Chemical analysis, antibacterial, and antioxidant activities of FWMPH
In general, chemical compositions (protein, fat, ash, and moisture) of FWMPH in feed determines the energy requirement and growth performance of broiler chicks. FWMPH exhibited the 72.52 ± 1.61% protein content, suggests that FWMPH could be utilized as a protein supplement in broiler diets. FWMPH had the 13.43 ± 0.39% moisture, 7.67 ± 1.11% fat and 6.06 ± 0.63% ash content.
FWMPH demonstrated excellent antibacterial efficacy against E. coli, S. aureus, and S. enteritidis. Antibacterial potentials of FWMPH may improve the storage stability of feed and maintain healthier gut flora of broiler chicks. This can also be interpreted as preventing the spread of salmonella in feed, a food- and water-borne disease that is regularly seen in broilers. In addition to antibacterial activities, FWMPH exhibited the antioxidants activities, including, DPPH (531.31 ± 19.62 µmol Trolox equivalent/g of FWMPH), TEAC (382.49 ± 37.72 µmol Trolox equivalent/g of FWMPH), FRAP (404.76 ± 49.23 µmol Trolox equivalent /g of FWMPH), metal chelating activity (479.33 ± 81.69 µmol EDTA/g of FWMPH). FWMPH also displayed the iron-binding (76.81 ± 1.97%), and ACE-inhibition activity (35.17 ± 2.41%). Hence, FWMPH is the multi-biofunctional protein hydrolysate that could endorse biological and nutritional functions in the broiler chicken feed. It has been stated that the incorporation of multi-biofunctional protein hydrolysate as a protein ingredient in animal diet could improve gut health, feed efficiency, growth performance, and abilities to resist infectious diseases in animals (Hou et al. 2017). Therefore, FWMPH can be explored as biofunctional protein ingredient in the in poultry feed that could help in healthy growth performance of broiler chicks.
Daily feed intake (FI), protein intake (PI), and body weight gain (BWG) of broiler chicks
Figure 1 (a, b, and c, respectively) depicts daily FI, PI, and weekly BWG of broiler chicks fed with different diets (SPSD, FMP-25, FMP-50, FMP-75, and FMP-100). Protein is an important nutrient required for the growth and development of broiler chicks. Moreover, the protein quality influences the overall development and growth performance of broilers birds. In the present study, feed consumption (daily FI and PI) and BWG of all diet group chicks increased with the feeding period until the end of the feeding trial (6 weeks). The result demonstrates that the palatability and acceptability of the diet incorporated with FWMPH were better than the standard diet. Higher BWG by all group chicks might be due to higher digestibility of feed and a higher conversion rate into body mass. Further, maximum feed consumption by all diet groups was observed from the 3rd to the 6th week. Generally, during the growing phase (3rd week onwards), chicks required more feed energy for overall growth. Among the all-diet groups, the highest feed consumption showed by FMP-25 diet group chicks (P < 0.05), and the lowest consumption was presented by FMP-100 diet group chicks (P < 0.05). FWMPH at a higher level (100%) might contribute to the off-smell, off-taste, and off-color to feed, which resulted in reduced feed palatability and lower feed consumption by the FMP-100 diet group chicks. In general, chicken broilers are selective about feed characteristics like aroma, appearance, and taste. The sense of taste is essential to ensure adequate ingestion of nutrients and allow the broiler to select pleasant ingredients and discard those that are unpleasant (Cuevas et al. 2005). Further, SPSD, FMP-50, and FMP-75 showed no significant difference for feed consumption (P > 0.05). This result implied that the palatability of the FMP-50 and FMP-75 diets is similar to the SPSD diet. Therefore, the soy protein can be replaced with 50% or 75% FWMPH in the broiler diet. The average feed intake of SPSD, FMP-25, FMP-50, FMP-75, and FMP-100 was found to be 502.20, 528.70, 501.82, 492.60, and 455.70 g/chick, respectively.
The reliable indicator of growth is BWG as it sheds light on how the feed has influenced the development of broilers (Lovell 1989).
BWG is a good indicator for the determination of diet influence on growth of broiler chicks (Lovell 1989). In general, all FWMPH containing diet chicks exhibited higher body weight gain than SPSD group chicks throughout the feeding period. This might be because the FWMPH provides a high level of quality protein or essential amino acids than the soy protein (SPSD), resulting in higher body weight gain and development of chicks. It has been stated that meat protein is a high-quality protein due to a good balance of essential amino acids, higher digestibility, and biological value (Ravindran 2015; FAO 2013). Meat meals and fish meals have been used as high-quality animal protein sources in poultry diets (Oluba et al. 2019). Generally, broiler chicks require quality dietary protein for their better growth performance. The mean weekly body weight gain of SPSD, FMP-25, FMP-50, FMP-75, and FMP-100 during the 6 weeks was 640.44, 691.03, 747.11, 821.16, 736.14 g/chick, respectively. Among the FWMPH diet groups, FMP-75 presented the highest body weight gain compared to other FWMPH diet group counterparts (P < 0.05). The results indicated that a 75% FWMPH diet could help in gut development and rapid muscle growth at a higher extent and resulted in the highest weight gain of broiler chicks. On the other hand, FMP-25 diet group chicks showed the lowest body weight gain among the FWMPH diet groups though had higher feed consumption throughout the feeding study (P < 0.05). This might be associated with the lower content of FWMPH as a protein source in the diet when compared to other FWMPH diet counterparts. No significant difference in the body weight gain of chicks fed with FMP-50 and FMP-100 diets was noticed except in the 6th week (P > 0.05). Therefore, FWMPH at 75% level enhanced the bodyweight of the broiler chicks, compared to other diet groups.
FCR, FER, and PER
FCR, FER, and PER of broiler chicks fed with different diets are presented in Fig. 1d. Broiler chicks fed with the FMP-75 diet showed the lowest FCR (1.96) among the all diet group counterparts (P < 0.05). This result indicated the presence of a sufficient amount of good quality proteins in the FMP-75 diet, which resulted in the higher body weight gain of broiler chicks. Lower the FCR, higher the feed quality, and high weight gained by the broiler chicks. Besides, SPSD (2.57) and FMP-25 (2.55) diet groups exhibited the highest FCR values (P < 0.05), followed by FMP-50 (2.23) and FMP-100 (2.18). A higher FCR value represents low feed quality. Hence, SPSD and FMP-25 diets imply the low-quality feed among all the diet group counterparts. FCR values of SPSD and FMP-25 diet group and FMP-50 and FMP-100 diet presented no significant difference (P > 0.05). The FMP-75 broilers revealed the maximum FER (0.51) and PER (2.32) values, implying that a higher weight gain was obtained from the FMP-75 diet when compared to other diet groups. FMP-100 diet chicks had 0.46 FER and 2.09 PER values, followed by FMP-50 (0.45 and 2.04), FMP-25 (0.39 and 1.78), and SPSD (0.39 and 1.77), respectively. However, no significant difference was noticed for FER and PER values between FMP-100 and FMP-50 and between FMP-25 and SPSD diet groups (P > 0.05). Maliwan et al. (2017) also observed that Korat chickens had a higher feed intake but exhibited poor FCR, PER, and energy efficiency ratio values. Overall, FMP-75 diet had the potential to enhance the broiler’s growth performance when compared to other diet counterparts. Therefore, 75% FWMPH could be used as a quality protein in broiler feed.
Hematology and serum biochemistry
The impacts of the different diets (SPSD, FMP-25, FMP-50, FMP-75, and FMP-100) on the hematological parameters of the broiler chicks are depicted in Table 2. All the experimental groups exhibited the hematology values within the normal reference range (Borsa et al. 2006; Cafe et al. 2012). The WBC, RBC, HGB, HCT, MCV, MCH, MCHC and LYM were in the range of 18.36–20.95 103/µL, 2.47–2.92 106/µL, 11.28–13.16 g/dL, 25.11–29.73%, 129.48-131.48 fL, 39.58–41.37 pg, 30.03–30.92 g/dL and 4.12–4.29%, respectively. FMP-75 fed broilers presented higher RBC (2.92 106/µL) and HGB (13.16 g/dL) values among all diet groups (P < 0.05). Accessible heam-iron in diet might have enhanced the RBC and HGB content. All FWMPH fed diet groups exhibited a significantly higher content of WBC, HCT, and MCH than SPSD (P < 0.05). Among the FWMPH fed groups, FMP-50 broilers displayed a slightly higher content of WBC. No significant difference in MCH values was noted among all FWMPH fed groups (P > 0.05). Furthermore, FMP-50 and FMP-75 diet groups exhibited the highest value of HCT compared to all diet groups. MCV was not significantly different across all diet groups. FMP-25 group showed slightly higher values of MCHC and LYM than others counterparts (P < 0.05). The lowest value of MCHC was noted in the SPSD diet group (P < 0.05), however, FMP-50, FMP-75, and FMP-100 diet groups did not show any significant difference in MCHC (P > 0.05). FMP-75 and FMP-100 diet groups presented the lowest content of LYM when compared to FMP-25, FMP-50, FMP-100 and SPSD diet group chicks (P < 0.05). In general, the comparable hematological parameters suggested that all group broilers chicks were healthy.
The serum biochemical profile of the broiler chicks is shown in Table 3. Generally, toxicity of feed mostly detected by the serum biochemical profile. Serum biochemical indices were influenced by different diet groups and are within the normal range established for broilers by Borsa et al. (2006) and Cafe et al. (2012). SGPT, SGOT, and ALP act as enzyme markers, and their levels in serum indicate the function of the liver. SGPT, SGOT, and ALP levels of all diet groups were in the range of 15.96–20.52 (U/L), 177.00-190.58 (U/L), and 167.50-179.01(U/L), respectively. FMP-100 diet group presented the slightly higher level of SGPT, SGOT, ALP, albumin, and cholesterol levels, followed by the FMP-75 diet groups (P < 0.05). The result revealed that the replacement of 100% soya protein by FWMPH in feed faintly boosted the levels of SGPT, SGOT, ALP, and cholesterol levels. Besides, lowest level of SGOT, ALP, and albumin were shown by the FMP-25 diet group (P < 0.05). However, SPSD and FMP-50 diet groups showed no significant albumin level difference (P > 0.05). Serum protein albumin synthesis gives the availability of protein and micro-nutrient in feed. SPSD diet group displayed the lowest SGPT and cholesterol levels (P < 0.05). The highest glucose level was found in the FMP-75 and FMP-100 groups, followed by the FMP-50 and FMP-25 diet groups (P < 0.05). No significant difference in glucose level between FMP-75 and FMP-100, and FMP-50 and FMP-25 was noted (P > 0.05). The highest creatinine level was noticed in FMP-50 to FMP-100 diet groups (P < 0.05), followed by FMP-25 and SPSD diet groups. No significant difference was noticed in triglycerides concentration in SPSD and FWMPH fed chicks (P > 0.05). FWMPH fed diet groups showed a slightly higher urea level compared to the SPSD diet group (P < 0.05). Conversely, the SPSD diet group presented a slightly higher level of total protein level when compared to FWMPH fed diet group counterparts (P < 0.05). Among the FWMPH fed groups, the FMP-100 showed a slightly higher protein value, followed by FMP-75 and FMP-50, respectively. Overall, it can be inferred from the hematological and serum biochemical parameters that the FWMPH did not have any adverse effects on the growth performance of broiler chicks. Therefore, 75% FWMPH can be used as a biofunctional protein ingredient in feed to maintain the nutrition status of broiler chicks.
Liver biochemistry and antioxidant activities of broiler chicks fed with different diets are shown in Table 4. Liver is considered to be a model organ to find the toxicity of diet in broiler chicks. Despite this, the liver involves in the metabolism of fat, carbohydrate, protein, vitamins, and minerals. The liver of FMP-100 diet group chicks presented the highest level of SGPT, SGOT, and ALP, followed by FMP-75 and FMP-50 diet groups (P < 0.05). A similar trend for SGPT, SGOT, and ALP levels was noticed in the serum biochemical profile (Table 3). Slightly higher SGPT, SGOT, and ALP in the FMP-100 diet group may be due to a higher level of FWMPH. The FMP-25 diet had the lowest level of SGOT and ALP activity (P < 0.05), whereas, the SPSD diet group showed the lowest SGPT activity (P < 0.05). Furthermore, the liver of the FMP-75 and FMP-100 diet group exhibited the highest cholesterol level, followed by the FMP-50, FMP-25, and SPSD broilers (P < 0.05). The outcome demonstrated the presence of 75% FWMPH in the diet slightly enhanced the cholesterol levels. Conversely, SPSD and FMP-25 diet groups displayed the lowest content of triglycerides levels and highest found in the FMP-75 and FMP-100 diet groups (P < 0.05). Further, the highest total protein level was noted in the FMP-100 diet group, followed by the FMP-75 and FMP-50 diet groups, and lowest in the SPSD diet group (P < 0.05). The antioxidant capacity of FWMPH in broiler chicks was measured using SOD and CAT activities in the liver. FMP-75 diet group had the highest SOD and CAT activity, followed by FMP-100 and FMP-50, respectively. Since FWMPH had an antioxidant potential, it was reflected in the in vivo broiler experiment and it can be concluded that a 75% FWMPH diet increased antioxidant capacity. However, as compared to the other diet groups, the SPSD diet group had the lowest SOD and CAT activities. Hence, substitution of 75% FWMPH in poultry feed could enhance the antioxidant potential in the broiler chick liver.
Relative organ weights and histopathology studies
The relative organ weights (g/100 g BW) of broiler chicks fed with different diets are shown in Table 5. The relative organ weights of broiler chicks also reveals the toxicity of feed. In the present study, FMP-75 and SPSD diet group chicks exhibited the highest and lowest relative organ weight of lungs (P < 0.05). The findings suggest that the FMP-75 diet may have aided proper or regular organ development and growth, owing to the inclusion of high-quality proteins (FWMPH) than the SPSD diet. The results were in line with the body weight gain of the broiler chicks (Fig. 1c). Further, no significant difference in relative organ weight of lung was noticed among the SPSD, FMP-25, and FMP-50 diet groups (P > 0.05). Tang et al. (2012) revealed that the organ weights of broilers were impacted by feeding the fermented ingredients. No significant difference in the liver weight of all diet groups was noticed (P > 0.05). Among all diet groups, FMP-75 showed the highest relative organ weight of heart, while, lowest presented by FMP-25 and FMP-100 diet groups (P < 0.05). Besides, FMP-25 and FMP-100 diet groups, and SPSD and FMP-50 diet groups revealed the no significant difference in heart organ weight (P > 0.05). The gizzard of SPSD and FMP-75 diet-fed broiler chicks exhibited higher relative weight in comparison to other groups (P < 0.05), however, no significant difference was found between them (P > 0.05). FMP-100 showed the lowest relative organ weight for kidneys, while other groups dint not have a significant difference (P > 0.05). In general, slight variations in relative organ weights all diet groups of chicks was observed but no adverse effect on the growth of lungs, liver, heart, gizzard, and kidney in broiler chicks. Overall, the FMP-75 diet group showed better growth of all studied organs compared to other diet group counterparts. Therefore, the FMP-75 diet could be feasible for the better growth and health of vital organs of broiler chicks.
The histological structure of the lungs, liver, heart, intestine, gizzard, and kidney are presented in Fig. 2. Diet can impact the structural characteristics of internal organs. No significant structural deviations in lungs, liver, heart, intestine, gizzard, and kidney of diet groups fed with FWMPH were noted. The result suggested that incorporation of FWMPH in broiler diets at all concentrations showed no significant toxic effects on vital organs of broiler chicks. Moreover, FWMPH and SPSD diet groups displayed maximum resemblances in the histological investigation of vital organs. The alveoli and bronchioles grew normally in all diet groups, indicating that there were no notable alterations in lung anatomy. In all diet groups, the histological structure of the liver exhibited normal hepatocytes, integrated architecture, portal veins, and no obvious symptoms of edema or hyperemia. Liver histopathology is well recognized examination for the investigation of feed toxicity in animal. All diet groups showed normal liver parenchyma structure with maintained portal and triad architecture. Across the all diet groups, the kidney revealed typical tubular structure nephrocytes and bowmen's capsules, with no noticeable alterations. The heart displayed striated muscle fibers with no gangrene or degeneration in all diet groups. The gizzard muscle of SPSD, FMP-25, FMP-50, and FMP-100 diet group chicks were striated, completely homogeneously stained, and showed normal architecture. However, the gizzard of the FMP-100 diet group showed the thickened lining with prominent folds and roughened, showing a bark-like appearance. Itakura (1981) reported that young chicks fed with the fish meal had mild lesions, which are natural. Those lesions had no relation to the fish meal consumed. The structure of villi showed minor ruptures across all the diet groups, but it is not influenced by the diet treatment. Overall, the result ascertained that incorporation of FWMPH in broiler chick’s diet had no organ toxicity. Thus, the relative organ weight and the histopathological studies demonstrated that the addition of FWMPH up to 75% as a protein source in the broiler diet did not affect the normal growth of vital organs and did not have any toxicology effect on the growth performance of experimental broiler chicks. Thus, 75% of FWMPH could be a substitute in the standard broiler feed as an alternative dietary protein source.