Effects of probiotics on fish growth, digestive enzymes, antioxidant enzymes and immune response parameters.

1. Jannatul Ferdush
Exam Roll: 81405
MS session: 2020-2021
Reg. No.: 2016-814-260
Fisheries management Branch
Department of Fisheries
University of Dhaka
Aquaculture and Fisheries 6 (2021) 283–288
Contents lists available at ScienceDirect
Aquaculture and Fisheries
journal homepage: www.keaipublishing.com/en/journals/aquaculture-and-fisheries
Effects of dietary supplementation of Lactobacillus plantarum and
Bacillus subtilis on growth performance, survival, immune response,
antioxidant capacity and digestive enzyme activity in olive flounder
(Paralichthys olivaceus)
Yangzhen Li , Yingming Yang , Limin Song , Jialin Wang , Yuanri Hu , Qian Yang , Peng Cheng , Jianhai Li
Impact factor: 3.69 (2022)
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2. 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion
Literature
Review
Presentation Outline
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3. 3
1.Introduction
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4. In aquaculture, Growth and disease resistance are the main parameters of
commercial importance.
 Probiotics is a long lasting, less costly and less time consuming solution and
widly used in aquaculture due to their beneficial impacts on,
 growth performance and health status
 enhance innate immune response
 suppress pathogenic microorganisms
 L. plantarum and B. subtilis can
improve growth performance,
strengthen immune responses,
modulate activities of antioxidant
and digestive enzymes in aquatic
species (Akhter et al., 2015)
 Olive flounder (Paralichthys
olivaceus) is a commercially
important marine flatfish
species in China, South
Korea and Japan.
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5. 12/10/2022
The overall objective of the study Research gap
 Little information is available on the
beneficial effects of L. plantarum and
B. subtilis during the whole growth
period (from juvenile to commercial
size) under commercial rearing
environment.
To find out the information about
probiotics administration strategy for
improving quality and productivity in
olive flounder.
The specific objectives of the study
To evaluate the growth performance and survival
To investigate the immunological indices, antioxidant ability
To evaluate the digestive enzyme activity
1
2
3
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5

6. 12/10/2022 6
2.Materials and Methods

7. Feed formulation
Fish collection
and
maintenance
Estimation of
Growth
performance
Sample collection
(blood, liver,
intestine and
stomach)
Assessment of
parameters
Overview of materials and methods
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8. 2.1 Diet preparation
8
Diet
Basal diet without
probiotics
Experimental diet
supplemented with
Lactobacillus plantarum
and Bacillus subtilis
Composition Percentage(%)
Crude protein 45.0
Crude fiver 10.0
Crude fat 3.0
Crude ash 16.0
Calcium 4.0
Lysine 2.5
Total phosphorus 1.5-3.0
Table-1 The proximate composition of basal diet on
dry matter basis provided by manufacturer.
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9. 9
Collection of Olive flounder juveniles( approximately four months old)
Fish were fed two times daily
Randomly distributed into three groups and each group consists of three
replicates
Each tank was supplied with filtered and sterilized sea water
Water temperature was controlled at 10–24 ⸰C, the salinity at 28–30ppt,
the dissolved oxygen at 6–8 mg/L, water exchange rate at 500% per day.
2.2 Fish grow-out maintenance
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10. According to, (Crane et al, 2020)
Weight gain rate (WGR):
𝑾𝟏−𝑾𝟎
𝑾𝟎
× 100%
Specific growth rate(SGR):
( 𝒆
𝒍𝒏 𝑾𝟏
−𝒍𝒏 𝑾𝟎
𝑻
−𝟏)
× 𝟏𝟎𝟎%
Survival rate:
𝑵𝟏
𝑵𝟎
×100%
Where,
 W0 is the initial body weight
 W1 is the harvest body weight
 N1 is fish number at harvest of each tank
 N0 is fish number at stocking of each tank
 T is days of grow-out period
2.3 Growth performance and Survival rate
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11. 2.4 Sample collection procedure
Three starved fish were randomly selected after anesthetized
Approximately 1 mL of whole blood samples were drawn from
the caudal vein using syringes
After clotting the sample at room temperature, Serum was
separated by centrifugation (1500 × g, 10 min, 4 ◦C) and stored
at -80 ◦C until use
Fresh liver, stomach and middle intestine tissue samples were
collected and then stored at -80 ◦C
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12. 2.5 The assay of immune ,antioxidant and digestive parameter
Immune
parameter
• Lysozyme (LYS), Acid phosphatase (ACP),Immunoglobulin M (IgM),
Complement protein 3 (C3) and Complement protein 4 (C4)
Antioxidant
parameter
• Catalase (CAT), Superoxide dismutase (SOD), Glutathione
peroxidase (GSH-Px), Total antioxidant capacity (T-AOC) and
Malondialdehyde (MDA)
Digestive
parameter
• Proteinase, Amylase and Lipase
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13. 13
50 μL diluted samples (1:4) and 100 μL horseradish peroxidase (HRP) conjugated antibody-
enzyme reagent were added
Incubated at 37 ◦C for 60 min and then washed with washing solution for 5 times.
After drying, tetramethyl benzidine (TMB) chromogenic substrate was pipetted and blended
gently
Again after incubation at 37 ◦C for 15 min in darkness, the enzymatic reaction was stopped by
adding 50 μL stopping buffer
Then the absorbance at 450 nm was assayed using a Rayto RT-6100 microplate reader
2.5 Continue…
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14. 2.6 Statistical analysis
All statistical analyses were carried out
using SPSS 25.0 software. The results
were presented as mean ± standard
error (SE).
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Normality of all the data and the
homogeneity of variances among
different groups were analyzed by
using Shapiro-Wilk test and Levene’s
test respectively.
One-way analysis of variance (ANOVA)
was performed to determine significant
differences in various parameter indices,
where significance level was set as P-
value < 0.05.

15. 12/10/2022 15
3.Results and discussion

16. Table 2 Initial body weight (IBW), harvest body weight
(HBW), weight gain rate (WGR), specific growth rate
(SGR) and survival rate (SR) of olive flounder. Values are
expressed as mean ± SE. Values with different
superscript letters in the same row show significant
differences (P < 0.05).
Index Diet
L. plantarum B. subtilis Control
IBW (g)
HBW (g)
WGR (%)
SGR
SR (%)
18.54 ± 0.71a
370.7 ± 3.77b
1896.06 ± 66.57b
0.99 ± 0.012b
73.89 ± 2.84a
18.22 ± 0.84a
397.50 ± 3.11a
2085.08 ± 64.10a
1.02 ± 0.010a
66.85 ± 3.56ab
18.87 ± 0.89a
353.29 ± 2.76c
1836.72 ±72.85c
0.98 ± 0.014c
61.94 ± 3.68b
3.1. Growth performance parameters and survival
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 The growth performance
parameters were significantly
enhanced by dietary
supplementation feed than
control.
 B. subtilis supplementation feed
show great superiority for growth
improvement in olive flounder.

17. 3.2. Immunological indices
Index Diet
L. plantarum B. subtilis Control
LYS (U/L) 5.99 ± 0.11a 4.32 ± 0.08b 3.69 ± 0.07c
ACP (U/L)
IgM (μg/mL)
C3 (μg/mL)
C4 (μg/mL)
11.13 ± 0.22a
46.94 ± 0.41a
150.27 ± 5.09a
195.21 ± 4.21a
7.95 ± 0.25b
42.02 ± 0.17b
132.51 ± 3.06b
133.23 ± 0.69b
7.81 ± 0.15b
37.62 ±0.58c
98.43 ±0.87c
123.76±2.3b
Table3 Effects of L. plantarum and B. subtilis
supplementation in diets on immune response. Values are
expressed as mean ± SE (n = 9). Values with different
superscript letters in the same row show significant
differences (P < 0.05).
LYS: lysozyme; ACP: acid phosphatase IgM:
immunoglobulin M; C3: complement protein 3; C4:
complement protein 4.
 Serum immunological indices
are significantly higher for L.
plantarum.
 LYS is a bacteriolytic enzyme
which is one of the most
important serum factors
because it can activate the
cellular defenses (Harikrishnan
et al, 2010).
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18. 3.3. Hepatic antioxidant capacity
L. plantarum B. subtilis Control
SOD (U/g) 1390.7±57.0b 1590.05±158a 1046.73±93b
CAT (U/g) 538.5 ±34.53a 590.22 ± 42.20a 408.45±30.1b
GSH-Px(U)
TAOC(U/)
MDA(U/g)
1.20 ± 0.11a
133.92±11.5a
80.15 ± 2.63b
1.19 ± 0.08a
128.54 ± 15.36a
75.56 ± 2.26b
0.96 ± 0.15a
80.01±16.10b
106.98±5.50a
Antioxidant enzymes can remove
excessive ROS and protect the cells
from lipid peroxidation.
The lower value of MDA the better the
conditions of liver
 Supplemented feed had elevated the
activities of SOD,CAT, GSH-Px and
T-AOC and lowered contents of MDA
in the liver.
Table 4 Effects of L. plantarum and B. subtilis supplementation
in diets on liver antioxidant indices. Values are expressed as
mean ± SE (n = 9). Values with different superscript letters in
the same row show significant differences (P < 0.05).
SOD: superoxide dismutase; CAT: catalase, GSH-Px: glutathione
peroxidase, T-AOC: total antioxidant capacity; MDA:
malondialdehyde.
18

19. 3.4.1. protease enzyme activity
Fig. 1. Protease activities in liver, stomach and intestine.
Different superscripts indicate significant differences (P
< 0.05) between treatments in the same viscus. 19
 For stomach and intestine
protease activity were
significantly higher in L.
plantarum and B. subtilis than
control.
 For liver B. subtilis
supplemented feed was higher
L. plantarum

20. 3.4.2. Lipase activity
 Lipase activity in dietary B.
subtilis supplementation group
was substantially (significant in
intestine and stomach, and
insignificant in liver) higher
than L. plantarum group.
Fig 2. Lipase activities in liver, stomach and intestine.
Different superscripts indicate significant differences (P <
0.05) between treatments in the same viscus.
20

21. 3.4.3. Amylase activity
 In liver the improvement levels of
both supplemented feed was
negligible.(p>0.05)
 In stomach and intestine amylase
activity was significantly higher in
both supplemeted feed than
control.
 But L. plantarum activity was not
significant than control.
Fig. 3. Amylase activities in liver, stomach and
intestine. Different superscripts indicate significant
differences (P < 0.05) between treatments in the same
viscus.
21

22. 12/10/2022 22
4. Conclusion

23. Separate supplementation of L. plantarum and B. subtilis in diet is more
beneficial for disease resistance (high survival) and growth performance
respectively.
Feed supplemented with B. subtilis and L. plantarum had a beneficiary
effects on innate immune indices, antioxidant and digestive activities.
The beneficiary compounded supplementation of L. plantarum and B.
subtilis in feed can be a great interest in fish farming industries, feed and
additive manufacturers and even aquaculture researchers.
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24. 12/10/2022 24
Thesis title
Effects of probiotics on digestive enzymes,
antioxidant enzymes and immune defense in Gulsha
Tengra (Mystus cavasius)

25. Received: 5 March 2020 | Revised: 26 October 2020 | Accepted: 29 October 2020
DOI: 10.1111/are.15009
 Growth performances, water quality, digestive enzyme and antioxidant enzymes in
liver spleen and kidney were improved.
 16S rRNA indicated that, modulate gut microbial communities and significantly
increase functions relating to nitrogen metabolism, phosphorylation and proteinases.
O R I G I N A L A R T I C L E
Effects of Clostridium butyricum on growth, digestive enzyme activity, antioxidant
capacity and gut microbiota in farmed tilapia (Oreochromis niloticus)
Mingqing Zhang1 | Bo Dong2 | Xingxing Lai1 | Zhuang Chen2 | Liping Hou1 | Rui Shu3 | Yanhua Huang4 | Hu Shu1
Key Findings
12/10/2022 25

26. Probiotics and Antimicrobial proteins (2021) 13:1723–1733
https://doi.org/10.1007/s12602-021-09787-3
The Effect of Lactococcus lactis subsp. lactis PTCC 1403 on the Growth
Performance, Digestive Enzymes Activity, Antioxidative Status, Immune
Response, and Disease Resistance of Rainbow Trout (Oncorhynchus
mykiss)
Sakineh Yeganeh1 · Milad Adel2 · Ahmad Nosratimovafagh1 · Mahmoud A. O. Dawood3
 Dietary Lactococcus lactis subsp. lactis PTCC 1403 enhanced the feed
efficiency, digestive enzyme activity, antioxidative status, and immunity of
rainbow trout.
 Increased resistance against infection.
Key Findings
Published online: 1 June 2018
# Springer Science+Business Media, LLC, part of Springer Nature 2018, corrected publication June/2018
12/10/2022 26

27. Aquaculture Nutrition
Volume 2022, Article ID 8968494, 17 pages
https://doi.org/10.1155/2022/8968494
Hindawi
Research Article
Effects of Single and Mixture Probiotic Supplements on Growth,Digestive Activity,
Antioxidative Status, Immune and Growth- Related Genes, and Stress Response of
Juvenile Red Sea Bream (Pagrus Major)
Ronick Spenly Shadrack ,1,2 Ishikawa Manabu,2 Shunsuke Koshio,2 Saichiro Yokoyama,2
Yukun Zhang,1 Kumbukani Mzengereza ,1 Mohammed Fouad El Basuini ,3,4
and Mahmoud A. O. Dawood 5,6
 Mixed probiotic bacteria strain containing commercial strain has significant potential
to improve growth performance, feed utilization, and immune response in red sea
bream.
Key Findings
12/10/2022 27

28. Probiotics and Antimicrobial Proteins (2019) 11:545–558
https://doi.org/10.1007/s12602-018-9409-8
Dietary Supplementation of Probiotic Bacillus subtilis Affects
Antioxidant Defenses and Immune Response in Grass Carp Under
Aeromonas hydrophila Challenge
Ying Tang1,2 & Lei Han1,3 & Xiaoxuan Chen1,2 & Mengqi Xie1,2 & Weiguang Kong1,4 & Zhixin Wu1,2
Published online: 13 April 2018
# Springer Science+Business Media, LLC, part of Springer Nature 2018
Key Findings
 Bacillus subtilis CH9 strain, isolated and identified used as a probiotic to
protect grass carp against pathogenic strain of Aeromonas hydrophila, and
improved fish antioxidant defences and immune response.
12/10/2022 28

29. Aquaculture 294 (2009) 306–313
Contents lists available at ScienceDirect
Aquaculture
journal homepage: www.elsevier.com/ locate/aqua-online
Effect of dietary probiotic Pediococcus acidilactici on antioxidant
defences and
oxidative stress status of shrimp Litopenaeus stylirostris
Mathieu Castex ⁎, Pierrette Lemaire, Nelly Wabete, Liet Chim
Key Findings
 Probiotic supplementation in shrimps' diet is an effective way to reduce the
susceptibility of shrimps to bacterial pathogens.
 Oxidative stress reduced in the shrimps fed with the probiotic diet.
 Lower the load of Vibrio in the shrimps' haemolymph and higher survival rates.
12/10/2022 29

30. Aquaculture 16 (2020) 100237
Contents lists available at ScienceDirect
Aquaculture
journal homepage: www.elsevier.com/ locate/aqrep
Mst Rubia Banua,*, Shammee Aktera, Md Reazul Islamb, Md Nurunnabi Mondola, Md Amzad Hossainc
Key Findings
 The growth performance of gulsa tengra in terms of final body weight, weight
gain, percent weight gain, and specific growth rate was significantly higher.
 Higher fish survivability, proximate composition.
 FCR(food conversion ratio) was lowered in supplemented feed.
Probiotic yeast enhanced growth performance and disease
resistance in freshwater catfish gulsa tengra, Mystus cavasius
12/10/2022 30

31. Journal of Oceanology and Limnology
Vol. 35 No. 1, P. 89-97, 2017
http://dx.doi.org/10.1007/s00343-016-5130-1
Effects of small peptides, probiotics, prebiotics, and synbiotics on
growth performance, digestive enzymes, and oxidative stress in
orange-spotted grouper
Epinephelus coioides ,juveniles reared in artificial seawater
WANG Tao (王涛), CHENG Yongzhou (成永洲), CHEN Xiaoyan (陈晓艳), LIU Zhaopu (刘兆普), LONG Xiaohua (隆
小华) **
Key Findings
 Digestive enzyme activities significantly enhanced by the small peptide
supplement.
 Significantly improved growth rates of grouper under artificial seawater
conditions.
 Dietary supplements did not improve antioxidant capacity under low-salinity
stress conditions.
12/10/2022 31

32. Received: 3 September 2018 | Revised: 28 January 2019 | Accepted: 9 February 2019
DOI: 10.1111/anu.12904
ORIGINAL ARTICLE
Dietary supplementation effects of Pediococcus acidilactici as probiotic
on growth performance, digestive enzyme activities and immunity
response in zebrafish (Danio rerio)
Mojtaba Mohammadi Arani1,2 | Amir Parviz Salati1 | Omid Safari3 |Saeed Keyvanshokooh1
Key Findings
 The inclusion of P. acidilactici in the diet is an effective way not only to reduce
both the production period and costs, but also to improve fish health.
 It is suggest that 4 × 106 cfu of the probiotic P. acidilactici in each gram of the
zebrafish diet improved growth performance, digestive enzyme and immune
response.
12/10/2022 32

33. Probiotics and Antimicrobial Proteins (2019) 11:938–945
https://doi.org/10.1007/s12602-018-9421-z
Effects of Dietary Clostridium butyricum on the Growth,Digestive Enzyme
Activity, Antioxidant Capacity, and Resistance to Nitrite Stress of Penaeus
monodon
Yafei Duan1 & Jiasong Zhang1 & Jianhua Huang1,2 & Shigui Jiang1
Published online: 1 June 2018
# Springer Science+Business Media, LLC, part of Springer Nature 2018, corrected publication June/2018
Key Findings
 Clostridium butyricum is a good probiotic and its optimum dosage is 1.0% (w/w) for
shrimp aquaculture.
 Dietary supplementation with C. butyricum improved the growth and intestine
digestive enzyme activity, intestine antioxidant capacity and nitrite stress resistance
capacity in Penaeus monodon, with no effects on the shrimp body composition.
12/10/2022 33

34. Aquaculture 531 (2021) 735852
Contents lists available at ScienceDirect
Aquaculture
journal homepage: www.elsevier.com/ locate/aqua-online
Key Findings
Lactic acid bacteria supplementation enhanced the liver and intestine antioxidant
status and significant improvement in the digestive enzyme activity.
 Increase the generation of probiotic bacteria and decrease the abundance of aquatic
pathogens.
The positive effects of single or conjoint administration of lactic acid
bacteria on Channa argus: Digestive enzyme activity, antioxidant capacity,
intestinal microbiota and morphology
Yidi Konga, Min Lia,c,d,e, Guosheng Chub, Hongjian Liub, Xiaofeng Shana,c,d,e,Guiqin Wanga,c,d,e, Guanghong Hanf,
12/10/2022 34

35. Thank You
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