EFFECT OF LIPID ENHANCEMENT ON BIOCHEMICAL AND REPRODUCTIVE PARAMETERS OF MUDSKIPPER (Pseudapocryptes elongatus, Curvier 1816) BROODSTOCK VIA DIETARY LIPID .pdf

CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES
DAO DUY TUNG
EFFECT OF LIPID ENHANCEMENT ON BIOCHEMICAL
AND REPRODUCTIVEPARAMETERSOF MUDSKIPPER
(Pseudapocryptes elongatus, Curvier 1816) BROODSTOCK
VIA DIETARY LIPID
THESIS
AQUACULTURE ADVANCED PROGRAM
Can Tho, 2024

CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES
DAO DUY TUNG
EFFECT OF LIPID ENHANCEMENT ON BIOCHEMICAL
AND REPRODUCTIVEPARAMETERSOF MUDSKIPPER
(Pseudapocryptes elongatus, Curvier 1816) BROODSTOCK
VIA DIETARY LIPID
THESIS
AQUACULTURE ADVANCED PROGRAM
Supervisor : A/Prof.Dr. Hua Thai Nhan
Can Tho, 2024

1
EFFECT OF LIPID ENHANCEMENT ON BIOCHEMICAL AND
REPRODUCTIVE PARAMETERS OF MUDSKIPPER (Pseudapocryptes
elongatus, Curvier 1816) BROODSTOCK VIA DIETARY LIPID
Dao Duy Tung1*
and Hua Thai Nhan1
1
College of Aquaculture and Fisheries – Can Tho University
*
Email: daoduytungst1412@gmail.com
ABSTRACT
The researchaims to determinethesuitable lipid contentin the diet for optimal growth,
potential maturation, and biochemistry composition of the mudskipper Pseudapocryptes
elongatus. The experiment are arranged according to 4 different treatments including the
treatment lipid 6% is a control treatment; Lipid 9%; 12% and 15%. Added to the fish oil,
plant oil-simply and ARA feeds corresponded to the treatments of the feed by mixing it and
dried at 70°C for approximately 2 days and then stored in the fridge during the study. The
results showed that the lipid content in the feed did not affect the survival rate of the
mudskipper. The lipids in the feed composition affect the chemical composition in the
muscles of the mudskipper. The lipid content in the liver of mudskipper is highest at 12%
(76,13±0,51) and the lowest is the control treatment (73.96±.,45). The lipid content in the
muscles of the mudskipper highestat 12% (3,47±0,20), the lowestin the treatmentlipid 9%
(2.28±0,39) and there was no significant difference (p>0,05). The HSI of mudskipper after
90 days of study ranged from 6.71-7.97%, the highest in the treatment 2: 9% (7.97±0.78).
Lipids have an effect on the accumulation of energy and chemical composition in muscles
and lipids in the liver of mudskipper. The highest GSI in this study was at 9% (0.35±0.21).
From theresults ofthe currentstudy, thelipidcontentof 9% in thefeed, it was enhancement
reproduction of and lipid content of 12% in the feed, it was enhanced biochemical of
mudskipper. Thecontroltreatmenthadthesmallesteggsize(averagelength41,45±5,4 μm).
All lipid supplementation treatments resulted in significantly larger egg sizes compared to
the control. The results of histological observation show that the development stage of
mudskipper is mainly detected in stage III and The treatment 15% lipid is a treatment
yielded the largest eggs.
Keyworks: Pseudapocryptes elongatus, mudskipper, Mudskipper broodstock, biology, dietary
lipid.

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1. Introduction:
In recent year aquaculture becoming increasingly important to the global economy and
enhancing the production efficiency of economically valuable fish species, enhancing the
efficiency and quality of aquaculture products has become a top priority. Mudskipper
(Pseudapocryptes elongatus), a species of high economic value in Southeast Asia
distributedin estuaries, mudflats,andtidal areas in India, China, Malaysia,andtheMekong
Delta (Rainboth, 1996) and is one of 34 species belonging to the Gobiidae family
distributed in East and West Africa, the South Pacific islands, and southern Australia
(Murdy, 1989). Mudskipper P. elongatus is a euryhaline species with accessory respiratory
organs and is an opportunistic feeder, living in mudflats and able to withstand harsh
environmental conditions (Ishimatsu et al., 2007).
In the past, mudskipperwas a species of low economicvalue inAsian countries (Takita
et al., 1999). However, in recent years, China has successfully bred and developed
monoculture and polyculture pond models for the related species Boleophthalmus
pectinirostris, another Gobiidae species, with yields reaching 750 to 975 kg/ha (Hong and
Zhang, 2004). According to a report by Hong et al. (2007), there are around 13,000 ha of
ponds culturing this fish species inChina. InVietnam, mudskipperhas becomeahigh-value
economic species in recent years (100,000 VND/kg in 2008) and has potential for
developmentof farming in the coastalprovinces of the MekongDelta. Mudskipperfarming
is developing rapidly and provides products mainly for domestic consumption. However, a
major obstacle is that successful hatchery production of mudskipper seeds has not been
achieved. Mudskipper P. elongatus seed stocks are currently obtained from wild capture
and supplied to farmers in the coastal provinces of the Mekong Delta. This has led to
increasedfishingpressureon wild mudskipperseed, particularlyin SocTrang and BacLieu
provinces (Truong Hoang Minh et al., 2010). Currently, information on the technical and
economic aspects of mudskipper culture is limited. Therefore, this study has been
conducted and is presented in this paper has been the subject of numerous studies aimed at
optimizing cultivation processes and improving product quality. Among the factors
affectingthe growthand maturityof mudskipper, nutrition, particularlythecontentandtype
of lipids in the diet, occupies an important position.
Lipids not only provide energy but are also a source of essential fatty acids, directly
influencing growth, reproductive capacity, and overall health of the fish. However, the use

3
of lipids in mudskipper diets needs to be carefully considered, as inappropriate quantities
and types of lipids can lead to health issues and reduced reproductive efficiency.
The research by Hien et al. (2014) demonstrated that different lipids do not affect the
survival rate of mudskipperbut can influencegrowth and chemicalcompositionof the fish,
highlighting the importance of selecting appropriate types of lipids in the diet for
mudskipper. Similarly, the study by Tu et al. (2014) emphasized determining the protein
and lipid needs in the diet to improve growth and meat quality of mudskipper.
Within the framework of this research topic “ study on the effect of differentlipids on
the maturation ability of mudskipper (Pseudapocryptes elongatus)”. We aim to explore
further the impact of different types of lipids on the maturity and product quality of
mudskipper, thus, proposing an optimal nutrition regime, contributing to the improvement
of mudskipper cultivation efficiency. Understanding the relationship between diet and
reproductivecapacityof mudskipperwillnot only be beneficialfor the aquacultureindustry
but also support the conservation and sustainable development of aquatic resources.
2. MATERIALS AND METHODOLOGY
2.1. Time and place study
The study was performed at January-Jun 2024 in AQUAFISH INNOVATION LAB,
the College of Aquaculture and Fisheries - Can Tho University.
Subjects of study: mudskipper (Pseudapocryptes elongatus, Curvier 1816).
2.2. Materials
The culture tank includes composite tanks (250L) with square tanks, scale, volume of
tank is 200L, biological filter tanks and oxygen system, salt water, net racket, plastic pipe,
salinity meter and some related materials, dryer, iodine, refractometer, DO meter.
2.3. Broodstock source
Broodstock source: Broodstock at size 35 individual/kg were collected from ponds in
Ca Mau provinces and were used for study. The broodstock were transferred to the lab,
College of Aquaculture and Fisheries, CanTho University and were arranged at density 20
individuals/tank.

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2.4. Experimental set-up
The treatment were conducted randomly in tanks 250L in RAS (Recirculating
Aquaculture System) and with 4 treatments various lipid including: 6% (treatment 1 -
Control treatment), 9% (treatment - 2), 12% (treatment - 3) and 15% (treatment - 4). The
feed usedin this studywere commercialpellets of LONG THANGwith thesizeabout 2mm
and it have protein 44%, fiber 3%, moisture 11%, Lysine 1,2% and Phosphorus 1%. The
amount of lipid-rich chemicals added to the feed including fish oil, plant oild-simply, ARA
respectively of each treatments added to the feed and being dried and stored in the fridge
during the study. The experimentwere set up randomly with different content lipid per diet.
The treatment with lipid content of 6% have not supplements lipid was the control
treatment.
The fish were arranged at density about 20 individuals/tank. The experiment consists
of 4 square tanks 250L, a recirculating aquaculture system operating continuously during
the study. A recirculating aquaculture system consists of 6 culture tanks and 1 biological
filter tank (20L), about 5L the substrate to put into the biological filtration tank and then
starts operating the system. Water from 4 culture tanks culture through the settling system
to settle suspended matter and waste of mudskipper, then water from settling system flows
through the biological filtration system and returns to the culture tank. Each tank has a
bundle of plastic wire to serve as a substrate for mudskipper to shelter. Salinity in all
treatments were maintained at 25‰.
Table:1 Chemical compositions (%dried matter) of the diets lipid
T1: Control
treatment
(6%)
T2: 9% T3: 12% T4: 15%
Moisture 8,46±0,15 4,30±0,01 3,94±0,08 3,92±0,25
Ash 13,15±0,05 12,75±0,02 12,58±0,00 12,25±0,06
Lipid 7,19±1,38 8,49±1,64 13,27±0,35 14,44±0,94
Protein 43,27±0,53 42,69±0,07 43,38±0,53 43,84±0,20
Notes: The values shown above are average ± standard deviation
2.5. Feeding and environmental management.
Fish were feed twice a day (8 AM and 16:30 PM with the amount of feed from 3% of
body weight). Monitor and record the activities of the fish daily: feed consumed and left
over, swimming, catch bait, the number of dead fish. Observe daily to adjust suitable feed

5
content. Regularly check and maintain the water quality in the tank in good condition for
fish growth throughout the experiment through environmental norms.
Environmental parameters during the treatment such as temperature, pH, dissolved
oxygen, water were measured every two days. The temperature is measured twice a day
(morning 7-8 am, afternoon 14-15 pm), pH and dissolved oxygen are measured by a meter.
The water environment indicator: temperature, pH, DO are measured twice a day by
Hanna 98196 indicator (Rumani).
2.6. Determination of chemical composition parameters and reproductive parameters
+ Chemical composition analysis of the diet
Moisture, crude, protein, and ash from experimental diets were determined using
standard methods (AOAC, 2000). Briefly, moisture was determined by oven-drying at
105°C for more than 12h for constant weight. Ash was determined to use a muffle furnace
at 550°C for 5h. The Crude protein (Ntimes6,25) of the samples was measured by the
Kjeldahl method after vitriol acid digestion using an automatic Kjeldahl analyzer (Velp
UDK 159). Crude lipid in the dites was determined by the Soxhlet Extraction.
Calculation Moisture
- Weight of wet sample:
mw=W1-T
- Weight of dry sample:
md=W2-T
- Moisture:
% Moisture =
𝑊1–𝑊2
𝑊1−𝑇
× 100
Where: W1: weight of the cup and sample before drying (g); W2: cup and sample
weight after drying (g); (T): weight of aluminum cup (g)
Calculation ash
% Total ash =
𝐖𝟑−𝐓
𝑾𝟏−𝑻
x 100
Where: W3: Sample weight after calcination (g); W1; Cup weight (g).

6
Calculation protein
%N = (V-Vo) x 0.0014
%CP = %N x 6.25 (%CP: % crude protein)
Where:Vo: Volume of H2SO4 0.1Nfor zero sample;V: Volume of H2SO4 0.1Nused for
titration; m: Weight of sample (g) %Dr: % dry matter (% dry matter = 100% - moisture
%);0.0014: g nitrogen equivalence to 1 mL H2SO4 0.1N used for titration.
Calculation lipid
%Lipid = (W2-W1) x 100 / (Wm x %Dr)
Where: W2: Weight of wool tube and sample after calcined (g); W1: wool tube weight
and sample after drying (g); Wm: weight of the sample (g).
+ Reproductive parameters of mudskipper
The development of the gonads were an examined and determined after the end of the
experiment (90 days). Broodstock samples were collected from the culture tanks, 3
individual/treatment and 4 individual from the control treatment. Before dissecting
mudskipper, parameters such as body weight (g), length (cm), gonad weight (g), liver
weight (g), somatic weight (g), and intestine length (cm) were carefully recorded. Then,
each fish was dissected to collect the gonads and liver, while at the same time observing
and recording the color of the ovaries. The development, gonad index (GSI), and Energy
accumulation index (HSI) were calculated using the following formulas:
- Histological Method were perform of Lima (2009) in lab histological.
The tissue preparation process begins by fixing the sample in a neutral buffered
formalin solution for 24 hours. Next, the sample is rinsed with water and immersed in 70%
alcohol for refrigerated storage. Before processing, the sample needs to be trimmed into a
block with a thickness of 5-7mm and placed in a cassette. The tissue processing is carried
out on an automatic tissue processor, involving stages of dehydration by immersing the
sample in increasing concentrations of alcohol solutions from 70% to 100%, clearing the
sample with xylene, and finally paraffin infiltration at 57-60°C. The duration of these steps
depends on the thickness of the sample.
After paraffin infiltration, the sample was embedded into a solid block using a mixture
of paraffin and beeswaxin a 7:3 ratio. This block was refrigerated to solidify, then removed
from the mold. Using a microtome blade, the sample block is cut into thin sections of 2µm

7
thickness. The sections were floated on a warm water bath at 45-50°C to allow the paraffin
to spread evenly. Next, an albumin adhesive was smeared onto a lame, and one end of the
lame was dipped into the water bath at a 45° angle to allow the section to adhere to the
lame. The lame was then dried at 45-50°C. The next step is to stain the sample according
to the Hematoxylin & Eosin (H&E) staining protocol. After staining, the lame is mounted
with Entellan mounting medium, and a coverslip is lowered onto the sample at a 45° angle
to prevent air bubbles. Finally, the sample was observed under a microscope at 10X, 40X,
and 100X (oil immersion) magnifications. Awell-prepared sample was have the cell nuclei
stainedpurple-blue(Hematoxylin)andotherstructures stainedpink(Eosin). Representative
images of the sample are captured.
- Survival rate (%):
Survival rate =100× (
𝑭𝑵
𝑰𝑵
)
Where: FN: Number of fish at the end of the treatment; IN: Number of fish at the
initual of the treatment, ( Akatsu., 1983)
Where: FW: Mean wight at the end of the treatment; IW: Mean weight at the initual
of the wxperiment; T: Time in days (Jauncey and Ross, 1982)
- Gonadosomatic index (%):
GSI = (GW/BW)*100
Where: GW is gonadal weight (g); BW is the total body weight of the rafter (g).
- Hepatosomatic index (%):
HSI = (LB/BW)*100
Where: LW is the weight of cod liver (g); BWis the total body weight of the rafter (g).
- Lipid Efficiency Ratio (LER):
LER = (Wt – Wo)/Lipid intake
Where: Wo is the initial weight of the fish (g); Wt is the final weight of the fish (g).
2.7. Analysis data
The collected data were calculated with average values and standard deviations using
Microsoft Excel 2017 software, comparing differences in survival rate, growth in length

8
and weight of fry according to analytical methods. One-factor ANOVA with Duncan test
using SPSS 22 statistical software at significance level (P<0,05).
3. RESULTS AND DISCUSSION
3.1. Environmental factors
Through the study result, it is possible to clearly show the difference of environmental
indicators between the morning and afternoon. In the morning, the environmental factors
have a trend to be more stable. Most of the measured pH values ranged from 4,5 to 6,1.
From this values, indicatingthat pH values is lowing, but with mudskipperit the still within
the acceptable. The temperature index was also quite suitable, with most values ranging
from 26°C to 29,9°C, meetingthe needs of aquaticspecies.AccordingtoDuong Nhut Long
et al. (2005), giant gourami raised for commercial purposes in earthen ponds at
temperatures of 27,5 – 34,5°C and pH = 6,6 - 8 still grew well. Similarly, according to
Nguyen Thi Ngoc Anh et al. (2010a) intensive mudskipper farming in recirculating tanks,
with average temperatures ranging from 28,4 – 29,9°C during the rearing period, the fish
still developed well and achieved relatively high survival rates (60,3 – 76,67%).
Additionally, the dissolved oxygen (DO) levels in the morning were relatively abundant,
from 4,8 mg/L to 6,95 mg/L, sufficient to supply oxygen for respiration and metabolic
processes of aquatic species.
However, in the afternoon, environment factor have significant changes. First, the pH
values fluctuated more widely, ranging from 4,1 to 7,4, with many values above 6,0 and
even reaching 7,4, shows that pH have a tends to rise in the afternoon. According to Aston
(1981), for tropical fish, the optimum temperature range is between23-32°;C. Boyd (1995)
suggests that temperatures and pH between 25-30°C, and 7,0-8,5 are suitable for many
shrimp and fish. This pH variation could have adverse effects on aquatic species if it is
sudden changed for a long time. Additionally, the temperature in the afternoon also
increased, mostly ranging from 28°C to 29,9°C, with many values above 29°C, posing a
risk of heat stress for the aquatic stock. This may be due to the process of decomposition of
excess feed and the excretion of accumulated fish in the tank.
Noticeable, the dissolved oxygen levels in the afternoon decreased significantly
compared to the morning. Many recorded DO values were below 5,0 mg/L, lower than the
safe thresholdfor the survivaland developmentof aquatic species. This could be due to the
respirationprocesses anddecompositionof organicmatter in the ponds consumingoxygen,
while the impact of sunlight was insufficient to replenish the lost oxygen. According to

9
Boyd (1993) and Dang Ngoc Thanh (1979), the metabolic activity of aquatic organisms
will reach a good level when the oxygen content in the culture medium ranges from 3 to 7
mg/L. Therefore, the oxygen content in these tanks completely satisfies the respiratory,
metabolic and development needs of the rafters.
Overall, it can be seen that the farming environment in the afternoon more changes
compared to the morning, with greater fluctuations in pH, temperature, and dissolved
oxygen levels. This necessitates close monitoring and control of environmental factors in
the afternoon to ensure suitable farming conditions for aquatic species. Additionally, the
fluctuations in temperature and oxygen levels between morning and afternoon are also an
issue that needs attention. Excessivechanges in these factors can cause stress, affecting the
growth and health of the aquatic stock. Therefore, maintaining a stable environment
throughout the day has become more important than ever.
The lipid content in the muscles of the mudskipper was highest at treatment lipid
12%, the lowest in the treatment lipid 9% and there was no significant difference
(p>0,05).
Table 2: Environmental factors after 90 days
Environmental factor Treatment Lipid (%)
T1: 6% T2: 9% T3: 12% T4: 15%
Temperature
(℃)
Morning 26,9-28,8 26,1-29,7 26-29,5 26,3-29,3
Afternoon 28,04-29,7 27,9-30 28-29,9 27,5-29,8
pH Morning 4,5-5,8 4,5-5,9 4,5-6,1 4,6-5,8
Afternoon 4,1-7,4 4,2-5,8 4,1-5,2 4,6-5,8
DO (mg/L) Morning 4,88-6,98 5,0-5,72 4,8-6,23 5,03-6,95
Afternoon 4,46-6,75 4,46-6,59 4,42-6,75 4,58-6,54
3.2. Determine chemical composition of broodstock muscle
Based on the results of the analysis of the chemical composition in the muscles and
liver of the mudskipper in Table 2 for 90 days, the results showed that the moisture content
of the fish tended to decrease gradually with the increase in the lipid content in the feed,
then increasedslightly in the 4th experiment(77,47±2,58). The moisturecontent of the fish
was the lowest in the treatment12% lipid (76,47±1,52)and significantlydifferent(P<0,05)
compared to other treatments. Similar to moisture, the lipid content in the muscles of
mudskipper after 90 experiments gradually increased with the increase in the lipid content

10
in the feed. The lipid content in the muscle was highest in the treatment 12% lipid
(3,47±0,20), the lowestin the treatment 9% lipid (2,28±0,39)and there was no statistically
significant difference (p>0,05) compared to the other treatments. Tran Thi Be (2014) also
founded that the lipid content in the muscles of mudskipper fluctuates gradually with the
lipid content in the feed (2,27-3,37). The protein content in the muscles of the mudskipper
increased slightly from the treatment 6% lipid (62,86±0,99) to the treatment 9% lipid
(63,93±0,40) and gradually decreased from the treatment 9% lipid (63,93±0,40) to the
treatment 15% lipid (61,07±0,15) and there was a statistically significant difference
(p<0,05) between the treatments.
Table 3: Chemical Composition of broodstock muscle (% dr matter) at the end of
experiment
T1: 6% T2: 9% T3: 12% T4: 15%
Moisture 86,24±0,03a
78,83±0,79b
76,47±1,52a
78,39±0,43a
Ash 18,26±0,22a
19,81±1,11b
18,98±1,23a
19,50±1,35a
Lipid 2,48±0,11a
2,28±0,39a
3,47±0,20a
2,37±0,63a
Protein 62,86±0,99a
63,93±0,40a
62,41±0,7a
61,07±0,15b
Values in the same row with different superscript letters (a, b) are not significantly different
(p>0,05)
When considering the lipid content in the liver of mudskipper through experiments,
significant fluctuations can be observed. The treatment 6% had the lowest lipid content
(73,96±1,45), then increased slightly to 74,02±0,65 in the treatment 9%. Noticeable, the
treatment 12% showed a spike, reaching the highest level of 76,13±0,51, an increase of
2,11% comparedto the treatment 9%. However, whenit increasedto 15%, the lipid content
decreased slightly to 75,7±0,65. This shows that increasing the lipid content by 15% does
not bring a positive effect. Therefore, it can be concluded that the lipid content in the liver
of the mudskipper tends to increase when the lipid in food is increased by 6% to 12%, with
the 12% experiment can be considered as the optimal point. This result is consistent with
the study by Nguyen et al. (2018), which showed that the lipid content in fish liver varies
according to farming conditions and diet. The study of Tran and Le (2020) also reported
that the lipid content in the liver of mudskipper ranges from 70% to 78% depending on the
season and stage of development. To better understand the significance of this difference,
it is necessary to perform a statistical analysis as proposed by Pham et al. (2019) in the
study of lipid fluctuations in brackish water fish species. Furthermore, Le and Vo (2021)

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emphasize the importance of considering environmental and physiological factors when
assessing the lipid content of fish liver.
Figure 1: The content lipid in liver of mudskipper
3.4. Reproduction parameters
After 90 days, it can be seen that Over 90 days of experiments, survival in the
treatments achieve 100% between experiments with different lipid content. Therefore, the
different lipid content in the feed does not affect the survival rate of the mudskipper. This
result is similar to the results of the study of La Thuy An 2014 studies on mudskipper P.
elongatus with different lipid content of the feed (3,6,9,12 and 18%) did not affect to
survival rate of mudskipper.
Basedon table 5 shows the growth and maturity (GSI) of the mudskipperafter 90 days,
experiment 3 (12%) had the longest average length (22,57±3,78 cm) while experiment 4
(15%) achieved the highest average weight (23,00±3,43g). The treatment 4 had the lowest
GSI (0,27±0,20) but the highest LER (29,04±3,28) compared to all other treatment. It is
noticeable that treatment 2 (9%) achieved the highest HSI (7,97±0,78) after 90 days.
Table 4: Reproduction parameters
Time Indicartors Treatments
T1: Control
treatment (6%)
T2: 9% T3: 12% T4: 15%
73,96±1,45 74,02±0,65
76,13±0,51 75,70±0,65
64
66
68
70
72
74
76
78
80
82
6 % 9 % 1 2 % 1 5 %
%
Lipid
in
liver
Lipid in food (%)
THE CONTENT LIPID IN LIVER OF
MUDSKIPPER
% Lipid

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Initial Weight (g)
Length (cm)
HSI
GSI
20,39±2,89
18,6±0,6
7,65±2,43
0,29±0,2
After
90 days
Length (cm)
Weight (g)
GSI
HSI
LER
Survival
rate (%)
19,87±0,72
22,56±2,91
0,27±0,20
6,71±2,66
36,2±0,77
100%
20,33±0,40
22,16±1,29
0,35±0,21
7,97±0,78
18,4±2,00
100%
22,57±3,78
21,25±0,97
0,29±0,04
7,66±0,43
15,9±0,80
100%
20,37±1,22
23,00±3,43
0,12±0,07
7,30±1,67
8,07±1,43
100%
Notes: The values shown above are averages ± standard deviations
Gonadsomatic Index (GSI) and Hepatosomatic index (HSI) after 90 days
Based on the results of the study, it can be clearly seen that the difference between the
experiments can be clearly seen. The treatment lipid 9% recorded the highest GSI value of
(0,35±0,21). Besides, the treatment lipid 12% also brought positive results with a GSI of
(0,29±0,04), significantly higher than the control treatment without lipid supplementation.
However, the treatment lipid 15% seemed to negative effective (0,12±0,07) lowest
compared to other treatment. This proves that the lipid-deficient environment has
significantly reduced the reproductive activity of the mudskipper.
The HSI result of the 90-day of mudskipper in Figure 2 showed that the treatment 9%
lipid recorded the highest HSI value of 7,97±0,78, significantly higher than the other
treatments. This shows that this treatment with a lipid content 9% is effective enhancing
the reproduction for mudskipper and at the the control treatment had the lowest HSI value
(6,71±2,66), lower than other treatment

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Figure 2: Gonadsomatic Index (GSI) and Hepatosomatic index (HSI)
3.7. Lipid efficiency ratio (LER)
The results showed that the experiments had a large difference in LER value between
the treatment lipid 6% and other treatment. The LER value of the 6% experiment
(36,2±0,77), which is significantly superior to the treatment lipid 9% (18,4±2,00) and is
twice as high as the treatment 12% (15,9±0,80). This shows that the treatment 6% ratio is
used extremely effectively during the growth of the study mudskipper. This suggests that
the mudskipper cannot effectively use the excess lipid source and can negatively affect the
growth process. In addition, when comparing the other experiments, it show that the LER
value of the 9% (18,4±2,00) experiment is significantly higher than that of the treatment
12% (15,9±0,80) and 15% (8,07±1,43) experiments. This suggests that a lipid ratio of 9%
is also a good option for higher lipid use efficiency than the other two treatments.
3.8. Gonalal development stage
The control treatment had the smallest egg size (average length 41,45±5,4 μm). All
lipid supplementation treatments resulted in significantly larger egg sizes compared to the
control. The 15% lipid treatment yielded the largest eggs (average length 93,44±10,93 μm)
is not significant difference compared to the treatment 6 and 9% lipid (p>0,05) and it have
significant difference compared to the treatment 12% lipid (p<0,05). Similarly to egg size,
the control treatment had the smallest egg size (average length 13,87±2,12 μm). All lipid
supplementation treatments resulted in significantly larger egg nuclei compared to the
control. There were significant differences in egg and egg sizes between the control and
0,27±0,20
0,35±0,21
0,29±0,04
0,12±0,07
6,71±2,66
7,97±0,78
7,66±0,43
7,30±1,67
6
6.2
6.4
6.6
6.8
7
7.2
7.4
7.6
7.8
8
8.2
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
6 % 9 % 1 2 % 1 5 %
HSI
(%)
GSI
(%)
Treatments lipid (%)
GSI AND HSI
GSI HIS

14
lipid supplementation treatments. Among the lipid supplementation treatments, the
differences were not as pronounced, but the 15% lipid treatment have trend to result in the
largest egg and egg sizes.
The results of histological observation in Figure 4 show that the development stage of
rcsa crab is mainly detected in stage III. The results of observing the histological in this
study showed that the gonads of the mudskipper developed well with a dense amount of
eggs. This result shows that high lipid content tends to promote better development of
gonads.
Table 5: Eggs and cell in the control treatment-6% lipid
Treatment Length egg diameter Length cell diameter
6% 41,45±5,4a
13,87±2,12a
9% 87,55±12,28a
34,61±5,03a
12% 84,8±11,55b
33,89±3,85b
15% 93,44±10,93b
40±6,98a
Values in the same column with different superscript letters (a, b) are not significantly different
(p>0,05)
A B
C D

15
Figure 3: Representative histology section of vary (control treatment 40x) (A);
Representativehistologysection of vary (treatment lipid 9%, 40x) (B); male gonads of
treatment lipid 12% (40x) (C); Representative histology section of vary (treatment
lipid 15%, 40x) (D)
3.9. The liver of mudskipper
Figure 4: Histology of liver in the control treatment (40x) (A); Histology of liver in
the treatment 9% lipid (40x) (B); Histology of liver in the treatment 12% lipids (40x)
(C); Histology of liver in the treatment 15% lipid (40x) (D)
In the control treatment, the liver structure is normal with liver cells lined up close to
each other. When the lipid content is increased to 9%, small gaps between liver cells begin
to appear. At the treatment 12% lipids, this gap becomes more pronounced and liver cells
begin to deform. By the treatment 15% lipid, the liver structure was seriously broken with
many large gaps and strong deformation of liver cells. This suggests that an increase in the
lipid content in the feed leads to an increasing accumulation of lipids in the liver, causing
significant changes in liver tissue structure. High lipid content in food can cause excessive
lipid accumulation in the liver of catfish, leading to changes in liver tissue structure and
potentially adversely affecting liver function.
A B
C D

16
4. CONCLUSION AND RECOMMENDATION
4.1. Conclusion
Based on the study results, the lipid content in the feed did not affect the survival rate
of the mudskipper. The lipids in the feed composition effect the chemical composition in
the muscles of the mudskipper. The lipid content in the liver of mudskipper is highest at
12% (76,13±0,51) and the lowest is the control treatment (73,96±1,45). The HSI of
mudskipper after 90 days of study ranged from 6,71-7,97%, the highest in the treatment 2:
9% (7,97±0,78). The lipid content in the muscles of the mudskipper highest at 12%
(3,47±0,20), the lowest in the treatment lipid 9% (2,28±0,39) and there was no significant
difference (p>0,05).
Lipids have an effect on the accumulation of energy and chemical composition in
muscles and lipids in the liver of mudskipper. The highest GSI in this study was at 9%
(0,35±0,21). The results of histological observation show that the development stage of
mudskipperis mainlydetectedin stageIII and thetreatment15% lipid is a treatmentyielded
the largest eggs
4.2. RECOMMEND
Should studies the impact of dietary lipid content on the disease resistance of
mudskipper broodstock and provide a more detailed analysis of fatty acid composition in
fish tissues across different lipid treatments.
4.3. ACKNOWLEDGMENT
I would like to express my deepest gratitude to Assoc. Prof. Dr. Hua Thai Nhan, who
has diligently guided and instructed me throughout the process of conducting this research.
His support and direction have significantly contributed to the success of this project.
I would also like to convey my appreciation to my colleagues in the laboratory who
enthusiastically guided and assistedme during the experimental process. Their support and
shared experiences have been invaluable to me.
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