The effects of dietary nucleotide mixture on growth performance, haematological and immunological...

SHORT COMMUNICATION

The effects of dietary nucleotide mixture on growth

performance, haematological and immunological

parameters of Nile tilapia

Margarida Maria Barros1, Igo Gomes Guimar~aes2, Luiz Edivaldo Pezzato1, Ricardo de Oliveira

Orsi1, Ademir C Fernandes Junior1, Caroline Pelegrina Teixeira1, Luciana Francisco Fleuri3 &

Carlos Roberto Padovani3

1Faculty of Veterinary and Animal Science, S~ao Paulo State University, AquaNutri, Botucatu, SP, Brazil2LAPAQ, Federal Goias University, Jatai, GO, Brazil3Bioscience Institute, S~ao Paulo State University, Botucatu, SP, Brazil

Correspondence: M M Barros, Faculty of Veterinary Medicine and Animal Science, Breeding and Animal Nutrition Department,

S~ao Paulo State University, PO Box 560, Botucatu, SP 18618-970, Brazil. E-mail: [email protected]

Nucleotides are low molecular biochemical com-

pounds that have numerous essential physiological

and biochemical functions, such as building mono-

meric units of nucleic acids, chemical energy

transference, biosynthetic pathways, biological reg-

ulators and coenzyme components (Gil 2002;

Gatlin & Li 2007). Although nucleotides are sub-

stances that are synthesized endogenously, dietary

nucleotides supplementation may have beneficial

effects on fish growth performance (Li, Neill &

Gatlin 2007; Keyvanshokooh & Tahmasebi-kohy-

ani 2012), immune response (Sakai, Taniguchi,

Mamoto, Ogawa & Tabata 2001; Leonardi, Sandino

& Klempau 2003; Li, Lewis & Gatlin 2004;

Choudhurya, Pala, Sahua, Kumara, Dasb & Muk-

herjeeb 2005; Li & Gatlin 2006) and intestinal

morphology (Burrells, William, Southage & Wads-

worth 2001; Welker, Lim, Aksoy & Klesius 2011).

Functional proprieties of dietary nucleotides may

have particular importance on the modulation of

the fish immune status. Indeed, it has been

reported to enhance macrophage activity, natural

killer cells, serum complement, lysozyme, phagocy-

tosis (Sakai et al. 2001; Ringo, Olsen, Vecino,

Wadsworth & Song 2012), as well as the antibody

production in Nile tilapia (Ramadan, Afifi, Mousta-

fa & Samy 1994); rainbow trout (Burrells et al.

2001; Leonardi et al. 2003) and hybrid striped

bass (Li et al. 2004). However, the optimum

dietary nucleotides supplementation level needs to

be accurately estimated, as high dietary concentra-

tion of these compounds may compromise growth

and protein accretion (Peres & Oliva-Teles 2003;

Oliva-Teles, Guedes, Vachot & Kaushik 2006).

Aeromonas hydrophila has been described as an

important bacterial disease in Brazil, resulting in a

significant economic impact on fish production

(Garcia, Pilarski, Onaka, Moraes & Martins 2007).

Therefore, it is important to develop sustainable

and effective strategies to mitigate the effect of this

diseases outbreak. One of these strategies is the

use of potential functional nutrients (Hardy &

Barrows 2002), namely nucleotides, which may

have beneficial effects on immune status modula-

tion of Nile tilapia.

AccelerAid� (FormilVet, Barueri, S~ao Paulo,

Brazil) is a dietary commercial nucleotide mixture

successfully used in terrestrial animals. In broilers,

this additive proved to enhance intestinal mucosa

proliferation after damage caused by coccidiosis

challenge (Pel�ıcia, Zavarize, Ducatti, Stradiotti, Pezz-

ato, Araujo, Mituo, Madeira & Sartori 2011). How-

ever, in non-challenged situation, this additive has

no effect on growth performance and intestinal

morphology of broilers (Zavarise, Sartori, Pel�ıcia,

Pezzato, Araujo, Stradiotti & Madeira 2011).

Thus, this study was undertaken to evaluate the

effects of a commercial dietary nucleotide mixture

© 2013 John Wiley & Sons Ltd 1

Aquaculture Research, 2013, 1–7 doi:10.1111/are.12229

on growth performance, haematological profile

and immune response in Nile tilapia. The effect of

a subsequent acute cold-water stress and A. hydro-

phila challenge was also evaluated.

A practical diet, formulated based on soybean

meal, corn, broken rice, fish meal, wheat mid-

dlings, corn gluten meal and soybean oil, contain-

ing 32% crude protein and 18 000 kJ kg�1 crude

energy (National Research Council (NRC) 1993;

Furuya, Furuya, Boscolo, Feiden, Cyrino, Pezzato

& Barros 2010), was supplemented with a com-

mercial nucleotide mixture (AccelerAid – contain-

ing 22.5% of total nucleotides) at 0; 0.5; 1.0; 2.0;

and 4.0 g kg�1.

Two hundred and eighty Nile tilapia fingerlings

(mean initial body weight: 4.74 � 0.03 g) were

randomly stocked into 40 250-L aquaria (7 fish/

aquarium, in octuplicate per dietary treatment).

The aquaria were supplied with 6.6 L min�1

dechlorinated tap water running through a biologi-

cal filter. Water temperature was 26.0 � 0.7°C; pHat 6.8 � 0.7; dissolved oxygen 5.8 � 0.5 mg L�1

and ammonia (NH3) 138.0 lg L�1. Fish were fed to

apparent satiation, four times a day (08:00; 11:00;

14:00; 17:00 hours) for 60 days. At the end of the

feeding trial, two fish per tank were sample for

whole-body composition analysis and a blood

sample from the caudal vein of eight anaesthetized

fish per treatment was collected for haematological

and immunological analysis. Then, remaining

fish were submitted to cold-induced stress and to

A. hydrophila challenge. Fish from the same treat-

ment were pooled and divided into 12 groups of 2

fish each; 6 of these groups were transferred to the

cold-water challenge and the other 6 groups

transferred to the bacterial challenge.

The water-cold challenge system was equipped

with 30 plastic aquaria (40 L) and a cooling

system; water temperature was gradually

decreased (2.3°C/day), from 23 to 16°C, and

maintained at 16°C for another 2 days. At the

end, a blood sample was collected from the caudal

vein of eight anaesthetized fish per treatment for

determination of total red blood cell count and

total leucocyte count, haemoglobin, haematocrit,

total plasma protein and albumin concentration,

according to Barros, Ranzani-Paiva, Pezzato,

Falcon and Guimar~aes (2009).

Bacterial challenge was performed in another

independent system, equipped with 24 plastic

aquaria (40 L), maintained at 26°C. Fish

were challenged by intraperitoneal injection with

A. hydrophila, from a virulent outbreak of haemor-

rhagic septicaemia of Nile tilapia. Twenty-four

hours after challenge, fish was fed the same exper-

imental diet that was assigned during the growth

trial, for 15 days. Fish mortality was recorded

twice a day. After the challenge period, blood sam-

ples were collected from six fish per treatment for

determination of haematological parameters and

burst respiratory activity. Burst respiratory activity

was measured through the production of hydrogen

peroxide (H2O2) and nitric oxide (NO) in monocyte

culture according to Secombes (1990).

Data of growth performance were analysed by

one-way analysis of variance (ANOVA) using the

general linear model (GLM). Data of haematologi-

cal parameters were analysed by two-way ANOVA

using the GLM to test the effects of the dietary

levels of nucleotide and time points of stress and

challenge tests and their interactions. If there was

a significant F-test and normality, subsequent

comparisons of treatment means were performed

using Bonferroni’s Multiple Range test. However, if

there was a significant F-test and no normality,

subsequent comparisons of the treatment means

were performed using Dunn’s Multiple Range test.

Differences were considered to be significant at the

0.05 probability level. All analyses were performed

using the SAS Institute (2004) statistical software

program.

Nucleotide mixture supplementation did not

affect weight gain, feed conversion ratio and pro-

tein efficiency ratio, but it significantly increased

feed intake, attaining a maximum with 1.0 g kg�1

supplementation level (Table 1).

Dietary nucleotide level did not affect haemato-

logical parameters before or after the cold-induced

stress. However, irrespectively of the dietary treat-

ment, cold-induced stress significantly increased

Hb, MCV and MCHC (Table 2). Total leucocytes

count was highest for 1.0 g kg�1 nucleotide diet,

mainly due to a significant increase in lympho-

cytes count, but it was not significantly different

from control diet. Cold-induced stress did not affect

total leucocytes count, increasing significantly

neutrophils and monocytes counts, but decreasing

significantly the lymphocytes counts, indepen-

dently of the dietary nucleotide level (Table 3).

Plasma metabolites were not affected by nucleotide

level, but cold-stress induced a significant reduc-

tion in plasma protein and albumin levels, regard-

less of the nucleotide mixture supplementation

(Table 4).

© 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–72

Nucleotide in growth performance and health of Nile tilapia M M Barros et al. Aquaculture Research, 2013, 1–7

The supplementation of 0.5 g kg�1 nucleotide

mixture induced a significantly higher peripheral

blood monocytes H2O2 generation than that of 0.0

and 1.0 g kg�1 nucleotide diets. However, NO pro-

duction was not affected by the dietary nucleotide

level. After such challenge, fish fed diets supple-

mented with 1.0 g kg�1 showed a higher H2O2

when compared with fish fed 2.0 g kg�1, and a

higher NO was observed in fish fed 2.0 g kg�1.

Aeromonas hydrophila challenge stimulated a

higher production of H2O2 by monocytes. How-

ever, a higher increase in NO, after bacterial

challenge, was observed only in fish fed diets sup-

plemented with 2.0 g kg�1 compared with fish not

submitted to bacterial challenge (Table 5). Neither

the number of days to first mortality nor the

cumulative mortality, 15 days post challenge, was

affected by nucleotide supplementation (Table 6).

The potential influence of the dietary nucleotides

on growth and voluntary feed intake in fish is

still not consistent. Some studies sustained a

chemo-attractive effect of exogenous nucleotides,

associated with the presence of some substances as

adenosine monophosphate, inosine monophos-

phate, uridine monophosphate and adenosine

diphosphate (Mackie 1973; Rumsey, Winfree &

Hughes 1992; Peres & Oliva-Teles 2003; Li &

Gatlin 2006; Oliva-Teles et al. 2006; Gatlin & Li

2007). However, depending on the nucleotide mix-

ture composition, incorporation level or fish stage,

exogenous nucleotides may have marginal or neg-

ative effect on growth and feed intake (Tacon &

Cooke 1980; Choudhurya et al. 2005; Oliva-Teles

et al. 2006; Li et al. 2007). Indeed, growth-stimu-

latory effect of nucleotide mixture depends on the

free adenine content, a potent inhibitor of feed

intake and growth (Rumsey et al. 1992) and on

fish stage, as larvae may have higher demand for

nucleotide due to high rate of cell replication

(Borda, Martinez-Puig & Cordoba 2003). This may

be the case of this study. Inclusion of 0.0225%

nucleotides (0.10% nucleotide mixture) led to a

significant higher feed intake, but growth or feed

efficiency were similar to that observed for control

diet.

Haematological parameters of Nile tilapia are

within the normal range for this species (Barros,

Pezzato, Falcon & Guimar~aes 2006; Weiss &

Wardrop 2010) and were unaffected either by the

nucleotide dietary supplementation or by cold-

stress. Similarly, yeast RNA supplementation had

no effect on haematological values of Labeo rohita

or Catla catla (Choudhurya et al. 2005; Jha, Pal,

Sahu, Kumara & Mukherjeea 2007).

Haemotopoietic cells and lymphocytes have lim-

ited capacity for de novo synthesis of nucleotides,

and so under particular conditions, such as stress,

endogenous production of nucleotides may impair

maturation, activation and proliferation of lym-

phocyte (Gatlin & Li 2007). For Nile tilapia, leuco-

cytes and lymphocytes counts were increased with

1.0% nucleotide mixture supplementation, a trend

that was maintained after the cold-stress. This

modulation action of dietary nucleotides on leuco-

cytes and lymphocytes have been previously

reported (Leonardi et al. 2003; Tahmasebi-Kohy-

ani, Keyvanshokooh, Nematollahi, Mahmoudi &

Pasha-Zanoosim 2011; Kenari, Mahmoudi, Soltani

& Abediankenari 2013). Jha et al. (2007), also

described an increase in leucocyte associated with

a higher lysozyme activity and phagocyte capacity

of fish fed 0.4–0.8% nucleotide diets.

Several factors could affect white blood cell dif-

ferentiation, among which, stress is a significant

Table 1 Weight gain (WG), daily feed intake (DFI), feed conversion ratio (FCR), protein efficiency ratio (PER) and net

protein utilization (NPU) of Nile tilapia fed diets supplemented with nucleotide mixture

Treatment (g kg�1) WG* (g fish�1) DFI† (g fish�1) FCR‡ PER§ (%)

0.0 110.9 � 12.7 121.9 � 6.2b 1.11 � 0.11 2.93 � 0.26

0.5 114.0 � 9.5 123.4 � 7.0b 1.08 � 0.06 2.98 � 0.18

1.0 125.7 � 12.6 135.9 � 7.9a 1.09 � 0.07 2.98 � 0.18

2.0 120.4 � 10.6 130.4 � 3.1ab 1.09 � 0.08 2.97 � 0.21

4.0 114.8 � 8.00 127.3 � 6.7ab 1.11 � 0.06 2.91 � 0.15

Values are mean � SD (n = 6); different letters within a column indicate significant differences at P < 0.05.

*WG = final weight � initial weight.

†DFI = MFI, where MFI is the mean feed intake by fish during the trial.

‡FCR = group feed intake/group weight gain.

§PER = weight gain/protein intake.

© 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–7 3

Aquaculture Research, 2013, 1–7 Nucleotide in growth performance and health of Nile tilapia M M Barros et al.

one, impairing the interleukin release and reduc-

ing leucocyte differentiation (Tripp, Maule, Schreck

& Kaattari 1987). The response observed after

cold-induced stress, with lower leucocyte and

lymphocyte and higher neutrophil was previously

observed for Nile tilapia (Falcon, Barros, Pezzato,

Sampaio & Hisano 2007; Barros et al. 2009).

Although cold-induced stress significantly

reduced total plasma protein and albumin content,

regardless of the nucleotide supplemented level,

plasma globulin and the albumin:globulin ratio

were maintained. Reduced plasma protein and

albumin levels, after cold-induced stress, are prob-

ably caused by the metabolism reduction at low

temperature. Albumin and globulin are essential

for healthy immune system (Tahmasebi-Kohyani

et al. 2011). Maintenance of plasma globulin con-

centration and A:G ratio suggest that immune

function was not compromised by cold-stress and

that dietary nucleotide supplement had a marginal

effect.

Similar results were observed for C. catla (Jha

et al. 2007), but the opposite was also observed

for other fish species (Choudhurya et al. 2005;

Kenari et al. 2013). Difference on the nucleotide

action on immune system modulation may be con-

ditioned by the species, life stage and composition

of nucleotide supplement.

The main role of nucleotide in fish health is to

enhance innate and specific immune response (Li

& Gatlin 2006), which may improve infection

resistance. Aeromonas hydrophila challenge stimu-

lates the production of oxygen reactive compounds

in all treatments. No clear effect of nucleotides on

the production of oxygen reactive compounds was

observed, but supplementation at the level of 0.5%

increased H2O2 production. Similar trend was

previously described for salmonids by Burrells,

William and Forno (2001) and red drum (Li et al.

2007). However, a positive effect of nucleotide on

burst respiratory activity was observed for L. rohita

(Choudhurya et al. 2005), Cyprinus carpio (Sakai

et al. 2001), hybrid striped bass (Morone chrys-

ops 9 M. saxatilis) (Li et al. 2004) and C. catla

(Jha et al. 2007). Different results could be related

to the different protocols used to evaluate leuco-

cyte burst respiratory activity. Thus, further stud-

ies are necessary to elucidate which pathway the

immunostimulants activate during the burst

respiratory process.

Nucleotides have been report to enhance resis-

tance to different pathogens (Ramadan et al.Table

2Haem

atologicalparametersofNiletilapia

feddiets

supplemen

tedwithnucleotidemixture

andsubmittedto

cold-inducedstress

Nucleotide(g

kg�1)

RBC

(106lL

�1)

Haematocrit(%

)Haemoglobin

(gdL�1)

MCV

(fL)

MCHC

(%)

Before

After

Before

After

Before

After

Before

After

Before

After

0.0

1.97�

0.19

1.98�

0.29

29.14�

2.03

29.14�

3.08

6.98�

0.48

7.54�

0.72

148.38�

14.02

148.72�

20.16

23.98�

1.25

25.92�

0.65

0.5

2.03�

0.15

2.07�

0.28

28.78�

2.92

30.29�

3.89

6.93�

0.44

7.80�

0.61

141.51�

9.60

146.76�

15.19

24.22�

1.94

25.98�

2.37

1.0

2.06�

0.16

2.09�

0.12

29.57�

2.17

33.07�

2.69

7.28�

0.42

8.22�

0.87

143.26�

5.43

158.65�

15.31

24.65�

1.30

24.89�

2.27

2.0

1.99�

0.40

1.99�

0.23

29.07�

3.91

30.86�

2.76

7.14�

1.37

7.95�

0.51

148.22�

18.15

155.50�

12.43

24.52�

2.68

25.84�

1.38

4.0

2.30�

0.43

1.99�

0.24

31.57�

2.39

30.36�

2.84

8.04�

0.54

7.68�

0.61

140.57�

23.24

153.18�

8.32

25.54�

2.06

25.35�

0.66

Period

2.07�

0.29

2.03�

0.23

29.63�

2.79

30.74�

3.18

7.27b�

0.81

7.84a�

0.67

144.39b�

14.86

152.57a�

14.57

24.58b�

1.88

25.60a�

1.60

Nucleotide

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

Period9

Nucleotide

ns

ns

ns

ns

ns

Values

are

mean�

SD(n

=8);within

each

dietary

treatm

ent,differentGreek

lettersindicate

significantdifferences(P

<0.05)before

andafter

thecold-inducedstress.



Table 3 Differential leucocyte count of Nile tilapia fed diets supplemented with nucleotide mixture and submitted to

cold-induced stress

Nucleotide

(g kg�1)

Leucocytes (cells lL�1) Lymphocytes (cells lL�1) Neutrophils (cells lL�1) Monocytes (cells lL�1)

Before After Before After Before After Before After

0.0 1.69ab � 0.63 1.42 � 0.63 1.63ab � 0.61 1.19 � 0.55 0.37 � 0.19 1.54 � 0.66 0.25 � 0.24 0.74 � 0.34

0.5 1.29b � 0.33 1.31 � 0.46 1.23b � 0.33 1.06 � 0.34 0.48 � 0.15 2.00 � 1.29 0.15 � 0.06 0.50 � 0.23

1.0 2.04a � 0.50 1.73 � 0.66 1.94a � 0.50 1.47 � 0.55 0.68 � 0.43 2.10 � 0.95 0.35 � 0.15 0.49 � 0.32

2.0 1.15b � 0.30 1.20 � 0.38 1.08b � 0.27 0.95 � 0.35 0.34 � 0.17 1.79 � 0.89 0.39 � 0.36 0.65 � 0.31

4.0 1.66b � 0.55 1.20 � 0.35 1.57ab � 0.53 1.07 � 0.36 0.51 � 0.39 0.87 � 0.28 0.44 � 0.26 0.46 � 0.15

Period 1.57 � 0.55 1.37 � 0.52 1.49a � 0.53 1.15b � 0.45 0.47b � 0.3 1.66a � 0.93 0.32b � 0.25 0.57a � 0.28

Nucleotide P < 0.01 ns P < 0.01 ns ns ns ns ns

Period 9

Nucleotide

ns ns ns ns ns ns ns ns

Values are mean � SD (n = 8); different lowercase letters within a column indicate significant differences between the same period

of stress (before or after) in the different diets; different Greek letters within a row indicate sign.

Table 4 Total plasma protein (TPP), globulins, albumin (ALB), and albumin to globulin ratio (A:G) of Nile tilapia fed

diets supplemented with nucleotide mixture and submitted to cold-induced stress

Nucleotide

(g kg�1)

TPP (g dL�1) ALB (g dL�1) Globulins (g dL�1) A:G

Before After Before After Before After Before After

0.0 3.26 � 0.54 2.84 � 0.27 1.17 � 0.17 0.85 � 0.32 2.09 � 0.48 1.99 � 0.30 0.58 � 0.14 0.45 � 0.23

0.5 3.27 � 0.19 2.92 � 0.41 0.94 � 0.19 0.79 � 0.06 2.33 � 0.30 2.02 � 0.37 0.42 � 0.13 0.40 � 0.06

1.0 3.16 � 0.25 2.92 � 0.25 1.03 � 0.10 0.95 � 0.13 2.13 � 0.21 1.97 � 0.21 0.49 � 0.07 0.48 � 0.09

2.0 3.03 � 0.44 2.90 � 0.31 0.99 � 0.20 0.86 � 0.15 2.04 � 0.31 2.04 � 0.19 0.49 � 0.10 0.42 � 0.06

4.0 3.43 � 0.30 3.04 � 0.38 1.02 � 0.24 0.87 � 0.14 2.41 � 0.48 2.17 � 0.30 0.46 � 0.22 0.40 � 0.07

Periods 3.23a � 0.36 2.90b � 0.31 1.03a � 0.19 0.86b � 0.17 2.20 � 0.37 2.04 � 0.27 0.49 � 0.14 0.43 � 0.11

Nucleotide ns ns ns ns ns ns ns ns

Period 9

Nucleotide

ns ns ns ns

Values are mean � SD (n = 8); different Greek letters within a row indicate significant differences at P < 0.05 for cold-induced

stress effect comparing values before and after stress; ns, not significant.

Table 5 Oxygen reactive species production (H2O2 and NO) by peripheral blood monocytes of Nile tilapia fed diets

supplemented with nucleotide mixture and challenged with Aeromonas hydrophila

Nucleotide (g kg�1)

H2O2 (nmol 105 cells�1) NO (lmol 105 cells�1)

Before After Before After

0.0 0.31 � 0.14bA 2.50 � 0.24abB 0.56 � 0.21aA 0.78 � 0.34bA

0.5 1.06 � 0.56aA 2.60 � 0.08abB 0.47 � 0.25aA 0.96 � 0.24bA

1.0 0.33 � 0.16bA 2.75 � 0.57aB 0.55 � 0.16aA 0.98 � 0.29bA

2.0 0.44 � 0.19abA 2.05 � 0.20bB 0.79 � 0.13aA 2.61 � 1.58aB

4.0 0.92 � 0.03abA 2.37 � 0.08abB 0.41 � 0.09aA 0.95 � 0.25bA

Period 0.61 � 0.43 2.45 � 0.36 0.55 � 0.21 1.26 � 0.97

Nucleotide P < 0.01 P < 0.01 ns P < 0.05

Period 9 Nucleotide P < 0.05 P < 0.05

Values are mean � SD (n = 6); different lowercase letters within a column indicate significant differences between the same period

of stress (before or after) in the different diets; different capital letters within a row indicate significant differences among the same

diet in different period of stress (before or after); ns, not significant.



1994; Burrells et al. 2001; Choudhurya et al.

2005; Jha et al. 2007). In this study, cumulative

mortality tended to be lower in fish fed 0.5–2.0%

of nucleotides, but tend to be higher for the high-

est nucleotide supplemented level. This effect could

be related to an overload of nitrogen compounds

affecting metabolism, which may increase infection

susceptibility. H2O2 production by bloodstream

monocytes at 0.5 g kg�1 correlates well with the

survival data. Reduced survival rate was also

observed for channel catfish fed diets supplemented

with mixture of nucleotides (0.9 and 2.7%; Welker

et al. 2011).

The results of this study demonstrated that the

commercial nucleotide tested had an attractant

effect, but did not affect growth performance or

feed utilization efficiency. Haematological profile

and innate immune response were also not influ-

enced by the dietary nucleotides, but Nile tilapia

resistance to A. hydrophila tended to be improved.

Acknowledgments

We are grateful to The S~ao Paulo State Research

Support Foundation – FAPESP, Brazil for support-

ing this research (Proc. 08/53270-7). We like to

thank Dra. Fabiana Pilarski, CAUNESP – Jabotica-

bal for her assistance with the bacterial challenge

and Dra. Helena Peres for her professional revision

of the manuscript.

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Table 6 Mean number of days to first mortality and

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lenge with Aeromonas hydrophila

Nucleotide

(g kg�1)

Days to first

mortality

Cumulative

mortality (%)

0.0 5.60 � 2.63 65.00 � 22.36

0.5 6.20 � 3.55 42.86 � 12.20

1.0 6.10 � 3.11 46.43 � 26.73

2.0 4.10 � 2.86 55.00 � 20.92

4.0 6.30 � 2.82 70.00 � 20.92

Values are mean � SD (n = 6).



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Keywords: Nile tilapia, nucleotides, growth

performance, haematological and immunological

parameters, stress and challenge tests



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