Mulberry leaves are rich in protein, fiber, minerals, and other nutrients, making them an excellent source of feed with high biological yield, comprehensive nutritional value, good palatability, and high digestibility. They can serve as a new type of protein feed resource to address feed shortages (Geng et al., 2024). Mulberry leaves grow in regions with diverse climatic conditions worldwide, ranging from temperate to tropical areas. The annual yield of mulberry leaves can reach 25 to 30 tons per hectare, with a protein content of 18-25% (on a dry matter basis) (Guha et al., 2010; Iqbal et al., 2012; Wang et al., 2022). Moreover, the digestibility of protein in mulberry leaves for animals can reach 75-85% (on a dry matter basis) (Chan et al., 2016; Chen et al., 2018). Analysis reveals that mulberry leaves possess a relatively high crude fat content and low crude fiber content, positioning them as a valuable source of high-quality protein for livestock feed. Additionally, it is also rich in protein, containing 218.6 g/kg (Trabi et al., 2017). They are rich not only in amino acid content but also in diversity. Some essential amino acids, which may be lacking in other feeds, are present in sufficient quantities in mulberry leaves, meeting the amino acid requirements of livestock and poultry and helping to balance amino acid ratios (Leterme et al., 2005). Mulberry leaves are also high in minerals, with 100 g containing 2.699% calcium and 3.101% potassium (Batiha et al., 2023). The vitamin content in mulberry leaves is also abundant, especially in B-complex and C vitamins, which are beneficial for improving the immune systems of animals (Chen et al., 2021). Additionally, mulberry leaves contain various physiologically active substances such as polysaccharides, flavonoids, steroids, volatile oils, and alkaloids, which play important physiological roles (Liu et al., 2001). Multiple studies have demonstrated that incorporating mulberry leaves as a feed ingredient can significantly improve the health status, productivity, meat quality, and flavor of ruminant animals. Supplementing 600 g/d of mulberry leaf feed in a rice straw-based diet can significantly increase the dry matter intake of beef cattle (Tan et al., 2012). Moreover, adding 2 g of air-dried mulberry leaves per head in the daily ration does not adversely affect the feed intake of sheep (Chen et al., 2015). According to Liu et al. (2001), supplementing ammonia-treated rice straw with varying levels of mulberry leaf powder as a replacement for rapeseed meal can enhance feed intake in lambs and improve their growth performance. These findings suggest that mulberry leaf products can be added as a feed ingredient in the diets of ruminant animals.

In recent years, the use of unconventional plant feed ingredients has become increasingly popular, with beneficial plant-based feed ingredients being widely adopted in animal husbandry, especially in intensive farming systems (Hashem et al., 2020; Zhao et al., 2022a). Reports indicate that supplementing animal diets with plant feed can improve animal productivity and immune response while reducing oxidative stress and inflammation (Zhao et al., 2023a). These substances act on the gut microbiota by neutralizing free radicals and preventing their interaction with cellular DNA, while also enhancing nutrient digestion and absorption, thereby exerting immunomodulatory effects (Yu et al., 2023a). Therefore, adding feed ingredients containing natural plant nutrients such as polyphenols to animal diets is considered an optimal alternative for improving animal productivity (Yu et al., 2023b; Zhao et al., 2023b). In organic farms, a diverse grassland with high plant diversity ensures adequate intake of polyphenolic compounds. Achieving such results in intensive farming requires supplementing feed with polyphenol-rich food additives (Huang et al., 2018). This strategy has been supported by research conducted on some farms where the addition of natural plant extract antioxidants to animal feed has improved the quality of animal products (Ji et al., 2018). Additionally, these natural plant extracts play an important role in the hindgut of animals. Therefore, a comprehensive exploration of the effects of natural plant components on the blood and hindgut health of ruminant animals in intensive farming is a focal point of the modern livestock industry. Considering these factors, the aim of this study is to evaluate the effects of supplementing fattening Hu sheep with mulberry leaf powder and retrograde diets on blood parameters and hindgut microbiota.

Adding food, industrial by-products, or unconventional feed products to animal diets is becoming an important approach in the development of animal husbandry today. For example, adding apple pomace (Maslovarić et al., 2017), citrus peel (Zhao et al., 2022b), and other vegetable and fruit waste to feed has become increasingly common (Sahoo et al., 2021). Previous studies have demonstrated that mulberry leaves are rich in various bioactive compounds, including flavonoids and phenolic acids, which contribute to their efficacy as a high-quality immune modulator (Cui X. et al., 2023). These bioactive components, such as flavonoids, can effectively improve the health status of the host through pathways like the PI3K-Akt signaling pathway and the AGE-RAGE pathway (Lv et al., 2022). Additionally, many studies have reported that mulberry leaves also have a certain growth-promoting effect, which will be an important focus of our future research (Ding et al., 2021; Mengistu et al., 2020).

In the animal biological system, the redox system plays a crucial role in the host’s immune and health status. GSH-Px, MDA, T-AOC, T-SOD, and CAT are the main indicators used to evaluate the antioxidant capacity of animals. Among these, GSH-Px reduces toxic hydrogen peroxide and organic peroxides to relatively harmless water and corresponding alcohols, playing a role in clearing peroxides within cells (Czyżowska et al., 2023; Sharma et al., 2021). CAT, on the other hand, is another antioxidant enzyme responsible for breaking down hydrogen peroxide (a type of oxygen free radical) into water and oxygen, thus protecting cells from oxidative stress damage, especially in tissues with active aerobic metabolism such as the liver and lungs (Kang et al., 2013). In this study, there was an increasing trend in GSH-Px and CAT after adding mulberry leaves to the diet, indicating that mulberry leaves have a positive effect on improving the immune status of sheep. This finding is consistent with previous research results (Jin et al., 2022; Li et al., 2022; Shi et al., 2022). This effect may be attributed to the various bioactive components abundant in mulberry leaves, similar to the significant antioxidant capacity reported previously for polysaccharides and flavonoids in mulberry leaves (Kim et al., 2020; Liao et al., 2017). Our current research does not offer a comprehensive analysis of antioxidant indicators. While some indicators show an improving trend, this does not fully substantiate the antioxidant effects of ML. It merely suggests that ML does not negatively impact the antioxidant system of Hu sheep. The specific effects will be explored in greater detail in future studies.

During the growth and development of animals, the concentrations of AKP and GPT reflect the growth and remodeling of bone and muscle tissues (Eriksson et al., 2016; Sobhani et al., 2021; Wang et al., 2017). In bone tissue, AKP is primarily secreted by osteoblasts and plays a critical role in promoting the deposition of calcium and phosphate, thereby facilitating bone mineralization and formation. AKP catalyzes the hydrolysis of phosphate esters, converting organic phosphate esters into inorganic phosphate and alcohols or phenols, a process essential for bone mineralization (Ansari et al., 2022). GPT catalyzes the transamination reaction between glutamate and pyruvate, converting glutamate to α-ketoglutarate and pyruvate to alanine. This process is essential for amino acid metabolism and nitrogen transport (Wei et al., 2022). In this study, the addition of mulberry leaves to the animal diet resulted in increased concentrations of AKP and GPT, suggesting that mulberry leaves may influence certain blood hormones to regulate the host’s development. However, the specific mechanisms underlying this effect warrant further investigation. The concentration of TP also reflects the metabolism within the animal’s body. A slight decrease in TP levels may indicate metabolic adjustments within the animal’s body (Mapfumo and Muchenje, 2015; Rogaly et al., 1982). In this study, the observed decrease in TP levels may be associated with the liver and kidney functions of Hu sheep. However, it remains unclear whether ML have a beneficial or detrimental effect on TP regulation. Some studies suggest that a moderate reduction in TP may be linked to immune system modulation, potentially lowering hyperactive immune responses and reducing inflammation-related damage (Azzini et al., 2022; Fuellen et al., 2023). Although there are indirect reports indicating that supplementing Hu sheep diets with ML may enhance protein utilization efficiency in intensive farming, the specific mechanisms underlying this effect remain unexplored and require further investigation (Ariyarathne et al., 2021; Laroche et al., 2022). This suggests that the addition of mulberry leaves to the diet has the potential to influence the host’s physiological processes. However, further investigation is needed to determine whether all these effects are indeed beneficial (Cui W. et al., 2023).

In this study, we focused on the influence of mulberry leaves on the composition of the intestinal microbiota. At the phylum level, there was a significant increase in the abundance of Campylobacterota, which may be due to the relative decrease in the abundance of other phyla. At the genus level, Campylobacterin the intestine may play a role in food degradation and digestion, potentially secreting enzymes that break down complex food components and facilitate nutrient absorption in the host (Burnham and Hendrixson, 2018). However, most studies identify Campylobacter as a harmful pathogen responsible for animal diseases. The observed increase in abundance in this study might be linked to the host’s nutrient metabolism. Campylobacter possesses unique metabolic pathways, primarily utilizing amino acids and organic acids as carbon and energy sources, instead of relying on carbohydrate fermentation. This adaptation enables their survival in low-oxygen environments, such as the gut and foodborne microbial niches (Stahl et al., 2012). Moreover, there are few reports regarding Mailhella, but it may contribute to maintaining the diversity and stability of the intestinal microbiota, interacting with other microbial communities to collectively maintain normal intestinal function. The significant increase in the abundance of Campylobacterota, Campylobacter, and Mailhella in this study may be related to changes in certain indicators in the blood, as demonstrated in the correlation analysis. Meanwhile, the observed decrease in the abundance of Alloprevotella, Roseburia, and Prevotellaceae UCG-003 may be attributed to changes in the microbial network. Additionally, their changes are strongly correlated with immune metabolic capacity (Shen et al., 2022; Ye et al., 2022; Yu et al, 2023c). Finally, we observed a significant increase in the complexity of the microbial network in the hindgut following the addition of mulberry leaves to the diet. More complex microbial networks are known to possess greater resistance to adverse external factors (Yu et al., 2023b; Yu et al., 2024), further supporting the notion that mulberry leaves can modulate intestinal microbiota structure and enhance host health.

This study offers new insights into the use of ML in ruminant production. The inclusion of ML can elevate the levels of AKP and GPT in the blood, and showing potential to enhance the host’s antioxidant capacity and improve the hindgut microbiota structure of Hu sheep. These findings are significant for the development of alternative feed resources. ML is a promising feed ingredient that benefits both animal health and environmental sustainability. Further investigation into ML’s impact on rumen fermentation, metabolism, and microbial communities in ruminants will support its wider adoption in ruminant production.