Aquaculture research, tập 42, số 7, 2011

Aquaculture Research, 2011, 42, 899^905 doi:10.1111/j.1365-2109.2010.02626.x Effects of ration level on growth, nitrogenous excretion and energy budget of juvenile yellow catfish, Pelteobagrus fulvidraco (Richardson) Lei Zhang1, Zhigang Zhao2, Dongmei Xiong1,Wei Fang1, Bo Li1, Qixue Fan1, Kai Yang1 & Xiaoyin Wang3 College of Fisheries, Huazhong Agricultural University,Wuhan, Hubei, China The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, Shandong, China College of Science, Huazhong Agricultural University,Wuhan, Hubei, China Correspondence: Qixue Fan, College of Fishery, Huazhong Agricultural University, Wuhan, Hubei, 430070, China E-mail: fanqixue@ mail.hzau.edu.cn; Xiaoyin Wang, College of Science, Huazhong Agricultural University, Wuhan, Hubei, 430070, China E-mail: wxywxq@163.com Abstract Growth, nitrogenous excretion and energy budget of juvenile yellow cat¢sh, Pelteobagrus fulvidraco (initial body weight 1.17 Æ 0.28 g) at various levels (50%, 60%, 70%, 80%, 90% and 100% satiation per day) were investigated with feeding diet containing 40% protein Speci¢c growth rate of yellow cat¢sh increased (2.79^3.34% day) signi¢cantly (Po0.05) with ration level (RL) increasing Feed conversion e⁄ciency, feed protein retention e⁄ciency and feed energy retention e⁄ciency increased with the increase in RL, peaked at 70% of satiation, and then decreased at higher ration, with the ranges of 78.97^97.28%, 31.31^37.93% and 26.55^31.88% respectively Both nitrogenous excretion (u, mg g À day À 1) and faecal production (f, mg g À day À 1) increased signi¢cantly with the increased RL, and ranged between 0.94^ 1.38 and 0.69^1.24 mg g À day À respectively Apparent digestibility coe⁄cients in dry matter, protein, energy decreased signi¢cantly as ration increased, with ranges of 54.42^69.64%, 78.24^89.90% and 69.66^82.07% respectively Energy budgets of juvenile yellow cat¢sh at satiation RL was: 100C 30F18U133R129G or 100A 54R146G Keywords: ration level, growth, nitrogenous excretion, energy budget, yellow cat¢sh Introduction Yellow cat¢sh, Pelteobagrus fulvidraco (Richardson) is a teleost ¢sh belonging to Siluriformes, Bagride, Pel- r 2011 Blackwell Publishing Ltd teobagrus, is restricted to freshwater habitats, found mostly in the east of Asia It is one of the favourite food ¢sh in China because of its palatability and nutritional quality of its £esh and its high economic importance It has become one of the most important freshwater aquaculture species in South China (Han, Jia, Xia, Mao & Wang 2003; Jiang, Song,Ye, Cai, Yang & Huang 2004; Lee & Lee 2005) In addition, the products from yellow cat¢sh are available in overseas markets in recent years With the increasing stock of cultured yellow cat¢sh, the large numbers of juveniles provided for large-scale aquaculture are mainly from arti¢cial breeding In the arti¢cial breeding of yellow cat¢sh, especially during the period of using arti¢cially formulated feed, one key issue is how to optimize ration to support fast growth and maximize feed conversion and to reduce feed waste and water pollution Suitable ration not only enhanced protein synthesis and energy allocation for improving ¢sh growth and feed conversion e⁄ciency (FCE) but also minimized aquaculture pollution and production cost A great deal of past research work have published on dealing with the determination of optimum ration level (RL) of different ¢sh species which has recently been reported by Ahmed (2010) Thus, it is necessary to investigate the e¡ects of RL on yellow cat¢sh growth, nitrogenous excretion, faecal production and energy budget Although many aspects of yellow cat¢sh have been investigated in the past, including biology (Liu 1997), culture technology (Han et al 2003), breeding (Zhang, Luo & Li 2000; Pan, Ding, Ge,Yan, Hao, Chen & Huang 2008) and nutrition (Jiang et al 2004; Lee & 899 Growth, nitrogenous excretion and energy budget of yellow cat¢sh L Zhang et al Lee 2005); however, little is known about the bioenergetics of this species (Yang & Yao 2006), therefore, the present investigation was undertaken to work out the various parameters such as growth, nitrogenous excretion, faecal production and energy budget of juvenile yellow cat¢sh in relation to ration Materials and methods Experimental ¢sh Juvenile yellow cat¢sh, P fulvidraco for the experiment were obtained from the arti¢cial breeding by the research group at the National Research Center of Freshwater Fisheries Engineering (Wuhan, China) Fish with the same batch and in apparent good health were collected from breeding ponds About 4000 ¢sh were transferred into eight indoor concrete ponds with about 500 ¢sh per pond (2.1m  1.6 m  1.1m, water volume 3.0 m3) and acclimated for weeks During this period, aeration was provided continuously except for the feeding time, dissolved oxygen was maintained above mg L À 1, water temperature was 28.5 Æ 1.3 1C, and ¢sh were subjected to a natural photoperiod regime (20 June 2008^17 July 2008) During feeding, the ¢sh were fed with test diet in the form of pellets (Table 1) The acclimated ¢sh of similar body size and showing normal feeding behaviour were collected from the concrete ponds, and then randomly stocked into a circular ¢breglass tanks (80 cm in diameter, water volume 300 L) for further acclimation for week A preliminary feed trial was conducted to estimate the maximal ration during this period, which would provide experimental evidence for designing the RLs used in the growth experiment During the acclimation period, enough pelleted feed (Table 1) was provided to satiation as judged by visual inspection twice a day at 07:00 and 18:00 hours, respectively, as described earlier (Wang, Xie & Ma 2001) Experimental diet The experimental diet contains 40% crude protein and gross energy value of 16.70 kJ g À Chromium (III)oxide was added as an inert indicator for digestibility determinations Formulation and chemical composition of the diet are shown in Table The diet was made into 1^2 mm pellets using a pellet press, oven-dried at 60 1C and stored at 1C for use 900 Aquaculture Research, 2011, 42, 899–905 Table Formulation (percentage of wet weight) and chemical composition of the experimental diet Contents (g 100 g À 1) Ingredients White fishmeal (Peru)à Fish oil a-starch Soybean mealw Wheat flour Soybean oil Immune polysaccharidez Mineral premix‰ Vitamin premixz Vitamin Ck Choline chloride Chromic oxide Chemical composition (% dry matter) Dry matter Crude protein Crude lipid Ash Gross energy (kJ g À 1) 35.00 1.00 5.00 36.00 17.00 1.00 0.10 3.40 0.30 0.10 0.10 1.00 94.38 39.88 4.49 7.12 16.70 ÃWhite ¢shmeal is made of cod by-product from Peru wSoybean meal as a by-product was obtained after soaked-oil soybean without squeeze zImmune polysaccharide was from the Bide Biotechnology Co Ltd (Guangdong, China) ‰Han et al (2004), mineral premix (mg kg À diet): NaCl, 500; MgSO4 Á 7H2O, 7500; NaH2PO4 Á 2H2O, 12500; KH2PO4, 16 000; Ca(H2PO4)2 Á H2O, 100 000; FeSO4, 1250; C6H10CaO6 Á 5H2O, 1750; ZnSO4 Á 7H2O, 176.5; MnSO4 Á 4H2O, 81; CuSO4 Á 5H2O, 15.5; CoSO4 Á 6H2O, 0.5; KI, 1.5; starch, 22.5 zHan et al (2004), vitamin premix (mg kg À diet): thiamine, 20; ribo£avin, 20; pyridoxine, 20; cyanocobalamine, 2; folic acid, 5; calcium pantothenate, 50; inositol, 100; niacin, 100; biotin, 5; starch, 3226; ascorbic acid, 111; Vitamin A, 110; Vitamin D3, 20; Vitamin E (DL -a-tocopherol acetate), 100; Vitamin K3 (menadione sodium bisulphite), 10 kCoated vitamin C was from the Sunhy Biology Company (Wuhan, China) Growth experiment Six RLs were tested in the growth experiment: 50%, 60%, 70%, 80%, 90% and 100% of satiation, with triplicates for each RL containing 50 ¢sh for each group Fish in the control group were hand-fed to apparent satiation, 100% of satiation The daily feed supplied was recorded, and the uneaten feed was collected after active feeding for 40 by pipetting and then oven-dried at 70 1C Potential loss of uneaten feed was determined by placing feed in water for 40 and then collecting, drying and weighing The proportion of the feed remaining was calculated and this value was used to adjust the amount of feed intake Ration levels of ¢sh in the other ¢ve experimental r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 899^905 Aquaculture Research, 2011, 42, 899^905 Growth, nitrogenous excretion and energy budget of yellow cat¢sh L Zhang et al groups was determined based on the average feed consumption of ¢sh in the control group The trial was conducted in a recirculatory system with running water consisting of 18 circular ¢breglass tanks (80 cm in diameter, 300 L, water exchange rate 6.0 L À 1) Fifteen to 20% water in the system was replaced daily Nine hundred ¢sh which had been starved for1day were captured, blotted of excess water and was individually weighed, then were placed into individual experimental tanks at the start of the growth experiment During the experiment, ¢sh (mean initial body weight 1.17 Æ 0.28 g) in each tank were subjected to the six prescribed RLs ranging from 50% of satiation to 100% of satiation twice a day at 07:00 and 18:00 hours respectively Faeces were collected twice a day (09:00 and 20:00 hours) throughout the whole experimental period by pipetting, oven-dried at 70 1C, weighed, homogenized and stored at À 20 1C for biochemical analysis In addition, another 180 ¢sh were sampled for measurement of initial body composition and energy content Ammonia-N and urea-N excretion were measured once every10 days During the measurement (ammonia of 0.31^2.28 mg L À and urea of 0.07^ 0.52 mg L À 1), the water £ow was stopped for 24 h (Xie, Cui,Yang & Liu 1997).Water was sampled before and after this period The ammonia-N and urea-N were determined using the method of Chaney and Marbach (1962) and converted into energy by multiplying with energy factors 24.83 kJ g À for ammonia and 23.03 kJ g À for urea (Elliott 1976) Water temperature, dissolved oxygen and pH were monitored on daily basis Water temperature was 28.6 Æ 1.6 1C, dissolved oxygen was maintained above 6.5 mg L À and pH was 7.1^7.6, and the experiment was conducted at the natural photoperiod conditions with similar light intensity for all tanks during this period The growth experiment lasted 60 days All ¢sh were weighed after day of starvation and 10 ¢sh from each tank were sampled randomly for biochemical analysis Chemical analysis Dry matter content of diet and initial and ¢nal ¢sh samples were determined by oven-drying at 105 1C Nitrogen contents of ¢sh, diet and faeces were analyzed using the Kjeldahl method; and crude protein contents were calculated (N-Kjeldahl  6.25) The contents of crude lipid were measured for ¢sh and diet by ether extraction (34.6 BP) Ash content of ¢sh and diet were measured by combustion at 550 1C in mu¥e furnace Gross energy contents of ¢sh, faeces and diet were measured by oxygen bomb calorimeter (Phillipson microbomb calorimeter, Gentry Instruments, Aiken, SC, USA) The contents of Cr2O3 for the diet and faeces were determined as described using the method of Bolin (Bolin, King & Klosterman 1952) Triplicate samples were measured for each variable and the mean of triplicate determination was taken as the result when the relative deviation was o2% Statistical analysis Statistical analyses were performed using STATISTICA 6.0 for windows A one-way ANOVA followed by Duncan’s multiple range tests was used for six RLs to determine the signi¢cant di¡erences (Po0.05) among the treatments Kolmogorov^Smirnov and Levene’s test were used to test the homogeneity and normality, and the arcsine transformation was used to the data when they were in percentage Data were expressed as mean Æ SE of triplicates Results Speci¢c growth rate (SGR) of juvenile yellow cat¢sh increased signi¢cantly (Po0.05) with the increase in RLs, and ranged between 2.79% and 3.34% day (Table 2), but there were no signi¢cant di¡erences (P40.05) among 70^100% of satiation ration Feed conversion e⁄ciency (FCE), feed protein retention ef¢ciency (PRE) and feed energy retention e⁄ciency (ERE) of ¢sh increased with RL, and peaked at 70% of satiation, and then decreased at higher ration, and ranged between 78.97^97.28%, 31.31^37.93% and 26.55^31.88% respectively The contents of dry matter, protein, lipid, ash and energy in the body of juvenile yellow cat¢sh at di¡erent RLs at the beginning and end of growth experiment are shown in Table 3.With the increase in RLs, dry matter, lipid and energy contents of ¢sh increased, whereas ash contents of ¢sh declined The contents of dry matter and energy at 100% of satiation ration were signi¢cantly (Po0.05) higher than those fed at other RLs The protein content was not signi¢cantly (P40.05) Both nitrogenous excretion and faecal production increased signi¢cantly (Po0.05) and ranged between the increased RLs, with ranges of 0.94^1.38 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 899^905 901 Aquaculture Research, 2011, 42, 899–905 Growth, nitrogenous excretion and energy budget of yellow cat¢sh L Zhang et al Table Growth, survival and conversion e⁄ciency of Pelteobagrus fulvidraco fed di¡erent ration levels (RL)à RL 50 Feeding rate Initial body weight Final body weight SGR FCE PRE ERE Survival 2.27 1.16 6.17 2.79 94.28 37.93 26.55 97.33 60 Æ Æ Æ Æ Æ Æ Æ Æ 0.02a 0.06 0.25a 0.11a 2.59bc 1.32b 0.92a 2.67a 2.38 1.20 6.91 2.93 90.82 35.80 28.13 98.67 70 Æ Æ Æ Æ Æ Æ Æ Æ 0.01b 0.10 0.07b 0.14ab 1.92b 0.88b 0.53ab 0.67a 80 2.59 1.23 8.09 3.14 97.28 37.83 31.88 100.00 Æ Æ Æ Æ Æ Æ Æ Æ 0.02c 0.02 0.04c 0.04bc 0.82c 0.45b 0.28c 0.00a 2.67 1.19 8.42 3.26 89.60 34.95 30.92 98.67 90 Æ Æ Æ Æ Æ Æ Æ Æ 0.04d 0.07 0.18cd 0.07c 2.30b 1.70ab 1.00bc 0.67a 2.85 1.21 8.72 3.29 89.01 34.30 30.43 98.67 100 Æ Æ Æ Æ Æ Æ Æ Æ 0.02e 0.02 0.28cd 0.03c 1.79b 0.58ab 0.53bc 1.33a 3.08 1.19 8.77 3.34 78.97 31.31 28.70 98.67 Æ Æ Æ Æ Æ Æ Æ Æ 0.02f 0.05 0.27d 0.09c 4.26a 1.76a 1.71ab 1.33a ÃMean values (mean Æ SE of three replicates) in the same row with di¡erent letters are signi¢cantly di¡erent (Po0.05) Feeding rate (FR) total feed intake  100/f[(initial body weight1¢nal body weight)/2]  feeding daysg Speci¢c growth rate (SGR) 100  [ln (¢nal body weight) À ln (initial body weight)]/days of the experiment Feed conversion e⁄ciency (FCE) 100  (¢nal body weight À initial body weight)/feed intake Feed protein retention e⁄ciency (PRE) 100  [(¢nal body weight  ¢nal protein content) À (initial body weight  initial protein content)]/(feed intake  protein content) Feed energy retention e⁄ciency (ERE) 100  [(¢nal body weight  ¢nal energy content) À (initial body weight  initial energy content)]/(feed intake  energy content) Table Body composition (%) and energy content (kJ g À 1) of Pelteobagrus fulvidraco fed di¡erent ration levels (RL, % per day)à RL Initial body composition Dry matter Protein Lipid Ash Energy 22.0 13.8 3.6 3.5 4.8 Æ Æ Æ Æ Æ 0.2 0.1 0.1 0.1 0.1 50 24.0 15.6 4.2 3.8 4.7 60 Æ Æ Æ Æ Æ 0.5a 0.2 0.1a 0.1b 0.1a 24.5 15.4 5.2 3.7 5.1 70 Æ Æ Æ Æ Æ 0.2a 0.0 0.3b 0.1ab 0.1b 24.6 15.3 5.4 3.6 5.4 80 Æ Æ Æ Æ Æ 0.3a 0.3 0.3b 0.1ab 0.1c 24.8 15.3 5.9 3.6 5.6 90 Æ Æ Æ Æ Æ 0.2a 0.3 0.4b 0.0ab 0.0c 24.7 15.2 5.8 3.6 5.6 100 Æ Æ Æ Æ Æ 0.1a 0.0 0.2b 0.1ab 0.0c 25.8 15.5 6.1 3.5 5.9 Æ Æ Æ Æ Æ 0.3b 0.0 0.3b 0.0a 0.1d ÃMean values (mean Æ SE of three replicates) in the same row with di¡erent letters are signi¢cantly di¡erent (Po0.05) and 0.69^1.24 mg g À day À respectively (Table 4) Apparent digestibility coe⁄cients in dry matter (ADCd), protein (ADCp), energy (ADCe) of yellow cat¢sh decreased signi¢cantly (Po0.05) as ration increased, with ranges of 54.42^69.64%,78.24^89.90% and 69.66^82.07% respectively Energy budgets at each RL for juvenile yellow cat¢sh were shown in Table The proportions of energy intake lost in faecal production and excretion were 17.93^30.34% and 7.03^7.69%, respectively, and both proportions tended to increase with an increase in RL As ration increased, the proportion of energy intake spent in metabolism decreased and was highest at 50% of satiation ration and ranged between 33.27% and 47.11%, whereas the proportions of energy intake stored in body as growth energy increased with the RL, peaked at 70% of satiation, and then decreased at higher ration with a ranged between 26.55% and 31.88% 902 Discussion Relatively high (97.33^100.00%) survival of juvenile yellow cat¢sh was obtained in the present study at the end of the trial Many authors reported optimum feeding regimes based on the relationship between RL and SGR or FCE (Hogendoorn 1983; Gˇnther, Galvez-Hidalgo, Ulloa-Rojas, Coppoolse & Verreth 1992; Cortes & Gruber 1994; Sun, Chen, Huang, Wang & Yan 2006) Usually, growth^ration relationship has a decelerating pattern and FCE shows a domed curve as ration increases, so growth and FCE were not maximized at the same RL (Xie et al.1997; Sun et al 2006) In this case, the optimum feeding strategy would be that ¢sh are fed at an intermediate not maximum ration to obtain both rapid growth and high FCE Present study indicated that, as ration increased, growth showed a decelerating curve, but the di¡erences were not signi¢cant at 70%, 80%, 90% and 100% of r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 899^905 Aquaculture Research, 2011, 42, 899^905 Growth, nitrogenous excretion and energy budget of yellow cat¢sh L Zhang et al Table Nitrogenous excretion (u, mg g À day À 1), faecal production (f, mg g À day À 1) and apparent digestibility coe⁄cients in dry matter (ADCd, %), protein (ADCp, %), energy (ADCe, %) of Pelteobagrus fulvidraco fed di¡erent ration levels (RL, % per day)à RL u F ADCd ADCp ADCe 50 0.94 0.69 68.12 89.90 80.71 60 Æ Æ Æ Æ Æ a 0.04 0.03a 0.96c 0.68d 0.91c 1.04 0.77 69.64 89.69 82.07 70 Æ Æ Æ Æ Æ ab 0.02 0.04a 0.53c 0.33d 0.48c 0.98 0.78 67.12 88.86 80.33 80 Æ Æ Æ Æ Æ ab 0.01 0.02a 0.53c 0.53d 0.77c 1.14 0.93 62.74 86.14 74.75 90 Æ Æ Æ Æ Æ b 0.06 0.04b 1.52b 1.03c 1.52b 100 1.15 1.11 56.17 81.07 71.58 Æ Æ Æ Æ Æ b 0.03 0.00c 1.13a 1.16b 1.72ab 1.38 1.24 54.42 78.24 69.66 Æ Æ Æ Æ Æ 0.10c 0.10c 0.81a 0.66a 1.30a ÃMean values (mean Æ SE of three replicates) in the same row with di¡erent letters are signi¢cantly di¡erent (Po0.05) ADCd 5100  (1 À Cr2O3 in the diet/Cr2O3 in the faeces) ADCp 5100  (1 À Cr2O3 in the diet  crude protein content in the faeces/Cr2O3 in the faeces  crude protein content in the diet) ADCe 5100  (1 À Cr2O3 in the diet  gross energy content in the faeces/Cr2O3 in the faeces  gross energy content in the diet) Table Energy budgets of Pelteobagrus fulvidraco fed di¡erent ration levels (RL, % per day)à RL 50 Food energy (kJ g À day À 1) 0.404 Æ 0.010 As a percentage of food energy (%) Growth energy 26.55 Æ 0.92a Faeces energy 19.29 Æ 0.91a Excretion energy 7.04 Æ 0.16a Metabolism energy 47.11 Æ 1.62c As a percentage of assimilated energy (%) Metabolism energy 63.93 Æ 1.56b Growth energy 36.07 Æ 1.56a 60 70 80 90 100 0.432 Æ 0.007 0.421 Æ 0.002 0.468 Æ 0.014 0.473 Æ 0.008 0.539 Æ 0.025 28.13 17.93 7.27 46.67 Æ Æ Æ Æ 0.53ab 0.48a 0.09ab 0.80c 62.39 Æ 0.79b 37.61 Æ 0.79a 31.88 19.67 7.03 41.41 Æ Æ Æ Æ 0.28c 0.77a 0.05a 0.78b 56.49 Æ 0.51a 43.51 Æ 0.51b 30.92 25.25 7.34 36.49 Æ Æ Æ Æ 1.00bc 1.52b 0.20ab 1.64a 54.09 Æ 1.58a 45.91 Æ 1.58b 30.43 28.42 7.36 33.79 Æ Æ Æ Æ 0.53bc 1.72bc 0.08ab 1.64a 52.56 Æ 1.32a 47.44 Æ 1.32b 28.70 30.34 7.69 33.27 Æ Æ Æ Æ 1.71ab 1.30c 0.20b 1.08a 53.74 Æ 2.01a 46.26 Æ 2.01b ÃMean values (mean Æ SE of three replicates) in the same row with di¡erent letters are signi¢cantly di¡erent (Po0.05) C F1U1R1G or A R1G, where C, food energy; A, assimilated energy; F, faeces energy; U, excretion energy; R, metabolism energy, calculated by di¡erence R C À F À U À G and G, growth energy satiation; however, FCE showed a domed curve and peaked at 70% of satiation The whole-body composition of ¢sh is often used as an indicator of ¢sh quality Several factors, including growth and diet are known to a¡ect the body composition of ¢sh Body composition is also signi¢cantly a¡ected by feeding rate (Panda, Mishra & Samantaray 1999; Adebayo, Balogun & Fagbenro 2000; Khan, Jafri & Chadha 2004; Ahmed 2007) The present results revealed that crude lipid content increased when ash content decreased with increased rations, consistent with the previous studies (Shimeno, Shikata, Hosokawa, Masumoto & Kheyyali 1997; Abdelghany & Ahmad 2002; Han, Xie, Lei, Zhu & Yang 2004; Cho, Lee, Park & Lee 2006) When ¢sh were fed at higher rations, lipid accumulation was prevalent One of possible reason is that there is a trend for ration to determine growth and more food energy at high RL could be converted into ¢sh fat (Shearer, Silverstein & Dickho¡ 1997; Han et al 2004) The decline in ash content with RLs could be caused by re- latively a lower proportion of bones compared with other tissues (Rasmussen & Ostenfeld 2000; Han et al 2004) However, in the present studies crude protein content of ¢sh was not signi¢cantly a¡ected by di¡erent RLs, which were similar to previous studies (Huisman1976; Grayton & Beamish1977; Reinitz 1983; Storebakken & Austreng 1987a, b) Based on the above results, it is recommended that 70% of satiation ration per day (actual 68% of satiation ration), about 2.59% body weight per day with 10.3 g protein kg À day À and 432.5 kJ digestible energy kg À day À 1, would be useful for optimum RL for maximum growth and FCE, and excellent body composition with the formulated feed (Table 1) The result is higher than the RLs reported for other cultured ¢sh species in terms of body weight/day which was recently reported byAhmed (2010) Nitrogenous excretion and faecal production are important indexes, which can estimate water pollution in the intensive culture In the present study, both nitrogenous excretion and faecal production of r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 899^905 903 Growth, nitrogenous excretion and energy budget of yellow cat¢sh L Zhang et al juvenile yellow cat¢sh increased with increased ration Higher rations could be result in water pollution because of more nitrogenous excretion and faecal production in the yellow cat¢sh intensive culture Some studies have reported that apparent energy digestibility coe⁄cients showed no signi¢cant di¡erences related to RL (Allen1980; Hogendoorn1983) In the present study, apparent digestibility coe⁄cients in dry matter, protein, energy of yellow cat¢sh decreased signi¢cantly with increased of ration, which is an agreement with the values reported on African cat¢sh and Nile tilapia by Henken, Kleingeld and Tijssen (1985) and Xie et al (1997) In the present study, the estimate of food energy (C), growth energy (G), faeces energy (F) and excretion energy (U) should be accurate Although the estimation of allowed errors might be a¡ected by nutrient leaching problems for faeces energy and by the estimation of initial energy content of experimental ¢sh from the mean values of the control ¢sh for growth energy, and the remaining component, metabolism energy (R), was estimated indirectly by di¡erence R C À F À U À G as theoretical energy budgets should be balanced (Sun & Chen 2009) The proportion of food energy intake channelled into growth showed the highest value at an intermediate ration (70% of satiation) and was lower at below or above RLs The food energy lost in metabolism decreased with increasing RLs This could be due to the limited food resources at low RLs Fish kept swimming to enhance the chances of obtaining feed, increasing the energy cost for activity (Han et al 2004).When increasing RLs to 80% of satiation, ¢sh activity decreased, and the metabolism energy were gradually stabilize Based on the information available on energy budgets for 14 ¢sh species, Cui and Liu (1990) calculated the following average energy budget for ¢sh fed at satiation ration: 100A 60R140G In the present study, energy budget of juvenile yellow cat¢sh at satiation ration was: 100A 54R146G The proportion of assimilated energy spent in growth for juvenile yellow cat¢sh was slightly higher than the average value, consistent with the results for southern cat¢sh (Silurus meridionalis) (Xie & Sun 1993) and Chinese longsnout cat¢sh (Leiocassis longirostris Gˇnther) (Han et al 2004), but di¡ers from the values reported for Nile tilapia (Oreochromis niloticus) of R (76%), G (24%) (Xie et al 1997) and Gibel carp (Carassius auratus gibelio) of R (75%), G (25%) (Zhu, Xie & Cui 2000), which were carnivores ¢sh This seemed to suggest that yellow cat¢sh has moderate growth e⁄ciency and metabolic expendi- 904 Aquaculture Research, 2011, 42, 899–905 ture, and the results were accordant with the fact that the yellow cat¢sh is an omnivorous ¢sh species Acknowledgments The authors are grateful for the project support by the National Key Technology R&D Program of China (Grant No 2007BAD37B02) References Abdelghany A.E & Ahmad M.H (2002) E¡ects of feeding rates on growth and production of Nile tilapia, common carp and silver carp polycultured in fertilized ponds Aquaculture Research 33, 415^423 Adebayo O.T., Balogun A.M & Fagbenro O.A (2000) E¡ect of feeding rates on growth, body composition and economic performance of juvenile clariid cat¢sh hybrid (, Clarias gariepinus  < Heterobranchus bidorsalis) Journal of Aquaculture in theTropics 15,109^117 Ahmed I (2007) E¡ect of ration size on growth, body composition, and energy and protein maintenance requirement of ¢ngerling Indian major carp, Labeo rohita (Hamilton) Fish Physiology and Biochemistry 33, 203^ 212 Ahmed I (2010) Response to the ration levels on growth, body composition, energy, and protein maintenance requirement of the Indian cat¢sh (Heteropneustes fossilisBloch 1974) Fish Physiology and Biochemistry 36, 1133– 1143 Allen J.R.M (1980) The estimation of natural feeding rate of the three-spined stickleback, Gasterosteus aculeatus L (Pisces) PhD thesis, University of Wales, Aberystwyth, 341pp Bolin D.W., King R.P & Klosterman E.W (1952) A simpli¢ed method for determination of chromic oxide (Cr2O3) when used as an inert substance Science 116, 634^635 ChaneyA.L & Marbach E.P (1962) Modi¢ed reagents for determination of urea and ammonia Clinical Chemistry 8, 130^132 Cho S.H., Lee S.M., Park B.H & Lee S.M (2006) E¡ect of feeding ratio on growth and body composition of juvenile olive £ounder Paralichthys olivaceus fed extruded pellets during the summer season Aquaculture 251,78^84 Cortes E & Gruber S.H (1994) E¡ect of ration size on growth and gross conversion e⁄ciency of young lemon sharks, Negaprion brevirostris Journal of Fish Biology 44, 331^341 Cui Y.B & Liu J.K (1990) Comparison of energy budget among six teleosts ^ I Food consumption, faecal production and nitrogenous excretion Comparative Biochemistry and Physiology 96A, 163^171 Elliott J.M (1976) Energy losses in the waste products of brown trout (Salmo trutta L.) Journal of Animal Ecology 45, 561^580 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 899^905 Aquaculture Research, 2011, 42, 899^905 Growth, nitrogenous excretion and energy budget of yellow cat¢sh L Zhang et al Grayton B.D & Beamish F.W.H (1977) E¡ect of feeding frequency on food intake, growth and body composition of rainbow trout (Salmo gairdneri) Aquaculture 11,159^172 Gˇnther J., Galvez-Hidalgo N., Ulloa-Rojas J., Coppoolse J & Verreth J.A.J (1992) The e¡ect of feeding level on growth and survival of jaguar guapote (Cichlasoma managuense) larvae fed Artemia nauplii Aquaculture 107, 347^358 Han D., Xie S.Q., Lei W., Zhu X.M & YangY.X (2004) E¡ect of ration on the growth and energy budget of Chinese longsnout cat¢sh, Leiocassis longirostris Gˇnther Aquaculture Research 35, 866^873 Han Q., Jia K.S., Xia W.F., Mao X.J & Wang W.B (2003) Trial on the cage culture of Pelteobagrus fulvidraco Fisheries Science 22, 32^33 (in Chinese with English abstract) Henken A.M., Kleingeld D.W & Tijssen P.A.T (1985) The e¡ect of feeding level on apparent digestibility of dietary dry matter, crude protein and gross energy in the African cat¢sh, Clarias gariepinus (Burchell,1822) Aquaculture 51,1^11 Hogendoorn H (1983) Growth and production of African cat¢sh, Clarias lazera (C and V.) III Bioenergetic relations of body weight and feeding level Aquaculture 35,1^17 Huisman E.A (1976) Food conversion e⁄ciencies at maintenance and production levels for carp, Cyprinus carpio L., and rainbow trout, Salmo gairdneri Richardson Aquaculture 9, 259^276 Jiang R., Song X.H., Ye Y.T., Cai C.F.,Yang C.G & Huang H.Z (2004) Optimum dietary protein and energy-protein ratio of yellow cat¢sh Pelteobagrus fulvidraco Richardson Journal of Dalian Fisheries University 19, 252^257 (in Chinese with English abstract) Khan M.A., Jafri A.K & Chadha N.K (2004) Growth and body composition of rohu, Labeo rohita (Hamilton), fed compound diet:Winter feeding and rearing to marketable size Journal of Applied Ichthyology 20, 265^270 Lee O.K & Lee S.M (2005) E¡ects of the dietary protein and lipid levels on growth and body composition of bagrid catsh, Pseudobagrus fulvidraco Aquaculture 243,323^329 Liu S.P (1997) A study on the biology of Pseudobagrus fulvidraco in Poyang lake Chinese Journal of Zoology 32, 10^16 (in Chinese with English abstract) Pan J.L., Ding S.Y., Ge J.C., Yan W.H., Hao C., Chen J.X & Huang Y.H (2008) Development of cryopreservation for maintaining yellow cat¢sh Pelteobagrus fulvidraco sperm Aquaculture 279, 173^176 Panda S., Mishra K & Samantaray K (1999) E¡ect of feeding rate on the growth performance of Channa punctatus (Bloch) fry, and protein and energy requirement for their maintenance and maximum growth Aquaculture 7, 37^42 Rasmussen R.S & Ostenfeld T.H (2000) E¡ect of growth rate on quality traits and feed utilization of rainbow trout (On- corhynchus mykiss) and brook trout (Salvelinus fontinalis) Aquaculture 184, 327^337 Reinitz G (1983) Relative e¡ect of age, diet and feeding rate on the body composition of young rainbow trout (Salmo gairdneri) Aquaculture 35,19^27 Shearer K.D., Silverstein J.T & Dickho¡ W.W (1997) Control of growth and adiposity of juvenile Chinook salmon (Oncorhynchus tshawytscha) Aquaculture 157, 311^323 Shimeno S., Shikata T., Hosokawa H., Masumoto T & Kheyyali D (1997) Metabolic responses to feeding rates in common carp, Cyprinus carpio Aquaculture 151, 371^ 377 Storebakken T & Austreng E (1987a) Ration level for salmonids I Growth, survival, body composition and feed conversion in Atlantic salmon fry and ¢ngerlings Aquaculture 60,189^206 Storebakken T & Austreng E (1987b) Ration level for salmonids II Growth, feed intake, protein digestibility, body composition, and feed conversion in rainbow trout weighing 0.5^1.0 kg Aquaculture 60, 207^221 Sun L.H & Chen H.R (2009) E¡ects of ration and temperature on growth, fecal production, nitrogenous excretion and energy budget of juvenile cobia (Rachycentron canadum) Aquaculture 292,197^206 Sun L.H., Chen H.R., Huang L.M.,Wang Z.D & YanY (2006) Growth and energy budget of juvenile cobia (Rachycentron canadum) relative to ration Aquaculture 257, 214^ 220 Wang C.F., Xie C.X & Ma J (2001) The feeding rhythm and the daily feeding rates of Pelteobagrus fulvidkraco in early life history Chinese Journal of Fisheries 14, 66^68 (in Chinese with English abstract) Xie S.Q., Cui Y.B.,YangY.X & Liu J.K (1997) Energy budget of Nile tilapia (Oreochromis niloticus) in relation to ration size Aquaculture 154, 57^68 Xie X.J & Sun R.Y (1993) Pattern of energy allocation in the southern cat¢sh (Silurus meridionalis) Journal of Fish Biology 42,197^207 Yang Y.O & Yao F (2006) E¡ect of ration level on nitrogen and energy budget of yellow cat¢sh Hubei Agricultural Sciences 45, 653^655 (in Chinese with English abstract) Zhang G.Q., Luo X.N & Li B (2000) Critical techniques for arti¢cial propagation of Pelteobagrus fulvidraco (Richardson) Journal of Dalian Fisheries University 15, 293^299 (in Chinese with English abstract) Zhu X.M., Xie S.Q & Cui Y.B (2000) E¡ect of ration size on growth and energy budget of Gibel carp Oceanologia et Limnologia Sinica 31, 471^479 (in Chinese with English abstract) r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 899^905 905 Aquaculture Research, 2011, 42, 906^917 doi:10.1111/j.1365-2109.2010.02633.x Real-time quantification of the immune gene expression in rainbow trout fed different forms of probiotic bacteria Lactobacillus rhamnosus Akshaya Panigrahi1,2, Kiron Viswanath3 & Suichi Satoh1 Department of Marine Biosciences,Tokyo University of Marine Science and Technology, Minato,Tokyo, Japan Central Institute of Brackish Water Aquaculture, Chennai,Tamil Nadu, India Faculty of Biosciences and Aquaculture, Bodo University College, Bodo, Norway Correspondence: A Panigrahi, Central Institute of BrackishWater Aquaculture,75, Santhom High Road, R.A Puram, Chennai-28,Tamil Nadu, India E-mail: panigrahi@ciba.res.in Abstract Whether it is better to use viable or non-viable probionts in aquaculture is still a matter of debate In this study, the molecular immunomodulation in rainbow trout Oncorhynchus mykiss induced by viable or killed forms of the probiont Lactobacillus rhamnosus JCM 1136 was investigated Three forms of this probiont: (1) heat-killed (HK), (2) live spray (LI) and (3) freezedried (FD) were incorporated into a basal (control) diet for rainbow trout O mykiss The LI and FD diets are referred to as viable diets A rearing trial, in triplicate, was conducted for 30 days, with the control and probiotic diets as treatments The cytokine genes such as the tumour necrosis factor (TNF), transforming growth factor (TGF-b), interferon (IFN) and immune gene Immunoglobulin (Ig) found in tissues from the kidney and spleen were assessed for their expression pattern by real-time polymerase chain reaction The tested immune genes were up-regulated in the treatment groups, sometimes even in many folds like in the case of the Ig gene The TNF gene was found to be highly (Po0.05) up-regulated (5000-fold) in groups fed both viable forms (LI, FD) With regard to the TGF-b gene, the spleen of the HK and FD groups showed signi¢cant up-regulation of 20- and 30-folds respectively The IFN gene was upregulated (Po0.05) in all treatments, but more in the viable diet treatments Kidney and spleen tissues showed similar expression patterns, i.e all of these genes were up-regulated more with the viable diets that with the control, and in most cases, the viable diets induced a higher expression of the immune genes than the HK diet 906 Keywords: lactic acid bacteria, rainbow trout, probiotics, cytokines, gene expression Introduction Probiotics are de¢ned as living microorganisms that, when administered in adequate amounts, confer a health bene¢t on the host The ever-increasing use of probiotics in aquaculture for alternative health management makes it essential to unravel the underlying mechanism Probiotics can keep pathogens at bay through competitive exclusion or host immunity enhancement In aquaculture, de¢ned probiotics are used as remediation agents in a number of ways such as enrichment of larval food, inclusion in the diet or addition to the water to maintain a balanced population Several probiotic species including the lactic acid bacteria (LAB) have been evaluated for various candidate aquaculture species (Gatesoupe 1991, 1994, 1999; Robertson, Reijula, Jarvis,Veijalainen & Hintikka 1997; Ringo & Gatesoupe1998; Robertson, Dowd & Burrells et al 2000; Vershuere, Rombaut, Sorgeloos & Verstraete 2000; Balcazar,Vendrell, De Blas, Ruiz-zarzuela, Girones & Muzquiz 2007; Salinas, Myklebust, Esteban, Olsen, Meseguer & Ringo 2008) Probiotics in aquaculture act as non-speci¢c immunostimulants (Anderson1992; Balcazar, De Blas, Ruiz-zarzuela, Vendrell, Calvo, Marquez, Girones & Muzquiz 2007), help improve water quality (Vadstein, Oie, Olsen, Salvesen, Skjermo & Skjak-braek 1993; Skjermo & Vadstein 1999), enhance larval survival and growth (Skjermo & Vadstein 1999) and help supplement nutrition (Ronnestad, Thorsen & Finn r 2011 Blackwell Publishing Ltd Aquaculture Research, 2011, 42, 906^917 Immune gene expressions with probiotic feeding A Panigrahi et al 1999) Further, several studies (Noh, Han,Won & Choi 1994; Moriarty 1998) have reported the use of functional feeds including probionts to induce growth and feed utilization The probiotic bacteria Bacillus sp in viable form have been proven to be bene¢cial for the growth and survival of shrimp (Rengpipat, Phianpak, Piyatiratitivorakul & Menasveta 1998) Culture viability is one of the important criteria in the standardization of probiotics A concentration of 107 CFU g À at the time of consumption is considered to be functional (Gomes & Malcata 1999) in humans The information available on the potential probiotic Lactobacillus rhamnosus JCM 1136 provides convincing evidence of its safety (GRAS generally regarded as safe) This probiotic bacterium is known to improve immune response and disease resistance in rainbow trout (Nikoskelainen, Ouwehand, Bylund, Salminen, Lilius 2003; Panigrahi, Kiron, Kobayashi, Puangkaew, Satoh & Sugita 2004; Panigrahi, Kiron, Puangkaew, Kobayashi, Satoh & Sugita 2005) and tilapia (Pirarat, Kobayashi, Katagiri, Maita & Endo 2006) Fructooligosachharides (FOS) as a prebiotic component in addition to the probiotic bacteria are also known to modulate lipid metabolism and improve the bioavailability of essential minerals There is an urgent need to know the density of probiotics, and for the same reason, it is important to understand the method of administration and dosage as that of the form of bacteria used Probiotic and cytokine gene interaction as reported previously (Panigrahi, Kiron, Satoh, Hirono, Kobayashi, Sugita, Puangkaewa & Aoki 2007) indicates that there is a strain-speci¢c up-regulation of interleukins (IL-b1), tumour necrosis factor (TNF) and transforming growth factor (TGF-b) in the spleen and kidney tissue of probiotic fed rainbow trout However, it is unlikely that only the viable form of probionts can be immunostimulating Previous studies have illustrated that the viable form exhibits a better e¡ect of elevating the cellular and humoral immune response than that of the heat^killed (HK) form In this study, the gene expression has been quanti¢ed following the interaction of macrophages with living and killed probionts provided through feed The present study aimed to investigate the immune gene expression induced by feeding di¡erent forms of the probiont L rhamnosus JCM 1136 to rainbow trout The in£uence of the non-viable HK bacteria is compared with that of the viable form [as viable sprayed and freeze-dried (FD)] of L rhamnosus on the immune gene [TNF,TGF-b, interferon (IFN) and immunoglobulin (Ig)] expression in the primary immune tissues Materials and methods Source, maintenance and forms of bacterial strains The bacterium L rhamnosus JCM 1136 was obtained from the Japan Collection of Microorganisms (JCM), Institute of Physical and Chemical Research (Riken), Japan, in a FD form Man, Rogosa and Share (MRS) media (De Man, Rogosa & Sharpe 1960) were used to revive the culture for experimentation and to preserve for subsequent use Following mass culture, L rhamnosus was harvested by centrifuging at 16500 g for 10 and washing with sterile peptone water (NaCl, 0.85% and polypeptone, 0.1%) to remove the media Three di¡erent forms of bacteria were incorporated into the diet either as live spray, a heat-inactivated form, or a FD form The viable bacteria were HK in a continuously stirred water bath at a temperature of 75 1C for 60 The non-viability was checked by plating on MRS agar three times The FD form was prepared by keeping the bacterial suspension for 60 h at À 20 1C in a REL 206 freeze-drier (Kyowa Vacuum Tech., Tokyo, Japan) The FD form was vacuum packed before preserving at À 20 1C until further use The FD powder and suspension form were enumerated for bacterial number per gram and per microlitre of the product respectively Diet formulation and probiotic incorporation The basal diet was formulated by incorporating prebiotic (10% FOS) components used for feeding the control group of animals FOS are naturally occurring non-digestible carbohydrates, which, in combination with the probionts, promote long-term health via gastrointestinal immunity The experimental diet was formulated with 50% defatted ¢sh meal (DFM) as the protein source and linseed oil as the lipid source The DFM was autoclaved to reduce the possible microbial load For the preparation of the diets, the ingredients, as described in one of our earlier papers (Panigrahi et al 2005), were mixed mechanically (ACM-50 LAT, Aikohsha, Tokyo, Japan) and sterilized water was added before pelletizing (AEZ12 M, Shimadzu, Kyoto, Japan) The pellets were dried in a REL 206 freeze-drier and stored at À 20 1C until further use The live spray (LI) diet was prepared by gently spraying a bacterial suspension (100 mL kg À feed) of the lactic acid bacterial strain taken on the basal diet and slowly mixing it in a drum mixer; the ¢nal mix was air dried on a clean bench for 12 h In the same way, mixing a similar amount of a HK bacterial r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 906^917 907 Aquaculture Research, 2011, 42, 1050–1055 Dietary protein requirement of Siganus rivulatus A Y El-Dakar et al were chemically analysed according to AOAC (1990) Speci¢c growth rate (SGR), feed conversion ratio (FCR), PE ratio and apparent protein utilization (APU) were calculated as Table Survival and growth of rabbit¢sh juveniles o¡ered diets with various protein levels for 49 days Diet no SGR ¼ 100  ðln FBWÀ ln IBWÞ tÀ1 where FBW is ¢nal body weight (g), IBW is initial body weight (g) and t is in days FCR ¼ ½weight of feed offered to fish ðgފ ½weight gain of fish ðgފ PE ¼ P30 P40 P50 P60 PSE Survival (%) 93 93 90 93 100 97 1.75 Initial weight (g) 1.46 1.49 1.48 1.51 1.49 1.51 0.01 Final weight (g) 9.85d 15.23c 23.24b 27.66a 29.58a 28.17a 1.81 SGRà 3.90d 4.74c 5.61b 5.93a 6.10a 5.98a 0.20 Values in the same row with di¡erent superscripts are signi¢cantly di¡erent from each other PSE, pooled standard error; SGR, speci¢c growth rate ½protein retained ðgފ ½protein offered ðgފ  100 Gross energy content of the experimental diets and ¢sh samples were calculated by using factors of 23.62, 17.56 and 39.5 kJ g À of protein, carbohydrate and lipid level respectively (NRC 1993) Digestible energy (DE) content was calculated from standard physiological fuel values as 16.72, 17.62 and 37.62 kJ g À of protein, carbohydrate and lipid respectively (Garling & Wilson 1976) Table Total and daily feed intake, protein intake, feed conversion and protein e⁄ciency of juvenile rabbit¢sh offered diets with various protein levels for 49 days Diet no P10 TFI (g) TPI (g) FCRà PEw APUz P20 c P30 bc 57.06 61.77 5.72e 12.40d 6.81a 5.74b 1.47a 1.11b 24.13a 17.85b P40 a P50 a P60 b PSE c 67.75 65.08 62.77 55.83 20.35c 26.04b 31.42a 33.51a 3.14c 2.50cd 2.24d 2.10d b b bc 1.07 1.01 0.90 0.80c 17.72b 16.22bc 15.34bc 13.22c 1.91 2.34 0.45 0.06 0.92 ÃFCR 5TFI/¢sh weight gain Statistical analysis All statistical analyses were performed according to Snedecor and Cochran (1982) using M-STATC software (MSTAT-C 1988) Data were analysed using ANOVA to determine signi¢cant di¡erences among treatment means at a level of signi¢cance a 0.05 Di¡erences among individual means were evaluated using Duncan’s multiple-range test Results Survival was ! 90% in all treatments with no signi¢cant di¡erences observed among treatments The least amount of dietary protein necessary to promote optimal growth was 40% Fish o¡ered diets with 440% protein did not grow faster than ¢sh o¡ered the 40% protein diet (Table 2) There were signi¢cant di¡erences in growth rate (Po0.05) among ¢sh in treatments P40, P30, P20 and P10, where growth rate was directly correlated with dietary protein content Total feed intake (TFI) was signi¢cantly greater (Po0.05) in P20 than all other treatments and TFI of the P30 group was signi¢cantly greater (Po0.05) than that of P10 and P60 but did not di¡er from TFI 1052 P20 ÃSGR (ln ¢nal weight À ln initial weight)/(no of days)  100 ½weight gain ðgފ  100 ½protein offered ðgފ APU ¼ P10 wPE ¢sh weight gain/TPI  100 zAPU total protein gain/TPI  100 Values in the same row with di¡erent superscripts are signi¢cantly di¡erent from each other PSE, pooled standard error; TFI, total feed intake per ¢sh; TPI, total protein intake per ¢sh; FCR, feed conversion ratio; PE, protein e⁄ciency; APU, apparent protein utilization of the P40 and P50 groups (Table 3) Alternatively, total protein intake was greatest in P50 and P60, and decreased sequentially as protein level in the feed decreased Feed conversion increased as dietary protein increased until P40 and then growth relative to dietary protein did not change (Table 3) Protein e⁄ciency was greatest in ¢sh o¡ered diet P10 and least for ¢sh o¡ered diet P60 There were no signi¢cant differences (P40.05) in PE among treatments P20, P30, P40 and P50 although an increasing trend in PE was observed with the decreasing dietary protein proportion between P60 and P10 Apparent protein utilization followed the same trend as PE Per cent dry matter of the ¢sh at harvest was significantly less (Po0.05) in treatment P10 than in P20, which was in turn signi¢cantly less than in treatment P30 There were no signi¢cant di¡erences (P40.05) in dry matter proportion of ¢sh among r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1050^1055 Aquaculture Research, 2011, 42, 1050^1055 Dietary protein requirement of Siganus rivulatus A Y El-Dakar et al Table Body composition of rabbit¢sh juveniles o¡ered diets with various protein levels for 49 days Diet P10 P20 P30 P40 P50 P60 PSE % Dry matter 24.76c 26.47b 27.28a 27.37a 27.68a 27.90a 0.26 % Crude protein 63.56a 60.87ab 59.40b 57.86b 60.47b 58.73b 0.57 c b b b ab % Ether extract 20.19 22.01 22.45 22.80 23.36 24.62a 0.37 % Ash 15.79 18.31 17.98 18.53 16.50 16.68 0.45ns Values for protein, ether extract and ash are as a per cent of dry weight Values in the same row with di¡erent superscripts are signi¢cantly di¡erent from each other PSE, pooled standard error treatments P30, P40, P50 and P60 (Table 4) The proportion of protein in ¢sh bodies from treatments P20, P30, P40, P50 and P60 were similar to each other Per cent protein content in ¢sh body from P10 was not di¡erent from per cent protein in ¢sh from P20 but signi¢cantly greater than per cent protein in all other treatments Conversely, the lipid proportion of the ¢sh body was greatest in treatments P50 and P60 and least in ¢sh from treatment P10 (Table 4) Discussion Growth of marbled spinefoot rabbit¢sh increased with an increase in dietary protein up to a level of 40% protein in the diet and then levelled o¡ and even decreased slightly at a protein level of 60% Although gross and DE were estimated based on published values rather than empirically determined, values were not used as absolute numbers but rather to compare among trials Accordingly, although DE was not signi¢cantly di¡erent among treatments, it did decrease slightly with the increasing protein content and thus growth appears to be a¡ected more by dietary protein level than by energy levels It is possible that by modifying energy content of the diet, we would modify the optimal protein requirement as suggested by Cowey (1979) Suggested optimal values of P/DE range between17 and 26 MJ kg À (NRC 1993), which in the present work correspond to diets with protein levels of 30% and 40% It is thus possible that by increasing DE in the diets, optimal protein in the diet would be o40% but 430% Tacon et al (1990), Bwathondi (1982) and Ismail, Wahyuni and Panggabeam (1986) reported that 31% protein was optimal for S canaliculatus growth but their diets contained 8% lipids Further studies of nutritional protein to en- ergy ratios in diets of siganids are necessary in order to formulate optimal diets for the species A model of SGR of the ¢sh plotted against dietary protein content exhibited a second-order polynomial growth function (y À 0.0014x210.1373x12.63; r2 50.9956), thus indicating a decrease in growth at high dietary protein levels Various ¢sh exhibit various growth patterns with respect to dietary inputs Some show better regression coe⁄cients when a broken line analysis is used (see Baker 1986) and some respond better to a second-order polynomial regression analysis (Tacon & Cowey 1985), as observed for rabbit¢sh in the present study In both methods of analysis, however, 40% dietary protein appears to be the minimum requirement for optimal growth of juvenile S rivulatus when dietary energy density is approximately 17 MJ kg À These results are in agreement with Shalaby (1998) who found that the growth curve of S rivulatus did not reach a plateau until 45% CP, in diets containing 25^45% protein level and 16^18 MJ kg À DE Daily feed intake decreased as dietary protein level increased, similar to results reported by most investigations of dietary protein requirements of various ¢sh However, DPI did not vary signi¢cantly among ¢sh o¡ered 20% and 50% protein diets, suggesting that ¢sh were regulating feed intake according to protein intake These results coupled with the fact that PE and APU were not signi¢cantly di¡erent among treatments from 20% to 50% protein suggest that at these protein levels, dietary DE of approximately 17 MJ kg À is acceptable (see Lazo et al 1998) A high PE at10% protein and a low PE at 60% protein indicate that the P/DE ratio at these protein levels is not optimal for growth, an observation further supported by the polynomial shape of the SGR to protein model Crude protein content of ¢sh carcass was signi¢cantly greater in siganids o¡ered the10% protein diet than in siganids o¡ered the diets with greater protein content Consequently, APU was much greater in the 10% protein treatment than in other treatments Such high APU is probably because of the low P/DE ratio and thus very little protein was used for energy production or transformed to glycogen or fat for energy storage In treatments where ¢sh were o¡ered diets with a P/DE ratio of 11g MJ À or greater, per cent carcass protein was similar among treatments Other experiments on various ¢sh species found no e¡ect of dietary protein level on carcass protein levels (see for example Lazo et al 1998; Salhi, Bessonart, Chediakb, Bellagamba & Carnevia 2004; Jana, Garg, r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1050^1055 1053 Dietary protein requirement of Siganus rivulatus A Y El-Dakar et al Barman, Arasu & Patra 2006), but in those experiments P/DE was high in all diets tested Carcass lipids were least in ¢sh o¡ered the 10% protein diet and most in ¢sh o¡ered the 60% protein diet, but similar in all other treatments Again, these results suggest that at low dietary P/DE ratios lipids in the diet are used for energy requirements while at high P/DE ratios lipids are stored in the body Fish in the present work were reared under optimal salinity and temperature conditions for the species (see Saoud, Kreydiyyeh, Chalfoun & Fakih 2007; Saoud, Mohanna & Ghanawi 2008) and thus dietary protein and energy requirements observed are suitable for growth under these conditions However, S rivulatus are very euryhaline and reasonably eurythermal (Saoud et al 2007; Saoud, Mohanna et al 2008) and will not always be reared in optimal environments As such, their protein and energy requirements will change and diets will need to be selected for various environmental conditions Results of the present study suggest that S rivulatus juveniles grow best when o¡ered a diet with at least 40% crude protein and a protein to DE of 23.5 g MJ À These levels of protein and energy should serve as a basis for further investigations such as dietary energetic needs, digestibility coe⁄cients of various dietary ingredients and protein to energy requirements of siganids A successful rabbit¢sh aquaculture industry will depend on the availability of such information and the development of proper diets for the species Acknowledgments Special thanks are due to Joly Ghanawi for helping with the literature search and reviewing the manuscript The authors are also grateful to the Lebanese National Council for Scienti¢c Research for funding Dr Saoud during the period of the present project References AOAC (Association of O⁄cial and Analytical Chemists) (1990) O⁄cial Methods of Analysis of the AOAC, 15th edn Association of O⁄cial and Analytical Chemists, Arlington,VA, USA Baker D.H (1986) Problems and pitfalls in animal experiments designed to establish dietary requirements for essential nutrients Journal of Nutrition 116, 2339^2349 Brett J.R & Groves T.D.D (1979) Physiological energetic In: Fish physiology, Bioenergetics and Growth (ed byW.S Hoar, D.J Randall & J.R Brett), pp 279^352 Academic Press, NewYork, NY, USA 1054 Aquaculture Research, 2011, 42, 1050–1055 Bwathondi P.O.J (1982) Preliminary investigations on rabbit¢sh, Siganus canaliculatus cultivations in Tanzania Aquaculture 27, 205^210 Carumbana E.E & Luchavez J.A (1979) A comparative study of the growth rates of Siganus canaliculatus, Siganus spinus and Siganus guttatus reared under laboratory and seminatural conditions in Southern Negros Oriental, Philippines SillimanJournal 26,187^209 Catacutan M.R & Coloso R.M (1995) E¡ect of dietary protein to energy ratios on growth, survival, and body composition of juvenile Asian seabass, Lutes calcarifer Aquaculture 131,125^133 Cowey C.B (1979) Protein and amino acid requirements of ¢n¢sh In: Fin¢sh Nutrition and Fishfeed Technology, Vol (ed by J.E Halver & K Tiews), pp 3^16 Schr Bundesforschungsanst Fisch., Hamburg, Germany Cowey C.B., Pope J.A., Adron J.W & Blair A (1972) Studies on the nutrition of marine £at¢sh; the protein requirements of plaice (Pleuronectes platessa) British Journal of Nutrition 28, 447^456 El-Sayed A.-F.M (2004) Protein nutrition of farmed tilapia: searching for unconventional sources In: Proceedings of the 6th International Symposium on Tilapia in Aquaculture (ed by R.B Bolivar, G.C Mair & K Fitzsimmons), pp 364–378 Central Luzon State University, Manila, Philippines Garling D.L Jr & Wilson R.P (1976) Optimum dietary protein to energy ratio for channel cat¢sh ¢ngerling, Ictalurus punctatus Journal of Nutrition 106,1368^1375 Gatlin D.M (1995) Review of red drum nutrition In: Nutrition and Utilization Technology in Aquaculture (ed by C.M Lim & D.J Sessa), pp 41^49 AOCS Press, Champaign, IL, USA Hara S., Duray M.N., Parazo M.M & Taki Y (1986) Yearround spawning and seed production of the rabbit¢sh, Siganus guttatus Aquaculture 59, 259^272 Ismail W.,Wahyuni I.S & Panggabeam T (1986) Preliminary study on the combination of feed for siganids Panelition Perikanan 10, l–5 Jana S.N., Garg S.K., Barman U.K., Arasu A.R & Patra T.B.C (2006) E¡ect of varying dietary protein levels on growth and production of Chanos chanos (Forsskal) in inland saline groundwater: laboratory and ¢eld studies Aquaculture International 14, 479^498 Juario J.V., Duray M.N., Duray V.M., Nacario J.F & Almendras J.M.E (1985) Breeding and larval rearing of the rabbit¢sh, Siganus guttarus (Blach) Aquaculture 44, 91^101 Lam T.J (1973) Siganids: their biology and mariculture potential Aquaculture 3, 325^354 Lazo J.P., Davis D.A & Arnold C.R (1998) The e¡ects of dietary protein level on growth, feed e⁄ciency and survival of juvenile Florida pompano (Trachinorus carolinus) Aquaculture 169, 225^232 Lovell R.T (1980) Practical ¢sh diets In: Fish FeedTechnology Aquaculture Development and Coordination Programme (ADCP/REP/80/1I), pp 333–350 FAO, Rome, Italy r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1050^1055 Aquaculture Research, 2011, 42, 1050^1055 Dietary protein requirement of Siganus rivulatus A Y El-Dakar et al MSTAT-C (1988) MSTAT-C, A Microcomputer Program for the Design, Management, and Analysis of Agronomic Research Experiments Michigan State University, East Lansing, MI, USA National Research Council (NRC) (1993) Nutrient Requirements of Fish National Academy Press,Washington, DC, USA Parazo M.M (1990) E¡ect of dietary protein and energy level on growth, protein utilization and carcass composition of rabbit¢sh, Siganus guttatus Aquaculture 86, 41^49 Popper D & Gundermann N (1975) Some ecological and behavioral aspects of siganid populations in the Red Sea and Mediterranean coasts of Israel in relation to their suitability for aquaculture Aquaculture 6, 127^141 Salhi M., Bessonart M., Chediakb G., Bellagamba M & Carnevia D (2004) Growth, feed utilization and body composition of black cat¢sh, Rhamdia quelen, fry fed diets containing di¡erent protein and energy levels Aquaculture 231, 435^444 Saoud I.P., Kreydiyyeh S., Chalfoun A & Fakih M (2007) In£uence of salinity on survival, growth, plasma osmolality and gill Na^K-ATPase activity in the rabbit¢sh Siganus rivulatus Journal of Experimental Marine Biology and Ecology 384, 183^190 Saoud I.P., Ghanawi J & Lebbos N (2008) E¡ects of stocking density on survival, growth, size variation and condition index of the rabbit¢sh Siganus rivulatus Aquaculture International 16,109^116 Saoud I.P., Mohanna C & Ghanawi J (2008) E¡ects of temperature on survival and growth of juvenile spinefoot rabbit¢sh (Siganus rivulatus) Aquaculture Research 39, 491^497 Snedecor G.W & CochranW.G (1982) Statistical Methods,6th edn Iowa State University Press, Ames, IA, USA Shalaby S.M (1998) Nutrition requirements of rabbit¢sh, Siganus rivulatus, ¢ngerlings PhD thesis, Alexandria University, Egypt Tacon A.G.J & Cowey C.B (1985) Protein and amino acid requirements In: Fish Energetics: New Perspectives (ed by P Tytler & P Calow), pp 155^183 The John Hopkins University Press, Baltimore, MD, USA Tacon A.G.J., Rausin N., Kadari M & Cornelis P (1990) The food and feeding of marine ¢n¢sh in £oating net cages at the National Seafarming Development Centre, Lampung, Indonesia: rabbit¢sh, Siganus canalicultus (Park) Aquatic Fish Management 21, 375^390 Tucker J.W (1998) Marine Fish Culture Kluwer Academic Publishing, Hingham, MA, USA Wee K.L & Tacon A.G.J (1982) A preliminary study on the dietary protein requirement of juvenile snakehead Bulletin of the Japanese Society of Scienti¢c Fisheries 48, 1463^ 1468 Woodland D.J (1983) Zoogeography of the siganidae (Pisces): an interpretation of distribution and richness patterns Bulletin of Marine Science 33,713^717 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1050^1055 1055 Aquaculture Research, 2011, 42, 1056^1060 doi:10.1111/j.1365-2109.2010.02659.x SHORT COMMUNICATION Larval release and attachment modes of the hydroid Ectopleura larynx on aquaculture nets in Norway Christina Carl1,2Ã, Jana Guenther1 & Leif Magne Sunde1 Centre for Research-Based Innovation in Aquaculture Technology, SINTEF Fisheries and Aquaculture,Trondheim, Norway Institute for Chemistry & Biology of the Marine Environment, Carl von Ossietzky University Oldenburg, Oldenburg, Germany Correspondence: C Carl, School of Marine and Tropical Biology, James Cook University, Angus Smith Drive, Douglas, Qld 4811, Australia Email: tine.carl@jcu.edu.au ÃPresent address: C Carl, School of Marine and Tropical Biology, James Cook University, Townsville, Qld, Australia Introduction In the ¢n¢sh industry, clean cage nets are essential for the health of the stock (Braithwaite & McEvoy 2005) The accumulation of biofouling, the unwanted attachment of marine organisms on submerged surfaces, decreases the water £ow through nets Subsequently, the water quality within sea cages is reduced, which may a¡ect ¢sh health negatively (Cronin, Cheshire, Clarke & Melville 1999; de Nys & Guenther 2009) Over the last decade, the hydroid Ectopleura larynx (syn Tubularia larynx) has become one of the most common fouling organisms in the Norwegian ¢sh farming industry, causing increasing problems for farmers (Guenther, Carl & Sunde 2009) The rapid growth of E larynx on aquaculture nets requires ¢sh farmers to clean their nets regularly, often on a fortnightly basis during the peak of the biofouling season between July and November (Guenther et al 2009) To reduce biofouling, the majority of Norwegian salmon farmers use copperbased coatings on nets, combined with regular underwater high-pressure washing (200^300 bar) with rotating discs (Olafsen 2006) Anecdotal observations suggest that hydroids grow back faster once the ¢rst washing has taken place (B Jensen, salmon farm manager, pers comm.) Despite the dominance of E larynx and its associated problems for the ¢sh farming industry, only a few studies have focused on hydroids on aquaculture nets These have examined the settlement and 1056 successional development of hydroids on aquaculture nets (Greene & Grizzle 2007; Guenther et al 2009) and the increase in drag force on the netting due to hydroids and other fouling organisms (Swift, Fredriksson, Unrein, Fullerton, Patursson & Baldwin 2006) Given the increasing occurrence of hydroiddominated biofouling in the Norwegian aquaculture industry (Guenther et al 2009), there is a need to identify the factors in£uencing the fouling community, particularly hydroids, on sea cages Understanding these factors and the link between E larynx and its response to the underwater washing of nets could allow control over this problematic fouling organism in an aquaculture environment Therefore, the speci¢c aims of this study were to identify the impact of the underwater washing on this fouling organism by determining the number of released actinulae, juveniles and polyps during the underwater washing Furthermore, the ability of polyps to release actinulae, after polyps were cut from the hydrorhiza (see description in Fig 1), was determined Finally, strategies of E larynx to maintain their attachment to the net were classi¢ed to determine whether the net material facilitated the colonization and attachment Materials and methods To investigate the response of E larynx to the underwater washing of cage nets and quantify the number of actinulae, juveniles and polyps of E larynx in the r 2010 Blackwell Publishing Ltd Aquaculture Research, 2011, 42, 1056^1060 Larval release and attachment modes of Ectopleura larynx C Carl et al to the water temperature at Sunde The number of released actinulae was counted after 24 h To qualitatively determine the strategies of E larynx to maintain their attachment to the net, eight samples of fouled ¢sh cage netting (8  cm, 15 mm halfmesh) from 10 to 12 m depth at a commercial salmon farm in Reitholmen (63136.95 N; 09107.48 E), Mid-Norway, were cut out in December 2007 and preserved in 1% formalin in seawater Hydroids were examined under a compound microscope and photographed using a Nikon DS camera Figure Diagram of Ectopleura larynx (modi¢ed by Hayward & Ryland 1990) water column, plankton samples were collected m downstream from a sea cage at a commercial salmon farm at EdÖya (63139.31 0N; 08141.05 E), Mid-Norway, in December 2007 Plankton samples were collected 40 before (n 3) and during (n 3) a ¢rst washing cycle in the morning and again 40 before (n 3) and during (n 3) a second washing cycle at noon, when a di¡erent part of the cage net was washed The samples collected 40 before the washing cycles provided information on the natural release of larvae and polyps by E larynx The water column was vertically sampled from to10 m water depth using a 100 mm mesh sized plankton net (KPT Naturfag) with an opening diameter of 30 cm The sampled volume was estimated by assuming that a water column of10 m height was sampled All plankton samples were preserved in1% formalin in seawater, and the actinulae, juveniles and polyps of E larynx in each plankton sample were identi¢ed and counted using a compound microscope Given the occurrence of several values in the data, the mean sums of all E larynx propagules (actinulae, juveniles and polyps) before and during each washing cycle were statistically compared using paired t-tests (SPSS version 16) The signi¢cance level of the t-test was adjusted accordingly for each of the two analyses (a 50.025) Data were log-transformed to meet the assumptions of normality and homogeneity of variances (Levene’s test) To quantify the number of released actinulae of cut polyps as a potential for colonization, adult E larynx were collected from a commercial salmon farm at Sunde (63130.21 0N; 09111.59 E), Mid-Norway, in October 2008 Thirty polyps were cut o¡ using scissors and placed individually in Petri dishes with 10 mL of 0.45 mm ¢ltered seawater at 12 1C, which was similar Results All the mean numbers of propagules (actinulae, juveniles and polyps) of E larynx increased several-fold during the washing cycles (Fig 2) Polyps increased the most in comparison with the initial mean numbers before the underwater washing More than 300 polyps were found in the water column during the ¢rst washing cycle, whereas none were found before the ¢rst washing cycle in the morning (Fig 2a) In general, the mean numbers of all propagules (actinulae, juveniles and polyps) were higher during the ¢rst washing cycle than during the second one (Fig 2b) The amount of E larynx propagules in the water column increased signi¢cantly during the ¢rst underwater washing (t 5.81, d.f 54, P 0.004) and also during the second washing cycle (t 6.25, d.f 54, P 0.003) Furthermore, cut polyps of E larynx released a mean number of 3.6 Æ 0.7 actinulae within 24 h under laboratory conditions The growth of E larynx on salmon cage nets was examined, and three strategies were determined to maintain their attachment to the nets (Fig 3) First, the hydrophytons grew around threads and often intertwined, creating compact tufts of hydrophytons around the thread The chitinous perisarc of the hydrophytons made the tuft in£exible, and the only way to detach the hydroids from the netting was to break it Second, the hydrophytons grew between loose nylon ¢laments and threads The hydrophytons were strongly attached to the net, because the loose nylon ¢lament functioned as a strap that secured the hydroid to the netting Third, some hydrophytons also incorporated nylon ¢laments into their chitinous perisarc (Fig 3) Discussion This study demonstrates that the underwater washing of sea cage nets resulted in higher numbers of r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1056^1060 1057 Larval release and attachment modes of Ectopleura larynx C Carl et al Aquaculture Research, 2011, 42, 1056^1060 Figure Numbers of actinulae, juveniles and polyps of the hydroid Ectopleura larynx collected from to 10 m depth at a commercial salmon farm at EdÖya, Norway, before and during (a) the ¢rst washing cycle in the morning, and (b) the second washing cycle at noon, when a di¡erent part of the net was washed Data are mean Æ SE Figure Overview of the three strategies of the hydroid Ectopleura larynx (H) to maintain their attachment to the net (N) (a) Winding of the hydrophyton around threads; (b) growth of the hydrophyton between loose nylon ¢laments and threads; and (c) incorporation of nylon ¢laments into the chitinous perisarc Matching schematic representations are shown below the photographs Arrows indicate loose nylon ¢laments of the thread (a^c) have the same magni¢cation Scale bar 500 mm E larynx actinulae, juveniles and polyps in the water column Strong currents of approximately 235 m s À created by underwater high-pressure washers (AKVAgroup ASA, Bryne, Rogaland, Norway) not only exceed the critical value of cm s1 (Pye¢nch & Downing 1949) to dislodge settled actinulae and 1058 juveniles from the substratum but also cause damage to the hydroids The strong currents induce the gonophores to burst and release actinulae These actinulae then encounter available space for settlement and growth on the cleaned nets and other underwater structures as soon as the current speed returns r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1056^1060 Aquaculture Research, 2011, 42, 1056^1060 Larval release and attachment modes of Ectopleura larynx C Carl et al to the ambient velocity Pye¢nch and Downing (1949) found that 440% of actinulae settled within h after their release under laboratory conditions Furthermore, detached polyps of E crocea (syn Tubularia crocea) continued to release actinulae (Rungger 1969), and this study showed that cut polyps of E larynx were also capable of releasing an average of 3.6 actinulae within 24 h Because the spread of hydroids within and between farms may be enhanced by the use of underwater high-pressure washers, this ¢nding could be of concern for other aquaculture sites in the vicinity of farms cleaning their nets with this method After settlement, E larynx ¢rst establishes an extensive system of hydrophytons (Pye¢nch & Downing 1949) to spread the colony and reduce the risk of mortality (Gili & Hughes 1995) This study showed that current net constructions with multi-¢lament nylon threads provided a structure for maintaining the attachment of E larynx on the nets, such as the growth under loose ¢laments The hydroid is fastened to the thread and their removal may be hindered Therefore, fragments of the hydroids may remain on the washed netting and a fast and extensive regrowth of E larynx may be facilitated The loose nylon ¢laments may be created during the cleaning of the fouled net with high-pressure washers This procedure strains the netting and possibly decreases the strength of ¢laments The results of this study contribute to an improved understanding of the factors in£uencing the abundance of hydroids on aquaculture nets, and to possible solutions to control and reduce their growth more e⁄ciently The current underwater washing is only a temporary measure to control E larynx as a fouling organism and e¡ective long-term solutions are needed (Guenther, Misimi & Sunde 2010) Future underwater washers may be modi¢ed to collect the cut polyps of hydroids to reduce the number of actinulae released in the water column Furthermore, to hinder the growth of hydroids between loose ¢laments and threads, which maintains their attachment on the nets, netting threads could be made of a single ¢lament or have a coating enhancing the cohesion of the nylon ¢laments, which can also withstand the high pressure used during the underwater washing This might also increase the e⁄ciency of the cleaning by reducing the numbers of fragments of hydroids on the nets after the washing procedure Thus, the possibility of fast and extensive re-growth is minimized The removal and onshore cleaning of nets is not popular with Norwegian salmon farmers due to operational lo- gistics (Guenther et al 2010) Further studies are also needed to determine powerful strategies to kill remaining fragments on the nets after the washing and to develop strategies for the complete removal of hydroids from the nets Acknowledgements We thank T Dempster, K Tangen and three anonymous reviewers for comments on earlier versions of this manuscript, employees at the commercial salmon farms for their support during sample collection and J.-M Gili for the identi¢cation of the hydroid species Financial support was provided by the Norwegian Research Council and the Fishery and Aquaculture Research Fund (Project number 164719) and the Centre for Research-Based Innovation in Aquaculture Technology (CREATE) References Braithwaite R.A & McEvoy L.A (2005) Marine biofouling on ¢sh farms and its remediation Advances in Marine Biology 47, 215^252 Cronin E.R., Cheshire A.C., Clarke S.M & Melville A.J (1999) An investigation into the composition, biomass and oxygen budget of the fouling community on tuna aquaculture farm Biofouling 13, 279^299 de Nys R & Guenther J (2009) The impact and control of biofouling in marine ¢n¢sh aquaculture In: Advances in MarineAntifouling Coatings andTechnologies (ed by C Hellio & D.Yebra), pp.177^221.Woodhead Publishing Limited, Cambridge, UK Gili J.M & Hughes R.G (1995) The ecology of marine benthic hydroids Oceanography ^ Marine Biology Annual Review 33, 351^426 Greene J.K & Grizzle R.E (2007) Successional development of fouling communities on open ocean aquaculture ¢sh cages in the western Gulf of Maine, USA Aquaculture 262, 289^301 Guenther J., Carl C & Sunde L.M (2009) The e¡ects of colour and copper on the settlement of the hydroid Ectopleura larynx on aquaculture nets in Norway Aquaculture 292, 252^255 Guenther J., Misimi E & Sunde L.M (2010) The development of biofouling, particularly the hydroid Ectopleura larynx, on commercial cage nets in Mid-Norway Aquaculture 300,120^127 Hayward P.J & Ryland J.S (1990) The marine fauna of the British Isles and North-West Europe.Volume I Introduction and Protozoans toArthopods Clarendon Press, Oxford r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1056^1060 1059 Larval release and attachment modes of Ectopleura larynx C Carl et al Olafsen T (2006) Cost analysis of di¡erent antifouling strategies SINTEF Fiskeri og Havbruk report, SFH80 A066041, ISBN: 82-14-03947-9 (in Norwegian) Pye¢nch K.A & Downing F.S (1949) Notes on the general biology of Tubularia larynx Ellis & Solander Journal of the Marine Biological Association of the United Kingdom 28, 21^43 Rungger D (1969) Autotomy in Tubularia crocea and its ecological and physiological signi¢cance Pubblicazioni della Stazione Zoologica di Napoli 37, 95^139 1060 Aquaculture Research, 2011, 42, 1056^1060 Swift M.R., Fredriksson D.W., Unrein A., Fullerton B., Patursson O & Baldwin K (2006) Drag force acting on biofouled net panels Aquacultural Engineering 35, 292^299 Keywords: biofouling, ¢sh farming, antifouling technologies, hydroids r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1056^1060 Aquaculture Research, 2011, 42, 1061^1065 doi:10.1111/j.1365-2109.2010.02688.x SHORT COMMUNICATION Effects of mechanical perturbation at various times during incubation on egg survival, hatching and malformation rates in the rainbow trout Oncorhynchus mykiss, and the influence of post-ovulatory oocyte ageing Sylvain Milla1,2, Elisabeth Sambroni2, Patrick Kestemont1 & Bernard Jalabert2 The University of Namur (FUNDP), Unite¤ de Recherche en Biologie des Organismes (URBO), Namur, Belgium INRA, UR1037, SCRIBE (Station Commune de Recherches en Ichtyophysiologie Biodiversite¤ et Environnement), Campus de Beaulieu, Fish Reproduction Research Group, Rennes, France Correspondence: S Milla,The University of Namur (FUNDP), Unite¤ de Recherche en Biologie des Organismes (URBO),61rue de Bruxelles, 5000 Namur, Belgium E-mail: sylvain.milla@fundp.ac.be It has long been known that mechanical disturbance of salmonid eggs during incubation may lead to high mortalities Even if a consensus states di¡erential sensitivity along the incubation period, some works gave rise to con£icting results Some authors mentioned that the period of egg sensitivity begins from 1to days post fertilization (pf) to the eyed stage (Davis 1953; Laird & Wilson 1979) But others also highlighted a mortality response to handling in the ¢rst hour of egg incubation (Billard 1976; Jensen & Alderdice 1983) Then, a stable high sensitivity was observed within the range of 40^100 degree  days pf in rainbow trout Oncorhynchus mykiss (Walbaum) at 10 1C (Jensen & Alderdice 1983) while an increasing sensitivity was pointed out between 20 and 80 degree  days at the same temperature (Dwyer, Fredenberg & Erdahl 1993) Most of the previous work have used automatized procedures to assess egg sensitivity (Post, Power & Kloppel 1974; Jensen & Alderdice 1983, 1989; Dwyer et al 1993), but few studies have performed similar perturbations to those usually encountered in husbandry practices or have investigated their impact on hatching and fry malformation rates Also, dead eggs, which turn white within the ¢rst days of incubation, are often the starting r 2010 Blackwell Publishing Ltd point of fungal development thus contaminating live eggs Importantly, because rainbow trout females not usually spawn in aquaculture systems, ¢sh are commonly inspected for ovulation and manually stripped by the farmer Thus, monitoring ¢sh and stripping are critical as post-ovulatory ageing of oocytes may occur if females are not stripped in time Indeed, mature oocytes of rainbow trout are released during ovulation into the coelomic cavity where they remain immersed in ovarian £uid until manual stripping is performed by the farmer Prolonged oocyte retention results in a decrease of egg quality marked by lower embryo survival and higher malformation rates (Azuma, Ohta, Oda, Muto, Yada & Unuma 2003; Aegerter & Jalabert 2004) Therefore, such eggs may also be expected to be more sensitive to perturbations during incubation The aim of the present study was (1) to assess changes in egg sensitivity to mechanical handling throughout incubation; and (2) to investigate the in£uence of post-ovulatory ageing on egg sensitivity to handling Experiments were carried out at the INRA SCRIBE ¢sh laboratory For each experiment, ¢ve to six males 1061 Mechanical perturbation during trout incubation S Milla et al Aquaculture Research, 2011, 42, 1061^1065 10 cm) at a number of 100^200 eggs incubator À Water temperature, monitored daily to precisely assess incubation stage, was maintained close to 10 1C Incubation trays were covered with grey rigid plastic to protect eggs against light At increasing incubation times, eggs in two or three trays were submitted to a10-s rotating movement, thus simulating the type of mild perturbation exerted in a tray with live eggs when some white eggs are taken among them Egg and six to 18 females were used Ovulation was checked twice weekly by manual inspection to assess retention times and egg and sperm collection were performed as described previously (Labbe¤ & Maisse 2001; Aegerter & Jalabert 2004) Fertilization was performed as described previously in Aegerter, Jalabert and Bobe (2005) with minor modi¢cations After hardening (about min), eggs were poured into small incubation trays (about 10 cm  10 cm  Table Percentage of one-cell, two-cell and abnormal eggs h post fertilization when the eggs were handled between and h post fertilization Handling time CO 15 30 45 1h 75 90 105 % One-cell % Two-cell % Abnormal 52 38 56 27 12 62 29 51 39 55 32 13 58 30 60 25 12 44 30 10 Handling time 2h 3h 4h 5h 6h 7h 8h % One-cell % Two-cell % Abnormal 65 24 11 51 35 13 53 35 10 60 33 54 34 10 62 27 62 28 N 77^118 eggs per sampling time CO, control ¢sh 30 * g Egg whitening (%) 25 20 * f 15 10 * e * d * * c a b a D D 10 D D D D D D D 8h D 7h 6h 4h 5h 3h 15 ' 30 ' 45 ' 60 ' 75 ' 90 ' 10 5' 2h Handling time Figure Percentage of egg whitening according to handling time The egg whitening is calculated either in manipulated eggs (black columns, N 5131^189 eggs in duplicate per sampling time) or non-manipulated eggs (grey columns, N 5130^183 eggs tray À 1, from 34 to trays per sampling time) Di¡erent letters indicate signi¢cant di¡erences between handling time (Po0.05) The asterisk indicates signi¢cant di¡erence between manipulated and unmanipulated eggs (Po0.05) 1062 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1061^1065 Aquaculture Research, 2011, 42, 1061^1065 Mechanical perturbation during trout incubation S Milla et al handling was performed on the following time course: every 15 between and h, every hour between and h, every day between and 10 days pf When whitening occurred on some eggs (usually within15 after handling), all white eggs were removed 24 h after the handling Hatching and malformation rates at the end of yolk resorption were calculated for each handling time For each handling between and h, about 100 eggs were sampled h pf and immediately immersed in Stockard ¢xative for 8^10 days Afterwards, egg samples were coloured in Giemsa solution (dilution 1/16 in acetate bu¡er 105 0.2 M, pH 4.2), and then observed under a binocular microscope in order to monitor the percentage of embryos at stages one-cell, two-cell (¢rst cleavage completed) and the percentage of abnormal eggs h pf The e¡ect of post-ovulatory oocyte ageing was investigated in a second experiment performed on the eggs from three groups of females (six females in each group) characterized by various post-ovulatory retention times: 0^3 days, 7^10 days and 15^18 days Statistical analyses were performed using STATISTICA software (StatSoft, Tulsa, OK, USA) The e¡ect of handling time on egg stage (one-cell vs two-cell vs (a) 100 Hatching rate (%) 95 90 85 * * 80 75 * 70 8h D D D D D D D D D D 10 C O 6h 7h 5h 4h ' 60 ' 75 ' 90 10 ' 5' 2h 3h ' 45 30 15 ' 65 Handling time 18 (b) * 16 Abnormal larvae (%) 14 12 10 8h D D D D D D D D D D 10 C O 7h 6h 4h 5h ' 60 ' 75 ' 90 10 ' 5' 2h 3h ' 45 30 15 ' Handling time Figure Hatching rate (a) and percentage of abnormal larvae at yolk resorption (b), according to the handling time N 65^114 eggs or larvae in duplicate per sampling time The asterisk indicates signi¢cant di¡erence when compared with the unhandled control (CO) (Po0.05) r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1061^1065 1063 Mechanical perturbation during trout incubation S Milla et al Aquaculture Research, 2011, 42, 1061^1065 0-3 days 80 d 7-10 days Egg whitening after handling (%) 15-18 days 70 60 cd 50 40 abc ab 30 bc c ab 20 ab a bc bc 10 ab a a a a a ab ab b abc b ab a a abc a D2 D3 D4 D5 D6 D7 D8 D9 D10 Handling time Figure Percentage of egg whitening according to the handling time and the period of post-ovulatory ageing N 5112^ 218 eggs in triplicate per sampling time Di¡erent letters indicate signi¢cant di¡erences between handling time for each retention period (Po0.05) abnormal) at the expected time of ¢rst cleavage (8 h at 10 1C) was checked by the w2-test The e¡ects of handling time on egg whitening, hatching rate and fry malformation rate were analysed by a one-way analysis of variance (ANOVA) followed by a Duncan post hoc test A two-way ANOVA was carried out to check the in£uence of oocyte ageing Each incubation tray was used as a statistical unit The data were arcsin-root transformed to satisfy the ANOVA conditions (normality and equal variance) The level of signi¢cance in all tests was Po0.05 No signi¢cant di¡erences in the percentage of onecell, two-cell and abnormal eggs could be detected between control eggs and eggs handled from to h (Table 1) Concerning egg mortality (characterized by egg whitening), no e¡ect could be observed when handling was performed before days pf Thereafter, the percentage of whitened eggs in handled eggs, increased from days pf to reach a maximum of 22% at days pf (Fig 1) Also, the percentage of whitened eggs in control trays (no handling) exhibited very low mortality during the seven ¢rst days (o0.04%), followed by an increase in mortality at 8^10 days pf, but to a lower extent than in handled eggs (Fig 1) The hatching rate followed a similar pattern: stable when handling was carried out in the three ¢rst days pf and lower in the 4^9 days pf period (only signi¢cant for 7^9 days period, Fig 2a) Moreover, the handling at day pf signi¢cantly resulted in higher 1064 malformation percentage at the end of yolk resorption when compared with controls (Fig 2b, Po0.05) In the second experiment, post-ovulatory oocyte ageing signi¢cantly in£uenced egg mortality in response to handling (Po10 À 4), without signi¢cant interactions between handling time and duration of ageing (Fig 3) Similar to the ¢rst experiment, the percentage of whitened eggs following handling increased from days until days pf in controls and then declined In the case of eggs from oocytes aged 7^10 days post ovulation, the percentage of whitened eggs following handling signi¢cantly rose 2.6-fold compared with control females (0^3 days) (Po10 À 4) and the maximum was also reached at days pf In the case of eggs from oocytes aged 15^18 days post ovulation, the average mortality percentage was nearly threefold higher than that measured in eggs from oocytes aged 7^10 days post ovulation, with a peak at day pf Our data show that moderate handling, similar to the kind of disturbance promoted by the removal of white eggs from the trays, does not signi¢cantly a¡ect the time to ¢rst embryonic cleavage (TFC) which has been shown to be an indicator of egg quality in trout (Aegerter, Le Callennec, Bonnet & Jalabert 2008) Moreover, no signi¢cant e¡ect of such handling could be found before days pf However, a critical period clearly appears between days pf and days pf at 10 1C, with a peak of egg sensitivity at days pf The high increased mortality rate appears to be asso- r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1061^1065 Aquaculture Research, 2011, 42, 1061^1065 ciated with lowered hatching rate and hatched fry abnormalities This period of sensitivity is probably linked to speci¢c stages in embryonic development Indeed, it corresponds to 1/2^2/3 epiboly, when the blastoderm begins to thin and overgrow the yolk, a period already known as critical in embryo development (Jensen & Alderdice 1989) Finally, this sensitivity to handling is also greatly in£uenced by oocyte post-ovulatory ageing Generally, this is accompanied by a lower egg quality marked by higher malformation rates and a decrease in egg survival (Azuma et al 2003) Our results bring additional arguments, which suggest that post-ovulatory ageing negatively interferes with development success Nevertheless, it does not alter the critical period (4^9 days post hatching) to handling during egg incubation In conclusion, our results support that eggs can be submitted to the kind of mild perturbation promoted by the removal of some white eggs from incubation trays within the ¢rst days pf at 10 1C, whereas such manipulation should be avoided between and days pf Acknowledgment The authors thank Cecile Melin for the excellent technical assistance References Aegerter S & Jalabert B (2004) E¡ects of post-ovulatory oocyte ageing and temperature on egg quality and on the occurrence of triploid fry in rainbow trout, Oncorhynchus mykiss Aquaculture 231, 59^71 Aegerter S., Jalabert B & Bobe J (2005) Large scale RealTime PCR analysis of mRNA abundance in rainbow trout eggs in relationship with egg quality and post-ovulatory Mechanical perturbation during trout incubation S Milla et al ageing Molecular Reproduction and Development 72, 377^385 Aegerter S., Le Callennec C., Bonnet E & Jalabert B (2008) Time to ¢rst embryonic cleavage in the rainbow trout (Oncorhynchus mykiss): parental e¡ects and correlations with the quality of subsequent development Cybium 32, 226^227 Azuma T., Ohta H., Oda S., Muto K., Yada T & Unuma T (2003) Changes in fertility of rainbow trout eggs retained in coelom Fisheries Science 69, 131^136 Billard R (1976) Sensibilite¤ des oeufs de truite arc-en-ciel aux chocs me¤caniques pendant la phase de durcissement Bulletin Franc° ais de la PeŒche et de la Pisciculture 263,45^49 Davis H.S (1953) Culture and Diseases of Game Fishes University of California Press, Berkeley, CA, USA, 322pp Dwyer W., Fredenberg W & Erdahl D.A (1993) In£uence of electroshock and mechanical shock on survival of trout eggs North American Journal of Fisheries Management 13, 839^843 Jensen J.O.T & Alderdice D.F (1983) Changes in mechanical shock sensitivity of coho salmon (Oncorhynchus kisutch) eggs during incubation Aquaculture 32, 303^312 Jensen J.O.T & Alderdice D.F (1989) Comparison of mechanical shock sensitivity of eggs of ¢ve Paci¢c salmon (Oncorhynchus) species and steelhead trout (Salmo gairdneri) Aquaculture 78, 163^181 Labbe¤ C & Maisse G (2001) Characteristics and freezing tolerance of brown trout spermatozoa according to rearing water salinity Aquaculture 201, 287^299 Laird M & Wilson R (1979) A method for improving the survival of ¢sh eggs during transportation Fisheries Management 3,129^131 Post G., Power D.V & Kloppel T.M (1974) Survival of rainbow trout eggs after receiving physical shocks of known magnitude Transactions of the American Fisheries Society 103, 711^716 Keywords: rainbow trout, handling, egg mortality, fry malformations r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1061^1065 1065 Aquaculture Research, 2011, 42, 1066 doi:10.1111/j.1365-2109.2011.02898.x ERRATA In Aquaculture Research 42:5, the following errors were published on page 677: Abstract This paper presents the preliminary results of different trials carried out with two species of mysids from Gran Canaria: Leptomysis lingvura (G.O Sars, 1866) and Paramysis nouvel This sentence was incorrect and should have read: This paper presents the preliminary results of different trials carried out with two species of mysids from Gran Canaria: Leptomysis lingvura (G.O Sars, 1866) and Paramysis nouveli (Labat, 1953) Line 20: We found that the ratio, DHA:EPA, was 0.85 0.02 and 0.89 0.01; the ratio, DHA: AA, 6.25 0.26 and 4.74 0.14; and the ratio, EPA:AA, 7.32 0.26 and 5.32 0.2, 1066 respectively, for P nouveli and L lingvura in cultures and these ratios not signi¢cantly di¡er (P40.05) from organisms in the wild This sentence was incorrect and should have read: We found that the ratio, DHA:EPA, was 0.85 Æ 0.02 and 0.89 Æ 0.01; the ratio, DHA: AA, 6.25 Æ 0.26 and 4.74 Æ 0.14; and the ratio, EPA:AA, 7.32 Æ 0.26 and 5.32 Æ 0.2, respectively, for P nouveli and L lingvura in cultures and these ratios not signi¢cantly di¡er (P40.05) from organisms in the wild We apologize for these errors Reference Herrera A., Go¤mez M., Molina L., Otero F & Packard T (2011) Rearing techniques and nutritional quality of two mysids from Gran Canaria (Spain) Aquaculture Research 42,677^ 683 doi:10.1111/j.1365-2109.2010.02786.x r 2011 Blackwell Publishing Ltd [...]... formulated diet Aquaculture Research 40,166^171 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 918^930 Aquaculture Research, 2011, 42, 931^942 doi:10.1111/j.1365-2109.2010.02661.x Aquaculture development and scenarios of change in fish trade and market access for the poor in Cambodia Martin L van Brakel1 & Lindsay G Ross2 1 World Fish Center, Penang, Malaysia 2 Institute of Aquaculture, University... Dijk H & Willers J.M (1979) Adjuvanticity of lactobacilli Di¡erential e¡ect of viable r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 906^917 Aquaculture Research, 2011, 42, 906^917 Immune gene expressions with probiotic feeding A Panigrahi et al and killed bacteria Clinical Experimental Immunology 37, 367^375 Bols N.C., Brubacher J.L., Ganassin R.C & Lee L.E.J (2001) Ecotoxicology and innate... Apart from fortifying the host defence mechanism, gut micro£ora plays a key role in the expression of host genes that regulate metabolic and physiological r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 906^917 Aquaculture Research, 2011, 42, 906^917 Immune gene expressions with probiotic feeding A Panigrahi et al mechanisms Earlier ¢ndings (Panigrahi et al 2007) on the forms of probionts and... with distilled water instead of 50% seawater (Sugita et al 2002) and poured into chloroform-killed plates (4.5 mL plate À 1) The plates were incubated at r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 906^917 Aquaculture Research, 2011, 42, 906^917 Immune gene expressions with probiotic feeding A Panigrahi et al Table 1 Primers selected for the expression study of selected cytokine genes... European Cytokine Network 2, 361^366 Vadstein O., Oie G., Olsen Y., Salvesen I., Skjermo J & SkjakBraek G (1993) A strategy to obtain microbial control dur- r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 906^917 Aquaculture Research, 2011, 42, 906^917 Immune gene expressions with probiotic feeding A Panigrahi et al ing larval development of marine ¢sh In: Proceedings of the First International... Cheers C (1995) Di¡erential induction of macrophage-derived cytokines by live and dead intracellular bacteria in vitro Infection and Immunity 63,720^723 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 906^917 917 Aquaculture Research, 2011, 42, 918^930 doi:10.1111/j.1365-2109.2010.02660.x Water quality in a serial-use raceway and its effect on the growth of South African abalone, Haliotis midae... South African Journal of Marine Science 5, 523^529 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 918^930 Aquaculture Research, 2011, 42, 918^930 Basuyaux O & Mathieu M (1999) Inorganic nitrogen and its e¡ect on growth of the abalone Haliotis tuberculata Linnaeus and the sea urchin Paracentrotus lividus Lamarck Aquaculture 174, 95^107 Bower C.E & Bidwell J.P (1978) Ionization of ammonia in... performed in triplicate PCR Values with di¡erent letters are statistically signi¢cant at (Po0.05) as determined by one-way ANOVA followed by Duncan’s test r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 906^917 Aquaculture Research, 2011, 42, 906^917 Immune gene expressions with probiotic feeding A Panigrahi et al Figure 3 Spleen TNF a mRNA level in three forms of probiont Lactobacillus rhamnosus... abalone À 1) and food conversion ratio in three replicate serial-use raceways as a function of the tank position and the average £ow index 922 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 918^930 Aquaculture Research, 2011, 42, 918^930 Water quality in an abalone serial-use raceway M A Naylor et al Table 3 Basic statistics for selected water quality variables from water samples taken in tank... were cyclical variations in the pH, dissolved oxygen concentration and the concentration of FAN (Fig 4) r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 918^930 923 Water quality in an abalone serial-use raceway M A Naylor et al Aquaculture Research, 2011, 42, 918^930 8.2 Dissolved oxygen (mg L–1) Dissolved oxygen (mg L–1) 8.2 y = 8.07x–0.04 8.0 7.8 7.6 7.4 7.2 0 1 2 3 4 5 6 8.0 7.8 7.6 y =