Effects of Aeration Flow Rate in the Culture Medium on the Growth Performance and Egg Production of Copepod Oithona similis Fed with Fermented Organic Diet

. The availability of copepod Oithona similis as live food organism for shrimp and marine fish larvae is strongly influenced by the optimum feed and environment conditions. Optimization of dissolved oxygen (DO) for O. similis can be carried out by providing proper aeration in culture media. Feeding with fermented organic diet besides phytoplankton is expected to support the individual growth, metabolism, and reproduction of O. similis. The aim of this study was to examine the effect of different aeration flow rates on the growth performance and egg production of O. similis and to determine the optimum aeration flow rate. Completely Randomized Design Experiment used in this study with 4 treatments and 4 replications. The treatment was O. similis culture with different aeration flow rate of 0.00; 22.00; 45.67; 66.67 mL.second -1 . The results showed that the difference aeration flow rate significantly affected (p <0.05) the growth performance and egg production of O. similis . The aeration flow rate of 45.67 mL.second -1 provided the best growth performance with total density of 81.25 ± 2.99 ind.mL -1 ; population specific growth rate 0.220 ± 0.002 day -1 ; and egg production28.40 ± 0.48 eggs.ind -1 . the aeration flow rate culture for O. similis is


Preparation of fitoplankton culture and fermented organic feed
C. calcitrans was provided by the Life Feed Laboratory, Laboratory of Brackish Water Aquaculture Development Research Center (BWADRC) Jepara, Central Java, Indonesia. This phytoplankton was cultured at sterile sea water at 25-28°C, 28-30 ppt of salinity, and pH 8-9. The sea water was sterilized by adding 60 mg.L -1 of Sodium Hypochlorite (NaClO) for 10-30 minutes, and neutralized with 80 mg.L -1 of Sodium Thiosulphate (NaS2O3) for 24 hours with continuous aeration. The culture was carried out in 60 L plastic tube containing 40 L sterilized seawater using a modified Walne media at a dosage of 0.5 ml.L -1 with a 24 hours light photoperiod and at 1500 to 1800 lux and controlled aeration. The volume of inoculant of C. calcitrans was 10% from the total volume of medium culture (Lee et al., 1996). The density of C. calcitrans was observed every day by taking a sample of the microalgae and then counted under a microscope (Olympus CH20) 10x magnification with a hemocytometer (Improved Neubauer volume 0.0025 mm 3 ).
The fermented organic feed used the combination of organic materials of tofu flour, rice bran and fish flour in powder form with an average diameter range of 50-100 µm [4]. The content of fermented organic feed protein was 30%. The plastic tube was filled 10 mL of EM4 probiotic containing Lactobacillus casei, Saccharomyces cerevisiae, and Bacillus sp. Then, the mixture was filled with 200 mL sterilized water and 25 mL molasses for 1 kg of mixed organic feed. The incubation was conducted for 48 hours before being used.

Culture conditions of Oithona similis
O. similis was obtained from the culture collection of the Marine Culture Development Research Center MCDRC) in Lampung, Indonesia [3]. The O. similis culture was carried out in a 15 L of volume container with 8 L of sterile seawater at 19.44 ppt of salinity [3], at 27-28 °C, pH 8, with a innitial stocking density of 1 ind.mL -1 [8] for 20 days. The density of C. calcitrans given to O. similis was 0.01 mg.ind -1 [8]. Based on [4] research, the dosage of fermented organic feed was 0.5 g.L -1 of culture media with the combinations of 50% phytoplankton cell and 50% of fermented organic feed [9]. Aeration flow rate treatments were established at 0.00; 22.00; 45.67; and 66.67 mL. second -1 by using flow meter (Matheson FM-1050-VIA Part No. 7642T-603). The population of O. similis at the nauplii, copepodite, and adult stages was observed under a microscope (Olympus CYK41). Each stadia is determined based on its morphology [10].

Design of experiment
A completely randomized design with 4 treatments and 4 replications was carried out in this study. The treatments were A (O. similis cultured with an aeration flow rate of 0.00 mL.second -1 ) B (22.00 mL.second -1 ), C (45.67 mL.second -1 ), and D (66.67mL.second -1 ). The density of O. similis was calculated every 4 days to obtain the total population, growth performance, and egg production. Calculation of total O. similis density consists of nauplii, copepodites, adult and female eggs laying densities. Calculation of the amount of O. similis was observed carefully using a microscope, magnifying glass, Petri dish, and dropper drops with adequate lighting. Samples were taken from 100 mL O. similis culture from each treatment. Egg production was calculated by comparing the abundance of eggs and the number of females laying eggs. Calculation of the number of eggs was done by randomly isolating O. similis to lay eggs from each treatment (n = 2). The egg-laying female was observed under a 10x -40x magnification microscope. Egg abundance was calculated by multiplying the number of egg sacs by the average number of eggs per bag [11].

Data collection and statistical analysis
The density of O. similis was counted every 4 days during 20 days observations. Amount of 50 mL sub sample was taken from the culture medium to calculate the number of total O. similis, population specific growth rate (r) and egg production. Growth performance was calculated using a formula by [12]: Where r is population growth (day -1 ), Nt is the final density of O. similis, No is an initial density of O. similis and t is days needed to achieve maximum growth.
Egg production is the number of eggs produced by the female O. similis on average during their lifetime according to [11]: Where s is the amount of egg sac, e is the average amount of eggs per sac and n is the number of ovigerous females (ind).
Data were analyzed by One Way Analysis of Variance (ANOVA) to determine the effect of different aeration flow rate on growth performance and egg production of O. similis. Least Significant Different (LSD) test (α=0.05) by using SPSS 16 was conducted when the treatment had a significant effect. The optimum point of aeration rate flow is determined using polynomial orthogonal analysis with the MAPLE program 2016.

Water quality in the difference aeration flow rate of O. similis culture medium
The treatment of different aeration flow rates of O. similis culture media provides different dissolved oxygen content ( Figure 1). The measurement results of Dissolved Oxygen (DO) content in each treatment showed digression from the first day of culture to the last day of the observation (20 th day). The treatment of 0.00 mL.second -1 aeration flow rate showed the lowest DO (2.50 mg.L -1 ) compared to the other three treatments. The DO measurement results at the end of each observation were as follows: aeration flow rate of 66.67 mL.second -1 produced the highest DO (4.41 ± 0.06 mg.L -1 ), 45.67mL.second -1 aeration flow rate produced DO of 4.18 mg.L -1 , and 22.00mL.second -1 aeration flow rate produced DO of 3.74 mg. L -1 .    The different aeration flow rates had a different effect on the population of O. similis for 20 days of observation ( Figure 3). The 0.00 mL.second -1 aeration flow rate provides the lowest density compared to other aeration flow rates. 45.67 mL.second -1 aeration flow rate is proven to provide the highest density compared to aeration flow rates of 66.67 and 22.00 mL.second -1 O. similis density increased from the first observation, namely the 4 th day, and continued to increase until the 20 th day observation. The correlation between aeration flow rate and total density shows a quadratic relationship patterned equation y = -0.0307x 2 + 2.8801x + 19.752 with R² = 0.9668, which means that 96.68% of total O. similis density is influenced by aeration flow rate factors and 3.32% is influenced by factors other. The O. similis total density response curve for the aeration flow rate (Figure 4) shows the optimum point of the aeration flow rate at 45.90mL.second -1 .

Egg production of O. similis
The average histogram of O. similis egg production at the end of the observation (Figure 7) showed that culture medium with an aeration flow rate of 45.67 mL.second -1 gave the highest egg production (28.40 ± 0.48 egg.ind -1 ) which was significantly different (p <0.05) with all three other treatments. The aeration flow rates of 66.67 and 22.00 mL.second -1 generated egg production which was not significantly different (p> 0.05) but was significantly different from the aeration flow rate of 0.00 mL.second -1 (7.05 ± 1.84 egg.ind -1 ). The results of water quality measurement from each different aeration flow rate show a range which is still suitable for O. similis life. The correlation of aeration flow rate and DO showed a simple linear correlation, where DO content will increase in line with the increase in aeration flow rate in O. similis culture media (Figure 2). This proves that there was a closeness in the relationship between the flow rate of aeration and DO. The results showed that the difference in aeration flow rate has a significant effect on total density, specific population growth rate, and reproduction of O. similis. Treatment of C (aeration flow rate 45.67 mL.second -1 ) with DO 4.18 mg.L -1 produced the highest of total density, population specific growth rate and egg production among the other treatments. It was suspected that the magnitude of the aeration flow rate has provided environmental conditions that in accordance with the needs and supports the growth and reproduction of O. similis. In addition, the movement of water generated by the aeration flow rate provided suitable conditions for the movement of O. similis in the water column. The environmental conditions of culture media that did not support will affect the growth and SR of Oithona sp. [13]. In nature, the factors that influence the population of copepoda (Oithona sp.) beside feed availability [14] are fish larvae (predators), other copepods (competitors), and abiotic factors, i.e. temperature, DO and salinity [15].
The observation during the study showed that the difference in the aeration flow rate had a different effect on the water movement in the O. similis culture media. Physically, treatment B showed a quieter water movement, but DO was lower than treatment C and D. This will relatively affect the growth and reproduction of O. similis because oxygen was also needed by phytoplankton Chaetoceros calcitrans. The presence of fermented organic food (feed organic fermented) added will also affect the DO content in the O. similis culture media. It was predicted that in treatment B the DO content was still insufficient for O. similis to grow and reproduce.
Treatment D, water movement was too strong that suspected as the stress factor for O. similis. These stress conditions will affect the use of a certain amount of energy to recover [16]. Copepode O. similis required sufficient energy to maintain its life so that the energy that should be allocated for growth was disrupted. This strong water movement causes y = -0.0105x 2  deposits of organic feed and other impurities at the bottom to stir up. The movement of O. similis to defend itself from the flow due to the movement of a large aeration flow rate also reduces the energy that is supposed to grow and metabolize. Treatment A where culture media without aeration showed the lowest DO (2.50 mg.L -1 ). The DO content was still feasible for the life of O. similis even though it provided the lowest growth and egg production performance. [17] stated that copepods could no longer tolerate DO <1.1 ppm as well as [18] stated that the minimum DO concentration that was still tolerated by copepod was 2 ppm. This is in accordance with the results of the research by [19] that copepods from the species Calanipeda aqvaedulcis and Arctodiaptomussalinus can grow well with a range of DO 1-8 ppm.
The values of temperature, pH and ammonia content in O. similis culture media (Table 1) are presumed to support the life of O. similis especially for its growth and egg production. The measurement results of temperature, pH and ammonia during the study of all treatments were still at a reasonable level for the life of O. similis. The optimum temperature range of copepod is 25ºC [15]; 25-30ºC [33]. Temperature plays an important role in the rate of metabolism and egg production [14]. Temperature affects the size of the female, which also affects egg production [20]. [22] reported copepod nauplii grow well at pH 7.7-8.5 and could tolerate ammonia ≤ 1.3 ppm. Low temperatures will increase total body length and prolong the growth and maturation of copepods [34]. In addition, temperature also affects the quality of copepods' nutrition. As an example, PUFA (Polyunsaturated Fatty Acid) content will increase at low temperature [33].
Based on the specific population growth rate, the optimum aeration flow rate for O. similis culture was 45.70mL.second -1 . In the optimum conditions will provide suitable and preferred environment to the high density, population specific growth rate and egg production of O. similis.

Conclusions
The difference in aeration flow rate have a significant effect (p <0.05) on growth performance and egg production of Oithona similis where 45.67mL.second -1 aeration flow rate showed the best growth and egg production performance (total density of 81.25 ± 2.99 ind.mL -1 ; specific population growth rate of 0.220 ± 0.002 day -1 , and egg production of 28.40 ± 0.48 egg.ind -1 . Based on the specific population growth rate, the optimum aeration flow rate for O. similis culture was 45.70mL.second -1 .