Journal of Fisheries science and Technology – No 4/2018

Journal of Fisheries science and Technology

No. 4 - 2018

LIST OF CONTENTS
JOURNAL OF FISHERIES SCIENCE AND TECHNOLOGY
No. 4, 2018
Effects of rearing water and tank on larval survival rate of white-striped cleaner shrimp
Lysmata amboinensis
Luc Minh Diep, Phung The Trung, Vu Dinh Chien

2

Role of antibiotics in chilled storage of sperm in grass carp (Ctenopharyngodon idella)
Le Minh Hoang, Dinh Van Khuong

7

Effects of feeding rate on density, biomass and protein compositions of oligochaete
(Limnodrilus hoffmeisteri Claparede, 1862)
Truong Thi Bich Hong, Nguyen Dinh Mao, Le Minh Hoang

13

Fish oil extraction from yellowfin tuna heads by enzymatic hydrolysis method
Nguyen Thi My Huong, Bui Truong Bich Ngan

19

Larviculture of slipper lobsters in the genus Ibacus and Thenus: a review
Kaori Wakabayashi

27

Voluntary feed intake and transition of ingesta in the gastrointestinal tract of juvenile cobia
(Rachycentron canadum) fed different diets
Nguyen Van Minh, M. Espe, Pham Duc Hung,
Pham Thi Anh, Ivar Rønnestad

34

Protect and enhance the resources by using artificial reef at coastal areas in central of Vietnam
Pham Viet Tich, Tran Duc Phu, Nguyen Trong Luong, Tran Van Hao

44

Assessing on coastal fishing activities and marine resources in Tuy An district, Phu Yen
province
Tran Duc Phu, To Van Phuong

53

Selenium deficiency, toxicity and its requirement in marine fish: A research review
Pham Duc Hung

60

Photoperiod manipulation in the induced breeding of the rabbit fish (Siganus guttatus)
Pham Quoc Hung, Hua Thi Ngoc Dung, Augustine Arukwe

69

Impact of trawling speed on vertical opening of trawl net by modelling method
Nguyen Huu Thanh

78

Can aqui-s help as an aneasthetic in long-distance live transportation of spiny lobsters
(Panulirus ornatus and P. homarus)?
Le Anh Tuan, Tran Bao Chan

84

Research on the fitness between the mesh size and the length of threadfin bream (Nemipterus
sp.) in stow net fishery
Nguyen Trong Luong, Vu Ke Nghiep

93

Effect of stocking density on performance of goldlined rabbitfish Siganus lineatus and the
environmental quality in a closed culture system

Luong Cong Trung

102


Journal of Fisheries science and Technology

No. 4 - 2018

EFFECTS OF REARING WATER AND TANK ON LARVAL SURVIVAL
RATE OF WHITE-STRIPED CLEANER SHRIMP Lysmata amboinensis
Luc Minh Diep¹, Phung The Trung¹, Vu Đinh Chien²
Received: 7.Nov.2017; Revised: 8.Jan.2018; Accepted: 29.Mar.2018

ABSTRACT
The white-striped cleaner shrimp Lysmata amboinensis is a favorite ornamental species in Vietnam and
worldwide, but the rearing conditions for larvae of this species has not been studied yet. Therefore, this study
was conducted to determine proper conditions for larval rearing of white-striped cleaner shrimp Lysmata
amboinensis. The experiment was designed as completely randomized design with 9 treatments, including 3
types of rearing water (disinfected water using chlorine, green-water and biofilter-water) and 3 types of tank
(upwelling, Weis and Kreisel tank). Each treatment had 3 replicates, resulting in a total of 27 experimental
units. The experimental units were tanks filled with 5L of one of three types of rearing water. The results showed
that larval survival was similar among three different water types. Larval survival was higher in Kreisel tanks
than in upwelling and Weis tanks. There was no interactive effect between rearing water and tank type on the
survival rate of the cleaner shrimp larvae. Therefore, disinfected water (lower operation cost) and Kreisel tank
are recommended for rearing of white-striped cleaner shrimps.
Keywords: Lysmata amboinensis, white-striped cleaner shrimp, Kreisel, Weis.

I. INTRODUCTION
The demand of ornamental organisms has
been rising rapidly during the last decades
with a total annual value of 200-300 million
USD [2; 7]. There are many marine species
such as finfish, starfish, jellyfish, mollusk
and crustacean that are cultured in aquarium
nowadays. Among ornamental species, whitestriped cleaner shrimp Lysmata amboinensis
is one of the favourite ornamental species as
they have attractive appearance and behavior
[5]. This species also has high trading value.

For example, the price per individual typically
varies from 65-85 USD [8]. However, most
of them are caught from coral reefs with
unsustainable methods, causing high pressure
to natural environment [3].
Although Lysmata amboinensis has high
market demand and value, there is a lack of
studies on the broodstock culture and efforts in
rearing larvae are, unfortunately, unsuccessful
[8]. Therefore, research on white-striped
shrimp production that includes artificial seed
production is, no doubt, contributing to satisfy
local and global market demand.
However, seed production of white-striped
¹ Institute of Aquaculture, Nha Trang University
² Aquaculture master student, Nha Trang University

2 • NHA TRANG UNIVERSITY

shrimp, as also for other marine crustacean
species, is still facing great challenges. This
is because the development of crustacean
larvae consists of many stages with
complex morphological and physiological
characteristics [3]. Furthermore, during early
larval stages, Lysmata are weak swimmers
and sensitive to environmental conditions,
resulting in a very low survival rate. Therefore,
the proper rearing water and tank design may
considerably increase the survival.

More generally, there are 3 water systems
in rearing crustacean larvae that are static
water, raceway water and biofiltered water.
Static water is only proper to culture larvae
at low density at laboratory scale for some
research purposes such as determination of
larval characteristics or requirements [1; 9;
10]. Raceway water and biofiltered water
could maintain and improve water quality but
it is difficult to operate the system for long
time [1; 6]. Besides, the larvae could be reared
in some types of tanks such as normal tank,
upwelling tank, Weis tank and Kreisel tank
that have been introduced and recommended
to rear ornamental crustacean larvae [4; 10].
Howerver, a proper rearing tank and water
system for rearing Lysmata amboinensis larvae


Journal of Fisheries science and Technology
had not been reported.
This experiment was designed to determine
the effects of rearing water and types of rearing
tank on white-striped cleaner shrimp larval
mortality. Based on the results, larval rearing
performance of Lysmata amboinensis could be
improved with proper rearing tank and water
treatment.
II. MATERIALS AND METHODS
1. Experimental design

The experiment was conducted indoor with
a completely randomized design that included
2 factors, rearing water and tank. There were 3

No. 4 - 2018
types of water and 3 types of tank, resulting a
total of 9 treatments (see detail in Table 1). Each
treatment had 3 replicates with a total of 27 trial
units.
Experimental units were 5 liter volume tanks
with 3 diferent designed systems (see Figure 1).
The water inlet and outlet of each tank covered
by nets with a mesh size of 100 µm to filter trash
and keep the larvae from escaping. Water in
rearing tanks was exchanged continuously by
a pump that located in a 200 liter volume sum
tank. There were 3 storage tanks for 3 systems
of water treatment including disinfected water,

Table 1. Detail of the experiment treatments

green water and biofiltered water. Each water
system consisted of 9 tanks that included 3
upwelling tanks, 3 Weis tanks and 3 Kreisel
tanks connected to the storage tank. Disinfected
marine water was use for disinfected water
system. The water was disinfected by chlorine a
at 30 ppm concentration, strongly aerated for 1
day then exposure under sunlight for another day
before use. The microalgae Nannochloropsis

oculata were used for green-water system
with an initial density of 0.8 × 106 cells per
mL. Biofilter-water system used orchid net as
biofilter material.
The larvae used in the experiment were
collected from 4 shrimp females. All 4 females
were at the same spawning stage. The stocking
density of larvae was 5 Zoeas 1 (larvae at stage
Zoea 1) per liter (25 individuals per tank).

Figure 1. The experimental design and operation
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Journal of Fisheries science and Technology
2. Experimental monitoring
Water temperature, salinity, pH and
total ammonia nitrogen in each tank were
measured and adjusted daily to meet the larval
requirements.
A diet of enriched rotifer was used in all
stages of the shrimp larvae. The rotifer were
enriched by DHA Protein Selco at 200 ppm
concentration before feeding shrimp larvae.
The density of rotifer was maintained at 20
individuals per ml by supplying new rotifer
daily to compensate for the number of rotifers
that had been eaten. From larval stage Zoea
3, they were fed by a mixture of rotifer, early

hatched nauplii and artificial feed. The rotifer
was supplied at the same density as in previous
stages. Early-hatched nauplii Artemia were
supplied at the density of 1 individual per
milliliter tank water per day. A mixture of
artificial feed, including 25% Frippak, 25%
Lansy and 50% V8-zoea was also used.
3. Data collection
Specific stage and accumulated larval
survival rates were calculated for each tank and
treatment based on the number of remaining
larvae. Specific survival rate in a stage n
was the percentage of survived larvae after
completing the transformation to stage n + 1
and the number of larvae at beginning of stage
n. Accumulated survival was the percentage of
survived larvae when finishing the experiment
and the initial number of stocking larvae.
The successfully transformed larvae of
a stage in a tank were determined when they
completely transformed to next stage with no
larvae of the previous stage left.
4. Data analysis
Data are presented as mean ± SD. Results
were compared by analysis of variance with
two factors (two-way ANOVA) followed by
the Duncan’s test when significant differences
were found at the p < 0.05. Data analyses were
performed with SPSS 20.0 for Windows.
III. RESULTS AND DISCUSSIONS

The survival rate of L. amboinensis larvae
did not differ among three types of water
(disinfected water, green-water and biofilter-

4 • NHA TRANG UNIVERSITY

No. 4 - 2018
water) (p > 0.05). The survival rates of the
larvae were 71.1 ± 11.6% in disinfected water
system, 67.6 ± 14.3% in green-water system
and 68.4 ± 12.1% in biofilter-water system for
zoea 1 then decreased to 61.3 ± 20.1%, 58.6
± 23.7% and 54.4 ± 15.7% for zoea 2 stage,
44.2 ± 23.6%, 39.1 ± 30.2% and 31.5 ± 25.4%
for zoea 3 stage, respectively. However, all of
this difference was not statistically significant
among the three water types.
Tank type significantly affected the survival
rate of the larvae (p < 0.05). The shrimp larvae
in later stages had significant higher survival
rate in Kreisel tanks than that in upwelling
tanks and Weis tanks (p < 0.05). Some other
studies on ornamental shrimp larval rearing
such as Calado et al. (2008) also reported that
different tank type affected significantly on
the survival rates of Lysmata seticaudata, L.
debelius and Stenopus hispidus [4].
In disinfected water system, the survival rate
of larvae in zoea 4 stage was 25.3% in Kreisel
tank, 9 times higher than that in upwelling

tank (2.7%) and almost 20 times higher than
that in Weis tank (1.3%). This result could be
seen in Figure 2 where Kreisel treatment was
shown significant higher survival rate of larvae
compare to the other two treatments.
Survival rate of shrimp larvae was generally
highest in Kreisel treatment (p < 0.01, see
figures 2, 3 and 4), except for larvae reared in in
biofilter-water system (p > 0.05) whose survival
rates only higher in Kreisel in zoe 5, but not in
previous stages. Note that although the survival
rates of larvae in tank types showed a dependence
on the rearing water, the interaction between two
factors was not significant (p > 0.05). The result
of no interaction between tank types and rearing
water could be because of the low sample size
(only 3 replicates per treatment).
In general, results from all water systems
types showed that the higher survival rate of
larvae reared in Kreisel suggests that this tank
type could be a potential and proper option for
rearing L. amboinensis larvae. Also, there is
no need to treat rearing water in advance by
making green-water or using biofilters. The


Journal of Fisheries science and Technology
disinfected marine water with low operation
cost should be used for white-striped cleaner
shrimp larval rearing.

IV. CONCLUSION
There was no significant effect of rearing
water system (disinfected, green and biofilter

No. 4 - 2018
water) on larval survival rate of white-striped
cleaner shrimp Lysmata amboinensis.
Types of tank significantly affected on
the larval survival rate. Generally, highest
larval survival rate occurred in Kreisel tank
treatments.

Figure 2. Accumulated survival rate (left) and stage-based survival rate (right) of the larvae in
disinfected water treatments
Z1 – Z6 indicate stages of the larvae from Zoea 1 to Zoea 6

Figure 3. Accumulated survival rate (left) and stage-based survival rate (right) of the larvae in
green-water treatments
Z1 – Z6 indicate stages of the larvae from Zoea 1 to Zoea 6

Figure 4. Accumulated survival rate (left) and stage-based survival rate (right) of the larvae in
biofilter-water treatments
Z1 – Z6 indicate stages of the larvae from Zoea 1 to Zoea 6

NHA TRANG UNIVERSITY • 5


Journal of Fisheries science and Technology
Disinfected water (with low preparation
and operation costs) and Kreisel tank should

be used in rearing Lysmata amboinensis.

No. 4 - 2018
ACKNOWLEDGEMENTS
This research was carried out under a
project of Nha Trang University funded by
The Ministry of Education and Training of
Vietnam.

REFERENCES
1. Calado, R., Martin, C., Santos, O. and Narciso, L., 2001. Larval development of the Mediterranean cleaner
shrimp Lysmata seticaudata (Risso, 1816) (Caridea; Hippolytidae) fed on different diets: Costs and benefits
of mark-time molting. Larvi'01 Fish and Crustacean Larviculture Symposium. European Aquaculture Society
(Special Publication), 30: 96-99.
2. Calado, R., Figueiredo, J., Rosa, R., Nunes, M.L., Narciso, L., 2005. Effects of temperature, density, and
diet on development, survival, settlement synchronism, and fatty acid profile of the ornamental shrimp Lysmata
seticaudata. Aquaculture, 245: 221 – 237.
3. Calado, R., 2008. Marine ornamental shrimp. Biology Aquaculture and Conservation. Wiley-Blackwell.
4. Calado, R., Pimentel T., Vitorino, A., Dionisio, G., Dinis, M.T., 2008. Technical improvements of a rearing
system for the culture of decapod crustacean larvae with emphasis on marine ornamental species. Aquaculture,
258: 264 – 269.
5. Calado, R., Vitorino, A. Lopes da Silva, T., Dinis, M.T., 2009. Effect of different diets on larval production,
quality and fatty acid profile of the marine ornamental shrimp Lysmata amboinensis (de Man 1888) using wild
larvae as a standard. Aquaculture Nutrition, 15: 484–491.
6. Ritar, J., 2001. The experimental culture of phyllosoma larvae of southern rock lobster (Jasus edwardsii) in
a flow-through system. Aquacultural Engineering, 24: 149-156.
7. Tziouveli, K., 2006. Studies on aspects of Reproductive biology - Broodstock conditioning and Larval
rearing of the ornamental cleaner shrimp Lysmata amboinensis. AIMS@JCU NEWS, 2(4): 4-4.
8. Tziouveli, V. & Smith, G., 2009. Sexual maturity and environmental sex determination in the white-striped
cleaner shrimp Lysmata amboinensis. Invertebrate Reproduction and Development, 53(3): 155-163.

9. Zhang, D., Lin, J. and Creswell, R., 1997. Larviculture and effect of food on larval survival and development
in golden corral shrimp Stenopus scutellatus. Journal of Shellfish Research, 16(2): 367-369.
10. Zhang, D., Lin, J. and Creswell, R., 1998. Ingestion rate and feeding behavior of the peppermint shrimp
Lysmata wurdemanni on Artemia nauplii. Journal of World Aquaculture Society, 29: 97-103.

6 • NHA TRANG UNIVERSITY


Journal of Fisheries science and Technology

No. 4 - 2018

ROLE OF ANTIBIOTICS IN CHILLED STORAGE OF SPERM IN
GRASS CARP (Ctenopharyngodon idella)
Le Minh Hoang¹, Dinh Van Khuong¹
Received: 30.Nov.2018; Revised: 20.Dec.2018; Accepted: 25.Dec.2018

ABSTRACT:
The objective of the present study was to evaluate the effect of antibiotics on chilled storage sperm motility of grass carp (Ctenopharyngodon idella). The extenders were used in this study were HBSS (Hanks’ balanced salt solution), Modified HBSS, CCSE-2 (common carp sperm extender), Kurokuda-1 and Kurokuda-2.
The dilution ratios were 1:1, 1:3 and 1:5 (sperm:extender). Two antibiotics Cephalexin and Amoxcelin were
used in this study at the concentration of 50, 100 or 150 ppm. The experiments were conducted in a refrigerator at the temperature of 4ºC. The results showed that the sperm motility was the highest and activated to day
9 when Kurokuda-2 was used as the extender at the dilution ratio of 1:3. The sperm motility can be maintained
until day 13 by adding 25ppm Cephalexin combined with 25ppm Amoxceline to extender.
Keywork: Grass carp, Ctenopharyngodon idella, sperm, chilled storage, extender, antibiotic.

I. INTRODUCTION
Chilled storage of fish sperm is a useful biotechnique that facilitates hatchery operations.
It reduces the need of frequent collection of
sperm from males, enables transportation
of sperm to distant locations and prevents

problems related to asynchrony in gamete
production between males and females.
Sperm chilled storage is affected by extenders,
dilution ratio, temperature, and antibiotics
(Le et al. 2011; Le et al. 2014). However, the
presence of microorganisms in chilled samples
may decrease fertilization and lower cell and
viability (Segovia et al. 2000). To address
this issue,, antibiotics are commonly added
to chilled storage of sperm, but the effect of
antibiotics on the chilled sperm storage of the
grass carp, an important freshwater species in
aquaculture, has not been tested.
Grass carp has a rapid growth rate and
a low requirement for protein from food.
The production of grass carp has a low cost
compared to other freshwater fish. Grass carp
can be cultured in integration to land farm to
maximize the use of resources such as food,
wastes and water. Grass carp is a favorite fish
of many Asian countries. In response to the
need of aquaculture of the grass carp, artificial
seed production of this species has been
¹ Institute of Aquaculture, Nha Trang University

investigated (FAO, 2004-2017).
There has been many studies investigating
on the preservation of fish sperm such as of
common carp, (Cyprinus carpio) (Alavi et al.,
2007), sturgion Acipenseridae (Alavi et al.,

2006), trout and salmon (Billard et al., 1992).
However, there has no study investigating the
preservation of grass carp sperm. This was the
reason we conducted the study “The role of
antibiotics in chilled storage of sperm in grass
carp (Ctenopharyngodon idella).
II. Materials and methods
All experiments were carried out at the
laboratory of the Department of Fisheries
Biology, Institute of Aquaculture, Nha Trang
University.
1. Fish handing and sperm collection
Sperm was collected from grass carps
during the spawning season between March
and May 2018 without hormonal stimulation.
The males were anesthetized with Methlylene
glycol mono ester (Merk, Germany) at the
concentration of 200 ppm before sperm
collection. Sperm was collected by abdominal
massage and put it into a 1.5 ml dry Eppendorf
tubes. Handling was done with care to avoid
contamination with urine and feces in samples
designated for chilled storage as these can lead
to the activation of spermatozoa. The samples
were immediately placed on crushed ice until
NHA TRANG UNIVERSITY • 7


Journal of Fisheries science and Technology
use for experiment after collection.

2. Evaluation sperm motility
The sperm motility was immediately
determined after sperm collection. The
percentage of sperm exhibiting rapid, vigorous,
forward movement was estimated under the
microscope by diluting the sperm in distilled
water at a ratio of 1:100 (sperm: distilled water).
Only samples with motility equal to or greater
than 80% were used for experiments. Motility
was checked using a light microscope at 400×
magnification and was expressed as percentage
of motile spermatozoa. An activating medium
of distilled water was used to estimate motility.
Sperm was diluted in distilled water at the ratio

No. 4 - 2018
of 1:100 (1µl sperm to 99 µl distilled water).
Then, 1µl was put on a glass slide without a
cover glass and observed at 400× magnification
under a microscope.
3. Effect of extenders on motile sperm
To determine the optimal extender, sperm
was diluted at a ratio of 1:3 (sperm:extender)
with Hanks balanced solution (HBSS),
Common carp sperm extender (CCSE-2), Kura
Kuro’s 1 (Ku1), Kura Kuro’s 2 (Ku2) and
Modified (Table 1). Diluted sperm was stored
in a refrigerator at 4ºC, storage treatments
were replicated three times. The percentage of
motile sperm in each tube was tested at 2-4 day

intervals until sperm stopped moving.

Table 1. Composition of extenders for chilled storage of sperm of grass carp in 50ml distilled water

Ku2: Kuro Kura’s2

4. Effect of dilution ratio on sperm motility
To determine the optimal dilution, sperm was
diluted in HBSS, CCSE-2, Ku1, Ku2, Modified
at the ratio of 1:1, 1:3 và 1:5 (sperm:extender).
Mixtures were placed in 1.5ml Eppendorf tubes
and stored in a refrigerator at 4ºC. Treatments
were replicated three times. The spermatozoa
motility was tested at 2-4 day intervals until
spermatozoa stopped moving. Sperm was not
diluted with extender was used as the control
samples.
5. Effect of antibiotics on sperm motility
To determine optimal antibiotics for chilled

8 • NHA TRANG UNIVERSITY

sperm storage of grass carp, the sperm was
diluted in Kura Kuro’s 2 at a ratio of 1:3
combined with antibiotics Cephalexin with
Amoxcelin at the concentrations of 50, 100 or
150 ppm. All treatments had three replicates and
stored in a refrigerator at 4ºC. The percentage
of motile sperm in each tube was tested at 2-4
day intervals until sperm motility ceased. The

sperm samples without antibiotic were used as
the control treatment.
6. Data analysis
Data were expressed as mean ± standard
error (SE). One-way ANOVA were performed


Journal of Fisheries science and Technology
using SPSS version 22.0. Differences with a
probability value (P) of 0.05 (P<0.05) were
considered significant.

No. 4 - 2018
III. Results and discussion
1. Effect of extenders on sperm motility

Figure 1. Sperm motility (%) in various extender Ku1, Ku2, HBSS, CCSE-2 and Modified
Control: No extender. Different alphabets indicate statiscial significance at p<0.05.

Sperm was stored in Ku2 retained its movable
spermatozoa longer than other extenders (Figure
1). Specifically, Ku2 sperm remained motile
for 9 days (3.22%), while sperm was stored in
CCSE-2, HBSS and Modified remained motile
only for 5 days with motility as 4.33%, 8.67%
and 18.78%, respectively. Sperm stored in later
extenders was immotile at the day 7. Sperm not

stored in extender, on the other hand, was not
active at the day 5.

At the day 9 the duration of sperm motility
in extender Ku2 retained 51.67s. However,
sperm which was stored in CCSE-2, HBSS and
Modified had a duration of sperm motility of
18.89s, 54.56s and 58.22s, respectively at the
day 5 (Figure 2).

Figure 2. Duration of sperm motility (s) in various extenders Ku2, HBSS, CCSE-2 và Modified
Control: No extender. Different alphabets indicate statistical significance at p<0.05.

2. Effect of dilution ratios on sperm motility
The most motile sperm was observed when
sperm stored in Ku2 at the ratio of 1:3 (9.67%),
which remained motile for 9 days and 7 days
at the ratios of 1:1 and 1:5 (9.67% and 10%,
respectively) (Figure 3).

Sperm stored in Kura Kuro’s 2 (Ku2) at the
ratio of 1:3 remained the duration of sperm motility
better than that of 1:1 and 1:5. The duration of sperm
motility at the ratio 1:3 retained 51.67s at the day 9
and at the ratio of 1:1 and 1:5 reached at the day 5
was 59.33s and 51s, respectively (Figure 4).
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No. 4 - 2018

Figure 3. Sperm motility (%) at various dilution ratios in Kura Kuro’s 2 extender
Control: No dilution. Different alphabets indicate statistical significance at p<0.05.

Figure 4. Duration of sperm motility (s) at various dilution ratios in Kuro’s 2 (Ku2) extender.
Control: No dilution. Different alphabets indicate statistical significance at p<0.05.

3. Effect of antibiotics on sperm motility
Sperm stored in Kura Kuro’s 2 at the ratio of
1:3 and with an addition of 25ppm Cephalexin
and 25ppm Amoxcelin had a higher motility
than those stored in other extenders and the
controls (no extender and without antibiotic). It
remained motile for 13 days (6.89%), whereas
the treatment without antibiotic sperm was
immobile after 9 days (Figure 5).
The duration of sperm motility in the
treatment of combination between 25ppm
Cephalexin and 25 ppm Amoxcelin reached
3.59s at the day 13. However, it remained 3.07
s and 3.19s at the day 11 in the treatment of
only Cephalexin or Amoxcelin respectively
(Figure 6).
10 • NHA TRANG UNIVERSITY

The addition of antibiotics either to the
undiluted sperm or to the storage diluent
usually improves storage duration, and this
addition can be one of the most important
parameters for chilled storage of sperm (Billard
et al., 2004; Bobe et al., 2009). According

to previous studies, a combination of 50 IU/
penicilin and 50 IU/streptomycin for carp
semen without dilution at 4ºC showed that
motile and fertilization capacity of sperm can
be remained more than 18 days (Saad et al.
1988). With same concentration, similar results
were obtained for sperm storage of atlantic cod
Gadus morha and haddock Melannogrammus
aeglefinus (DeGraaf and Berlinsky, 2004).
Paddlefish Polyodon spathula sperm storage


Journal of Fisheries science and Technology

No. 4 - 2018

Figure 5. Sperm motility (%) at the different antibiotics such as Cephalexin, Amoxcelin and
thei combination. Control 1: No extender, Control 2: Without antibiotic Different alphabets
indicate statistical significance at p<0.05.

Figure 6. Duration of sperm motility treated with different antibiotics as Cephalexin, Amoxcelin
and their combination. Control 1: No extender, Control 2: Without antibiotic. Different alphabets
indicate statistical significance at p<0.05.

was also improved by adding a combination
of antibiotic penicilllin/streptomycin (Brown
and Mims, 1995). In African catfish (Clarias
gariepinus), however, addition of 25 to 50 IU/
ml penicillin + 25 to 50 µg/ml streptomycin
did not improve sperm quality during short

term storage and doses of 100 IU/ml + 100 µg/
ml were toxic for the cells whereas addition
of gentamycine sulfate at 1 mg/ml did not
improve the motility of these stored sperms
(Christensen and Tiersch, 1996).
IV. CONCLUSION AND RECOMMENDATION
1. Conclusion
The highest sperm motility and duration

of sperm motility were obtained after chilled
storage at 4ºC in a dilution ratio of 1:3
(sperm:Ku2) containing 25 ppm Cephalexin +
25 Amoxcelin. It reamained the lifetime until
the day 13.
2. Recommendation
In this study, addition of two commonly
used antibiotics Cepalexin and Amoxcelin
prolonged the survival of sperm for three days
compared to untreated sperms. It remains to
be tested whether using other antibiotics may
improve the chilled sperm storage of the grass
carp for a longer duration.

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No. 4 - 2018

REFERENCES
1. FAO 2004-2017. Cultured Aquatic Species Information Programme. Ctenopharyngodon idellus. Cultured
Aquatic Species Information Programme. Text by Weimin, M. In: FAO Fisheries and Aquaculture Department
[online]. Rome. Updated 1 January 2004. [Cited 30 May 2017]. .
2. Alavi S.M.H, Cosson J.(2006). Sperm motility in fishes. (II) Effects of ions and osmolality: A reveiw. Cell
biol Int.1(30).
3. Alavi S.M.H, Rodina M., Policar T., Kozak P., Psenicka M., Linhart O.(2007). Semen of Perca fluviatilis:
Sperm volume and density, seminal plasma indices and effects of dilution ratio, ions and osmolality on sperm
motility. Theriogenology.2(68).
4. Billard R., Cosson J.(1992). Some problems related to the assessment of sperm motility in freshwater fish.
Journal Experimental Zoology.2(261).
5. Billard R., Cosson J., Noveiri S.B., and Pourkazemi M.(2004). Cryopreservation and short-term storage of
sturgeon sperm, a review. Aquaculture.236:p1-9.
6. Bobe J., and Labbe C.(2009). Chilled storage of sperm and eggs, in Methods in Reproductive Aquaculture:
Marine and Freshwater Species, Cabrita, E., Robles, V. and Herrasez, P., Editors. CRC Press, Taylor Francis
Group.p 219-235.
7. Boitano S., Omoto C.K.(1991). Membrane hyperpolarization activates trout sperm without an increase in
intracellular pH. Journal Cell Science.98(3):p 343-349.
8. Rana K. J., Muiruri R. M., McAndrew B. J., Gilmour A. N. N.(1990). The influence of diluents, equilibration
time and prefreezing storage time on the viability of cryopreserved Oreochromis niloticus (L.) spermatozoa.
Aquaculture Research.21(1):25-30.

12 • NHA TRANG UNIVERSITY


Journal of Fisheries science and Technology

No. 4 - 2018

EFFECTS OF FEEDING RATE ON DENSITY, BIOMASS AND

PROTEIN COMPOSITIONS OF OLIGOCHAETE
(Limnodrilus hoffmeisteri Claparede, 1862)

Truong Thi Bich Hong¹, Nguyen Dinh Mao¹, Le Minh Hoang¹
Received: 30.Oct.2018; Revised: 5.Dec.2018; Accepted: 26.Dec.2018

ABSTRACT
L. hoffmeisteri is an aquatic invertebrate, belonging to the class Oligochaeta and family Tubificidae, used
as an important live food for feeding larval stages of freshwater species. This study was carried out to provide
scientific knowledge for L.hoffmeisteri culture as well as optimal feeding ration affected on density, biomass
and protein compositions. L. hoffmeisteri was cultured under flow-through in concreted trench system (160 x
25 x 20 cm) with mud bottoms for 5 weeks. They were fed a mixture of 33.3% soybean meal, 33.3% corn meal
and 33.3% rice bran at feeding rations of 5%, 10%, and 15% of body mass.
The results showed that different feeding rations significantly effect on the density, biomass and protein
compositions of L. hoffmeisteri. Specifically feeding ration of 15% resulted in the highest density (64 ± 5
individual/cm²), biomass (133.90 ± 9.24 mg/cm²), protein (% of dry biomass) (52.34 ± 1.35 %). Conversely,
the lowest density (5± 1 individual/cm²) and biomass (10.24 ± 1.18 mg/cm²) were recorded in the control
treatment (not fed). The lowest protein (% of dry biomass) (45.76 ± 1.18 %) was recorded in the treatment with
feeding ration of 5 %. In conclusion, feeding at 15% of body mass/day displayed as a suitable ration for L.
hoffmeisteri.
Keyworms: L. hoffmeisteri worms, feeding rations, culture

I. INTRODUCTION
L.hoffmeisteri is one of many species
of aquatic worms that is widely distributed
throughout the world [5], tolerating a wide
variety of environmental conditions. In
Vietnam, these worms can be found in fish
ponds, river and wastewater ditches [8].
L. hoffmeisteri is a small species with

body size about 20-35 mm long and plays an
important role to freshwater aquaculture [9].
Furthermore, this species in high in nutritional
values (5575 cal g- on a dry weight basis [2])
and highly digestible for aquatic animals.
L. hoffmeisteri is mainly used as food in
aquarium fishes and have been reported as an
important live food in larval rearing of many
commercially important fishes, particularly for
catfish and another fish such as gray eel-catfish
and crab [10,11].
In Vietnam, current total supply of these
worms mainly comes from wild caught source
which is unreliable and insufficient to meet
the demand. Information related to culture
¹ Institute of Aquaculture, Nha Trang University

of L. hoffmeisteri in Vietnam is not known.
Therefore, the present study was undertaken
to determine the effects of feeding ratio on
density, biomass and protein compositions of
L. hoffmeisteri worms.
II. MATERIALS AND METHODS
1. Experimental worms and system
L. hoffmeisteri worms were collected
from waste water ditch of Vinh Ngoc district,
Khanh Hoa province in Vietnam. The collected
worms were rinsed and cultured at the Nha
Trang University Laboratory. The worms
were cultured in a flow-through system over

2 month period to achieve quantity (450g) for
experiments.
All experiments were conducted from
early November to mid-December 2015 for
5 weeks. The worms were cultured in indoor
concrete culverts (160 cm x 25 cm x 10 cm)
system to protect from rain and sunlight. Prior
to the experiment, the culverts were with clean
freshwater. Each culvert was connected to
flow-through-system. Substrate was made by
mud layer of 1 cm thickness.
NHA TRANG UNIVERSITY • 13


Journal of Fisheries science and Technology
2. Experimental design
Four treatments of feeding rations were used
in this experiment including the 0% (negative
control, not fed), 5.0%, 10.0% and 15.0% of
body mass. With 6 replicates each. All worms
were fed daily with the same mixture feed at
08:00 am. Ingredients of mixture feed were
33.3% soybean meal, 33.3% corn meal and
33.3% rice bran. A sample of feed was sent
to the Biotechnology institute - Nha Trang
University of for analysis (Table1).

3. Inoculation of L. hoffmeisteri Oligochaete
Water flow was adjusted one day before
inoculation of worms to the culverts. The

collected L. hoffmeisteri worms were
inoculated at the density of 5 individual/cm²
and spread over the media homogeneously as
much as possible in each of the culvert.
4. Periodic supply of feed
The supply of feed was done following a
date of worms’ inoculation. The amount of
food was changed once every 7 days. When
feeding, water flow was stopped. Amount of
food was spread throughout the culvert. Then,
the water flow was reopened after 30 minutes.
5. Methods of data collection and Statistical
Analysis
5.1. Water quality
Continuous water flow was maintained to
keep the dissolved oxygen in suitable level (>3
mg L-1) for development L. hoffmeisteri. Water
temperature (ºC), dissolved oxygen (mg L-1)
and pH of the culture culverts were measured
twice a day at 8:00 and 14:00. Using a portable
dissolved oxygen meter (Model YSI Pro20,
USA).

14 • NHA TRANG UNIVERSITY

No. 4 - 2018
5.2. Sampling
Worm samples were collected after 7, 14,
21, 28 and 35 days of inoculation. Each sample
involved water and media (4x2 cm²) from

five randomly selected sites of each culvert.
They were rinsed off with clean water . After
that, unwanted particles was removed by using
forceps and dropper. Finally, L. hoffmeisteri
oligochaetes were dried with blotting tissue.
They were weighted by Mettler Electric balance
(KD-TBED 320) to the nearest 0.0001g. Number
of individuals was recorded for each sample to
calculate average biomass and density.
The biomass quality of L. hoffmeisteri worms
was analyzed as biochemical content (% of dry
weight) and fatty acid. Samples were dried at
80ºC in the incubator and kept in vacuum bag
until analysis. Total protein of L. hoffmeisteri
was determined by “Kjeldahl method”;
moisture content and ash in the sample L.
hoffmeister worms were determined by “AOAC
950.46 – 1995” and “AOAC 923.03 – 1995”,
respectively. Fatty acids were determined by
gas chromatography (GC) and processed by
software GC A.08.03 ChemStation (Agilent
Technologies © Inc., Santa Clara, USA)
5.3. Statistical analysis
Data were presented as mean values
± standard deviation. One-way ANOVA
was applied to analyze the differences of
density, biomass and protein compositions
of the worms. Differences were regarded as
statistically significant when significance level
less than 0.05.

III. RESULTS AND DISCUSSION
1. Results
1.1. Water quality
Water temperature in the experiments
were ranged from 29 to 31ºC in which the
average temperature was 30.2 ± 0.8ºC. pH
in the treatments was ranged from 6.8 – 7.8.
Fluctuation of temperature and pH were
negligible and did not affect growth and
development of the L. hoffmeisteri populations.
The dissolved oxygen was ranged from
3.5–5.0 mg L-1. Fluctuation of dissolved
oxygen was negligible and suitable for


Journal of Fisheries science and Technology
growth, development and reproduction of the
L. hoffmeisteri populations. This is because
normal development of the embryo of
species of tubificids requires a minimum
oxygen content of 2.5-7.0 mg L-1 [7]. The
culture system was provided the high dissolved
oxygen content (≈ 3mg L-1) not only maintained
the highest worm density but also ensured the
highest fecundity [6].
1.2. Effect of feeding rations on biomass of L.
hoffmeisteri population
During the experiment, the biomass of L.
hoffmeisteri population in the negative control
increased very slowly. Conversely, the biomass

of L. hoffmeisteri population when this species
fed at the 5%, 10% and 15% feeding rate of this
food had increased rapidly. In the first 2 weeks,

No. 4 - 2018
the biomass of L. hoffmeisteri population were
not affected by feeding rate and showed quite
similar value between treatments (those of 10%,
15% were respectively 53.87 ± 11.47, 54.39 ±
6.86 mg/cm²). However, on the day of sampling
(21, 28, 35) the feeding rate had affected the
biomass of L. hoffmeisteri population. At the
35th day, the treatment that L. hoffmeisteri
population were fed at ration of 15% had highest
biomass (133.90 ± 9.24 mg/cm²), followed by
the ration of 10% and 5% (111.41 ± 7.52, 88.37
± 10.42 mg/cm², respectively). Conversely,
lowest biomass (10.23 ± 1.18 mg/cm²) of this
species were recorded in control treatment (not
fed) and significantly lower than those in other
treatments (P<0.05) (Figure 1).

Figure 1: Biomass of L. hoffmeisteri populations at different feeding rations

1.3. Effect of feeding rations on density of L.
hoffmeisteri
Density of L. hoffmeisteri population in
the control treatment was increased slightly
in the 3rd week after that it gradually reduced
in the 5th week. Conversely, L. hoffmeisteri

population density of the other treatments had
increased continuously throughout the entire
experimental period. From 3rd weekend to
5th weekend, the feeding rations had affected
the population density of L. hoffmeisteri
population. At 5th week, The treatment that
L. hoffmeisteri were fed at feeding ration of
15% had highest density (64 ± 5 individual/
cm²) and significantly higher than those in

other treatments (P<0.05). Conversely, lowest
density (10 ± 3 individual/cm²) was recorded
in control treatment (not fed) (Figure 2).
1.4. Effect of feeding rations on biochemical
ingredients of L.hoffmeisteri
The biochemical ingredients of dry L.
hoffmeisteri were showed in Table 2
Protein ingredient was highest (52.34
± 1.35 %) in treatment that L. hoffmeisteri
population were fed at ration 15% of body
mass/day and significantly higher than those
in other treatments (P<0.05). Conversely, the
lowest protein ingredient (45.76 ± 1.18 %)
was recorded when feeding L. hoffmeisteri
population with ration 5% of body mass/day.

NHA TRANG UNIVERSITY • 15


Journal of Fisheries science and Technology

No. 4 - 2018

Figure 2: Density of L. hoffmeisteri populations at different feeding rations
Table 2: The biochemical ingredients of dry L. hoffmeisteri

Lipid ingredient was also highest (17.08 ± 0.83
%) in treatment that L. hoffmeisteri population
were fed at ration 15% of body mass/day.
However, there was no significant difference
between the treatment 15% and the two
treatment 10% and 0% (51.97 ± 1.94, 48.93
± 2.79 % respectively) (P>0.05). The lowest
lipid ingredient (13.00 ± 2.00) was recorded
when feeding L. hoffmeisteri population with
ration 10 % of body mass/day and significantly
lower than those in other treatments (P<0.05)
(Table 2)
The fatty acids ingredients (mg/g) of dry
L. hoffmeisteri population were significant
different between the treatments. The HUFA
was highest (4.58 ± 0.18 mg/g) in the treatment
that L. hoffmeisteri population were fed at the
ration 15% of body mass/day, followed by the
ration 5% of body mass/day (4.17 ± 0.17 mg/g)
and significantly higher than those in other
treatments (P<0.05). The lowest HUFA (3.37 ±
0.18 mg/g) was recorded when this species fed
at the 10% ration (Table 3).
Saturated fatty acids (SFA) and unsaturated

16 • NHA TRANG UNIVERSITY

fatty acids with a double bond (MUFA)
were highest (2.51 ± 0.11, 3.26 ± 0.16 mg/g,
respectively) in the treatment 15% of body
mass/day, and significantly higher than those in
other treatments (P<0.05). Docosa hexaenoic
acid (DHA) was quite similar between
treatments. DHA was also highest (2.21 ± 0.20
mg/g) in the treatment with the ration of 15%
but no significantly higher than those in other
treatments (P>0.05) (Table 3).
The ingredient percentage of SFA, MUFA
and HUFA were highest in treatment that L.
hoffmeisteri population was fed at the ration
15% of body mass/day. Conversely, lowest
SFA, MUFA and HUFA (11.51 ± 0.97, 16.06 ±
0.85, 24.43 ± 1.02, respectively) were recorded
when feeding this species with ration 5 % of
body mass/day) and significantly lower than
those in other treatment (P<0.05). While, the
ingredient percentage of PUFA, EPA and DHA
were quite similar and no significant differences
were found between treatments (Table 4).
The Table 4 showed that, the percentage
of total fatty acids received little attention in


Journal of Fisheries science and Technology

No. 4 - 2018

Table 3: The fatty acids (mg/g) of dry L. hoffmeisteri

Table 4: The percentage of total fatty acids in dry L. hoffmeisteri

previous studies of L. hoffmeisteri. Average
free amino acid concentration of L.hoffmeisteri
is 7.78 (nmol/mg) [3].
2. Discussion
Limitted information on the culture of L.
hoffmeisteri is available in the literature. Our
study, presents a culture system that is different
from that of other L. hoffmeisteri studies in the
literature, most having used glass beakers with
substrates that consisted of a mixture of mud
and organic matter in laboratory condition [12].
The study was to test whether large individuals
of L. hoffmeisteri produce more eggs and/or
cocoons than small individuals and to assess
the influence of two granulometric fractions
of sand on the reproduction and growth of L.
hoffmeisteri under laboratory conditions [4].
Under laboratory conditions, the space was
very small. Each experimental unit consisted of
600 ml glass beakers [12]. The experiment was
conducted in 250-mL beakers containing 100
mL of sand, 100 mL of water [4]. Therefore,
density and wet weight of the L. hoffmeisteri
population increased slowly. These results

indicate that the production system that we
describe is more efficient and can produce a
larger mass of L. hoffmeisteri more quickly
than previously described systems in laboratory
conditions.

This study showed that feeding rate
significantly effected on L. hoffmeisteri density
and biomass. However, feeding rate had little
effect L. hoffmeisteri protein compositions.
In general, density and biomass were highest
at the 15% feeding ration and statistically
with other feeding ration (0, 5, 10 %). Protein
compositions was also highest at the 15%
feeding ration but no statistically with 0, 10 %
feeding ration. Little information on the effect
of ration on L. hoffmeisteri density, biomass and
protein compositions is available. In previous,
L. hoffmeisteri culture studies in laboratory
condition, olygochaete were provided low
organic matter or high organic matter [12].
Other oligochaete culture studies, oligochaete
were provided an excess of food [6,1].
IV. CONCLUSION
5th weekend, Biomass and density of L.
hoffmeisteri population were highest (133.90
± 9.24 mg/cm², 64 ± 5 individual/cm²,
respectively) in the treatment when were fed
at ration 15 % of body mass/day, followed at
the 10 % ration (111.41 ± 7.52 mg/cm², 54 ±

4 individuals/cm²). The lowest density (5± 1
individual/cm²) and biomass (10.24 ± 1.18 mg/
cm²) were recorded in the control treatment
(not fed).
The protein compositions of L. hoffmeisteri
NHA TRANG UNIVERSITY • 17


Journal of Fisheries science and Technology
population were significanty different between the
treatments. Protein ingredient was highest (52.34 ±
1.35 %) in treatment when were fed at ration 15%
of body mass/day, followed at the 10% and 0%
ration. The Protein ingredient was lowest (45.76 ±
1.18 %) at the 5% of feeding ration.

No. 4 - 2018
V. ACKNOWLEDGEMENTS
We would like to thank the Ministry of
Education & Training who supported finance
for this study.

REFERENCES
1. Ahamed, M.T và M.F.A.Mollah. 1992. Effects of various levels of wheat bran and mustard oil cake in the
culture media on Tubificid production. Aquaculture 107: 107-113
2. Giere, O. and Fannkuche, O.P, 1982. Biology and ecology of marine oligochaete, a review. In M.Barnes (ed),
Oceanography and Marine Biology, Abendeen University Press.
3. Graney, R.L., Keilty, T.J, Giesy, P. 1986. Free amino acid pools of five species of freshwater oligochaetes
Can.J.Fish. Aquaculture, 43: 600-607.
4. Haroldo L and Alves R G, 2011. Influence of body weight and substrate granulometry on the reproduction

of L. hoffmeisteri (Oligochaeta: Naididae: Tubificinae).
5. Kathman, R. D. and Brinkhurst R. O. 1998. Guide to the freshwater oligochaetes of North America. Aquatic
Resources Center, College Grove Tennessee.
6. Marian. M. P and T.J. Pandian, 1984. Culture and harvesting techniques for tubifex tubifex, Aquaculture,
42, 303-315.
7. Poddubnaya, T.L., 1980. Life cycles of mass species of Tubificidae. In: R.O. Brinkhurst and D.G. Cook
(Editors), Aquatic Oligochaete Biology. Plenum, New York, NY, pp. 175-184
8. Thai Tran Bai, 2005, Invertebrate, Education publishers (in Vietnamese).
9. The Marine Life Information Network, 2003. Information on the biology of species and the ecology of
habitats found around the coasts and seas of the British Isles.
10. Tran Ngoc Hai, Le Quoc Viet, Ly Van Khanh and Cao My An, 2011, Effects of different diets on the growth
and survival rates of grey-ell catfish (Plotosus canius), Can Tho University Journal of scientific, 18b, 254-261.
11. Tran Duy Khoa, Ngo Quoc Huy and Tran Ngọc Hai, 2011. Study on broodstock culture, spawning and
rearing of rice crab (Somanniathelphusa germaini), Can Tho University Journal of scientific, 17b, 70-76.
12. Warucha Kanchana-Aksorn1and Saran Petpiroon, 2008. Study on Limnodrilus hoffmeisteri Population
Response to Different Organic Enrichment in Laboratory Condition.

18 • NHA TRANG UNIVERSITY


Journal of Fisheries science and Technology

No. 4 - 2018

FISH OIL EXTRACTION FROM YELLOWFIN TUNA HEADS
BY ENZYMATIC HYDROLYSIS METHOD
Nguyen Thi My Huong¹, Bui Truong Bich Ngan¹
Received: 9.Nov.2018; Revised: 15.Dec.2018; Accepted: 25.Dec.2018

ABSTRACT

A study on the fish oil extraction from yellowfin tuna heads by hydrolysis method using Protamex enzyme
was carried out. The parameters of hydrolysis process, fish oil yield and chemical quality of tuna head oil were
determined. The study results showed that a considerable amount of oil can be extracted from yellowfin tuna
heads. The suitable parameters of enzymatic hydrolysis process for recovering fish oil from yellowfin tuna
heads were the water/material ratio of 0.5/1, Protamex concentration of 0.5%, hydrolysis temperature of 55°C
and hydrolysis time of 1h. High quality of the yellowfin tuna head oil was obtained from enzymatic hydrolysis.
This study suggested that the yellowfin tuna heads generated from tuna processing industry could be utilized
as a good source for oil recovery. Tuna head oil could be used as a valuable ingredient both in food and aquaculture feed.
Key words: Enzymatic hydrolysis, fish oil extraction, oil recovery, yellowfin tuna head.

I. INTRODUCTION
Tuna is a valuable source of food and plays
an important role in the economy of many
countries. Tuna generally is processed as raw
fish flesh and marketed as loins. Viet Nam tuna
products are exported to the U.S., EU, Japan,
ASEAN and other markets (VASEP, 2016). A
large amount of by-products consisting of head,
bone, viscera, skin and dark muscle is generated
from the tuna processing industry (Herpandi et
al., 2011). Tuna by-products are perishable due
to their high protein and fat contents. Increasing
environmental pollution has emphasized the
need for better utilization of tuna by-products.
Therefore, using the tuna head to recover fish
oil is very important to reduce environmental
problems. The tuna head oil is an excellent
source of omega-3 fatty acids, which are mainly
composed of eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) (Nguyen Thi

My Huong, 2013). These fatty acids play an
essential role in human health and nutrition.
Lipid extraction from many sources and by
different methods have been extensively studied
(Salam et al., 2005; Gbogouri et al., 2006;
Batista et al., 2009; Khoddami et al., 2012;

Ramakrishnan et al., 2013). Fish oil is usually
extracted from whole fish or fish by-products
by chemical process (Mahmoud et al., 2008;
Norziah et al., 2009), by cooking and pressing
(Chantachum et al., 2000), or by enzymatic
process (Batista et al., 2009; Khoddami et
al., 2012; Ramakrishnan et al., 2013). Among
the mentioned methods, the enzymatic
hydrolysis method used for oil extraction has
many advantages, such as the mild hydrolysis
conditions, low energy requirement, no use
of solvent. The low hydrolysis temperatures
minimize the oxidation of polyunsaturated
fatty acids. Enzymatic tissue disruption may
be a valid alternative technique for releasing
natural lipids from fish. During the enzymatic
hydrolysis, the combination between lipid and
protein was broken down, which lead to fish oil
release much easier from fish by-product (Qiyuan et al., 2016).
The purpose of this study was to determine
the suitable hydrolysis conditions for oil
recovery from yellowfin tuna heads using
Protamex and to value the chemical quality

of tuna head oil with various parameters,
including free fatty acid, acid value, peroxide
value, iodine value and saponification value.

¹ Faculty of Food Technology - Nha Trang University

NHA TRANG UNIVERSITY • 19


Journal of Fisheries science and Technology
II. MATERIALS AND METHODS
1. Materials
Yellowfin tuna (Thunnus albacares) heads
were provided by Thinh Hung, a seafood
processing company in Nha Trang, Vietnam.
Yellowfin tuna heads were stored with crushed
ice at 0 - 4°C in a polystyrene box and
transported immediately to the laboratory of
Nha Trang university. After their arrival, they
were washed and ground. The minced tuna
heads were packed in plastic bags, frozen and
stored at -20°C until their use (approximately
a month).
2. Enzyme
The enzyme used for the hydrolysis of
yellowfin tuna heads was Protamex, which
was produced by Novozymes (Denmark).
Protamex is a Bacillus protease complex. The
declared activity of Protamex is 1.5 AU/g.
Optimal working conditions of Protamex are at

pH 5.5-7.5 and 35-60°C.
3. Determination of suitable hydrolysis conditions
for oil extraction from yellowfin tuna head
3.1. Determination of suitable water/material
ratio
In order to determine the suitable water/
material ratio for oil recovery, the minced
tuna heads were hydrolyzed by using 0.5%
Protamex in 2h at temperature of 50°C, pH
6.5 with water/material ratios of 0/1, 0.25/1,
0.5/1, 0.75/1 and 1/1. After hydrolysis, the
enzyme was inactivated by heat treatment at
90°C for 10 minutes in a water bath. Then,
the mixture was filtered through a mesh to
remove the solid fraction (bones). The filtrate
was centrifuged at 10000 rpm at 4°C for 30
minutes. After centrifugation, the following
four fractions were formed: the oil fraction on
the top, the emulsion and the liquid protein
hydrolysate in the middle and the sludge on
the bottom. The oil fraction was recovered,
then weighed to calculate the percentage of
recovered oil. The acid value and peroxide

20 • NHA TRANG UNIVERSITY

No. 4 - 2018
value of oil were determined. From obtained
results, the suitable water/material ratio was
selected.

3.2. Determination of suitable enzyme
concentration
With the suitable water/material ratio
identified and hydrolysis conditions as above,
the minced tuna heads were hydrolyzed with
0.1%, 0.3%, 0.5%, 0.7% and 0.9% Protamex.
After hydrolysis, the same steps as above were
carried out. The suitable enzyme concentration
was selected.
3.3. Determination of suitable hydrolysis
temperature
With the suitable water/material ratio and
enzyme concentration identified, the minced
tuna heads were hydrolyzed in 2h at pH 6.5
and temperature of 45°C, 50°C, 55°C and
60°C. After hydrolysis, the same steps as
above were carried out. The suitable hydrolysis
temperature was selected.
3.4. Determination of suitable hydrolysis time
With the suitable water/material ratio
and enzyme concentration identified, the
minced tuna heads were hydrolyzed at pH
6.5 and suitable hydrolysis temperature
identified in 0.5h; 1h; 2h; 3h and 4h. After
hydrolysis, the same steps as above were
carried out. The suitable hydrolysis time
was selected.
4. Chemical analyses
Lipid content was determined according to
the method of Folch et al. (1957). The free fatty

acid content, acid value, peroxide value, iodine
value, saponification value were determined
according to American Oil Chemists’ Society
AOCS (1997).
5. Oil recovery
The oil obtained was weighed using a
digital balance (Precisa-Model XT 2200c).
The percentage of recovered oil from
yellowfin tuna head was calculated as
follows:


Journal of Fisheries science and Technology
6. Statistical analysis
The experiments were carried out in
triplicates. The obtained data were subjected
to one way analysis of variance (ANOVA),
followed by the Duncan’s multiple range
test to determine the significant difference
between samples at P<0.05 level using the
SPSS 15.0 programme.

No. 4 - 2018
III. RESULTS AND DISCUSSION
1. Determination of suitable hydrolysis conditions
for oil extraction from yellowfin tuna head
1.1. Determination of suitable water/material
ratio
The influence of water/material ratio on the
oil recovery, acid value and peroxide value of

tuna head oil is shown in Figure 1.

Figure 1. The influence of water/material ratio on the oil recovery (a), acid value (b) and
peroxide value (c) of yellowfin tuna head oil.

Enzymatic hydrolysis resulted in formation
of four phases: an oily phase, emulsion phase,
aqueous phase and sludge. The results indicated
that the water/material ratio had a significant
effect on the oil recovery (Figure 1a). The oil
recovery reached the highest value (54.4%)
with water/material ratio of 0.5/1. Qi-yuan et al
(2016) showed that the maximum oil recovery
from mackerel viscera was 78.66%. The oil
recovered from salmon heads using Bromelain
and Protex were 65% and 88%, respectively
(Mbatia et al, 2010).
The oil recovery reduced with the increase
in water/material ratio from 0.5/1 to 1/1. Mbatia
et al. (2010) also reported that an increase
in water/material ratio during the hydrolysis
resulted in a decrease in oil yield. Decrease in
oil yield with increasing water/material ratio
during the hydrolysis could have been due to
emulsion formation (Mbatia et al., 2010).
The acid value (Figure 1b) and peroxide
value (Figure 1c) of the tuna head oil tended
to increase with the raise of water/material
ratio. However, the increases in acid value and
peroxide value of oil were not significant. The

oil extracted from yellowfin tuna heads had
the highest acid value (3.20 mg KOH/g) and
the highest peroxide value (2.28 meq O2/kg)
when water/material ratio was 1/1. The acid
value indicates the formation of free fatty acids
because of oil hydrolysis. Ahmed et al (2017)
showed that the acid values of the oil extracted
from bigeye tuna by-products ranged from 4 to
7.4 mg KOH/g. The peroxide value of the oil
extracted from sardine tissue was 2.78 meq O2/
kg (Pravinkumar et al., 2015).
The study indicated that the enzymatic
hydrolysis using Protamex with water/material
ratio of 0.5/1 has brought the highest percentage
of oil recovery. Therefore, the water/material
ratio of 0.5/1 was suitable for oil recovery from
yellowfin tuna head.
1.2. Determination of suitable enzyme
concentration
During the enzymatic extraction of oil from
the yellowfin tuna heads with Protamex, enzyme
concentration plays an important role in the
recovery of oil. Figure 2 indicate the influence
of enzyme/material ratio on the release of oil,
acid value and peroxide value of tuna head oil.
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Journal of Fisheries science and Technology

No. 4 - 2018

Figure 2. The influence of enzyme concentration on the oil recovery (a), acid value (b) and
peroxide value (c) of yellowfin tuna head oil

The results demonstrated that increasing
enzyme concentration increased the oil
recovery from tuna heads (Figure 2a). The
oil recovery increased strongly (P<0.05)
from 39.9% to 54.1% with the increase of
the enzyme concentration from 0.1 to 0.5%.
However, there were no significant differences
in oil recovery among the samples with the
enzyme concentrations of 0.5%, 0.7% and
0.9%. Ramakrishnan et al (2013) also indicated
that increasing the enzyme concentration
increased the oil recovery from mackerel
head. For the 0.5% enzyme concentration
and 1 hour hydrolysis time, the oil recovery
from mackerel head was 55.82%. When the
enzyme concentration was increased from
0.5% to 1%, the oil recovery increased from
55.82 to 56.96%. Mbatia et al. (2010) stated
that maximum oil recovery from salmon heads
was achieved when 0.5% Bromelain was used.
A higher enzyme concentration did not result
in further increase in oil recovery.
There were not significant differences in
acid values (Figure 2b) and peroxide values

(Figure 2c) of the fish oil obtained among
the samples with the different enzyme
concentrations. This mean that the enzyme
concentration did not significantly affect on
the acid value and peroxide value of the oil
extraced from yellowfin tuna heads. The acid
values of the oil extracted from hilsa fish (Hilsa
ilisha) by-products ranged from 4.16 to 12 mg
22 • NHA TRANG UNIVERSITY

KOH/g (Salam et al., 2005). According to
Khoddami et al (2012), the peroxide value of
the oil extracted from tuna (Euthynnus affinis)
head was 7.31 meq O2/kg.
According to the results in this study, the
highest oil recovery was obtained with enzyme
concentration of 0.5%. A higher enzyme
concentration did not improve the oil recovery
as well as acid value and peroxide value.
Therefore, the enzyme concentration of 0.5%
was suitable for the oil extraction in order to
reduce the cost associated with the enzyme.
1.3. Determination of suitable hydrolysis
temperature
The influence of hydrolysis temperature on
the oil recovery, acid value and peroxide value
of tuna head oil is demonstrated in Figure 3.
The results indicated that the hydrolysis
temperature had a significant effect (P<0.05)
on the oil recovery (Figure 3a). Increasing the

hydrolysis temperature led to increase the oil
recovery from tuna heads. The oil recovery
increased sharply from 48.6% to 59% with the
hydrolysis temperatures in a range of 45-55°C.
The highest oil recovery (59%) was achieved
at 55°C. However, with the hydrolysis
temperature of 60°C, the oil recovery from
tuna head decreased to 50.9%. This may be due
to decreasing the activity of enzyme Protamex
at 60°C. Deepika et al (2014) showed that the
highest oil recoveries from the salmon gut,
heads and frame were 80.01%, 59.92% and


Journal of Fisheries science and Technology

No. 4 - 2018

Figure 3. The influence of hydrolysis temperature on the oil recovery (a), acid value (b) and
peroxide value (c) of yellowfin tuna head oil

78.58%, respectively.
The acid value (Figure 3b) and peroxide
value (Figure 3c) of the tuna head oil slightly
increased from 3.06 to 3.31 mg KOH/g and
from 1.69 to 2.35 meq O2/kg, respectively
when the temperature increased from 45°C to
60°C. The higher extraction temperatures led
to fish oil with higher acid value. Increasing
extraction temperature can cause faster lipid

degradation to form free fatty acids. Deepika et
al. (2014) reported that the oil extracted from
salmon heads and frame at 30°C and 40°C
had low acid values (0.33-2.10 mg KOH/g).
However, the acid values of the oil extracted
at 30°C and 40°C from salmon gut were 12.91

and 17.49 mg KOH/g, respectively. Deepika
et al. (2014) also showed that the peroxide
value of all oil samples extracted at different
temperatures and reaction time were between
0.28-2.65 meq/kg.
The results showed that the suitable
temperature for oil extraction from yellowfin
tuna heads was 55°C.
1.4. Determination of suitable hydrolysis time
During the enzymatic extraction of oil
with protease, the hydrolysis time plays an
important role in the oil recovery from the tuna
head and quality of oil (acid value and peroxide
value). The influence of hydrolysis time on the
oil recovery, acid value and peroxide value of

Figure 4. The influence of hydrolysis time on the oil recovery (a), acid value (b) and
peroxide value (c) of yellowfin tuna head oil

tuna head oil is shown in Figure 4.
The results indicated that there was a
significant increase in oil recovery during the
first hour, followed by a decrease during the

next 3 hours (Figure 4a). The oil recovery from
tuna heads was 34% after 0.5h of hydrolysis
and reached the highest value (63.7%) after 1h
of hydrolysis. However, when the hydrolysis
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Journal of Fisheries science and Technology
time prolonged over 1h, the free oil recovery
decreased significantly. After 4h of hydrolysis,
the oil recovery only attained 43%.
These results implied that the hydrolysis
time of 0.5h was not sufficient to release the
oil. That led to a low percentage of oil recovery.
The hydrolysis time of 1h was sufficient to
release a large amount of free oil from tuna
heads. The decrease in amount of free oil after
1h may be due to interaction of released oil
with hydrolyzed proteins during hydrolysis
as showed by Šližyte et al. (2005). Mbatia
et al. (2010) also reported the initial stage of
hydrolysis could be sufficient to release the
lipids. The longer hydrolysis time did not
improve the oil recovery. According to Dumay
et al. (2009), it is not beneficial to perform a
long hydrolysis to obtain the highest oil release.
Indeed, the tissue disruption obtained at the
beginning of the proteolysis appears sufficient
to release the lipids.

The acid values (Figure 4b) and peroxide
values (Figure 4c) of tuna head oil were not
significantly different among the samples with

No. 4 - 2018
the hydrolysis time from 0.5 to 3h. The highest
acid value and peroxide value were obtained
after 4h of hydrolysis. This may be due to the
hydrolysis and oxidation of released oil with
the long time of hydrolysis.
The study results suggested that the suitable
hydrolysis time for oil extraction was 1h.
In brief, the suitable hydrolysis conditions
for oil extraction from yellowfin tuna heads in
this study were the water/material ratio of 0.5/1,
enzyme concentration of 0.5%, hydrolysis
temperature of 55°C and hydrolysis time of 1h.
2. Chemical quality of the oil extracted from
yellowfin tuna heads
Yellowfin tuna heads were hydrolyzed with
the suitable hydrolysis conditions determined,
the oil recovery from yellowfin tuna heads
was 63.7 ± 0.8%. In order to assess the quality
of oil extracted from yellowfin tuna heads,
some chemical properties including free fatty
acid content, acid value, peroxide value,
iodine value and saponification value were
determined. Chemical quality of yellowfin
tuna head oil is shown in Table 1.

Table 1. Chemical quality of oil extracted from yellowfin tuna heads

The free fatty acid content in oil is one
of the most important quality parameters to
evaluate the quality of oil because the free fatty
acid are more susceptible to oxidation than
esterified fatty acids (Ahmed et al.,2017). The
lower free fatty acid content ensures higher
grade quality with fewer changes for further
oxidation. As quality specifications for crude
fish oil, Bimbo (1998) reported that the free
fatty acid content should range between 1 and
7% but usually ranges between 2 and 5%. The
result in this study (Table 1) demonstrated that
the amount of free fatty acid in the yellowfin
tuna head oil was low (1.56%). This value was
much lower than that of the oil extracted from
24 • NHA TRANG UNIVERSITY

head of tuna Euthynnus affinis (4.08%) studied
by Khoddami et al. (2012).
The acid value is a measure of the lipid
hydrolysis that had occurred in the oil and
is defined as the number of milligrams of
potassium hydroxide required to neutralize the
free fatty acids in 1g of oil. The acid value of
yellowfin tuna head oil was found to be 3.12
mg KOH/g, which is below the acceptable
limit of 7-8 mg KOH/g reported by Bimbo and
Crowther (1991).

The peroxide value is commonly used to
determine the rancidity of oil and is expressed
in milli equivalent of active oxygen per kg of
oil. The maximum limit of peroxide value of


Journal of Fisheries science and Technology
crude oil is 8 meq O2/kg to be acceptable for
human consumption (Boran et al., 2006). The
oil extracted from yellowfin tuna head had a
peroxide value of 2.24 meq O2/kg, which was
still within the acceptable quality limit. This
indicated that the extracted fish oil had low
lipid oxidation rate. According to Khoddami et
al. (2012), the peroxide value of oil from head
of tuna Euthynnus affinis was 7.31 meq O2/ kg.
Bimbo (1998) reported that the peroxide value
of crude fish oil was between 3 to 20 meq O2/kg.
The iodine value is a measure of degree of
unsaturation of the oil and is defined as grams of
iodine absorbed by 100 g of oil. Yellowfin tuna
head oil had a iodine value of 177 g I2/100g,
which was higher than that of mackerel oil (134
g I2/100g) (Zuta et al., 2003). This indicated that
the oil from yellowfin tuna head contains a high
amount of unsaturated fatty acids.
Saponification is the process of breaking
down a neutral oil into glycerol and fatty
acids by alkali treatment. Saponification
value represents the number of milligrams of

potassium hydroxide required to saponify 1 g

No. 4 - 2018
of oil. The oil extracted from yellowfin tuna
heads had a saponification value of 185 mg
KOH/g, which was similar to that of sardine
oil (186.85 mg KOH/g) reported by NoriegaRodríguez et al. (2009). Saponification values
of the hilsa fish oils from different parts were
found to be arranged from 180.28 to 194
(Salam et al., 2005).
IV. CONCLUSION
The effects of the hydrolysis conditions on
the extraction of oil from the yellowfin tuna
heads were studied. The suitable parameters
for oil extraction from yellowfin tuna heads
were the water/material ratio of 0.5/1, enzyme
concentration of 0.5%, hydrolysis temperature
of 55°C and hydrolysis time of 1h. With these
suitable hydrolysis conditions, the oil recovery
from yellowfin tuna heads was 63.7%. The oil
obtained after enzymatic hydrolysis had a good
quality with acid value of 3.12 mg KOH/g and
peroxide value of 2.24 (meq O2/kg). Tuna head
oil could be used as a valuable ingredient both
in food and aquaculture feed.

REFERENCES
1. Ahmed, R., Haq, M., Cho, Y.J., Chun, B.S. 2017. Quality evaluation of oil recovered from by-products of
bigeye tuna using supercritical carbon dioxide extraction. Turkish Journal of Fisheries and Aquatic Sciences.
17: 663-672.

2. AOCS, 1997. Official methods and recommended practices of the American Oil Chemists’ Society. 5th
Edition. AOCS Press, Champaign USA.
3. Batista, I., Ramos, C., Mendonça, R., Nunes, M.L., 2009. Enzymatic hydrolysis of sardine (Sardina
pilchardus) by-products and lipid recovery. Journal of Aquatic Food Product Technology. 18:120-134.
4. Bimbo, A.P., 1998. Guidelines for characterizing food-grade fish oils. Inform 9: 473-483.
5. Bimbo, A. P., Crowther, J.B., 1991. Fish oil: processing beyond crude oil. Infofish International. 6: 20-25.
6. Boran, G., Karaςam, H., Boran, M. 2006. Changes in the quality of fish oil due to storage temperature and
time. Food Chemistry. 98: 693-698.
7. Chantachum, S., Benjakul, S., Sriwirat, N., 2000. Separation and quality of fish oil from precooked and non
- precooked tuna heads. Food Chemistry. 69, 289-294.
8. Deepika, D., Vegneshwaran, V.R., Julia, P., Sukhinder, K.C., Sheila, T., Heather, M., Wade, M. 2014.
Investigation on oil extraction methods and its influence on omega-3 content from cultured salmon. Journal of
Food Processing & Technology.5: 1-13.
9. Dumay, J., Allery, M., Donnay-Moreno1, C., Barnathan, G.,Jaouen J., Carbonneau, M.E., Bergé, J.P., 2009.
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