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Diet development and evaluation for juvenile abalone, Haliotis asinina: animal and plant protein sources
Aquaculture 219 (2003) 645 – 653 www.elsevier.com/locate/aqua-online
Diet development and evaluation for juvenile abalone, Haliotis asinina: animal and plant protein sources Myrna N. Bautista-Teruel a,*, Armando C. Fermin a, Shunsuke S. Koshio b,1 a
Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Iloilo 5021, Philippines Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20, Kagoshima 890-0056, Japan
b
Received 4 March 2002; received in revised form 6 August 2002; accepted 7 August 2002
Abstract Growth studies were conducted to determine the suitability of animal and plant protein sources in the diet of abalone, Haliotis asinina. Juvenile abalone with mean initial weight and shell length of 0.69 F 0.04 g and 11.4 F 0.35 mm, respectively, were fed practical diets for 84 days at a temperature range of 28 – 31 jC. The practical diets contained 27% crude protein from various sources such as fish meal (FM), shrimp meal (SM), defatted soybean meal (DSM), and Spirulina sp. (SP). A formulated diet (diet 1) served as the control. The diets were fed to abalone at 2 – 5% body weight once daily at 1600 h. Weight gain (WG), increase in shell length (SL), specific growth rate (SGR), protein efficiency ratio (PER) and feed conversion ratio (FCR) were evaluated. Highest weight gain (WG: 454%) was attained with abalone fed diet 2 with protein sources coming from a combination of FM, SM, and DSM. This value was, however, not significantly different ( P < 0.05) from those fed diets 4 and 1 (Control diet) with protein sources coming from FM, SM, SP and FM, DSM, SM, respectively. Abalone fed diet 3, which used both plant protein sources, DSM and SP, showed significantly lower WG (327%). Survival was generally high ranging from 85% to 100% for all treatments. The SGR showed the same trend as the percent weight gain. The FCR and PER obtained, however, were not significantly different for all treatments. The amino acid profile of diets 1, 2, and 4 simulated that of the abalone protein, which could have been a contributing factor to the higher growth rate of abalone fed these diets. Diet 3, which contained only plant protein sources, showed
*
Corresponding author. Tel.: +63-33-3362965; fax: +63-33-3351008. E-mail addresses: [email protected] (M.N. Bautista-Teruel), [email protected] (S.S. Koshio). 1 Tel.: +81-99-286-4182; fax: +81-99-286-4184. 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 4 1 0 - 6
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relatively lower methionine values compared with the abalone muscle tissue. Although abalone are considered herbivorous animals, results of this study indicate that a combination of dietary plant and animal protein sources was necessary to attain the best growth rate. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Protein; Haliotis asinina; Spirulina sp.; Abalone; Defatted soybean meal
1. Introduction The decreasing commercial catch and the high market demand for abalone in both the domestic and export markets have stimulated great interest in the development of its aquaculture. As early as 1881, Japanese scientists led the initiation of biological studies that became the basis for the successful controlled reproduction attempts for Haliotis gigantea and Haliotis discus. In the United States, studies on controlled seed production and culture were particularly focused on the red abalone Haliotis rufescens (Hahn, 1989). The aquaculture of tropical and subtropical abalone species such as the Haliotis asinina and the Haliotis diversicolor supertexta begun in Thailand and Taiwan (Jayarabhand and Paphavasit, 1996). Interest on the other species like Haliotis varia and Haliotis ovina were not as developed as the former two species mainly because of the smaller sizes of the latter that keeps them less attractive to consumers or export buyers. H. asinina is the largest among the tropical species (Singhagraiwan and Doi, 1993). Artificial feeding for abalone culture has been practiced in countries like Japan, USA, and Australia. Feeding experiments in Taiwan showed that growth of juveniles fed artificial diets was 65% greater than those juveniles fed macroalgae. Further, animals fed artificial diets had higher body weight per shell length and a relatively higher protein content in their meat compared to animals fed seaweed. Since abalone are generally characterized by slow and heterogenous growth rates, proper nutrition must be provided to make a successful culture. Artificial feed for abalone must contain sufficient protein and essential amino acids in order to satisfy their nutrient requirements. Protein, which is essential for growth of abalone, is the most expensive component in their diet (Fleming et al., 1996). It is necessary that the protein supplied in the diet be palatable, digestible and possess an appropriate balance of amino acids, before the inclusion levels are determined. Thus, protein sources should first be evaluated before incorporation into practical diets. The most commonly used protein sources in abalone feed diets are fish meal, defatted soybean meal, and casein (Guzman and Viana, 1998). Fish meal is the only sole protein source that can support good growth performance whereas soybean meal and casein needs to be fed in combination with other protein sources in order to support good growth (Fleming et al., 1996). A combination of abalone viscera silage and soybean meal as protein sources in abalone, Haliotis fulgens diet was found to be effective in supporting good growth (Guzman and Viana, 1998). Uki et al. (1985a,b), in their work with various protein sources for abalone, found casein to be the most suitable protein. The reason for better growth was attributed to protein quality due to differences in digestibility of each protein source by juvenile abalone. However, because of its relatively high cost, casein is
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not practical for use in abalone feeds. Spirulina, likewise, was shown to have a very good potential as a protein source for abalone Haliotis midae diets (Britz et al., 1994). Plant protein sources such as field peas, faba beans, yellow lupins defatted soyflour and vetch were tested and used by Vandepeer et al. (1999) for Haliotis laevigata. Their results showed that all these legumes were well digested by various abalone species. This study focused on the development and evaluation of practical diets for the culture of juvenile abalone, H. asinina, with emphasis on determining the suitability of protein sources, such as fish meal, shrimp meal, defatted soybean meal, and Spirulina sp. for incorporation in formulated diets.
2. Materials and methods 2.1. Experimental animals Abalone, H. asinina, juveniles with initial mean weight and shell length of 0.69 F 0.04 g and 11.4 F 0.35 mm, respectively, were used as the experimental animals. They were harvested from natural spawnings at the mollusc hatchery of the SEAFDEC, Aquaculture Department, Tigbauan, Iloilo, Philippines. The abalone were acclimated in the laboratory for a week in a 500-l fiberglass tank filled with seawater and fed with commercial pellets. 2.2. Diet preparation Four practical diets were formulated to contain either FM, SM, DSM (diet 1); FM, DSM (diet 2); DSM, SP (diet 3); FM, SM, SP (diet 4) at 27% crude protein. The source of energy from carbohydrate came from wheat flour while that of lipid was from a 1:1 mixture of squid oil and soybean oil. Mineral and vitamin mixes were added to the diet. Seaweed and wheat flour served as binders. Composition of experimental diets is shown in Table 1. Diet 1, a slightly modified formulation used and tested to give good growth in a previous experiment, served as the control. Procedures for diet preparation were patterned after Bautista-Teruel and Millamena (1999). 2.3. Proximate and amino acid analysis Proximate analysis of the different diets and abalone carcasses (Table 2) were conducted using standard methods (AOAC, 1985). Moisture was determined using a moisture balance, crude protein by semimicro Kjeldahl, crude fat by soxhlet extraction, and crude fiber was obtained in a fat-free material sample by dilute acid and alkali treatment. Ash content was determined in a muffle furnace at 550 jC and nitrogen-free extract (NFE) was calculated by difference. Amino acid analysis was done on the various diets and abalone muscle tissue using an HPLC amino acid analyzer employing a Shimadzu Shim-pack ISC-071/S1504; 4.0 mm 15 cm column packing with a flow rate of 0.5 ml/min. The flow rate of the reaction reagent was 0.3 ml/min; column temperature at 550 jC with wavelength of ex
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Table 1 Percentage (dry weight basis) composition of the formulated diets (g/100 g diet) Ingredients (g/100 g diet)
Diet number 1
2
3
4
Fish meal Shrimp meal Defatted soybean meal Spirulina Rice bran Wheat flour Seaweed Squid oil Soybean oil Vitamin mixa Mineral mixa Dicalcium phosphate Butylatedhydroxy toluene
10.00 15.00 20.00 – 15.00 20.00 9.00 0.50 0.50 3.00 4.00 2.95 0.05
7.50 7.50 35.00 – 10.00 20.00 9.00 0.50 0.50 3.00 4.00 2.95 0.05
– – 35.00 20.00 5.00 20.00 9.00 0.50 0.50 3.00 4.00 2.95 0.05
10.00 10.00 – 20.00 20.00 20.00 9.00 0.50 0.50 3.00 4.00 2.95 0.05
a Vitamin and mineral mixes, commercial brand. h-carotene (3.0 MIU kg 1), cholecalciferol (0.6 MIU kg 1), thiamin (3.60 g kg 1), riboflavin (7.20 g kg 1), pyridoxine (6.60 g kg 1), cyanocobalamine (0.02 g kg 1), a-tocopherol (16.50 g kg 1), menadione (2.40 g kg 1), niacin (14.40 g kg 1), pantothenic acid (4.00 g kg 1), biotin (0.02 g kg 1), folic acid (1.20 g kg 1), inositol (30.00 g kg 1), Stay C (100.00 g kg ). Mineral mix: P (12.00%), Ca (12.00%), Mg (1.50%), Fe (0.15%), Zn (0.42%), Cu (0.21%), K (7.50%), Ge (0.0001%), Co (0.011%), Mn (0.160%), Se (0.001%), Mo (0.0005%), Al (0.0025%), I (0.04%), B (0.0001%), Ni (0.0001%).
348 nm and em 450 nm. Reaction reagents used were o-pthalaldehyde/sodium carbonate buffer and sodium hypochlorite. The HPLC system consisted of the following: pump LC-3A; step gradient unit, SGR-1A; oven CTO-2A; fluorescence detector, FLD; and chromatopac, C-RIB. The following buffers were used: 0.07 M sodium citrate (pH 3.19); 0.07 M sodium citrate (pH 3.80); 0.20 M sodium citrate (pH 8.48); and 0.50 M sodium hydroxide. 2.4. Feeding experiment Twenty abalone juveniles were stocked in 12 60-l fiberglass tanks containing 40-l of filtered seawater. Shelters of two halved polyvinyl chloride pipes (65 mm diameter) were Table 2 Proximate composition (%) of diets Diet number
Crude protein Crude fat NFEa Estimated metabolizable energy(kcal/kg)b a
1
2
3
4
27.84 3.13 43.55 3137
27.30 3.27 42.30 3078
27.72 2.81 43.68 3109
26.97 3.04 42.62 3057
NFE—nitrogen free extract, given by difference. Computed based on standard physiological fuel values of 9 kcal/g lipid and 4 kcal/g protein and carbohydrate (Brett and Groves, 1979). b
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provided in each tank. Four dietary treatments with three replications were randomly allocated to different tanks. A flow-through water system was maintained at approximately 300 –350 l/h with gentle aeration provided. Excess feed that settled on the tank bottom was siphoned out, dried and weighed to get the true value of the feed consumed by the abalone. Water temperatures ranged from 28 to 31 jC and salinity varied from 28xto 32x . Ranges of dissolved oxygen, nitrite, ammonia, and pH in the rearing tanks were at 5.0 –5.6 mg/l; 0 – 0.05 mg/l; 0– 0.40 and 8.3 – 8.4, respectively. Diets were fed to abalone at 2 –5% of their body weight once in the late afternoon at about 1600 h for 90 days. Shell length increase (SL), weight gain (WG), survival (SURV), and feed conversion ratio (FCR) were determined every 20 days until the 84th day. Water stability of the diets was conducted according to the method of Hastings et al. (1971). Diet stability was determined at 6, 12, and 24 h interval. Percent water stability was computed as: %water stability ¼ ðB AÞð%dry matterÞ=Að%dry matterÞ 100 where B is the final weight of feed and A is the initial weight of feed. 2.5. Statistical analysis All data were analyzed by one-way analysis of variance (ANOVA) and Duncan’s Multiple Range Test on the SAS Package for the IBM-PC (SAS Institute, 1988). Differences among treatment means were considered significant at P < 0.05.
3. Results At the end of the 84-day feeding trial, the highest weight gain (453.8%) was attained with abalone fed diet 2 with protein sources coming from a combination of FM, SM, and DSM. This value was, however, not significantly different from those fed diets 4 (420.6%) with protein sources from FM, DSM, SP and the control diet (400%) containing protein sources from FM, SM, and DSM. Abalone fed diet 3, which contained DSM and SP, had significantly lower weight gain of 326.5%. Similarly, the specific growth rates and shell Table 3 Initial and final weight, shell length, and SGR of abalone fed formulated diets for 84 daysa Treatment
Initial weight (average, g)
Initial SL (average, mm)
Final weight (average, g)
Final SL (average, mm)
Weight gain (%)b
SGRc
1 2 3 4
0.73 F 0.08 0.65 F 0.03 0.68 F 0.03 0.68 F 0.01
11.2 F 0.27 11.5 F 0.48 11.5 F 0.28 11.4 F 0.37
3.65 F 0.19 3.60 F 0.10 2.90 F 0.28 3.54 F 0.17
35.4 F 0.31 34.5 F 0.58 33.2 F 0.91 35.4 F 0.86
400.0 F 49.8a 453.8 F 42.8a 326.5 F 392b 420.6 F 20.6a
0.83 F 0.09a 0.89 F 0.02a 0.75 F 0.07b 0.86 F 0.03a
a Means of three replicate groups with the same superscript in each column are not significantly different ( P < 0.05). b Weight gain: [(final weight initial weight)/initial weight] 100. c SGR (specific growth rate) = 100 (ln average final weight ln average initial weight)/number of days.
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Table 4 Shell length increase, total feed intake, FCR, percent survival of abalone fed formulated diets for 84 days and percent water stability of pellets Treatment
Shell length increase (%)
Total feed intake (g)
FCRa
PERb
SURV (%)
Percent water stability of pellets (24 h)c
1 2 3 4
216 F 2.45a 200 F 3.52a 188 F 2.12b 210 F 2.38a
365.36 391.68 285.06 329.16
1.01 1.00 0.97 0.86
3.90 4.17 4.14 4.58
95 95 85 85
64 63 65 65
Means of replicate groups F S.E.M. with the same superscript in each column are not significantly different ( P < 0.05). a FCR = dry weight of feed (g)/wet weight gain (g). b PER = wet weight gain/protein intake. c %Water stability=(final dry weight of feed/initial dry weight %dry matter)/100.
length increase of abalone fed diets 1, 2, and 4 were significantly higher compared to those fed diet 3 (Table 3). No significant differences were observed in the efficiency of protein (PER) and feed conversion (FCR) for all treatments (Table 4). Survival was generally high, ranging from 85% to 95%. All diets prepared were found to be water stable retaining at 64% of dry mass after the 24 h test for stability (Table 4). Amino acid composition of diets 1, 2, and 4 had profiles that were very similar to that of abalone tissue. Diet 3, which contained all plant – protein sources, showed some lower level of methionine (Table 5) as compared to other diets.
Table 5 Amino acid composition of formulated diets and abalone meat (g/100 g protein)a Amino acid
Alanine Arginine Aspartic Cystine Glutamic Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine a
Diet number
Abalone tissue
1
2
3
4
2.68 5.28 7.05 0.35 8.99 3.65 1.35 2.05 5.09 6.14 4.40 2.42 2.27 1.74 3.60 0.98 2.34 4.26
2.79 5.12 6.11 0.59 8.10 2.70 1.25 1.97 5.60 5.99 4.32 2.72 2.12 1.10 3.59 0.65 2.29 4.28
2.38 5.09 6.07 0.36 8.72 2.99 1.24 1.86 5.17 5.78 2.97 2.18 2.55 1.21 2.79 0.43 1.52 3.41
2.40 5.22 7.08 0.11 7.98 3.15 1.38 2.35 5.42 5.22 4.42 2.35 2.70 1.42 3.38 1.01 2.05 4.48
Means of two replicate samples.
3.72 5.48 6.23 0.42 9.56 3.71 1.78 2.51 5.95 5.90 4.09 3.29 3.41 2.30 3.69 0.55 2.97 4.90
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4. Discussion Abalone fed formulated diets with protein sources from a combination of animal and plant protein origin such as fish meal, shrimp meal, defatted soybean meal, and Spirulina sp. showed better performance in terms of growth rate per day, weight gain, and increase in shell length compared to those fed diets with protein sources of plant origin alone. Since all diets were made isonitrogenous at 27%, the results would signify that the quality and physiological availability of protein in the combined animal and protein sources may have contributed significantly to the positive effect of these diets on the growth of abalone. The amino acid profile of the various diets tested (Table 5) showed some low levels of certain amino acids such as methionine in the formulated diet, which contained only the plant protein sources. The low level of this essential amino acid (methionine) in diet 3 with protein sources coming solely from plant origin might have contributed to the lower growth rate of abalone fed this diet. The use of plant protein source alone in a formulated diet may need some supplementation of crystalline amino acids or may be used in combination with other animal protein sources to compensate for the low levels of some essential amino acids such as methionine. Murai et al. (1986, 1989) reported that supplementing soybean meal diets with either coated and uncoated methionine significantly improved growth and feed efficiency of common carp fingerlings. Guzman and Viana (1998) have successfully demonstrated the effectivity of the use of a combination of soybean meal and abalone viscera silage as protein sources in the diet of abalone, H. fulgens. In the study conducted by Britz (1996), abalone, H. midae fed fish meal and Spirulina spp. based diets produced a significantly higher length increment and specific growth rate compared to those fed diets containing soya oil cake, torula yeast, and casein alone. Faster growth rate of abalone with formulated diets containing combination of plant and animal protein sources has also been documented in studies for other Haliotis species (Hahn, 1989; Morrison and Whitington, 1991; Nie, 1992). Another concern in the use of plant protein source is the presence of antinutritional factors, which if not removed or processed may likewise contribute to the low growth of fish. In this study, however, plant protein sources used were properly processed and were assumed not to contain any of these antinutritional factors. The higher growth rate obtained when abalone were fed diets containing animal protein sources in combination with plant protein sources may be attributed to the fact that some limiting amino acids in plant protein source may be compensated by the availability of that amino acid in the animal protein source, resulting in a better profile of the diet formulation and better growth for the abalone. The amino acid profile of a diet is at its best when it simulates that of the tissue muscle of the animal being fed. The profile of animal protein source like fish meal is considered to be an excellent one, which can support good growth performance used alone or in combination with other plant protein sources. Generally, animal protein sources like fish meal are highly digestible and contain attractants that make them an attractive ingredient for any fish diet. However, the high demand for fish meal may soon result in an unaffordable price and availability of good quality sources may also be a problem. It is best therefore that fish meal or any animal protein source be used in combination with plant protein source to compensate for the limiting amino acids of
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each other, thus may result in faster growth rate of the animal. Plant protein sources are cheaper but are generally deficient in some of the sulfur-containing amino acid such as methionine. Thus, these plant protein sources are best used in combination with animal protein sources. The values obtained for the feed conversion ratio for all dietary treatments were generally low ranging between 0.9 and 1.0 and were not significantly different ( P > 0.05). This would indicate that there was high efficiency of feed conversion to body weight in all treatments. The more efficient feed conversion but lower growth rate of abalone fed the all plant protein-based diet compared to those fed the fish meal based diet or the animal – plant combination diet can be explained perhaps by the lower feed consumption of abalone fed diet 3. However, this finding does not conform with the results of Uki et al. (1986) wherein they reported poorer growth and high FCR values for H. discus hannai fed fish meal based diets compared with casein-based diets. The source and processing of fish meals used in these two different studies probably may account for the difference in results. The ability of H. discus hannai and H. asinina to digest fish meal cannot be discounted. Viana et al. (1993), on the other hand, reported that fish meal fed abalone had similar growth rates and FCR values with those abalone fed casein as protein source. This is likewise in conformity with the work of Britz (1996), wherein abalone, H. midae fed fish meal and Spirulina-based diet exhibited good growth and high efficiency of feed conversion to body weight. The efficiency of protein utilization as evidenced by the PER values (4.0 – 4.6) was not significantly different for all treatments. This would indicate that the proteins from each diet were efficiently utilized by abalone regardless of the protein source. The lower growth rates obtained with abalone fed diets with plant origin as the sole protein sources might be due to lower levels of some essential nutrients that could not have been sufficient for deposition of tissue muscle. Although the essential amino acid content was used as a determining factor for the nutritive value of the different protein sources, further work has to be done to test the availability of these amino acids to abalone. It has been reported that apparent amino acid availability and true amino acid availability vary within and among the various protein sources (Wilson, 1989).
Acknowledgements The authors would like to thank Ms. Mae F. Mallare and Mr. Narciso Entusiasmo for their assistance in the conduct of the study, Ms. Florence Jarder for the proximate analyses and Dr. O.M. Millamena for the review of the manuscript.
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Brett, J.R., Groves, T.D., 1979. Physiological energetics. In: Hoar, W.S., Randall, D.J., Brett, J.R. (Eds.), Fish Physiology, vol. 8. Academic Press, London, pp. 280 – 344. Britz, P.J., 1996. The suitability of selected protein sources for inclusion in formulated diets for the South African abalone Haliotis midae. Aquaculture 140, 63 – 73. Britz, P.J., Hecht, T., Kanuer, J., Dixon, M.G., 1994. The development of an artificial feed for abalone farming. S. Afr. J. Sci. 90, 7 – 8. Fleming, A.E., Van Barneveld, R.J., Hone, P.W., 1996. The development of artificial diets for abalone. A review and future directions. Aquaculture 140, 5 – 53. Guzman, J.M., Viana, M.T., 1998. Growth of abalone Haliotis fulgens fed diets with and without fish meal, compared to a commercial diet. Aquaculture 136, 313 – 321. Hahn, K., 1989. Nutrition and growth of abalone. In: Hahn, K. (Ed.), Handbook of Culture of Abalone and Other Marine Gastropods. CRC Press, Boca Raton, FL, pp. 135 – 180. Hastings, W.H., Meyers, S.P., Butler, D.P., 1971. A commercial process for water-stable fish feeds. Feedstuffs 43 (47), 38. Jayarabhand, P., Paphavasit, N., 1996. A review of the culture of tropical abalone with special reference to Thailand. Aquaculture 140, 159 – 168. Morrison, J., Whitington, J., 1991. Development of an artificial food for abalone. Austasia Aquac. 5, 53 – 54. Murai, T., Ogata, H., Kosutarak, P., Arai, S., 1986. Effects of amino acid supplementation and methanol treatment on utilization of soy flour by fingerling carp. Aquaculture 56, 197 – 206. Murai, T., Daozun, W., Ogata, H., 1989. Supplementation of methionine to soy flour diets for fingerling carp. Aquaculture 77, 373 – 385. Nie, Z.Q., 1992. A review of abalone culture in China. In: Shepeherd, S.A., Tegner, M.J., Guzman del Proo, S.A. (Eds.), Abalone of the World Biology, Fisheries and Culture. Fishing News Books, Oxford, pp. 592 – 602. Singhagraiwan, T., Doi, M., 1993. Seed production and culture of a tropical abalone, Haliotis asinine, Linne. Eastern Marine Fisheries Development Center (Thailand) and Japan International Cooperation Agency. 32 pp. Uki, N., Kemuyama, A., Watanabe, T., 1985a. Nutritional evaluation of various protein sources in diets for abalone, Haliotis discus hannai. Bull. Jpn. Soc. Sci. Fish. 51, 1835 – 1839. Uki, N., Kemuyama, A., Watanabe, T., 1985b. Development of semi-purified diets for abalone. Bull. Jpn. Soc. Sci. Fish. 51, 1825 – 1833. Uki, N., Kemuyama, A., Watanabe, T., 1986. Effect of heat treatment of dietary protein sources on their protein quality for abalone. Bull. Jpn. Soc. Sci. Fish. 52, 1199 – 1204. Vandepeer, M.E., Van Barneveld, R.J., Hone, P.W., Havenhand, J.N., 1999. The digestibility of raw, autoclave and phytase treated legumes in greenlip abalone, Haliotis laevegata. Book of Abstracts. Proceedings of the 6th Annual Abalone Aquaculture Workshop. World Aquaculture Society Conference, Sydney, Australia, April 26 – May 2, 1999, p. 788. Viana, M.T., Lopez, L.M., Salas, A., 1993. Diet development of juvenile abalone, Haliotis fulgens. Evaluation of two artificial diets and macroalgae. Aquaculture 117, 149 – 156. Wilson, R.P., 1989. Amino acids and proteins. In: Halver, J.E. (Ed.), Fish Nutrition, 2nd ed. Academic Press, San Diego, pp. 112 – 151.
Diet development and evaluation for juvenile abalone, Haliotis asinina: animal and plant protein sources Myrna N. Bautista-Teruel a,*, Armando C. Fermin a, Shunsuke S. Koshio b,1 a
Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Iloilo 5021, Philippines Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20, Kagoshima 890-0056, Japan
b
Received 4 March 2002; received in revised form 6 August 2002; accepted 7 August 2002
Abstract Growth studies were conducted to determine the suitability of animal and plant protein sources in the diet of abalone, Haliotis asinina. Juvenile abalone with mean initial weight and shell length of 0.69 F 0.04 g and 11.4 F 0.35 mm, respectively, were fed practical diets for 84 days at a temperature range of 28 – 31 jC. The practical diets contained 27% crude protein from various sources such as fish meal (FM), shrimp meal (SM), defatted soybean meal (DSM), and Spirulina sp. (SP). A formulated diet (diet 1) served as the control. The diets were fed to abalone at 2 – 5% body weight once daily at 1600 h. Weight gain (WG), increase in shell length (SL), specific growth rate (SGR), protein efficiency ratio (PER) and feed conversion ratio (FCR) were evaluated. Highest weight gain (WG: 454%) was attained with abalone fed diet 2 with protein sources coming from a combination of FM, SM, and DSM. This value was, however, not significantly different ( P < 0.05) from those fed diets 4 and 1 (Control diet) with protein sources coming from FM, SM, SP and FM, DSM, SM, respectively. Abalone fed diet 3, which used both plant protein sources, DSM and SP, showed significantly lower WG (327%). Survival was generally high ranging from 85% to 100% for all treatments. The SGR showed the same trend as the percent weight gain. The FCR and PER obtained, however, were not significantly different for all treatments. The amino acid profile of diets 1, 2, and 4 simulated that of the abalone protein, which could have been a contributing factor to the higher growth rate of abalone fed these diets. Diet 3, which contained only plant protein sources, showed
*
Corresponding author. Tel.: +63-33-3362965; fax: +63-33-3351008. E-mail addresses: [email protected] (M.N. Bautista-Teruel), [email protected] (S.S. Koshio). 1 Tel.: +81-99-286-4182; fax: +81-99-286-4184. 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 4 1 0 - 6
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relatively lower methionine values compared with the abalone muscle tissue. Although abalone are considered herbivorous animals, results of this study indicate that a combination of dietary plant and animal protein sources was necessary to attain the best growth rate. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Protein; Haliotis asinina; Spirulina sp.; Abalone; Defatted soybean meal
1. Introduction The decreasing commercial catch and the high market demand for abalone in both the domestic and export markets have stimulated great interest in the development of its aquaculture. As early as 1881, Japanese scientists led the initiation of biological studies that became the basis for the successful controlled reproduction attempts for Haliotis gigantea and Haliotis discus. In the United States, studies on controlled seed production and culture were particularly focused on the red abalone Haliotis rufescens (Hahn, 1989). The aquaculture of tropical and subtropical abalone species such as the Haliotis asinina and the Haliotis diversicolor supertexta begun in Thailand and Taiwan (Jayarabhand and Paphavasit, 1996). Interest on the other species like Haliotis varia and Haliotis ovina were not as developed as the former two species mainly because of the smaller sizes of the latter that keeps them less attractive to consumers or export buyers. H. asinina is the largest among the tropical species (Singhagraiwan and Doi, 1993). Artificial feeding for abalone culture has been practiced in countries like Japan, USA, and Australia. Feeding experiments in Taiwan showed that growth of juveniles fed artificial diets was 65% greater than those juveniles fed macroalgae. Further, animals fed artificial diets had higher body weight per shell length and a relatively higher protein content in their meat compared to animals fed seaweed. Since abalone are generally characterized by slow and heterogenous growth rates, proper nutrition must be provided to make a successful culture. Artificial feed for abalone must contain sufficient protein and essential amino acids in order to satisfy their nutrient requirements. Protein, which is essential for growth of abalone, is the most expensive component in their diet (Fleming et al., 1996). It is necessary that the protein supplied in the diet be palatable, digestible and possess an appropriate balance of amino acids, before the inclusion levels are determined. Thus, protein sources should first be evaluated before incorporation into practical diets. The most commonly used protein sources in abalone feed diets are fish meal, defatted soybean meal, and casein (Guzman and Viana, 1998). Fish meal is the only sole protein source that can support good growth performance whereas soybean meal and casein needs to be fed in combination with other protein sources in order to support good growth (Fleming et al., 1996). A combination of abalone viscera silage and soybean meal as protein sources in abalone, Haliotis fulgens diet was found to be effective in supporting good growth (Guzman and Viana, 1998). Uki et al. (1985a,b), in their work with various protein sources for abalone, found casein to be the most suitable protein. The reason for better growth was attributed to protein quality due to differences in digestibility of each protein source by juvenile abalone. However, because of its relatively high cost, casein is
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not practical for use in abalone feeds. Spirulina, likewise, was shown to have a very good potential as a protein source for abalone Haliotis midae diets (Britz et al., 1994). Plant protein sources such as field peas, faba beans, yellow lupins defatted soyflour and vetch were tested and used by Vandepeer et al. (1999) for Haliotis laevigata. Their results showed that all these legumes were well digested by various abalone species. This study focused on the development and evaluation of practical diets for the culture of juvenile abalone, H. asinina, with emphasis on determining the suitability of protein sources, such as fish meal, shrimp meal, defatted soybean meal, and Spirulina sp. for incorporation in formulated diets.
2. Materials and methods 2.1. Experimental animals Abalone, H. asinina, juveniles with initial mean weight and shell length of 0.69 F 0.04 g and 11.4 F 0.35 mm, respectively, were used as the experimental animals. They were harvested from natural spawnings at the mollusc hatchery of the SEAFDEC, Aquaculture Department, Tigbauan, Iloilo, Philippines. The abalone were acclimated in the laboratory for a week in a 500-l fiberglass tank filled with seawater and fed with commercial pellets. 2.2. Diet preparation Four practical diets were formulated to contain either FM, SM, DSM (diet 1); FM, DSM (diet 2); DSM, SP (diet 3); FM, SM, SP (diet 4) at 27% crude protein. The source of energy from carbohydrate came from wheat flour while that of lipid was from a 1:1 mixture of squid oil and soybean oil. Mineral and vitamin mixes were added to the diet. Seaweed and wheat flour served as binders. Composition of experimental diets is shown in Table 1. Diet 1, a slightly modified formulation used and tested to give good growth in a previous experiment, served as the control. Procedures for diet preparation were patterned after Bautista-Teruel and Millamena (1999). 2.3. Proximate and amino acid analysis Proximate analysis of the different diets and abalone carcasses (Table 2) were conducted using standard methods (AOAC, 1985). Moisture was determined using a moisture balance, crude protein by semimicro Kjeldahl, crude fat by soxhlet extraction, and crude fiber was obtained in a fat-free material sample by dilute acid and alkali treatment. Ash content was determined in a muffle furnace at 550 jC and nitrogen-free extract (NFE) was calculated by difference. Amino acid analysis was done on the various diets and abalone muscle tissue using an HPLC amino acid analyzer employing a Shimadzu Shim-pack ISC-071/S1504; 4.0 mm 15 cm column packing with a flow rate of 0.5 ml/min. The flow rate of the reaction reagent was 0.3 ml/min; column temperature at 550 jC with wavelength of ex
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Table 1 Percentage (dry weight basis) composition of the formulated diets (g/100 g diet) Ingredients (g/100 g diet)
Diet number 1
2
3
4
Fish meal Shrimp meal Defatted soybean meal Spirulina Rice bran Wheat flour Seaweed Squid oil Soybean oil Vitamin mixa Mineral mixa Dicalcium phosphate Butylatedhydroxy toluene
10.00 15.00 20.00 – 15.00 20.00 9.00 0.50 0.50 3.00 4.00 2.95 0.05
7.50 7.50 35.00 – 10.00 20.00 9.00 0.50 0.50 3.00 4.00 2.95 0.05
– – 35.00 20.00 5.00 20.00 9.00 0.50 0.50 3.00 4.00 2.95 0.05
10.00 10.00 – 20.00 20.00 20.00 9.00 0.50 0.50 3.00 4.00 2.95 0.05
a Vitamin and mineral mixes, commercial brand. h-carotene (3.0 MIU kg 1), cholecalciferol (0.6 MIU kg 1), thiamin (3.60 g kg 1), riboflavin (7.20 g kg 1), pyridoxine (6.60 g kg 1), cyanocobalamine (0.02 g kg 1), a-tocopherol (16.50 g kg 1), menadione (2.40 g kg 1), niacin (14.40 g kg 1), pantothenic acid (4.00 g kg 1), biotin (0.02 g kg 1), folic acid (1.20 g kg 1), inositol (30.00 g kg 1), Stay C (100.00 g kg ). Mineral mix: P (12.00%), Ca (12.00%), Mg (1.50%), Fe (0.15%), Zn (0.42%), Cu (0.21%), K (7.50%), Ge (0.0001%), Co (0.011%), Mn (0.160%), Se (0.001%), Mo (0.0005%), Al (0.0025%), I (0.04%), B (0.0001%), Ni (0.0001%).
348 nm and em 450 nm. Reaction reagents used were o-pthalaldehyde/sodium carbonate buffer and sodium hypochlorite. The HPLC system consisted of the following: pump LC-3A; step gradient unit, SGR-1A; oven CTO-2A; fluorescence detector, FLD; and chromatopac, C-RIB. The following buffers were used: 0.07 M sodium citrate (pH 3.19); 0.07 M sodium citrate (pH 3.80); 0.20 M sodium citrate (pH 8.48); and 0.50 M sodium hydroxide. 2.4. Feeding experiment Twenty abalone juveniles were stocked in 12 60-l fiberglass tanks containing 40-l of filtered seawater. Shelters of two halved polyvinyl chloride pipes (65 mm diameter) were Table 2 Proximate composition (%) of diets Diet number
Crude protein Crude fat NFEa Estimated metabolizable energy(kcal/kg)b a
1
2
3
4
27.84 3.13 43.55 3137
27.30 3.27 42.30 3078
27.72 2.81 43.68 3109
26.97 3.04 42.62 3057
NFE—nitrogen free extract, given by difference. Computed based on standard physiological fuel values of 9 kcal/g lipid and 4 kcal/g protein and carbohydrate (Brett and Groves, 1979). b
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provided in each tank. Four dietary treatments with three replications were randomly allocated to different tanks. A flow-through water system was maintained at approximately 300 –350 l/h with gentle aeration provided. Excess feed that settled on the tank bottom was siphoned out, dried and weighed to get the true value of the feed consumed by the abalone. Water temperatures ranged from 28 to 31 jC and salinity varied from 28xto 32x . Ranges of dissolved oxygen, nitrite, ammonia, and pH in the rearing tanks were at 5.0 –5.6 mg/l; 0 – 0.05 mg/l; 0– 0.40 and 8.3 – 8.4, respectively. Diets were fed to abalone at 2 –5% of their body weight once in the late afternoon at about 1600 h for 90 days. Shell length increase (SL), weight gain (WG), survival (SURV), and feed conversion ratio (FCR) were determined every 20 days until the 84th day. Water stability of the diets was conducted according to the method of Hastings et al. (1971). Diet stability was determined at 6, 12, and 24 h interval. Percent water stability was computed as: %water stability ¼ ðB AÞð%dry matterÞ=Að%dry matterÞ 100 where B is the final weight of feed and A is the initial weight of feed. 2.5. Statistical analysis All data were analyzed by one-way analysis of variance (ANOVA) and Duncan’s Multiple Range Test on the SAS Package for the IBM-PC (SAS Institute, 1988). Differences among treatment means were considered significant at P < 0.05.
3. Results At the end of the 84-day feeding trial, the highest weight gain (453.8%) was attained with abalone fed diet 2 with protein sources coming from a combination of FM, SM, and DSM. This value was, however, not significantly different from those fed diets 4 (420.6%) with protein sources from FM, DSM, SP and the control diet (400%) containing protein sources from FM, SM, and DSM. Abalone fed diet 3, which contained DSM and SP, had significantly lower weight gain of 326.5%. Similarly, the specific growth rates and shell Table 3 Initial and final weight, shell length, and SGR of abalone fed formulated diets for 84 daysa Treatment
Initial weight (average, g)
Initial SL (average, mm)
Final weight (average, g)
Final SL (average, mm)
Weight gain (%)b
SGRc
1 2 3 4
0.73 F 0.08 0.65 F 0.03 0.68 F 0.03 0.68 F 0.01
11.2 F 0.27 11.5 F 0.48 11.5 F 0.28 11.4 F 0.37
3.65 F 0.19 3.60 F 0.10 2.90 F 0.28 3.54 F 0.17
35.4 F 0.31 34.5 F 0.58 33.2 F 0.91 35.4 F 0.86
400.0 F 49.8a 453.8 F 42.8a 326.5 F 392b 420.6 F 20.6a
0.83 F 0.09a 0.89 F 0.02a 0.75 F 0.07b 0.86 F 0.03a
a Means of three replicate groups with the same superscript in each column are not significantly different ( P < 0.05). b Weight gain: [(final weight initial weight)/initial weight] 100. c SGR (specific growth rate) = 100 (ln average final weight ln average initial weight)/number of days.
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Table 4 Shell length increase, total feed intake, FCR, percent survival of abalone fed formulated diets for 84 days and percent water stability of pellets Treatment
Shell length increase (%)
Total feed intake (g)
FCRa
PERb
SURV (%)
Percent water stability of pellets (24 h)c
1 2 3 4
216 F 2.45a 200 F 3.52a 188 F 2.12b 210 F 2.38a
365.36 391.68 285.06 329.16
1.01 1.00 0.97 0.86
3.90 4.17 4.14 4.58
95 95 85 85
64 63 65 65
Means of replicate groups F S.E.M. with the same superscript in each column are not significantly different ( P < 0.05). a FCR = dry weight of feed (g)/wet weight gain (g). b PER = wet weight gain/protein intake. c %Water stability=(final dry weight of feed/initial dry weight %dry matter)/100.
length increase of abalone fed diets 1, 2, and 4 were significantly higher compared to those fed diet 3 (Table 3). No significant differences were observed in the efficiency of protein (PER) and feed conversion (FCR) for all treatments (Table 4). Survival was generally high, ranging from 85% to 95%. All diets prepared were found to be water stable retaining at 64% of dry mass after the 24 h test for stability (Table 4). Amino acid composition of diets 1, 2, and 4 had profiles that were very similar to that of abalone tissue. Diet 3, which contained all plant – protein sources, showed some lower level of methionine (Table 5) as compared to other diets.
Table 5 Amino acid composition of formulated diets and abalone meat (g/100 g protein)a Amino acid
Alanine Arginine Aspartic Cystine Glutamic Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine a
Diet number
Abalone tissue
1
2
3
4
2.68 5.28 7.05 0.35 8.99 3.65 1.35 2.05 5.09 6.14 4.40 2.42 2.27 1.74 3.60 0.98 2.34 4.26
2.79 5.12 6.11 0.59 8.10 2.70 1.25 1.97 5.60 5.99 4.32 2.72 2.12 1.10 3.59 0.65 2.29 4.28
2.38 5.09 6.07 0.36 8.72 2.99 1.24 1.86 5.17 5.78 2.97 2.18 2.55 1.21 2.79 0.43 1.52 3.41
2.40 5.22 7.08 0.11 7.98 3.15 1.38 2.35 5.42 5.22 4.42 2.35 2.70 1.42 3.38 1.01 2.05 4.48
Means of two replicate samples.
3.72 5.48 6.23 0.42 9.56 3.71 1.78 2.51 5.95 5.90 4.09 3.29 3.41 2.30 3.69 0.55 2.97 4.90
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4. Discussion Abalone fed formulated diets with protein sources from a combination of animal and plant protein origin such as fish meal, shrimp meal, defatted soybean meal, and Spirulina sp. showed better performance in terms of growth rate per day, weight gain, and increase in shell length compared to those fed diets with protein sources of plant origin alone. Since all diets were made isonitrogenous at 27%, the results would signify that the quality and physiological availability of protein in the combined animal and protein sources may have contributed significantly to the positive effect of these diets on the growth of abalone. The amino acid profile of the various diets tested (Table 5) showed some low levels of certain amino acids such as methionine in the formulated diet, which contained only the plant protein sources. The low level of this essential amino acid (methionine) in diet 3 with protein sources coming solely from plant origin might have contributed to the lower growth rate of abalone fed this diet. The use of plant protein source alone in a formulated diet may need some supplementation of crystalline amino acids or may be used in combination with other animal protein sources to compensate for the low levels of some essential amino acids such as methionine. Murai et al. (1986, 1989) reported that supplementing soybean meal diets with either coated and uncoated methionine significantly improved growth and feed efficiency of common carp fingerlings. Guzman and Viana (1998) have successfully demonstrated the effectivity of the use of a combination of soybean meal and abalone viscera silage as protein sources in the diet of abalone, H. fulgens. In the study conducted by Britz (1996), abalone, H. midae fed fish meal and Spirulina spp. based diets produced a significantly higher length increment and specific growth rate compared to those fed diets containing soya oil cake, torula yeast, and casein alone. Faster growth rate of abalone with formulated diets containing combination of plant and animal protein sources has also been documented in studies for other Haliotis species (Hahn, 1989; Morrison and Whitington, 1991; Nie, 1992). Another concern in the use of plant protein source is the presence of antinutritional factors, which if not removed or processed may likewise contribute to the low growth of fish. In this study, however, plant protein sources used were properly processed and were assumed not to contain any of these antinutritional factors. The higher growth rate obtained when abalone were fed diets containing animal protein sources in combination with plant protein sources may be attributed to the fact that some limiting amino acids in plant protein source may be compensated by the availability of that amino acid in the animal protein source, resulting in a better profile of the diet formulation and better growth for the abalone. The amino acid profile of a diet is at its best when it simulates that of the tissue muscle of the animal being fed. The profile of animal protein source like fish meal is considered to be an excellent one, which can support good growth performance used alone or in combination with other plant protein sources. Generally, animal protein sources like fish meal are highly digestible and contain attractants that make them an attractive ingredient for any fish diet. However, the high demand for fish meal may soon result in an unaffordable price and availability of good quality sources may also be a problem. It is best therefore that fish meal or any animal protein source be used in combination with plant protein source to compensate for the limiting amino acids of
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each other, thus may result in faster growth rate of the animal. Plant protein sources are cheaper but are generally deficient in some of the sulfur-containing amino acid such as methionine. Thus, these plant protein sources are best used in combination with animal protein sources. The values obtained for the feed conversion ratio for all dietary treatments were generally low ranging between 0.9 and 1.0 and were not significantly different ( P > 0.05). This would indicate that there was high efficiency of feed conversion to body weight in all treatments. The more efficient feed conversion but lower growth rate of abalone fed the all plant protein-based diet compared to those fed the fish meal based diet or the animal – plant combination diet can be explained perhaps by the lower feed consumption of abalone fed diet 3. However, this finding does not conform with the results of Uki et al. (1986) wherein they reported poorer growth and high FCR values for H. discus hannai fed fish meal based diets compared with casein-based diets. The source and processing of fish meals used in these two different studies probably may account for the difference in results. The ability of H. discus hannai and H. asinina to digest fish meal cannot be discounted. Viana et al. (1993), on the other hand, reported that fish meal fed abalone had similar growth rates and FCR values with those abalone fed casein as protein source. This is likewise in conformity with the work of Britz (1996), wherein abalone, H. midae fed fish meal and Spirulina-based diet exhibited good growth and high efficiency of feed conversion to body weight. The efficiency of protein utilization as evidenced by the PER values (4.0 – 4.6) was not significantly different for all treatments. This would indicate that the proteins from each diet were efficiently utilized by abalone regardless of the protein source. The lower growth rates obtained with abalone fed diets with plant origin as the sole protein sources might be due to lower levels of some essential nutrients that could not have been sufficient for deposition of tissue muscle. Although the essential amino acid content was used as a determining factor for the nutritive value of the different protein sources, further work has to be done to test the availability of these amino acids to abalone. It has been reported that apparent amino acid availability and true amino acid availability vary within and among the various protein sources (Wilson, 1989).
Acknowledgements The authors would like to thank Ms. Mae F. Mallare and Mr. Narciso Entusiasmo for their assistance in the conduct of the study, Ms. Florence Jarder for the proximate analyses and Dr. O.M. Millamena for the review of the manuscript.
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