Animal Feed Science and Technology 303 (2023) 115689

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Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Effect of substitution of poultry by-product meal by bovine

by-product meal on overall performance for Litopenaeus vannamei
Escarlt López-Ortiz a, Eulalio Arambul-Muñoz b, Aurora Tinajero c,
Oscar Basílio del Rio-Zaragoza c, Luis Alonso Galindo-Valdez c, María Teresa Viana c, *
a
Facultad de Ciencias Marinas, Licenciatura en Biotecnología en Acuacultura, Universidad Autónoma de Baja California (UABC), km 107 carretera
Tij/Eda, 22860 Ensenada, Baja California, Mexico
b
CONACYT, Estancia posdoctoral en el Instituto de Investigaciones Oceanológicas (IIO), UABC, Mexico
c
Instituto de Investigaciones Oceanológicas, UABC, Mexico

A R T I C L E I N F O A B S T R A C T

Keywords: The effect of partial to the total substitution of poultry by-product meal for bovine by-product
Alternative protein meal (BBM) was evaluated under fish-free basis diets for the white shrimp Litopenaeus vanna­
Shrimp mei. For this purpose, four isoproteic (46.5%) and isolipidic (8.5%) diets without any fish meal
Aquafeed
and fish oil added, different inclusion levels of BBM were included (0%, 13.6%, 27.2%, and
Nutrition
Sustainability
40.7%) equivalent to Control, L-BBM, M-BBM, and H-BBM, were evaluated. In triplicate groups,
three hundred and sixty shrimp were randomly distributed (0.47 g ± 0.006) in 12 ponds (30
shrimp per unit). After seven weeks of experimentation, the biological indices, hemolymph
chemistry, apparent digestibility (%), and fatty acid profile were evaluated. It was observed that
the H-BBM treatment improved biological indices compared to the Control. In addition, signifi­
cant differences were found, such as higher final weight (2.66 ± 0.13), weight gain (2.2 ± 0.1),
and specific growth rate (3.4 ± 0.1). In addition, the lower feed intake observed in the H-BBM
resulted in lower feed conversion (1.5 ± 0.12) than the Control group (2.8 ± 0.12). On the
contrary, in survival, the H-BBM treatment presented the lowest percentage (88.9 ± 4.6), while
the control diet had the highest survival (100%). Regarding the apparent digestibility (%), it was
observed that the H-BBM treatment presented the highest percentage (91.5 ± 0.3). At the same
time, the Control group had the lowest (85.8 ± 0.6), finding significant differences among
treatments with BBM compared to the Control group. In addition, a low protein concentration
was observed in the M-BBM treatment for hemolymph chemistry. At the same time, bovine meal
showed significant differences among treatments, particularly in the H-BBM treatment, resulting
in higher cholesterol and triglycerides levels. Therefore, it is concluded that BBM is a protein
source with a high prospect of being included in shrimp diets, resulting in better benefits in its
performance with higher feed conversion efficiency and digestibility. However, further research
on its effect on lipid metabolism is recommended.

Abbreviations: PBM, Poultry by-product meal; BBM, Bovine by-product meal; FM, Fish meal; FO, Fish oil.
* Corresponding author.
E-mail address: viana@uabc.edu.mx (M.T. Viana).

https://doi.org/10.1016/j.anifeedsci.2023.115689
Received 26 January 2023; Received in revised form 22 May 2023; Accepted 23 May 2023
Available online 24 May 2023
0377-8401/© 2023 Elsevier B.V. All rights reserved.
E. López-Ortiz et al. Animal Feed Science and Technology 303 (2023) 115689

1. Introduction

The high demand for protein sources has led to using non-conventional sources, such as fishmeal (FM). However, poultry by-
product meal (PBM) has progressively replaced the FM with excellent outcomes (Parés et al., 2014; Badillo et al., 2016; Man­
riquez-Patiño et al., 2021), resulting in high PBM demand and, consequently, a price increase. However, other renderer’s sources could
also be used. As an example, bovine by-product meal (BBM) is a protein source containing around 50% crude protein (CP) and 10%
crude fat (CG), allowing its use as an alternative not only for FM but also for PBM in diets for white shrimp Litopenaeus vannamei.
However, even if the benefits exist, it is calculated that 75% of the fishmeal and fish oil is used within the aquaculture sector
(Auchterlonie, 2018; Hulefeld et al., 2018). However, the use of FM depends on its price and availability, so its use will be sharply
reduced. According to Hua et al. (2019), shrimp diets contain a 10% FM at their maximum level, whereas plant protein diets and PBM
rapidly undertake the feed market for shrimp.
According to FAO (2020), the fishmeal (FM) price varies between USD 1800 /MT (2013) to USD 1700 /MT (2020), whereas the fish
oil (FO) is between USD 2300 /MT and USD 2000 /MT during the similar years. In contrast, BBM could reach half of PBM’s price (USD
900 /MT).
Since the previous decade, as stated before, FM and FO have been strongly reduced from the formulated feeds for aquatic species
(Salin et al., 2018; Malcorps et al., 2019). Therefore, this gap has allowed the industry to look for new alternatives with zootechnical
improvements resulting in efficient and sustainable productions (Naylor et al., 2009). In the first instance (Gasco et al., 2018),
vegetable-origin sources, especially cereals, legumes, and oilseeds, were proposed as an alternative to reduce FM inclusion. However,
even if feed manufacturers regularly use them, antinutritional factors have been observed when using high levels of vegetable protein
sources —such as soybean meal, reducing the overall performance and health parameters, besides its digestibility (Gatlin et al., 2007;
Bell and Waagbø, 2008; Hardy, 2010; Karapanagiotidis et al., 2019). Consequently, enormous efforts have been made to increase its
digestibility by reducing the antinutritional factors by treating the plant-origin sources, including grains, such as protein isolates, and
using enzymes and additives, among others (Karlsen and Skov, 2022).
Poultry by-product meal is the leading animal renderer product sold in the market. However, its demands overgrow as the primary
protein ingredient in most manufactured animal production feed, particularly pets and aquaculture. Nevertheless, even if the poultry
industry grows steadily, it is at a different amount than what is needed to cover the market. Further, even if its performance in fish is
acceptable, some production still needs to be better controlled (Rocker et al., 2021; Volpato et al., 2022).
Bovine meat and bone meal are produced from the renderers of animal production and are considered inedible, not desired for
direct human consumption. The raw material comes specifically from cattle. BBM is processed similarly to the PBM produced. The BBM
represents more than 10% of the total raw material processed in the production of meat for human consumption (García and Phillips,
2009). This product is efficiently processed to eliminate any possible trace of pathogens. The most widely used treatment method is the
so-called dry extraction process, in which the water is removed from the ground material by drying before the fat is separated from the
mass. According to Skovgaard (2012), sterilization should be carried out following the EU by-product regulation at 133 ◦ C for 20 min,
which can be done before (pre-sterilization) or after (post-sterilization) the drying process, where the drying is carried out at atmo­
spheric pressure with a temperature between 100 ◦ C and 120 ◦ C.
Several researchers have carried out bioassays to determine the inclusion levels of BBM, with promising results for aquatic species,
such as fish (Wei et al., 2006; Goda et al., 2007; Li et al., 2010; Kader et al., 2011; Lee et al., 2012; Suloma et al., 2013; Moutinho et al.,
2017). Apart from the BBM, there is a market for plasma protein extracted from bovine whey and plasma (Amer et al., 2019). Bovine
plasma contains soluble proteins with high levels of essential amino acids, salts, lactose, and vitamin B (dos Santos Barbosa et al.,
2010). The latter is essential as functional proteins increase the immune system, promoting health and higher growth (Henrichs et al.,
2020). However, BBM is a renderer protein blend from the meat industry producing protein meal with promising properties containing
around 50–52% crude protein (CP), resulting in a completely different product.
Historically, BBM has been withdrawn from the market for health reasons after being associated with the spongiform syndrome
produced by a prion in the cattle´s brain. Although the battle and sanitary control had led a long battle, today, it is considered a reliable
product many species can use as feed (Denton et al., 2005). However, it should be noted that transmission from terrestrial organisms
(bovine) to humans through aquatic organisms has never been proven (Horigan et al., 2018). However, this fact has led to this
by-product acquiring little demand. In addition, BBM can cost up to half of PBM and up to 25% of what FM costs (CONAFAB, 2021).
Research is needed to identify lower-cost products (BBM) to maintain the same or better nutritional quality than traditional
products (FM and FO) and products that are already being used (PBM, SBM) in shrimp feed (Rivas-Vega et al., 2006; Álvarez et al.,
2007; Suárez et al., 2009; Forster et al., 2019). However, SBM or other vegetable products should be constantly monitored since their
use is limited, especially in carnivorous species, since they have nutritional weaknesses such as deficiencies of essential amino acids,
palatability, and nutrient availability (Toko et al., 2008). For this reason, blending of ingredients (animal of vegetable origin) has been
constantly suggested to allow nutritional supplementation in cultured organisms and, at the same time, reduce costs in the diet
formulation (Davis and Arnold, 2000; Hu et al., 2008; Ye et al., 2011). Diets with BBM inclusion have had contradictory results on
shrimp zootechnical response variables. For example, some recommended the inclusion of animal products with equally favorable
results replacing traditional products (Forster et al., 2003; Hernández et al., 2004, 2008; Ye et al., 2011), while others suggest the
combination of mixtures of various products to counteract deficiencies for better responses (Ye et al., 2011).
Earlier in our lab, it was proven that PBM could efficiently substitute FM if the LC-PUFAs (DHA and EPA), cholesterol, and
phospholipids are added to the diets of shrimp and fish aquafeeds (Parés et al., 2014; Badillo et al., 2016; Manriquez-Patiño et al.,
2021).
However, based on the background of the application opportunities of bovine by-products to create a portfolio from different

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ingredients to aquafeed formulation, the present investigation aims to evaluate the effect of gradually substituting PBM by BBM on
survival, growth performance, and hemolymph biochemistry parameters of white shrimp Litopenaeus vannamei.

2. Materials and Method

2.1. Shrimp and experimental units

A batch of 10,000 white shrimp, Litopenaeus vannamei organisms was obtained and transported to the facilities of the Instituto de
Investigaciones Oceanológicas de la Universidad Autónoma de Baja California (IIO-UABC, Mexico). The juveniles of Litopenaeus
vannamei were provided by the FITMAR commercial hatchery in Sinaloa, Mexico, and characterized to represent a Mexican genetic line
resistant to local pathogens (P é rez-Enríquez et al., 2018). Upon arrival at the IIO, they were received in a recirculation system (RAS)
composed of two 1,000-L circular tanks, a pump, a biofilter, and a protein skimmer. Shrimp were fed during the acclimatization period
(10% of total biomass) with commercial diets (41% protein, 9% fat). When the organisms reached a size of 0.47 ± 0.006 g, they were
randomly distributed in 12 circular tanks containing 500 L, with a final density of 30 organisms per tank (0.5 m2). Shrimp were fed
experimental diets four times a day (8:00, 11:00, 14:00, and 17:00 h) for seven weeks, starting with a feeding rate of 10%, increasing or
decreasing according to their demand. Water temperature (27.8 ± 0.3 ◦ C), salinity (33 ± 0.5 ppt), and dissolved oxygen (6.2 ± 0.4
mgL− 1) were monitored daily (YSI-55, YSI Inc, Yellow Springs, OH, USA). In addition, nitrogenous compounds such as ammonium
(0.05 ± 0.02 mgL− 1) and nitrite (0.3 ± 0.01 mgL− 1) were measured once a week (API test kits, Mars Fishcare Inc., Chalfont, PA, USA).

2.2. Diets Preparation and Experimental Design

Four diets were formulated to contain 46.5% CP and 8.5% CF using BBM to replace PBM in a completely fish-free formulation, using
microalgae oil and cholesterol to reach the optimum levels, as shown in Table 1. The microalgae oil contained 17% DHA, and it was
equally added to each diet (5%). The experimental diets were prepared in the IIO - UABC, following the internal protocols. First, the
dietary ingredients were pulverized at 0.5 mm (Inmimex M-300, Mexico) and sieved (Kemutek-Gardner K300, USA). Subsequently,

Table 1
Ingredients and proximate composition (g Kg− 1 on a dry matter basis, DM) of four isoproteic and isolipidic diets formulated to contain different
bovine by-product meal (BBM) levels fed white-leg shrimp Litopenaeus vannamei for 50 days.
Ingredients, g Kg− 1, DM Dietary bovine meal levels

Control L-BBM M-BBM H-BBM

Bovine by-product meala 0 135.9 271.6 406.9

Poultry by-product meala 300 200 100 0
Soybean 65% ADMb 200 200 200 200
Wheat flourc 98.9 61.0 23.3 0
Gelatind 80 80 80 80
Corn glutene 40 40 40 40
Beef tallowf 0 2.0 6.0 8.0
Maizena™ g (corn starch) 170 170 168 154
Metionineg 10 10 10 10
Rovimixh 30 30 30 30
Stay Ch 5.0 5.0 5.0 5.0
Phospholipidsi 10 10 10 10
Cholesterolj 5.0 5.0 5.0 5.0
DHA-Nature™ 17%k 50 50 50 50
Sodium benzoatel 1.0 1.0 1.0 1.0
BHTl 0.1 0.1 0.1 0.1
Proximate composition, (%, DM)
Crude Protein 46.21 46.02 47.71 46.71
Crude Lipid 8.5 8.6 9.0 9.1
Ash 8.8 12.0 13.9 16.7
NFE 36.5 33.4 29.4 27.5
Acid insoluble ash 8.0 8.4 7.4 7.3
Gross energy content (kJ) 20.5 20.0 19.9 19.4

L-BBM (low inclusion); M-BBM (medium inclusion); H-BBM (high inclusion).

NFE (g kg− 1) = 100% − (crude protein+crude lipid+moisture+ash);
a
Scoular de México S. de R.L. de C.V. Originally from the USA (National Renderers Association); bSoycomilk ADM; cMolinera del Valle S.A. de C.V.,
México; d Progel Mexicana SA de CV, Léon, Guanajuato, México; e Ingredión México S.A. de C.V., México; f Kindly donated by Grasas y Derivados de
Tijuana; g Maizena, Unilever Food Solutions, México; h DSM Nutritional Products México, S.A. de C.V., contains in g kg ρ-aminobenzoic acid 1.45;
biotin 0.02; myo-inositol 14.5; nicotinic acid 2.9; Capantothenate 1.0; pyridoxine-HCl 0.17; riboflavin 0.73; thiamine-HCl 0.22; menadione 0.17;
α-tocopherol 1.45; cyanocobalamine 0.0003; calciferol 0.03; L-ascorbyl-2- phosphate-Mg 0.25; folic acid 0.05; choline chloride 29.65; retinol 0.015;
NaCl 1.838; MgSO4・7H2O 6.85; NaH2PO4・2H2O 4.36; KH2PO4 11.99; Ca(H2PO4)2・2H2O 6.79; Fe-citrate 1.48; Ca-lactate 16.35; AlCl3・6H2O
0.009; ZnSO4・7H2O 0.17; CuCl2 0.0005; i Comercializador Costa bella S. de R.L. de C.V., Ensenada BC, México; j Mitsui & Co, México; k ADM; l
Sigma Aldrich

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E. López-Ortiz et al. Animal Feed Science and Technology 303 (2023) 115689

they were mixed with a vertical mixer-cutter (Robot Coupe R-60, USA) until a homogeneous mass was obtained. Next, micronutrients
were incorporated into the bulk blend. At last, the mixture of oils from each experimental diet was added and mixed. Finally, the water
was added separately with the cooked starch and gelatin until the desired texture was achieved. Then the diets were mixed (Robot-
Coupe, model R10, USA), granulated to 5 mm in a meat grinder (Tor-O-Rey, model M32–5, Mexico), and dried at 60 ◦ C in a forced air
oven for 24 h. Once the diets were dry, they were kept refrigerated (4 ◦ C) throughout the experiment. The four diets were formulated to
contain progressively different concentrations of BBM: Control [BBM (0%)], L-BBM [low bovine (13.6%)], M-BBM [medium bovine
(27.2%)], H-BBM [high bovine (40.7%)].
After a 50-day feeding trial, fish were counted and individually weighed to assess performance in terms of the following metrics:

Relative weight gained (RWG, %) = ((final body weight – initial body weight) x 100)/initial weight⋅

Specific growth rate (SGR, %/day) = 100 x ((ln Wf – ln Wi) /days of feeding)⋅

FCR (Feed Conversion Ratio) = total feed consumed / wet weight gained⋅

Hepatosomatic index (HSI, %) = (hepatopancreas weight/body weight) x 100⋅

Thermal growth coefficient (TGC) = [(final weight1/3 – initial weight1/3)/(T◦ C x days)]x1000

Survival (Sv, %) = (Nf/Ni)×100

Wf and Wi were the final and initial shrimp weights, respectively, while Nf and Ni correspond to the final and initial number of
shrimps, respectively.
From day seven of the experimental procedure, feces were individually collected daily per experimental unit (three per treatment)
and frozen to determine apparent digestibility. In addition, feces were collected by siphoning the bottom of the tank after each feeding
(9:00, 12:00, and 15:00 h). Feces samples were filtered and sieved, rinsed with distilled water, and placed in Eppendorf® tubes to store
at − 18 ◦ C per experimental unit.
At the end of the trial, the organisms were handled with care to minimize stress. Three organisms per tank of each treatment were
sacrificed (euthanized by hypothermia) and subsequently stored at − 80 ◦ C before analysis, according to the protocols of the ethics
committee from the UABC. For hemolymph samples, three shrimps per tank were cooled down (22◦ ) with gel ice to extract the he­
molymph samples (approximately 150–200 µL per shrimp). Then the hemolymph was withdrawn from the ventral sinus with sterile
syringes of 1 mL. The syringes were previously washed with an anticoagulant solution (900 mM sodium chloride, 20 mM potassium
chloride, 20 mM HEPES, and 20 mM EDTA, at a pH of 7.3), and a drop was left to prevent clotting during hemolymph extraction.
Hemolymph was centrifuged at 7000 rpm for 5 min, and then, the supernatant was removed and immediately frozen at − 80 ◦ C until
further analysis. Shrimps were immediately sacrificed.

2.3. Chemical analysis

All the experimental diets were analyzed for proximate composition in triplicate samples according to the AOAC (2015) and shown
in Table 1. In summary, the diets´dry weight was determined by drying the samples at 60 ◦ C for 24 h. Then samples were ashed in a
muffle furnace at 550 ◦ C for 6 h to determine the ash. Next, crude protein was analyzed by the micro-Kjeldahl method (UDK 129, Velp,
Italy), and protein content was calculated by nitrogen conversion (%N x 6.25). Finally, the Soxhlet method analyzed the lipid content
according to the AOAC using petroleum ether as the solvent carrier.
Apparent digestibility was determined using the acid-insoluble ash as an internal marker. The content (%) of acid-insoluble ash of
the experimental diets and the feces collected during the bioassay was determined. The methodology described by Tejada de
Hernández (1992) with some modifications (Montaño-Vargas et al., 2002) was used, representing the siliceous compounds in the
nutrients, principally those from vegetable origin unable to be absorbed through the intestinal tract. In summary, the stored feces were
weighed and placed in porcelain crucibles in the furnace oven at 550 ◦ C for 6 h. After the calcination process, they were weight and
placed in 50 mL beakers; 25 mL of HCl (2 N) were added, covered with a watch glass, and placed on an electric iron at 100 ◦ C until they
reached their boiling point. Then, samples were allowed to stay there for 5 min, and the content was cooled for 5 min. Then, the content
of the beakers was filtered through a 47 mm diameter GF-F glass fiber filter (Whatman®) previously weight to a constant dry weight. A
flask connected to a vacuum pump was used to filter the samples with hot distilled water to rinse all possible ash remainder in the glass
beakers. Then, the filter was placed in aluminum foil and ashed again in a muffle at 550 ◦ C for 6 h. Once the samples were calcinated,
they were weighed, and the ashes were calculated according to Tejada de Hernández (1992):
(AW) − (CW)
%IA = X100
DMW

Where IA: Acid-insoluble ash; AW: Ash weight (g); CW: Crucible weight (g), and DMW: Dry matter weight.
Fatty acids were analyzed from the experimental diets and muscle and hepatopancreas tissues according to the methodology
adapted from Folch et al. (1957) using dichloromethane instead of chloroform. First, the crude oil obtained from the diets was
separated to further methylate following the method described by Castro-Gómez et al. (2014). Then, the muscle and hepatopancreas
were directly methylated using the transmethylation methodology described by Parrish et al. (2014), using dichloromethane and
methanol as solvents. Next, fatty acid methyl esters (FAME) were analyzed by gas chromatography equipped with a flame ionization

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E. López-Ortiz et al. Animal Feed Science and Technology 303 (2023) 115689

detector (Agilent GC 6880, Agilent Technologies, Santa Clara, CA, USA) using nitrogen as carrier gas. The GC column
(60 m × 0.25 mm with 0.25 µm film thickness; Agilent 122–2362 dB-23) conditions were: initial oven temperature 50 ◦ C for 1 min,
50–140 ◦ C at 30 ◦ C min− 1, maintained at 140 ◦ C for 5 min, from 140 ◦ C to 240 ◦ C for 4 ◦ C min− 1, and finally at 240 ◦ C for 20 min. The
injector and detector temperatures were maintained at 230 ◦ C and 260 ◦ C, respectively. FAMEs were identified and quantified by
comparing retention times between a standard mixture (37-component FAMEs, PUFA 1, and PUFA 3, Supelco/Sigma-Aldrich, St.
Louis, MO, USA) using a C:19 as an internal marker to calculate the concentration.
For the chemistry from the extracted hemolymph, total protein was analyzed using Biuret reagent (Sigma-Aldrich) and measured at
540 nm. The standard curve was obtained using bovine serum albumin (BSA), and the protein was reported as protein equivalent to
BSA. Glucose was quantitatively determined with a kit (Valtek Diagnostics, Santiago de Chile) in which glucose is oxidized to gluconic
acid and hydrogen peroxide by glucose oxidase. The hydrogen peroxide then reacts with p-hydroxybenzoic acid and 4-amino-antipy­
rine in peroxidase. The colored product is measured spectrophotometrically at 505 nm. Further, the triglycerides were estimated after
hydrolysis with a specific lipase that produces fatty acids and glycerol.
In summary, the reaction consisted of the enzyme glycerol kinase phosphorylated glycerol. Then, the glycerol-1-phosphate is
oxidized by the glycerol-phosphate oxidase to generate hydrogen peroxide, which is then determined by the 4-amino-antipyrine and 3,
5-dichloro-2-hydroxy-acid benzenesulfonic acid in the presence of peroxidase, a reaction measured at 520 nm (Valtek diagnostics,
Santiago de Chile). Finally, cholesterol was determined by an enzymatic method in which cholesterol esterase hydrolyzes cholesterol,
and free cholesterol is then oxidized by cholesterol oxidase, producing hydrogen peroxide that is quantified by the chromogenic system
containing p-hydroxybenzoic acid and 4-amino antipyrine in the presence of peroxidase and is measured at 505 nm (Valtek di­
agnostics, Santiago de Chile). Absorbance readings were performed in a microplate reader (Multiskan GO, Thermo Scientific).

2.4. Statistical analysis

The homoscedasticity and normality of the data were verified using the Leven test and the Kolmogorov-Smirnov test, respectively.
The zootechnical performance variables were analyzed using a one-way analysis of variance (ANOVA) and a Tukey test when a sig­
nificant difference was detected in the treatments (IBM SPSS Statistics V23.0.0 Copyright IBM Corporation 1989, 2011, USA). The
differences were considered significant at 95%. Then a polynomial (quadratic) regression was performed among treatments. The
significance value of P < 0.05 was applied for all statistical tests (IBM SPSS Statistics V23.0.0 Copyright IBM Corporation 1989, 2011,
USA).

3. Results

The proximate composition of the experimental diets is shown in Table 1. The crude protein and fat contents were 46–47.7 and
8.5–9.1%, respectively. The proximate composition of treatments M-BBM and H-BBM resulted in slightly higher values than in the
Control and L-BBM dietary treatments. Ash content increased along the BBM was included, from 8.8 in the Control group up to 16.7 for

Table 2
Fatty acid composition (% of total fatty acids) of formulated diets containing different inclusion levels of bovine by-product meal in replacement of
poultry by-product meal.
Dietary treatments

Fatty acid Control L-BBM M-BBM H-BBM

12:0 0.41 0.39 0.41 0.43

14:0 11.20 11.31 11.71 12.95
16:0 19.87 21.73 19.89 20.50
18:0 6.65 7.01 8.51 10.16
20:0 0.20 0.22 0.20 0.20
∑
SFA 38.32 40.66 40.71 44.23
14:1 0.33 0.23 0.32 0.43
16:1 3.14 2.97 2.26 1.87
18:1n9 27.07 26.20 26.88 26.83
20:1n9 0.29 0.28 0.25 0.22
∑
MUFA 30.84 29.67 29.71 29.34
18:2n6 15.64 14.90 11.00 8.96
20:4n6 0.41 0.45 0.62 0.48
∑
PUFAn6 16.05 15.35 11.61 9.43
18:3n3 1.09 1.05 0.91 0.84
20:3n3 0.48 0.45 0.29 0.19
20:5n3 2.29 2.10 2.73 2.55
22:6n3 10.33 10.15 12.94 12.16
∑
PUFAn3 14.19 13.75 16.88 15.74
∑
PUFA`s 30.24 29.10 28.49 25.17
Others 0.61 0.57 1.09 1.25
∑ ∑ ∑ ∑ ∑
SFA, MUFA, PUFA n6, PUFA n3, PUFA are the sum of saturated, monounsaturated, polyunsaturated, polyunsaturated n3 and poly­
unsaturated n6 respectively. L-BBM (low inclusion); M-BBM (medium inclusion); H-BBM (high inclusion).

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the H-BBM. Whereas the fatty acid content of diets shown in Table 2 resulted in apparent differences in certain fatty acids, decreasing
the linoleic acid (18:2n6) along the BBM was incorporated. While the linolenic acid (18:3n3) decreased along the BBM was
incorporated.
Regarding the overall performance, survival resulted in significant differences. A higher survival was observed among the Control
diet (100%), a survival that decreased evenly up to 88.9 ± 4.6 for the H-BBM. Significant differences were observed in all parameters
concerning treatments (P < 0.05). After seven weeks of the experimental procedure, the overall performance resulted in differences
(Table 3). The H-BBM treatment presented the highest significant SGR (3.43 ± 0.09%/day) and TGC (0.44 ± 0.016). However, the
highest level of BBM resulted in the lowest FCR (1.51 ± 0.12) (P < 0.05). The relative weight gain increase (%) was significantly
higher (457.05 ± 27.0) for treatment H-BBM compared to the Control (258.6 ± 6.6). Apart from the ANOVA test, a polynomial
regression was carried out to observe the correlation between overall performance and dietary treatments (Fig. 1), showing a low
correlation between the HI and the treatments (R2 = 0.245), a medium correlation between the treatments and the Final Weight (R2 =
0.478), SGR (R2 = 0.419) and the FCR (R2 = 0.455).
The Apparent Digestibility significantly (P < 0.05) improved as long as the BBM increased in the dietary treatments (Table 4).
Further, a high correlation was observed between Apparent Digestibility (R2 = 0.986) and the applied treatments (Fig. 2). The highest
percentages of Apparent Digestibility were observed in the H-BBM with significant differences (P < 0.05) in all treatments versus the
Control (Table 3). Regarding hemolymph chemistry, including bovine by-product meal resulted in significant differences (P < 0.05)
among treatments. Significant differences (P < 0.05) were observed in all hematological and blood chemistry parameters. The highest
protein and glucose concentrations were recorded in L-BBM and the lowest in M-BBM. However, the highest cholesterol and tri­
glycerides were observed in H-BBM and the lowest in the Control treatment (Table 5).
The fatty acid content in hepatopancreas tissue resulted in evident differences (Table 6) where saturated fatty acids such as 14:0
and 16:0 were accumulated, and the oleic acid (18:1n9). However, the linoleic acid (18:2n6) was significantly reduced, whereas the
total PUFAn6 was lower in the H-BBM than in the Control group.

4. Discussion

The present work has demonstrated that BBM can replace PBM for shrimp aquafeeds, improving overall performance. A consid­
erable number of studies have shown the effectiveness of PBM in diets for aquaculture and in particular for shrimp, and can even
completely replace FM (Amaya et al., 2007; Shapawi et al., 2007; Markey et al., 2010; Parés et al., 2014; Badillo et al., 2016; Hossain
et al., 2017; Mariquez-Patiño et al., 2021). However, even if PBM is a cheaper alternative to FM, the feed industry’s short supply of raw
materials is scrutinized. Therefore, the results shown here are significant, considering having other raw materials to reduce costs
without struggling with the performance quality of shrimp production by using BBM in their feed. Further, BBM, equal to PBM, comes
from rendering processes of terrestrial animal production, representing a contribution to the goal of sustainability of aquafeeds. Being
recycled products, they contribute to a circular economy, potentially reducing the carbon footprint of feeds compared to other protein
sources such as FM and those of vegetable origin.
Here, with increasing levels of BBM inclusion, it was observed that performance improved significantly (P < 0.05). However, even
if the growth rate failed to achieve the expected desirable growth, we should notice that shrimp were grown at 27.8 ◦ C. Therefore, the
TGC was also estimated to consider the temperature. Besides, the shrimps used in the present work come from a Mexican genetic line
project chosen to deal with pathogens along the Mexican coast. Usually, the genetic lines used for several hatchery producers come
from the USA (Florida or Texas), where growth is chosen with apparent consequences on survival due to illness (Perez-Enriquez et al.,
2018). Therefore, despite the inconveniences, the highest inclusion level of BBM performs better than those from the Control group.
Nevertheless, the present growth rates (SGR) obtained here are higher than those reported by Chien et al. (2023) and slightly lower
than those reported by Huang et al. (2023), both using L. vannamei.
Forster et al. (2003) concluded that meat and bone meal had lower digestibility in shrimp with fishmeal. However, product quality
could be responsible for the differences obtained. The BBM used here comes from the North American Renderers Association and is

Table 3
Overall performance of white leg juveniles of Litopenaeus vannamei, fed with four diets containing four different bovine by-product meal (BBM); 0%,
13.6%, 27.2%, and 40.7% in replacement of poultry-byproduct meal (PBM) for 50 days.
Dietary treatments

Control L-BBM M-BBM H-BBM P value

IBW (g) 0.47 ± 0.006 0.47 ± 0.002 0.47 ± 0.004 0.478 ± 0.001 0.237
FBW (g) 1.70 ± 0.04c 2.27 ± 0.059b 1.73 ± 0.040c 2.66 ± 0.132a 0.001
RWG (%) 258.61 ± 6.62c 383.84 ± 11.44b 267.26 ± 7.36c 457.05 ± 26.97a 0.001
SGR (%) 2.55 ± 0.03c 3.15 ± 0.04b 2.60 ± 0.04c 3.43 ± 0.09a 0.001
FCR 2.76 ± 0.12a 1.93 ± 0.07b 2.65 ± 0.09a 1.51 ± 0.12c 0.001
HI 6.32 ± 0.41 5.73 ± 0.22 8.06 ± 2.77 6.64 ± 1.25 0.748
TGC 0.303 ± 0.005c 0.39 ± 0.007b 0.30 ± 0.006c 0.44 ± 0.016a 0.001
Sv (%) 100 ± 0a 97.78 ± 3.50ab 96.67 ± 4.43ab 88.89 ± 4.6b 0.043

L-BBM (low inclusion); M-BBM (medium inclusion); H-BBM (high inclusion). Mean ± SD values in each row with different superscripts were
significantly different (P < 0.05, Tukey’s test). IBW= Initial body weight, FBW= final body weight, RWG= relative weight gain, SGR= specific
growth rate, FCR= conversion ratio, HI= hepatosomatic index, TGR= thermal growth coefficient and Sv= survival.

6
E. López-Ortiz et al. Animal Feed Science and Technology 303 (2023) 115689

Fig. 1. Polynomial (quadratic) regression analysis between levels of beef meal in the diet and weight gain, specific growth rate (SGR), feed con­
version factor (FCR), hepatosomatic index (HI). The markers indicate the values shown in Table 3. The lines indicate the best-fit polynomial
regression equations and the R2 values indicate the power of the model.

Table 4
Apparent digestibility coefficient calculated using acid insoluble ash as
internal marked in diets containing four different bovine by-product
meal (BBM); 0%, 13.6%, 27.2%, and 40.7% in replacement of
poultry-byproduct meal (PBM) for 50 days, fed to Litopenaeus vannamei
(n = 3).
Dietary treatment Apparent digestibility (%)

Control 85.76 ± 0.55b

L-BBM 90.20 ± 0.55a
M-BBM 91.31 ± 0.88a
H-BBM 91.51 ± 0.34a
P value 0.001

L-BBM (low inclusion); M-BBM (medium inclusion); H-BBM (high in­

clusion). Mean ± SD values in each row with different superscripts
were significantly different (P < 0.05, Tukey’s test).

produced under a certified quality process. In this study, growth, SGR, and FCR also resulted in significant differences (P < 0.05) from
the Control group. Apart from the protein content, the fatty acids could play an essential role in protein sparing effect.
Therefore, BBM can successfully replace PBM without adverse effects observed in the cultivated organism’s performance or health
indicators (Hernández et al., 2004; Cruz-Suárez et al., 2007; Guo et al., 2007). Apparent digestibility from diets resulted in better
figures at higher BBM replacement. One could say this is a surprising result to get a better apparent digestibility in all BBM treatments
than PBM (from an AD of 86 for the Control group, up to 91% in any of the BBM diets) considering the high ash content. However, BBM
comes from meat and bone by-products, containing a high amount of collagen converted into gelatin, which is characteristic of high

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E. López-Ortiz et al. Animal Feed Science and Technology 303 (2023) 115689

Fig. 2. Polynomial (quadratic) regression analysis between levels of beef meal in the diet and apparent digestibility (AD). The markers indicate the
values shown in Table 4. The lines indicate the best-fit polynomial regression equations and the R2 values indicate the power of the model.

Table 5
Hemolymph biochemistry parameters of white shrimp Litopenaeus vanamei fed on poultry meal balance with different percentages of bovine by-
product meal inclusion (Mean ± SD). Three samples per experimental unit were analyzed.
Treatment Parameters

Protein Glucose Cholesterol Triglycerides

(gdL− 1) (mgdL− 1) (mgdL− 1) (mgdL− 1)

Control 7.03 ± 0.34a 9.78 ± 0.33b 7.71 ± 0.72b 8.77 ± 0.23b

L-BBM 7.16 ± 0.06a 17.74 ± 2.67a 9.39 ± 2.04b 11.82 ± 0.52b
M-BBM 4.18 ± 0.75b 10.57 ± 1.88b 19.91 ± 8.38b 11.17 ± 2.04b
H-BBM 5.98 ± 0.59ab 14.55 ± 1.10ab 40.80 ± 4.43a 29.52 ± 1.88a
P value 0.001 0.009 0.001 0.001

L-BBM (low inclusion); M-BBM (medium inclusion); H-BBM (high inclusion). Mean ± SD values in each row with different superscripts were
significantly different (P < 0.05, Tukey’s test).

digestibility and nutritious properties (Sweers et al., 2022). According to GME (2020), skin and bones are presumed to contain up to
29.4% gelatin.
Further, Siccardi et al. (2006) reported that BBM resulted in as high as 99.7 apparent energy digestibility. Therefore, BBM benefits
the shrimp diets, primarily due to its high digestibility and probably for the amino acid profile from the bovine meat. After applying the
polynomial (quadratic) regression between the inclusion levels of bovine meal in the diets, a high positive correlation was observed (R2
= 0.986), reflected in a better SGR and FCR. Therefore, a high ash content could negatively affect the diet water stability. However,
considering the H-BBM treatment group presented a higher feed conversion ratio, the diet stability cannot be speculated.
Moreover, the authors of the present work failed to find references on the adverse effect of high ash content on digestibility and
nutrient absorption. Even if ash levels in diets could interfere with the physical properties of pellets, having a proper amount of starch
to gelatinize the pellets should not be a problem. Further, the high ash content in the present work fails to show detrimental effects on
shrimp.
Therefore, it is inferred that the favorable response from feeds with high percentages of BBM inclusion in shrimp is due to the high
quality of nutrients, contrary to earlier reports on shrimp and some fish species fed with BBM (Millamena, 2002; Tan et al., 2005;
Hossain et al., 2017). Those reports concluded this could be for the fatty acid content (Table 5). Here we used DHA and EPA from
microalgae to substitute those contained only in the FM or fish oil, not supplied, besides cholesterol. It is widely known that LC-PUFAs
are essential for shrimp, and when they are not supplied, growth is compromised (Araújo et al., 2019). According to several authors,
combining different protein sources should overcome the requirements that primary sources do not initially contain (Borgeson et al.,
2006; Kader et al., 2010). Docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids are essential in shrimp. These long-chain
polyunsaturated acids (LC-PUFA) are generally available in marine by-products (Turchini et al., 2009; Araujo et al., 2019).
The microalgae oil rich in DHA and EPA has been proven affordable when fish oil is not supplied in the diet. Araújo et al. (2019)
concluded that combining beef tallow with microalgae oil (rich in DHA and EPA) resulted in better performance than using fish oil in
shrimp diets, and microalgae oil resulted in a cheaper combination. Shrimp has not the capacity to synthesize LC-PUFAs. Chen et al.
(2014) mention that EPA and DHA should be included when shrimp are grown at salinities higher than 20 ppm. The diets used in the
present study were formulated with DHA (5 g Kg− 1, DM), and it was observed that the percentages of FA in the hepatopancreas are

8
E. López-Ortiz et al. Animal Feed Science and Technology 303 (2023) 115689

Table 6
Fatty acid (% of total fatty acids) composition of juveniles of Litopenaeus vannamei hepatopancreas fed with diets fed the different Bovine by-product
meal in replacement of poultry by-product meal.
Fatty acid Dietary treatments

Control L-BBM M-BBM H-BBM P value

14:0 3.28 ± 0.39b 3.27 ± 0.05b 4.53 ± 0.05a 4.40 ± 0.15a 0.04
16:0 18.54 ± 0.28c 23.07 ± 0.51a 19.72 ± 0.23bc 20.86 ± 0.54b 0.001
18:0 8.55 ± 0.67ab 6.85 ± 0.13c 8.85 ± 0.32a 7.65 ± 0.22ab 0.027
20:0 0.37 ± 0.06 0.24 ± 0 0.53 ± 0.11 0.29 ± 0.05 0.083
∑
SFA 30.76 33.44 33.65 33.21 -
14:1 0.06 ± 0.01b 0.10 ± 0.01ab 0.22 ± 0.06a 0.19 ± 0.01ab 0.033
16:1 1.64 ± 0.12 2.05 ± 0.13 1.77 ± 0.03 2.09 ± 0.07 0.035
18:1n9 26.53 ± 1.11b 33.72 ± 0.49a 28.90 ± 0.16b 33.54 ± 0.33a 0.001
20:1n9 1.05 ± 0a 0.77 ± 0.01b 0.80 ± 0.06b 0.79 ± 0.05b 0.006
∑
MUFA 29.31 36.66 31.7 36.62 -
18:2n6 13.36 ± 0.22a 11.11 ± 0.20b 10.57 ± 0.06b 8.33 ± 0.06c 0.001
20:4n6 5.30 ± 0.40a 1.91 ± 0.17b 4.29 ± 0.30a 1.76 ± 0.27b 0.001
∑
PUFAn6 18.67 13.01 14.87 10.09 -
18:3n3 0.65 ± 0.07 0.57 ± 0.07 0.55 ± 0.14 0.60 ± 0.02 0.887
20:3n3 3.45 ± 0.42a 1.22 ± 0.09bc 2.25 ± 0.08b 0.99 ± 0.18c 0.001
20:5n3 1.94 ± 0.10ab 1.66 ± 0.05b 2.14 ± 0.11a 2.28 ± 0.71a 0.005
22:6n3 11.44 ± 0.44b 10.04 ± 0.10c 11.44 ± 0.25b 12.67 ± 0.08a 0.001
∑
PUFAn3 17.49 13.5 16.39 16.56 -
∑
PUFAs 36.16 26.51 31.26 26.65 -
Others 3.75 ± 0.01 3.34 ± 0.09 3.37 ± 0.20 3.49 ± 0.17 0.232
∑ ∑ ∑ ∑ ∑
Values represent means ± standard error. SFA, MUFA, PUFA n6, PUFA n3, PUFA are the sum of saturated, monounsaturated, poly­
unsaturated, polyunsaturated n3 and polyunsaturated n6 respectively. abc Different letters mean statistical differences among experimental diets,
according to Tukey´s test (P < 0.05)

statistically different (P < 0.05) both in ARA (C20:4n-6), EPA (C20:5n-3) and DHA (C22:6n-3) among treatments. In addition, the
accumulation of LC-PUFAs increased, directly correlating to the inclusion of DHA, which indicates that selective accumulation plays an
essential role in the accumulation of PUFAs (Glencross and Rutherford, 2010; Tocher, 2015).
Regarding the rest of the fatty acid content, the significant reduction in n6 fatty acids, particularly linoleic acid, reflects its lower
content in the BBM. Linoleic acid is associated with an inflammatory process when supplied in high quantities. Therefore, the BBM can
also contribute to the shrimp´s overall health. However, even if the total cholesterol and triglycerides content in hemolymph were
higher at a higher amount of BBM in H-BBM dietary treatment, all levels are within the expected range for healthy and stress-free
shrimp (Mercier et al., 2006). Therefore, it is reasonable to observe an increase in hemolymph values in the H-BBM diet, being the
treatment with the best performance in weight gain and better digestibility, among others. It is precisely the use of these parameters
that are recognized to assess the state of health of an organism in the face of nutritional and environmental changes (Lu et al., 2020).
However, those authors reported that under stress conditions, the cholesterol level in hemolymph could be reduced from 21.1 to
17.1 mg/dL, and there is still not a piece of evidence that 40.8 mg/dL as here registered in shrimp fed H-BBM could result in phys­
iological problems.
Further, shrimps cannot synthesize cholesterol, so its level in the hemolymph could be controlled. In conclusion, the results of the
present study provide clear evidence that L. vannamei can be fed diets based on BBM in substitution of PBM without affecting the
growth performance and health in FM-free diets. In addition, including BBM improved the percentages of apparent digestibility of the
organisms, outperforming the productive yields of PBM. Our results on the percentages of FA accumulation in the hepatopancreas of
the organisms suggest that L. vannamei can efficiently assimilate PUFAs and MUFAs in higher percentages concerning the PBM from the
Control diet. However, the hemolymph chemistry shows that the H-BBM diet resulted in higher cholesterol accumulation, an essential
nutrient of shrimp. Therefore, it will be interesting to know more about fat metabolism using BBM to adjust diet cholesterol or fat
content, which could reduce the feed cost.
Likewise, it is observed that the high accumulation of glucose, cholesterol, and triglycerides did not directly influence the pro­
ductive yields of the organisms studied. The increase in glucose is reasonable from 9.8 to 14.6 mgdL− 1 (Control vs. H-BBM) in terms of
growth increase. It is precisely the use of these parameters that are recognized to assess the state of health of an organism in the face of
nutritional and environmental changes. However, these changes between treatments do not indicate a deterioration in the state of
health of the organisms among the range obtained.
Nevertheless, future research suggests complementing fat metabolism using gene expression to estimate the cause of the high
amount of cholesterol and triglycerides. Therefore, it is concluded that BBM is a protein source with a high possibility of being included
in shrimp diets replacing the PBM, resulting in better benefits in its performance. However, further research on its effect on lipid
metabolism is recommended.

Declaration of Competing Interest

The authors declare no conflict of interest.

9
E. López-Ortiz et al. Animal Feed Science and Technology 303 (2023) 115689

Acknowledgments

This project was also financed by UABC (22a/403/1/C/10/22). In addition, shrimps were kindly donated by FITMAR, SA de CV,
Mazatlán Sinaloa, México.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.anifeedsci.2023.115689.

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