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FISHERIES SCIENCE 2006; 72: 310–321

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Growth, stress tolerance and non-specific immune response of Japanese flounder Paralichthys olivaceus to probiotics in a closed recirculating system

Yousuke TAOKA, 1 Hiroto MAEDA, 2a * Jae-Yoon JO, 3 Min-Jee JEON, 3 Sungchul C. BAI, 3 Won-Jae LEE, 4 Kazuya YUGE 5 AND Shunsuke KOSHIO 6

1 The United Graduate School of Agricultural Sciences, Faculty of Fisheries, Kagoshima University, Kagoshima 890-0065, 2 Laboratory of Microbiology, Faculty of Fisheries, Kagoshima University, Kagoshima 890-0056, Japan, 3 Department of Aquaculture, Pukyong National University, Busan 599-1, 4 Department of Microbiology, Pukyong National University, Busan 599-1, Korea, 5 DSM Nutrition Japan KK, Tokyo 143-0016, and 6 Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Kagoshima 890-0056, Japan

ABSTRACT: Effects of probiotics on growth, stress tolerance and non-specific immune response in Japanese flounder Paralichthys olivaceus were evaluated in a closed recirculating system. Survival and growth of flounder treated by supplying commercial probiotics either in the diet (the probiotic diet group), or into the rearing water (the water supply group), were higher compared to the untreated group (the control group). Water quality parameters, pH, NH 4 -N, NO 2 -N and PO 4 -P showed lower con- centration in the probiotic diet group compared with the control group and the supply group. Plasma lysozyme activity in the probiotic diet group and the water supply group was significantly higher (P < 0.05) than that in the control group. In heat shock stress tests, flounder in the probiotics-treated groups showed greater heat tolerance (measured by 50% lethal time, LT50) than the control group. Pathogen challenge tests with Vibrio anguillarum (2 × 10 7 c.f.u./mL) resulted in significantly higher survival in the probiotics-treated groups than the control group. Results indicated that probiotics sup- plied in the rearing water and the diet of fish enhanced the stress tolerance and the non-specific immune system of Japanese flounder, providing them a higher resistance against stress conditions and pathogens.

KEY WORDS: closed recirculating system, flounder, lysozyme, non-specific immune response, probiotics, water quality.

INTRODUCTION

In intensive aquaculture, growth and proliferation of microorganisms in the rearing habitat are accel- erated by excessive feeding. 1 Successful aquacul- ture relies on adequate microbial control to prevent the proliferation of pathogenic bacteria that often results in fish mortality. Bacteria can accumulate in fish by ingestion or through the body surfaces and is transported by the body fluids into the tissues. This may be one of the causes of indigenous microflora formation and bacterial infection in fish.

*Corresponding author: Tel: 81-59-231-9566. Fax: 81-59-231-9557. Email: maeda@bio.mie-u.ac.jp a Present address: 1577 Kurimamachiya, Tsu, Mie, 514-8507 JAPAN Received 22 November 2004. Accepted 24 October 2005.

Antibiotics had been frequently used to cure bacterial infection and prevent fish mortality in aquaculture systems. However, the use of antibiot- ics has become limited because certain pathogens have developed resistance to the drugs. This out- come gave rise to the use of probiotics as method for preventing fish disease and in improving aquaculture conditions and fish health. Generally, probiotics are described as live microbial feed additives that improve the microbial condition of the host animal’s gastrointestinal tract. 2 Probiotics include components of microbial cells that stimu- late the immune system against pathogens. 3 Such immunostimulants enhance the defense system of host organisms against pathogens by enhance- ment of phagocytosis, antibody production, chemiluminescent response and superoxide anion production. 4 Probiotics are inoculated into the rearing water to improve culture conditions or

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incorporated in the food of fish through diet. 57 Probiotics composed of lactic acid bacteria are widely used to improve the health of humans and livestock. 8 In black tiger shrimp, scallop, flounder, Atlantic salmon, rainbow trout and turbot, the growth and survival plus water and sediment qual- ity of the farm environment were improved, and the microbial flora in fish intestine, rearing water and feces were stabilized by addition of probiot- ics. 813 The influence of microbial flora from the rearing water on the gastrointestinal flora of the cultured animal is widely recognized. 14 Flounder is one of the most highly valued species in Japanese and Korean aquaculture and its culture has increased in scale. The fish, however, have been affected by vibriosis caused by Vibrio anguil- larum. 15 The bacteria act as an opportunistic pathogen in various juvenile fishes. However, the application of probiotics in flounder is still not enough to produce healthier fish and improve the immune defense system. Therefore, this study was conducted to investigate the effects of probiotics on growth performance, stress tolerance, non- specific immune response and the most effective method of application of probiotics in the culture of Japanese flounder Paralichthys olivaceus. In addition, experiments were conducted to investi- gate if probiotic treatment would enhance toler- ance to stress and resistance to pathogens.

MATERIALS AND METHODS

Experimental animals

One thousand Japanese flounder with a mean weight of 15 g were obtained from Uljin Marine Hatchery in Korea and transported to the Depart- ment of Aquaculture, Pukyong National University. The fish population was conditioned in aerated 1 m 3 tanks at 20–22°C for 1 month prior to the experiment. Two weeks before the start of the experiment, the fish were transferred into a closed recirculating system at a density of 20 individuals per aquarium. During the conditioning period, the fish were fed with the control diet twice daily at 1% of body weight (Table 1). The recirculating system was equipped with four 120-L aquaria and one 450-L filter tank treated with NH 4 Cl to accelerate nitrification.

Experimental design

Three treatments with three simultaneous replica- tions were used: (i) a control group fed with the

Table 1

Compositions

and

proximate

analyses

of

experimental diets

 
 

Diets

Ingredients (%)

Control

Probiotic diet

Fish meal Probiotics Soy bean meal α-starch Fish oil Soy bean lecithin Corn oil Vitamin mix. Mineral mix. Taurine IMP Betain Alanine α-cellulose CMC 1 Total

54.9

54.9

1.0

20.0

20.0

3.4

3.4

5.2

5.2

3.2

3.2

2.1

2.1

3.0

3.0

5.0

5.0

0.6

0.6

0.1

0.1

0.6

0.6

0.4

0.4

1.0

0.5

0.5

100

100

Approximate composition (% wet weight)

 

Pooled SEM 2

Moisture

11.3 ± 0.05 50.6 ± 0.09 12.5 ± 0.90 14.5 ± 0.05

13.6 ± 0.31 46.5 ± 0.18 13.5 ± 0.06 14.1 ± 0.46

1.35

Protein

2.37

Fat

0.50

Ash

0.27

Values are means ± standard error (n = 3). menadione, 3.17; DL-α-tochophenol acetate, 26.67; thiamin- HCl, 4.0; riboflavin, 13.33; pyridoxine-HC, 3.17; biotin, 0.33; inositol, 267.0; nicotinic acid, 53.33; Ca panthothenate, 18.67; folic acid, 1.0; choline chloride, 545.83; vitamin A-palmitate, 12.83; calcifenol, 0.67; ascorbyl-2-phosphate-Mg, 4.67; cellulose, 45.33 (g/kg premix). NaCl, 36.76; MgSO 4 ·7H 2 O, 137.0; NaHPO 4 ·2H 2 O, 77.06; KH 2 PO 4 , 239.80; Ca(H 2 PO 4 )·2H 2 O, 6.34; Fe citrate, 29.70; Ca lactate, 461.80; Al (OH) 3 , 0.56; ZnSO 4 ·7H 2 O, 3.58; CuSO 4 , 0.14; MnSO 4 ·7H 2 O, 0.696; Ca(IO 3 ) 2 , 0.16; CoSO 4 ·7H 2 O, 2.17; cellulose, 4.234 (g/kg premix). 1 CMC; carboxymethyl cellulose. 2 SEM; standard error of mean.

control diet and reared in water without probiotics; (ii) a probiotic diet group fed with a diet containing 1% probiotics and reared in water without probiot- ics; and (iii) a water supply group fed with the con- trol diet and reared in water with probiotics. Each group was raised in separate recirculating systems to avoid inter-treatment contamination. The composition of the experimental diets is presented in Table 1. The commercial probiotics (Alchem Poseidon; Alchem- Korea CO., LDT, Wonju, Korea) contained Bacillus subtilis (>1.6 × 10 7 c.f.u./g), Lactobacillus acidophilus (>1.2 × 10 8 c.f.u./g), Clostridium butyricum (>2.0 10 7 c.f.u./g) and Saccharomyces cerevisiae

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(>1.6 × 10 7 c.f.u./g). The probiotics were added in corn oil at 40°C and mixed with the other ingredi- ents at a proportion of 1% using a food mixer. Water was added at 40% to dry weight of the mix- ture to facilitate pelleting by a meat chopper. After pelleting, diets were dried with an electric fan at room temperature. In the water supply group, 10 g of the probiotics suspended in 100 mL rearing sea water and aerated vigorously for 12 h was intro- duced into the rearing system every 10 days. The experimental fish were fed twice daily at 1% of body weight and reared for 50 days at water tem- perature of 17 ± 2°C and salinity of 33–35 psu. Shortly before feeding, the hands of feeder were sterilized with 70% ethanol to prevent contamina- tion of the control diet with the probiotics. Excess food and feces were removed a few hours after feeding.

Sample collection

During the 50 days of feeding, the body weight of fish was collectively measured every 10 days. At 50 days of rearing, moisture attached to body sur- face of sampled fish was removed in a net and the fish was quickly placed in a preweighed small ves- sel with seawater to record the body weight and the body length was measured. Livers were dis- sected out from six fish in each group, and weighed to calculate the hepatosomatic index (HSI). Growth performance indicators; survival rate, Fulton’s condition factors, specific weight gain (SWG) and specific growth rate (SGR) were calculated. At the end of rearing trial, three fish were sampled from each recirculating system per treatment group for approximate analysis of fish body compositions, and the data from the three fish were pooled. Blood samples from four fish in each group were obtained from the caudal vein by using a heparin- ized syringe. Each blood sample was centrifuged at 5000 × g for 10 min. The supernatant was then taken as a plasma sample and stored at 20°C until analysis. At the end of experiment, skin mucus of four fish in each group was sampled according to Aranishi and Nakane. 16 Fish were anesthetized with 0.5% (w/v) MS-222 (ethyl-3-aminobenzoate methane- sulforic acid salt) solution for 20 min then immersed in 10 mM sodium phosphate buffer (PBS, pH 7.5) containing 115 mM NaCl for 1 min. Skin mucus was collected by wiping with polyeth- ylene gloves. The sample was homogenized in four volumes of PBS and centrifuged at 15 000 × g for 30 min at 4°C. The supernatant was stored at 20°C until analysis.

Analytical methods

Water quality

Rearing water samples (1 L) were collected every

5 days with a polyethylene bottle, and then stored

in a freezer at 20°C until analysis. At the time of

sampling, pH, dissolved oxygen (DO), salinity and water temperature were checked by using a port- able DO meter (KDO5151, Kasahara Chemical Instrument, Saitama, Japan) and a portable pH meter (Pinpoint pH meter, American Marine INC, Ridgefield, USA). Urea-nitrogen was analyzed by the method of Newell et al. 17 NH 4 -N, NO 2 -N and PO 4 -P were analyzed by the method of Strickland and Parsons. 18 NO 3 -N was analyzed with a DREL 2000 (HACH company, Loveland, Colorado, USA). Chemical oxygen demand (COD) was analyzed by potassium permanganate–iodine titration.

Proximate analyses of the feed and carcass

Proximate analyses of the whole body of flounder

and the test diets were carried out. Crude protein, ash content and moisture were analyzed according

to the method of the Association of Official Analyt-

ical Chemists. 19 Crude fat was analyzed by using

the Soxtec system 1046 (Foss-Tecator, Höganäs, Sweden) after freeze-drying the sample.

Monitoring of non-specific immune responses

Protein concentrations in the plasma and skin mucus were determined according to Lowly et al. 20 Lysozyme activity of fish plasma and skin mucus were determined by turbidometric assays. 21,22 Plasma (100 µL) was added to 900 µL of Micrococ- cus lysodeikticus (lyophilized cell, Sigma-ALDRICH Chemical company, St. Louis, Mo, USA) aqueous solution and incubated at 25°C. The absorbance was read at 530 nm with a spectrophotometer (OPRON-3000, Hanson Technology Co., Ltd, Korea) at 0.5 and 4.5 min after reaction. Fish mucus (0.5 mL) was added to 2.5 mL of M. lysodeikticus aqueous solution and incubated at 25°C. The absorbance was read and recorded at 530 nm with

a spectrophotometer 20 min after reaction. The

M. lysodeikticus aqueous solution was prepared by adding M. lysodeikticus to 50 mM PBS (for plasma sample) and 5 mM (for mucus sample), and was adjusted to the absorbance of 0.7 and 0.6 at 530 nm, respectively. PBS and all apparatus used for lysozyme activity assay were autoclaved for 15 min at 121°C The unit of lysozyme activity (U) was defined as the amount of enzyme that caused a decrease in absorption of 0.001/mg protein.

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Stress tolerance tests

After feeding for 50 days, flounder in each treat-

ment were subjected to heat shock stress tests. Six flounder were transferred from each rearing sys- tem to individual test glass aquaria with a volume

of 30 L. Water temperature was maintained at 30°C

with a thermostatically controlled glass heater, based on preliminary experiments. The test was conducted until half of the test population died. The number of surviving fish was recorded and the 50% lethal time (LT50) was calculated. The blood

from the caudal vein of surviving fish was collected with a heparinized syringe and plasma sample was stored at 20°C until analysis. All measurements were conducted in duplicate. The tolerance to exposure to air in each treat- ment was also examined according to the method

of Koshio et al. 23 A net with small mesh was spread

out on a plastic container, with a slight sag. Five

flounder from each group were placed in the net

for 105 min. Before exposure to air, moisture on the body surface of the flounder was removed using

a napkin. After exposure to air, the flounder

were immediately returned to sea water. Survival and upright curvature or stiffness of fish body was recorded. The air dive test was conducted in duplicate.

Pathogen challenge test

The flounder in each treatment were also chal- lenged with the pathogenic bacteria Vibrio anguil- larum which had been cultured and maintained using PPESII medium consisting of proteose pep- tone (1 g/L), polypeptone (2 g/L), bacto soytone (1 g/L), bacto yeast extract (1 g/L) and ferric citrate (0.1 g/L), adjusted to pH 7.8. Seven flounder were immersed for 60 min in a suspension of V. anguillarum at 2 × 10 7 c.f.u./mL according to Austin et al. 24 Before and after immersion, a blood sample was obtained from the caudal vein by a heparinized syringe. The flounder were reared for

14 days and fed with the control and the probiotic diets. In the water supply group, probiotics were added to the rearing water in the same proportion as the rearing experiment. Fish survival rate was monitored every 2 days. After 14 days, all surviving fish from each aquarium were sampled for plasma and fish skin mucus analyses. The challenge test was carried out in triplicate.

Analysis of data

All experimental data were analyzed using one way-analysis of variance (ANOVA) followed by Duncan’s multiple range test (SPSS Version. 10.0 software, SPSS, Inc, Chicago, IL). The change of the lysozyme activity and the plasma protein concen- tration in heat shock stress test were subjected to a Student’s t-test to check the differences.

RESULTS

Rearing experiment

The results of approximate analysis of whole body flounder are presented in Table 2. No significant differences were observed in the moisture, protein and ash contents of the fish between treatment groups (P > 0.05). The fat content, however, was significantly lower in the probiotic diet group than the control and the water supply groups (P < 0.05). The control group had the highest fat content among the three treatments (P < 0.05). There were no observable differences in water quality among groups. Water temperature ranged 14–19°C. During 50 days of culture, pH in the water supply group was significantly lower than the con- trol and probiotic diet groups. NH 4 -N in the control group was significantly higher compared to the probiotic diet group. In NO 2 -N, the probiotic diet group was significantly lower than the control and the water-supply groups. PO 4 -P in the probiotic diet group was significantly lower than the control

Table 2

Whole body approximate composition of P. olivaceus after 50 days of culture

 
 

Treatment groups

 

Control

Probiotic diet

Water supply

Pooled SEM

Moisture

78.5 ± 0.01 a 16.8 ± 0.05 a 2.36 ± 0.01 c 4.1 ± 0.25 a

79.1 ± 0.61 a 16.1 ± 0.79 a 1.16 ± 0.07 a 4.1 ± 0.79 a

79.1 ± 0.13 a 16.5 ± 0.01 a 1.83 ± 0.06 b 4.0 ± 0.20 a

0.41

Protein

0.46

Fat

0.54

Ash

0.41

mg/L

g/Lmg/L

m

mg/L

mg/mL

mg/L

pH

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(a) Urea-N

(c)

(e)

(g)

0.5

0.4

0.3

0.2

0.1

0

0.5

0.4

0.3

0.2

0.1

0

24

20

16

12

8

4

0

8

7.8

7.6

7.4

7.2

7

6.8

0.4 0.3 0.2 0.1 0 24 20 16 12 8 4 0 8 7.8 7.6 7.4

0

10

20

30

Days

NO 2 -N

40

50

0 8 7.8 7.6 7.4 7.2 7 6.8 0 10 20 30 Days NO 2 -N

0

10

20

30

Days

PO 4 -N

40

50

30 Days NO 2 -N 40 50 0 10 20 30 Days PO 4 -N 40

0

10

20

Days

pH

40

50

50 0 10 20 30 Days PO 4 -N 40 50 0 10 20 Days pH

0 10

20

30

Days

40

50

(b) NH 4 -N

(d)

(f)

1.4

1.2

1.2

1

0.8

0.6

0.4

0.2

0

0

10

20

30

40

50

 

Days

 

NO 3 -N

30

30

24

18

12

6

0

0

10

20

30

40

50

 

Days

 

COD

8

6

6

4

2

0

0 10

20

30

40

50

Days

Fig. 1 Change of water quality parameters in a closed recirculat- ing system after 50 days of cul- ture. (a) urea-N, (b) NH 4 -N, (c) NO 2 -N, (d) NO 3 -N, (e) PO 4 -N, (f) chemical oxygen demand (COD), (g) pH. ( ) control, ( ), probiotic diet, ( ) water supply. Error bars represent mean ± standard devia- tion (n = 3).

group. There was no significant difference in NO 3 - N and COD among groups (Fig. 1, Table 3). Addition of probiotics in the rearing water pro- duced the best growth of flounder. Average body weights and total length in the water supply group were significantly greater than in the other two groups (Fig. 2, Table 4). The probiotics-treated

groups showed significantly greater survival rate as compared to the control group at the end of rearing experiment (Fig. 2). Although there was no signifi- cant difference in HSI and condition factor among groups, HSI in the probiotics-treated groups was higher than the control group, particularly the pro- biotic diet group (Table 4). SGR values in the water

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Table 3

Water quality parameters of rearing water during 50 days of culture

 
 

Treatment groups

 
 

Control

Probiotic diet

Water supply

Pooled SD

Pooled SE

pH NH 4 -N NO 2 -N NO 3 -N PO 4 -P COD†

7.7 ± 0.2 a 0.24 ± 0.22 a 0.15 ± 0.08 b 13.7 ± 6.4 a 13.0 ± 3.9 b 3.5 ± 1.7 a

7.6 ± 0.2 a 0.12 ± 0.10 b 0.08 ± 0.08 a 16.4 ± 7.9 a 10.2 ± 3.0 a 3.7 ± 1.9 a

7.4 ± 0.3 b 0.15 ± 0.25 ab 0.14 ± 0.16 b 14.7 ± 8.1 a 11.7 ± 4.6 ab 3.2 ± 1.6 a

0.24

0.02

0.21

0.02

0.11

0.01

7.51

0.78

1.72

0.18

3.98

0.44

Values are means ± standard deviation (n = 11). Different superscripts indicated statistically different (P < 0.05, Duncan’s multiple range test). COD, chemical oxygen demand. SD, standard deviation; SE, standard error.

(a)

33 30 27 24 21 18 15 0 10 20 30 40 50 Average body
33
30
27
24
21
18
15
0
10
20
30
40
50
Average body weight (g/fish)

Days

(b) Control Probiotic diet Water supply 100 80 60 40 20 0 0 10 20
(b)
Control
Probiotic diet
Water supply
100
80
60
40
20
0
0
10
20
30
40
50
Survival rate (%)

Days

Fig. 2 Experiment holding P. olivaceus for 50 days in a closed recirculating system. (a) average body weight, (b) survival. Error bars represent mean ± standard deviation (n = 3).

supply group were significantly higher than the other two groups. SWG and WG in the water supply group were significantly higher than the control and the probiotic diet groups, and the probiotic diet group also showed a significantly higher value than the control group. Skin mucus protein concentration in the control group was significantly higher than in the water supply group (Fig. 3a). Plasma protein at 30 and 50 days increased compared to those at 0 days in all groups. At 30 days, plasma protein in the probi- otic diet group was significantly higher than that in the control. Finally, the plasma protein concentra- tion was not significantly different between treat- ment groups (Fig. 3b). Skin mucus lysozyme activity in the probiotic diet group was significantly higher than the activity observed in the control group and the water supply group (Fig. 3c). Plasma lysozyme activity was sim- ilar among groups (0.5 min incubation). At 4.5 min incubation, however, the water supply group showed significantly greater activity than the con- trol group. The probiotics-treated groups in gen- eral showed a higher trend compared to the control group (Fig. 3d).

Tolerance to stress

LT50 was not significantly different between groups, although the probiotics-treated groups showed slightly higher tolerance than the control group (Fig. 4). Plasma lysozyme activity (0.5 min incubation) significantly decreased in the control group and the probiotic diet group. The probiotic diet group showed significantly higher activity compared to the control group, but the water sup- ply group showed significantly higher activity com- pared to the probiotic diet group. Plasma lysozyme

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Table 4

Growth and feeding performance in P. olivaceus after 50 days of culture

 
 

Treatment groups

 

Control

Probiotic diet

Water supply

± SEM

0 day Initial mean weight (g) Initial mean length (cm) Condition factor HIS (%) § 50 days

16.6 ± 2 13.0 ± 0.6 0.75 ± 0.03 1.24 ± 0.19

16.6 ± 2 13.0 ± 0.6 0.75 ± 0.03 1.24 ± 0.19

16.6 ± 2 13.0 ± 0.6 0.75 ± 0.03 1.24 ± 0.19

Mean

weight (g) §§ length (cm)

28.1 ± 0.61 a 15.1 ± 1.25 a 12.7 ± 0.6 a 80.0 ± 1.3 a 0.52 ± 0.03 a 0.96 ± 0.03 a 1.96 ± 0.05 a

29.2 ± 0.61 a 15.2 ± 0.94 a 13.7 ± 0.5 b 87.9 ± 2.1 b 0.55 ± 0.01 a 0.86 ± 0.07 a 3.39 ± 1.40 a

32.1 ± 0.5 b 16.1 ± 1.44 b 16.3 ± 0.1 c 107.3 ± 1.9 c 0.63 ± 0.01 b 0.85 ± 0.01 a 2.36 ± 0.76 a

1.97

Mean

1.97

WG at individual (g)

1.64

SWG (%) SGR (%) Condition factor HIS (%)

12.30

0.05

0.05

0.94

Values with the different superscript in the same row are significantly different (P < 0.05). Standard error, calculated from the mean square error of the ANOVA. Condition factor = (body weight/body length 3 ) × 100 (n = 6). § HIS (%) = liver weight/body weight (n = 6). Weight gain, WG (g) = (Final body weight – initial body weight)/n (n = 3). Specific weight gain, SWG = (WG/initial weight) × 100 (n = 3). Specific growth ratio, SGR (%) = [(In final body weight – In initial body weight)/time in days] × 100 (n = 3). §§ n = 3. n = 10.

(a)

15 a 12 ab b 9 6 3 0 Control Probiotic diet Water supply Mucus
15
a
12
ab
b
9
6
3
0
Control
Probiotic diet
Water
supply
Mucus protein (mg/L)
(c) b 300 250 a 200 a 150 100 50 0 Control Probiotic diet Water
(c)
b
300
250
a
200
a
150
100
50
0
Control Probiotic diet Water
supply
Lysozyme activity
(unit/mg-protein)

(b)

50 b 40 a a a a a 30 20 10 0 Control Probiotic diet
50
b
40
a
a
a
a
a
30
20
10
0
Control
Probiotic diet
Water
supply
(d)
700
ab
b
600
a
a
500
a
a
400
300
200
100
0
Control
Probiotic diet
Water
supply
Lysozyme activity
(unit/mg-protein)
Plasma protein (mg/L)

Fig. 3 Protein concentration

and lysozyme activity in plasma

and skin mucus. (a) skin mucus

protein, (b) plasma protein on

day 0 ( ), day 30 ( ) and day 50

), (c) skin mucus lysozyme activity, (d) plasma lysozyme activity at incubation time of 0.5 min ( ) and 4.5 min ( ). Error bars represent mean ± standard

deviation. Bars denoted by the

same letter are not significantly different (P > 0.05, Duncan’s mul- tiple range test) (n = 4).

(
(

Effects of probiotics on Japanese flounder

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Table 5

Lysozyme activity and plasma protein concentration of P. olivaceus after heat shock

317

Control

Probiotic diet

Water supply

Responses

Before

After

Before

After

Before

After

Lysozyme activity (units/mg protein)

0.5 min incubation 4.5 min incubation Plasma protein (mg/mL)

354 ± 31 429 ± 67 28.92 ± 1.67

117 ± 22* a 443 ± 3 a 28.42 ± 0.84 a

413 ± 49 518 ± 84 30.09 ± 1.67

297 ± 12* b 406 ± 20 a 35.12 ± 0.10* b

405 ± 37 527 ± 59 28.80 ± 1.88

400 ± 0 c 557 ± 13 b 27.65 ± 3.08 a

The test was conducted twice and value are mean ± standard deviation (n = 4). *the value is significantly different compared to that before heat shock (P < 0.05). Values with the same letters are not significantly different (P > 0.05, Duncan’s multiple range test).

70 a 60 50 40 a 30 a 20 10 0 Control Probiotic diet Water
70
a
60
50
40
a
30
a
20
10
0
Control
Probiotic diet
Water supply
Time of death (min)

Fig. 4 The 50% lethal time for P. olivaceus submitted to heat shock test (30°C). Bars denotd by the same letter are not significantly different (P > 0.05, Duncan’s multiple range test). Error bars represent mean ± standard devia- tion (n = 2).

activity (4.5 min incubation) in the water supply group was significantly higher than the control and the probiotic diet groups. Following heat shock, lysozyme activity in the supply group did not sig- nificantly decrease, although those in the control group and the probiotic diet group significantly decreased. Plasma protein was significantly higher in the probiotic diet group after heat shock than in the control and the water supply groups (Table 5). The control group was greatly affected by the air dive test, evidenced by the greater upright curva- ture or stiffness of the body compared to the pro- biotics-treated group. There was no significant difference in recovery rate among groups, although the water supply group showed 100% recovery rate (Fig. 5).

Tolerance to pathogens

The pathogenic bacteria V. anguillarum affected only the fish in the control group whose popula- tion decreased to 86 on day 4 and 76 on day 6 (Fig. 6). However, mortality was not observed

a a a 100 a 80 60 b 40 b 20 0 Control Probiotic diet
a
a
a
100
a
80
60
b
40
b
20
0
Control
Probiotic diet
Water supply
Percentage (%)

Fig. 5 Responses of P. olivaceus after air dive test. ( ) stiffness/curvature, ( ) recovery rate. Bars denoted by the same letter are not significantly different (P > 0.05, Duncan’s multiple range test) (n = 3).

100 80 60 40 20 0 0 2 4 6 8 10 12 14 Days
100
80
60
40
20
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Fig. 6 Survival of P. olivaceus exposed to Vibrio anguil- larum at 2 × 10 7 c.f.u./mL (n = 3).

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Fig. 7 Lysozyme activity and protein concentration in plasma and skin mucus 14 days after

exposure to Vibrio anguillarum at

2 × 10 7 c.f.u./mL. (a) plasma pro- tein, (b) plasma lysozyme activity

at incubation time of 0.5 min ( )

and 4.5 min ( ), (c) skin mucus

protein, (d) skin mucus lysozyme

activity. Error bars represent mean ± standard deviation. Bars

denoted by the same letter are

not significantly different (P >

Control

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0.05, Duncan’s multiple range

diet

supply

test) (n = 4).

between day 7 and day 14. All fish in probiotics- treated groups survived throughout the rearing period and the survival rate in the probiotics- treated group was significantly higher than that in the control group. The plasma protein concentration was similar among groups, although the water supply group showed a higher level (Fig. 7a). The plasma lysozyme activity of flounder was not significantly different among groups (Fig. 7b). Mucus protein was significantly higher in the control group than in the probiotics-treated groups (Fig. 7c). The skin mucus lysozyme activity in the probiotic diet group was significantly higher than the water sup- ply and the control groups (Fig. 7d).

DISCUSSION

The decrease of pH in the water supply group was distinct compared to the control and the probiotic diet groups. The difference may be due to acid pro- duction by bacteria, because commercial probiot- ics used in this study included acid-producing bacteria. Rengpipat et al. 9 reported that probiotics (Bacillus) added to shrimp diets did not affect water quality. However, when Bacillus was added

to the rearing water, the water quality slightly improved as compared to the case without Bacil- lus. 5,25 The inoculation of probiotics to the rearing water in the present experiment did not remark- ably influence water quality, but the addition of probiotics to the diet affected some water quality parameters, such as NH 4 -N, NO 2 -N, and PO 4 -P. In NH 4 -N and PO 4 -P, the differences may be due to the nitrogen and phosphorus metabolism because nitrogen and phosphorus excretion of fish are affected by components of diet, digestibility and digestive enzyme activity. De Schrijver and Ollevier 26 reported that protein digestion in juve- nile turbot was improved by feed supplement of probiotic bacteria V. proteolyticus, and that supple- ment with V. proteolyticus resulted in a signifi- cantly increased proportion of protein of low molecular weight in the gut. Several probiotic bac- teria excrete extracellular enzymes such as amylase and protease, and these enzymes may affect diges- tion in the gastrointestinal tract. 2729 The introduction of probiotics into the rearing water appeared to enhance growth and survival rate of Japanese flounder. Growth trials showed that the introduction of probiotics to the rearing water accelerated the growth of flounder and improved the survival significantly. However, a sig-

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nificant difference in growth rate was not observed between the control group and the probiotic diet group. These results are not in agreement with the findings of Byun, 11 who concluded that the growth rate of flounder was improved by feeding with a probiotic diet. The survival rate in the probiotic diet group was significantly higher than that in the control group. Rengpipat et al. 9 showed that the growth and the survival rates of black tiger shrimp were improved by feeding with probiotic diet. This disagreement between the findings of Byun, 11 Rengpipat et al., 9 and our findings may be due to the concentration of probiotics added in diet, the bacterial strains, and the species of organisms examined. Enhancement of lysozyme activity was observed in the probiotics-treated groups, especially in the water supply group. Lysozyme has an important role in non-specific immune defense system and is contained in the mucus on the fish body surface, and in plasma and liver. Lysozyme has an anti- biotic ability and is released by leukocytes. It can damage bacterial cell walls, especially of Gram-positive and some Gram-negative bacte- ria. 30,31 Stress conditions induce variation of lysozyme activity. Some studies suggested that lysozyme activity was modified by intensity and duration of the stress. 32,33 Caruso and Lazard 34 reported that plasma lysozyme activity in sheatfish Silurus glanis decreased under persistent stress, random modification of environmental light, and holding. Yehuda et al. 35 also reported that negative correlation was observed between lysozyme activity and cortisol. Cortisol is secreted as a response to stress. Plasma lysozyme activity in tila- pia Oreochromis nilotocus stressed by social pres- sure was lower than that of unstressed fish. 34 However, there have been few data available to explain the effect of stress on lysozyme activity. Demers and Bayne 36 reported that simultaneous increases in cortisol and lysozyme activity were induced by acute stress in rainbow trout, Onco- rhynchus mykiss (Wallbaum). In the present study, lysozyme activity in the control was lower than the probiotic-treated groups. These results indicated improvement in antibiotic activity and modifica- tion of the stress conditions of fish. Probiotic treat- ment affected the protein concentration and lysozyme activity in fish skin mucus at the end of this experiment. As an immune defense system, skin mucus has an important role and contains some enzymes to prevent the invasion of patho- gens, such as lysozyme, lectine, and protease. Pro- biotic treatment has been shown by this study to improve lysozyme activity of plasma and skin mucus, and to increase plasma protein. These results indicate that the non-specific immune

system of fish treated with probiotics is partially improved. Fish excretes mucus under the stressful conditions, and protein concentration in the skin mucus is an indicator for amounts of skin mucus excreted. In the present study, the concentration of skin mucus protein in the control group was signif- icantly higher than that in the water supply group. In the case of red sea bream Pargus major, oral administration of lactoferrin as an immunostimu- lant increased the secretion of skin mucus, lectin and lactoferrin, but did not affect the lysozyme activity of skin mucus. 37 In the heat shock and exposure-to-air stress tests, the stress parameters indicated that stress tolerance of fish is enhanced by probiotics. Mock and Peters 33 and Weyts et al. 38 reported that lysozyme activity decreased when fish were stressed due to handling and exposure to polluted water. In the heat shock stress test, the control group showed lower lysozyme activity compared to those of the probiotics-treated groups, especially the water supply group. In addition, lysozyme activity in the water supply group did not signifi- cantly decrease by heat shock, although those in the control group and the probiotic diet group decreased significantly. This result indicated that the water supply group is not stressed as much by heat shock stress as were the control and probiotic diet groups, from the viewpoint of lysozyme activ- ity after heat shock stress. The present study showed that probiotic treat- ment enhances disease tolerance in cultured fish. Increased resistance to the pathogen by probiotics has been widely reported. Growth inhibition against pathogens by Carnobacterium was demon- strated. 39 Black tiger shrimp fed with a probiotic diet had greater tolerance to V. harveyi, and phago- cytosis and phagocytic activity in hemolymph were activated. 9,40 The tolerance of rainbow trout Onco- rhynchus mykiss to furunculosis was enhanced when fed with a diet including the probiotic L. rhamnosus. 41 In Atlantic cod Gadus morhua, tol- erance to V. anguillarum was measured by feeding with lactic acid bacteria (Carnobacterium diver- gens) supplemented in the diet. 42 Robertson et al. 12 reported that Atlantic salmon Salmo salar and rain- bow trout O. mykiss, Walbaum fed with Carnobac- teria spp. supplemented in the diet were more tolerant to disease. However, adverse effects of pro- biotics were also observed. The highest mortality was observed when Atlantic salmon S. salar was fed a probiotic diet containing lactic acid bacteria. 43 Skin mucus lysozyme activity in the probiotics- treated groups showed significantly better results than that of the control groups. Takahashi et al. 21 suggested that fish mucus included antibacterial materials and lysozyme or non-specific defense

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factors, based on the relationship between bacteria number attached to the fish body surface and that of the other parts with stressed fish. Fevolden et al. 44 found a negative relationship between lysozyme activity and mortality in rainbow trout, meaning that enhanced lysozyme levels correlated with significantly higher mortality following ex- posure to Aeromonas salmonicida. Probiotics orally administrated colonize the wall of intestine and several probiotics can produce pathogen- inhibitory substances (bacteriocin). Invading pathogens or opportunistic bacteria were sup- pressed by these substances. Carnobacterium could stay in the intestine of rainbow trout by con- tinuous feeding of probiotics. 12 In the case of newly hatched turbot, bacteria introduced to rearing water colonized and became a major part of the autochthonous flora of the gut of the larvae. 45 From the enhancement of disease resistance in the pathogen challenge test, it was supposed that the immune system is comprehensively improved by probiotics treatment. In the rearing trial and the pathogen challenge test, results showed higher concentration of skin mucus protein in the control group compared to the probiotics-treated groups. Stress indicators in the air dive test also supported that probiotics-treatment may improve tolerance to stress. Generally, stress can affect the growth of fish. Skin mucus protein in the water supply group showed the lowest value. This result may explain why the growth rate in the water supply group was significantly higher than the probiotic diet group. From the viewpoint of non-specific parameters, mucus secretions, air dive test, and the pathogen challenge test, it has been indicated that probiotic treatment may activate fish immune response, and improve the resistance to disease and tolerance to stress. In this study, the improvement of flounder health by probiotics treatment was indicated by enhanced survival rate and growth rate. Tolerance to stress in heat shock and air exposure, and resis- tance to pathogens were enhanced by probiotics treatment. Moreover, the introduction of probiot- ics to rearing water seemed to be more effective than oral administration, from the viewpoint of the growth rate. This study indicated that stronger and healthier flounder would be produced if probiotics were used. However, the mechanisms of probiotic action on cultured fish are still complicated and unknown. Thus, more detailed knowledge should be accumulated and further research is required.

ACKNOWLEDGMENTS

Sincere thanks to Professor T. Sakata and Dr. T. Yoshikawa, Laboratory of Microbiology, Faculty of

Fisheries, Kagoshima University, and Professor S. Teshima, Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, for advice. Thanks to Professors IB. Kim and YJ. Chang, Department of Aquaculture, Pukyong National University, and Professors MJ. Formacion, EB. Seraspe and CA. Saclauso, Institute of Aquaculture, College of Fisheries and Ocean Sciences, Univer- sity of the Philippines, for their instruction. Thanks to Dr. XJ. Wang and Mr. S. Choi for advices on test diets, to Dr. JW. Lee, Dr. L. Peng, Dr. Muslim and staff of Pukyong National University, Busan, Korea, and to JH. Kim for support. This study was sup- ported in part by the exchange student program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).

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