Antibiotics have been extensively employed in poultry breeding to optimize growth performance and enhance health. However, misuse and/or overuse of antibiotics have resulted in increased emergence and dissemination of drug-resistant bacteria [1]. Furthermore, research has revealed that excessive antibiotic treatment may affect host energy metabolism and cause health problems [2]. The incorporation of antibiotics into animal feed has been subjected to regulatory restrictions in several countries, including China, the European Union and the United States [3,4]. This restriction poses a challenge to the development of the livestock and poultry industries, necessitating the exploration of alternative feed additives capable of enhancing the growth performance and immune capacity of livestock and poultry. Previous investigations have provided evidence that plant-based feed additives can improve growth performance, immunological function, and microbial regulation. Abdelatty et al [5] observed that dietary supplementation with Manjiang red leaf powder had favorable effects on gut morphology and function in broilers. Farahat et al [6] found that plant extracts altered the intestinal morphology and VH/CD ratio while increasing the Lactobacillus population. Therefore, plant-based feed additives are expected to become viable alternatives to antibiotics and are increasingly being studied. Intestinal health is a crucial determinant of chicken growth performance, and the intestinal tract is an essential immune organ that maintains body homeostasis via selective barrier functions. Therefore, ensuring intestinal health remains a top priority in animal farming [7]. The symbiotic equilibrium between the gut microbiota and intestines influences the health status, growth performance, and developmental processes of chickens [7]. However, variations in intestinal morphology and microbiota composition have been observed among broilers at different stages of development [8]. Consequently, it is vital to explore how feed additives impact chicken health and growth performance through changes in intestinal health.

Cordyceps militaris solid medium (CMM) is a byproduct derived from the artificial cultivation of Cordyceps militaris. The culture technology of Cordyceps militaris has developed rapidly, and artificial large-scale cultivation has been realized. A large amount of CMM remains after Cordyceps militaris is harvested, with more than 1 kg of CMM produced for every 1 kg of Cordyceps militaris harvested [9]. Most CMM is currently treated as waste, resulting in resource waste and environmental pollution. Cordyceps militaris is a fungus that can be used as both a medicine and food; it contains a variety of nutrients and active ingredients, such as cordycepin, cordycepic acid and cordyceps polysaccharide, and has a variety of biological activities [10]. CMM also contains valuable active components such as cordycepin and cordyceps acid [11]. Several reports have shown that CMM possesses biological activity and functional benefits. For example, Cheng et al. found that the addition of CMM to feed at a 0.5% ratio affected the production performance and egg quality of laying hens [12]. Moreover, Hsieh et al demonstrated that the addition of 1% CMM to broiler meals improved intestinal health and growth performance and also enhanced antioxidant capacity [13]. Moreover, Wang et al reported increased egg production and reduced cholesterol levels in laying hens supplemented with 2% CMM in feed [14]. Dai et al. found that adding CMM to the diet at 0.01% to 0.03% could improve the performance and carcass quality of finishing pigs [15]. These findings suggest that CMM as a feed additive exhibit promising efficacy, underscoring the importance of further research and development in this area. However, the addition of CMM as a feed additive varies significantly across studies, which hinders the application of CMM in livestock and poultry farming. It is also not clear whether CMM has a regulatory effect on chicken intestinal flora. Thus, in this study, three doses of CMM in feed (0.02%, 0.2%, and 2%) were used to systematically compare the effects of different doses of CMM on the growth performance, intestinal morphology, immune capacity and intestinal microbes of broilers to provide a basis for determining the effective dose of CMM as a feed additive.

The experimental procedure received approval from the Lanzhou Animal Husbandry and Veterinary Medicine Animal Ethics Committee and was performed in strict compliance with the guidelines for the management of experimental animals in Gansu Province (approval number: 2023-007).

In intensive livestock and poultry farming, antibiotics are commonly used to combat bacterial infections and diseases caused by pathogenic microorganisms. However, excessive and unnecessary use of prophylactic antibiotics is prevalent, leading to the rapid emergence of drug-resistant bacteria and environmental contamination [17]. Moreover, concerns about food safety have been raised due to the presence of drug residues. Several countries have implemented comprehensive prohibitions on the use of antibiotics in animal food products. Hence, alternative feed additives that substitute for antibiotics and are both safer and more environmentally friendly are needed.

In our study, we assessed the impact of different amounts of CMM supplement on the growth performance of broilers. The results indicated that the ADG of broilers supplemented with CMM was greater than that of the Control group and promoted the growth of broilers, but only at Low and Mid CMM concentrations (p<0.05). However, the ADFI and FCR in the High CMM group were significantly greater than those in the Control group. These findings indicate that the effect of CMM on the growth performance of broilers is not positively correlated with dose in the range from 0.02% to 2%. Cheng et al. also proposed that the effect of 0.5% CMM in feed on production performance and egg quality of laying hens was better than that of 1% CMM, indicating that higher doses of CMM do not result in the best performance [12]. In contrast, Wang reported that 2% CMM had a better effect on egg production in laying hens than did 1% CMM. The beneficial effect of CMM on growth performance may be due to the active ingredients contained in CMM. The active ingredient content should be considered when choosing the dosage of CMM as a feed additive, to achieve more effective production.

The developmental status of immune organs, including the thymus, spleen, and bursa, can serve as a direct reflection of immune competence in chickens and is a crucial indicator for assessing their growth status [18]. Our results showed that a Low CMM concentration significantly increased the bursa and thymus indices at 14 and 28 d, whereas a Mid CMM concentration significantly increased the bursa index at 14 d but did not alter other indices compared with those of the Control group (p>0.05). Notably, the spleen index of the High CMM group decreased (p>0.05), probably due to excessive dosage, which is consistent with the result that High CMM does not increase growth performance. These results highlight the importance of an appropriate CMM dosage in promoting the development of immune organs and initiating immune responses. Serum immunoglobulins (IgA, IgM, and IgG) play key roles in evaluating animal immune status and serve as the most important indicators of the humoral immune response in animals [19]. When bacteria or viruses infiltrate the body, they can bind to specific antigens, triggering the activation of humoral immunity. This immune response works to eliminate bacteria, viruses, and other pathogenic substances, playing crucial protective and defensive roles in the body [20]. Throughout the trial period, all the immunoglobulin indices were either better than or similar to those of the Control group, possibly because CMM-active components promote the development of immune cells and improve the immune response [21].

The biochemical indices in serum are influenced by the body’s metabolism and health status. Changes in these indicators reflect alterations in physiological processes [22]. The TP and ALB levels in serum are indicators of physiological health and homeostasis maintenance in animals [23]. Our results indicated that CMM did not significantly increase TP or ALB levels. In addition, the slight decrease in AST and ALT levels related to CMM suggests that CMM has no significant effect on liver function, as increased activity of these two enzymes generally indicates liver cell damage [24]. An increase in TG indicates elevated fat levels in blood; no notable changes in TG levels were observed in our study in response to CMM supplementation, but there was a slight decrease in TCH content that was not statistically significant. These findings indicate that CMM had no effect on blood lipid. Additionally, CMM did not significantly alter CREA or UREA levels in serum; CREA serves as an indicator of the glomerular filtration rate, and elevated levels of both of these markers indicate renal function impairment [25], implying that CMM did not induce renal damage in broilers. Thus, CMM has no discernible effect on the serum indices or health status of broilers.

In intensive poultry farming, several environmental factors and pathogens can weaken the immune system of poultry, affecting their intestinal health. Stimulation of the intestinal mucosa activates immune responses and the release of cytokines, such as the proinflammatory factors IL-1β, IL-6, IL-2, and TNF-α as well as the anti-inflammatory factor IL-10, all of which influence immune responses [26]. Our further analysis revealed that the TNF-α, IL-1β, IL-6 and IL-2 mRNA levels in the jejunum tissues of the CMM group were downregulated, whereas the levels of IL-10 and IFN-γ tended to increase. This finding is consistent with the results of Calik, who reported similar immune function regulation by vitamin E and selenium [27], indicating a suppression of IL-1β and TNF-α levels and an increase in IL-4 and IL-10 levels in the jejunum of broilers. The small intestine serves as a pivotal site for digestion and nutrient assimilation, and positive changes in VH and CD can positively affect the intestinal absorption of animals and indicate their status, [28]. In our study, the addition of low and high concentrations of CMM to the diet improved intestinal morphology, regulated VH and CD, and affected the intestinal digestion and absorption capacity of chickens to some extent. The effect of CMM addition on feed conversion may also be attributed to changes in intestinal morphology.

The gut microbial environment serves as a barrier against foreign pathogens, and modulation of the gut flora can impact metabolism and the immune response in animals [29]. Mao et al reported that fermented dandelion inhibited the proliferation of harmful bacteria and increased the abundance of beneficial bacteria, maintaining the flora balance and modulating immune capacity [30]. As a prospective feed supplement, the effects of CMM on intestinal flora need to be investigated. In our study, the addition of CMM increased the number of unique OTUs and α diversity in chickens. The findings indicated that incorporating a low concentration of CMM led to increased ACE and Chao 1 index values, indicating increased richness in the cecal flora. However, no level of CMM addition exerted any discernible effect on the Shannon and Simpson indices. This collective outcome suggests an overall improvement in the intestinal flora. Moreover, classification of the intestinal flora at the phylum level in broilers supplemented with CMM remained largely unchanged, maintaining the fundamental structure of intestinal flora. However, it may affect the abundance of microbes at lower taxonomic levels such as classes, orders, families, and genera. Firmicutes and Bacteroidetes are the main components of the intestinal flora, and it is generally speculated that an impaired intestinal barrier decreases the Firmicutes/Bacteroidetes ratio [31]. The incorporation of CMM increased the Firmicutes/Bacteroides ratio, which is similar to the results of Kong et al [32], in which the addition of glycerol monolaurate increased the Firmicutes abundance. Deferribacteres are bacteria that generate energy through obligate or facultative anaerobic metabolism and maintain iron balance in the intestine. We found that the abundance of Deferribacteres in the Low CMM group significantly increased at 14 d. Another study reported that the administration of probiotics resulted in an increase in the abundance of Deferribacteres [33]. Moreover, the Mid CMM group showed a reduced proportion of Proteobacteria at 14 and 28 d. Notably, Proteobacteria include various pathogenic bacteria, such as E. coli, which can secrete histamine [34], thereby inducing allergic or toxic reactions and promoting the occurrence of gastrointestinal disease [35]. This finding suggests that CMM can modulate gastrointestinal health.

At Days 14 and 28, CMM decreased the proportion of Bacteroides in all groups. Wu et al [36] observed a negative correlation between Bacteroides and immunoglobulins, suggesting that CMM may positively affect immune function. After 28 d of CMM feeding, the proportion of Lachnospira colonizing the gut increased. Lachnospira can produce nutrients such as butyrate, which provides energy to colon epithelial cells, maintaining immune homeostasis and promoting health [37]. Synergistes, a recently discovered genus, are widely distributed in anaerobic environments, including in soil and the intestinal tract [38]. Our study revealed that Low CMM concentrations increased the proportion of Synergistes bacteria at 14 d, whereas all CMM doses increased the proportion at 28 d, which is similar to findings that alfalfa flavones increased the abundance of intestinal Synergistes bacteria [39]. Furthermore, Li et al [40] found a negative correlation between syntrophic bacterial abundance and IL-1β, IL-6, and TNF-α levels, which is consistent with our findings suggesting that CMM may modulate inflammatory factors by altering intestinal microorganisms. During the 28-d feeding cycle, Oscillospiraceae were significantly enriched at all doses. This bacterial family is recognized for its capacity to synthesize short-chain fatty acids (SCFAs), particularly butyrate, through the degradation of different structural carbohydrates [41]. Moreover, Oscillospiraceae has been recognized as a promising candidate for developing probiotics [42]. In addition, Megamonas, classified under Firmicutes, has the capacity to ferment diverse carbohydrates, thereby promoting SCFA production in the intestine. We found that the proportion of Megamonas in the Low and Mid CMM groups increased at 14 and 28 d. Previous studies have shown that chicken samples fed phenylpyruvate have a relatively high proportion of Megamonas [43]. We also found that a Low CMM concentration increased the proportion of Faecalibacterium, which has been shown to be positively correlated with upregulation of IL-10 expression and can improve SCFA production and enhance intestinal immunity [44].

In summary, the addition of 0.02% CMM to the base diet improved the growth performance, immunity parameters, intestinal morphology, and intestinal microbes of yellow-feathered broilers. This provides foundational support for the utilization of CMM as a poultry feed additive.