Sparing effect of pond water on vitamins in shrimp diets

Sparing effect of pond water on vitamins in shrimp diets

Aquaculture 258 (2006) 388 – 395 www.elsevier.com/locate/aqua-online Sparing effect of pond water on vitamins in shrimp diets Shaun M. Moss a,⁎, Ian ...

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Aquaculture 258 (2006) 388 – 395 www.elsevier.com/locate/aqua-online

Sparing effect of pond water on vitamins in shrimp diets Shaun M. Moss a,⁎, Ian P. Forster a , Albert G.J. Tacon b a

The Oceanic Institute, 41-202 Kalanianaole Highway, Waimanalo, Hawaii 96795, USA b Halliday Place, Kaneohe, Hawaii 96744, USA

Received 7 August 2003; received in revised form 27 March 2006; accepted 4 April 2006

Abstract A 10-wk experiment was conducted to determine whether shrimp pond water has a sparing effect on vitamins, trace minerals, and protein levels in diets fed to juvenile Pacific white shrimp, Litopenaeus vannamei. Twenty-four 52-L aquaria were stocked with 0.7-g shrimp at a density of 24 shrimp/aquaria (100 shrimp/m2 equivalent). Shrimp were exposed to flow-through seawater from one of two sources: clear well water from a seawater aquifer or organically rich water from a pond used for intensive shrimp culture. In addition, four diets were evaluated in each of the two water sources (three replicates/treatment), including: 1) a 35%-protein diet with vitamin and trace mineral premixes, 2) the same 35%-protein diet minus the vitamin premix, 3) the 35%-protein diet minus the trace mineral premix, and 4) a 25%-protein diet with vitamin and trace mineral premixes. Shrimp grown in well water without vitamins in their diet had a significantly lower (Pb 0.05) final weight, growth rate, and survival, and a significantly higher FCR, than shrimp grown in well water with vitamins. However, there was no significant difference in final weight, growth rate, survival, or FCR between pond-water reared shrimp with and without vitamins, indicating that removal of vitamins from the diet of pond water-reared shrimp had no effect on shrimp performance. In contrast to vitamins, there was no sparing effect of pond water on trace minerals or protein levels. As expected, growth rates of shrimp reared in pond water were greater than those in well water for each of the four diets. The largest difference in growth rate was seen with the 35%-protein diet minus vitamins. Shrimp fed this diet grew 306% faster in pond water than in well water. It appears that the growth enhancing effect of pond water is more pronounced when shrimp are fed diets of inferior quality. Results from this study indicate that pond water has a sparing effect on vitamins in shrimp diets, and microbes likely contributed significantly to this effect. By exploiting endogenously produced microbes and associated detritus, shrimp farmers and feed manufacturers can reduce substantially vitamin levels in shrimp feeds, resulting in reduced feed costs without compromising shrimp growth, survival, or FCR. © 2006 Elsevier B.V. All rights reserved. Keywords: Vitamins; Minerals; Protein; Shrimp; Diet; Pond water; Sparing effect

1. Introduction Although qualitative requirements for vitamins and minerals have been elucidated for several commercially important penaeid shrimp species, quantitative requirements for these nutrients are not well defined (Conklin, ⁎ Corresponding author. Tel.: +1 808 259 3110; fax: +1 808 259 9762. E-mail address: [email protected] (S.M. Moss). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.04.008

1997; Davis and Lawrence, 1997). Difficulties in quantifying water-soluble vitamin requirements for shrimp exist because of challenges associated with delivering these nutrients to aquatic organisms. Shrimp are slow eaters and feed pellets can remain submerged in water for several hours prior to ingestion. Once a pellet is located, it is grasped by the shrimp's pereiopods and transferred to the mouthparts (Hindley and Alexander, 1978). Small particles are placed directly in a pre-oral cavity, whereas larger items are held to the mouthparts by the third maxillipeds for

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further manipulation (Alexander et al., 1980). As a result of prolonged submersion and handling by the shrimp, watersoluble vitamins can leach out of the feed, thus making inferences about quantitative requirements difficult (Goldblatt et al., 1980; Gadient and Schai, 1994). Similarly, there are difficulties in quantifying mineral requirements because of the way shrimp eat and their ability to use minerals dissolved in seawater (Davis et al., 1990; Davis and Lawrence, 1997). Despite a paucity of quantitative information about dietary vitamin and mineral requirements for penaeid shrimp, vitamin and mineral supplements typically are added to commercial shrimp feeds (Akiyama et al., 1992). These feeds often are over-fortified with vitamins to mitigate concerns about vitamin loss associated with feed processing and storage (especially ascorbic acid), as well as from leaching during feeding (Castille et al., 1996; Conklin, 1997). Similarly, shrimp feeds typically contain a significant amount of minerals from premixes and other feed ingredients, such as binders (Davis and Lawrence, 1997). There are disadvantages in using shrimp feeds containing excessive amounts of vitamins and minerals. Vitamin premixes can account for as much as 15% of total feed ingredient cost, so the inclusion of excessive vitamins can be costly (Akiyama et al., 1992). In addition, over-fortification of certain vitamins (e.g. riboflavin, niacin, and vitamin B6) can result in reduced shrimp growth (Deshimaru and Kuroki, 1979; Catacutan and De la Cruz, 1989; Conklin, 1997), and this can negatively impact production and profitability for the farmer. With regard to over-fortification of minerals, this can also increase feed cost, enhance phosphorus pollution, and reduce the bioavailability of other minerals (Davis and Lawrence, 1997). Also, excessive inclusion of certain minerals (e.g. iron) can reduce shrimp growth (Deshimaru and Yone, 1978), and certain minerals may accumulate to toxic levels in minimal- or zero-water exchange systems (Alcivar-Warren and Meehan, 2001). Penaeid shrimp cultured in extensive and semiintensive production systems depend on natural pond biota as a direct nutritional source (Moss, 2002). Food items consumed by shrimp in these systems are similar to those in the wild and include plant and animal matter, as well as microbes and detritus. These food items contain vitamins and minerals that, if available in sufficient quantities, could preclude the need for nutrient supplements in exogenously supplied shrimp feeds (Phillips, 1984; Brown et al., 1999). In fact, the sparing effect of natural pond biota on vitamins and minerals has been documented for the tiger prawn, Penaeus monodon, reared under extensive culture conditions (Triño and Sarroza, 1995). Growth and survival were not

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significantly different between shrimp fed two different diets; one with vitamin and mineral supplements and one without supplements. It is important to note that this study was conducted in extensive ponds stocked at 7.5 shrimp/m2, so there was abundant natural pond biota and insignificant grazing pressure by the shrimp. However, under intensive culture conditions, many meiofaunal and macrofaunal prey become rare or absent during the growout period (Hopkins et al., 1988; Visscher et al., 1988), and this could reduce or eliminate the potential sparing effect of natural pond biota on vitamins and minerals, as well as other nutrients. The primary objective of this study was to determine whether natural pond biota had a sparing effect on vitamins and trace minerals in diets fed to juvenile Pacific white shrimp, Litopenaeus vannamei, reared under intensive culture conditions. A secondary objective was to determine whether natural pond biota had a sparing effect on protein levels. Although previous research has shown a proteinsparing effect of natural pond biota on postlarval shrimp (Otoshi et al., 2001), it is unclear whether this effect occurs in larger shrimp. 2. Materials and methods 2.1. Experimental design and protocols The indoor laboratory used in this experiment was equipped with 52-L, glass aquaria (76 cm×31 cm ×31 cm) that received flow-through seawater from one of two sources; clear well water pumped from a seawater aquifer or pond water pumped from a 337-m2 round pond used for intensive shrimp culture. Well water was the source water for the round pond, so any differences in water quality between well and pond water resulted from inputs into and management strategies affecting the round pond. Previous studies indicate that water from the round pond can contain high concentrations of suspended organic matter, including microalgae and microbial–detrital aggregates, whereas well water typically has low concentrations of organic matter and is devoid of microalgae (Moss and Pruder, 1995; Otoshi et al., 2001; Divakaran and Moss, 2004). Water in the pond was mixed by a 1-HP paddlewheel that operated nightly, and pond water was transferred into the laboratory by a 1/4-HP submersible pump placed 0.5 m below the water surface at the outer perimeter of the pond. The round pond had a mean water depth of 1 m. (For more details about the round pond, see Wyban and Sweeney, 1989). Twenty-four 52-L aquaria were stocked with 0.68-g (SD + 0.02 g) Pacific white shrimp, L. vannamei, at a density of 24 shrimp/aquaria (100 shrimp/m2 equivalent). Shrimp were produced at the Oceanic Institute

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Table 1 List of the eight treatments evaluated in this study Treatment

Diet

Water source

W/35 P/35 W/35-V P/35-V W/35-M P/35-M W/25 P/25

35% protein + vitamins + minerals 35% protein + vitamins + minerals (35% protein + minerals) − vitamins (35% protein + minerals) − vitamins (35% protein + vitamins) − minerals (35% protein + vitamins) − minerals 25% protein + vitamins + minerals 25% protein + vitamins + minerals

Well Pond Well Pond Well Pond Well Pond

(Waimanalo, Hawaii, USA) and were free of specific pathogens listed by the U.S. Marine Shrimp Farming Program (U.S. Marine Shrimp Farming Program, 2003), including those pathogens that are OIE (International Office of Epizootics) notifiable (OIE, 2002). Eight treatments (Table 1; three replicates/treatment) were evaluated for 10 weeks, and consisted of: 1) shrimp maintained in well water and fed a 35%-protein diet with vitamin and mineral premixes (W/35); 2) shrimp maintained in pond water and fed the same 35%-protein diet (P/ 35); 3) shrimp maintained in well water and fed the 35%protein diet minus the vitamin premix (W/35-V); 4) shrimp maintained in pond water and fed the 35%-protein diet minus the vitamin premix (P/35-V); 5) shrimp maintained in well water and fed the 35%-protein diet minus the mineral premix (W/35-M); 6) shrimp maintained in pond water and fed the 35%-protein diet minus the mineral premix (P/35-M); 7) shrimp maintained in well water and fed a 25%-protein diet with vitamin and mineral premixes (W/25); and 8) shrimp maintained in pond water and fed the same 25%-protein diet (P/25). Prior to the start of the experiment, shrimp were stocked into the aquaria (treatments were randomly assigned to the 24 aquaria), reared in well water, and fed a commercial shrimp diet containing 35% protein and 2.5% squid (Rangen, Inc., Buhl, Idaho, USA). At the start of the experiment, aquaria received either pond or well water and shrimp were fed one of the four diets identified above. All shrimp were fed by hand at 0800, 1100, 1400, and 1700 h, and the amount of feed added to each aquarium was adjusted daily in an attempt to keep a slight excess of feed on aquarium bottoms. It was assumed that, if excess feed accumulated on the aquarium bottom prior to each morning feeding, shrimp were not food limited. Individual shrimp were weighed at the beginning and end of the 10-week experiment. Feed conversion ratio (FCR) was estimated for each aquarium and was calculated by dividing the weight of the feed added to each aquarium throughout the trial by shrimp wet weight gain. Estimated wet weight gain of dead shrimp was included in the FCR

calculation because dead shrimp did not remain in the aquaria very long (14 h maximum) and they were largely intact at the time they were removed and weighed. FCR values are likely to be overestimated because no attempt was made to collect, dry, and re-weigh uneaten feed pellets prior to their removal from the aquaria. Uneaten feed, feces, and exuviae were removed by siphoning aquarium bottoms immediately prior to each morning feeding. No effort was made to clean the glass walls of the aquaria. Water flow rates through the aquaria ranged from 0.36 to 0.54 L/min and all aquaria were aerated by airstones and a regenerative blower. Water temperature was measured daily and dissolved oxygen (DO) concentration was measured weekly for each aquarium using a YSI Model 57 oxygen meter (Yellow Springs Instrument Company, Yellow Springs, Ohio, USA). In addition, pH, salinity, chlorophyll a concentration, and total ammonia nitrogen (TAN) concentration were measured weekly for each aquarium. pH was measured using an Accumet AP61 pH meter (Fischer Scientific, Pittsburgh, Pennsylvania, USA), salinity was measured using a temperature-compensated refractometer (Aquatic EcoSystems, Inc., Apopka, Florida, USA), chlorophyll a concentration was estimated using the method of Strickland and Parsons (1972), and TAN concentration was estimated by the automated analysis method of Solorzano (1969) using a Technicon Auto-Analyzer II (Technicon Industry Systems, Tarrytown, New York, USA). 2.2. Diet preparation Four diets were evaluated in this experiment (Tables 2 and 3). Diets were prepared by mixing all major dry feed ingredients for 15 min in a Model D-300 Hobart food mixer (Hobart Manufacturing Corporation, Troy, Ohio, USA). A warm (∼60 °C) aqueous solution of sodium phosphate, potassium phosphate, choline chloride, and trace mineral premix was then added to the dry ingredient mix to bring Table 2 Proximate composition of experimental diets (values are on an as-fed basis) Diet

Moisture (%) Crude protein (%) (N*6.25) Crude lipid (%) Ash (%) Gross energy (cal/g)

35

35-M

35-V

25

6.3 36.5 9.4 6.3 4589

6.8 36.1 9.2 6.2 4552

6.3 36.1 9.3 6.4 4583

6.6 26.6 8.7 4.7 4449

35=35% protein+vitamins+minerals; 35-M=(35% protein+vitamins)− minerals; 35-V=(35% protein+minerals)−vitamins; 25=25% protein+ vitamins+minerals.

S.M. Moss et al. / Aquaculture 258 (2006) 388–395 Table 3 List of ingredients and corresponding concentrations (g/kg as fed) used to formulate each of the four diets evaluated in this study Ingredient (g/kg as fed)

Diet

Fishmeal — Norse LT 94® Squid mealb Soybean mealc Wheat, wholed Vital wheat glutenc Brewers yeaste Krill hydrolysatef Soy lecithing Menhaden fish oilh Cholesterol-FG (64% active)i Potassium phosphate, dibasic j Calcium phosphate, monobasic j Sodium phosphate, dibasic j Mineral premixk Vitamin premixl Choline chloride (52% active)m Vitamin C (35% active)m Chromic oxide a

35

35-M

35-V

25

245 25 95 470 40 30 20 20 30 2.3 5.6 5.6 5.6 0.6 4.0 1.2 0.7 0.0

245 25 95 470 40 30 20 20 30 2.3 5.6 5.6 5.6 0.0 4.0 1.2 0.7 0.0

245 25 95 476 40 30 20 20 30 2.3 5.6 5.6 5.6 0.6 0.0 0.0 0.0 0.0

165 0 0 690 40 0 20 20 35 2.3 5.6 5.6 5.6 0.6 4.0 1.2 0.7 5.0

a

SSF Sildolje-og Sildemelindustriens Forskningsinstitutt. Agribrands Purina Mexico S.A. de C.V., Mexico (by courtesy of). c Land-o-Lakes, Seattle, WA. d Hawaii Flour Mill, Honolulu, HI. e Williams Bio-Products, Decatur, IL (by courtesy of). f Specialty Marine Products, Vancouver, Canada (by courtesy of). g Central Soy, Co. Inc., Fort Wayne, IN (by courtesy of). h Omega Protein, Reedville, VA. i Solvay Pharmaceuticals B.V., The Netherlands. j ICN Biomedicals, Inc. Aurora, OH. k OI mineral premix (LV99.1) to supply the following elements (mg kg− 1 diet): zinc (as sulfate) 72 mg, iron (as sulfate) 36 mg, manganese (as sulfate) 12 mg, copper (as sulfate) 24 mg, cobalt (as chloride) 0.6 mg, iodine (as iodate) 1.2 mg, chromium (trivalent, as chloride) 0.8 mg, selenium (as selenate) 0.2 mg, and molybdenum (as molybdate) 0.2 mg. l Provided the following (mg/kg diet, except as noted): B12 0.096; Niacin 80; Riboflavin 60; Pantothenic acid 180; Menadione 40; Folic acid 6; Biotin 0.60; Thiamin 40; Pyridoxine 60; Inositol 400; Vitamin A 6000 IU; Vitamin D3 2000 IU; Vitamin E 250 IU and Astaxanthin 60. Prepared for OI by Roche Vitamins, Belvedere, NJ (by courtesy of). m Roche Vitamins, Belvedere, NJ (by courtesy of). b

the moisture content of the resulting mash to ∼34–35%. The mash was then blended for an additional 15 min. Half of the supplemental oil and lecithin and all of the cholesterol were blended in a Model K5SS KitchenAid mixer (KitchenAid, St. Joseph, Michigan, USA), added to the mash, and mixed for an additional 15 min. The resulting mash was passed through a Hobart grinder fitted with a 3mm diameter die. The resulting moist pellets were dried in a forced-air oven at ambient temperature (∼28–32 °C) until the moisture content was below 10%. The vitamin premix and vitamin C source (when present) were emulsified with the remaining oil and lecithin in a KitchenAid mixer and

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this mixture was added to the dry, cooled pellets by top coating using a Hobart D-300 food mixer with a whisk beater. Finished pellets were then stored in plastic bins at 19–20 °C until used. 2.3. Statistical analyses Final weight, growth rate, survival, FCR, feed input, and water quality (temperature, DO concentration, pH, salinity, chlorophyll a concentration, and TAN concentration) initially were analyzed using two-way ANOVA with diet (four) and water source (two) as the two main factors. If there was a significant diet × water source interaction, one-way ANOVA was used to compare treatment effects. Tukey's post hoc test was used to determine differences among treatment means at an experimental error rate (α _ ) of 0.05. Survival data were transformed using the square root of the arcsine prior to analysis. Final weight, growth rate, and FCR data were transformed using the natural log transformation prior to analysis. All statistical analyses were performed using SigmaStat version 2.03 (SPSS Inc., Chicago, Illinois USA). 3. Results There was no significant interaction, water source, or diet effect on water temperature, DO concentration, pH, and salinity. Water temperature ranged from 25.8 to 27.6 °C, DO concentration ranged from 4.9 to 6.8 mg/L, pH ranged from 7.1 to 7.9, and salinity ranged from 32 to 35 ppt for all aquaria over the 10-week experiment. Although there was no significant interaction or diet effect on chlorophyll a and TAN concentration, there was a significant water source effect. Mean chlorophyll a concentration was significantly greater (P b 0.001) in pond water (139.1 μg/L, SD ± 65.4 μg/L) than well water (below detectable limits). This observation is consistent with previous studies indicating that water from the OI round pond can contain high concentrations of microalgae, whereas well water essentially is devoid of microalgae (Moss and Pruder, 1995; Otoshi et al., 2001; Divakaran and Moss, 2004). Similarly, mean TAN concentration was significantly greater (P b 0.05) in pond water (12.2 μM, SD ± 10.2 μM) than well water (2.6 μM, SD ± 2.3 μM). With regard to shrimp performance, there was a significant interaction effect (P b 0.05) on final weight, growth rate, survival, FCR, and feed input. Because of this result, analyses of the two main factors (diet and water source) were not reported. Removal of vitamins from the diet of well water-reared shrimp negatively impacted shrimp performance. Shrimp in the W/35-V treatment had a

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Table 4 Mean (SD) final weight, growth rate, survival, FCR, and feed input for shrimp fed four diets in well or pond water for 10 weeks Treatmenta W/35 P/35 W/35-V P/35-V W/35-M P/35-M W/25 P/25

Final weight (g) b

5.64 (0.99) 13.83 (0.64)c 3.76 (0.23)a 13.28 (0.44)c 5.18 (0.85)a,b 13.43 (0.17)c 4.57 (0.64)a,b 13.84 (0.19)c

Growth rate (g/wk) b

0.50 (0.10) 1.31 (0.06)c 0.31 (0.03)a 1.26 (0.04)c 0.45 (0.08)a,b 1.28 (0.02)c 0.39 (0.06)a,b 1.32 (0.02)c

Survival (%) b

88.3 (5.8) 88.3 (10.4)b 41.7 (15.3)a 98.3 (2.9)b 76.7 (11.5)a,b 93.3 (5.8)b 90.0 (10.0)b 83.3 (20.2)b

FCR

Feed input (g) b

2.48 (0.38) 1.34 (0.10)c 5.42 (1.68)a 1.33 (0.08)c 2.96 (0.23)b 1.38 (0.09)c 3.02 (0.11)b 1.56 (0.34)c

212.6 (16.5)b 309.1 (3.8)c 129.3 (12.6) a 328.9 (6.0)c 200.5 (4.9)b 329.2 (6.5)c 210.4 (12.2)b 328.2 (7.5)c

Values in columns with different superscripts are significantly different (Tukey's post hoc test; ( α _ ) = 0.05; n = 3). a 35% = 35% protein; V = vitamin premix; M = mineral premix; 25% = 25% protein.

significantly lower (P b 0.05) final weight, growth rate, and survival, and a significantly higher FCR, than shrimp in the W/35 treatment (Table 4). However, there was no significant difference in final weight, growth rate, survival, or FCR between shrimp in the P/35-Vand P/35 treatments, indicating that removal of vitamins from the diet of pond water-reared shrimp had no effect on shrimp performance. In contrast to vitamins, there was no sparing effect of pond water on trace minerals or protein levels. Final weight, growth rate, survival, and FCR were not significantly different between the W/35-M and W/35 treatments, and between the P/35-M and P/35 treatments (Table 4). Similarly, there was no significant difference in final weight, growth rate, survival, or FCR between the W/35 and W/25 treatments, and the P/35 and P/25 treatments. As expected, growth rates of shrimp reared in pond water were greater than growth rates of shrimp in well water for each of the four diets. Shrimp fed the 25%protein diet with vitamin and mineral premixes grew 238% faster in pond water than in well water, and this difference was significant (P b 0.05, Table 4). The growth enhancing effect of pond water was less pronounced (but significant, P b 0.05) with the higher protein diet. Shrimp fed the 35%-protein diet with vitamin and mineral premixes grew 162% faster in pond water than in well water. The largest difference in growth rate was seen with the 35%-protein diet minus vitamins. Shrimp fed this diet grew 306% faster in pond water than in well water. In addition to improved growth rates, FCR of shrimp reared in pond water were lower than in well water for each of the four diets. FCR of shrimp fed the 25%- and 35%protein diets with vitamin and mineral premixes were 48% and 46% lower respectively, in pond water than in well water, and these differences were significant (P b 0.05, Table 4). Similar to growth rates, the largest difference in FCR was seen with the 35%-protein diet minus vitamins. Shrimp fed this diet exhibited an FCR that was 75% lower in pond water than in well water.

Because feed consumption rates varied among aquaria, total feed input was significantly different among treatments (P b 0.05, Table 4). Shrimp in the W/35-V treatment received significantly less feed over the study period than in any other treatment. This was due, in large part, to the relatively low survival in this treatment (41.7%). However, on a per shrimp basis, feed rate (% body weight/day) was similar among treatments and ranged from 3% to 8% throughout the study, depending on the size of the shrimp. Importantly, shrimp were not food limited because feed rates were adjusted daily to keep a slight excess of feed remaining on aquarium bottoms every morning. There was a significant negative correlation (P b 0.05, r= 0.94, n = 8) between feed input and FCR, and a significant positive correlation (P b 0.05, r= 0.96, n =8) between feed input and final weight. 4. Discussion Results from this study corroborate earlier work regarding the growth enhancing effect of shrimp pond water on juvenile L. vannamei (Leber and Pruder, 1988; Moss et al., 1992; Moss, 1995; Divakaran and Moss, 2004). This penaeid species is particularly adept at exploiting organic matter produced autochthonously in shrimp growout ponds, including particles less than 5.0 μm (Moss et al., 1992). In this study, mean growth rate of shrimp in the P/35 treatment was 162% greater than in the W/35 treatment. In a similar study using a lower protein diet (30% crude protein), growth rate of shrimp in pond water was 300% greater than in well water (Divakaran and Moss, 2004). In contrast, in studies using higher protein diets (45– 52% crude protein), the growth enhancing effect of pond water was not as pronounced and ranged from 48% to 62% (Leber and Pruder, 1988; Moss, 1995). These data suggest that the growth enhancing effect of pond water plays a more significant role when shrimp are fed diets of inferior quality. This inference is further corroborated by results in the present study where the largest difference in growth rate

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(306%) between pond- and well water-reared shrimp occurred when shrimp were fed the poorest diet (35%protein diet minus vitamins). In addition to enhanced growth, pond water-reared shrimp had a lower FCR than well water-reared shrimp for each of the four diets. This likely resulted from the assimilation of natural pond biota which supplemented exogenous feed. In addition, pond water is known to affect the abundance and species composition of gut microflora (Moss et al., 2000) and can stimulate digestive enzyme activity in juvenile L. vannamei (Moss et al., 2001; Divakaran and Moss, 2004). These effects also may have contributed to improved growth and FCR of pond waterreared shrimp in this study. Shrimp in the W/35-V treatment had a significantly lower final weight, growth rate, and survival, and a significantly higher FCR, than shrimp in the W/35 treatment. Previous studies evaluating vitamin-deficient diets in “clear water” systems also resulted in inferior shrimp performance. For example, Fenneropenaeus indicus fed a vitamin-deficient diet grew slower, exhibited a higher FCR, and had a lower survival than F. indicus fed a diet with vitamin supplements (Reddy et al., 1999). In another study, a diet deficient in vitamin C (ascorbyl-2-polyphosphate, AAPP) was fed to L. vannamei in a recirculating system and resulted in 11% survival after four weeks (Castille et al., 1996). In contrast, survival of shrimp fed a diet with moderate to high concentrations of AAPP ranged from 92% to 100%. Interestingly, in the presence of natural pond biota, shrimp exhibited N80% survival and a growth rate of 2.0 g/wk when reared in earthen ponds and fed a diet with no supplemental vitamin C (Lawrence and Lee, 1997). It was suggested that natural pond biota provided the shrimp with a sufficient amount of vitamin C to mitigate potential deleterious effects of a vitamin C-deficient feed pellet. It is possible that shrimp adjust their food selection behavior to compensate for nutritional imbalances in prepared diets by differentially selecting among available food resources (Mayntz et al., 2005), although research is needed to determine if shrimp exhibit this type of feeding strategy. In the present study, there was no significant difference in final weight, growth rate, survival, or FCR between shrimp in the P/35-V and P/35 treatments, suggesting that shrimp compensated for the vitamin-deficient diet by consuming natural pond biota that had sufficient vitamins to meet their needs. Pond water used in this study came from an outdoor round pond managed for intensive shrimp culture, and this water typically contains high concentrations of particulate organic matter in the form of bacteria, microalgae, and microbial-detrital aggregates (Moss and Pruder, 1995; Divakaran and Moss, 2004). In this study, chlorophyll a

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concentrations ranged from 37.0 to 221.4 μg/L in the pond water treatments, indicating the presence of a microalgal bloom in the outdoor round pond. The role of microalgae and other microbes as potential suppliers of essential nutrients to marine detritivores has received some attention, including their role in providing vitamins and minerals (Phillips, 1984). For example, Cowley and Chrzanowski (1980) reported that the inclusion of salt marsh yeasts in Bcomplex vitamin deficient diets fed to fiddler crabs mitigated the negative effects of the vitamin deficiency, and other microbial sources for these vitamins include bacteria (Kutsky, 1981) and microalgae (Carlucci and Bowes, 1971). In another study, Brown et al. (1999) determined the vitamin content of microalgae commonly used in aquaculture, including members of the genera Nannochloropsis, Chaetoceros, and Thalassiosira. These genera are frequent members of the microalgal community in the round pond used in this study (Wyban and Sweeney, 1993; Otoshi et al., 2001). Based on a comparison of vitamin content in microalgae with published vitamin requirements for marine fish and shrimp, Brown et al. (1999) concluded that microalgae contain ascorbate, folates, and cobalamin in excess of the animal's requirements, and most contain adequate amounts of α-tocopherol, thiamine, riboflavin, pyridoxine, and biotin. These observations corroborate earlier work by De Roeck-Holtzhauer et al. (1991, 1993) who also reported high vitamin content in microalgae commonly used in aquaculture. Despite these published data, it is not clear if microalgal vitamins are bioavailable to the consuming animal and more research is needed in this area. Additional research also is needed to quantify vitamins and minerals in other microbes inhabiting aquaculture ponds, including bacteria and protozoans, which are potential food resources for shrimp (Decamp et al., 2002; Moss, 2002). Recently, Tacon et al. (2002) determined the nutrient composition of particulate organic matter, or “microbial floc”, produced autochthonously in experimental, zerowater exchange tanks used for shrimp research. These particles contain a number of important macro- (calcium, phosphorus, potassium, and magnesium) and micro-minerals (copper, iron, manganese, and zinc), as well as a suite of amino and fatty acids, and appear to play an important role in shrimp nutrition. The benefits of establishing and managing microbial floc in commercial shrimp culture are now well documented (McIntosh, 1999; Browdy et al., 2002; Burford et al., 2003), and this approach is becoming an important management strategy worldwide (Rosenberry, 2003). In summary, results from this study indicate that there is a significant sparing effect of organically rich pond water on vitamins in shrimp diets. These results provide insight into the important role of pond microbes in shrimp

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