Replacement of fish meal with poultry by-product meal in practical diets for Litopenaeus vannamei, and digestibility of the tested ingredients and diets

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Aquaculture 272 (2007) 466 – 476 www.elsevier.com/locate/aqua-online

Replacement of fish meal with poultry by-product meal in practical diets for Litopenaeus vannamei, and digestibility of the tested ingredients and diets Lucia Elizabeth Cruz-Suárez ⁎, Martha Nieto-López, Claudio Guajardo-Barbosa, Mireya Tapia-Salazar, Ulrike Scholz, Denis Ricque-Marie Programa Maricultura, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Cd. Universitaria Apdo. Postal F-56, San Nicolás de los Garza, Nuevo León 66451, México Received 17 January 2007; received in revised form 28 April 2007; accepted 30 April 2007

Abstract The use of poultry by-product meal-pet food grade (PBM-PFG, 66% crude protein) as a substitute for a fish meal blend (FM, a 50/50 mix of American menhaden meal and Mexican sardine meal, 65% average crude protein), in a control diet containing 35% crude protein, was evaluated in Pacific white shrimp. The replacement levels (w/w) were: 35, 50, 65 and 80%, with 13.7, 19.6, 25.5 and 31.4% PBM-PFG inclusion in the test diets, respectively. Two commercial feeds from Mexico, containing 30 and 35% protein, were used as additional controls. The diets were fed to shrimp (450 mg initial weight) in a 4 week feeding trial in order to evaluate growth, feed consumption, feed conversion ratio (FCR), total biomass, survival, protein efficiency ratio (PER) and nitrogen retention efficiency (NRE). In addition, apparent protein, dry matter and energy digestibility coefficients (PDC, DMDC, EDC) of all diets, as well as those of the ingredients, FM and PBM, were determined using the in vivo chromic oxide method and shrimp of 1.6 to 2 g initial weight. Digestibility coefficients were similar and greater than 80% for all diets. PDC, DMDC and EDC were high and similar for FM and PBM-PFG. Survival along the growth trial, FCR, PER and NRE were not affected by any of the test diets, but there was a slight linear negative effect of replacement level on consumption and growth, which seemed related to energy over formulation rather than a palatability problem. The commercial control diets, which did not contain PBM-PFG, gave results for growth that were equal to or lower than those of the test diets. Results of this experiment showed that PBM-PFG can adequately replace w/w up to 80% of FM in commercial diets for the white shrimp L. vannamei. © 2007 Elsevier B.V. All rights reserved. Keywords: Poultry by-products meal; Fish meal replacement; Shrimp; Growth trial; Digestibility

1. Introduction Fish meal is one of the primary proteins used in fish feeds, because of its known nutritional and palatability ⁎ Corresponding author. Tel./fax: +52 8 3526380. E-mail addresses: [email protected], [email protected] (L.E. Cruz-Suárez). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.04.084

characteristics. The demand for fish feeds is rising by some 5% a year because production from aquaculture is rising. Nevertheless, the supply of this ingredient cannot increase and prices have increased to a historic high level. In order to sustain aquafeed industry, a great part of nutritional research has been focused on the search for alternative proteins. One of the most promising alternatives is poultry by-product meal-pet food grade (PBM-PFG).

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PBM consists of ground rendered clean parts of the carcass of slaughtered poultry such as necks, feet, undeveloped eggs and intestines, exclusive of feathers, except in the amounts as might occur unavoidably in good processing practices. Variation in the composition of this protein meal could be largely due to variability in raw material composition and quality. The rendering processes involve the application of heat, the extraction of moisture and the separation of fat; the time and temperature of the cooking process are critical and also determine the quality of the finished product. The meals produced from by-products have historically been viewed as products with high variability in their biochemical composition, with high levels of ash and low digestibility. Nevertheless, in modern rendering facilities, those problems have been reduced by the use of computerized process control. The rendering process and conditions used (time / temperature) are sufficient to destroy most pathogenic microorganisms, with a minimum effect on digestibility of amino acids: “All rendering system technologies include the collection and sanitary transport of raw material to a facility where upon it is ground into a consistent particle size, conveyed to a cooking vessel whether of continuous or batch configuration. Cooking is accomplished generally via steam at temperatures of 245° to 290 °F (approx: 120 °C to 145 °C) for 40 to 90 min depending upon the system type. Most of North American rendering systems currently are continuous units. Regardless of the type of cooking, the melted fat is separated from the protein and bone solids and a portion of the moisture is removed. Most importantly cooking is similar to a sterilization process functioning to inactivate microorganisms to include bacteria, viruses, protozoa and parasitic organism” (Pearl, 2005). Federal laws in US and Canada prohibit renderers from accepting and processing animals infected with avian influenza. Additionally, there are current existing factors that result in even higher inedible raw material quantities being generated; these include further processing, prepacked/table ready meat products, which leave increasing amounts of inedible portions at the processing locations (Pearl, 2005). A variety of ingredients which include poultry byproducts mixed with other by-products or ingredients are commercially available and a distinction must be made. The label of PBM shall include the raw material used and must guarantee minimum crude protein, minimum crude fiber, minimum phosphorus and minimum and maximum calcium. The calcium level shall not exceed the actual level of phosphorus by more than 2.2 times. Currently the PBM is mainly used in pet foods

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because of its palatability, quality protein and critical amino acids, essential fatty acids, vitamins and minerals. There have been a few studies carried out on shrimp to test the replacement of FM with PBM; the main differences among these studies are the shrimp species, the quality and the quantity of FM replaced and the type of PBM tested, the protein content and the composition of the control diets, and the methodology of replacement used (on weight basis, on protein basis, on amino acids basis, on gross nutrient basis, or without nutrient compensation). Tan et al. (2003) and Zhu and Yu (2002) conducted studies with PBM on L. vannamei using diet formulations with 40 to 41% protein, 7.5% lipids, and containing 40% and 22% anchovy meal, respectively. These authors found a maximum FM replacement level of 80%. Cheng et al. (2002) evaluated the effects of two types of PBM (regular PBM and low-ash PBM-PFG) as a substitute for FM in diets (35% CP, 24.5% FM) on growth and body composition of juvenile shrimp, L. vannamei at 33.3, 66.7 and 100% on an equal weight (w/w) basis. Additionally, the PBM and PBM-PFG were defatted, and fish oil was added back so that their oil contents were the same as the original. No body weight differences were observed between shrimp fed diets based on PBM and PBM-PFG (P = 0.188) or between shrimp fed PBM with and without fish oil supplementation (P = 0.549). However, shrimp fed the 33.3 or 66.7% FM replacement diets grew faster than those fed the 100% FM replacement diet (P = 0.018 and P = 0.048, respectively). They showed that up to 66.7% FM could be replaced by PBM without affecting shrimp growth. Adding fish oil to defatted PBM diets did not affect shrimp body composition. Davis and Arnold (2000) and Samocha et al. (2004) evaluated the use of co-extruded soybean/poultry byproduct meal with egg supplement and a flash-dried PBM, replacing a menhaden meal included at 30% in a control diet (32% protein and 8% lipid). Yield parameters were improved or not affected with 80 and 100% replacement level of the menhaden meal. Menasveta and Yu (2002) reported that P. monodon grew significantly faster when 60% of FM was replaced with a PBM; in this study the control diet contained 40% FM which was replaced on a weight basis by PBM at 20, 40 and 60% levels. Phuong and Yu (2003) and Yang et al. (2004) showed that PBM did not affect the immune response on Macrobrachium nipponenese. In most of these studies the digestibility of test ingredients and the amino acid composition were not analyzed. General research work suggests that dietary FM could be partially replaced w/ w by PBM for substantial feed cost savings without loss in performance.

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Nutrient digestibility data of PBM measured from shrimp are very scarce. Tan and Yu (2002) reported PBM-PFG protein and dry matter digestibility coefficients of 90% and 76% from L. vannamei, respectively. The present study was conducted with the aim of determining the effect of replacement of a 50/50 mix of Mexican sardine meal and American menhaden meal with PBM-PFG in a practical shrimp feed (35% CP, and 39% FM), using two commercial pelleted Mexican feeds as controls. The chemical composition (proximate composition, energy, cholesterol, phospholipids and amino acid profile), the protein and energy digestibility coefficients and the in vitro protein digestibility of the test ingredients were also determined. 2. Materials and methods 2.1. Test ingredients The test ingredients used were a PBM-PFG, provided through the National Renderers Association (Premium Pro Poultry Protein ®, Griffin Industries Inc., Bastrop, Texas), and a blend of 50% Mexican sardine meal (Alimentos Marinos) and 50% American menhaden meal (Select Omega), both commonly used in Mexican shrimp feeds, and named as fish meal (FM). Other feed ingredients were obtained from a local feed company. The test ingredients were analyzed in terms of proximate composition (see below), total cholesterol (Courchaine et al., 1959; Zlatkis et al., 1953 as reported in Kates, 1987), phospholipids (Kates, 1987), soluble protein, and gross energy content (Parr calorimeter). Amino acid compositions were determined (HPLC) by Degussa Laboratory, Germany. Pepsin in vitro digestibility values were determined by an AOAC modified method (pepsin was diluted at 0.02% instead of 0.2%) and also by the Torry modified method (pepsin diluted at 0.0002%). 2.2. Experimental diets The diets were formulated to meet the recommendations by Akiyama et al. (1999). The control diet was formulated to contain about 9% lipid and 35% crude protein. Four other diets, replacing 35, 50, 65 and 80% of the FM with PBM-PFG on an equal weight (w/w) basis, were also formulated. The experimental diets also contained 1% chromic oxide as an inert marker. Additionally, two different reference diets were prepared for digestibility determination of PBM-PFG and FM, according to the method of Cho and Slinger (1979). One diet contained the ingredients of diet 1, minus the FM and with the remaining ingredients maintained at the

same proportions as in diet 1, except for chromic oxide. The second reference diet was prepared in the same way based on diet 5, minus the PBM-PFG (Table 1). For the growth trial, two Mexican commercial feeds were evaluated as additional controls. 2.3. Diet preparation All macro-ingredients (FM, PBM-PFG, shrimp meal etc.) were mixed in a Kitchen Aid mixer of 5 L capacity to obtain a homogenous mixture. Of this, a small proportion was removed and mixed with the microingredients (vitamin mix, chromic oxide etc.), and then incorporated into the remaining macro-ingredients and mixed again. The oil-based ingredients (fish oil, lecithin) were then added and finally, water (30%) to obtain a firm paste. For pelleting, a Torrey meat grinder with 1.6 mm diameter holes was used. The mixed diets were passed through the meat grinder at a temperature of 70– 75 °C at a rate of 40 min/kg diet. The resulting spaghetti-like strands were dried in a convection oven at 100 °C for 8 min, then allowed to cool overnight at room temperature, and conserved in plastic bags at 4 °C. 2.4. Chemical analysis The shrimp diets, test ingredients and shrimp carcasses were analyzed for proximate composition as follows: moisture (AOAC, 1997, method #930.15), crude protein (Tecator, 1987), crude lipid Soxhlet (Tecator, 1983), ash (AOAC, 1997, method #942.05) and fiber (AOAC, 1997, method #962.09). Nitrogen free extract (NFE) was calculated by the difference. Gross energy was measured in diets, test ingredients and feces with a Parr calorimeter (Parr, 1992a,b). Chromic oxide and nitrogen in the experimental diets and feces were analyzed using the same hydrolysis procedure (Bolin et al., 1952), and modified micro-Kjeldahl method (Nieto-López et al., 1997) using Tecator equipment. Diet leaching in seawater at 28 °C and 35 g L− 1 (3 replicates per diet) was simulated by a method recommended by David Smith from CSIRO, Cleveland Australia (2000, personal communication). A 3 g sample of pellets was weighed in a sieve (#40 mesh) that was then fixed in the mouth of a 250 mL plastic bottle, containing 200 mL seawater (the pellets being immersed under the water upwelling through the mesh screen). After agitating the bottles in a water bath for 1 h (30 rpm, 28 °C), the sieves were drained for a few minutes, and then dried before being weighed again.

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Table 1 Ingredient composition of test and reference diets (g/kg as is) Diet

1

2

3

4

5

Reference

Reference

% FM substitution

0%

35%

50%

65%

80%

Diet 1

Diet 2

PBM-PFG a Menhaden meal b Sardine meal c Soft wheat d Shrimp meal e Kelp meal f Soy meal g Lecithin h Fish oil i Vitamin mix j Mineral mix k ETQ (Dresquin) l Check mold m Vitamin C n Chromic oxide o

0.00 196.05 196.05 435.38 37.50 36.00 30.00 35.00 18.43 2.50 2.00 0.30 0.30 0.50 10.00

137.23 127.43 127.43 440.55 37.50 36.00 30.00 29.74 18.53 2.50 2.00 0.30 0.30 0.50 10.00

196.05 98.02 98.02 441.43 37.50 36.00 30.00 29.08 18.30 2.50 2.00 0.30 0.30 0.50 10.00

254.86 68.62 68.62 442.4 37.50 36.00 30.00 28.41 18.00 2.50 2.00 0.30 0.30 0.50 10.00

313.67 39.21 39.21 443.56 37.50 36.00 30.00 27.75 17.50 2.50 2.00 0.30 0.30 0.50 10.00

– – – 725.32 61.91 59.44 49.53 57.78 30.42 2.50 2.00 0.30 0.30 0.50 10.00

– 57.28 57.28 652.55 54.78 52.59 43.82 40.54 25.56 2.50 2.00 0.30 0.30 0.50 10.00

a

Poultry by-product meal, pet food grade, Griffin Industries Inc., Bastrop, Texas, 66.3% crude protein (CP). Special select menhaden meal, Omega Protein Company, Louisiana, 62.8% CP. c Mexican sardine meal, Alimar, Sonora, 65% CP. d Soft wheat: Triticum aestivum, 14.04% CP. e Chilean pelagic shrimp meal, 37.8% CP. f Kelp meal: Macrocystis pyrifera, Productos del Pacífico, Baja California, 12.4% CP. g Dehulled soybean meal, solvent extracted, Proteinas Naturales, Nuevo León, 46.3% CP. h Lecithin: ADM. i Fish oil: Industrias Barda, Mexico. j Vitamin mix: Vit. A, 4000 IU/g; B1, 24,000 mg/kg; B2, 16,000 mg/kg; DL Ca pantotenate, 30,000 mg/kg; B6, 30,000 mg/kg; B12, 80 mg/kg; C, 60,000 mg/kg; K3, 16,000 mg/kg; D3, 3200 IU/g; E, 60,000 mg/kg; H, 400 mg/kg; niacin, 20,000 mg/kg; folic acid, 4000 mg/kg. k Mineral mix: Co, 2000 mg/kg; Mn, 16,000 mg/kg; Zn, 40,000 mg/kg; Cu, 20,000 mg/kg; Fe, 1 mg/kg; Se, 100 mg/kg; I, 2000 mg/kg. l Antioxidant: ETQ66%, Dresquin, México D.F. m Antifungic: Check mold 50%, Dresquin, México D.F. n Vitamin C: Stay-C® by Roche, L-ascorbyl-2-polyphospate, 35% active C. o Cr2O3: Impex Continental, batch 52-0305. b

Loss of dry matter (LDM %) and loss of crude protein (LCP %) of the experimental diets were determined employing the following formulas (Cruz-Suárez et al., 2001): %LDM ¼ ½ðWeight of feedðdry wtÞbefore leaching weight of feedðdry wtÞafter leachingÞ =weight of feedðdry wtÞ before leaching⁎100 %LCP ¼ ½ð%crude protein in the feed⁎100Þ  ð%crude protein in the feed after leaching⁎ð100  %LDMÞÞ=%crude protein in the feed:

flow-through rate of 350 ml per min. All tanks contain a double bottom, covered with black stocking, and an internal recirculating “air lift” system. The facility is designed so that possible water quality variations affect all tanks simultaneously. Water quality parameters of salinity (30 to 24‰) and temperature (27 to 31 °C) were measured daily, pH (7.8 to 8.1), NH3 + NH4+(0 to 0.5 mg/L), NO2 (0.25 to 0.5 mg/L) and NO3 (20 to 80 mg/L) were recorded weekly. The parameters remained well within the optimum for L. vannamei throughout the trial. 2.6. Experimental animals and experimental designs

2.5. Bioassay facility and water quality parameters The growth and digestibility trials were conducted at the same time, in a closed recirculation artificial seawater system. The experimental facility contains 54 (60 L) experimental fiber glass tanks, each continuously fed with synthetic marine water (Fritz, Dallas, TX) at a

Approximately 800 L. vannamei of 450 mg average body weight and a further 400 L. vannamei of 1.5 g were obtained from Pecis Industries, Yucatan and acclimated to the conditions of the bioassay facilities in 500 L holding tanks, prior to the growth and digestibility trials.

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For the 28 days growth trial, 280 juvenile shrimp, with a mean initial weight of 460 mg were used. Ten animals per tank were distributed into 28 60 × 30 × 35 cm fiber glass tanks by weighing individually on a digital balance after blotting excess water on a moist cloth. Care was taken to distribute animals of the same size range in all tanks. Dietary treatments were then randomly assigned to the tanks using a four block design (4 replicates). The day after distributing the animals, any dead shrimp was replaced, and feeding on the respective diets was initiated. Remaining feed and any mortality was recorded every morning and the tanks cleaned of remaining feed and feces. The shrimp were initially fed at 10% of the biomass of each tank and the ration was adjusted to actual consumption every day, thus reducing uneaten feed to a minimum. Uneaten feed was estimated visually every morning in each tank as a percentage of the feed fed the previous day. The shrimp were fed twice daily, once after cleaning in the morning and again in the afternoon. The weighed feed strands were broken into small pieces to insure a minimum of one pellet per shrimp at each feeding. Individual weight was determined at 0, 14 and 28 days of the experiment. The shrimp were weighed individually on a digital balance (1 mg precision), after blotting on a moist cloth. Biomass per tank is the sum of the individual weights of the shrimp present in each tank. This variable reflects the effects of growth and survival. Individual weight gain (%) is the increase in weight with respect to individual initial mean weight. This variable was calculated per tank from initial mean weight and final mean weight. Individual weight gain = [(individual mean final weight − individual mean initial weight) / individual mean initial weight] × 100. Survival (%) is the final number of shrimp per tank expressed as a percentage of the initial number of animals. Survival = (Final number of shrimp / Initial number of shrimp) × 100. Individual feed consumption (IFC) was estimated every morning from the weighed ration fed, the percentage of uneaten feed recorded the next morning and the number of animals present on that day in the tank. For each tank, reported consumption was the sum of estimated individual daily feedP consumption throughout the 28 day growth trial. IFC ¼ 28 1 (consumption in the tank on day i / number of shrimp in tank on day i). A value adjusted for the pre-prandial loss of dietary dry matter was calculated as follows: IFC adj. = IFC ⁎ (1 − LDM / 100). Feed conversion ratio (FCR) is the amount of feed consumed (as fed) per unit weight gained (live weight).

FCR = estimated individual feed consumption / increase in mean individual weight. FCR adjusted for preprandial nutrients losses in water was expressed as FCR adj. = FCR (1 − LDM / 100). Protein efficiency ratio (PER) is the increase in weight with respect to protein consumed. This variable was calculated for each tank from the initial mean weight and the final mean weight, as well as the amount of protein consumed. PER = (final individual mean weight − initial individual mean weight) / (individual consumption ⁎ protein concentration in the feed). The values were also given with an adjustment for LCP. PER adj. = PER / (1 − LCP / 100). Nitrogen retention efficiency (NRE) is the nitrogen deposition with respect to ingested nitrogen. This variable was calculated for each tank from the initial nitrogen content of the shrimp as stocked and the final nitrogen content of shrimp in the tank. NRE = [(individual mean final weight ⁎ final body nitrogen) − (initial individual mean weight ⁎ initial body nitrogen)] / nitrogen consumed. Values adjusted for LCP were calculated as follows. NRE adj. = NRE / (1 − LCP / 100). Nitrogen content of shrimp was determined at the beginning of the growth trial on a sample of the shrimp used to stock the tanks, and at the end on four shrimp per tank. Shrimp were freeze-dried to allow proper grinding and sample homogenization, and the nitrogen content was then determined using the Kjeldahl method (Tecator, 1987), calculating the final result as a percent of wet weight. In vivo apparent protein, dry matter and energy digestibility coefficients (PDC, DMDC and EDC) of the diets were determined using 4 replicate tanks with 10 shrimp each (1.6 to 2 g initial weight). The shrimp were fed twice a day at a fixed daily ration of 10% of the biomass and feces collection started after acclimation of the shrimp to the feed for two days. After removing uneaten feed, feces were collected at 90 and 120 min after feeding for 7 days to collect at least 1.5 g feces per tank (wet weight). The feces were collected by siphoning and were immediately rinsed with distilled water several times and frozen. DMDC, PDC and EDC of diets were calculated using the following equations (Maynard et al., 1981): %DMDC ¼ 100  100⁎ð% Cr in diet=% Cr in fecesÞ %PDC ¼ 100  100⁎ð%Cr in diet=%CP in dietÞ ⁎ð%CP in feces=%Cr in fecesÞ %EDC ¼ 100  100⁎ð%Cr in diet=GE in dietÞ ⁎ðGE in feces=%Cr in fecesÞ

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where chrome (%Cr), crude protein (%CP) and gross energy (GE) concentrations are given in % of dry matter. Additionally, digestibility coefficients (DC) were adjusted for losses due to leaching before feed ingestion, using the following equations, in which dry matter and protein intake values are corrected for leaching by the last term of the expression (Cruz-Suárez et al., 2001):

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The parameters per tank (final biomass, growth, survival, feed consumption, FCR, PER, NRE, and digestibility coefficients) were also submitted to one-way ANOVAs to determine if significant differences exist among the different treatments, followed by SNK tests to identify where differences occurred. 3. Results

%DMDC adj: ¼ 100  100⁎ð%Cr in diet=%Cr in fecesÞ ⁎ð1=ð1  % LDM=100Þ

%PDC adj: ¼ 100  100⁎ð%CPfeces=%CrfecesÞ ⁎ð%Crdiet=%CPdietÞ ⁎ð1=ð1  %LCP=100ÞÞ: Ingredients DMDC, PDC and EDC were evaluated according to the substitution principle, using the method of Cho and Slinger (1979) and mathematical expressions in agreement with Forster (1999), and Bureau and Hua (2006): %IDMDC ¼ ð100⁎%DMDCexpdiet ⁎%DMexpdiet ð100  %TIÞ⁎%DMDCrefdiet ⁎%DMrefdiet Þ =ð%TI⁎%DMTI Þ

%IPDC ¼ ð100⁎%PDCexpdiet ⁎%CPexpdiet ð100  %TIÞ⁎%PDCrefdiet ⁎%CPrefdiet Þ =ð%TI⁎%CPTI Þ %IEDC ¼ ð100⁎%EDCexpdiet ⁎GEexpdiet  ð100  %TIÞ ⁎%EDCrefdiet ⁎GErefdiet Þ=ð%TI⁎GETI Þ where: % TI is the % inclusion of the test ingredient dry matter (in % of the ingredient dry matter mixture), % DMTI, %CPTI and GETI are the concentrations of dry matter, crude protein and GE in the test ingredient (in % of dry matter). The apparent differences with the mathematical expressions given by Forster, or Bureau and Hua (op. cit) are solved when considering that TI is given in % of the mixture dry matter instead of as used. 2.7. Statistical analysis Individual weight was used for statistical comparison (analysis of variance, ANOVA) of the replicate groups inside each treatment, thus validating the replicates. Individual weight was then analyzed statistically using one-way ANOVA and Student–Newman–Keuls multiple range tests (SNK).

Chemical compositions of PBM-PFG and FM were relatively similar (Table 2). The PBM-PFG had higher gross energy, crude lipid, fiber, phospholipid and soluble protein contents, and less ash than the FM. The amino acid profile was very similar with slightly higher levels in FM except for arginine and tryptophan, which had higher levels in PBM-PFG. Lysine was the most limiting amino acid in PBM. Pepsin digestibility was higher for FM than for PBM-PFG (91% vs 81.5% with pepsin at 0.02%). The proximal composition of the diets, LDM and LP after 1 h immersion in seawater, as well as diets water absorption, are presented in Tables 3a and 3b. The crude protein content was equal for all experimental diets at 34.6% ± 0.25, while the crude lipid and fiber content Table 2 Chemical composition and in vitro protein digestibility of the poultry by-product meal-pet food grade (PBM-PFG) and fish meal blend (% as is)

Moisture Protein Lipid Ash Fiber NFE Arg Lys Met Met-Cys Val Leu Iso Phe His Trp Thr Cholesterol Phospholipids Soluble protein (% of total protein) Gross energy kcal/g analyzed Pepsine Torry digestibility 0.0002 % Pepsine digestibility 0.02%

PBM-PFG

FM blend

4.41 66.3 12.6 12.0 0.97 3.73 4.41 3.97 1.24 1.96 3.01 4.48 2.44 2.47 1.48 0.63 2.48 0.6 3.7 18.3 5.1 63.2 81.5

4.58 65.0 8.95 17.3 0 0 3.89 5.30 1.72 2.23 3.3 4.86 2.81 2.64 1.61 0.55 2.70 0.86 2.98 12 4.5 84.5 91.0

Mean for 2 analyses, except for amino acids, which were analyzed 1 time.

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Table 3a Proximate analysis of test and reference diets (as fed), gross energy content, loss of dry matter (LDM), loss of crude protein (LCP) after 1 h immersion in sea water, and water absorption (WA) Diet

1

2

3

4

5

Reference

Reference

Probability

Replacement

0%

35%

50%

75%

80%

diet 1

diet 2

ANOVA

Moisture (%) Protein (%) Lipid (%) Ash (%) Fiber (%) NFE (%) Energy kcal/g Protein/Energy ratio (mg/kcal) LDM (%) LCP (%) WA (%)

5.32 34.5 8.60 11.8 1.80 38.0 4.4 79 8.10 a 13.9 ab 125 b

4.95 34.2 8.63 11.1 2.13 38.9 4.4 77 8.77 ab 11.4 a 131 b

4.41 34.6 9.51 10.7 2.90 37.9 4.5 77 10.4 c 12.5 ab 135 c

2.69 34.9 9.73 10.4 2.64 39.6 4.6 76 9.68 bc 12.4 ab 134 bc

4.13 34.6 9.45 10.3 2.87 38.6 4.6 75 10.9 c 15.4 b 117 a

4.80 15.9 9.07 7.2 2.86 60.2 4.3 37 13.5 18.5 128

3.23 21.2 7.51 8.4 3.44 56.3 4.5 47 12.7 12.9 105

0.002⁎ 0.084 0.002⁎

⁎ Differences are highly significant at a probability of b0.01. Different letters in the same row display differences according to SNK multiple range test (P = 0.05).

increased slightly with increasing PBM-PFG inclusion level (8.60 to 9.45 and 1.80 to 2.87%, respectively). The ash content of the experimental diets decreased with increasing replacement level (from 11.8% in diet 1 to 10.3% in diet 5). Dietary gross energy slightly increased with the PBM-PFG inclusion level from 4.4 to 4.6 kcal/g then the dietary crude protein (mg)/gross energy (kcal) ratio diminished from 79 to 75. Regarding leaching, LDM increased (P = 0.002) with increasing FM replacement level (from 8.10 to 10.9%). Loss of crude protein was not significantly affected (P = 0.084). Water absorption capacity also displayed significant differences for the different diets (P = 0.002), the capacity to absorb water increasing with increasing FM replacement level. Only diet 5 (80% FM replacement) displayed reduced water absorption of 117%, differing significantly from diets 1 to 4 (131% average). Commercial diets (Table 3b) displayed proximal composition values compatible with those certified in their label. 3.1. Growth trial results The results of the growth trial at 28 days are presented in Table 4. Feed consumption displayed highly significant differences (P b 0.001). The animals on diet 5 (80% replacement) displayed significantly lower feed consumption than those on diet 1. Nevertheless, the lowest feed consumption was with the commercial diets 6 and 7. The values obtained for growth also presented significant differences among treatments (P b 0.0001). Although multiple mean comparisons by SNK do not separate the experimental diets in different subsets

(Table 4), growth fell gradually from 447% for diet 1 to 357% for diet 5; the use of a regression analysis of the replicated data for diets 1 to 5, with the FM replacement level as the independent factor, allows to show a significant (P = 0.015) linear diminution of weight gain (%weight gain = 447.3 − 1.0236 ⁎ %PBM-PFG inclusion level), indicating how much growth potential may be sacrificed for inclusion of PBM. The commercial diets 6 and 7 led to the lowest growth overall, especially diet 7. Total biomass per tank showed the same tendencies as growth (ANOVA P b 0.001). The shrimp fed diets 1 to 5 showed a gradual diminution of the final biomass, while the shrimp fed diet 7 displayed significantly lower biomass than those fed the experimental diets. Survival and FCR (adjusted for leaching or not) did not differ for any of the treatments. Survival was high for all treatments, while the FCR averaged 1.7.

Table 3b Proximate analysis of the commercial diets (as fed), gross energy content, loss of dry matter (LDM), loss of crude protein (LCP) after 1 h immersion in sea water, and water absorption (WA) Diet

6 (Commercial)

7 (Commercial)

Moisture (%) Protein (%) Lipid (%) Ash (%) Fiber (%) NFE (%) Energy (kcal/g) Prot/energy (mg/kcal) LDM (%) LCP (%) WA (%)

8.5 31.1 8.5 10.1 2.0 39.9 4.3 72 4.3 9.4 80.4

6.0 36.6 10.1 10.6 2.4 34.1 4.5 81 12.6 13.0 93.8

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Table 4 Response of juvenile L. vannamei (mean initial weight 0.46 g ± S.D. 0.10 g) to practical diets containing increasing levels of PBM-PFG replacing a FM blend (28 days) Treatment

Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

Diet 7

Prob

Replacement

0%

35%

50%

65%

80%

Control 1

Control 2

ANOVA

Weight (g)

2.51 ± 0.42 c 24.4 ± 3.7 c 3.67 ± 0.64 c 3.38 ± 0.59 c 1.80 ± 0.17 1.66 ± 0.16 447 ± 83 c 97.5 ± 5.0

2.23 ± 0.16 c 21.7 ± 0.6 bc 3.44 ± 021 bc 3.14 ± 0.19 cd 1.94 ± 0.12 1.77 ± 0.10 395 ± 26 bc 97.5 ± 5.0

2.34 ± 0.19 bc 22.8 ± 2.5 c 3.47 ± 0.29 bc 3.11 ± 0.24 cd 1.85 ± 0.19 1.66 ± 0.17 420 ± 42 c 97.5 ± 5.0

2.21 ± 0.18 bc 21.6 ± 1.9 bc 3.13 ± 0.21 bc 2.83 ± 0.19 cd 1.79 ± 0.08 1.62 ± 0.07 382 ± 32 bc 97.5 ± 5.0

2.11 ± 0.16 abc 20.5 ± 1.8 bc 2.87 ± 0.17 ab 2.56 ± 0.15 bc 1.75 ± 0.10 1.56 ± 0.10 357 ± 40 bc 97.5 ± 5.0

1.83 ± 0.14 ab 17.9 ± 2.2 ab 2.36 ± 0.24 a 2.26 ± 0.24 ab 1.71 ± 0.08 1.64 ± 0.08 308 ± 31 ab 97.5 ± 5.0

1.73 ± 0.14 a 15.6 ± 1.3 a 2.33 ± 0.23 a 2.04 ± 0.20 a 1.84 ± 0.14 1.61 ± 0.12 273 ± 33 a 90 ± 0.0

P = 0.010⁎

Biomass (g) IFC (g) IFC adj. (g) FCR FCR adj. % Weight gain Survival (%)

P b 0.001⁎ P b 0.001⁎ P b 0.001⁎ P = 0.323 P = 0.405 P b 0.001⁎ P = 0.230

Means of four replicates ± standard deviation. ⁎ Differences are highly significant at a probability of ≤ 0.01 Different letters in the same row display differences according to SNK multiple range test (P = 0.05).

3.2. Protein efficiency ratio and nitrogen retention efficiency PER was similar with all diets except (P = 0.003) for the shrimp fed commercial diet 6 (30% CP) displaying a higher value (1.88 ± 0.08) than all other treatments (Table 5). When the values were adjusted for loss of protein, diet 6 remained the diet with the highest value and thus highest efficiency (P = 0.010). Regarding NRE of shrimp, a similar pattern was found. Before correcting for loss of protein, the animals fed diet 6 differed significantly from all other treatments (P = 0.006), while corrected values led to a less pronounced difference, diet 6 retaining the highest value, but significantly different only from shrimp fed diets 2 and 7.

3.3. Digestibility results The PDC, DMDC and EDC of the experimental diets showed significant differences, but mainly due to the lower values found for diet 3 (Table 6a). When leaching was corrected for, the differences remained virtually the same with P = 0.08 for PDCadj. and P = 0.008 for DMDCadj. Values adjusted for leaching were slightly lower than non-corrected values, reflecting that nutrients lost in the water are not anymore considered as being ingested and retained by the shrimp. The digestibility values for the reference diets are given in Table 6b. Reference diet 2 (for PBM-PFG digestibility determination) had lower values than reference diet 1 (for FM digestibility determination) for all the variables.

Table 5 Protein efficiency ratio and net protein utilization of shrimp fed on the different diets Diet

1

2

3

4

5

6

7

Prob.

Replacement

0%

35%

50%

65%

80%

Commercial

Commercial

ANOVA

PER

1.62 ± 0.17 a 1.88 ± 0.20 ab 0.28 ± 0.03 a 0.33 ± 0.03 ab

1.51 ± 0.09 a 1.71 ± 0.10 a 0.26 ± 0.01 a 0.30 ± 0.01 a

1.58 ± 0.16 a 1.80 ± 0.18 ab 0.28 ± 0.03 a 0.31 ± 0.03 ab

1.60 ± 0.07 a 1.83 ± 0.08 ab 0.28 ± 0.01 a 0.32 ± 0.01 ab

1.65 ± 0.11 a 1.95 ± 0.12 ab 0.29 ± 0.02 a 0.34 ± 0.02 ab

1.88 ± 0.08 b 2.08 ± 0.09 c 0.32 ± 0.01 b 0.35 ± 0.01 b

1.49 ± 0.11 a 1.71 ± 0.13 a 0.26 ± 0.03 a 0.30 ± 0.03 a

PER adj. NRE NRE adj.

Means of four replicates ± standard deviation. ⁎Differences are highly significant at a probability of ≤0.01. Different letters in the same row display differences according to SNK multiple range test (P = 0.05).

0.003⁎ 0.010⁎ 0.006⁎ 0.018

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Table 6a Apparent protein, dry matter, and energy digestibility coefficients of diets (PDC, DMDC and EDC respectively) and coefficients adjusted for losses due to leaching before ingestion (mean of four replicate values ± standard deviation) Diet

1

2

3

4

5

Replacement

0%

35%

50%

65%

80%

PDC

88.9 ± 2.0 ab 85.5 ± 2.6 ab 90.0 ± 1.9 bc 87.1 ± 2.3 ab 84.3 ± 2.9b

89.2 ± 0.8 ab 85.3 ± 1.1 ab 88.6 ± 0.9 ab 87.8 ± 0.9 ab 83.9 ± 1.2b

86.9 ± 0.6 a 83.0 ± 0.7 a 87.4 ± 1.4 a 85.0 ± 0.7 a 81.0 ± 0.8a

90.5 ± 0.7 b 87.8 ± 1.1 b 91 ± 0.6 c 89.1 ± 0.8 b 86.5 ± 1.2b

89.3 ± 2.6 ab 86.7 ± 1.6 b 90.3 ± 0.9 bc 87.4 ± 3.1 ab 85.0 ± 1.8b

DMDC EDC PDC adj. DMDC adj.

ANOVA

0.06 0.009⁎

IPDC IDMDC IEDC IPDC adj. IDMDC adj.

FM

PBM-PFG

ANOVA

87.9 ± 2.7 81.4 ± 6.7 90.4 ± 4.7 94.5 ± 3.3 81.4 ± 6.7

90.4 ± 4.4 90.8 ± 5.1 93.3 ± 2.63 96.5 ± 5.4 90.2 ± 5.5

0.373 0.068 0.261 0.563 0.088

0.001⁎ 0.08 0.008⁎

⁎Differences are highly significant at a probability of ≤0.01. Different letters in the same row display differences according to SNK multiple range test (P = 0.05).

The protein, dry matter and energy digestibility of the ingredients (FM and PBM-PFG), as well as the values adjusted for protein and dry matter losses, are presented in Table 7. No significant differences were observed in protein and energy digestibility, while differences for dry matter digestibility were at the limit of significance, in favor of PBM-PFG. Digestibility coefficients were generally higher for PBM-PFG than for FM, without or with adjustment for leaching, while adjustment increased protein digestibility coefficients but had no effect on dry matter digestibility coefficients of both meals. 4. Discussion The FM substitution w/w with PBM-PFG affected slightly the feed physical properties, reducing water stability and increasing water absorption capacity. This could be explained because the higher lipid and fiber content of PBM-PFG in comparison with the FM. In fact

Table 6b Apparent protein, dry matter, and energy digestibility of diets adjusted for losses due to leaching before ingestion (mean of four replicate values ± standard deviation)

PDC DMDC EDC PDC adj. DMDC adj.

Table 7 Apparent protein, dry matter, and energy digestibility of ingredients (IPDC, IDMDC and IEDC respectively), as obtained by standard determination and adjusted for losses due to leaching before ingestion (mean of four replicate values ± standard deviation)

Reference diet 1

Reference diet 2

90.5 ± 0.4 88.2 ± 0.8 90.1 ± 1.3 88.5 ± 0.5 86.4 ± 1.0

87.3 ± 2.4 84.7 ± 4.7 88 ± 3.7 85.4 ± 2.8 s82.4 ± 5.4

the high lipid content of PBM-PFG represents a potential technical problem that has to be managed at the feed mill plants. It is well known that high lipid content in the diets diminish the pellet hardness due to the reduction in the compression capacity of the press pellet machine. The use of PBM-PFG in place of FM had a gradual negative effect on feed consumption and growth, leading to differences that reached significance (as detected by SNK tests) at 80% replacement. As survival, FCR, PER and NRE were not affected by the experimental diets and the differences found for growth and total biomass were directly related to differences in feed consumption, no anti-nutritional effect could be attributed to PBM-PFG, nor a lysine deficiency, as expected from the amino acid profile comparison vs FM. Digestibility coefficients of dietary protein, dry matter and energy were not affected significantly by the replacement of the FM with PBM-PFG. The lower digestibility coefficients obtained at 50% replacement (diet 3) were the effect of a chromic oxide analytical error, because a chromic oxide content slightly higher than 1% was measured in this diet. When examined on their own, PBMPFG and FM displayed high digestible coefficients for protein, dry matter and energy, but with higher coefficients for PBM-PFG, the difference reaching significance for dry matter digestibility. The higher digestible energy content of PBM in comparison to FM and the lack of energy compensation on the replacement strategy used in the present study could explain the lower feed consumption observed at 65 and 80% replacement levels. Then, it is possible that, with similar protein/energy dietary ratios, the 100% replacement could be made without affecting performance. This is in agreement with the results reported by Davis and Arnold (2000) and Samocha et al. (2004), who also found that L. vannamei did not demonstrate palatability problems when a PBM product was used to replace 80 or 100% FM in the diet, neither any difference in terms of survival or growth. Our results are also in the line of those reported by Tan et al. (2003), Zhu and Yu (2002) and Cheng et al. (2002) with 80,

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80 and 66% w/w replacement levels, respectively. The favorable response of shrimp to FM replacement with PBM-PFG used in the present study is probably due to the high quality of the raw materials and processing technology used to produce both ingredients and their similar nutrient composition and availability. The FM replacement has a variety of advantages, including a reduction of the ash content of the diets, no need to use additional attractants or supplemental amino acids, a good provision and contribution of cholesterol and phospholipids content in the diet which are expensive nutrients, and finally the reduced cost of PBM-PFG compared to FM. The cost-effectiveness of substituting PBM-PFG for FM varies depending on the local cost of the ingredient, the price difference between FM and the PBM-PFG and the rate of substitution (Yu, 2006). In the conditions of the present study, 50 to 65% replacement of FM would lead to a cost reduction of about 10 to 14 % per ton of feed, which makes it quite interesting considering the global need to reduce shrimp production costs. Finally, all the experimental diets (35% CP) performed at least equally well to the commercial diets with 31 and 35% protein content, showing that a high inclusion of PBM-PFG in the formula can lead to the same or better results than those currently obtained with commercial Mexican feeds formulas. 5. Conclusions PBM-PFG is an excellent alternative protein source for L. vannamei; it is a source of highly digestible protein (90%), which possesses an EAA profile very similar to that of FM, provides good quantities of phospholipids and cholesterol and contains lower level of ash than FM; it is readily available, at a price lower than FM and it is a sustainable source of animal protein. PBM-PFG was in the present study on white shrimp an effective substitute on a weight basis for a blend of good quality fish meals, up to an 80% replacement level (i.e. 31% PBM-PFG) in diets with a moderate 35% crude protein level. Moreover, although no significant differences were shown by SNK mean comparisons, a regression analysis indicates a slight linear negative effect on feed consumption and growth, possibly due to an excess of energy in the diets supplemented with PBM-PFG. Using replacement strategies that consider the higher digestible energy and lower lysine content of PBM-PFG, the replacement rate could be total. FM substitution with PBM-PFG results in important savings in feed cost and additionally contributes to the sustainability of aquaculture.

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