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 4°C 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 PO₄^3- 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 26-35^oC recommended by Dhert (1996). According to Nguyen Van Hai (2008) rotifers grow best at 28⁰C.

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).

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

• 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.

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.

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

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