Aquaculture Volume 354-355 Issue None 2012 (Doi 10.1016/j.aquaculture.2012.04.028) Zhi Yong Ju Dong-Fang Deng Warren Dominy - A Defatted Microalgae (Haematococcus Pluvialis) Meal As A
Defatted microalgae meal as a protein ingredient to partially replace fishmeal in diets of Pacific white shrimp. DMM was a by-product of astaxanthin production from Haematococcus pluvialis and contained 40.3% crude protein and 0.9% crude lipid. After an 8-week feeding trial, shrimp fed the diet with 12.5% of the fishmeal protein replaced showed a significantly higher growth rate and lower feed conversion ratio.
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Aquaculture Volume 354-355 Issue None 2012 [Doi 10.1016%2Fj.aquaculture.2012.04.028] Zhi Yong Ju; Dong-Fang Deng; Warren Dominy -- A Defatted Microalgae (Haematococcus Pluvialis) Meal as A
Defatted microalgae meal as a protein ingredient to partially replace fishmeal in diets of Pacific white shrimp. DMM was a by-product of astaxanthin production from Haematococcus pluvialis and contained 40.3% crude protein and 0.9% crude lipid. After an 8-week feeding trial, shrimp fed the diet with 12.5% of the fishmeal protein replaced showed a significantly higher growth rate and lower feed conversion ratio.
Aquaculture Volume 354-355 Issue None 2012 (Doi 10.1016/j.aquaculture.2012.04.028) Zhi Yong Ju Dong-Fang Deng Warren Dominy - A Defatted Microalgae (Haematococcus Pluvialis) Meal As A
Defatted microalgae meal as a protein ingredient to partially replace fishmeal in diets of Pacific white shrimp. DMM was a by-product of astaxanthin production from Haematococcus pluvialis and contained 40.3% crude protein and 0.9% crude lipid. After an 8-week feeding trial, shrimp fed the diet with 12.5% of the fishmeal protein replaced showed a significantly higher growth rate and lower feed conversion ratio.
A defatted microalgae (Haematococcus pluvialis) meal as a protein ingredient to
partially replace shmeal in diets of Pacic white shrimp (Litopenaeus vannamei,
Boone, 1931) Zhi Yong Ju , Dong-Fang Deng, Warren Dominy Aquatic Feeds and Nutrition Department, Oceanic Institute, 41202 Kalanianaole Hwy, Waimanalo, Hawaii 96795, USA a b s t r a c t a r t i c l e i n f o Article history: Received 21 July 2011 Received in revised form 5 April 2012 Accepted 10 April 2012 Available online 2 May 2012 Keywords: Microalgae Fishmeal replacement Shrimp Growth Pigmentation This trial evaluated the effects of partial replacement of shmeal protein by a defatted microalgae meal (DMM) in a shrimp diet. The DMM was a by-product of astaxanthin production from Haematococcus pluvialis and contained 40.3% crude protein (CP) and 0.9% crude lipid (CL). Test diets were prepared by using DMM (3, 6, 9 and 12% in a diet) to replace 12.5%, 25%, 37.5% or 50% of a shmeal protein in a control diet (32.3% CP & 8.9% CL). All test diets had similar protein and lipid levels. Each diet was randomly assigned to four tanks (12 juvenile shrimp per tank) in an indoor ow through seawater laboratory. After an 8-week feeding trial, shrimp fed the diet with 12.5% of the shmeal protein replaced showed a signicantly higher growth rate and lower feed conversion ratio than the shrimp fed the control diet (Pb0.05). The other three DMM- added diets had a similar effect on the growth of shrimp as the control diet (P>0.05). Shrimp fed the four DMM-added diets appeared redder and contained higher free and esteried astaxanthins than shrimp fed the control diet. The results indicated that DMM could be a valuable alternative protein and pigmentation ingredient in shrimp feed. 2012 Elsevier B.V. All rights reserved. 1. Introduction Many species of microalgae have been used to produce nutraceutical or biofuel products (Acien-Fernandez et al., 2003; Mata et al., 2010; Singh and Gu, 2010). For example, Haematococcus pluvialis (astaxanthin), Dunaliella salina (-carotene) and Muriellopsis sphaerica (lutein) have been used for carotenoid production; Odeontella aurita, Phaedactylum tricomutum and Isochrysis galbana have been used for algae oil or omega-fatty acid production; and Chlorella, Nannochloropsis, Chaetoceros and Dunaliella species have been used for biodiesel production or development (Acien-Fernandez et al., 2003; Jin and Melis 2003; Mata et al., 2010). These production activities can result in huge amounts of defatted microalgae by-products. The defatted microalgae meal (DMM) may contain many valuable nutrients for animal feed, such as proteins, carbohydrates, minerals, water-soluble vitamins, and bioactive compounds even though most of the lipid and lipid-soluble nutrients have been removed (Ju et al., 2009). Researchers (Cuzon et al., 1981; Hanel et al., 2007; Ju et al., 2009) found that microalgae proteins could be promising substitutes for shmeal protein or as a valuable additive in aquafeeds. Application of many species of microalgae in shrimp feed is still in the research stage and only a few have been tested in shrimp trials (Cuzon et al., 1981; Hasan and Chakrabarti, 2009; Ju et al., 2009). The feeding trials on shrimp revealed that some microalgae had enhancing growth effects. Cuzon et al. (1981) reported that lipid free fraction of Spirulina meal promoted growth of Penaeus japonicas and suggested that the microalgae might contain an unknown growth factor. Nakagawa and Gomez-Diaz (1995) found that whole Spirulina meal signicantly improved growth, survival and feed utilization in giant freshwater shrimp (Macrobrachium rosenbergii). They suggested that the improved growth and feed utilization was probably due to the enhancement of protein assimilation. Ju et al. (2009) reported enhanced shrimp growth by inclusion of 9% whole microalgae meal of Thalassiosira weissogii or of Nannochloropsis in the control diet. The same study found that it was the defatted meal fraction not the lipid fraction fromboth microalgae that resulted in enhanced shrimp growth. The enhanced shrimp growth effects were not attributed to the contribution of macronutrients from whole or defatted microalgae, as their dietary inclusions decreased crude proteinandcrude lipidcontents and gross energy value (Ju et al., 2008, 2009). It is unclear what factors or compounds contained in the microalgae played a role in improving shrimp growth. In feeding trials with sh, however, microalgae have been found to increase sh digestibility, physiological activity, stress response, starvation tolerance, disease resistance, and carcass quality (Becker, 2004; Mustafa and Nakagawa, 1995). Shrimp are omnivorous organisms and microalgae are their natural foods (Moss et al., 2006; Otoshi et al., 2001; Takeuchi et al., 2002). H. pluvialis meal or its DMM, has never been investigated as Aquaculture 354355 (2012) 5055 Corresponding author. Tel.: +1 808 259 3128; fax: +1 808 259 7936. E-mail address: zyju@oceanicinstitute.org (Z.Y. Ju). 0044-8486/$ see front matter 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.04.028 Contents lists available at SciVerse ScienceDirect Aquaculture j our nal homepage: www. el sevi er . com/ l ocat e/ aqua- onl i ne a protein ingredient in shrimp feed. However, H. pluvialis meal has frequently beenaddedindiets to test for pigmentationof shrimp, salmon and trout (Chien and Shiau, 2005). Astaxanthin production from this microalga has reached industrial scale worldwide (Krichnavaruk et al., 2008). A company in Hawaii annually produces 70 tons of H. pluvialis meal. The H. pluvialis meal is usually treated with supercritical CO 2 to extract the fat-soluble astaxanthin for humannutraceutical andcosmetic applications due to its strong antioxidant properties (Guerin et al., 2003). The remaining DMM (>80% biomass) has been treated as a waste by-product (Krichnavaruk et al., 2008). The extraction method using CO 2 does not contaminate the by-product and no toxic chemicals or toxic effects have been reported for the H. pluvialis algae in animal applications (Satoh et al., 2009). Therefore, DMM may have potential as an alternative protein ingredient to replace shmeal in shrimp feed. Additionally, the left-over astaxanthin in DMM may have a pigmentation effect on shrimp. The objective of this study is to determine the effects of partial replacement of shmeal with the defatted H. pluvialis meal on growth performance, nutritional composition and pigmentation of juvenile shrimp. 2. Materials and methods 2.1. Dry microalgae meal (DMM) and chemicals The DMM was obtained from Cyanotech Corporation (Kona, Hawaii, USA). It is an industrial by-product from dried H. pluvialis after extraction by super-critical CO 2 for astaxanthin. The organic solvents, including acetone, acetonitrile, and methanol were purchased from Fisher Scientic (Boston, Massachusetts, USA). The analytical chemicals triethylamine (TEA) and hydrochloric acid for amino acid analyses were obtained from Fisher Scientic; and phenyl isothiocyanate (PITC) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, Missouri, USA). The DMM was nely ground and stored at 4 C before use. 2.2. Diet formulation and preparation A control test diet was formulated to contain 32% crude protein (CP) and 8.9% crude lipid (CL) notated as D-0% (Table 1). The other four test diets were formulated by adding DMM to replace 12.5%, 25.0%, 37.5%, and 50.0% of the shmeal protein in the control diet, corresponding inclusion amounts of DMM were 3.0%, 6.0%, 9.0% and 12.0% of diet biomass, respectively. These four test diets were named: D-12.5%, D-25.0%, D-37.5% and D-50.0% (Table 1). Wheat starch and menhaden sh oil were adjusted to balance the diets. The test diets were prepared according to methods described by Ju et al. (2008). A commercial shrimp feed (Rangen, Inc., Buhl, Idaho, USA) with 40% CP and 5% squid meal was included in the trial as a commercial control diet. Finished pellets were then stored in plastic bins at 1920 C until fed within 3 months. 2.3. Feeding trial The growth trial was carried out in an indoor ow through system with 24 glass aquaria (52-L; 76 cm31 cm31 cm). The tanks were aerated and received a constant supply of seawater (1 L min 1 ). Each aquarium was stocked with juvenile shrimp (~1.0 g), at a density of 12 shrimp per aquarium (50 shrimp m 2 ). Each diet (one control, four DMM-added, and one commercial feed) was randomly assigned to four tanks. All shrimp were fed by hand 4 times daily at 09:30, 11:30, 14:00, and 16:15 h for 8 weeks. The shrimp were initially fed 12% and 18% of the initial stocked weight in week 1 and week 2, respectively. The feeding rate was adjusted weekly in a range of 6% to 10% based on body weight obtained bi-weekly or by using an estimated growth rate established in the laboratory. Uneaten feed, feces, and molts were removed by siphoning the aquaria immediately prior to the morning feeding. Water temperature (~25 C) was monitored daily. Dissolved oxygen (DO; 6.306.85 mg L 1 ), water pH (7.667.86) and salinity (32.4832.84 ppt) were monitored weekly. Total ammonia nitrogen level was measured weekly lower than the detectable level (b0.08 mg L 1 ) by the automated analysis method of Solorzano (1969). Feed conversion ratio (FCR), percent weight gain (PWG), and specic growth rate (SGR) were calculated as follows: PWG % W 2 W 1 =W 1 X100; SGR %=day ln W 2 =W 1 =tX100; FCR Total dried feed g fed= W 2 W 1 : where the W 2 is nal weight (g) of shrimp, W 1 is initial weight (g) and t is the number of days for the growth trial. All surviving shrimp in each tank were collected after the 8-week feeding and samples were used for analysis of proximate composition and astaxanthin content. Whole shrimp body samples were freeze-dried using a 5-L freeze-drier (EC Apparatus Inc, Holbrook, New York, USA). The dried samples were nely groundusinga grinder (IKAWorks Inc, Wilmington, North Carolina, USA), and stored at 20 C until used for analysis. 2.4. Sample analysis Proximate composition was analyzed following the methods by AOAC(2000). Moisture was determinedby drying a 2 g of representative sample in an oven with air circulation at 105 C for 1624 h. Crude protein (CP) was determined from total nitrogen by a LECO's FP-528 Nitrogen/Protein Determinator (LECO Cooperation, St. Joseph, MI, USA) using a multiplication factor of 6.25. Total crude lipid (CL) was determined by ethyl-ether extraction using an Accelerated Solvent Extractor (Dionex Corporation, Bannockburn, Illinois, USA). Ash was determined by incineration of a representative 0.5 g sample in an oven at 550 C for 6 h. Mineral content was analyzed by an inductively coupled plasma atomic emission spectroscopy (Model Atomscan 16, Thermo Jarrel Ash, Franklin, Massachusetts, USA). Gross energy was determined using bomb calorimetry (Parr 1261 Calorimeter, Parr Inst. Table 1 Formulation (%) of test diets with graded levels of defatted microalgae meal (Haematococcus pluvialis) replacing shmeal in the control diet. Diet ingredient Diet with replacement level of shmeal protein D-0% D-12.5% D-25.0% D-37.5% D-50.0% Menhaden shmeal a 15.00 13.12 11.24 9.36 7.48 Defatted microalgae meal b 0.00 3.00 6.00 9.00 12.00 Whole wheat (hard red winter) c 37.80 36.25 34.71 33.16 31.61 Soybean meal (47%) d 25.00 25.00 25.00 25.00 25.00 Squid meal e 6.00 6.00 6.00 6.00 6.00 Dicalcium phosphate f 4.50 4.50 4.50 4.50 4.50 Soy lecithin (liquid) g 2.00 2.00 2.00 2.00 2.00 Cholesterol f 0.12 0.12 0.12 0.12 0.12 Potassium chloride f 2.50 2.50 2.50 2.50 2.50 Calcium carbonate f 1.00 1.00 1.00 1.00 1.00 Magnesium oxide f 1.60 1.60 1.60 1.60 1.60 Mineral/vitamin premix #1 h 0.23 0.23 0.23 0.23 0.23 Mineral/Vitamin Premix #2 h 0.21 0.21 0.21 0.21 0.21 Stay C-35 i 0.04 0.04 0.04 0.04 0.04 Menhaden oil j 2.00 2.18 2.35 2.53 2.71 Soybean oil k 2.00 2.00 2.00 2.00 2.00 a Kodiak Fishmeal Company, Kodiak, Alaska, USA. b Cyanotech Corporation, Kona, Hawaii, USA. c Hawaiian Flour Mill, 703 N Nimitz Hwy, Honolulu, HI 96817, USA. d Land-o-Lakes, Seattle, WA, USA. e Inual, Santiago, Chile. f Sigma Aldrich, 3050 Spruce St., St. Louis, Mo 63103, USA. g Central Soya Company Inc, Fort Wayne, Indiana, USA. h Industrias de Nata, Aguadulce, Panama. i DSM Nutritional Products, Inc., 45 Waterview Boulevard, Parsippany, NJ 07054, USA. j Omega Protein Inc., Reedville, Virginia, USA. k ConAgra Foods Inc., Omaha, Nebraska, USA. 51 Z.Y. Ju et al. / Aquaculture 354355 (2012) 5055 Co., Moline, Illinois, USA). Amino acid (AA) proles of the samples were analyzed using an Agilent 1200 HPLC equipped with Agilent 1200 Series diode array detectors (Santa Clara, California, USA), following the methoddescribedby Ju et al. (2008). Free andesteriedastaxanthins in shrimp samples were analyzed using a modication of the HPLC method described by Lin et al. (2005). 2.5. Statistical analysis Data was analyzed by using one way analysis of variance (ANOVA), to determine if signicant (Pb0.05) differences existed among treatment means. Means separations were performed using Fishers Protected Least Signicant Difference Test. All statistical analyses were carried out using computer software (SigmaStat for Windows v3.5, Systat Software, Inc., San Jose, California, USA). 3. Results and discussion 3.1. Growth performance and nutritional compositions of shrimp The DMM contained 40.3% of CP and 12.8% of ash, but a very low content of CL (0.9%; Table 2). As formulated, the ve test diets were similar in nutrient compositions (Table 2). Pellet water stability showed only minor changes (92.7% to 94.1%; P>0.05); suggesting that the DMM inclusions did not signicantly affect the pellet water stability. After the 8-week feeding trial, the test diets and the commercial feed resulted in a high survival (87.5% to 100%; P>0.05; Table 3). Shrimp fed the D-12.5% showed a signicantly (Pb0.05) higher growth rate (1.25 g/wk) than that (1.11 g/wk) of the shrimp fed the D-0% (control diet). Shrimp fed the remaining three DMM-added diets (D-25.0%, D-37.5% and D-50.0%) had similar growth rates (1.13 to 1.22 g/wk) to shrimp fed the control diet. Effects of the test diets on PWG and SGR followed a similar trend as the results above. These results suggest that DMM could replace 50% of shmeal protein in the control diet without negatively affecting shrimp growth. A low inclusion level of DMM in the control diet actually improved the shrimp growth. Previous research has documented that inclusion of more than 20% microalgae into a sh feed resulted in poor palatability and thus affected growth (Hasan and Chakrabarti, 2009). However, for the current study no test was performed to examine if the high inclusion of microalgae affected the palatability of shrimp. Based on feeding observations and results of the similar growth and FCR for shrimp fed the ve test diets in this study, the inclusion levels (3% to 12%) did not seem to affect the shrimp palatability. Conversely, a higher replacement of shmeal by the algae meal may result in reduced feed intake of shrimp. This will need to be conrmed in future research. Furthermore, the ve test diets in this study resulted in signicantly higher growth rates than the commercial feed (0.74 g/wk; Table 3) even though the commercial feed had a higher CP content (39.9%) than the CP content (31.3 to 32.3%) in the ve test diets (Table 2). The ve test diets also showed a better feed conversion ratio (2.13 to 2.28:1) than the commercial feed (2.63:1; Pb0.05; Table 3). Based on the results of this study, we do not have a clear explanation on why the performance of shrimp fed the test diets was better than the shrimp fed the commercial diet. The essential amino acid proles are similar between the control diet and the commercial diet except that the commercial diet had relatively lower histidine (0.85%) than the test diets (1.11.25%; Table 4). The commercial feed contained lower potassium (0.91%) and magnesium (0.20%) levels than the potassium and magnesium content (2.032.24%; 1.171.25%) in the ve test diets. There is very limited information available for the nutritional requirements of histidine and the minerals by Pacic white shrimp (Kuhn et al., 2010). These warrant further investigation. The DMMproteinshowed a similar essential AAprole to menhaden shmeal protein (Table 4). As a result, the replacement of the shmeal protein with DMM protein did not cause a signicant change in essential AA composition for the test diets (Table 4). This may be the reason that no adverse effect was observed on the shrimp growth when shrimp were fed the diets containing different levels of DMM. The low replacement of shmeal (12.5%) by DMM or low inclusion (3%) of DMM in the control diet signicantly enhanced shrimp growth. This may be due to some bioactive compounds in the DMM (Cuzon et al., 1981; Hanel et al., 2007; Ju et al., 2009) or from some of the health benets of DMM (Hayashi and Hayashi, 1996; Hayashi and Katoh, 1994; Jensen et al., 2001). These bioactive compounds in the microalgae could be growth hormones or insulin-like growth factors (Guillaume et al., 1989), micronutrients such as vitamins and minerals (Brown, 2002; Spolaorea et al., 2006); and compounds inducing gene expression, such as free amino acids and free fatty acids (Clarke et al., 2002; Distel et al., 1992; Fafournoux et al., 2000). The benecial effects from microalgae biomass could enhance the immune system, resulting in anti-viral, anti- inammatory, phagocytosis, and anti-stress effects in aquatic animals (Hayashi and Hayashi, 1996; Hayashi and Katoh, 1994; Jensen et al., Table 2 Proximate composition (dried sample, n=1) of the ve test diets, a commercial feed, and a defatted microalgae meal (DMM; Haematococcus pluvialis). Composition Test Diets DMM D-0% D-12.5% D-25.0% D-37.5% D-50.0% Commercial feed Proximate (%) Dry matter 92.8 92.1 92.6 92.7 93.3 89.8 94.5 Ash 14.9 14.9 14.7 14.5 14.4 9.1 12.8 Crude protein 32.3 32.2 31.4 31.7 31.3 39.9 40.3 Crude lipid 8.9 9.1 9.3 9.4 9.3 8.5 0.9 Crude ber 1.5 1.8 2.1 2.4 2.8 2.7 9.6 Gross energy (cal/g) 4045 4042 4097 4112 4162 4349 4082 Macro minerals (%) Phosphorus 1.71 1.63 1.49 1.44 1.41 1.40 0.95 Potassium 2.09 2.06 2.05 2.03 2.24 0.91 0.43 Calcium 2.49 2.30 2.13 2.01 1.92 2.33 0.48 Magnesium 1.15 1.17 1.19 1.23 1.25 0.20 0.82 Sodium 0.12 0.11 0.11 0.11 0.11 0.40 0.20 Micro-minerals (ppm) Boron 15 16 16 17 17 10 23 Copper 32 35 29 47 15 24 11 Iron 557 577 594 672 797 259 1287 Manganese 62 72 70 59 65 142 87 Zinc 106 113 132 137 146 120 396 52 Z.Y. Ju et al. / Aquaculture 354355 (2012) 5055 2001; Mustafa and Nakagawa, 1995). The improvement seen in the growth rates might also be associated with physiological conditions such as increasing protein assimilation, lipid metabolism, and liver function (Nakagawa, 2011). However, high microalgae inclusion increased the ber content in aquafeed (Table 2). High ber content in diets could increase passage rate of feed in the gut, reducing the availability of nutrients, decrease feed digestibility, and reduce animal growth (Sudaryono et al., 1996). Thus, the increased ber content from high DMM inclusion might counteract the microalgae benecial effects (Table 2). This may explain why only 3% DMM inclusion achieved a signicantly enhanced shrimp growth effect, and suggests that feed manufacturers may consider using the DMM by-product as a feed additive for better shrimp growth performance. Previous growth studies on sh found that the growth performance of sh progressively decreased when diets were incorporated with algal meal (1520%), and total replacement of shmeal by algal meal generally showed very poor growth responses (Hasan and Chakrabarti, 2009). Using DMMto replace shmeal protein did not affect the proximate and mineral composition of the shrimp product (Table 5). But shrimp fed the commercial diet had signicantly lower CL than shrimp fed the ve test diets. This indicated less efciency of nutrient digestibility or retention for the shrimp fed the commercial feed. 3.2. Pigmentation of shrimp The harvested raw Pacic white shrimp turned to a pink or red color after freeze-drying at 50 C. It was observed that the shrimp increased in redness with elevated DMM inclusions in the diets. HPLC analysis exhibited signicantly increased contents of free and esteried astaxanthin in shrimp fed the diet with the levels of DMM inclusion (Fig. 1). Specically esteriedastaxanthincontents were muchincreased in shrimp fed the high DMM-added diets in comparison to shrimp fed the control diet. Therefore, the DMM from H. pluvialis used as an ingredient for shrimp feed can improve the quality of shrimp. In general, whole H. pluvialis dried meal contained approximately 15 to 20% lipid and 2% astaxanthin (Lorenz and Cysewski, 2000). The DMM was determined to contain 0.9% lipid (Table 2) and 0.05% astaxanthin. Therefore, the four test diets with the elevated levels of DMM inclusion were estimated to be supplemented at 15, 30, 45 and 60 mg of astaxanthin per kg of diet. These amounts of astaxanthin supplementation fromDMMresulted in signicant pigmentation effects for shrimp as shown in Fig. 1. The control diet was not supplemented with astaxanthin; however the vitamin and mineral mix ingredients contained -carotene (Table 1). Previous tests (Chien and Shiau, 2005; Ju et al., 2011; Pan et al., 2001) revealed that the optimumsupplemental amount of astaxanthin in shrimp diets were from 75 to 100 mg/kg diet for enhanced color. Currently many diets or commercial feeds for shrimp and sh are being formulated containing 50 to 60 mg/kg of synthetic astaxanthin. The synthetic astaxanthin is free astaxanthin and not preferred by consumers (Rodriguez et al., 2010). The natural astaxanthins from H. pluvialis meal are mainly composed of esteried astaxanthins (~95%), and generally are the accepted pigments in animal products (Barbosa et al., 1999; Coral-Hinostroza and Bjerkeng, 2002; Lorenz and Cysewski, 2000). 4. Conclusion In a summary, this study demonstrated that 1) the DMMcan be used as a feed additive (3% in a diet) to stimulate shrimpgrowth andimprove feed utilization; 2) replacement of 50% shmeal protein by the DMM does not have any adverse effect on shrimp based on the growth performance and nutritional composition of shrimp; 3) the DMM can also improve shrimp quality by enriched accumulation of astaxanthin, which is benecial to animal and human health. The results from this study provide important informationregarding the potential application of defatted microalgae as a valuable alternative protein and natural pigment source for shrimp culture. More studies will be needed to test this by-product at higher replacement levels for shmeal and its effects on shrimp palatability and digestibility. Table 3 Effects of replacing shmeal with a defatted microalgae meal (Haematococcus pluvialis) in the control diet on growth and survival of juvenile shrimp, Litopenaeus vannamei (n=4). Diet Initial weight Final weight Growth rate Survival FCR 1 PER 1 TFF 1 PWG 1 SGR 1 (g) (g) (g/wk) (%) (g/g) (g/g) (g) (%) (%/day) D-0% 1.12 a2 10.04 b 1.11 b 91.7 a 2.28 c 1.36 b 880.1 b 796.4 b 3.91 b D-12.5% 1.06 a 11.06 c 1.25 c 95.8 a 2.13 a 1.40 c 974.6 c 943.4 c 4.11 c D-25.0% 1.09 a 10.12 b 1.13 b 97.9 a 2.21 b 1.44 c 932.7 b 828.4 b 3.93 b D-37.5% 1.10 a 10.88 bc 1.22 bc 87.5 a 2.27 c 1.39 b 922.6 bc 889.1 bc 4.07 c D-50.0% 1.08 a 10.28 b 1.15 b 95.8 a 2.22 b 1.44 c 929.9 b 851.9 b 3.96 b Commercial feed 1.08 a 7.03 a 0.74 a 100.0 a 2.63 a 0.95 a 748.1 a 550.9 a 3.18 a SEM 0.05 0.61 0.08 3.1 0.14 0.07 37.1 31.8 0.18 1 FCR=feed conversion ratio, PER=protein efciency ratio, TFF=total feed fed, PWG=percent weight gain, and SGR=specic growth rate. 2 Means not sharing a superscript in the same column are signicantly different (Pb0.05, n=4). Table 4 Amino acid proles (n=1) of the shmeal, a defatted microalgae meal (Haematococcus pluvialis), test diets and a commercial feed. Amino acids Men. meal 1 DMM 1 D-0% D-12.5% D-25.0% D-37.5% D-50.0% Com. feed 1 (g/100 g sample) No-essential AA Ala 4.71 3.61 1.68 1.69 1.74 1.70 1.68 2.18 Asp+ASN 5.81 3.32 3.95 4.03 4.04 3.99 4.04 3.53 Cys 0.87 0.34 0.78 0.76 0.74 0.66 0.71 1.22 Glu+Gln 6.68 3.64 4.06 3.58 3.25 3.34 3.55 3.82 Gly 4.93 2.78 1.83 2.17 2.22 2.18 2.42 2.95 Pro 2.69 1.76 1.48 1.68 1.72 1.77 1.81 2.29 Ser 2.17 1.62 1.14 1.26 1.38 1.46 1.54 1.66 Tyr 2.12 1.45 0.99 1.09 1.12 1.16 1.14 1.22 Subtotal 29.98 18.51 15.91 16.25 16.22 16.26 16.90 18.85 Essential AA Arg 5.57 2.49 2.38 2.32 2.28 2.22 2.21 2.99 His 1.78 0.96 1.25 1.23 1.17 1.14 1.10 0.85 Ile 2.82 1.99 1.43 1.41 1.38 1.31 1.15 1.68 Leu 4.55 3.06 2.23 2.02 1.98 1.97 1.82 2.55 Lys 5.83 2.22 1.85 1.88 1.89 1.82 1.75 2.25 Met 1.08 0.60 0.64 0.69 0.68 0.67 0.67 0.77 Phe 2.98 1.84 1.60 1.57 1.54 1.52 1.38 1.71 Thr 2.86 2.01 1.00 1.00 0.96 0.96 0.97 1.47 Val 3.53 2.69 2.17 2.20 2.23 2.16 2.17 2.84 Subtotal 31.01 17.86 14.56 14.32 14.11 13.77 13.22 17.10 Total 60.99 36.37 30.47 30.57 30.32 30.03 30.12 35.95 1 Men. meal =menhaden meal; DMM=defatted microalgae meal, Com. feed=commercial feed. 53 Z.Y. Ju et al. / Aquaculture 354355 (2012) 5055 Acknowledgments Support for this research, through grant agreement No. 59-5320- 2-712 from the United States Department of Agriculture, Agricultural Research Service, is gratefully acknowledged. The authors also wish to thank Valerie L. Harmon, Director of Cultivation of Cyanotech in Kona of Hawaii for providing the defatted microalgae meal for this study. Ward Kashiwa, Gavin Nagaue, Richard Lee, David Anderson, and Jarryd Maeda are acknowledged for their technical assistance with the feeding trial and sample analyses and Lindon Hansink for her editorial work on the manuscript are also appreciated. References Acien-Fernandez, F.G.A., Hall, D.O., Guerrero, E.C., Rao, K.K., Grima, E.M., 2003. Outdoor production of Phaeodactylum tricornutum biomass in a helical reactor. Journal of Biotechnology 103, 137152. AOAC (Association of Ofcial Analytical Chemists), 2000. Ofcial Methods of Analysis, 17th ed. 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Composition Content in whole shrimp body (MeanSD) D-0% D-12.5% D-25.0% D-37.5% D-50.0% Com. feed 1 Proximate (%) Dry matter 93.30.9 a2 93.50.5 a 94.70.2 a 93.70.9 a 94.40.3 a 93.90.4 a Ash 10.30.5 a 10.50.3 a 10.70.4 a 10.50.3 a 10.40.5 a 10.40.2 a Crude protein 71.80.6 a 72.11.4 a 71.41.3 a 70.51.0 a 71.60.3 a 71.71.6 a Crude lipid 6.70.4 b 6.90.2 b 6.80.1 b 6.70.4 b 6.90.4 b 6.00.2 a Macro-minerals (%) Phosphorus 1.10.0 b 1.10.0 b 1.10.0 b 1.10.0 b 1.20.1 b 0.90.0 a Potassium 1.30.0 a 1.30.0 a 1.20.0 a 1.20.0 a 1.30.1 a 1.20.0 a Calcium 2.70.4 a 2.40.5 a 2.40.5 a 2.80.5 a 2.60.6 a 2.80.1 a Magnesium 0.30.0 a 0.30.0 a 0.20.0 a 0.30.0 a 0.30.0 a 0.20.0 a Sodium 1.00.0 a 1.00.0 a 1.00.0 a 0.90.0 a 1.00.1 a 1.00.0 a Micro-minerals (ppm) Boron 2.40.5 a 2.40.5 a 2.40.3 a 2.30.1 a 2.70.3 a 2.50.3 a Copper 102.34.9 a 103.25.3 a 102.46.4 a 99.84.8 a 100.76.7 a 100.24.4 a Iron 16.45.1 a 14.51.6 a 16.35.6 a 15.11.7 a 16.91.8 a 15.92.7 a Manganese 1.30.1 a 1.50.1 b 1.40.1 ab 1.40.1 ab 1.50.1 b 1.50.1 b Zinc 52.312.7 a 46.95.4 a 44.91.5 a 47.65.1 a 53.815.3 a 45.81.7 a 1 Com. feed=commercial feed. 2 Means not sharing a superscript in the same row are signicantly different (Pb0.05, n=4). 0 10 20 30 40 50 60 D-0% D-12.5% D-25.0% D-37.5% D-50.0% A s t a x a n t h i n
c o n t e n t
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