Tohid Mokarrami, Bahman Navidshad *1, Sadegh Karimzadeh, Ali Kalantari Hesari, Farzad Mirzaei Aghjehgheshlagh
Abstract: (25 Views)
Introduction and Objective: The poultry industry has faced two major challenges in recent years: first, the increasing demand for antibiotic-free products due to concerns about antimicrobial resistance, and second, the need to improve production efficiency and bird health without the use of chemical growth promoters. In this context, bioactive peptides (short fragments of proteins, typically 2–20 amino acids) released through enzymatic hydrolysis have emerged as one of the most promising alternatives. Blood meal from slaughterhouses is a valuable protein source rich in essential amino acids, but its direct use in poultry diets is limited due to poor palatability and low digestibility. Enzymatic hydrolysis of blood meal can convert large proteins into short-chain, low-molecular-weight peptides, which not only have higher bioavailability but also acquire new functional properties such as immunomodulation and antioxidant activity. The present study aimed to evaluate the effects of different levels of bioactive peptides derived from enzymatic hydrolysis of blood meal (BMP) on antibody titers against Newcastle disease virus, serum oxidative stress parameters (TAC and MDA) and metabolic parameters, as well as the population of two major ileal bacterial groups (Lactobacillus and Escherichia coli) in broiler chickens.
Materials and Methods: First, bioactive peptides were prepared from slaughterhouse blood meal using enzymatic hydrolysis with Alcalase (enzyme-to-substrate ratio 1:20, temperature 50°C, pH 8, for 2 hours). The hydrolysate was then heat-inactivated and lyophilized. Molecular weight distribution analysis by HPLC showed that over 62% of the peptides were in the range of 180–500 Daltons, which facilitates intestinal absorption via the PepT1 transporter and may enhance its biological activity. A total of 400 one-day-old broiler chicks (Ross 308) were distributed in a completely randomized design with 5 treatments, 5 replicates, and 16 birds per replicate. Experimental treatments were: 1) basal diet without additive (control), 2) basal diet + 0.2% BMP, 3) basal diet + 0.4% BMP, 4) basal diet + 0.6% BMP, and 5) basal diet + 0.8% BMP. Diets were formulated according to Ross 308 requirements over four feeding periods. On day 35, blood samples were collected from two birds per replicate, and antibody titers against Newcastle disease virus were measured by hemagglutination inhibition (HI) test. Serum biochemical parameters (glucose, cholesterol, triglycerides, total protein, albumin, liver enzymes AST and ALT, HDL, and uric acid) and oxidative stress indices including total antioxidant capacity (TAC) by FRAP method and malondialdehyde (MDA) by thiobarbituric acid method were measured. On day 38, ileal contents were cultured for Lactobacillus counts (on MRS Agar under anaerobic conditions) and Escherichia coli counts (on Chromogenic Agar under aerobic conditions). Data were analyzed using SAS software with the GLM procedure in a completely randomized design, and means were compared by Tukey's test at P < 0.05.
Results: BMP supplementation significantly increased antibody titers against Newcastle disease virus (P < 0.0001). The control group had the lowest titer (5.8 log₂), while 0.4%, 0.6%, and 0.8% BMP treatments produced titers of 7.7, 8.4, and 8.5 log₂, respectively, which were significantly higher than the control (P < 0.05). No significant differences were observed among the three higher levels, indicating a threshold level of approximately 0.4% for maximal immune stimulation. Serum TAC and uric acid levels decreased linearly with increasing BMP (P < 0.0001), with the lowest values recorded in the 0.8% BMP treatment (TAC from 2.48 to 1.40 mmol/L, and uric acid from 5.46 to 2.54 mmol/L). MDA levels showed a decreasing trend (from 2.56 to 1.92 nmol/mL) but this reduction was not significant (P = 0.106). None of the other metabolic parameters (glucose, cholesterol, triglycerides, liver enzymes, albumin, total protein, HDL) were affected by BMP (P > 0.05). Ileal populations of Lactobacillus and Escherichia coli also did not change significantly (P = 0.1834 and P = 0.8670, respectively), although numerically Lactobacillus increased in BMP treatments.
Conclusion: The present study comprehensively demonstrated that bioactive peptides derived from enzymatic hydrolysis of blood meal (BMP), with a predominance of peptides in the 180–500 Dalton molecular weight range, possess strong immunomodulatory and antioxidant effects in broiler chickens. Dietary supplementation with BMP at levels of 0.4% to 0.8% significantly increased antibody titers against Newcastle disease virus, indicating improved vaccine response. The decreases in serum uric acid and total antioxidant capacity, along with a downward trend in malondialdehyde, suggest alterations in systemic oxidative status rather than a definitive reduction in oxidative stress. Importantly, these effects occurred without significant changes in ileal microflora populations (Lactobacillus and Escherichia coli) and without disturbing basal metabolic parameters or liver enzymes, indicating that the main mechanism of BMP action is through direct host pathways (rather than through microbiota modulation). Importantly, these effects were observed without significant changes in the populations of ileal Lactobacillus and Escherichia coli and without disruption of basic metabolic parameters or liver enzyme activities. Therefore, the effects of BMP do not appear to be necessarily mediated through alterations in the bacterial populations examined in the ileum, suggesting that direct mechanisms involving host immune responses and antioxidant status may also contribute to the observed effects.From a practical perspective, BMP can be used as a safe, effective, and sustainable feed additive in broiler diets, particularly in antibiotic-free production systems, to reduce oxidative stress and enhance immune system efficiency. The optimal level based on the findings of this study is 0.4% of the diet. Future research should investigate molecular mechanisms (such as gene expression of cytokines and antioxidant enzymes via the Nrf2 pathway) as well as evaluate the effect of BMP under real pathogen challenge conditions.
Materials and Methods: First, bioactive peptides were prepared from slaughterhouse blood meal using enzymatic hydrolysis with Alcalase (enzyme-to-substrate ratio 1:20, temperature 50°C, pH 8, for 2 hours). The hydrolysate was then heat-inactivated and lyophilized. Molecular weight distribution analysis by HPLC showed that over 62% of the peptides were in the range of 180–500 Daltons, which facilitates intestinal absorption via the PepT1 transporter and may enhance its biological activity. A total of 400 one-day-old broiler chicks (Ross 308) were distributed in a completely randomized design with 5 treatments, 5 replicates, and 16 birds per replicate. Experimental treatments were: 1) basal diet without additive (control), 2) basal diet + 0.2% BMP, 3) basal diet + 0.4% BMP, 4) basal diet + 0.6% BMP, and 5) basal diet + 0.8% BMP. Diets were formulated according to Ross 308 requirements over four feeding periods. On day 35, blood samples were collected from two birds per replicate, and antibody titers against Newcastle disease virus were measured by hemagglutination inhibition (HI) test. Serum biochemical parameters (glucose, cholesterol, triglycerides, total protein, albumin, liver enzymes AST and ALT, HDL, and uric acid) and oxidative stress indices including total antioxidant capacity (TAC) by FRAP method and malondialdehyde (MDA) by thiobarbituric acid method were measured. On day 38, ileal contents were cultured for Lactobacillus counts (on MRS Agar under anaerobic conditions) and Escherichia coli counts (on Chromogenic Agar under aerobic conditions). Data were analyzed using SAS software with the GLM procedure in a completely randomized design, and means were compared by Tukey's test at P < 0.05.
Results: BMP supplementation significantly increased antibody titers against Newcastle disease virus (P < 0.0001). The control group had the lowest titer (5.8 log₂), while 0.4%, 0.6%, and 0.8% BMP treatments produced titers of 7.7, 8.4, and 8.5 log₂, respectively, which were significantly higher than the control (P < 0.05). No significant differences were observed among the three higher levels, indicating a threshold level of approximately 0.4% for maximal immune stimulation. Serum TAC and uric acid levels decreased linearly with increasing BMP (P < 0.0001), with the lowest values recorded in the 0.8% BMP treatment (TAC from 2.48 to 1.40 mmol/L, and uric acid from 5.46 to 2.54 mmol/L). MDA levels showed a decreasing trend (from 2.56 to 1.92 nmol/mL) but this reduction was not significant (P = 0.106). None of the other metabolic parameters (glucose, cholesterol, triglycerides, liver enzymes, albumin, total protein, HDL) were affected by BMP (P > 0.05). Ileal populations of Lactobacillus and Escherichia coli also did not change significantly (P = 0.1834 and P = 0.8670, respectively), although numerically Lactobacillus increased in BMP treatments.
Conclusion: The present study comprehensively demonstrated that bioactive peptides derived from enzymatic hydrolysis of blood meal (BMP), with a predominance of peptides in the 180–500 Dalton molecular weight range, possess strong immunomodulatory and antioxidant effects in broiler chickens. Dietary supplementation with BMP at levels of 0.4% to 0.8% significantly increased antibody titers against Newcastle disease virus, indicating improved vaccine response. The decreases in serum uric acid and total antioxidant capacity, along with a downward trend in malondialdehyde, suggest alterations in systemic oxidative status rather than a definitive reduction in oxidative stress. Importantly, these effects occurred without significant changes in ileal microflora populations (Lactobacillus and Escherichia coli) and without disturbing basal metabolic parameters or liver enzymes, indicating that the main mechanism of BMP action is through direct host pathways (rather than through microbiota modulation). Importantly, these effects were observed without significant changes in the populations of ileal Lactobacillus and Escherichia coli and without disruption of basic metabolic parameters or liver enzyme activities. Therefore, the effects of BMP do not appear to be necessarily mediated through alterations in the bacterial populations examined in the ileum, suggesting that direct mechanisms involving host immune responses and antioxidant status may also contribute to the observed effects.From a practical perspective, BMP can be used as a safe, effective, and sustainable feed additive in broiler diets, particularly in antibiotic-free production systems, to reduce oxidative stress and enhance immune system efficiency. The optimal level based on the findings of this study is 0.4% of the diet. Future research should investigate molecular mechanisms (such as gene expression of cytokines and antioxidant enzymes via the Nrf2 pathway) as well as evaluate the effect of BMP under real pathogen challenge conditions.
Keywords: Antioxidant capacity, Bioactive peptide, Blood meal hydrolysate, Humoral immunity, Ileal microflora.
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