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Culturing tiny rotifer Brachionus angularis with Chlorella

Nguyen Huu Yen Nhi, Nguyen Thi Bich Hanh1 and Trinh Thi Lan

Department of Aquaculture, Faculty of Agriculture and Natural Resources, An Giang University, Viet Nam National University Ho Chi Minh City, 18 Ung Van Khiem, Dong Xuyen ward, Long Xuyen city, An Giang province, Viet Nam
nhynhi@agu.edu.vn
1 Laboratory of An Giang University, Viet Nam National University Ho Chi Minh City, 18 Ung Van Khiem, Dong Xuyen ward, Long Xuyen city, An Giang province, Viet Nam

Abstract

The experiment on rearing Brachinonus angularis with dried Chlorella was conducted from May to August 2019 at An Giang University to evaluate the use of dried algae instead of fresh algae in rotifer culture as feed for shrimp larvae and small fish. The initial stocking density was 200 individual/ml and fed 60,000 individuals of algae per one rotifer. The four treatments in a randomized design with 3 replications were ratios of: cultured Chlorella (CC) and reconstituted Chlorella (RC) as follows: 75CC:25RC; 50CC:50RC; 25CC:75RC; 0CC:100RC. Environmental factors were monitored and adjusted to create favorable conditions for the development of the rotifers.

The rate of multiplication of the rotifers increased over the first 4 days subsequently declining to negligible numbers after 9 days. At all stages of the incubation the rate of mulitiplication of the rotifers decreased as the freshly cultured Chlorella was replaced by those reconstituted from the dried product. It is possible to use dried Chlorella algae to replace fresh Chlorella algae to feed B. angularis rotifers but results are better with the freshly cultivated algae.

Key words: algae, fish, hatcheries


Introduction

The rotifers commonly called wheel animals or wheel animalcules, make up a phylum (Rotifera) of microscopic and near-microscopic pseudocoelomate animals. Most rotifers are around 0.1- 0.5 mm long (although their size can range from 50 μm to over 2 mm). Some rotifers are free swimming and truly planktonic, others move by inchworming along a substrate, and some are sessile, living inside tubes or gelatinous holdfasts that are attached to a substrate. About 25 species are colonia, either sessile or planktonic. Rotifers are an important part of the freshwater zooplankton, being a major food source and with many species also contributing to the decomposition of organic matter. Zooplankton plays an vital role in the food chain of fish as animal food, which supply amino acids, fatty acids, vitamins, minerals, etc. (Watanabe et al 1983, Lubzens et al 1989, Dhont and Dierckens 2013, Dhert et al 2014). Brachionus, which is the most known form of all rotifers, serve as an idea starter diet for early larval stages of many fish and prawn species in marine as well as freshwater. Species of the genus Brachionus (Brachionidae: Rotifera) are well represented in different water bodies worldwide (Pejler 1977). Depending on the mouth size of the cultured organisms, small (50-100 micron length) or large (100-200 micron length) rotifers are used.

Brachionus angularis is a small-sized (86 μm) (Ogata et al 2011) and freshwater rotifer species (Mostary et al 2007). Its monogenetic reproduction, round shape and micro-algal diet are similar to those of the brackish water rotifer Brachionus plicatilis (Koiso et al 2009), which has been playing an important role in the field of saltwater aquaculture as a starter food for larval rearing. B. angularis has a smaller size than that of B. plicatilis (Fukusho and Iwamoto 1981), and only a few studies (Ogata et al 2010) have investigated the use of B. angularis as a live feed for larval fish.There have been many studies on rearing rotifers, mainly Brachionus plicatilis (Snell and Carrillo 1984). This species lives well in brackish water environment (Kostopoulou et al 2012) and the large size makes it unsuitable to use for freshwater fish. In larval rearing procedures for the endogenous cyprinid, silver barb (Hypsibarbus malcolmi) by using B. angularis as a live food is shown that H. malcolmi larvae feed on B. angularis, the larvae got 100% survival rate and increased in body size after feeding. This research also indicated that B. angularis not only has a morphologically edible size as a live food but also is nutritionally valuable for fish larval (Ogata et al 2010, 2011). B. angularis was also used as the starter food for the marble goby (Oxyeleotris marmoratus) (Senoo et al 1994). Fry of other species with the small mouth size such as striped catfish (Pangasius hypophthalmus), basa catfish (Pangasius bocourti), Malayan leaffish (Pristolepis fasciata), tire trackeel (Mastacembelus favus) and orange-fin loach (Botia modesta) also need the small live feed as B. angularis. Actually, freshwater rotifers are relatively less studied as a source of live feed, probably because small cladocerans and copepods are preferred as food items in freshwater aquaculture. However, development of culture techniques for freshwater zoo planktons with a smaller size of B. angularis than that of cladocerans and copepods have been useful in the culture of fish larvae with a small mouth size. The morphological characteristics of B. angularis make an appropriate live food for small-mouthed larvae of freshwater fish.

At the farm, fry fish have a very high loss rate during the first 30 days after stocking into the nursery pond. There are many reasons affecting the survival rate of fry, such as poor quality breed, incorrect management during transport and release of the fry, and losses caused by predators (wild fish, tadpoles ...), and especially lack of natural food sources or providing food sources not suitable for the small mouth size of the fry. Combining rotifers and other natural food sources may improve fry survival rate, thereby reducing costs and increasing income for producers.

Brachionus angularis is a small-sized freshwater species, suitable for the mouth sizes of many young fish (Ogata and Kurokura 2012). However, the cultivation of Brachionus angularis biomass has not been stable, and the biomass yield is not high enough to meet the needs of farmers rearing fry fish. According to Mostary et al (2007), Tran Suong Ngoc (2012) and Tran Thi Thuy (2017), rotifers B. angularis achieved the highest biomass when feeding fresh Chlorella. To achieve a high and stable biomass of chlorella for the consumption of B. angularis, there must be plenty of space and conditions for algae culture. Moreover, algae bloom and die when they reach a high density so sometimes there will be no fresh algae available for B. angularis. Therefore, this project evaluates the ability to use dry Chlorella as a substitute for fresh algae to produce B. angularis in order to help the rearing farms to be flexible in producing freshwater rotifer, and improve the biomass culture process of B. angularis to meet the natural feed demand of aquatic seed production.


Materials and methods

The experiment was conducted at An Giang University, Vietnam National University Ho Chi Minh City from May to August 2019.

Dried Chlorella algae were obtained from Hunan Nutramax Inc, China, the commercial name of which is Chlorella powder. They were first soaked in 100 mL water per gram to expand the algae, after that the density was determined. Fresh Chlorella were cultured in composite tanks (2m3) and in plastic bags (60 litres) with walne medium (modified from Laing 1991) (Photo 1). The algae were cultured for one week then collected by centrifugation and stored at 4C prior to feeding them to the rotifers (Brachinonus angularis).

Photo 1. Culturing Chlorella as feed for B. angularis

Twelve plastic containers (60 L) were fitted with an aeration system and arranged in a sheltered house to limit changes in temperature and avoid invasion of predators. At the beginning of the experiment, running water de-chlorination was supplied into the twelve rearing plastic containers. The environmental factors were checked and ensured that dissolved oxygen (DO) reached 2-3 mg/l; the aeration was adjusted to avoid strong air bubbles which can break the rotifer bodies.

Treatments and design

The four treatments in a randomized design with 3 replications were ratios of: cultured Chlorella (CC) and reconstituted Chlorella (RC) as follows:

75CC:25RC; 50CC:50RC; 25CC:75RC; 0CC:100RC

The addition of Chlorella in all the treatments was at the rate of 60,000 individuals per unit of B. angularis rotifer/day. The initial stocking density of B. angularis was 200 individuals/ml.

Photo 2. The breed of rotifer was multiplied (b) and cultured in twelve
plastic containers of the experiment (a)
Measurements

pH, temperature and concentrations of NO2-and PO43- were measured daily by test kits (Sera GmbH, Germany).

Multiplication of B. angularis was monitored every day by taking 3 samples from each container and counting the rotifer under the microscope (10X objective). The density of rotifers was calculated as:

…. N/v where N = total number of rotifers counted and v = volume of the sample

The density of rotifers was determined by transferring 1 ml of sample by pipette to a “Sedgwick Rafter” counting chamber. The rotifers were dyed with lugol solution (1g Iodine + 2g KI + distilled water to 100ml). Counts were made of the individuals that absorbed the dye. The rotifers that did not take the dye color (had died) and were not counted.

Statistical analysis

Growth data and water quality parameters were analysed using the General Linear Model (GLM) in the ANOVA program of the Minitab software version 16.2.0 (Minitab 2010).


Results and discussion

Environmentall factors

Water environmental factors did not vary among treatments during the cultvation of the rotifers (Table 1) and for temperature were were within the range of 26-35oC recommended by Dhert (1996). According to Nguyen Van Hai (2008) rotifers grow best at 280C.

Table 1. Environmental parameters during the experiment

Treatments

Temperature
(oC)

pH

NO2-
(mg/L)

PO43-
(mg/L)

75CC:25RC

26.4

8.53

-

0.45

50CC:50RC

26.6

8.53

-

0.45

25CC:75RC

26.6

8.50

-

0.45

0CC:100RC

26.6

8.50

-

0.45

SEM

0.103

0.030

-

0.028

p

0.462

0.793

-

1.000

According to Le Ngoc Ha (2009), Brachionus angularis grows best at pH 8 while Tran Ngoc Hai and Tran Thi Thanh Hien (2000) suggested the optimal pH for rotifers as between 7.5 and 8.5. Swimming and respiratory activity of rotifers are almost unchanged at pH range of 6.5 - 8.5 but declines at pH below 5.6 or above 8.7 (Nogrady 1993). Hoff and Snell (1987) suggested that the optimal pH range for B.calyciflorus was from 6.0 to 8.0 with the upper and lower limits of 9.5 and 4.5, respectively.

Nitrite (NO2-) was not detected during the experiment in accordance with requirements of B. angularis which is very sensitive to NO2- levels. PO4 3- did not vary across treatments.

The development of rotifer B. angularis

The initial density of the rotifers was set at 200/ml in accordance with the findings of Tran Suong Ngoc (2012). Their subsequent multiplication increased with time over the first 4 days subsequently declining to negigible numbers after 9 days (Figure 1). At all stages of the incubation the rate of mulitiplication of the rotifers decreased as the freshly cultured Chlorella were replaced by those reconstituted from the dried product (Figure 2).

Table 2. The growth of rotifers (individuals/ml) of B. angularis fed with Chlorella of different origins [freshly cultured (CC) or reconstituted from dried material (RC)] and incubated for up to 9 days

Time

75CC:25RC

50CC:50RC

25CC:75RC

0CC:100RC

SEM

p- value

Initial

200

200

200

200

-

ns

Day 1

313.7a

220.0b

212.3b

209.3b

3.51

<0.001

Day 2

714.3a

458.3b

416.3b

415.3b

18.8

<0.001

Day 3

1588a

801.7b

766bc

649.3c

32

<0.001

Day 4

2073.7a

1212.7bc

1246.3b

1065.7c

39.3

<0.001

Day 5

1940.3a

991b

986.3b

799c

38.3

<0.001

Day 6

1727.3a

766.7b

640.7b

647.7b

53.7

<0.001

Day 7

910.7a

355b

209c

233c

14.5

<0.001

Day 8

358a

195.7b

103b

140.3b

30.1

0.002

Day 9

51

30.7

16.3

21.3

8.7

0.089

abc Means within rows without common superscript differ at p<0.05



Figure 1. Effect of duration of the incubation on
multiplication of the rotifers
Figure 2. Effect of source of Chlorella (freshly cultured or reconstituted
from dried Chlorella) on multiplication of the rotifers


Conclusions


Acknowledgements

The authors are grateful for the financial support for this research from An Giang Department of Science and Technology and An Giang University, Vietnam National University-Ho Chi Minh. The authors would also like to thank the Department of Aquaculture, Faculty of Agriculture and Natural Resources of An Giang University for infrastructure support.


References

Dhert 1996 Rotifers, p. 49-78. In: Lavens, P. & Sorgeloos, P. (Eds.). Manual on the production and use of live food for aquaculture. FAO Fisheries Technical Paper 361. FAO, Rome.

Dhert P, King N and O'brien E 2014 Stand-alone live food diets, an alternative to culture and enrichment diets for rotifers. Aquaculture, 431, 59-64.

Dhont J and Dierckens K 2013 Rotifers, Artemia and copepods as live feeds for fish larvae in aquaculture. In: Allan G, Burnell G (Eds.), Advances in aquaculture hatchery technology. Woodhead Publishing. Cambridge.

Fukusho K and Iwamoto H 1981 Polymorphosis in size of rotifer, Brachionus plicatilis, cultured with various feeds. Bull. Nat. Res. Inst. Aquaculture, 2:1-10.

Hoff and Snell 1987 Plankton Culture Manual. Dade City, FL: Florida. Aqua Farms Inc.

Koiso M, Yoshikawa M, Kuwada H and Hagiwara A 2009 Effect of maternal diet on survival and life history parameters of next generations in the rotifer brachionus plicatilis sp complex. Nippon Suisan Gakkaishi 75, 828–833.

Kostopoulou V, Carmona M J and Divanach P 2012 The rotifer Brachionus plicatilis: an emerging bio-tool for numerous applications. Journal of Biological Research, 17.

Laing I 1991 Cultivation of marine unicellular algae. MAFF Laboratory Leaflet Number 67. Directorate of Fisheries Research Lowestoft, UK. 31pp.

Le Ngoc Ha 2009 Effect of pH on life cycle of freshwater rotifer ( Branchionus angularis). Bachelor thesis specialized in aquaculture, fisheries department, Can Tho University. (in Vietnamese)

Lubzens E, Tandler A and Minkoff G 1989 Rotifers as food inaquaculture. Hydrobiologia, 186(1), 387-400.

Minitab 2010 Minitab 16 Statistical Software 16.2.0.

Mostary S, Rahman M S, Hossain M A 2007 Culture of rotifer Brachionus angularis Hauer feeding with dried Chlorella. University Journal of Zoology, Rajshahi University. 26, 73-76.

Nguyen Van Hai 2008 Effect of pH and temperature on the development of freshwater rotifers ( Brachionus angularis). Bachelor thesis specialized in aquaculture, fisheries department, Can Tho University. (in Vietnamese)

Nogrady T 1993 Rotifera, SPB Academic publishing, 139p.

Ogata Y, Morioka S, Sano K, Vongvichith B, Eda K, Kurokura H and Khonglaliane T 2010 Growth and morphological development of laboratory-reared larvae and juveniles of Laotioan indigenous cyprinid Hypsibarbus malcolmi. Ichthyol 57, 389–397

Ogata Y, Tokue Y, Yoshikawa T, Hagiwara A, and Kurokura H 2011 A Laotian strain of the rotifer Brachionus angularis holds promise as a food source for small-mouthed larvae of freshwater fish in aquaculture. Aquaculture, 312(1-4), 72-76.

Ogata Y and Kurokura H 2012 Use of the freshwater rotifer Brachionus angularis as the first food for larvae of the Siamese fighting fish Betta splendens. Fisheries science. 78, 109-112.

Pejler B 1977 On the global distribution of the family Brachionidae (Rotifera). Arch. Hydrobiol. Suppl., 53:255-307.

Senoo S, Kaneko M, Cheah S H, Ang K J 1994 Study of artificial seed production of marble goby.1. Egg development, hatching, and larval development of marble goby Oxyeleotris marmoratus under artificial rearing conditions. Fish. Sci. 60, 1–8.

Snell T W and Carrillo K 1984 Body size variation among strains of the rotifer Brachionusplicatilis. Aquaculture. 37: 359-367.

Tran Ngoc Hai and Tran Thi Thanh Hien 2000 Lecture on techniques for raising natural food. Can Tho University. (in Vietnamese)

Tran Suong Ngoc 2012 Study on biological characteristics, rearing and using freshwater rotifers (Brachionus angularis). Doctoral thesis of Fisheries, Can Tho University. (in Vietnamese).

Tran Thi Thuy 2017 Culturing freshwater rotifers (Brachionus angularis) used as initial food for aquaculture farmers. Project of Science and Technology department in An Giang province.

Watanabe T, Tamiya T, Oka A, Hirata M, Kitajima C, Fujita S 1983 Improvement of dietary value of live foods for fish larvae by feeding them on ω3 highly unsaturated fatty acids and fat-soluble vitamins. Nippon Suisan Gakkaishi 49:471–479


Received 27 March 2020; Accepted 13 April 2020; Published 1 May 2020

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