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The culture and early weaning of larval haddock (Melanogrammus aeglefinus) using a microparticulate diet
Aquaculture 201 Ž2001. 61–72 www.elsevier.comrlocateraqua-online
The culture and early weaning of larval haddock žMelanogrammus aeglefinus / using a microparticulate diet H.J. Hamlin 1, L.J. Kling ) School of Marine Science, UniÕersity of Maine, 5763 Rogers Hall, Orono, ME 04469-5763, USA Received 17 February 2000; accepted 25 January 2001
Abstract In order to effectuate haddock, Melanogrammus aeglefinus, as a practical aquaculture candidate, they must be able to successfully wean onto a formulated diet. This weaning should be accomplished soon after hatching in order to limit financial bottlenecks associated with the high cost of live feed production. Two experiments were designed to determine the earliest point at which haddock could be successfully weaned onto a formulated food. The first examined start weaning at 14, 21, 28, and 35 days post-hatch Ždph. at 8.58C. Control animals were given only live feeds throughout the experiment. Samples of larvae were taken weekly for length and dry weight analysis. Haddock did not wean as effectively as cod onto the commercial diet ŽBiokyowae., and survival was compromised for all treatments examined. There were no differences in survival for any of the weaning periods investigated with averages ranging from 2.5% to 6.3%. However, the survival of the control group was significantly higher at 37.9%. The second experiment examined weaning at later stages and at an increased temperature. This experiment examined start weaning periods of 30, 35, and 42 days post-hatch. The 42-day start weaning period yielded results similar to those obtained with the live feed control, however survival and growth was compromised for both the 30- and 35-day weaning periods. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Haddock; Melanogrammus aeglefinus; Weaning; Larval culture; Microparticulate diet
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Corresponding author. Tel.: q1-207-581-2735; fax: q1-207-581-2744. E-mail address: [email protected] ŽL.J. Kling.. 1 Current address: Mote Marine Laboratory, 1600 Ken Thompson Pkwy, Sarasota, FL 34236, USA.
0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 5 5 7 - 9
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1. Introduction Haddock Ž Melanogrammus aeglefinus. have been an integral part of the groundfish industry on the North Atlantic seaboard for decades. Despite declines in natural stocks, haddock have remained one of the most highly sought after species of fish in the United States and Canada. Although the commercial potential of haddock for aquaculture has been well documented ŽHenry, 1997; Waterman, 1995., in order to be considered a practical aquaculture candidate, they must be able to successfully wean onto a formulated diet. This has presented many challenges since, unlike cod, there is no precedent for the intensive culture of haddock, and rearing attempts beyond larval stages on even live feeds have been met with limited success ŽDowning and Litvak, 1999.. Although there have been a number of recent developments in the production and rearing of marine fish larvae ŽRosenlund, 1997., the culture of the majority of currently investigated species is still based on the utilization of live feeds for a significant portion of the larval period. This can create significant financial barriers to the practical production of these larvae for commercial aquaculture. Sea bass, for example, are not usually weaned before 40–45 days post-hatch, with the cost of live feeds representing up to 79% of production costs ŽLe Ruyet et al., 1993.. Consequently, any practice that can decrease dependence on live feeds would significantly reduce financial bottlenecks in the rearing process, making the cost-effective production of marine larvae an attainable goal. Considerable effort has been made working with and developing formulated feeds ŽYufera et al., 2000.. Adron et al. Ž1974. were the first to rear plaice through metamorphosis using only formulated food, with Bromely Ž1977., Appelbaum Ž1985., Dabrowski et al. Ž1986., Ottera˚ and Lie Ž1991., and Cahu and Zambonino Infante Ž1994. following with other larval marine fish species. In each case, formulated diets resulted in reduced growth and survival compared to live food controls. Holt Ž1993., Hart and Purser Ž1996. and Rosenlund et al. Ž1997. reported improved growth and survival of several larval marine fish with the combined feeding of live and manufactured diets Žco-feeding.. Holt Ž1993. achieved 60% survival in red drum from hatch to metamorphosis on a commercial diet when supplied in combination with live food for the first 5 days. Previously, red sea bream had been successfully weaned only after 21–25 days post-hatch Ždph.. Hart and Purser Ž1996. achieved significantly higher survival in greenback flounder Ž Rhombosolea tapirina, Gunther. weaned prior to metamorphosis at 23 dph using a 10-day co-feeding regime, than was achieved weaning after metamorphosis, i.e. after 50 dph. Bromley Ž1978. found the limited use of live food to be critical for successful weaning during early larval turbot stages, but was unimportant for larger larvae. Similarly, Canavate and Fernandez-Dıaz ˜ ´ ´ Ž1999. found enhanced growth and survival in earlyweaned senegal sole Ž Solea senegalensis. larvae co-fed with live food. The purpose of this paper is to describe the successful culture of haddock, and determine the earliest point at which the larvae can be successfully weaned onto a microparticulate ŽMP. diet. This paper describes two experiments. The first examines early weaning from 14 to 35 days post-hatch at 8.58C, and the second examines weaning from 30 to 42 days post-hatch at 10.58C.
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2. Materials and methods 2.1. Eggs and disinfection Eggs for both experiments were obtained from the Department of Fisheries and Oceans laboratories in St. Andrews, Canada, in the morula stage of development. The broodstock were maintained on an 8-week advanced photoperiod and temperature cycle. Eggs were incubated in 135-l circular tanks in complete darkness at 7.5–8.58C. Constant aeration and high flow rates kept the eggs in motion throughout the incubation period. Eggs for experiment 1 were disinfected 2 days prior to hatching with 200 ppm glutaraldehyde for 10 min, were measured volumetrically Ž318 eggsrml average., and were placed into 22-l experimental vessels. Eggs for experiment 2 were incubated and disinfected identically to those in experiment 1, and were then transferred to 135-l rearing tanks where the larvae remained until the start of the experiment. 2.2. Experimental tanks and system design Experimental tanks consisted of blue, 22-l plastic enclosures partially immersed in a water bath. The tanks were supplied with water at the surface of the tank from a recirculating system of synthetic sea water. The water exited through a mesh covered cylinder at the base of the tank. No green water was added to any of the tanks at any point in the experiment. Microparticulate food was administered to the tanks by hand and by automated feeders. Tanks were aerated by air pumped through perforated plastic tubing near the base of the tank, thereby keeping the larvae in constant motion. Lighting was maintained at 8 lumenrft 2 for a 24-h L:Oh D photoperiod, and salinity remained at 32 ppt throughout the experiment. For experiment 1, initial flow rates were 230 mlrmin but were increased to 330 mlrmin as the larvae increased in size Ž; 9.5 mm. to maintain water quality: ) 7 mgO 2rl and - 0.005 mgNH 3rl. For experiment 2, flow rates remained at 350 mlrmin throughout the experiments with similar water quality parameters. 2.3. Measurements and analysis Images of the larvae were captured under a dissecting microscope using a Targa plus frame grabber ŽMedia Cybernetics, Silver Springs, MD, USA.. Standard lengths ŽHardy, 1978. were measured to the nearest 0.01 mm using image analysis software ŽImage Pro Plus, Truevision, Indianapolis, IN, USA.. Larvae were freeze-dried and measured to the nearest "2 mg for dry weight analysis. All statistical procedures used SASrSTATe software ŽSAS Institute, 1985.. A completely random design with subsampling was used for each of the experiments. Normality of the data ŽShapiro and Wilk, 1965. and homogeneity of variance ŽSnedecor and Cochran, 1993. were tested to ensure the assumptions for analysis of variance were met. Analysis of variance was conducted for length, dry weight, and survival ŽSnedecor and Cochran, 1993.. Survival data were arcsin transformed prior to analysis. Treatment differences were determined at P F 0.05, and Duncan’s multiple range test ŽSnedecor
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H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72
and Cochran, 1993. was used to distinguish differences among treatment means Ž P F 0.05.. 2.4. Experiment 1 Each experimental tank contained 120 larvaerl Ž2640 larvaertank. at the start of the experiment Žhatching.. All tanks were supplied with enriched ŽDHA Selcoe, INVE Aquaculture, Grantsville, UT, USA. rotifers Ž Brachionus plicatilis. until the introduction of microparticulate diet ŽBiokyowae, Kyowa Hakko Kogyo, Tokyo, Japan. at 14 MP, 21 MP, 28 MP, or 35 MP dph ŽFig. 1.. A live feed control was fed only rotifers
Fig. 1. Feeding regimes of haddock larvae in experiments 1 ŽA. and 2 ŽB.. Rotifer Ø Ø Ø Ø Ø Ø Ø Ø Ø ; Artemia; --- Ø Ø --- Ø Ø ---; MP . For additional explanation, see text.
H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72
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until day 21, at which point Artemia ŽGreat Salt Lake. enriched with DHA Selcoe were introduced Ž21A.. There were four replicates per treatment, and each treatment was co-fed, either with microparticulate diet or Artemia, for 1 week. Larvae fed rotifers received 17rmlrfeeding with four feedingsrday for a total of 1.5 million rotifersrday, and larvae fed Artemia received 5rmlrfeeding with four feedingsrday for a total of 400,000rday. The microparticulate diet was offered as 250- or 400-mm particles, and Artemia were given at the third instar stage. During the 1-week co-feeding period, larvae were fed rotifers at 0800 and 2000 h; those given MP diet were fed every 2 h during the 24-h photoperiod, while the control was fed Artemia at 0800, 1200, 1600, and 2000 h. After the 1-week co-feeding period, experimental tanks were given only microparticulate food, and control tanks were given only Artemia. After hatching Žday 0., 100 larvae were sampled from an extra experimental vessel for initial length and dry weight analysis. Starting at 14 dph, 20 larvaertank were sampled each week until 42 dph for length and dry weight. At 42 dph, only 10 larvaertank were sampled due to low survival in some of the tanks. Survival percentages were calculated at 47 dph. Temperature was maintained at 8.58C. 2.5. Experiment 2 Twenty-five days post-hatch larvae previously fed rotifers and Artemia were transferred by beaker from 135-l rearing vessels into 22-l experimental vessels. The fish were then acclimated for 5 days prior to the start of the experiment, which began at 30 dph at which time they averaged 8.85 mm in length. These larvae were reared similarly to the live feed control animals in experiment 1, and consequently were fed exclusively live feeds prior to the start of the experiment. The larvae were weaned at 30 MP, 35 MP, or 42 MP dph with a 1-week co-feeding period ŽFig. 1.. During the co-feeding period, microparticulate diet was offered every 2 h and Artemia were given at 0800, 1200, 1600, and 2000 h in successively smaller amounts until the end of the co-feeding period. The animals were offered 700 and 1000 mm Biokyowae microparticulate diet. The control was fed Artemia only throughout the experiment, which ended at 59 dph. The temperature was maintained at 10.58C.
3. Results 3.1. Experiment 1 Survival was significantly reduced by early weaning ŽTable 1.. There were no significant differences in survival among treatments weaned at 14, 21, 28, or 35 dph; survival for these treatments ranged from 2.5% to 6.3%. Average survival for control animals was significantly higher at 37.9%, indicating a superiority of live feeds over the earlier microparticulate weaning regimes. There were no differences in length between any of the treatments sampled throughout the experiment, and the growth dispensation
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Treatment Žday start-weaned.
14
21
28
35
Control
Initial
4.75 0.031 6.66"0.23 0.120"0.013 7.49"0.28 0.222"0.041 8.37"0.43 0.502"0.173 9.87"0.54 0.976"0.262 11.19"1.26 1.384"0.682 b 2.8"1.1b
4.75 0.031 6.37"0.42 0.109"0.020 7.33"0.41 0.214"0.050 8.59"0.43 0.661"0.409 9.66"0.48 1.020"0.050 11.46"1.05 1.714"0.286 b 6.3"2.7 b
4.75 0.031 6.42"0.40 0.111"0.014 7.34"0.26 0.192"0.032 8.65"0.42 0.676"0.143 10.22"0.67 1.132"0.264 12.48"0.81 2.187"0.160 b 2.6"1.8 b
4.75 0.031 6.79"0.20 0.131"0.014 7.62"0.30 0.237"0.043 9.05"0.28 0.814"0.150 10.03"0.49 1.130"0.181 12.22"1.05 2.246"0.580 b 2.6"1.7 b
4.75 0.031 6.76"0.33 0.118"0.011 7.84"0.49 0.278"0.070 9.04"0.57 0.580"0.318 10.19"0.68 1.200"0.280 13.43"1.41 3.563"0.600 a 37.9"3.6 a
Day 14 Day 21 Day 28 Day 35 Day 42 Day 47
Length Žmm. Dry weight Žmg. Length Žmm. Dry weight Žmg. Length Žmm. Dry weight Žmg. Length Žmm. Dry weight Žmg. Length Žmm. Dry weight Žmg. Length Žmm. Dry weight Žmg. Survival 1
Values are means"standard error of four replicates. Means within a row with the same superscript are not significantly different Ž P F 0.05.. Growth measurements are on a per larvae basis. 1 Survival Ž%. s ŽTotal number of survivors at the end of the experiment.rŽTotal number of larvae at start y number of larvae sampled throughout experiment..
H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72
Table 1 The effect of weaning age on survival and growth Žlength, dry weight. of haddock larvae ŽExperiment 1.
H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72
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appeared to be similar within the treatments as well. No differences in weight were seen until the 42-day sampling period, at which point the control was significantly heavier than all other treatments with an average of 3.56 and 1.88 mg, respectively. Hence, the feeding of solely live feeds resulted in a significant increase in weight and survival vs. any of the treatments fed live feeds followed by exclusively formulated diets. As it was not possible to accurately obtain daily mortality data, no quantitative information about periods of punctuated mortality in relation to weaning times could be established. However, qualitative estimates indicate a decrease in numbers shortly after the larvae were given nothing but formulated diet. In these experiments, the larvae did appear to be eating the diet approximately 3–4 days after its introduction as illustrated by gut coloration within the tank and microscopic evaluation. However, no information regarding amounts consumed could be collected. Consequently, it is possible the larvae were either not consuming enough of the diet, or the diet was nutritionally inadequate. The microparticulate diet did create issues with tank hygiene, as uneaten food particles sedimented and collected at the bottom of the tank, and to a less extent, along the sides. The bottom was siphoned daily and water quality parameters were never compromised. Occasionally a gelatinous growth, isolated as bacterial but unidentified further was observed in some of the tanks. Although it did not appear to have an effect, it is unclear what impact this may have had. 3.2. Experiment 2 Survivals Žfrom 30 to 59 dph. for the 30, 35, 42, and control treatment were 35%, 51%, 65%, and 73%, respectively ŽTable 2.. Survival was significantly reduced for both the 30- and 35-day weaning periods, while introducing the microparticulate diet at day 42 yielded results similar to those obtained with the live feed control. The dry weight of the 30-day start weaning period was comparable to the control, while the 35- and 42-day start weaning intervals exhibited significantly greater weights than either of the other treatments. There were no differences in length between any of the treatments. Morts were collected, counted, and evaluated for signs of damage. The 42-day start weaning treatment exhibited the greatest growth dispensation followed closely by the 35-day start weaning treatment. The 30-day treatment and the control contained fish that were relatively similar in size. There was a 10% discrepancy in the totals of fish counted at
Table 2 The effect of weaning times on haddock survival and growth Žlength, dry weight. to 59 dph. Experiment 2 Treatment Žday start weaned. Length Žmm. Dry weight Žmg. Survival Ž%.1
30
35 a
29.9"2.2 0.65"0.13 a 35.1"3.6 a
42 a
33.5"1.8 0.90"0.10 b 50.7"5.3 b
Control a
33.0"3.9 0.82"0.03 b 64.5"3.96 c
28.8"2.0 a 0.57"0.11a 72.9"7.07 c
Values are means"standard error of four replicates. Means within a row with the same superscript are not significantly different Ž P F 0.05.. Growth measurements are on a per larvae basis. 1 Survival Ž%. s ŽTotal number of survivors at the end of the experiment.rŽTotal number of larvae at start y number of larvae sampled throughout..
68 H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72 Fig. 2. Daily mortality of haddock from 30 to 59 days post-hatch. Experiment 2. 30 MP, 35 MP, 42 MPs microparticulate diet introduced at 30, 35 and 42 days post-hatch, respectively. Controls Artemia fed throughout the experiment. For additional explanation, see text.
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the end vs. what was expected from mortality data. Although cannibalism was never witnessed in the tank, there were many acts of fatal aggression, which appeared to account for the majority of mortality. The morts examined showed no visible signs of disease Ži.e., no abnormal gill tissue, lesions, enlarged or otherwise abnormal organs, or excessively frayed fins., had food in their guts, and were not emaciated. The graph of daily mortality ŽFig. 2. shows periods of punctuated mortality following the discontinuation of the live feeds. This phenomenon is most dramatic at the earlier weaning periods and gets progressively less pronounced at later weaning periods. At the end of the experiment, the largest fish in each treatment were sacrificed and examined for signs of cannibalism, however, no evidence of cannibalism was evident in the dissection.
4. Discussion One of the objectives of these experiments was to establish a successful culture technique for rearing larval haddock. We were able to achieve the greatest survival reported to date, with an average of 38.9% through 47 dph for the live feed controls. These numbers are comparable to results we have had with cod larvae ŽBaskervilleBridges and Kling, 2000.. The other objective of these experiments was to determine an adequate protocol for the early weaning of larval haddock onto a formulated feed. As it was evident after the first experiment that haddock could not be effectively weaned onto microparticulate feeds during the early time periods examined, we made the decision to increase the temperature and focus on later periods. We did this with the intention of investigating earlier periods if the later interval testing proved successful. We were then able to adequately wean haddock by 42 days post-hatch with minimal risks to growth and survival. Since the 30- and 35-day start weaning periods at this higher temperature proved unsuccessful, we felt it unnecessary to investigate intervals prior to those examined in this experiment. Although we were able to wean haddock commensurate to live feed control animals by 42 dph, this is not considered early weaning, and more work will need to be done to determine the limiting factor in the weaning process. Although there have been a number of studies over the years focusing on the development of an adequate diet that could, ideally, replace live feeds while yielding similar results, there has been limited success. Whether or not this is due to nutritional, or possibly physical barriers or inadequacies, remains unclear. It has been reported that haddock prefer smaller, less mobile prey than cod ŽAuditore et al., 1994; Kane, 1984., but it is uncertain how well they will accept an inert food item. Since fish larvae are predominantly visual feeders ŽGerking, 1994., it has been theorized that larvae prefer moving prey over suspended inert particles, and consequently, many larvae will either reject the non-motile food and eventually starve to death, or will delay feeding behavior, which introduces risks to growth and development ŽLe Ruyet et al., 1993.. In tanks where the water is stagnant or has little motion, the lack of movement of microparticles could be of considerable concern ŽBengston, 1993.. However, in tank systems with a great deal of motion such as these, concern is limited since gross movements of the
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microparticles and live feeds are at the mercy of the water current and move in much the same manner in the water column. The larvae are also in motion, which increases predator–prey contact rates. It is unclear if haddock larvae can detect subtle movements of prey such as cilia undulations of rotifers or fine movements of Artemia and it is, therefore, difficult to discern the significance of such movements as an attractant for the larvae. Braid Ž1977. obtained adequate growth and survival of striped bass larvae with heat killed Artemia, which negates movement of the prey as the sole attractant for this species. Appelbaum Ž1989. provided recommendations for the successful rearing of marine larvae using inert diets, in which he suggested that raising the temperature of the water increases larval metabolism and activity level, potentially improving their readiness to accept inert diets. In the first experiment survival was extremely low even at the 35 dph start weaning period. Increasing the temperature from 8.58C to 10.58C may have increased the ability of haddock to accept the microparticulate diet. An increase in temperature also advances the developmental stage of the larvae Žincreasing number of degree-days., and it is possible that this advanced developmental state affords the larvae a more favorable physiological condition in which to assimilate the diet. This lab has found the differentiation of structures, which results in a digestive system similar to the adult to begin at 35 days or 10 mm, and is well developed by 53 days or 15 mm. Since a widening of the food spectrum accompanies the transformation from larval to adult stages ŽForstner et al., 1983. it may prove difficult to wean haddock prior to this critical developmental period. In the second experiment, survival from 30 to 59 dph ranged from 35% to 73%, which is not high for this time period. In general, the greatest mortality is experienced at earlier larval stages, and we would expect mortality at these later stages to be minimal. A great deal of this mortality may have been due to aggression andror cannibalism. The dead were siphoned and counted daily and exhibited no visual signs of disease. The dead fish had food in their guts and exhibited no signs of starvation. Frequently, however, we observed aggressive acts such as fish grasping others by the head and shaking them to death. This occurred in all of the treatments but was more pronounced in tanks fed exclusively with formulated feeds. At the end of the experiment, the largest fish in each tank were sacrificed to evaluate their digestive tracts for signs of cannibalism and no evidence was found, although it would take only a few hours or less for evidence to be digested ŽFolkvord, 1993.. There are few instances in which predatory fish do not practice cannibalism ŽDavis, 1985.. Although we did not observe cannibalism at these early stages, we are not convinced that it did not occur since there was a 10% discrepancy between expected Žusing starting numbers minus daily mortality. and actual survival. There was a large growth dispensation in all of the treatments, although replicates varied somewhat. Size variation may be a principal cause of aggressive or agonistic behavior, which can have the same end result as cannibalism ŽHecht and Pienaar, 1993.. The weights of the fish were greatest for the 35- and 42-day start weaning periods vs. the 30-day start weaning period and the control. For the control animals, this situation may have been created as a consequence of remaining on a prey item that is most likely smaller than they would normally be ingesting.
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Acknowledgements Maine Agricultural and Forest Experiment Station external publication a2476. This work was supported by the University of Maine and University of New Hampshire Sea Grant College Program as well as the Maine Agricultural and Forest Experiment Station. Appreciation is extended to Jacqueline Hunter for her technical assistance in the production of live feeds, and to the Department of Fisheries and Oceans for providing eggs used in the experiments.
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Hecht, T., Pienaar, A.G., 1993. A review of cannibalism and its implications in fish larvaculture. J. World Aquacult. Soc. 24, 246–261. Henry, R., 1997. Overview of recent developments in haddock culture in New Brunswick. AAC Spec. Publ. 2, 37–40. Holt, G.J., 1993. Feeding larval red drum on microparticulate diets in a closed recirculating water system. J. World Aquacult. Soc. 24, 225–230. Kane, J., 1984. The feeding habits of co-occurring cod and haddock larvae from Georges Bank. Mar. Ecol. Prog. Ser. 16, 9–20. Le Ruyet, J.P., Alexandre, J.C., Thebaud, L., Mugnier, C., 1993. Marine fish larvae feeding: formulated diets ´ or live prey? J. World Aquacult. Soc. 24, 211–224. Ottera, ˚ H., Lie, O., 1991. Weaning trials with cod Ž Gadus morhua L.. fry on formulated diets. Fiskeridir Skr., Ser. Ernaer. 4, 85–94. Rosenlund, G., 1997. Recent developments in the production of marine fish. AAC Spec. Publ. 2, 25–30. Rosenlund, G.J., Stoss, C., Talbot, 1997. Co-feeding marine fish larvae with inert and live diets. Aquaculture 155, 183–191. SAS Institute, 1985. SAS User’s Guide: Statistics, version. 5 edn. SAS Inst., Cary, NC. Shapiro, S.S., Wilk, M.B., 1965. An analysis of variance test for normality Žcomplete samples.. Biometrika 52, 591–611. Snedecor, G.W., Cochran, W.G., 1993. Levene’s Test of Homogeneity of Variance. Statistical Methods. 8th edn. Iowa State Univ. Press, Ames. Waterman, M.P., 1995. Haddock: northern aquaculture’s new rising star? Aquacult. Mag., 30–36, MarchrApril. Yufera, M., Fernandez-Dıaz, ´ ´ C., Pascual, E., Sarasquete, M.C., Moyano, F.J., Dıaz, ´ M., Alarcon, ´ F.J., Garcıa-Gallego, M., Parra, G., 2000. Towards an inert diet for first-feeding gilthead seabream Sparus ´ aurata L. larvae. Aquacult. Nutr. 6, 143–152.
The culture and early weaning of larval haddock žMelanogrammus aeglefinus / using a microparticulate diet H.J. Hamlin 1, L.J. Kling ) School of Marine Science, UniÕersity of Maine, 5763 Rogers Hall, Orono, ME 04469-5763, USA Received 17 February 2000; accepted 25 January 2001
Abstract In order to effectuate haddock, Melanogrammus aeglefinus, as a practical aquaculture candidate, they must be able to successfully wean onto a formulated diet. This weaning should be accomplished soon after hatching in order to limit financial bottlenecks associated with the high cost of live feed production. Two experiments were designed to determine the earliest point at which haddock could be successfully weaned onto a formulated food. The first examined start weaning at 14, 21, 28, and 35 days post-hatch Ždph. at 8.58C. Control animals were given only live feeds throughout the experiment. Samples of larvae were taken weekly for length and dry weight analysis. Haddock did not wean as effectively as cod onto the commercial diet ŽBiokyowae., and survival was compromised for all treatments examined. There were no differences in survival for any of the weaning periods investigated with averages ranging from 2.5% to 6.3%. However, the survival of the control group was significantly higher at 37.9%. The second experiment examined weaning at later stages and at an increased temperature. This experiment examined start weaning periods of 30, 35, and 42 days post-hatch. The 42-day start weaning period yielded results similar to those obtained with the live feed control, however survival and growth was compromised for both the 30- and 35-day weaning periods. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Haddock; Melanogrammus aeglefinus; Weaning; Larval culture; Microparticulate diet
)
Corresponding author. Tel.: q1-207-581-2735; fax: q1-207-581-2744. E-mail address: [email protected] ŽL.J. Kling.. 1 Current address: Mote Marine Laboratory, 1600 Ken Thompson Pkwy, Sarasota, FL 34236, USA.
0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 5 5 7 - 9
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1. Introduction Haddock Ž Melanogrammus aeglefinus. have been an integral part of the groundfish industry on the North Atlantic seaboard for decades. Despite declines in natural stocks, haddock have remained one of the most highly sought after species of fish in the United States and Canada. Although the commercial potential of haddock for aquaculture has been well documented ŽHenry, 1997; Waterman, 1995., in order to be considered a practical aquaculture candidate, they must be able to successfully wean onto a formulated diet. This has presented many challenges since, unlike cod, there is no precedent for the intensive culture of haddock, and rearing attempts beyond larval stages on even live feeds have been met with limited success ŽDowning and Litvak, 1999.. Although there have been a number of recent developments in the production and rearing of marine fish larvae ŽRosenlund, 1997., the culture of the majority of currently investigated species is still based on the utilization of live feeds for a significant portion of the larval period. This can create significant financial barriers to the practical production of these larvae for commercial aquaculture. Sea bass, for example, are not usually weaned before 40–45 days post-hatch, with the cost of live feeds representing up to 79% of production costs ŽLe Ruyet et al., 1993.. Consequently, any practice that can decrease dependence on live feeds would significantly reduce financial bottlenecks in the rearing process, making the cost-effective production of marine larvae an attainable goal. Considerable effort has been made working with and developing formulated feeds ŽYufera et al., 2000.. Adron et al. Ž1974. were the first to rear plaice through metamorphosis using only formulated food, with Bromely Ž1977., Appelbaum Ž1985., Dabrowski et al. Ž1986., Ottera˚ and Lie Ž1991., and Cahu and Zambonino Infante Ž1994. following with other larval marine fish species. In each case, formulated diets resulted in reduced growth and survival compared to live food controls. Holt Ž1993., Hart and Purser Ž1996. and Rosenlund et al. Ž1997. reported improved growth and survival of several larval marine fish with the combined feeding of live and manufactured diets Žco-feeding.. Holt Ž1993. achieved 60% survival in red drum from hatch to metamorphosis on a commercial diet when supplied in combination with live food for the first 5 days. Previously, red sea bream had been successfully weaned only after 21–25 days post-hatch Ždph.. Hart and Purser Ž1996. achieved significantly higher survival in greenback flounder Ž Rhombosolea tapirina, Gunther. weaned prior to metamorphosis at 23 dph using a 10-day co-feeding regime, than was achieved weaning after metamorphosis, i.e. after 50 dph. Bromley Ž1978. found the limited use of live food to be critical for successful weaning during early larval turbot stages, but was unimportant for larger larvae. Similarly, Canavate and Fernandez-Dıaz ˜ ´ ´ Ž1999. found enhanced growth and survival in earlyweaned senegal sole Ž Solea senegalensis. larvae co-fed with live food. The purpose of this paper is to describe the successful culture of haddock, and determine the earliest point at which the larvae can be successfully weaned onto a microparticulate ŽMP. diet. This paper describes two experiments. The first examines early weaning from 14 to 35 days post-hatch at 8.58C, and the second examines weaning from 30 to 42 days post-hatch at 10.58C.
H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72
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2. Materials and methods 2.1. Eggs and disinfection Eggs for both experiments were obtained from the Department of Fisheries and Oceans laboratories in St. Andrews, Canada, in the morula stage of development. The broodstock were maintained on an 8-week advanced photoperiod and temperature cycle. Eggs were incubated in 135-l circular tanks in complete darkness at 7.5–8.58C. Constant aeration and high flow rates kept the eggs in motion throughout the incubation period. Eggs for experiment 1 were disinfected 2 days prior to hatching with 200 ppm glutaraldehyde for 10 min, were measured volumetrically Ž318 eggsrml average., and were placed into 22-l experimental vessels. Eggs for experiment 2 were incubated and disinfected identically to those in experiment 1, and were then transferred to 135-l rearing tanks where the larvae remained until the start of the experiment. 2.2. Experimental tanks and system design Experimental tanks consisted of blue, 22-l plastic enclosures partially immersed in a water bath. The tanks were supplied with water at the surface of the tank from a recirculating system of synthetic sea water. The water exited through a mesh covered cylinder at the base of the tank. No green water was added to any of the tanks at any point in the experiment. Microparticulate food was administered to the tanks by hand and by automated feeders. Tanks were aerated by air pumped through perforated plastic tubing near the base of the tank, thereby keeping the larvae in constant motion. Lighting was maintained at 8 lumenrft 2 for a 24-h L:Oh D photoperiod, and salinity remained at 32 ppt throughout the experiment. For experiment 1, initial flow rates were 230 mlrmin but were increased to 330 mlrmin as the larvae increased in size Ž; 9.5 mm. to maintain water quality: ) 7 mgO 2rl and - 0.005 mgNH 3rl. For experiment 2, flow rates remained at 350 mlrmin throughout the experiments with similar water quality parameters. 2.3. Measurements and analysis Images of the larvae were captured under a dissecting microscope using a Targa plus frame grabber ŽMedia Cybernetics, Silver Springs, MD, USA.. Standard lengths ŽHardy, 1978. were measured to the nearest 0.01 mm using image analysis software ŽImage Pro Plus, Truevision, Indianapolis, IN, USA.. Larvae were freeze-dried and measured to the nearest "2 mg for dry weight analysis. All statistical procedures used SASrSTATe software ŽSAS Institute, 1985.. A completely random design with subsampling was used for each of the experiments. Normality of the data ŽShapiro and Wilk, 1965. and homogeneity of variance ŽSnedecor and Cochran, 1993. were tested to ensure the assumptions for analysis of variance were met. Analysis of variance was conducted for length, dry weight, and survival ŽSnedecor and Cochran, 1993.. Survival data were arcsin transformed prior to analysis. Treatment differences were determined at P F 0.05, and Duncan’s multiple range test ŽSnedecor
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H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72
and Cochran, 1993. was used to distinguish differences among treatment means Ž P F 0.05.. 2.4. Experiment 1 Each experimental tank contained 120 larvaerl Ž2640 larvaertank. at the start of the experiment Žhatching.. All tanks were supplied with enriched ŽDHA Selcoe, INVE Aquaculture, Grantsville, UT, USA. rotifers Ž Brachionus plicatilis. until the introduction of microparticulate diet ŽBiokyowae, Kyowa Hakko Kogyo, Tokyo, Japan. at 14 MP, 21 MP, 28 MP, or 35 MP dph ŽFig. 1.. A live feed control was fed only rotifers
Fig. 1. Feeding regimes of haddock larvae in experiments 1 ŽA. and 2 ŽB.. Rotifer Ø Ø Ø Ø Ø Ø Ø Ø Ø ; Artemia; --- Ø Ø --- Ø Ø ---; MP . For additional explanation, see text.
H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72
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until day 21, at which point Artemia ŽGreat Salt Lake. enriched with DHA Selcoe were introduced Ž21A.. There were four replicates per treatment, and each treatment was co-fed, either with microparticulate diet or Artemia, for 1 week. Larvae fed rotifers received 17rmlrfeeding with four feedingsrday for a total of 1.5 million rotifersrday, and larvae fed Artemia received 5rmlrfeeding with four feedingsrday for a total of 400,000rday. The microparticulate diet was offered as 250- or 400-mm particles, and Artemia were given at the third instar stage. During the 1-week co-feeding period, larvae were fed rotifers at 0800 and 2000 h; those given MP diet were fed every 2 h during the 24-h photoperiod, while the control was fed Artemia at 0800, 1200, 1600, and 2000 h. After the 1-week co-feeding period, experimental tanks were given only microparticulate food, and control tanks were given only Artemia. After hatching Žday 0., 100 larvae were sampled from an extra experimental vessel for initial length and dry weight analysis. Starting at 14 dph, 20 larvaertank were sampled each week until 42 dph for length and dry weight. At 42 dph, only 10 larvaertank were sampled due to low survival in some of the tanks. Survival percentages were calculated at 47 dph. Temperature was maintained at 8.58C. 2.5. Experiment 2 Twenty-five days post-hatch larvae previously fed rotifers and Artemia were transferred by beaker from 135-l rearing vessels into 22-l experimental vessels. The fish were then acclimated for 5 days prior to the start of the experiment, which began at 30 dph at which time they averaged 8.85 mm in length. These larvae were reared similarly to the live feed control animals in experiment 1, and consequently were fed exclusively live feeds prior to the start of the experiment. The larvae were weaned at 30 MP, 35 MP, or 42 MP dph with a 1-week co-feeding period ŽFig. 1.. During the co-feeding period, microparticulate diet was offered every 2 h and Artemia were given at 0800, 1200, 1600, and 2000 h in successively smaller amounts until the end of the co-feeding period. The animals were offered 700 and 1000 mm Biokyowae microparticulate diet. The control was fed Artemia only throughout the experiment, which ended at 59 dph. The temperature was maintained at 10.58C.
3. Results 3.1. Experiment 1 Survival was significantly reduced by early weaning ŽTable 1.. There were no significant differences in survival among treatments weaned at 14, 21, 28, or 35 dph; survival for these treatments ranged from 2.5% to 6.3%. Average survival for control animals was significantly higher at 37.9%, indicating a superiority of live feeds over the earlier microparticulate weaning regimes. There were no differences in length between any of the treatments sampled throughout the experiment, and the growth dispensation
66
Treatment Žday start-weaned.
14
21
28
35
Control
Initial
4.75 0.031 6.66"0.23 0.120"0.013 7.49"0.28 0.222"0.041 8.37"0.43 0.502"0.173 9.87"0.54 0.976"0.262 11.19"1.26 1.384"0.682 b 2.8"1.1b
4.75 0.031 6.37"0.42 0.109"0.020 7.33"0.41 0.214"0.050 8.59"0.43 0.661"0.409 9.66"0.48 1.020"0.050 11.46"1.05 1.714"0.286 b 6.3"2.7 b
4.75 0.031 6.42"0.40 0.111"0.014 7.34"0.26 0.192"0.032 8.65"0.42 0.676"0.143 10.22"0.67 1.132"0.264 12.48"0.81 2.187"0.160 b 2.6"1.8 b
4.75 0.031 6.79"0.20 0.131"0.014 7.62"0.30 0.237"0.043 9.05"0.28 0.814"0.150 10.03"0.49 1.130"0.181 12.22"1.05 2.246"0.580 b 2.6"1.7 b
4.75 0.031 6.76"0.33 0.118"0.011 7.84"0.49 0.278"0.070 9.04"0.57 0.580"0.318 10.19"0.68 1.200"0.280 13.43"1.41 3.563"0.600 a 37.9"3.6 a
Day 14 Day 21 Day 28 Day 35 Day 42 Day 47
Length Žmm. Dry weight Žmg. Length Žmm. Dry weight Žmg. Length Žmm. Dry weight Žmg. Length Žmm. Dry weight Žmg. Length Žmm. Dry weight Žmg. Length Žmm. Dry weight Žmg. Survival 1
Values are means"standard error of four replicates. Means within a row with the same superscript are not significantly different Ž P F 0.05.. Growth measurements are on a per larvae basis. 1 Survival Ž%. s ŽTotal number of survivors at the end of the experiment.rŽTotal number of larvae at start y number of larvae sampled throughout experiment..
H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72
Table 1 The effect of weaning age on survival and growth Žlength, dry weight. of haddock larvae ŽExperiment 1.
H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72
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appeared to be similar within the treatments as well. No differences in weight were seen until the 42-day sampling period, at which point the control was significantly heavier than all other treatments with an average of 3.56 and 1.88 mg, respectively. Hence, the feeding of solely live feeds resulted in a significant increase in weight and survival vs. any of the treatments fed live feeds followed by exclusively formulated diets. As it was not possible to accurately obtain daily mortality data, no quantitative information about periods of punctuated mortality in relation to weaning times could be established. However, qualitative estimates indicate a decrease in numbers shortly after the larvae were given nothing but formulated diet. In these experiments, the larvae did appear to be eating the diet approximately 3–4 days after its introduction as illustrated by gut coloration within the tank and microscopic evaluation. However, no information regarding amounts consumed could be collected. Consequently, it is possible the larvae were either not consuming enough of the diet, or the diet was nutritionally inadequate. The microparticulate diet did create issues with tank hygiene, as uneaten food particles sedimented and collected at the bottom of the tank, and to a less extent, along the sides. The bottom was siphoned daily and water quality parameters were never compromised. Occasionally a gelatinous growth, isolated as bacterial but unidentified further was observed in some of the tanks. Although it did not appear to have an effect, it is unclear what impact this may have had. 3.2. Experiment 2 Survivals Žfrom 30 to 59 dph. for the 30, 35, 42, and control treatment were 35%, 51%, 65%, and 73%, respectively ŽTable 2.. Survival was significantly reduced for both the 30- and 35-day weaning periods, while introducing the microparticulate diet at day 42 yielded results similar to those obtained with the live feed control. The dry weight of the 30-day start weaning period was comparable to the control, while the 35- and 42-day start weaning intervals exhibited significantly greater weights than either of the other treatments. There were no differences in length between any of the treatments. Morts were collected, counted, and evaluated for signs of damage. The 42-day start weaning treatment exhibited the greatest growth dispensation followed closely by the 35-day start weaning treatment. The 30-day treatment and the control contained fish that were relatively similar in size. There was a 10% discrepancy in the totals of fish counted at
Table 2 The effect of weaning times on haddock survival and growth Žlength, dry weight. to 59 dph. Experiment 2 Treatment Žday start weaned. Length Žmm. Dry weight Žmg. Survival Ž%.1
30
35 a
29.9"2.2 0.65"0.13 a 35.1"3.6 a
42 a
33.5"1.8 0.90"0.10 b 50.7"5.3 b
Control a
33.0"3.9 0.82"0.03 b 64.5"3.96 c
28.8"2.0 a 0.57"0.11a 72.9"7.07 c
Values are means"standard error of four replicates. Means within a row with the same superscript are not significantly different Ž P F 0.05.. Growth measurements are on a per larvae basis. 1 Survival Ž%. s ŽTotal number of survivors at the end of the experiment.rŽTotal number of larvae at start y number of larvae sampled throughout..
68 H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72 Fig. 2. Daily mortality of haddock from 30 to 59 days post-hatch. Experiment 2. 30 MP, 35 MP, 42 MPs microparticulate diet introduced at 30, 35 and 42 days post-hatch, respectively. Controls Artemia fed throughout the experiment. For additional explanation, see text.
H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72
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the end vs. what was expected from mortality data. Although cannibalism was never witnessed in the tank, there were many acts of fatal aggression, which appeared to account for the majority of mortality. The morts examined showed no visible signs of disease Ži.e., no abnormal gill tissue, lesions, enlarged or otherwise abnormal organs, or excessively frayed fins., had food in their guts, and were not emaciated. The graph of daily mortality ŽFig. 2. shows periods of punctuated mortality following the discontinuation of the live feeds. This phenomenon is most dramatic at the earlier weaning periods and gets progressively less pronounced at later weaning periods. At the end of the experiment, the largest fish in each treatment were sacrificed and examined for signs of cannibalism, however, no evidence of cannibalism was evident in the dissection.
4. Discussion One of the objectives of these experiments was to establish a successful culture technique for rearing larval haddock. We were able to achieve the greatest survival reported to date, with an average of 38.9% through 47 dph for the live feed controls. These numbers are comparable to results we have had with cod larvae ŽBaskervilleBridges and Kling, 2000.. The other objective of these experiments was to determine an adequate protocol for the early weaning of larval haddock onto a formulated feed. As it was evident after the first experiment that haddock could not be effectively weaned onto microparticulate feeds during the early time periods examined, we made the decision to increase the temperature and focus on later periods. We did this with the intention of investigating earlier periods if the later interval testing proved successful. We were then able to adequately wean haddock by 42 days post-hatch with minimal risks to growth and survival. Since the 30- and 35-day start weaning periods at this higher temperature proved unsuccessful, we felt it unnecessary to investigate intervals prior to those examined in this experiment. Although we were able to wean haddock commensurate to live feed control animals by 42 dph, this is not considered early weaning, and more work will need to be done to determine the limiting factor in the weaning process. Although there have been a number of studies over the years focusing on the development of an adequate diet that could, ideally, replace live feeds while yielding similar results, there has been limited success. Whether or not this is due to nutritional, or possibly physical barriers or inadequacies, remains unclear. It has been reported that haddock prefer smaller, less mobile prey than cod ŽAuditore et al., 1994; Kane, 1984., but it is uncertain how well they will accept an inert food item. Since fish larvae are predominantly visual feeders ŽGerking, 1994., it has been theorized that larvae prefer moving prey over suspended inert particles, and consequently, many larvae will either reject the non-motile food and eventually starve to death, or will delay feeding behavior, which introduces risks to growth and development ŽLe Ruyet et al., 1993.. In tanks where the water is stagnant or has little motion, the lack of movement of microparticles could be of considerable concern ŽBengston, 1993.. However, in tank systems with a great deal of motion such as these, concern is limited since gross movements of the
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H.J. Hamlin, L.J. Kling r Aquaculture 201 (2001) 61–72
microparticles and live feeds are at the mercy of the water current and move in much the same manner in the water column. The larvae are also in motion, which increases predator–prey contact rates. It is unclear if haddock larvae can detect subtle movements of prey such as cilia undulations of rotifers or fine movements of Artemia and it is, therefore, difficult to discern the significance of such movements as an attractant for the larvae. Braid Ž1977. obtained adequate growth and survival of striped bass larvae with heat killed Artemia, which negates movement of the prey as the sole attractant for this species. Appelbaum Ž1989. provided recommendations for the successful rearing of marine larvae using inert diets, in which he suggested that raising the temperature of the water increases larval metabolism and activity level, potentially improving their readiness to accept inert diets. In the first experiment survival was extremely low even at the 35 dph start weaning period. Increasing the temperature from 8.58C to 10.58C may have increased the ability of haddock to accept the microparticulate diet. An increase in temperature also advances the developmental stage of the larvae Žincreasing number of degree-days., and it is possible that this advanced developmental state affords the larvae a more favorable physiological condition in which to assimilate the diet. This lab has found the differentiation of structures, which results in a digestive system similar to the adult to begin at 35 days or 10 mm, and is well developed by 53 days or 15 mm. Since a widening of the food spectrum accompanies the transformation from larval to adult stages ŽForstner et al., 1983. it may prove difficult to wean haddock prior to this critical developmental period. In the second experiment, survival from 30 to 59 dph ranged from 35% to 73%, which is not high for this time period. In general, the greatest mortality is experienced at earlier larval stages, and we would expect mortality at these later stages to be minimal. A great deal of this mortality may have been due to aggression andror cannibalism. The dead were siphoned and counted daily and exhibited no visual signs of disease. The dead fish had food in their guts and exhibited no signs of starvation. Frequently, however, we observed aggressive acts such as fish grasping others by the head and shaking them to death. This occurred in all of the treatments but was more pronounced in tanks fed exclusively with formulated feeds. At the end of the experiment, the largest fish in each tank were sacrificed to evaluate their digestive tracts for signs of cannibalism and no evidence was found, although it would take only a few hours or less for evidence to be digested ŽFolkvord, 1993.. There are few instances in which predatory fish do not practice cannibalism ŽDavis, 1985.. Although we did not observe cannibalism at these early stages, we are not convinced that it did not occur since there was a 10% discrepancy between expected Žusing starting numbers minus daily mortality. and actual survival. There was a large growth dispensation in all of the treatments, although replicates varied somewhat. Size variation may be a principal cause of aggressive or agonistic behavior, which can have the same end result as cannibalism ŽHecht and Pienaar, 1993.. The weights of the fish were greatest for the 35- and 42-day start weaning periods vs. the 30-day start weaning period and the control. For the control animals, this situation may have been created as a consequence of remaining on a prey item that is most likely smaller than they would normally be ingesting.
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Acknowledgements Maine Agricultural and Forest Experiment Station external publication a2476. This work was supported by the University of Maine and University of New Hampshire Sea Grant College Program as well as the Maine Agricultural and Forest Experiment Station. Appreciation is extended to Jacqueline Hunter for her technical assistance in the production of live feeds, and to the Department of Fisheries and Oceans for providing eggs used in the experiments.
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