Nguyễn Thị Nguyệt Huệ, Hồ Sơn Lâm, Đặng Trần Tú Trâm, Đào Thị Hồng Ngọc*, Đinh Trường

An,
Đỗ Hải Đăng, Đoàn Văn Thân

EFFECT OF DIETARY SELCO ON REPRODUCTIVE PERFORMANCE, EGG AND LARVAE

QUALITY OF CLOWFISH AMPHIRION OCELLARIS (CUVIER, 1830)
Nguyen Thi Nguyet Hue, Ho Son Lam, Dang Tran Tu Tram, Dao Thi
Hong Ngoc*, Dinh Truong An, Do Hai Dang, Doan Van Than.
Institute of Oceanography, VAST
*Email: [email protected]
Abstract. Broodstock nutrition is one of the most important research field in aquaculture. The
study was conducted to evaluate the influence of HUFA (A1-DHA Selco-Selco, Inve)
supplemented in the broodfish diets on reproductive performance, egg and larvae quality
of clownfish Amphirion ocellaris (Cuvier,1830). There were 5 treatments with 5 diets
supplemented Selco with levels: 0.0 (as the basal diet), 50 g, 100 g, 150 g and 200 g
Selco/kg feed, respectively. Each diet treatment was repeated in triplicate and the
experimental period was 12 months. The results showed that spawning frequency, re-
maturation and spawning, egg size and larvae were not affected by the addition of Selco
to broodstock diets. Diet supplemented with Selco 50 and 100 g/kg feed had positive
effects on absolute fecundity, egg loss rate (p<0.05). The hatching rate and survival rate
of 3 days posthatch larvae fish in the treatment supplemented Selco 100 g/kg feed were
the highest and had significantly difference (94.73±0.19 % and 94.56±0.93%,
respectively) (p<0.05). The malformation rate of fish larvae in the treatment
supplemented with Selco 150 g/kg feed was the lowest (0.55 ±0.022 %) but there were
no differences from the diet treatments supplemented 50 and 100 g Selco/kg feed. The
study showed that supplementation of Selco with 100 g Selco/kg feed in broodfish diet
had significantly increased absolute fecundity, hatching and survival rate of larvae.
Key words: Amphiprion ocellaris, Clownfish nemo, DHA Selco, HUFA.
1. Introduction
The success of the breeding marine ornamental fish depends on many factors such as:
culture system, culture technique, nutrition, stocking density, care regimes, environmental
factors, disease. In which, broodstock nutrition is one of the important factors contributing
to improving fecundity, hatching and survival rate in rearing, growth of fish species
(Fernández-Palacios et al., 2011). An improvement in broodstock nutrition has been
shown to greatly improve not only egg and sperm quality but also seed production
(Izquierdo et al., 2001). The fatty acid composition of fish eggs is directly affected by the
fatty acid content of the broodstock diets (Fernández-Palacios et al., 1995). There have
been numerous studies on effect of n-3 HUFAs (highly unsaturated fatty acids) supplied in
broodstock diets to fecundity fertilization and hatching rate of egg, larvae quality.
Requirement n-3 HUFAs in marine fish species such as eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) due to they cannot themselves synthesize these fatty acids
(Rainuzzo et al., 1997; Tocher, 2010). In addition the n-3 HUFAs, n-6 fatty acids such as
arachidonic acid (ARA) are also essential for growth, reproduction, and larval quality
(Bell et al., 2003; Furuita et al., 2003). Many studies have demonstrated that dietary lipids
and fatty acids enhance the reproductive performance of broodstock. Lipids and their fatty
acids in dietary composition affect reproductive development (Nzohabonayo et al., 2017),

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reproductive performance (Zakeri et al., 2011) as well as egg and larval quality (Noori et
al., 2019). Despite being given considerable attention over the past two decades,
understanding the requirements for EFA in marine fish remains complicated by the fact
that requirements may vary with species, environmental conditions and life stage (Tocher,
2010). It has also been suggested that future research focuses not only on the quantities of
DHA, EPA and ARA but also on the ratios between them as being critical in further
understanding their effects on fish growth (Bell et al., 2003; Tocher, 2010). In particular,
the lipid and fatty acid composition of broodstock diets have been shown to affect
reproduction and larval survival in marine species such as Gilthead sea bream (Sparus
aurata) (Fernandez-Palacios et al., 1995), European sea bass (Dicentrarchus labrax)
(Bruce et al., 1999), Japanese flounder (Paralichthys olvaceus) (Furuita et al., 2002),
crescent sweetlips (Plectorynchus cinctus) (Li et al., 2005), Chilean flounder
(Paralichthys adspersus) (Wilson, 2009) and yellowfin sea bream (Acanthopagrus latus)
(Zakeri et al, 2011). However, some studies have shown that excess n-3 HUFA can also
have a negative impact on egg quality (Furita et al., 2002; Li et al., 2005). Most of the
nutritional studies are on commercial fish, there are very few studies of ornamental fish,
especially nemo fish.
The aquarium trade is a rapidly growing industry worldwide with marine ornamental fish
sourced mainly from coral reefs. Marine ornamental fish is one of the potential fisheries
products in the domestic and overseas markets because of its abundant and beautiful
colors that attract the interest of aquarists and as a result, a large number of aquarium
businesses developed. However, the ornamental fish trade is still dependent on catches
from the wild. This causes considerable pressure on the ecosystem such as over-
exploitation of natural populations, damage to coral reefs and environmental degradation.
Therefore, it is necessary to develop marine ornamental fish cultivation to reduce pressure
on the ecosystem. Amphiprion sp. is known as the most popular tropical marine
ornamental fish with unique colouration and sequential hermaphroditic, social hierarchy
and monogamous characteristics and the clownfish Amphiprion ocellaris has become one
of most popular in the world. Nemo clownfish is a fish that reproduces continuously, with
a short vitellogenic period, so a complete diet will shorten the time to re-maturing,
increase the reproductive efficiency of the fish, improve the hatching rate of eggs and the
rate of growth, the survival of larvae, thereby increasing seed production (Ha Le Thi Loc,
2010). However, there is few research on the nutritional requirements and effects of
supplements on the reproductive quality of nemo fish. Therefore, this study was carried
out to evaluate the effect of HUFA (A1DHA Selco) on egg and larval quality as well as
hatching rate, survival rate and malformation rate of larvae.
2. Materials and methods
2.1. Experimental diets and feeding preparative
The HUFA used in this experiment was A1 DHA Selco with five diets: 0 g/kg feed
(control), 50, 100, 150 and 200 g/kg feed (with fatty acid as 1.11; 1.72; 2.22; 2.72 and
3.26% were mixed with basic diet). Basic diet ingredients include 70% shrimp meat, 30%

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Nguyễn Thị Nguyệt Huệ, Hồ Sơn Lâm, Đặng Trần Tú Trâm, Đào Thị Hồng Ngọc*, Đinh Trường An,
Đỗ Hải Đăng, Đoàn Văn Thân

mollusca, astaxanthin 150 mg/kg feed, vitamin E (α-tocopheryl acetate) 375 mg/kg feed.
Food was stored at -40oC and used within 1 month. The food was defrosted at room
temperature and cut into small pieces for the fish to eat.
2.2. Experimental design
Broodstock prepare. Twenty pairs of pre-mature nemo fishes from artificial breeding at
the Institute of Oceanography, Vietnam were selected and growth in a separated
experimental tank for six months prior to feeding trials. The fish were fed by fresh shrimp
and molluscs meat twice a day (at 8 h and 16 h), at a level of 5–10% of total biomass
weight. Food waste in the tank were siphoned after an hour of feeding. Breeding tanks
were cleaned daily. After six months, the fishes were matured and 15 pairs of the fishes
(male length of 5.24 ± 0.44 cm; female length of 0.67 ± 0.73 cm) were selected for the
feeding trial.
Experimental tank. There were 15 glass tanks and the pair of fish have been paired but
have never participated in spawning, without anemone were stocked in 120 liters/tank
with a clay pot as a substrate. The experiment was carried out in door with circulating
filter system for 12 months.
Management. The waste in the tank will be siphoned after about an hour of feeding. The
culture tank was cleaned daily and supplemented with fresh water to maintain salinity as
well as evaporation throughout the experiment period.
2.3. Data collection and analyses
2.3.1. Reproductive performance parameters
Absolute fecundity (The number of eggs per spawning per female). The total number of
eggs after fish lay within 4h were counted directly through a magnified image of the entire
egg nest using the Canon PowerShot A2200HD 14.1 megapixels camera.
Spawning frequency = Number of times the fish spawn during the entire experimental
period/month (number of spawning times/month).
Egg loss rate (%) = The total number of eggs before hatching × 100/number of eggs laid
on the first day. The total number of eggs at the time before hatching will be counted
directly by eye through a magnified image of the entire egg nest with using the Canon
PowerShot A2200HD 14.1 megapixels camera
2.3.2. Evaluation of the quality of eggs
Diameter of eggs. After 40-60 minutes of spawning, 5 eggs/nest were randomly taken
using a pinwheel and were put into 1.5 mL Eppendorf tubes containing 4% formol fixation
solution. The diameter of the eggs was measured by light microscopy using a calibrated
ocular micrometer.
Hatching rate of eggs (%) = Number of newly hatched larvae/ The total number of eggs
before hatching × 100%. The number of newly hatched larvae were determined indirectly

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through the number of eggs at the time before hatching minus the number of remaining
unhatched eggs. The number of remaining unhatched eggs were sinked eggs at the bottom
tank and left unhatched eggs on the substrate.
2.3.3. Evaluation of the quality of larvae
Survival rate of 3 day posthatch larvae fish (%) = (number of fish hatched - number of
dead fish) x 100/number of hatched fish. Every day, dead fish were siphoned on the entire
bottom of the tank. The dead fish were counted for 3 days from the time the eggs hatch.
The rate of malformation of newly hatched fish larvae (%): After 12 h, all newly hatched
fish larvae were dead, weak, lying on the bottom, or swimming close to the bottom,
lethargic were siphoned. These samples were fixed in 4% formol and observed using light
microscope. Malformation larvae were larvae with abnormal shapes (curved body,
crooked body, short body and short mouth). Malformation rate of newly hatched larvae
(%) = Number of malformed fish x100/total number of newly hatched fish.
2.3.4. Statistical analysis
The performances of reproduction, egg quality was statistically analysed by using one-
way analysis of variance (ANOVA), followed by Duncan’s multiple range test using
SPSS 18 for Windows. Significant differences were based on the P <0.05 level. The data
are presented as mean ± SE.
3. Results
3.1. Spawning of broodstock in the experimental
The results showed that the maturation process in female fish started with the appearance
of enlarged abdomen and expanded oviduct from the genital hole of female, then the
parent pair of fish together cleaned the egg laying area, and spawned at 11 a.m-15.30 p.m
and the eggs were cared by the male broodstock during incubation time. The colour of
egg will changed from orange on the first day to silver eye on the seventh day, and the
eggs hatched at 6.00-8.00 p.m on the eighth day.
3.2. Survival of broodstock during the experimental period
During the experiment, there were no fish died in the treatments. This result showed that
the A1 DHA Selco supplemented in the diet did not affect the survival rate of nemo
broodstock, this result again confirmed the feed used in the experiment (inherited from
previous studies) were suitable for the growth and development of memo fish.
3.3. Reproductive efficiency of broodstock
The re-maturation time and spawning frequency of nemo broodfish were not affect by the
Selco supplemented diet (p>0.05) (Table 1), but it had significantly positive effects on the
fecundity and the egg loss rate.

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Nguyễn Thị Nguyệt Huệ, Hồ Sơn Lâm, Đặng Trần Tú Trâm, Đào Thị Hồng Ngọc*, Đinh Trường An,
Đỗ Hải Đăng, Đoàn Văn Thân

Table 1. Effect of Selco on the productive efficiency (mean value ± SE).

Reproductive Selco supplementation (g/kg feed)
efficiency
0 50 100 150 200
Re-maturation and
spawning 13.41±0.198a 13.24±0.103a 12.99±0.214a 12.93±0.203a 13.20±0.201a
(day/spawning)

Spawning frequency
2.24±0.032 a 2.27±0.018 a 2.31±0.036 a 2.32±0.038 a 2.28±0.034 a
(nests/month)

Fecundity (eggs/nest) 430.00±2.605a 503.31±4.024b 512.08±4.901b 472.94±3.487c 441.81±5.359a

Egg loss rate (%) 14.82±0.158a 12.74±0.164bc 12.62±0.151b 13.50±0.248c 14.45±0,228a

Note: Letters after value on the same line showed significant difference (p<0.05).
The results showed that the number of eggs per spawning of fish in the treatment added
with Selco at 50-150 g/kg feed was higher (472.94-503.31eggs) than not supplemented
(430 eggs) or supplemented at 200 g/kg of feed (441.18 eggs). Although the number of
eggs depends on the size of the female fish and the number of eggs may improve in the
next spawning, the difference of the eggs number may be due to diet. This result is
contrary to the study on seabream (Acanthopagrus latus), supplementing with HUFA with
concentrations of 6.67; 4.26 and 2.92%, respectively, the reproductive performance of fish
was significantly improved (p<0.05). Accordingly, the relative fecundity was highest in
broodstock fed the 6.67% HUFA diet (1.642.477±1534 eggs), followed by the fish fed the
supplemented diet 4.26%. HUFA (1.473,557±1846 eggs) and the lowest was
1.20.020±1073 eggs while the diet supplemented with 2.92% HUFA (Zakeri et al., 2011).
However, the results in Paralichthys olvaceus showed that egg production was highest in
fish fed the highest n-3 HUFA level (6.2%) but egg quality parameters, such as percentage
of buoyant eggs, hatching rate and percentage of normal larvae were significantly higher
in the group fed the lowest n-3 HUFA diet (2.1%) (Furuita et al., 2002), whereas in Sparus
aurata, adding 1.6% had a significantly higher number of eggs than adding 1.3; 2.18;
3.15%, the results of this research also showed that fish fecundity was reduced and yolk
sac hypertrophy appeared in the newly larvae hatched from the broodstock fed the highest
HUFA (3.15%) (Fernandez-Palacios et al., 1995). Thus, the effectiveness of HUFA
depends mainly on the fish species studied.
After 12 months of the experiment, the time of re-maturation and spawning and spawning
frequency of nemo broodstock were not statistically significant different between
treatments (p> 0.05). The fecundity of nemo fish broodstock fed diets containing different
levels of Selco for 12 months (Fig. 1). The results showed that the number of eggs were
increased in the diets with Selco supplemented at 50-150 g/kg of feed and there were
differences between groups without Selco and 200 g/kg of feed and the highest number of
eggs was 100 g/kg of feed added (p<0.05).

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Figure 1. Effect of A1DHA Selco on fecundity.

Table 1 also shown that the addition of Selco to broodstock also improved the loss rate of
nemo eggs (p < 0.05). The egg loss rate was significantly reduced in groups were fed
Selco at 50-150 g/kg of feed (12.74-13.50%) compared with 14.82% in the control group
and 14.45% in the diet supplemented Selco at 200 g/kg feed. However, statistical analysis
of broodstock spawning data by month also showed that the supplementation of Selco
reduced the percentage of eggs lost (Fig. 2). The egg loss rate of the Selco-supplemented
treatments were lower than in the control (12.00-15.91 and 14.27-16.02%, respectively),
in which the loss rate was lowest in the treatment supplemented with 100 g/kg of feed
(12.00-13.89%), but there were no difference between treatments. Thus, this supplement
has made an important contribution to improving the reproductive efficiency of nemo
broodstock.
Hatching rate can be considered as an indicator to evaluate the egg quality of aquatic
products. The hatching rate of nemo broodstock in the groups of fish supplemented with
Selco were significantly different from that of the group without A1DHA Selco (p<0.05),
in which the highest hatching rate was the supplemented diet of 100 g/kg of feed (Fig. 3).
The effect of HUFA on hatching rate of eggs was also mentioned in research on flame
angelfish (Centropyge loriculus), the broodstock supplemented with 3.6% n-3 HUFA had
a higher hatching rate than other groups supplemented with 1.8%, 2.9% and markedly
improved compared with the control (Callan et al., 2012). According to Zakeri et al.
(2011), the study on seabream (Acanthopagrus latus) also showed that the diet
supplemented with high HUFA concentration (6.67%) had a higher hatching rate
(59.81±2.4%) than fish groups supplemented 4.26% and 2.92% (43.55±2.7% and
39.1±1.9%, respectively) (p<0.05).

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Nguyễn Thị Nguyệt Huệ, Hồ Sơn Lâm, Đặng Trần Tú Trâm, Đào Thị Hồng Ngọc*, Đinh Trường An,
Đỗ Hải Đăng, Đoàn Văn Thân

Figure 2. Effect of A1DHA Selco on egg loss rate.

Figure 3. Hatching rate (mean ± SE) of nemo fish.

3.4. Effect of A1DHA Selco on egg and larval quality
Diets supplemented with Selco at different levels did not affect the eggs diameter and the
size of the 3 days posthatch larvaes fish, but it did improve the hatching rate, the survival
rate of 3 days posthatch larvae fish, and decreased malformation rate (p<0.05) (table 2).
The diameter of nemo eggs was larger in the Selco 150 g/kg feed diet (2.49±0.081 mm) than
in the other groups (1.97-2.41 mm), but no correlation was found between the egg diameter
and dietary Selco levels (p>0.05). Similarly, HUFA supplementation in the broodstock diets
had no positive effect on larvae size.

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Table 2. Seed quality of nemo fish

Seed quality Selco supplementation (g/kg feed)
parameters
0 50 100 150 200
Egg length (mm) 1.97 ±0.486a 2.38± 0.084a 2.39 ±0.089a 2.49± 0.081a 2.41 ±0.054a
Egg width (mm) 1.6 ±0.408a 1.25 ±0.027a 1.33 ±0.118a 1.28 ±0.037a 1.22 ±0.006a
Larvae size (mm) 2.55 ±0.775a 3.44 ±0.009a 3.50 ±0.024a 3.51 ±0.009a 3.56 ±0.032a
Hatching rate (%) 92.45 ±0.231a 93.85 ±0.147b 94.73 ±0.189c 94.33 ±0.224bc 92.57 ±0.272a
Survival rate of
92.273±0.146ab 92.728±0,208bd 94.556±0.93c 93.072±0.158d 91.906±0.124a
larvae (%)
Larvae malformation
0.72 ±0.036a 0.60 ±0.025ab 0.61 ±0.022ab 0.55 ±0.022b 0.96 ±0.048c
rates (%)
Note: Letters after value on the same line showed significant difference (p<0.05).
The malformation rate of nemo fish in the experiment was significantly affected by the
concentration of Selco supplement (Fig. 4). The results showed that the malformation rate
of fish was lowest in the treatment supplemented with 150 g Selco (0.55%), followed by
the treatment 50 and 100 g Selco (about 0.6%) and this rate was quite high in the control
(0.72%) and the highest at Selco 200 g/kg feed (0.96%) (p <0.05). However, monthly data
analysis showed that the malformation rate of nemo fish did not differ much (Fig. 4). The
study on seabream (Acanthopagrus latus) showed that the fertilization rate and the
malformation rate of fish larvae were not significantly different between the
concentrations of HUFA n-3 supplemented (p>0.05) (Zakeri et al., 2011).
The survival rate of 3 days posthatch larvae was different between the experimental
treatments (p<0.05) (Fig. 5). The survival rate of 3 days posthatch larvae fish was highest in
the treatment supplemented Selco with 100 g/kg feed (94.55%), followed by the fish groups
supplemented with 50 and 150 g Selco/kg feed (92.72 and 93.07%, respectively) and the
lowest in the group supplemented with 200 g Selco/kg (91.90%).
According to the results of this research, it was shown that the optimal HUFA
concentration requirement for nemo broodstock to improve reproductive performance was
100 g/kg of feed (equivalent to fatty acids of 2.22%). The concentration of HUFA
supplemented for broodstock in this study may be different from other studies, this
difference may be influenced by species, experimental conditions.
Nutrition study (n-3 HUFA) for European seabream broodstock showed that egg quality,
hatching rate and survival rate of 3 days posthatch larvae of fish were improved with
increasing n-3 HUFA concentration 1.6%. Two nutritional studies of nemo broodstock
also showed that diets supplemented with vitamin E and astaxanthin also improved
hatching, survival and malformed rate of lavae (Dao et al., 2018; Nguyen et al., 2020).
Specifically, the optimal vitamin E concentration supplemented to broodstock nemo
showed the highest hatching rate, survival rate and lowest malformation rate (89.24%;
94.48%; 0.67%, respectively) (Dao et al., 2018). Similarly, 150 mg/kg feed of Astaxanthin

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Nguyễn Thị Nguyệt Huệ, Hồ Sơn Lâm, Đặng Trần Tú Trâm, Đào Thị Hồng Ngọc*, Đinh Trường An,
Đỗ Hải Đăng, Đoàn Văn Thân

supplementation also improved survival, hatching and malformed rate of lavae (92.14%;
93.57% and 0.55%, respectively) (Nguyen et al., 2020).

Figure 4. Effect of A1DHA Selco on larvae malformed rate.

Figure 5. Effect of Selco on survival rate of 3 days posthatch larvae.

Conclusions
The addition of different levels of Selco to the nemo broodstock feed before and during
spawning period did not affect the re-maturation time, egg diameter and size of the
broodstock nemo but improved significantly absolute fecundity, hatching rate,
malformation rate and survival rate of 3 days posthatch larvae fish.

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The present study showed that the suitable fatty acid concentration of A1DHA Selco
supplemented with 100 g/kg feed for nemo broodstock significantly affected reproductive
performance and egg quality.
Acknowledgments: The study was supported by the Vietnam Academy of Science and
Technology, grant number UDSPTM.04/21-22. This paper is a contribution to celebrate
the 100 years Anniversary of the Institute of Oceanography, Vietnam Academy of Science
and Technology.
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 HỘI NGHỊ BIỂN ĐÔNG 2022
Nha Trang, 13-14/09/2022

ẢNH HƯỞNG CỦA CHẾ ĐỘ ĂN BỔ SUNG SELCO ĐẾN HIỆU QUẢ SINH SẢN,
CHẤT LƯỢNG TRỨNG VÀ ẤU TRÙNG CỦA CÁ KHOANG CỔ NEMO
AMPHIRION OCELLARIS (CUVIER, 1830)
Nguyễn Thị Nguyệt Huệ, Hồ Sơn Lâm, Đặng Trần Tú Trâm, Đào
Thị Hồng Ngọc*, Đinh Trường An, Đỗ Hải Đăng, Đoàn Văn Thân
Viện Hải dương học, Viện Hàn lâm KHCNVN
*Email: [email protected]
Tóm tắt. Dinh dưỡng cho cá bố mẹ là một trong những lĩnh vực quan trọng trong nuôi trồng thủy
sản. Nghiên cứu được thực hiện để tìm ra ảnh hưởng của việc bổ sung HUFA (sản
phẩm thương mại A1 DHA Selco gọi tắt Selco, Inve) đến hiệu quả sinh sản, chất lượng
trứng và ấu trùng của cá khoang cổ nemo Amphiprion ocellaris (Cuvier, 1830). Thí
nghiệm được bố trí 5 nghiệm thức với 5 chế độ ăn có bổ sung Selco vào thức ăn cá bố
mẹ lần lượt là 0 g, 50 g, 100 g, 150 g và 200 g Selco/kg thức ăn. Mỗi nghiệm thức có
ba lần lặp, thời gian thí nghiệm là 12 tháng. Kết quả thí nghiệm cho thấy tần suất sinh
sản, thời gian tái thành thục sinh dục, kích thước trứng và ấu trùng không bị ảnh hưởng
của việc bổ sung Selco vào các chế độ ăn cá bố mẹ (p>0,05). Bổ sung Selco 50 và 100
g/kg thức ăn có tác động tích cực đến sức sinh sản tuyệt đối, tỷ lệ hao hụt trứng
(p<0,05). Tỷ lệ nở và tỷ lệ sống của ấu trùng 3 ngày tuổi ở nghiệm thức bổ sung Selco
100g/kg thức ăn là cao nhất và sai khác có ý nghĩa (lần lượt là 94,73±0,19% và
94,56±0,93%) (p<0,05). Tỷ lệ dị hình của ấu trùng cá ở nghiệm thức bổ sung Selco
150g/kg thức ăn là thấp nhất (0,55 ±0,022%) nhưng không có sai khác với nghiệm thức
bổ sung 50 và 100 g Selco/kg thức ăn. Nghiên cứu đã chỉ ra việc bổ sung Selco ở mức
100 g/kg thức ăn vào chế độ ăn cho cá bố mẹ có thể nâng cao hiệu quả sinh sản và chất
lượng ấu trùng của cá khoang cổ nemo.
Từ khóa: Amphiprion ocellaris, cá khoang cổ nemo, DHA Selco, HUFA

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