EGF has been considered a powerful bioactive molecule since it was first discovered in 1962; however, initial extraction methods resulted in low yields. Today, this is no longer the case as EGF is mass produced by utilizing a variety of expression systems. However, results from other studies [10, 12–16, 22] have revealed that different types of systems yield remarkably different expression levels of EGF, and even when utilizing the same system, levels of EGF can vary depending on the conditions. In addition, protein production in nisin-induced L. lactis expression systems could be increased by optimizing various factors [23]. In the present study, gEGF was expressed with a nisin-induced L. lactis expression system, and nisin concentration and induction time were optimized for yield. Our results showed that both the concentration of nisin and induction time could affect the expression level of gEGF. As the concentration of nisin increased, the gEGF yield increased initially, followed by a decrease at 10 ng/ml. While a threshold concentration of nisin is required for complete induction, excess concentrations could inhibit the growth of the host strain due to innate antibacterial properties. Further, as the induction time increased, the expression level of EGF increased gradually before decreasing at 12 h, perhaps due to excess lactate accumulation, which disrupts cell metabolism, during cell growth [24, 25]. The concentration of gEGF in the supernatant was 2.67 µg/ml, which was similar to or higher than the results of other studies [12, 14, 16, 22].
EGF promotes cell proliferation and differentiation. Despite minor differences in the amino acid sequences of EGF from different species, similarity of the EGF receptor (EGFR) structure ensures that EGF is not highly species-specific. For example, hEGF has been shown to function in rats [26], mEGF in pigs [27], and pEGF in chickens [21], although some anomalies have been reported. Lax et al. showed that the binding ability of mEGF to chicken EGFR was 100 times lower than that to human EGFR; further, the binding ability of TGF-alpha to chicken EGFR was equal to or higher than that to human EGFR. These results highlight the differences in the binding ability of EGF compared to that of EGFR in different species [28], indicating that EGF of a given species may have different effects for another species. The proliferation of human dermal fibroblasts has been shown to be promoted by hEGF [29]. Therefore, in the current study, a chicken embryo fibroblast line, UMNSAH/DF-1, was used to detect the biological activity of gEGF in vitro. Our results showed that gEGF could promote the proliferation of DF1 cells. Moreover, the ability to promote proliferation increased initially with gEGF concentration before decreasing at 5 µg/ml, indicating that the proliferation-promoting effect of gEGF was concentration-dependent.
EGF has been reported to promote growth performance, improve immune function, and stimulate intestinal development or repair in animals [16, 17, 30, 31]. Wang et al. showed that 14 days of rearing early-weaned piglets with LL-pEGF significantly increased the final body weight and average daily gain of these piglets, compared to the control group [31]. Bedford et al. showed that the supernatant of recombinant Lactococcus lactis could promote growth performance in early-weaned piglets, while the fermentation product, which expressed EGF, could not, possibly due to bacterial overload [16]. However, since previous researches have focused on EGF in mammals [17, 32, 33], there are no studies concerning gEGF or its biological activity. Therefore, it was necessary and meaningful to investigate these activities in relation to growth performance, immune function, and the intestinal development of broilers.
The effect of gEGF on the immune function of broilers was evaluated by measuring the immune organ index, serum IgA and IgG, and intestinal mucosal sIgA. Very few studies have investigated the effects of EGF on immune function. Dietary supplementation of recombinant pEGF was shown to effectively increase the level of IgA in the jejunum mucosa of weaned piglets [34–36]. Further, the level of serum IgA in weaned piglets increased significantly after 18 days of treatment with pEGF, while the same treatment significantly increased levels of IgA in the jejunum mucosa after 28 days [17]. In our study, serum IgA increased significantly during 2 weeks of gEGF treatment, which was similar to the effect of pEGF in piglets. However, the level of sIgA only increased in the mucosa of the duodenum, and not in the mucosa of the jejunum or ileum. This result may be due to degradation of gEGF along the gastrointestinal tract as degradation rates of EGF in the small intestinal lumen of weaned pigs has been shown to increase from the proximal to the mid and distal regions [37], which resulted in relatively high levels of gEGF in the proximal intestinal tract. In addition, we found that the thymus and spleen indices increased significantly in broilers treated with gEGF for 2 weeks, indicating that immune function improved in phenotypes. Overall, these results suggest that gEGF could improve immune function in broilers.
The effect of gEGF on the intestinal development of broilers was evaluated by measuring villus height, crypt depth, and VH/CD in the mucosa of the duodenum, jejunum, and ileum. Intestinal villus with greater height has a larger area for nutrient absorption, leading to enhanced absorption, while shallower crypts lead to higher maturation rates of intestinal epithelial cells and better absorption. The VH/CD value could thus reflect the function of the small intestine, with higher values implying better nutrient absorption in the intestine [38]. Our results clearly showed that gEGF promoted the development of the intestinal villus in the duodenum, jejunum, and ileum following 2 weeks of gEGF treatment; however, in the case of crypt depth, gEGF could only promote development in the duodenum. Thus, gEGF effectuated a pronounced effect on the villus, in contrast to the findings of Thompson et al. indicating that EGF had greater effects on the crypt [39]. Moreover, gEGF was found to be strongly associated with the development of the duodenum, which could also be due to varied degradation rates within different regions of the intestine [36].
This study evaluated growth performance by measuring the final body weight, average daily gain, daily feed intake, and the gain:feed ratio. Most indices of growth performance were improved during the first week of gEGF treatment. The current study found that an increase in the gain:feed ratio occurred both in the P-LL and gEGF-P-LL groups compared with that in the Control group within the first week, suggesting that there may be some components of the fermentation product of LL-pNZ8149 affecting growth performance. However, the gain:feed ratio of the gEGF-P-LL group was significantly higher than that of the other two groups following 2 weeks of gEGF treatment, indicating that gEGF was an important factor in the growth of broilers. In addition, these results indicated that gEGF significantly improved the growth performance of broilers via increasing the gain:feed ratio. Kim et al. showed that mEGF exerted beneficial effects on the growth performance of broiler chickens prior to challenge with Eimeria, while enhancing the expression of the nutrient transporter gene xCT1 [21]. Bedford et al. found that the supernatant of the EGF-LL fermentation product increased levels of sucrase and alkaline phosphatase by day 8 of treatment [16]. Wang et al. reported that sucrase in 3 intestinal segments, aminopeptidase A in the duodenum and jejunum, and aminopeptidase N and dipeptidase Ⅳ in the duodenum were significantly higher in the group treated with LL-pEGF than in the control group [30]. Thus, enhanced expression of nutrient transporters and increased levels of digestive enzymes caused by EGF may have contributed to the improved growth performance of broilers and early-weaned piglets. Bedford et al. showed that the gain:feed ratio of early-weaned piglets did not increase following 2 weeks of treatment with the supernatant of recombinant bacteria [16]. Therefore, further study will be required to investigate if the growth- promoting effect of gEGF is transient. However, overall, our results suggested that gEGF may be utilized as a growth promotant in broiler chickens.
Antibiotics such as virginiamycin have been used as growth promotants in broiler chickens for decades [40] because initially, they inhibited pathogenic bacteria; reduced the use of nutrients by bacteria; increased the synthesis of vitamins and other growth factors; and promoted nutrient absorption by reducing the thickness of intestinal epithelial cells [41]. However, long-term use of antibiotics resulted in reduction of sensitivity to the drug, often leading to complete resistance to this therapy [42, 43]. Moreover, continual use of antibiotics destroyed normal microbiota along with the targeted flora, causing serious damage to intestinal microecology [44, 45]. In addition, abuse of antimicrobials in commercial animals could lead to finished livestock and poultry products containing antibiotic residue in the form of prototypes or metabolites, which might alter the intestinal flora of consumers or result in allergic reactions, threatening the health of consumers. At the same time, overuse of antibiotics increased the incidence of drug-resistant bacterial diseases in both humans and animals. Thus, the current study trialed gEGF as an alternative to antibiotics and found that this therapy significantly promoted the growth performance of broiler chickens, via increased food intake and weight gain, improved nutrient absorption, and enhanced intestinal and immune functioning. Therefore, gEGF should be trialed as an alternative growth promotant because it exhibited non-toxic side effects and did not elicit an antibacterial effect that could influence the natural intestinal flora of consumers, and thereby might avoid some of the problems associated with chicken and food safety. However, the limitation of this study was that the chickens were only grown for two weeks and therefore further research should be conducted by following similar birds to harvest age to see if this therapy does ultimately yield larger healthier birds.