Report to Aquafauna Bio-Marine Inc., September 1998

SUMMARY

A trial was conducted to evaluate two Schizochytrium-based test products (AlgaMac-3000, Schizotein) as Artemia enrichments for rearing Atlantic halibut (Hippoglossus hippoglossus L.) larvae. The rearing performance of halibut (in terms of survival. growth and metamorphosis) receiving the test diets was compared to those receiving a UK control feeding protocol (AlgaMac-2000-enriched and Super Selco enriched Artemia, fed in a 1: I ratio). The 3 experimental enrichments comprised Schizotein-only (at 0.6g/l), AlgaMac-3000 only (at 0.6g/l) and AlgaMac-3000 (0.6g/l) + Super Selco. Four replicate tanks per diet (110 larvae per tank) were established on the experimental diets on day 9 post-first feeding (307° days post hatch) and run until day 37 PFF (626°d). At day 37 PFF the replicated groups were pooled into two tanks and the larvae continued to receive the experimental diets for a further 15 days, until 54 PFF (839° d). No statistics were applied to the data from the final, non-replicated phase of the trial (days 37-54 PFF). Larvae were sampled for weight measurement on days 9, 26, 37 and 54 PFF. All surviving halibut were scored for eye migration and pigmentation characteristics on day 54 PFF. For data pooled from all treatments, the average survival rate over the period day 9-37 PFF was 52.6% and for the period 38-54 PFF, 98.4%. Diet treatment exhibited clear effects on the growth rates and metamorphosis characteristics of the halibut larvae. The mean dry weight of all halibut at the outset of the study was 1.24±0.06mg and at the conclusion was 37.12±16.55mg, an overall specific growth rate of 7.481%/day. All groups showed a reduction in specific growth rate during the final phase of the trial (days 38-54 PFF), however this was particularly marked in the Schizotein treatment. Likewise, Schizotein larvae had poorer eye migration and a greater proportion of ambi-colouration than all other treatments on day 54 PFF. Larvae receiving AlgaMac-3000, (either in isolation, or together with Super Selco) exhibited better eye migration and pigmentation characteristics than all other treatments. The AlgaMac-3000 only treatment contained the highest proportion of "perfectly" pigmented halibut (65.9%). However, a deceleration in growth rate was observed within this treatment during the latter part of the trial (days 38-54 PFF), which may have been indicative of lipid shortage during metamorphosis.

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 BACKGROUND

Schizochytrium-based products are now widely used by commercial UK marine fish hatcheries for the enrichment of live prey. As reported by Gara et al (in press), a standardized feeding protocol for larval Atlantic halibut is currently in use, involving the presentation of AlgaMac-2000 enriched Artemia in combination with Artemia enriched using proprietary lipid emulsions, such as Selco products (Inve Aquaculture NV). The rationale for this dual enrichment approach is to combine the DHA-boosting attributes of AlgaMac-2000 with high energy provision via the lipid emulsion (metamorphosis having been identified as an energy-demanding process for halibut). However, limitations have been identified with the method, namely the association between prolonged presentation of lipid emulsions and albinism of the halibut juveniles (Gara et al, in press).

Methods are therefore being sought to avoid the pigmentation problems associated with the available emulsions, without incurring the mortality penalty that occurs if emulsions are completely withdrawn. In this respect, the development by Monsanto of new Schizochytrium-based products, containing higher lipid contents, provides an opportunity to simplify the halibut feeding regime (ie: requirement for only one enrichment type). Such products may also enable better pigmentation characteristics to be achieved, through greater dietary input of DHA. In conjunction with these new lipid-rich products, the availability of de-fatted cell preparations may enable both the protein and lipid components of live prey to be manipulated.

The aim of the present study was to determine whether the rearing performance (survival rate, growth rate, metamorphosis attributes) of Atlantic halibut could be improved by using a Schizochytrium-based preparation (AlgaMac-3000), with higher lipid content than the commercially available product, AlgaMac-2000. The trial also incorporated an experimental de-fatted cell preparation (Schizotein), to evaluate whether it is beneficial at any stage of the feeding larval phase to restrict dietary lipid input. The test products supplied by Aquafauna Bio-Marine were compared against a standardized halibut feeding protocol. In the absence of any information on preferred enrichment protocols for these test products, we adopted the basic technique developed for AlgaMac-2000. AlgaMac-3000 was tested both alone and in combination with a lipid emulsion (Super Selco ), to investigate whether the lipid content of the experimental Schizochytrium product was sufficient to sustain halibut larvae through metamorphosis.

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METHODS

On 12^th May 1998, 110 Atlantic halibut larvae (9 days post-first feeding, 307° days post-hatch) were stocked into each of sixteen experimental rearing tanks (4 tanks per treatment) containing 801 5um-filtered, UV sterilized sea water and microalgae, Nannochloris atomus. The larvae were retained in this replicated tank system for 28 days (37 PFF, 626° d). During this period, 20% of the tank water was exchanged, via a surface inflow, on alternate days and microalgae was added after each exchange. On day 28, the replicate tanks from each treatment were pooled into single tanks (501) and the larvae were reared for a further 15 days, until day 54 PFF (839° d). During this phase of the trial the larvae received a continuous water flow and microalgae was withdrawn. Salinity, oxygen levels and water temperature were monitored daily. The mean temperature (± standard deviation) over the period from 9 to 37 PFF was 11.8±0.1° C and from 38 to 54 PFF was 14.1±0.1° C.

Daily ration levels were adjusted according to consumption, with the aim of minimizing levels of residual prey.

Artemia preparation and enrichment protocols were as follows:

Brine shrimp Artemia cysts (EG grade, Inve Aquaculture NV) were decapsulated and incubated for 18 hours in aerated sea water (27° C, 34° /o o ), that had been filtered to 5um and UV sterilized. After harvesting, the newly hatched nauplii were set up in aerated seawater in 5 liter polyethylene containers, at a density of 150,000 individuals per liter, at 27° C. These nauplii were retained in sea water only until 18 hours prior to use. Enrichments were blended in freshwater for 1 minute before adding to the nauplii in the 5 liter containers. AlgaMac-2000 was applied in a single dose at a concentration of 0.5g/l, 18 hours prior to feeding. AlgaMac-3000 and Schizotein were applied at a concentration of 0.6g/l, 18 hours prior to feeding. Super Selco was applied in two doses of 0.3g/l, 18 hours and 12 hours prior to feeding.

Samples of enriched Artemia (25,000 per sample) were collected on three occasions during the trial and stored in liquid nitrogen, awaiting lipid analysis.

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A sample of forty halibut larvae was taken from the source population at the start of the trial (9 PFF, 307° days) for analysis of wet and dry weights. The mean wet weight of these larvae was 15.93±1.62mg (n=40). On day 17 (26 PFF, 507° d) five larvae from each experimental tank were removed for measurement of wet and dry weights, while on day 28 (37 PFF, 626° d) 10 individuals per tank were removed for this purpose. Weights of halibut in the two treatment groups were compared directly on each sampling day. Specific growth rates (SGR, %/d) were calculated according to the equation:

SGR = 100 x (1n(w_i) - 1n(w_t)

Where w_t is the final weight (at time t) , w_i is the initial weight and t is the time interval in days.

Within-group growth variability was measured using the coefficient of variation for wet weight (%), which was calculated for each sampling day according to the equation:

CV = 100 x s.d./x

Where s.d. is the sample standard deviation and x is the sample mean.

At the conclusion of the trial (54 PFF, 839° D) eye migration and distribution of pigment were classified for individual fish using the methods described by Gara et al (in press). Each fish was examined and scores relating to eye migration and pigmentation were assigned by visual inspection. Eye migration was assessed by laying the larva flat on it's blind side and awarding a score as follows:

0:Blind side eye not visible.

I: Blind side eye just visible.

2:Full diameter of blind side eye visible, but lens not visible.

3: Complete migration, both eyes on ocular side with lenses visible.

Pigmentation was scored separately for the ocular and blind sides of the body using a 5 point scoring system.

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RESULTS

The survival rates of halibut larvae from the 4 treatment groups, measured on day 37 PFF and day 54 PFF are shown in Table 1. The mean survival rate (±st. dev.) during the 28 day period from days 9 to 37 PFF was 52.61 ± 16.68%. No significant variation was seen in percentage survival over this period (F_3:12=0.33, p>0.05), although those reared on Schizotein showed a lower rate than all other treatments. From days 38 to 54 PFF a mean survival rate of 98.45% was seen. and this was similar in all groups (Table 1).

Table 1. Percent survival (±st. dev.) of halibut larvae reared on 4 experimental diets, measured on days 37 and 54 post first feeding (626' and 839d).

Wet weight data are presented for the benefit of commercial hatchery operators, who are not equipped to carry out dry weight measurements. The mean wet weight (± st. dev.) of halibut larvae at the beginning of this trial (day 0) (9 PFF, 307° d) was 15.93±1.62mg. On day 17 (26 PFF, 507° d) this had increased to 44.92±19.20mg. At this time there were significant differences between the weights of halibut in the different treatment groups (F_3:10=5.74, P<0.05) with larvae reared on the AlgaMac-3000 + SS regime having the highest mean weight (Fig. 1). The increase in wet weight over this period represents a mean specific growth rate (SGR) of 6.15±0.65%/day and there were no significant differences between treatment groups (F_3:10=5.74,P>0.05). SGR values for each treatment group are shown in Table2. 

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Figure 1. The increase in mean wet weight (± standard effort) of halibut larvae reared on 4 experimental diets over the trial period, from 9 to 54 days post-first feeding.

Table 2. Mean wet weight specific growth rates (%/d) (± standard deviation) of halibut larvae reared on 4 experimental diets during the first two time intervals in this study, and from 9 to 37 PFF.

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Figure 2. Mean wet weight of halibut larvae reared on 4 experimental diets on day 28 (37 PFF, 626° d). Vertical bars represent one standard deviation.

On day 54 PFF (839° d) the mean wet weight of experimental halibut was 190.18±79.16mg. Table 3 presents the mean weights of halibut from each treatment, the corresponding SGR values for the period 38-54 PFF, and SGRs for the entire trial period day 9 to 54 PFF. On day 54 PFF the wet weights of halibut reared on Schizotein-enriched Artemia were highly significantly lower than those of all other treatment groups (F_3:76=10.51, P<0.001, Fig. 1). In this case the individual fish was treated as the replicate for the purpose of analysis as there was only one tank per treatment.

Table 3. Mean wet weights on day 54 PFF of halibut larvae reared on 4 experimental diets, specific growth rates (%/d) over the final non-replicated period of the trial (days 38-54 PFF) and over the entire 45 day study period. Standard deviations in parenthesis.

Within-group growth variability, as assessed using the coefficient of variation for wet weight, increased substantially in all treatments during the first time interval of the trial, days 9-26 PFF, (Figure 3). Larvae in the Schizotein treatment exhibited the highest growth variability on day 26 PFF, while those in the control treatment (AlgaMac-2000 + SS) had the lowest. Coefficient of variation had decreased overall by the end of the replicated phase of the trial, on day 37 PFF, although the inter-treatment differences in CV were greater at this time (Figure 3). The highest growth variability was seen in the AlgaMac-3000 + SS treatment group, while the AlgaMac-3000 and AlgaMac-2000 + SS groups had similarly low CVs.

Figure 3. The coefficient of variation for wet weight (%) in the four treatment groups of halibut from day 9 to 37 post first feeding.

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Figure 4. Mean dry weights of halibut from the 4 treatment groups on day 37 PFF (626° d). Vertical bars represent one standard deviation.

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Table 4. Dry weight specific growth rates (1/o/d) (± standard deviation) of experimental halibut over the first three time intervals of this study and over the entire study period from 9 - 54 days PFF.

Between-treatment differences in dry weight SGR followed the same trends as the wet weight SGR data. The

As final metamorphosis data was collected at the conclusion of this trial, following the pooling of all replicate tanks on day 37 PFF, there is no data on the variability in the parameters under investigation and no statistical analyses were undertaken. Eye migration at the conclusion of the trial (54 PFF, 839° d) was much poorer for halibut reared on the Schizotein diet than for those from all other treatments (Figure 5). The two

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Figure 5. Percent frequency distribution of halibut eye migration categories (54 PFF, 839° d). 0: blind side eye not visible; 1: blind side eye just visible; 2: full diameter of blind side eye visible, but lens not visible, 3: complete migration, both eyes on ocular side with lenses visible.

Figure 6. Percent frequency distribution of halibut pigment categories (54 PFF, 839° d). 

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