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Fish roe as a major component in start-feed for marine fish larvae
Aquaculture, 79 (1989) 353-362 Elsevier Science Publishers B.V., Amsterdam -
353 Printed in The Netherlands
Fish Roe as a Major Component in Start-Feed for Marine Fish Larvae 0. GARATUN-TJELDSTIZI’, I. OPSTAD2, T. HANSEN3 and I. HUSE’ ‘University of Bergen, Department of Biochemistry, Arstadveien 19, N-5000 Bergen (Norway) 21nstitute of Marine Research, Austevoll Marine Aquaculture Station, N-5392 Storebe (Norway) 31nstitute of Marine Research, Matre Aquaculture Station, N-5198 Matredal (Norway)
ABSTRACT Garatun-Tjeldste, O., Opstad, I., Hansen, T. and Huse, I., 1989. Fish roe as a major component in start-feed for marine fish larvae. Aquaculture, 79: 353-362. Marine fish larvae, cod (Gadus morhua L.) and plaice (Pleuronectes platessa), were start-fed in laboratory tanks using diets containing fish roe aa a major component, and compared to larvae start-fed on other types of diets (Calanus diets, yeast-based diets, microencapsulated diets and several experimental diets from various commercial sources). Although metamorphosis did not occur due to a high mortality rate, It is concluded that roe-based start diets were consumed by the larvae, and gave better growth rates and longer survival time than the other dry feeds. Roe diets were also tested in start-feeding of salmon fry, which showed negligible mortality and, in some cases, a higher growth rate than fry on a commercial salmon diet.
INTRODUCTION
Marine fish larvae are, with a few exceptions (Adron et al., 1974; E. Moxness, personal communication, 1987)) dependent upon a supply of live feed for growth and development during the start-feeding period. To our knowledge, there are no reports of good and reliable artificial start-feeds for marine fish larvae. We are at present trying to develop artificial start-feeds for cod, plaice, turbot and halibut by modifying the cod roe diet of Molvik et al. (1984). We have also performed start-feeding experiments with several diets based upon other materials ( Garatun-Tjeldsto et al., 1984; Opstad et al., 1984). Although metamorphosis did not occur, due to a high mortality rate, it was concluded that roe-based feeds, fortified with peptone and coated with hard fat or stearol, are capable of initiating development of the gut and filling of the swim bladder, and supporting growth of cod larvae (Garatun-Tjelsto et al., 1987). In this paper we present data from start-feeding experiments with roe-based
0044-8486/89/$03.50
0 1989 Elsevier Science Publishers B.V.
354
diets compared to various diets from commercial sources, and it is suggested that fish roe may be a possible major component in experimental start-feeds for marine fish larvae. MATERIALS AND METHODS
Eggs, larvae and roe for incorporation in diets Cod larvae (Gardus morhua L.) were obtained at Austevoll Aquaculture Station by natural spawning of broodstock in large pens. Fertilized eggs were collected, hatched in tanks, and transferred to the rearing units at 2-4 days after hatching (Huse et al., 1984; Opstad et al., 1989). Plaice larvae (Pleuronectes platessa) were obtained from eggs of the broodstock at Austevoll Aquaculture Station. Salmon fry (Salmo salar) were obtained from eggs of the broodstock at Matre Aquaculture Station. Cod roe for feed incorporation was obtained from local fishermen, or at the fish market. Capelin roe for incorporation in the start-feeds was washed in salt water during the industrial production, and frozen in blocks of 20 kg for storage. Thawing and rinsing in one volume of fresh water, and sieving to remove salt, were done with minor weight losses. Herring roe to be used as a feed ingredient was washed in fresh water during the industrial production before freezing and storage. Preparation of diets Several procedures and diets have previously been described (Huse, 1981; Huse et al., 1982; Molvik et al., 1984; Opstad et al., 1984; Garatun-Tjeldsto et al., 1984,1987). Heat denaturation of feed materials was performed by boiling in steam at 100” C, by autoclaving at 120” C, or in a microwave oven to specified temperature. Dehydration of feed materials was peiformed by vacuum, by a spray drier, or in an air stream. Materials dried in vacuum were frozen on metal in l-cm layers and dried at an oven temperature of 45 oC for 12 to 16 h. Roe materials dried in an air stream were prepared with antioxidants (in most cases ethoxyquin, 40 mg/kg wet roe), plated on perforated shelves and dried in a gentle air stream at 60°C. Materials dried by spray technique were prepared with antioxidants and sprayed through a nozzle at 150 oC, with an outlet temperature of 50’ C. Grinding and mixing of dry feed materials were done in a ball mill flushed with nitrogen, or in an ultracentrifugal pin mill (Retsch ZMl ) . Granulates and particles predominantly in the range of 75-300 pm were obtained, using the coating material as a glue. Particle formation of feeds based
upon spray-dried herring roe and fine powders was obtained by including 2% Tylose C6000 ( a binder produced by Hoechst AG, Frankfurt) in the dry feed, adding 20% of water, pelleting the mixture, allowing it to dry in a gentle air stream at 25’ C, and grinding the pellets. Vitamins were included before drying, in the case of gentle drying methods, or applied together with the coating materials (Molvik et al., 1984). In most cases the vitamins added to 1 kg wet roe included the following: 20 mg thiamine-HCl, 45 mg riboflavin, 20 mg pyridoxine, 40 mg DL-pantothenol, 40 mg nicotinic acid, 5 mg folic acid, 0.03 mg cyanocobalamine, 80 mg inositol, 0.25 mg biotin, 3.0 g ascorbic acid, 3.5 g ascorbylpalmitate, 10 mg menadione, 100 mgp-aminobenzoic acid and 100 mg cantaxanthine, stirred in 25 ml of distilled water, with 100 mg tocopherylacetate in 1 ml of cod liver oil. The herring roe washed in fresh water was denatured by autoclaving in batches of 40 kg, and suspended in one volume of fresh water; the mixture included ethoxyquin at 40 mg/kg. Approximately 50% of the material passed through a nylon sieve (24 mesh), and was spray-dried, resulting in a very electrostatic fine powdered meal used in feed processing. Capelin roe-based diets were used for start-feeding of salmon. The roe was thawed, rinsed in fresh water, and ethoxyquin (40 mg/kg) was added, before air drying at 60 oC. The material contained approximately 20% fat after drying. The diets were prepared from 1 kg dried roe, 3 g lecithin, 0.4 g inositol, 30 g stearol, 0.7 g ascorbic acid, and other vitamins at 1.5 times the amount recommended by NRC (1978). One batch of this mixture was ground and pelleted with 20 g tylose C6000, another was used without grinding and tylose was omitted. The batch containing whole roe could be used directly as start-feed for salmon larvae, but the particle size of whole roe is too large for the smaller marine larvae. Oxytetracycline (0.15 g/kg wet roe) was included in the diets referred as II.85 and III.85 in Fig. 3. Some properties of the commercial feeds are presented with the results. Experimental design and evaluation of results Feeding experiments with cod and plaice larvae were carried out as previously described (Opstad et al., 1984), in 200-l conical laboratory tanks supplied with microfiltered (5 pm) UV-irradiated sea water, and a flow-rate of at least two shifts per day. The feeds designated by the number 83 were fed by hand three times each day, and the other feeds were given automatically by feeder disks during the light period of 16 h. A typical feeding experiment with 17 tanks included one unfed group, 4-12 groups fed on diets produced by us, groups on live feeds, and groups on com-
356
mercial feed sources. Thus, the data from each experiment could be interpretated after pooling in rough categories (roe feeds versus commercial feeds or unfed groups). RESULTS
Survival data for salmon, cod and plaice Salmon fry have been brought beyond the start-feeding period with almost no mortality, and excellent growth response, when start-fed on a cod roe diet (Molvik et al., 1984). We have reproduced this result, using herring roe (unpublished), and by using diets based on capelin roe, as shown in the present study. No marine larvae were brought beyond metamorphosis when fed artificial dry feeds. However, the roe-based diets were.shown to be ingested, and the survival time of cod larvae was extended from 14 days (unfed) to 35 days (roe fed). The survival time of plaice larvae was extended from 16 to 42 days. Start-feeding of salmon with roe-based diets Salmon fry were used as a reference organism able to accept artificial startfeeds. We compared growth and survival of salmon fry start-fed on a commercial dry diet (TESS elite G-0.6, Skretting A/S, Norway), with diets based upon ground (less than 650 pm), and unground capelin roe. The survival was excellent using all feeds. The specific growth rates, calculated according to Houde and Schekter (1981)) are given in Table 1. At least during the later phase of the start-feeding period (days 38-49 after commencement of first feeding), we found that whole capelin roe gave a better specific growth response than the commercial dry diet. Normalization of survival curves for cod larvae Each set of simultaneous experiments included a starvation group for estimation of the yolk sack period. These fish typically demonstrated mass death at day 14. Cod larvae fed on collected zooplankton or cultured rotifers were brought beyond metamorphosis, showing that it is possible to obtain some survival (2-10% typically) under laboratory conditions. During the first days after transfer of the cod larvae to the rearing units, we observed some mortality of unpredictable magnitude, but the survival curves were stable with a plateau from day 5 to day 13. To minimize the problem of statistical uncertainty of the initial samples we defined the mean of four observations in this plateau area as 100% reference value (Garatun-Tjeldsto et al., 1984).
357 TABLE 1 Specific growth rate of salmon fry start-fed on a commercial diet and on two diets based on capelin roe (average of four sets of experiments) Days after commencement of first feeding Period of calculation:
O-28 (early phase)
38-49 (late phase
Diet
Specific growth rate (average) % W/day+ SD
% W/day? SD
0.81 f 0.16 0.97~0.1 0.99 + 0.03
1.52 f0.24 2.16k0.13 1.96kO.l
Capelin roea ground Capelin roe* whole TESS elite G-O.6
Days O-28, temperature 7°C; days 38-49, temperature 10.5-113°C. “Diets coated with stearol.
SURVIVAL OF COD LARVAE ROE FEED 15 EXP.)
10
TIME
(DAYS
.a0
SURVIVAL OF COD LARVAE LIVE FEED (2 EXP.)
30 AFTER
HATCHING)
Fig. 1. Normalized survival of cod larvae on three feeding regimes, with reference (100%) to the mean of four observations between day 5 and day 14 after hatching. From day 12 to day 25 after hatching the observed survival of larvae fed roe diets typically exceeded that of larvae fed on live feeds (Garatun-Tjeldstar et al., 1984). In the later phase this relationship was reversed (Fig. 1 ), and the population of larvae fed artificial feeds died.
Comparison of larvae fed on roe-based and commercial diets Some comparisons of cod larvae fed on the roe-based diets and the commercial diets are shown in Fig. 2 and 3. Within each group of simultaneous feeding experiments, we always found some roe diets (circles) which gave better growth and survival than the best commercial feed materials (crosses). Typically, in the later phase of each experiment, the average survival and growth of larvae fed roe feeds (whole lines) exceeded the survival and growth of larvae fed the
358
I
IO +w CALANUS (GRANULATED)
100
EXP.
20
30
COD LARVAE (III.641 ROE DIETS 0 -2 EXP. COMMERCIAL +-I 6 EXP. (MODIFIED CARP TYPE)
+
?
:,
E 2
: +
:p I
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0 K
8 %
-3
COD LARVAE (L84) ROE D,EIS o -6 EXP. CQMMERClAL +- - 3 EXP. WODIFIED CARP TYPE)
A,’ ‘\
i
bt
‘+ 0
I
10
20
30 TIME
10 (DAYS
AFTER
20
30
HATCHING)
Fig. 2. Weight and survival of cod larvae start-fed diets based upon cod roe, compared to Calanus diets and modified carp-type diets from commercial sources. (Lines: mean value of roe diets; broken lines: mean value of other diets. Circles: best roe diet; crosses: best commercial or Calanus diet.)
commercial diets (broken lines). We observed the same tendency on growth and extended survival in experiments with start-feeding of plaice larvae (Fig. 4). The commercial feeds referred to in Fig. 2 (I.84 and 111.84) were of the carpfeed type, experimentally modified by the manufacturer for start-feeding of marine fish larvae. The other commercial feeds have been used successfully in shrimp rearing, and were recommended by the manufacturers for weaning and experiments with start-feeding of marine fish larvae. Cod larvae fed on the nylon encapsulated feeds (Fig. 3, 11.85) died before the unfed group. The microspheres (Fig. 3, III.85 ) and the yeast-based microspheres (Fig. 4, III.87 ) showed good buoyancy properties. According to the manufacturer, the contents were adjusted to simulate the nutritional value of natural plankton. These diets did not produce much pollution.
I’
7 0
__---
_’
0 ’
_---
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-\,+ ‘\
1’
10
_. 20
10
30
COD LARVAE (1185) ROE DIETS o 6 EXP. COMMERCIAL + - - 2 EXP. INYLON CAPSULES1
20
30
COD LARVAE (rIL65) ROE DIETS o 2 EXP. COMMERCIAL + 2 EXP. (SPHERES So-qm)
10
20
30
COD LARVAE (IIL651 ROE DIETS o 4 EXP. COMMERCIAL + i - 2 EXP. (GRANULATED)
1 Oo(-------
2
++ 0
5
5 ;
50
iz
.:
‘*,+ .. 0
0 0
4 _-_k
v
E0
+ 10 20
10
30 TIME
(DAYS
20
AFTER
.,’ $OO ‘\+
‘\
‘.+
30
10
20
30
HATCHING)
Fig. 3. Weight and survival of cod larvae start-fed with diets based upon cod roe (11.85) and cod roe mixed with raw cod muscle (111.85)) compared to commercial microencapsulated diets and granulated “larval diets” from commercial sources. (Lines: mean value of roe diets; broken lines: mean value of commercial diets. Circles: best roe diet; crosses: best commercial diet.)
DISCUSSION
We have seen in the experiment with salmon as a reference organism, that roe-based diets are acceptable as good starter diets, at least for some species, and are significantly better than a good commercial diet when the larvae are large enough to ingest whole unground roe particles. Grinding, pelleting, and crushing of the pellets, which is necessary in production of start-feeds for the smaller marine larvae, reduced the performance of the roe diets. This may indicate that the roe-based diets have a potential for improvement by microencapsulation techniques and adjustment of particle properties. It was previously concluded from start-feeding of cod larvae that roe-based starter feeds fortified with peptone and coated with hard fat or stearol, are
360
1501-----1
I
10 PLAICE LARVAE tIII.87) ROE DIETS o-2 EXP. COMMERCIAL +. t EXP. (FLAKES) UNFED EXP.
A
20
30
PLAICE LARVAE (111.87) ROE DIETS o 2 EXP. COMMERCIAL+-1 U[P. IYEAST-BASED SPHERES) UNFED A I EXP.
I
A I
L!!Yi I I
I
I
i,
\
\
‘.
10
TIME
(DAYS
AFTER
\
2;
36
HATCHING)
Fig. 4. Weight and survival of plaice larvae start-fed with diets based upon herring roe (111.87)) compared to commercial flake diets, and yeast-based microspheres. (Circles: roe diets; crosses: commercial diets.)
capable of initiating development of the gut and filling of the swim bladder, and supporting larval growth (Garatun-Tjeldste et al., 1987 ) . We feel that strictly “paired” experiments with variation of one factor each time are not possible to perform. As an example, variation of the fat content in the diet may simultaneously influence energy content, leaching properties, tank hygiene, particle buoyancy, feed clogging, swelling and several other factors involved in the experimental situation. One approach, open for interpretation of the data, is pooling of the experiments into rough categories (roe feeds versus commercial or live feeds) and see whether the conclusions point in the same direction. From this approach we conclude that roe-based start-feeds were consumed by cod and plaice larvae, and gave better growth and longer survival times than several commercial diets. This study was based on more than 150 experiments with modified diets (in-
361
eluding roe from eight species), but no larvae were brought to metamorphosis. With the exception of the positive effects of choosing roe fortified with peptone and coated with hard fat as a major feed component, we have not been able to recognise any trends in the material. Copepod nauplii are assumed to be the natural food of cod larvae. Dry feeds based upon adult Cu2unu.s that had been supplemented with vitamins, fortified with peptone (either raw or heat denatured), lyophilized, ground and coated with lipid, did not support growth (11.83). Rearing cod larvae on natural plankton in enclosed ponds in extensive systems has resulted in a survival of 30~50% beyond metamorphosis (Kvenseth and 0iestad, 1984). In intensive laboratory evironments, only about 10% survival is achieved by rearing on live feed and no survival on artificial feeds. One of the crucial goals for further research must be to establish the causes of this high mortality: whether they be factors associated with the artificial diets, feeding procedures, infection, population density, or due to the laboratory environment. ACKNOWLEDGEMENT
The work was supported by The Norwegian Fisheries Research Council (NFFR) , project V.108.002 and V.108.010, and by the Norwegian Department of Fisheries, project Eff.141/85. Preparation of feed materials was done in cooperation with The Norwegian Herring Oil and Meal Industry Research Institute (NFFR-projects 111.108.013 and V.108.020). The technical assistance of Anita Janne Andersen and Inger Ottesen is gratefully acknowledged. REFERENCES Adron, J.W., Blair, A. and Cowey, C.B., 1974. Rearing plaice (Pleuroneetes platessa) larvae to metamorphosis using an artificial diet. Fish. Bull., 72: 353-357. Garatun-Tjeldste, O., Isaksen, E., Raae, A., Teigland, M., Thomassen, J., Walther, B.T., Klungseyr, L., Opstad, I., Jensen, P., Strand, B. and Huse, I., 1984. Experiments with start-feeding of cod (Gadus morhuu L.) larvae in the laboratory. In: Acts of the Norwegian-French Workshop on Aquaculture, 4-8 December 1984. IFREMER, Brest, France, pp. 269-286. Garatun-Tjeldste, O., Thomassen, J., Klungseyr, L., Opstad, I., Strand, B. and Huse, I., 1987. Artificial start-feed for cod larvae (Gadus morhua L.) based upon cod roe. Sarsia, 72: 373-374. Houde, E.D. and Schekter, R.C., 1981. Growth rates, rations and cohort consumption of marine fish larvae in relation to prey concentrations. Count. Meeting, Int. Count. Explor. Seas, 178: 441-453. Huse, I., 1981. An attempt to start feed cod larvae with artificial diets. Count. Meeting, Int. Count. Explor. Seas, F 14: l-4 (mimeo). Huse, I., Opstad, I., Klungseyr, L. and Walther, B.T., 1982. Endocrine factors affecting the first food uptake by cod larvae. Count. Meeting, Int. Count. Explor. Seas, F 17: l-7 (mimeo). Huse, I., Jensen, P. and Strand, B., 1984. Intensive production of cod fry. In: Acts of the Norwegian-French Workshop on Aquaculture, 4-8 December 1984. IFREMER, Brest, France, pp. 179-192. Kvenseth, P. and Oiestad, V., 1984. Large-scale rearing of cod fry on the natural production in an
362 enclosed pond. In: E. Dahl, D.S. Danielsen, E. Moxness and P. Solemdal (Editors), The Propagation of Cod (Gadus morhua L.). An International Symposium, 14-18 June 1983, at Fladevigen Biological Station, Arendal, Norway, Vol. 2, pp. 645-655. Molvik, G., Hjelmeland, K., Rings, E. and Raa, J., 1984. Properties of a new diet for fish larvae, including cod (Gadus morhua L.). In: E. Dahl, D.S. Danielsen, E. Moxness and P. Solemdal (Editors), The Propagation of Cod (G&US morhua L. ) . An International Symposium, 14-18 June 1983, at Fledevigen Biological Station, Arendal, Norway, Vol. 1, pp. 203-211. National Research Council, 1978. Nutrient Requirements of Laboratory Animals, no. 10. National Academy Press, Washington, DC. Opstad, I., Strand, B., Huse, I., Garatun-Tjedsts, O., Klungseyr, L., Thomassen, J. and Walther, B., 1984. Laboratory studies on the use of dry diets, rotifers (Bruchionusplicatili O.F. Mtiller) for start feeding cod larvae (Gadus morhuu L. ) . In: Acts of the Norwegian-French Workshop on Aquaculture, 4-8 December 1984. IFREMER, Brest, France, pp. 257-267. Opstad, I., Strand, B., Huse, I. and Garatun-Tjeldster, O., 1989. Laboratory studies on the use of rotifers (Bruchionus plicatilis O.F. Mtiller) as start-feed for cod larvae (G&us morhua L.). Aquaculture, 79: 345-351. Ring@, E., Olsen, R.E. and Bse, B., 1987. Initial feeding of wolf fish (Anarhichus lupus L.) fry. Aquaculture, 62: 33-43.
353 Printed in The Netherlands
Fish Roe as a Major Component in Start-Feed for Marine Fish Larvae 0. GARATUN-TJELDSTIZI’, I. OPSTAD2, T. HANSEN3 and I. HUSE’ ‘University of Bergen, Department of Biochemistry, Arstadveien 19, N-5000 Bergen (Norway) 21nstitute of Marine Research, Austevoll Marine Aquaculture Station, N-5392 Storebe (Norway) 31nstitute of Marine Research, Matre Aquaculture Station, N-5198 Matredal (Norway)
ABSTRACT Garatun-Tjeldste, O., Opstad, I., Hansen, T. and Huse, I., 1989. Fish roe as a major component in start-feed for marine fish larvae. Aquaculture, 79: 353-362. Marine fish larvae, cod (Gadus morhua L.) and plaice (Pleuronectes platessa), were start-fed in laboratory tanks using diets containing fish roe aa a major component, and compared to larvae start-fed on other types of diets (Calanus diets, yeast-based diets, microencapsulated diets and several experimental diets from various commercial sources). Although metamorphosis did not occur due to a high mortality rate, It is concluded that roe-based start diets were consumed by the larvae, and gave better growth rates and longer survival time than the other dry feeds. Roe diets were also tested in start-feeding of salmon fry, which showed negligible mortality and, in some cases, a higher growth rate than fry on a commercial salmon diet.
INTRODUCTION
Marine fish larvae are, with a few exceptions (Adron et al., 1974; E. Moxness, personal communication, 1987)) dependent upon a supply of live feed for growth and development during the start-feeding period. To our knowledge, there are no reports of good and reliable artificial start-feeds for marine fish larvae. We are at present trying to develop artificial start-feeds for cod, plaice, turbot and halibut by modifying the cod roe diet of Molvik et al. (1984). We have also performed start-feeding experiments with several diets based upon other materials ( Garatun-Tjeldsto et al., 1984; Opstad et al., 1984). Although metamorphosis did not occur, due to a high mortality rate, it was concluded that roe-based feeds, fortified with peptone and coated with hard fat or stearol, are capable of initiating development of the gut and filling of the swim bladder, and supporting growth of cod larvae (Garatun-Tjelsto et al., 1987). In this paper we present data from start-feeding experiments with roe-based
0044-8486/89/$03.50
0 1989 Elsevier Science Publishers B.V.
354
diets compared to various diets from commercial sources, and it is suggested that fish roe may be a possible major component in experimental start-feeds for marine fish larvae. MATERIALS AND METHODS
Eggs, larvae and roe for incorporation in diets Cod larvae (Gardus morhua L.) were obtained at Austevoll Aquaculture Station by natural spawning of broodstock in large pens. Fertilized eggs were collected, hatched in tanks, and transferred to the rearing units at 2-4 days after hatching (Huse et al., 1984; Opstad et al., 1989). Plaice larvae (Pleuronectes platessa) were obtained from eggs of the broodstock at Austevoll Aquaculture Station. Salmon fry (Salmo salar) were obtained from eggs of the broodstock at Matre Aquaculture Station. Cod roe for feed incorporation was obtained from local fishermen, or at the fish market. Capelin roe for incorporation in the start-feeds was washed in salt water during the industrial production, and frozen in blocks of 20 kg for storage. Thawing and rinsing in one volume of fresh water, and sieving to remove salt, were done with minor weight losses. Herring roe to be used as a feed ingredient was washed in fresh water during the industrial production before freezing and storage. Preparation of diets Several procedures and diets have previously been described (Huse, 1981; Huse et al., 1982; Molvik et al., 1984; Opstad et al., 1984; Garatun-Tjeldsto et al., 1984,1987). Heat denaturation of feed materials was performed by boiling in steam at 100” C, by autoclaving at 120” C, or in a microwave oven to specified temperature. Dehydration of feed materials was peiformed by vacuum, by a spray drier, or in an air stream. Materials dried in vacuum were frozen on metal in l-cm layers and dried at an oven temperature of 45 oC for 12 to 16 h. Roe materials dried in an air stream were prepared with antioxidants (in most cases ethoxyquin, 40 mg/kg wet roe), plated on perforated shelves and dried in a gentle air stream at 60°C. Materials dried by spray technique were prepared with antioxidants and sprayed through a nozzle at 150 oC, with an outlet temperature of 50’ C. Grinding and mixing of dry feed materials were done in a ball mill flushed with nitrogen, or in an ultracentrifugal pin mill (Retsch ZMl ) . Granulates and particles predominantly in the range of 75-300 pm were obtained, using the coating material as a glue. Particle formation of feeds based
upon spray-dried herring roe and fine powders was obtained by including 2% Tylose C6000 ( a binder produced by Hoechst AG, Frankfurt) in the dry feed, adding 20% of water, pelleting the mixture, allowing it to dry in a gentle air stream at 25’ C, and grinding the pellets. Vitamins were included before drying, in the case of gentle drying methods, or applied together with the coating materials (Molvik et al., 1984). In most cases the vitamins added to 1 kg wet roe included the following: 20 mg thiamine-HCl, 45 mg riboflavin, 20 mg pyridoxine, 40 mg DL-pantothenol, 40 mg nicotinic acid, 5 mg folic acid, 0.03 mg cyanocobalamine, 80 mg inositol, 0.25 mg biotin, 3.0 g ascorbic acid, 3.5 g ascorbylpalmitate, 10 mg menadione, 100 mgp-aminobenzoic acid and 100 mg cantaxanthine, stirred in 25 ml of distilled water, with 100 mg tocopherylacetate in 1 ml of cod liver oil. The herring roe washed in fresh water was denatured by autoclaving in batches of 40 kg, and suspended in one volume of fresh water; the mixture included ethoxyquin at 40 mg/kg. Approximately 50% of the material passed through a nylon sieve (24 mesh), and was spray-dried, resulting in a very electrostatic fine powdered meal used in feed processing. Capelin roe-based diets were used for start-feeding of salmon. The roe was thawed, rinsed in fresh water, and ethoxyquin (40 mg/kg) was added, before air drying at 60 oC. The material contained approximately 20% fat after drying. The diets were prepared from 1 kg dried roe, 3 g lecithin, 0.4 g inositol, 30 g stearol, 0.7 g ascorbic acid, and other vitamins at 1.5 times the amount recommended by NRC (1978). One batch of this mixture was ground and pelleted with 20 g tylose C6000, another was used without grinding and tylose was omitted. The batch containing whole roe could be used directly as start-feed for salmon larvae, but the particle size of whole roe is too large for the smaller marine larvae. Oxytetracycline (0.15 g/kg wet roe) was included in the diets referred as II.85 and III.85 in Fig. 3. Some properties of the commercial feeds are presented with the results. Experimental design and evaluation of results Feeding experiments with cod and plaice larvae were carried out as previously described (Opstad et al., 1984), in 200-l conical laboratory tanks supplied with microfiltered (5 pm) UV-irradiated sea water, and a flow-rate of at least two shifts per day. The feeds designated by the number 83 were fed by hand three times each day, and the other feeds were given automatically by feeder disks during the light period of 16 h. A typical feeding experiment with 17 tanks included one unfed group, 4-12 groups fed on diets produced by us, groups on live feeds, and groups on com-
356
mercial feed sources. Thus, the data from each experiment could be interpretated after pooling in rough categories (roe feeds versus commercial feeds or unfed groups). RESULTS
Survival data for salmon, cod and plaice Salmon fry have been brought beyond the start-feeding period with almost no mortality, and excellent growth response, when start-fed on a cod roe diet (Molvik et al., 1984). We have reproduced this result, using herring roe (unpublished), and by using diets based on capelin roe, as shown in the present study. No marine larvae were brought beyond metamorphosis when fed artificial dry feeds. However, the roe-based diets were.shown to be ingested, and the survival time of cod larvae was extended from 14 days (unfed) to 35 days (roe fed). The survival time of plaice larvae was extended from 16 to 42 days. Start-feeding of salmon with roe-based diets Salmon fry were used as a reference organism able to accept artificial startfeeds. We compared growth and survival of salmon fry start-fed on a commercial dry diet (TESS elite G-0.6, Skretting A/S, Norway), with diets based upon ground (less than 650 pm), and unground capelin roe. The survival was excellent using all feeds. The specific growth rates, calculated according to Houde and Schekter (1981)) are given in Table 1. At least during the later phase of the start-feeding period (days 38-49 after commencement of first feeding), we found that whole capelin roe gave a better specific growth response than the commercial dry diet. Normalization of survival curves for cod larvae Each set of simultaneous experiments included a starvation group for estimation of the yolk sack period. These fish typically demonstrated mass death at day 14. Cod larvae fed on collected zooplankton or cultured rotifers were brought beyond metamorphosis, showing that it is possible to obtain some survival (2-10% typically) under laboratory conditions. During the first days after transfer of the cod larvae to the rearing units, we observed some mortality of unpredictable magnitude, but the survival curves were stable with a plateau from day 5 to day 13. To minimize the problem of statistical uncertainty of the initial samples we defined the mean of four observations in this plateau area as 100% reference value (Garatun-Tjeldsto et al., 1984).
357 TABLE 1 Specific growth rate of salmon fry start-fed on a commercial diet and on two diets based on capelin roe (average of four sets of experiments) Days after commencement of first feeding Period of calculation:
O-28 (early phase)
38-49 (late phase
Diet
Specific growth rate (average) % W/day+ SD
% W/day? SD
0.81 f 0.16 0.97~0.1 0.99 + 0.03
1.52 f0.24 2.16k0.13 1.96kO.l
Capelin roea ground Capelin roe* whole TESS elite G-O.6
Days O-28, temperature 7°C; days 38-49, temperature 10.5-113°C. “Diets coated with stearol.
SURVIVAL OF COD LARVAE ROE FEED 15 EXP.)
10
TIME
(DAYS
.a0
SURVIVAL OF COD LARVAE LIVE FEED (2 EXP.)
30 AFTER
HATCHING)
Fig. 1. Normalized survival of cod larvae on three feeding regimes, with reference (100%) to the mean of four observations between day 5 and day 14 after hatching. From day 12 to day 25 after hatching the observed survival of larvae fed roe diets typically exceeded that of larvae fed on live feeds (Garatun-Tjeldstar et al., 1984). In the later phase this relationship was reversed (Fig. 1 ), and the population of larvae fed artificial feeds died.
Comparison of larvae fed on roe-based and commercial diets Some comparisons of cod larvae fed on the roe-based diets and the commercial diets are shown in Fig. 2 and 3. Within each group of simultaneous feeding experiments, we always found some roe diets (circles) which gave better growth and survival than the best commercial feed materials (crosses). Typically, in the later phase of each experiment, the average survival and growth of larvae fed roe feeds (whole lines) exceeded the survival and growth of larvae fed the
358
I
IO +w CALANUS (GRANULATED)
100
EXP.
20
30
COD LARVAE (III.641 ROE DIETS 0 -2 EXP. COMMERCIAL +-I 6 EXP. (MODIFIED CARP TYPE)
+
?
:,
E 2
: +
:p I
50 k
0 K
8 %
-3
COD LARVAE (L84) ROE D,EIS o -6 EXP. CQMMERClAL +- - 3 EXP. WODIFIED CARP TYPE)
A,’ ‘\
i
bt
‘+ 0
I
10
20
30 TIME
10 (DAYS
AFTER
20
30
HATCHING)
Fig. 2. Weight and survival of cod larvae start-fed diets based upon cod roe, compared to Calanus diets and modified carp-type diets from commercial sources. (Lines: mean value of roe diets; broken lines: mean value of other diets. Circles: best roe diet; crosses: best commercial or Calanus diet.)
commercial diets (broken lines). We observed the same tendency on growth and extended survival in experiments with start-feeding of plaice larvae (Fig. 4). The commercial feeds referred to in Fig. 2 (I.84 and 111.84) were of the carpfeed type, experimentally modified by the manufacturer for start-feeding of marine fish larvae. The other commercial feeds have been used successfully in shrimp rearing, and were recommended by the manufacturers for weaning and experiments with start-feeding of marine fish larvae. Cod larvae fed on the nylon encapsulated feeds (Fig. 3, 11.85) died before the unfed group. The microspheres (Fig. 3, III.85 ) and the yeast-based microspheres (Fig. 4, III.87 ) showed good buoyancy properties. According to the manufacturer, the contents were adjusted to simulate the nutritional value of natural plankton. These diets did not produce much pollution.
I’
7 0
__---
_’
0 ’
_---
0
I’+;
-\,+ ‘\
1’
10
_. 20
10
30
COD LARVAE (1185) ROE DIETS o 6 EXP. COMMERCIAL + - - 2 EXP. INYLON CAPSULES1
20
30
COD LARVAE (rIL65) ROE DIETS o 2 EXP. COMMERCIAL + 2 EXP. (SPHERES So-qm)
10
20
30
COD LARVAE (IIL651 ROE DIETS o 4 EXP. COMMERCIAL + i - 2 EXP. (GRANULATED)
1 Oo(-------
2
++ 0
5
5 ;
50
iz
.:
‘*,+ .. 0
0 0
4 _-_k
v
E0
+ 10 20
10
30 TIME
(DAYS
20
AFTER
.,’ $OO ‘\+
‘\
‘.+
30
10
20
30
HATCHING)
Fig. 3. Weight and survival of cod larvae start-fed with diets based upon cod roe (11.85) and cod roe mixed with raw cod muscle (111.85)) compared to commercial microencapsulated diets and granulated “larval diets” from commercial sources. (Lines: mean value of roe diets; broken lines: mean value of commercial diets. Circles: best roe diet; crosses: best commercial diet.)
DISCUSSION
We have seen in the experiment with salmon as a reference organism, that roe-based diets are acceptable as good starter diets, at least for some species, and are significantly better than a good commercial diet when the larvae are large enough to ingest whole unground roe particles. Grinding, pelleting, and crushing of the pellets, which is necessary in production of start-feeds for the smaller marine larvae, reduced the performance of the roe diets. This may indicate that the roe-based diets have a potential for improvement by microencapsulation techniques and adjustment of particle properties. It was previously concluded from start-feeding of cod larvae that roe-based starter feeds fortified with peptone and coated with hard fat or stearol, are
360
1501-----1
I
10 PLAICE LARVAE tIII.87) ROE DIETS o-2 EXP. COMMERCIAL +. t EXP. (FLAKES) UNFED EXP.
A
20
30
PLAICE LARVAE (111.87) ROE DIETS o 2 EXP. COMMERCIAL+-1 U[P. IYEAST-BASED SPHERES) UNFED A I EXP.
I
A I
L!!Yi I I
I
I
i,
\
\
‘.
10
TIME
(DAYS
AFTER
\
2;
36
HATCHING)
Fig. 4. Weight and survival of plaice larvae start-fed with diets based upon herring roe (111.87)) compared to commercial flake diets, and yeast-based microspheres. (Circles: roe diets; crosses: commercial diets.)
capable of initiating development of the gut and filling of the swim bladder, and supporting larval growth (Garatun-Tjeldste et al., 1987 ) . We feel that strictly “paired” experiments with variation of one factor each time are not possible to perform. As an example, variation of the fat content in the diet may simultaneously influence energy content, leaching properties, tank hygiene, particle buoyancy, feed clogging, swelling and several other factors involved in the experimental situation. One approach, open for interpretation of the data, is pooling of the experiments into rough categories (roe feeds versus commercial or live feeds) and see whether the conclusions point in the same direction. From this approach we conclude that roe-based start-feeds were consumed by cod and plaice larvae, and gave better growth and longer survival times than several commercial diets. This study was based on more than 150 experiments with modified diets (in-
361
eluding roe from eight species), but no larvae were brought to metamorphosis. With the exception of the positive effects of choosing roe fortified with peptone and coated with hard fat as a major feed component, we have not been able to recognise any trends in the material. Copepod nauplii are assumed to be the natural food of cod larvae. Dry feeds based upon adult Cu2unu.s that had been supplemented with vitamins, fortified with peptone (either raw or heat denatured), lyophilized, ground and coated with lipid, did not support growth (11.83). Rearing cod larvae on natural plankton in enclosed ponds in extensive systems has resulted in a survival of 30~50% beyond metamorphosis (Kvenseth and 0iestad, 1984). In intensive laboratory evironments, only about 10% survival is achieved by rearing on live feed and no survival on artificial feeds. One of the crucial goals for further research must be to establish the causes of this high mortality: whether they be factors associated with the artificial diets, feeding procedures, infection, population density, or due to the laboratory environment. ACKNOWLEDGEMENT
The work was supported by The Norwegian Fisheries Research Council (NFFR) , project V.108.002 and V.108.010, and by the Norwegian Department of Fisheries, project Eff.141/85. Preparation of feed materials was done in cooperation with The Norwegian Herring Oil and Meal Industry Research Institute (NFFR-projects 111.108.013 and V.108.020). The technical assistance of Anita Janne Andersen and Inger Ottesen is gratefully acknowledged. REFERENCES Adron, J.W., Blair, A. and Cowey, C.B., 1974. Rearing plaice (Pleuroneetes platessa) larvae to metamorphosis using an artificial diet. Fish. Bull., 72: 353-357. Garatun-Tjeldste, O., Isaksen, E., Raae, A., Teigland, M., Thomassen, J., Walther, B.T., Klungseyr, L., Opstad, I., Jensen, P., Strand, B. and Huse, I., 1984. Experiments with start-feeding of cod (Gadus morhuu L.) larvae in the laboratory. In: Acts of the Norwegian-French Workshop on Aquaculture, 4-8 December 1984. IFREMER, Brest, France, pp. 269-286. Garatun-Tjeldste, O., Thomassen, J., Klungseyr, L., Opstad, I., Strand, B. and Huse, I., 1987. Artificial start-feed for cod larvae (Gadus morhua L.) based upon cod roe. Sarsia, 72: 373-374. Houde, E.D. and Schekter, R.C., 1981. Growth rates, rations and cohort consumption of marine fish larvae in relation to prey concentrations. Count. Meeting, Int. Count. Explor. Seas, 178: 441-453. Huse, I., 1981. An attempt to start feed cod larvae with artificial diets. Count. Meeting, Int. Count. Explor. Seas, F 14: l-4 (mimeo). Huse, I., Opstad, I., Klungseyr, L. and Walther, B.T., 1982. Endocrine factors affecting the first food uptake by cod larvae. Count. Meeting, Int. Count. Explor. Seas, F 17: l-7 (mimeo). Huse, I., Jensen, P. and Strand, B., 1984. Intensive production of cod fry. In: Acts of the Norwegian-French Workshop on Aquaculture, 4-8 December 1984. IFREMER, Brest, France, pp. 179-192. Kvenseth, P. and Oiestad, V., 1984. Large-scale rearing of cod fry on the natural production in an
362 enclosed pond. In: E. Dahl, D.S. Danielsen, E. Moxness and P. Solemdal (Editors), The Propagation of Cod (Gadus morhua L.). An International Symposium, 14-18 June 1983, at Fladevigen Biological Station, Arendal, Norway, Vol. 2, pp. 645-655. Molvik, G., Hjelmeland, K., Rings, E. and Raa, J., 1984. Properties of a new diet for fish larvae, including cod (Gadus morhua L.). In: E. Dahl, D.S. Danielsen, E. Moxness and P. Solemdal (Editors), The Propagation of Cod (G&US morhua L. ) . An International Symposium, 14-18 June 1983, at Fledevigen Biological Station, Arendal, Norway, Vol. 1, pp. 203-211. National Research Council, 1978. Nutrient Requirements of Laboratory Animals, no. 10. National Academy Press, Washington, DC. Opstad, I., Strand, B., Huse, I., Garatun-Tjedsts, O., Klungseyr, L., Thomassen, J. and Walther, B., 1984. Laboratory studies on the use of dry diets, rotifers (Bruchionusplicatili O.F. Mtiller) for start feeding cod larvae (Gadus morhuu L. ) . In: Acts of the Norwegian-French Workshop on Aquaculture, 4-8 December 1984. IFREMER, Brest, France, pp. 257-267. Opstad, I., Strand, B., Huse, I. and Garatun-Tjeldster, O., 1989. Laboratory studies on the use of rotifers (Bruchionus plicatilis O.F. Mtiller) as start-feed for cod larvae (G&us morhua L.). Aquaculture, 79: 345-351. Ring@, E., Olsen, R.E. and Bse, B., 1987. Initial feeding of wolf fish (Anarhichus lupus L.) fry. Aquaculture, 62: 33-43.