Effects of a new generation of fish protein hydrolysate on performance, intestinal microbiology, and immunity of broiler chickens

Amir Hossein Alizadeh-Ghamsari1, Amir Reza Shaviklo2,*, Seyyed Abdullah Hosseini1
Author Information & Copyright
1Department of Animal and Poultry Nutrition and Physiology, Animal Science Research Institute of Iran, Agricultural Research, Education and Extension Organization (AREEO), Karaj 3146618361, Iran
2Department of Animal Processing, Animal Science Research Institute of Iran, Agricultural Research, Education and Extension Organization (AREEO), Karaj 3146618361, Iran
*Corresponding author: Amir Reza Shaviklo, Department of Animal Processing, Animal Science Research Institute of Iran, Agricultural Research, Education and Extension Organization (AREEO), Karaj 3146618361, Iran. Tel: +98-26-34256001, E-mail:

© Copyright 2023 Korean Society of Animal Science and Technology. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Aug 13, 2022; Revised: Oct 09, 2022; Accepted: Nov 01, 2022

Published Online: Jul 30, 2023


This study was conducted to evaluate the effects of co-dried fish protein hydrolysate (CFPH) on broilers performance, intestinal microbiology, and cellular immune responses. Five hundred one-day-old (Ross 308) male broilers were allocated to four treatments with five replicates of 25 birds in a completely randomized design. The experimental treatments included four levels of CFPH (0% as the control, 2.5%, 5%, and 7.5%) in the isonitrogenous and isocaloric diets. During the experiment, body weight (BW) and feed intake (FI) were periodically recorded in addition to calculating average daily gain (ADG), feed conversion ratio (FCR), liveability index, and European broiler index (EBI). In addition, cellular immune responses were evaluated at 30 days of age. On day 42, ileal contents were obtained to examine the microbial population. Based on the findings, Dietary supplementation of 5 and 7.5% CFPH increased the percentage of the thigh while decreasing the relative weight of the gizzard compared to the control group. The highest relative length of jejunum was observed in birds receiving 2.5 and 5% CFPH, and its highest relative weight belonged to birds fed with 5% CFPH. The number of coliforms, enterobacters, and total gram-negative bacteria in the intestines of birds receiving CFPH was less than that of the control group. In general, the application of CFPH in broiler nutrition can decrease the level of soybean meal in diet and it can be considered as a new protein supplement in poultry production. It is suggested to study the incorporation of this new supplement in other livestock’s diets.

Keywords: Hydrolyzed fish protein; Growth performance; Broiler chicken; Intestinal microbial population; Immune response


The processing operations of fish and aquatic products generate a considerable amount of rest raw materials called by-products. Fish by-products are regularly converted into fish meals and consumed as a protein source in aquatic farming, poultry, and livestock nutrition. Mincing, cooking, pressing, drying, and milling, which are applied in traditional fish-meal production, are overpriced and complex procedures, and the high temperature utilized for processing fish meal could impair the digestibility of the final product [1,2]. Therefore, during the past decades, a large quantity of scientific documents have been published regarding the properties and potential applications of fish proteins recovered from fish by-products for animal nutrition [1,3,4].

Fish protein hydrolysate (FPH) is a functional product developed from the whole fish or fish by-products using the protein hydrolysation method in which fish proteins are broken into smaller parts, namely, peptides and amino acids [5]. FPH is available in liquid, paste, and dried forms in the market. Liquid FPH contains up to 90% of moisture and is highly unstable for long-time storage; therefore, its transportation is not economic. Thus, the dried form of this product is preferred because of less-challenging storage, longer shelf-life, and easier transport. On the other hand, one of the problems of dried-form FPH production is the separation of a considerable amount of water from liquid FPH which is a tough and expensive task [6,7].

Drying of hydrolyzed proteins is a serious challenge in the operation of these products [2]. The co-drying of hydrolyzed fish protein with dry agricultural by-products is an innovation in the use of agricultural and fishery by-products for animal feed. Several researchers have used this method on a laboratory scale to co-dry liquid hydrolyzed protein and use the product in animal feed. This method can be economical if produced on a semi-industrial or industrial scale [8-11]. Thus, this offers a profitable and novel method for converting the by-products of fish into useful protein-hydrolysate materials in order to feed birds, aquaculture, and other livestock [12].

Meanwhile, dependence on soybean imports from major producer countries for animal farming including broilers husbandry, has led many livestock producers to replace local protein products with soy protein. The use of local protein products, including fish protein ingredients from fishery by-products, is an alternative to partial substitution of soybean meal in livestock diet [13]. Co-drying of FPH to develop new sources of value-added animal protein as a feed supplement is a new approach for feeding animals with local agricultural and fisheries by-products [2].

Although some studies have reported the positive influence of FPH on the growth performance of broiler chickens [14-16], but limited applied studies have addressed the application and evaluation of co-dried fish protein hydrolysate (CFPH), produced in a feasible method, in the animal nutrition. Further, more data are required to be published about the immunological and microbiological effects of these products on commercial poultry species. Accordingly, this research focused on the production of a novel protein supplement based on FPH and its effects on the performance, intestinal microbiology, and cellular immunity of broiler chickens. The results of this study may be employed to valorize the application of a new generation of FPH in the animal feeding industry and may help to reduce the amount of soy bean meal in diets.


Experimental procedures relating to chicken rearing and care in the current study were reviewed and approved by the Animal Ethics Board of the Animal Science Research Institute of Iran (Certificate No. 47-13-13-083-990566- 22 September 2020).

Preparation of co-dried fish protein hydrolysate

The CFPH was developed from whole Kilka fish (Clupeonella spp.) using an enzymatic process on a pilot scale. The alcalase, a proteolytic enzyme, was used to produce FPH [6]. The whole fish protein solution (without phase separation) was stabilized by formic acid (CH2O) to obtain a pH value of 4.5. After stabilizing the pH, a 50% fish protein solution, 20% rice bran, 29% defatted sesame seeds (Sesamum indicum L.), and 1% feed grade calcium bentonite clay were applied to prepare a semidry material. The semidry FPH was co-dried in a vacuum oven-dryer at 60°C for 4 h. The dried samples were milled to pass through a 0.4-mm sieve and stored at ambient temperature (25°C) until included in the diets.

Analysis of co-dried fish protein hydrolysate

The dry matter, ash, crude fiber, ether extract, crude protein, calcium, available phosphorus, pH, theAA compositions and total carbohydrate of CFPH were determined according to AOAC [17]. The total volatile basic nitrogen (TVB-N) content was estimated via the method described by Goulas and Kontominas [18]. The fatty acid (FA) compositions of CFPH were analyzed by gas chromatography (GC, 6890 series, Agilent Technologies, Wilmington, DE, USA) following the procedure described in previous research [19]. The chemical compositions of the CFPH are provided in Table 1.

Table 1. Chemical compositions of CFPH used in this experiment1)
Item (g/kg)
 Dry matter 936.6
 Crude protein 457.2
 Ether extract 216.5
 Total carbohydrate 155.4
 Crude fiber 10.0
 Ash 97.5
 Calcium 14.9
 Available phosphorus 6.5
 Total volatile basic nitrogen 0.154
 pH 4.69
 Metabolisable energy (MJ) 14.76
Amino acid profile (g/kg)
 Aspartic acid 30.0
 Glutamic acid 58.8
 Histidine 8.6
 Serine 13.9
 Arginine 28.0
 Glycine 17.4
 Threonine 14.3
 Alanine 17.2
 Tyrosine 12.3
 Tryptophan 4.2
 Cysteine 7.7
 Methionine 8.3
 Valine 15.8
 Phenylalanine 14.0
 Isoleucine 12.8
 Leucine 23.0
 Lysine 17.2
Fatty acid profile (% of total fatty acids)
 Myristic acid (C14:0) 1.83
 Myristoleic acid (C14:1) 0.31
 Palmitic acid (C16:0) 16.83
 Palmitoleic acid (C16:1) 1.50
 Margaric acid (C17:0) 0.50
 Stearic acid (C18:0) 5.36
 Oleic acid (C18:1) 26.70
 Linoleic acid (C18:2) 46.94
 SFA 9.50
 PUFA 72.64

1) Chemical compositions of CFPH determined in a laboratory based on AOAC [17] methods, and the amount of metabolisable energy was calculated based on the previous results [19].

CFPH, co-dried fish protein hydrolysate; SFA, saturated fatty acid; PUFA, polyunsaturated fatty acid.

Download Excel Table
Birds, housing, and rearing

The project was conducted at the Research Poultry House affiliated with the Agricultural Research, Education, and Extension Organization (AREEO) of Iran (Karaj, Alborz, Iran). Five hundred one-day-old male broiler chickens (Ross 308) were acquired from an industrial hatchery (with an initial body weight [BW] of 40 ± 0.5 g) and randomly distributed in 20 floor pens. The initial temperature of the farm was kept at 33 ± 2°C and regularly declined (2.4°C weekly) to reach a persistent temperature of 21–23°C at the age of 28 days. Throughout the experiment, the lighting regime and relative humidity were maintained in 23:1 h of light/darkness and 50%–60%, respectively. The access of chickens to water and feed was unlimited during the experiment.

Diet formulation and experimental design

Before formulating diets, the chemical composition of the main feed ingredients, including maize and soybean meal, was analyzed according to the AOAC procedures [17], and the data were applied to formulate the experimental diets. Four levels of CFPH (0% as the control, 2.5%, 5%, and 7.5%) were included in the dietary treatments (Table 2). All diets contained a similar amount of protein and energy and were formulated based on Ross 308 recommendations [20].

Table 2. Feed ingredients and nutrient compositions of experimental diets
Item CFPH (%)
Days 1–10 Days 11–24 Days 25–42
0 2.5 5 7.5 0 2.5 5 7.5 0 2.5 5 7.5
Ingredients (g/kg)
 Maize grain 544.5 541.6 536.9 532.1 615.2 614.5 608.2 603.4 656.9 657.8 653.3 650.3
 Soybean meal (44% CP) 399.0 374.0 348.0 320.0 330.0 305.0 280.0 253.0 290.0 263.0 237.0 210.0
 Soybean oil 11.0 10.0 10.0 10.0 11.0 9.5 9.5 9.5 12.7 10.5 10.5 9.5
 CFPH - 25.0 50.0 75.0 - 25.0 50.0 75.0 - 25.0 50.0 75.0
 Limestone 13.0 13.0 13.0 13.0 13.0 13.0 13.0 13.0 11.5 11.5 11.0 11.0
 Dicalcium phosphate 18.5 18.5 18.5 18.5 17.8 17.8 17.8 17.8 16.5 16.5 16.5 16.5
 Sodium chloride 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.3 2.5 2.5 2.2 2.5
 Bicarbonate sodium 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
 Vitamin-mineral premix1) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
 DL- Methionine 99% 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.1 2.1 2.1 2.1
 L- Lysine HCL 1.4 1.7 2.1 2.6 1.3 1.6 2.0 2.5 1.2 1.2 1.2 1.7
 L- Threonine 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.6 0.6 0.6 0.6
 Washed sand (inert filler) - 4.5 9.8 17.1 - 1.9 7.8 14.3 - 3.3 9.6 14.8
Nutrient composition2)
 Metabolizable energy (MJ/kg) 12.0 12.0 12.0 12.0 12.4 12.4 12.4 12.4 12.6 12.6 12.6 12.6
 Crude protein (g/kg) 225.2 225.8 225.9 225.3 201.3 201.0 201.5 201.2 185.6 185.3 185.0 185.0
 Calcium (g/kg) 9.5 9.4 9.7 9.5 9.2 9.1 9.3 9.4 8.0 8.1 8.2 8.3
 Available phosphorus (g/kg) 4.5 4.6 4.7 4.8 4.4 4.4 4.6 4.7 3.8 3.9 4.0 4.1
 Sodium (g/kg) 1.9 1.9 2.0 2.0 2.1 2.1 2.2 2.5 1.7 1.7 1.7 1.8
 Lysine (g/kg) 13.7 13.7 13.7 13.7 11.9 11.9 11.9 11.9 10.5 10.5 10.2 10.3
 Methionine + Cystine (g/kg) 9.5 9.6 9.7 9.7 9.0 9.2 9.5 9.4 8.5 8.5 8.9 8.9

1) To provide vitamins and minerals per kilogram of diet: vitamin A, 17500 IU; vitamin E, 35 mg; vitamin D3,3900 IU; vitamin K3, 4.8 mg; riboflavin, 7.4 mg; vitamin B12, 1.7 mg; niacin, 56 mg; thiamine, 2.96 mg; biotin, 0.17 mg; pyridoxine, 4.55 mg; folic acid, 1.8 mg; ethoxyquin, 0.124 mg; pantothenic acid, 17.7 mg; choline chloride, 486.5 mg; cyanocobalamin, 0.025 mg; Zn-sulfate, 83 mg; Fe-sulfate, 39.5 mg; iodine (calcium iodate), 1.25 mg; Cu-sulfate, 19 mg; Mn-sulfate, 158 mg; selenium (sodium selenite), 0.30 mg.

2) Determined by the main ingredient analysis, then the results were used for calculating the nutritional compositions of diets.

CFPH, co-dried fish protein hydrolysate; CP, crude protein.

Download Excel Table
Performance traits and organ weight

Performance variables, including feed intake (FI) and BW, were recorded periodically. The difference between given and residual feed was considered as FI. Mortality was daily recorded, and data related to average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were corrected based on this parameter. At the end of the experiment (42 days of age), 30 birds from each treatment (six birds/pen which had close weight to the average of the pen) were chosen, and then weighed and slaughtered after 4 h of fasting. Chickens were slaughtered based on the European Union legislation on the protection of animals used for scientific purposes (Directive 2010/63/EU). Next, the slaughtered birds were plucked, and visceral organs were excised to determine the relative weight of the carcass and organs. At 42 days of age, the European broiler index (EBI) of broiler chicks (including the sacrificed birds) was calculated by the following formula [21]:

EBI = Livability ( % ) × average daily gain ( g ) Feed coversion ratio ( g / g ) × 10

Different parts of the small intestine, including jejunum, duodenum, and ileum, were sectioned, intestinal digesta was removed, and intestinal segments were washed in neutral-buffered saline and blotted dry on paper towels. Then, the relative weight and length of each segment were calculated by dividing the weight and length of each section by live BW, respectively [22].

Cellular immunity

At 30 days of age, the cutaneous basophilic hypersensitivity (CBH) reaction to phytohemagglutinin P (PHA-P, Sigma Chemical, St. Louis, MO, USA) was assessed by intra-dermally injection of 100 μg of PHA-P (dissolved in a 0.1 mL of sterile saline) into the toe web of the left foot of three birds from each pen based on the method described by previous researchers [23]. The difference between skin thickness before and 12 or 24 h after injection was considered as the CBH reaction to PHA-P.

Moreover, at 30 days of age, the DNCB reaction was determined by calculating the difference between skin thickness on the right side of the lateral abdomen in three birds per pen (other than those treated with PHA-P) before and 24 or 48 h after the challenge with 0.1 mL of the prepared DNCB (2,4-dinitrochlorobenzene, Merck, Darmstadt, Germany) solution [24]. The average of the three repeat measurements for each bird was used for the analysis.


Ileal samples were subjected to microbial analyses based on the previously published method [25]. Concisely, each sample of the ileal content (approximately 1 g) was homogenized, followed by serial dilutions. A 10 μL sample of each serial dilution was inoculated using bacteria-specific agars, including MacConkey agar (MC agar, Neogen, Lansing, MI, USA) for the enumeration of total coliforms, total enterobacters [25,26], total yeast and molds and total gram-negative bacteria [27,28]. Furthermore, Rogosa agar (Becton, Dickinson, Franklin Lakes NJ, USA) was employed for total lactobacilli [24]. At the end of incubation, the number of colonies was counted as units per gram of the sample.

Statistical analysis

The GLM procedures of SAS 9.1 software [29] were utilized to analyze data in a completely randomized design. Values in percentage were transformed to arcsine. The pen of birds functioned as the experimental unit. Duncan’s multiple range test was applied for comparing the means [30]. Orthogonal contrasts were performed to determine linear and quadratic relationships among treatments. Differences were considered to be statistically significant at p < 0.05.


Growth performance

As shown in Table 3, the dietary inclusion of CFPH linearly and quadratically affected BW and ADG at 10 and 24 days of age (p < 0.05). At the age of 10 days, the BW and ADG of the control group were higher than those of the groups consuming CFPH. However, at 24 days of age, only the BW of 5% CFPH-received birds was negatively affected compared to the control (p < 0.05). At 42 days of age, no significant difference was observed between experimental treatments regarding BW and ADG (p > 0.05).

Table 3. Performance of broiler chicks fed with a diet consisting of co-dried fish protein hydrolysate
Parameters Co-dried fish protein hydrolysate (%) SEM p-value
0 (control) 2.5 5 7.5 Linear Quadratic
BW (g)
 d 10 289.11a 238.32b 197.22c 212.61bc 9.13 < 0.001 0.002
 d 24 1137.02a 1125.41a 1006.55b 1105.10a 16.37 0.045 0.024
 d 42 2744.03 2747.06 2614.22 2705.31 55.02 0.216 0.338
ADFI (g/d/bird)
 d 1–10 28.82 28.11 25.84 27.45 0.31 0.172 0.263
 d 11–24 97.86a 96.93a 86.02b 90.01b 1.16 0.001 0.102
 d 25–42 180.68 186.77 193.43 191.42 21.08 0.623 0.639
 d 1–42 4909.02 4999.20 4944.70 4979.54 34.20 0.654 0.683
ADG (g/d/bird)
 d 1–10 24.91a 19.83b 15.72c 17.26bc 0.50 < 0.001 0.002
 d 11–24 60.56ab 63.36a 57.80b 63.74a 0.52 0.049 0.044
 d 25–42 89.27 90.09 89.31 88.90 2.14 0.319 0.439
 d 1–42 64.38 65.98 61.29 63.45 1.34 0.326 0.472
FCR (g/g)
 d 1–10 0.98c 1.18b 1.31a 1.29ab 0.04 0.001 0.010
 d 11–24 1.61 1.52 1.48 1.41 0.09 0.109 0.209
 d 25–42 2.02 2.07 2.16 2.15 0.05 0.086 0.089
 d 1–42 1.79 1.82 1.89 1.84 0.04 0.094 0.084
Livability (d 1–42, %) 99.99 99.96 99.93 99.95 0.108 0.112 0.207
EBI (d 1–42) 359.62 362.38 324.05 344.66 8.26 0.091 0.191

a-c Means in the same row with different superscripts vary significantly (p < 0.05).

BW, body weight; ADFI, average daily feed intake; ADG, average daily gain; FCR, feed conversion ratio; EBI, European broiler index; SEM, pooled standard error of the mean.

Download Excel Table

In the period of 11–24 days of age, the ADFI was linearly decreased by the dietary supplementation of CFPH (p < 0.05); however, no significant effect was detected in this regard during 1–10, 24–25, and 1–42 days of age (p > 0.05). The dietary addition of CFPH in the period of 1-10 days had a linear and quadratic negative effect on the FCR (p < 0.05), but this effect was not significant in the period of 11–24, 25–42, and 1–42 days of age (p > 0.05). Moreover, liveability and EBI were not influenced by adding different levels of CFPH into the diet.

Carcass characteristic

The carcass yield and relative weight of the breast, back and neck, heart, liver, pancreas, bursa of Fabricius, spleen, and abdominal fat were not affected by dietary treatments (Table 4). However, the addition of 5% CFPH to the diet linearly and quadratically increased the relative weight of the thigh compared to the control group (p < 0.05), which was not significantly different from the other levels of this product (p > 0.05). Based on the findings, the relative weight of gizzard in the group receiving 7.5% CFPH demonstrated a significant linear decrease when compared to the control (p < 0.05).

Table 4. Carcass yield and organs relative weight (g/100 g of live BW) of 42 day-old broiler chicks fed with a diet containing co-dried fish protein hydrolysate
Parameters (g/100 g of live BW) Co-dried fish protein hydrolysate (%) SEM p-value
0 (control) 2.5 5 7.5 Linear Quadratic
Carcass yield 73.40 73.80 74.40 75.80 0.481 0.080 0.630
Breast 24.70 23.70 21.80 25.00 0.783 0.780 0.119
Thigh 17.9b 18.7ab 19.40a 18.50ab 0.187 0.050 0.010
Back and neck 21.40 22.20 23.30 22.51 0.276 0.061 0.123
Heart 0.40 0.42 0.48 0.43 0.012 0.092 0.104
Liver 2.01 2.13 1.97 2.02 0.053 0.566 0.857
Pancreas 0.17 0.18 0.20 0.16 0.007 0.731 0.129
Gizzard 4.95a 2.65ab 2.86a 2.31b 0.084 0.009 0.355
Bursa of Fabricious 0.045 0.045 0.042 0.052 0.003 0.630 0.604
Spleen 0.076 0.110 0.094 0.094 0.006 0.470 0.166
Abdominal fat 0.93 0.82 1.28 1.02 0.062 0.112 0.478

a,b Means in the same row with different superscript differ significantly (p < 0.05).

BW, body weight; SEM, pooled standard error of the mean.

Download Excel Table
Intestinal development

The relative weight and length of the duodenum and ileum were not affected (p > 0.05) by the dietary inclusion of CFPH (Fig. 1). Meanwhile, the relative weight and length of the jejunum were linearly influenced by the dietary supplementation of CFPH (p < 0.05) so that the highest relative length was observed in the birds that received 2.5 and 5% CFPH, and the highest relative weight belonged to the group fed with 5% CFPH, which was significantly different from the control (p < 0.05).

Fig. 1. Relative weight (g/100 g of live BW) and length (cm/100 g of live BW) of small intestinal sections in 42 day-old broiler chicks fed with diets including different levels of co-dried fish protein hydrolysate. a,bColumns with the same pattern and different superscripts shows significant difference (p < 0.05). BW, body weight.
Download Original Figure
Immune responses

The addition of CFPH to the broiler diet had no significant effect on the hypersensitivity reaction induced by the injection of DNCB and PHA-P at 30 days of age (Fig. 2).

Fig. 2. Cell-mediated immunity by the response of skin to DNCB and toe web swelling by PHA-P in broilers fed with a diet containing co-dried fish protein hydrolysate at 30 days of age. PHA-P, phytohemagglutinin; DNCB, 2, 4-dinitrochlorobenzene.
Download Original Figure
Ileal microbial population

The results (Fig. 3) indicated that the total number of lactobacilli and yeasts and molds was not affected by experimental treatments (p > 0.05). Meanwhile, the effects of increasing the level of CFPH on coliforms, enterobacters, and the total gram-negative bacteria population were linearly significant (p≤0.05) so that the highest number of these three bacterial species was observed in the control group, and their number decreased by an increase in the CFPH level.

Fig. 3. Cecal microbial populations (Log 10 CFU/g) of 42 day-old broiler chicks fed with a diet consisting of co-dried fish protein hydrolysate. a-cColumns with the same pattern and different superscripts shows significant difference (p < 0.05). BW, body weight.
Download Original Figure


The dietary inclusion of CFPH up to the level of 7.5% exerted no significant impact on the productive traits of broilers, including BW, FI, ADG, FCR, EBI, live-ability, and carcass percentage at 42 days of age. This finding is consistent with the results of some other researchers. For instance, Al-Marzooqi et al. [31] reported that fish-derived recycled protein could be substituted for part of soybean meal in broiler diets without adversely affecting performance. Similarly, Ramírez et al. [32] concluded that the hydrolyzed protein obtained from a mixture of several fish waste in the broiler diets had no negative effect on the growth performance and meat quality of broilers, and thus it can be replaced with part of the plant proteins in the poultry diet as a reliable feed source. On the other hand, Ramírez et al. [33] found that the hydrolyzed protein of fish in a quail diet has a positive effect on the meat quality and growth performance of birds and can be replaced with some of the common plant proteins in the diet. In another study, the addition of 5% hydrolyzed salmon protein to the diet improved the performance of broilers compared to controls [14].

Meanwhile, in the present study, the addition of FPH to the diet caused a decrease in the BW at the age of 10 days. As a possible reason for this phenomenon, it can be supposed that since the FPH contains fish waste (including undigested contents of the fish digestive tract, intestinal tissue, and the like), the low digestibility of this product at an early age resulted in decreasing broiler performance in the first ten days of life. It is noteworthy that these negative effects diminished concurrent with growing and improving the digestive capacity of the bird [34] so that no significant difference in BW was observed between experimental groups at 42 days of age. Another reason is that according to the nature of FPH, part of energy supply in diets containing this product is of fish oil origin. Specifically, the amount of fish oil in the aforementioned test diets was 0%, 0.525%, 1.05% and 1.57% respectively. In the early days of age, the secretion of bile FAs is insufficient [35], which can cause a decrease in the ability to dietary fat absorption and a decrease in performance. Along with increasing age and the improvement of the bird’s absorption ability, the weight loss was compensated; but probably due to the lower weight at the age of 10 days, the expected improvement in performance traits such as weight, feed consumption and FCR was not observed. Other reasons for the difference in the results of the present study in terms of performance with former findings are probably variations in fish species, the type of processing applied for hydrolysis, and the other existing components in the final product such as fillers or moisture-absorbing compounds. During 1–24 days, the FI of the groups receiving 5 and 7.5% CFPH was lower than that of the birds fed with 2.5% CFPH and the control; this may be related to the lower weight of birds in both latter groups.

In the current study, carcass components (e.g., carcass yield, breast, back, and neck) and most internal organs were not affected by CFPH supplementation. The ineffectiveness of dietary sources containing fish oil (e.g., CFPH containing a 21% ether extract) on the relative weight of internal organs has also been reported by other researchers [36]. At the same time, relative weight of thigh tended to improve by increasing the dietary level of CFPH and the highest relative weight of thighs were observed in the birds received 5% of CFPH. Thigh muscles are of the places for fat storage in poultry [37] and the CFPH oil is more absorbable at the end of the rearing period, therefore, it may result in increase of fat storage in the thigh and its relative weight.

The dietary inclusion of 7.5% CFPH caused a significant reduction in the relative weight of the gizzard compared to the control (p < 0.05), which is in conformity with the results of Kiflay et al. [38]. Researchers have previously found that aquatic-based products (including fish meal) have the potential to erode the gizzard [39]. The lesions can range from small scratches on the gizzard to severe erosion and bleeding. While processing aquatic products, histidine or histamine in the body parts of fish can react with lysine to produce a compound called gizzerosine. Gizzerosine is not a biogenic amine, but the ability to stimulate acid production by this compound is 10 times stronger than histamine, which can lead to gizzard erosion [40]. In the present experiment, although no obvious lesions were observed in the gizzards of the CFPH-fed chickens, it can be assumed that the occurrence of this phenomenon in a mild and subclinical manner is the reason for the relative weight loss of this organ in birds receiving the highest level of CFPH.

The effect of experimental treatments on the relative weight and length of the jejunum was significant (p < 0.05) so that the highest length was observed in chickens that received 2.5 and 5% CFPH, and the highest weight was found in 5% CFPH-fed birds, which represents a significant difference from the control group. Few reports have evaluated the effects of a supplementing diet with hydrolyzed fish protein on the gastrointestinal characteristics of broilers. A recent study reported an increase in the ratio of villus length to crypt depth in the jejunum of chickens receiving hydrolyzed fish protein [41]. It might indirectly have a positive impact on the relative weight and length of this segment of the intestine. In contrast, Saki et al. [42] demonstrated that increasing the level of fish-based products from 2.3% to 8.7% in the diet exerted no significant effect on the relative weight and length of different parts of the intestine at 42 days of age. Discrepancies in the results can be related to modifications in processing methods, as well as the level of product consumption in the diet.

The density and composition of the intestinal microbial population are among the main characteristics of the intestinal ecosystem that help maintain its health and regulate host function. The reduction in the number of harmful bacteria (specific coliforms) as a result of the use of hydrolyzed fish protein in the present study is in line with the findings of Some other studies [1,41]. Organic acids in processed foods can eradicate undesirable bacterial species either directly by preventing penetration into the cell membrane or indirectly by acidifying the gastrointestinal tract environment [43]. Reduction in the number of coliforms in our experiment can be related to the preventing penetration into the cell membrane, as, the number of lactobacilli was not significantly affected by experimental treatments in the current study. Similar to our results, Al-Khalaifah and Al-Nasser [44] reported the ineffectiveness of consuming a food source containing fish oil on the population of lactobacilli in the ileal contents of broiler chickens. In this regard, Seidavi and Simões [45] found that the dietary inclusion of a source containing fish oil up to 2% did not have a negative effect on the population of beneficial gastrointestinal bacteria and helped maintain the health of birds.

A hypersensitivity reaction is a test in which the proliferation of a set of cells related to the cellular immune system, especially T lymphocytes, is stimulated and indirectly evaluated via the intradermal injection of compounds such as PHA-P and DNCB [46]. In the present experiment, cellular immune responses were not affected by CFPH supplementation, which contradicts the findings of Al-Khalaifah and Al-Nasser [44]. These researchers concluded that the dietary addition of food sources containing omega-3 and omega-6 FAs (e.g. fish oil) intensified the hypersensitivity reaction to the subcutaneous injection of PHA-P. The difference in the results is probably due to the dissimilarity in the processing method and the filler materials used in the experimental fish by-product.


In general, it is possible to feed broilers with CFPH up to the level of 7.5% without adverse effects on performance and immunity. Efficient utilization of fish by-products and agricultural residues is obtained by co-drying of such rest raw materials for animal nutrition. The application of CFPH in broiler nutrition can decrease the level of soybean meal in feed formulation and it can be considered as a new protein feed supplement in animal production especially in developing countries where the animal production is dependent on importation of plant protein sources. It is also suggested to incorporate this new supplement in other livestock’s diets.

Competing interests

No potential conflict of interest relevant to this article was reported.

Funding sources

Not applicable.


Support provided by Guilan Science and Technology Park (GSTP) is truly acknowledged.

Availability of data and material

Upon reasonable request, the datasets of this study can be available from the corresponding author.

Authors’ contributions

Conceptualization: Shaviklo AR.

Data curation: Alizadeh-Ghamsari AH, Shaviklo AR.

Formal analysis: Hosseini SA.

Methodology: Alizadeh-Ghamsari AH, Hosseini SA.

Software: Hosseini SA.

Validation: Alizadeh-Ghamsari AH.

Investigation: Shaviklo AR.

Writing - original draft: Alizadeh-Ghamsari AH.

Writing - review & editing: Alizadeh-Ghamsari AH, Shaviklo AR, Hosseini SA.

Ethics approval and consent to participate

Experimental procedures relating to the chicken rearing and care in the current study were reviewed and approved by the Animal Ethics Board of Animal Science Research Institute of Iran (Certificate No. 47-13-13-083-990566- 22 Sep. 2020).



Shabani A, Boldaji F, Dastar B, Ghoorchi T, Zerehdaran S. Preparation of fish waste silage and its effect on the growth performance and meat quality of broiler chickens. J Sci Food Agric. 2018; 98:4097-103


Etemadian Y, Ghaemi V, Shaviklo AR, Pourashouri P, Sadeghi Mahoonak AR, Rafipour F. Development of animal/ plant-based protein hydrolysate and its application in food, feed and nutraceutical industries: state of the art. J Clean Prod. 2021; 278:123219


Shavandi A, Hou Y, Carne A, McConnell M, Bekhit AEA. Marine waste utilization as a source of functional and health compounds.In In: Toldrà F, editor.editor Advances in food and nutrition research. Cambridge, MA: Academic Press. 2019; p. p. 187-254


Shaviklo AR, Etemadian Y. Overcoming current challenges in commercial applications of fish protein isolates in food and feed systems: a review. J Food Sci Technol. 2019; 56:4775-84


Damodaran S, Parkin KL. Fennema’s food chemistry. 5th ed. Boca Raton: CRC Press. 2017


Kristinsson HG, Rasco BA. Fish protein hydrolysates: production, biochemical, and functional properties. Crit Rev Food Sci Nutr. 2000; 40:43-81


Petrova I, Tolstorebrov I, Eikevik TM. Production of fish protein hydrolysates step by step: technological aspects, equipment used, major energy costs and methods of their minimizing. Int Aquat Res. 2018; 10:223-41


Fagbenro OA, Jauncey K. Chemical and nutritional quality of dried fermented fish silages and their nutritive value for tilapia (Oreochromis niloticus). Anim Feed Sci Technol. 1994; 45:167-76


Goddard JS, Al-Yahyai DSS. Chemical and nutritional characteristics of dried sardine silage. J Aquat Food Prod Technol. 2001; 10:39-50


Goddard JS, Perret JSM. Co-drying fish silage for use in aquafeeds. Anim Feed Sci Technol. 2005; 118:337-42


Palkar ND, Koli JM, Gund DP, Patange SB, Shrangdher ST, Sadawarte RK, et al. Preparation of co-dried fish silage by using fish market waste and its comparative study. Int J Pure Appl Biosci. 2018; 6:1567-77


Hou Y, Wu Z, Dai Z, Wang G, Wu G. Protein hydrolysates in animal nutrition: industrial production, bioactive peptides, and functional significance. J Anim Sci Biotechnol. 2017; 8:24


Shaviklo AR, Alizadeh-Ghamsari AH, Etemadian Y, Rafipour F. The effects of dietary fish protein hydrolysate-based supplementation on sensory properties and meat quality of broiler chicken. Turk J Vet Anim Sci. 2021; 45:1041-51


Mahata ME, Dharma A, Ryanto HI, Rizal Y. Effect of substituting shrimp waste hydrolysate of Penaeus merguensis for fish meal in broiler performance. Pak J Nutr. 2008; 7:806-10


Opheim M, Sterten H, Øverland M, Kjos NP. Atlantic salmon (Salmo salar) protein hydrolysate - effect on growth performance and intestinal morphometry in broiler chickens. Livest Sci. 2016; 187:138-45


Thuy NT, Ha NC. Effects of inclusion of protein hydrolysis from Tra catfish by-product waste water in the diets on apparent ileal digestibility and total tract retention coefficients of local chickens. Livest Res Rural Dev. 2017; 29:55


AOAC [Association of Official Analytical Chemists] International. Official methods of analysis of AOAC International. 18th ed. Arlington, VA: AOAC International. 2005


Goulas AE, Kontominas MG. Effect of salting and smoking-method on the keeping quality of chub mackerel (Scomber japonicus): biochemical and sensory attributes. Food Chem. 2005; 93:511-20


Long SF, Kang S, Wang QQ, Xu YT, Pan L, Hu JX, et al. Dietary supplementation with DHA-rich microalgae improves performance, serum composition, carcass trait, antioxidant status, and fatty acid profile of broilers. Poult Sci. 2018; 97:1881-90


Aviagen. Ross 308: broiler nutrition specifications 2019 [Internet]. 2019[cited 2020 Apr 18]


Marcu A, Vacaru-Opriş I, Dumitrescu G, Ciochina LP, Marcu A, Nicula M, et al. The influence of genetics on economic efficiency of broiler chickens growth. Anim Sci Biotechnol. 2013; 46:339-46


Metzler-Zebeli BU, Magowan E, Hollmann M, Ball MEE, Molnár A, Witter K, et al. Differences in intestinal size, structure, and function contributing to feed efficiency in broiler chickens reared at geographically distant locations. Poult Sci. 2018; 97:578-91


Corrier DE, DeLoach JR. Evaluation of cell-mediated, cutaneous basophil hypersensitivity in young chickens by an interdigital skin test. Poult Sci. 1990; 69:403-8


Verma J, Johri TS, Swain BK, Ameena S. Effect of graded levels of aflatoxin, ochratoxin and their combinations on the performance and immune response of broilers. Br Poult Sci. 2004; 45:512-8


Khalaji S, Zaghari M, Hatami KH, Hedari-Dastjerdi S, Lotfi L, Nazarian H. Black cumin seeds, Artemisia leaves (Artemisia sieberi), and Camellia L. plant extract as phytogenic products in broiler diets and their effects on performance, blood constituents, immunity, and cecal microbial population. Poult Sci. 2011; 90:2500-10


Proietti PC, Dal Bosco A, Hilbert F, Franciosini MP, Castellini C. Evaluation of intestinal bacterial flora of conventional and organic broilers using culture-based methods. Ital J Anim Sci. 2009; 8:51-63


Engberg RM, Hedemann MS, Steenfeldt S, Jensen BB. Influence of whole wheat and xylanase on broiler performance and microbial composition and activity in the digestive tract. Poult Sci. 2004; 83:925-38


Graham BD, Selby CM, Teague KD, Graham LE, Vuong CN, Latorre JD, et al. Development of a novel in ovo challenge model for virulent Escherichia coli strains. Poult Sci. 2019; 98:5330-5


SAS. SAS/STAT® 9.1 user’s guide. Cary, NC: SAS Institute. 2004


Duncan DB. Multiple range and multiple F tests. Biometrics. 1955; 11:1-42


Al-Marzooqi W, Al-Farsi MA, Kadim IT, Mahgoub O, Goddard JS. The effect of feeding different levels of sardine fish silage on broiler performance, meat quality and sensory characteristics under closed and open-sided housing systems. Asian-Australas J Anim Sci. 2010; 23:1614-25


Ramírez JCR, Espain JII, Leyva RG, Ulloa JA, Ulloa PR. Use of biological fish silage in broilers feed: effect on growth performance and meat quality. J Anim Plant Sci. 2016; 27:4293-304


Ramírez JCR, Ibarra JI, Romero FA, Ulloa PR, Ulloa JA, Matsumoto KS, et al. Preparation of biological fish silage and its effect on the performance and meat quality characteristics of quails (Coturnix coturnix japonica). Braz Arch Biol Technol. 2013; 56:1002-10


Lilburn MS, Loeffler S. Early intestinal growth and development in poultry. Poult Sci. 2015; 94:1569-76


Ravindran V, Abdollahi MR. Nutrition and digestive physiology of the broiler chick: state of the art and outlook. Animals. 2021; 11:2795


Seidavi A, Asadpour L, Dadashbeiki M, Payan-Carreira R. Effects of dietary fish oil and green tea powder supplementation on broiler chickens immunity. Acta Sci Vet. 2014; 42:1-13


Guo Y, Guo X, Deng Y, Cheng L, Hu S, Liu H, et al. Effects of different rearing systems on intramuscular fat content, fatty acid composition, and lipid metabolism-related genes expression in breast and thigh muscles of Nonghua ducks. Poult Sci. 2020; 99:4832-44


Kiflay S, Temesgen G, Taye M. Effect of fish offal meal on feed intake, growth performances and carcass characteristics of Hubbard Classic broiler chicken breeds. J Agric Environ Sci. 2018; 371-86


Okazaki T, Noguchi T, Igarashi K, Sakagami Y, Seto H, Mori K, et al. Gizzerosine, a new toxic substance in fish meal, causes severe gizzard erosion in chicks. Agric Biol Chem. 1983; 47:2949-52


Contreras M, Zaviezo D. Causes of gizzard erosion and proventriculitis in broilers. Poult Inf Prof. 2007; :1-4


Shabani A, Boldaji F, Dastar B, Ghoorchi T, Zerehdaran S, Ashayerizadeh A. Evaluation of increasing concentrations of fish waste silage in diets on growth performance, gastrointestinal microbial population, and intestinal morphology of broiler chickens. Anim Feed Sci Technol. 2021; 275:114874


Saki AA, Abbasinezhad M, Ghazi S, Tabatabai MM, Ahmadi A, Zaboli K. Intestinal characteristics, alkaline phosphatase and broilers performance in response to extracted and mechanical soybean meal replaced by fish meal. J Agric Sci Technol. 2012; 14:105-14


Ashayerizadeh O, Dastar B, Samadi F, Khomeiri M, Yamchi A, Zerehdaran S. Study on the chemical and microbial composition and probiotic characteristics of dominant lactic acid bacteria in fermented poultry slaughterhouse waste. Waste Manag. 2017; 65:178-85


Al-Khalaifah H, Al-Nasser A. Dietary supplementation with various fat oils affect phytohemagglutinin skin test in broiler chickens. Front Immunol. 2020; 11:1735


Seidavi A, Simões J. Evaluation of dietary fish oil plus green tea supplementation on the gizzard, ileum and cecum microflora in broiler chickens. Arch Zootec. 2015; 64:397-402


Selvaraj RK, Shanmugasundaram R, Rengasamy R. The use and abuse of immune indices in nutritional immunology studies. J Appl Poult Res. 2016; 25:284-91