Reproductive hormones, as a key component of the endocrine system, are critical for physiological processes such as skeletal development, muscle growth, and fat metabolism in young livestock (Yuting et al., 2011). Androgens have been shown to accelerate protein synthesis, promote muscle growth, and regulate glucose and lipid metabolism, thereby improving growth performance (Schiffer et al., 2017). In contrast, estrogen plays a pivotal role in skeletal maturation, epiphyseal closure, and the cessation of linear growth (Li, 2024). Letrozole (LE), a third-generation aromatase inhibitor, works by specifically binding to the aromatase enzyme, thereby suppressing the conversion of androgens to estrogens and increasing androgen levels within the organism. A study by Rezaei et al. (2020) demonstrated that administering LE at a dosage of 0.25 mg/kg significantly increased serum testosterone (T) levels in goats, which correlated with enhanced average daily gain (ADG) and carcass weight. Furthermore, the microbiota has been shown to play significant roles in the reproductive endocrine system by interacting with hormones such as estrogen, androgen, and insulin, while also influencing the ruminal microbial structure and metabolic function (Rosales-Nieto et al., 2021; Tian, 2022). In ruminants, steroid hormones reach the rumen either through transmural diffusion across the ruminal wall or via saliva (Han, 2010), and these hormones subsequently modulate ruminal metabolic activity (Li et al., 2022).

Currently, research on the mechanism by which LE regulates growth performance in ruminants has primarily focused on endocrine pathways, while its effects on rumen microbial communities and metabolic products remain unclear. Therefore, this study used Hu sheep as the experimental subjects. By supplementing diets with varying levels of LE, we investigated its effects on growth performance, serum hormone levels, rumen fermentation parameters, rumen microbial community structure, and metabolomic characteristics. The aim was to elucidate the rumen microbial mechanisms through which LE regulates growth in Hu sheep, thereby providing a theoretical basis for the rational application of LE in ruminant production.

The efficient degradation of feed in ruminants largely depends on the homeostasis of the ruminal internal environment, which is closely associated with rumen pH, NH₃-N concentration, and VFA levels (Castro-Montoya et al., 2011). Rumen pH serves as a critical physiological indicator of this balance, and deviations from the normal range (typically 5.5–7.5) can disrupt fermentation processes (Qiu et al., 2022). In the present study, rumen pH values across all lamb groups remained within the normal physiological range following LE supplementation, indicating that LE administration did not significantly affect ruminal fermentation.

The NH₃-N in the rumen is derived from the microbial breakdown of non-fiber feed components. It serves as a key nitrogen substrate in microbial anabolism, supporting the growth and proliferation of bacteria and other microorganisms essential for microbial protein synthesis (Jefferson et al., 2022). Receptors for steroid sex hormones, such as T, E₂, and progesterone (P), have been identified in the cytoplasm and nuclei of rumen bacteria. The binding of these hormones to their receptors can influence ruminal metabolism in ruminants (Han, 2006). Chen et al. (1999) demonstrated that daidzein supplementation via duodenal infusion in male water buffaloes increased blood T levels and significantly elevated ruminal concentrations of both NH₃-N and TVFA. The increase in ruminal NH₃-N concentration following LE-induced elevation in plasma T is likely mediated by T binding to microbial hormone receptors. This suggests that elevated T may interact with hormone receptors on rumen microbes, modulating the microbial community and thereby enhancing NH₃-N production.

Short-chain fatty acids (SCFAs) in the rumen are primarily synthesized through microbial fermentation of feed. The VFAs, a major component of SCFAs, serve as the primary energy source for ruminants, providing 70-80% of their total energy needs (Kumar et al., 2024). The present study demonstrated that LE supplementation induced a significant shift in the ruminal A:P, indicating a broader modulatory effect of LE on rumen fermentation. Propionate is a critical contributor to weight gain in ruminants, with its adequate availability being essential for maintaining healthy growth (Zhang et al., 2022). A highly significant linear increase in ruminal propionate concentration was observed with increasing doses of LE, likely due to T-enhanced microbial carbohydrate degradation. However, this shift in ruminal fermentation did not result in improved growth performance, such as ADG, in lambs. This lack of response may be attributed to the relatively short trial duration, suggesting that further investigation is needed to determine the optimal dosage and duration of LE supplementation for maximizing growth performance.

The rumen of ruminants harbors a complex consortium of symbiotic microorganisms, including bacteria, archaea, ciliates, fungi, and viruses (Firkins and Yu, 2015). Among these, the phyla Firmicutes and Bacteroidetes are the most abundant, containing numerous fibrolytic genera such as Succiniclasticum, Prevotella, and Ruminococcus, which form the core ruminal microbiota (Mizrahi et al., 2021). These microbes play a critical role in degrading plant fibers and polysaccharides, fermenting them into intermediates such as succinate, lactate, and fumarate, which are then converted into end products including VFAs, microbial protein, and other nutrients that provide energy to the host (Schwab and Broderick, 2017). Consequently, the rumen microbiota is central to the digestive and metabolic processes of ruminants. A key finding of this study was the significant alteration in the core ruminal microbiota following LE supplementation, specifically a decrease in the relative abundance of Bacteroidetes and an increase in Firmicutes compared to the CON group. Furthermore, the correlation analysis between the growth performance of Hu sheep lambs and rumen microbiota revealed a significant positive correlation between the relative abundance of Firmicutes and the average daily gain (ADG) of lambs. Given that Firmicutes are involved in the conversion of dietary fiber to VFAs (Ko et al., 2020), while Bacteroidetes are critical for polysaccharide breakdown, this shift suggests a potential modification in the ruminal metabolic landscape. Previous studies have linked a higher Firmicutes-to-Bacteroidetes (F/B) ratio with improved growth performance (Bauman et al., 2011; Huangfu, 2023). Our correlation analysis between the rumen microbiota and growth parameters in lambs produced similar findings, further supporting this relationship. The observed increase in TVFA and propionate levels may be attributed to the elevated relative abundance of Firmicutes. At the genus level, Succiniclasticum abundance was consistently higher in all treatment groups compared to the CON group. This genus is known for its ability to degrade fiber to produce succinate, which is then converted to propionate, providing energy to the host. Therefore, the increase in Succiniclasticum abundance likely contributed to the elevated propionate concentration observed in this study.

The growth, development, and metabolic processes of animals are regulated by various factors, with hormones and growth factors playing pivotal roles (Mei, 2024). In male animals, T, as a primary reproductive hormone, not only exerts hormonal effects but also regulates critical physiological processes such as appetite control, protein metabolism, and lipid metabolism (Yutong et al., 2025). During osteoblast differentiation, T helps maintain bone growth and ameliorate osteoporosis (Vanderschueren et al., 2014). Endogenous T has also been found to suppress the response of melanin-concentrating hormone (MCH) neurons to glucose, thereby reducing post-fasting plasma glucose levels, enhancing induced satiety, and increasing feed intake (Fukushima et al., 2015). It has further been shown to promote protein synthesis in skeletal muscle and facilitate skeletal growth, contributing to improved overall growth performance in animals (McClure et al., 2000; Zhang et al., 2019). Research indicates that sustained T circulation in lambs has both direct and indirect effects on muscle protein synthesis and bone growth, which collectively enhance growth performance (Schanbacher et al., 1980). A study by Li et al. (2019) found that laying hens supplemented with 0.5 mg/d of LE for 6 weeks showed a significant increase in body weight. Similarly, Rezaei et al. (2020) demonstrated that LE supplementation in goats significantly increased ADG and F:G, positively affecting growth performance. However, in the present study, no significant changes in growth performance were observed in lambs following LE supplementation. Existing research indicates that T plays a role in regulating both protein and lipid metabolism, promoting protein anabolism while also stimulating lipid catabolism (Guo, 2015). Lipid metabolism is particularly crucial for the short-term improvement of animal growth performance. In this trial, although LE supplementation increased circulating T concentrations in lambs, it did not result in enhanced ADG. It is hypothesized that the weight gain from T-stimulated muscle protein synthesis may have been counteracted by concurrent lipid catabolism, leading to no net improvement in overall growth performance.

Animal growth is regulated by GH, and elevated T levels can stimulate GH secretion, which, in turn, plays a key role in various developmental and metabolic processes such as protein synthesis, growth promotion, and lipolysis (Song et al., 2023). In contrast, INS exerts opposing effects to GH in lipogenesis, enhancing tissue glucose uptake and fatty acid synthesis by promoting fatty acid synthase (FAS) expression (Guo, 2015). As a highly selective cytochrome P450 aromatase inhibitor, LE effectively blocks the conversion of T to E₂, thereby elevating systemic T levels. This action is hormonally selective and does not interfere with the normal secretion of corticosteroids or thyroid hormones (Liu et al., 2024). Fu (2023) demonstrated that oral administration of varying doses of LE (0.1, 1.0, and 10 mg/kg/d) to female juvenile mice reduced ovarian E₂ levels and increased T levels. Similarly, Ortiz-Carrera et al. (2019) observed a significant increase in serum T levels following supplementation with 2.5 mg/d LE in male goats, with serum E₂ levels remaining unchanged. These findings align with the results of the present study.

Plasma biomarkers such as TP, ALB, and GLB—which reflect protein assimilation, transport and repair, and immune function, respectively (Peng et al., 2020)—were analyzed. All concentrations were within normal physiological ranges, and no significant differences were observed among groups. This suggests that LE supplementation did not have an overt impact on these parameters under the current experimental conditions. Therefore, the potential immunomodulatory effects of LE in Hu lambs warrant further investigation under different conditions or with more targeted immune challenges.

During metabolic processes, organisms generate various highly reactive oxygen species (ROS), reactive nitrogen species (RNS), and singlet oxygen. When present in excessive concentrations, these molecules can damage intracellular macromolecules, such as DNA and proteins, contributing to the pathogenesis of numerous diseases (Pisoschi and Pop, 2015). It has been suggested that androgens help mitigate oxidative stress in organisms. A study by Wang (2023) found that T deficiency exacerbated oxidative damage in the hippocampal region of male APP/PS1 mice, a model of Alzheimer’s disease. Additionally, Yao (2022) reported that during heat stress, castrated beef cattle showed significantly lower activities of T-AOC, SOD, and GSH-Px compared to partially castrated and sham-operated groups. Chen (2016) observed that LE supplementation in bulls increased T-AOC and reduced MDA content in seminal plasma. As an aromatase inhibitor, LE works by inhibiting aromatase activity, thus blocking the conversion of androstenedione (AD) and T to estrogens, which indirectly elevates androgen levels in muscle tissue (Bai et al., 2015). In the present study, LE supplementation did not significantly affect most plasma antioxidant parameters (T-AOC, CAT, SOD, GSH-Px) or MDA levels in Hu lambs. However, a notable decrease in NO concentration was recorded 4 h after supplementation in the T1 and T2 groups. The discrepancy between these results and previous findings may be attributed to the varying stress conditions inherent to the experimental models.

In summary, dietary LE supplementation appears to enhance the ruminal fermentation pattern and fiber degradation capacity by modulating the microbial community (Figure 13). This aligns with previous research identifying E₂, P, and T in ruminant rumen fluid, with T shown to promote ruminal fermentation (Han, 2006). Additionally, Silva et al. (2024) reported greater ruminal microbial diversity in male ruminants compared to females. Alpha-diversity indices (including Observed OTUs, Chao1, and Simpson) reflect community richness and diversity. In this study, Group T3 exhibited a significantly higher number of OTUs compared to the CON group, with an even more pronounced increase compared to Group T1. Furthermore, all alpha-diversity indices (OTUs, Chao1, Simpson) showed significant enhancement with increasing LE doses. These findings collectively suggest that LE supplementation increased both the diversity and richness of the ruminal microbiota in lambs, likely due to LE’s inhibition of androgen aromatization, which indirectly shaped the ruminal microbial environment.

Under the experimental conditions, LE supplementation increased lambs’ concentrations of NH₃-N, propionic acid, and isovaleric acid, elevated the relative abundance of the Firmicutes, reduced the relative abundance of the Bacteroidetes, altered rumen fermentation patterns, and promoted an increase in plasma testosterone concentration. In comparison, LE supplementation at a dose of 0.2 mg/kg BW yielded superior effects.