30 reproductive performance of hatchery bred donkeys ear abalone, haliotis asinina, linne, fed natural and diets bautista teruel2001

Aquaculture Research, 2001, 32 (Suppl 1), 249±254 Reproductive performance of hatchery-bred donkey's ear abalone, Haliotis asinina, Linne, fed natural and arti®cial diets M N Bautista-Teruel, O M Millamena & A C Fermin Aquaculture Department, South-east Asian Fisheries Development Center, Tigbauan, Iloilo, Philippines Correspondence: Myrna N Bautista-Teruel, Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Iloilo, Philippines, 5021 Tel +63-33 336-2965 Fax: +63-33 335-1008 E-mail: mbt@aqd.seafdec.org.ph Abstract Introduction Hatchery-bred donkey's ear abalone, Haliotis asinina, Linne broodstock were given diets consisting of natural food, seaweed (SW), Gracilariopsis bailinae, D1; combination of SW and arti®cial diet (AD), D2; and AD alone, D3 Equal numbers of : female and male abalone were stocked in 24 units, 60 L tanks with eight replicate tanks per dietary treatment Reproductive performance, e.g number of spawnings, instantaneous fecundity and egg hatching rates, was monitored over 270 days The mean number of spawnings was not signi®cantly different among treatments The mean instantaneous fecundity and percent hatching rates were signi®cantly higher in abalone fed D2 or D3 compared to those given D1 Survival of abalone broodstock fed D1 was, however, signi®cantly higher at 88% than those fed either D2 or D3 at 75% Fatty acid analysis showed that the n-3/n-6 fatty acid ratios of abalone hepatopancreas re¯ected those of their diets Mature abalone ovary had n-3/n-6 fatty acid ratio of 1.3 A higher amount of essential nutrients in the arti®cial diet such as protein, lipid and the highly unsaturated fatty acids, e.g 20 : 4n-6, 20 : 5n-3, 22 : 6n-3 in abalone fed D2 or D3, may have in¯uenced the increased reproductive performance The decreasing commercial catch, and the high price that abalone command in both the domestic and export markets, have stimulated great interest in the development of its aquaculture (FAO 1998) A major constraint to development of abalone aquaculture is the declining supply of seed from the wild due to overexploitation and habitat destruction (Jarayabhand & Paphavasit 1996) Although spontaneous spawning of abalone is reported to occur year-round (Singhagraiwan & Doi 1992; Singhagraiwan & Doi 1993; Capinpin, Encena & Bayona 1998), production of good quality larvae is very inconsistent Thus, there is a need to develop a reliable technique for abalone broodstock development through dietary manipulation Nutrition of broodstock has a profound effect on gonadal maturation and fecundity (Watanabe 1988), and plays a major role in reproductive success in both ®sh and crustaceans (Yu, Sinnhuber, & Hendricks 1979; Takeuchi, Ishii, & Ogino 1981; Teshima & Kanazawa 1983; Watanabe, Itoh, Murakami, Tukashima, Kitajima, & Fujita 1984) There are no published studies on the in¯uence of nutrition on the reproductive performance of abalone broodstock, despite their importance in abalone aquaculture Thus, this study was undertaken to investigate the effects of natural and arti®cial diets on reproduction of the donkey's ear abalone, Haliotis asinina, Linne Keywords: broodstock, nutrition, abalone, Haliotis asinina ã 2001 Blackwell Science Ltd 249 Reproductive performance of abalone M N Bautista-Teruel et al Aquaculture Research, 2001, 32 (Suppl 1), 249±254 Table Percentage composition of arti®cial abalone broodstock diet (g kg±1diet) Ingredients Amount (g kg±1 diet) Fish meal Shrimp meal Defatted soybean meal Rice bran Wheat ¯our Seaweed (Gracilaria sp.) (powdered) Tuna oil Soybean oil Vitamin mix* Mineral mix** Dicalcium phosphate Butylatedhydroxy toluene 100 100 200 119.5 200 170 5 30 40 30 0.5 Crude protein Crude fat Nitrogen-free extract Estimated energy (kcal kg±1)2 Natural diet1 Arti®cial diet 17.56 0.48 35.38 2161 25.40 4.38 47.59 3315 Seaweed, Gracilariopsis bailinae Computed based on standard physiological fuel values of kcal g±1 lipid and kcal g±1 protein and carbohydrates (Brett & Groves 1979) *Vitamin and mineral mixes, commercial brand Vitamin A (3.0 M.I.U kg±1), Vitamin D3 (0.6 M.I.U kg±1), Vitamin B1 (3.60 g kg±1), Vitamin B2 (7.20 g kg±1), Vitamin B6 (6.60 g kg±1), Vitamin B12 (0.02 g kg±1), Vitamin E (16.50 g kg±1), Vitamin K3 (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), Stable C (100.00 g kg±1) **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%) Materials and Methods Experimental diets Dietary treatments consisted of a natural diet, seaweed (Gracilariopsis bailinae) (D1), combination of natural and arti®cial diets (D2), and arti®cial diet (D3) Seaweed was given every days, ad libitum The combination diet was given alternately each day Arti®cial diet was a modi®cation of a previous formulation of Bautista-Teruel & Millamena (1999) in that the amount of some ingredients, e.g shrimp meal, tuna and soybean oils, was reduced while adding more of the others, e.g rice bran, seaweed The composition and proximate composition of the natural and arti®cial diets are shown in Tables and 2, respectively Treatments were arranged in a completely randomized experimental design Abalone broodstock fed diets D2 and D3 were initially fed D1 and gradually switched to the arti®cial diet by the second week of culture Formulated diet was given at 3±5% of broodstock 250 Table Proximate composition (%) of natural and arti®cial diet for broodstock abalone, Haliotis asinina, Linne biomass once daily at 16.00±17.00 hours Excess diet was removed and the feeding rate was adjusted based on weight gain after each sampling, which was done every weeks Mortalities were recorded daily Abalone culture and rearing system Experiments were conducted at the wet laboratory of the SEAFDEC Aquaculture Department in Iloilo, the Philippines Abalone, Haliotis asinina, adults with initial mean body weight of 24.5 T 0.12 g and shell length of 48.5 T 0.68 mm were obtained from the mollusc hatchery at SEAFDEC, Tigbauan, Iloilo Gonadal maturity of the female abalone was judged according to the criteria developed by Singhagraiwan & Doi (1993) Abalone having immature gonads were used in the study Forty-eight male and female abalone were stocked at a : ratio into 24 units, 60-L ®breglass tanks with eight replicate tanks per dietary treatment Halved PVC pipes were provided in each tank as shelter for abalone Sand-®ltered seawater was supplied in a ¯ow-through system equipped with adequate aeration Tanks were cleaned of faeces and excess diet whenever necessary Abalone broodstock were checked for spawning each day in the early morning Eggs were siphoned and counted in 40-L ®breglass tanks located just under the spawning tanks Instantaneous fecundity was computed as the total number of spawned eggs per g body weight of abalone Percent hatching rate was computed as the total number of larvae divided by the total number of spawned eggs multiplied by 100 ã 2001 Blackwell Science Ltd, Aquaculture Research, 32 (Suppl 1), 249±254 Aquaculture Research, 2001, 32 (Suppl 1), 249±254 Reproductive performance of abalone M N Bautista-Teruel et al Table Reproductive performance of abalone, Haliotis asinina, Linne, broodstock fed natural diet alone, combination of natural and arti®cial diets, and arti®cial diet alone Treatment1 Parameter D1 Mean number of spawnings Mean instantaneous fecundity Mean hatching rate (%) Broodstock survival (%) Mean frequency of spawning/month Mean spawning time interval (days) 8.0 T 0.6a 4077 T 67.8 39.49 T 8.2a 88 T 7.6a 1±2 19 T 1.8 D2 a 9.0 5478 46.22 75 1±2 17 D3 T T T T 1.8a 45.8b 7.3b 12.5b T 1.3 9.5 5050 45.65 75 1±2 16 T T T T 1.2a 74.1b 11.2b 12.5b T 0.7 Treatment means with different superscripts are signi®cantly different (P < 0.005) D1 = Seaweed, Gracilariopsis bailinae (natural diet) (given alone) D2 = Seaweed (natural diet) + arti®cial diet given alternately D3 = arti®cial diet (given alone) Table Comparison of fatty acid composition (percentage of total lipid) of natural and arti®cial diets for abalone, Haliotis asinina, Linne, with mature ovary of the wild abalone Fatty acid Natural diet Arti®cial diet Mature ovary1 14 : 15 : 16 : 16 : 1n-7 18 : 18 : 1n-7 18 : 2n-6 18 : 3n-3 18 : 4n-3 20 : 4n-6 20 : 5n-3 20 : 5n-6 22 : 5n-3 22 : 6n-3 Total n-3 Total n-6 n-3/n-6 6.6 2.3 29.2 1.4 1.5 15.5 10.9 16.8 7.0 2.1 1.8 0.9 3.2 0.7 28.5 14.4 2.0 16.5 1.5 20.1 2.3 1.8 22.8 13.4 7.5 1.2 2.4 3.9 0.8 0.8 3.8 17.2 16.5 1.0 12.9 3.8 22.9 1.3 2.4 20.5 10.8 5.9 1.5 3.1 5.1 1.2 2.3 5.2 20.0 15.1 1.3 methods (AOAC 1995) Fatty acid composition of the diets and abalone muscle tissues were determined by gas-liquid chromatography (GLC) Total lipids were extracted using the method of Bligh & Dyer (1959) Methyl esters of constituent fatty acids were prepared by the saponi®cation-transesteri®cation method of Metcalfe, Schmitz, & Pelka (1966) Fatty acid composition of replicate samples was analysed using a Shimadzu GC 9A (Shimadzu Co., Tokyo, Japan) gas chromatograph with a ¯ame ionization detector (FID) ®tted with 15% diethylene glycol succinate column on chromosorb support The gas chromatograph was operated isothermally at 190 °C with a 30 mL min±1 ¯ow of nitrogen Fatty acids were identi®ed using authentic standards and published values for fatty acid marine oils (Ackman & Burgher 1965) and quanti®ed with an electronic integrator (Shimadzu Chromatopac CR-2AX) Results are presented as the weight percentage of total fatty acids Statistical analysis Water temperature (26±30 °C) and salinity (32± 35 g L±1) were monitored daily Ammonia nitrogen (0±0.08 mg L±1), nitrite nitrogen (0.0±0.17 mg L±1) and dissolved oxygen (4.6±7.1 mg L±1) were monitored weekly Water quality remained within ranges suitable for abalone culture in tanks Data were analysed using analysis of variance (Gomez & Gomez 1984) and Duncan's multiple range test (P ` 0.05) to test signi®cant differences among treatment means (Duncan 1955) Arcsin transformation of percentage data was done prior to statistical analysis Replicate tanks were considered units of observation for ANOVA testing Chemical analyses Results Natural and formulated diets were analysed for proximate composition according to standard The effects of the different diets on reproductive performance of broodstock abalone are shown in Mature ovary from wild abalone ã 2001 Blackwell Science Ltd, Aquaculture Research, 32 (Suppl 1), 249±254 251 Reproductive performance of abalone M N Bautista-Teruel et al Aquaculture Research, 2001, 32 (Suppl 1), 249±254 Table Fatty acid composition (percentage of total lipid) of hepatopancreas of abalone, Haliotis asinine, Linne, fed natural diet alone, combination of natural and arti®cial diets and arti®cial diet alone Hepatopancreas Discussion Fatty acids D1 D2 D3 14 : 15 : 16 : 16 : 1n-7 18 : 18 : 1n-7 18 : 2n-6 18 : 3n-3 18 : 4n-3 20 : 4n-6 20 : 5n-3 20 : 5n-6 22 : 5n-3 22 : 6n-3 Total n-3 Total n-6 n-3/n-6 9.3 4.1 21.2 5.8 5.7 17.9 9.4 13.5 5.3 2.1 1.8 0.8 2.8 0.7 24.1 12.3 2.0 12.2 7.2 22.1 1.9 6.4 19.2 10.5 8.3 0.9 3.2 2.8 0.6 1.2 2.5 15.7 14.3 1.1 14.0 6.3 23.2 2.2 9.2 18.6 8.9 5.2 1.2 2.4 3.7 0.7 0.9 3.1 14.1 12.0 1.2 D1 = Seaweed, Gracilariopsis bailinae (natural diet) (given alone) D2 = Seaweed (natural diet) + arti®cial diet given alternately D3 = arti®cial diet (given alone) Table Mean number of spawnings ranged from 8.0 T 0.6 to 9.5 T 1.2 and were not different among treatments (P > 0.05) Spawning occurred once or twice every weeks The time interval between spawnings varied from 15 to 35 days The mean instantaneous fecundity (5478 T 45.8, D2; 5050 T 74.1, D3) and percent hatching rates (46.22 T 7.3, D2; 45.65 T 11.2,D3) were higher (P < 0.05) in abalone fed D2 or D3 compared to those fed D1 (mean instantaneous fecundity = 4077 T 67.8; percent hatching rate = 39.49 T 8.2) Survival of abalone broodstock fed D1 was however, signi®cantly higher (88%) than those fed either D2 or D3 (75%) Table shows the fatty acid pro®les of the natural and arti®cial diets The natural diet had higher n-3/n-6 ratio (2.0) compared to arti®cial diet (1.0) Mature abalone ovary from the wild abalone had n-3/n-6 ratio of 1.3, whereas those of the hepatopancreas were more of a re¯ection of their diets (Table 5) Highly unsaturated fatty acids 252 (HUFA), e.g 20 : 4n-6, 20 : 5n-3, 22 : 6n-3, were present in relatively higher amount in the arti®cial diets as compared with those found in the natural diet The higher reproductive performance in terms of total number of spawnings, instantaneous fecundity and hatching rates of abalone Haliotis asinina fed the combination of natural and arti®cial diets or the arti®cial diet alone would suggest that the nutritional quality of the broodstock diet in¯uences reproduction Although it is possible to mature and spawn abalone with natural diet, the provision of an effective arti®cial diet fed alone or in combination with the natural diet can achieve better reproductive performance of the abalone broodstock This may result in the enhancement of production of quality seeds for abalone hatcheries Dietary nutrients, especially in terms of proteins, lipids, and fatty acids, e.g 20 : 4n-6; 20 : 5n-3, 22 : 6n-3, which are essential in reproduction and that are insuf®cient in the natural diet, may have been compensated by feeding the arti®cial diet as a supplement or as total food for the abalone Proteins that are associated with essential fatty acids and are usually mobilized to the gonads are considered important components for reproductive functions (Harrison 1991) Highly unsaturated fatty acids (HUFAs) are important nutrients for reproductive functions of ®sh and crustaceans (Millamena, Primavera, Pudadera & Caballero 1986; Millamena 1989; Millamena & Quinitio 2000) Watanabe, Takeuchi, Ogino & Kawabata (1977) have also demonstrated that essential fatty acids play as important a role in reproductive physiology in ®sh as in higher animals The results obtained in red sea bream fed essential fatty acidde®cient diets for months before spawning indicated that the total egg production and hatchability were signi®cantly in¯uenced by the level of these essential fatty acids in the diet (Watanabe et al 1984) The n-3/n-6 fatty acid ratios of the hepatopancreas re¯ected those of their diets at 2.0 (D1), 1.1 (D2) and 1.2 (D3) (Table 5) Cahu, Fauvel, & Aquacop (1986) and Millamena (1989) reported a correlation between dietary fatty acid composition, tissue fatty acid pattern and quality of eggs in shrimp, Penaeus monodon Dietary HUFAs and n-3/ n-6 ratio that simulate that of mature ovaries have ã 2001 Blackwell Science Ltd, Aquaculture Research, 32 (Suppl 1), 249±254 Aquaculture Research, 2001, 32 (Suppl 1), 249±254 Reproductive performance of abalone M N Bautista-Teruel et al been reported to improve reproductive performance and hatchability of eggs in the tiger shrimp (Millamena 1989) This could partly explain why better reproductive performance was obtained with the combination of natural and arti®cial diet or arti®cial diet given solely The type of macroalgae consumed can offer different proportions of nutritionally important PUFA (Dunstan, Baillie, Barrett, & Volkman 1996; Mai, Mercer, & Donlon 1996) In this experiment, the amount of PUFA provided by the natural diet, which is the seaweed Gracilariopsis bailinae, may not have been as suf®cient as the one provided in the arti®cial diet The essential nutrients such as proteins, lipids, and fatty acids, which are limiting in the natural diet may have been compensated by the amounts present in the arti®cial diet, thus better reproductive performance resulted in abalone fed D2 or D3 The lower survival of the abalone fed either D2 or D3 may indicate that more spawnings that were undergone by the abalone fed these diets might have contributed to more stressful activity, thus leading to higher mortalities Feeding abalone broodstock with suitable arti®cial diet may enhance their reproductive performance Acknowledgements The authors wish to acknowledge the assistance of Ms M Mallare and Mr N Entusiasmo in the conduct of the study; Ms F Jarder for the proximate analysis; Ms A Asutilla for the water analysis; Ms I Borlongan for the use of the gas chromatograph; and Mr R Sanares for the statistical analysis of the data The Organizing Committee of The Ninth International Symposium on Nutrition and Feeding in Fish through the Chairman, Dr T Watanabe, and South-east Asian Fisheries Development Center through the Chief, Dr R Platon, are likewise gratefully acknowledged for funding support References Ackman K.G & Burgher R.G (1965) Cod liver oil fatty acids as secondary reference standards in GLC of polyunsaturated fatty acids of animal origin: analysis of the dermal oil of the Atlantic leatherback turtle Journal of the American Oil Chemists' Society 42, 38±42 AOAC (1995) In: Of®cial Methods of Analysis of the Association of Of®cial Analytical Chemists 16th edn (ed by S Williams) Association of Of®cial Analytical Chemists, Arlington, VA Bautista-Teruel M.N & Millamena O.M (1999) Diet development and evaluation for juvenile abalone, Haliotis asinina: protein/energy levels Aquaculture 178, 117±126 Bligh E.G & Dyer W.J (1959) A rapid method of total lipid extraction and puri®cation Canadian Journal of Biochemistry and Physiology 37, 912±917 Brett J.R & Groves T.D (1979) Physiological energetics In: Fish Physiology, Vol (ed, by W.S Hoar, & D.J.Randall), pp 599±675 Academic Press, New York Cahu C & Fauvel C (1986) Effect of Food Fatty Acid Composition of Penaeus vannamei Broodstock on Egg Quality Council Meeting of the International Council for the Exploration of the Sea (ICES), Copenhagen, Denmark 8pp Capinpin E., Encena V & Bayona N (1998) Studies on the reproductive biology of donkey's ear abalone, Haliotis asinina, Linne Aquaculture 166, 141±150 Duncan D.B (1955) Multiple range and multiple F-tests Biometrics 11, 1±42 Dunstan G.A., Baillie H.J., Barrett S.M & Volkman J.K (1996) Effect of diet on the lipid composition of wild and cultured abalone Aquaculture 140, 115±127 FAO (1998) Yearbook of Fisheries Statistics, Vol 83 FAO Fisheries Series, no 51, 179 FAO, Rome Gomez K.A & Gomez A.A (1984) Statistical Procedures for Agricultural Research The International Rice Research Institute, Los Banos, Laguna, The Philippines Harrison K.E (1991) Crustacean Reproduction Nutrition In: The Crustacean Nutrition Newsletter World Aquaculture Society International Working Group on Crustacean Nutrition (ed by J D Castell & K.E Corpron), Halifax, N.S., Canada, pp 62±70 Jarayabhand P & Paphavasit N (1996) A review of the culture of tropical abalone with special reference to Thailand Aquaculture 140, 159±168 Mai K., Mercer J.P & Donlon J (1996) Comparative studies on the nutrition of two species of abalone, Haliotis tuberculata L and Haliotis discus hannai Ino V The role of polyunsaturated fatty acids of macroalgae in abalone nutrition Aquaculture 139, 77±89 Metcalfe L.D., Schmitz A.A & Pelka J.R (1966) Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis Analytical Chemistry 38, 514 Millamena O.M (1989) Effect of fatty acid composition of broodstock diet on tissue fatty acid patterns and egg fertilization and hatching in pond-reared Penaeus monodon Fabricius broodstock Asian Fisheries Science 22, 127±134 Millamena O.M., Primavera J.H., Pudadera R.A & Caballero R.V (1986) The effect of diet on the reproductive performance of pond-reared Penaeus monodon Fabricius broodstock The First Asian Fisheries Forum, 1, 593±596 ã 2001 Blackwell Science Ltd, Aquaculture Research, 32 (Suppl 1), 249±254 253 Reproductive performance of abalone M N Bautista-Teruel et al Aquaculture Research, 2001, 32 (Suppl 1), 249±254 Millamena O.M & Quinitio E (2000) The effects of diets on reproductive performance of eyestalk ablated and intact mud crab Scylla serrata Aquaculture 181, 81±90 Singhagraiwan T & Doi M (1992) Spawning pattern and fecundity of the donkey's ear abalone, Haliotis asinina, Linne, observed in captivity Thai Marine Fisheries Research Bulletin 3, 61±69 Singhagraiwan T & Doi M (1993) Seed Production and Culture of Tropical Abalone Haliotis asinina, Linne The Research Project of Fishery Resource Development in the Kingdom of Thailand, The Eastern Marine Fisheries Development Center, Department of Fisheries, Ministry of Agriculture and Cooperatives, Thailand Takeuchi M., Ishii S & Ogino T (1981) Effect of dietary vitamin E on growth, vitamin E distribution, and mortalities of the fertilized eggs and fry in Ayu, Plecoglossus altivelis Bulletin of the Tokai Regional Fish Laboratory 104, 111±122 Teshima S & Kanazawa A (1983) Variation in lipid composition during the ovarian maturation of the prawn Bulletin of the Japanese Society of Scienti®c Fishery 49, 957±962 Watanabe T (1988) Fish Nutrition and Mariculture The 254 General Aquaculture Course Kanagawa International Fisheries Training Center, JICA, Kanagawa Prefecture, Kanagawa, Japan Watanabe T., Itoh A., Murakami A., Tukashima Y., Kitajima C & Fujita S (1984) Effects of nutritional quality of diets given to broodstock on the verge of spawning on reproduction of red sea bream Bulletin of the Japanese Society of Scienti®c Fishery 50, 1023± 1028 Watanabe T., Ohhashi S., Itoh A., Kitajima C & Fujita S (1984) Effects of nutritional composition of diets on chemical components of red sea bream broodstock and eggs produced Bulletin of the Japanese Society of Scienti®c Fishery 50, 503±515 Watanabe T., Takeuchi T., Ogino C & Kawabata T (1977) Effect of alpha-tocopherol de®ciency on carp VII The relationship between dietary levels of linoleate and alpha-tocopherol requirement Bulletin of the Japanese Society of Scienti®c Fishery 43, 935±946 Yu T.C., Sinnhuber R.D & Hendricks J.D (1979) Reproduction and survival of rainbow trout fed linolenic acid as the only source of essential fatty acids Lipids 14, 572±575 ã 2001 Blackwell Science Ltd, Aquaculture Research, 32 (Suppl 1), 249±254 ... Aquaculture Research, 2001, 32 (Suppl 1), 249±254 Reproductive performance of abalone M N Bautista-Teruel et al Table Reproductive performance of abalone, Haliotis asinina, Linne, broodstock fed natural. .. Research, 2001, 32 (Suppl 1), 249±254 Table Fatty acid composition (percentage of total lipid) of hepatopancreas of abalone, Haliotis asinine, Linne, fed natural diet alone, combination of natural. .. composition (percentage of total lipid) of natural and arti®cial diets for abalone, Haliotis asinina, Linne, with mature ovary of the wild abalone Fatty acid Natural diet Arti®cial diet Mature ovary1