3. Discussion

3.1 Effects of Acidifiers with Different Administration Routes on Growth Performance of Broilers

Since the strict ban on feed antibiotics in China’s livestock industry was implemented in July 2020, a variety of feed additives have been widely developed, including probiotics, enzyme preparations, plant extracts, and acidifiers. These additives differ in physiological functions and action mechanisms, but all aim to replace growthpromoting antibiotics in feed, so as to maintain animal growth performance under antibioticfree feeding conditions. Among them, acidifiers have been extensively applied in animal husbandry due to their high efficiency, pollutionfree property and no residue.

The present results showed that acidifiers supplemented via different routes significantly improved the European Production Efficiency Factor of broilers, with no significant difference compared with the antibiotic group, which was consistent with previous studies. Acidifiers could effectively enhance broiler growth performance by reducing intestinal pH, inhibiting the proliferation of harmful intestinal bacteria, and improving the digestibility and utilization of dietary nutrients. The acidifier used in this trial was mainly composed of formic acid and ammonium formate. As one type of shortchain organic acid, formic acid exhibits strong antibacterial and bactericidal abilities and is widely used in the feed market. Excessive supplementation of formic acid has disadvantages such as strong corrosiveness and pungent odor, while appropriate addition of formic acid and its ammonium salt can improve diet palatability and promote feed intake. Moreover, free formic acid can be released in vivo, exerting bactericidal effects and reducing gastrointestinal pH.

This study indicated that dietary acidifier supplementation significantly reduced mortality and tended to decrease the overall feed-to-gain ratio, with an effect comparable to antibiotics, suggesting that dietary addition was superior to drinking water addition in improving growth performance. After adding acidifier to drinking water in this trial, the pH value of drinking water decreased to approximately 3.9. Previous studies have demonstrated that watersoluble acidifiers could increase average daily gain and feed conversion ratio of broilers by regulating lipase activity and reducing the abundance of intestinal harmful bacteria. Accordingly, acidifier added via drinking water also showed a good antibioticreplacement effect in the present study. However, excessively acidic drinking water might reduce palatability, thereby weakening its efficacy compared with dietary supplementation.

In addition to the above effects, dietary acidifier supplementation could also enhance the activities of total intestinal protease and small intestinal protease. The superior effect of dietary acidifier over water supplementation might be related to the retention time in the gastrointestinal tract. A longer retention time facilitates better regulation of intestinal function, higher protease activity and nutrient absorption capacity, consequently resulting in better growth performance.

3.2 Effects of Acidifiers with Different Administration Routes on Intestinal Morphology of Broilers

The small intestine is the main site for digestion and absorption of dietary nutrients in broilers. Villus height and crypt depth are key morphological indicators reflecting the contact area between nutrients and intestinal epithelium. Increased villus height enlarges the intestinal absorption area; shallower crypt depth indicates a higher maturation rate of intestinal epithelial cells; an elevated villus height/crypt depth (V/C) ratio further implies improved intestinal development and enhanced absorption function.

The present results showed that dietary acidifier supplementation exerted a better improving effect on the morphological structure of duodenum, jejunum and ileum at 21 and 42 days of age, whereas the beneficial effects of wateradded acidifier were mainly concentrated in the duodenum, indicating that watersupplemented acidifier mainly acted on the anterior segment of the small intestine. A number of recent studies have reported that dietary acidifier supplementation effectively increases villus height and decreases crypt depth in the duodenum, jejunum and ileum, and the improvement degree varies with the type and supplemental level of acidifiers. Different organic acids can stimulate the proliferation of intestinal epithelial cells, bind with inorganic salts in the intestine to form organic acid complexes, increase villus height and effective absorption area, and ultimately promote growth performance.

Most previous studies on drinking water acidifiers focused on their effects on villus height and crypt depth of the jejunum, while few reported beneficial impacts on the posterior small intestine. The above findings are consistent with the present results: dietary acidifier improved the morphology of multiple intestinal segments, while water supplementation showed a limited range of action. The possible reason is that dietary acidifier passes through the digestive tract more slowly than wateradded acidifier, leading to longer action time and wider effective range, thus achieving better improvement in intestinal morphology.

The acidic intestinal microenvironment created by acidifiers can effectively prevent the invasion of harmful bacteria to the intestinal mucosa, reduce intestinal fermentation of feed, decrease chyme viscosity, improve digestibility, and promote the growth and repair of intestinal mucosa. Other studies have shown that supplementation of 0.25% organic acid in drinking water reduced intestinal pH, fecal moisture and duodenal chyme viscosity in broilers; likewise, 0.25% dietary organic acid also significantly decreased duodenal pH and chyme viscosity. Nevertheless, few reports have compared the differences in intestinal pH and chyme viscosity between the two supplementation modes.

3.3 Effects of Acidifiers with Different Administration Routes on Goblet Cell Number and Tight Junction Protein Expression in the Intestine

Goblet cells are widely distributed among small intestinal epithelial cells and serve as important markers of intestinal barrier function. Goblet cells secrete a large amount of mucin, which forms a lubricating mucus layer covering the epithelial surface after releasing into the intestinal lumen, constituting the first line of defense for maintaining intestinal integrity and health. Intestinal tight junctions are highly dynamic barrier structures that selectively regulate the permeability of water, ions and nutrients between adjacent cells. The integrity of tight junction proteins plays a vital role in stabilizing intercellular connection and maintaining intestinal epithelial integrity. Therefore, goblet cell number and tight junction protein expression are important evaluation indexes for intestinal barrier function and intestinal development.

Acidifier supplementation can increase the content of shortchain fatty acids in the intestine. Shortchain fatty acids not only provide energy for intestinal cells but also regulate intestinal cellular metabolism. The present results showed that both supplementation routes of acidifier tended to increase the number of ileal goblet cells. Notably, dietary acidifier supplementation significantly increased goblet cell density in the jejunum, showing a better effect than antibiotics. Previous studies have demonstrated that acidifiers exert an equal or even better promoting effect on goblet cells of piglets compared with antibiotics, which is consistent with our results. It has also been reported that acidifiers can enhance poultry immune function, while antibiotics may disrupt the dynamic balance of intestinal immune cells and induce gut microbiota disorder, further confirming that acidifiers are superior to antibiotics in regulating intestinal barrier function.

Further detection of tight junction protein expression in jejunal mucosa revealed that dietary acidifier supplementation significantly upregulated the relative mRNA expression of claudin3, which was consistent with the results of intestinal morphology analysis. Studies have shown that acidifiers can repair intestinal injury, strengthen intercellular tight junctions and enhance intestinal barrier function by upregulating the abundance of tight junction proteins in porcine small intestinal epithelial cells.

In conclusion, acidifiers can improve intestinal barrier function by increasing tight junction protein expression and goblet cell number. Dietary acidifier supplementation could further optimize such beneficial effects, resulting in better intestinal development than drinking water supplementation.

4. Conclusions

① Both dietary and drinking water supplementation of acidifiers exerted comparable growthpromoting effects to antibiotics in broilers, and showed superior effects on intestinal development.② Dietary acidifier supplementation presented better improvements in growth performance and intestinal development of broilers than drinking water supplementation.