirement and whether daily P gain per unit body weight
can change with increasing body mass or growth rate
Analysis of data (Fig 7), however, shows that the absolute
phosphorus content in fish varies linearly with body weight
previous findings that report P content in salmonids to be
weight for rainbow trout through different growing stages
(Bureau et al 2003)
Estimation of minimal dietary P level based on
verte-bral ash or P content analysis was also found to be
(Table 1) Because data on dietary available P were
pro-vided in only nine of 23 studies using vertebral P content
as a response criterion, it is difficult to make a conclusive
statement as regards vertebral P content as the most
strin-gent response criterion
During P deficiency, plasma and urinary P levels are
reduced (Rodehutscord 1996; Lall 2003), suggesting that
plasma P can be used as a response criterion for
estimat-ing P requirement of fish Plasma P concentration can vary
in relation to dietary input, mobilization from tissues, time
of sampling etc Thus, the use of this criterion as an index
of P requirement is less reliable and especially so for
cross-study comparisons From the results of the meta-analysis
(Tables 1 & 2), it was found that plasma P levels as an
index holds well for interstudy comparisons in a single cies data set (Rbt), only when the data are expressed interms of available P in the diet and the postprandial time
spe-of sampling does not vary drastically across studies.This meta-analysis shows that the P requirement esti-mates as minimal dietary P levels tend to vary depending
on the response criterion and the levels are higher when weuse whole-body or vertebral P concentration as the crite-rion than when we use WG as the response criterion WG
confi-dence limits) followed by whole-body P concentration; and
con-fidence limits) for the data sets ‘All’ and ‘All-Rbt’ ever, all response criteria analysed were found to fit better
val-ues Thus, under a given set of physiological and mental conditions, in terms of precision and variance, itcan be deduced that criteria based on whole-body P con-centration were found to be the most stringent of the fourdifferent response variables analysed
environ-Apart from these four widely used response criteria, fewother potentially effective response variables have also beeninvestigated, as summarized in Table 2 Scales are a majorsite of mineral metabolism, and resorption of scales takesplace during food deprivation (Lall 2003) Based on this,scale ash or P content has been used as a non-invasiveresponse for determining P requirement of fish in few stud-ies, but with limited success On the other hand, urinary Pexcretion has been found to be an effective and relativelyquick method for studying the P requirement and P status
of fish, especially in large fish (Rodehutscord et al 2000;Sugiura et al 2000a) Lately, gene expression analyses of Ptransporters in pyloric caeca, intestine and kidney have alsobeen employed to analyse the P status and estimate mini-mal dietary P levels in rainbow trout (Sugiura et al 2007).However, it is not clear whether such response criteria areconsistent and reliable to be useful in determining Prequirement of fish
In studies aiming at quantifying the requirement of anynutrient, the use of diets made of refined ingredients isideal Diets made of non-refined practical ingredients may
be used provided the basal diet contains very low levels ofthe nutrient under investigation (NRC 2011) Therefore,under conditions where the P concentration in the basaldiet itself is high enough to almost meet the P requirement
of the fish, the probability of overestimating the minimaldietary P level required is high (Fig 6) Hence, it is better
to avoid diets containing ingredients that are inherently
Trang 14rich in available phosphorus for quantifying the dietary P
requirement of fish
The unit or mode of expressing minimal dietary P levels
required to fish is very crucial and needs close attention It
was suggested that as the feed gain ratio and hence the P
intake per unit WG are determined by the digestible energy
content of the diet, expressing minimal dietary P levels as g
1996) Analysis of data to find the best way of expressing P
However, although a direct comparison is not possible,
takes into account the variations that may arise due to the
difference in fish size, P intake and growth rate, thus
pro-viding an estimate in absolute terms
From this meta-analysis, it can be concluded that there
exists considerable homogeneity of data on the P
require-ment of rainbow trout in flow-through system and all other
species when dealt with in terms of available P level in the
diets However, this needs confirmation through a
mixed-effect approach Among the response criteria analysed,
WG was more reliable and whole-body P was the most
stringent; therefore, using whole-body P as a variable
(whole-body P balance, retention, deposition or daily P
gain) could be more efficient in determining the P
require-ment of fish Environrequire-mental factors such as water P
con-centration and available P concon-centration of basal diet can
affect the estimation of the minimal dietary P level The
results of this meta-analysis could have been more robust if
more data on dietary available P were available, especially
for species other than rainbow trout Further studies on P
requirement of fish should necessarily provide data on
available P levels in the diets and basic information on feed
intake, macronutrient composition, DE and levels of other
minerals in the diets, as well as water P levels Similar work
on the analysis of available data on requirements of fish
for other essential macro- and microminerals is warranted
We acknowledge the contribution of Professor Daniel
Paris), Dr Inge Geurden, Dr Stephanie Fontagne and Dr
Genevieve Corraze (INRA St-Pee-sur-Nivelle), Professor
Dominique Bureau (Univ Guelph, Canada) and Professor
Santosh Lall (NRC, Halifax, Canada) for their valuable
suggestions This work was carried out as a part of thePhD programme funded by INRA under the INRA-WURAquaculture platform This work is also a contribution to
an EU-funded project (‘ARRAINA: Advanced ResearchInitiatives for Nutrition & Aquaculture’, KBBE-2011-288925)
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1
Institute, Sichuan Academy of Animal Science, Chengdu, China
This study was conducted to investigate the effect of dietary
phosphorus on the intestine and hepatopancreas antioxidant
capacity of juvenile Jian carp (Cyprinus carpio var Jian)
were fed with diets containing graded concentrations of
available phosphorus, namely 1.7 (control), 3.6, 5.5, 7.3, 9.2
intestine and hepatopancreas, content of malondialdehyde
(MDA), protein carbonyl (PC) and glutathione (GSH),
capacity of anti-superoxide anion (ASA) and anti-hydroxyl
radical (AHR), and glutathione reductase (GR), catalase
(CAT), glutathione S-transferase (GST), superoxide
dismu-tase (SOD) and glutathione peroxidase (GPx) activities were
qua-dratic responses occurred in MDA content and ASA, GST,
GPx and AHR activities in intestine, GSH content and CAT
results indicate that optimal level of dietary phosphorus
pre-vented oxidative damage and increased antioxidant enzyme
activities in the intestine and hepatopancreas of juvenile Jian
carp The phosphorus requirement estimated from MDA
Jian carp (Cyprinus carpio var Jian), phosphorus
Received 5 August 2011, accepted 12 February 2012
Correspondence: Xiao-Qiu Zhou, Animal Nutrition Institute, Sichuan
Agricultural University, Ya’an 625014, China E-mail: xqzhouqq@tom.
Mitra et al (2008) reported that digestive and brush borderenzyme activities in the intestine of Labeo rohita may berelated to the structure and function of digestive organ
Furthermore, intestinal function was found to correlatewith antioxidant status (Shoveller et al 2005) Study fromour laboratory demonstrated that structural integrity offish enterocyte is associated with oxidative damage degree(Chen et al 2009) Our previous study also indicated thatfish fed phosphorus-deficient diet induced putrescence andexfoliation of intestinal epithelium cell, putrescence and dis-integration of pancreatic gland alveolus in Jian carp (Cypri-
information is available regarding the effects of phosphorus
on antioxidant status of digestive organs, Xuan et al
(2000) reported that the antioxidant function of cytes in hypophosphatemic cows significantly decreased Itsuggested that dietary phosphorus deficiency may lead toantioxidant status reduction of fish digestive organs, whichneeds to be investigated
erythro-Reactive oxygen species (ROS) from mitochondria andother cellular sources have been traditionally regarded astoxic by-products of metabolism with the potential to causeoxidation to lipid and protein (Freeman & Crapo 1982)
To protect against the potential damage of ROS, cells sess a complete set of antioxidant defences that contained .doi: 10.1111/j.1365-2095.2012.00955.x .2013 19; 250–257
pos-Aquaculture Nutrition
Trang 19enzymatic and non-enzymatic systems (Sies 1986)
Enzy-matic antioxidant defences include superoxide dismutase
(SOD), glutathione peroxidase (GPx), catalase (CAT); as
well as non-enzymatic antioxidants represented by
(vita-min E) and other antioxidants (Valko et al 2007) There is
no information about the lipid peroxidation (LP) and
pro-tein oxidation caused by phosphorus deficiency in fish
SOD is the first enzyme to respond against oxygen radicals
and enzymatic degradation of superoxide anion radical to
SOD activity decreased in the erythrocytes of
hypophos-phatemic cows (Xuan et al 2000) Moreover, phosphorus
is the component of NADPH, which participates in the
reduction of GSSG to GSH as reducing potential and the
formation of active CAT tetramers (Salvemini et al 1999)
The reduced GSH acts as a substrate or cofactor for some
enzymatic reactions of the glutathione-dependent enzymes
such as GPx, glutathione S-transferase (GST) and
glutathi-one reductase (GR) (Elia et al 2006) Ogawa et al (1989)
found that GSH content of red blood cell decreased when
cows were fed with phosphorus-deficient diet Another
study on cows showed that GPx activity decreased in the
hypophosphatemic erythrocytes (Xuan et al 2000) In this
sense, phosphorus improved the structure and function of
intestine and hepatopancreas in fish may be partly related
to the improvement of antioxidant defence, which warrants
further investigation
The same growth trial was used in this study as Xie et al
(2011) This study was conducted firstly to determine the
possible effects of phosphorus on free radical-scavenging
ability and antioxidant activity in fish, which was a part of
larger research investigating the effects of phosphorus on
digestive and absorptive capacity in Jian carp (Xie et al
2011) The results would provide partial theoretical
evi-dence for the effect of phosphorus on digestive and
absorp-tive capacity in fish
The basal diet (Table 1) was formulated to contain 314.8 g
crude protein per kg diet and 51.0 g crude lipid per kg diet
starch were used as protein, lipid and carbohydrate
sources, respectively Lysine, methionine, pantothenic acid,
pyridoxine, inositol, thiamine, riboflavin, iron and zinc
were supplemented to meet the requirements of juvenile
Jian carp according to our laboratory’s studies (Zhou et al.2008; He et al 2009; Jiang et al 2009; Tang et al 2009;Wen et al 2009; Li et al 2010; Ling et al 2010; Huang
except phosphorus met or exceeded the requirements ofcommon carp (NRC 1993) The basal diet (control) wassupplemented with graded levels of monosodium phosphate
The corresponding available phosphorus levels in the diets
Table 1 Composition and nutrients content of the basal diet
starch to 1 kg.
897.97 g.
3
mix-ture): each treatment group containing monosodium phosphate
0 g, 178.33 g, 346.67 g, 516.67 g, 686.67 g and 863.33 g, tively Each monosodium phosphate mixture was diluted with corn starch to 1 kg.
determined according to the method of the AOAC (1998), and the total phosphorus in each experimental diet were 5.24, 7.34,
of available phosphorus was calculated based on the digestibility
of basal diet as determined by digestibility trial.
Trang 20were 1.7 (control), 3.6, 5.5, 7.3, 9.2 and 11.0 g kg diet,
respectively, which were also the same as our previous
study (Xie et al 2011) Diets were prepared by thoroughly
mixing all the ingredients Distilled water was included to
achieve a proper pelleting consistency, and the mixture was
further homogenized and extruded through a 2-mm die
The noodle-like diets were dried using an electrical fan at
All experimental protocols were approved by the Animal
Care Advisory Committee of Sichuan Agricultural
Univer-sity Juvenile Jian carp were supplied by Tong Wei
Hatch-ery (Sichuan, China) Fish were acclimatized to the
experimental environment for 4 weeks Exactly 1200 fish
con-nected to a closed recirculating water system with
continu-ous aeration Water was drained through biofilters so as to
reduce ammonia concentration and remove solid
sub-stances Water quality was the same as previously described
(Xie et al 2011) The experimental units were maintained
under a natural light and dark cycle For the feeding trial,
fish were fed with their respective diets six times daily from
1 to 4 weeks and four times daily from 5 to 9 weeks Fish
were fed to apparent satiation and uneaten feed were
removed by siphoning after 30 min of feeding
After feeding for 9 weeks, 15 fish were collected from each
aquarium 12 h after the last feeding The intestine and
hepatopancreas were quickly removed, frozen in liquid
and hepatopancreas samples were homogenized in 10
vol-umes (w/v) of ice-cold physiologic saline and centrifuged at
for antioxidant parameters analysis The protein content of
intestine and hepatopancreas was determined by the
method of Bradford (1976)
Malondialdehyde (MDA), a product of LP, was
mea-sured as thiobarbituric acid reactive substance (Livingstone
car-bonyl residues were determined according to the method
described by Armenteros et al (2009) The intestine and
hepatopancreas protein carbonyls content were calculated
from the peak absorbance at 370 nm, using an absorption
-scaveng-ing ability) and anti-hydroxy radical capacity ing ability) were determined using the method described byJiang et al (2010) SOD and glutathione peroxidase (GSH-Px) activity were measured according to the methoddescribed by Zhang et al (2008) CAT activity was mea-sured by the decomposition of hydrogen peroxide (Aebi1984) GST activity was measured by monitoring the for-mation of an adduct between GSH and 1-chloro-2, 4-dini-trobenzene (CDNB) (Lushchak et al 2001) GR activitywas assayed as previously described by Lora et al (2004)
(OH-scaveng-GSH content was determined by the formation of nitrobenzoate followed spectrophotometrically at 412 nmwith a minor modification (Vardi et al 2008)
5-thio-2-All data were subjected to a one-way analysis of variance
sig-nificant differences among treatment groups, and P < 0.05was considered to be statistically significant The parame-ters with significant differences were subjected to a linear
or quadratic regression model The quadratic equation used
was the content of MDA, PC and GSH and activities ofantioxidant enzymes in intestine and hepatopancreas;
of the quadratic terms, when the regression model was
Malondialdehyde, PC, SOD, ASA, anti-hydroxyl radical(AHR), CAT, GST, GPx, GR and GSH in intestine areshown in Table 2 Results indicated that MDA content
phosphorus levels and showed quadratic responses to
increased Both ASA capacity and GST activity
.
Trang 21(P > 0.05) Both AHR capacity and GPx activity were
higher for fish fed diet containing available
SOD activity was the lowest for fish fed the basal diet
activity showed quadratic responses to the increasing
The CAT activity was higher for fish fed diet containing
with a further increase in dietary phosphorus concentration
available phosphorus per kg diet, then followed by 9.2 and
11.0 g available phosphorus per kg diet, and there was no
Malondialdehyde, PC, SOD, ASA, AHR, CAT, GST,
GPx, GR and GSH in hepatopancreas are shown in
Table 3 Both MDA and PC were the highest for fishfed the basal diet, and the lowest for fish fed diet contain-
MDA and PC content showed quadratic responses toincreasing dietary available phosphorus concentrations
capacities of ASA and AHR were the highest when dietary
responses to increasing dietary available phosphorus
decreased with increasing dietary phosphorus levels up to
with a further increase in dietary phosphorus concentration
were with further increasing dietary available phosphorus
quadratic responses to increasing dietary available
0.05) GST activities were higher for fish fed diets
diet
Available P in the diet
Trang 22significantly (P< 0.05) increased with dietary phosphorus
then plateaued thereafter and showed quadratic responses
to increasing dietary available phosphorus supplementation
Dietary phosphorus requirement estimated by the quadratic
shown in Fig 1
Our previous study showed that dietary phosphorus isessential for maintaining normal growth of Jian carp (Xie
phospho-rus improved growth and development of fish intestine and
enzymes and brush border membrane enzymes (Xie et al
2011) Our laboratory study demonstrated that structuralintegrity of fish enterocyte is associated with oxidativedamage degree (Chen et al 2009) Oxygen, an obligate fuelfor aerobic life, has been shown to be toxic through its del-eterious reactive species, called ROS, which can cause oxi-dative stress and lead ultimately to cell and organismdeath, and markedly affect the physiology of the cell (Jans-sens et al 2000) Most components of cellular structureand function are likely to be the potential targets of ROS,and the most susceptible substrates for oxidation are poly-unsaturated fatty acids in the biomembrane, which undergoperoxidation rapidly (Zhang et al 2004) MDA is one ofthe most widely assayed end products of both enzymaticand non-enzymatic LP reactions (Requena et al 1996) Thepresent study showed that optimal level of phosphorus sig-nificantly declined MDA content in intestine and hepato-
depressed Protein oxidation damage can be induced byROS (Berlett & Stadtman 1997) and LP end products such
as MDA and 4-hydroxynonenal (Negre-Salvayre et al
2008) The content of protein carbonyl is the most widelyused biomarker for oxidative damage to proteins (Berlett &
Stadtman 1997) In the present study, we observed thatoptimal level of phosphorus significantly decreased protein
Figure 1 Quadratic regression analysis of malondialdehyde content
in intestine for juvenile Jian carp fed diets containing graded levels
of phosphorus for 9 weeks The dietary phosphorus requirement of
Trang 23carbonyl content in hepatopancreas, which took a similar
trend with LP, and was positively related to MDA
showed a pattern consistent with that in hepatopancreas,
which suggesting that protein oxidation in intestine was
also depressed by phosphorus supplementation
Superoxide anions as a species of oxygen radicals are
toxic by-products of respiration and, with a relatively long
half-life, can cause a wide range of oxidative damage
within the cell and might imply a higher toxicity of the
compound (Bai & Cederbaum 2001) Hydroxyl radicals are
considered to be the most reactive radical of oxygen in
bio-logical systems; because of its high reactivity, it can react
at a high rate with most molecules in the cell, including
DNA, protein, lipid and so on (Kohen & Nyska 2002) In
our study, we observed that optimal level of phosphorus
significantly increased the capacity of ASA and AHR in
intestine and hepatosomatic tissue, which suggesting the
beneficial effect of phosphorus on superoxide
radical-scav-enging ability and hydroxyl radical-scavradical-scav-enging ability
However, the detailed way was not clear and need further
investigation
As the first enzyme to respond against oxygen radicals,
& Di Giulio 1991) Interestingly, the present results showed
that optimal level of phosphorus can promote the activity
of SOD in fish intestine, while SOD activity decreased in
hepatopancreas with phosphorus supplementation Catalase
is one of the central enzymes involved in scavenging the
high level of ROS by breaking down hydrogen peroxide
(Li et al 2008) In our study, we observed that optimal
level of phosphorus significantly increased the activities of
CAT in intestine and hepatopancreas Phosphorus is the
component of NADPH, which is required for the
forma-tion of active CAT tetramers (Salvemini et al 1999)
Fur-thermore, the reduction of GSSG to GSH is catalysed by
GSSG reductase, which also uses NADPH as reducing
potential (Salvemini et al 1999) In the present study, we
found that GSH content in intestine and hepatopancreas
significantly increased with optimal level of phosphorus
supplement Similar observation has been reported in cows
(Ogawa et al 1989) The GSH acts as substrate or cofactor
for some enzymatic reactions of the glutathione-dependent
enzymes such as GST, GPx and GR (Elia et al 2006) In
this study, we observed that optimal level of phosphorus
significantly increased the activities of GST and GR in
intestine and hepatopancreas and the activity of GPx in
intestine, while pattern of GPx activity in hepatopancreas
was properly opposite with that in intestine To our
knowl-edge, no studies have been reported about the effect of tary phosphorus on activities of GST, GPx and GR in fish.Xuan et al (2000) found that the activity of erythrocytesGPx was decreased significantly in hypophosphatemiccows In a word, our results indicated that phosphoruscould increase enzymatic antioxidant capacity and GSHcontent in intestine and hepatopancreas of fish, which may
die-be related to its role as the component of NADPH ever, little information is available about the relationshipbetween phosphorus and fish antioxidant enzyme activities,and the detailed way of phosphorus-mediated antioxidantstatus warrants further study
How-The dietary phosphorus requirement estimated by thequadratic regression model based on MDA content in
diet, whichwas slightly higher than the requirement for SGR in Jian
In conclusion, phosphorus could promote the dant defence in intestine and hepatopancreas of fish byincreasing the free radical-scavenging ability, enzymaticantioxidant capacity and GSH content, thus protecting thestructure and function of these organs Therefore, thisstudy provides partial theoretical evidence for the improve-ment of carp digestive and absorptive capacity by phospho-rus Nevertheless, the specific molecule mechanism thatphosphorus mediates antioxidant defence in fish needs fur-ther investigation
antioxi-This study was supported by National Department Public
(201003020) and Program for New Century Excellent entsin University (NCET-08-0905) The authors would like
Tal-to thank the personnel of these teams for their kind tance
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1
Nutrition Research Group, School of Biomedical and Biological Sciences, The University of Plymouth, Plymouth, Devon,
UK
A nutrition trial with striped catfish (Pangasianodon
hypophthalmus) juveniles was undertaken to evaluate the
effect of replacing dietary fishmeal (FM) protein with corn
gluten meal (CGM) A diet with FM as the main protein
source was used as the control diet (FM) Five
for-mulated to progressively replace 20% (CGM20), 40%
(CGM40), 60% (CGM60), 80% (CGM80) and 100%
(CGM100) of FM protein Fifteen fish per tank (initial
80-litre fibreglass tanks connected to a closed recirculation
triplicate for 12 weeks The final weight and specific growth
rate (SGR) of fish fed diets CGM20, CGM40 and CGM60
were not significantly different compared to fish fed the
FM diet Feed intake (FI) tended to decrease with
increas-ing dietary CGM level Striped catfish fed FM, CGM20
and CGM40 had significantly lower feed conversion ratio
(FCR) compared with fish fed CGM80 and CGM100
the CGM80 and CGM100 diets was significantly lower
ammo-nia-nitrogen (TAN) excretion increased with elevated
die-tary CGM inclusion The viscerosomatic index (VSI) of
fish fed the CGM80 and CGM100 diets were significantly
treat-ments The crude lipid content in the final body
composi-tion of the striped catfish was elevated significantly with
increasing dietary CGM levels Fish fed the CGM80 and
CGM100 diets displayed haematocrit levels significantly
haemo-globin content in fish was significantly higher in fish fedCGM20 and lower at CGM100 compared to fish fed the
FM diet The results of the present trial indicated that theoptimum level of FM protein replacement with CGMdetermined by quadratic regression analysis was 25.1% onthe basis of maximum SGR
Pangasianodon hypophthalmus, Striped catfish, total nia excretion
ammo-Received 14 October 2011; accepted 14 February 2012 Correspondence: Betu¨l Gu¨roy, Department of Aquaculture, Armutlu Vocational College, University of Yalova, Yalova, 77500, Turkey.
E-mail: bguroy@yalova.edu.tr
In recent years, the aquaculture output of the striped fish (Pangasianodon hypophthalmus), generally known aspangasius, has grown rapidly with production output in
cat-2008 exceeding over 1 million MT (Phan et al 2009;
Pos-ma 2009) The Pos-market for this fish continues to grow, ticularly in the United States where catfish are a popularfood fish; indeed, in 2010 alone, over 45 000 MT of panga-sius were imported to the USA (Globefish 2011) Vietnam
par-is by far the largest pangasius producer, but Thailand,China, Bangladesh and Cambodia also culture pangasius
on a commercial scale (FAO 2010)
Fishmeal (FM) is the primary protein source used inaquafeeds because of its well-balanced essential amino acid(EAA) profile, essential fatty acids, energy and minerals It
is also highly palatable and highly digestible to most finfishspecies (Hertrampf & Piedad-Pascual 2000) The global .doi: 10.1111/j.1365-2095.2012.00953.x .2013 19; 258–266
Aquaculture Nutrition
Trang 27production of FM has reached a plateau with total annual
production at approximately 6 million metric tons (Tacon
& Metian 2008) As the aquaculture industry is projected
to continue expanding, FM must be used more strategically
as the required aquafeed production volumes increase
Thus, the use of alternative less-expensive and more
sus-tainable protein sources is considered a high priority in the
aquafeed industry Plant ingredients are now widely used at
appreciably high levels in fish feeds owing to the current
market scarcity and rapid increase in the cost of FM
(En-gin & Carter 2005; Glencross et al 2007; Tacon & Metian
2008)
Corn gluten meal (CGM) is considered to be a
cost-effective alternative protein source for aquafeed owing to
its high content of available protein with a reasonably
well-balanced amino acid profile, high digestibility, competitive
price and steady supply compared to other vegetable
pro-tein sources (Hertrampf & Piedad-Pascual 2000) There
have been numerous studies on the utilization of CGM in
aquafeeds for various fish species such as the gilthead
seab-ream (Sparus aurata) (Robaina et al 1997; Pereira &
Oliva-Teles 2003), turbot (Psetta maxima) (Regost et al
1999), Japanese flounder (Paralichthys olivanceus) (Kikuchi
(Lewis & Kohler 2008) and puffer (Takifugu fasciatus)
(Zhong et al 2011) However, no information is presently
available regarding the effects of substituting FM with
CGM in striped catfish diets The influence of replacing
dietary FM on feed performance and growth is species
dependent Therefore, the aim of the present study was to
evaluate the effects of total and partial replacement of FM
with CGM on feed efficiency, growth response and carcass
composition of striped catfish Additionally, analyses of
total ammonia-nitrogen (TAN) excretion and selected
hae-matological parameters were also undertaken
The experiments were carried out at the Aquaculture and
Fish Nutrition Research Aquarium, University of Yalova,
Turkey Four hundred fish were obtained from a local
commercial aquarium, Istanbul, Turkey, and acclimated to
laboratory conditions for 6 weeks during which they were
fed a commercial diet (Camli Feed Company: crude protein
into 18 80-L (15 fish per tank) fibreglass tanks containing
aerated recirculated freshwater Fish were fed experimentaldiets twice a day (08:00 and 17:00) until apparent satiation
light: dark photoperiod Fish were weighed individually atthe beginning and the end of the feeding trial and bulk-weighed fortnightly during the experimental period Water
pH was maintained between 6.8 and 7.5, dissolved oxygen
diets were formulated A diet with FM as the main proteinsource was used as the control diet (FM) The experimentaldiets were formulated to produce diets in which 20(CGM20), 40 (CGM40), 60 (CGM60), 80 (CGM80) and
100 (CGM100)% of FM protein was replaced by that ofCGM protein The proximate composition of the FM andCGM used is shown in Table 1 Fish oil and corn oil wereused at a 1:1 ratio The full dietary formulations are dis-played in (Table 2) Dietary ingredients were mixed in afood mixer (model no: IBT-22; Dirmak Food Equipment,Turkey) with warm water until a soft slightly moist consis-tency was achieved This was then cold-press-extruded(PTM P6 extruder; Yalova, Turkey) to produce a 2-mmpellet The moist pellets were then fan-dried and stored fro-
the amino acid composition of the experimental diets areshown in Tables 2 and 3, respectively
Six fish samples were randomly taken from the initial pool
at the beginning of the experiment, and three fish fromeach tank were sampled at the end of the trial to conductwhole-body proximate analysis Analysis of moisture, crudeprotein and ash in diets and the whole body of fish were
) of meal (FM) and corn gluten meal (CGM)
Trang 28performed according to standard AOAC (2000) procedures.
constant weight was obtained Ash content was determined
after acid digestion using the Gerhardt system Crude fibre
was determined by acid/alkali hydrolysis recovering filtered
residue and ignition of the dried sample for 3 h Dietary
and whole-body lipids were extracted according to the
procedure of Folch et al (1957) with chloroform/methanol
(2:1 v/v) (AOAC 2000) Nitrogen-free extract (NFE) wascalculated by taking the sum values for moisture, crudeprotein, lipid, ash and crude fibre and then subtracting thisvalue from 100 (Olvera-Novoa et al 1994) Dietary grossenergy was calculated using the conversion factors of
To analyse amino acids, 60 mg of diet was hydrolysed in
10 ml of 6 M HCl in screw-capped tubes The tubes were
hydrolysates were rotary-evaporated to dryness under
buffer at pH 2.2 The amino acids were separated byion-exchange chromatography on a sodium column anddetected following postcolumn derivatization with ninhy-
Identifica-tion and quantificaIdentifica-tion of the detected AA was performedusing external standards after adjustments by linear regres-sion The standard AA was purchased from Sigma-Aldrich
Co, USA, as a synthetic mixture of AAs
After the feeding trial, fish were starved for a period of
3 days to ensure evacuation of food from the gut On themorning of the fourth day, tanks were thoroughly cleanedand fish in all tanks were fed the appropriate diet to appar-ent satiation Thirty minutes postprandial, water flow toeach aquarium was discontinued, uneaten food removed,and a baseline TAN excretion level analysed using the
(DR-2800; HACH, Loveland, CO, USA) At 6 h dial, a further sample was taken and analysed and theTAN levels determined by subtracting the baseline value
postpran-Blood was sampled from three fish per tank at the end ofthe trial Samples were taken from the caudal arch using a32-gauge needle and 2-mL syringe Haematocrit levels weredetermined by drawing fresh blood into microhaematocrittubes and centrifugation at 3600 g for 6 min in a microhae-matocrit centrifuge Haematocrit values were measured and
Haemoglobin levels were estimated using Sahli’s method
weight basis) of experimental diets
0.411 mg; folic acid: 0.685 Agromey Feed Mill Company, I˙zmir,
Turkey.
0.274 mg; Se: 0.0274 mg; Ca: 125 mg; K: 189 mg, Agromey Feed
Mill Company, I˙zmir, Turkey.
Trang 29Erythrocyte counts were determined by diluting whole blood
in Dacies solution (1/50 dilution) and enumeration in a
haemocytometer To determine differential leucocyte levels
(cell identifications based on the descriptions of
Mungkorn-karn & Termtachartipongsa 1994), blood smears were
pre-pared, stained with Giemsa (BDH) and mounted in DPX
(BDH) A minimum of 100 cells per sample were counted
from six fish per dietary group, and values were expressed as
a percentage of the total leucocytes (Merrifield et al 2010)
Growth performance, in terms of specific growth rate
(SGR), feed conversion ratio (FCR), protein efficiency
ratio (PER) and net protein utilization (NPU) were
deter-mined using the following formulae:
The somatic indexes were calculated on three fish per
tank according to the following formulae:
))
where FBW is final body weight (g), IBW is initial body
weight (g), FI is feed intake (g), WG is weight gain (g), T
is time (days), PI is dietary protein intake (g), PG is protein
gain (g), FL is fish length (cm), GW is gut weight (g), LW
is liver weight (g) and VW is viscera weight (g)
All data were subjected to a one-way analysis of variance
treatments Duncan’s multiple-range test was performed torank the groups using Statgraphics 4.0 (ManugisticsIncorporated, Rockville, MD, USA) statistical software(Zar 2001) Quadratic regression analysis using the GLMmethod was conducted to analyse the SGR of the fish inresponse to FM protein replacement by CGM The rela-tionship between TAN excretion rates and dietary FM pro-tein replacement levels, as a dietary percentage, wasdescribed by linear regression of the form y = a + bx,where y is the excretion rate of TAN and x is the dietary
FM replacement levels (Gu¨roy et al 2012) Regressionanalysis was used to describe the relationships between FMprotein replacement levels and TAN excretion rates forindividual tanks in each treatment All values were consid-ered significant at the 5% level
The growth performance and feed utilization parametersfor juvenile striped catfish fed increasing levels of CGM areshown in Table 4 Survival rate was 100% for all treat-ments Mean final weight ranged from 28.48 g (CGM20) to16.37 g (CGM100) and was significantly lower in fish fedCGM100 than the other diets Additionally, the growth offish fed diets CGM20, CGM40 and CGM60 was not signif-icantly different compared to fish fed the FM diet Qua-dratic regression analysis indicated that a maximum growthoccurred at 25.1% replacement of FM protein by CGMprotein (Fig 1), which is effectively a replacement of12.5% FM by weight basis
Feed intake (FI) tended to decrease with increasing tary CGM level, and fish fed diets FM, CGM20, CGM40and CGM60 had significantly higher FI values comparedwith fish fed the CGM80 and CGM100 diets The fish fed
die-FM, CGM20 and CGM40 displayed significantly lower
Table 3 Amino acid composition of experimental diets (g/100 g N)
Trang 30FCR compared with fish fed CGM80 and CGM100
than that of CGM20, CGM40 and CGM60, no significant
The PERs of fish fed the CGM100 (1.16) and CGM80
(1.79) diets were significantly lower than those of all other
for fish fed the CGM100 diet was significantly lower
Total ammonia-nitrogen (TAN) excretion data are
pre-sented in Table 4 and Fig 2 Replacing FM with CGM
in the striped catfish diets tended to increase nitrogen
excretion The TAN of the CGM100 diet was significantly
higher than that of the other diets The TAN of fish fed
replacement levels (x; %) and the mean TAN excretion
Data on biometric parameters of fish fed the experimentaldiets are displayed in Table 5 The condition factor (CF)was higher for fish fed with the FM and CGM20 diets thanthose fed diets CGM80 and CGM100 The viscerosomaticindex (VSI) of striped catfish fed the CGM80 and CGM100
other diets, and the same trend was observed, inversely, forthe dress-out (DO) content
Total and partial replacement of dietary FM by CGMsignificantly affected the whole-body proximate composi-tion of the striped catfish (Table 6) No significant differ-ences were found in the body protein levels of catfish fedthe various test diets and control diets at the end of theexperiment, but the crude lipid content of the experimentalfish increased significantly with increasing dietary CGMlevels The data were also analysed by linear regressionwith either dietary FM protein replacement levels or bodylipid levels as a variable The relationship between dietary
FM protein replacement levels (x; %) and the body lipid
The haematological parameters of striped catfish fed theexperimental diets are shown in Table 7 The PCV (%) infish significantly decreased with CGM inclusion beyond60% replacement of FM protein (i.e diets CGM80 andCGM100) Compared to the control group, fish fedCGM20 contained significantly higher haemoglobin levels,whereas fish fed CGM100 displayed significantly lower hae-
differ-ential leucocyte proportions were not affected by dietarytreatment
This is the first study where CGM has been evaluated as a
FM substitute in diets for striped catfish The results showthat low-level inclusion of CGM as a partial substitute for
FM supports good growth performance, which could be
Figure 1 The quadratic relationship between specific growth rate
(SGR) and dietary corn gluten meal (CGM) protein replacement
level in striped catfish.
.
Trang 31economically beneficial with respect to striped catfish
pro-duction The quadratic regression analysis of SGR
indi-cates that optimum growth performance occurred at 25.1%
replacement of FM protein by CGM protein However,
there was an inverse relationship between high CGM
inclu-sion level and the SGR of striped catfish, which resulted in
significantly lower growth performance and weight gain in
has been studied as an alternative protein source for FM in
finfish diets and appears to be a promising protein source
when incorporated at moderate levels: that is, up to 10%
(14.8% FM protein replacement), 18%, 20% (20% FM
protein replacement) and 40% (60% FM protein
replace-ment) CGM can be utilized in diets for puffer (Zhong et al
2011), sunshine bass (Lewis & Kohler 2008), turbot
(Regost et al 1999) and gilthead seabream (Pereira &
Oliva-Teles 2003), respectively, without negatively
impact-ing growth performance The present findimpact-ings are in
agree-ment with those reported in other species and suggest that
CGM could be used as a partial substitute for FM in
striped catfish diets
As feed intake (FI) values were significantly depressed
with increasing FM substitution in the present trial, the
could be due to lower dietary available energy It is well
known that dietary inclusion of high plant protein levelsaffects palatability, and recently, reduced striped catfish FIhas been reported with increasing dietary soybean meal lev-els (Phumee et al 2011) Lower FI is directly proportional
to the protein intake, which in turn affects growth rate andnutrient utilization
This may help explain the reductions in growth mance observed in the present study However, it wouldappear that amino acid imbalances caused by the replace-ment of FM with CGM also contributed to reduced growth
perfor-in high CGM diets Lysperfor-ine appears to be the first limitperfor-ingamino acid in the present study with the lysine concentration
of the CGM80 (2.80%) and CGM100 (1.81%) diets farbelow that of the FM diet (8.60%) and also below musclelysine levels (the only comparable indication of lysinerequirement level of striped catfish) reported previously byPhumee et al (2011) Lysine is often one of the first limitingamino acids in fish feeds when FM is replaced by plant pro-tein sources and can lead to reduced growth as well aschanges in carcass composition (Li et al 2009) Indeed, inthe present study, striped catfish fed diets containing thehigher levels of CGM exhibited significantly elevated carcasslipid levels A possible reason for this is that the lysine reduc-tion level related to increasing CGM inclusion in the dietsalong with other EAA imbalances, such as lowered arginine,led to a disproportionate use of dietary energy from aminoacid deamination and subsequent metabolism of ketogenicmetabolites It is well known that a lack of lysine leads to alower N retention in fish and the remaining amino acids arechannelled along different metabolic pathways (gluconeo-genesis and ketogenesis); this was further supported by thefact that the highest fat deposition and viscerosamatic indi-ces were recorded in catfish fed with CGM80 and CGM100where the lysine imbalance was most evident
An excessive (up to 14.35% N for CGM100) provision
of dietary leucine was present in diets rich in CGM, whichmay also pose specific metabolic problems This level wasfar beyond the levels of the FM diet level [7.53%], previ-ously reported pangasius muscle [7.7%] (Phumee et al.2011) and the known requirements for related fish species
Figure 2 The relationship regression between dietary FM protein
replacement levels and TAN excretion for striped catfish.
Trang 32(NRC 2011) It is important that the ‘ideal protein’ concept
is also applied to diet formulations for fish (Wilson 2002)
as established for many other terrestrial farmed animals
and that deficiencies and excesses of specific amino acids
are minimized FM is often utilized as a baseline for
com-parisons, but FM itself is not the ideal protein source for
fish Dietary crude ash levels were reduced with increasing
CGM as a consequence of the decreasing FM contribution
as a dietary mineral source Thus, the reduced dietary ash
content in high-inclusion CGM diets may also have had a
negative contribution towards growth performance
Feed conversion ratio in fish fed the diets with CGM
protein replacement level in excess of 60% was significantly
higher than in the other dietary groups including the FM
group When the CGM protein replacement level increased
from 0% to 60%, the PER increased significantly, but with
the increase in CGM protein replacement level from 80%
to 100%, the PER decreased significantly The reasons for
the decline in feed utilization and PER could be a decrease
in feed intake Similar results were observed in gilthead sea
bream (Robaina et al 1997), turbot (Regost et al 1999)
and Japanese flounder (Kikuchi 1999)
There is no information concerning the effects of
differ-ent protein sources on TAN excretion of striped catfish
Ammonia is produced in fish as an end product of protein
catabolism, and excretion of ammonia is found to be high
when protein synthesis is low (Engin & Carter 2001, 2005)
Therefore, increased TAN excretion can be an indicator ofreduced protein synthesis, expressed as lower growth andprotein retention In the present study, TAN excretionincreased with the dietary inclusion level of CGM; thehighest TAN excretion was observed in the CGM100 diet,but there were no significant differences between theCGM0 and CGM40 diets Results on TAN excretion cor-related well with those of growth and nutrient utilization,
in that reduced growth performance, owing to amino acidimbalances and reduced FCR, resulted in elevated TANexcretion levels Fish fed the FM, CGM20 and CGM40diets had the best growth and nutrient utilization, and fishfed the FM and CGM20 diets exhibited the lowest amount
of TAN excretion Compared to fish fed the FM diet, anincrease in TAN excretion in response to elevated levels ofplant meals has previously been reported in rainbow trout(Oncorhynchus mykiss) (Yang et al 2011), gilthead seab-ream (Robaina et al 1995) and Asian seabass (Latescalcarifer) (Tantikitti et al 2005) fed with soybean mealand European seabass (Dicentrarchus labrax) fed dietCGM (Ballestrazzi et al 1994) and other multiple plantprotein sources (Kaushik et al 2004)
In terms of general health status, haematological teristics showed that the haemoglobin and haematocrit lev-
Haemoglobin and haematocrit levels are important tors of fish health; the present results indicated that adecrease in haematological values coincided with inferiorgrowth performance, especially in the CGM100 group con-
decreased haematocrit and haemoglobin values in Japaneseflounder fed high dietary CGM levels We hypothesize thatthis might be due to lower iron levels in the CGM richdiets compared to FM based diets as CGM iron levels areappreciably lower then FM iron levels (Cheng & Hardy2003; Bragado´ttir et al 2004) Signs of iron deficiency such
as reduced growth, poor feed conversion, and repressedhaematocrit and haemoglobin are well declared in fish fedhigh plant protein diets (Lall 2002)
Table 6 Whole-body composition of striped catfish after 12 weeks of feeding on experimental diets
Figure 3 The relationship linear regression between dietary FM
protein replacement levels and body lipid levels of striped catfish.
.
Trang 33This study shows that 60% of FM protein could be replaced
by CGM in juvenile striped catfish without causing any
adverse effects on growth and feed utilization A quadratic
equation according to regression analysis of SGR against
dietary fishmeal replacement level indicated that the optimal
level of dietary CGM protein replacement for maximum
growth was in the order of 25.1% It is suggested that
sup-plementary crystalline L-lysine could enhance the nutritional
value of CGM diets and thereby increase FM replacement
levels to over 60% if the requirement for this first limiting
amino acid was met Further studies are needed to verify the
actual requirement of lysine in diets for striped catfish
Indeed, it would be most preferable to characterize the
digestible EAA requirements for this species in respect of the
major protein sources such as FM and plant proteins to
achieve optimum EAAs in practical diets This could also be
valuable in terms of using complimentary sources of protein
concentrates to temper deficiencies or excesses of EAAs and
attaining the ‘ideal protein’ balance
CGM clearly offers much potential for inclusion in diets
for striped catfish, and this preliminary study provides a
basis for more extensive investigations
We would like to thank Agromey Feed Mill Company,
Otuz petshop (www.otuz.com), Kartal Chemical
Incorpo-rated, Cargill (Istanbul, Turkey), and DSM Nutritional
Products (Turkey) for providing feed ingredients This
research has been supported by Yalova University
Scien-tific Research Projects Coordination Department Project
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Trang 351 1 2 2 3 1 1
Department of Marine Bio-Materials and Aquaculture/Feeds and Foods Nutrition Research Center, College of Fisheries
Gangwon-do, Korea
Two feeding trials were carried out to determine the
opti-mum feeding rates in juvenile olive flounder, Paralichthys
olivaceus, at the optimum rearing temperature Fish
com-mercial diet at the feeding rates of 0%, 3.0%, 4.0%,
feeding trials lasted for 2 weeks Results from experiment 1
indicated that weight gain (WG) and specific growth rate
(SGR) of fish fed to satiation were significantly higher than
those of fish fed at other feeding rates while feed efficiency
(FE) and protein efficiency ratio (PER) of fish fed at
indi-cated that the optimum feeding rates in 5.0 and 20 g
respectively Broken line analysis of WG suggested the
5.0 and 20 g fish, respectively Therefore, these results
for 20 g size of juvenile olive flounder
at the optimum rearing temperature
Paralichthys olivaceus
Received 29 July 2011; accepted 22 February 2012 Correspondence: S.C Bai, Department of Marine Bio-Materials and Aquaculture/Feeds and Foods Nutrition Research Center, College of Fisheries Biology, Pukyong National University, 599-1 Daeyeon-3-dong, Nam-gu, Busan 608-737, Korea E-mail: scbai@mail.pknu.ac.kr
Feed intake is perhaps the principal factor affecting growthrate of fish (Li et al 2004) Several studies have shown thatgrowth correlates to food intake (Silverstein et al 1999;Mihelakakis et al 2002; Cho et al 2007; Kim et al 2007;Wang et al 2007; Ozorio et al 2009) Adequate supply ofnutritionally balanced feed is very important to ensureoptimum growth, survival, improved immunity to disease,good dress-out yield, and desirable organoleptic properties
of flesh These in turn increase profitability of an ture venture Inadequate supply of diet leads to reducedgrowth and survival of fish Conversely, overfeedingincreases fish production cost and reduces profitability due
aquacul-to increased cost of feed and deterioration of water quality,which can eventually reduce growth of fish Food applica-tion rate does not necessarily equal food consumption rate
in large scale commercial systems, since a significant andunknown portion of administered food may not be eaten(Yamada 1985) Hence, optimum feeding rates should bedetermined in fish to optimize profitability and productquality
Optimum feeding rate varies with feeding frequency,nutrient content of feed, fish species, fish size, water tem-perature and other water quality parameters, etc Channel
diet when both were fed at approximately 75% of satiationbut weight gain (WG) of fish was not different when thefish were fed to satiation (Minton 1978) This suggests a
.
Aquaculture Nutrition
Trang 36relationship between feeding rate and nutrient (protein)
content of diet A somewhat similar observation was made
by Li & Lovell (1992a,b) in the same species Channel
cat-fish, I punctatus, fed twice daily consumed more feed and
had faster growth rates than fish receiving a single daily
meal (Collins 1971; Andrews & Page 1975), indicating the
dependence of feeding rate on feeding frequency Hung &
Lutes (1987) and Hung et al (1989, 1995) stated that the
optimum feeding rates for growth of fish could be affected
by fish size Hung et al (1993) reported the optimum
feed-ing rate for 30 g white sturgeon, Acipenser transmontanus,
feeding rates for the same species were reported to be
and 0.25–0.5 kg sizes, respectively (Hung & Lutes 1987;
Hung et al 1989) The optimum feeding rate of white
suggest that optimum feeding rates have to be defined for
specific species at different growth stages and set conditions
Olive flounder, Paralichthys olivaceus, is one of the most
commercially important marine aquaculture species in
Korea, ranking first among the marine finfish aquaculture
production in the country in 2009 According to FAO
(2011) olive flounder contributed approximately 50%
(54 674 metric tons) of the 109 516 metric tons of marine
finfish aquaculture production in Korea in 2009, a huge
increase from 249 metric tons in 1989 and 21 368 metric
tons in 1999 This sharp increase in production is
attrib-uted to various policies by the government to encourage
aquaculture production in the face of the dwindling capture
production of this species
Due to the importance of olive flounder in Korea,
vari-ous studies have been conducted in this species (Wang
2008; Sun et al 2007a,b; Yoo et al 2007) However,
opti-mum feeding rates for the entire growth stages in this
spe-cies are yet to be determined Kim et al (2007) determined
the optimum feeding rate in juvenile olive flounder reared
high; hence, it could be better to determine the feeding rate
over a shorter growth period, as the feeding rate could
eas-ily change with size Furthermore, Cho et al (2006)
reported the optimum feeding rate in 17 g juvenile olive
flounder in terms of the percentage of satiation (95%)
on farm considering that the farmers may not have to carry
out the feeding trial to determine the satiation point Onthe contrary, knowing the weight of fish, the feed allow-ance can easily be estimated if the optimum feeding rate isreported in percent body weight per day Recently, we car-ried out a series of studies to determine the optimum feed-ing rates in olive flounder at various growth stages As apart of the series, here we report the optimum feeding rates
in 5 and 20 g juvenile olive flounder reared at the optimumtemperature, based on growth performance, whole-bodyproximate composition, serological characteristics and his-tological changes in fish
Commercial feed used in these feeding trials was supplied
by Suhyup Feed Company Limited (Uiryeong, sangnamdo, Korea) Pellet sizes with the correspondingproximate composition were selected according to the sizes
Gyeong-of fish, as done on farm and in line with the company mendation Proximate composition of feed used in the feed-
used
Fish for experiment 1 were collected from Hampyeong,Cheonnam while those for experiment 2 were obtained
Table 1 Proximate composition of the experimental diets for 5 and
20 g juvenile olive flounder, Paralichthys olivaceus (dry matter basis)
Composition Experiment 1 (5 g)
Experiment 2 (20 g)
Trang 37from Jeju-do, both in Korea Prior to the start of the
experiment, fish were fed the respective commercial diets
acclimate them to the experimental diets and conditions
were randomly distributed into each of 28 aquaria in
exper-iment 1 and 18 aquaria in experexper-iment 2 Each aquarium
was then randomly assigned to one of four replicates of
seven respective feeding rates: 0%, 3.0%, 4.0%, 4.25%,
aquarium was randomly assigned to one of three replicates
of six respective feeding rates: 0%, 1.0%, 2.0%, 3.0% and
were hand-fed three times a day at 08.00, 13.00 and
18.00 h with each daily ration divided into three portions
For satiation feeding, an excess amount of feed was
weighed out daily and fish were fed as much as they would
ingest (within 20 min) at each feeding time Feeding was
done slowly and carefully to ensure total ingestion of feed
The remaining feed was weighed and its weight subtracted
from the initial weight Total body weight in each
aquar-ium was determined at the end of the first week and the
amounts of diet fed to fish were adjusted accordingly
Experiment 1 was conducted by using a semi-recirculating
system with twenty-eight 30 L rectangular aquaria receiving
tank while a flow-through system with eighteen 50 L
aqua-ria receiving filtered seawater from the center tank was
used in experiment 2 All the experimental aquaria were
maintained at 12 : 12 (light : dark) The seawater
heaters in the center tank during the whole experimental
period and supplemental aerations were provided to
in both
in the feeding trials and both trials lasted for 2 weeks
At the end of the feeding trial, fish were starved for 24 h,
counted Weight gain, specific growth rate (SGR), feed
effi-ciency (FE), protein effieffi-ciency ratio (PER), survival and
whole-body proximate composition were calculated and
measured Blood analysis was also carried out on fish in
experiment 2, as fish in experiment 1 were too small for
blood to be obtained easily Blood samples were obtainedfrom the caudal vessels of three individual fish per aquar-ium by using a heparinized syringe and pooled by treat-ment groups for the determination of hematocrit (HCT)(packed cell volume; PCV), hemoglobin (Hb), glutamic ox-aloacetic transaminase (GOT), glutamic pyruvic transami-nase (GPT), blood glucose and serum total protein.Hematocrit was determined using the microhematocritmethod and hemoglobin (Hb) was measured by the cyanm-ethemoglobin procedure using Drabkin’s reagent (Brown1980) An Hb standard prepared from human blood(Sigma Chemical, St Louis, MO, USA) was used Serumsamples were prepared from blood on clotting by centrifu-
commercial clinical investigation kits (NeoDin Co., Ltd.,Korea) and the specific methods employed are as follows:the biuret method for serum total protein, the enzymaticmethod for blood glucose and the Reitman-Frankelmethod for GOT and GPT Three fish from each aquariumwere used to analyze whole-body proximate composition.Proximate composition analyses of experimental diets andfish body were performed by the standard methods of
(1995) Samples of diets and fish were dried to constant
soxh-let extraction using Soxtec system 1046 (Foss, Hoganas,
after acid digestion
Clinical signs of fish were carefully monitored dailythroughout these feeding trials At the end of the feedingtrials tissues were obtained from the hepatopancreas, kid-ney and anterior intestine of three randomly selected fishfrom each tank for histological examination Tissues werefixed in Bouins solution and processed for a routine histo-logical examination
Analytical Software; St Paul, MN, USA) to test for thedietary treatments When a significant treatment effect wasobserved, a Least Significant Difference (LSD) test wasused to compare means Treatment effects were considered
Trang 38estimate the optimum feeding rate in olive flounder
(Rob-bins et al 1979)
At the end of 2 weeks of feeding trial in experiment 1, WG
significantly higher than those of fish fed at other feeding
There were no significant differences in these parameters
significantly higher than those of the starved fish but
signif-icantly lower than those of fish fed at other feeding rates
SGR, which were significantly lower than those of fish fed
the experimental diet at all feeding rates Feed efficiency
fed to satiation However, there were no significant
differ-ences in these parameters among the other treatments,
except for the unfed fish, which had no values for these
parameters, as they received no feed Survival of the unfed
fish was significantly lower than those for other treatments
no significant differences in survival of fish fed the
experimental diet at all feeding rates Broken line analysis
of WG indicated that the optimum feeding rate in 5.0 g
juvenile olive flounder, Paralichthys olivaceus, could be
Table 3 shows the whole-body proximate composition of
5 g juvenile olive flounder fed the experimental diet at
dif-ferent feeding rates There was a trend towards decreasedwhole-body moisture and increased whole-body crude lipidwith feeding rate, although there was a drop in crude lipidcontent of fish fed to satiation Moisture and crude ash ofthe unfed fish were significantly higher than those in other
signifi-cantly lower than those of fish fed at 0%, 3%, 4% and
whole-body crude protein of fish in all treatments
Histological changes in the hepatopancreas, kidney andanterior intestine of 5 g juvenile olive flounder fed theexperimental diet for 2 weeks are shown in Fig 2 Thehepatopancreas of the unfed fish appeared to be in criticalcondition The nucleus of the cells of the hepatopancreasappeared to be highly contracted while the capillary lookedexpanded There was a reduction in the number of pancre-atic zymogen granules There was also expansion of blood
Trang 39Table 3 Whole-body proximate composition (g kg ) of 5 g juvenile olive flounder, Paralichthys olivaceus
Figure 2 Histological changes of the hepatopancreas, kidney and anterior intestine of 5 g juvenile olive flounder, Paralichthys olivaceus, fed
hepa-tic cord; It, interstitial tissue; Mf, mucosal fold; Ml, muscularis; P, pancreas; Rt, renal tubule; Se, serosa; Sm, submucosa; Zg, zymogen granule.
Trang 40cells in the glomerulus and a decrease in the number of
macrophages in the pancreas in the unfed fish The
appeared to have thickened in these fish These conditions
were all observed more in the unfed fish than in fish fed at
Weight gain, SGR, FE, PER and survival of 20 g juvenile
olive flounder, P olivaceus, fed the experimental diet at
varying feeding rates for 2 weeks are shown in Table 4
Weight gain and SGR of fish fed at 3.5% body weight (BW)
significantly higher than those of the unfed fish and fish fed
sig-nificantly higher at each feeding rate than at the preceding
one for the other treatments Feed efficiency of fish fed at
were no significant differences in FE among fish fed at
of fish fed to satiation and those fed at 3.0% and
signifi-cant differences in this parameter among fish fed at 3.0%
and those fed to satiation Protein efficiency ratio for fish
for other treatments except for the unfed fish, which had no
values There were no significant differences in survival of
fish fed at all feeding rates Broken line analysis of WG
showed that the optimum feeding rate in 20 g juvenile olive
Whole-body proximate composition of 20 g fish fed the
experimental diet at different feeding rates is shown in
Table 5 Moisture, crude protein and crude ash were icantly higher while crude lipid content was significantlylower in the unfed fish than in fish fed the experimentaldiet at all feeding rates These parameters fluctuated for thefed fish
signif-Effects of feeding rates on serological characteristics of
20 g juvenile olive flounder, P olivaceus, are depicted in
significantly higher than those of fish fed at 1.0%, 2.0%
differences in HCT among the unfed fish, fish fed at
transaminase of the unfed fish was significantly higher thanthose of all the fed ones Although there were significantdifferences in GOT of the fed fish, there were no cleartrends in this parameter Glutamic pyruvic transaminaseincreased and decreased after a peak value, which was
Figure 3 Broken line analysis of weight gain of 20 g juvenile olive flounder, Paralichthys olivaceus, fed the experimental diet for
.