Feed is a major input cost in poultry production, accounting for up to 70% of the total production costs (Thirumalaisamy et al., 2016). Soybean meal and corn are the main ingredients in commercial poultry diets that provide crude protein and energy. However, the current feedstuffs supply cannot fulfill the needs of livestock production (Cao and Li, 2013). A low-protein diet is based on the theory of protein amino acid nutritional balance and reduces protein levels and nitrogen emissions by adding appropriate types and quantities of industrial amino acids, which could decrease production costs. However, the inappropriate energy levels with low protein diet can lead to lipid metabolism disorders and oxidative stress in the liver of laying hens (especially for late-laying hens), decreasing egg yield and quality (Ampong et al., 2020; Scappaticcio et al., 2022; Zutphen et al., 2016).

When laying hens enter the late-laying stage, large amounts of lipids accumulate in the liver. Oxidative stress is the key factor of liver lipid accumulation, resulting in liver function reduction, which may lead to hen tissue damage. This phenomenon leads to a decrease in the production performance and egg quality of laying hens in the late laying period (Liu et al., 2018). The gastrointestinal tract is the major site of food digestion and nutrient absorption (Yan et al., 2017). The gut microbiota profoundly affects many metabolic processes in the host, including energy homeostasis, lipid metabolism, and immune system development (Sonnenburg and Bäckhed, 2016; Sommer and Bäckhed, 2016). The cecum is the main site for intestinal microorganisms, and manipulation of the cecum microbiota of chickens might increase their productivity. Most chicken microbiota studies have focused on microbial communities in the cecum.

The gut microbiota is closely related to hepatic function via the gut-liver axis (Konturek et al., 2018). In the gut-liver axis, the liver and gut functions regulate each other, and they are both affected by diet (Ringseis et al., 2020; Albillos et al., 2020). Dietary composition is the most crucial factor for maintaining the growth of microbes, and changing the ingredients of the diet can directly affect the intestinal microbiota in the host (Abdollahi et al., 2018; Crisol-Martínez et al., 2017; Beam et al., 2021). Additionally, diet energy levels that do not fulfill the nutritional needs will lead to oxidative stress in the liver (Tan et al., 2018). At present, there are few reports on the effects of low-protein diets with different energy levels on liver oxidative stress and intestinal composition in the late laying period. In this study, we conducted a correlation analysis of liver antioxidant capacity and intestinal microbial composition in laying hens during the late laying period. To achieve this objective, first, we fed late laying hens different energy levels with the same low-protein diet content. Next, we detected the antioxidant capacity of the liver and performed a metagenomic gene sequencing analysis of the gut microbiomes of the three groups of laying hens. Finally, evaluated the differences in liver antioxidant capacity and gut microbiome composition of these three groups of laying hens to understand the optimal dietary energy density of laying hens.

HFD is one of the important causes of obesity and liver oxidative stress (Milagro et al., 2006; Chu et al., 2024). MDA is a soluble degradation product of lipids, and indirectly reflects the degree of cell damage. Oxidative stress occurs in tissues, and the cell membrane of tissue cells will produce MDA through an oxidation reaction. MDA is the final product of lipid peroxidation, and the MDA level increased significantly with the increase in energy (Chen et al., 2018). According to Lasker et al. (2019), oxidative stress induced by a high-energy diet significantly increased MDA content in rats. Hosseinian and Hasanzadeh (2021) increased dietary energy to 3,450 kcal/kg, increased serum MDA concentration and decreased T-AOC level in pigeons. Our results support this view. We also detected the contents of T-AOC, T-SOD, GSH, and GSH-Px, which play an important role in maintaining the internal redox balance; they are important indicators for evaluating the body ‘s antioxidant capacity (Li et al., 2022). T-AOC is an integrative index used to reflected the functional status of the body ‘s antioxidant system (Miao et al., 2017). In the liver, according to our results, the HL diet significantly increased the liver MDA level and decreased the T-AOC level. This result prove that the high-energy diet has an adversely affected on the liver oxidation ability of aged laying hens.

Microbes that colonize the intestine are called the gut microbiota, regarded as an organ that contributes to the regulation of host metabolism. The gut microbiota might also affect production efficiency by altering the shape of intestinal tissue in poultry. According to our results, at the genus level, Bacteroides and Parabacteroides play key roles in the host immune system. Bacteroides can protect the intestinal tract from pathogens and reduce the production of lipopolysaccharides in intestinal microorganisms (Yoshida et al., 2018). Parabacteroides can help the body regulate metabolism, relieve inflammation, and regulate the immune system (Fu et al., 2019). Some studies have found that Parabacteroides can protect against inflammation and obesity (Cui et al., 2022; Wang et al., 2022). Alistipes have protective effects against liver fibrosis but are pathogenic in colorectal cancer and cardiovascular diseases (Rau et al., 2018; Moschen et al., 2016; Zuo et al., 2019). Polansky et al. Polansky et al. (2015) reported that in the cecal microbiota of chickens, Alistipes is a propionate producer. In research conducted by Oliphant and Allen-Vercoe (2019) also found that Alistipes can benefit the host gut and produce acetate in the cecum. Alistipes has a protective role, and a decrease in its abundance correlates with an increase in hepatic encephalopathy recurrence (Sung et al., 2019). Alistipes grows particularly well in the gut microbiota of patients who are obese, suggesting that Alistipes is associated with obesity. Therefore, the LL group had the lowest risk of liver cirrhosis. Desulfovibrio can act as opportunistic pathogens or induce asymptomatic infections in the host gastrointestinal (GI) tract (Urata et al., 2008). Desulfovibrio infections are often associated with inflammation (Ma et al., 2018). Gibson et al. (2010) postulated a role for Desulfovibrio in ulcerative colitis (UC). Our results showed Desulfovibrio abundance in the HL group; thus, a high energy diet was not conducive to host gut health. At the species level, Bacteroides fragilis was the lowest in the LL group. Bacteroides fragilis is a Gram-negative anaerobic bacterium that colonizes the host’s lower gastrointestinal tract. Although Bacteroides fragilis occupies a eminently low proportion of the gut microbiome, it benefits the immune system. Decreases in Bacteroides fragilis can lead to ulcerative colitis and Crohn’s disease (Zamani et al., 2017), which are associated with an imbalance in the intestinal flora. Furthermore, Desulfovibrio piger increased with an increase in energy. Loubinoux et al. (2002) found that the number of Desulfovibrio piger significantly increased in patients with inflammatory bowel disease. Doumatey et al. (2020) have reported that Desulfovibrio piger improves hyperglycemia and reduces insulin resistance.

As the core of the liver-gut axis, intestinal flora is involved in the regulation of liver diseases. We found that liver oxidation indexes were closely related to some microorganisms in the cecum. In our research, M.woesei was significantly correlated with the MDA level; Phocaeicola sp. Sa1YUN3 was significantly correlated with the T-AOC level. In this study, M.woesei increased with an increase in energy. Notably, M. woesei participates in the regulation of gut metabolism, such as methane metabolism. Additionally, in this study, M.woesei had higher intestinal content in chickens with higher abdominal fat content (Xiang et al., 2021). Our research also found that as the MDA level increased, M.woesei content increased. According to our results, Phocaeicola sp. Sa1YUN3 was highly abundant in the HL group. Sun et al. found Phocaeicola sp. could improved colitis in mice. Choi et al. (2023) found that the HF diet increased the relative abundance of Phocaeicola, supported by our findings.

As an important metabolic organ of the body, the liver can maintain oxidative balance by scavenging excessive reactive oxygen species in the body. Intestinal flora imbalance can cause oxidative stress (Vajro et al., 2013). Intestinal microorganisms are closely related to the liver through the gut–liver axis. This result has been further confirmed in our experiments. As an important component of the diet, energy affects not only affect intestinal composition, but also affect liver function. We found that high-energy–low-protein diets can cause oxidative stress in the liver.

In this study, a high-energy-low-protein diet caused oxidative stress in laying hens. According to our results, the HL group was enriched in the infectious disease viral pathway at the KEGG L2 level, indicating that feeding a high-energy-low-protein diet affected the intestinal health of chickens adversely. Our experiment further demonstrated that high-energy diets cause oxidative stress in laying hens through the gut-liver axis. This research might indicate that high energy adversely affects the production of laying hens.