THE EFFECT OF VITAMIN C ( L-ASCORBYL MONOPHOSPHATE-Mg ) ON THE DEFORMITY PERFORMANCE OF HUMPBACK GROUPER ( Cromileptes altivelis ) LARVAE

This study was aimed to get a standard protocol for vitamin C application in enrichment of Artemia nauplii and to examine the impact of vitamin C on humpback grouper larval deformity performance. Vitamin C in the form of L-ascorbyl-2-monophosphateMg (AMP-Mg) was used for the enrichment and incorporated into Artemia nauplii at a given level of 0-1.5 g/L with enrichment period of 4, 6, and 24 hours for each level. The effect of AMP on larval deformity was tested using the following treatments: unenriched both Artemia nauplii and commercial diet, un-enriched Artemia nauplii and experimental diet containing 2.5 g AMP-Mg/kg diet, enriched Artemia nauplii obtained from the protocol and commercial larval diet. Analysis of vitamin C content in Artemia nauplii was carried out using HPLC and larval deformities were observed using staining method, visual examination, or X-ray depending on larval age. The results showed that the best enrichment method of AMP in Artemia nauplii is achieved at the dose of 0.9 g/L for 6 h. The lowest percentage of abnormalities was found in larvae fed with enriched Artemia nauplii and commercial diets, suggesting that vitamin C has an important role in controlling larval deformities.


INTRODUCTION
Humpback grouper (Cromileptes altivelis) is one of important food fish widely cultured in Southeast Asia notably in Indonesia, the Philippines, and Taiwan.Seed production technology of this species has been successfully developed for more than a decade (Sugama et al., 2001;Kawahara et al., 2000), however, problems particularly incidences of larval deformities and larval mass mortalities are still experienced.Improvements in larval nutrition are then necessary to solve these problems.
Vitamin C (ascorbic acid, AA) is an essential vitamin in fish larval diets and has been shown to give a pronounced effect in reducing stress and increasing disease resistance (Merchie et al., 1995a;1995b, 1997;Gapasin et al., 1998;Moe, 2004).Besides that, inadequate amount of dietary vitamin C results in a number of deformity signs, such as scoliosis, distorted/ twisted gill filaments, opercular deformity, and short snout (Gapasin et al., 1998;Dabrowski, 1990;Soliman et al., 1986).The requirement of vitamin C in fish is only obtained from exogenous supply of vitamin C source as fish lack the enzyme L-gulonolactone oxidase (EC 1.1.3.8) which is responsible for synthesis of vitamin C de novo (Wilson, 1973).To satisfy the need, the supply of dietary vitamin C is usually given through the formulated diet or enrichment in live feed.Extensive studies of dietary vitamin C requirement have been done for juvenile and adult stages of several marine

THE EFFECT OF VITAMIN C (L-ASCORBYL MONOPHOSPHATE-Mg) ON THE DEFORMITY PERFORMANCE OF HUMPBACK GROUPER (Cromileptes altivelis) LARVAE
fish (Sealey et al., 1999;Wilson et al., 1973;Ai et al., 2004;Wang et al., 2003;Dabrowski et al., 1990) and the recommended values ranged from 20 to 50 mg of ascorbic acid kg -1 diet (NRC, 1993).Nevertheless, few data on vitamin C requirement for first feeding larvae particularly in improving larval deformity are available.Merchie et al. (1996) reported that ascorbic acid concentration in un-enriched Artemia that can reach up to 500 mg equivalent of ascorbic acid per gram dry matter was adequate to satisfy the nutritional requirement of larvae.Meanwhile, ascorbic acid concentration of approximately 1,400 μg/g DW in enriched Artemia was sufficient to improve growth and larval performance of African catfish (Merchie et al., 1997).Another study (Gapasin et al., 1998) reported that milkfish larvae fed with HUFA and ascorbic acidenriched live food had significantly lower opercula deformity incidence and exhibited higher growth than those given un-enriched live food.Those results suggest that the requirement varies to some degree with fish species, size, diet and its preparations, the form of vitamin C and experimental conditions.However, there is no information about the effect of vitamin C on larval stage of groupers, particularly humpback grouper.
On the other hand, due to the highly unstable of vitamin C and its loss activity upon processing and storage, the stable forms of ascorbic acid derivatives were used; they are ascorbyl palmitate (AP), L-ascorbyl-2-monophosphate-Mg (AMP-Mg), L-ascorbyl-2-monophosphate-Na/Ca (AMP-Na/Ca), L-ascorbyl-2polyphosphate (APP), ascorbyl-2-sulphate, etc.Of the derivatives, ascorbyl-2-monophosphate seems recognized to be very stable.
Thus, the present study was aimed to investigate the effect of vitamin C (in the form of AMP-Mg) to control deformities on humpback grouper larvae.Incorporating AMP-Mg into larval grouper was conducted through live food enrichment.For this purpose, Artemia nauplii enrichment with AA was conducted and further investigated as a tool to deliver vitamin C to the larvae.

Artemia Enrichment
Decapsulated Artemia cysts (INVE, Great Salt Lake Prime Gold, Prime Artemia, Midvale, UT, USA) were cultured in white polycarbon-ate cones filled with 100 L of sea water (34 ± 1 ppt) with aeration system at 28 o C-33 o C. At 24 hours, newly hatched Artemia were removed from the hatching cones.Two liters of Artemia nauplii were sampled, three-times rinsed in distilled water, filtered and freeze dried for dry weight (DW) and AA analyses.After that, the rest of Artemia nauplii were cultured in 24 L of seawater with aeration for ± 12 h.After 12 h, Artemia nauplii were divided and placed into 12 of 2-L of seawater-filled plastic bottles for enrichment protocol treatments.Treatments were conducted by enriching the nauplii with L-ascorbyl-2-monophosphate-Mg (AMP-Mg) at the level of 0, 0.6, 0.9, 1.2, and 1.5 g/L and enrichment period of 4, 6, and 24 h.To ensure the presentation of appropriate particle size for Artemia nauplii, AMP was blended for 30 s in seawater before added to Artemia.Enriched Artemia nauplii were removed and washed as previously described.Artemia nauplii samples were freeze-dried for 24 h and prepared for AA analysis.
Total AA is detected using High-Performance Liquid Chromatography (HPLC) with Lichrocart 250-4 column and mobile phase 0.05 M KH 2 PO 4 pH 2.8, flow rate 0.5 mL/min., at room temperature.The product was detected using UV detector at 245 nm (Shiau & Hsu, 1994).Factorial statistical analysis was conducted using the MSUSTAT program (MSUSTAT).Values of (P<0.05) were considered significantly different.

Larval Culture
Eggs of humpback grouper at a density of 100 eggs/L were stocked into three 5m 3concrete tanks filled with seawater (salinity 34±1 ppt).During the first 10 days, the larvae were reared without water exchange.Larval rearing feeding regime followed the method described in Table 1.
At day 20 th , the larvae were started fed with the following treatments: (A) un-enriched both Artemia nauplii and commercial larval diet (control); (B) un-enriched Artemia nauplii and vitamin C-enriched experimental diet (containing 2.5 g AMP/kg, prepared in the laboratory; provided in Table 2); and (C) vitamin C-enriched Artemia nauplii (based on the result of the enrichment protocol experiment) and unenriched commercial larval diet.Samples of larvae were taken started on day 30 th and collected every 10 days for stain-ing.The staining procedure followed the method described by Potthoff (1983).Samples of 90-days and 5-months old juveniles were taken for visual examination and X-ray analysis.At the end of the experiment, the juveniles were harvested and calculated to quantify their deformity.

Artemia Enrichment
Ascorbate level in Artemia nauplii given in different concentrations of AMP-Mg and enrichment period is shown in Figure 1.The endogenous level of AA in un-enriched Artemia nauplii was 713.1 μg g -1 DW.In Figure 1(a), it is clearly seen that with increasing concentration of AMP-Mg at each enrichment period, the ascorbate level of Artemia nauplii significantly increased (P<0.05) and reached a maximum level at 0.9 g AMP-Mg/L enrichment.Interestingly, the period of AMP-Mg enrichment also influenced the endogenous AA level of Artemia nauplii.Short period of enrichment into Artemia nauplii exhibited the increased endogenous AA level from 0.72% to 1%, 1.14%, 1.05%, and 1.4%, respectively in each AMP-Mg concentration.Prolonged enrichment to 6 h showed a similar trend of AA level except for that at enrichment dose of 1.5 g AMP-Mg/L.Unlike those in 4 and 6h of enrichment period, the endogenous AA level of 24 h-enriched Artemia nauplii decreased in each concentration.The highest ascorbate level of Artemia was attained at 6 h of enrichment period.This suggested that the best enrichment method of AMP-Mg in Artemia nauplii was achieved at a dose of 0.9 g/L for 6 h (P<0.05) and this result was used as the protocol for humpback grouper larval rearing.The result in the present study was different from that reported by Smith et al. (2004).Their study showed that the maximum enrichment using ascorbyl-2-phosphate was attained at rates of 8 1.2 g/L for 24 h giving a 60-fold increase in Artemia nauplii AA levels.The discrepancy is

Tot al 100
The effect of vitamin C (L-ascorbyl monophosphate-Mg) ... (Sophia Lasma Sagala) possibly due to differences in Artemia size used for enrichment, the form of vitamin C derivatives, and experimental conditions.Of all, it can be concluded that AMP-Mg could be assimilated as AA in Artemia nauplii in a dose and period dependant.

Larval Performance
The deformity performance of humpback grouper larvae determined using staining method is given in Figure 2. It is seen that deformities in humpback grouper larvae were still found and humpback grouper larvae fed with un-enriched Artemia nauplii and commercial diet (control diet) exhibited the highest percentage incidence of deformities during 50 days of culture.In addition, larvae fed with un-enriched Artemia and AA-enriched experimental diet gave lower deformities than those in fed control diet, while larvae fed with AA-enriched Artemia and un-enriched com-mercial diet gave the lowest.This suggested that vitamin C played a role in controlling larval abnormality.
Based on the staining method, the different trial groups presented a variety of deformities.The most frequent deformities found were vertebral deformities occurred at the backbone which could not be observed visually (Figure 3).
Vertebral deformities observed consisted of lordosis, scoliosis, kyphosis, and abnormal on the backbone (Figure 3A-E).Deformities in fusion on dorsal spine and epurals, and branching of the dorsal proximal radial were also found during the trials.Although feed had contained AA supplementation, deformities were still found; this may be attributed to the insufficient requirement of AA concentration for tissue and bone matrix of the larvae or to the reason that AA supplementation did not totally eliminate After three months of culture, deformity of the juvenile was measured visually and a similar trend to those with staining method was observed (Figure 4).However, the result in the trial 1 of 90-day-old larvae was on the contrary to those in the trial 2 of 90-day-old larvae and in the staining specimens.This may be ascribed to the eggs coming from different broodstock or different spawned-egg period contributing to different quality of larvae which deformity was not apparent during early larval stage.Visual examination was also conducted to 150-day-old juveniles and a similar trend as those in the staining results was obtained.
Visually, deformities found in the juveniles were mostly open operculum (opercula deformity), short body, bent head, and discharged epithelia.A similar finding that an opercula deformity observed in ascorbate deficient milkfish larvae was reported by Gapasin et al. (1998).They reported that the opercula deformity incidence was significantly lower in the larvae fed with HUFA + vitamin C-enriched rotifers and Artemia than those fed with HUFAenriched or un-enriched rotifers and Artemia.Another study by Fraser & de Nys (2010) reported that deficiency of vitamin C induced spinal deformities like 'broken back' syndrome and lordosis and opercula deformity in barramundi juvenile.The addition of vitamin C (in the form of free ascorbic acid) significantly reduced the spinal deformities and completely lowered the opercula deformity incidence but not affect the incidence of jaw deformities in the juvenile.X-ray examination was only applied to 150day-old samples to visualize the bone/skeletal structure of juveniles.In accordance with that in the staining result, the deformities observed dominantly occurred on the back bone (Figure 5).The effect of vitamin C (L-ascorbyl monophosphate-Mg) ... (Sophia Lasma Sagala) Interestingly, after 5 months (150 days) of survival rate of the larvae did not differ among the treatment groups in each trial (Figure 6), suggesting that vitamin C did not affect the survival rate of the larvae.The find-ing was in accordance with the previous results reported by Gapasin et al. (1998).Cahu et al. (2003) also reviewed that the enrichment ascorbic acid did improve neither the survival nor the growth.In the current study, it is clear that vitamin C supplementation in larval diet was highly important and revealed low percentage of deformities.The results were in accordance to the previous studies both for larval and juvenile of marine species (Merchie et al., 1995b(Merchie et al., , 1996(Merchie et al., , 1997;;Gapasin et al., 1998;Moe et al., 2004;Dabrowski et al., 1990;Soliman et al., 1986;Wilson, 1973;Sealay et al., 1999;Wilson et al., 1973;Ai et al., 2004;Wang et al., 2003).The requirement of this micronutrient varied to different species.In addition, Dabrowski et al. (1996) reported that larval stages need a higher level of vitamin C than juvenile stages of African catfish.The study of Fraser & de Nys (2010) suggested that vitamin C had a role in ontogeny of skeletal deformities in barramundi larval.

CONCLUSIONS
1.The optimum enrichment method of Lascorbyl-2-monophosphate in Artemia nauplii was at a dose of 0.9 g/L for 6 h.

Figure 3 .
Figure 3. Main skeletal deformities developed in humpback grouper larvae (stained specimens): A. lordosis, B. scoliosis, C. kyphosis, D and E. abnormal on back bone, F. fusion on dorsal spine, G. fusion on epurals, H. branching of the dorsal proximal radial

2.
Humpback grouper larvae fed with vitamin C-enriched feed exhibited low percentage of deformity, indicating that vitamin C has an important role in controlling abnormality of C. altivelis larvae.

Table 1 .
Feeding regime to humpback grouper larvae