Growth performance
The performance of broilers during various phases of raising is displayed in Table 2. No significant differences were noted in ADFI, ADWG, and FCR between two diet groups in the starter (0 to 10 d), and grower (11 to 24 d) periods. In addition, adding MFBAS in water did not improve ADFI and ADWG; consequently, no betterment was found for FCR in the entire period (0 to 36 d). Similar to the current findings, no effect of dietary supplementation of the potato antimicrobial peptide (AMP) on the performance of pigs has been reported (Jin et al., 2009). However, Ohh et al. (2009) published that adding an increasing level of antimicrobial peptide (AMP) isolated from solanum tuberosum to the diet caused an increase in the overall weight gain (Ohh et al., 2009). The current outcomes may be related to the environmental factors of the trial. It has been shown that the effect of feed additives such as probiotics or plant extracts on performance in a pollution-free environment is less (Gunal et al., 2006; Konjufca et al., 1997). Moreover, this discrepancy in results might be due to the concentration of dietary supplementation of MFBAS, or the method of action of the MFBAS.
Interestingly, ADWG was affected in the finisher period that was better in birds that received MFBAS compared to control. In the finisher period, weight gain observed in broilers fed with MFBAS may be due to the improvement in balance and quality of the microflora, small intestinal morphology, and mucosal immune system (Jin et al., 2008; Ohh et al., 2010; Tang et al., 2009). Wang et al. (2009) found that birds fed with antimicrobial peptide (purified from the pig gut) had better weight gain and FCR than birds fed a non-supplemented diet (Bao et al., 2009; Wang et al., 2009).
Ileal and Cecal microbial population
At 10 d of age, the colony-forming units of lactobacillus acidophilus bacteria and total aerobic bacteria in the gut were not considerably influenced by two diet groups (Tables 3). Although ileal of chicks received MFBPs had slightly lower colony-forming units of coliforms than the control chicks, at 10 d. In the second stage of microflora counting (24 d), experiment supplementation with MFBPs had no significant influence on CFU of total aerobic bacteria in the small intestine and cecum, although there was a tendency for numbers of coliforms to be less in the birds receiving MFBPs. In this experiment, drinking water containing 50 ppm MFBPs had the potential to diminish toxic gut bacteria like coliforms. Recent studies proved that the dietary addition of antimicrobial peptides purified from potato tubers has decreased coliform count in excreta and cecal microbiome of broilers (Ohh et al., 2010, 2009). Inconsistency with present results, Karimzadeh et al. (2016) found that birds fed diets containing canola bioactive peptides (CBP) had lower gram-negative bacteria in Ileum and cecal digesta in comparison with control birds (Karimzadeh S, 2016). In addition, Tanmay Paul et al (2015) showed that bioactive peptides isolated from chicken feathers have reduced multiple-antibiotic-resistant (MAR) Staphylococcus aureus count in the in vitro (Paul et al., 2015). The possible reason for the bactericidal activity of the MFBPs is due to bacterial cell membrane disruption, intracellular leakage, and finally bacterial lysis (Brogden, 2005). Moreover, MFBPs can interact with immune cells and enhance the immune response in the face of inflammation, for example, stimulation of cytokine release, and neutralization of LPS-induced septic effects (Elsbach, 2003). However, the effect of MFBPs on decreasing gram-negative bacteria needs to be further investigated.
Morphology of the small intestine
The effects of mixed feather bioactive peptides on gut morphology in 10 and 24 d are shown in tables 4 and 5 respectively. Generally, an increase in villus height has been defined along with the development of the surface area and the increase in the intestinal capacity for absorption (Samanya and Yamauchi, 2002). In the current study, intestinal villus height of the duodenum, and ileum in chickens that received 50 ppm of MFBPs were higher than (P<0.001) control group at 10 d which indicates an increased absorptive capacity (Table 4).
Moreover, the addition of 50 ppm of MFBPs to the drinking water of chickens increased crypt depth when compared to birds of the control group at 10 days of age. In agreement with the finding of our research, the use of antibacterial peptides isolated from pig's small intestine in the diet of broiler chickens increased villus height of the duodenum and jejunum (Bao et al., 2009). In another research, chickens fed at 90 mg/kg AMP diet had greater villus height in the intestine than chickens fed the diet without supplementation of AMP, while dietary treatments had no significant effects on crypt depth of the intestine. (Choi et al., 2013). In addition, MFBPs had a significant effect (P < 0.0001) on the muscle layer thickness in the intestine at 10d. The jejunal epithelium thickness was markedly decreased (P<0.001) using MFBPs in 10d birds, but no effect on the duodenum and ileum. Reduced jejunal epithelium thickness could be due to lower mucin secretion from goblet cells and endogenous protein losses. It has been found that a decrease in the activity of harmful microbes on the surface of the brush border reduces damage to enterocytes in the gut (Hughes, 2003). At 24 d of age, the villus height (duodenum, jejunum, and ileum) and VH: CD (duodenum, jejunum, and ileum) of chickens that received the 50 ppm of MFBPs were not different from control birds. The mixed feather bioactive peptides (MFBPs) supplement at 50 ppm to water significantly decreased (P < 0.001) Villus diameter in the duodenum and increased (P < 0.0001) crypt depth in the duodenum at 24d. Also, the epithelium thickness (duodenum and ileum) was less (P<0.05; Table 5) in chickens that received 50 ppm of MFBPs compared with chickens fed the diet without supplementation of MFBPs at 24d. Silva and Smithard (2002) have been suggested that the decrease in the thickness of the unstirred layer in the small intestine leads to improve nutrient absorption (Silva and Smithard, 2002). In the current research, supplementation of broiler water with 50 ppm of MFBPs increased muscle layer thickness (jejunum) compared to the control group at 24d, suggesting an increased absorptive capacity.
Effect of MFBAS supplemented to diet on serum lipid parameters
The in vivo anticholesterol activity of MFBAS at 10 and 24 days of age was studied. Values of TG, HDL, LDL, VLDL, and experimental groups are shown in Table 6. At d 10 of the experiment, a significant diminish in serum LDL (24.75%), and VLDL (26.9%) levels were noted in the MFBAS group compared to those of the control group. Interestingly, the application of MFBAS for 10 days to MFBAS -fed birds, at a dose of 50 ppm, assisted a significant diminution (p<0.05) in serum TG (26.39%), TC (17.89%) level compared to those of the control group. However, there were no significant differences between serum lipid parameters for birds fed MFBAS and control groups at d 10 of the trial. Indeed, the anticholesterol effects of the MFBAS could be caused by some potent bioactive peptides achieved by the enzymatic hydrolysis of the chicken feathers. In fact, the hypothesis of this anticholesterol action may be due to the high bile acid-binding capacity of peptides and cholesterol excretion in the feces or diminish the micellar solubility of cholesterol in the epithelial cells and finally decrease the blood cholesterol level (Nagaoka et al., 1999; Sugano et al., 1990). Another hypothesis on the cholesterol-lowering mechanism is that some peptides can inhibit enzymes that conduct cholesterol biosynthesis such as the HMG-CoA reducta (Soares et al., 2015). In this regard, Liu et al. (2012) also reported that Rhopilema esculentum protein hydrolysate reduces the plasma cholesterol and triglyceride level in rats fed with high-fat diet (Liu et al., 2012). More research is needed to illuminate the mechanism of action of mixed feather bioactive peptides (MFBPs) from chicken feathers proteins on the cholesterol-lowering.
Meat Quality
The findings on meat quality of leg and breast are presented in Fig 1. The high extent of polyunsaturated fatty acids in poultry meat make it sensitive to oxidative deterioration. (Jimenez et al., 2002). The level of oxidative degradation of lipid by reactive oxygen species (ROS) can be measured by estimating the MDA rate (Wan et al., 2017). In this study, the application of MBPs reduced the (p<0.05) content of MDA in the thigh tissue which displays the effective function of bioactive peptides in the prevention of lipid peroxidation and membrane damage. Therefore, the anti-cholesterol activity of MFBAS could decrease the amount of MDA in the meat; this process resulted in improving the oxidative status and reducing cellular oxidative damage. About 44% of chicken feather protein (keratin) is composed of the amino acid (Dunn, 2004). Cystine has an S-S bond which could play as a natural antioxidant. Organic compounds of sulfhydryl (–SH) and sulphenic acids (–SOH) may be formed under microbial fermentation of chicken feathers which can be considered as powerful antioxidants. Several studies have been demonstrated that herbal products rich in polyphenols can reduce lipid peroxidation in the meat due to the absorption of produced free radicals. (Cao et al., 2012; Gouveia and Castilho, 2013). It is proven that the leg muscle is more sensitive to oxidation than breast meat due to its unsaturated fat level (Higgins et al., 1998; Jensen et al., 1997). Hence, breast meat had lower MDA content than leg muscle in this trial. Similarly, Botsoglou et al. have shown that the MDA values in thigh samples were higher than breast samples of broilers (Botsoglou et al., 2002).