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Weaning of hatchery-reared greenback flounder (Rhombosolea tapirina Günther) from live to artificial diets: Effects of age and duration of the changeover period
Aquaculture 145 (1996) 171-181
Weaning of hatchery-reared greenback flounder ( RhombosoZea tapirina Gtinther) from live to artificial diets: Effects of age and duration of the changeover period Piers R. Hart *, G. John Purser Depurtment
ofAquaculture, University of Tasmcmia. PO Box 1214, Launceston, Tus. 7250, Austruliu Accepted 25 April 1996
Abstract The greenback flounder (Rhombosolea fupirina) is a potential culture species in Australia, but poor survival has been recorded during weaning from live Artemiu to artificial diets. This paper describes the results of three experiments on the effects of fish age and the duration of the changeover period from live to artificial food on weaning survival and subsequent growth. Fish weaned for IO days from Day 50 post-hatch or later had higher mortality (P < 0.05) than fish weaned earlier, but no differences in final weights or lengths (P > 0.05) were observed between fish weaned on different days post-hatch. Weaning from Artemia to artificial diets was shown to be possible from Day 23 post-hatch, with 82.2% survival after a IO-day overlap. An overlap of 5 days between Artemia and artificial diets resulted in a reduction in final length (P < 0.05) compared with overlaps of 10 or 20 days, but no difference in survival (P > 0.05). Overlaps of 20 days resulted in significantly heavier final weights (P < 0.05) than either 10 or 5 days. Weaning from Day 23 post-hatch with a 20-day overlap would therefore appear to result in the best growth and highest survival rate, but a IO-day overlap may result in reduced Artemiu cost with minimal effect on performance. Keywords: Age; Artificial diets; Greenback flounder; Larvae;
Rhombosolrcr
tupirinu ; Weaning
* Corresponding author. 0044-8486/96/$15.00 PII
SOO44-8486(96)0
Copyright 0 1996 Elsevier Science B.V. All rights reserved. 1343-9
172
P.R. Hart, GJ. Purser/Aquuculture
145 (1996) 171-181
1. Introduction The greenback flounder (Rhombosolea tupirina) is being investigated as a potential culture species in southern Australia. Important parameters affecting larval rearing success have been identified (Hart, 1994; Hart and Purser, 1995; Hart et al., 1996), but poor survival has been encountered during the changeover from live to artificial feeds (Hart, 1994). Live feeds continue to be essential for the first feeding of marine fish larvae because they lead to increased feeding, stimulate enzyme secretion, and result in consistently good growth and survival. However, the production and use of live feeds is expensive. There can also be a number of undesirable side effects associated with live feeds: (1) bacteria associated with zooplankton cultures can be detrimental to fish larvae; (2) the nutritional values of live feeds can be highly variable; (3) metabolites from zooplankton increase the load on fish rearing systems; (4) fine-mesh outlet screens are required to retain zooplankton so low water flow rates are necessary to avoid screen blockages and resultant tank overflows. For these reasons it is considered highly desirable to use artificial diets as early as possible in the rearing process. There have been very few successful attempts to feed the larvae of marine fish on artificial diets from first feeding (Adron et al., 1974; Applebaum, 1985; Walford et al., 1991). It appears that the main problems with artificial diets are their unacceptability to the larvae, low digestibility, and problems associated with lack of buoyancy and instability in water (Bengston, 1993; Jones et al., 1993). It is known that the digestive enzyme system of early larvae is poorly developed and that the digestion of food is assisted by the autolysis of natural prey resulting from the enzymes contained within that prey (Lauff and Hofer, 1984; Hjelmeland et al., 1988; Munilla-Moran and Stark, 1989; Munilla-Moran et al., 1990). The formation of the stomach and the commencement of peptic enzyme production appears to be critical to successful digestion of conventional artificial diets (Segner et al., 1993). Crawford (1984) weaned newly metamorphosed greenback flounder ( rhombosolea tupirina) juveniles (approximately 50 days at 14.5”C post-hatch) on to 0.5 mm trout crumbles. Complete mortality was observed when a direct transfer was used, but 70% survival was achieved with a gradual changeover of 14-28 days. This paper presents the results of a series of experiments designed to investigate the effects of (a) fish age and size, and (b) duration of the changeover period from live to artificial diets, on growth and survival .of greenback flounder during weaning. The aims were to determine the optimum age and size at which to commence weaning, and to determine the duration of the changeover period required for successful weaning from live to artificial feeds.
2. Materials and methods All experiments were conducted in a set of nine black hemispherical fibreglass tanks of 25 1 capacity. The tanks were within a recirculation system incorporating mechanical and biological filtration equipment. A 24-h light regime was provided by overhead
P.R. Hart, GJ. Purser/Aquaculture Table 1 Weaning schedule
for lo-day
weaning
145 (1996) 171-181
of R. tapirinu (adapted
Days from start of weaning
Artemia (No. ml-
l-2 3-4 5-6 7-8 9-10 11-18 19-32 33+
10 5 5 5 2.5 0 0 0
173
from Devresse et al., 1991)
’ day- ‘)
Artificial
food (e m- 3 dav- ‘)
24 24 24 40 72 80 100 10% biomass
fluorescent tubes, giving a light intensity of 650-750 lux at the water surface. Temperature and salinity were maintained at 15-17°C and 34-36%0, respectively. Flow rates were set at 30 1 h-l tank-‘. Larvae were initially reared in 200-l black hemispherical tanks prior to transfer into the experimental tanks. Larvae were fed twice daily with enriched rotifers to Day 14 post-hatch, and with Artemia nauplii (instar II) from Day 9 or Day 10 post-hatch until weaning. The changeover period from live to artificial diets followed a schedule adapted from Devresse et al. (1991) (Table 1) where Arremiu was gradually reduced and artificial diet increased over a period of 10 days. This schedule was either extended to 20 days or reduced to 5 days in the experiment described in Sect. 2. The artificial feed used in all experiments was a 1: 1 mixture of Biodiet No. 1 (Bioproducts Inc., Oregon, USA; particle size 300-500 ym, proximate composition 43% protein, 14.5% fat) and Skretting 0.6G (T. Skretting A/S, Stavanger, Norway; particle size 600 km, proximate composition 51.7% protein, 17.3% fat). Feeding with artificial feed was carried out three times daily at equal intervals during an 8-h period, while Artemia was introduced once daily, just after the last presentation of artificial feed for the given day. All experiments were carried out with three, randomly selected tanks assigned to each treatment. Measurements of the individual wet weights and total lengths of 20 fish from each tank were recorded at regular intervals during the experiments using an electronic balance (kO.5 mg) and calipers (k 0.05 mm). Growth rate was calculated using the formula Weight or length increase (mg or mm) Growth rate (mm day-’
or mg day- ‘) =
Duration of experiment
(days)
The base of each tank was siphoned daily to remove dead fish, uneaten food and faeces. Dead fish were counted in order to assess mortality. At the termination of the experiment, the survivors were counted and a random sample of 30 fish from each tank was individually weighed and measured. There was usually only a small discrepancy between the initial numbers of fish and the numbers recovered, either dead or alive. Survival at the end of the experiment was calculated by the formula Live fish remaining Actual survival
(%) =
at the end of the experiment
Initial number of fish
x 100
174
P.R. Hurt, GJ. Purser/Ayuuculture
145 (1996) 171-181
2.1. Age and size at weaning 2.1.1. Weaning after metamorphosis: Days 43, 51 and 59 post-hatch (688, 816 and 944”days, respectively) Each tank was stocked with 225 metamorphosed flounder at 33 days post-hatch (528”days, length 16.5 f 0.3 mm, weight 0.063 f 0.003 g; i * se., n = 50). Larvae had previously been fed with live feeds enriched with a mixture of microalgae, Pavfova lutheri and Zsochrisis (Tahitian strain), and Frippak Booster (microencapsulated high HUFA enrichment from Sanofi Aquaculture, Paris, France). All fish were fed at a rate of 10 Artemia ml-’ day-’ until weaning began; a feed rate of 10 Artemia ml- ’ day- ’ equates to approximately 1000 Artemia fish-’ day-’ with 10 fish 1-l. A lo-day weaning schedule was employed in this experiment (Table 1). Weaning commenced on Day 43 post-hatch for fish in three randomly selected tanks (Group 1). Weaning of the fish in a second set of three tanks commenced on Day 5 1 (Group 2) and fish in the third set of tanks were weaned from Day 59 post-hatch (Group 3) onwards. The experiment was terminated on Day 89 post-hatch (46 days from the start of the experiment). 2.1.2. Weaning before metamorphosis: Day 23 post-hatch (368”days) An experiment was undertaken to investigate whether weaning could be initiated before metamorphosis was completed, and what effect this would have on growth and survival. Three batches of 107 larvae at 23 days post-hatch (initial length 8.71 f. 0.21 mm, initial weight 0.016 + 0.001 g; i + s.e., n = 30) were transferred to three 25-l tanks. The larvae had previously been fed with live feeds enriched with Frippak Booster. These larvae had completed stomach differentiation, but were still undergoing eye migration (Hart, 1994). Weaning was carried out using the IO-day schedule (Table 1) and the experiment was terminated on Day 58 post-hatch (35 days from the start of the experiment). 2.2. Length of the changeover
period
To investigate the effect of different weaning periods on growth and survival, each tank was stocked with 200 fish at 28 days post-hatch (448”days, initial length 7.86 * 0.2 mm, weight 0.0073 k 0.001 g; j;_f s.e., n = 50). These larvae had previously been fed with live feeds enriched with Frippak Booster. Weaning periods of 5, 10 and 20 days were randomly assigned to each of the nine tanks, giving three replicates for each treatment. The experiment was terminated on Day 76 post-hatch (48 days from the start of the experiment). 2.3. Statistical analyses All statistical analyses were carried out using one-way analysis of variance (ANOVA) and Fisher’s test for least significant differences (LSD) at the 95% confidence level. Homogeneity of variance was tested using Bartlett’s test, and normality of distribution was tested using a Shapiro-Wilk W-test on the residuals of the replicate means.
P.R. Hart, GJ. Purser/Aquaculture
175
I45 (1996) 171-181
Percentages were transformed using arc sine square-root transformation. The JMP@ 2.0 statistical package for Macintosh computers was used for all statistical analyses.
30
25 -E 5 .!z F! f 20
(A)
15
I
5'0
40
0.35
60
7’0
8’0
9’0
-1
0.3
-
Group
1
-----o-
Group
2
-o-
Group
3
0.25 ^M 2
0.2
.g S 0.15
0.1
/
0.05 40
50
I
I
I
I
60
70
80
90
Days
posthatch
Fig. 1. Growth ( f f s.e.; n = 3) in (A) length and (B) weight of Rhomhosofea from Day 42 (Group I), Day 50 (Group 2) or Day 58 (Group 3) post-hatch.
tapirina weaned for 10 days
P.R. Hart, G.J. Purser/Aquaculture
176
145 (1996) 171-181
3. Results 3.1. Age and size at weaning 3.1 .I. Weaning ajler metamorphosis: Days 42, 50 and 58 post-hatch There were no significant differences (P > 0.05) in final mean lengths or weights between treatments (Fig. l), but early weaning resulted in the best survival (Fig. 2). Growth rate appeared to be fairly constant throughout the experiment (5 mg day-’ and 0.23 mm day-’ ) with the exception of fish weaned from Day 50 (Group 2), which showed a reduction during the weaning period. This may have been the result of unrepresentative sampling on Day 54 post-hatch. The survival rate of fish weaned from Day 42 post-hatch (58.2 f 3.8%; X f s.e., n = 3) (Group 1) was significantly (P < 0.05) greater than that of fish weaned from Day 58 post-hatch (38.1 + 2.7%) (Group 3). Fish weaned from Day 50 post-hatch (Group 2) showed intermediate survival (50.9 f 6.5%) not significantly different (P > 0.05) from Groups 1 and 3. Mortalities occurred in the same pattern in all groups, increasing rapidly approximately 5 days after the last provision of Artemia and levelling off 14-20 days later. 3.1.2. Weaning before metamorphosis: Day 23 post-hatch Weaning on Day 23 post-hatch resulted in higher survival (82.2 + 4.3%) than recorded in any other weaning experiment. Daily mortality showed a slight increase at
100
1 90-
80-
‘;;i .5
e
5040-[7--
Group
1
3 0 -:
-
Group
2
2 0 _I
v
Group
3
:
lo-
o -I 40
-1
5’0
6lO
Days
7’0
s10
90
posthatch
Fig. 2. Mean survival (n = 3) of Rhomhosolea tupirina weaned for 10 days from Day 42 (Group 1). Day 50 (Group 2) or Day 58 (Group 3) post-hatch. Points sharing a common lowercase letter are not significantly different (P > 0.05). * Survival here is survival observed daily. Actual calculated survival is given in the text. Maximum s.e. was f4.35% (Group I), f7.19% (Group 2) and +5.95% (Group 3) over the experimental period.
P.R. Hart, GJ. Purser/Aquaculture
177
145 (I991.5) 171-181
approximately 15 days after the last feeding with Artemia, but it remained lower than recorded in previous experiments. Weight increased rapidly after weaning (0.8 mg day- I >, but the rate of increase in length (0.18 mm day- ’ ) showed little change during 20
*-
17.5
5
days
--+--
10 days
-o-
20 days b
z
15
5 L: 3 212.5
10 (A) 7.5 3'0
4lO
I SO
--I6'0
70
80
Days posthatch
0.125
u
5 days
-
10 days
V
20 days
Days posthatch Fig. 3. Growth (X + s.e.; n = 3) in (A) length and (B) weight of Rhombosofea tapirim weaned over 5, 10 or 20 days. Points sharing a common lowercase letter are not significantly different (P > 0.05). Points with no visible error bars have very small s.e.
P.R. Hart, GJ. Purser/Aquaculture
178
145 (1996) I7I-181
100
15
;
20
‘;;i .t
day wean
,
5
50
t
cz
days
-
10 days
-o-
20
days
15
0 20
30
50
40
Days
60
70
80
posthatch
Fig. 4. Mean survival rates (n = 3) of Rhombosolea tapirina weaned over 5, 10 or 20 days. * Survival here is survival observed daily. Actual calculated survival is given in the text. Maximum s.e. was f5.24% (5-day weaning), f 2.93% (IO-day weaning) and f 2.67% (20-day weaning) over the experimental period.
the experiment. Final weight 14.9 * 0.1 mm, respectively.
and length
on Day 58 post-hatch
were 0.045 f g and
3.2. Length of the changeover period The length of the weaning period had a significant effect on growth; longer weaning periods resulted in faster growth (Fig. 3). The final length and weight of fish weaned over a 20-day period (17.9 + 0.4 mm, 0.21 mm day-‘; 0.104 f 0007 g, 2.0 mg day-‘) were significantly greater (P < 0.05) than those of fish weaned over 5 days (14.2 k 0.3 mm, 0.13 mm day-‘; 0.051 it 004 g, 0.9 mg day-‘) or 10 days (16.0 f 0.3 mm, 0.17 mm day-‘; 0.071 + 004 g, 1.3 mg day- ‘). Growth rates in each treatment were reduced during or just after the changeover period, but subsequently increased in fish weaned over 10 or 20 days. Fish weaned over 5 days showed a continuing low growth rate after weaning. Overall survival was high in this experiment (69.6 f 2.2%) and there were no significant differences (P > 0.05) in survival among treatments (Fig. 4). Mortalities began to occur earliest in fish weaned over a 5-day period, but otherwise mortality was similar in all three treatments and remained fairly constant following the last provision of Artemia as feed. An observed, but unquantified, effect of the long changeover period was the occurrence of possible vitamin C deficiency symptoms such as shortened opercula and spinal deformities.
P.R. Hart, GJ. Pursrr/Aquuculture
145 (1996) 171-181
179
4. Discussion The results of the present study show that it is possible to wean the larvae of the greenback flounder onto a dry diet after a period of feeding with enriched live diets (rotifers and Artemia spp.). Feeding with live feeds once daily during the weaning period, following an 8-h period of feeding artificial diets alone (Gatesoupe, 1983), within the weaning protocol described by Devresse et al. (19911, proved to be a successful weaning method. 4.1. Age and size at weaning The present study showed that flounder over 50 days old suffered higher mortality at weaning than did younger fish (Fig. 2). Applebaum (1985) showed that this is also the case with sole (Solea solea). The digestive system of younger fish may lack specialisation to any particular diet, but as the fish grow there may be physiological adaptations to suit particular diets. It has been shown that the type of food consumed by larval fish affects the digestive capacity and enzyme composition of the gut (Hofer, 1985; Segner et al., 1993). Segner et al. (1993) provided evidence that the type of feed given to larval fish can affect their digestive physiology indefinitely. The major difference between the digestive tracts of larval and juvenile fish is the lack of a functional stomach in early larvae (Segner et al., 1993). This, rather than a lack of digestive enzymes, may be the reason for the inability of larvae to digest artificial diets if these are provided as the first feed. Flounder larvae at 23 days post-hatch were weaned successfully in the present study. The stomach of flounder is fully differentiated by Day 20 at 15°C (Hart, 1994), although the larvae may have to reach a minimum size rather than age for stomach development. This size appears to be around 8-10 mm. Weaning of flounder larvae that had reached a length of around 8-10 mm on Day 23 post-hatch resulted in a survival rate of over 80% without appearing to reduce growth substantially. In a commercial setting this would result in considerable savings in Artemia compared with post-metamorphosis weaning, leading to reduced costs of larval production. Person-Le Ruyet et al. (1993) have stated that the live prey biomass used for the production of sea bass (Dicentrarchus labrax) would be reduced by 60% if weaning was carried out at Day 25 rather than Day 35 post-hatch. Gatesoupe and Luquet (198 1/ 1982) recorded a significant reduction in growth when sole were weaned onto a moist pellet before metamorphosis, but this was offset by a significant reduction in the number of Artemia nauplii required per metamorphosed juvenile as compared with weaning after metamorphosis. 4.2. The duration of the weaning period The duration of the weaning period had no effect on the survival of flounder (Fig. However, a 5-day weaning period resulted in reduced growth (Fig. 3). Increasing weaning period to 20 days improved growth significantly, presumably owing to greater length of time the larvae spent feeding on Artemia. However, extending weaning period beyond 10 days increases the costs associated with the provision
4). the the the of
180
P.R. Hart, GJ. Purser/Aquacuhre
145 (1996) 171-181
Artemia and delays the onset of mortality without improving survival rate. The same phenomenon has been observed with sole (Bromley and Sykes, 1985). The increased growth rate might offset the increase in production costs, but further research is required to quantify this. Gatesoupe and Luquet (198 l/1982) obtained better growth rates of sole by using an abrupt change rather than a gradual changeover from live Artemia nauplii to artificial food, but this was associated with survival of only 15-28%. Crawford (1984) recorded 100% mortality when attempting abrupt weaning of flounder, so this was not attempted in the present experiment. In addition to increased costs of production associated with extended weaning periods there was an apparent vitamin C deficiency. Person-Le Ruyet (1990) mentions that turbot (.Scophthalmus maximus) also suffer from vitamin C deficiency, and this may be a general problem with flatfish, possibly due to a high requirement for this vitamin. The results of the study show that weaning of greenback flounder larvae onto an artificial diet can be achieved effectively between Days 23 and 50 post-hatch, with a 10 or 20 day overlap during which the provision of live feed is reduced and the supply of artificial diet increased.
References Adron, J.W., Blair, A. and Cowey, C.B., 1974. Rearing of plaice (Pleuronectes pkaressa) larvae to metamorphosis using an artificial diet. Fish. Bull., 72: 353-357. Applebaum, S.. 1985. Rearing of the Dover sole (Solea solea) through its larval stages using artificial diets. Aquaculture, 49: 209-221. Bengston, D.A.. 1993. A comprehensive program for the evaluation of artificial diets, J. World Aquacult. Sot., 24: 285-293. Bromley, P.J. and Sykes, P.A., 198.5. Weaning diets for turbot (Scophrhatmus muximus L.), sole (Soiea sofea L.) and cod (G&us morha L.). In: C.B. Cowey, A.M. Mackie and J.G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press, London, pp. 191-211. Crawford, C.M., 1984. An ecological study of Tasmanian flounder. Ph.D. Thesis, University of Tasmania, Hobart, 181 pp. Devresse, B., Candreva, P., L6ger, P. and Sorgeloos, P., 1991. A new artificial diet for the early weaning of seabass (Dicentruchus fabrar) larvae. In: P. Lavens, P. Sorgeloos, E. Jaspers and F. Ollevier (Editors), Larvi ‘91 Symposium on Fish and Crustacean Larviculture, European Aquaculture Society, Special Publication No. 15, Gent, pp. 178-182. Gatesoupe, F.J., 1983. Weaning of sole (S&n solea) before metamorphosis achieved with high growth rates. Aquaculture, 32: 401-404. Gatesoupe, F.J. and Luquet, P., 1981/1982. Weaning of the sole (Solea solea) before metamorphosis. Aquaculture. 26: 359-368. Hart, P.R., 1994. Factors affecting the early life history stages of hatchery-reared greenback flounder (Rhombosofea tapirha Ginther 1879). Ph.D. Thesis, University of Tasmania, Launceston, 231 pp. Hart, P.R. and Purser, G.J., 1995. Effects of salinity and temperature on eggs and yolk sac larvae of the greenback flounder (Rhombosolea tapirinn, Glinther, 1862). Aquaculture, 136: 221-230. Hart, P.R., Hutchinson, W.G. and Purser, G.J., 1996. Effects of photoperiod, temperature and salinity on hatchery-reared larvae of the greenback flounder (Rhombosolea tapirina Giinther, 1862). Aquaculture, in press. Hjelmeland, K.. Pederson, B.H. and Nilssen, E.M., 1988. Trypsin content in intestines of herring larvae, CIupea harengus, ingesting polystyrene spheres or live crustacea prey. Mar. Biol., 98: 331-33.5. Hofer, R., 1985. Effects of artificial diets on the digestive processes of fish larvae. In: C.B. Cowey, A.M. Mackie and J.G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press, London, pp. 213-216.
P.R. Hart, GJ. Purser/Aquaculiure
145 (1996) 171-181
181
Jones, D.A., Kamarudin, MS. and Le Vay, L., 1993. lhe potential for replacement of live feeds in larval culture. .I. World Aquacult. Sot., 24: 199-209. Lauff, M. and Hofer, R., 1984. Proteolytic enzymes in fish development and the importance of dietary enzymes. Aquaculture, 37: 335-346. Munilla-Moran, R. and Stark, J.R., 1989. Protein digestion in early turbot larvae, Scophthulmus marimus CL.). Aquaculture, 8 1: 3 15-327. Munilla-Moran, R., Stark, J.R. and Barbour, A., 1990. The role of exogenous enzymes in digestion in cultured turbot larvae (Scophthdmus muximus L.). Aquaculture, 88: 337-350. Person-Le Ruyet, J., 1990. Sole and turbot culture. In: G. Barnabt (Editor), Aquaculture. Vol. 2. Ellis Horwood, New York, pp. 687-734. Person-Le Ruyet, J., Alexandre, J.C., Thebaud, L. and Mugnier, C., 1993. Marine fish larvae feeding: Formulated diets or live prey? J. World Aquacult. Sot., 24: 2 1 l-224. Segner, H., RI&h, R., Verreth, J. and Witt, U., 1993. Larval nutritional physiology: Studies with Ckzrius gariepinus, Coregonus lauaretus and Scophthalmus maximus. J. World Aquacult. Sot., 24: 12 1- 134. Walford, J., Lim, T.M. and Lam, T.J., 1991. Replacing live foods with microencapsulated diets in the rearing of seabass (Lates calcarifer) larvae: Do the larvae ingest and digest protein-membrane microcapsules? Aquaculture, 92: 225-235.
Weaning of hatchery-reared greenback flounder ( RhombosoZea tapirina Gtinther) from live to artificial diets: Effects of age and duration of the changeover period Piers R. Hart *, G. John Purser Depurtment
ofAquaculture, University of Tasmcmia. PO Box 1214, Launceston, Tus. 7250, Austruliu Accepted 25 April 1996
Abstract The greenback flounder (Rhombosolea fupirina) is a potential culture species in Australia, but poor survival has been recorded during weaning from live Artemiu to artificial diets. This paper describes the results of three experiments on the effects of fish age and the duration of the changeover period from live to artificial food on weaning survival and subsequent growth. Fish weaned for IO days from Day 50 post-hatch or later had higher mortality (P < 0.05) than fish weaned earlier, but no differences in final weights or lengths (P > 0.05) were observed between fish weaned on different days post-hatch. Weaning from Artemia to artificial diets was shown to be possible from Day 23 post-hatch, with 82.2% survival after a IO-day overlap. An overlap of 5 days between Artemia and artificial diets resulted in a reduction in final length (P < 0.05) compared with overlaps of 10 or 20 days, but no difference in survival (P > 0.05). Overlaps of 20 days resulted in significantly heavier final weights (P < 0.05) than either 10 or 5 days. Weaning from Day 23 post-hatch with a 20-day overlap would therefore appear to result in the best growth and highest survival rate, but a IO-day overlap may result in reduced Artemiu cost with minimal effect on performance. Keywords: Age; Artificial diets; Greenback flounder; Larvae;
Rhombosolrcr
tupirinu ; Weaning
* Corresponding author. 0044-8486/96/$15.00 PII
SOO44-8486(96)0
Copyright 0 1996 Elsevier Science B.V. All rights reserved. 1343-9
172
P.R. Hart, GJ. Purser/Aquuculture
145 (1996) 171-181
1. Introduction The greenback flounder (Rhombosolea tupirina) is being investigated as a potential culture species in southern Australia. Important parameters affecting larval rearing success have been identified (Hart, 1994; Hart and Purser, 1995; Hart et al., 1996), but poor survival has been encountered during the changeover from live to artificial feeds (Hart, 1994). Live feeds continue to be essential for the first feeding of marine fish larvae because they lead to increased feeding, stimulate enzyme secretion, and result in consistently good growth and survival. However, the production and use of live feeds is expensive. There can also be a number of undesirable side effects associated with live feeds: (1) bacteria associated with zooplankton cultures can be detrimental to fish larvae; (2) the nutritional values of live feeds can be highly variable; (3) metabolites from zooplankton increase the load on fish rearing systems; (4) fine-mesh outlet screens are required to retain zooplankton so low water flow rates are necessary to avoid screen blockages and resultant tank overflows. For these reasons it is considered highly desirable to use artificial diets as early as possible in the rearing process. There have been very few successful attempts to feed the larvae of marine fish on artificial diets from first feeding (Adron et al., 1974; Applebaum, 1985; Walford et al., 1991). It appears that the main problems with artificial diets are their unacceptability to the larvae, low digestibility, and problems associated with lack of buoyancy and instability in water (Bengston, 1993; Jones et al., 1993). It is known that the digestive enzyme system of early larvae is poorly developed and that the digestion of food is assisted by the autolysis of natural prey resulting from the enzymes contained within that prey (Lauff and Hofer, 1984; Hjelmeland et al., 1988; Munilla-Moran and Stark, 1989; Munilla-Moran et al., 1990). The formation of the stomach and the commencement of peptic enzyme production appears to be critical to successful digestion of conventional artificial diets (Segner et al., 1993). Crawford (1984) weaned newly metamorphosed greenback flounder ( rhombosolea tupirina) juveniles (approximately 50 days at 14.5”C post-hatch) on to 0.5 mm trout crumbles. Complete mortality was observed when a direct transfer was used, but 70% survival was achieved with a gradual changeover of 14-28 days. This paper presents the results of a series of experiments designed to investigate the effects of (a) fish age and size, and (b) duration of the changeover period from live to artificial diets, on growth and survival .of greenback flounder during weaning. The aims were to determine the optimum age and size at which to commence weaning, and to determine the duration of the changeover period required for successful weaning from live to artificial feeds.
2. Materials and methods All experiments were conducted in a set of nine black hemispherical fibreglass tanks of 25 1 capacity. The tanks were within a recirculation system incorporating mechanical and biological filtration equipment. A 24-h light regime was provided by overhead
P.R. Hart, GJ. Purser/Aquaculture Table 1 Weaning schedule
for lo-day
weaning
145 (1996) 171-181
of R. tapirinu (adapted
Days from start of weaning
Artemia (No. ml-
l-2 3-4 5-6 7-8 9-10 11-18 19-32 33+
10 5 5 5 2.5 0 0 0
173
from Devresse et al., 1991)
’ day- ‘)
Artificial
food (e m- 3 dav- ‘)
24 24 24 40 72 80 100 10% biomass
fluorescent tubes, giving a light intensity of 650-750 lux at the water surface. Temperature and salinity were maintained at 15-17°C and 34-36%0, respectively. Flow rates were set at 30 1 h-l tank-‘. Larvae were initially reared in 200-l black hemispherical tanks prior to transfer into the experimental tanks. Larvae were fed twice daily with enriched rotifers to Day 14 post-hatch, and with Artemia nauplii (instar II) from Day 9 or Day 10 post-hatch until weaning. The changeover period from live to artificial diets followed a schedule adapted from Devresse et al. (1991) (Table 1) where Arremiu was gradually reduced and artificial diet increased over a period of 10 days. This schedule was either extended to 20 days or reduced to 5 days in the experiment described in Sect. 2. The artificial feed used in all experiments was a 1: 1 mixture of Biodiet No. 1 (Bioproducts Inc., Oregon, USA; particle size 300-500 ym, proximate composition 43% protein, 14.5% fat) and Skretting 0.6G (T. Skretting A/S, Stavanger, Norway; particle size 600 km, proximate composition 51.7% protein, 17.3% fat). Feeding with artificial feed was carried out three times daily at equal intervals during an 8-h period, while Artemia was introduced once daily, just after the last presentation of artificial feed for the given day. All experiments were carried out with three, randomly selected tanks assigned to each treatment. Measurements of the individual wet weights and total lengths of 20 fish from each tank were recorded at regular intervals during the experiments using an electronic balance (kO.5 mg) and calipers (k 0.05 mm). Growth rate was calculated using the formula Weight or length increase (mg or mm) Growth rate (mm day-’
or mg day- ‘) =
Duration of experiment
(days)
The base of each tank was siphoned daily to remove dead fish, uneaten food and faeces. Dead fish were counted in order to assess mortality. At the termination of the experiment, the survivors were counted and a random sample of 30 fish from each tank was individually weighed and measured. There was usually only a small discrepancy between the initial numbers of fish and the numbers recovered, either dead or alive. Survival at the end of the experiment was calculated by the formula Live fish remaining Actual survival
(%) =
at the end of the experiment
Initial number of fish
x 100
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2.1. Age and size at weaning 2.1.1. Weaning after metamorphosis: Days 43, 51 and 59 post-hatch (688, 816 and 944”days, respectively) Each tank was stocked with 225 metamorphosed flounder at 33 days post-hatch (528”days, length 16.5 f 0.3 mm, weight 0.063 f 0.003 g; i * se., n = 50). Larvae had previously been fed with live feeds enriched with a mixture of microalgae, Pavfova lutheri and Zsochrisis (Tahitian strain), and Frippak Booster (microencapsulated high HUFA enrichment from Sanofi Aquaculture, Paris, France). All fish were fed at a rate of 10 Artemia ml-’ day-’ until weaning began; a feed rate of 10 Artemia ml- ’ day- ’ equates to approximately 1000 Artemia fish-’ day-’ with 10 fish 1-l. A lo-day weaning schedule was employed in this experiment (Table 1). Weaning commenced on Day 43 post-hatch for fish in three randomly selected tanks (Group 1). Weaning of the fish in a second set of three tanks commenced on Day 5 1 (Group 2) and fish in the third set of tanks were weaned from Day 59 post-hatch (Group 3) onwards. The experiment was terminated on Day 89 post-hatch (46 days from the start of the experiment). 2.1.2. Weaning before metamorphosis: Day 23 post-hatch (368”days) An experiment was undertaken to investigate whether weaning could be initiated before metamorphosis was completed, and what effect this would have on growth and survival. Three batches of 107 larvae at 23 days post-hatch (initial length 8.71 f. 0.21 mm, initial weight 0.016 + 0.001 g; i + s.e., n = 30) were transferred to three 25-l tanks. The larvae had previously been fed with live feeds enriched with Frippak Booster. These larvae had completed stomach differentiation, but were still undergoing eye migration (Hart, 1994). Weaning was carried out using the IO-day schedule (Table 1) and the experiment was terminated on Day 58 post-hatch (35 days from the start of the experiment). 2.2. Length of the changeover
period
To investigate the effect of different weaning periods on growth and survival, each tank was stocked with 200 fish at 28 days post-hatch (448”days, initial length 7.86 * 0.2 mm, weight 0.0073 k 0.001 g; j;_f s.e., n = 50). These larvae had previously been fed with live feeds enriched with Frippak Booster. Weaning periods of 5, 10 and 20 days were randomly assigned to each of the nine tanks, giving three replicates for each treatment. The experiment was terminated on Day 76 post-hatch (48 days from the start of the experiment). 2.3. Statistical analyses All statistical analyses were carried out using one-way analysis of variance (ANOVA) and Fisher’s test for least significant differences (LSD) at the 95% confidence level. Homogeneity of variance was tested using Bartlett’s test, and normality of distribution was tested using a Shapiro-Wilk W-test on the residuals of the replicate means.
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Percentages were transformed using arc sine square-root transformation. The JMP@ 2.0 statistical package for Macintosh computers was used for all statistical analyses.
30
25 -E 5 .!z F! f 20
(A)
15
I
5'0
40
0.35
60
7’0
8’0
9’0
-1
0.3
-
Group
1
-----o-
Group
2
-o-
Group
3
0.25 ^M 2
0.2
.g S 0.15
0.1
/
0.05 40
50
I
I
I
I
60
70
80
90
Days
posthatch
Fig. 1. Growth ( f f s.e.; n = 3) in (A) length and (B) weight of Rhomhosofea from Day 42 (Group I), Day 50 (Group 2) or Day 58 (Group 3) post-hatch.
tapirina weaned for 10 days
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145 (1996) 171-181
3. Results 3.1. Age and size at weaning 3.1 .I. Weaning ajler metamorphosis: Days 42, 50 and 58 post-hatch There were no significant differences (P > 0.05) in final mean lengths or weights between treatments (Fig. l), but early weaning resulted in the best survival (Fig. 2). Growth rate appeared to be fairly constant throughout the experiment (5 mg day-’ and 0.23 mm day-’ ) with the exception of fish weaned from Day 50 (Group 2), which showed a reduction during the weaning period. This may have been the result of unrepresentative sampling on Day 54 post-hatch. The survival rate of fish weaned from Day 42 post-hatch (58.2 f 3.8%; X f s.e., n = 3) (Group 1) was significantly (P < 0.05) greater than that of fish weaned from Day 58 post-hatch (38.1 + 2.7%) (Group 3). Fish weaned from Day 50 post-hatch (Group 2) showed intermediate survival (50.9 f 6.5%) not significantly different (P > 0.05) from Groups 1 and 3. Mortalities occurred in the same pattern in all groups, increasing rapidly approximately 5 days after the last provision of Artemia and levelling off 14-20 days later. 3.1.2. Weaning before metamorphosis: Day 23 post-hatch Weaning on Day 23 post-hatch resulted in higher survival (82.2 + 4.3%) than recorded in any other weaning experiment. Daily mortality showed a slight increase at
100
1 90-
80-
‘;;i .5
e
5040-[7--
Group
1
3 0 -:
-
Group
2
2 0 _I
v
Group
3
:
lo-
o -I 40
-1
5’0
6lO
Days
7’0
s10
90
posthatch
Fig. 2. Mean survival (n = 3) of Rhomhosolea tupirina weaned for 10 days from Day 42 (Group 1). Day 50 (Group 2) or Day 58 (Group 3) post-hatch. Points sharing a common lowercase letter are not significantly different (P > 0.05). * Survival here is survival observed daily. Actual calculated survival is given in the text. Maximum s.e. was f4.35% (Group I), f7.19% (Group 2) and +5.95% (Group 3) over the experimental period.
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145 (I991.5) 171-181
approximately 15 days after the last feeding with Artemia, but it remained lower than recorded in previous experiments. Weight increased rapidly after weaning (0.8 mg day- I >, but the rate of increase in length (0.18 mm day- ’ ) showed little change during 20
*-
17.5
5
days
--+--
10 days
-o-
20 days b
z
15
5 L: 3 212.5
10 (A) 7.5 3'0
4lO
I SO
--I6'0
70
80
Days posthatch
0.125
u
5 days
-
10 days
V
20 days
Days posthatch Fig. 3. Growth (X + s.e.; n = 3) in (A) length and (B) weight of Rhombosofea tapirim weaned over 5, 10 or 20 days. Points sharing a common lowercase letter are not significantly different (P > 0.05). Points with no visible error bars have very small s.e.
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145 (1996) I7I-181
100
15
;
20
‘;;i .t
day wean
,
5
50
t
cz
days
-
10 days
-o-
20
days
15
0 20
30
50
40
Days
60
70
80
posthatch
Fig. 4. Mean survival rates (n = 3) of Rhombosolea tapirina weaned over 5, 10 or 20 days. * Survival here is survival observed daily. Actual calculated survival is given in the text. Maximum s.e. was f5.24% (5-day weaning), f 2.93% (IO-day weaning) and f 2.67% (20-day weaning) over the experimental period.
the experiment. Final weight 14.9 * 0.1 mm, respectively.
and length
on Day 58 post-hatch
were 0.045 f g and
3.2. Length of the changeover period The length of the weaning period had a significant effect on growth; longer weaning periods resulted in faster growth (Fig. 3). The final length and weight of fish weaned over a 20-day period (17.9 + 0.4 mm, 0.21 mm day-‘; 0.104 f 0007 g, 2.0 mg day-‘) were significantly greater (P < 0.05) than those of fish weaned over 5 days (14.2 k 0.3 mm, 0.13 mm day-‘; 0.051 it 004 g, 0.9 mg day-‘) or 10 days (16.0 f 0.3 mm, 0.17 mm day-‘; 0.071 + 004 g, 1.3 mg day- ‘). Growth rates in each treatment were reduced during or just after the changeover period, but subsequently increased in fish weaned over 10 or 20 days. Fish weaned over 5 days showed a continuing low growth rate after weaning. Overall survival was high in this experiment (69.6 f 2.2%) and there were no significant differences (P > 0.05) in survival among treatments (Fig. 4). Mortalities began to occur earliest in fish weaned over a 5-day period, but otherwise mortality was similar in all three treatments and remained fairly constant following the last provision of Artemia as feed. An observed, but unquantified, effect of the long changeover period was the occurrence of possible vitamin C deficiency symptoms such as shortened opercula and spinal deformities.
P.R. Hart, GJ. Pursrr/Aquuculture
145 (1996) 171-181
179
4. Discussion The results of the present study show that it is possible to wean the larvae of the greenback flounder onto a dry diet after a period of feeding with enriched live diets (rotifers and Artemia spp.). Feeding with live feeds once daily during the weaning period, following an 8-h period of feeding artificial diets alone (Gatesoupe, 1983), within the weaning protocol described by Devresse et al. (19911, proved to be a successful weaning method. 4.1. Age and size at weaning The present study showed that flounder over 50 days old suffered higher mortality at weaning than did younger fish (Fig. 2). Applebaum (1985) showed that this is also the case with sole (Solea solea). The digestive system of younger fish may lack specialisation to any particular diet, but as the fish grow there may be physiological adaptations to suit particular diets. It has been shown that the type of food consumed by larval fish affects the digestive capacity and enzyme composition of the gut (Hofer, 1985; Segner et al., 1993). Segner et al. (1993) provided evidence that the type of feed given to larval fish can affect their digestive physiology indefinitely. The major difference between the digestive tracts of larval and juvenile fish is the lack of a functional stomach in early larvae (Segner et al., 1993). This, rather than a lack of digestive enzymes, may be the reason for the inability of larvae to digest artificial diets if these are provided as the first feed. Flounder larvae at 23 days post-hatch were weaned successfully in the present study. The stomach of flounder is fully differentiated by Day 20 at 15°C (Hart, 1994), although the larvae may have to reach a minimum size rather than age for stomach development. This size appears to be around 8-10 mm. Weaning of flounder larvae that had reached a length of around 8-10 mm on Day 23 post-hatch resulted in a survival rate of over 80% without appearing to reduce growth substantially. In a commercial setting this would result in considerable savings in Artemia compared with post-metamorphosis weaning, leading to reduced costs of larval production. Person-Le Ruyet et al. (1993) have stated that the live prey biomass used for the production of sea bass (Dicentrarchus labrax) would be reduced by 60% if weaning was carried out at Day 25 rather than Day 35 post-hatch. Gatesoupe and Luquet (198 1/ 1982) recorded a significant reduction in growth when sole were weaned onto a moist pellet before metamorphosis, but this was offset by a significant reduction in the number of Artemia nauplii required per metamorphosed juvenile as compared with weaning after metamorphosis. 4.2. The duration of the weaning period The duration of the weaning period had no effect on the survival of flounder (Fig. However, a 5-day weaning period resulted in reduced growth (Fig. 3). Increasing weaning period to 20 days improved growth significantly, presumably owing to greater length of time the larvae spent feeding on Artemia. However, extending weaning period beyond 10 days increases the costs associated with the provision
4). the the the of
180
P.R. Hart, GJ. Purser/Aquacuhre
145 (1996) 171-181
Artemia and delays the onset of mortality without improving survival rate. The same phenomenon has been observed with sole (Bromley and Sykes, 1985). The increased growth rate might offset the increase in production costs, but further research is required to quantify this. Gatesoupe and Luquet (198 l/1982) obtained better growth rates of sole by using an abrupt change rather than a gradual changeover from live Artemia nauplii to artificial food, but this was associated with survival of only 15-28%. Crawford (1984) recorded 100% mortality when attempting abrupt weaning of flounder, so this was not attempted in the present experiment. In addition to increased costs of production associated with extended weaning periods there was an apparent vitamin C deficiency. Person-Le Ruyet (1990) mentions that turbot (.Scophthalmus maximus) also suffer from vitamin C deficiency, and this may be a general problem with flatfish, possibly due to a high requirement for this vitamin. The results of the study show that weaning of greenback flounder larvae onto an artificial diet can be achieved effectively between Days 23 and 50 post-hatch, with a 10 or 20 day overlap during which the provision of live feed is reduced and the supply of artificial diet increased.
References Adron, J.W., Blair, A. and Cowey, C.B., 1974. Rearing of plaice (Pleuronectes pkaressa) larvae to metamorphosis using an artificial diet. Fish. Bull., 72: 353-357. Applebaum, S.. 1985. Rearing of the Dover sole (Solea solea) through its larval stages using artificial diets. Aquaculture, 49: 209-221. Bengston, D.A.. 1993. A comprehensive program for the evaluation of artificial diets, J. World Aquacult. Sot., 24: 285-293. Bromley, P.J. and Sykes, P.A., 198.5. Weaning diets for turbot (Scophrhatmus muximus L.), sole (Soiea sofea L.) and cod (G&us morha L.). In: C.B. Cowey, A.M. Mackie and J.G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press, London, pp. 191-211. Crawford, C.M., 1984. An ecological study of Tasmanian flounder. Ph.D. Thesis, University of Tasmania, Hobart, 181 pp. Devresse, B., Candreva, P., L6ger, P. and Sorgeloos, P., 1991. A new artificial diet for the early weaning of seabass (Dicentruchus fabrar) larvae. In: P. Lavens, P. Sorgeloos, E. Jaspers and F. Ollevier (Editors), Larvi ‘91 Symposium on Fish and Crustacean Larviculture, European Aquaculture Society, Special Publication No. 15, Gent, pp. 178-182. Gatesoupe, F.J., 1983. Weaning of sole (S&n solea) before metamorphosis achieved with high growth rates. Aquaculture, 32: 401-404. Gatesoupe, F.J. and Luquet, P., 1981/1982. Weaning of the sole (Solea solea) before metamorphosis. Aquaculture. 26: 359-368. Hart, P.R., 1994. Factors affecting the early life history stages of hatchery-reared greenback flounder (Rhombosofea tapirha Ginther 1879). Ph.D. Thesis, University of Tasmania, Launceston, 231 pp. Hart, P.R. and Purser, G.J., 1995. Effects of salinity and temperature on eggs and yolk sac larvae of the greenback flounder (Rhombosolea tapirinn, Glinther, 1862). Aquaculture, 136: 221-230. Hart, P.R., Hutchinson, W.G. and Purser, G.J., 1996. Effects of photoperiod, temperature and salinity on hatchery-reared larvae of the greenback flounder (Rhombosolea tapirina Giinther, 1862). Aquaculture, in press. Hjelmeland, K.. Pederson, B.H. and Nilssen, E.M., 1988. Trypsin content in intestines of herring larvae, CIupea harengus, ingesting polystyrene spheres or live crustacea prey. Mar. Biol., 98: 331-33.5. Hofer, R., 1985. Effects of artificial diets on the digestive processes of fish larvae. In: C.B. Cowey, A.M. Mackie and J.G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press, London, pp. 213-216.
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Jones, D.A., Kamarudin, MS. and Le Vay, L., 1993. lhe potential for replacement of live feeds in larval culture. .I. World Aquacult. Sot., 24: 199-209. Lauff, M. and Hofer, R., 1984. Proteolytic enzymes in fish development and the importance of dietary enzymes. Aquaculture, 37: 335-346. Munilla-Moran, R. and Stark, J.R., 1989. Protein digestion in early turbot larvae, Scophthulmus marimus CL.). Aquaculture, 8 1: 3 15-327. Munilla-Moran, R., Stark, J.R. and Barbour, A., 1990. The role of exogenous enzymes in digestion in cultured turbot larvae (Scophthdmus muximus L.). Aquaculture, 88: 337-350. Person-Le Ruyet, J., 1990. Sole and turbot culture. In: G. Barnabt (Editor), Aquaculture. Vol. 2. Ellis Horwood, New York, pp. 687-734. Person-Le Ruyet, J., Alexandre, J.C., Thebaud, L. and Mugnier, C., 1993. Marine fish larvae feeding: Formulated diets or live prey? J. World Aquacult. Sot., 24: 2 1 l-224. Segner, H., RI&h, R., Verreth, J. and Witt, U., 1993. Larval nutritional physiology: Studies with Ckzrius gariepinus, Coregonus lauaretus and Scophthalmus maximus. J. World Aquacult. Sot., 24: 12 1- 134. Walford, J., Lim, T.M. and Lam, T.J., 1991. Replacing live foods with microencapsulated diets in the rearing of seabass (Lates calcarifer) larvae: Do the larvae ingest and digest protein-membrane microcapsules? Aquaculture, 92: 225-235.