Aquaculture Nutrition 2013 19; 845–859 doi: 10.1111/anu.12029 1,2 Skretting Aquaculture Research Centre, Stavanger, Norway; University, Wageningen, the Netherlands This article focuses on understanding the role of vital wheat gluten on the structural parameters of extruded fish feed and its correlation to the physical and functional properties Gluten–soy protein concentrate blends with five gluten concentrations (0–200 g kgÀ1) were produced An abrupt reduction in oil uptake was observed with the 200 g gluten kgÀ1 blend Inclusion of gluten from 100 to 200 g kgÀ1 resulted in unacceptable product properties Sinking of feed pellets with and 50 g gluten kgÀ1 was 100%, whereas only 36% of pellets with 200 g gluten kgÀ1 sank We suspect that this is due to a relationship between morphological structure and oil impregnation during coating of feeds The addition of gluten at 200 g kgÀ1 gave a smoother and non-porous outer surface Pellets without gluten had a larger number of cells that were smaller than 200 lm (P < 0.05) compared with pellets with 100 and 200 g gluten kgÀ1 More spherical cell shapes (P < 0.01) and a compact structure were favoured in the presence of gluten The closed porosity increased (P < 0.05), whereas interconnectivity between pores decreased (P < 0.01), with increasing gluten content from to 200 g kgÀ1 The effects of the addition of gluten are probably related to the filmforming properties of gluten KEY WORDS: extrusion, fish feed, microstructure, physical quality, soy protein concentrate, wheat gluten Received July 2012; accepted 26 November 2012 Correspondence: Skretting ARC, PO Box 48, 4001 Stavanger, Norway E-mail: vukasin.draganovic@skretting.com In an effort to increase formulation flexibility in the production of modern salmonid feeds and to enhance the sustainability of feeds by replacement of fish protein with ª 2012 John Wiley & Sons Ltd 2 Laboratory of Food Process Engineering, Wageningen plant protein, vital wheat gluten has been shown to have high potential as a feed ingredient (Gatlin et al 2007) Among plant proteins, soy protein concentrate (SPC) is still the primary alternative ingredient to fish meal due to its availability and competitive prices, but the high concentration of carbohydrates in SPC remains a concern (Gatlin et al 2007) In this respect, vital wheat gluten may have good potential due to its high protein content and nutrient digestibility (Sugiura et al 1998; Robaina et al 1999), its lower level of indigestible fibres and absence of anti-nutritional factors Compared with fish meal, wheat gluten is low in methionine and especially low in lysine, whereas it is higher in cysteine content (Allan et al 2000) It has been reported previously that in salmonids, supplementation with lysine (Davies et al 1997; Cheng et al 2003) or a combination of lysine and methionine (Pfeffer & Henrichfreise 1994) is required for diets containing wheat gluten to maintain fish growth Wheat gluten is also extensively used in food applications due to its functionality (Day et al 2006) and availability in large quantities (Domenek et al 2004) Feed pellets obtained after extrusion should have a welldefined porosity that allows sufficient oil absorption capacity leading to specific sinking rates and durability (Glencross et al 2010) In a commercial fish feed manufacturing operation, incorporation of gluten was shown to significantly influence the physical properties of feed, such as oil infusion For example, in high-fat feeds (>300 g kgÀ1 added fat), gluten can be used only in small amounts ( 0.05) except for ALP, which was significantly decreased in tolerance test group (D5) compared with the control group (D1) (P < 0.05) No significant differences were also found in RBC, haemoglobin (Hb), haematocrit (Hct) and thrombocytes (PLT) among all treatments (P > 0.05) Serum MDA contents decreased with the increasing of dietary lutein level, although no significant difference was observed probably due to the large variability There was no significant difference in SOD Aquaculture Nutrition 19; 936–945 ª 2013 John Wiley & Sons Ltd Table Colour parameters in carapace and plastron skin of soft-shelled turtle fed the diets supplemented with lutein (means Æ SEM) Diet no (Lutein content, mg kgÀ1) D1 (1.16) D2 (70.3) D3 (132) D4 (239) D5 (3410) L* 48.8 Æ 0.59c 69.8 Æ 0.59ab Carapace Plastron a* Carapace Plastron b* Carapace Plastron H(o) Carapace Plastron C* Carapace Plastron 45.8 Æ 0.41b 71.0 Æ 0.56bc À0.60 Æ 0.21a 4.75 Æ 0.42b 45.9 Æ 0.37b 71.7 Æ 0.46c 46.1 Æ 0.43b 71.5 Æ 0.47bc 44.4 Æ 0.62a 68.5 Æ 0.58a À0.03 Æ 0.01ab 2.19 Æ 0.33a 0.23 Æ 0.19b 2.79 Æ 0.56a À0.38 Æ 0.16ab 2.96 Æ 0.53a À0.05 Æ 0.01ab 1.91 Æ 0.52a 14.9 Æ 0.31a 12.7 Æ 0.66a 18.3 Æ 0.20b 19.2 Æ 0.50b 19.6 Æ 0.26c 23.7 Æ 0.56c 20.3 Æ 0.30c 24.9 Æ 0.55 cd 20.2 Æ 0.49c 26.3 Æ 0.47d 93.4 Æ 0.82b 69.3 Æ 1.65a 91.2 Æ 0.53a 81.3 Æ 1.48b 90.3 Æ 0.38a 85.7 Æ 1.16b 90.2 Æ 0.42a 84.9 Æ 0.73b 89.9 Æ 0.64a 84.0 Æ 1.18b 14.9 Æ 0.30a 13.9 Æ 0.71a 18.3 Æ 0.20b 19.6 Æ 0.52b 19.6 Æ 0.25c 23.9 Æ 0.59c 20.3 Æ 0.30c 25.1 Æ 0.55cd 20.3 Æ 0.49c 26.6 Æ 0.48d Within the same row, values with different superscripts are significantly different (P < 05) H = arctan (b*/a*) CÃ ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi aÃ2 þ bÃ2 Hue values 90 60 D5 D4 D3 D5 D4 D3 D2 150 D2 30 D1 D1 15 180 20 15 C* 10 5 y = 0.04x + 15.1 R² = 0.96 y = –2×10–5x + 20.7 R² = 10 25 y = 1×10–4x + 24.8 R² = 20 Carapace X = 142 and 143 30 y = 0.08x + 12.8 R² = 0.99 25 Yellow value b* 120 30 10 15 20 25 30 C* Figure Mean hue (H°) and C* values of the carapace and plastron skin area of soft shelled turtle in five lutein treatments (D1-5) Hue being an angle is a circular variable where 0° indicates a red hue and 90° denotes a yellow hue and 180° shows green hue Chroma (C*) is the saturation of skin color The black points (●) represent the mean value of carapace skin (24 turtles, n = 48) and the white points (○) repreent the mean value of the plastron skin (24 turtles, n = 24) Final body weight of the tested soft-shelled turtles was at least eightfolds of the initial body weight, and all groups of turtles showed very low FCR (0.82–0.98) during weeks feeding trial in the present study, which is comparable with Aquaculture Nutrition 19; 936–945 ª 2013 John Wiley & Sons Ltd 500 1000 1500 Plastron 2000 2500 3000 3500 4000 Dietary lutein content (mg kg–1) Figure Relationship between yellow values b* and dietary lutein contents for soft-shelled turtle based on broken-line analysis, where x represent the optimal dietary lutein content for the skin color in carapace and plastron of soft-shelled turtle the performance of commercial practice Lutein supplementation in diets has increased the growth performance of soft-shelled turtle, which is in line with the results of  et al (2011) in Wels catfish (Silurus glanis) and  ZaTKov A Wang et al (2012) in yellow catfish (Pelteobagrus fulvidra et al (2011) showed that the SGR of juve co) ZaTKov A nile Wels catfish (yearlings: about 14–25 g) was increased significantly when being fed with Alga diets enriched in lutein (26.4–35.9 mg kgÀ1); the results of Wang et al (2012) revealed that 76.3 mg kgÀ1 xanthophylls (mainly lutein) enhanced the growth performance and feed 18 Tissue lutein content (mg kg–1) 16 Skin y = 1.75Ln(x) – 0.96 R2 = 0.90 P = 0.01 Muscle 14 12 10 y = 1.12Ln(x) – 0.51 R2 = 0.90 P = 0.01 –2 400 800 1200 1600 2000 2400 2800 3200 3600 Dietary lutein content(mg kg–1) BCF Figure Correlations of lutein deposition in skin and muscle with dietary lutein contents for soft-shelled turtle 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 A Muscle Skin a B D1 b D2 B b D3 B b B c D4 D5 Experimental diets Figure Bioaccumulation factors in muscle and skin of softshelled turtle fed experimental lutein diets Within the same column, means with different superscripts are significantly different (P < 0.05) efficiency of juvenile yellow catfish (21 g) However, several reports indicated that lutein did not improve the growth performance in fish or poultry Olsen & Baker (2006) observed that lutein at the level of 23 mg kgÀ1 of diet did not increase the growth of adult Atlantic salmon Inclusion of 50–300 mg kgÀ1 xanthophyll (mainly lutein) did not affect the growth of goldfish (7.8 Æ 0.3 g) after 10 weeks of rearing and hybrid catfish (61 Æ 0.9 g) after 20 days of feeding (Leng et al 2010; Shi et al 2010) In most of the poultry studies, xanthophyll (or lutein) did not increase the growth performance (Haq & Bailey 1995; Perez-Vendrell et al 2001) The growth promotion function of carotenoid for animals is still under controversy now In shrimp, many studies have reported that carotenoids like astaxanthin (from Haematococcus pluvialis) (Niu et al 2009; Parisenti et al 2011), b-carotene (from Dunaliella salina) (Supamattayaa et al 2005), astaxanthin and capsanthin (extracted from pepper) (Arredondo-Figueroa et al 2003) supplement induced higher weight gain and survival In fish, growth promotion was observed in first-feeding fry of Atlantic salmon (Salmo salar) when fed diets supplemented with 30 mg kgÀ1 astaxanthin (Torrissen 1984; Christiansen et al 1995), and dietary astaxanthin (12.5–92.9 mg kgÀ1) for maternal rainbow trout (Oncorhynchus mykiss) broodstock improved the SGR of fry offspring (Bazyar Lakeh et al 2010) However, most of previous studies did not found that the carotenoid could significantly affect the growth and survival of grown-up fish (Bell et al 2000; Amar et al 2001; Wang et al 2006) As for poultry, no growth promotion was also reported for carotenoids (Perez-Vendrell et al 2001) These results suggested that most carotenoids might affect the growth performance in different mechanism on shrimp, fish and poultry Carotenoids might have a positive effect on growth of most of shrimp species and some of fish species mainly during the first developmental stages In our knowledge, there are few reports published for the effect of carotenoid on aquatic reptile except for recent study of Chen & Table Effects of dietary lutein on serum biochemistry parameters in soft-shelled turtles (means Æ SEM) (n = 6) Diet no (Lutein content, mg kgÀ1) D1 (1.16) SOD(U mLÀ1) MDA(nmol mLÀ1) ALT (U LÀ1) AST (U LÀ1) ALP (U LÀ1) Protein (g LÀ1) Triglycerides (mmol mLÀ1) Cholesterol (mmol mLÀ1) UN (mmol LÀ1) Glucose (mmol LÀ1) 203 2.94 63.2 162 649 33.4 3.40 8.11 2.02 8.52 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 7.4 1.02 3.52 12.1 32.1b 1.70 0.71 0.56 0.24 0.43 D2 (70.3) 197 2.87 71.0 177 591 39.6 3.51 8.47 1.82 7.81 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 12.9 0.98 9.00 26.6 46.9ab 3.50 0.55 0.88 0.09 0.52 D3 (132) 198 2.41 58.8 153 546 36.9 2.96 7.63 1.73 9.16 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 15.1 0.80 3.69 17.3 44.0ab 2.96 0.34 0.24 0.11 0.80 D4 (239) 203 1.45 62.7 162 548 35.3 4.17 8.00 1.79 8.55 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 6.3 0.61 8.01 22.8 39.4ab 1.35 0.47 0.68 0.10 0.50 D5 (3410) 215 1.41 62.0 143 490 36.5 3.24 7.13 2.21 7.59 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 8.6 0.56 6.44 12.9 44.9a 2.25 0.16 0.40 0.21 0.40 Within the same row, values with different superscripts are significantly different (P < 05) SOD, serum superoxide dismutase; MDA, malondialdehyde; ALT, alanine aminotransferase; AST, aspartate transaminase; ALP, alkaline phosphatase; UN, urea nitrogen Aquaculture Nutrition 19; 936–945 ª 2013 John Wiley & Sons Ltd Table Red blood cell count, haemoglobin concentration (Hb), haematocrit (Hct) and thrombocytes count (PLT) of soft-shelled turtles fed various levels of dietary lutein (means Æ SEM) (n = 6) Diet no (Lutein content, mg kgÀ1) D1 (1.16) RBC(91012 LÀ1) Hb (g LÀ1) Hct (%) PLT(9109 LÀ1) 0.22 93.8 31.00 55.7 Æ Æ Æ Æ 0.02 5.92 1.49 5.27 D2 (70.3) 0.22 92.5 30.67 62.0 Æ Æ Æ Æ 0.02 6.32 0.32 10.12 D3 (132) 0.22 101 31.40 62.6 Æ Æ Æ Æ D4 (239) 0.01 3.47 0.49 6.85 0.24 106 33.17 65.2 Æ Æ Æ Æ D5 (3410) 0.01 2.81 1.33 3.04 0.22 99.7 30.83 50.2 Æ Æ Æ Æ 0.01 2.96 1.10 5.96 RBC, red blood cell count; Hb, haemoglobin; Hct, haematocrit; PLT, thrombocytes Huang (2011), in which b-carotene has increased the growth of juvenile soft-shelled turtles The results of the present study also showed the growth promotion of lutein on hatchling soft-shelled turtles Niu et al (2009) declared that the species, the developmental stage, the source, the inclusion level of the pigments and the duration of feeding must be taken into account to understand the different effect of carotenoids on growth performance Therefore, considering the first stage of turtles used in this study, lutein is helpful to promote the growth for juvenile soft-shelled turtle, and the optimal dietary lutein content for the good growth performance of juvenile soft-shelled turtle was 162 mg kgÀ1 Almost all animals are able to enzymatically convert carotenoids into vitamin A (Gross 1991) Soft-shelled turtles are also capable of converting b-carotene to vitamin A (Chen & Huang 2011) It has been also reported that lutein can be converted to vitamin A in several freshwater fish (Li et al 2011), while the ability of converting lutein into vitamin A in turtles is still undiscovered Moreover, the carotenoids have been considered the antioxidants and been utilized to neutralize the free radicals released during the oxidation process within the living cells, which is also proved in this study The serum MDA contents decreased with the supplemented level of dietary lutein The functions of lutein as vitamin A precursor and antioxidant might be beneficial for promoting growth in the first development stage of turtles plastron colour is yellowish Yellowness is one of the characteristic coloration for wild soft-shelled turtles The inclusion of lutein in artificial diet turned the skin colour both in carapace and plastron into yellow hue Lutein also significantly increased the yellowness values b* both in carapace and in plastron skin This is in accordance with several previous studies of lutein on goldfish (Leng et al 2010), hybrid catfish (Shi et al 2010) and yellow catfish (Ding et al 2010) It is well known that the effectiveness of carotenoid source in terms of deposition and pigmentation is species specific and that all fish species not possess the same pathways for the metabolism of carotenoids (Pavlidis et al 2006) In present study, the optimal dietary lutein levels for the desired yellowness skin colour in carapace and plastron of juvenile soft-shelled turtle were 142 and 143 mg kgÀ1, respectively Leng et al (2010) reported that dietary 150 mg kgÀ1 xanthophyll (mainly as lutein) increased the redness and decreased the lightness of goldfish skin In the study of Yuangsoi et al (2011), dietary 50 mg kgÀ1 lutein had also improved the redness of skin in fancy carp The capability of converting lutein into astaxanthin was reported on fancy carp and goldfish (Hirao et al 1963; Katayama et al 1973) The metabolism and converting lutein into other style of carotenoids of soft-shelled turtles were not reported yet In general, the metabolism and degradation of carotenoids, including lutein, have not been well understood in animals until now (Li et al 2011) In contrast to the other chelonian families, the soft-shelled turtles had a soft and pliable shell, both in the carapace and plastron (Pough et al 2004) The epidermis of the carapace and plastron had a thick corneous layer Like other animals, pigment cells of soft-shelled turtles were mainly present in the superficial dermis or beneath the basal layer of the epidermis (Alibardi & Toni 2006) The coloration patterns in plastron and carapace of soft-shelled turtle were different from each other, the carapace colour is yellowish green, and the Considering that animal feed stands at the front of the food chain, the carryover of lutein in human food chain could also be advantageous for human health, especially because of its positive effect on defending adult macula degeneration (Baker & G€ unther 2004) In the present study, the highest lutein treatment (D5, 3410 mg kgÀ1) was designed as a tolerance level group, which is almost 14-fold of D4 and 21-fold of optimum dietary level for highest growth performance, to assess the safety of lutein for the Aquaculture Nutrition 19; 936–945 ª 2013 John Wiley & Sons Ltd target animal There were no mortality in all treatments and no treatment-related significant adverse effects of lutein in serum biochemistry parameters and haematology parameters except for ALP, which was significantly decreased in tolerance level group compared with the control group ALP catalyses the dephosphorylation of phosphorylated organic compounds and also involves in bone formation and in membrane transport activities (Lan et al 1995) The elevated serum ALP was related to the activities of osteoblastic cell and liver function and was the most common index of bone disease in clinical use, such as osteomalacia and rickets (Magnusson et al 1999) The decreased serum ALP for animal generally does not have clinical significance Although the growth of soft-shelled turtle in the tolerance level group (D5) was lower than that of D3, it was not different from that of the control group (D1) In the present study, no any significant negative effects were found on physiological and antioxidant system for softshelled turtles even treated by extremely high level of lutein (25.3 mg kgÀ1 bw dayÀ1) We concluded that the tested lutein is safe for 4.81 g juvenile soft-shelled turtles after the 8-week continuously oral test As for the bioaccumulation of lutein in tissue of turtles, the highest lutein accumulation level (14 mg kgÀ1) was found in skin of D5 treatment In recent years, lutein has become the subject of intense investigations for the public food because of its potential health benefits There has been a significant effort by researchers to elucidate safety of lutein (Ravikrishnan et al 2011) The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has reviewed lutein as a food additive and allocated a group acceptable daily intake (ADI) of to mg kgÀ1 bw dayÀ1 for lutein from Tagates erecta (JECFA 2006) Therefore, as for our studies, it seems impossible to beyond the ADI for the public to eat the turtle muscle or skin even if in the highest dietary lutein level group So, supplementation of lutein in turtle’s feed is also safe for the public interest In conclusion, dietary lutein supplementation is an efficient and safe way to improve the growth performance and skin yellowness of juvenile soft-shelled turtle During 8-w growth trial, the optimal dietary lutein content for the highest SGR of juvenile soft-shelled turtle was 162 mg kgÀ1; the optimal dietary lutein levels for the desired skin colour in carapace and plastron of juvenile soft-shelled turtle were 142 and 143 mg kgÀ1, respectively It is generally safe to use lutein in soft-shelled turtles feed, and the tolerance level is almost 21-fold of optimum content for highest SGR Further investigations on the conversion of lutein to vitamin A and other carotenoids, the metabolism and degradation of lutein in turtles are desirable This work was funded by the Special Fund for Agro-Scientific Research in the Public Interest (201203015); Natural Science Foundation of Hebei Normal University (L2008B08, L2009Y08); and National Natural Science Foundation of China (31272315) The authors are grateful to Peipei Zhang and Huaixia Mu for their turtle husbandry help Alibardi, L & Toni, M (2006) Skin structure and cornification proteins in the soft-shelled turtle Trionyx spiniferus Zoology (Jena), 109, 182–195 Amar, E.C., Kiron, V., Satoh, S & Watanabe, T (2001) Influence of various dietary synthetic carotenoids on bio-defence mechanisms in rainbow trout, Oncorhynchus mykiss (Walbaum) Aquacult Res., 32, 162–173 Arredondo-Figueroa, J.L., Pedroza-Islas, R., Ponce-Palafox, J.T & Vernon-Carter, E.J (2003) Pigmentation of Pacific white shrimp (Litopenaeus vannamei, Boone, 1931) with esterified and saponified carotenoids from red chili (Capsicum annuum) in comparison to astaxanthin Rev Mex Ing Quim., 2, 101–108 Association of Official Analytical Chemist (AOAC) (2006) Official Methods of Analysis of AOAC International AOAC International, Gaithersburg, MD, USA Baker, R & G€ unther, C (2004) The role of carotenoids in consumer choice and the likely benefits from their inclusion into products for human consumption Trends Food Sci Technol., 15, 484–488 Bazyar Lakeh, A.A., Ahmadi, M.R., Safi, S., Ytrestøyl, T & Bjerkeng, B (2010) Growth performance, mortality and carotenoid pigmentation of fry offspring as affected by dietary supplementation of astaxanthin to female rainbow trout (Oncorhynchus mykiss) broodstock J Appl Ichthyol., 26, 35–39 Bell, J.G., McEvoy, J., Tocher, D.R & Sargent, J.R (2000) Depletion of tocopherol and astaxanthin in Atlantic salmon (Salmo salar) affects autoxidative defense and fatty acid metabolism J Nutr., 130, 1800–1808 Breithaupt, D.E (2007) Modern application of xanthophylls in animal feeding - a review Trends Food Sci Technol., 18, 501–506 Chen, L.P & Huang, C.H (2011) Effects of dietary b-carotene levels on growth and liver vitamin A concentrations of the softshelled turtle, Pelodiscus sinensis (Wiegmann) Aquacult Res., 42, 1848–1854 Christiansen, R., Lie, O & Torrissen, O.J (1995) Growth and survival of Atlantic salmon, Salmo salar L., fed different dietary levels of astaxanthin First-feeding fry Aquacult Nutr., 1, 189–198 Commission Internationale de l’ Eclairage (CIE) (1978) Recommendations on Uniform Color Spaces, Color Difference Equations, Psychometric Color Terms Supplement No to CIE Publication No 15, Colorimetry, Bureau Central de la CIE, Paris Ding, X.F., Ye, Y.T., Jiang, R., Cai, C.F., Zhu, G.Y & Wang, J.T (2010) Effects of feed pigments on carotenoids, lutein content and tyrosinase activity in the skin and serum of Pelteobagrus fulvidraco J Fish China, 34, 1728–1735 Aquaculture Nutrition 19; 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280, 206–210 Yang, Z.C., Niu, C.J & Sun, R.Y (1999) Review of the biology study on Chinese soft-shelled turtle Chin J Zool., 34, 41–44 Yuangsoi, B., Jintasataporn, O., Areechon, N & Tabthipwon, P (2011) The pigmenting effect of different carotenoids on fancy carp (Cyprinus carpio) Aquacult Nutr., 17, e306–e316  I., SergejevovA,  M., Urban, J., Vachta, R & STys,   ZaTKov A, D & MasojIDek, J., (2011) Carotenoid-enriched microalgal biomass as feed supplement for freshwater ornamentals: albinic form of Wels catfish (Silurus glanis) Aquacult Nutr., 17, 278–286 Aquaculture Nutrition doi: 10.1111/anu.12040 1 2013 19; 946–954 1,2,3 Program in Biotechnology, Chulalongkorn University; Department of Microbiology, Chulalongkorn University; The Center of Excellence for Marine Biotechnology (CEMB), Faculty of Science, Chulalongkorn University, Bangkok, Thailand The effect of Bacillus S11 (BS11)- and/or Bacillus P11 (BP11)-supplemented feeds on the growth performance, survival, immunoenhancement and disease resistance of cultured Pacific white shrimp, Litopenaeus vannamei, was evaluated Four feeding treatments of (i) regular feed (control), (ii) BS11-supplemented feed, (iii) BP11-supplemented feed and (iv) BS11-and BP11-supplemented feed were prepared and used for shrimp cultivation in closed recirculating cement tanks (~400 L) in two trials, one for juvenile and PL-30 shrimp at 60 and 90 days, respectively The results showed that BS11 gave a higher probiotic potential than BP11 for both age groups of L vannamei in cultivation, because the average weight and survival of shrimp fed BS11-supplemented feed were significantly higher (P < 0.05) than those of the control and the other two groups The survival of shrimp fed either BS11-or both BS11-and BP11-supplemented feed was significantly higher (P < 0.05) than that of the control group In addition, the highest total haemocyte and granular haemocyte counts and phenoloxidase activity were found in shrimp fed with the BS11-supplemented feed After challenge with Vibrio harveyi 639 (~107 CFU mLÀ1) by immersion, the lowest cumulative death (%) and disease resistance were clearly found in shrimp fed with the BS11-supplemented feed KEY WORDS: Bacillus P11, Bacillus S11, Bacillus subtilis, Litopenaeus vannamei, Pacific white shrimp, probiotic bacterium Received September 2012; accepted 20 December 2012 Correspondence: Sirirat Rengpipat, Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand E-mail: sirirat@sc.chula.ac.th During 2004–2008, the great loss of the disease-related black tiger shrimp, Penaeus monodon Fabricus (Decapoda: Penaeidae), caused a change in the major farmed species from P monodon to the Pacific white shrimp, Litopenaeus vannamei (Decapoda: Penaeidae), a prominent species from the western hemisphere Currently, L vannamei are the most cultivated and exported shrimp from Thailand and are economically important For example, in the first quarter of 2010, the revenue from exported Thai shrimp exceeded US$ 628 million was reported (Fisheries statistics of Thailand (2001–2010) However, the drawback of the lower shrimp yield per crop obtained from L vannamei aquaculture has recently been compounded with several problems in the shrimp aquaculture industry, such as a lack of efficient broodstocks, feed and environmental management (Valderrama & Engle 2004; Alam et al 2007; Boyd et al 2008; Leung & Dudgeon 2008) The limited genetic diversity may adversely affect the physiology and health of L vannamei Also, juvenile shrimp can easily become ill from chronic infection of various pathogens in situ during their culture in earthen ponds (Brock & LeaMaster 1992; Owens et al 1998; Lightner 2003; NACA & FAO 2008a,b, 2009a,b; Flegel 2009; Walker & Winton 2010) Antibiotics have been used as either growth promoters or therapeutic agents for aquaculture since 1950 However, prolonged antibiotic use causes antibiotic residues in the shrimp meat is a current serious concern (Schwarz et al 2001; Akinbowale et al 2006) In addition, there are many reports illustrating the possibility of transferring resistant genes amongst bacteria (Van den Bogaard & Stobberingh 2000; Witte 2000; Schwarz et al 2001; Holmstrom et al 2003) Food safety starting from the raw materials of the food chain is required Protein supply from ª 2013 John Wiley & Sons Ltd shrimp is still a major source of dietary protein for human consumption due to the rapid increase in the human population Hence, alternative methods to prevent the deleterious effects of pathogen infections in aquaculture by the application of probiotics have been practiced and are now accepted as biological agents Probiotic microorganisms are living forms which, when are consumed in adequate amounts, confer a health benefit for the host (Reid et al 2003) Bacillus S11 (BS11) (Rengpipat et al 1998; Powedchagun et al 2011) and Bacillus P11 (BP11) (Utiswannakul et al 2011), both designated as members of Bacillus subtilis, were originally isolated from healthy P monodon broodstocks’ intestines and have been demonstrated to reduce the mortality of P monodon after challenge with the luminescent-causing pathogenic bacteria, Vibrio harveyi BS11 and BP11 are a part of the intestinal bacterial community of P monodon and are grouped as an autochthonous strain and recognized as bacterial probiotics Moreover, after the administration of BS11 or BP11, the growth and survival of P monodon shrimp in aquaculture were improved, whilst their immune ability was enhanced (Rengpipat et al 2000; Utiswannakul et al 2011) Therefore, the objectives of this study were to evaluate the effects of BS11 and BP11, established probiotics of P monodon, on the growth performance, survival, immunoenhancement and disease resistance of the Pacific white shrimp L vannamei, as both a commercially important aquaculture species and a representative of a phylogenetically different but related genus to start to examine how broad the probiotic effects might be Bacillus S11 and Bacillus P11 previously isolated from the gastrointestinal tract of a P monodon broodstock caught in the Gulf of Thailand and the Andaman sea of Thailand, respectively Both demonstrated to be effective probiotics for P monodon (Rengpipat et al 1998, 2000; Utiswannakul et al 2011) and a member of Bacillus subtilis (Powedchagun et al 2011; Utiswannakul et al 2011) were prepared by culturing in tryptic soy broth (TSB; Difco, Sparks, MD, USA) with a shaking incubator at 200 g, 37 °C for 24 h V harveyi 639, a pathogenic strain isolated from P monodon dying of luminescent disease, was kindly provided by the Shrimp Culture Research Center, Charoen Pokphan Feedmill, Samutsakorn, Thailand It was cultured in TSB or tryptic soy agar (TSA, Difco, Sparks, MD, USA) con- Aquaculture Nutrition 19; 946–954 ª 2013 John Wiley & Sons Ltd taining 2% (w/v) NaCl at 30 °C for 24 h Identity of V harveyi 639 was confirmed as described previously (Baumann & Schubert 1984) Presumptive concentrations, as colony-forming units (CFU) mLÀ1, of Vibrio sp P were determined using spread plates of thiosulphate citrate bile sucrose agar (TCBS, Difco, Sparks, MD, USA) All bacteria were routinely checked for their purity and characterized and identified using the database APILAB plus, comprised of the API 50 CHE and API 20 E strips test (bioMerieux, Marcy-I’Etoile, France) Formulated shrimp feed consisted of (on a dry weight basis) 220 g kgÀ1 ground fish, 40 g kgÀ1 ground shrimp, 300 g kgÀ1 ground soy bean, 60 g kgÀ1 wheat gluten, 40 g kgÀ1 ground squid, 30 g kgÀ1 fish oil, 10 g kgÀ1 cholesterol, 10 g kgÀ1 lecithin, 20 g kgÀ1 vitamin complex (Complet DV; Codel (Thailand) Co Ltd Nonnthaburi, Thailand), 20 g kgÀ1 minerals complex (Cal-Plus; Codel (Thailand) Co Ltd.), 230 g kgÀ1 wheat flour and 20 g kgÀ1 cellulose These ingredients were mixed and extruded, heated at 110 °C for 10 and then kept at 80 °C for 60 After this, the diet was kept at 20 °C until use (maximum storage time of 2–3 weeks) Proximate compositions of the feeds (by dry weight) were protein 360 g kgÀ1, fat 60 g kgÀ1, carbohydrate 335 g kgÀ1, crude fibre 54 g kgÀ1, ash 73 g kgÀ1 and moisture 118 g kgÀ1 After incubation at 37 °C for 24 h with shaking at 200 g, fresh BS11 or BP11 cells (~1010 CFU gÀ1) were centrifuged and washed three times with sterile normal saline solution (NSS) and wet cells thoroughly mixed with feed at : and : 4, respectively (~2.5% of bacteria by weight), and then air-dried for 1–2 h at 28 °C and stored in clean, plastic bags at °C until use Shrimp feed was prepared twice weekly, and each batch was analysed for BS11, BP11 and total bacterial counts (as CFU gÀ1) on TSA after incubated at 37 °C for 24 h Juvenile or postlarva (PL-15) L vannamei were obtained from a shrimp farm in Pathumthani and Chachoengsao provinces, Thailand, respectively Shrimp were acclimatized in concrete tanks with flat bottoms (each measuring 80 74 87 cm (width x length x height)) with a closed recirculating water system of 400 L for ~14 days during which time they were fed regular feed three times daily at a dose of 5% body weight per day (Menasveta et al 1989) The culture water salinity level was initially at 20 mg LÀ1 Shrimp were placed randomly in test tanks, which were covered afterwards to reduce the light intensity The initial juvenile shrimp weights of the four treatment groups were regular feed (control; 2.44 Æ 0.52 g), BS11-supplemented feed (2.39 Æ 0.54 g), BP11-supplemented feed (2.48 Æ 0.52 g) and BS11- and BP11-supplemented feed (2.49 Æ 0.49 g), with no significant weight difference (P ! 0.05) between the four groups Duplicated tanks (50 shrimp per tank) of each individual treatment group were performed For the postlarva shrimp, the initial postlarvae shrimp (PL-30) weights of the three treatments were not significantly different, being regular feed (control; 0.28 Æ 0.09 g), BS11-supplemented feed (0.28 Æ 0.08 g) and BS11-and BP11-supplemented feed (0.29 Æ 0.09 g) Individual treatments were comprised of three replicates, and each tank contained 70 shrimp Shrimp were fed three times daily at 5% their wet body weight per day Water samples (100 mL) were collected from the centre of each tank, along with shrimp faeces (~100 mg), and one live shrimp every 30 days for bacterial determination, starting from the first day of the feeding trials Water quality was monitored in terms of the pH, dissolved oxygen, temperature, salinity, ammonium, nitrite and alkalinity levels, as described by Strickland & Parsons (1972) Shrimp were dissected using surgical scissors, and the hepatopancreas and intestines were removed for microbial enumeration and identification Bacterial determinations were made using serial dilution in sterile NSS, followed by plating on plate TSA (with 10 g LÀ1 NaCl) and TCBS After 24–48 h of incubation at 37 °C, colonies were counted and recorded Bacteria were re-examined using Gram staining, spore staining and selected biochemical tests as described by Sneath (1986) In both experiments, the shrimp live weight and survival were measured every 30 days for 60 (Juvenile shrimp) or 90 (PL-30 shrimp) days At the end of the cultivation period, on day 60 (Juvenile shrimp) or 90 (PL-30 shrimp), the immunity indices from the shrimp’s haemolymph were determined Haemolymph from each shrimp was collected from the ventral sinus cavity using a 26 1/2-gauge needle and a 1-mL syringe containing 40 g LÀ1 sodium citrate as anticoagulant solution Haemolymph (0.2 mL) was collected from nine randomly selected shrimp, three shrimp per tank, into anticoagulant solution at a : (v/v) ratio and then mixed gently A 20-uL suspension was applied to the haemocytometer, and the total number of haemocytes and granular haemocytes were counted using light microscopy at 400 magnification and calculated as the number of cells mLÀ1 haemolymph Haemolymph mixed with anticoagulant solution a : (v/ v) ratio was centrifuged at 1,100 g/min at o C for 10 The haemocyte pellet was collected and suspended in 500 lL cacodylate buffer (CAC buffer) (Smith & Soderh€ all 1991), then sonicated for 10 s and centrifuged at 20,600 x g/min at °C for 20 The haemocyte lysate supernatant (HLS) was collected for determination of the haemocyte phenoloxidase activity L-3,4-dihydroxyphenylalanine (L-DOPA; Sigma) was used as substrate (Soderh€ all 1981), and trypsin, as the elicitor (Smith & Soderh€ all 1991) A total of 200 uL of HLS was incubated with 200 uL of g LÀ1 trypsin (1000–2000 unit mgÀ1) (SigmaAldrich, St Louis, MO, USA) in CAC buffer at room temperature for 30 and added with 200 uL of L-DOPA (3 g LÀ1 in CAC buffer).Each reaction mixture was further diluted with 600 uL of CAC buffer and mixed, and the optical density was measured at 490 nm Absorbance measurements were made against a blank consisting of CAC buffer, L-DOPA and elicitor to control for spontaneous oxidation of the substrate alone One unit (U) of enzyme activity was defined as an increase in absorbance of 0.001 minÀ1 mg proteinÀ1 (Soderh€ all & Unestam 1979) The protein content in the HLS was measured by the Bradford method (Bradford 1976), using bovine serum albumin as the standard protein After 60 and 90 days of shrimp culture (for juvenile and PL-30 shrimp, respectively), shrimp were collected randomly from each treatment and were transferred to fibreglass tanks (~40 L) at 10 shrimp per tank Each challenge test used a factorial design and so included 24 or 18 aquaria for juvenile and PL-30 shrimp, respectively, with Aquaculture Nutrition 19; 946–954 ª 2013 John Wiley & Sons Ltd triplicate replicates of individual treatments Shrimp were immersed in a V harveyi 639 suspension at ~107 CFU mLÀ1 with no water exchange for 3–5 days During these immersion tests, the tank water and dead shrimp were collected and examined for the presence of V harveyi every day from each tank V harveyi from the hepatopancreas and intestine of each shrimp were isolated and identified by the characteristic of green and luminescent colonies on TCBS, Gram staining and oxidase test These were compared with the original V harveyi 639 isolate, to confirm the V harveyi strain The effect of BS11 and/or BP11 on the shrimp growth, survival and V harveyi 639 disease resistance was evaluated using analysis of variance (ANOVA) and Duncan’s multiple range test at P < 0.05 (Statistical Analysis System 1983) Differences were considered significant when P < 0.05 After 30 and 60 days of culture, the live weight of the juvenile shrimp fed the BS11-supplemented feed was significantly higher (P 0.05) from that of the control or two other supplemented feeds (Fig 1a), and after 60 days, the live weight of the shrimp fed with BS11-supplemented feed ranged from 1.52-fold higher than the control group to 1.46- and 1.40-fold higher than the BP11 and BP11 plus (a) BS11 feed groups, respectively (data not shown) Concurrently, the highest survival rate was found in shrimp fed with BS11-supplemented feed (P 0.05), although this was not statistically higher than that for shrimp fed with the BS11-and BP11-supplemented feed (Fig 2a) No significant difference (P ! 0.05) in the total weight or survival of shrimp fed with either regular (control) or BP11-supplemented feed were found The apparent probiotic effect of BS11 on L vannamei was then assayed on younger shrimp, PL-30 (initial weight of ~0.28–0.29 g) for 90 days However, the BP11-supplemented feed was omitted due to the low probiotic response seen in the juvenile shrimp, and so only three treatment groups were evaluated After 90 days of culture, the same trend of results as that seen for the juvenile shrimp was found That is, the most pronounced total weight as well as the highest survival level was detected in shrimp fed with the BS11-supplemented feed (Figs 1b and 2b) The total weight was significantly higher (P 0.05) than that in the control or the BS11-and BP11-supplemented feed groups at all 30-day periods (30, 60 and 90 days culture treatment) Although shrimp fed with the BS11- and BP11-supplemented feed showed a higher total weight and survival after V harveyi exposure than the control shrimp (P 0.05), it was not statistically significant (except for the total weight at 90 days) and was significantly lower than that seen in the BS11-fed shrimp (Figs 1b and 2b) These results clearly indicated that BS11 isolated from P monodon guts can act as an effective probiotic bacterium for L vannamei as well as in the previously reported P monodon (Rengpipat et al 1998, 2000) Whether this (b) Figure Live weight (g) over each 30-day period of juvenile L vannamai (a) fed with regular feed (control), BS11-, BP11- or BS11- and BP11-supplemented feeds after 60 days of culture, or PL-30 L vannamai (b) fed with regular feed (control), BS11-supplemented or BS11and BP11-supplemented feeds for 90 days of culture Data are shown as the mean Æ SD and are derived from (a) 50 or (b) 90 shrimp Means not sharing the same lowercase letter at the same time of culture are significantly different (P 0.05) Aquaculture Nutrition 19; 946–954 ª 2013 John Wiley & Sons Ltd (a) (b) Figure Survival (%) of (a) juvenile L vannamai fed with regular feed (control), BS11-, BP11-supplemented or BS11- and BP11-supplemented feeds after 60 days of culture, and (b) PL-30 L vannamai fed with regular feed (control), BS11-supplemented or BS11- and BP11supplemented feeds after 30, 60 or 90 days of culture Data are shown as the mean Æ SD and are derived from (a) duplicate or (b) triplicate independent repeats Means not sharing the same lowercase letter at the same time of culture are significantly different (P 0.05) reflects the close relationship between the genera Penaeus and Litopanaeus, which are both classified in the Family Penaeidae (Perez Farfante & Kensley 1997), or a broader probiotic activity of BS11 is unclear and awaits further testing However, it clearly does not hold for BP11 as previously reported to be an active probiotic in P monodon (Rengpipat et al 2000); it appears to potentially not be effective in L vannamei In addition, these results clearly support the possibility of using an allochthonous strain as a probiotic, as documented earlier that Lactobacillus plantarum MRO3.12 from the digestive tract of wild P merguiensis (Kongnum & Hongpattarakere 2012), Bacillus subtilis E20 from fermented soybean (Tseng et al 2009) and Lactobacillus plantarum 7–40 (NTU102) from homemade Korean-style cabbage pickles (Chiu et al 2007) could all increase the survival, disease resistance and immune ability of L vannamei However, the residential time of individual probiotic bacteria strains, as well as potentially competitive mixed strains, in the shrimp guts after a single or multiple meal consumption should be evaluated to compare their efficiency for use From this study, BS11 appeared to provide a better probiotic function for L vannamei than BP11 Moreover, an enhanced effect of BS11 on the shrimp growth was found using the BS11-and BP11-supplemented feed This could be due to the quicker specific growth rate of BS11 (0.9 hÀ1) compared with BP11 (0.83 hÀ1) (data not shown) Likewise, BS11 could retard BP11 growth in the shrimp gut and was observed to clearly grow faster when their coculture was evaluated in vitro (data not shown) Therefore, prior to the use of mixed probiotic cultures, the ability of each strain to stay alive and be maintained at least at the minimal required level in the host animal for optimal probiotic activity should be examined and evaluated for each proposed combination The water quality values for the shrimp cultures in the concrete tanks were similar for ammonium (0.0–0.5 mg LÀ1), alkalinity (110–130 mg LÀ1), dissolved oxygen (6.1–6.7 and 6.1–6.9 mg LÀ1), nitrite (0.0–0.5 mg L À1), pH (7.89–8.4 and 7.7–8.4) and salinity (19–20 and 18–20 mg LÀ1) levels, but were slightly different for temperature (28.8 –29.8 and 25.3–29.9 °C) However, all of these water quality parameters are considered safe for shrimp culture (Menasveta et al 1989; Boyd 1990) BS11 and/or BP11 at ~10 CFU mLÀ1 was found in all culture water taken from the concrete tanks of shrimp fed with BS11-, BP 11or BS11- and BP11-supplemented feed in both experiments during the shrimp culture period, compared with less than ~10 CFU mLÀ1 of Bacillus spp in the culture water from the control group (data not shown) After challenge with V harveyi 639 (~107 CFU mLÀ1) by immersion for days, the cumulative mortality (%) of 60-day cultured juvenile shrimp was high for shrimp fed the regular feed (control; 55.0 Æ 7.1%) and lower in the shrimp fed with the BP11-supplemented feed (42.5 Æ 10.6%) (Fig 3a) However, at the same time point, the mortality was significantly lower in the shrimp fed with the BS11-and BP11 (35.0 Æ 7.1%)-supplemented and BS11 (20.0 Æ 0.0%)-supplemented feed, respectively (Fig 3a) The cumulative mortality then increased more markedly Aquaculture Nutrition 19; 946–954 ª 2013 John Wiley & Sons Ltd over the next two days in the control and BP11-supplemented feed fed shrimp, reaching 100% and ~83.3%, respectively, compared with the much lower mortality level and rate of increase seen in shrimp fed the BS11-and BP11supplemented and BP11-supplemented feed at ~56.7% and ~30% mortality, respectively The same trend in the cumulative mortality following challenge with V harveyi was also observed in the 90-day cultured PL-30 shrimp, where three days after challenge a cumulative mortality of 53.3 Æ 5.8%, 40.0 Æ 10.0% and 23.3 Æ 5.8% in the shrimp fed regular feed (control), BS11-and BP11-supplemented and BS11-supplemented feed, respectively (Fig 3b) Both the 60-day cultured juvenile shrimp and the 90-day cultured PL-30 shrimp that were age groups fed regular and probiotic feeds and then mock challenged (no V harveyi in the tanks) were healthy, and no mortality was detected over the same time five-day time period (data not shown) Amongst all the treatments in PL-30 shrimp, the lowest Vibrio spp (CFU g À1) in the guts of shrimp after challenge by V harveyi 639 for days was found in the BS11supplemented feed group (Table 1) These indicated the ability of BS11 to compete and stay on the gut surface of shrimp Before and after challenge of L vannamei by V harveyi 639, the total haemocyte and granulocyte counts as well as the phenoloxidase activity of shrimp fed with the BS11-supplemented feed were significantly higher (P < 0.05) than (a) Table Bacteria counts (CFU gÀ1) in the guts of 90-day cultured PL-30 L vannamei after challenge by V harveyi 639 for days Bacteria counts (x 106 CFU gÀ1)a Feed type Vibrio spp Regular BS11-supplemented BS11- & BP11-supplemented 3.73 Æ 0.60 1.15 Æ 0.06 2.22 Æ 0.02 Probiotic Bacillus ND 1.18 Æ 0.03b 0.13 Æ 0.003b5 g) (Fig 4e), whereas the total haemocyte and granular haemocyte levels are more influenced in the younger shrimp (live weight of < g) (Fig 4b,d) The effects of BS11 were found to share several modes of action on the Pacific white shrimp, L vannamei, as that reported previously, such as an increase in the growth and survival rates (Rodrıguez et al 2007; Wang 2007; G omez et al 2008; Zhou et al 2009; Yang et al 2010), competitive exclusion of pathogenic bacteria Figure Mean total haemocyte (a, b) and granular haemocyte (c, d) counts (cell mLÀ1) and phenoloxidase activity (U minÀ1 mg proteinÀ1) (e, f) before and after challenge for days by Vibrio harveyi 639 of juvenile L vannamai after fed regular feed (control), BS11-, BP11- or BS11- and BP11-supplemented feeds after 60 days of culture, or PL-30 L vannamai after fed with regular feed (control), BS11-supplemented or BS11- and BP11-supplemented feeds for 90 days of culture Data are shown as the mean Æ SD and are derived from nine shrimp Means not sharing the same lowercase letter at the same time of culture are significantly different (P 0.05).* indicates significant difference at P 0.05 between the same treated shrimp group before and after challenge by Vibrio harveyi 639 (Balc azer et al 2007; Tseng et al 2009), enhancement of the immune response of the host shrimp species (Gullian et al 2004; Chiu et al 2007; Li et al 2009) and improving its intestinal microbial balance (Thompson et al 1999) In addition, as a member of Bacillus subtilis, BS11 is classified as a risk group I microorganism that is not known to cause disease in humans or adverse effects on the environment (U.S Department of Health & Human Services 1986; Frommer et al 1989) Therefore, as well as their safe properties for use in P monodon, as previously reported (Powedchagun et al 2011), it is possible to use BS11 as an alternative safe probiont for L vannamei However, to evaluate their possible utilization for industrial shrimp farming, field trials in earthen ponds should be performed Aquaculture Nutrition 19; 946–954 ª 2013 John Wiley & Sons Ltd Bacillus subtilis (BS11)-supplemented feed could clearly provide some benefits in terms of the growth performance, survival, immunoenhancement and disease resistance of the Pacific white shrimp, Litopenaeus vannamei The authors sincerely thank Robert Butcher for revising the manuscript and providing many valuable suggestions and comments This research was supported by the Thai Government Stimulus Package (TKK 2555) under the Project for Establishment of Comprehensive Center for Innovative food, Health products and Agriculture (PERFECTRA); the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission (FW643A); and CU Graduate School Thesis Grant from Chulalongkorn University Akinbowale, O.L., Peng, H.H & Barton, M.D (2006) Antimicrobial resistance in bacteria isolated from aquaculture sources in Australia J Appl Microbiol., 100, 1103–1113 Alam, S.M.N., Pokrant, B., Yakupitiyage, A & Phillips, M.J (2007) Economic returns of disease affected extensive shrimp farming in southwest 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ỉverland, M., Sứrensen, M., Storebakken, T., Penn, M., Krogdahl,  A & Skrede, A (2009) Pea protein concentrate substituting fish meal or soybean meal in diets for Atlantic salmon (Salmo salar)Effect on growth performance, nutrient digestibility, carcass composition, gut health, and physical feed quality Aquaculture, 288, 305311 Aquaculture Nutrition 19;... salmonid feeds Aquaculture, 159, 177202 Trater, A.M., Alavi, S & Rizvi, S.S.H (2005) Use of non-invasive X-ray microtomography for characterizing microstructure of extruded biopolymer foams Food Res Int., 38, 709719 Warburton, S.C., Donald, A.M & Smith, A.C (1992) Structure and mechanical properties of brittle starch foams J Mater Sci., 27, 14691474 Aquaculture Nutrition doi: 10.1111/anu.12030 1,2 2013 19;... edls@mail.sysu.edu.cn; ganlian@ scau.edu.cn Grass carp (Ctenopharyngodon idella) has a long history in aquaculture and is one of the most important species cultured in inland water bodies in China After silver carp, grass carp currently has the largest production in freshwater aquaculture globally It constitutes 7.18% of the world aquaculture production (FAO 2010) Low dissolved oxygen is a type of stress frequently... (Oncorhynchus mykiss) for maximum growth Aquaculture, 235, 569586 FAO ed (2010) 2008 FAO Year Book Annuaire:Fishery and Aquaculture Statistics FAO, Rome Foss, A., Vollen, T & Iestad, V (2003) Growth and oxygen consumption in normal and O2 supersaturated water, and interactive effects of O2 saturation and ammonia on growth in spotted wolffish (Anarhichas minor Olafsen) Aquaculture, 224, 105116 Furuya, W.M.,... bidyanus: I Digestibility of alternative ingredients Aquaculture, 1 86, 293 310 Anderson, R.A., Conway, H.F & Peplinski, A.J (1970) Gelatinization of corn grits by roll cooking, extrusion cooking and steaming Starch - St arke, 22, 130135 Badrie, N & Mellowes, W.A (1991) Texture and microstructure of cassava (Manihot esculenta Crantz) flour extrudate J Food Sci., 56, 13191322 Barrett, A.H & Ross, E.W (1990) Correlation... and intraperitoneal fat White muscle from both sides of the fillets without skin and liver were dissected and frozen immediately in liquid nitrogen and stored at 70 C until Aquaculture Nutrition 19; 860869 ê 2013 John Wiley & Sons Ltd used The plasma was separated by centrifugation and also stored at 70 C until analysed Diets and fish samples (including white muscle and liver) were analysed... separation Treatment effects and interactions were considered significant at P < 0.05 Mean total ammonia nitrogen concentrations under HO and LO groups were 0.61 ặ 0.15 mg L1 and Aquaculture Nutrition 19; 860869 ê 2013 John Wiley & Sons Ltd 0.59 ặ 0.11 mg L1 respectively Total ammonia nitrogen concentrations in high oxygen tanks were higher than in low oxygen tanks, but there was no difference... level The results indicated that AST, ALT, TG, GLU, urea and GLDH of grass carp fed at low dissolved oxygen were significantly higher than those of grass carp fed at high dis- Aquaculture Nutrition 19; 860869 ê 2013 John Wiley & Sons Ltd 4.06 18.9 4.59 366 2.74 85.6 1.48 28.7 1.99 179 IBW FBW FI WG SGR Survival FCR NR PER LR ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.02 1.17 0.28 37.6 0.14 7.29 0.08B 4.42 0.29 4.36... (g)/lipid fed (g) 13 Oxygen level Lysine level g kg1 LO Table 3 Effect on growth and nutrients retention in grass carp with supplemental lysine at different oxygen levels Aquaculture Nutrition 19; 860869 ê 2013 John Wiley & Sons Ltd 13 LO 10.4 2.90 2.57 2.02 0.20 0.17 0.13 0.02 10.3 2.99 2.45 1.95 0.19 0.17 0.18 0.03 ặ ặ ặ ặ 1.7 2.6 0.4 0.4 ặ ặ ặ ặ ặ ặ ặ ặ 644 ặ 0.3 82.5 ặ 6.0 95.0 ặ 0.4