Organic acids work best in Nile tilapia (Oreochromis niloticus)
under suboptimal conditions
Kim T. Tran-Ngoc1,2, Son T. Huynh2, Thinh H. Nguyen2, Arjen Roem1, Johan W. Schrama1, Johan A.J. Verreth1
1Aquaculture
and Fisheries Group, Wageningen UR, the Netherlands
2Nong Lam University, Ho Chi Minh City, Vietnam
[email protected] [email protected]
22 September 2016
Introduction
2
Previous study: effect of oxygen and soybean meal
on Nile tilapia
Specific growth rate
2.4
%bw.d-1
2.3
2.3
2.2
2.2
2.1
2.1
2.0
2.0
1.9
1.8
3
Literature reviews
4
SO, HOW DOES THE OA
WORKS AGAINST
PATHOGEN BACTERIA???
AND, WHAT ARE THE
BENEFITS TO THE
FISH???
DIET + ORGANIC ACIDS
pH and buffering capacity
INTESTINE
harmful bacteria intake
Epithelial cell proliferation
feed hygiene
Nutrient absorption/digestibility
Mineral availability
FECES
STOMACH
harmful bacteria
enzyme activity
mineral solubility
Phosphorus load
Microbiota load
Beneficial microbiota
Colonization of harmful bacteria
Gut health
Risk of cross infection
Water quality
5
Source: Wing-Keong Ng and Chik-Boon Koh, 2016
Introduction: Hypothesis and Objectives
Low DO Level
Organic Acids
INTESTINAL
HEALTH
DIGESTION
Soybean Meal Based Diet
NILE TILAPIA
INTESTINAL
MORPHOLOGY
6
Organic acids
pKa - the pH at which the acid is half dissociated. The
higher value of pKa, the more antimicrobial activity
Antimicrobial activity  reduced
survival of pathogens in GI/
balancing the microbial population
ACID
PKA
FORMIC ACID
Propionic acid
4.88
BUTYRIC ACID
Butyric acid
4.82
ACETIC ACID
Acetic acid
4.76
MALIC ACID
Sorbic acid
4.76
CITRIC ACID
Lactic acid
3.86
Formic acid
3.75
Malic acid
3.40
Citric acid
3.13
Fumaric acid
3.03
Tartaric acid
2.98
PH AND PKA
1
2
3
STOMACH
ANTERIOR GUT
POSTERIOR GUT
Reducing the pH in gut 
stimulating the activity of
digestion enzymes
4
5
6
7
8
9
10
7
Potassium diformate & Calcium butyrate
FORMIC
ACID
Potassium diformate (KDF)*
BUTYRIC
ACID
(Coated) Calcium butyrate
Butyrate
Formic
Acid (35.4%)
Formate (34.6%)
Potassium (30%)
AQUAFORMTM, ADDCON, NORWAY
*KDF: crystalline, granular compound
Calcium
Butyrate
BUTIPEARLTM, KEMIN, USA
8
Experimental diets
Diets
Ingredients
Soybean meal
Rice bran
Cassava
Corn gluten meal
Fish oil
Soybean oil
DL-Methionine
Di-Calcium phosphate
Vitamin and Mineral Premix
Chromic oxide
Inclusion
%
52
12
10
15
3
3
1
1
2
1
Diet 1
Diet 2 (KDF)
Diet 3 (CAB)
Diet 4
(KDF+CAB)
Control
0.2% potassium diformate
0.2% Calcium butyrate
0.4% mixture of KDF and
CAB (1:1)
CT
KDF
CAB
KDF+CAB
Dry matter
91.8
91.8
91.6
91.7
Crude protein
Ash
Gross energy (MJ. Kg-1)
38.2
7.7
20.5
38.4
7.6
20.8
38.4
7.6
20.8
38.6
7.5
20.6
Chromic oxide
1.3
1.3
1.2
1.4
9
Materials and Method
Fish
480 juvenile male Nile tilapia ~ 30g:
40 fish/ tank x 3 replicate
Housing
Two time periods: of each five weeks
-
normoxia in the first period
-
hypoxia in the second period
- Period 1: Normoxia (DO ~ 80% saturation):
aeration is provided through air stones supplied in
the water storage tanks and fish-tanks
- Period 2: Hypoxia (DO ~ 40% saturation): the
twelve fish-tanks are not aerated.
10
Measurement of gut morphology on Nile tilapia
Measurable and countable method are extended from semi-quantitative
method developed at Wageningen University
Lamina propria
1.
Number of goblet cell (GC)
in µm2 of villi
2.
Number of eosinophilic
granulocytes (EG) in µm2 of villi
3. Thickness of lamina propria in
µm
Eosinophilic
granulocytes
Goblet cells
4. Thickness of submucosa in µm
Submucosa
11
Results: Organic acids improved the intestinal morphology
THE NUMBER OF GOBLET CELLS_PROXIMAL INTESTINE
At hypoxia
KDF
Control
25
20
10-3 cells µm-1
Normoxia
Hypoxia
15
GC
10
5
0
Control
KDF
CAB
THE THICKNESS OF LAMINA PROPRIA - PROXIMAL
KDF+CAB
INTESTINE
Goblet cells (10-3
cells µm-1)
Control KDF CAB KDF+CAB
Normoxia
12
11 10**
9
Hypoxia
20 14** 13***
11
80
µm
70
60
Normoxia
50
Hypoxia
40
30
LP (µm)
Normoxia
Hypoxia
Control KDF
56
48
68
51
CAB KDF+CAB
43
51**
44*
58***
20
10
12
0
Control
KDF
CAB
KDF+CAB
Results: Organic acids improved the intestinal morphology
700
CAB treatment
THE THICKNESS OF SUBMUCOSA _DISTAL INTESTINE
Hypoxia
Normoxia
600
500
400
µm
Normoxia
Hypoxia
300
200
100
1
0
Control
KDF
CAB
KDF+CAB
THE NUMBER OF EOSINOPHILIIC
GRANULOCYTES_PROXIMAL INTESTINE
0.9
0.8
Eosinophilic
granulocytes (10-3 Control
cells µm-1)
Normoxia
0.3
Hypoxia
0.9
KDF
CAB KDF+CAB
0.7
10-3 cells µm-1
SM (µm) Control
KDF
CAB KDF+CAB
Normoxia
501
498
518
508
Hypoxia
666
489*** 389*** 423***
0.6
Normoxia
0.5
Hypoxia
0.4
0.3
0.2
0*
0.4
0.3
0.3
0.1
0.3
0.1
13
0
Control
KDF
CAB
KDF+CAB
Conclusions
The enteritis like symptoms were induced in fish fed high level of SBM
and under low dissolved oxygen (at the control treatment)
OA did not significantly improve intestinal morphology under normoxia.
However, it has a beneficial impact under hypoxia Environmental
condition can alter the effect of OA on the intestinal morphology.
OA supplementation in diets  highly recommended whenever fish
are being culture under challenging conditions
The synergistic effect in improvement of intestinal morphology were
not found in the combination between formic and butyric acid.
14
Thank you
I would like to acknowledge João Sendão
for his comments to this presentation
15