Journal of Fisheries science and Technology – No 4/2018

Journal of Fisheries science and Technology – No 4/2018 present the content effects of rearing water and tank on larval survival rate of white-striped cleaner shrimp Lysmata amboinensis; larviculture of slipper lobsters in the genus Ibacus and Thenus a review; assessing on coastal fi shing activities and marine resources in Tuy An district, Phu Yen province...

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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

Role of antibiotics in chilled storage of sperm in grass carp (Ctenopharyngodon idella)

Le Minh Hoang, Dinh Van Khuong

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

Fish oil extraction from yellowﬁ n tuna heads by enzymatic hydrolysis method

Nguyen Thi My Huong, Bui Truong Bich Ngan

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

Kaori Wakabayashi

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

Protect and enhance the resources by using artiﬁ cial reef at coastal areas in central of Vietnam

Pham Viet Tich, Tran Duc Phu, Nguyen Trong Luong, Tran Van Hao

Assessing on coastal ﬁ shing activities and marine resources in Tuy An district, Phu Yen province

Tran Duc Phu, To Van Phuong

Selenium deﬁ ciency, toxicity and its requirement in marine ﬁ sh: A research review

Pham Duc Hung

Photoperiod manipulation in the induced breeding of the rabbit ﬁ sh (Siganus guttatus)

Pham Quoc Hung, Hua Thi Ngoc Dung, Augustine Arukwe

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

Nguyen Huu Thanh

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

Research on the fi tness between the mesh size and the length of threadfi n bream (Nemipterus sp.) in stow net fi shery

Nguyen Trong Luong, Vu Ke Nghiep

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

Luong Cong Trung

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¹ Institute of Aquaculture, Nha Trang University

² Aquaculture master student, Nha Trang University

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 bioﬁ lter-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 ﬁ lled 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 fi nfi sh, starfi sh, jellyfi sh, mollusk

and crustacean that are cultured in aquarium

nowadays Among ornamental species,

white-striped 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 artiﬁ cial seed

production is, no doubt, contributing to satisfy

local and global market demand

However, seed production of white-striped

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 bioﬁ ltered 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 bioﬁ ltered water could maintain and improve water quality but

it is difﬁ cult 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

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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

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 ﬁ lter 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 bioﬁ ltered 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 Bioﬁ lter-water system used orchid net as bioﬁ lter 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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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 artiﬁ cial 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

artiﬁ cial feed, including 25% Frippak, 25%

Lansy and 50% V8-zoea was also used

3 Data collection

Speciﬁ c stage and accumulated larval

survival rates were calculated for each tank and

treatment based on the number of remaining

larvae Speciﬁ c 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 ﬁ nishing 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 signiﬁ cant 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 bioﬁ

lter-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 bioﬁ lter-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 signiﬁ cant among the three water types

Tank type signiﬁ cantly affected the survival rate of the larvae (p < 0.05) The shrimp larvae

in later stages had signiﬁ cant 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 signiﬁ cantly 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 signiﬁ cant 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

ﬁ gures 2, 3 and 4), except for larvae reared in in bioﬁ lter-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 signiﬁ cant (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 bioﬁ lters The

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disinfected marine water with low operation

cost should be used for white-striped cleaner

shrimp larval rearing

IV CONCLUSION

There was no signiﬁ cant effect of rearing

water system (disinfected, green and bioﬁ lter

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

green-water treatments

bioﬁ lter-water treatments

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Disinfected water (with low preparation

and operation costs) and Kreisel tank should

be used in rearing Lysmata amboinensis.

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 beneﬁ ts

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 proﬁ le 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 proﬁ le 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 ﬂ ow-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 Shellﬁ sh 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.

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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 ity of grass carp (Ctenopharyngodon idella) The extenders were used in this study were HBSS (Hanks’ balanced salt solution), Modiﬁ ed 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

motil-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 ﬁ sh sperm is a useful

bio-technique 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 ﬁ sh 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 ﬁ sh

of many Asian countries In response to the

need of aquaculture of the grass carp, artiﬁ cial

seed production of this species has been

investigated (FAO, 2004-2017)

There has been many studies investigating

on the preservation of ﬁ sh 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

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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×

magniﬁ cation 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

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× magniﬁ cation 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 Modiﬁ ed (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, Modiﬁ ed

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

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

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using SPSS version 22.0 Differences with a

probability value (P) of 0.05 (P<0.05) were

considered signiﬁ cant

III Results and discussion

1 Effect of extenders on sperm motility

Figure 1 Sperm motility (%) in various extender Ku1, Ku2, HBSS, CCSE-2 and Modiﬁ ed

Control: No extender Different alphabets indicate statiscial signiﬁ cance at p<0.05.

Sperm was stored in Ku2 retained its movable

spermatozoa longer than other extenders (Figure

1) Speciﬁ cally, Ku2 sperm remained motile

for 9 days (3.22%), while sperm was stored in

CCSE-2, HBSS and Modiﬁ ed 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

Figure 2 Duration of sperm motility (s) in various extenders Ku2, HBSS, CCSE-2 và Modiﬁ ed

Control: No extender Different alphabets indicate statistical signiﬁ cance at p<0.05.

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 Modiﬁ ed had a duration of sperm motility of 18.89s, 54.56s and 58.22s, respectively at the day 5 (Figure 2)

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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Figure 3 Sperm motility (%) at various dilution ratios in Kura Kuro’s 2 extender

Control: No dilution Different alphabets indicate statistical signiﬁ cance 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 signiﬁ cance 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)

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 aegleﬁ nus (DeGraaf and Berlinsky, 2004) Paddleﬁ sh Polyodon spathula sperm storage

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was also improved by adding a combination

of antibiotic penicilllin/streptomycin (Brown

and Mims, 1995) In African catﬁ sh (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

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 signiﬁ cance at p<0.05.

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

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 signiﬁ cance at p<0.05.

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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 ﬁ shes (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 ﬂ uviatilis:

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 ﬁ sh 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 inﬂ uence of diluents, equilibration

time and prefreezing storage time on the viability of cryopreserved Oreochromis niloticus (L.) spermatozoa

Aquaculture Research.21(1):25-30

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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 Tubiﬁ cidae, used

as an important live food for feeding larval stages of freshwater species This study was carried out to provide scientiﬁ c knowledge for L.hoffmeisteri culture as well as optimal feeding ration affected on density, biomass and protein compositions L hoffmeisteri was cultured under ﬂ ow-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 signiﬁ cantly effect on the density, biomass and protein compositions of L hoffmeisteri Speciﬁ cally 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 ﬁ sh

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 ﬁ shes and have been reported as an

important live food in larval rearing of many

commercially important ﬁ shes, particularly for

catfi sh and another fi sh such as gray eel-catfi sh

and crab [10,11]

In Vietnam, current total supply of these

worms mainly comes from wild caught source

which is unreliable and insufﬁ cient to meet

the demand Information related to culture

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.

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 ﬂ ow-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

ﬂ ow-through-system Substrate was made by mud layer of 1 cm thickness

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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)

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

ﬁ ve 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 signiﬁ cant when signiﬁ cance 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

3 Inoculation of L hoffmeisteri Oligochaete

Water ﬂ ow 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 ﬂ ow was stopped Amount of

food was spread throughout the culvert Then,

the water ﬂ ow was reopened after 30 minutes

5 Methods of data collection and Statistical

Analysis

5.1 Water quality

Continuous water ﬂ ow 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)

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growth, development and reproduction of the

L hoffmeisteri populations This is because

normal development of the embryo of

species of tubiﬁ cids 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 ﬁ rst 2 weeks,

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 signiﬁ cantly 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 signiﬁ cantly 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 signiﬁ cantly 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

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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 signiﬁ cant 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 signiﬁ cantly

lower than those in other treatments (P<0.05)

(Table 2)

The fatty acids ingredients (mg/g) of dry

L hoffmeisteri population were signiﬁ cant

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 signiﬁ cantly 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

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 signiﬁ cantly 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 signiﬁ cantly 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 signiﬁ cantly lower than those in other treatment (P<0.05) While, the ingredient percentage of PUFA, EPA and DHA were quite similar and no signiﬁ cant differences were found between treatments (Table 4) The Table 4 showed that, the percentage

of total fatty acids received little attention in

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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 inﬂ uence 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 efﬁ cient and can produce a

larger mass of L hoffmeisteri more quickly

than previously described systems in laboratory

conditions

This study showed that feeding rate

signiﬁ cantly 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

Trang 18

population were signiﬁ canty 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

V ACKNOWLEDGEMENTS

We would like to thank the Ministry of Education & Training who supported ﬁ nance for this study

4 Haroldo L and Alves R G, 2011 Inﬂ uence of body weight and substrate granulometry on the reproduction

of L hoffmeisteri (Oligochaeta: Naididae: Tubiﬁ cinae).

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 Tubiﬁ cidae 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 catﬁ sh (Plotosus canius), Can Tho University Journal of scientiﬁ c, 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 scientiﬁ c, 17b, 70-76.

12 Warucha Kanchana-Aksorn1and Saran Petpiroon, 2008 Study on Limnodrilus hoffmeisteri Population

Response to Different Organic Enrichment in Laboratory Condition

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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 fi sh oil extraction from yellowfi n tuna heads by hydrolysis method using Protamex enzyme was carried out The parameters of hydrolysis process, fi sh 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 yellowfi n tuna heads The suitable parameters of enzymatic hydrolysis process for recovering fi sh oil from yellowfi n 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 yellowfi n tuna head oil was obtained from enzymatic hydrolysis This study suggested that the yellowfi n 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 culture feed.

aqua-Key words: Enzymatic hydrolysis, ﬁ sh oil extraction, oil recovery, yellowﬁ n 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 ﬁ sh

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 ﬁ sh or ﬁ sh 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 fi sh During the enzymatic hydrolysis, the combination between lipid and protein was broken down, which lead to fi sh oil release much easier from fi sh by-product (Qi-

yuan et al., 2016).

The purpose of this study was to determine the suitable hydrolysis conditions for oil recovery from yellowﬁ n 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 saponiﬁ cation value

¹ Faculty of Food Technology - Nha Trang University

Trang 20

1 Materials

Yellowﬁ n tuna (Thunnus albacares) heads

were provided by Thinh Hung, a seafood

processing company in Nha Trang, Vietnam

Yellowﬁ n 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

yellowﬁ n 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 yellowﬁ n 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 ﬁ ltered through a mesh to

remove the solid fraction (bones) The ﬁ ltrate

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

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 identiﬁ ed 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 identiﬁ ed, 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, saponiﬁ cation 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 yellowﬁ n tuna head was calculated as follows:

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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 signiﬁ cant difference

between samples at P<0.05 level using the

SPSS 15.0 programme

III RESULTS AND DISCUSSION

1 Determination of suitable hydrolysis conditions for oil extraction from yellowﬁ n tuna head

1.1 Determination of suitable water/material ratio

The inﬂ uence 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 inﬂ uence of water/material ratio on the oil recovery (a), acid value (b) and

peroxide value (c) of yellowﬁ n 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 signiﬁ cant

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 signiﬁ cant The

oil extracted from yellowﬁ n 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 yellowﬁ n tuna head

1.2 Determination of suitable enzyme concentration

During the enzymatic extraction of oil from the yellowﬁ n tuna heads with Protamex, enzyme concentration plays an important role in the recovery of oil Figure 2 indicate the inﬂ uence

of enzyme/material ratio on the release of oil, acid value and peroxide value of tuna head oil

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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 signiﬁ cant 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 signiﬁ cant differences in

acid values (Figure 2b) and peroxide values

(Figure 2c) of the ﬁ sh oil obtained among

the samples with the different enzyme

concentrations This mean that the enzyme

concentration did not signiﬁ cantly affect on

the acid value and peroxide value of the oil

extraced from yellowﬁ n tuna heads The acid

values of the oil extracted from hilsa ﬁ sh (Hilsa

ilisha) by-products ranged from 4.16 to 12 mg

KOH/g (Salam et al., 2005) According to Khoddami et al (2012), the peroxide value of the oil extracted from tuna (Euthynnus afﬁ nis)

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 inﬂ uence 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 signiﬁ cant 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

Figure 2 The inﬂ uence of enzyme concentration on the oil recovery (a), acid value (b) and

peroxide value (c) of yellowﬁ n tuna head oil

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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 ﬁ sh 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 yellowﬁ n 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 inﬂ uence of hydrolysis time on the oil recovery, acid value and peroxide value of

Figure 3 The inﬂ uence of hydrolysis temperature on the oil recovery (a), acid value (b) and

Figure 4 The inﬂ uence of hydrolysis time on the oil recovery (a), acid value (b) and

tuna head oil is shown in Figure 4

The results indicated that there was a

signiﬁ cant increase in oil recovery during the

ﬁ rst 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

Trang 24

time prolonged over 1h, the free oil recovery

decreased signiﬁ cantly After 4h of hydrolysis,

the oil recovery only attained 43%

These results implied that the hydrolysis

time of 0.5h was not sufﬁ cient to release the

oil That led to a low percentage of oil recovery

The hydrolysis time of 1h was sufﬁ cient 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 sufﬁ cient to release the

lipids The longer hydrolysis time did not

improve the oil recovery According to Dumay

et al (2009), it is not beneﬁ cial to perform a

long hydrolysis to obtain the highest oil release

Indeed, the tissue disruption obtained at the

beginning of the proteolysis appears sufﬁ cient

to release the lipids

The acid values (Figure 4b) and peroxide

values (Figure 4c) of tuna head oil were not

signiﬁ cantly different among the samples with

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 yellowﬁ n 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 yellowﬁ n tuna heads

Yellowﬁ n tuna heads were hydrolyzed with the suitable hydrolysis conditions determined, the oil recovery from yellowﬁ n tuna heads was 63.7 ± 0.8% In order to assess the quality

of oil extracted from yellowfi n tuna heads, some chemical properties including free fatty acid content, acid value, peroxide value, iodine value and saponifi cation value were determined Chemical quality of yellowfi n tuna head oil is shown in Table 1

Table 1 Chemical quality of oil extracted from yellowﬁ n 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

esteriﬁ ed fatty acids (Ahmed et al.,2017) The

lower free fatty acid content ensures higher

grade quality with fewer changes for further

oxidation As quality speciﬁ cations for crude

ﬁ sh 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 yellowﬁ n

tuna head oil was low (1.56%) This value was

much lower than that of the oil extracted from

head of tuna Euthynnus afﬁ nis (4.08%) studied

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

Trang 25

crude oil is 8 meq O2/kg to be acceptable for

human consumption (Boran et al., 2006) The

oil extracted from yellowﬁ n 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 ﬁ sh oil had low

lipid oxidation rate According to Khoddami et

al (2012), the peroxide value of oil from head

of tuna Euthynnus afﬁ nis was 7.31 meq O2/ kg

Bimbo (1998) reported that the peroxide value

of crude ﬁ sh oil was between 3 to 20 meq O2/kg

The iodine value is a measure of degree of

unsaturation of the oil and is deﬁ ned as grams of

iodine absorbed by 100 g of oil Yellowﬁ n 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 yellowﬁ n tuna head contains a high

amount of unsaturated fatty acids

Saponiﬁ cation is the process of breaking

down a neutral oil into glycerol and fatty

acids by alkali treatment Saponiﬁ cation

value represents the number of milligrams of

potassium hydroxide required to saponify 1 g

of oil The oil extracted from yellowﬁ n tuna heads had a saponiﬁ cation value of 185 mg KOH/g, which was similar to that of sardine oil (186.85 mg KOH/g) reported by Noriega-

Rodríguez et al (2009) Saponiﬁ cation values

of the hilsa ﬁ sh 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 yellowﬁ n tuna heads were studied The suitable parameters for oil extraction from yellowﬁ n 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 yellowﬁ n 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 Ofﬁ cial 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 ﬁ sh oils Inform 9: 473-483

5 Bimbo, A P., Crowther, J.B., 1991 Fish oil: processing beyond crude oil Infoﬁ sh International 6: 20-25

6 Boran, G., Karaςam, H., Boran, M 2006 Changes in the quality of ﬁ sh oil due to storage temperature and time Food Chemistry 98: 693-698

7 Chantachum, S., Benjakul, S., Sriwirat, N., 2000 Separation and quality of ﬁ sh 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 inﬂ uence 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

Trang 26

Optimization of hydrolysis of sardine (Sardina pilchardus) heads with Protamex: enhancement of lipid and

phospholipid extraction Journal of the Science of Food and Agriculture 89: 1599 - 1606

10 Folch, J., Lees, N., Sloane-Stanley, G.H., 1957 A simple method for the isolation and puriﬁ cation of total lipids from animal tissues, J Biol Chem 226: 497-509

11 Gbogouri, G.A., Linder, M., Fanni, J., Parmentier, M 2006 Analysis of lipids extracted from salmon

(Salmon salar) heads by commercial proteolytic enzymes, European Journal of Lipid Science and Technology

108:766-775

12 Herpandi, N H., Rosma, A., Wan Nadiah, W.A 2011 The Tuna Fishing Industry: A new Outlook on Fish Protein Hydrolysates Comprehensive Reviews in Food Science and Food Safety, 10: 195-207

13 Khoddami, A., Arifi n A, Bakar J, Ghazali HM 2009 Fatty acid profi le of the oil extracted from fi sh waste

(head, intestine and liver) (Sardinella lemuru) World Applied Sciences Journal 7(1):127-131.

14 Khoddami, A., Arifﬁ n, A.A., Bakar, J., Ghazali, H.M 2012 Quality and fatty acid proﬁ le of the oil extracted

from ﬁ sh waste (head, intestine and liver) (Euthynnus aﬁ nis) African Journal of Biotechnology 11:1683-1689.

15 Mbatia, B., Adlercreutz, D., Adlercreutz, P., Mahadhy, A., Mulaa, F., Mattiasson, B., 2010 Enzymatic oil extraction and positional analysis of ω-3 fatty acids in nile perch and salmon heads Process Biochemistry 45: 815-819

16 Mahmoud, A.K., Linder, M., Fanni, J., Parmentier, M 2008 Charaterisation of the lipid fractions obtained

by proteolytic and chemical extractions from rainbow trout (Oncorhynchus mykiss) roe Process Biochemistry

43: 376-383

17 Nguyen Thi My Huong, 2013 Protein and lipid recovery from tuna head using industrial protease Journal

of Science and Development 11 (8): 1150-1158

18 Noriega-Rodríguez, J A., Ortega-García, J., Angulo-Guerrero, O., García, H S., Medina-Juárez, L A.,

Gámez-Meza, N., 2009 Oil production from sardine (Sardinops sagax caerulea) CyTA - Journal of Food, 7:

173-179

19 Norziah, M.H., Nuraini, J., Lee, K.Y 2009 Studies on the extraction and characterization of ﬁ sh oil from wastes of seafood processing industry As J Food Ag-Ind 2: 959-973

20 Pravinkumar, M., Eugien, L X., Viswanathan, C., & Rafﬁ , S M., 2015 Extraction of ﬁ sh body oil from

Sardinella longiceps by employing direct steaming method and its quantitative and qualitative assessment Journal of Coastal Life Medicine,3, 962-966.

21 Qi-yuan, L., Jun-qing, Q., Xiao-ge, W 2016 Optimization of enzymatic ﬁ sh oil extraction from mackerel

viscera by response surface methodology Int Food Res J.23, 992-997.

22 Ramakrishnan, V.V., Ghaly, A.E., Brooks, M.S., udge SM 2013 Extraction of oil from mackerel ﬁ sh processing waste using Alcalase enzyme Enzyme Engineering 2:1-10

23 Salam, K A., Motahar Hossain, A.K.M., Khurshid Alam, A.H.M., Pervin, F., Absar, N., 2005 A Comparative analysis on physico-chemical characteristic of oil extracted from six different parts of

Hilsa fish (Hilsa ilisha) Journal of Biological Sciences 8: 810-815.

24 Šližyte R, Dauksas E, Falch E, Storro I, Rustad T 2005 Yield and composition of different fractions

obtained after enzymatic hydrolysis of cod (Gadus morhua) by-products Process Biochem 40: 1415-1424.

25 VASEP 2016 Bản tin thương mại thủy sản Số 48

26 Zuta, P.C, Simpson, K.B., Chan, M.H., and Phillips, L.2003 Concentrating PUFA from mackerel processing waste, JAOCS 80: 933-936

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Ibacus AND Thenus: A REVIEW

of lobster larvae

Keywords: Phyllosoma, Scyllaridae, Seed production, Lobster aquaculture, Gelatinous zooplankton

I INTRODUCTION

Slipper lobsters are the crustaceans in the

family Scyllaridae (Achelata, Decapoda)

This family includes more than 80 species

which are distributed in four subfamilies:

Arctidinae (including the genera Arctides,

Scyllarides), Ibacinae (Ibacus, Parribacus,

Evibacus), Theninae (Thenus), and Scyllarinae

(13 genera) (Holthuis, 1991; Webber and

Booth, 2007; WoRMS, 2018) Except the

species in Scyllarinae which are normally

less than 10 cm in body length, the slipper

lobsters are of commercial interest (Holthuis,

1991) Particularly in the Northwest and

Western Central Paciﬁ c, the slipper lobsters

are account for 15–50% of total lobster catch

(Vijayakumaran and Radhakrishnan, 2011;

FAO, 2018) Currently these lobsters are fully

exploited from the natural environments It

has been reported that the lobster populations

at several locations have been dramatically

declined probably due to over-exploitation

(Deshmukh, 2001; Radhakrishnan et al.,

2005) Juvenile production and the subsequent

farming are still in the research level and have

desired from the viewpoints of both food

production and resource conservation

The early life cycle of slipper lobsters is

similar to that of spiny lobsters in the family

Palinuridae The females of slipper lobsters

brood the fertilized eggs on pleopods until the larvae hatch The planktonic larva of slipper lobsters, so-called “phyllosoma”, is a zoeal phase which has an extremely ﬂ attened body

(Phillips and Sastry, 1980; Sekiguchi et al., 2007; Palero et al., 2014) As it grows, the

appendages develop at successive moults At the ﬁ nal stage, phyllosoma has rudimental gills at the basal parts of pereiopods (Phillips

and Sastry, 1980; Sekiguchi et al., 2007; Palero et al., 2014; Vijayakumaran and

Radhakrishnan, 2011) The ﬁ nal-stage phyllosoma metamorphosed into the postlarval phase, named “nisto”, which corresponds to the puerulus of spiny lobsters and the megalopa

of the brachyuran crabs (Martin, 2014; Palero

et al., 2014) The nisto settles into a benthic habitat (Sekiguchi et al., 2007; Vijayakumaran

and Radhakrishnan, 2011) It possesses the undeveloped mouthparts and is considered

a non-feeding (Mikami and Kuballa, 2007) Finally it reaches the juvenile phase after a single moult and then starts eating

Larvae of Ibacus and Thenus hatch in a more advanced condition compared with the larvae of the other species of scyllarids (Baisre,

1994; Booth et al., 2005) The newly hatched phyllosomas of Ibacus and Thenus lobsters

possess four fully segmented pereiopods (1st

to 4th pereiopods) and incompletely developed 5th pereiopods, whereas the phyllosomas of

Scyllarides, Arctides, and Parribacus have

three fully segmented pereiopods (1st to 3rd

¹ Graduate School of Biosphere Science, Hiroshima University,

Kagamiyama 1-4-4, Higashihiroshima, Hiroshima 739-8528,

Japan; email: kaoriw@hiroshima-u.ac.jp

LARVICULTURE OF SLIPPER LOBSTERS IN THE GENUS

Trang 28

pereiopods) The larval size and the duration of

the former groups are much larger and shorter

than those in the latter groups Lobsters in

Ibacus and Thenus (Fig 1) seem to be more

ideal species in aquaculture

Here, our knowledge on larviculture of these lobsters is reviewed with a special attention to the dietary items for phyllosomas

Figure 1 Selected species of the slipper lobsters in the genus Ibacus and Thenus

(A) Ibacus ciliatus (von Siebold, 1824), Karato ﬁ sh market, Yamaguchi, Japan; (B) Ibacus novemdentatus Gibbes, 1850, off Ainan, Kochi, Japan; (C) Thenus orientalis (Lund, 1793), Binh Thuan, Vietnam; (D)

Thenus australiensis Burton and Davie, 2007, Shark Bay, Western Australia.

II HISTORY OF LARVICULTURE

TRIALS

1 Ibacus spp

Saisho and Nakahara (1960) described the

larval development of Ibacus ciliatus for the

ﬁ rst time and achieved to observe the 1st to

4th stages of phyllosoma Dotsu et al (1966)

also obtained the newly hatched phyllosomas

of I ciliatus as well as Ibacus novemdentatus

and cultured them until the 3rd and 4th stages,

respectively These two trials were the pioneer

works on the larval development of slipper

lobsters in anticipation of seed production

Artemia nauplii and ﬁ sh larvae which are

commonly used for ﬁ sh and crustacean

larviculture were applied in these studies, but

none of phyllosomas completed the planktonic

phase

Later, Takahashi and Saisho (1978) have

achieved the complete larval development

from hatching to metamorphosis of both I

ciliatus and I novemdentatus Phyllosomas of

I novemdentatus were demonstrated to take 7

instars and those of I ciliatus to take 7 or 8

instars before metamorphosing into the nisto

stage Finely chopped clam ﬂ esh was mainly

used as larval diet in their trials Matsuda et al

(1988) and Mikami and Takashima (1993) also

reported the completion of I ciliatus larval

development in which phyllosomas were fed

with Artemia nauplii for the earlier stages

and ﬁ nely chopped mussel ﬂ esh for the later

stages Matsuda et al (1988) tested diverse

items including fi sh meat, clam, mussel, abalone, squid, krills, and moon jellyfi sh, and found out that bivalve fl esh and moon jellyfi sh were the items on which phyllosomas preyed

most actively Most recently, Wakabayashi et

al (2012, 2016) reported the complete larval

development of these lobsters with feeding jellyﬁ sh (Fig 2) Jellyﬁ sh is known as one of

the natural diets of phyllosomas (e.g Booth et al., 2005; Sekiguchi et al., 2007; Wakabayashi

et al., in press) Growth rates of phyllosomas

fed on jellyﬁ sh were not inferior to those fed

on clams reported by Takahashi and Saisho

(Wakabayashi et al., 2012, 2016) Wakabayashi

et al (2012) demonstrated that different methods of rearing (static water vs recirculating

water) did not result in a signiﬁ cant difference

Trang 29

of duration and size at each developmental

stage throughout the phyllosomal phase of

I novemdentatus However, survival rate in

recirculating water was remarkably lower,

which could be caused by multiple factors

including interference between

The complete larval development of

the Australia species Ibacus peronii was also achieved by Marinovic et al (1994) Phyllosomas were fed with Artemia nauplii

and then mussel ovaries as they grew This species passes through 6 instars before metamorphosing

Figure 2 Complete larval development from newly hatched phyllosoma to the ﬁ rst juvenile stage of

Ibacus novemdentatus Gibbes, 1850 Scale bar: 5 cm This ﬁ gure is reproduced after Wakabayashi and

Tanaka (2012) with a permission from the Japanese Society of Systematic Zoology.

2 Thenus spp

Taxonomy of this genus was recently revised

(Burton and Davie, 2007) As the only species

Thenus orientalis was recognized before the

revision, the earlier studies on the larviculture

were also represented by a single species

Ito (1988) for the ﬁ rst time cultured the newly

hatched larvae of Thenus lobsters in Australian

(described as T orientalis Form A and B,

currently identiﬁ ed as either one of Thenus

parindicus and Thenus australiensis) He used

Artemia nauplii and clam ﬂ esh; however, the

larvae did not survive until the metamorphosis

Mikami and Greenwood (1997) achieved

the complete larval development of the both

Thenus species and conﬁ rmed that these species

take four instars before metamorphosing into the nisto stage Phyllosomas preyed on fresh clam ﬂ esh could develop into the juvenile stage, while those fed on defrosted clam ﬂ esh

did not survive They used Artemia nauplii

enriched with a commercial product of Selco

as supplemental diet together with clam ﬂ esh although the presence of supplemental diet did

not affect the results Hải et al (2012) worked

on larval development of T orientalis (no

detailed information of species identiﬁ cation was given) in Vietnam They mainly used

Trang 30

Artemia nauplii and fresh oyster ﬂ esh as larval

diet and blood cockle ﬂ esh was also used as

a supplemental diet Metamorphosis was not

observed, but it was noticed that the larvae

preyed on the supplemental diet for longer

period grew and survived better than those had

lesser opportunity of preying on blood cockle

ﬂ esh

In India, the completion of larval

development of Thenus unimaculatus (former

T orientalis in India) was described by

Kizhakudan et al (2004) and Kizhakudan and

Krishnamoorthi (2014) Phyllosomas were

fed with fresh chopped clam ﬂ esh and live

ctenophores The phyllosomas at the earlier

stages likely prefer the clam ﬂ esh to ctenophores,

whereas those at the later stages are opposite

Recently, phyllosomas of T australiensis

with a conﬁ rmation of species identiﬁ cation

were reared in tanks and the complete larval

development was described by Wakabayashi

and Phillips (2016) Moon jellyﬁ sh was used as

the sole diet for phyllosomas which successfully

metamorphosed into the nisto stage, though the

juveniles showed an abnormal form

III IMPORTANCE OF SIZE AND

MOTILE CHARACTERS IN DIET FOR

PHYLLOSOMAS

Each trial in the previous papers had

different rearing conditions, and those

differences probably inﬂ uenced the results

of phyllosomal growth and survival more

or less Even considering this, the previous

observations clearly show that the choice of

food items makes a critical difference of results

in growth of phyllosomas

A known information can tell us that

the major natural diet of slipper lobster

phyllosomas are likely gelatinous zooplankton

They have been often found in association with

gelatinous zooplankton in the wild (Shojima,

1963; 1973; Thomas, 1963; Herrnkind et al.,

1976; Barnett et al., 1986; Ates et al., 2007;

Wakabayashi et al., 2017 a, b) Anatomical

and molecular approaches demonstrated that

digestive organs of wild-caught scyllarid

phyllosomas contained gelatinous zooplankton

tissues including cnidarian jellyﬁ sh and

larvaceans (Sims and Brown, 1968; Suzuki et al., 2006, 2007) In the laboratory, a variety of

food items including gelatinous zooplankton were tested as the diet materials for slipper lobster phyllosomas as mentioned above (see also table 5.1 in Mikami and Kuballa, 2007) The phyllosomas do accept a diverse type of food, it may be because they are opportunistic feeders as suggested in spiny lobsters (Jeffs, 2007)

Among the ﬁ ve previous trials with I ciliatus, phyllsomas fed on fresh bivalves or

jellyﬁ sh successfully metamorphosed into the

nisto stage, whereas those fed on Artemia and

ﬁ sh larvae did not (Table 1) Dotsu et al (1966) observed that newly hatched phyllosoma of

I ciliatus likely had a difﬁ culty of catching Artemia nauplii They used ﬁ sh larvae (> 3.0 mm in total length) instead of Artemia

nauplii (< 1.0 mm in total length) and found out that phyllosomas preferred large sized

ﬁ sh larvae (Sebastes achycephalus nigricans,

7.0 mm in total length) followed by middle

(Sebastes innermis, 5.0 mm in total length) and small (Sebastiscus marmoratus, 3.0 mm

in total length) items However, the survival of phyllosomas fed on those ﬁ sh larvae was not

improved from the trials with Artemia nauplii

(Table 1), which causes were not discussed by the authors Hải et al (2012) mentioned in their paper that their colleagues found out that both

rotifer and Artemia nauplii were not adequate food items for Thenus lobster phyllosomas

because those animals were too small and swimming too fast, respectively Mikami and Kuballa (2004) also pointed out that size and

nutritional quality of Artemia nauplii is not ideal for Thenus lobster phyllosomas Bivalve

ﬂ esh and jellyﬁ sh are more ideal items rather

than rotifer, Artemia and ﬁ sh larvae for a success of long-term larval rearing of Ibacus and Thenus phyllosomas, in size and motile

points of view

Growth increment of I ciliatus phyllosomas

normally ranges between 5.6% and 8.2% of total length per day at any developmental

Trang 31

stages in the three previous successful trials

regardless of rearing environment (Table 1)

The average value of daily growth increment

is 6.6–6.7% At least for I ciliatus, this may

be useful as an indicator to maintain a quality

of rearing environment for a successful

larval development A high survival rate of

phyllosomas from hatching to settlement (ca

60%) can be expected when using an individual

rearing system to avoid the mortality due to

cannibalism (Wakabayashi et al 2016)

IV ACKNOWLEDGEMENTS

The author expresses her gratitude to Dr

Pham Quoc Hung (Nha Trang University)

and Dr Motohiko Sano (Tokyo University

of Marine Science and Technology) for

allowing meto have the opportunity of writing

this review The gratitude is extended to Mr Quan Nguyen Hong (Hiroshima University / Research Institute for Aquaculture No.2) and

Mr Hiroki Sugiura (Hiroshima University) for their assistance of translating Vietnamese

reference and providing a photograph of Thenus orientalis in this paper, respectively This work

was partly supported by the JSPS Core-to-core Program B Asia-Africa Science Platforms (Building up an international research network for successful seed production technology development and dissemination leading South-East Asian region, coordinated by Dr Motohiko Sano) and JSPS KAKENHI Grant-in-Aid for Young Scientists (B) (Grant number 17K15310) to the author

3 Barnett B.M., Hartwick R.F., Milward N.E., 1986 Descriptions of the nisto stage of Scyllarus demani

Holthuis, two unidentiﬁ ed Scyllarus species, and the juvenile of Scyllarus martensii Pfeffer (Crustacea: Decapoda: Scyllaridae), reared in the laboratory; and behavioural observations of the nistos of S demani, S martensii and Thenus orientalis (Lund) Aust J Mar Freshwater Res., 37: 595–608.

4 Booth J.D., Webber W.R., Sekigushi H., Coutures E 2005 Diverse larval recruitment strategies within Scyllaridae N.Z J Mar Freshwater Res., 39: 581–592

5 Webber W.R., Booth J.D., 2007 Taxonomy and evolution In: Lavalii KL, Spanier E (ed) The Biology and Fisheries of Slipper Lobsters, CRC Press, Boca Raton, Florida, USA: 25–52

6 Burton T.E., Davie P.J.F., 2007 A revision of the shovel-nosed lobsters of the genus Thenus (Crustacea:

Trang 32

Decapoda: Scyllaridae), with descriptions of three new species Zootaxa, 1429: 1–38.

7 Deshmukh V.D., 2001 Collapse of sand lobster ﬁ shery in Bombay waters Indian J Fish., 48: 71–76

8 Dotsu Y., Seno K., Inoue S., 1966 Rearing experiments of early phyllosomas of Ibacus ciliatus (von Siebold) and I novemdentatus Gibbes (Crustacia: Reptantia) Bull Fac Fish Nagasaki Univ., 21: 181–194

(In Japanese with English abstract)

9 FAO, 2018 FishStatJ – software for ﬁ shery statistical time series In: FAO Fisheries and Aquaculture Department (online)

10 Hải T.N., Nhứt T.M., Khoa T.N.D., 2012 Preliminary results on rearing of sand lobster (Thenus orientalis)

larvae with different feeding regimes Tạp chí Khoa học, 21b: 133–140 (In Vietnamese with English abstract)

11 Herrnkind W., Haluskey J., Kanciruk P., 1976 A further note on phyllosoma larvae associated with medusae Bull Mar Sci., 26: 110–112

12 Holthuis L.B., 1991 Marine Lobsters of the World FAO Fish Synop., 13: 1–292

13 Ito M., 1988 Mariculture-related laboratory studies on the early life histories of the scyllarid lobster

(Crustacea: Decapoda: Scyllaridae): two forms of Thenus Leach; and Scyllarus demani Holthuis M.Sc thesis,

James Cook University of North Queensland, Queensland, Australia Pp 31–53

14 Kizhakudan J.K., Krishnamoorthi S., 2014 Complete larval development of Thenus unimaculatus Burtone

& Davir, 2007 (Decapoda, Scyllaridae) Crustaceana, 87: 570–584

15 Kizhakudan J.K., Thirumilu P., Rajapackiam S., Manibal C., 2004 Captive breeding and seed production

of scyllarid lobsters – opening new vistas in crustacean aquaculture Mar Fish Infor Serv., 181: 1–4

16 Marinovic B., Lemmens J.W.T.J., Knott B., 1994 Larval development of Ibacus peronii Leach (Decapoda:

Scyllaridae) under laboratory conditions J Crustac Biol., 14: 80–96

17 Martin J.W., 2014 Introduction to the Decapoda In: Martin JW, Olsen J, Høeg JT (ed) Atlas of Crustacean Larvae, Johns Hopkins University Press, Baltimore, Maryland, USA: 230–234

18 Matsuda H., Yamashita T., Tsujigado A., 1988 Seed production of Ibacus ciliatus – II Annual Report of

the Mie Prefecture Fisheries Research Institute, 1988: 70–73 (In Japanese)

19 Mikami S., Greenwood, J.G., 1997 Complete development and comparative morphology of larval Thenus orientalis and Thenus sp (Decapoda: Scyllaridae) reared in the laboratory J Crustac Biol., 17: 289–308.

20 Mikami S., Kuballa A.V., 2007 Factors important in larval and postlarval molting, growth, and rearing In: Lavalii KL, Spanier E (ed) The Biology and Fisheries of Slipper Lobsters, CRC Press, Boca Raton, Florida, USA: 91–110

21 Mikami S., Takashima F., 1993 Development of the proventriculus in larvae of the slipper lobster, Ibacus ciliatus (Decapoda, Scyllaridae) Aquaculture, 116: 199–217.

22 Palero F., Clark P.F., Guerao G., 2014 Achelata In: Martin JW, Olsen J, Høeg JT (ed) Atlas of Crustacean Larvae, Johns Hopkins University Press, Baltimore, Maryland, USA: 272–278

23 Phillips B.F., Sastry A.N., 1980 Larval ecology In: Cobbs JS, Phillips BF (ed) The Biology and Management

of Lobsters, Vol II Ecology and Management, Adacemic Press, New York, USA: 11–57

24 Radhakrishnan E.V., Deshmukh V.D., Manisseri M.K., Rajamani M., Kizhakudan J.K., Thangaraja R.,

2005 Status of the major lobster ﬁ sheries in India N.Z J Mar Freshwater Res., 39: 723–732

25 Saisho T., Nakahara K., 1960 On the early development of phyllosomas of Ibacus ciliatus (von Siebold) and Panulirus longipes (A Milne Edwards) Mem Fac Fish Kagoshima Univ., 9: 84–90.

26 Sekiguchi H., Booth J.D., Webber W.R., 2007 Early life histories of slipper lobsters In: Lavalii KL,

Trang 33

Spanier E (ed) The Biology and Fisheries of Slipper Lobsters, CRC Press, Boca Raton, Florida, USA: 69–90.

27 Shojima Y., 1963 Scyllarid phyllosomas’ habit of accompanying the jelly-ﬁ sh (preliminary report) Bull Jap Soc Sci Fish., 29: 349–353

28 Shojima Y., 1973 The phyllosoma larvae of Palinura in the East China Sea and adjacent waters – I Ibacus novemdentatus Bull., Seikai Reg Fish Lab., 43: 105–115 (In Japanese)

29 Sims Jr H.W., Brown Jr C.L., 1968 A giant scyllarid phyllosoma larva taken north of Bermuda (Palinuridae) Crustaceana, Suppl 2: 80–82

30 Suzuki N., Murakami K., Takeyama H., Chow S., 2006 Molecular attempt to identify prey organisms of lobster phyllosoma larvae Fish Sci., 72: 342–349

31 Suzuki N., Murakami K., Takeyama H., Chow S., 2007 Eukaryotes from the hepatopancreas of lobster phyllosoma larvae Bull Fish Res Agen., 20: 1–7

32 Takahashi M., Saisho T., 1978 The complete larval development of the scyllarid lobsters, Ibacus ciliatus (von Siebold) and Ibacus novemdentatus Gibbes in the laboratory Mem Fac Fish Kagoshima Univ., 27:

305–353

33 Thomas L.R., 1963 Phyllosoma larvae associated with medusae Nature, 198: 208

34 Vijayakumaran M., Radhakrishnan E.V., 2011 Slipper lobsters In: Fotedar RK, Phillips BF (ed) Recent Advances and New Species in Aquaculture, Blackwell, West UK: 85–114

35 Wakabayashi K., Tanaka Y., 2012 The jellyﬁ sh rider: phyllosoma larvae of spiny and slipper lobsters associated with jellyﬁ sh TAXA, Proc Jap Soc Syst Zool., 33: 5–12 (In Japanese with English abstract)

36 Wakabayashi K., Sato R., Ishii H., Akiba T., Nogata Y., Tanaka Y., 2012 Culture of phyllosomas of Ibacus novemdentatus (Decapoda: Scyllaridae) in a closed recirculating system using jellyﬁ sh as food Aquaculture,

330–333: 162–166

37 Wakabayashi K., Nagai S., Tanaka Y., 2016 The complete larval development of Ibacus ciliatus from hatching to the nisto and juvenile stages using jellyﬁ sh as the sole diet Aquaculture, 450: 102–107

38 Wakabayashi K., Phillips B.F., 2016 Morphological descriptions of laboratory reared larvae and post-larvae

of the Australian shovel-nosed lobster Thenus australiensis Burton and Davie, 2007 (Decapoda, Scyllaridae)

Crustaceana, 89: 97–117

39 Wakabayashi K., Tanaka Y., Phillips B.F., in press Culture of slipper lobster larvae (Decapoda: Achelata: Scyllaridae) fed jellyﬁ sh as food In: Radhakrishnan EV, Achamveetil G, Phillips BF, Muralidharan C (ed) Lobsters: Biology, Fisheries and Aquaculture, Springer

40 WoRMS, 2018 Scyllaridae Latreille, 1825 Accessed at http://www.marinespecies.org/aphia.php?p=taxdetails&id=106795 on 2018-12-29

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VOLUNTARY FEED INTAKE AND TRANSITION OF INGESTA IN THE

GASTROINTESTINAL TRACT OF JUVENILE COBIA (Rachycentron canadum)

FED DIFFERENT DIETS

Nguyen Van Minh¹, M Espe², Pham Duc Hung¹, Pham Thi Anh¹, Ivar Rønnestad³

Received: 15.Oct.2018; Revised: 18.Dec.2018; Accepted:25.Dec.2018

ABSTRACT

This study aimed to evaluate the palatability of a plant based protein diet (BL/A), with high inclusion levels of plant protein sources but balanced in lysine to arginine ratio (1.1), compared to two locally commercial pellets CD1 (Uni-President, Ltd.) and CD2 (INVE, Ltd.), and the transition of ingesta in juvenile cobia Juvenile cobia were fed to satiety with each of the three diets had equal feeding rate of 5.3-5.4±0.3% BW (for a meal)

No differences in stomach ﬁ lling occurred between cobia fed the PBD diet and those ﬁ sh fed the CD1 or CD2 diet Gastric evacuation rates in cobia were performed as an exponential relationship, and were estimated

as the function V T =V 0 e -b(x) (V T , volume of feed at time T; V 0 , volume of feed at time 0; b, the instantaneous evacuation rate; and x, time postfeeding; R²>0.95) Between 77 to 80% of the stomach contents were evacuated

to the lower parts of the gastrointestinal tract at 8 h, and most of consumed feed (98%) was emptied out of the stomach at 16 h postfeeding This was supported by the fact that cobia had good appetite in the 2 nd feeding of the day Time required for the return of appetite in cobia was within 8 h after feeding to satiation.

Key words: Cobia, lysine, arginine, evacuation

I Introduction

Cobia, Rachycentron canadum Linnaeus

(1766), is the only species of the family

Rachycentridae, and is widely distributed

in subtropical, tropical and temperate

areas, except for the central and eastern

Pacific (Briggs, 1960) This species

has many favorable production- related

characteristics, such as rapid growth, and

thus is regarded as a good candidate species

for aquaculture Under optimal feed and

temperature condition cobia fingerlings

can reach the marketable size of 4-6 kg

(Chou, Su, & Chen, 2001) or even 6-10 kg

(Su, Chien, & Liao, 2000) within a year

Further, cobia is highly marketable prized

because of its high quality with white, firm

and good flavored flesh that is also suitable

for the sashimi industry (Chou et al., 2001)

However, since cobia was only recently

introduced into aquaculture documentation

on the nutritional requirement of the species

is still limited Cobia culture is hampered

by a lack of good feeding protocols and nutritionally optimized diets

Chou and coworkers reported that protein concentration of 445 g kg-1 dry matter diet would give maximum growth in cobia, while optimum dietary lipid for juvenile cobia was found to be 57.6 g kg-1 dry matter (Chou et al., 2001) Replacement of fishmeal

by plant protein sources, the nowadays dominant protein ingredient in aquaculture diets, shows promising results In cobia, up

to 400 g kg-1 of fishmeal can be replaced with soybean meal without negatively affect growth and feed conversion ratios (Chou et al., 2004; Zhou, Mai, Tan, & Liu, 2005) Plant ingredients may not well balanced

in indispensable amino acids profiles that consequently reduce growth performance in fish (Rumsey, Siwicki, Anderson, & Bowser, 1994) Amongst in the indispensable amino acids in fish, lysine and arginine concentrations and/or its proportion in the diets are often taken into consideration when fishmeal protein is replaced by plant

² Institute of Marine Research (IMR), Bergen, Norway

³ Department of Biological Sciences, University of Bergen,

Bergen, Norway

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protein sources in aquafeeds (Venero, Davis,

& Lim, 2008) Concentrations of lysine and

arginine are often low in gluten or

corn-based proteins and in casein (Venero et

al., 2008) In addition to protein turnover,

lysine and arginine are involved in a range

of metabolic and physiological functions

(Harpaz, 2005; Walton, Cowey, & Adron,

1984) Lysine affects collagen synthesis, as

its hydroxylation product, hydroxylysine,

is necessary for formation of the

intermolecular crosslinks in collagen (Eyre,

1980; Piez & Likins, 1957) Arginine is the

precursor for synthesis of nitric oxide, urea,

polyamines, proline, glutamate, creatine

and/or agmatine (Hird, 1986; Wu & Morris,

1998) Further, arginine participates in the

regulation of extra-endocrine signaling

pathways including AMP-activated

protein kinase (AMPK) and the target of

rapamycin, TOR (Jonsson et al., 2006; Yao

et al., 2008), as well as immune functions

(Li, Yin, Li, Kim, & Wu, 2007; Wu, Jaeger,

Bazer, & Rhoads, 2004) and reproductive

performance in mammals (Mateo et al.,

2007) Additionally, lysine and arginine

are assumed to share and/or compete for

the same trans-membrane carrier systems

The metabolism and utilization of one

of the amino acid affects the other and

may give negative effects on fish growth

(Berge, Sveier, & Lied, 2002) Although,

mechanism of absorption, metabolism and

utilization of lysine and arginine in cobia is

yet to be determined

In cobia the requirement of lysine and

arginine requirement for maximized weight

gain, specific growth rate and protein

efficiency ratio is reported to be 23.8 and

28.2 g kg-1 diet, respectively (Ren, Ai,

& Mai, 2012; Zhou, Wu, Chi, & Yang,

2007) Plant based protein diets may lead

to imbalance in lysine to aginine ratio, and

thus resulting in poor palatability, reduced

palatability and/or digestibility, that

consequently reduce growth performance

in fish (Dabrowski, Arslan, Terjesen, &

Zhang, 2007; Nguyen, Jordal, Buttle, Lai,

& Rønnestad, 2013; Nguyen, Rønnestad, Buttle, Lai, & Espe, 2014) Understanding the rate of digestion in association with gastric evacuation rate may help to predict the return of appetite (Riche, Haley, Oetker, Garbrecht, & Garling, 2004), and figure out appropriate feeding strategies for better feed intake and feed efficiency by administering food as soon as appetite has returned (Grove, Loizides, & Nott, 1978; Lee, Hwang, & Cho, 2000) In the present study cobia with a plant based protein diet (BL/A), with high inclusion levels of plant protein sources but balanced in lysine to arginine ratio (1.1), compared to two locally commercial pellets CD1 (Uni-President, Ltd.) and CD2 (INVE, Ltd.) The aim of this study is to evaluate the palatability of these diets, and feed intake and the transition of ingested feed in the gastrointestinal tract of juvenile cobia postfeeding

II Materials and methods

1 Experimental diets

Two locally commercial diets pellets CD1 (Uni-President, Ltd.) and CD2 (INVE, Ltd.), and a plant based protein diet (BL/A) produced and extruded by EWOS Innovation AS, Norway were used

in the present experiment (Table 1) The CD1 diet contained 480 g protein and 74

g lipid kg-1 dry matter, while the CD2 diet contained 550 g protein and 95 g lipid kg-1

dry matter) The BL/A diet contained 206 g

kg-1 of fishmeal, krill meal and fish protein concentrate, while the rest of the dietary protein was a blend of plant ingredient (730 g kg-1; wheat, soy protein concentrate, sunflower meal and pea protein concentrate) blended to balance the dietary amino acids towards anticipated requirements (NRC, 2011) (Table 1) Appropriate amount of crystalline lysine and arginine were added

in the BL/A diet in a balanced ratio and fulfill the requirements of juvenile cobia (Ren et al., 2012; Zhou et al., 2007) The pellet size was 1.6 mm

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2 Experimental ﬁ sh and water-circulation

tanks

Juvenile cobia (500 juveniles of 3.0-5.0 g

body weight), purchased from a local hatchery

in Nha Trang, Vietnam, were transported

and acclimatized in a ﬁ berglass tank (5 m³)

at the Center for Aquatic Animal Health and

Breeding Studies (Nha Trang University) for

a period of one week During acclimatization,

the ﬁ sh were fed ad libitum by hand at 8:00

and 17:00 with a pellet diet (480 g protein and

160 g lipid kg-1 diet) produced at the University

of Nha Trang After the acclimatization period,

cobia were sorted out and ﬁ sh of similar BW

(8.0±0.1 g) were used for the experiment

The ﬁ sh were randomly distributed into the

experiment tanks

The experimental tanks used were

rectangular ﬁ berglass tanks (0.4x0.5x0.6 m),

with 110 L water ﬁ lled, setting under a water

recirculation system with continuous aeration

Each of the diets were randomly assigned to

three tanks Input water from a ﬁ ltered ﬁ berglass

tank (1.0x1.0x2.0 m) went through plastic

pipes to rearing tanks (0.2 L second-1) Output

water from the rearing tanks was collected by

perpendicular pipes (Ø 27 mm) in the middle

of each tank Output water was then ﬁ ltrated

in a fi berglass tank (1.0x1.5x 2.0 m), before it were pumped back in to the fi ltered fi berglass tank (for input water) Seawater was pumped into a reservoir (24 m³), and was desedimented and chloride treated before coming into the recirculation system Water in the recirculation system was renewed every 2-3 days depended

on environmental parameter analyses In experiment I, water temperature was 30.5±2.3

°C (mean±SD), salinity was at 30±3.1 g L-1,

pH at 7.8-8.3, oxygen at 3.8±0.5 mg L-1 and

NH3≤0.1 mg L-1 While, these parameters for water in experiments II and III were 29.2±2.8

°C, salinity was 28±3.1 g L-1, pH 7.8-8.3, oxygen 4.6±0.5 mg L-1, NH3≤0.03 mg L-1 The experimental tanks were covered by a ﬁ shing-net on the top to prevent any cobia jumping out

of the experimental system

3 Feeding trial and sampling procedure

Feeding trial: One hundred and eighty

juvenile cobia (8.0±0.1 g) were distributed in to

ﬁ fteen tanks (12 individuals/tank) and starved for 24 h The juvenile cobia were randomly

assigned to the three diets Cobia were fed ad lib by hand at the morning meal at 8:00 for

sampling during 24 h periprandial Fifty four unfed cobia were also included as a reference (control group)

Table 1 Formulation (g kg −1 dry matter basis) of the experimental diets

a Fish meal, krill meal and ﬁ sh protein concentrate (in order of inclusion high to low)

b Soya protein concentrate, pea protein concentrate, wheat protein, sunﬂ ower meal and wheat gluten

c Micronutrients include vitamin premix, trace element premix Compositions of micronutrients were added to fulﬁ ll the requirement of Atlantic salmon according to National Research Council (1993); Crystalline lysine (78%; DSM Ltd.co.) and arginine (100%; EVONIK industries)

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Sampling procedure: Prior to exposure to

any sampling, juvenile cobia were anesthetized

by MS-222 solution (0.4 g L-1) Individual

body weight and total length were measured

to the nearest 0.1 g and 0.1 cm Six fed cobia

the CD1-, CD2- and BL/A diet, were dissected

for collection of ingesta and chyme from the

stomach, midgut and hindgut at just before

feeding and at each of the following time

postfeeding 0.25, 0.5, 1, 2, 4, 8, 16 and 24 h

Six unfed cobia from the control group were

also dissected for collection of chyme in the

GI-tract at the above sampling points Therefore, control fi sh had fasted for 48 h at the time of the fi nal sampling The fi sh’s GI-tract was dissected and carefully separated in stomach, midgut and hindgut to avoid loss of content (Fig 1) Chyme and ingesta in these segments were carefully collected and transferred onto pre-weight aluminum foils The collected contents in the GI-tract were dried at 105 °C in the oven (Clayson Laboratory Apparatus Pty Ltd.) for 24 h for determining dry weight basic

Fig 1 Schematic diagram showing the dissection of cobia for collecting samples.

A, the juvenile cobia with body cavity opened; B stomach (a); midgut (b); hind gut (c).During dissection, the gut was carefully stretched

out, then the hindgut was identifi ed from the GI terminus to the fi rst folded-gut site, and the midgut was identifi ed between the hindgut and the outlet of the stomach (pylorus).

4 Statistical analysis

Data was analyzed by the statistical

program SPSS for Windows (IBM® SPSS®

Statistics version 24) Values are given as tank

means ± SEM (standard error of the mean)

ANOVA was used to test any differences

between dietary treatments If differences were

obtained (p<0.05), the Tukey's test was used

to evaluate the differences between treatments

Prior to applying ANOVA, a Levene's test was

done for testing the homogeneity of variances

of the dependent variables

III Results and discussions

Juvenile cobia showed high appetite when

they were offered the two commercial diets, and

the plant-based protein test diet with balanced

lysine to arginine ratio (BL/A) Analysis of

the contents from the stomach indicated that

juvenile cobia had a feeding rate of 5.3±0.3%

BW for CD1-, and BL/A diet, and slightly higher

for the CD2 diet (5.4±0.4% BW) No signiﬁ cant

differences in stomach ﬁ lling occurred between cobia fed the BL/A diet and the two commercial diets Dry matter in the stomach of unfed cobia was stable as a minimum level (1.88-2.83 mg

or 0.03-0.04% BW) within the time of the experiment Signiﬁ cantly higher stomach ﬁ lling

in fed cobia compared to unfed cobia indicated the good palatability of the plant-based protein diet and both the commercial diets

Gastric evacuation rates in juvenile cobia fed three diets (Fig 2) could be ﬁ tted by the exponential function YT=V0 e-b(x) (VT, volume

of feed at time T; V0, volume of feed at time 0; b, the instantaneous evacuation rate; and x,

time postfeeding; R²>0.95) One hour after a

single meal, most of the ingesta was still in the stomach (89; 88 and 91% estimated from dry matter basic for CD1-, CD2- and BL/A diet, respectively), with only a small fraction transferred to the midgut (MG) and hindgut (HG) Stomach was gradually emptying, and

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36-41 % of ingested feed was transferred

to the further parts of the GI-tract at the 4 h

after a meal Between 77 to 80% of stomach

contents was evacuated to the lower parts of

the GI-tract at 8 h, and most of consumed feed

(98%) was emptied out of the stomach around the 16 h postfeeding (Fig 2) Based on gastric evacuation results at 8 h postfeeding, it could be inferred that the return of appetite in cobia was within this period after being fed to satiation

Data are presented as means (n=6) at selected time points after feeding Sampling started from time 0/ just after cobia fed to statiety Vertical bar indicates

±SEM The upper graph (insert) shows calculated gastric evacuation based on exponential ﬁ t for each diet The equation for the relationship between stomach content (Y) over time (x) postpradial in cobia fed the CD1 diet was Y = 0.526e -0.233x (R² = 0.9914); CD2 diet, Y = 0.536e -0.23x (R² = 0.9891); and BL/A diet, Y = 0.520e -0.23x (R² = 0.9503).

Fig 2 Stomach ﬁ lling (dry mass) in juvenile cobia fed different diets postfeeding.

Dry contents of chyme in the MG gradually

increased and peaked at 4-6 h postfeeding, and

then gradually declined to the level close to the

unfed cobia at 16 h postfeeding (Fig 3) No

signiﬁ cant differences in the chyme content

(dry mass) in midgut of juvenile occurred

between cobia fed the BL/A diet and the two

commercial diets

At the 0.5 h postfeeding, content of the

chyme in the HG rapidly increased to the

highest level observed during the study, and

stabilized at this level within the 4-16 h, followed by a rapid decrease to the minimum level similarly to unfed cobia around the 24 h postfeeding (Fig 3)

It should be noted that there was a methodological challenge regarding sampling the complete contents of the GI-tract The pyloric caeca is a complex compartment, and despite the relatively large appearance the intraluminal volume of each caecum was very small and impossible to empty The chyme

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Data are presented as means (n=6) at selected time points after feeding Sampling started from time 0/ just after cobia fed to statiety Vertical bar indicates ±SEM.Fig 3 Chyme content (dry mass) in midgut of juvenile cobia fed different diets postfeeding.

Data are presented as means (n=6) at selected time points after feeding Sampling started from time 0/ just after cobia fed to statiety Vertical bar indicates ±SEM.

Fig 4 Chyme content (dry mass) in hindgut of juvenile cobia fed different diets postfeeding.

stored in the caeca appeared to be relatively

small when stripping was tested, but these

trials resulted in crushed tissue and unreliable

and mixed matter (tissue and chyme) Also, the

remaining content from GI-tract in 24-h and

48- h starved cobia shows that there was still

some leftover chyme (unfed, Figs 2, 3, 4) The

composition of this is not known, but might probably be indigestible matter with some bile due to the yellow color

In the present experiment, cobia had consumed 5.3-5.4% body weight (BW) when they were ﬁ rst offered the CD1-, CD2- and BL/A diet This indicated good palatability

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of all three diets when compared to the

recommendations made by Sun and coworkers

(2006) that feeding rate should be from 9%

BW day-1 in cobia 10–20 g (41); and reduced

to 2–3% BW day-1 in cobia of 100–200 g BW

for better growth and feed efﬁ ciency (42)

Replacement of ﬁ shmeal by plant protein

sources in the diets may lead to imbalance in

lysine to aginine ratio, and thus resulting in

reduced palatability and/or digestibility, that

consequently reduce growth performance in

ﬁ sh (Nguyen et al., 2013; Nguyen et al., 2014;

Rumsey et al., 1994) In order to maximize

growth and feed utilization in ﬁ sh fed

plant-based protein feed, a blend of plant protein

ingredients is formulated in combination with

supplementation of crystalline amino acids

By doing so, dietary amino acid proﬁ les fulﬁ ll

the requirement and/or mimic the amino acid

proﬁ les of the ﬁ shmeal-based diets (Espe,

Lemme, Petri, & El-Mowaﬁ , 2006; Espe,

Mowaﬁ , & Ruohonen, 2012) Understanding

the rate of digestion in association with gastric

evacuation rate may help to predict the return

of appetite (Riche et al., 2004), and ﬁ gure out

appropriate feeding strategies for better feed

intake and feed efﬁ ciency by administering food

as soon as appetite has returned (Grove et al.,

1978; Lee et al., 2000) Several mathematical

models have been proposed to estimate gastric

evacuation rate, for example linear model

(Bromley, 1988; Tyler, 1970), exponential

model (He & Wurtsbaugh, 1993; Riche et

al., 2004; Stubbs, 1977), square root or linear

model (Jobling, 1987; Lambert, 1985; Pandian,

1967) Though, there is still controversial as to

which model would be the most appropriate

applicable one due to the variation of factors

affecting gastric evacuation rate For example,

Jobling (1987) proposed that small particles of

a low energy density, e.g zooplankton, were

exponentially evacuated, while large particles

of high energy density, e.g ﬁ sh prey, were

linearly evacuated (Jobling, 1987) Plotting the

gastric evacuation curves for the data obtained

in the pilot experiment indicates an exponential

relationship between the stomach content and

the time postfeeding in cobia (Fig 2) These

ﬁ ndings were in line with the model proposed that cobia show gastric evacuation rate in an exponential function (He & Wurtsbaugh, 1993; Riche et al., 2004; Stubbs, 1977) However, feed makers were not available in the present study and cobia were fed pellets to satiety only one meal, thus the precision of the estimated model is limited Further studies using inert indicators such as titanium dioxide (TiO2)

or ferric oxide (Fe2O3) (Riche et al., 2004; Richter, Luckstadt, Focken, & Becker, 2003)

in combination with different diet composition and feeding regimes are required to accurately estimate the gastrointestinal transit kinetics in cobia

The evacuation time of the ingesta through the GI-tract is in association with the absorption of nutrients following feeding (Dabrowski, 1983; Fletcher, 1984; Talbot, Higgins, & Shanks, 1984) Generally, cold water ﬁ sh require longer time to achieve complete digestion than warm water ﬁ sh species, consequently warm water

ﬁ sh show shorter evacuation time of ingesta though the GI-tract compared to cold water

ﬁ sh (Smith, 1989) Atlantic salmon showed gut transit time of 60 h (Talbot et al., 1984), while

this in hybrids sarotherodon, Oreochromiss niloticus x Sarotherodon areus was 24 h (Ross

& Jauncey, 1981) Time required for gastric

evacuation in common dab, Limanda limanda, and black rockﬁ sh, Sebastes melanops was

15 h and 76 h, respectively (Brodeur, 1984; Fletcher, 1984) In the present experiment, about 80% of the stomach content had been evacuated to the lower part of the GI-tract at the 8 h postfeeding The return of appetite is closely related to the GI emptying (Huebner

& Langton, 1982; Sims, Davies, & Bone, 1996; Vahl, 1979) Hunger in satiety feeding

ﬁ sh recovers when 80-90% of the stomach content has been evacuated (Grove et al., 1978; Riche et al., 2004; Valen, Jordal, Murashita, & Rønnestad, 2011), as orexigenic signals in the GI-tract may increase when most of the content

in the stomach evacuates, while anorexigenic signals decrease accordingly (Valen et al.,

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