Probiotics on larvae macrobranchium

The Effects of Probiotics on Culture Conditions of
Freshwater Prawn (Macrobrachium rosenbergii) Larvae
Dang Thi Hoang Oanh, Tran Thi Tuyet Hoa and Nguyen Thanh Phuong
Institute for Marine Aquaculture, College of Agriculture,
Can Tho Unversity, Can Tho, Vietnam

Abstract
Probiotics CP Bio-dream was used at the concentration of 1g/m3 in larval rearing water of giant
freshwater prawn (Macrobrachium rosenbergii) applying modified static green water system. Four
trials (i) no probiotics; (ii) use probiotics one every day; (iii) use probiotics one every five days and
(iv) use probiotics one every 10 days, were set up to evaluate the influence of probiotics on water
parameters, growth of pathogenic bacterial population, and the development of larvae and its
sensitivity to bacterial pathogens. Water temperature, pH, dissoved oxygen, NH3 and H2S were found
better in trials with probiotics. Moreover, daily usage of probiotics in trial 2 was the best among trials.
The first postlarvae was found in this trial at day 18th, the total Vibrio bacterial count was the lowest
and postlarvae were less sensitive to pathogenic pathogen.

1. Introduction
Giant freshwater prawn (Macrobrachium rosenbergii deMan, 1879) is an important commercial
species thank to its delicious meat and lower cholesterol and high protein content. In Vietnam, giant
freshwater prawn is distributed mainly in the Southern region where average temperature does not
change much between night and day times and seasons. Increasing demand of this species for
domestic and export markets has reduced remarkably its natural capture. Rapid development of prawn
farming with large scale, high stocking density and intensive feeding have motivated the need of good
quality prawn seed supply. However, to produce healthy prawn seed, which has ability to resist with
pathogens and less sensitive in extreme environmetal conditions is still concern of aquaculturists.
Recently, the use of probiotics to improve and maintain healthy environment for prawn culture has
become popular. Probiotics was used to supply beneficial bacterial strains to rearing water that will
help to increase microbial species composition in the environment and to improve water quality.
Probiotics is considered to be able to make cultured animals more healthy by inhibiting the growth of
pathogenic bacteria in the same habitat. This has led to new strategy for prevention of disease

outbreak and improvement of seed quality. However, effectivenees of probiotics in aquaculture is still
a debate due to different observations in different areas and cultured species (Maeda 1999).
This study aimed to figure out the roles of probiotics in improving and maitaining water quality in
larval rearing water of giant fresh water prawn (Macrobrachium rosenbergii) applying modified static
green water system. Our main purposes were to evaluate the influence of probiotic on water
parameters, the growth of pathogenic bacterial population, the development of larvae and its
sensitivity to bacterial pathogens. The experiment was conducted in prawn experimental hatchery at
Institute for Marine Aquaculture, Can Tho University.

1


2. Materials and Methods
Larval rearing experiment
Healthy female broodstocks carrying eggs, shining body and no sign of diseases were chossen.
Weight of brooders ranged from 20-30 g/individual. Broodstock were kept in concecre tank and fed
by small crustaceans. Larvae from these brooders were reared in composite tanks of 90 litres with full
aeration. There was no water exchange during the experiment and water salinity was maintaind at 12
o
/oo. Chlorella spp was maintained at the density of 750,000-1,5000,000 cells/ml to create green water.
Probiotics used in the study was CP Bio-dream supplied by CP Group. Probiotics was applied to the
tank in early morning at the concentration of 1g/m3.
Experimental tanks were set up randomly with 4 trial: (i) no probiotics; (ii) use probiotics one every
day; (iii) use probiotics one every five days and (iv) use probiotics one every 10 days. Each trial has 3
replicates. Each tank was filled 60 litres of water and larvae was stock at the density of 50
individual/litre
Artemia nauplii were used to feed the larvae 3 times per day during the first 4 stages, and 1 time in the
evening for the remaining stages. Artificial feed (called custard) was fed 3 times per day since stage 4
to replace the artemia nauplii. Different size of custard was feed according to different developmental
stages of larvae.

Water temperature was measured every day both in the morning and in the afternoon using
thermometer. pH and dissoved oxygen were measured every two day using pH meter and Oxy meter.
Water sample for NH3 and H2S were collected every five days in the morning. H2S concentration was
measured following Metylene blue method. NH3 concentration was measured following Salicylate
method using DR2000 spectrophotometer.
Total Vibrio bacteria in prawn larvae and rearing water were counted every 5 days applying plate
counting method on TCBS (Thiosulphate Citrate Bile-salt Sucrose) agar. Larval developmental stages
were recorded every day by collecting 5 random samples. Stage development of the larvae was
determined as described by Uno and Soo (1969). Lengths of the larvae were measured at stage 1, 4, 8,
11 from the eye talk to the tail and were calculated by the formular:
L = ∑ Li/i
Where: L: average length of the larvae
Li: length of each measured larvae
i: number of larvae measured
As soon as 90 % of larvae reached the postlarvae stage, salinity acclimation was done to allow
postlarvae sustain in freshwater. Number of postlarvae were counted and the metamorphosis
percentage and survival rate of larvae were calculated by the following formulars:
Metamorphosis percentage = 100 x A/B
Survival rate = (A+B) × 100/C
Where: A is number of postlarvae
B is number of larvae
C is number of larvae from the beginning

2


Challenge experiment
Sensitivity of rearing postlarvae to Vibriosis were examined by challenge test applying immersion
method with pathogenic Vibrio harveyi strain number 84. This strain was isolated from prawn
postlarvae in Long My hatchery. Fifty postlarvae were kept in 1 litre capacity glass bottles static

freshwater water at 25 °C and aeration. There was no water exchange during the challenge test.
Bacterial cultures were grown in 10 ml of heat infusion broth at 28 °C, centrifuged at 13,000 rpm for
3 minutes, whereafter pellets were resuspended in 10 ml of sterile 0.9 % (w/v) NaCl. The optical
density of bacterial cells was estimated using spetrophotometer method. Bacterial suspension was
added to the challenge bottle at the concentration of 106 cell ml-1. Prawn behaviours and disease
symptoms were observed for 7 days post challenge and the number of dead animals was recorded
daily. Before and after challenge water and prawn samples were subjected for bacteriological
examination.
Data analysis
Recorded data were analyzed using Microsoft Excel 97.

3. Results and Discussion

32

Figure 1(a): Average temperature in the morning

25

22

22
19

Rearing time

24

16

25

22

19

16

13

10

7

4

23

26

13

24

28

10

25

7

26

30

4

Average temperature

27

1

28

1

Average temperature

Water temperature ranged from 25-27 oC in the morning and 26-31 oC in the afternoon throughout the
experiment (Figure 1).

Rearing day

Figure 1(b): Average temperature in the afternoon

Optimal temperature in prawn larval rearing water could range from 25-30 oC (Thang 1993). In
optimal range of temperature, the higher temperature the quicker prawn parvae will grow (New and
Singholka 1985). Optimal temperature for larvae development ranged from 26-31 oC. Temperature of

rearing water under 24-26 oC larvae will not grow well and development stage will be longer. If water
temperature is higher than 33 oC prawn larvae will not survive.
Dissolved oxygen (DO) concentration during the experiment ranged from 6.22-7.44 ppm in the
morning (Figure 2a) and 7.25-7.62 ppm in the afternoon (Figure 2b). In rearing water, DO is the
product of photosynthesis process and is diffused from the air. DO concentration in water is
influenced by water temperature, respiration of aquatic animal and organic mater load. At low DO
concentration prawn larvae will not survive. DO concentration in rearing water should be at least 5
ppm and as near saturation level as possible (New and Singholka 1985). Stable DO concentration
during the experiment was good for larval development. This can also be explained due to stable
temperature and full aeration throughout the experiment.

3


Dissolved Oxygen (ppm)

Dissolved Oxygen (ppm)

8.0
7.0
6.0
5.0
1

2

3

4

5

6

7

8

7.8
7.6
7.4
7.2
7.0
1

Sampling time

2

3

4

5

6

7

8

Sampling time

Figure 2a: Dissoved oxygen measured in
the morning

Figure 2b: Dissoved oxygen measured in
the afternoon

Water pH is an important element for prawn larval development. Fluctuation of pH during the
experiment pH is performed in figure 3. It is ranged from 7.79-7.95 in the morning and 7.88-8.13 in
the everning. There is no significant different of pH between sampling time (at P<0.05). In green
rearing water system, pH fluctuates according to the development of algae. The degradation of
organic matter also reduces pH. It is normally low in the early morning and high in the afternoon. H2S
tocixity will increase when water pH is low, whereas, tocixity of NH3 will increase when water pH is
high. Low pH usually causes gill damage, soft shell and molting difficulty in prawn larvae (Thang
1993).

Morning
Afternoon

8.2
8.1

pH

8.0
7.9
7.8
7.7

7.6
1

2

3

4

5

6

7

Sampling time

Figure 3 : Fluctuation of pH during the experiment
According to New and Singholka (1985) fresh and marine water resources use for prawn hatchery
should have pH ranges from 7-8.5. If water pH is lower than 5, prawn larvae become less active, come
to water surface or the tank’s wall and die if there is no improvement of water pH. Sudden change of
water pH will make the larvae stress and die.
Ammonia (NH3) concentration ranged from 0.03-0.243 ppm throughout the experiment. NH3
concentration in prawn rearing tank is mainly resulted from degradation of protein in remaining food
and shrimp excrement and faeces. Toxicity of NH3 fluctuates depending on water pH and temperature,
especially if water pH increase NH3 tocixity will also increase.

4

NH 3 concentration (ppm)

Trial 1

0.30
Trial 2

0.20

Trial 3

0.10

Trial 4

0.00
1

2

3

4

5

Sampling time

Figure 4 : Fluctuation of NH3 during the experiment
In our experiment, NH3 concentration increased after each sampling time (Figure 4). However, at the

fifth sampling time, NH3 concentration droped at four trial. This can be explained due to the cleaning
of tank bottom. At trial 2 (use probiotics everyday), NH3 concentration was the lowest and quite stable
compared to other trials. NH3 is a limit factor in larval rearing environment of most aquatic species.
Prawn larval is very sensitive with the variation of NH3. The daily administration of probiotics
showed a possibility of maintaning healthy environment for prawn larval in the improved green water
system.

H2 S concentration (ppm)

Hydrogen sulfide (H2S) concentration increased along with rearing period and ranged from 0.00730.1933 ppm (Figure 5).

0.25

Trial 1
Trial 2
Trial 3
Trial 4

0.20
0.15
0.10
0.05
0.00
1

2

3

4

5

Samplingtime

Figure 5 : H2S fluctuation during the experiment
H2S concentration pattern appeared as same as NH3, which was reduced at the last sampling time and
trial 2 was the lowest and more stable. Tocixity of H2S is also influenced by water pH and
temperature and its toxic concentration for prawn larval is 0.09 ppm (Thang 1995).
Total Vibrio bacterial count was high at the first sampling time and varied a lot in trials. Vibrio
bacterial count reduced remarkably in the following sampling times. The highest count was 410
CFU/ml in trial 1 and the lowest was 10 CFU/ml in trial 2 (Table 1a). Shariff et al. 1992 considered
that Vibrio bacteria in rearing water at the concentration of 102 cell m-1 will cause diseases.

5


Table 1a : Total Vibrio count in rearing water
Sampling time
1 (15/11/99)
2 (21/11/99)
3 (26/11/99)
4 (01/12/99)
5 (07/12/99)

Number of Vibrio bacteria (cfu/ml)
Trial 2
Trial 3
2,663
5,430

33
33
490
0
3
37
10
40

Trial 1
363
0
0
0
410

Trial 4
7
33
267
0
183

Table 1b : Total Vibrio count in larval prawn tissue
Number of Vibrio bacteria (cfu/ml)
Trial 2
Trial 3
0
17
23

10
2,887
0
30
67
293
3,480

Sampling time
1 (15/11/99)
2 (21/11/99)
3 (26/11/99)
4 (01/12/99)
5 (07/12/99)

Trial 1
67
0
5,657
67
43

Trial 4
10
0
1,187
3
57

Percentage of metamorphosis was different in 4 trials from the fifth day of larval development as

shown below:
Trial 1: stage IV (19.98 %), stage V (80.02 %)
Trial 2: stage IV (13.32 %), stage V (86.68 %).
Trial 3: stage IV (100%)
Trial 4: stage IV (100%)
From the sixth day, there were always 2-3 developmental stage observed in each trial. The first post
larva was seen in the 18th day in trial 2 and the 19th in trial 1. From the 20th day postlarvae were seen
in all trials. 90-95 % of larvae reached postlarvae stage on day 25-26th. According to Thang (1995), it
will take 15-60 days for prawn larvae to pass 11 molting times. At 26-30 oC, it will take prawn larvae
19 days to reach postlarval stage. At the earlier stages the larvae molts more frequent (1-2 days/stage)
than the later stages (2-3 days/stage),
Length of prawn larvae at different developmental stages are given in figure 7. Uno and Soo (1969)
found that prawn larvae at the first and 11th stages has a normal length of 2 mm and 7 mm,
respectively. The highest was measured from prawn in trial 2.

Length of larvae
(mm)

Trial 1
Trial 2

6
5
4
3
2
1
0

Trial 3

Trial 4

1

2

3

Developmental stage
Figure 6. Length of larvae

6

4


Percentage (%)

Number of larvae reached postlarval stages (54.75 %) and survival rate (56.16 %) was the highest at
trial 2 (Figure 7).
Postlarvae stage

60
50
40
30
20
10
0

Survival rate

I

II

III

IV

Trial
Figure 7 : Number of larvae reached postlarval stages and survival rate
Challenge test: Vibrio count in water resource was negative. Five days post challenge, dead prawn
were observed and recorded as given in table 2. The highest mortality was found in trial 1 (32 %) and
the lowest was in trial 2 (4%). The administration of the probiotics did not show any possitive effects
on the quality of postlarvae. Further study is needed to confirm the hypothesis that probiotics can be
used to prevent disease outbreak and improve seed quality.
Table 2. Number of dead prawn in challenge experiment
Trial
Day post challenge
Number of dead animal
I
5
10
II
5
1
III
5
2

IV
5
5

Percentage (%)
40
4
8
20

4. Conclusions
Water temperature, pH, dissoved oxygen, NH3 and H2S were found better in trials with probiotics.
Moreover, daily usage of probiotics in trial 2 was the best among trials. The first postlarvae was found
in this trial at day 18th, the total Vibrio bacterial count was the lowest and postlarvae were less
sensitive to pathogenic pathogen. However, it is considered that bacterial count and water parameter
should be examined a day after applying probiotics instead of five day interval to get more acurate
information on the effect of probiotic in rearing water.

Literature Cited
Maeda, M. 1999. Microbial Processes in Aquaculture. National Research Institute of Aquaculture.
Nansei, Mie 516-0193, Japan
New M.B. and Singholka, 1985. Freshwater prawn farming. A manual for the culture of
Macrobrachium rosenbergii. FAO fish.Tech.Pap. (225) Rev. 1: 118 p.
Thang, NV. 1993. Study on biological and reproductive characteristics of freshwater prawn
(Macrobrachium rosenbergii de Man 1879) in the Southern part of Viet Nam. Ph.D. thesis (in
Vietnamese).
Thang, NV. 1995. Freshwater prawn farming, Agricultural Publishing House. HoChiMinh city (in
Vietnamese).
Shariff M., Subasinghe RP and Arthus JR, 1992. Diseases in Asian Aquacultute. P.P. 157-162.
Uno, Y. and Soo KC, 1969. Larval development of Macrobrachium rosenbergii reared in the

laboratory. J. Tokyo Univ. Fish., 55(2): 79-90

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