Aquatic animal health management has become a crucial component in the goal of increasing catfish aquaculture productivity. Additionally, hybrid catfish (Clarias gariepinus × C. macrocephalus) has been promoted as a highly profitable freshwater fish in Asia. Interestingly, the crucial diseases induced by Aeromonas hydrophila have been reported to greatly impede catfish production. To overcome this challenge, the aim was to investigate the effects of the oral administration of potentially synbiotic chitosan (CH) and Acinetobacter KU011TH (AK) on the growth performance, immunological responses, and disease resistance of hybrid catfish against A. hydrophila.

1. Synbiotic Effects of the Chitosan and Acinetobacter KU011TH Mixture on
Growth Performance, Immune Responses and Disease Resistance against
Aeromonas hydrophila of Hybrid Catfish
(Clarias gariepinus x C. macrocephalus)
Pisey Say1,2, Sukkrit Nimitkul3, Anurak Bunnoy1,2, Uthairat Na-Nakorn4,5 and
Prapansak Srisapoome1,2
1 Laboratory of Aquatic Animal Health Management, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Chatuchak,
Bangkok, Thailand.
2 Center of Excellence in Aquatic Animal Health Management, Department of Aquaculture Faculty of Fisheries, Kasetsart University,
Chatuchak, Bangkok, Thailand.
3 Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Chatuchak, Bangkok, Thailand.
4 Laboratory of Aquatic Animal Genetics, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, 50 Paholayothin Rd,
Ladyao, Chatuchak, Bangkok, Thailand.
5 Academy of Science, The Royal Society of Thailand, Bangkok, Thailand.
IFS 2022
10th International Fisheries
Symposium 2022

2. 2
Key trends
in
aquaculture
W
e
r
Fee
ds
Drugs
Gen
etic
Diseases
Genetics
+ Seeds
Environ+
Culture
system
Diets
I. Introduction
(Luis et al., 2019; Tacon et al., 2020; FAO, 2022)

3. 1. Hybrid catfish (C. macrocephalus x C. gariepinus)
3
(Senanan et al., 2004; Abid et al., 2013; FAO, 2021; Nguyen et al., 2022)
➢ Great proportion production
➢ Strongly biological resistance to
infectious pathogen, and
environments.
0
40
80
120
160
200
0
40
80
120
160
200
2014 2015 2016 2017 2018 2019
Values
(x10
6
$)
Productions
(x10
3
t)
Years
Thailand catfish aquaculture
Total productions Total values
0
40
80
120
160
200
0
40
80
120
160
200
2014 2015 2016 2017 2018 2019
Values
(x10
6
$)
Productions
(x10
3
t)
Years
Catfish aquaculture total productions in Thailand
Total productions Total values
➢ Aeromonas septicemia disease
- Tremendous concerns

4. 2. Strategies of applications
4
(Gibson and Roberfroid, 1995)

5. 5
- Sources: the crustaceans, insects, and fungi
- polysaccharide linked by β-(1,4)-glycosidic
bonds
- Application: biomedical and pharmaceutical field,
antimicrobial, antioxidant and immunostimulant
(Peter, 1995; ; Xing et al., 2005; Huang et al., 2006; Jayakumar et al., 2011; Bunnoy et al., 2019; Cen et al., 2021)
✓ Acinetobacter KU011TH
- Isolated from mucus of bighead catfish
- Gram (-) coccobacillus
- Improvement in growth and disease resistance
B. Synbiotics
- A potential application
- Improving aquaculture
+ Chitosan

6. 2. Study objectives
6
❑ To evaluate the effects of the synbiotics (chitosan
and Acinetobacter KU011TH) on growth performance,
immune responses and disease resistance against
Aeromonas hydrophila of hybrid catfish under long-
term application during winter.
❑ Improving catfish production and disease
resistance during critical winter periods

7. 7
Synbiotic application will enhance growth
performance, immune-responses and disease
resistance to A. hydrophila of hybrid catfish.
Ho: 0 effects Ha:> 1 effects
Hypothesis

8. 1. Experimental animals
8
Water 200 L
30 fishes
100 ppm
Formalin/5 mins
Volume 5,000 L
5.70 ± 0.41 g/fish,
8.80 ± 0.88 cm/fish
Feed 10% of BW
I. II. Materials and Methods
(Ethics ID: ACKU61-FIS-004)
Bangkok
Suphanburi
N
S

9. 9
2. Experimental feeding design
3 4
Weeks
Days
0 1 2
Syn F
Boost
Syn F
1st meal: 9-10 AM
2nd meal: 4-5 PM
Note:
F: Feed
Syn: Synbiotics
Syn
Table Formulated feeds top mixing
Treat-
ments
Feed intake
(g; 2-10%/BW)
NaCl
(mL/kg)
Chitosan
(mL/kg)
Probiotic
(CFU/kg)
A (Ctrl) 1,000 200 - -
B 1,000 200 20 -
C 1,000 200 20 1x108
D 1,000 200 20 1x109
E 1,000 200 20 1x1010

10. 10
2.1 Probiotic preparation
4 oc
0.085%
NaCl
Growth 12 h, 37 oc Culture 6 h, 35 oc
5
Enrich 24 h, 35 oc
4
3
OD: 600 nm Spin at 2,500 rpm/ 10 mins
2
1
-80 oc
(Bunnoy et al., 2019)

11. 3. Experimental design
Treatment
Experimental Plot
1 2 3 4
1 A4 B1 E3 A3
2 C4 A2 D2 B3
3 B4 E1 A1 C2
4 D3 C1 C3 D1
5 E4 D4 B2 E2
5 treatments x 4 replicates
11
Completely randomized design
(CRD)

12. 12
4. Data collection
Blood
1) Hematocrit
20 μL
Spin 12,000
rpm (2mins)
2) Blood Count
2.5 μL
+ Natt-Herrick
497.5 μL
3) Spin 8,500
rpm/10 mins
1-NBT; 2-Lysozyme; 3-ACH50 ;
4-Bactericidal activity
serum
-20 oc
weeks
0 1 2 3 4
Water quality
Growth parameters
Challenge test
Immune parameters
November December January February
Feed
1
2
3
4

13. 13
4.1 Respiratory burst dye reduction assay
(Dehler et al., 2017)
0.85%
NaCl: 250
μL
Serum
10 μL
NBT:
14.29 μL
OD: 540 nm
NBT, OD:540 nm = Abs Test – Abs Blank
1
2
3
Incubate 2 h/37 OC

14. 14
4.2 Lysozyme activity assay
(Myrnes & Johansen, 1994; Kreutz et al., 2011)
pH 6.2
10 μL
Serum
10 μL
M. lysodeikticus
(2 μg/mL Saline)
250 μL
Substrate
hydrolysis
0 & 5 min Abs
OD: 540 nm
Units/ml enzyme = [(Δ(0 - 5 mins) Abs Test –
Δ(0 - 5 mins) Abs Blank)
(df)]/(0.001)(0.1)
1
2
3

15. 15
OD
540 nm
4.3 Alternative complement pathway (ACH50) assay
(Al-Dohail et al., 2009)
Sheep blood
(2x108 cell/mL PBS)
100 μL
D.W
100 μL
Serum
200 μL
PBS. pH
7.4
100 μL
Centrifuge 1,500G, 5mins/RT
Incubate
1:30 h/RT
Complement value (ACH50) (Unit/mL serum) = (1/B) x Dilution factor x 0.2
1
2
3
4
1
0
0
%
0
%
0

16. 16
Incubate
1 h/ 37OC
4.4 Bactericidal activity
Incubate
12 h, 37 oc
Enrich cell
24 h, 35 oc
3 OD: 600 nm
Abs: 1 ,
4x108
CFU/mL
2,500 rpm/
10 mins
dilution 1:9 =>
1x103
CFU/mL
Serum
100 μL
A. hydrophilla
100 μL
Incubate
1 H/ 37OC
2
1
100 μL
(Anyanwu & Chah, 2015)

17. 17
4.5 Challenge test
Standard curve
1×108
CFU/mL
Micrococcus
lysodeikticus
200 μL
Survival rate
24 h/14 days
4 oc
0.85%
NaCl
- Water 80 L
- 10 fish
10%/2 days
3 4
February
4

18. 5. Statistic analysis
18
One-way ANOVA
& DMRT
Time (weekly)
Trearment 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
TA1
TA2
TA3
TA4
TB1
TB2
TB3
TB4
TC1
TC2
TC3
TC4
TD1
TD2
TD3
TD4
TE1
TE2
TE3
TE4
Data colloection table
Survival analysis
Kaplan–Meier
Growth
parameters
Immune
parameters
Survival rates

19. 19
Tab 1. Say et al. (2022)
Treatments p-
value
Growth parameters A (Control) B C D E
Initial length (IL; cm) 8.87 ± 0.26a
8.78 ±0.25a
8.75 ± 0.20a
9.03 ± 0.19a
8.70 ± 0.10a
0.212
Final length (FL; cm) 25.93 ± 0.61a
26.28 ± 0.22a
26.65 ± 0.72a
26.28 ± 0.41a
25.93 ± 0.50a
0.054
Initial weight (IW; g) 5.49 ± 0.32a
5.67 ± 0.48a
5.72 ± 0.59a
5.74 ± 0.31a
5.90 ± 0.42a
0.765
Final weight (FW; g) 150.63 ± 13.80ab
165.28 ± 11.28b 156.80 ± 22.82b 155.03 ± 10.93b 128.67 ± 3.66a 0.044
Length gain (LG; cm) 17.06 ± 0.79a
17.50 ± 0.34a
17.91 ± 0.82a
17.25 ± 0.50a
17.23 ± 0.41a 0.345
Weight gain (WG; g)
145.15 ± 18.01a
159.61 ± 9.31a
151.08 ± 27.84a
149.29 ± 9.40a
122.77 ± 6.27a
0.061
Average daily gain
(ADG;
g/individual/day)
1.20 ± 0.14a
1.31 ± 0.07a
1.27 ± 0.19a
1.23 ± 0.07a
1.02 ± 0.05a
0.063
Specific growth rate
(SGR; %/day)
1.25 ± 0.05ab
1.28 ± 0.06b
1.25 ± 0.04b
1.24 ± 0.03b
1.15 ± 0.03a
0.013
Feed conversion ratio
(FCR)
1.81 ± 0.02
a
1.68 ± 0.30a
1.82 ± 0.23a
1.90 ± 0.11a
1.98 ± 0.06a
0.052
Accumulative
mortality (%)
7.78 ± 5.09a
7.78 ± 7.70a
5.56 ± 6.94a
6.67 ± 3.85a
0.83 ± 1.67a
0.161
I.
III. Results
1. Growth performance analysis

20. Fig 1. Say et al. (2022)
(a)
(b)

21. Fig 2. Say et al. (2022)
21
2. Hematological analysis

22. 22
3. Serum immunological analysis
Fig 3. Say et al. (2022)

23. Fig 4. Say et al. (2022)
ab
b
b
ab
a
d
cd
bc
b
a
100
80
60
40
20
Survival
rate
(%)
23
A (Control)
B (Chitosan+)
C (Chitosan+10^8 Acinetobacter)
D (Chitosan+10^9 Acinetobacter)
E (Chitosan+10^10 Acinetobacter)
100
80
60
40
20
Survival
rate
(%)
4. Disease resistance against A. hydrophila analysis
b
ab
a

24. The prebiotics and synbiotics have been the ongoing key strategy on
aquatic production and health aspects.
1. Fish’s growth performance
(Atassi et al., 2010 ; Chenoll et al., 2011; Abid et al., 2013; Ismail et al., 2020; Salam et al., 2021)
I. IV. Discussion
Environment
Bio-
metabolism
- The prebiotics and synbiotics (CH +
Acinetobacter KU011TH: 108-9 CFU/kg):
no adverse effects growth
24
- The synbiotics (CH + Acinetobacter
KU011TH: 1010 CFU/kg): inhibited
growth

25. 2. Fish’s health and immunological status
(Munir et al., 2018; Knoop et al., 2018; Yang et al., 2021; Liang et al. 2021; Mohammadi et al., 2022)
I. IV. Discussion
Adaptive
- Lymphoid
cells and
- Immunoglo-
bulins
Innate
- Nonspecific humoral factors:
antimicrobial peptides,
complement peptides, and
- Cellular responses:
phagocytosis
•GI
enzymes
•MAMPs
•PAMPs
•PRPs
•GI
microbes
25
- Bunnoy et al. (2019) examined that probiotics positively increased catfish
growth and health performance, as results in a significant high survivals in
bighead catfish.

26. - Synbiotic group C and D cannot contribute better growth
performance and the adverse effects were found in the group E
of 1×1010CFU/kg feed during winter.
- Synbiotics were driven the immunomodulation of serum immune
factors.
- Prebiotic chitosan and synbiotic group C could improve disease
resistance against A. hydrophila.
* Synbiotic chitosan and Acinetobacter KU011TH at 1×108
CFU/kg feed is recommended for optimizing production and
health performances during winter.
I
. V. Conclusions and summaries
26
effective doses or times, cost effectiveness, and the biological mechanisms

27. “Product Development of a Novel Probiotics, Acinetobacter clariasiae for Disease
Prevention and Growth Promotion of Catfish”
(Project ID: RGU6280008)
Faculty of Fisheries,
Graduate School,
Kasetsart University
Graduate School,
National University of
Battambang

28. 28