Effect of L-carnitine enrichment on the rotifer and influences on clownfish larvae

Enrichment has shown considerable influence on the population growth, reproduction and individual growth of rotifer (Brachionus plicatilis Müller, 1786) and its utility on the clownfish larvae (Amphiprion ocellaris Cuvier 1830). Using the culture medium of microalgae (Nannochloropsis salina D.J. Hibberd 1981) as the control, rotifers were enriched by dissolving in S-presso (for short term enrichment) or L-carnitine supplements. The study was conducted in three replicates for five and 10 days of batch culture. On day-4, the population density of rotifers exposed to 1, 10 and 100 mg l -1 L-carnitine was significantly increased (p<0.001) by 43, 39 and 54%, respectively, compared to the control. The population density in these three treatments also increased on day-2, day-3 and day-5 compared to the 1000 mg L treatment (p<0.05) and control (p<0.05). The body length (p<0.001) and width (p<0.05) were significantly reduced in the 1000 mgL treatment compared to the control. The mean values of total unsaturated fatty acids (PUFA's, HUFA's and n-3 fatty acids) and the ratio of n-3/n-6, DHA/EPA, EPA/AA were significantly higher (p<0.05) in the rotifers enriched with L-carnitine compared to S-presso and algae. Larvae fed using rotifer enriched with Lcarnitine gained the maximum growth (65.7±1 mg), followed by those enriched with S-presso (42.2±0 mg). In L-carnitine treatment, metamorphosis took only 9 days, followed by the control. In L-carnitine and S-presso units, the clownfish larvae took only three days for the first pigmentation compared to four days in N. salina (control) and 10 days with S-presso. Rotifers enriched with L-carnitine showed the highest survival rate (70 %) of the clownfish followed by the control (68%) and S-presso (52%). The results suggested that L-carnitine could be a positive factor to enhance the rotifer production and also its utilization in clownfish larval culture.


Introduction
Rotifers are microscopic metazoans and are the most important live food of fish larvae especially during the early period of exogenous feeding (Hagiwara et al., 2001). The nutrients available in the rotifer (Brachionus plicatilis Müller, 1786) include highly unsaturated fatty acids (mainly 20:5n-3 and 22:6n-3) that are essential for survival of marine fish larvae (Zhang et al., 2002). Watanabe et al. (1983) have shown that high quality feed i.e. a mixture of algae and other enrichment, facilitate the transfer of essential fatty acids and other dietary components from algae via the The experiment in triplicates was maintained with filtered seawater at 30 ± 2 °C, 26 ± 2 % salinity and 12 hr/12 hr day/night period. The specific growth rate was calculated according to Rico-Martinez and Dodson (1992). The initial density was 250 individual/ml and it was monitored daily by counting the number of rotifers in 3 ml/L samples at each treatment. The egg ratio was calculated using the Equation 1 as described by (Zhang et al., 2002) Egg ratio = amictic egg numbers/rotifers (Equation 1)

Body size determination
At the last day of the culture trial, the Lorica length and width were measured using a microscope (Leica model) at 100 × magnification with computed length analysis software to determine the body size of a rotifer. One-egg carrying rotifers were randomly measured (n=120 per treatment). The rotifers were fixed with 6.5 % HCl as described by Fu et al. (1991), for easy measurement without a significant shrinkage.

Storage and analysis
After bio-encapsulation, these enriched rotifers were collected, washed properly and stored in vials under -80 °C. Total lipids were extracted from the enriched rotifers using Bligh and Dyer method (1959) at A to Z Pharmaceuticals Laboratory in Chennai, India, and the fatty acid analysis were done using Gas Chromatography (Chrompak CP-9001 GC).

Larval feeding
The capacity of each larval rearing tank was 100 l, well-aerated under 26-32 °C temperature, pH, 8.0-8.2, salinity 24-28 % and ammonia <0.01 ppm. The rotifer density was maintained at 8-10 individuals/ml. Sixty larvae were measured to the nearest 0.05-0.15 mg individually and introduced to each larval-rearing tank. Every morning, tanks were clearly checked and dead larvae were counted and removed along with the debris settled in the bottom adopting a siphoned method. Approximately 20 % water exchange was maintained at the experimental tanks to avoid rotifers settlement at the bottom. To keep the rotifer population at 8-10 individuals/ml, the enriched feed was supplied on 8-10 am and 4-6 pm. The experiment was continued for a period of 15 days and the final wet weight was obtained. Throughout the experimental period, the records on rotifer growth in terms of weight (mg), % survival and number of days taken for pigmentation and metamorphosis under different treatments were obtained.

Analysis of growth and culture evaluation
Larval dry weight was calculated by weighing larvae at the size of 3.5 mm on prewashed and pre-weighed fibreglass filters following the method proposed by Zhang et al. (2002). The specific growth rate was calculated from the slope of the linear regression of log-transformed dry weight against age (Albentosa et al., 1997). The final biomass was calculated with the dry weight and the larval density was obtained at the end as described by (Cahu et al., 1998). The dissolved oxygen level (mg/l) and pH were measured daily. The ammonia and nitrite level at each tank were analyzed as described by Albentosa et al. (1997).

Statistical analysis
The values on population density were transformed to a logarithmic scale and the egg ratio to the square root for statistical analysis. One-way ANOVA was conducted and the significant differences among treatments on each day by Duncan's multiple range test (DMRT) at p=0.05 (Zar, 1999). All statistical analyses were performed using SPSS 20.0 statistical software package and Figures were made using Origin 6.1 software.

Effects on growth population
The rotifers enriched with 1000 mgl -1 L-carnitine showed 100 % mortality at the end of 10 th day of culture. The population density of other 6 groups (treated with 0-10 mgl -1 L-carnitine) except for rotifers enriched with higher level of 100 and 1000 mg l -1 Lcarnitine, showed a clear population increase after 5 and 10 days of culture ( Figure  1). However, the 100 and 1000 mgl -1 treatments showed a decreasing trend in population density. Compared to the first 5 days, the specific growth rate of rotifers in all treatments decreased during the last 5 days of the experiment. The population densities between treatments were significantly different at different culture trials (p<0.05). On the day-4, the population density of the rotifers exposed to 1, 10 and 100 mgl -1 L-carnitine increased by 43, 39 and 54 %, respectively, (p<0.001) compared to the control. The same treatments also showed an increase in population density of rotifers in day-2, day-3 and day-5 compared to treatment with 1000 mg l -1 L-carnitine and the control (p<0.05). From day-2 to day-5 of the experiment, the rotifers enriched with 1000 mg l -1 L-carnitine showed 33-35 % decrease in population density (p<0.01) compared to the untreated control ( Figure  2). Figure 2. Population density (ind.ml -1 ) and specific growth rate (day -1 ) of B. plicatilis enriched with eight levels of L-carnitine (mg l -1 after 5 days) individual culture. Vertical lines indicate the standard error. Note: concentrations of L-carnitine (mg l -1 ) in a=0, b=0.001, c=0.01, d=0.1, e=1.0, f=10, g=100, and h=1000. Figure 3 illustrates the egg ratio variation in B. plicatilis enriched with five levels of Lcarnitine during five days of batch culture. The 1 and 10 mgl -1 treatments showed a significantly lower egg ratio on day-4 (p <0.01) compared to the control. The egg ratio of all groups decreased after 24 hrs followed by an increase in L-carnitine enriching levels.

Effects on the body size
The 1000 mgl -1 L-carnitine treatment significantly increased (p<0.001) the body length of rotifers with a marginal reduction in the width (p<0.05), compared to the control. In the other three treatments (1, 10 and 100 mg l -1 L-carnitine), a larger body size was recorded ( Figure 4). The results showed that in 1 mgl -1 L-carnitine treatment, body length of rotifers increased significantly (p<0.05) but not their width (p>0.05). However, at 100 mg l -1 treatment resulted in a significantly larger body width (p<0.05) and marginally higher body length. There were no significant differences in the changes in the body size in 10 mg l -1 L-carnitine treatment and the control (p>0.05).

Fatty acid analysis of enriched encapsulated rotifers
The L-carnitine enriched rotifer was found to be superior to other diets in terms of fatty acid content, with the ration of 1:7:0.5 (Table 1). The ratio for other diets namely, N. salina-enriched rotifers 0.4:8.4:0.2 and S-presso-enriched rotifers 0.2:7.0:0.05.  Table 2 shows the fatty acid profile of rotifers enriched with L-carnitine, S-presso and N. salina. The enrichments effects are statistically significant (p<0.01). The mean values of total unsaturated fatty acids, PUFA's, HUFA's and n-3 fatty acids and their ratios such as n-3/n-6, DHA/EPA, EPA/AA were significantly higher (p<0.05) in the rotifers enriched with L-carnitine compared with other diets. An exceptionally high PUFA content was recorded in rotifers enriched with S-presso (46.55) and the ratio of EPA/AA was high in rotifers enriched with L-carnitine (118.321). The high PUFA content in rotifers enriched with L-carnitine was mainly due to the contribution of 18:3n-3 to the total PUFA content. The total unsaturated fatty acid, n-3 fatty acids, HUFA, DHA, EPA, AA levels and their ratio are show in Table 3. The number of days taken for the first pigmentation of clownfish larvae, i.e. from translucent colour to dull black colour, differed among treatment. In the L-carnitine and S-presso treatments, the larvae took only three days for the first pigmentation, whereas four days were taken in the N. salina treatment ( Table 4). The number of days taken by the clownfish larvae to show the initial signs on metamorphosis changed with different feeds (Table 4), i.e. nine days in L-carnitine treatment followed by 10 days in N. salina; and 10 days in S-presso.  day-7 and day-8 were less than 5 % during metamorphosis. In N. salina treatment, less than 30 % mortality was recorded from day-4 to day-10. Larvae fed at S-presso attained the maximum growth 56.92±0.2 mg, followed by L-carnitine (48.74±1 mg), and those fed with an algae-enriched rotifers recorded the maximum growth of 39.41±3 mg. Observations made on the number of days taken for first pigmentation indicates that L-carnitine and S-presso take only 3-4 days followed by N. salina (4-5 days).

Discussion
The L-carnitine supplement (1500 mgl -1 ) has shown a significant response on the metabolism of treated European sea bass fry (Santulli et al., 1990). The present study showed similar results demonstrating that supplemented L-carnitine in culture medium has resulted in significant responses in reproduction and individual growth of enriched rotifers. The mechanical and chemical receptors of rotifers are sensitive to environmental stimulation and are in direct contact with external medium (Clement et al., 1983). Thus, it is possible that rotifers exposed to a medium enriched with L-carnitine are affected directly. This study was not conducted in an axenic condition and hence, other associated microorganisms that may interact in the trophic relationship were also exposed to supplemented L-carnitine in culture medium (Lubzens et al., 1985).
The supplemented of L-carnitine to the culture may change the normal interaction between the microenvironment and rotifers, which could be either detrimental or beneficial. In the present study, 38-50 % improvement in population density was detected in rotifers treated with 1-100 mgl -1 L-carnitine. Rotifers exhaust endogenous lipid through reproduction (anabolism) and respiration (Olsen et al., 1993). In the individual culture trial, rotifers enriched with 1000 mg l -1 of L-carnitine showed 100 % mortality on day-10. Oie and Olsen (1997) reported that higher food ration resulted in higher growth rate, and then rotifers respond by a higher egg ratio. Normally, high reproduction causes smaller size as more energy is allocated for reproduction over somatic growth (Duncan, 1989). As observed in this study, increased body size of clownfish larvae when fed with rotifers in the 1-100 mg l -1 Lcarnitine treatment may be the response to the protein sparing action produced by exogenous L-carnitine stimulation. In this treatment, rotifer did not show a higher egg ration compared to the treatment with 1000 mg l -1 L-carnitine however, they achieved a larger body size and improved population growth.
The nutritional quality of rotifers was improved by feeding them on algae or, on emulsified Pollock or cattle fish oil prior to their transfer into fish tanks (Watanabe et al., 1983). The relationship between the n-3 and n-6 fatty acid series, and more recently, the DHA/EPA, EPA/AA, and DHA: EPA:AA ratios seem to be indicators of the best survival and growth of marine fish larvae (Sargent et al., 1997). Selco, Super Selco and Topal emulsions (Artemia systems, Belgium) are also used for enrichment. The S-presso, one of the enrichment diets in the present study have a good source of HUFA's (32.2 %) and total unsaturated fatty acids (76.78 %) is also used as a short term enrichment. Results presented in Table 1 showed that L-carnitine DHA is higher than EPA and AA is present in higher levels. The subsequent slow increase of fish larval growth rate was due to an incorporation of n-3 fatty acids and sufficient period after enrichment to serve as a live feed for marine fish larvae (Olsen et al., 1993). The increase in n-3 HUFA levels of enriched rotifers fed on capelin oil or by low temperature crystallization separation for rotifer enrichment reached a maximum between 6 and 12 hrs of enrichment and did not change significantly thereafter (Kissil and Koven, 1990).
In the present study, rotifer fed with N. salina gave a very good fatty acid profile especially HUFA, and n-3 fatty acids, the n-3/n-6, DHA/EPA and EPA/AA ratios. In turbot (Scophthalmus maximus Linnaeus, 1758), the dietary deficiencies in AA have resulted in high mortality and obvious pathology (Bell et al., 1985), while Castell et al. (1994) reported a positive effect of AA on survival of turbot from levels ranging from 0.5-1.0%. The total HUFA fraction in the enriched rotifers revealed that rotifers enriched with S-presso contained the highest percentage of HUFA fraction among the tested feed, where the major contribution is from the DHA and EPA fatty acids, which the marine fish is lacking (Sargent et al., 1997). The rotifers enriched with Spresso had more 20:4n-6, which has an essential function in producing eicosanoids, compared to N. Salina, however, showed lower level than in L-carnitine. Arachidonic acid is essential for certain marine fin fishes (Castell et al., 1994). The DHA:EPA:AA ratio, which is important for marine finfish larvae was promising in rotifers enriched with L-carnitine (1:7:0.5) followed by those enriched with N. salina (0.4:8.4:0.2). Except in oil-enriched rotifers, the DHA level is generally low but the EPA levels are high. In marine finfish larvae, the conversion of EPA to DHA is possible to meet the requirement of DHA. The results suggested a strong possibility of retro-conversion of DHA to EPA in rotifers, as the high content of DHA in S-presso was not reflected after enrichment of rotifers. After enriching with S-presso, rotifers should be handled with care as if not, it could result in rotifer releasing out or vomiting the enrichment taken in.
Clown fish larvae can be reared up to metamorphosis with enriched rotifers. Two main live feeds are used for marine fish seed production, rotifers and brine shrimp nauplii (Dhaneesh et al., 2012). The major source, n-3 HUFA for live organisms can also vary substantially (Sargent et al., 1997). Clownfish larvae can be weaned on to a formulated dry feed for seven days after hatch with no significant reduction in survival, although the optimum time for weaning on a dry formulated feed was between 15 and 20 days after hatch (Ajith Kumar and Balasubramanian, 2009).
The enrichment products, S-presso and micro alga N. salina, had EFA such as 20:5n-3, 22:6n-3 and PUFA such as 20An-6, vitamins such as B 12, C and A that could be utilized and easily incorporated into this feeding regime. The long-chain highly unsaturated fatty acids (HUFA's), particularly EPA and DHA, are important in the nutrition of young marine fish (Watanabe et al., 1983). The DHA and EPA arachidonic acid (AA, 20An-6) has also been recognized as essential for marine fish (Castell et al., 1994). In the present study, a significant difference was observed in the growth and survival of clownfish larvae fed with DHA-enriched live food (high DHA/EPA ratio) i.e. S-presso, L-carnitine and N. salina. Baker et al. (1998) also noted a direct relationship between normal pigmentation and levels of DHA in the diet of summer flounder larvae, where the DHA/EPA ratio in the enriched live food was high. In the present study, however, pigmentation success was significantly higher in clownfish larvae fed with rotifers containing L-carnitine. The fatty acid profile of rotifers enriched with L-carnitine and S-presso showed higher levels of DHA compared to that of N. salina. Recent research has shown that the n-6 HUFA, arachidonic acid (20An-6 or AA) was also important for growth, survival and stress resistance flounder (Baker et al., 1998). In milkfish (Chanos chanos Forsskal, 1775), the effects of DHA-enriched live food on growth may not be readily discussed over a short period but rather after extended rearing (Gapasin and Duray, 2001). The DHA must be present in the diet to maximize the survival of larvae of damselfish (Acanthochromis polyacanthus Bleeker, 1855; Southgate and Kavanagh, 1999). The AA was nutritionally more important in tropical species than in temperate species. Nannochloropsis salina contained relatively high levels (4.6 %) of AA (Thrush et al., 1993). In the present study, the rotifer enriched with L-carnitine and N. salina satisfied the AA requirement of clownfish larvae. Sargent et al. (1997) stated that both the amount and proportions of DHA, EPA and AA are important in marine fish nutrition and suggested that the optimum ratio may vary with species but, would be in the range of 10:5:1 for DHA:EPA:AA. Free tyrosine and free phenylalanine have been reported to increase or to be maintained at constant levels around first feeding in European seabass (Dicentrarchus labrax Linnaeus, 1758; Ronnestad et al., 1998), Asian seabass (Lates calcarifer Bloch, 1790; Sivaloganathan et al., 1998), and Senegalese sole (Solea senegalensis Kaup, 1858;Parra et al., 1999).

Conclusion
Supplementation with L-carnitine can affect rotifers directly and indirectly. The rotifers enriched with L-Carnitine showed positive and significant responses to population growth, reproduction and individual growth under the optimum Lcarnitine concentration. The optimum concentration of supplemented L-carnitine in the culture medium is 1 mg l -1 . Other biological and abiotic environments may be considered when the rotifer is enriched by L-carnitine in a normal culture medium. The rotifers enriched with L-carnitine as a feed may further influence the growth of clownfish larvae, in some aspects. The present study also revealed that commercial L-carnitine and S-presso are promising as long-and short-term rotifer enrichments, respectively. The microalgae N. salina is also good supplement in terms of its fatty acid profile but not comparable in growth performance to the L-carnitine and Spresso.