The poultry industry plays a crucial role in ensuring global food security and nutritional supply by providing high-quality protein in the form of affordable and nutritious chicken meat and eggs (1). Global per capita meat consumption is increasing, with poultry accounting for 70% of the total meat consumption; over 66 billion broilers are slaughtered worldwide annually (2). Antibiotics have been used for poultry farming since the late 1940s. Consequently, the widespread use of antibiotics as feed additives has become a common practice (3). The extensive use of antibiotics has resulted in the accumulation of antibiotic-resistant bacteria with selection of antibiotic resistance genes in the environment, posing potential risks to ecosystems and human health (4, 5). The intake of antibiotics by food-producing animals and the resulting antibiotic residues in food have been recognized as major causes of the rapid spread of antimicrobial resistance in humans (6, 7). The continuous intake of antibiotics can result in an imbalance in the gut microbiota (8) and induce several intestinal diseases (9, 10). The 2019 Global Burden of Disease Report estimated that 14 million people died from infection-related causes, making it the second leading cause of death after ischaemic heart disease, with bacterial pathogens responsible for 7.7 million deaths, and antibiotic resistance in bacteria causing 1.3 million deaths (11). Therefore, there is an urgent need to develop green, natural, and non-toxic feed additives.

Antimicrobial peptides (AMPs) are small peptides produced by the innate immune system and are characterized by structural diversity and broad-spectrum antimicrobial activity (12, 13). As dietary additives, AMPs can improve broiler growth performance, immune organ indices, and gut microbiota (14–16). The bactericidal capacity of AMPs generally depends on their ability to interact with microbial cell membranes and cell walls. Most AMPs selectively bind to negatively charged cell membranes/cell walls owing to their highly positive charge and hydrophobic primary structure, resulting in non-enzymatic destruction of the cell membrane, thus making it difficult for bacteria to develop resistance (17–19). For example, the antimicrobial peptides magainin 2 and PGLa are synergistically inserted into the phospholipid bilayer to form pores that inactivate microorganisms (20). Some peptides can enter the cell without disrupting the cell membrane and kill bacteria by inhibiting crucial intracellular functions (21). Antimicrobial peptides such as cathelicidins can induce the production of intracellular reactive oxygen species (ROS), thereby damaging bacterial molecules and leading to slow growth or cell death (22). The antimicrobial peptide Mastoparan X reduced the serum levels of inflammation-related genes IL-6 and LITNF in broilers, while increasing the mRNA expression levels of the antioxidant genes CAT, HMOX1, and SOD1 (23). Sublancin, an antimicrobial peptide, helps alleviate necrotizing enteritis induced by Clostridium perfringens (24).

The Isalo scorpion cytotoxic peptide (IsCT) (25) is a short natural cytotoxic peptide. IsCT has demonstrated good in vitro antimicrobial activity against both Gram-negative and Gram-positive bacteria (26); moreover, it has shown significant antimicrobial effects against multidrug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and other pathogens (27). Studies have indicated that IsCT improves gut barrier function, immune function, and gill health in grass carps (28–30).

This study aimed to explore the effects of different doses of IsCT supplementation in the diet of yellow-feathered broilers on their growth performance, serum immune indicators, and immune organ indices. Moreover, using 16S rDNA high-throughput sequencing analysis of the caecal microbiota, we investigated the effects of IsCT on the gut microbiota of yellow-feathered broilers, thus providing a theoretical basis for the application of antimicrobial peptides as novel feed additives in livestock and poultry farming.

The antimicrobial peptide Plectasin improves the growth performance of broiler chickens, which may be mediated by the inhibition of harmful bacteria in the intestine (14). Another study found that Microcin C7 increased the ADG of broilers and reduced their feed-to-gain ratio (31). Additionally, research indicated that AMPs promoted broiler growth (32). This study found that adding 150 and 200 mg/kg IsCT to the diet significantly increased the final body weight and ADG of yellow-feathered broilers at 60 days of age. Moreover, 150 mg/kg IsCT significantly reduced the ADFI of yellow-feathered broilers. These findings suggest that supplementing broiler diets with IsCT can improve growth performance, possibly by enhancing intestinal barrier function and immune performance, thereby improving nutrient digestion and absorption. Similar additives include essential oils, probiotics and polysaccharide. These additives improve gut health, thereby mediating enhanced growth performance (33–35). What differs in certain aspects is that IsCT possesses a small molecular weight (only 13 amino acids) and a simple composition, allowing it to be more easily metabolized without leaving residues in the animal’s body.

Immune organ indicators are commonly used to evaluate the immune performance of poultry (36), and an increase in the immune organ index often indicates good immune organ development (37). The spleen is involved in both humoral and cellular immune responses, making it an important immune organ in poultry (38). Studies have found that adding Mastoparan X to the diet of bacteria-challenged chickens can increase their relative spleen weight (23). Another study indicated that feeding broilers the antimicrobial peptide Gal-13 significantly increased the weight of their spleens and thymuses (39). This study found that adding 150 mg/kg IsCT to the diet significantly increased the spleen index of yellow-feathered broilers, which is consistent with previous findings, indicating that IsCT has a positive effect on spleen development in broilers.

According to previous studies, the addition of antimicrobial peptides to feed can effectively improve animal serum indicators (40, 41). Studies have found that adding bioactive peptides from sesame meal to the diet of broilers can increase the concentration of serum albumin (42), which is consistent with the results of this study. Another study indicated that supplementing the diet with the antimicrobial peptide LLv raised serum IgA and IgM levels in broilers at 21 and 42 days of age (43). Additionally, it has been found that antimicrobial peptides increased serum IgA and IgG levels in Gushi chickens (44). In this study, IsCT significantly increased serum IgA, IgM, and IgG levels, which is consistent with previous literature. ALT is not only closely related to liver fat accumulation (45, 46) but also plays an important role in the intermediate metabolism of amino acids and glucose (47). ALT is closely associated with metabolic health in animals and serves as a marker of liver disease (48). Some studies have shown that dietary lysozymes and ZnB significantly reduce ALT levels (49). In this study, we found that IsCT reduced serum ALT content in broilers, indicating an improvement in liver function. Additionally, ALT levels increased significantly in broilers fed with antibiotics. Research has shown that long-term use of antibiotics can have adverse effects on the liver, including significant increases in ALT levels (50–52). Based on previous studies, we speculated that the elevated serum ALT levels in the antibiotic group of broilers in this study were caused by long-term antibiotic feeding.

The gut microbiota is a key contributor to maintaining intestinal homeostasis and host health, and has been shown to play a role in nutrient absorption, metabolism, immune system maturation, and the intestinal mucosal barrier (53, 54). Indicators reflect the richness and evenness of species within a single sample (55). Studies have shown that the Chicken NK-2 peptide effectively reduces the reproductive capacity of *Eimeria* parasites in the host’s intestines (14). Another study showed that the antimicrobial peptide cLFchimera restored the microbial community balance in the ileum of birds disrupted by antibiotics (16). Through α-diversity analysis, it was found that IsCT reduced the richness of the caecal microbiota in broilers, which may be due to the inhibitory effect of IsCT on certain genera. β-diversity is an important indicator of differences in species composition between different samples (56). Through β-diversity analysis, it was found that the composition of the microbiota in the IsCT group differed significantly from that in the CON group, suggesting that the antimicrobial peptide IsCT had some impact on the caecal microbiota structure of broilers. In the caecum of broilers, Bacteroidetes and Firmicutes are the two major bacterial phyla. They play important roles in the gut microbial community, and are closely associated with host nutrient absorption, immune regulation, and health status. Intestinal-associated Bacteroidetes can degrade complex polymers, thereby facilitating food digestion and nutrient acquisition (57). The study found that at the phylum level, the abundance of Bacteroidetes in the caecal microbiota of broilers increased, whereas the abundance of Firmicutes decreased, which may improve the intestinal immune status, enhance mucosal health, and potentially reduce excessive energy absorption, thereby positively impacting the health and growth performance of broilers (58, 59). Notably, some species of the genus Bacteroides secrete short-chain fatty acids that can influence gut and brain functions (60). The metabolites produced by these microorganisms may induce autism spectrum disorder (61). However, the molecular mechanisms by which Bacteroidetes affect the host remain to be explored. Additionally, LDA analysis showed that at the phylum level, the relative abundances of Actinobacteriota and Patescibacteria were significantly higher in the IsCT100 group than in the other groups. Although Actinobacteriota constitute a relatively small proportion of the gut microbiota, they play a crucial role in maintaining intestinal homeostasis (62). Patescibacteria typically coexist symbiotically with other microbes, with certain Patescibacteria forming mutualistic relationships with Actinobacteria, suggesting that they play a special ecological role in the gut (63).

This study concluded that adding IsCT to the diet of yellow-feathered broiler chickens could improve their growth performance, immune function, and gut microbiota, with an optimal supplementation level of 150 mg/kg.

We aimed to investigate the effects of Isalo scorpion cytotoxic peptide (IsCT) on the growth, immune performance, and intestinal microbiota of yellow-feathered broilers. A total of 240 yellow-feathered broilers were randomly assigned to six groups, with different doses of IsCT added to the basal diet of each group. The results showed an increase in immunoglobulin levels and daily weight gain and reduction in daily feed intake and feed conversion ratio. Additionally, the abundance of beneficial bacteria increased whereas that of harmful bacteria decreased in the caecum of the chickens. Therefore, IsCT helps improve the growth and immune performance of yellow-feathered broilers and optimizes the intestinal microbiota.