Impact of partial substitution of fish meal by methylated soy protein isolates on the nutritional, immunological, and health aspects of Nile tilapia, Oreochromis niloticus fingerlings

Journal Pre-proof Impact of partial substitution of fish meal by methylated soy protein isolates on the nutritional, immunological, and health aspects of Nile tilapia, Oreochromis niloticus fingerlings

Shimaa A. Amer, Shaimaa A.A. Ahmed, Rowida E. Ibrahim, Naif A. Al-Gabri, Ali Osman, Mahmoud Sitohy PII:

S0044-8486(19)32767-X

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734871

Reference:

AQUA 734871

To appear in:

aquaculture

Received date:

17 October 2019

Revised date:

15 December 2019

Accepted date:

15 December 2019

Please cite this article as: S.A. Amer, S.A.A. Ahmed, R.E. Ibrahim, et al., Impact of partial substitution of fish meal by methylated soy protein isolates on the nutritional, immunological, and health aspects of Nile tilapia, Oreochromis niloticus fingerlings, aquaculture (2019), https://doi.org/10.1016/j.aquaculture.2019.734871

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© 2019 Published by Elsevier.

Journal Pre-proof Impact of partial substitution of fish meal by methylated soy protein isolates on the nutritional, immunological, and health aspectsofNile tilapia, Oreochromisniloticusfingerlings Shimaa A. Amera*,Shaimaa A.A. Ahmedb , Rowida E. Ibrahimb, Naif A. Al-Gabric, Ali Osmand, and Mahmoud Sitohyd a

Department of Nutrition & Clinical Nutrition, Faculty of Veterinary Medicine, Zagazig University, 44511, Zagazig,

Egypt. b

Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Zagazig University, 44511, Zagazig,

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Egypt. Pathology department, Faculty of Veterinary Medicine, Thamar University, Dahamar, Yemen. Biochemistry Department, Faculty of Agriculture, Zagazig University, 44511, Zagazig, Egypt.

*

Corresponding authors:

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Shimaa A. Amer

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E-mail: [email protected], [email protected]

Zagazig, Egypt.

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Address: Department of Nutrition & Clinical Nutrition, Faculty of Veterinary Medicine, Zagazig University, 44511,

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ORCID ID: 0000–0002–8349–0425

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Journal Pre-proof ABSTRACT The impact of partial replacement of fish meal with methylated soy protein isolates (MSPI) on fish performance was assessed in 10 weeks feeding trial using Nile tilapia,Oreochromisniloticusfingerlings (n = 225) with average initial weight 18.51± 0.05 gm/fish. The effects on the growth, histopathological condition of certain organs, economic efficiency, immune status, and disease resistance to Aeromonashydrophila were evaluated. The fingerlings were randomly segregated into five experimental groups with varying percentages of fish meal with substituted MSPI: 0%, 22%, 44%, 66%, and 88% (MSPI0, MSPI22, MSPI44, MSPI66, and MSPI88). The results of final body weight, body weight gain, daily body weight gain, protein efficiency ratio,and SGR were uniquely

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increased by the treatment MSP22, while increasing the level of MSPI more than 22% resulted in linearly

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proportional decreases in these parameters over the respective control. The broken-line regression analysis using feed conversion ratio and body weight gain data revealed that the maximum dietary replacement of fishmeal with

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MSPI was 66%. Alterations in the histomorphological structures of the liver and intestine were observed with an

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increased level of replacement.The total weight of surviving fish, phagocytic index, IgM level, and lysozyme activity post bacterial challenge were significantly increased by MSPI substitution in fish meal. The survival

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percentage after the bacterial challenge was increased by MSPI inclusion, the highest survival percentage was recorded in the MSPI66 group (95.55%) compared to MSPI0 (66.66%).MSPI22 increased the total return, net profit,

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and performance index %. It could be concluded that the maximum dietary replacement of fishmeal with MSPI Oreochromis niloticus fingerlings was 66%. The growth performance was decreased by increasing the substitution

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percentage of fish meal by MSPI. MSPI was shown to be a good immune-modulating substance and improved gut health. MSPI substitution at 88% is not recommended due to the bad effect on the hepatic and intestinal tissues.

Keywords: Oreochromisniloticus, Methylated soy protein isolates, Growth performance, histopathology, Disease resistance, Aeromonashydrophila

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1.

Introduction Fish meal, with a balanced amino acid profile, is the primary source for high-quality protein in fish feeds

(Bendiksen, Johnsen, Olsen, Jobling, 2011). However, the increasing demand and cost of fish meal, has necessitated the search for a cheaper substitute or at least partial supplement for fish meal (Tacon, Metian, 2008). Compared to other plant-based proteins, soy proteins are an economical alternative, containing high-quality protein content and a suitable amino acid profile, little fiber and carbohydrate content (NRC, 2011), and is a contender for total or partial replacement of fish meal. Methionine- and lysine-supplemented diets in Nile tilapia could completely replace a fish

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meal (El-Saidy, Gaber, 1997; El‐ Saidy, Gaber, 2002). Although substitution with soybean meal could reduce

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production costs for fisheries (Haard, Dimes, Arndt, Dong, 1996), factors like the presence of protease inhibitors, phytic acid, saponin, allergens, low methionine and lysine concentrations, phytoestrogens, etc., in soy may hinder

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growth and feed utilization in fishes (Francis, Makkar, Becker, 2001; Gatlin III, Barrows, Brown, Dabrowski,

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Gaylord, Hardy, Herman, Hu, Krogdahl, Nelson, 2007). The adverse effects of soy meal substitution on growth and nutrient assimilation in several species have been shown in previous studies (Lim, Kim, Ko, Song, Oh, Kim, Kim,

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Lee, 2011; Silva-Carrillo, Hernández, Hardy, González-Rodríguez, Castillo-Vargasmachuca, 2012; Ye, Liu, Wang, Wang, 2011; Zhang, Ji, Wu, Han, Qin, Wang, 2016; Zhou, Song, Shao, Peng, Xiao, Hua, Owari, Zhang, Ng, 2011).

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Another line of studies has also shown the effective substitution of fish meal with up to 60% soy protein without significant adverse effects on growth with supplemental amino acids (Hernández, Martínez, Jover, García, 2007;

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Kader, Bulbul, Koshio, Ishikawa, Yokoyama, Nguyen, Komilus, 2012; Silva-Carrillo, Hernández, Hardy, GonzálezRodríguez, Castillo-Vargasmachuca, 2012; Zhang, Ji, Wu, Han, Qin, Wang, 2016). It can be elucidated that the supplementation of a few essential amino acids that are deficient in soy meal should be considered when used as a replacement for fish meal, along with other processing techniques to reduce or remove anti-nutritional factors to avoid nutritional imbalance (Francis, Makkar, Becker, 2001; Murai, 1992; Rumsey, Hughes, Winfree, 1993). Soy product processing can reduce the harmful effects of anti-nutritive factors on growth and improve nutrient uptake (de la Barca, Ruiz‐ Salazar, Jara‐ Marini, 2000). The isolated soy protein, obtained by processing of defatted soybean meal, is devoid of several anti-nutritive factors, heat resistant oligosaccharides, and antigens (Bureau, Harris, Cho, 1998; Cromwell, 2000). Methylation of native soy protein produces methylated soy protein (MSPI), where free negatively charged carboxyl groups are neutralized and the net positive charge on the protein is increased; this improves its antimicrobial activity (AbdelShafi, Osman, Enan, El-Nemer, Sitohy, 2016; Chobert, Sitohy, Billaudel, Dalgalarrondo, Haertlé, 2007; Sitohy,

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Journal Pre-proof Osman, 2010). This enhanced positive charge on the modified protein alongside with the increased hydrophobicity caused by grafting the methyl groups on the carboxylate groups may be the direct reasons of this emergent antimicrobial activity(Mahgoub, Osman, Sitohy, 2011; Omar, Amer, Mohamed, Osman, Sitohy, 2018; Osman, Mahgoub, Sitohy, 2014; Sitohy, Osman, 2018; Sitohy, Mahgoub, Osman, 2011; Sitohy, Mahgoub, Osman, ElMasry, Al-Gaby, 2013) which is expected to play a crucial role in the fish gut protecting it from different microbial infection. This is particularly true when the prevalent physiological pH-range inside fish stomach is usually 3.5-4.5, where MSPI is totally soluble and the ester groups are also stable under the acidic conditions.MSPI has previously been found to be nutritionally safe in toxicity studies (acute and sub-acute) in male Wistar Albino rats (Sitohy,

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Osman, Gharib, Chobert, Haertlé, 2013).

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Significant economic losses in aquaculture are caused by the bacterium Aeromonashydrophila(Romero et al., 2012), capable of infecting several fish species (Janda, Abbott, 2010). Aeromonas infections are usually tackled by

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using antibiotics, which in turn triggers the emergence of drug-resistant bacteria and causes hazards to human

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health. The current study aims to assess the impact of partial substitution of MSPI in the fish diet on the general health and nutritional aspects like growth, body composition, fish health, histopathological status, and immune

Material and methods

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response before or after A. hydrophila challenge in Oreochromisniloticusfingerlings.

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2.1. Fish and experimental conditions

This study was conducted at the Department of Fish Disease and Management, Faculty of Veterinary Medicine, Zagazig University, Egypt. All animal procedures were conducted following Egyptian laws of animal experiments and with approval from Egypt's Veterinary Organization, Ethical Committee for Animal Experiments (Approval no: ZU-IACUC/2/F/156/2019) and maximum efforts were made to minimize suffering. Healthy Oreochromis niloticus fingerlings (n = 225), average initial weight 18.51± 0.05gm/fish, were purchased from the Abbassa Fish Hatchery, Sharkia Province, Egypt. Oreochromis niloticus fingerlings were accustomed to the experimental diet in a lead-in period of 14 days to adapt to the laboratory conditions during which they were fed the control diet followed by a treatment period of 10 weeks with twice-daily feeding till satiety by hand at 9 am and 4 pm (Obirikorang, Amisah, Fialor, Skov, 2015). The fish health status was checked before the experiment according to CCoA (2005). Fish were stocked in fifteen monitored static water glass aquaria (80 cm × 40 cm × 30 cm) with a daily water exchange of about 25%. Water parameters were kept within recommended ranges during the observation

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Journal Pre-proof period (pH = 7.2 ± 0.5; ammonia = 0.02 ± 0.001 mg/L; nitrite = 0.017 ± 0.001 mg/L; water temperature = 24 ±2°C; photoperiod 12:12 light : dark) according to APHA (1998).Fish were observed daily for signs of disease or mortality and whole water changes were given to the aquaria twice weekly. At the end of the trial, all experimental groups were intraperitoneally inoculatedwith pathogenic A. hydrophila at a dose of 0.1 mL cell suspension containing 1.5×107 cells/mL by using McFarland standard tubes. The isolate was previously isolated from moribund fish at the Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Zagazig University and confirmed to be pathogenic for O. niloticus. A. hydrophila was identified by conventional biochemical tests and VITEK 2-C15 automated system for bacterial identification (BioMérieux, France) according to manufacturer 's instructions as

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described by (Scheidegger, Fracalanzza, Teixeira, Cardarelli-Leite, 2009; Zhou, Song, Shao, Peng, Xiao, Hua, Owari,

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Zhang, Ng, 2011) at Microbiology and Immunology Department, National Research Centre (NRC), Dokki, Giza,

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Egypt. Fish mortalities and clinical signs were observed for two weeks according to Lucky (1977).

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2.2. Preparation of methylated soy protein, Diet preparation, and experimental design

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Methylated soy protein isolate (MSPI) was prepared by interacting soy protein isolate (5%, w/v in methanol >99.5%) with methanol in the presence of HCl (50 MR) under ice-bath conditions with continuous stirring for 10 h

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according to the procedure of Sitohy, Chobert, Haertlé (2001), and the esterification extent was colorimetrically quantified by the reaction with hydroxylamine hydrochloride (Bertrand-Harb, Chobert, Dufour, Haertle, 1991).

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Five isoenergetic diets with similar protein content (Table 1) were formulated with five concentrations of MSPI substituted in fish meal: 0%, 22%, 44%, 66%, and 88% (MSPI0, MSPI22, MSPI44, MSPI66, and MSPI88), with lysine and methionine supplementation to compensate the amino acid deficiency in the higher MSPI diets. The zero percentage represented the control group. Each treatment group was subdivided into three replicates (15 fingerlings/ replicate), and each aquarium representing a replicate. Diets were prepared in the form of 2 mm pellets and formulated according to standard measures of Oreochromisniloticus by assessing for dry matter, crude protein, ether extract, crude fiber, and ash (NRC, 2011). The digestible energy (DE) content of the diets was measured according to Santiago, Bañes-Aldaba, Laron (1982). Proximate chemical analysis of fish and diets were done according to AOAC (2000).

2.3. Growth performance parameters and proximate body composition

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The initial fish weights were recorded at the beginning of the experiment then fish body weights and feed intake were recorded every two weeks. Total weight gain, average daily gain (ADG), specific growth rate (SGR), and feed conversion ratio (FCR) were calculated according to (Castell, Tiews, 1980). The protein efficiency ratio (PER) was determined according to(Stuart, Hung, 1989). Total weight gain (g/fish) = (WT–WI), where WT = final weight of fish in grams and WI = initial weight of fish in grams; ADG (g/fish/day) = total gain/ experimental days; SGR (%/day) = 100 × (ln WT – ln WI)/time in days [where ln is the natural logarithm]. FCR = total feed intake (g)/total gain (g); PER = total gain (g) / protein intake (g); percentage (%) survival = (number of fish in each group post 10

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weeks feeding period / initial number of fish) × 100. The total weight of surviving fish per aquarium was recorded

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after bacterial challenge.

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The procedure for determining the fish approximate body compositionwas carried out after 10 weeks feeding period according to the Official Analytical Chemists protocol(AOAC, 2000).Six fish per group were sampled,

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frozen at –20 °Ctill analyzed. The Frozen whole fish were thawed, dried in the hot air oven, blended and analyzed for determination of moisture, crude protein, ether extract, and ash content. Moisture content was estimated by

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drying the samples to constant weight at 85 C in a drying oven (GCA, model 18EM, Precision Scientific Group, Chicago, IL, USA). Nitrogen content was measured using a micro Kjeldahl apparatus (Labconco, Labconco

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Corporation, Kansas, MO, USA), and crude protein were estimated by multiplying nitrogen content by 6.25. Lipid

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content was determined by ether extraction in a multiunit extraction Soxhlet apparatus (Lab-Line Instruments, Inc., Melrose Park, IL, USA) for 16 h, and Total ash was determined by combusting dry samples in a muffle furnace (Thermolyne Corporation, Dubuque, IA, USA) at 550 C for 6 h.

2.4. Histology and morphometric methods

The tissue specimens of the intestine and liver were collected from each group at the end of the feeding trial and fixed in 10% neutral buffered formalin. The specimens were dehydrated in ascending grades of ethyl alcohol (70–100%), cleared in xylene, and embedded in paraffin. Tissue sections of 5 µ thicknesses were prepared with the help of microtome (Leica®, Wetzlar, Germany) and stained with hematoxylin and eosin (H&E) and alcian blue stain. Slides were examined for morphometric analysis and photographed using the AmScope digital camera-attached Ceti England microscope for histopathological examination (Suvarna, Layton, Bancroft, 2013). Morphometric analyses were done

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Journal Pre-proof using 20 images per animal captured at 40X and 400X for villus height and width, crypt depth, lymphocytes and Goblet cells counting using AmscopeToupView 3.7 software (AmScope, United States).

2.5. Sample collection and laboratory analysis

Blood samples were collected at two experimental points; at the end of the feeding trial and 14 days post fish experimental challenge. Whole blood samples with EDTA was taken for measurement of phagocytic capacity. The

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white blood cells were separated from peripheral blood of the tested fish belonging to different experimental groups.

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Heat-inactivated Candida albicans, which was isolated and identified at the Department of Bacteriology, Mycology,

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and Immunity, Faculty of Veterinary Medicine, Zagazig University, was used to determine the phagocytic capacity of the phagocytic cells in each experimental group (Wilkinson, 1977). The slides were air-dried and stained

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sequentially with Leishman's stain and viewed under oil immersion at 100x. Approximately 100 cells were counted from random fields of view and the numbers of phagocytic cells were recorded. The percent phagocytosis and the

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phagocytic index were calculated according to the formula described by Rashid, Nakai, Muroga, Miyazaki (1997) after counting at least 100 phagocytic cells either phagocytizing or not for the calculation of the percent

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phagocytosis and by counting at least 100 bacteria that were phagocytized by certain number of phagocytic

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cells/macrophages for the phagocytic index calculation. Phagocytic index= Total no. of phagocytized bacteria/ No. of phagocytic cells phagocytizing bacteria % Phagocytosis = (No. of phagocytic cell phagocytizing bacteria/ Total no. of phagocytic cells counted) ×100 Separate blood samples were collected without anticoagulants, allowed to clot at room temperature or in the refrigerator for an hour and the serum was separated by centrifuging at 3000 RPM for 15 min. The serum was transferred into dry, sterile, labeled, stoppered vials and used for determination of the IgM and Complement component 3 using MyBiosource Co. ELISA kits (Cat. Nos. MBS282651 and MBS005953) according to the manufacturer’s instructions. Lysozyme activity was spectrophotometrically measured according to (Ellis, 1990). Liver function tests (alanine aminotransferase and aspartate aminotransferase) and kidney function tests (urea and creatinine) were measured using kits from Spin react (Esteve De Bas,Girona,Spain) according to Murray (1984), Burtis, Ashwood (1994), Kaplan (1984), and Fossati, Prencipe, Berti (1983), respectively. Nitric oxide levels were measured according to (Sun, Zhang, Broderick, Fein, 2003)). The growth hormone was measured using the

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Journal Pre-proof MyBiosource Co. ELISA kit (Cat. No. MBS266317) according to the manufacturer’s instructions. All samples were taken in triplicate.

2.6. Economic efficiency

Collective efficiency measures were calculated according to El-Telbany, Atallah (2000), Dunning, Daniels

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(2001). It includes total return, total costs, variable costs, net profit. Performance index (PI) was calculated

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according to North, Bell (1984).

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2.7. Statistical analysis

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All data are expressed as a mean±standard error (SE). All data were verified for normality after transformation (ASIN). ANOVA test was applied based on polynomial orthogonal contrasts. Linear and quadratic regression

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equations were calculated using SPSS Version 17 for Windows (SPSS Inc., Chicago, Illinois, USA) to determine the effect of fish meal substitution with MSPI(with amino acid supplementation) in Oreochromis niloticus diets on

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growth, health,histopathological examination of specific organs,economic efficiency, and the immune response of fish challenged with A. hydrophila. The regressions were considered significant when P < 0.05.Post-hoc Tukey’stest was used to determine differences among means based on linear regression. Statements of statistical significance are based on P < 0.05 unless otherwise stated. The broken-line regression with Tukey's test was considering data on body weight gain and FCR for quantifying the maximum acceptance of Oreochromis niloticus to dietary MSPI supplementation(Shearer, 2000).

3.

3.1.

Results

Methylated soy protein (MSPI)

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Journal Pre-proof The MSPI prepared for this study was made by esterification of about 83% of free carboxyl groups with methanol in the presence of hydrochloric acid, and had an isoelectric point of around 8.

3.2.

Growth performance, survivability, and fish whole-body composition

Table 2 shows the effects of the partial replacement of fishmeal with MSPI on the growth performance and the percentage survival of the bacterial challenged fish. The results of final body weight, body weight gain, daily body weight gain, protein efficiency ratio, and SGR were uniquely increased by the treatment MSP22 (34.68g±0.72,

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16.41g±0.76, 0.23g±0.01, 1.86±0.18, and 0.92±0.03 respectively), while increasing the level of MSPI more than

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22% resulted in linearly proportional decreases in these parameters over the respective control “MSPI0”.

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Differences in the total feed intake and feed conversion ratio in MSPI22 to MSPI88 groups were non-significant (P>0.05) compared to the MSPI0 group. With the bacterial challenge, the total weight of surviving fish was quadratically

(P=0.01)

by MSPI

inclusion

(338.12g±37.23,

463.60g±40.96,

456.43g±16.43,

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increased

446.18g±23.47, and 422.70g±12.45 for MSPI0, MSPI22, MSPI44, MSPI66, MSPI88 respectively). The percentage

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survival after the bacterial challenge was increased linearly and quadratically (P < 0.05) by MSPI inclusion, the highest survival percentage was recorded in the MSPI66 group (95.55%±4.44). The results of the broken-line

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regression method using FCR and body weight gain data revealed that the maximum dietary replacement of

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fishmeal with MSPI was 66% (Figs.1 and 2)

The dry matter and crude protein content changes in the fish body as compared to MSPI0 were non-significant (P>0.05) in MSPI22–MSPI66, while they decreased linearly and quadratically (P < 0.05) in MSPI88 group. Fat content in the MSPI66 group (120±6.1g/kg) was decreased linearly (P = 0.005) compared to the MSPI0 group (157 ±9.3 g/kg). Ash content was increased quadratically (P=0.004) by increasing the substitution percentage of fish meal by MSPI (Table 2).

3.3. Histopathological findings

The liver sections showed; normal histomorphological structures including central vein in the MSPI0 group;nearly normal with mild welling hepatocytes which compensatory narrowing of sinusoidsin the MSPI22 group; marked congested hepatic blood vessels with mild fatty hepatocytes were noticedin the MSPI44-66 groups,

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Journal Pre-proof while liver sections from fish fed the highest level of MSPI “MSPI88” showed marked congested central vein and swelling hepatocytes with disappears of sinusoids (Figure 3). The intestinal sections showed: normal intestinal villi structures in MSPI0; tall villi with notable goblet cell metaplasia in MSPI22; normal tall, thin villi with partially destructed tips in MSPI44; thick villi with numerous broad tips in MSPI66, and normal tall villi with some fusion in MSPI88 (Figure 4).

3.4. Morphometric measures of the intestines

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The effects of MSPI substitution in the fish meal on the morphometric intestinal features are presented in Table

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3. The groups displayed increasing villous length linearly (P < 0.05) with increasing MSPI substitution, with

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MSPI88 showing the longest villi (P = 0.001). The crypt depth was linearly decreased (P = 0.002) in MSPI88 while being non-significant (P>0.05) in groups MSPI22–MSPI66. Goblet cell numbers were linearly increased (P = 0.00)

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3.5. Blood biochemical parameters

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in MSPI22 versus MSPI0 and MSPI44–MSPI88 groups.

As shown table 4 and 5, the growth hormone level was increased quadratically (P=0.005) in MSPI22, MSPI44,

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and MSPI66 groups before the bacterial challenge (5.07±1.22, 4.21±1.28, 4.64±0.75ng/mL respectively) compared to the MSPI0 group (1.59±0.05 ng/mL) while after bacterial challenge, it was increased linearly and quadratically (P=0.006, P=0.03 respectively) with MSPI inclusion. ALT levels were decreased both linearly and quadratically (P < 0.05) with increasing the percentage MSPI inclusion before and after the bacterial challenge. AST level was increased linearly (P < 0.05) in the MSPI88 group (10.6±0.0U/L) compared to MSPI0 (10.1±0.1U/L) before the bacterial challenge and it was not significantly differ (P>0.05) with MSPI inclusion after bacterial challenge. Urea level was decreased linearly (P < 0.05) with increasing the percentage of MSPI inclusion before the bacterial challenge, while with the bacterial challenge, urea level was decreased both linearly and quadratically(P < 0.05) with increasing the percentage of MSPI inclusion. Creatinine levels were non-significantly differ (P>0.05) with MSPI inclusion.

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Journal Pre-proof 3.6. Immunological parameters

As shown in Tables 4 and 5, the percent phagocytosis and phagocytic index before the bacterial challenge and the phagocytic index after bacterial challenge were increased both linearly and quadratically (P < 0.05)with increasing MSPI substitution levels. The percent phagocytosis was increased linearly (P < 0.05) with increasing MSPI substitution levels after bacterial challenge (76.0±2.9, 75.3±3.2, 82.7±2.6, and 80.0%±2.9 for MSPI22, MSPI44, MSPI66, and MSPI88 respectively compared to 65.0%±2.9 for MSPI0). Before the bacterial challenge, the IgM levels were not significantly affected (P>0.05) by MSPI substitution while the lysozyme activity, complement 3

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and nitric oxide levels were increased quadratically (P < 0.05) in MSPI22-MSPI66 groups (Table 4). When tested

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post bacterial challenge, the levels of IgM, complement 3, and lysozyme activity were increased both linearly and

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quadratically (P < 0.05) with MSPI inclusion. The blood level of nitric oxide was increased linearly (P < 0.05) with MSPI inclusion (55.1±2.4, 73.7±2.1, 61.3±5.5, 75.9±6.5, 68.3±7.1 µmol/l for MSPI0, MSPI22, MSPI44, MSPI66,

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and MSPI88 respectively (Table 5).

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A. hydrophila challenged fish showed clinical signs ranged from mild erythema to severe acute septicemia among the challenged fish of different treatment groups. Behaviorally, the fish in the MSPI0 group were sluggish in

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response to external stimuli, and a trend of depression, restlessness, and only moderate responses to stimuli were observed in the MSPI22 and MSPI44 groups as well. The MSPI66 and MSPI88 groups were comparatively more

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active and displayed normal swimming behavior with good responses to stimuli. Clinical symptoms like hemorrhage (especially at the base of fins, operculum, and mouth), along with loss of scales, exophthalmia, and irregular ulcers on the lateral surface of the trunk were also observed in the MSPI0 group.

3.7. Economic efficiency

The effects of MSPI substitution in the fish meal with amino acid supplementation on the economic efficiency of the experimental diets are presented in Table 6. The results of the total return, net profit, feed cost, total cost, and performance index were decreased linearly with increasing the replacement percentage (P < 0.05). The highest total return, net profit, and performance index (P < 0.05) were recorded in MSPI22 (17.81±0.43 LE, 10.25±0.66 LE, and 19.49%±2.75 respectively). The lowest feed and total costs were

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Journal Pre-proof observed in MSPI88 (3.23±0.29 and 6.23±0.29 LE respectively). The results of the economic efficiency and feed cost/kg gain were not significantly affected (P>0.05) by MSPI inclusion.

4.

Discussion The current study was conducted to investigate the effect of partial substitution of fishmeal with MSPI in the

fish diet on the general health and nutritional aspects like growth, body composition, fish health, histopathological status, and immune response before or after A. hydrophila challenge in Oreochromis niloticus fingerlings. As seen in this study, the body weight, body weight gain, protein efficiency ratio, specific growth rate were decreased linearly

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(P≤0.05) by increasing the substitution percentage of fish meal by MSPI. The maximum dietary replacement of

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fishmeal with MSPI was 66% according to the broken-line regression analysis which indicated a high tolerance of

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Oreochromis niloticus fingerlings to dietary MSPI supplementation. The lower levels of MSPI substitution (especially MSPI22, 22%) yielded better results, while higher ones (MSPI88, 88% substitution) showed reduced

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growth. Similarly, Marković, Poleksić, Lakić, Živić, Dulić, Stanković, Spasić, Rašković, Sørensen (2012) reported decreased weight gain, feed intake and poor feed conversion in carp fed diet with complete fish meal replacement

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with a mixture of plant protein: full fat extruded and toasted soybean meal. This may be attributed to soy products palatability, digestive disturbance, and intestinal dysfunction especially with increasing the inclusion level that led to

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decrease the feed intake and consequently poor feed conversion ratio and decreased fish weights and specific growth

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rates, as also reported in (Francis, Makkar, Becker, 2001; Gatlin III, Barrows, Brown, Dabrowski, Gaylord, Hardy, Herman, Hu, Krogdahl, Nelson, 2007; Riche, Williams, 2011). Riche, Williams (2011) have earlier reported no significant growth effects in the Florida pompano Trachinotus carolinus with up to 80% substitution of soy protein isolate in fish meal. In another study, white shrimp fed on soy protein concentrate and soy meal protein with amino acid supplementation instead of fish meal had no significant effects on body weight, weight gain, feed utilization, feed conversion, and protein efficiency ratio, attributing this to the relatively more digestible soy meal composition (Xie et al., 2016). Xu, Wang, Zhao, Luo (2012) demonstrated that substitution of fish meal with soy protein isolate up to 62.5% and 100% (along with crystalline amino acid supplementation) did not significantly affect the growth performance of juvenile Amur sturgeons. Along similar lines, it has been reported that fish meal could be replaced with soy proteins, without compromising growth performance, up to 27.3% in silvery-black porgy juveniles (Yaghoubi, Mozanzadeh, Marammazi, Safari, Gisbert, 2016), 40% in gilt-head sea bream (Venou, Alexis, Fountoulaki, Haralabous, 2006), and 60% in sharp snout sea bream (Hernández, Martínez, Jover, García, 2007) with amino acids supplementation. Being the most common first-

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Journal Pre-proof limiting amino acids in soy-based fish diets (Mambrini et al., 1999), methionine and lysine supplementation along with soy protein substitutions had considerable impact. The fish whole body composition did not show a significant difference in total dry matter and crude protein content in MSPI22–MSPI66 but declined in MSPI88, as reported in(Omar, Amer, Mohamed, Osman, Sitohy, 2018). Body fat was decreased in MSPI66–MSPI88, which may be due to low protein and fat retention in plant proteinbased diets as compared to regular fish meal diet (Dias, Alvarez, Arzel, Corraze, Diez, Bautista, Kaushik, 2005). Earlier studies have shown that lipid deposition and fatty acid bioconversion, along with serum and liver lipid levels, are affected by dietary protein sources (Aoyama, Fukui, Takamatsu, Hashimoto, Yamamoto, 2000; Dias, Alvarez,

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Arzel, Corraze, Diez, Bautista, Kaushik, 2005; Lindholm, Eklúnd, 1991; Terasawa, Hirano, Wada, Takita,

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Nakamura, Innami, 1994). Soy-based diets could also result in lower body fat, serum cholesterol, and triglyceride levels compared to whole fish meal diets (Dias, Alvarez, Arzel, Corraze, Diez, Bautista, Kaushik, 2005; Kaushik,

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Coves, Dutto, Blanc, 2004; Kaushik, Cravedi, Lalles, Sumpter, Fauconneau, Laroche, 1995).

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Assessment of the intestinal and hepatic histological structure in fish fed MSPI Provide valued material about digestive capability and possible health effects of MSPI(Caballero, Izquierdo, Kjørsvik, Montero, Socorro,

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Fernández, Rosenlund, 2003; Diaz, Escalante, Garcia, Goldemberg, 2006). In our study, alterations in the histological structure of the intestine with an increased level of replacement are responsible for the decreased

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weights of fish fed the highest MSPI levels. Normal intestinal structures were observed in MSPI22-66 groups as reported in our previous research (Omar, Amer, Mohamed, Osman, Sitohy, 2018), while mild alterations were

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observed in the intestinal structure of MSPI88.Also, Marković, Poleksić, Lakić, Živić, Dulić, Stanković, Spasić, Rašković, Sørensen (2012) reported some changes in the intestinal structure of carp fed diet with complete fish meal replacement that is suggestive of SBM prompted enteritis. By increasing the level of substitution, alterations in the histological structure of the liver were observed. The normal structure was observed in MSPI22 while by increasing the level of MSPI, congested hepatic blood vessels, congested central vein, mild fatty hepatocytes, and swelling hepatocytes with disappears of sinusoids were observed.Robaina, Izquierdo, Moyano, Socorro, Vergara, Montero, Fernandez-Palacios (1995) also reported that increasing the level of dietary soy led to increased liver fat deposition and decreased glycogen deposits. These results were verified by the significant increase in the serum level of AST in the highest replacement percentage of fishmeal with MSPI (MSPI88). Elevated serum AST may indicate liver dysfunction or damage (Amer, Metwally, Ahmed, 2018; Amer, Osman, Al-Gabri, Elsayed, Abd El-Rahman, Elabbasy, Ahmed, Ibrahim, 2019; Omar, Amer, Mohamed, Osman, Sitohy, 2018). Xu, Wang, Zhao, Luo (2012) similarly hinted at increased liver dysfunction in

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Journal Pre-proof juvenile Amur sturgeon due to increasing soy protein isolate inclusion in diet, showing increased serum ALP levels. These results were consistent with previous observations in rainbow trout (Escaffre, Kaushik, Mambrini, 2007; Yamamoto, Suzuki, Furuita, Sugita, Tanaka, Goto, 2007). The increased weight of surviving fish in the current study post-infection in MSPI22–MSPI88 over MSPI0 is due to increase the survival percentage. Bacterial infections often cause huge economic losses owing to high mortality rates and fatal symptoms like hemorrhagic septicemia and ulcers (Zhou et al., 2015). Our experiments with bacterial challenge confirmed the immune-modulating effects of methylated soy protein isolate and showed that fish resistance could be improved to help fight infections like those of A. hydrophila by using an MSPI-

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inclusive fish diet. Severe adverse effects like hemorrhages at the base of fins, operculum, and mouth, loss of

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scales, exophthalmia, and lateral surface ulcers causing high mortality seen in control group MSPI0 could be

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rescued with a gradual increase in the percentage of MSPI in the diet. This further explains MSPI’s immunepotentiating effect that was reflected by blood immunomodulatory indices represented by high serum levels of

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IgM, lysozyme activity, complement component 3, and percent phagocytosis and phagocytic index (Amer, Metwally, Ahmed, 2018; Amer, Osman, Al-Gabri, Elsayed, Abd El-Rahman, Elabbasy, Ahmed, Ibrahim, 2019;

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Omar, Amer, Mohamed, Osman, Sitohy, 2018) in increased MSPI groups. Antimicrobial nitric oxide levels (Bogdan, Röllinghoff, Diefenbach, 2000) post-infection was also significantly increased in MSPI22 and MSPI66

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groups. Similarly, Zhao, Song, Xie, Ge, Liu, Xia, Yang, Wang, Zhu (2016)also reported high nitric oxide activity

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upon fish meal substitution with soybean hydrolysate in yellow catfish diets. Immunomodulatory effects of soybean hydrolysate against lymphocyte proliferation and macrophagic phagocytosis stimulation have also been previously shown (Kong et al., 2008). Regarding the economic value of the experimental diets, the feed costs decreased by increasing the level of substitution as follows MSPI0>MSPI22 >MSPI44 >MSPI88. However, the decreased growth due to the increased replacement percentage resulted in decreased total return, net profit, and performance index. The highest total return, net profit, and performance index were recorded in MSPI22.

5.

Conclusion From the aforementioned results, the maximum dietary replacement of fishmeal with MSPI Oreochromis

niloticus fingerlings was 66%. The growth performance was decreased by increasing the substitution percentage of fish meal by MSPI. MSPI was shown to be a good immune-modulating substance able to improve immune responses of A. hydrophila challenged fish and overall gut health. Moreover, MSPI inclusion increased the total

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Journal Pre-proof weight of fish surviving after bacterial challenge. MSPI substitution at 22% increased the performance index, net profit, and total return. MSPI substitution at 88% is not recommended for poor growth and hepatic and intestinal structural alterations.

Acknowledgment The authors acknowledge fish diseases and the management department and Nutrition & Clinical Nutrition department, Faculty of Veterinary Medicine, Zagazig University, Egypt for their cooperation.

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Conflict of interest

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The authors declare that they have no conflict of interests. This research did not receive any specific grant from

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funding agencies in the public, commercial, or not-for-profit sectors.

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Figure captions

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Fig. 1. Assessment of the maximum dietary fishmeal replacement with MSPI for Oreochromis niloticus fingerlings by

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means of broken-line regression analysis using the body weight gain.

Fig. 2. Assessment of the maximum dietary fishmeal replacement with MSPI for Oreochromis niloticus fingerlings by

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means of broken-line regression analysis using feed conversion ratio.

Fig. 3. Representative photomicrograph of H&E stained sections from fish's liver showing (a) Normal histomorphology structures including central vein (arrow) (X400). (b) Nearly normal with mild swelling hepatocytes which associated with compensatory narrowing of sinusoids (star) (X400). (c) Marked congested hepatic blood vessels and sinusoids (arrows). (d,e) Low (square) and high magnification of the congested all hepatic blood vessels (arrow) with mild fatty hepatocytes (X 100, 400). (f) Marked congested central vein (arrow) and swelling hepatocytes with disappears of sinusoids spaces (star) (X400). (a= MSPI0, b=MSPI22, c= MSPI44, d,e= MSPI66, f= MSPI88) Fig. 4. Representative photomicrograph of H&E stained sections from fish's intestine at the end of the feeding period showing: (a) Normal intestinal villous structures X40. (b,c) Apparently tall and branched villi with marked goblet cell metaplasia and partially destructed tips (arrows) 40X. (d) Apparently tall, separate and thin villi (arrow) with a few fusion villi (star) X40. (e) Thick and short villi with numerous broad tips (arrow) 40X. (f) Apparently normal intestinal

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Journal Pre-proof villous height and broad tips (arrows) with some fusions (star). 40X. (A = MSPI0, B, C = MSPI22, D = MSPI44, E = MSPI66, F = MSPI88)

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Journal Pre-proof Table 1 Proximate chemical composition of the experimental diets (g/kg):

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Ingredients MSPI0 MSPI22 MSPI44 MSPI66 MSPI88 Fish meal 65% 180 140 100 60 20 MSPI 40 80 120 160 SBM 44% 255 212 192.3 175.5 162 Ground corn 243 263 273 278 280.5 Corn gluten 60% 110 110 110 110 110 Wheat bran 90 110 110 110 50 Wheat 50 50 50 50 110 Fish oil 60 60 62 66 70 Dical.ph. 0 2.5 7.5 12.5 17 Caco3 0 0 1.5 3 5 Lysine 0 0 1 2 2 Methionine 0 0.5 0.7 1 1.5 Vitamin &mineral 12 12 12 12 12 premix# Chemical composition g/kg ## Crude Protein 336 336 336 336 336 Fat 94.6 92.5 92.1 93.5 94.9 Crude Fiber 37.4 36.9 35.5 34.1 32.9 NFE* 386 394 394 392 389 DE kcal/kg 2907 2913 2907 2913 2918 Lysine 18.3 17.2 17.4 18.4 17.2 Methionine 7.1 7.1 6.9 6.8 6.9 Calcium 10.4 9.1 9 8.9 8.9 AP** 9.1 8.8 8.8 8.8 8.8 *Nitrogen free extract. ** availablephosphorus. #premix: 1Each 1kg of premix contain: vit A 550000 IU, vit D 110000 IU, vit E 11000 mg, vit K 484 mg, vit C 50 gm, vit B1 440 mg, vit B2 660 mg, vit B3 132oo mg, vit B5 1100 mg, vit B6 1045 mg, vit B9 55 mg, Choline 110000 mg, Biotine 6.6 mg, iron 6.6 gm, copper 330 mg, Mn 1320 mg, Zn 6.6 gm, Se 44 mg, iodine 110 mg. ## According to NRC (2011)

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Journal Pre-proof Table 2 The effect of partial replacement of fish meal with methylated soy protein isolates on the growth performance of Oreochromisniloticus fingerlings: Parameters

Initial wt./fish (g) Final BW/fish (g)

MSPI0

MSPI22

18.62±0.22 33.89±1.01

MSPI44 18.56±0.10

18.26±0.03

ab

ab

34.68±0.72

a

a

18.47±0.04

32.62±0.38

abc

0.20±0.006

abc

Regression analysis*

MSPI88

31.11±0.32

bc

0.18±0.005

bc

linear

Quadratic

18.62±0.05

0.53

0.13

30.23±0.43

c

0.00

0.26

0.17±0.008

c

0.00

0.20

0.22±0.01

Total BWG/ fish (g)

15.27±0.81ab

16.41±0.76a

14.06±0.42abc

12.64±0.31bc

11.74±0.57c

0.00

0.20

Total FI/fish (g)

32.42±3.61

26.51±1.32

29.23±1.33

27.91±2.50

29.00±1.62

0.46

0.26

FCR

2.16±0.36

1.63±0.15

2.08±0.05

2.20±0.14

2.48±0.19

0.09

0.12

PER

1.45±0.22

1.86±0.18

1.43±0.04

1.36±0.08

1.21±0.08

0.05

0.17

0.00

0.14

66.66±7.69

Total Wt. of survival fish after bacterial challenge (g)

338.12±37.23

Whole body composition (g/kg)

263±5.1a

0.81±0.02 a

463.60±40.96

93.33±3.84

259±4.2a

a

456.43±16.43

266±3.2a

0.74±0.01

bc

95.55±4.44

0.70±0.03 a

c

93.33±3.84

a

0.005

0.02

446.18±23.47

422.70±12.45

0.12

0.01

254 ±6.1a

227±5.3b

0.00

0.004

0.001

0.002

Crude Protein *

653±6.2

671±13.02

Fat *

157±9.3a

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Dry Matter#

88.88±5.87

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Survival % after bacterial challenge

0.92±0.03

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b

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0.85±0.02

abc

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SGR (%/day)

a

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Daily BWG/ fish (g)

ab

0.23±0.01

MSPI66

167±7.1a

153±3.04a

120±6.1b

147±3.5ab

0.005

0.56

Ash*

113±4.1

102±7.3

102±3.01

107±3.2

123 ±3.01

0.10

0.004

653±23.05

a

Jo ur

a

a

638±16.1

a

524±24.01

b

*The regressions were considered significant when P < 0.05. BW: body weight. BWG: Body weight gain. FI: feed intake. FCR: Feed conversion ratio. PER: Protein efficiency ratio. SGR: Specific growth rate. #

Dry matter on fresh basis. * on dry matter basis.

22

Journal Pre-proof Table 3 The effect of partial replacement of fishmeal with methylated soy protein isolates on the morphometric measures of the intestine (µm) of Nile tilapia fingerlings at the end of thefeeding period..

Parameters

MSPI0

Villus height

169.0±19.0

Villus width

81.5±6.1

Crypt depth

92.2±6.1

a

63.0±3.8

b

Goblet cells

MSPI22 b

163.57±19.1

MSPI44 b

88.3±15.8 62.8±13.1

179.8±16.1

ab

237.9±12.8

92.5±8.7

ab

103.6±10.9

MSPI66

a

61.2±6.0

9.60±0.67

ab

58.2±7.7

c

ab

16.0±2.7

c

Jo ur

na

lP

re

-p

ro

of

*The regressions were considered significant when P < 0.05.

23

Linear

Quadratic

0.001

0.19

57.6±9.6

0.058

0.11

48.4±6.1

b

0.002

0. 23

14.2±2.1

c

0.00

0.45

268.0±37.1

69.1±9.9

ab

Regression analysis*

MSPI88 a

Journal Pre-proof Table 4 Impact of partial replacement of fish meal with methylated soy protein isolates on the immune status, blood levels of growth hormone and nitric oxide, and kidney and liver function tests of Oreochromisniloticus fingerlings at the end of the feeding period. Parameters

MSPI0

MSPI22

MSPI44

MSPI66

MSPI88

Regression analysis* linear

Immunological tests IgM (µg/mL) Lysozymes (µg/mL) Complement 3 (µg/mL) Phagocytic %

Quadratic

249±9.2 134.3±5.4

254±11.2 140.7±3.3

252±5.6 143.3±6.7

239±8.1 105.0±2.3

0.79 0.69

0.08 0.00

55.3±2.2

72.6±1.5

73.6±9.7

57.7±6.0

53.7±1.4

0.3

0.01

43.7±0.3c

52.7±0.7b

53.0±1.5b

57.3±0.7a

56.7±0.9a

Phagocytic index

1.97±0.0c

2.98±0.1b

3.19±0.1ab

3.53±0.0a

3.43±.07a

0.00 0.00

0.001 0.00

Growth hormone (ng/mL) NO (µmol/l) Liver function tests ALT (U/L)

1.59±0.05

5.07±1.22

4.21±1.28

4.64±0.75

1.63±0.20

0.89

0.005

55.3±2.2

72.6±1.5

73.6±9.7

57.7±6.0

53.7±1.4

0.3

0.01

12.0±0.1a

11.7±.0b

12.3±0.1a

11.4±0.0c

10.9±0.0d

0.00

0.00

AST (U/L)

10.1±0.1b

10.1±0.0b

9.3±0.1c

9.1±0.0c

10.6±0.0a

0.03

0.97

Kidney function tests Urea (mg/dL) 7.14±0.1ab

8.52±0.86a

5.67±0.39b

5.55±0.58b

5.49±0.15b

0.00

0.34

0.52±0.0

0.55±0.0

0.50±0.0

0.52±0.0

0.19

0.4

ro

-p

re

lP

0.53±0.00

na

Creatinine (mg/dL)

of

237 ±2.0 106.7±2.4

Jo ur

*The regressions were considered significant when P < 0.05.

24

Journal Pre-proof Table 5 Impact of partial replacement of fish meal with methylated soy protein isolates on the immune status, blood levels of growth hormone and nitric oxide, and kidney and liver function tests of Oreochromisniloticus fingerlings after the bacterial challenge with Aeromonashydrophila Parameters

MSPI0

MSPI22

MSPI44

MSPI66

MSPI88

Regression analysis* linear

234±6.2c

273±2.8a

247±2.0bc

278±3.5a

261 ±5.5ab

0.00

0.01

13.1±1.0c

20.4±0.4a

15.0±0.1bc

21.0±1.6a

18.5±0.5ab

0.004

0.03

103.7±3.2b

127.7±2.4ab

109. 3±1.8b

133.0±5.7a

112.5±2.5bc

0.02

0.01

65.0±2.9b

76.0±2.9a

75.3±3.2a

82.7±2.6a

80.0±2.9a

0.04

0.15

Phagocytic index

4.2±.057c

4.7±0.05b

4.6±0.05b

5.3±0.05a

5.1±0.05a

0.00

0.02

Growth hormone (ng/mL) NO (µmol/l)

1.57±0.07c

7.94±0.05a

3.24±1.08bc

8.55±1.55a

6.14±0.08ab

0.006

0.03

55.1±2.4b

73.7±2.1a

61.3±5.5ab

75.9±6.5a

68.3±7.1ab

0.04

0.15

12.1±0.05a

11.0±0.011b

10.7±0.03c

10.2±0.02d

10.1±0.04d

0.00

0.00

7.4±0.0

7.6±0.2

7.9±0.0

0.14

0.15

8.39±0.0a

8.88±1.3a

5.01±0.1b

4.20±0.0b

0.02

0.01

0.63±0.0

0.64±0.0

0.63±0.0

0.3

0.26

0.62±0.01

0.62±0.0

ro

-p

Jo ur

Creatinine (mg/dL)

7.9±0.0

re

AST (U/L) 7.5±0.0 Kidney function tests Urea (mg/dL) 6.51±0.0ab

lP

Liver function tests ALT (U/L)

of

Lysozymes (µg/mL) Complement 3 (µg/mL) Phagocytic %

na

Immunological tests IgM (µg/mL)

Quadratic

*The regressions were considered significant when P < 0.05.

25

Journal Pre-proof Table 6 The effect of partial replacement of fishmeal with methylated soy protein isolates on the economic efficiency of the experimental diets of Oreochromisniloticusfingerlings at the end of the feeding period. Parameters

MSPI22

MSPI44

MSPI66

MSPI88

Regression analysis* Linear

Quadratic

10.25±0.66

7.15±0.16

7.51±0.55

6.53±1.19

0.056

0. 37

15.68±1.13ab

17.81±0.43a

14.36±0.19ab

14.34±0.69ab

12.76±0.94b

0.003

0.19

Feed cost (LE)

4.66±0.07a

4.56±0.22a

4.22±0.19a

3.83±0.151ab

3.23±0.29b

0.00

0.19

Total cost (LE)

7.66±0.07a

7.56±0.22a

7.22±0.19a

6.83±0.15ab

6.23±0.29b

0.00

0.19

Feed cost (LE) /kg gain

45.74±9.77

27.05±3.15

52.54±2.24

51.53±10.26

52.59±7.32

0.13

0.64

EE

1.73±0.28

2.28±0.26

1.70±0.09

2.12±0.56

0.64

0.95

11.77±3.72ab

19.49±2.75a

7.62±0.40b

7.27±1.16b

0.02

0.58

PI %

ro

Total return (LE)

of

8.02±1.19

1.95±0.06

-p

Net profit (LE)

MSPI0

8.25±1.90b

re

*The regressions were considered significant when P < 0.05.

Jo ur

na

lP

EE: economic efficiency, PI%: performance index %. LE: Egyptian pound

26

Journal Pre-proof Highlights 1. Dietary MSPI supplementation increased weight and survival of Oreochromis niloticus” 2. The maximum dietary replacement of fishmeal with MSPI was 66% 3. MSPI improved the immune responses of Aeromonas. hydrophila challenged fish. 4. MSPI substitution at 22% increased the performance index, net profit, and total return.

Jo ur

na

lP

re

-p

ro

of

5. MSPI substitution at 88% induced poor growth, hepatic- intestinal structural changes.

27

Figure 1

Figure 2

Figure 3

Figure 4