3.1. Protein expression verification
EGF is a member of the growth promoting factor family, which can participate in a series of physiological processes, such as promoting cell division and growth, stimulating the synthesis and secretion of some extracellular macromolecules, embryo development, tissue regeneration, regulating the function of endocrine system and wound healing, etc. We constructed and optimized a chEGF sequence and transformed it into BL21 (DE3). Fig.1 shows protein expression detected by western-blot, the protein is about 30 kDa which consistent with the target protein. Then the chEGF protein was fed to broilers to detect its effect on the digestive function of broilers.
3.2. Effect on digestive enzyme
Alkaline protease is a common indicator to evaluate the growth performance for neonate animal and human beings. Chicken ALP in duodenum, jejunum and ileum from each group were measured at 21, 28, 35 and 42 day old broiler by sandwich ELISA (Fig.2). The results show that ALP in duodenum from 21-day-old broilers seem to decrease due to the disturbance of antibiotics. The supplement of EGF could improve the clinical performance in this stage. Although ALP in jejunum and ileum from EGF group has similar performance with those from ND and CTC groups, both 21-day-old and 42-day-old broilers secreted more ALP in duodenum with chEGF supplement. Interestingly, chEGF supplement didn’t promote these expression at a high level all the time. Thus, proper supplement can be used for modulating chicken intestinal health.
With the help of bile salts, lipases, as a group of fat-splitting enzymes, hydrolyze triglycerides into fatty acid and glycerol molecules and execute initial fat digestion. Associated with the feed efficiency, lipases in chicken can be utilized to evaluate digestive ability. The level in the intestinal juice during different growth phase is a dynamic process. However, diet supplement such as EGF and antibiotic may break the equilibrium and regulate intestinal health. We found that alternation of lipases in the chicken ileum is obvious. Compared with normal diet, the expression of lipases in CTC group was unstable at the 28 and 35 days, but upregulated in EGF group at 35 days with significant data (Fig.3).
Due to the hydrolysis of starch, α-amylase is always used as a main indicator to access the sugar digestive ability. Different from proteases and lipases, α-amylase in each sample collection time own an increasing tendency. Especially, the level of α-amylase in duodenum and jejunum among 35-day-old broliers was significantly upregulated via EGF supplement (Fig.4).
3.3. Effect on the expression of duodenal and jejunal EGFR in situ hybridization
Through evaluating fluorescent mean density under the dark field, we found the expression of EGFR in duodenum significantly increasing via EGF supplement compared with normal diet and antibiotic diet groups (Fig.5).
3.4. Effect on morphology of intestinal barrier
Different from antibiotics, chEGF is a natural protein existing in the animal host. Fig.6 shows that at early time (21 days) post-administration chEGF in the duodenum, the ratio of villus to crypts was decreased because of the decreasing height of villus. Most intestinal stem cells usually origin from the crypts, which means chEGF supplement may promote the stem cell proliferation by activating EGF associated signaling pathway and create a pool to adapt nutrition uptaken. Interestingly, it is obvious that chEGF functioned with spatial-temporal diversity. A long period chEGF supplement can slightly increase the ratio (V/C) in the ileum, but there is nearly no alternation with a short supplement. The long period antibiotics’ supplement can mitigate or destroy the length of villus at whole intestines.
3.5. Growth performance in a pilot assay
According to previous research, experimental evidences proved that EGF is a potential alternative supplement of antibiotics. A pilot assay to evaluate their growth-promoting role was conducted with white feather broilers. Consistant vaccine process and feeding administration were performed with limited EGF containing drinking water (100g/ton) as shown in Table 2. We found that European Efficiency Index (EEI), a most important indicator of broiler productivity, was significantly upregulated via supplement of EGF in this pilot involving more than 167,000 broiler (Fig. 7).
Table 2 Growth Performance of a broiler farm in Shandong, China
Nursery Number
|
total weight
|
Number
|
average weight
|
feeding weight
|
feed conversation rate (FCR)
|
Ratio of Survival
|
Fattened days
|
1
|
46337.07
|
17460
|
2.654
|
68040
|
1.468
|
94.47
|
45
|
2
|
44185.05
|
17234
|
2.564
|
66200
|
1.498
|
93.25
|
44
|
3
|
47367.56
|
17690
|
2.678
|
70120
|
1.48
|
95.71
|
45
|
4
|
47769.31
|
17816
|
2.681
|
72080
|
1.509
|
96.4
|
46
|
5
|
46590.74
|
17596
|
2.648
|
70720
|
1.518
|
95.21
|
46
|
6
|
45444.26
|
17719
|
2.565
|
69560
|
1.531
|
95.45
|
46
|
7
|
44205.02
|
17600
|
2.512
|
67760
|
1.533
|
94.81
|
45
|
8
|
44926.16
|
17775
|
2.527
|
69280
|
1.542
|
95.75
|
45
|
9
|
33204.85
|
13349
|
2.487
|
52280
|
1.574
|
94.39
|
45
|
10
|
35470.97
|
13433
|
2.641
|
54240
|
1.529
|
94.96
|
45
|