Biotic and Abiotic Factors Affecting Productive Performance of Rotifers (Brachionus Plicatilis) Population Produced Under 3 Days Batch Culture System

INTRODUCTION

Rotifer (Brachionus plicatilis) is a cyclically parthenogenetic organism that appears regularly and typically in saltwater lakes and coastal lagoons. Several studies revealed that B. plicatilis was a complex species and at least three sympatric species (Brachionus plicatilis, Brachionus rotundiformis and Brachionus ibericus) have been observed. The two species B. plicatilis and B. rotundiformis are used worldwide, alone or in conjunction with other types of food to feed early developmental stages of marine finfish and crustaceans under hatchery conditions. Further, rotifer transmits adequate supplies of micro and macro-nutrients, vitamins and even antibodies to fish larvae (Lubzens et al., 2001). However, among them B. plicatilis was widely used as live feed in aquaculture. Moreover, B. plicatilis are currently essential for intensive culture of marine larval fish in many hatcheries throughout the world (Fielder, et al., 2000). Rotifer size varies in response to different environmental conditions. The fish larvae select the rotifers according to their size, and this resulted in increasing growth rate, and reduces mortality (Qie et al., 2011).

Diet is regarded as the most important criterion that could affect growth and quality of rotifers. Moreover, B. plicatilis cultures are often influenced by variety of food types as diets. Rotifers density, production, fecundity, and growth rate were dependent on food availability and quality (Ferreira et al., 2011). Microalgae are the first found to be an excellent food that could provide best rotifer quality and quantity for marine fish larvae (Yin and Zhao, 2008). The microalga Chlorella, Nannochloropsis, and Tetraselmis have often been used as exclusive food in culturing B. plicatilis (Lubzens et al., 1997). However, Ashour (2011) reported that Nannochloropsis may be the best microalgal species used as rotifer feed. Rotifer quality is often enhanced by different enrichment techniques. For example, it has been suggested that the replacement of baker’s yeast supplemented with oils rich in n-3 HUFA can be used as an alternative food for rotifer culture. However, using yeast could deteriorate water quality (Nhu, 2004).

A large number of researches have been reported that temperature, salinity and food types have variable effects on productivity of different strains of rotifers (Miracle and Serra, 1989, Fulks and Main, 1991). On the other hand, the density of rotifers in the water column has a significant effect on the feeding success of marine fish larvae by influencing the probability of encounter (Fielder et al., 2000).

            The aims of the present study were to:

1-  Investigate the effect of different algal diets (Nannochloropsis oculata or Spirulina platensis) as a single diet or a supplemented diet with baker’s yeast (Saccaromysis cervicates) on rotifer population, as increase in number.

2-  Investigate the effect of different water salinities, temperatures, and initial rotifer culture densities on rotifer population growth during a batch culture of rotifer (72 hrs.).

MATERIAL AND METHODS

MATERIALS

The experiments were conducted in Fish Rearing Laboratory, El-Max Research Station, Aquaculture Division, National Institute of oceanography and Fisheries (NIOF) Alexandria, Egypt. The culture water used in these experiments were obtained from a saline ground water (30±2 ppt).The rotifers B. plicatilis (L - type with average length ~ 200 μm) were obtained from a routine culture at the marine Finfish hatchery of NIOF. The present study was divided into two phases using patch culture system, each one was continued for 72 hrs. The necessary dilution to the desired initial density of rotifer was accomplished using sterilized and filtered seawater. The algal cell density was determined with a haemocytometer and the rotifer density with a 1- ml glass pipette using Sedgwick-Rotifer chamber. Rotifer culture treatments were conducted in special jars (30 liters) with three replicates for each treatment .

Phase1: study the effect of different food type on rotifer productive performance and its fatty acids content:

Four treatments were conducted, from different algal species and yeast, as food for rotifer; (1) Nannochloropsis oculata (N) at 15 X10⁴ cell/ind/day (Wendy and Kevan 1991), (2) Nannochloropsis oculata + dried yeast (NY) at 0.5 g/10⁶ ind., (3) dried Spirulina (Spirulina platensis) (S) at 0.5 g/10⁶ ind.and (4)  dried Spirulina (0.5 g/10⁶ ind )+dried yeast  (SY) (0.5 g/10⁶ ind).  The culture conditions during the experiment were: salinity 25 ppt, temperature 30^oC, pH 7.8 and desolved oxegen(DO) was 4 mg/l. The initial rotifer density was conducted at 100 ind. /ml (fulks and main, 1991).

Phase 2: Effect of salinity, temperature and initial density on rotifer productive performance:

Depending on the results of the  phase 1, the treatment NY which possessed the highest rotifer final number (population), as well as achieved the highest polyunsaturated fatty acid, Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) percent.  Accordingly, this treatment was selected and candidate to investigate the effects of different salinity, temperature and initial density on rotifer productive performance.

Salinity experiment was conducted at three levels; 25, 27, and 29 ppt. Each level was tested under controlled conditions of temperature 26±1°C, DO 4 mg/l, and pH 7.8. The initial rotifer density was conducted at 100 ind. /ml.

Temperature experiment was conducted at four levels; 26, 28, 30, 32 ^oC. Each level was tested under controlled conditions of salinity 25 ppt, DO 4 mg/l, and PH 7.8. The initial rotifer density was conducted at 100 ind./ml.

The previously recorded optimum water quality parameters of Salinity 25‰, temperature 28 ^oC and the best food type of NY were subsequently provided to test the best initial rotifer cultured densities from between 50, 100, 150 and 200 ind. /ml.

Experimental methodology:

Productive performance of rotifer: At the end of the experiments, as well as at the end of every day during experiments, 1 ml of rotifer culture from each treatment was collected five times and counted to determine the average

final number of rotifer, as a population of rotifers B. plicatilis (Ashour, 2011).

Fatty acids analysis: Preparation of fatty acids methyl ester from total lipids was performed according to the procedure of Radwan (1978). All analysis for identification of fatty acids fractions were performed on gas chromatography instrument, model HP (Hewlett Packard) 7890 GC equipped with a flame ionization detector.  The conditions for fatty acid analysis were:

Water quality: During this experiment, temperature was kept at 30±0.5°C, salinity 25 ppt, and pH 7.45 – 7.8. Dissolved oxygen concentrations was 4 mg/l at the starting time and decreased after three days of culturing to 2.3, 2.4, 2.6, and 2.4 mg/l for treatments, S, SY, N, and NY, respectively. Total ammonia concentrations at the starting time varied between 0.54–0.55 mg/l and increased after three days of culturing to 0.78, 0.76, 0.78, and 0.77 mg/l for treatments, S, SY, N, and NY, respectively.

Statistical Analyses:

At the termination of the experiments, Total Number of Rotifer (millions) in days 1, 2, and 3 , Final Density (ind/ml), Total Increment of rotifer (Millions in 3 days/30 litres), Average Daily Increment (Millions/30 litres/ day), and Average Increment per day (%) were subjected to One-way analysis of variance (ANOVA) using the software Package (SPSS Version 16) to test the significance between treatments (water temperature, water salinity, feeding types, and rotifer density). The differences between Rotifer culture system treatments were performed using LSD test with 0.05 significance level according to Steel and Torrie (1980).

Growth performance, and feed utilization parameters

Means of weight gain, percentage weight gain, average daily gain (ADG) and specific growth rate (SGR % / day) were calculated according to the following equations:

Weight gain = W1- W0.

Percentage weight gain (WG %) = {(W1 - W0) / W0} x 100.

Average daily gain (ADG) = weight gain/experimental period (d).

Specific growth rate (SGR %) = {(In W1 - In W0)/T)}x 100.

Where:

W0: Mean initial weight (g).

W1: Mean final weight (g).

T: Time in days between weightings.

Means of feed conversion ratio (FCR), protein efficiency ratio (PER) and Protein Productive Value (PPV %) were calculated according to the following equations:

Feed conversion ratio (FCR) = Feed intake (g) / Weight gain (g).

Protein efficiency ratio (PER) = Weight gain (g). / Protein intake (g).

Protein Productive Value (PPV %) = {(BP1– BP0)/CP} x 100.

Where:

BP0: Initial body protein content (g)

BP1: Final body protein content (g)

CP: Protein intake (g)

Condition Factor:

k=  FW / L3 X 100

Fulton’s condition factor K with W  whole body wet weight In grams and L  length in cm; the factor 100 is used to bring K close to unity.

RESULTS:

Food type:

The results of the effect of different food types (N, NY, S, and SY) on rotifers (B. Plicatilis) population showed that there were significant differences between all treatments (P≤0.05), as showed in Table 1.

In the first day, data showed that NY diet achieved the highest significant total number of rotifer (2.25±0.042X10⁶/30L), followed by SY (2.18±0.157X10⁶/30L), and N (1.86±0.170X10⁶/30L), while the lowest (1.80±0.085X10⁶/30L) was achieved by S diet. In the second day, our results showed that NY achieved the highest significant total number of rotifer (3.06±0.085X10⁶/30L), followed by SY (2.88±0.212X10⁶/30L), and S (2.52±0.099X10⁶/30L), while the lowest value was recorded by N diet (2.52±0.099X10⁶/30L).while, the results obtained at the third day observed that the highest significant total number of rotifer was achieved by NY (4.74±0.169X10⁶/30L) followed by SY(4.50±0.127X10⁶/30L), and N(3.60±0.255X10⁶/30L), but the lowest was achieved by S diet (3.12±0.084X10⁶/30L), as shown in table (1).On the other hand, the highest significant final density (Ind./ml) of B. plicatilis was achieved by NY diet  (158±5.656, Ind./ml), followed by SY (150±4.243 Ind./ml), and N (120±8.485Ind./ml),while the lowest was achieved by S (104+2.828 Ind./ml).Similar trend was observed in the daily increment percentage,, the highest significant daily increment percentage (%) was achieved by NY (72%), SY (66.7 %), and N (46.7 %), while the lowest was achieved by S diet (36 %).

Table 1: Effect of different food types on the productive performances of rotifers B. plicatilis

* N: N. oculata; NY:N. oculata + Yeast; S: S. platensis; and SY:S. platensis+Yeast   

   ** Initial Density was 50 Ind./ml.;     ***   The volume of the rotifer tank is 30 liter of water culture.

The results of fatty acids profile of B. plicatilis feed on N, NY, S, and SY were shown in Table 2. Rotifer fed on S diet achieved the highest total saturated fatty acids (TSFA) percentage (52.30%), followed by SY (38.66%) and N (37.93%), while the lowest TSFA was achieved by NY (24.18%). Palmitic acid C16:0 was the main fatty acid (FA) presented in all treatments; it was achieved (28.13, 21.85, 26.80, and 10.91%) at S, SY, N, and NY diet, respectively. The second main FA obtained in SFA was C18:0, it was 5.02, 10.05, 6.04, and 10.91% at S, SY, N, and NYdiet, respectively. Furthermore, rotifer fed on S achieved the highest total monounsaturated fatty acids (TMUFA) percentage (20.78%), followed by NY (6.18%) and N (5.12%), while the lowest TMUFA was achieved by SY diet (4.57%). Moreover, rotifer fed on S diet achieved the lowest total polyunsaturated fatty acids (TPUSFA) percentage (6.23%), followed by SY (7.93%) and N (11.79%), while the highest TPUSFA was achieved by NY (13.65%). As well as, rotifer fed on NY achieved the highest DHA percentage (10.99%) and EPA (2.66%), comparing to rotifer fed on N (8.60 and 2.08%) and S (0.64 and 1.85%), respectively. Further, rotifer fed on SY did not possessed any DHA percent, while possessed EPA about 1.83% of total fatty acids.On the other hand, rotifer fed on NY recorded the highest omega3(ΣU-3) (13.66%), comparing to N (10.99%), S (2.49%) and SY (1.83%), while did not achieved any omega6 (ΣU-6) percentage, comparing to SY (6.10%), S (3.74%), and N (0.80%).

 EPA/DHA ratio was high (2.89) in rotifer fed on S diet, comparing to rotifer fed on N (0.24) and NY (2.24), while DHA/EPA was low (0.35)in rotifer fed on S diet, comparing to rotifer fed on N (4.14) and NY (4.12), as shown in Table 2.

Table 2: Fatty acid profiles (% of total FA) of different food types used as rotifer feed.

     ND: non detected
Management parameters during the second experimental phase :

Effect of Salinity ;

The results of the effect of different water salinities (25, 27, and 29 ppt) on rotifers (B. Plicatilis) population showed that there were significant differences between all treatments (P≤0.05), as showed in Table 3. In the first day, data showed that salinity 25 ppt recorded the highest significant total number of rotifer (2.365±0.495X10⁶/30 L), followed by 27 ppt (2.100±0.042 X 10⁶/30 L), while the lowest significant total number of rotifer (2.ooo±0.014X10⁶/30 L L) was recorded by 29 ppt. In the second day, our results showed that salinity 25 and 27 ppt achieved the highest significant total number of rotifer (3.285±0.021X10⁶/30 L) and (3.240±0.042X10⁶/30 L respectively). The lowest significant total number of rotifer, 2.130±0.042X106/30 L was achieved by 29 ppt. As well as, the results obtained from the third day showed similar trends, salinity 25 and 27 ppt possessed the highest significant total number of rotifer (3.285±0.021X 10⁶/30 L) and (3.240±0.042X10⁶/30 L respectively), while the lowest significant total number of rotifer (2.130±0.042X10⁶/30 L) was achieved by 29 ppt, as shown in Figure 2. On the other hand, the highest significant final density of B. plicatilis was achieved by salinity 25 ppt (1.865±0.007Ind./ml) and 27 ppt (1.795±0.007Ind./ml), respectively, while the lowest was achieved by 29 ppt (1.320±0.042Ind./ml). Furthermore, the highest daily Increment percentage (%) was achieved by salinity 25 ppt (41.33%) and 27 ppt (40.00%), respectively, whereas the salinity 29 ppt achieved the lowest daily Increment (29.34%).

                  Table3: Effect of different salinities on the productive performance of rotifers B. plicatilis

 * Initial density was 50 Ind./ml. **The volume of the rotifer tank is 30 liter of water culture. ***means in within same column

 sharing same superscript are not significantly different (p ≤ 0.05)

     Table 4: Effect of different water temperatures on the productive performance of rotifers B.  plicatilis

* Initial Density was 50 Ind./ml. ** The volume of the rotifer tank is 30 liter of water culture. ***means in within same column

   sharing same superscript are not significantly different (p ≤ 0.05)