Development and Application of Soybean Based Diets for Pacific White Shrimp 
Litopenaeus vannamei 
 
by 
 
Daranee Sookying 
 
 
 
 
 
A dissertation submitted to the Graduate Faculty of 
Auburn University 
in partial fulfillment of the 
requirements for the Degree of 
Doctor of Philosophy 
 
Auburn, Alabama 
December 13, 2010 
 
 
 
 
 
Keywords: Soybean, Alternative proteins, Soy protein concentrate,  
Feed formulation, Pacific white shrimp 
 
 
Copyright 2010 by Daranee Sookying 
 
 
Approved by 
 
Donald A. Davis, Chair, Professor of Fisheries and Allied Aquacultures 
Claude E. Boyd, Professor of Fisheries and Allied Aquacultures 
Chhorn E. Lim, Affiliate Professor of Fisheries and Allied Aquacultures  
James A. Stoeckel, Assistant Professor of Fisheries and Allied Aquacultures 
 
 
 
ii 
 
Abstract  
 
Soybean meal is a readily available feedstuff that can be used in shrimp feed 
formulations. Soybean meal and its products have been found to be an acceptable protein 
ingredient with good digestibility for shrimp. Compared to marine animal meal, soybean 
meal has lower nutrient content in terms of protein, essential amino acids, highly 
unsaturated fatty acids and minerals. However, these shortages can be adjusted with 
supplements or blending with other products. Various mixtures of feed ingredients (e.g., 
animal by-product, vegetable protein, and plant protein concentrate) in association with 
soybean meal are options that provide more balanced nutrients than using soybean meal 
as a sole ingredient.  There is little information on the use of diets containing high levels 
of soybean meal in combination with other ingredients. Therefore, a series of feeding 
experiments were conducted at the Alabama Department of Conservation and Natural 
Resources Marine Resource Division, Claude Peteet Mariculture Center in Gulf Shores, 
Alabama between May 2007 and September 2009, to evaluate the use of high levels 
soybean meal as a main protein source in combination with other potential ingredients in 
formulated diets for L. vannamei.  
Results from these studies demonstrated that formulated diets containing 
approximately 36% protein and 8% lipid with balanced amino acid profiles can be 
formulated using high levels of soybean meal as the primary protein source. Good
 
iii 
 
results were demonstrated when using high levels of soybean meal in combination with   
poultry by-product meal, distiller?s dried grains with solubles, pea meal or soy protein 
concentrates (SPC). Although better results were obtained at lower levels of inclusion, 
using soy protein concentrate as a substitute for soybean meal at inclusion of 20% and 
greater resulted in a reduction in mean final weights and an increase in FCR. The 
supplementation of methionine or fish soluble to the diets containing high level of SPC 
(40%) did not enhance growth of L. vannamei.  
Overall, results from these studies reveal that use of high levels of soybean meal 
as a main protein source in combination with other potential ingredients (poultry by-
product meal, distiller?s dried grains with solubles, pea meal or soy protein concentrates 
(SPC) in formulated diets for L. vannamei is viable as long as essential nutrients in diet 
are properly balanced to meet shrimp nutritional requirements. 
 
 
 
 iv 
Acknowledgements 
 
 This dissertation is dedicated to Professor Donald A. Davis for persevering with 
me as my advisor throughout the time it took me to complete this research and write the 
dissertation. Doing the research under his supervision was one of the most important and 
formative experiences in my life. I am grateful as well to Professor Claude E. Boyd, Dr. 
Chhorn E. Lim, and Dr. James A. Stoeckel for coordinating and generously giving their 
time and expertise to better my work that made it possible for me to complete my degree. 
It is a pleasure to thank my father who educates and advices me in many ways and 
for whom I have become. Heartily thanks to my mother for her invaluable love, 
encouragement, and enlightenments. I also would like to make a special reference to my 
middle sister and her husband for their warmly welcomed home and open hearted when I 
needed a resting place as well as someone to share my good and bad moments. Special 
thanks to my youngest sister for devoting herself to support our family while I was away 
from home.  
Especially, I offer my gratitude and deep appreciation to friends, colleagues, 
students, teachers, and the staffs of Swingle hall whose friendship, hospitality, assistance, 
knowledge, and supports in any respect during the completion of the project. I thank them 
for their contribution and their good-natured support. I also would like to extend my 
gratitude to the American Soybean Association and NOAA for assisting in the support of 
this research. 
 
 v 
Table of Contents 
 
Abstract ............................................................................................................................... ii 
Acknowledgements ............................................................................................................ iv 
List of Tables .................................................................................................................... vii 
List of Figures .................................................................................................................... xi 
I. Introduction ......................................................................................................................1 
II. Pond Production of Pacific White Shrimp (Litopenaeus vannamei) Fed High Levels of 
Soybean Meal in Combination with Poultry By-product Meal, Fish Meal, Distiller?s 
Dried Grains with Solubles and Pea Meal 
 
 Abstract ....................................................................................................................8 
 Introduction ............................................................................................................10 
 Materials and Methods ...........................................................................................15 
 Results ....................................................................................................................21 
 Discussion ..............................................................................................................23 
 References ..............................................................................................................29 
III. Replacement of Solvent Extracted Soybean Meal by Soy Protein Concentrate for 
Pacific White Shrimp (Litopenaeus vannamei) reared under clear water conditions 
 
 Abstract ..................................................................................................................53 
 Introduction ............................................................................................................55 
 Materials and Methods ...........................................................................................57 
 Results ....................................................................................................................60 
 
 vi 
 Discussion ..............................................................................................................62 
 References ..............................................................................................................68 
IV. Use of Soy Protein Concentrate in Practical Diets for Pacific White Shrimp 
(Litopenaeus vannamei) reared under various culture conditions 
 
 Abstract ..................................................................................................................82 
 Introduction ............................................................................................................84 
 Materials and Methods ...........................................................................................87 
 Results ....................................................................................................................95 
 Discussion ..............................................................................................................97 
 References ............................................................................................................102 
V. Summary and Conclusions..........................................................................................121 
Literature Cited ................................................................................................................124
 
 vii 
List of Tables 
 
Chapter II 
Table 1 Feeding regime for Litopenaeus vannamei post larvae, based on mean shrimp 
weight determined every three days, percent body weight and feed types utilized 
throughout a 22-day nursery period .......................................................................41 
 
Table 2 Water quality variables observed over a 22-day nursery period for L. vannamei 
post larvae, nursed in a recirculating system composed of six, 6,000-L nursery 
tanks .......................................................................................................................42 
 
Table 3 Mean production parameters of L. vannamei post larvae (initial weight of         
1.5 mg), stocked at a density of 52 PL L-1 and nursed for 22 days in a 
recirculating system composed of six, 6,000-L nursery tanks. ..............................43 
 
Table 4 Ingredient composition (g 100 g-1 dry weight) of four experimental diets utilizing 
soybean-based diets in combination with 10% poultry by-product meal (PBM), 
fish meal (FM), distiller?s dried grains with soluble (DDGS) or ground pea meal 
(PM) as a protein source. .......................................................................................44 
 
Table 5 Nutrient composition (dry matter basis) of four experimental diets containing 
soybean meal in combination with 10% poultry by-product meal (PBM), fish 
meal (FM), distiller?s dried grains with solubles (DDGS) or ground pea meal 
(PM) .......................................................................................................................45 
 
Table 6 Amino acid profile of four experimental diets containing soybean meal in 
combination with 10% poultry by-product meal (PBM), fish meal (FM), distiller?s 
dried grains with solubles (DDGS) or ground pea meal (PM) ..............................46 
 
Table 7 Feeding regime for L. vannamei reared in the grow-out 0.1-ha ponds during     
18-week experimental period, based on estimated growth rate of 1.5 g wk-1 and 
assumed survival rate of 70% ................................................................................47 
 
Table 8 Summary of water quality variables monitored over an 18-week growth trial for 
L. vannamei, fed experimental diets containing high levels of soybean meal in 
various combinations and cultured in 0.1-ha ponds. Values represent the mean     
? standard deviation and values in parenthesis represent minimum and maximum 
readings... ...............................................................................................................48
 
 viii 
Table 9 Mean production parameters at 18 week experimental period of juvenile Pacific 
white shrimp, L. vannamei (initial weight of 40 mg), fed experimental diets 
containing high levels of soybean meal in combination with poultry by-product 
meals (PBM), fish meal (FM), distiller?s dried grains with solubles (DDGS) or 
ground pea meal (PM), and cultured in 0.1-ha ponds ............................................49 
 
Table 10 Water quality variables monitored over a 10-week growth period of juvenile    
L. vannamei (initial weight of 2.13 g), fed experimental diets containing high 
levels of soybean meal in combination with poultry by-product meals (PBM), fish 
meal (FM), distiller?s dried grains with solubles (DDGS) or ground pea meal 
(PM), and reared under a semi-closed recirculating system. Values represent the 
mean ? standard deviation and values in parenthesis represent minimum and 
maximum readings .................................................................................................50 
 
Table 11 Mean production parameters at 10 week experimental period of L. vannamei 
(initial weight of 2.13?0.16 g), fed diets containing high levels of soybean meal in 
combination with poultry by-product meals (PBM), fish meal (FM), distiller?s 
dried grains with solubles (DDGS) or ground pea meal (PM), and a commercial 
feed used as reference diet, and reared under a semi-closed recirculating      
system ....................................................................................................................51 
 
Chapter III 
 
Table 1 Ingredient composition (g 100 g-1 dry weight) of six experimental diets 
containing increasing percentages of SPC (0, 5, 10, 20, 40 and 40% supplemented 
with methionine) as a substitute for SE-SBM........................................................73 
 
Table 2 Nutrient composition (dry matter basis) of six experimental diets containing 
increasing percentages of SPC (0, 5, 10, 20, 40 and 40% supplemented with 
methionine) as a substitute for SE-SBM ................................................................74 
 
Table 3 Ingredient composition (g 100 g-1 dry weight) of six experimental diets 
containing increasing percentages of SPC (0, 5, 10, 20, 40, 40% supplemented 
with methionine and 40% with fish solubles) as a substitute for SE-SBM ...........75 
 
Table 4 Nutrient composition (dry matter basis) of seven experimental diets containing 
increasing percentages of SPC (0, 5, 10, 20, 40, 40% supplemented with 
methionine and 40% with fish solubles) as a substitute for SE-SBM ...................76 
 
Table 5 Water quality variables for growth trials with juvenile L. vannamei reared     
under clear water system for both trials. Values represent the mean ? standard 
deviation and values in parenthesis represent minimum and maximum       
readings ..................................................................................................................77 
 
 
 
 
 ix 
Table 6 Growth performance of L. vannamei (initial weight of 0.12?0.01 g) reared under 
clear water system in polyethylene tanks during 55-day experimental period   
(first trial), fed diets containing increasing percentages of SPC (0, 5, 10, 20, 40 
and 40% supplemented with methionine) as a substitute for SE-SBM .................78 
 
Table 7 Growth performance of L. vannamei (initial weight of 0.14?0.05 g) reared under 
clear water system in polyethylene tanks during 53-day experimental period 
(second trial), fed diets containing increasing percentages of (0, 5, 10, 20, 40, 
40% supplemented with methionine and 40% with fish solubles) as a substitute 
for  SE-SBM, compare to a commercial reference diet .........................................79 
 
Chapter IV 
 
Table 1 Feeding regime for Litopenaeus vannamei post larvae, based on mean shrimp 
weight determined every three days, percent body weight and feed types utilized 
throughout a 15-day nursery period.. ...................................................................108 
 
Table 2 Water quality variables observed over a 15-day nursery period for L. vannamei 
post larvae, nursed in a recirculating system composed of six, 6,000-L nursery 
tanks. ....................................................................................................................109 
 
Table 3 Mean production parameters of L. vannamei post larvae (initial weight of 1.0 
mg), stocked at a density of 52 PL L-1 and nursed for 15 day in a recirculating 
system composed of four, 6,000-L nursery tanks ................................................110 
 
Table 4 Ingredient composition (g 100 g-1 as is) of four experimental diets designed to 
contain increasing percentage of 0, 4, 8 and 12% soy protein concentrate (SPC)  
as a substitute for soybean meal and used for Litopenaeus vannamei growth   
trials......................................................................................................................111 
 
Table 5 Nutrient composition of the experimental diets and a commercial reference diet 
used for L. vannamei reared under different culture conditions ..........................112 
 
Table 6 Water quality variables monitored during 10 week growth period of juvenile L. 
vannamei, fed experimental diets containing increasing level of SPC at 0, 4, 8 and 
12% as a substitute for soybean meal, and reared under a clear water recirculating 
system. Values represent the mean ? standard deviation of daily and weekly 
determinations. Values in parenthesis represent minimum and maximum   
readings ................................................................................................................113 
 
Table 7 Water quality variables monitored during 10-week growth period of juvenile L. 
vannamei (initial weight of 1.0 g), fed experimental diets containing increasing 
level of SPC at 0, 4, 8 and 12% as a substitute for soybean meal, and reared under 
a semi-closed recirculating system. Values represent the mean ? standard 
deviation of daily and weekly determinations. Values in parenthesis represent 
minimum and maximum readings .......................................................................114 
 
 x 
Table 8 Feeding regimes for L. vannamei reared in the grow-out pond during an 18-week 
experimental period, at estimated survival rate of 75%. ......................................115 
 
Table 9 Summary of water quality variables monitored over an 18-week growth trial for 
L. vannamei, fed experimental diets with varying level of SPC and cultured in 
0.1-ha ponds. Values represent the mean ? standard deviation of daily and weekly 
determinations. Values in parenthesis represent minimum and maximum    
readings ................................................................................................................116 
 
Table 10 Mean (n = 3) production parameters at 10-week experimental period of            
L. vannamei (initial weight of 0.94?0.04 g), fed diets containing increasing level 
of SPC at 0, 4, 8 and 12% as a substitute for soybean meal, and reared under a 
clear water recirculating system...........................................................................117 
 
Table 11 Mean production parameters at 10-week experimental period of L. vannamei 
(initial weight of 0.99?0.03 g), fed diets containing increasing level of SPC at 0, 
4, 8 and 12% as a substitute for soybean meal, and a reference commercial diet, 
and reared under a green water recirculating system ...........................................118 
 
Table 12 Mean (n = 4) production parameters at 18 week experimental period of juvenile 
Pacific white shrimp, L. vannamei (initial weight of 10 mg), fed experimental 
diets containing increasing level of SPC at 0, 4, 8 and 12% as a substitute for 
soybean meal, and cultured in 0.1-ha production ponds. .....................................119 
 
 
 
 
 
 
 
 
 xi 
List of Figures 
 
Chapter II 
Figure 1 Relationship between water temperature (?C) and mean weight gain (g) over an 
18-week growth trial for L. vannamei (initial weight of 40mg), fed experimental 
diets containing high levels of soybean meal in combination with poultry          
by-product meals (PBM), fish meal (FM), distiller?s dried grains with solubles 
(DDGS) or ground pea meal (PM), and cultured in 0.1-ha ponds .........................52 
 
Chapter III 
 
Figure 1 Mean final weight (g) and FCR of L. vannamei post larval (initial weight of 
0.12?0.01 g) reared under clear water system in polyethylene tanks during 55-day 
experimental period (first trial), fed diets containing increasing percentages of 
SPC (0, 5, 10, 20, 40 and 40% supplemented with methionine) as a substitute for 
SE-SBM .................................................................................................................80 
 
Figure 2 Mean final weight (g) and FCR of L. vannamei post larval (initial weight of 
0.14?0.05 g) reared under clear water system in polyethylene tanks during 53-day 
experimental period (second trial), fed diets containing increasing percentages of 
(0, 5, 10, 20, 40, 40% supplemented with methionine and 40% with fish solubles) 
as a substitute for SE-SBM, compare to a commercial reference diet ...................81 
 
Chapter IV 
 
Figure 1 Mean final weight (g) and FCR of L. vannamei juveniles fed diets containing 
increasing level of SPC at 0, 4, 8 and 12% as a substitute for soybean meal, and 
reared under a clear water recirculating system (a), a green water semi-closed 
recirculating system (b), and production pond conditions (c) .............................120 
 
 
 
 
 
 
 
 
 
 1 
CHAPTER I 
INTRODUCTION 
 
Pacific white shrimp, Litopenaeus vannamei (Boone) is native to the eastern 
Pacific Ocean from Sonora, Mexico to Northern Peru. Currently, it is the most popular 
cultured shrimp species and has experienced a dramatic increase in aquaculture 
production from 186,113 tonnes in 1999 to 2,296,630 tonnes in 2007 (FAO, 2009). The 
industry growth has been paralleled by an increase in shrimp feed production. The 
increases in demand and limitations of supply have resulted in some ingredients 
becoming less available and more costly, especially fish meal. Fish meal and other 
marine ingredients, are considered desirable ingredients in shrimp feed because of their 
nutrient content and palatability. In commercial feeds, fishmeal typically accounts for 20 
to 30% of shrimp feed formulation (Tacon and Metain, 2008). The cost of fish meal has 
generally increased over time as a result of the uncertainty of availability and large 
fluctuations in the price. Furthermore, there are growing social and environment concerns 
regarding the long term sustainability of the use of fishmeal. In addition to feed prices 
increasing, the market value for shrimp has declined because of increased production and 
limited demand. This has resulted in a reduction in the profit margin for shrimp farmers. 
When margins were good, feed manufacturers could afford to use expensive ingredients 
and over formulate a diet. However, as the margin decreased feeds must become more 
cost effective. Feed costs can account for as much as 
 
 2 
40% to 60% of production costs (Hertrampf and Piedad-Pascual, 2000). Feed costs and 
feed management both influence the investment in feeds. Reducing or removing costly 
protein sources through the use of a combination of less expensive and more economical 
protein sources could result in substantial saving in feed cost. Practical diets using plant 
based protein sources to replace fish meal has become an interesting alternative which 
could reduce these problems.  
Use of renewable plant protein sources have become the focus of protein 
substitution studies in shrimp feeds around the world because of their acceptable protein 
level, suitable amino acid content, economic opportunity, and consistent quality 
(Watanabe, 2002). Formulated diets are designed to contain sufficient levels of nutrients 
to meet requirements using plant based protein sources for which production can be 
expanded and are often more cost effective. Feeding plant-based proteins to shrimp 
requires that the ingredients possess certain nutritional characteristics, such as low levels 
of fiber, starch (especially insoluble carbohydrates), and antinutrients. They must also 
contain a relatively high protein content, favorable amino acid profile, high nutrient 
digestibility, and reasonable palatability (Gatlin et al., 2007; Naylor et al., 2009).  
Ten indispensable amino acids which are required for growth and maintenance of 
shrimp are arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, 
threonine, tryptophan, and valine (Guillaume, 1997; Kanazawa, 1990). These amino 
acids should satisfy shrimp requirements to support optimum growth performance. Fish 
meal is considered ideal fish and shrimp production because of its high level of essential 
amino acids. On the other hand, plant protein sources contain lower levels of some 
essential amino acids (Tacon, 1994). Thus, the essential amino acid balance must be 
 
 3 
considered when diets are formulated to contain plant protein sources to replace fish 
meal. In general, the, amino acid profile of soybean meal is comparable with that of fish 
meal, albeit is lower in sulfur amino acids, i.e., methionine and cystine (Peres and Lim, 
2008).  
Soybean meal is often considered as the most reliable ingredient and cost 
effective source in shrimp feed because of its high protein content, high digestibility, 
relatively well-balanced amino acid profile, reasonable price and steady supply (Amaya 
et al., 2007a, b; Davis and Arnold, 2000). The protein digestibility was found higher in 
soybean protein than that in the marine animal meals (Akiyama, 1989). However, the 
inclusion of soybean meal at high levels or as a sole protein source has resulted in 
reduced performance of the shrimp (Lim and Dominy, 1990). This could be the results of 
imbalanced amino acid profiles or deficiencies of other dietary nutrients that were not 
taken into account. 
Fish meal is utilized as a protein source but it also provides lipids, essential fatty 
acids, minerals and vitamins to the diet. Consequently, there will be most likely a need to 
use a variety of feed ingredients in association with soybean meal to provide a better 
balanced nutrient profile.  Utilization of various potential protein sources in shrimp feeds 
such as animal by-product and other plant sources have been evaluated under different 
rearing condition (Amaya et al., 2007a, b; Cruz-Suarez et al., 2001; Lim and Dominy, 
1990; Piedad-Pascual et al., 1990; Ray et al., 2009; Sudaryono et al., 1995). One of those 
ingredients that are considered a promising alternative for the substitution of fish meal in 
shrimp feeds is poultry by-product meal (Amaya et al., 2007a; Davis and Arnold, 2000; 
Markey, 2007; Samocha et al., 2004). Distiller?s dried grains with solubles (DDGS) is 
 
 4 
also a potential protein source for shrimp feed because of its low cost and consistent 
supply as a co-product of the bio-ethanol production which is expected to increase 
rapidly in the next decade. Several studies reported the successful use of DDGS as an 
alternative protein source in fish and crustacean feeds without causing negative impact on 
growth performance (Cheng and Hardy, 2004; Coyle et al., 2004; Lim et al. 2007, 2009; 
Robinson and Li, 2008; Stone et al., 2005; Thompson et al., 2008; Webster et al., 1991, 
1992; Wu et al., 1994). Pea meal is also another widely used feed ingredient, mostly in 
livestock because of its high energy, moderate protein level (22 ? 26% crude protein), 
amino acid profile, and low cost (Borlongan et al., 2003). Several studies indicated that 
feed pea is another potential ingredient in fish and shrimp feeds (Bautista-Teruel et al., 
2003; Booth et al., 2001; Borlongan et al., 2003; Burel et al., 2000; Carter and Hauler, 
2000; Cruz-Suarez et al., 2001; Davis et al., 2002; Gomes et al., 1995; Gouveia and 
Davies, 2000). Because of the limitation in nutrient component of most ingredients, more 
than one ingredient is required for balanced feed formulations. Therefore, shrimp diets 
containing soybean meal as a main protein source should be combined with other 
alternative protein ingredients, i.e., poultry by-product meal, DDGS, and pea meal. 
Soybean and its products are acceptable protein sources with good digestibility 
for shrimp. However, soybean meal is deficient in the essential amino acids (EAAs) such 
as methionine, lysine, and tryptophan as well as essential fatty acids and minerals (Lim 
and Dominy, 1990). Methionine is one of the ten essential or indispensable amino acids 
that are dietary essential for shrimp (Millamena et al., 1996). Thus, supplementation of 
sulfur amino acids, i.e., methionine or cystine, in soybean based diet to meet the shrimp 
requirement is recommended to provide a good growth response (Akiyama, 1989). Low 
 
 5 
levels of methionine found in soybean meal can also be countered by mixing with other 
protein sources and or the supplementation of synthetic methionine. Several studies had 
reported successfully replacing fish meal with soybean meal with a methionine 
supplement in Milkfish (Davis et al., 1995; Shiau et al., 2007). Conversely, diet 
containing only soybean protein with a methionine supplement was poorly utilized by red 
drum (Reigh and Ellis, 1992). McGoogan and Gatlin (1997) suggested that diets 
containing soybean meal with low levels or no fish meal may have palatability problems. 
Thus, the inclusion of attractants or palatability enhancers, e.g., fish soluble, may be 
considered. A reduction in feed intake was reported in largemouth bass fed diets with 
increased soybean meal levels (Kubitza et al., 1997; Cho et al., 1974). Similar results 
were observed in red drum (Davis et al., 1995; Reigh and Ellis, 1992) and Pacific white 
shrimp (Lim and Dominy, 1990). 
There are other issues of using soybean meal as an alternative to fishmeal besides 
nutritional factor such as the presence of nutrient inhibitors.  Raw soybean contains anti-
nutritional factors such as trypsin inhibitors, lectins, oligosaccharides, antigens, and 
saponins that may affect the digestion and reduce nutrient availability to shrimp 
(Dersjant-Li, 2002). However, the effect of some of these anti-nutrients can be reduced 
by heat process (New, 1987). The processes of producing solvent extracted soybean meal 
(SE-SBM), typically referred to as soybean meal, is initiated  by removing the hulls then 
the beans are rolled into flakes to remove oil using a solvent, and toasted to inactivate 
trypsin inhibitors. Soybean meal, containing about 48% crude protein, is widely used in 
shrimp feeds. However, this product still contains some anti-nutritional factors such as 
trypsin inhibitors and lectins (Dersjant-Li, 2002; Venero et al., 2008).   
 
 6 
Another disadvantage is that, due to low protein content (48%) of SE-SBM, its 
use in nutrient dense diets may be restricted as a result of insufficient ?room? in the 
formulation for all necessary components. Soy protein concentrate is produced from high 
quality, dehulled soybean seeds by removing most of the oil, water soluble non-protein 
constituents, and many of the anti-nutritional factors. It contains approximately 65% 
crude protein in soy protein concentrate (SPC) which is similar to that found in fish meal, 
and considerably higher than that of SE-SBM (Liu, 1997; Paripatananont et al., 2001). 
Most feed manufacturers prefer a reasonable level of a high protein ingredient in their 
formulations to allow room to manipulate other ingredients. Hence, soy protein 
concentrate could be used as an alternative ingredient to replace fish meal in shrimp diets. 
The uses of SE-SBM as a protein source in diets of L. vannamei has received 
considerable attention with good results (Alvarez et al., 2007; Lim and Dominy, 1990; 
Samocha et al., 2004). The use of soy protein concentrate in aquatic feed has been 
reported in many fish species and in shrimp (Forster et al., 2002; Kaushik et al., 1995; 
Mambrini et al., 1999; Paripatananont et al., 2001; Storebakken et al., 1998). However, 
there is limited information available on the utilization of SPC in L. vannamei feed.  
Because of the interest in reducing feed costs, considerable effort is being made in 
evaluating and demonstrating the applicability of plant based diets and to identify 
limiting factors in these feeds. Therefore, information is needed for the practical use of 
high levels of soybean meal and the use of plant based diets with soybean meal as a main 
protein source in combination with other potential ingredients as well as the practical use 
of soy protein concentrate in formulated diets for L. vannamei. 
 
 7 
The overall objective of this research is to demonstrate the feasibility of using 
soybean protein in practical feeds for the Pacific white shrimp (Litopenaeus vannamei) 
reared under both laboratory and field conditions. Three specific objectives were included 
to identify the response of L. vannamei to different treatments:  
1. To evaluate the response of L. vannamei to different protein sources as 
substitutes for fishmeal. 
2. To evaluate the response of L. vannamei to increasing levels of SPC reared 
under clear water condition. 
3. To evaluate the response of L. vannamei to the use of SPC in commercial feed 
formulations and reared under various culture conditions. 
 
 
 8 
CHAPTER II 
POND PRODUCTION OF PACIFIC WHITE SHRIMP (Litopenaeus vannamei) 
FED HIGH LEVELS OF SOYBEAN MEAL IN COMBINATION WITH 
POULTRY BY-PRODUCT MEAL, FISH MEAL, DISTILLER?S DRIED GRAINS 
WITH SOLUBLES OR PEA MEAL 
 
Abstract 
The objective of this study was to demonstrate the feasibility of four diets 
formulated to contain high levels of soybean meal in combination with either poultry by-
product, fish meal, distiller?s dried grains with solubles, or pea meal in production diets 
for L. vannamei. Outdoor ponds and tanks were utilized to evaluate the response of the 
shrimp over the culture period. The pond production trial was conducted in sixteen, 0.1-
ha ponds using four replicates per diet. Post larvae were obtained from commercial 
sources and nursed for three weeks. Juvenile shrimps (mean weight ? S.D., 0.038 ? 0.004 
g) were stocked at 35 shrimp m-2 and were cultured under standardized pond production 
conditions for 18 weeks. Daily feeding rates were predetermined using expected weekly 
growth of 1.5 g wk-1, a feed conversion ratio (FCR) of 1.2, and estimated survival rate of 
70% over an 18-week period. The same dietary treatments were offered to shrimp 
maintained in outdoor tanks (800 L) over a 12-week period. A total of 24 tanks, five 
tanks for each of the four test diets and four tanks for a commercial reference diet, were 
stocked with juvenile shrimp (initial weight of 2.1 g) obtained from production ponds at a 
 
 9 
density of 30 shrimp per tank. Daily feed inputs were calculated, based upon an expected 
weight gain of 1.5 g per week and an expected feed conversion ratio of 1.2. At the 
conclusion of these experiments, final production, final individual weight, survival, and 
FCR were evaluated. The results for the pond study demonstrated that final production 
ranged between 4,729 and 5,794 kg ha-1, mean final weight varied from 14.27 to 17.76 g, 
survival ranging from 74.6 to 99.8%, and FCR was between 1.22 and 1.46. There were 
no differences (P ? 0.05) or notable trends in shrimp production variables among 
different test diets. Shrimp reared in the outdoor tanks confirmed these findings. Average 
final weights ranged between 17.88 and 20.76 g, survival varied from 93.3 to 100.0%, 
and FCR was between 1.2 and 1.42. The results from the outdoor tank study also showed 
no differences (P ? 0.05) in final weight, survival, or FCR among test diets. Diet 
containing partial fishmeal showed no benefit on growth performance of L. vannamei 
comparing with diets containing high levels of soybean meal in combinations with 
poultry by-product, distiller?s dried grains with solubles, or pea meal. Therefore, these 
studies demonstrated that practical shrimp feeds contain no fish meal had no negative 
impact on final production, final weight, survival, and FCR of L. vannamei.    
 
 10 
Introduction 
World shrimp production has been rapidly increasing as aquaculture production 
has increased.  Pacific white shrimp Litopenaeus vannamei is the major cultured shrimp 
species which accounts for over 2.3 million tonnes in 2007 compared to 186,113 tonnes 
in 1999 (FAO, 2009). The expanded production has resulted in an increasing demand for 
shrimp feed and the ingredients used to make the feeds.  This same trend has occurred 
across aquaculture sectors which currently rely on fish meal as one of the primary protein 
sources.  
According to Tacon and Metian (2008), estimated production of commercial 
shrimp feed will be increased from 0.9 million tonnes (Mt) in 1995 to 9.2 Mt in 2020. 
Fish meal has often been used as the protein source in commercial shrimp feed because 
of its excellent sources of nutrients, e.g., balanced amino acid profiles, essential fatty 
acids, and mineral content (Tacon and Akiyama, 1997). The inclusion of fish meal 
generally averages 20% of shrimp feed formulation. These estimates suggest that the 
global demand for fish meal in marine shrimp feed is expected to increase from 0.38 Mt 
to 1.97 Mt from the year 2000 to 2015. The combination of high price and fluctuated 
supply of fish meal during the past few years resulted in an increasing of feed cost. Feed 
costs can account for as much as 40 to 60% of production costs (Hertrampf and Piedad-
Pascual, 2000), it is critical to minimize the cost of feeds relative to production output.  
In order to reduce feed cost, finding alternative protein sources to replace costly 
proteins such as fishmeal is necessary. There is considerable interest in optimizing the 
protein sources in shrimp feed using terrestrial animal and plant protein sources. This is 
because of their steady supply, consistent quality, and low cost which could contribute to 
 
 11 
reduced feed costs (Samocha et al., 2004; Watanabe, 2002) as compared to fish meal. The 
amino acid profiles in fish meal are considered highly suitable for fish and shrimp. 
Therefore, alternative protein sources used to replace fish meal must contain optimum 
amino acids profiles that support the shrimp nutritional requirements for growth and 
survival.  
Because of their acceptable protein level, suitable amino acid content, economic 
opportunity, and consistent quality, the use of renewable plant protein sources as the 
alternative protein sources to replace fish meal has become the focus of studies in shrimp 
feeds around the world. Plant protein sources and their products, such as soybean, pea, 
cotton seed, corn gluten, wheat gluten, and distiller?s dried grains soluble, have been 
successfully used in aquatic animal feeds (Smith et al., 1999; Alvarez et al., 2007; 
Samocha et al., 2004; Lim and Dominy, 1990; Mambrini et al., 1999; Carter and Hauler, 
1999; Kikuchi, 1999; Boonyaratpalin et al., 1998; McGoogan and Gatlin, 1997; Webster 
et al., 1991, 1993, 1995; Sudaryono et al., 1995, 1999; Kaushik et al., 1995; Allan et al., 
2000; Fonta?nhas-Fernandes, 1999; Watanabe et al., 1993). Among these plant protein 
sources, soybean meal is considered one of the most promising ingredients and widely 
used as a protein source in fish and shrimp feeds because of its acceptable amino acid 
profile, consistent composition, and consistent availability at a reasonable price (Forster 
et al., 2002). However, soybean meal has potential problems associated with insufficient 
levels of lysine and methionine, presence of anti-nutritional factors, and poor palatability 
(Dersjant-Li, 2002). Additionally, soybean meal contains lower levels of minerals such as 
phosphorus and lack of n-3 highly unsaturated fatty acids (HUFAs), e.g., eicopentaenoic 
acid (EPA) and docosohexaenoic acid (DHA) compared to fish meal (Fox et al., 2004). 
 
 12 
To overcome these possible problems when fish meal is removed from feed, a balanced 
nutrient profile in shrimp feed must be considered. Samocha et al. (2004) indicated using 
mixtures of complementary ingredients as a substitute for fish meal in shrimp diets, e.g., 
a co-extruded soybean poultry by-product meal with egg supplement, can increase 
nutrient utilization and facilitate processing of shrimp feed. Furthermore, Suarez et al. 
(2009) reported high survival (84 - 86.5%) and weight gain (0.98 g wk-1) of L. vannamei 
fed test diets containing a mixture of soybean meal and canola meal with combinations of 
fish meal ranging from 0 to 15% in a recirculation system under clear water conditions. 
Considerable research has demonstrated that substitution of fishmeal with 
soybean meal in combination with other ingredients in shrimp diets resulted in acceptable 
growth, survival, and FCR (Amaya et al., 2007a,b; Cruz-Saurez et al., 2001; Ray et al., 
2010; Lim and Dominy, 1990, 1992; Sudaryono et al., 1995; Samocha et al., 2004; 
Piedad-Pascaul et al., 1990; Smith et al., 2001). Amaya et al. (2007a) evaluated the diets 
containing 0 to 9% fish meal in combination with 16% of poultry by-product meal, a 
plant base diet containing 1% squid meal, and a commercial diet for L. vannamei reared 
under production pond conditions and an outdoor semi-closed recirculating system. The 
results from these studies demonstrated that shrimp growth and production were not 
different among shrimp feed diets with and without fish meal. Moreover, Roy et al. 
(2009) demonstrated that diets containing combinations of vegetable protein and a diet 
containing 10% fish meal resulted in similar weight gain, survival, and FCR of Pacific 
white shrimp reared in low salinity water under laboratory and outdoor tank conditions. 
Several studies demonstrated that diets containing partially substitution or no fish meal 
could be fed to shrimp without negative impact to growth and production when feeds 
 
 13 
were formulated to contain well balanced of nutrients for animal (Amaya et al., 2007a, b; 
Davis and Arnold, 1990; Davis, 2004). Conversely, Lim and Dominy (1990) and Molina-
Poveda et al. (2004) reported the decline in shrimp weight gain when the animals were 
fed with diets containing soybean as a total replacement of fishmeal compared to diets 
with partially fish meal. However, it should be noted that not all studies are in agreement 
about the successful uses of non fish meal diets for L. vannamei.  
Clearly the use of soybean meal in shrimp feed is feasible. However, there are 
several other plant protein sources that may be considered as alternative ingredients used 
in association with soybean meal to balance nutritional composition in feed formulation 
when non-fishmeal diets are formulated. Distiller?s dried grains with solubles (DDGS) 
which is a co-product of the ethanol production from grain has been considered as a 
protein source for aquatic animals as a result of its inexpensive cost per unit protein basis. 
The nutrient composition of DDGS varies with the type of the process as well as quality 
of raw material; however, on a dry basis, corn DDGS usually contains approximately 
30% protein and 10% fat (Bonnardeaux, 2007; Spiehs et al., 2002). Additionally, DDGS 
have become more available and less expensive ingredients as a result of an increasing in 
bio-ethanol production. Several studies have evaluated DDGS as another promising 
alternative protein sources in fish and crustacean diets such as rainbow trout (Cheng and 
Hardy, 2004,;Stone et al., 2005), sunshine bass (Thomson et al., 2008), tilapia (Wu et al., 
1994; Coyle et al., 2004; Lim et al., 2007), and channel catfish (Webster et al., 1991, 
1992, 1993; Robinson and Li, 2008; Lim et al., 2009), fresh water prawn (Tidwell et al., 
1993), and Pacific white shrimp (Lemos et al., 2009).  
 
 14 
Feed pea meal is also widely used as a feed ingredient, mostly in livestock feed, 
because of its nutrient profile with moderate protein level (22 ? 26% crude protein), 
amino acid balance, high energy, and low cost (Borlongan et al., 2003; Gatlin et al., 
2007). According to Gatlin et al. (2007), utilization of raw feed pea meal in fish feed is 
limited as a result of its high level of indigestible carbohydrate; however, heat processing 
or extrusion helps to break down starch and improve digestibility of feed pea. Several 
studies have reported the use of feed pea as a potential ingredient in feed for rainbow 
trout (Burel, 2000; Gomes et al., 1995), milkfish (Borlangan et al., 2003), salmon (Carter 
and Hauler, 1999), silver perch (Booth et al., 2001), sea bass (Gouveia and Davies, 
2000), and shrimp (Bautista-Teruel et al., 2003; Davis et al., 2002; Cruz-Suarez et al., 
2001).  
Another viable ingredient for replacing fish meal in feed is rendered by-products 
of terrestrial animal protein which generally contain 45 to 65% crude protein and are 
often good sources of indispensable amino acids, essential fatty acids, vitamins, and 
minerals along with good palatability (Samocha et al., 2004; Meeker, 2009). Poultry by-
product meal contains protein at a level similar to fish meal (ranges from 56.4 to 84.2%), 
but less expensive and slightly lower in some essential amino acids (Shiau, 2008; Yu, 
2008).   Earlier studies indicated that poultry by-product meal is a promising alternative 
for the substitution of fishmeal and soybean meal in aquatic animal feeds (Samocha et al., 
2004; Webster et al., 1999, 2000; Fowler, 1991; Garza, 2009; Saoud et al., 2008; Davis 
and Arnold, 2000; Amaya et al., 2007a, b; Markey, 2007). 
Many studies have been reported the use of alternative protein sources as 
replacements of fish meal in feeds. However, no one has yet evaluated the use of these 
 
 15 
potential sources together in practical diets for L. vannamei reared under outdoor 
conditions. Therefore, the objective of the present study was to evaluate the use of high 
levels of soybean meal in combination with pea meal, distiller?s dried grains solubles, 
poultry by-product meal, or fish meal as a protein source in feed for Pacific white shrimp 
reared under pond conditions and outdoor semi-closed system.  
 
Methods 
 A series of experiments were conducted at the Alabama Department of 
Conservation and Natural Resources Marine Resource Division, Claude Peteet 
Mariculture Center in Gulf Shores, Alabama. Two growth trials were carried out in 
parallel which included a pond trial and outdoor tank trial, during June through 
September 2007.  
 
Experimental animals  
Shrimp post larvae (PL) were received from a commercial hatchery, Shrimp 
Improvement System, Key West, FL. Dissolved oxygen (DO), temperature, salinity, and 
pH were randomly measured among the shipping bags using a YSI 556 MPS meter 
(Yellow Spring Instruments Co., Yellow springs, OH, USA). Two water samples were 
collected in order to determine the total ammonia-nitrogen (TAN) using a 
spectrophotometer (Spectronic 20 Genesys, Spectronic Instrument Inc. Rochester, NY, 
USA) following the Nesslerization method (APHA 1989). An oxygen tank with one air 
stone was operated to provide oxygen during the acclimation period. Newly hatched 
brine shrimp, Artemia salina (INVE Americas, Inc., Salt Lake City, UT, USA), were 
 
 16 
prepared one night before PL reception, and were fed to PL in the acclimation tank. PL 
were slowly acclimated and were released into the acclimation tank. Water quality 
variables were measured to control temperature and salinity change in the range of 4 C 
hr-1 and 2 ppm hr-1, respectively. After acclimation, PL were concentrated by drain 
harvesting into a 57-L concentration tank and quantified volumetrically. Six sub-samples 
were taking using a 60 ml beaker while the water in the concentration tank was mixing 
vigorously. Average numbers of PL per ml were then determined. Sixty shrimp were 
randomly collected and individually weighed to determine the average weight and predict 
the biomass for determination of feed inputs. 
The shrimp were divided and spread equally into six nursery tanks (6,000 L). The 
nursery system was stocked at a density of 33 postlarvae per liter. Each nursery tank was 
equipped with three air lifts and two air stones to help with water circulation and aeration 
which was provided by a common regenerative blower. The recirculating nursery system 
was composed of six culture tanks, a biological filter, a pressurized sand filter and a 1.5-
hp circulation pump (Aquatic Eco-systems, Apopka, Florida, USA). Feeds were applied 
on daily basis four times per day. Uneaten feed was siphoned out to maintain the water 
quality in the nursery tanks (Table 1). PL were raised in nursery system for a 3-week 
period. Water quality was monitored two times per day at 800 h and 1600 h., and TAN 
was measured biweekly (Table 2). Every three days, 60 PL per tank were randomly 
collected and sub-samples of 10 shrimps each were pooled weighed in order to evaluate 
growth and adjust daily feed rate. 
At the end of three weeks, each tank was partially drained to concentrate the 
shrimp. The shrimp were then collected by dip nets. Six sub-samples were taken from 
 
 17 
each tank to determine average weight of shrimp (g),  Once the number of shrimp per 
unit weight was determined, shrimp were spread evenly into 16 buckets each representing 
one production pond. Shrimp from each nursery tank were distributed across all ponds to 
minimize any bias from variation in nursery tanks. The shrimp were then transferred to 
the corresponding pond and stocked. Final population, weight gain (g), percentage 
survival, final weight (g), and feed conversion ratio for each nursery tank was determined 
(Table 3).  
 
Experimental diets 
Four experimental diets were formulated to contain soybean meal as the primary 
protein source with the inclusion of either 10% of poultry by-product meal, fish meal, 
distiller?s dried grains with solubles, or ground pea meal (Table 4). To further improve 
the amino acid balance of the plant-based diets, methionine at 1.7% of total protein and 
corn gluten meal at 4.8% of the diet were also included. The experimental diets were 
manufactured by Rangen Inc., under commercial feed manufacturing condition as a 
sinking extruded pellet. All experimental diets contain approximately 36% protein and 
8% lipid with lysine and methionine plus cystine contents of 5% and 3% of the total 
protein, respectively (Table 5, Table 6). Dietary treatments were tested in production 
ponds and outdoor tank system.  
 
Production pond Trial 
 Ponds used for the grow-out phase were approximately 0.1 ha in surface area, 
(rectangular 46 x 20 m) with a 1.0 m average depth. Each pond was equipped with a 20-
 
 18 
cm diameter standpipe, a concrete catch basin (3.0 x 2.0 x 0.5 m), and lined with 1.52 
mm thick high-density polyethylene lining (HDPE). The bottom of each pond was 
covered with a 25-cm deep layer of sandy-loam soil. After each culture cycle, ponds were 
dried and tilled in order to allow oxidation and mineralization of organic matter. 
Approximately three weeks before stocking, ponds were filled with brackish water from 
the intra-costal canal between Mobile and Perdido Bay. Incoming water was filtered 
through a three foot 250 ?m-mesh nylon filter sock (Micron Domestic Lace Mfg., Inc) to 
prevent the introduction of predators, as fish larvae, crab, or other planktonic organisms, 
but allow the introduction of primary productivity.. 
Two weeks before stocking juvenile pacific white shrimp, the combination of 
inorganic liquid fertilizers, 32-0-0 mixed with 10-34-0, were applied in a rate of 1,697 ml 
and 303 ml, respectively, per pond which provided 5.73 kg of N and 1.03 kg of P205 ha-1 
in order to promote natural productivity in all ponds, and the second fertilization was 
reapplied at half of the initial rate when the Secchi disk reading was greater than 40 cm. 
All ponds were provided aeration about 10 hp ha-1 using 1-hp paddlewheels aerators 
(Little John Aerator, Southern Machine Welding Inc. Quinton, AL) with either 1-hp or  
2-hp Aire-O2 aerators (Aire-O2, Aeration Industries International, Inc. Minneapolis, 
Minnesota) to maintain dissolved oxygen above 3 mg L-1, additional aerators were 
provided as needed when dissolved oxygen fell below 3 mg L-1. This study was a 
sustainable semi-intensive system with well-managed feeding, and minimal water 
exchanged. 
Juvenile L. vannamei (initial weight of 0.04 g) were collected from nursery 
system and stocked in the production ponds at the rate of 35 shrimp m-2. Four test diets 
 
 19 
were randomly assigned to 16 production ponds using four replicates per diet. Rations 
were divided to two feedings per day, early morning 800 h and late afternoon 1600 h. 
Feeding strategy for the first four weeks was based on previous studies which assumed 
that PL feed on primary productivity. Thus, during this period, a small amount of feed 
was applied to promote natural productivity in pond. Thereafter, feed input was 
calculated based upon an expected weight gain of 1.3 g wk-1, a feed conversion of 1.2, 
and a survival of 75% during the pond culture period (Table 7). 
Dissolved oxygen (DO), temperature, salinity, and pH were monitored three times 
a day, at sunrise (0500 h - 0530 h), during the day (1500 h - 1530 h), and at night (2000 h 
- 2200 h), using a YSI 556 MPS meter. Secchi disk reading and TAN were monitored 
once weekly. Water samples were taken in all ponds at the depth of 80 cm and TAN was 
determined using a spectrophotometer (Spectronic 20 Genesys, Spectronic Instrument 
Inc. Rochester, NY, USA) following the Nesslerization method (APHA 1989). Each 
week, sixty shrimps were sampling using two 5-foot cast nets (monofilament net, 1.22 m 
radius and 0.95 cm opening) to determine the average weight.  
The shrimp were harvested at the end of the 18-week study period. Feed was 
withdrawn 36 h before harvest in order to clear the shrimp gut. To harvest, about two 
thirds of the water was drained from ponds the night before harvest. The following day 
the remaining water was drained and the shrimp were pump harvested from the catch 
basin using a fish pump equipped with a 25-cm diameter suction pipe (Aqualife-Life 
pump, Magic Valley Heli-arc and Mfg, Twin Falls, Idaho, USA), and collected into the 
container truck. The shrimp were rinsed and weighed then approximately 150 shrimps 
 
 20 
from each pond were randomly selected to measure individual weight. Mean final weight, 
yields, percent survival, and FCR were determined. 
 
Outdoor tank system 
The outdoor tank system was a recirculating system consisted of a central 
reservoir (800 L) with a biological filter, a 0.33-hp sump pump, and 24, circular 
polyethylene tanks (0.85 m height x 1.22 m upper diameter, 1.04 m lower diameter). 
Another sump pump was used to move water from one of the production ponds to the 
central reservoir at a rate of 8 L min-1during the study period between 800 h and 1400 h 
to mimic a production pond setting. 
Juvenile L. vannamei (2.13 g initial weight) were collected from the research 
ponds and were size-sorted by hand. Juvenile shrimps were randomly selected and 
stocked at a rate of 30 shrimp per tank. Each tank and central reservoir were provided 
with two air stones connected to a 0.5-hp regenerative blower (Sweetwater Aquaculture 
Inc. Lapwai, ID, USA) to supply aeration. All tanks were covered by netting to prevent 
the shrimps from jumping out.  
Five test diets were randomly assigned amongst the 24 tanks (750 L). Four 
experimental diets used in production ponds were tested using five replicates per diet. 
The fifth diet, a reference commercial diet (Rangen Shrimp Grower, 35% protein, 8% 
lipid), was tested using four replicates. Juvenile shrimp were offered test diets twice per 
day at 0800 h and 1600 h. Daily ration of feed was calculated based upon an expected 
weekly growth of 1.5 g wk-1 and expected FCR of 1.2.  
 
 21 
 Dissolved oxygen concentration, temperature, salinity, and pH were monitored 
twice a day at 600 h and 1600 h using a YSI meter. Water samples were taken bi-weekly 
from central reservoir and two tanks to measure TAN with a spectrophotometer 
according to previously described methods.   
At the conclusion of the 10-week growth trial shrimp were counted and weighed 
individually. Mean final weight, final biomass, percent survival, and feed conversion 
ratio were determined.  
 
Statistical analysis 
The data (mean final weight, yields, percent survival, and feed conversion ratio) 
(FCR) were statistically analyzed using one-way analysis of variance to determine 
significant differences (P<0.05) among treatments, followed by the Student?Neuman?
Keuls multiple comparison test to determine significant differences among treatment 
means (Steel and Torrie, 1980). All statistical analyses were carried out using SAS (V9.1. 
SAS Institute, Cary, NC, USA). 
 
Results 
Nursery system 
  Average water quality variables throughout the 22-day nursery period were as 
follows: dissolved oxygen 6.02 ? 1.03 mg L-1; temperature, 26.08 ? 1.05 oC; pH 7.60 ? 
0.15; salinity 23.61 ? 1.71 ppt; and TAN 0.37 ? 0.04 mg L-1 (Table 2). Water quality 
conditions were maintained within acceptable limits for the culture of this species. At the 
conclusion of the shrimp nursery period, mean individual weight 0.038 ? 0.003 g, weight 
 
 22 
gain 0.036 ? 0.003 g, survival 57.94 ? 7.57%, and FCR 1.12 ? 0.06 were observed  
(Table 3). 
 
Production pond trial  
 The overall mean, standard deviation, and range of morning, afternoon, and night 
water quality variables observed throughout the 18-week growth trial are displayed in 
Table 8.  Water quality conditions were within a reasonable range for the culture of this 
species. However, high temperature readings (above 35.0 oC) and high salinity readings 
(above 32.0 ppt) were recorded related to high air temperature and drought condition 
Also, high dissolved oxygen readings (above 10 mg L-1) were observed in the afternoon 
due to photosynthetic activity. On the other hand, low dissolved oxygen readings (below 
2.5 mg L-1) were observed during periods that the phytoplankton died off (Boyd, 2007, 
2009). 
 Growth performance of L. vannamei reared under production pond conditions is 
summarized in Table 9. There were no differences (P > 0.05) in any of the production 
parameters. Mean final weight ranged from 16.86 g (fish meal treatment) to 15.98 g 
(PBM treatment).  Shrimp survival ranged from 86.6% (fish meal treatment) to 93.74% 
(PBM treatment). Yield ranged from 5,055 kg ha-1 (fish meal treatment) to 5,266 kg ha-1 
(DDGS treatment), and FCR ranged from 1.33 (DDGS treatment) to 1.38 (fish meal 
treatment). 
  
 
 
 
 23 
Outdoor tank trial 
 The overall mean, standard deviation, and range of morning and afternoon water 
quality variables observed over the 10-week growth trial were within acceptable limits 
for the culture of this species (Table 10). The outdoor tanks system had similar 
temperature (from 22.56 to 33.98 oC) and salinity readings (from 22.18 to 31.98 ppt) as 
compared to those observed in the pond water that was used as the system water supply.  
 Production results from the outdoor tanks were similar to those observed in the 
ponds.  There were no differences (P > 0.05) in growth performances of L. vannamei 
reared in the outdoor tank system. Biomass loading ranged from 548.3 g tank-1 (DDGS 
treatment) to 559.58 g tank-1(PBM treatment), mean final weight ranged from 18.53 g 
(DDGS treatment) to 19.00 g (pea meal treatment), percent survival ranged from 97.33% 
(pea meal treatment) to 98.67% (PBM, fish meal, DDGS treatments), and FCR ranged 
from 1.30 (PBM treatment) to 1.33 (DDGS treatment). Shrimp maintained on the 
commercial reference diets were not significantly different from those maintained on the 
test diets, albeit reference treatment had slightly higher biomass (579.83 g tank-1) and 
final weight (19.67 g), and lower FCR (1.24) (Table 11). 
  
Discussion 
Fish meal and other marine ingredients are excellent nutrient sources of essential 
nutrients for aquatic animals. These ingredients are from limited resources and will not 
support the continued expansion of aquaculture. The use of marine products will continue 
to be systematically replaced by terrestrial agriculture products for which production can 
be expanded. To reduce costs and improve the sustainability of shrimp feeds, fish meal 
 
 24 
must be replaced by other ingredients that are more cost effective and are from renewable 
resources. Soybean meal is the most widely used alternative ingredient to replace fish 
meal because of its lower cost per unit protein and capability to be produced in mass 
production. Moreover, the protein content and amino acid profile in soybean meal are 
easily balanced to make them comparable to those in fish meal. Several studies have 
demonstrated that soybean meal in combination with other ingredients is a viable 
alternative for partially or even totally replacement of fish meal from shrimp feeds  
(Davis and Arnold, 2000; Amaya et al, 2007a, b; Samocha et al., 2004; Lim and Dominy, 
1990; Molina-Poveda et al., 2004; Hernandez et al., 2004). These studies used nutrient 
balanced feed formulation, without causing negative impacts on growth performance of 
L. vannamei  
The results from the present study demonstrate that, L. vannamei fed formulated 
diets containing soybean meal as a basal ingredient in combination with either 10% 
poultry by-product meal, fish meal, distiller?s dried grains with solubles or ground pea 
meal resulted in similar yield, growth, survival, and feed conversion ratio of shrimp. 
Furthermore, treatments receiving diets with no fish meal showed no differences in 
performances compared to fish meal treatment or commercial reference treatment in both 
pond production and outdoor tank trials. The results from the present study confirm that 
fish meal in soybean meal-based diets can be totally replaced with either 10% poultry by-
product meal, DDGS or pea meal. 
 Similar results were observed by Roy et al. (2009) who tested these same dietary 
treatments with juvenile L. vannamei reared in inland low salinity waters of west 
Alabama. Similar growth, survival, weight gain (%) and FCR were observed among 
 
 25 
treatments in both laboratory and on farm outdoor tank trials. Shrimp fed diets containing 
either 10% poultry by-product meal, distiller?s dried grains with solubles, or pea meal 
performed as well as shrimp fed the fish meal diet.  Markey (2007) also reported no 
differences in growth when shrimp were offered a soybean based diet mixed with poultry 
by-product meal (PBM) at level of 0, 5, 10, and 15% PBM as a replacement for fish meal 
in diets for L. vannamei used in both production pond and an outdoor tank trials. They 
obtained excellent results with mean final weight ranging from 19.4 to 24 g, survival 
from 80 to 96.7%, and feed conversion ratio from 1.0 to 1.1.  Amaya et al. (2007a,b) fed 
diets containing 16% PBM in combination with soybean meal as a major protein source 
and the inclusion of corn gluten meal to replace fish meal (FM) in different levels at 9%, 
6%, 3%, and 0% fish meal for L. vannamei reared in production ponds and green water 
outdoor tanks. The results show no differences in mean net yield, mean final weight, 
survival, and FCR of the shrimp. Samocha et al. (2004) reported no differences on 
growth responses of Pacific white shrimp fed practical diets containing 0% to 100% co-
extruded soybean poultry by-product meal with egg supplement as a substitute for fish 
meal. Steffens (1994) also reported a viable use of poultry by-product meal in 
combination with feather meal at 27% inclusion as a partial replacement or with lysine 
and methionine supplementation at the complete replacement to fish meal in diets for 
rainbow trout. The results from this study confirm the viable use of soybean meal in 
combination with poultry by-product to substitute fish meal without affecting shrimp 
growth and production.    
 Distiller?s dried grains with soluble (DDGS) are also one of the potential 
ingredients which are co-product of ethanol production. DDGS up to 30% can be used in 
 
 26 
channel catfish diet when lysine is supplemented (Robinson and Li, 2008; Lim et al., 
2009). The results from this study are similar to the results reported by Robinson and Li 
(2008) that fish fed diet containing DDGS mixed with soybean meal and corn gluten 
meal had similar or higher weight gain and lower FCR compared to fish fed soybean 
meal-fish meal control diet. Likewise, Webster et al. (1991) reported the feasibility of 
DDGS used as a partial replacement for soybean meal with the inclusion of 10% fish 
meal in juvenile channel catfish diet. Fish fed diets contain 35% DDGS performed as 
well as fish fed control diet (0% DDGS. They also reported that, with lysine 
supplementation, up to 70% DDGS can be used in juvenile channel catfish diet. 
Additionally, Webster et al. (1992) demonstrated that fish meal in juvenile channel 
catfish diet can be totally replaced by 35% DDGS and soybean meal with supplemental 
lysine without adversely affecting their growth performance. Therefore, diet containing 
10% DDGS inclusion with soybean meal and corn gluten meal can be a potential 
alternative for L. vannamei diet.   
Pea meal has been successfully used in aquaculture feeds. The results of the 
present study show that shrimp fed 10% pea meal inclusion with soybean meal and corn 
gluten meal have equal performance to shrimp fed the other test diets. Similar result was 
reported by Cruz-Suarez et al. (2001) who successfully replaced soybean meal and wheat 
with 30% pea meal in diets for blue shrimp, L. stylirostris without affecting shrimp 
performance. Gouveia and Davies (2000) also demonstrated the feasibility of 40% pea 
seed meal inclusion to substitute fish meal in European sea bass diet. Another successful 
research of pea meal inclusion to replace soybean meal and whole wheat in L. vannamei 
diets was reported by Davis et al. (2002). The results after two 7-week growth trials 
 
 27 
under the laboratory showed no significant effects on shrimp performance when fed diets 
containing pea meal.   
Besides feed ingestion, growth, feed conversion ratio, and survival of shrimp can 
be affected by water condition, e.g., temperature, salinity, etc. Ponce-Palafox et al. (1997) 
indicated that juvenile P. vannamei had the best survival and growth between 
temperatures of 28 and 30 oC and salinities between 33 and 40 ppt. The mean temperature 
and salinity reading from this study were in acceptable range for the culture of 
Litopenaeus vannamei. However, vary temperature readings ranged from 22.56 to 33.98 
?C, and 24.97 to 35.68 ?C were observed in the outdoor tanks and production ponds, 
respectively. Additionally, obviously high salinity readings between 21.90 ? 32.78 ppt 
were recorded in this study compared to those earlier reported for this site for the 
production ponds.  
As demonstrated in Figure 1, weekly weight gain of shrimp from the pond study 
tended to increase as average temperatures moved from  28 C to 32 C then decrease when 
water temperature in ponds were greater than 32 ?C. This finding is similar to the results 
reported by Wyban et al. (1995), who reported decreasing growth of P. vannamei when 
pond temperature was below 23 oC and at 30 oC or greater. Mean survival rates of this 
study are excellent, 98.3% under tank trial and 90.3% under pond trial. Similar results 
had been reported by Bray et al. (1994), who found no differences on survival of L. 
vannamei across different salinities from 5 to 49 ppt. However, mean final weights of 
shrimp cultured at low salinity were significantly greater than at high salinity.  
Mean final weight of shrimp reared in a 10-week outdoor tank trial (15.98 ? 16.86 
g) are greater than shrimp reared in a 17-week production pond trial (18.5 ? 19.7 g) at the 
 
 28 
similar salinity, but temperature in ponds was 2 ?C greater than in tanks. These results 
suggested that the reduction of growth performance of shrimp observed from this study 
may affect by high temperature or the interaction of high water temperature. However, 
these factors showed no effect on survival of shrimp.  
In conclusion, the result from this study indicated that diets containing soybean 
meal as a primary protein source in combination with 10% of poultry by-product meal, 
fish meal, distiller?s dried grains solubles or pea meal are viable under a variety of 
research conditions. This is in agreement with Roy et al. (2009) who reported that high 
levels of soybean meal can be used for commercial shrimp feed formulations. 
Additionally, fish meal and animal protein can be replaced by plant protein via well 
balanced feed formulation.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 29 
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Warner-Smith, R., 2000. Replacement of fish meal in diets for Australian silver 
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186, 293-310. 
Alvarez, J.S., Hernandez-Llamas, A., Galindo, J., Fraga, I., Garcia, T., Villareal, H., 
2007. Substitution of fishmeal with soybean meal in practicaldiets for juvenile 
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 41 
Table 1. Feeding regime for Litopenaeus vannamei post larvae, based on mean shrimp 
weight determined every three days, percent body weight and feed types utilized 
throughout a 22-day nursery period.  
Day Mean weight  (mg)           Feed type Feed (% body weight) 
1 to 3 1.54 100 Artemia
a / PL, PL redib 
(1:2) 25 
4 to 7 2.53 PL redi, Cruc #0 (1:2) 25 
8 to 9 4.53 Cru # 0 25 
10 to 12 5.91 Cru # 0 15 
13 to 15 10.73 Cru # 0, Cru #1& #2 (1:1) 15 
16 to 22 18.20 Cru # 1& #2 15 
aINVE Americas, Inc. Salt Lake City, UT, USA. 
bPL Redi-reserve 400-600 microns. 50% Protein, produced by Zeigler Bros, Inc., 
Gardners, PA, USA. 
cCrumble feed, 45% protein, produced by Rangen Inc., Buhl, Idaho, USA. 
 
 
 
 
 
 
 42 
Table 2. Water quality variables observed over a 22-day nursery period for L. vannamei 
post larvae, nursed in a recirculating system composed of six 6,000-L nursery tanks.  
 Average Minimum Maximum Standard deviation CVa 
Temperature (?C) 26.08 23.41 28.54 1.05 4.03 
DO (mg L-1)b 6.02 3.32 7.83 1.03 17.06 
Salinity (ppt) 23.61 21.30 27.04 1.71 7.23 
pH 7.60 7.20 7.90 0.15 1.91 
TAN (mg L-1)c 0.37 0.32 0.45 0.04 10.69 
aCV = Standard deviation / mean x 100. 
bDissolved oxygen. 
cTotal ammonia-nitrogen. 
 
 
 
 
 
 
 
 
 
 43 
Table 3. Mean production parameters of L. vannamei post larvae (initial weight of 1.5 
mg), stocked at a density of 33 PL L-1 and nursed for 22 days in a recirculating system 
composed of six 6,000-L nursery tanks.
 Mean Minimum Maximum Standard 
deviation CV
a 
Final weight (mg) 38.09 33.91 44.46 3.68 9.66 
Weight gainb (mg) 36.59 32.41 42.96 3.68 10.06 
Survival (%) 57.94 47.00 66.24 7.57 13.07 
FCRc 1.12 1.01 1.17 0.06 5.53 
Yield (g tank-1) 4,353 4,146 4,836 263.80 6.06 
Standing cropd (kg m-3) 1.209 1.152 1.343 0.073 6.060 
aCV = Standard deviation / mean * 100. 
bWeight gain = Final weight - Initial weight. 
cFood conversion ratio = Total feed offered / biomass increase. 
dStanding crop (kg m-3) = (Yield/1,000)/3.6 m3. 
 
 
 
 
 
 44 
 Table 4. Ingredient composition (g 100 g-1 as is) of four experimental diets utilizing 
soybean-based diets in combination with 10% poultry by-product meal (PBM), fish meal 
(FM), distiller?s dried grains with soluble (DDGS) or ground pea meal (PM) as a protein 
source.  
Ingredient 10% PBM 10% FM 10% DDGS 10% PM 
Soybean meal  55.12 53.71 58.01 58.00 
Milo 24.81 26.19 16.34 15.33 
PBM 9.99 - - - 
FM - 10.01 - - 
DDGS - - 10.00 - 
PM - - - 10.00 
Corn gluten meal - - 4.83 4.83 
Dicalcium phosphate  2.90 2.90 3.38 3.42 
Fish oil  5.08 5.09 4.83 5.82 
Bentonite 1.50 1.50 1.50 1.50 
Squid meal  - - 0.50 0.50 
Vitamin premixa        0.33 0.33 0.33 0.33 
Mold inhibitor 0.15 0.15 0.15 0.15 
Mineral premixa 0.08 0.09 0.09 0.09 
Stay-C, 35% vit. C activity 0.02 0.02 0.02 0.02 
Copper sulfate 0.01 0.01 0.01 0.01 
Total 100 100 100 100 
Diets were formulated to contain 36% protein and 8% lipid. 
Diets were commercially manufactured by Rangen? (Angleton, TX, USA) using 
extrusion processing. 
aVitamin premix and mineral premix are proprietary products, thus their composition is 
not listed. 
 
 45 
Table 5. Nutrient composition (dry matter basis) of four experimental diets containing 
soybean meal in combination with 10% poultry by-product meal (PBM), fish meal (FM), 
distiller?s dried grains with solubles (DDGS) or ground pea meal (PM). 
Nutrient 10% PBM 10% FM 10% DDGS 10% PM 
Crude protein 35.9 35.5 38.2 36.1 
Moisture 8.85 8.11 8.43 8.43 
Fat 8.16 8.26 7.66 7.41 
Crude fiber 1.78 1.77 7.95 2.39 
Ash 8.40 9.59 6.68 7.90 
Diets were formulated to contain 36% protein and 8% lipid. 
Diets were commercially manufactured by Rangen? (Angleton, TX, USA) using 
extrusion processing. 
Diets were analyzed by New Jersey Feed Laboratory, Trenton, NJ, USA (Roy et al., 
2009). 
 
 
 
 
 
 
 
 
 46 
Table 6. Amino acid profile of four experimental diets containing soybean meal in 
combination with 10% poultry by-product meal (PBM), fish meal (FM), distiller?s dried 
grains with solubles (DDGS) or ground pea meal (PM). 
Amino acid 
Percent as is 
10% PBM 10% FM 10% DDGS 10% PM 
Methionine 0.54 0.61 0.62 0.60 
Cystine 0.53 0.54 0.62 0.61 
Lysine 2.19 2.08 2.18 2.07 
Phenylalanine 1.52 1.63 1.71 1.72 
Leucine 2.65 2.78 2.91 2.99 
Isoleucine 1.47 1.54 1.53 1.53 
Threonine 1.31 1.33 1.35 1.34 
Valine 1.27 1.33 1.30 1.29 
Histidine 0.81 0.87 0.90 0.88 
Arginine 2.42 2.41 2.54 2.51 
Glycine 1.71 1.91 1.7 1.56 
Aspartic Acid 3.61 3.8 3.9 3.83 
Serine 1.72 1.72 1.82 1.86 
Glutamic Acid 6.15 6.57 7.40 7.00 
Proline 2.03 1.84 2.06 2.06 
Hydroxyproline 0.24 0.15 0.15 0.07 
Alanine 1.91 1.82 1.86 1.75 
Tyrosine 1.08 1.10 1.20 1.21 
Total 33.16 34.03 35.75 34.88 
 
Diets were analyzed by New Jersey Feed Laboratory, Trenton, NJ,USA (Roy et al., 
2009). 
 
 47 
Table 7. Feeding regime for L. vannamei reared in the grow-out 0.1-ha ponds during an   
18-week experimental period, based on estimated growth rate of 1.5 g wk-1 and assumed 
survival rate of 70%. 
Week Population 
Feed input per day 
kg pond-1 kg ha-1 
1 35,000 0.24 2.36 
2 34,382 0.89 8.93 
3 33,765 3.00 30.00 
4 33,147 2.79 27.86 
5 32,529 8.36 83.65 
6 31,912 7.62 76.20 
7 31,294 8.05 80.47 
8 30,676 7.32 73.25 
9 30,059 7.18 71.77 
10 29,441 7.03 70.30 
11 28,824 7.57 75.71 
12 28,206 7.57 75.71 
13 27,588 6.50 65.00 
14 26,971 6.50 65.00 
15 26,353 6.50 65.00 
16 25,735 4.46 44.64 
17 25,118 5.71 57.14 
18 24,500 4.29 42.86 
 
 
 48 
Table 8. Summary of water quality variables monitored over an 18-week growth trial for 
L. vannamei, fed experimental diets containing high levels of soybean meal in various 
combinations and cultured in 0.1-ha ponds. Values represent the mean ? standard 
deviation and values in parenthesis represent minimum and maximum readings. 
Parameter 10% PBM 10% FM 10% DDGS 10% PM  
Temperature (?C) 
       am 
  
       noon 
  
       pm 
 
 
 
29.36?1.72 
(24.97, 33.08)  
31.55?1.91 
(26.76, 34.94)  
31.22?1.95 
(25.07, 34.96) 
  
29.48?1.76 
(25.21, 32.91)  
31.63?1.98 
(26.02, 35.65)  
31.40?2.03 
(25.47, 35.68) 
 
 
29.37?1.71 
(25.21, 32.87)  
31.49?1.95 
(26.48, 35.03)  
31.25?1.95 
(26.46, 35.40) 
  
29.33?1.76 
(25.01, 32.88)  
31.65?1.99 
(26.69, 35.28)  
31.42?2.01 
(25.37, 35.49)  
DOa (mg L-1) 
       am 
  
       noon 
  
       pm 
  
     Readings< 2.5(days) 
  
4.21?1.12 
(1.21, 10.69)  
10.25?3.07 
(2.22, 19.43)  
8.31?2.99 
(1.54, 17.80)  
5.0?3.8 
  
4.04?1.29 
(0.23, 13.43)  
10.92?3.35 
(2.46, 20.99)  
8.89?3.22 
(0.52, 17.65)  
9.0?6.1 
  
4.30?1.24 
(0.29, 13.84)  
10.51?3.54 
(2.14, 18.99)  
8.52?3.12 
(1.39, 19.41)  
4.5?3.1 
  
4.06?1.18 
(0.96, 12.63)  
11.98?3.38 
(1.48, 21.36)  
9.33?3.27 
(0.16, 19.99)  
6.0?3.2  
pH 
       am 
  
       noon 
  
       pm 
 
  
7.75?0.55 
(6.42, 9.20)  
8.49?0.54 
(7.15, 9.93)  
8.45?0.58 
(7.03, 9.82) 
  
7.79?0.47 
(6.95, 9.11)  
8.55?0.48 
(7.08, 9.46)  
8.51?0.50 
(7.19 ,9.47) 
  
7.70?0.46 
(6.93, 9.00)  
8.43?0.51 
(6.70, 9.52)  
8.42?0.54 
(7.10, 10.01) 
  
7.97?0.43 
(7.09, 9.04)  
8.67?0.43 
(7.05, 9.91)  
8.65?0.43 
(7.19, 9.81) 
Salinity (ppt) 
       am 
  
       noon 
  
       pm 
 
 
28.10?1.44 
(23.83, 31.25)  
28.07?1.40 
(23.92, 31.14)  
27.96?1.49 
(23.83, 31.15) 
 
27.78?2.02 
(22.35, 32.75)  
27.75?1.97 
(22.74, 32.58)  
27.69?2.02 
(22.70, 32.63) 
 
28.30?1.68 
(23.36, 32.72)  
28.25?1.66 
(23.42, 32.57)  
28.17?1.71 
(23.48, 32.58) 
 
27.97?1.67 
(21.93, 31.78)  
27.92?1.64 
(21.98, 31.52)  
27.84?1.70 
(21.90, 31.59)  
Secchi (cm) 
 
 
30.47?26.89 
(5, 110) 
 
27.37?23.23 
(5, 110) 
 
30.79?27.26 
(5, 110) 
 
25.53?22.94 
(5,110)  
TANb (mg L-1) 
 
 
0.72?1.59 
(0.00, 7.39) 
 
0.89?1.58 
(0.00, 6.03) 
 
0.93?1.95 
(0.00, 10.00) 
 
0.43?0.72 
(0.00, 2.95) 
aDissolved oxygen. 
bTotal ammonia-nitrogen. 
 
 49 
Table 9. Mean production parameters at 18 week experimental period of juvenile Pacific 
white shrimp, L. vannamei (initial weight of 40 mg), fed experimental diets containing 
high levels of soybean meal in combination with poultry by-product meals (PBM), fish 
meal (FM), distiller?s dried grains with solubles (DDGS) or ground pea meal (PM), and 
cultured in 0.1-ha ponds. 
Treatment Yield  (kg ha-1) Final weight  (g) FCRa Survival  (%) 
10% PBM 5187.4 15.98 1.33 93.74 
10% FM  5054.8 16.86 1.35 86.60 
10% DDGS 5265.5 16.28 1.33 92.20 
10% PM 5194.8 16.25 1.37 88.58 
P-valueb 0.8215 0.7724 0.8622 0.6146 
PSEc 73.9001 0.2825 0.0184 1.9943 
aFeed conversion ratio = Total feed offered / biomass increase. 
bAnalysis of variance was used to determine significant differences (P < 0.05) among 
treatment means (n=4). 
cPooled standard error of treatment means.  
 
 
 
 
 
 50 
Table 10. Water quality variables monitored over a 10-week growth period of juvenile L. 
vannamei (initial weight of 2.13 g), fed experimental diets containing high levels of 
soybean meal in combination with poultry by-product meals (PBM), fish meal (FM), 
distiller?s dried grains with solubles (DDGS) or ground pea meal (PM), and reared under 
a semi-closed recirculating system. Values represent the mean ? standard deviation and 
values in parenthesis represent minimum and maximum readings. 
 Temperature  (?C) DO (mg L-1)a pH Salinity  (ppt) TAN  (mg L-1)b 
am 27.83?1.73 (22.56, 30.91) 6.11?0.61 (3.77, 8.06) 7.84?0.15 (7.44, 8.44) 29.96?0.94 (23.86, 31.71) 0.13?0.27 (0.00, 1.19) 
pm 30.39?1.93 (25.19, 33.98) 6.07?0.59 (3.00, 7.73) 8.09?0.18 (7.61, 8.78) 29.90?1.13 (22.18, 31.98) 
 
 
aDissolved oxygen. 
bTotal ammonia-nitrogen. 
 
 
 
 
 
 
 51 
Table 11. Mean production parameters at 10 week experimental period of L. vannamei 
(initial weight of 2.13?0.16 g), fed diets containing high levels of soybean meal in 
combination with poultry by-product meals (PBM), fish meal (FM), distiller?s dried 
grains with solubles (DDGS) or ground pea meal (PM), and a commercial feed used as 
reference diet, and reared under a semi-closed recirculating system. 
Treatment Biomass (g tank-1) Final weight  (g) FCR Survival  (%) 
10% PBM 553.0 18.7 1.32 98.7 
10% FM 557.7 18.8 1.31 98.7 
10% DDGS 548.3 18.5 1.33 98.7 
10% PM 554.4 19.0 1.31 97.3 
Reference dietsa 579.8 19.7 1.25 98.3 
P-valueb 0.1032 0.0930 0.8722 0.1482 
PSEc 3.8095 0.1354 0.4486 0.0109 
aReference diets = 35% Protein, Rangen 35,0 (Buhl, Idaho).  
bAnalysis of variance was used to determine significant differences (p < 0.05) among 
treatment means (n=4). 
 cPooled standard error of treatment means. 
 
 
 
 
 52 
Figure 1. Relationship between water temperature (?C) and mean weight gain (g) over an 
18-week growth trial for L. vannamei (initial weight of 40mg), fed experimental diets 
containing high levels of soybean meal in combination with poultry by-product meals 
(PBM), fish meal (FM), distiller?s dried grains with solubles (DDGS) or ground pea meal 
(PM), and cultured in 0.1-ha ponds. 
 
 
 
 
 
 
 
y = -0.059x2 + 3.7905x - 59.707 
R? = 0.2403 
0.00
0.50
1.00
1.50
2.00
2.50
26.00 28.00 30.00 32.00 34.00 36.00
We
ekly
 grow
th 
(g)
 
Water temperature (?C) 
 
 53 
CHAPTER III 
REPLACEMENT OF SOLVENT EXTRACTED SOYBEAN MEAL BY SOY 
PROTEIN CONCENTRATE FOR PACIFIC WHITE SHRIMP  
(Litopenaeus vannamei) REARED UNDER CLEAR WATER CONDITION 
 
Abstract 
Solvent extracted soybean meal (SE-SBM) is one of the most efficient and widely 
used products at present; however, compare to fish meal which has a more optimal 
nutrient profile but higher price, soybean meal has lower nutrient contents such as 
protein, essential amino acids, essential fatty acids, and minerals which need be 
supplemented in feed formulation. As the nutrient density of the diet is increased the use 
of SE-SBM is inefficient as there is insufficient room in the formulation for other 
necessary components. Hence, higher protein ingredients are often required. Soy protein 
concentrate is a high protein ingredient that is produced from high quality, dehulled 
soybean seeds. By removing most of the oil, water soluble non-protein constituents, and 
many of the anti-nutritional factors, a variety of high protein products with varying 
characteristics can be produced. With increased availability and reduced prices of soy 
protein concentrate, which has higher protein content than SE-SBM, there is interest in 
using these meals in practical diet formulations. The objective of this study was to 
evaluate the growth response of Pacific white shrimp Litopenaeus vannamei fed practical 
diets containing different levels of soy protein concentrate (SPC) as a replacement of 
 
 54 
soybean meal. In the first trials juvenile shrimp (0.12?0.01 g, initial weight) were stocked 
at a density of 15 shrimp per tank in 24, (340 L) square tank with a common biological 
filter. Six dietary treatments containing increasing percentages of SPC (0%, 5%, 10%, 
20%, 40%, and 40% supplemented with DL-methionine) were offered to shrimp 2 times 
daily over a 55-day feeding trial using 4 replicates per treatment. In the second trial, 
juvenile shrimp (1.14?0.05 g, initial weight) were stocked under the same system and 
same conditions as the first trial. Diets were similar to those in the first experiment with 
the following modifications, crystalline DL-methionine was replaced with micro-
encapsulated methionine (diet 6). Diet 7 that contained 40% SPC with additional fish 
soluble, and a commercial reference diet were also included. The test diets were fed to 
juvenile shrimp 2 times daily over a 53-day feeding trial using 3 replicates per treatment.  
The results of these studies indicated that SPC in substitution of soybean meal at 
20% and greater caused a reduction in mean final weights and an increase in FCR. 
Survival was not different among treatments. Thus, the results from this study suggest 
that up to 10%of SPC could be included in soybean meal based diet without causing 
adverse effect. Moreover, the addition of methionine or fish soluble to the diets with high 
level of SPC (40%) did not enhance growth of L. vannamei. 
 
 
 
 
 
 
 
 55 
Introduction 
 Soybean has long been recognized as one of the most widely distributed oil seeds 
which produce a variety of products used in the animal feed industry. The use of soybean 
products, in the form of soybean meals or specialty soy products, have increased steadily 
over the past decade. This is a result of their consistent quality, especially with respect to 
protein and amino acid profile, and high digestibility. Soybean meal currently represents 
the predominant choice as an alternate protein source in commercial feed formulations 
for aquatic species.  
 However, the use of soybean meal in feeds for aquatic animals has limitations. 
Raw soybean seeds contain antinutritional factors that can cause negative impacts to 
shrimp digestive system and may influence its palatability (Forster et al., 2002). 
Additionally, soybean proteins are relatively low in sulfur amino acids (SAAs), e.g., 
methionine and cystine, as well as lack of n-3 highly unsaturated fatty acids (HUFAs), 
e.g., eicosapentaenoic acids (EPA) and docosahexaenic acids (DHA), which can have 
adverse effects on growth performance of shrimp (Lim and Dominy, 1990; Messina, 
1999; Akiyama, 1989). However, exposing soybean to heat process can reduce the 
presence of antinutritional factors (ANF) and improve its palatability (Brown et al., 2008; 
Akiyama, 1989). Moreover, SAAs and EFAs in feed can be adjusted through well 
balanced formulations by supplementing synthetic methionine and various oil sources of 
marine origin to meet shrimp requirement.  
 The use of soy products as alternative protein sources in shrimp diets has been the 
focus of numerous research projects. Solvent extracted soybean meal (SE-SBM) is one of 
the most efficient and widely used protein sources in the feed industry. This is primarily 
 
 56 
because of its favorable protein content, suitable amino acid profile, economic 
opportunity, and good digestibility. The uses of SE-SBM as a protein source in diets of  
L. vannamei has received considerable attention with good results (Alvarez et al., 2007; 
Samocha et al., 2004; Lim and Dominy, 1990). However, in comparison to fish meal 
which has a more optimal nutrient profile but higher price, soybean meal has a lower 
nutrient content in terms of protein, essential amino acids (e.g. methionine), essential 
fatty acids, and minerals which need be adjusted with supplements or blending with other 
products. As the nutrient density (protein and energy) of the diet is increased, the use of 
SE-SBM is inefficient as there is insufficient room in the formulation for other necessary 
components.   
 Soy protein concentrate (SPC) is another high protein product that is produced 
through a series of different extraction and precipitation process from high quality 
dehulled soybeans. By removing most of the oil, water soluble non-protein constituents, 
and many of the anti-nutritional factors, a variety of high protein products with varying 
characteristics can be produced. There is approximately 65% crude protein in soy protein 
concentrate (SPC) which is similar to level found in fish meal, and considerably higher 
than those of SE-SBM (Paripatananont et al., 2002; Liu, 1997). Many feed manufacturers 
prefer a reasonable level of a high protein ingredient in their formulations to allow room 
to manipulate other ingredients. Hence, soy protein concentrate could be used as an 
alternative ingredient to soybean meal in shrimp diets. These products are increasingly 
being used by the animal feed industry. As the availability of these product increases, the 
price is likely to be reduced making them more cost effective solutions. As SPC has 
 
 57 
higher protein content than SE-SBM, there is interest in using these meals in practical 
diet formulations.  
Although the use of soy protein concentrate in aquatic feed has been reported in 
many fish and shrimp species (Tibaldi et al., 2006; Refstie et al., 2001; Forster et al., 
2002; Paripatananont et al., 2002; Mambrini et al., 1999; Storebakken et al., 1998; 
Kaushik et al., 1995), there is limited information available on the utilization of SPC in L. 
vannamei diets. Both soybean meal and SPC are deficient in methionine. Hence, 
methionine supplement may be required in shrimp diets with high levels of SPC. In 
addition, feed palatability is always a concern when using non-marine ingredients such as 
SPC. This may be improved by adding attractive materials such as fish soluble.  
Therefore, the objective of this study is to evaluate the response of L. vannamei to 
increasing levels of SPC as replacement of SE-SBM in practical diets for L. vannamei, 
and to determine if methionine or fish soluble could improve their growth response.  
 
Materials and Methods 
A series of experiments were conducted at the Alabama Department of 
Conservation and Natural Resources, Marine Resource Division, Claude Peteet 
Mariculture Center in Gulf Shores, Alabama. Two growth trials were carried out to 
confirm the response of L. vannamei to increasing levels of SPC as replacement of SE-
SBM in clear water tank system. 
 
 
 
 
 58 
First trial 
 Experimental diets  
Six diets formulated to be isonitrogenous (approximately 35% crude protein with 
6% lipid) were tested using juvenile L. vannamei. Six experimental diets were formulated 
to contain vary levels of SPC (0, 5, 10, 20, 40 and 40% supplemented with crystalline 
DL-methionine) as replacement of SE-SBM (Table 1). The proximate composition 
including protein digestibility (using 0.0002% pepsin solutions) of experimental diets are 
listed in Table 2. Diets were prepared by mixing the ingredients in a mixer (Hobart, Troy, 
Ohio) for 30 minutes. Subsequently, hot water was added to the mixture until appropriate 
consistency for pelleting was obtained.  Diets were then passed through a meat grinder 
and a 3-mm mesh sieve. Pellets were air dried (<50 C) to a moisture content of less than 
10%. After drying, pellets were crumbled, packed in sealed plastic bags and stored in a 
freezer until use. 
Growth trial  
Juvenile shrimp were obtained from nursery system and were selected by hand-
sorting to a uniform size. Then juvenile shrimp (0.12?0.01 g, initial weight) were stocked 
into 24 square tanks (0.7x0.7x0.7 m, 340-L volume), under a recirculating system at a 
density of 15 shrimp per tank. Six experimental diets were randomly assigned with four 
replications (tanks) per treatment. Test diets were applied twice daily at 0800 and 1600 h 
for a 55-day experimental period. Feed input was pre-calculated on a weekly basis using 
an expected feed conversion ratio of 2:1 and a doubling in size until individual shrimp 
weighed one gram.  Thereafter, a growth rate of one gram per week was assumed. Each 
tank was covered with nylon netting, and was continuously aerated with two air stones. 
 
 59 
Shrimp were counted every two weeks to readjust the daily feed input. At the conclusion 
of the 10 week growth trial shrimp were counted and weighed individually. Percentage 
survival, mean final weight, biomass and feed conversion ratio were determined.  
 
Second trial 
 Experimental diets  
The second trial was a repeat of the first trial using the same increasing levels of 
SPC as replacements of SE-SBM except that micro-encapsulated methionine or fish 
soluble were used to replace crystalline DL-methionine (diet 6 and 7, respectively) (Table 
3). Experimental diets were formulated to be isonitrogenous (approximately 35% crude 
protein) with constant lipid content (6%) were tested using juvenile L. vannamei. A 
commercial diet (Rangen Shrimp Grower, 35% protein, 8% lipid) was used as a reference 
diet. The proximate nutrient composition of experimental diets are listed in Table 4. Diets 
were prepared and kept by the same processes as the first trial.  
Growth trial  
Juvenile shrimp were obtained from nursery system and were selected by hand-
sorting to a uniform size. Then juvenile shrimp (1.12?0.05 g, initial weight) were stocked 
into the same recirculating clear water system at a density of 15 shrimp per tank. Seven 
experimental diets and a commercial reference diet were randomly assigned to three 
replications tanks. Test diets were applied twice daily at 0800 and 1600 h for a 53-day 
experimental period. Based on the previous work, a growth of 0.8 g wk-1 and a FCR of 2 
were used to calculate the amount feed needed each week, as feed were pre-weighed on a 
weekly basis. Shrimp were counted every two weeks to readjust the daily feed input. At 
 
 60 
the conclusion of the growth trial shrimp were counted and weighed individually. 
Percentage survival, mean final weight, biomass, and FCR were determined.  
 
Water quality monitoring 
For both experimentals, dissolved oxygen (DO), temperature, salinity and pH 
were measured twice daily in the biological filter and two of the rearing tanks at the 0800 
and the 1600 h using a YSI 556MPS meter (Yellow Spring Instrument Co., Yellow 
Springs, OH, USA). Water samples were taken from the filter and two of the tanks to 
determine total ammonia-nitrogen (TAN) on a weekly basis using the Orion ammonia 
electrode probe (Thermo Fisher Scientific Inc., Waltham, MA, USA).  
 
Statistical analysis 
All data were statistically analyzed using one-way analysis of variance to 
determine significant differences (P<0.05), followed by the Student?Neuman?Keuls 
multiple comparison test to determine significant differences among treatment means  
(Steel and Torrie, 1980). All statistical analyses were carried out using SAS (V9.1. SAS 
Institute, Cary, NC, USA). 
 
Results 
First trial 
 Overall means, standard deviations, and ranges of morning and afternoon water 
quality variables observed over 55-day growth trial were within acceptable limits for the 
 
 61 
culture of this species (Table 5). Mean DO, temperature, salinity, pH, and TAN were  
6.38 mg L-1, 27.67 ?C, 22.1 ppt, 8.41 and 0.49 mg L-1, respectively. 
 Although there were no differences (P > 0.05) in survival, significant differences 
in biomass, mean individual weight, and FCR among treatments were observed in the 55-
day growth trial in L. vannamei fed the various diets (Table 6). Survival ranged from 98.3 
to 100%. Mean individual weight ranged from 5.50 to 6.38 g with diets containing SPC at 
20% or greater resulted in lower mean individual weight than diet containing lower level 
of SPC. Biomass ranged from 82.5 to 96.2 g tank-1. FCR ranged from 2.19 to 2.57 with 
diets containing SPC at 20% or higher resulted in greater FCR than diet containing SPC 
at 10% and lower (Figure 1). Methionine supplementation in diet with 40% SPC did not 
improve growth and FCR compare to diet with same level of SPC containing no 
methionine. 
   
Second trial 
 Overall means, standard deviations, and ranges of morning and afternoon water 
quality variables observed over 53-day growth trial were within acceptable limits for the 
culture of this species (Table 5). Mean DO, temperature, salinity, pH, and TAN were    
7.6 mg L-1, 26.5 ?C, 16.1 ppt, 7.7 and 0.10 mg L-1, respectively. 
 Results from this trial yielded similar results to the first trial with lower mean 
individual weight and higher FCR at SPC level of 20% or greater. However, there were 
no differences in mean individual weight and FCR at the end of a 53-day growth trial in 
L. vannamei fed diet containing increasing level of SPC as replacement of SE-SBM and 
diets with fish soluble or micro-encapsulated methionine supplement (Figure 3-2). 
 
 62 
Survival ranged from 77.8 to 100%. Mean individual weight ranged from 6.0 to 8.0 g 
with diets containing SPC at 20% or greater resulted in lower mean individual weight 
than diet containing lower level of SPC. Biomass ranged from 70.0 to 117.9 g tank-1. 
FCR ranged from 1.82 to 2.54 with diets containing 40% SPC exhibiting higher FCR 
than diets containing lower level of SPC. The inclusion of micro-encapsulated 
methionine or fish solubles in diet containing 40% SPC resulted in no beneficial effects 
on either mean final weight or FCR. Shrimp fed on the commercial reference (control) 
diets were not significantly different from those maintained on the test diets. 
 
Discussion 
 The results from the present study suggested that SPC can be included at 10% as 
replacement of SE-SBM in L. vannamei diet without causing adverse effect on growth 
performance of shrimp. In addition, lower biomass and higher feed conversion ratio were 
observed in diets containing SPC at 20% or greater in both growth trials. There were no 
differences in performance of shrimp fed experimental diets containing high level of SPC 
(40%) with and without methionine supplementation in both trials, or with the addition of 
fish solubles in the second trial. The results of this study are in agreement with 
Paripatananont et al. (2001) who evaluated diets containing SPC as a replacement of fish 
meal in diet for Penaeus monodon, reared under laboratory condition using diets 
containing 36% protein on dry weight basis. They found that 17.5% SPC can be 
incorporated in shrimp feed containing 12% SBM without affecting the growth of 
shrimp. However, significantly lower percent survival and weight gain, and higher FCR 
were observed in shrimp fed diets containing SPC at 26.5% or higher as replacements   
 
 63 
75 and 100% of fish meal in diets. With the same species, Forster et al. (2002) suggested 
an applicable level of 27.2% SPC. It should be noted, however, their diets contained fish 
meal and supplemented with indispensible amino acid.  
With rainbow trout, Mambrini et al. (1999) observed a decrease in growth when 
more than 50% fish meal in diets was replaced by SPC. In contrast, Kaushik et al. (1995) 
obtained no negative effect on the growth performance and nutrient utilization of rainbow 
trout fed diet containing SPC supplemented with L-methionine as a total replacement of 
fish meal. However, the same study report a reduction in growth rate of fish when up to 
50% of fish meal was replaced by soy flour. Likewise, with shrimp, Lim and Dominy 
(1990) reported a decrease in weight gain when L. vannamei were fed with diets 
containing 42%SE-SBM or higher. For juvenile American lobster, Floreto et al. (2000) 
indicated that dietary inclusion of SBM at levels of 50% or higher resulted in 
significantly lower body weight gain than in treatment fed diets contain less than 50% 
SMB. The same study also showed that amino acid supplementation, e.g., arginine, 
leucine, methionine, and tryptophan, resulted in growth enhancement when SBM level 
was less than 50%. In addition, low palatability was observed when treatment were fed 
diets containing 50% SBM or higher. However, in the present study there was no sign of 
poor palatability of shrimp fed experimental diets containing various level of SPC. 
 The poor performance of shrimp fed higher levels (20% or higher) of SPC as a 
substitute for SE-SBM observed in this study may be caused by imbalanced of essential 
amino acid, or the presence of anti-nutritional factors (Guillaume, 1997). For example, 
lower levels of methionine are found when diets are formulated to contain increasing 
 
 64 
levels of soy protein as a direct replacement for fish meal (Storebakken et al., 1998; 
Mambrini et al., 1999). 
  Supplementation of methionine in diets containing high levels of soy protein 
found to have beneficial effect on growth performance of aquatic animals. Kaushik et al. 
(1995) reported that supplementation of L-methionine in the diet containing SPC as a 
primary protein ingredient for rainbow trout resulted in similar growth performance to 
fish fed fish meal diet (Kaushik et al., 1995). Similar results were stated by Floreto et al. 
(2000) that diets supplemented with crystalline L-methionine resulted in enhancing body 
weight gain and shortening molting cycle period of juvenile American lobster.  Forster et 
al. (2002) suggested that SPC can be used to replace up to 75% fish meal when diets were 
supplemented with deficient indispensable amino acids. 
Since the essential amino acid requirements for shrimp have not been determined 
(Fox et al., 1995), the amino acid profile of shrimp tissue has been used as a reference for 
diet formulation to balance amino acid level in order to sustain optimal growth (Cuzon et 
al., 2004). Methionine is considered the first limiting amino acids in shrimp diets 
containing high levels of soybean protein (Akiyama et al., 1991). Soy protein concentrate 
contains approximately 1.4 g methionine per 100 g crude protein (Perkins et al., 1995) as 
compare to 2.6 g per 100 g crude protein in L. vannamei tail muscle (Lim, 1993) or 2.4 
and 3.5% of the dietary protein for methionine and methionine-cystine levels, 
respectively, for commercial shrimp production feeds (Akiyama et al., 1991). Millamena 
et al. (1996) reported the methionine requirement of P. monodon postlarvae at 0.89% of 
the diet or 2.4% of crude protein. In addition, the total sulfur amino acid requirement; 
 
 65 
methionine plus cystine, would be 1.3% of the diet or 3.5% of protein in a diet containing 
0.41% cystine.  
The result from this study suggested that methionine supplementation in soy 
based diets containing 40% SPC in substitution to SE-SBM showed no improvement in 
growth and survival of L. vannamei. Likewise, Refstie et al. (2001) obtained similar 
growth in Atlantic salmon fed diets containing 30% SPC with or without DL-methionine 
supplementation as a replacement of fish meal. Diet containing only soy protein or soy 
protein supplemented with methionine have also been shown to be poorly utilized by red 
drum (Reigh and Simon,1992). Therefore, lower mean final weight and higher FCR of 
shrimp fed diets containing 40% SPC may not be related to methionine deficiency.  
 Chemoattractants or feed stimulants have been used to enhance feed palatability 
and feed intake when diets are formulated with soybean protein as alternative to fish 
meal. Nunes et al. (2006) obtained good feeding response of L. vannamei fed a 
commercial diet containing fish solubles. Fish solubles, a by-product of fish meal and 
fish oil production,contain high levels of water soluble vitamins. Fish solubles have been 
used in poultry and swine feeds as a source of unidentified growth factors (Soares et al., 
1973). However, results obtained from our study suggested that addition of fish solubles 
in diet containing 40% SPC did not improve the performance of shrimp in terms of 
survival, growth and feed conversion ratio. Moreover, there was no indication of feed 
being rejected when shrimp were fed diets containing up to 40% SPC without the 
addition of fish solubles.  
The pepsin digestibility of crude protein in all dietary treatments ranged from 
81% to 89% with highest digestibility in diet containing 40% SPC. The digestibility 
 
 66 
values of our test diets are considered reasonable comparing to the apparent protein 
digestibility of soybean meal (90%) and of fishmeal (80.7%) reported for L. vannamei by 
Akiyama et al. (1989). Divakaran et al. (2000) also reported that the apparent digestibility 
of protein in soybean meal is at least 89% in L. vannamei. Therefore, protein digestibility 
of test diets is not a contributing factor to lower weight gain in treatment fed diets 
containing 20% SPC or higher.  
Soy protein concentrate is produced by exposing soybean to heat process to 
inactivate antinutritional factors (ANF) and improve its palatability (Brown et al., 2008, 
Akiyama, 1989). Refstie et al. (1999) reported low levels of trypsin inhibitors in all 
commercial soy products. Nonetheless, Storebakken et al. (1998) reported the reduction 
of N apparent digestibility when Atlantic salmon were fed SPC diets with the present of 
phytate. Non-starch polysaccharides (NSP) fractions I soybean have been reported to 
negatively affect the digestive system of Atlantic salmon (Refstie et al., 1999). 
Dabrowski et al. (1989) reported that a decrease of protein digestibility in rainbow trout 
fed soybean meal was due to trypsin inhibitors. However, Sessa and Lim (1991) indicated 
that L. vannamei can tolerate relatively high levels of trypsin inhibitors (TI) and levels of 
0.64-6.14 mg TI per g diet) had no effect on weight gain, FCR, and survival. It has also 
been suggested that ANF present in SBM diets may interfere with protein and lipid 
digestion and affect the palatability and nutrient absorption (Amaya, 2006; Dersjant-Li et 
al., 2002).  
Results from the present study suggested that 10% SPC in combination with SBM 
is acceptable and can be utilized as the primary protein source in shrimp diet without 
causing negative effect on biomass, growth, survival, and feed conversion ratio of            
 
 67 
L. vannamei reared under laboratory conditions. Furthermore, the results from this study 
confirmed that supplementation of DL-methionine, micro-encapsulated methionine or 
fish solubles to the diet containing 40% SPC are not required. However, it was not sure 
that the reduction in growth performance in treatments fed diets containing SPC at 20% 
or greater may cause by imbalanced nutrient content in feed or other unknown factors. 
There for, further research must be done to evaluate the nutrient composition of SPC use 
in shrimp feed formulation as well as utilization of SPC in outdoor production conditions. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 68 
References 
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Akiyama, D.M., Dominy, W.G., Lawrence, A.L., 1991. Penaeid shrimp nutrition for the 
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Alvarez, J.S., Hernandez-Llamas, A., Galindo, J., Fraga, I., Garcia, T., Villareal, H., 
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Litopenaeus vannamei reared in tanks or in ponds. Aquaculture 235, 513-551. 
 
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Dabrowski, K., Poczyczynski, P., K?ck, G., Berger, B., 1989. Effect of partially or totally 
replacing fish meal protein by soybean meal protein on growth, food utilization 
and proteolytic enzyme activities in rainbow trout (Salmo gairdneri). New in vivo 
test for exocrine pancreatic secretion. Aquaculture 77, 29-49. 
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Ricque-Marie, D., Tapia-Salazar, M., Gaxiola-Cort?s, M.G., Simoes, N. (Eds.), 
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M?xico. 
Divakaran, S., Velasco, M., Beyer, E., Forster, I., Tacon, A.G.J., 2000. Soybean meal 
apparent digestibility for Litopenaeus vannamei, including a critique of 
methodology. Avances en Nutrici?n Acu?cola V. Memorias del V Simposium 
Internacional de Nutrici?n Acu?cola, 19-22 Noviembre, 2000. M?rida, Yucat?n, 
M?xico. 
Forster, I.P., Dominy, W., Tacon, A.G.J., 2002. The use of concentrates and other soy 
products in shrimp feeds. In: Cruz-Su?rez, L.E., Ricque-Marie, D., Tapia-Salazar, 
M., Gaxiola-Cort?s, M.G., Simoes, N. (Eds.), Advaces en Nutricion Acuicola VI. 
Memorias del VI Simposium Internacional de Nutrcion Acuicola., 3 al 6 de 
Septiembre del 2002, Canc?n, Quintana Roo, M?xico. 
Fox, J.M., Lawrence, A.L., Li-Chan, E., 1995. Dietary requirement for lysine by juvenile 
Penaeus vannamei using intact and free amino acid sources. Aquaculture 131, 
279-290. 
 
 
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Floreto, E.A.T., Bayer, R.C., Brown, P.B., 2000. The effects of soybean-based diets, with 
and without amino acid supplementation, on growth and biochemical composition 
of juvenile American lobster, Homarus americanus. Aquaculture 189, 211-235. 
Guillaume, J., 1997. Protein and amino acids. In: D?Abramo, L.R., Conklin, D.E., 
Akiyama, D.M. (Eds.), Crustacean Nutrition. The World Aquaculture Society, 
Baton Rouge, LA, USA, pp. 26-50. 
Kaushik, S.J., Cravedi, J.P., Lalles, J.P., Sumpter, J., Fauconneau, B., Laroche, M., 1995. 
Partial or total replacement of fish meal by soybean protein on growth, protein 
utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh 
quality in rainbow trout, Oncorhynchus mykiss. Aquaculture 133, 257-274. 
Lim, C., Dominy, W., 1990. Evaluation of soybean meal as a replacement for marine 
animal protein in diets for shrimp (Penaeus vannamei). Aquaculture 87, 53-63. 
Lim, C., 1993. Effect of dietary pH on amino acid utilization by shrimp (Penaeus 
vannamei). Aquaculture 114, 293-303. 
Liu, K.S., 1997. Soybeans: chemistry, technology and utilization. Chapman & Hall, New 
York, NY, USA, 532 pp. 
Mambrini, M., Roem, A.J., Carvedi, J.P., Lalles, J.P., Kaushik, S.J., 1999. Effects of 
replacing fish meal with soy protein concentrate and of DL-methionine 
supplementation in high-energy, extruded diets on the growth and nutrient 
utilization of rainbow trout, Oncorhynchus mykiss. Journal of animal science 77, 
2990-2999. 
Messina, M.J., 1999. Legumes and soybeans: overview of their nutritional profiles and 
health effects. American Journal of Clinical Nutrition 70, 439S-450S. 
 
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Millamena, O.M., Bautista-Teruel, M.N., Kanazawa, A., 1996. Methionine requirement 
of juvenile tiger shrimp Penaeus monodon Fabricius. Aquaculture 143, 403-410. 
Nunes, A.J.P., S?, M.V.C., Andriola-Neto, F.F., Lemos, D., 2006. Behavioral response to 
selected feed attractants and stimulants in Pacific white shrimp, Litopenaeus 
vannamei. Aquaculture 260, 244-254. 
Paripatananont, T., Boonyaratpalin, M., Pengseng, P., Chotipuntu, P., 2001. Substitution 
of soy protein concentrate for fishmeal in diets of tiger shrimp Penaeus monodon. 
Aquaculture research 32, 369-374. 
Perkins, E.G., 1995. Composition of soybeans and soybean products. Practical Handbook 
of Soybean Processing and Utilization, pp. 9?28. 
Refstie, S., Svihus, B., Shearer, K.D., Storebakken, T., 1999. Nutrient digestibility in 
Atlantic salmon and broiler chickens related to viscosity and non-starch 
polysaccharide content in different soyabean products. Animal Feed Science and 
Technology 79, 331-345. 
Refstie, S., Storebakken, T., Baeverfjord, G., Roem, A.J., 2001. Long-term protein and 
lipid growth of Atlantic salmon (Salmo salar) fed diets with partial replacement 
of fish meal by soy protein products at medium or high lipid level. Aquaculture 
193, 91-106. 
Reigh, R.C., Ellis, S.C., 1992. Effects of dietary soybean and fish-protein ratios on 
growth and body composition of red drum (Sciaenops ocellatus) fed 
isonitrogenous diets* 1. Aquaculture 104, 279-292. 
 
 72 
Samocha, T.M., Davis, D.A., Saoud, I.P., DeBault, K., 2004. Substitution of fish meal by 
co-extruded soybean poultry by-product meal in practical diets for the Pacific 
white shrimp, Litopenaeus vannamei. Aquaculture 231, 197-203. 
Sessa, D.J., Lim, C., 1992. Effect of feeding soy products with varying trypsin inhibitor 
activities on growth of shrimp. Journal of the American Oil Chemists' Society 69, 
209-212. 
Soares Jr, J., D. Miller, et al. (1973). "A review of the chemical and nutritive properties 
of condensed fish solubles." Fishery Bulletin 71, 255-265. 
Steel, R.C.D., Torrie, G., 1980. Principles and procedures of statistics. A biometrics 
approach. McGraw-Hill Books, New York, New York, USA. 
Storebakken, T., Shearer, K.D., Roem, A.J., 1998. Availability of protein, phosphorus 
and other elements in fish meal, soy-protein concentrate and phytase-treated soy-
protein-concentrate-based diets to Atlantic salmon, Salmo salar. Aquaculture 161, 
365-379. 
Teshima, S., A. Kanazawa, et al. (1992). "Supplemental effects of methionine-enriched 
plastein in Penaeus japonicus diets* 1." Aquaculture 101(1-2): 85-93. 
Tibaldi, E., Hakim, Y., Uni, Z., Tulli, F., de Francesco, M., Luzzana, U., Harpaz, S., 
2006. Effects of the partial substitution of dietary fish meal by differently 
processed soybean meals on growth performance, nutrient digestibility and 
activity of intestinal brush border enzymes in the European sea bass 
(Dicentrarchus labrax). Aquaculture 261, 182-193. 
 
 
 
 
 
 73 
Table 1. Ingredient composition (g 100 g-1 dry weight) of six experimental diets 
containing increasing percentages of SPC (0, 5, 10, 20, 40 and 40% supplemented with 
methionine) as a substitute for SE-SBM. 
Ingredient 0% 5% 10% 20% 40% 40%+ Met 
Soybean meal solvent-extracted 58.8 52.2 45.5 32.5 6.2 6.2 
Soy concentrate 0.0 5.0 10.0 20.0 40.0 40.0 
Menhaden fish oil 4.3 4.3 4.3 4.3 4.3 4.3 
Soy  oil 0.0 0.0 0.1 0.1 0.1 0.1 
Corn starch 2.2 3.8 5.5 8.5 14.7 14.6 
Whole wheat 20.0 20.0 20.0 20.0 20.0 20.0 
Trace mineral premixa 0.5 0.5 0.5 0.5 0.5 0.5 
Vitamin premixa 2.0 2.0 2.0 2.0 2.0 2.0 
Stay C, 35% vit. C activity 0.1 0.1 0.1 0.1 0.1 0.1 
CaP-diebasic 3.0 3.0 3.0 3.0 3.0 3.0 
Soy lecithin 1.0 1.0 1.0 1.0 1.0 1.0 
Cholesterol 0.1 0.1 0.1 0.1 0.1 0.1 
Corn gluten meal 8.0 8.0 8.0 8.0 8.0 8.0 
DL-methionine 0.0 0.0 0.0 0.0 0.0 0.1 
Total 100 100 100 100 100 100 
aVitamin premix and mineral premix are proprietary products, thus their composition is 
not listed. 
 
 
 74 
Table 2. Nutrient composition (dry matter basis) of six experimental diets containing 
increasing percentages of SPC (0, 5, 10, 20, 40 and 40% supplemented with methionine) 
as a substitute for SE-SBM. 
Nutrient 0% 5% 10% 20% 40% 40%+ Met 
Crude protein 40.00  39.70  39.60  40.08  40.20  40.40  
Moisture 5.71  6.69  7.58  7.13  7.00  6.16  
Fat 6.69  6.45  6.43  6.15  5.88  5.95  
Crude fiber 2.69  2.81  2.87  2.86  2.79  2.96  
Ash 6.21  6.16  5.98  5.79  5.57  5.68  
Protein difestibility 
(0.0002% pepsin) 87.77 81.23 83.29 83.52 89.66  
Diets were formulated to contain 35% protein and 6% lipid. 
Diets were analyzed by New Jersey Feed Laboratory, Trenton, NJ, USA.  
 
 
 
 
 
 
 
 
 75 
Table 3. Ingredient composition (g 100 g-1 dry weight) of six experimental diets 
containing increasing percentages of SPC (0, 5, 10, 20, 40, 40% supplemented with 
methionine and 40% with fish solubles) as a substitute for SE-SBM. 
Ingredient 0% 5% 10% 20% 40% 40%+ Met 40%+  fish sol. 
Soybean meal solvent extracted  54.50 48.10 41.70 29.00 3.50 2.30 3.50 
Soy protein concentrate  0.00 5.00 10.00 20.00 40.00 40.00 40.00 
Menhaden fish oil  4.60 4.60 4.60 4.60 4.65 4.60 4.60 
Corn starch  0.19 1.59 2.99 5.69 11.14 11.39 10.79 
Whole wheat  27.81 27.81 27.81 27.81 27.81 27.81 27.81 
Trace mineral premixa  0.50 0.50 0.50 0.50 0.50 0.50 0.50 
Vitamin premixa  2.00 2.00 2.00 2.00 2.00 2.00 2.00 
Stay C, 35% vit. C activity 0.10 0.10 0.10 0.10 0.10 0.10 0.10 
CaP-diebasic  3.20 3.20 3.20 3.20 3.20 3.20 3.40 
Soy lecithin  1.00 1.00 1.00 1.00 1.00 1.00 1.00 
Cholesterol  0.10 0.10 0.10 0.10 0.10 0.10 0.10 
Corn gluten meal  6.00 6.00 6.00 6.00 6.00 6.00 6.00 
Micro-encapsulated methionine 0.00 0.00 0.00 0.00 0.00 0.02 0.00 
Fish solubles  0.00 0.00 0.00 0.00 0.00 0.00 1.00 
Total 100 100 100 100 100 100 100 
a Vitamin premix and mineral premix are proprietary products, thus their composition is 
not listed. 
 
 
 76 
Table 4. Nutrient composition (dry matter basis) of seven experimental diets containing 
increasing percentages of SPC (0, 5, 10, 20, 40, 40% supplemented with methionine and 
40% with fish solubles) as a substitute for SE-SBM. 
Nutrient 0% 5% 10% 20% 40% 40%+   Met 40%+  fish sol. 
Crude protein 36.80 37.30 35.90 35.80 35.80 40.40 37.10 
Moisture 7.35 6.90 7.39 8.96 11.04 6.16 7.54 
Fat 7.15 6.93 6.80 6.62 6.02 5.95 6.35 
Crude fiber 3.15 3.31 3.18 3.26 2.91 2.96 3.15 
Ash 6.68 6.49 6.47 6.26 5.82 5.68 6.04 
Diets were formulated to contain 35% protein and 6% lipid. 
Diets were analyzed by New Jersey Feed Laboratory, Trenton, NJ, USA.  
 
 
 
 
 
 
 
 
 
 77 
Table 5. Water quality variables for growth trials with juvenile L. vannamei reared under 
clear water system for both trials. Values represent the mean ? standard deviation and 
values in parenthesis represent minimum and maximum readings. 
Variable First Trial Second Trial 
DO (mg L-1)a 6.38 ? 0.50 7.6 ? 0.95 
Temperature (?C) 22.67 ? 1.46 26.5 ? 1.68 
Salinity (ppt)  22.06 ? 1.28 16.1 ? 0.54 
pH 8.41 ? 0.32 7.7 ? 0.20 
TAN (mg L-1)b 0.49 ? 0.19 0.10 ? 0.20 
 
aDissolved oxygen. 
bTotal ammonia-nitrogen. 
 
 
 
 
 
 
 
 
 
 
 78 
Table 6. Growth performance of L. vannamei (initial weight of 0.12?0.01 g) reared under 
clear water system in polyethylene tanks during 55-day experimental period (first trial), 
fed diets containing increasing percentages of SPC (0, 5, 10, 20, 40 and 40% 
supplemented with methionine) as a substitute for SE-SBM. 
Treatment Biomass  (g tank-1) Final weight  (g) FCRa Survival  (%) 
0% SPC 92.98ab 6.31ab 2.19a 98.33 
5% SPC 95.63a 6.38ab 2.17a 100.00 
10% SPC 96.23a 6.30a 2.14a 98.33 
20% SPC 86.70bc 5.78bc 2.42b 100.00 
40% SPC 86.60bc 5.77bc 2.42b 100.00 
40% SPC+Met 82.50c 5.50c 2.57b 100.00 
P-valueb 0.001 0.005 0.001 0.564 
PSEc 1.322 0.096 0.038 0.384 
aFeed conversion ratio = Total feed offered / biomass increase. 
bMeans (n = 4) not sharing a common superscript within a row are significantly different 
based on analysis of variance (P > 0.05) followed by Student Newman-Keuls multiple 
range test. 
cPooled standard error of treatment means. 
 
  
 
 
 79 
Table 7. Growth performance of L. vannamei (initial weight of 0.14?0.05 g) reared under 
clear water system in polyethylene tanks during 53-day experimental period (second 
trial), fed diets containing increasing percentages of (0, 5, 10, 20, 40, 40% supplemented 
with methionine and 40% with fish solubles) as a substitute for SE-SBM, compare to a 
commercial reference diet. 
Treatment Biomass  (g tank-1) Final weight  (g) FCRa Survival  (%) 
0% SPC 109.1 7.6 1.89 95.6a 
5% SPC 111.9 8.0 1.82 93.3a 
10% SPC 110.2 7.5 1.92 97.8a 
20% SPC 96.0 7.0 2.08 91.1ab 
40% SPC 90.1 7.1 2.10 84.4ab 
40% SPC+Met 70.0 6.0 2.54 77.8b 
40% SPC+fish sol. 85.7 6.7 2.21 84.4ab 
Reference dietb  117.9 7.9 1.84 100.0a 
P-valuec 0.9362 0.2081 0.3906 0.0028 
PSEd 10.4565 0.6148 0.3130 2.8868 
aFeed conversion ratio = Total feed offered / biomass increase. 
bDiets were commercially manufactured by Rangen? (Angleton, TX, USA) using 
extrusion processing to contain 35% Protein and 8% lipid. 
cMeans (n = 4) not sharing a common superscript within a row are significantly different 
based on analysis of variance (P > 0.05) followed by Student Newman-Keuls multiple 
range test. 
dPooled standard error of treatment means. 
 
 80 
Figure 1. Mean final weight (g) and FCR of L. vannamei post larval (initial weight of 
0.12?0.01 g) reared under clear water system in polyethylene tanks during 55-day 
experimental period (first trial), fed diets containing increasing percentages of SPC (0, 5, 
10, 20, 40 and 40% supplemented with methionine) as a substitute for SE-SBM.. 
 
 
 
 
 
 
 
ab ab a 
bc bc c 
  a   a   a 
  b    b 
  b 
0.00
0.50
1.00
1.50
2.00
2.50
3.00
0.00
2.00
4.00
6.00
8.00
10.00
12.00
0%SPC 5%SPC 10%SPC 20%SPC 40%SPC 40%SPC
+Met
FC
R 
Mea
n fina
l we
ight 
(g)
 
Final weight
FCR
 
 81 
Figure 2. Mean final weight (g) and FCR of L. vannamei post larval (initial weight of 
0.14?0.05 g) reared under clear water system in polyethylene tanks during 53-day 
experimental period (second trial), fed diets containing increasing percentages of (0, 5, 
10, 20, 40, 40% supplemented with methionine and 40% with fish solubles) as a 
substitute for SE-SBM, compare to a commercial reference diet. 
 
 
 
 
 
 
0
0.5
1
1.5
2
2.5
3
0
3
6
9
12
15
18
0%SPC 5%SPC 10%SPC 20%SPC 40%SPC 40%SPC
+Met
40%SPC
+Fish sol.
Reference
diet
FC
R 
Mea
n fina
l we
ight 
(g)
 
Final Weight
FCR
 
 82 
 
 
CHAPTER IV 
USE OF SOY PROTEIN CONCENTRATE IN PRACTICAL DIETS FOR 
PACIFIC WHITE SHRIMP (Litopenaeus vannamei) REARED UNDER  
VARIOUS CULTURE CONDITIONS 
 
Abstract 
 The objective of this project was to demonstrate the feasibility of diets formulated 
to contain increasing percentages of soy protein concentrate (SPC) in production diets for 
Litopenaeus vannamei reared under different culture conditions. A series of growth trials 
using different culture systems which included clear water recirculating system, a green 
water, semi-closed, recirculating system, and production ponds, were conducted at 
Claude Peteet Mariculture Center in Gulf Shores, Alabama. Four practical diets 
containing increasing percentages (0, 4, 8, and 12%) of SPC were utilized to evaluate the 
response of the shrimp over the culture period. The clear water trial was conducted in 
600-L polyethylene tanks using three replicates per diet. Juvenile L. vannamei (mean 
weight ? S.D., 0.94 ? 0.04 g) were stocked at a density of 15 shrimp m-2 and cultured for 
10 weeks. The same four dietary treatments and a commercial reference diet were offered 
to juvenile shrimp (initial weight of 1.0 ? 0.03 g) maintained in outdoor green water 
system over a 10 week period at a stocking density of 30 shrimp per tank. In addition, test 
diets were tested in 0.1-ha ponds using four replicates per diet. Post larvae were obtained 
from commercial sources and nursed for three weeks. Juvenile shrimps (mean weight ? 
 
 83 
S.D., 0.013 ? 0.001 g) were stocked at 35 shrimp m-2 and were cultured under 
standardized pond production conditions for 18 weeks. At the conclusion of these 
experiments; net yield, mean individual weight, survival, and FCR were evaluated. The 
results in green water tank trial and pond trial demonstrated the feasibility of soy protein 
concentrate inclusion up to 12% SPC in soybean based diet without causing negative 
effect on growth, feed conversion, survival and net yield. The results in clear water tank 
trial show no consistent trend in mean final weight among treatments, but no differences 
were observed in net yield, survival and FCR among treatments. Based on the results 
from the previous study as well as of this study results, SPC at a level up to 12% can be 
used in commercial feed formulations for L. vannamei. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 84 
Introduction 
 Protein is the most expensive component of the diet and is a major nutrient 
required for maintenance and growth of shrimp (Shiau, 1998). An insufficient daily 
intake of protein will result in reduction in growth or abnormality. The minimum protein 
requirements for shrimp to maintain optimal performance varied depending on age or size 
of shrimp, quality of dietary protein (essential amino acid profiles and digestibility), 
availability of alternative food sources, environmental parameters, and culture 
management (D?Abramo and Sheen, 1994; Lim and Akiyama, 1995; Venero, 2006). 
Generally, commercial shrimp feeds contain 30% to 50% crude protein, composed 
mostly of marine animal protein such as fish meal, shrimp meal and squid meal (Mente et 
al., 2002; Lim and Dominy, 1990). These ingredients have excellent nutritional value 
with high palatability, but are expensive and not always readily available (Lim and 
Persyn, 1989). Considerable research has been conducted to find alternative protein 
sources which are inexpensive, have a stable supply and are sustainable to replace marine 
animal protein in shrimp feed formulations.  
Among alternative protein ingredients, soybean meal is considered one of the 
most promising ingredients and widely used as a protein source in fish and shrimp feeds 
because of its acceptable amino acid profile, consistent composition, availability and 
reasonable price (Dersjant-Li, 2002; Forster et al., 2002; Hardy, 1999). Several studies 
have been conducted to evaluate the nutritional value of soybean products in shrimp diets 
(Lim and Dominy, 1990, 1992; Amaya et al., 2007a,b;  Cruz-Suarez et al., 2001; Ray et 
al., 2009; Sudaryono et al., 1995; Samocha et al., 2004; Piedad-Pascaul et al., 1990; 
Smith et al., 2001). However, the use of soybean meal as a sole protein ingredient in 
 
 85 
shrimp feed is limited because of its shortage in sulfur amino acids, highly saturated fatty 
acids, and mineral such as phosphorus and the presence of anti-nutritional factors such as 
trypsin inhibitors that may affect digestibility and palatability of feed (Akiyama, 1989; 
Dersjant-Li, 2002). Therefore, nutrient content of the feed should be well formulated to 
meet nutritional requirements of the shrimp when soy products are utilized. As the 
nutrient density (protein and energy) of the diet is increased the use of solvent-extracted 
soybean meal (44-48% protein) is inefficient as there is insufficient room in the 
formulation for all necessary components. Hence, higher protein ingredients are often 
required.  
Compared to solvent-extracted soybean meals, soy protein concentrate (SPC) 
contains higher protein and generally lower levels of antinutrients. Soy protein 
concentrate contains approximately 65% crude protein, a level similar to that found in 
fish meal, and considerably higher than SE-SBM (Paripatananont et al., 2002; Liu, 1997). 
Likewise, several anti-nutritional factors such as trypsin inhibitors and soluble 
oligosaccharide can be inactivated through the alcohol extraction process of SBM 
(Kaushik et al., 1995). Soy protein concentrate (SPC) is produced through a series of 
different extraction and precipitation process from high quality de-hulled soybeans. By 
removing most of the oil and water soluble non-protein constituents, a variety of high 
protein products with varying characteristics can be produced.  
Numerous published studies have indicated the feasibility of SPC inclusion in 
feeds for many fish and shrimp species such as European sea bass (Tibaldi et al., 2006), 
rainbow trout (Kaushik et al., 1995; Mambrini et al., 1999), Atlantic halibut (Berge et al., 
1999), Atlantic salmon (Refstie et al., 2001; Storebakken et al., 1998, 2000), tiger shrimp 
 
 86 
(Liu et al., 2000; Paripatananont et al., 2002), and Pacific white shrimp (Forster et al., 
2002). However, there is limited information on the utilization of SPC in combination 
with soybean meal in commercial feeds for L. vannamei. According to our previous 
studies which have evaluated increasing levels of SPC in soybean based diet for L. 
vannamei under laboratory conditions, inclusion of 20% SPC or higher in soybean based 
diet resulted in adverse effect on shrimp growth. Inclusion of lower levels of SPC in 
soybean based diet, however, resulted in good performance of shrimp. Thus, inclusion of 
SPC at levels from 0 to 12% in combination with soybean meal were used to evaluate 
feasibility of SPC use in commercial feed for L. vannamei.  
As the presence of natural foods can have a synergistic effect on the performance 
of the shrimp, studies were carried out through a series of growth trials under both 
laboratory and field conditions. Nutrition studies are often carried out under laboratory 
conditions where water quality management is easier and there is no natural productivity 
(Amaya et al., 2007a). On the other hand, the presence of natural food sources in green 
water tank system and in ponds may enhance shrimp growth and maintain acceptable 
water quality (Moss, 2000). Shrimp have the ability to harness food and nutrient particles 
suspended in water column and through benthic foraging, (Tacon et al., 2004). Hence, the 
evaluation of shrimp performance in the presence of natural feeds should be evaluated. 
Both pond and green water tank systems allow shrimp to have access to natural foods. 
However, green water tanks systems are less variable and less costly than ponds. Given 
there is still limited information regarding to the utilization of SPC under commercial 
conditions, this study was carried out to evaluate the feasibility of diets formulated to 
contain increasing percentages of SPC (0, 4, 8 and 12%) in production diets for L. 
 
 87 
vannamei reared under different culture environments, e.g., clear water tank system, 
green water tank system, and production ponds. 
 
Methods 
  A series of experiments were conducted at the Alabama Department of 
Conservation and Natural Resources Marine Resource Division, Claude Peteet 
Mariculture Center in Gulf Shores, Alabama. The growth trials of postlarvae L. vannamei 
were conducted under three rearing systems; a clear water recirculating tank system, a 
green water tank system and an outdoor pond system.  
 
Experimental animals  
Shrimp post larvae (PL) were obtained from a commercial hatchery, Shrimp 
Improvement System, Key West, FL. Dissolved oxygen (DO), temperature, salinity, and 
pH were randomly measured among the shipping bags using a YSI 556 MPS meter 
(Yellow Spring Instruments Co., Yellow springs, OH, USA). Two water samples were 
collected in order to determine the total ammonia-nitrogen (TAN) using the Orion 
ammonia electrode probe (Thermo Fisher Scientific Inc., Waltham, MA, USA). An 
oxygen tank with one air stone was operated to provide oxygen during the acclimation 
period. Newly hatched brine shrimp, Artemia salina (INVE Americas, Inc., Salt Lake 
City, UT, USA), were prepared one night before PL reception, and were fed to PL in the 
acclimation tank. PL were slowly acclimated to temperature and salinity prior to 
releasing into the acclimation tank. Water quality variables were measured to control 
temperature and salinity change in the range of 4 ?C per hour and 2 ppm per hour, 
 
 88 
respectively. After acclimation PL were concentrated by drain harvesting into a 57-L 
concentration tank and quantified volumetrically. Six sub-samples were taking using a 
60-ml beaker while the water in the concentration tank was mixing vigorously. Average 
numbers of PL per ml were then determined. Sixty shrimps were randomly collected and 
individually weighed to determine the average weight and predict the biomass for 
determination of feed input. 
The shrimp were divided and spread equally into four nursery tanks (6,000 L). 
The nursery system was stocked at a density of 54 postlarvae per liter. Each nursery tank 
was equipped with three air lifts and two air stones to help with water circulation and 
aeration which was provided by a common regenerative blower. The recirculating 
nursery system was composed of six nursery tank, a biological filter, a pressurized sand 
filter and a 1.5-hp circulation pump (Aquatic Eco-systems, Apopka, Florida, USA). Feeds 
were applied on daily basis four times per day. Uneaten feed was siphoned as needed to 
maintain water quality in the nursery tanks (Table 1). PL were reared in the nursery 
system for 3 weeks. Water Quality was monitored two times per day at 800 and 1600 h, 
and Total ammonia-nitrogen (TAN) was measured bi weekly using a spectrophotometer 
(Spectronic 20 Genesys, Spectronic Instrument Inc. Rochester, NY, USA) following the 
Nesslerization method (APHA, 1989) (Table 2). Every three days, 60 PL per tank were 
randomly collected and sub-samples of 10 shrimps each were pooled weighed in order to 
evaluate growth and adjust daily feed inputs. 
At the end of two weeks, each tank was partially drained to concentrate the 
shrimp. The shrimp were then collected by dip nets. Six sub-samples were taken from 
each tank to determine average wet weight of the shrimp (g). Once the number of shrimp 
 
 89 
per unit weight was determined, shrimp were harvested and small groups weighed 
(around 100g per group) and then distributed into one of 16 buckets (1 bucket per pond) 
each representing one production pond. Shrimp from each nursery tank were distributed 
across all ponds to minimize any bias from variation in nursery tanks. The shrimp were 
then transferred to the corresponding pond and stocked. Final population, weight gain (g), 
percentage survival, final weight (g), and feed conversion ratio for each nursery tank was 
then determined (Table 3).  
 
Experimental diets 
 Four experimental diets (Table 4) were formulated to contain graded levels of soy 
protein concentrate (SPC). The experimental diets were manufactured as a sinking 
extruded pellet by Rangen Inc. (Angleton, TX, USA) under commercial feed 
manufacturing condition. All experimental diets contain approximately 35% protein and 
8% lipid with lysine and methionine plus cystine contents of 5% and 3% of the total 
protein and squid meal at 0.5% of the diet was also included. The proximate composition 
including protein digestibility (using 0.0002% pepsin solutions) of the test diets are 
presented in Table 5. The basal diet was designed to contain 58% solvent extracted de-
hulled soybean meal which was replaced with graded levels of soy protein concentrate (0, 
4, 8 and 12%) for test evaluations. 
 
Clear water tank trials  
Juvenile shrimp obtained from the nursery system were selected by hand-sorting 
to a uniform size. Then juvenile shrimp (0.94?0.04 g, initial weight) were stocked at a 
 
 90 
density of 15 shrimp per tank. The clear water tank system was a semi-closed 
recirculating system consisted of 12 culture tanks (0.53 m height x 1.19 m diameter, 600-
L volume), a central reservoir, a biological filter and a circulation pump.  Four 
experimental diets were randomly assigned with three replications (tanks) per treatment. 
Test diets were offered to shrimp twice daily at 0800 and 1600 h for a 10-week 
experimental period. Feed input was pre-calculated on a weekly basis based on an 
expected feed conversion ratio of 1.5 and a growth rate of one gram per week. Shrimp 
were counted every two weeks to readjust the daily feed input. Nettings were used to 
cover all tanks to prevent shrimp from jumping out.   
During the 10-week growth trial, dissolved oxygen concentration, temperature, 
salinity, and pH were monitored twice daily in the central filter and two of the rearing 
tanks at 600 h and 1600 h using a YSI meter, (Yellow Spring Instrument Co., Yellow 
Springs, OH, USA). Water samples were taken bi-weekly from central reservoir and two 
tanks to measure Total ammonia-nitrogen (TAN) using the Orion ammonia electrode 
probe (Thermo Fisher Scientific Inc., Waltham, MA, USA). Water quality results are 
summarized in Table 6. 
At the conclusion of the 10-week growth trial shrimp were counted and weighed 
individually. Percentage survival, mean final weight, biomass and feed conversion ratio 
were determined.  
 
Green water tank trials 
Juvenile L. vannamei (1.0?0.03g initial weight) were collected from the research 
ponds and were size-sorted by hands. Juvenile shrimp were randomly selected and 
 
 91 
stocked at a rate of 30 shrimp per tank (38 shrimp per m3) in an outdoor recirculating 
system. The outdoor tank system consisted of a central reservoir (800 L) with a trickle 
filter, a 0.33-hp submersible pump, and 24, circular polyethylene tanks (0.85 m height x 
1.22 m upper diameter, 1.04 m lower diameter) designed to contain 800 L. A 0.33-hp 
submersible pump was used to move water from one of the production ponds to the 
central reservoir at a rate of 8 liters per minute between 0800 h and 1400 h to mimic a 
production pond setting. This exchange rate allowed a 100% water exchange in the 
recirculating system every six days. Each tank and central reservoir were provided with 
two air stones connected to a 0.5-hp regenerative blower (Sweetwater Aquaculture Inc. 
Lapwai, ID, USA) to supply aeration. Netting was used to cover all tanks to protect the 
shrimps from jumping out.  
During the 10-week growth trial, dissolved oxygen concentration, temperature, 
salinity, and pH were monitored twice a day in the central filter and two of the system 
tanks at 0600 h and 1600 h using a YSI meter, (Yellow Spring Instrument Co., Yellow 
Springs, OH, USA). Water samples were taken bi-weekly from central reservoir and two 
tanks to measure Total ammonia-nitrogen (TAN) using the Orion ammonia electrode 
probe (Thermo Fisher Scientific Inc., Waltham, MA, USA). Water quality results are 
summarized in Table 7. 
Five test diets were randomly assigned amongst the 24 tanks (750 L). Four 
experimental diets used in production ponds were tested using five replicates per diet, and 
the fifth diet, a reference commercial diet (Rangen Shrimp Grower, 35% protein, 8% 
lipid), was tested using four replicates. Juvenile shrimp were offered test diets twice per 
 
 92 
day at 0800 h and 1600 h. Daily ration of feed was calculated based upon an expected 
weekly growth rate of 1.3 g wk-1 and expected FCR of 1.2.  
At the conclusion of the 10 week growth trial shrimp were counted and weighed 
individually. Mean final weight, final net yield, percent survival, and feed conversion 
ratio were determined.  
 
Pond Production Trial 
 Juvenile L. vannamei (initial weight of 0.01?0 mg) were collected from nursery 
system and stocked in the production ponds at the rate of 35 shrimp m-2. Ponds used for 
the grow-out phase were approximately 0.1 ha in surface area, (rectangular 46 x 20 m) 
with a 1.0 m average depth. Each pond was equipped with a 20-cm diameter standpipe, a 
concrete catch basin (3.0 x 2.0 x 0.5 m), and lined with 1.52 mm thick high-density 
polyethylene lining (HDPE). The bottom of each pond was covered with a 25-cm deep 
layer of sandy-loam soil. After each culture cycle, ponds were dried and tilled in order to 
allow oxidation and mineralization of organic matter. Approximately three weeks before 
stocking, ponds were filled with brackish water from the intra-costal canal between 
Mobile and Perdido Bay, AL. Incoming water was filtered through a three foot 250 ?m-
mesh nylon filter sock (Micron Domestic Lace Mfg., Inc) to prevent the introduction of 
predators, as fish larvae, crab, or other planktonic organisms, but allow the introduction 
of phytoplankton in ponds. Two weeks before stocking juvenile shrimp, the combination 
of inorganic liquid fertilizers of 32-0-0 mixed with 10-34-0 were applied in a rate of 
1,697 ml and 303 ml per pond, which provided 5.73 kg of N and 1.03 kg of P205 ha-1, 
respectively, in order to promote natural productivity in all ponds. The second 
 
 93 
fertilization was reapplied at half of the initial rate when the Secchi disk reading was 
greater than 40 cm. All ponds were provided aeration about 10 hp ha-1 using 1-hp 
paddlewheels aerators (Little John Aerator, Southern Machine Welding Inc. Quinton, 
AL) with either 1-hp or 2-hp Aire-O2 aerators (Aire-O2, Aeration Industries International, 
Inc. Minneapolis, Minnesota) to maintain dissolved oxygen above 3 mg L-1, additional 
aerators were provided as needed when dissolved oxygen fell below 3 mg L-1. This study 
was a sustainable semi-intensive system which was well managed feeding, and 
minimized water exchanged. 
Four test diets were randomly assigned to 16 production ponds using four 
replicates per diet. Test diets were divided to two feedings per day, early morning 0800 h 
and late afternoon 1600 h. Feed inputs were based on historical results as well as current 
observations. A small amount of commercial feed was applied to promote natural 
productivity in pond water during the 1st to 4th weeks. Thereafter, feed input was 
calculated based upon an expected weight gain of 1.3 g wk-1, a feed conversion of 1.2, 
and a survival of 75% during the pond culture period (Table 8).  
During the experimental period, shrimp growth was monitored on a weekly basis 
by determining the average weight in a sample of 70 to 100 animals per pond. Sampling 
was carried out by capturing shrimp by seine during first two weeks and two 5 foot cast 
nets (monofilament net, 1.52 m radius and 0.95 cm opening) for the remaining of the 
culture period. 
Water quality variables, e.g., dissolved oxygen (DO), temperature, salinity, and 
pH were monitored three times a day, at sunrise (0500 h - 0530 h), during the day (1500 h 
- 1530 h), and at night (2300 - 2400 hr), using a YSI 556 MPS meter, (Yellow Spring 
 
 94 
Instrument Co., Yellow Springs, OH, USA). Secchi disk reading and TAN were 
monitored on a weekly basis. Water samples were taken from all ponds at the depth of 80 
cm in the water column and TAN was determined using the Orion ammonia electrode 
probe (Thermo Fisher Scientific Inc., Waltham, MA, USA). Water quality results for the 
pond trial are summarized in Table 9.  
The shrimp were harvested at the end of the 18-week study period, three feedings 
were stopped before harvest in order to clear the shrimp gut. The night before harvest, the 
water was drained down about two thirds of the ponds and aeration provided using 
paddlewheel aerators to keep shrimp alive and minimize erosion of the pond bottom. On 
harvest day, the remaining water was drained and the shrimp were pump harvested from 
the catch basin using a hydraulic fish pump equipped with a 25-cm diameter suction pipe 
(Aqualife-Life pump, Magic Valley Heli-arc and Mfg, Twin Falls, Idaho, USA). The 
pump was placed in the catch basin and shrimp pumped, de-watered and collected in a 
hauling truck. The shrimp were rinsed and weighed. Approximately 150 shrimps for each 
pond were randomly selected to measure individual weight and size distribution. At the 
conclusion, mean final weight, net yields, percent survival, and FCR were determined. 
 
Statistical analysis 
All data were statistically analyzed using one-way analysis of variance to 
determine significant differences (P<0.05) among treatments, followed by the Student?
Neuman?Keuls multiple comparison test to determine significant differences between 
treatment means (Steel, Torrie, 1980). All statistical analyses were carried out using SAS 
(V9.1. SAS Institute, Cary, NC, USA). 
 
 95 
Results 
Nursery system 
  Average water quality variables throughout the 15-day nursery period were as 
follows: dissolved oxygen 6.44 ? 1.45 mg L-1; temperature 26.22 ? 1.44 oC; pH 7.55 ? 
0.33; salinity 28.63 ? 5.03 ppt; and TAN 0.15 ? 0.14 mg L-1 (Table 2). Water quality 
conditions were maintained within acceptable limits for the culture of this species. At the 
conclusion of the shrimp nursery period, mean individual weight 0.013? 0.006 g, weight 
gain 0.012 ? 0.006 g, survival 58.07 ? 5.27%, and FCR 1.42 ? 0.04 were observed  
(Table 3). 
 
Clear water tank trial 
 Overall means, standard deviations, and ranges of morning and afternoon water 
quality variables observed over 10-week growth trial were within acceptable limits for 
the culture of this species (Table 6). The results of one replicate from treatment fed diet 
0% SPC was excluded from the statistical analysis because of the occurrence of algae in 
the tank which caused better performance of shrimp. Growth performance of L. vannamei 
reared under clear water recirculating system is summarized in Table 10.  There were no 
differences (P > 0.05) in survival, FCR, and net yield. However, significant differences in 
mean individual weight among treatments were observed in the 10-week growth trial in 
L. vannamei fed diet contain increasing percentage of 0, 4, 8, and 12% SPC in 
substitution to soybean meal as a protein source. Survival ranged from 88.9 to 97.8%. Net 
yield ranged from 77.1 to 100.6 g tank-1. FCR ranged from 2.65 to 3.61. Mean individual 
 
 96 
weight ranged from 5.3 to 7.2 g with the highest mean individual weight in shrimp fed 
diet containing % SPC (Figure 1a).  
 
Green water tank trial 
 The overall means, standard deviations, and ranges of morning and afternoon 
water quality variables observed over 10-week growth trial were within a reasonable 
range for the culture of this species(Table 7). Growth performance of L. vannamei reared 
under green water tank system is summarized in Table 11. Results indicated that there 
were no differences (P > 0.05) in survival, mean final weight, and FCR of L. vannamei 
fed diet containing increasing percentage of SPC in substitution to soybean meal as a 
protein source. However, significant differences in net yield among treatments were 
observed l. Survival ranged from 95.8 to 100.0%. Mean individual weight ranged from 
13.5 to 15.0 g. FCR ranged from 1.17 to 1.28. Net yields ranged from 397.2 to 431.6 g 
tank-1 with the highest yield in treatment fed 8% SPC. Shrimp maintained on the 
commercial reference diets showed significantly higher in net yield and lower FCR than 
shrimp fed on the test diets albeit; treatment fed diet containing 8% SPC had similar yield 
to treatment fed reference diet. Additionally, similar FCR to treatment fed reference diet 
was observed in treatment fed 8 and 12% SPC (Figure 1b). 
 
Production pond trial  
 The overall means, standard deviations, and ranges of morning, afternoon, and 
night water quality variables observed throughout the 18-week growth trial are displayed 
in Table 9.  Water quality conditions were within a reasonable range for the culture of 
 
 97 
this species under pond production conditions. High dissolved oxygen readings (above 10 
mg L-1) were observed in the afternoon due to photosynthesis activity. On the other hand, 
low dissolved oxygen readings (below 2.5 mg L-1) were observed due to the 
phytoplankton died off (Boyd, 2007, 2009).  
 General trends in production results from the pond trial were similar to those 
observed in the outdoor tanks. The results of one replicate from treatment fed diet 
containg 0% SPC was excluded from the statistical analysis because of low survival of 
shrimp. Growth performance of L. vannamei reared under production pond condition is 
summarized in Table 12. There were no differences (P > 0.05) in any of the measured 
production parameters. Treatment fed diet containing 4% SPC had slightly larger yield of 
5051 kg ha-1 compared with 8% SPC (4508 kg ha-1), 12% SPC (4479 kg ha-1), and 0% 
SPC (4190 kg ha-1), respectively. Likewise, treatment fed diet containing 4% SPC had 
numerally lower FCR at 1.30 compared with 12% SPC (1.48), 8% SPC (1.50), and 0% 
SPC (1.59), respectively. However, there were no differences in survival and mean final 
weight among treatments. Shrimp survival ranged from 86.7% (0% SPC) to 93.3% (12% 
SPC), and mean final weight ranged from 13.5 g (0, 8 and 12% SPC) to 15.7 g (4% SPC), 
but these differences were not significant. 
 
Discussion  
 The results from this study indicate that SPC at a level up to 12% may be include 
in diets containing high level of soybean meal without adversely affecting growth and 
survival of L. vannamei. Although, there is no consistent trend in mean final weight of 
shrimp results from the clear water tank trial, significant difference in mean final weight 
 
 98 
among treatments were observed. However, there were no differences in final biomass 
among treatments. This finding is similar to growth response of shrimp in green water 
tank trial or pond trial. Furthermore, the results from the previous study in clear water 
system with shrimp fed diets containing from 0 to 10% SPC also showed no difference in 
growth performance.  
 In general, high level of soybean proteins inclusions in shrimp feed, with the 
absence of expensive fish meal, are viable and result in acceptable performances of 
shrimp, when essential nutrients in diet are properly balanced to meet shrimp 
requirement. Similar results was reported by Kaushik et al. (1995) who found that diet 
containing SPC with L-methionine supplement could completely replace fish meal in 
rainbow trout diet without causing negative effect on growth performance and nutrient 
utilization of fish.  
 On the other hand, Lim and Dominy (1990) found the decrease in weight gain of 
L. vannamei fed diets containing 42% SE-SBM or higher. Likewise, Floreto et al. (2000) 
indicated that diets containing 50% soybean meal and higher resulted in significantly 
lower body weight gain in juvenile American lobster than treatment fed diets with lower 
level of soybean meal inclusion.  
 In the clear water tank system, shrimp growth is the result of the availability of 
compound feed entering the system to satisfy the nutritional requirements as there are 
little or no other food sources presented (Moss ,1995). There were no differences (P > 
0.05) in survival, FCR, and net yield. However, significant differences in mean individual 
weight among treatments were observed in the 10-week growth trial Mean final weight of 
shrimp fed 0% SPC was significantly higher than shrimp offered the diets with 4% SPC, 
 
 99 
but there were no differences in the growth performances among dietary treatments 
containing 0, 8,and 12% SPC or 4, 8 and 12% SPC. This does not follow the expected 
dose response and does not match previous experiments or results for final biomass. In 
addition, there was an evidence of the present of filamentous algae in 0% SPC treatment 
which may have serves as a potential supplemental food that could certainly improve 
growth performance of shrimp in this dietary treatment. Based on the results in clear 
water systems from the previous chapter which shrimp fed diets containing increasing 
level of SPC from 0 to 10% resulted in no differences in growth response, the differences 
observed in this trial is more likely caused by the extrinsic factors or random variation.  
In the green water tank trial, the results at the conclusion of the 10-week growth 
trial indicated that net yield in treatment fed diets containing higher level of SPC (4, 8 or 
12%) resulted in significantly better net yield than treatment fed diet without SPC (0% 
SPC). However, there were no differences (P > 0.05) in survival, mean final weight, and 
FCR for shrimp offered diets containing various levels of SPC.  Shrimp maintained on 
the commercial reference diets had significantly higher mean final weight than shrimp 
fed the test diets.  This could be the results of slight inadequacies of the plant based diet 
or simply variation across treatments. These results confirm that SPC at a level up to 12% 
could be used in shrimp feed formulations and that higher levels provided numerically 
better results. As has been previously reported growth performance of the shrimp was 
considerably better in the green water trial comparing to that of the clear water system, 
(1.44 g wk-1 versus 0.61 g wk-1, respectively). This is caused by the availability of 
additional nutrient from natural food organisms present in the water column and sediment 
 
 100 
in the green water tanks that serve as growth-enhancing factor in shrimp (Moss, 1995; 
Tacon et al., 2002). 
 Similar to the results to the green water tank trial, production parameters results 
from the semi-intensive pond trial over an 18-week culture period were not different (P > 
0.05) among treatments. Net yields (3,900 ? 5,051 kg ha-1) were in acceptable production 
range for semi-intensive shrimp production. Overall mean final weight (14.3 g) from this 
trail was found to be slightly lower than previous historical data from the same site.  This 
could be the results of overstocking of juvenile shrimp as percent survivals in several 
ponds were over 100%. Since, feed inputs were calculated based on the expected survival 
of 75% the higher than expected survival may have resulted in feed limitations to shrimp. 
Shrimp reared under the green water tank system which were obtained from the 
production ponds showed better performance in term of growth per week than those in 
production ponds, 1.44 g wk-1 comparing with 0.86 g wk-1, respectively. These 
observations may indicate that underfeeding may be one of the reasons for the reduction 
in shrimp growth. In addition, shrimp growth can be affected by unknown water quality 
conditions or shrimp genetics. 
 Based on the observed response of the shrimp from this experiment, there were no 
differences across the SPC treatments in all culture environments, except in the clear 
water system. Thus, we can conclude that the use of SPC at a level up to 12% is 
acceptable and promote growth in Pacific white shrimp. The pepsin digestibility of crude 
protein in all dietary treatments ranged from 76.9 to 80.5%. The digestibility values of 
our test diets are considered reasonable comparing to the apparent protein digestibility of 
soybean meal (90%) and of fishmeal (80.7%) reported for L. vannamei by Akiyama et al. 
 
 101 
(1989). Consequently, the results of this study confirm that the use of SPC in commercial 
feed formulations is possible regarding to promote SPC use as a high protein ingredient 
for shrimp feeds. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 102 
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161. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 108 
Table 1. Feeding regime for Litopenaeus vannamei post larvae, based on mean shrimp 
weight determined every three days, percent body weight and feed types utilized 
throughout a 15-day nursery period. 
Day Mean weight  (mg)           Feed type Feed (% body weight) 
1 to 3 1.01 100 Artemiaa / PL, PL redib (1:1) 25 
4 to 6 2.24 PL redi  , Cruc #0 (1:1) 25 
7 to 9 3.53 Cru # 0 25 
10 to 12 5.03 Cru # 0 15 
13 to 15 8.10 Cru # 0, Cru #1& #2 (1:1) 15 
aINVE Americas, Inc. Salt Lake City, UT, USA. 
bPL Redi-reserve 400-600 microns. 50% Protein, produced by Zeigler Bros, Inc., 
Gardners, PA, USA. 
cCrumble feed, 45% protein, produced by Rangen Inc., Buhl, Idaho, USA. 
 
 
 
 
 
 
 109 
Table 2. Water quality variables observed over a 15-day nursery period for L. vannamei 
post larvae, nursed in a recirculating system composed of six, 6,000-L nursery tanks.  
 Average Minimum Maximum Standard deviation CVa 
Temperature (?C) 26.22 23.30 29.50 1.44 5.50 
DO (mg L-1)b 6.44 2.75 9.71 1.45 23.06 
Salinity (ppt) 28.63 17.10 34.00 5.03 17.58 
pH 7.55 7.11 8.11 0.33 4.42 
TAN (mg L-1)c 0.15 0.01 0.49 0.14 95.57 
aCV = Standard deviation / mean x 100. 
bDissolved oxygen. 
cTotal ammonia-nitrogen. 
 
 
 
 
 
 
 
 110 
Table 3. Mean production parameters of L. vannamei post larvae (initial weight of 1.0 
mg), stocked at a density of 52 PL L-1 and nursed for 15 day in a recirculating system 
composed of four, 6,000-L nursery tanks. 
 Mean Minimum Maximum Standard deviation CVa 
Final weight (mg) 12.88 12.10 13.40 0.57 4.41 
Weight gainb (mg) 11.87 11.09 12.39 0.57 4.78 
Survival (%) 58.05 55.13 61.02 3.27 5.63 
FCRc 1.42 1.35 1.45 0.04 3.08 
Yield (g tank-1) 2,440 2,381 2,554 77.19 3.16 
Standing crop (kg m-3)d 0.678 0.661 0.709 0.021 3.164 
aCV = Standard deviation / mean x 100. 
bWeight gain = Final weight - Initial weight. 
cFood conversion ratio = Feed offered per shrimp / weight gain per shrimp. 
dStanding crop (kg m-3)= (Yield/1,000)/3.6 m3. 
 
 
 
 111 
Table 4. Ingredient composition (g 100 g-1 as is) of four experimental diets designed to 
contain increasing percentage of 0, 4, 8 and 12% soy protein concentrate (SPC) as a 
substitute for soybean meal and used for Litopenaeus vannamei growth trials. 
Ingredient 0% SPC 4% SPC 8% SPC 12% SPC 
Soybean meal (47%) 58.01 52.01 46.01 39.67 
Milo 16.34 18.34 20.34 22.67 
Corn distillers grain 10.00 10.00 10.00 10.00 
Corn gluten meal 4.83 4.83 4.83 4.83 
SPC - 4.00 8.00 12.00 
Dicalcium phosphate 3.38 3.38 3.38 3.38 
Fish oil  4.33 4.33 4.33 4.33 
Bentonite 1.50 1.50 1.50 1.50 
Squid meal  0.50 0.50 0.50 0.50 
Soy lecithin 0.50 0.50 0.50 0.50 
Vitamin premixa        0.33 0.33 0.33 0.33 
Mold inhibitor 0.15 0.15 0.15 0.15 
Mineral premixa 0.09 0.09 0.09 0.09 
Stay-C 35% (C) 0.02 0.02 0.02 0.02 
Copper sulfate 0.01 0.01 0.01 0.01 
Total 100 100 100 100 
Diets were formulated to contain 35% protein and 8% lipid and commercially 
manufactured by Rangen? (Angleton, TX, USA) using extrusion processing. 
aVitamin premix and mineral premix are proprietary products, thus their composition is 
not listed.  
 
 112 
Table 5. Nutrient composition of the experimental diets and a commercial reference diet 
used for L. vannamei reared under different culture conditions. 
Nutrient 
Proximate analyses (%) 
0% SPC 4% SPC 8% SPC 12% SPC Reference diet 
Crude protein 35.8 37.0 36.7 35.9 35.8 
Moisture  8.34 8.61 7.70 7.71 11.39 
Fat (AH) 9.34 8.51 8.69 8.49 9.47 
Crude fiber 2.87 3.06 2.91 2.96 2.57 
Ash 9.08 9.26 9.48 9.27 7.92 
Protein digestibility  
(0.0002% pepsin)  79.89 80.54 77.66 76.88  
Diets were formulated to contain 35% protein and 8% lipid. 
Diets were commercially manufactured by Rangen? (Angleton, TX, USA) using 
extrusion processing. 
Diets were analyzed by New Jersey Feed Laboratory, Trenton, NJ,USA. 
 
 
 
 
 113 
Table 6. Water quality variables monitored during 10 week growth period of juvenile L. 
vannamei (initial weight of 0.94 g), fed experimental diets containing increasing level of 
SPC at 0, 4, 8 and 12% as a substitute for soybean meal, and reared under a clear water 
recirculating system. Values represent the mean ? standard deviation of daily and weekly 
determinations. Values in parenthesis represent minimum and maximum readings. 
 Temperature (?C) DO (mg L-1)a pH Salinity (ppt) TAN (mg L-1)b 
am 26.29?1.16 (21.73, 28.34) 6.10?0.63 (4.58, 8.61) 7.82?0.29 (6.72, 8.36) 16.25?0.63 (12.67, 17.47) 0.04?0.02 (0.02, 0.10) 
pm 28.80?0.92 (25.37, 31.16) 5.98?0.56 (4.60, 7.15) 8.20?0.30 (7.33, 8.67) 16.25?0.67 (14.89, 17.49) 
 
 
aDissolved oxygen. 
bTotal ammonia-nitrogen. 
 
 
 
 
 
 
 
 
 
 
 114 
Table 7. Water quality variables monitored during 10-week growth period of juvenile L. 
vannamei (initial weight of 1.0 g), fed experimental diets containing increasing level of 
SPC at 0, 4, 8 and 12% as a substitute for soybean meal, and reared under a semi-closed 
recirculating system. Values represent the mean ? standard deviation of daily and weekly 
determinations. Values in parenthesis represent minimum and maximum readings. 
 Temperature  (?C) DO  (mg L-1)a pH Salinity (ppt) TAN (mg L-1)b 
am 27.16?1.27 (21.97, 29.61) 5.66?0.77 (3.71, 7.26) 7.93?0.36 (6.66, 8.51) 15.60?0.46 (14.49, 16.22) 0.23?0.20 (0.04, 0.70) 
pm 30.08?1.27 (27.24, 33.15) 5.55?0.68 (3.66, 6.72) 8.26?0.32 (7.19, 8.70) 15.51?0.42 (14.19, 16.20) 
 
 
aDissolved oxygen. 
bTotal ammonia-nitrogen. 
 
 
 
 
 
 
 
 
 115 
Table 8. Feeding regime for L. vannamei reared in the grow-out pond during an 18-week 
experimental period, at estimated survival rate of 75%. 
Day Week Population 
Feed input per day 
kg pond-1 kg ha-1 
1 0 35,000 1.00 10.00 
8 1 34,485 1.50 15.00 
15 2 33,971 3.00 30.00 
22 3 33,456 5.00 50.00 
29 4 32,941 5.86 58.57 
36 5 32,426 4.04 40.42 
43 6 31,912 6.60 66.04 
50 7 31,397 7.00 69.97 
57 8 30,882 6.88 68.82 
64 9 30,368 6.88 68.82 
71 10 29,853 6.18 61.78 
78 11 29,338 6.54 65.38 
85 12 28,824 4.84 48.35 
92 13 28,309 5.40 54.01 
99 14 27,794 6.19 61.94 
106 15 27,279 6.34 63.42 
113 16 26,765 6.50 65.00 
120 17 26,250 2.50 25.00 
 
 
 
 116 
Table 9. Summary of water quality variables monitored over an 18-week growth trial for 
L. vannamei, fed experimental diets with varying level of SPC and cultured in 0.1-ha 
ponds. Values represent the mean ? standard deviation of daily and weekly 
determinations. Values in parenthesis represent minimum and maximum readings. 
Parameter 0% SPC 4% SPC 8% SPC 12% SPC  
Temperature (?C) 
       am 
  
       noon 
  
       pm 
 
 
 
28.25?1.93 
(19.26, 31.70)  
30.52?2.57 
(23.98, 34.92)  
30.88?2.64 
(22.48, 34.09) 
  
28.52?1.95 
(21.37, 33.59)  
30.74?2.65 
(24.00, 35.38)  
30.30?2.76 
(22.76, 34.80) 
 
 
28.26?2.08 
(20.51, 31.67)  
30.69?2.66 
(23.99, 34.74)  
30.23?2.71 
(20.54, 34.17) 
  
28.24?2.09 
(21.14, 33.30)  
30.83?2.70 
(24.14, 36.03)  
30.37?2.84 
(21.82, 35.16)  
DOa (mg L-1) 
       am 
  
       noon 
  
       pm 
  
  Readings < 2.5 (days) 
  
5.36?1.63 
(0.78, 9.74)  
10.39?3.43 
(2.62, 23.12)  
7.94?2.27 
(1.40, 14.26)  
5?3.83 
  
5.20?2.06 
(0.35, 14.85)  
10.40?3.39 
(2.32, 21.61)  
8.16?2.47 
(1.77, 14.80)  
9?6.06 
  
5.48?1.79 
(1.69, 11.48)  
10.58?3.40 
(3.03, 27.09)  
8.14?2.44 
(1.66, 14.71)  
4.5?3.11 
  
5.36?1.75 
(1.57, 10.01)  
10.56?3.43 
(2.52, 21.88)  
8.13?2.51 
(1.63, 17.70)  
6?3.16  
pH 
       am 
  
       noon 
  
       pm 
 
  
8.03?0.46 
(6.47, 9.42)  
8.85?0.55 
(7.02, 10.14)  
8.65?0.50 
(6.91, 9.79) 
  
8.08?0.45 
(6.56, 9.12)  
8.82?0.49 
(7.55, 9.87)  
8.69?0.49 
(7.37, 9.74) 
  
8.12?0.51 
(6.41, 9.37)  
8.93?0.56 
(6.77, 10.14)  
8.75?0.56 
(6.77, 10.16) 
  
7.96?0.48 
(6.61, 9.20)  
8.88?0.54 
(7.33, 10.16)  
8.64?0.50 
(7.21, 10.08) 
Salinity (ppt) 
       am 
  
       noon 
  
       pm 
 
 
15.36?1.37 
(11.34, 18.41)  
15.56?1.27 
(11.14, 19.17)  
15.49?1.13 
(11.14, 19.17) 
 
15.42?1.24 
(12.70, 18.88)  
15.48?1.19 
(12.87, 19.49)  
15.52?1.25 
(12.89, 19.44) 
 
14.92?1.44 
(11.64, 18.75) 
 
14.84?1.33 
(11.65, 18.55)  
15.03?1.33 
(11.66, 18.88) 
 
15.51?1.38 
(12.73, 19.49)  
15.60?1.33 
(12.74, 19.85)  
15.59?1.37 
(12.75, 19.70)  
Secchi (cm) 
 
 
37.35?38.00 
(10, 110) 
 
38.16?36.12 
(10, 110) 
 
41.41?40.70 
(5, 110) 
 
36.90?36.73 
(10,110)  
TANb (mg L-1) 
 
 
0.69?1.18 
(0.01, 5.00) 
 
0.64?1.13 
(0.01, 4.00) 
 
0.73?1.39 
(0.01, 6.00) 
 
0.36?0.84 
(0.01, 4.00) 
aDissolved oxygen. 
bTotal ammonia-nitrogen. 
 
 117 
Table 10. Mean (n = 3) production parameters at 10-week experimental period of L. 
vannamei (initial weight of 0.94?0.04 g), fed diets containing increasing level of SPC at 
0, 4, 8 and 12% as a substitute for soybean meal, and reared under a clear water 
recirculating system. 
Treatment Biomass (g tank-1) Final weight (g) FCRa Survival (%) 
0% SPCb 100.6 7.2a 2.65 93.3 
4% SPC 78.2 5.3b 3.54 97.8 
8% SPC 77.1 5.8ab 3.61 88.9 
12% SPC 93.9 6.4ab 2.85 97.8 
P-valuec 0.159 0.044 0.130 0.713 
PSEd 7.302 0.350 0.289 6.443 
aFeed conversion ratio = Total feed offered / biomass increase. 
bResults with only 2 replicates. 
cMeans (n = 4) not sharing a common superscript within a row are significantly different 
based on analysis of variance (P > 0.05) followed by Student Newman-Keuls multiple 
range test. 
dPooled standard error of treatment means. 
 
 
 
 
 118 
Table 11. Mean production parameters at 10-week experimental period of L. vannamei 
(initial weight of 0.99?0.03 g), fed diets containing increasing level of SPC at 0, 4, 8 and 
12% as a substitute for soybean meal, and a reference commercial diet, and reared under 
a green water recirculating system. 
Treatment Biomass (g tank-1) Final weight (g) FCR Survival (%) 
0% SPC 398.9c 13.5b 1.28a 98.0 
4% SPC 397.2bc 13.7b 1.28a 96.7 
8% SPC 416.8ab 13.9b 1.22ab 100.0 
12% SPC 411.5bc 13.9b 1.23ab 98.7 
Reference dieta 431.6a 15.0a 1.17b 95.8 
P-valueb 0.001 0.008 0.002 0.152 
PSEc 5.448 0.263 0.018 1.170 
aReference diets = 35% Protein, Rangen 35,0 (Buhl, Idaho).  
bMeans (n = 5) not sharing a common superscript within a row are significantly different 
based on analysis of variance (P > 0.05) followed by Student Newman-Keuls multiple 
range test. 
cPooled standard error of treatment means. 
 
 
 
 
 
 
 119 
Table 12. Mean (n = 4) production parameters at 18 week experimental period of juvenile 
Pacific white shrimp, L. vannamei (initial weight of 10 mg), fed experimental diets 
containing increasing level of SPC at 0, 4, 8 and 12% as a substitute for soybean meal, 
and cultured in 0.1-ha production ponds. 
Treatment Yield  (kg ha-1) Final weight  (g) FCR Survival  (%) 
0% SPCa 4190.2 13.5 1.59 86.7 
4% SPC 5051.4 15.7 1.30 89.5 
8% SPC 4508.6 13.5 1.50 92.9 
12% SPC 4479.4 13.5 1.48 93.3 
P-valueb 0.137 0.363 0.136 0.861 
PSEc 234.378 1.020 0.071 6.019 
aResults with only 3 replicates.  
bAnalysis of variance was used to determine significant differences (p < 0.05) among 
treatment means (n=4). 
cPooled standard error of treatment means. 
 
 
 
 
 
 
 
 120 
Figure1. Mean final weight (g) and FCR of L. vannamei juveniles fed diets containing 
increasing level of SPC at 0, 4, 8 and 12% as a substitute for soybean meal, and reared 
under a clear water recirculating system (a), a green water semi-closed recirculating 
system (b), and production pond conditions (c). 
 
 
 
a 
b b b 
0.00
0.60
1.20
1.80
2.40
3.00
3.60
4.20
0.00
5.00
10.00
15.00
20.00
25.00
0%SPC 4%SPC 8%SPC 12%SPC
FC
R 
Me
an
 fin
al 
we
igh
t (g
) 
(a) 
Final weight
FCR
b b b b a 
a a ab ab 
b 
0.00
0.30
0.60
0.90
1.20
1.50
0.00
5.00
10.00
15.00
20.00
25.00
0%SPC 4%SPC 8%SPC 12%SPC Reference
diet
FC
R 
Me
an
 fin
al 
we
igh
t (g
) 
(b) 
0.00
0.30
0.60
0.90
1.20
1.50
1.80
0.00
5.00
10.00
15.00
20.00
25.00
0%SPC 4%SPC 8%SPC 12%SPC
FC
R 
Me
an
 fin
al 
we
igh
t (g
) 
(c) 
 
 121 
CHAPTER V 
SUMMARY AND CONCLUSIONS 
 
Increasing the use of plant-based ingredients and reducing the use of marine 
ingredients in shrimp feed formulations has been a primary goal of many researchers. 
Towards this goal, there is considerable interest in using soybean products in shrimp 
feeds because of their acceptable nutrient contents as well as low cost compared to 
marine animal protein sources. Soybean meal contains a moderate protein content, high 
digestibility, relatively well-balanced amino acid profile, and steady supply with a 
reasonable price. However, the use of soybean meal as a primary protein source in shrimp 
feed is limited because of its shortage in sulfur amino acids, highly unsaturated fatty 
acids, and minerals such as phosphorus as well as the presence of antinutritional factors 
such as trypsin inhibitors that may affect digestibility and palatability of feed. In addition, 
as the nutrient density (protein and energy) of the diet is increased, the use of solvent-
extracted soybean meal (44-48% crude protein) is inefficient as there is insufficient room 
in the formulation for all necessary components. Hence, higher protein ingredients are 
often required. Various mixtures of soybean meal and other protein sources or utilization 
of soybean products containing higher protein can provide solutions to more balanced 
feed formulations.  
Results from this study confirm that utilization of soybean meal is an acceptable 
ingredient in feed for Pacific white shrimp, as long as dietary nutrients are properly 
 
 122 
balanced to meet their requirements. For example, soybean meal-based diets with the 
inclusion of either 10% of poultry by-product meal, fish meal, distiller?s dried grains with 
solubles or ground pea meal resulted in excellent performance of shrimp reared under 
outdoor conditions. Shrimp fed soybean-based diets in combination with poultry meal or 
other plant based proteins produced similar performances to shrimp fed the diet 
containing fish meal or a commercial reference diet. Consequently, we can conclude that 
fish meal can be totally replaced by a combination of plant and animal protein sources 
(soybean meal and poultry by-product meal), or all plant protein sources (DDGS, or pea 
meal with the inclusion of corn gluten meal and squid meal) when diets are formulated to 
contain acceptable nutrient levels and proper balance of essential amino acids.  
The inclusion of soy protein concentrate as a high protein ingredient in 
combination with soybean meal and corn gluten meal in practical diets resulted in good 
performance of shrimp at an inclusion level of 10%. Increasing the inclusion levels of 
SPC to 20% or higher caused a reduction in shrimp growth.  The supplementation of DL-
methionine, micro-encapsulated methionine or fish solubles to the diets containing high 
level of SPC (40%) as a replacement for soybean meal had no effect on the growth 
performance of shrimp.  
Based on the observed response of the shrimp fed practical diets containing 
increasing level of SPC under various culture conditions, SPC inclusion at a level up to 
12% SPC in soybean-based diet caused no negative effect on growth, feed conversion, 
survival and yield of shrimp under both laboratory and field conditions. Consequently, 
the inclusion of SPC in shrimp feed formulation at a level of up to 12% is acceptable and 
promotes good growth in Pacific white shrimp. 
 
 123 
In conclusion, based on laboratory, outdoor tank and pond based research soybean 
meal can be used as the primary protein source in L. vannamei feed formulations 
provided that proper combination with other low cost protein sources such as poultry by-
product, distiller?s dried grains with solubles, pea meal or soy protein concentrates is 
made. To develop such feed formulations, it is critical that the ingredients are of high 
quality and the essential nutrients in diets are properly balanced to meet shrimp 
requirements. Such diets should facilitate reduced feed costs and promote the use of 
sustainable plant protein sources in shrimp production. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 124 
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