Probiotics as an environment-friendly approach to enhance red sea bream, Pagrus major growth, immune response and oxidative status

Accepted Manuscript Probiotics as an environment-friendly approach to enhance red sea bream, Pagrus major growth, immune response and oxidative status Mahmoud A.O. Dawood, Shunsuke Koshio, Manabu Ishikawa, Mabrouk El-Sabagh, M. Angeles Esteban, Amr I. Ibrahim PII:

S1050-4648(16)30513-7

DOI:

10.1016/j.fsi.2016.08.038

Reference:

YFSIM 4138

To appear in:

Fish and Shellfish Immunology

Received Date: 22 June 2016 Revised Date:

11 August 2016

Accepted Date: 14 August 2016

Please cite this article as: Dawood MAO, Koshio S, Ishikawa M, El-Sabagh M, Esteban MA, Ibrahim AI, Probiotics as an environment-friendly approach to enhance red sea bream, Pagrus major growth, immune response and oxidative status, Fish and Shellfish Immunology (2016), doi: 10.1016/ j.fsi.2016.08.038. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Probiotics as an environment-friendly approach to enhance red sea bream, Pagrus

2

major growth, immune response and oxidative status

3 1, 3,*

, Shunsuke Koshio 2, Manabu Ishikawa 2, Mabrouk El-Sabagh4,

4

Mahmoud A.O. Dawood

5

M. Angeles Esteban5, Amr I. Ibrahim6

7

1

8

Korimoto, Kagoshima 890-0056, Japan

9

2

RI PT

6

The United Graduate School of Agriculture Sciences, Kagoshima University, 1-21-24

Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, 4-50-

20, Kagoshima 890-0056, Japan

11

3

12

Egypt

13

4

Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Kafrelsheikh, Egypt

14

5

Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty of

15

Biology, Regional Campus of International Excellence “Campus Mare Nostrum”, University

16

of Murcia, 30100 Murcia, Spain

17

6

SC

10

Animal Health Research Institute(AHRI-DOKI), Egypt

TE D

18 19 20 21

EP

22 23

26

AC C

24 25

27

Correspondence:

28

[email protected]

29 30 31 32

M AN U

Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, 33516, Kafrelsheikh,

Mahmoud

A.O.

Dawood,

Fax.

+81

992864184,

E-mail:

ACCEPTED MANUSCRIPT Abstract

34

A usual strategy in modern aquaculture to combat production bottlenecks associated with

35

intensification is preventive health care through the use of consumer and environment-

36

friendly alternatives including probiotics. The current study evaluates the influence of

37

Lactobacillus rhamnosus (LR), a lyophilized probiotic bacterium, on health status and

38

performance of red sea bream (Pagrus major). Probiotics were incorporated in the diets at

39

four different concentrations: 0 (control diet, LR0), 102 (LR1), 104 (LR2) and 106 (LR3) cells

40

g-1 and diets were administered to the fish for a period of 8 weeks. After the feeding trial,

41

final body weight, body weight gain, specific growth rate, protease activity, protein

42

digestibility, Lactobacillus sp. intestinal count, and superoxide dismutase were significantly

43

higher in all probiotic-fed groups (P<0.05). In addition, lipid and dry matter digestibility,

44

reactive oxygen metabolites, biological antioxidant potential, and humoral and mucosal

45

immune parameters including (total serum protein, alternative complement pathway,

46

bactericidal and peroxidase activities) were also significantly elevated in fish fed probiotic

47

supplementations being the effects dose-dependent. All growth, feed utilization, immune and

48

oxidative parameters were significantly improved following probiotic administration. Present

49

results revealed that L. rhamnosus is a promising probiotic candidate employed to help red

50

sea bream protect themselves, thus promoting safe farming that would be less dependent on

51

chemotherapy against infectious diseases.

SC

M AN U

TE D

52

RI PT

33

Key words: Red sea bream; Probiotics; Environmental approach; Growth; Gut microbiota;

54

Oxidative status; Humoral immunity; Mucosal immunity

56 57 58 59

AC C

55

EP

53

ACCEPTED MANUSCRIPT 1.

Introduction

61

Great and fast development of aquaculture in recent decades has increased the interest in

62

studies focus on the physiological aspects of the stress on fish mainly due to the fact that

63

these situations could increase the risk of fish disease outbreaks [1]. Although application of

64

antibiotics and chemotherapeutics is considered quite effective, drug resistance and serious

65

environmental hazards are considered as negative impacts of their use. Furthermore, other

66

undesirable effects such as tissue accumulation, immunosuppression, development of

67

antibiotic resistant bacteria and/or destruction of environmental microbiota have also been

68

recorded [2]. For these reasons it is assume that a good way to avoid such problems, and

69

concomitantly to enhance the fish survival rate on farms, would be to use natural preventive

70

approaches [3].

71

Probiotic supplements have recently received extensive attention as an alternative method to

72

antibiotic treatment [4-10]. Probiotics have been described as efficient tools to control

73

pathogens and improve aquaculture production through different mechanisms [11-15]. In

74

fact, probiotics may boost the quality of both the fish (e.g. growth, well-being and health

75

status) and the fish environment [16]. Abundant efforts have been placed on probiotics’

76

capability to modulate fish humoral immunity as they have been shown potent modulators of

77

mucosal immunity even after compromised situations such as microbial infections or stress

78

[15,16]. However, at present, much more efforts are still needed in this important research

79

area.

80

There are many options for choosing a probiotic being the genus Lactobacillus the main

81

strain used in aquaculture feed additives [9,11,17-19]. In previous studies, different beneficial

82

effects

83

immunoregulatory stimulation, modulation and expression of cytokine genes and disease

84

resistance by dietary supplementation of Lactobacillus has been demonstrated in different

85

fish species like rainbow trout, Oncorhynchus mykiss [18,20-25]; Nile tilapia, Oreochromis

86

niloticus [26,27] and zebrafish, Danio rerio [28]. However, to the best of our knowledge, the

87

information regarding the effects of L. rhamnosus (LR) for red sea bream (Pagrus major) is

88

scarce. This fish species was selected for the present work due to its good taste, high market

89

value, rapid growth and strong resistance to stress. These properties make red sea bream a

90

great fish candidate for intensive aquaculture in many countries and it is one of the most

91

commercially important species in Japan [5,13]. Despite this, there have been some issues

EP

TE D

M AN U

SC

RI PT

60

on

growth

performance,

hematological

and

oxidative

status,

AC C

especially

ACCEPTED MANUSCRIPT facing this fish species at the fry stage, such as growth retardation, high mortality and low

93

feed utilization which have provoked important economic losses.

94

Taken into account all these considerations, the aim of the present study was to investigate

95

the effects of dietary administration of LR on different parameters of red sea bream including

96

growth performance, body composition, nutrient digestibility, gut microbiota, immune

97

response and oxidative status. These data could be the base for future incorporations of

98

probiotics not only to this important fish species, but also to other species sharing the same

99

feeding habits.

RI PT

92

100

2.

Materials and methods

102

2.1.

Probiotic preparation

103

Lactobacillus rhamnosus (LR) lyophilized bacteria was obtained from Morinaga Milk

104

Industry CO., LTD. (Kanagawa, Japan), and the concentration of LR in the dry product is

105

1×109 cells g-1. α-Cellulose powder used to adjust to the required concentrations of LR. The

106

confirmation of the bacterial strain was based on colony and cell morphology and Gram

107

staining. Briefly, the bacterium was cultured on to plates of DeMan, Rogosa and Sharpe agar

108

(MRS, Merck, Darmastadt, Germany) by cultivating it for 48 h at 37°C. The viability of

109

bacteria was determined by plate counting on MRS agar. Colony forming units (CFU g-1)

110

were determined for viable bacterial populations. The bacteria cells were stored at -20°C until

111

use [13].

TE D

M AN U

SC

101

EP

112

2.2.

Experimental diets

114

Detailed composition and proximate analysis of the basal diet used in the present study

115

described previously by Dawood et al. [5]. Experimental diets were prepared by

116

supplementing a basal formulated diet with LR at four different levels (0 as control, 102, 104

117

and 106 cells g-1). Accordingly, the experimental diets were named as LR0, LR1, LR2, and

118

LR3, respectively. Graded doses of LR were added, in 50 ml of soybean lecithin oil, to the

119

basal diet at the expense of α-Cellulose to obtain the required levels followed by mixing with

120

a blender. All the dietary ingredients of the experimental diets were mixed with water and

121

cold press and extruded in order to produce 1.6-2.1-mm pellets, which were dried at room

122

temperature under sterile conditions and stored at 4°C. To keep up LR viability, new batches

AC C

113

ACCEPTED MANUSCRIPT of feed were produced every two weeks. The total viable LR counts present in the diet were

124

determined by spread plating on MRS agar (de Man, Rogosa and Sharpe; MRS, Merck,

125

Darmstadt, Germany) and TSA (Trypto-Soya agar, Nissui Pharmaceutical Co. Ltd., Japan)

126

[18]. To do this, diet samples were first completely powdered and serially diluted with sterile

127

saline [phosphate-buffered saline (PBS, pH=7.4)]. The agar plates were inoculated with each

128

dilution and they were incubated anaerobically at 37 °C for two days. Colony forming units

129

(CFU g-1) were determined for viable bacterial populations. The control feed wasn’t

130

supplemented with LR probiotic.

RI PT

123

131

2.3.

Fish culture and feeding trial

SC

132

Red sea bream fry (mean weight 3.31 ± 0.01 g) were obtained from Akahoshi farm

134

(Kumamoto Prefecture, Japan) were acclimatized to the experimental conditions for 1 week.

135

During this period, a commercial diet (50% crude protein; Higashimaru, Japan) was supplied

136

to the fish. Before the feeding trial, health condition of fishes was checked visually through

137

their movements, infectious diseases symptoms and physical appearance all over the body

138

and fins of the fish. Thereafter, fish were randomly allocated into twelve 100-litre tanks

139

(twenty fish per tank and triplicate tanks per treatment) in a flow-through seawater system

140

where each tank was equipped with an inlet, outlet, and continuous aeration. All the tanks

141

were maintained under natural light/dark photoperiod. Water temperature, dissolved oxygen

142

content and pH were monitored daily and maintained at 22.1±1.8°C, 6.2±0.5 mg L-1 and

143

8±0.5, respectively [13]. During the rearing experiment (8 weeks), fish were hand fed to

144

apparent satiation twice a day (at 09.00 and 16.00 hours). Any uneaten feed left was removed

145

after feeding and dried using a freeze drier. Afterwards, the uneaten feed was weighted and

146

subtracted from the total feed intake. All fish were weighed in bulk at 2 weeks’ interval to

147

determine growth, check their health condition and ration was adjusted according to mean

148

fish weight.

AC C

EP

TE D

M AN U

133

149 150

2.4.

Determination of growth performance, feed utilization and survival rate

151

The growth performance parameters including weight gain (WG), specific growth rate

152

(SGR), condition factor (CF), feed intake (FI), feed and protein efficiency ratio (FER and

ACCEPTED MANUSCRIPT PER), protein gain (PG) and survival rate (SR) were calculated according to the following

154

formulae:

155

WG (%) = W2-W1,

156

SGR = 100 (ln W2-ln W1)/T,

157

CF = weigh of fish (g)/ (length of fish)3 (cm)3 ×100

158

FI (g/fish/8 weeks) = (dry diet given − dry remaining diet recovered)/ no. of fish

159

FER= WG/FI,

160

PER = WG/dry protein intake (g)

161

PG (g kg-1) = {(W2 × final whole body protein content (%)/100) − (W1 × initial whole body

162

protein content (%)/100)}/ WG ×1000

163

where W1 is the initial weight (g), W2 is the final weight (g), T is time (d) and WG is the

164

weight gain (g).

165

SR= (Nf / N0) ×100

166

where N0 is the initial number of fish and Nf is the final number of fish.

M AN U

SC

RI PT

153

167

2.5.

Sample collection and biochemical analysis

169

In order to collect the skin mucus, nine fish were collected as per methods described by

170

Dawood et al. [5], their surfaces were rinsed with distilled water and skin mucus was

171

consequently obtained from the body surface. Heparinized (1600 UI ml-1, Nacalai Tesque,

172

Kyoto, Japan) syringes were utilized to extract blood from the caudal vein from five fish in

173

each replicate tank (fifteen fish per treatment), for hematocrit and plasma analysis.

174

Additionally, blood was collected using non-heparinized disposable syringes in order to

175

collect serum. In order to assess protease activity, the digestive tracts were removed, then cut

176

into small pieces, rinsed with pure water and finally stored at -80°C.

177

Plasma biological antioxidant potential (BAP) and reactive oxygen metabolites (d-ROMs)

178

were measured spectrophotometrically with an automated analyzer (FRAS4, Diacron

179

International s.r.l., Grosseto, Italy) [29].

180

Total serum proteins were also measured spectrophotometrically with an automated analyzer

181

(SPOTCHEM™ EZ model SP-4430, Arkray, Inc. Kyoto, Japan) [30]. Protease activity was

182

measured using commercial kits (QuantiCleaveTM Protease Assay Kit, product number

183

23263, Thermo Fisher Scientific Inc., USA) according to the procedure outlined by the

AC C

EP

TE D

168

ACCEPTED MANUSCRIPT 184

manufacturer. Finally, the diets and fish whole body were analyzed for moisture, crude

185

protein, total lipid and ash, in triplicate, using standard methods [31].

186

2.6.

Total intestinal microbiota and Lactobacillus sp. bacteria count

188

The total intestinal microbiota and Lactobacillus sp. bacteria count (TBC and LBS,

189

respectively) were determined at the end of the feeding trial. Three fish choose by random

190

from each experimental groups were sampled 24 h after cease of feeding [13,32]. The whole

191

gastrointestinal tract was aseptically removed and samples were washed with PBS. Aliquots

192

of 100 µl of the gut solutions were spread onto triplicate TSA media to determine total

193

bacterial populations. DeMan, Rogosa and Sharpe (MRS, Merck, Darmstadt, Germany) were

194

also used to detect viable Lactobacillus sp. The agar plates inoculated with each dilution were

195

then incubated at 37 °C for two days. CFU ml-1 were determined for viable bacterial

196

populations [18].

M AN U

SC

RI PT

187

197

2.7.

Apparent digestibility coefficients

199

Digestibility trial was performed at the end of the feeding trial. The remaining fish from the

200

same treatment were randomly distributed into triplicate tanks and fed the corresponding

201

diets previously supplemented with chromium oxide (Wako Pure Chemical Industries, Ltd)

202

which acted as the inert marker at a level of 0.5% (Cr2O3, 5 g kg-1) [13].

TE D

198

203

2.8.

Immunological analysis

205

The serum alternative complement pathway (ACP) activity was determined according to

206

Sitja-Bobadilla et al. [33]. The serum dilution factor was plotted in logarithmic scale against

207

the percentage of rabbit red blood cells (RRBCs) lysed at each dilution. The dilution

208

corresponding to 50 % hemolysis ml-1 was expressed as ACH50.

209

Lysozyme activities of serum and mucus were determined with the turbidimetric assay, using

210

Micrococcus lysodeikticus (lyophilized cell, Sigma, USA) as specific substrate [34]. A unit of

211

enzyme activity was defined as the amount of enzyme that causes a decrease in absorbance of

212

0.001 min-1.

213

Serum and mucus bactericidal activities were measured using the plate counting method for

214

viable bacterial populations (CFU) according to Iida et al. [35]. The total peroxidase content

215

in serum was measured based on the modified protocol of Salinas et al. [36] using a plate

AC C

EP

204

ACCEPTED MANUSCRIPT 216

reader at 450 nm. The percentage reaction inhibition rate of enzyme with WST (Water

217

Soluble Tetrazolium dye) substrate and xanthine oxidase using a SOD Assay Kit (Dojindo

218

Molecular Technologies, Inc., Kumamoto, Japan) was used to measure the serum superoxide

219

dismutase (SOD) activity as instructed by the manufacturer [13].

220

2.9.

222

The data were subjected to statistical analysis using the package super ANOVA 1.11, Abacus

223

Concepts, Berkeley, California, USA. Differences between treatments were determined by

224

one-way analysis of variance (ANOVA) followed by Duncan's multiple range test. P value of

225

<0.05 was considered statistically significant.

226 227

3.

228

Overall, LR probiotic-enriched diets elevated the growth performance, feed utilization,

229

protease enzyme activity, digestibility, humoral and mucosal immune responses as well as the

230

oxidative status of red sea bream. Thus, this supplement could be useful for maintaining the

231

overall health status of red sea bream.

TE D

232

M AN U

Results

SC

Statistical analysis

RI PT

221

3.1.

234

Results of growth performance, nutrient utilization and survival of red sea bream specimens

235

fed tested diets for 8 weeks are shown in Table 1. Growth performance and nutrient

236

utilization of fish generally increased during the trial in all the experimental groups. The

237

highest values of FBW, BWG and SGR were obtained in fish fed LR-supplemented diets. FI,

238

FER, PER and PG of fish fed LR supplemented diets recorded high values, however, no

239

significant deviations were observed compared with the values found for control group

240

(P>0.05). The poorest growth performances and nutrient utilization were observed in LR free

241

group (control group).

242

Fish fed the different LR supplemented diets showed no abnormal symptoms and physical

243

appearance with survival rates between 90% and 98.33% although no significant differences

244

were observed among all groups (P>0.05).

245

Growth performance and feed utilization of fish

AC C

EP

233

ACCEPTED MANUSCRIPT 3.2.

Protease activity and apparent digestibility coefficients of fish

247

Protease activity (PA) in the digestive tract and apparent digestibility coefficients (ADC) of

248

red sea bream fed diets supplemented with LR for 8 weeks are presented in Table 2. PA and

249

ADC of protein values were significantly higher in fish fed LR-supplemented diets compared

250

with levels from fish of control group (P<0.05). ADC of lipids in fish fed LR3 diet showed a

251

higher significant difference respect to the values recorded from fish fed LR0 (control) and

252

LR1 diets (P<0.05), however, no significant differences were recorded for fish fed LR2 or

253

LR3 diets (P>0.05). Furthermore, ADC of dry matter was significantly enhanced in fish fed

254

LR2 and LR3 diets respect to the control fish.

SC

255

RI PT

246

3.3.

Intestinal microbial counts

257

The levels of total intestine bacteria (TBC) and Lactobacillus sp. (LBS) counts in fish fed

258

LR-supplemented diets are shown in Figure 1A and 1B. TBC counts were not significantly

259

affected after feeding fish with LR supplementation (P>0.05). However, fish fed LR-

260

supplemented diets showed relatively high TBC levels when compared with fish fed LR0 diet

261

(Fig. 1A). Additionally, supplementation of LR at 102, 104 or 106 cells g-1 significantly

262

increased Lactobacillus sp. count (LBS) when compared with fish fed LR-free diet (P<0.05)

263

with highest being in LR3 group (Fig. 1B).

264

TE D

M AN U

256

3.4.

Immunological parameters

266

The effects of dietary LR supplementation on the immunological parameters of red sea bream

267

are summarized in Figs. 2,4,5,6,7 and 8. Alternative complement pathway (ACP), peroxidase

268

activities, and total serum protein levels were significantly higher in fish fed LR2 and LR3

269

diets, respect to the values obtained for the other groups (P<0.05). With regard to sodium

270

oxide dismutase (SOD), this activity was significantly higher than control group (P<0.05) in

271

fish fed LR-supplemented diets. Serum bactericidal activity (BA) was significantly enhanced

272

in case of fish fed LR2 diet (P<0.05), however, no significant differences were detected

273

among the other groups (P>0.05). Hematocrit content was significantly higher in case of fish

274

fed LR3 diet when compared with LR0 and LR1 groups (P<0.05), however, no significant

275

differences were detected among the other groups (P>0.05). On the other hand, serum mucus

AC C

EP

265

ACCEPTED MANUSCRIPT 276

lysozyme and mucus BA activities were not enhanced significantly with LR supplementation

277

(P>0.05).

278

3.5.

Oxidative status

280

Oxidative status and the combined effects pattern of biological antioxidant potential (BAP)

281

and reactive oxygen metabolites (d-ROMs) of red sea bream fed with test diets are illustrated

282

in Table 3 and Figure 3. Dietary supplementation with LR at 106 cells g-1 showed enhanced

283

BAP value, however, no significant differences were observed among the other groups

284

(P>0.05). Furthermore, dietary supplementation with LR at 106 cells g-1 showed lowered d-

285

ROMs value when compared with the control group (P<0.05), although no significant

286

difference was observed among the other groups (P>0.05). The pattern of combined effects

287

of BAP and d-ROMs values of red sea bream fed with test diets is showed on Fig. 3. The

288

LR1 and LR3 groups were located in zone A, meanwhile LR0 and LR2 groups were in zone

289

C.

M AN U

SC

RI PT

279

290

4.

Discussion

292

The use of dietary supplements such as probiotics as an environment-friendly alternative

293

approach to antibiotics increased as the world aquaculture industry has grown dramatically

294

during the last years [9,37-39]. Common interest has led to the use of components (as

295

additives in diets) that could treat diseases without causing any negative impact on the

296

environment [3,40]. These components are represented by prebiotics, probiotics, and plant

297

extracts. In this study, we focus on the effects of probiotics. In connection with the earlier

298

studies [5,7,10,41-44], LR incorporated with red sea bream diets in the current study

299

modulated the intestinal microbiota by promoting the growth of beneficial microorganisms

300

(i.e. Lactobacillus sp.), enhanced immune responses particularly at the mucosal surface,

301

which is the first line of defense, and improved significantly the growth performance and

302

oxidative status.

303

In the present study, growth performance parameters (FBW, BWG and SGR) and feed

304

utilization (FI, FCR, PER and PG) of red sea bream improved with dietary supplementation

305

of LR. According to previous research, beneficial bacteria may enhance host nutrition by

306

stimulating digestive enzymes and aiding in the synthesis of vitamins, which may result in

AC C

EP

TE D

291

ACCEPTED MANUSCRIPT higher weight gain and feed utilization [19,45,46]. Additionally, probiotic cells located in the

308

intestine not only improve microbial balance but also, in the process, they further enhance

309

nutrient absorption and utilization [47,48]. Such improvements could be due to enhanced

310

protease activity and digestibility coefficients. Additional factors, including variations in

311

intestinal bacterial communities, may contribute to improve growth performance of fish [49].

312

Actually, previous research has shown that the growth and/or activities of some bacteria such

313

as lactic acid bacteria and Lactobacillus sp. can be value-added by probiotics [5,11,20,21].

314

Through improvements of the gut health, these beneficial bacteria could also promote feed

315

utilization and growth performance. To confirm this hypothesis, further analysis of the

316

intestinal bacterial communities should be done.

317

Aside from improving growth, probiotic supplementation also influenced feed utilization.

318

This is due to the fact that a probiont is provided, thus creating competition with endogenous

319

populations

320

implantation of live microbial dietary supplement in the digestive tract of the host [20,50].

321

There is positive correlation with the activities of digestive enzymes and the digestive

322

capacity of fish [10,51,52] which reveals the fish’s ability to obtain nutrients from food [53].

323

In the present study, there was a significant increase in intestine protease activities with

324

supplementation of dietary LR. This showed an improvement in digestibility coefficients of

325

red sea bream. The fish gut microbiota assisted in improving the health of the host;

326

additionally, on top of the establishment of digestive enzymes, normal gut flora was also

327

formed [49,54]. Thus, the better enzyme activities of groups treated with LR supplementation

328

might be associated with the manipulation of the red sea bream's gut flora towards a

329

potentially more beneficial microbial community. Supplementation of LR significantly

330

affected TBC and LBS counts in the present study, as further supported this hypothesis. In

331

the present study, intestine PA increased significantly with dietary LR supplementation.

332

Extracellular enzymes secreted by LR due to Gram-positive bacteria, especially the members

333

of genus Bacillus, partially improved digestive enzyme activities [55]. Additionally, the

334

activities of digestive enzymes could be enhanced by probiotic administration (LR) [19]. The

335

effects of digestive processes caused by probiotics can be seen through the increase of

336

beneficial microorganisms, microbial enzyme activity and through improvement of the

337

intestinal microbial balance, as well as improvement of the digestibility and absorption of

338

food and feed utilization [56,57]. Furthermore, enhanced digestibility coefficients of the

SC

RI PT

307

AC C

EP

TE D

M AN U

[3]. This process allows for the effective improvement of both the survival and

ACCEPTED MANUSCRIPT crude protein, lipid and dry matter contents were observed in the current study, indicating the

340

beneficial effects of LR supplementation on the digestibility coefficients and absorption

341

ability of red sea bream.

342

Dietary LR supplementation significantly enhanced hematocrit of red sea bream as a general

343

health response towards nutritional strategies. In the present experiment, hematocrit content

344

was significantly highest in fish fed LR3 diet. Other LR supplemented groups showed

345

intermediate values whereas control group showed the lowest value. This indicated that

346

dietary LR elevated the health status of fish. Similarly, Dawood et al. [37,40] and Panigrahi

347

et al. [23] reported the enhanced hematocrit level by the supplementation of probiotics in red

348

sea bream and rainbow trout diets, respectively.

349

There is considerable evidence in the importance of host microbiota to fish immunity

350

response [58,59]. In the current study, LR supplementation succeeded in improving certain

351

immunological parameters compared to the control fish. In aquatic animals, there have been

352

many studies reported about LR supplementation which succeeded in improving certain

353

immunological parameters, including ACP, LZY, BA, SOD, TSP and peroxidase content

354

[9,13,18,20-22,60]. In agreement with the mentioned studies, our results indicated that

355

supplementation of LR enhanced ACP, peroxidase, SOD and TSP. Although to the best of

356

our knowledge, the available data on the efficiency of probiotic on red sea bream innate

357

immune response are scarce. In agreement to our findings, Dawood et al. [11] stated that

358

immune response was significantly higher in red sea bream fed diets supplemented with L.

359

plantarum. Moreover, Nikoskelainen et al. [18] stated that immune response was

360

significantly higher in rainbow trout (O. mykiss) by potential probiotic bacteria (L.

361

rhamnosus). However, administration of dietary B. subtilis in cobia (Rachycentron canadum)

362

[61], Weissellaci baria in hybrid surubim (Pseudoplatystoma sp.) [62] and B. subtilis in

363

juvenile large yellow croaker (Larimichthys crocea) [63] had no remarkable effects on

364

immune parameters.

365

The serum alternative complement pathway (ACP) may have effector mechanisms like direct

366

killing of microorganisms by lysis, opsonization of microorganisms by phagocytosis,

367

chemotactic attraction to the site of inflammation and activation of leucocytes, processing of

368

immune complexes and induction of specific antibody responses by augmentation of the

369

localization of antigens to B lymphocytes and antigen presenting cells [20]. Total serum

370

protein (TSP) is considered one of the most important compounds in the fish blood and are

AC C

EP

TE D

M AN U

SC

RI PT

339

ACCEPTED MANUSCRIPT essential for sustaining a healthy immune system [23,37]. In the present study, the fish fed

372

with diets containing LR exhibited a significant increase in ACP and TSP. All of these results

373

confirmed that innate immunity improves in aquatic animals fed with diets containing L.

374

rhamnosus. Previous studies demonstrated that oral administration of LR to rainbow trout (O.

375

mykiss) activated humoral immune defenses [18,20]. Salinas et al. [36] stated that using a

376

combination of bacterial strains that balance each other and through this, inhabit different

377

areas within the gut microflora, could enhance or extend desirable effects on the host immune

378

response and health.

379

A good way to assess host resistance against pathogenic bacteria would be through

380

bactericidal activity (BA). The present study’s results showed that the highest serum and

381

mucus BA were found in fish fed LR supplemented diets. Similarly, Dawood et al. [5,12]

382

reported that dietary probiotics significantly increased the BA of red sea bream. Lysozyme

383

activities in fish have been reported to be modulated by several probiotic species, like in

384

rainbow trout (O. mykiss) by L. rhamnosus and L. plantarum supplementation [20,64]. In

385

agreement with previous studies, in this study probiotic LR affected the LZY of P. major.

386

The serum peroxidase activity is an important enzyme that utilizes oxidative radicals to

387

produce hypochlorous acid to kill pathogens. During oxidative respiratory burst, it is mostly

388

released by the azurophilic granules of neutrophils [36,37]. In the present study, the

389

peroxidase values were enhanced by the LR supplemented diets, confirming previous data in

390

sea bream [5,12,36]. Superoxide dismutase (SOD) catalyzes the dismutation of the highly

391

reactive −O2 to less reactive H2O2 and functions in the main antioxidant defense pathways in

392

response to oxidative stress [13]. In the present study, SOD activities improved significantly

393

in the treatment groups. Similar observations were reported in L. rohita fed diets

394

supplemented with B. subtilis singularly or in combination with L. plantarum [17].

395

One of the foremost barriers of fish is its skin and epidermal mucus. These are crucial as the

396

fish’s innate defense mechanisms [65,66] as they avert any direct exposure to pollutants and

397

stressors in the surrounding environment. Results of this study showed that higher mucus

398

immune activities were observed in all fish fed LR -supplemented diets compared to that of

399

fish fed the control diet. Similarly, oral administration of probiotics increased the mucosal

400

immune responses of skin mucus in red sea bream (P. major) [5,11-13] and gilthead

401

seabream (Sparus aurata) [36].

AC C

EP

TE D

M AN U

SC

RI PT

371

ACCEPTED MANUSCRIPT Aquatic animal species are exposed to different adverse conditions that can result with

403

oxidative stress [67]. It is a consequence of increased reactive oxygen species (ROS)

404

production, while it decreases in the antioxidant defence [25]. Using the free radical

405

analytical system, oxidative stress was determined by measuring d-ROMs and BAP in plasma

406

samples [5,11,12]. Recently, these tests have also been applied as a suitable tool for

407

evaluating the oxidative stress in fish [5]. In their previous researches, Ballerini et al. [68] and

408

Pasquini et al. [69] noted that higher values of d-ROMs in fish implicate that there is a more

409

oxidative condition, while higher BAP values mean stronger oxidation tolerance. Using these

410

parameters, our study showed that fish fed diets supplemented with LR were more tolerant of

411

oxidative stress indicating a higher health status. It has been reported that, probiotic-enriched

412

diets can increase plasma antioxidant levels, thus neutralizing ROS [70]. Similarly, Dawood

413

et al. [5] illustrated that dietary supplementation of L. plantarum also stimulated the oxidative

414

status of red sea bream. To date there remains a lack of explanation about how these additives

415

work to affect these parameters, so more studies are needed.

416

LR as probiotic cells that are administered in such a way as to enter the gastrointestinal tract

417

and to be kept alive, with the aim of improving health of the host [59]. The heightened

418

immune responses and oxidative status with supplementation of LR as observed in the

419

present study can be related to the generally improved health status of red sea bream. The

420

observed improvement of fish immune parameters like the complement, bactericidal and

421

peroxidase activities by probiotic bacteria may be helpful by giving new standpoints and

422

tools for screening new strains of probiotic bacteria and given doses for use in aquaculture.

423

The probiotic bacteria have to be administered at an optimal dose that may depend on the size

424

and species of experimental fish and used probiotic strain.

EP

TE D

M AN U

SC

RI PT

402

AC C

425 426

Conclusions

427

Red sea bream fed with LR-supplemented diet showed improvements in growth performance,

428

feed utilization, immune response and oxidative status, indicating that LR could act as a

429

growth promoting agent for red sea bream aquaculture. In terms of innate immunity, addition

430

of probiotics seemed to be beneficial for red sea bream, as all the tested parameters (ACP,

431

BA, SOD, TSP and peroxidase) were enhanced. Incorporation of 104 to 106 cells g-1 LR

432

would be a more suitable supplement to enhance growth and to replace the use of antibiotics

433

which are known to exert harmful effects on the environment and on fish and human health.

ACCEPTED MANUSCRIPT 434 435

Acknowledgements

436

Financial support from Egyptian Government is gratefully acknowledged. The authors wish

437

to thank Mrs. Amina Moss for her kind advices. Special thanks to Dr. Hongyi Wei for the

438

cultivation of LR.

RI PT

439

References

441

[1] Miranda C. D., Zemelman R. (2002). Bacterial resistance to oxytetracycline in Chilean

442

salmon farming. Aquaculture 212:31-47.

443

[2] MacMillan J. R. (2001). Aquaculture and antibiotic resistance: A negligible public health

444

risk?. World Aquaculture 32:49-50.

445

[3] Dawood M. A. O., Koshio S. (2016a). Recent advances in the role of probiotics and

446

prebiotics in carp aquaculture: A review. Aquaculture 454:243-251.

447

[4] Dawood M. A. O., Koshio S., Ishikawa M., Yokoyama S., El Basuini M. F., Hossain M.

448

S., Nhu T.H., Moss A.S., Dossou S., Wei H. (2015c). Dietary supplementation of β-glucan

449

improves growth performance, the innate immune response and stress resistance of red sea

450

bream, Pagrus major. Aquaculture Nutrition. doi: 10.1111/anu.12376

451

[5] Dawood M. A. O., Koshio S., Ishikawa M., Yokoyama S. (2015a). Interaction effects of

452

dietary supplementation of heat-killed Lactobacillus plantarum and β-glucan on growth

453

performance, digestibility and immune response of juvenile red sea bream, Pagrus major.

454

Fish & shellfish immunology 45:33-42.

455

[6] Dimitroglou A., Davies S. J., Sweetman J., Divanach P., Chatzifotis S. (2010). Dietary

456

supplementation of mannan oligosaccharide on white sea bream (Diplodus sargus L.) larvae:

457

effects on development, gut morphology and salinity tolerance. Aquaculture Research

458

41:245-251.

459

[7] Hoseinifar S. H., Mirvaghefi A., Amoozegar M. A., Sharifian M., Esteban M. Á. (2015).

460

Modulation of innate immune response, mucosal parameters and disease resistance in

461

rainbow trout (Oncorhynchus mykiss) upon synbiotic feeding. Fish & shellfish immunology

462

45:27-32.

463

[8] Pionnier N., Falco A., Miest J. J., Shrive A. K., Hoole D. (2014). Feeding common carp

464

Cyprinus carpio with β-glucan supplemented diet stimulates C-reactive protein and

AC C

EP

TE D

M AN U

SC

440

ACCEPTED MANUSCRIPT complement immune acute phase responses following PAMPs injection. Fish & shellfish

466

immunology 39:285-295.

467

[9] Talpur A. D., Munir M. B., Mary A., Hashim R. (2014). Dietary probiotics and prebiotics

468

improved food acceptability, growth performance, haematology and immunological

469

parameters and disease resistance against Aeromonas hydrophila in snakehead (Channa

470

striata) fingerlings. Aquaculture 426:14-20.

471

[10] Zhang C. N., Li X. F., Xu W. N., Zhang D. D., Lu K. L., Wang L. N., Liu W. B. (2015).

472

Combined effects of dietary fructooligosaccharide and Bacillus licheniformis on growth

473

performance, body composition, intestinal enzymes activities and gut histology of triangular

474

bream (Megalobrama terminalis). Aquaculture Nutrition 21:755-766.

475

[11] Dawood M. A. O., Koshio S., Ishikawa M., Yokoyama S. (2015b). Effects of heat killed

476

Lactobacillus plantarum (LP20) supplemental diets on growth performance, stress resistance

477

and immune response of red sea bream, Pagrus major. Aquaculture 442:29-36.

478

[12] Dawood M. A. O., Koshio S., Ishikawa M., Yokoyama S. (2016c). Effects of dietary

479

inactivated Pediococcus pentosaceus on growth performance, feed utilization and blood

480

characteristics of red sea bream, Pagrus major juvenile. Aquaculture Nutrition 22(4): 923-

481

932.

482

[13] Dawood M. A. O., Koshio S., Ishikawa M., Yokoyama S., El Basuini M. F., Hossain M.

483

S., Nhu T.H., Dossou S., Moss A.S. (2016a). Effects of dietary supplementation of

484

Lactobacillus rhamnosus or/and Lactococcus lactis on the growth, gut microbiota and

485

immune responses of red sea bream, Pagrus major. Fish & shellfish immunology 49:275-285.

486

[14] Iwashita M. K. P., Nakandakare I. B., Terhune J. S., Wood T., Ranzani-Paiva M. J. T.

487

(2015). Dietary supplementation with Bacillus subtilis, Saccharomyces cerevisiae and

488

Aspergillus oryzae enhance immunity and disease resistance against Aeromonas hydrophila

489

and Streptococcus iniae infection in juvenile tilapia Oreochromis niloticus. Fish & shellfish

490

immunology 43:60-66.

491

[15] Nayak S.K. (2010). Probiotics and immunity: a fish perspective. Fish & Shellfish

492

Immunology 29:2–14.

493

[16] Lazado C. C., Caipang C. M. A. (2014). Atlantic cod in the dynamic probiotics research

494

in aquaculture. Aquaculture 424:53-62.

AC C

EP

TE D

M AN U

SC

RI PT

465

ACCEPTED MANUSCRIPT [17] Giri S. S., Sukumaran V., Oviya M. (2013). Potential probiotic Lactobacillus plantarum

496

VSG3 improves the growth, immunity, and disease resistance of tropical freshwater fish,

497

Labeo rohita. Fish & shellfish immunolog 34:660-666.

498

[18] Nikoskelainen S., Ouwehand A. C., Bylund G., Salminen S., Lilius E. M. (2003).

499

Immune enhancement in rainbow trout (Oncorhynchus mykiss) by potential probiotic bacteria

500

(Lactobacillus rhamnosus). Fish & shellfish immunology 15:443-452.

501

[19] Suzer C., Çoban D., Kamaci H. O., Saka Ş., Firat K., Otgucuoğlu Ö., Küçüksari H.

502

(2008). Lactobacillus spp. bacteria as probiotics in gilthead sea bream (Sparus aurata, L.)

503

larvae: effects on growth performance and digestive enzyme activities. Aquaculture 280:140-

504

145.

505

[20] Panigrahi A., Kiron V., Kobayashi T., Puangkaew J., Satoh S., Sugita H. (2004).

506

Immune responses in rainbow trout Oncorhynchus mykiss induced by a potential probiotic

507

bacteria Lactobacillus rhamnosus JCM 1136. Veterinary immunology and immunopathology

508

102:379-388.

509

[21] Panigrahi A., Kiron V., Puangkaew J., Kobayashi T., Satoh S., Sugita H. (2005). The

510

viability of probiotic bacteria as a factor influencing the immune response in rainbow trout

511

Oncorhynchus mykiss. Aquaculture 243: 241–254.

512

[22] Panigrahi A., Kiron V., Kobayashi T., Puangkaew J., Satoh S., Sugita H. (2007).

513

Immune modulation and expression of cytokine genes in rainbow trout Oncorhynchus mykiss

514

upon probiotic feeding. Developmental and Comparative Immunology 31: 372–382.

515

[23] Panigrahi A., Kiron V., Satoh S., Watanabe T. (2010). Probiotic bacteria Lactobacillus

516

rhamnosus influences the blood profile in rainbow trout Oncorhynchus mykiss (Walbaum).

517

Fish Physiology and Biochemistry 36: 969–977.

518

[24] Nikoskelainen S., Ouwehand A.C., Salminen S., Bylund G. (2001). Protection of

519

rainbow trout Oncorhynchus mykiss from furunculosis by Lactobacillus rhamnosus.

520

Aquaculture 198: 229–236.

521

[25] Topic Popovic, N., Strunjak-Perovic, I., Sauerborn-Klobucar, R., Barisic, J., Jadan, M.,

522

Kazazic, S., Kesner-Koren, I., Prevendar Crnic, A., Suran, J., Beer Ljubic, B., Matijatko, V.

523

(2016). The effects of diet supplemented with Lactobacillus rhamnosus on tissue parameters

524

of

525

DOI: 10.1111/are.13074

AC C

EP

TE D

M AN U

SC

RI PT

495

rainbow

trout,

Oncorhynchus

mykiss

(Walbaum).

Aquaculture

Research.

ACCEPTED MANUSCRIPT [26] Pirarat N., Kobayashi T., Katagiri T., Maita M., Endo M. (2006). Protective effects and

527

mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimental

528

Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Veterinary Immunology and

529

Immunopathology 113: 339–347.

530

[27] Goncalves A.T., Maita M., Futami K., Endo M., Katagiri T. (2011). Effects of a

531

probiotic bacterial Lactobacillus rhamnosus dietary supplement on the crowding stress

532

response of juvenile Nile tilapia Oreochromis niloticus. Fisheries Science 77: 633–642.

533

[28] Qin C., Xu L., Yang Y., He S., Dai Y., Zhao H., Zhou Z. (2014). Comparison of

534

fecundity and offspring immunity in zebrafish fed Lactobacillus rhamnosus CICC 6141 and

535

Lactobacillus casei BL23. Reproduction 147: 53–64.

536

[29] Morganti P., Bruno C., Guarneri F., Cardillo A., Del Ciotto P., Valenzano F. (2002).

537

Role of topical and nutritional supplement to modify the oxidative stress. International

538

journal of cosmetic science 24:331-339.

539

[30] Tatsumi N., Tsuji R., Yamada T., Kubo K., Matsuda T. (2000). Spot chem. EZ SP-4430

540

no kiso teki kento. Journal of Clinical Laboratory Instruments and Reagents 23:427-433.

541

[31] AOAC (1998). Official methods of analysis of AOAC International. 16th edition.

542

Association of Official Analytical Chemists, Washington, DC., USA.

543

[32] Hoseinifar S. H., Soleimani N., Ringø E. (2014). Effects of dietary fructo-

544

oligosaccharide supplementation on the growth performance, haemato-immunological

545

parameters, gut microbiota and stress resistance of common carp (Cyprinus carpio) fry.

546

British Journal of Nutrition 112:1296-1302.

547

[33] Sitjà-Bobadilla A., Mingarro M., Pujalte M. J., Garay E., Alvarez-Pellitero P., Pérez-

548

Sánchez J. (2003). Immunological and pathological status of gilthead sea bream (Sparus

549

aurata L.) under different long-term feeding regimes. Aquaculture 220:707-724.

550

[34] Lygren B., Sveier H., Hjeltnes B., Waagbø R. (1999). Examination of the

551

immunomodulatory properties and the effect on disease resistance of dietary bovine

552

lactoferrin and vitamin C fed to Atlantic salmon (Salmo salar) for a short-term period. Fish &

553

Shellfish Immunology 9:95-107.

554

[35] Iida T., Takahashi K., Wakabayashi H. (1989). Decrease in the bactericidal activity of

555

normal serum during the spawning period of rainbow trout [Salmo gairdneri]. Bulletin of the

556

Japanese Society of Scientific Fisheries (Japan) 55:463-465.

AC C

EP

TE D

M AN U

SC

RI PT

526

ACCEPTED MANUSCRIPT [36] Salinas I., Abelli L., Bertoni F., Picchietti S., Roque A., Furones D., Esteban M. Á.

558

(2008). Monospecies and multispecies probiotic formulations produce different systemic and

559

local immunostimulatory effects in the gilthead seabream (Sparus aurata L.). Fish & shellfish

560

immunology 25:114-123.

561

[37] Dawood M. A. O., Koshio S., Ishikawa M., Yokoyama S. (2016b). Immune responses

562

and stress resistance in red sea bream, Pagrus major, after oral administration of heat-killed

563

Lactobacillus plantarum and vitamin C. Fish & shellfish immunology 54:266-275.

564

[38] De B. C., Meena D. K., Behera B. K., Das P., Mohapatra P. D., Sharma A. P. (2014).

565

Probiotics in fish and shellfish culture: immunomodulatory and ecophysiological responses.

566

Fish physiology and biochemistry 40:921-971.

567

[39] Ringø E., Dimitroglou A., Hoseinifar S.H. (2014) Prebiotics in finfish: an update. In:

568

Merrifield D, Ringø E (eds) Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics pp.

569

360-400. Wiley-Blackwell Publishing, Oxford, UK.

570

[40] Dawood M. A. O., Koshio S., Ishikawa M., Yokoyama S. (2015d). Effects of Partial

571

Substitution of Fish Meal by Soybean Meal with or without Heat-Killed Lactobacillus

572

plantarum (LP20) on Growth Performance, Digestibility, and Immune Response of

573

Amberjack, Seriola dumerili Juveniles. BioMed research international 2015.

574

[41] Akrami R., Nasri-Tajan M., Jahedi A., Jahedi M., Razeghi Mansour M., Jafarpour S. A.

575

(2015). Effects of dietary synbiotic on growth, survival, lactobacillus bacterial count, blood

576

indices and immunity of beluga (Huso huso Linnaeus, 1754) juvenile. Aquaculture

577

Nutrition 21:952-959.

578

[42] Van Doan H., Doolgindachbaporn S., Suksri A. (2014). Effects of low molecular weight

579

agar and Lactobacillus plantarum on growth performance, immunity, and disease resistance

580

of basa fish (Pangasius bocourti, Sauvage 1880). Fish & shellfish immunology 41:340-345.

581

[43] Zhang Q., Ma H., Mai K., Zhang W., Liufu Z., Xu W. (2010). Interaction of dietary

582

Bacillus subtilis and fructooligosaccharide on the growth performance, non-specific

583

immunity of sea cucumber, Apostichopus japonicus. Fish & shellfish immunology 29:204-

584

211.

585

[44] Zhang C. N., Li X. F., Xu W. N., Jiang G. Z., Lu K. L., Wang L. N., Liu W. B. (2013).

586

Combined effects of dietary fructooligosaccharide and Bacillus licheniformis on innate

587

immunity, antioxidant capability and disease resistance of triangular bream (Megalobrama

588

terminalis). Fish & shellfish immunology 35:1380-1386.

AC C

EP

TE D

M AN U

SC

RI PT

557

ACCEPTED MANUSCRIPT [45] Bairagi A., Ghosh K. S., Sen S. K., Ray A. K. (2002). Enzyme producing bacterial flora

590

isolated from fish digestive tracts. Aquaculture International 10:109-121.

591

[46] Ramirez R. F., Dixon B. A. (2003). Enzyme production by obligate intestinal anaerobic

592

bacteria isolated from oscars (Astronotus ocellatus), angelfish (Pterophyllum scalare) and

593

southern flounder (Paralichthys lethostigma). Aquaculture 227:417-426.

594

[47] Daniels C. L., Merrifield D. L., Boothroyd D. P., Davies S. J., Factor J. R., Arnold K. E.

595

(2010). Effect of dietary Bacillus spp. and mannan oligosaccharides (MOS) on European

596

lobster (Homarus gammarus L.) larvae growth performance, gut morphology and gut

597

microbiota. Aquaculture 304:49-57.

598

[48] Lara-Flores M., Olvera-Novoa M. A., Guzmán-Méndez B. E., López-Madrid W. (2003).

599

Use of the bacteria Streptococcus faecium and Lactobacillus acidophilus, and the yeast

600

Saccharomyces cerevisiae as growth promoters in Nile tilapia (Oreochromis niloticus).

601

Aquaculture 216:193-201.

602

[49] Dimitroglou A., Merrifield D. L., Moate R., Davies S. J., Spring P., Sweetman J.,

603

Bradley G. (2009). Dietary mannan oligosaccharide supplementation modulates intestinal

604

microbial ecology and improves gut morphology of rainbow trout, (Walbaum). Journal of

605

animal science 87:3226-3234.

606

[50] Glenn G. R., Roberfroid M. B. (1995). Dietary modulation of the human colonic

607

microbiota: introducing the concept of prebiotics. Journal of Nutrition 125:1401-1412.

608

[51] Dawood M. A. O., El-Dakar A., Mohsen M., Abdelraouf E., Koshio S., Ishikawa M.,

609

Yokoyama S. (2014). Effects of Using Exogenous Digestive Enzymes or Natural Enhancer

610

Mixture on Growth, Feed Utilization, and Body Composition of Rabbitfish, Siganus rivulatus.

611

Journal of Agricultural Science and Technology 4(3B).

612

[52] Perez-Casanova J. C., Murray H. M., Gallant J. W., Ross N. W., Douglas S. E., Johnson

613

S. C. (2006). Development of the digestive capacity in larvae of haddock (Melanogrammus

614

aeglefinus) and Atlantic cod (Gadus morhua). Aquaculture 251:377-401.

615

[53] Furne M., Hidalgo M. C., Lopez A., Garcia-Gallego M., Morales A. E., Domezain A.,

616

Sanz A. (2005). Digestive enzyme activities in Adriatic sturgeon Acipenser naccarii and

617

rainbow trout Oncorhynchus mykiss. A comparative study. Aquaculture 250:391-398.

618

[54] Ringø E., Gatesoupe F. J. (1998). Lactic acid bacteria in fish: a review. Aquaculture

619

160:177-203.

AC C

EP

TE D

M AN U

SC

RI PT

589

ACCEPTED MANUSCRIPT [55] Azokpota P., Hounhouigan D. J., Nago M. C., Jakobsen M. (2006). Esterase and

621

protease activities of Bacillus spp. from afitin, iru and sonru; three African locust bean

622

(Parkia biglobosa) condiments from Benin. African journal of Biotechnology 5:265.

623

[56] Lee S. Y., Lee B. H. (1990). Esterolytic and Lipolytic Activities of Lactobacillus Casei-

624

subsp-Casei LLG. Journal of food science 55:119-122.

625

[57] Bagheri T., Hedayati S. A., Yavari V., Alizade M., Farzanfar A. (2008). Growth,

626

survival and gut microbial load of rainbow trout (Onchorhynchus mykiss) fry given diet

627

supplemented with probiotic during the two months of first feeding. Turkish Journal of

628

Fisheries and Aquatic Sciences 8:43-48.

629

[58] Gómez G. D., Balcázar J. L. (2008). A review on the interactions between gut

630

microbiota and innate immunity of fish. FEMS Immunology & Medical Microbiology 52:145-

631

154.

632

[59] Merrifield D. L., Dimitroglou A., Foey A., Davies S. J., Baker R. T., Bøgwald J., Ringø

633

E. (2010). The current status and future focus of probiotic and prebiotic applications for

634

salmonids. Aquaculture 302:1-18.

635

[60] Kühlwein H., Merrifield D. L., Rawling M. D., Foey A. D., Davies S. J. (2014). Effects

636

of dietary β (1, 3)(1, 6)-D-glucan supplementation on growth performance, intestinal

637

morphology and haemato-immunological profile of mirror carp (Cyprinus carpio L.). Journal

638

of animal physiology and animal nutrition 98:279-289.

639

[61] Geng X., Dong X. H., Tan B. P., Yang Q. H., Chi S. Y., Liu H. Y., Liu X. Q. (2011).

640

Effects of dietary chitosan and Bacillus subtilis on the growth performance, non-specific

641

immunity and disease resistance of cobia, Rachycentron canadum. Fish & shellfish

642

immunology 31:400-406.

643

[62] Mouriño J. L. P., Do Nascimento Vieira F., Jatobá A. B., Da Silva B. C., Jesus G. F. A.,

644

Seiffert W. Q., Martins M. L. (2012). Effect of dietary supplementation of inulin and W.

645

cibaria on haemato-immunological parameters of hybrid surubim (Pseudoplatystoma sp).

646

Aquaculture Nutrition 18:73-80.

647

[63] Ai Q., Xu H., Mai K., Xu W., Wang J., Zhang W. (2011). Effects of dietary

648

supplementation of Bacillus subtilis and fructooligosaccharide on growth performance,

649

survival, non-specific immune response and disease resistance of juvenile large yellow

650

croaker, Larimichthys crocea. Aquaculture 317:155-161.

AC C

EP

TE D

M AN U

SC

RI PT

620

ACCEPTED MANUSCRIPT [64] Son V. M., Chang C. C., Wu M. C., Guu Y. K., Chiu C. H., Cheng W. (2009). Dietary

652

administration of the probiotic, Lactobacillus plantarum, enhanced the growth, innate

653

immune responses, and disease resistance of the grouper Epinephelus coioides. Fish &

654

shellfish immunology 26:691-698.

655

[65] Hernandez L. H. H., Barrera T. C., Mejia J. C., Mejia G. C., Del Carmen M., Dosta M.,

656

Sotres J. A. M. (2010). Effects of the commercial probiotic Lactobacillus casei on the growth,

657

protein content of skin mucus and stress resistance of juveniles of the Porthole livebearer

658

Poecilopsis gracilis (Poecilidae). Aquaculture Nutrition 16:407-411.

659

[66] Palaksha K. J., Shin G. W., Kim Y. R., Jung T. S. (2008). Evaluation of non-specific

660

immune components from the skin mucus of olive flounder (Paralichthys olivaceus). Fish &

661

shellfish immunology 24:479-488.

662

[67] Dawood M. A. O., Koshio S. (2016b) Vitamin C supplementation to optimize growth,

663

health and stress resistance in aquatic animals. Reviews in aquaculture. DOI:

664

10.1111/raq.12163

665

[68] Ballerini A., Civitareale C., Fiori M., Regini M., Betti M., Brambilla, G. (2003).

666

Traceability of inbred and crossbred Cinta Senese pigs by evaluating the oxidative stress.

667

Journal of Veterinary Medicine Series A 50:113-116.

668

[69] Pasquini A., Luchetti E., Marchetti V., Cardini G., Iorio E. L. (2008). Analytical

669

performances of d-ROMs test and BAP test in canine plasma. Definition of the normal range

670

in healthy Labrador dogs. Veterinary Research Communications 32:137-143.

671

[70] Martinez Cruz P., Ibanez A.L., Monroy Hermosillo O.A., Ramirez Saad H.C. (2012).

672

Use

673

doi:10.5402/2012/916845.

SC

M AN U

TE D

probiotics

in

aquaculture.

EP

of

AC C

674

RI PT

651

ISRN

Microbiology

2012,

916845.

ACCEPTED MANUSCRIPT 1

Table 1. Growth, nutrient utilization parameters and survival of red sea bream fed

2

experimental diets for 8 weeks.

3

LR1

LR2

LR3

In. wt.

3.31±0.012

3.3±0.01

3.31±0.01

3.31±0.02

FBW

16.36±0.46a

19.43±0.41b

19.7±0.36b

19.54±0.4b

BWG

395.31±15.1a 488.41±13.3b

495.81±11.4b 491.44±11.58b

SGR

2.86±0.05 a

3.16±0.04 b

3.19±0.03b

3.17±0.04b

FI

13.84±0.36a

14.18±0.22a

14.4±0.18ab

14.83±0.37ab

FER

0.94±0.05a

1.14±0.04ab

1.14±0.02ab

1.09±0.01ab

PER

1.88±0.1a

2.2±0.14ab

2.2±0.1ab

PG

159.62±5.7a

178.43±3.62ab 182.78±4.38b 178.17±4.22ab

SR

90±2.89

95±2.89

SC

RI PT

Parameter1,2 LR0

96.67±3.33

M AN U

98.33±1.67

2.12±0.02ab

4

1

5

significant different (P > 0.05).

6

2

7

SGR, specific growth rate (% day−1); FI, feed intake (g dry diet fish−1 56 days−1); FER, feed

8

efficiency ratio; PER, protein efficiency ratio; PG, protein gain; SR, survival (%).

12 13 14 15 16 17 18 19 20 21 22 23

TE D

11

EP

10

In. wt., initial weight (g); FBW, final weight (g); BWG, percentage of weight gain (%);

AC C

9

Values in the same column with the same superscript or absence of superscripts are not

ACCEPTED MANUSCRIPT 24

Table 2. Protease activity (PA) and apparent digestibility coefficients (ADC) of tested fish

25

fed experimental diets for 8 weeks.

26

Item1

LR0

PA (unit mg-1 protein)

0.027±0.003a 0.029±0.002b 0.032±0.002b 0.031±0.001b

ADC (% protein)

82.96±0.14a 85.21±0.41b 85.46±0.28b 85.87±0.86b

ADC (% lipid)

67.91±0.47a 68.89±1.9a

ADC (% dry matter)

58.41±0.28a 58.36±1.87a 63.75±0.83b 64.57±1b

1

28

significant different (P > 0.05).

LR2

LR3

RI PT

27

LR1

72.51±1.9ab

74.14±1.2b

SC

Values in the same column with the same superscript or absence of superscripts are not

29

AC C

EP

TE D

M AN U

30

ACCEPTED MANUSCRIPT

Table 3. Oxidative status of tested fish fed experimental diets for 8 weeks. Item1,2

LR0

BAP (µmol L-1)1

2182.67±77.62a 2499.33±112.1ab 2335±67.4a 2794±181.59b

d-ROMs (U. Carr)2

57.33±6.39b

41.67±5.49ab

LR3

47±4.16ab

38±3.79a

Values in the same column with the same superscript or absence of superscripts are not

significant different (P > 0.05).

EP

TE D

M AN U

SC

BAP, biological antioxidant potential; d-ROMs, reactive oxygen metabolites.

AC C

2

LR2

RI PT

1

LR1

ACCEPTED MANUSCRIPT

ab

ab

ab

RI PT

6.8 6.6 6.4 6.2 6 5.8 5.6 5.4 5.2 5

a

LR0

LR1

LR2

c

b

a

d

TE D

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

EP

Lactobacillus sp. count (log CFU g-1 intestine)

(B)

LR3

M AN U

Test diets

SC

Total bacterial count (log CFU g-1 intestine)

(A)

LR1

LR2

LR3

Test diets

AC C

LR0

Fig. 1. Effects of dietary L. rhamnosus (LR) supplementation on: (A) total bacterial count (log CFU g-1 intestine), (B) viable Lactobacillus sp. (LBS count, log CFU g-1 intestine) of red sea bream fed test diets for 8 weeks. Where LR0 (0), LR1 (102), LR2 (104), and LR3 (106) (cell LR g-1 diet), respectively. Values are means, with standard errors represented by vertical bars. Mean values with unlike letters were significantly different (P<0.05).

ACCEPTED MANUSCRIPT

a

a

LR0

LR1

ab

LR2

(B)

a

a

2.5

1.5 1 0.5 0

b

b

LR2

LR3

TE D

2

EP

Total serum protein (g dl-1)

3.5

LR3

M AN U

Test diets

3

b

RI PT

45 40 35 30 25 20 15 10 5 0

SC

Hematocrit level (%)

(A)

LR1

Test diets

AC C

LR0

Fig. 2. Effects of dietary L. rhamnosus (LR) supplementation on: (A) hematocrit levels (%), (B) total serum protein (g dl-1) of red sea bream fed test diets for 8 weeks. Where LR0 (0), LR1 (102), LR2 (104), and LR3 (106) (cell LR g-1 diet), respectively. Values are means, with standard errors represented by vertical bars. Mean values with unlike letters were significantly different (P<0.05).

ACCEPTED MANUSCRIPT

2900 (A)

2800

(B)

LR3

2600

LR1

2500 2400

LR2

(C)

2300 2200

(D)

LR0

2100 0

20

40

60

100

M AN U

d-ROMs (U.Carr)

80

SC

2000

RI PT

BAP (µ MOL L-1)

2700

Fig. 3. Oxidative stress parameters in red sea bream fed test diets for 8 weeks. Values are expressed as mean (n=3). Central axis based on mean values of reactive oxygen metabolites (dROMs), biological antioxidant potential (BAP) from each treatment. Zone (A): high BAP and low d-ROMs (good condition); Zone (B): high BAP and high d-ROMs (acceptable condition); Zone (C): low BAP and low d-ROMs (acceptable condition); Zone (D): low BAP and high d-

AC C

EP

diet), respectively.

TE D

ROMs (stressed condition). Where LR0 (0), LR1 (102), LR2 (104), and LR3 (106) (cell LR kg-1

ACCEPTED MANUSCRIPT

ab

c

RI PT

60 50

bc

a

40 30 20 10 0 LR1

LR2

LR3

M AN U

LR0

SC

ACP (ACH50 units ml-1)

70

Test diets

Fig. 4. Alternative complement pathway (ACP) of red sea bream fed test diets for 8 weeks. Where LR0 (0), LR1 (102), LR2 (104), and LR3 (106) (cell LR g-1 diet), respectively. Values are means, with standard errors represented by vertical bars. Mean values with unlike letters were

AC C

EP

TE D

significantly different (P<0.05).

ACCEPTED MANUSCRIPT

Mucus

LR0

LR1

LR2

SC

RI PT

50 45 40 35 30 25 20 15 10 5 0

LR3

M AN U

LZY (units ml-1)

Serum

Test diets

Fig. 5. Serum and mucus lysozyme activity (LZY) of red sea bream fed test diets for 8 weeks. Where LR0 (0), LR1 (102), LR2 (104), and LR3 (106) (cell LR g-1 diet), respectively. Values are means, with standard errors represented by vertical bars. Mean values with unlike letters were

AC C

EP

TE D

significantly different (P<0.05).

ACCEPTED MANUSCRIPT

b ab

ab a

LR0

LR1

LR2 Test diets

LR3

RI PT

8 7 6 5 4 3 2 1 0

Mucus

SC

BA (105 cfu ml-1)

Serum

M AN U

Fig. 6. Serum and mucus bactericidal activity (BA) of red sea bream fed test diets for 8 weeks. Where LR0 (0), LR1 (102), LR2 (104), and LR3 (106) (cell LR g-1 diet), respectively. Values are means, with standard errors represented by vertical bars. Mean values with unlike letters were

AC C

EP

TE D

significantly different (P<0.05).

ACCEPTED MANUSCRIPT

70 b

50

b

b

LR1

LR2

a

40 30 20 10 0 Test diets

LR3

SC

LR0

RI PT

SOD (U ml-1)

60

M AN U

Fig. 7. Superoxide dismutase (SOD) of red sea bream fed test diets for 8 weeks. Where LR0 (0), LR1 (102), LR2 (104), and LR3 (106) (cell LR g-1 diet), respectively. Values are means, with standard errors represented by vertical bars. Mean values with unlike letters were significantly

AC C

EP

TE D

different (P<0.05).

ACCEPTED MANUSCRIPT

2

b

b

LR2

LR3

a

a 1.5 1 0.5

LR0

LR1 Test diets

SC

0

RI PT

Peroxidase (OD at 450 nm)

2.5

M AN U

Fig. 8. Serum peroxidase of red sea bream fed test diets for 8 weeks. Where LR0 (0), LR1 (102), LR2 (104), and LR3 (106) (cell LR g-1 diet), respectively. Values are means, with standard errors represented by vertical bars. Mean values with unlike letters were significantly different

AC C

EP

TE D

(P<0.05).

ACCEPTED MANUSCRIPT

Highlights


Probiotics could be used as an alternative method to antibiotic treatment in red sea bream aquaculture. Probiotics studied enhance fish growth.




Probiotics studied enhance fish innate immunity and oxidative status.




Incorporation of 104 to 106 cells g-1 LR is a suitable supplement to enhance general

AC C

EP

TE D

M AN U

SC

performances of red sea bream.

RI PT