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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.54 No.6 pp.91-98
DOI : https://doi.org/10.14397/jals.2020.54.6.91

Effect of Different Culture Conditions on Nutritional Value of Moina macrocopa as a Live Feed for Fish Fry Production

Therese Ariane Neri1, Zuliyati Rohmah2, Bernadeth F. Ticar3, Byeong-Dae Choi1*
1Department of Seafood and Aquaculture Science/Institute of Marine Industry, Gyeongsang National University, Tongyeong 53064, Republic of Korea
2Faculty of Biology, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
3College of Arts and Sciences, Iloilo Science and Technology University, Burgos St., La Paz, Iloilo City 5000, Philippines
*Corresponding author: Byeong-Dae Choi Tel: +82-55-772-9142 Fax: +82-55-772-9149 E-mail: bdchoi@gnu.ac.kr
January 16, 2020 ; December 10, 2020 ; December 21, 2020

Abstract


The effects of supplementing ESP-FM (Erythrobactor sp.), freshwater Chlorella (Chlorella sp.), and baker’s yeast (Saccharomyces cerevisiae) on the nutritional value and mass production of Moina macrocopa, which is used as a live feed for fish fry production, was investigated. Consequently, the effects of feeding the enriched M. macrocopa to the nutritional composition of larval rockfish (Sebastes schlegel) and carp (Cyprinus carpio) was also investigated. Maximum density of M. macrocopa was reached within 15-21 days after inoculation (0.5 to 22 individual/mL), at various temperatures, and either decreased or remained almost constantly thereafter. Protein content and amino acids composition of M. macrocopa were found to be influenced by their respective diets while lipid and ash contents did not considerably change. M. macrocopa fed with baker’s yeast were low in n-3 HUFA content, and those fed on the freshwater Chlorella were high in the 18:2n-6 and 18:3n-3 HUFA content, and in cultures treated with ESP-FM were high content in n-3 HUFA. The utilization of M. macrocopa as a substitute fish feed for carp and rockfish showed the enrichment nutritional content.



초록


    Introduction

    Traditional live feeds used in fish culture has been lacking essential fatty acids and amino acids, particularly, docosahexaenoic acid (DHA) which is an essential fatty acid required for fish embryonic development (Cahu et al., 2003;Aragao et al., 2007). Fish fry production has always depended on the seed production of Artemia to provide necessary nutrition. Thus, study on alternative live feed for fish fry production is needed (Uppanunchai et al., 2015). Moina macrocopa, a Cladoceran, has been gaining attention as substitute for Artemia in the post-larvae production of freshwater prawns and fish fry (Alam et al., 1993;Qin & Culver 1996) due to M. macrocopa’s adaptability to the changes in culture condition and predation (Kushniryk et al., 2015;Manklinniam et al., 2018). For Cladocera to be efficient as a feed for larval fish, nutrient enrichment is necessary. Food, along with ambient culture temperature (Farhadian et al., 2012), is important factor for Cladoceran culture because their biochemical composition changes accordingly with their diet (Olsen et al., 1997). Correspondingly, nutritional requirements of fish larvae can be supplied with specific essential amino acids and fatty acids from nutrient-cultured M. macrocopa (Yoshimatsu et al., 1997;Koven et al., 2001;Kotani et al., 2016). In this study, we evaluated the use of M. macrocopa as potential nutrientenrichment dietary source for carp and rockfish. To assess the nutritional and dietary values, M. macrocopa was first cultured with different feeding treatments and under different temperatures. Growth rate, proximate composition, mineral contents, amino acids, and fatty acid compositions of M. macrocopa fed with different diets were inspected. Lastly, changes in fatty acid composition of rockfish and carp fish larva fed with the cultured M. macrocopa with optimum nutritional quality were assessed to check their nutritional relationship.

    Materials and Methods

    1. Monitoring and assessing M. macrocopa’s populations

    M. macrocopa were cultured with ESP-FM (Erythrobacter sp. Sπ-1, Livefood Laboratory, Je-Eun Co., Pusan, Korea), concentrated freshwater Chlorella (Chlorella sp.), and baker’s yeast (Saccharomyces cerevisiae) to investigate the best feeding condition of M. macrocopa culture. Adult M. macrocopa were stocked at 3 individual/mL. ESP-FM, concentrated Chlorella, and yeast (1.5 kg/20 m3 of water) were fed at 350 mL/day/ton (1 individual/mL) three or four times a day, respectively. To determine the optimum culture temperature, M. macrocopa were cultured in separate aquariums set at different temperatures, ranging from 18°C to 31°C. The Moina cultures were inspected daily for 21 days to determine the health of the M. macrocopa. A dissecting microscope was used to check the density of M. macrocopa. This process was repeated 10 times.

    2. Experimental fish and diets

    M. macrocopa culture with optimum nutritional quality composition from the first experiment was used as live feed of carp and rockfish. The live M. macrocopa were harvested from two 8 m3 tanks. The tanks had no aeration and no water exchange. The depth of water was monitored at 20 cm throughout the culture period. Each day, about 5 kg of M. macrocopa were collected using plankton net (55 μm mesh) and fed to fish two or three times a day. When densities reached 18 individual/mL and above, small amount of water was added to maintain the rate of growth. After 12 days, the M. macrocopa were inoculated to the next tank. Larval rockfish (Sebastes schlegeli Hilgendorf, 1880) and juvenile of carp (Cyprinus carpio L., 1758) were used in the feeding experiments. The juvenile carp was fed with M. macrocopa 3 days after complete absorption of egg yolk, while the larval rockfish was fed with M. macrocopa 20 days after hatching and after rotifer feeding was completed. The fry feeding experiment was conducted in a circular aquarium with a capacity of 8 m3. The tank is provided with overflow and continuous supply of clean water.

    3. Moisture, protein and lipid analysis

    The crude lipid content was determined according to Bligh & Dyer (1959) method, crude protein content (semi-micro Kjeldahl method) and total moisture (oven-drying method) of M. macrocopa were determined using AOAC (2009) methods 920.153 and 930.15, respectively.

    4. Amino acids analysis

    All samples were freeze-dried for 24 hr and milled. One-gram sample from each batch was hydrolyzed in flame sealed Pyrex tubes for 16 hr at 110℃ in 10 mL 6 N HCl under nitrogen. Hydrolyzed samples were filtered with 3G-4 glass filters and dried by rotary evaporator under vacuum. The residue was then diluted with deionized water and the evaporation was repeated three times. The residue was dissolved in citric acid buffer with a pH 2.2 for amino acid analysis (Biochrom 30 plus, Biochrom GmbH, Berlin, Germany).

    5. Ash and mineral analysis

    Two to three grams of the dried defatted samples were weighed (to 0.1 mg) porcelain dishes. Samples were charred at 250℃ for 1 hr, then ashed at 550℃ for 4 hr. After cooling, 1 mL 50% HNO3 was added. The sample was again charred, then ashed at 550℃ for 1 hr. The process was repeated until the ash is completely white. A blank was carried out to determine residue from HNO3. The ash was dissolved in deionized water, and then diluted to 100 mL in a volumetric flask. The diluted solution was then analyzed for 8 elements with an ICP-MS spectrometer (OPTIMA 5300DV, PerkinElmer, Waltham, MA, USA).

    6. Lipids extraction and preparation of fatty acid methyl esters

    Total lipids were extracted using the Bligh & Dyer (1959) method and determined gravimetrically. For a 50 mg of lipid, 1 mg internal standard (23:0 methyl ester), 1.5 mL 0.5 N NaOH solutions were placed in a screw-capped Teflon-lined tube. The tube was flushed with nitrogen, sealed, and heated for 5 min at 100℃. After cooling, 2 mL 12% BF3 methanol was added and blanket with N2. The tube was capped tightly, mixed, and heated for 20 min at 100℃. After cooling to 30-40℃, 1 mL isooctane was added, blanketed with N2, capped, and vortexed. The isooctane layer was separated into another tube. The aqueous layer was again extracted two times with 1 mL isooctane each. The combined isooctane extract was washed once with 3 mL water, dried over anhydrous Na2SO4, and concentrated for gasliquid chromatography.

    7. Gas-liquid chromatography (GLC)

    The fatty acids composition of the fatty acid methyl esters (FAME) were determined by GLC using a Shimadzu 17A (Shimadzu Co., Tokyo, Japan), fitted with FID and a flexible fused-silica open tubular column (30 m × 0.32 mm i.d., Omegawax-320). The injector and detector were held at 250 and 270℃, respectively, the column with the following program of column temperature: initially 185℃ for 8 min, then an increase in temperature of 3℃ per min to 230℃, and a final hold for 13 min. Helium was also used as the carrier gas at a pressure of 1.0 kg/cm2 with split ratio 1:50. The fatty acids were identified by comparison with ECL (equivalent chain length) values of standard FAME analyzed under the identical conditions, or by comparison with menhaden oil FAME (Choi et al., 1999).

    8. Lipid class determination

    The MK6 Iatroscan thin-layer chromatography/flame ionization detection (TLC/FID) analyzer (LSI Medience Co., Tokyo, Japan) was utilized for the quantitative analysis of the lipid classes from each sample after separation on silica-gel coated Chromarods-S III (Ackman, 1981). The general procedure was modified for partial scan and redevelopments as shown by Parrish (1987). One to 5 μL of a chloroform solution of the sample was applied to the Chromarods with Drummond disposable micropipets. After spotting, rods were placed in a constant humidity tank over a saturated sodium chloride solution for 10 min and then transferred immediately to the developing of solvent systems to enhance separation of different lipid classes. Total lipid on the Chromarod-S III was developed with hexane/diethylether/ formic acid (97:3:1, by vol) for 55 min. Developed rods were placed in an oven at 110℃ for 3 min to evaporate the solvents and then transferred to the scanning frame of the Iatroscan analyzer for each partial or complete scan.

    9. Statistical analysis

    Data are expressed as the means ± standard deviation (SDs). The statistical test was conducted using analysis of variance with SAS software for Windows (SAS ver. 9.2, SAS Institute, Cary, NC, USA). Duncan’s multiple-range tests were used to compare differences between samples (p<0.05).

    Results

    1. Population densities of Moina at various temperature and diets

    The influence of temperature on M. macrocopa culture are shown in Fig. 1. The slowest growth rate was observed at 18℃, with its maximum density at 22 individual/mL after 21 days of cultivation. The growth rate of M. macrocopa at 22℃ was slightly higher than at 18℃, showing 1.6 individual/mL after 5 days of culture. At the beginning, the culture in 31℃ has similar growth rate as the culture in 28℃, which began to decrease after reaching the maximum density of 15.2 individual/mL. The growth rate at 28℃ was higher than at 25℃ at the early stage of cultivation, showing the density of 20.5 individual/mL after 17 days while M. macrocopa population at 25℃ reached to 20.1 individual/mL after 19 days. The growth rate alternately increased or decreased depending on the period of culture.

    The effect of different diets on the growth rate of M. macrocopa is summarized in Fig. 2. After inoculation of 3 individual/mL in aquarium of 30 L capacity, the organisms were withdrawn 10 times at regular intervals using a pipette of 10 mL. The density per mL was averaged. The population density of M. macrocopa in the ESP-FM, Chlorella, and yeast diet progressed in increments. In the ESP-FM-fed aquarium, the density reached 21 individual/mL within 10 days from the start of feeding. The lowest growth rate was observed in culture fed with yeast. After 11 days of cultivation, the growth rate slowed down as population density decreased.

    2. Nutritional value of M. macrocopa

    The proximate composition of the M. macrocopa fed with Erythrobacter sp., freshwater Chlorella, and baker’s yeast are shown in Table 1. The crude lipid and ash content were similar for all samples. Table 2 demonstrated the mineral content of M. macrocopa. The three diets showed almost similar ash content at 1.02-1.03 g/100 g sample. Interestingly, the different diet experiments showed the different quantities of the minerals. Calcium and sodium content were relatively higher in cultures with yeast diet, 138.9 ppm, and about 7 times less in the ESP-FM diet, 19.4 ppm. Mg, K, Mn, Sr, and Zn are relatively low. In the Chlorella diet, the Zn content is relatively higher at 8.0 ppm than the other two diets. The phosphorus content was observed to be in the range of 4.4-5.6 ppm in all diets.

    Table 3 showed the changes in amino acids content of M. macrocopa growth corresponding to the different diets. The ESP-FM diet showed the highest total amino acids content at 3,639 mg/100 g sample. Chlorella and yeast diets resulted in 3,526 and 3,382 mg/100 g sample, respectively. Table 4 displayed the fatty acids composition of M. macrocopa fed with different diets. Fatty acid is a component of the cell wall and a key effect on its growth. The 16:1 and 18:1 fatty acid content were high in the yeast diet, while 18:2n-6 and 18:3n-3 fatty acid contents were prominent in the Chlorella diet. The present study showed that ESP-FM has the highest content of highly unsaturated fatty acids (HUFA) at 13.90%, but Chlorella has 0.40%, and yeast diet has 0.35%.

    The lipid class composition of M. macrocopa cultured as the live feed is shown in Fig. 3. The lipid fraction was composed of triglyceride (TG), cholesterol (CHOL), phospholipid and pigments (PL), and free fatty acids (FFA). The highest TG content was found in culture with the ESP-FM diet, at 19.0%, while the lowest was observed in the culture with Chlorella treatment diet, at 5.0%. Meanwhile, the lowest PL content was noted in culture with ESP-FM diet at 71.1%, while both Chlorella and yeast diet cultures both reached around 80%.

    3. Fatty acids composition of rockfish and carp larval

    Artemia is widely used as live feed, but without enrichment, it would lack of DHA and n-3 HUFA contents and become unsuitable as a live food. Table 5 showed the comparison of the fatty acids composition of the tissues of wild rockfish and rockfish fed by unenriched Artemia, enriched Artemia, and ESP-FM. The rockfish fed with unenriched Artemia contained 3.9% of DHA and 13.1% of n-3 HUFA, contrary to the report of Harel & Place (1998) that wild Artemia does not contain DHA. The rockfish fed with enriched Artemia contained DHA and n-3 HUFA equivalent to 8.4% and 17.2%, respectively. Those fed with ESP-FM cultured M. macrocopa had 12.6% of DHA and 23.2% of n-3 HUFA, which approximates the 12.0% DHA and 20.5% n-3 HUFA found in the tissues of wild rockfish. These results were confirmed by the remarkable agility and decreased infection and deformity of the cultivated rockfishes.

    The fatty acids composition in the tissues of juvenile carps is shown in Table 6. The linoleic acid (18:2n-6) content of the carp fed with commercial diet was very low at 1.9%, and the n-3 HUFA content (including DHA and EPA) was very high at 18.1%. It was presumed that the commercial diet was fortified with fish oil. In the carp fed with M. macrocopa with ESP-FM diet, the oleic acid (18:1n-9) was lower than the carp with natural diet. The EPA, DHA, and n-3 HUFA contents of carp fed with M. macrocopa were slightly higher than natural diet carp.

    Discussion

    M. macrocopa are resistant to extreme temperature and can easily withstand a daily variation from 5 to 31℃ (41-88⊞F). The higher temperature tolerance of M. macrocopa is a great advantage to aquacultures of live feeds at home and commercial tropical fish farmers who maintain their hatcheries above 18-2 2℃ (64-72⊞F). Ivleva (1969) said that their optimum temperature is 24-31℃. Therefore, for succeeding culture trials were set as 25℃ optimum temperatures. The optimum temperature of M. macrocopa was suitable with rockfish rearing temperature as describe by Mizanur and Bai (2014). A comparison of the production of Daphnia magna and M. macrocopa cultures fertilized with yeast and ammonium nitrate showed that the average daily yield of M. macrocopa was higher than Daphnia (Ivleva, 1969). Rice bran is a potential feed for Moina since it contains various nutrients such as protein (12-13%), lipid (16-20%), linoleic acid (6.35-6.85%), α-linolenate (0.2-0.27%), vitamin B, and minerals (6-9%), which are dominated by calcium and iron (Faria et al., 2012). Different culture condition of M. macrocopa showed various nutritional contents of M. macrocopa cultures. In contrast to their tolerance of low oxygen, Daphnia are highly sensitive to disturbances of the ionic composition of the culture medium, especially changes in the concentration of some cations (Hoff and Snell, 1997). The amino acids content determines the quality of protein source and its function. For example, the protein quality of algae is regarded as very good because its essential amino acid profile is close to that of the cultured fishes (Watanabe et al., 1983). The M. macrocopa culture in Chlorella diet proved the methionine and arginine contents were low and the lysine content was high, and in the yeast diet, the methionine content was low. On the other hand, the essential and non-essential amino acids were evenly contained in the ESP-FM diet, thus, the ESP-FM is regarded a suitable live food. Lysine concentration in M. macrocopa fed with cassava bran suspension (3.33 g/100 g protein) was lower compared to that of M. macrocopa fed with rice bran suspension (6.39 g/100 g protein), indicating a lysine deficiency that caused a decrease in embryo growth in the embryonic developmental cavity (Li et al., 2009). In fish embryos and larvae, however, depletion rates of both non-EAA and EAA are comparable, indicating no sparing selectivity between the metabolism of EAA and non-EAA. Therefore, larval fish dietary proteins need to contain higher proportions of EAA in order to compensate for their loss during metabolism.

    Fatty acids composition of feed is important to the survival and growth of intensively cultured fish fry (Kim et al., 2014). The Omega-3 highly unsaturated fatty acids (n-3 HUFA, above 20 carbons), are essential for the survival and growth of many species of fish. In Cladocera, it plays a role in increasing somatic growth, while HUFAs play important roles in reproduction, growth performance, and survival (Fereidouni et al., 2013). The high HUFA content of ESP-FM fed M. macrocopa is an indication that it is the most effective for the growth of M. macrocopa. Chlorella and yeast are inferior to ESP-FM because they lack the essential fatty acid DHA and that their fatty acid compositions were limited. The lack of natural DHA in live feeds is the reason why the feeds for mass culture of rotifer, Artemia and Moina are fortified with various nutrients. However, since these fortifiers did not give out any substantial effects, an easily digested lipid was used to enrich these live feeds. Live food rich in n-3 HUFA increase the survival rate of the juvenile fishes. In the absence of dietary n-6 and n-3 fatty acids, larval growth, learning, and visual acuity are impaired and severe structural and metabolic disorders can take place (Wacker & Creuzburg, 2007;Fereidouni et al., 2013).

    As the fatty acids contained in TG was mostly used as an energy source, the highest TG content in the ESP-FM diet is related to the fast growth rate of the live food in the ESP-FM diet. The CHOL content was relatively high in the Chlorella diet. Copeman et al. (2002) reported that feeding with rotifers having a low DHA/EPA ratio resulted in lower growth of gilthead sea bream and yellowtail flounder larvae. Therefore, the lower DHA/EPA ratio of frozen and enriched M. macrocopa might have caused the lower growth and stress tolerance of larvae. The primary beneficial effect of phospholipid was improved growth in both larvae and early juveniles, but also increased survival rates and decreased incidence of malformation in larvae, and perhaps increased stress resistance (Tocher et al., 2008). Copepods generally have higher proportion of polar lipids than Artemia, the latter being rich in triglycerides. So, not only are copepods a rich source of EFA, the EFA are also generally present as polar lipids. They are believed to be advantageous in terms of their uptake and assimilation by fish larvae (Kanazawa et al., 1983). According to the amino acid and fatty acid contents of the M. macrocopa treated with different diet, the M. macrocopa fed with ESP-FM was the best nutritional content of M. macrocopa cultures in this experiment.

    Fish culture in seawater requires a brackish water fish food, which can withstand the salt concentration in seawater, or a species that can adapt to seawater. On the other hand, the seed production of seawater fishes requires a brackish water tolerant M. macrocopa so that it can stay alive until it is eaten by the fry (Gordo et al., 1994). Several species of M. macrocopa were observed in a wide range of salinities. M. rectirostris and M. macrocopa were observed in up to 4 ppt (Ivleva, 1969). In this experiment M. macrocopa was used as rockfish larval feed and increased the nutritional content of the fish larva. M. macrocopa can survive in the rockfish tank salinity and can stay alive until being eaten by fish larva. M. macrocopa fed with ESP-FM showed a good promise as a complete substitute for Artemia in larval culture of rockfish. Carp was also used as experimental subject in determining the nutritional efficiency of M. macrocopa as a live feed. Carp is freshwater fish, which is most widely cultivated in Korea. Carp larvae are fed with wild M. macrocopa for 3-4 days upon complete absorption of egg yolk post hatch. Carp larvae fed with M. macrocopa has higher nutritional content than wild carp. It is therefore concluded that the ESP-FM cultured M. macrocopa should be the suitable live food for the juvenile carp. Regarding to the fatty acid composition of a juvenile carp, the content of 18:1n-7 was very high (32.01%), the contents of EPA, DHA, and other fatty acids also varied.

    The use of M. macrocopa as a live fish food can enrich the nutritional content of larval carp and rockfish. Lipid content of carp and rockfish fed with M. macrocopa showed higher value than natural fish. Omega-3 HUFA content of rockfish fed with M. macrocopa were higher in value than the one fed with Artemia. The high fatty acid composition of carp and rockfish showed that utilization of M. macrocopa as live fish food is better than the use of commercial feed.

    Figures

    JALS-54-6-91_F1.gif

    Population density of M. macrocopa fed with ESP-FM on various temperature.

    JALS-54-6-91_F2.gif

    Population density of M. macrocopa fed with ESP-FM, freshwater Chlorella and baker’s yeast at 25℃.

    JALS-54-6-91_F3.gif

    Lipid class of Moina macrocopa fed with ESP-FM, freshwater Chlorella and baker’s yeast at 25℃. FFA, free fatty acid; TG, triglycerides; CHOL, cholesterol; PL, phospolipids and pigments.

    Tables

    Proximate composition of M. macrocopa fed with ESP-FM, freshwater Chlorella and baker’s yeast (wet basis, %)

    Whole-body ash and mineral composition of M. macrocopa fed with ESP-FM, freshwater Chlorella and baker’s yeast

    Amino acids composition of M. macrocopa fed with ESP-FM, freshwater Chlorella and baker’s yeast

    Fatty acids composition of M. macrocopa fed with ESP-FM, freshwater Chlorella and baker’s yeast (%)

    Comparison of fatty acids profile of larval rockfish1 fed with different live feed (%)

    Comparison of fatty acids profile of juvenile carp1 fed with different diets (%)

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