With improved living quality, people are paying more attention to good taste while ensuring food provides nutrition. The balanced nutrition and taste requirements of ruminants for food are similar to humans’ dietary requirements. As described by the Pavlovian reaction, the secretion of digestive juices is influenced by appetite, and the digestion and absorption of feed are also affected (Tellez et al., 2016). Sweeteners play a crucial role in enhancing the feeding efficiency of ruminant animals due to their unique taste stimulation (Neumann et al., 2005). The sweetness of these additives stimulates the animals’ taste buds, transmitting signals to the cerebral cortex and the digestive system (Gaillard et al., 2017; Iwamura et al., 2020). This stimulation increases saliva, intestinal fluid, pancreatic fluid, and bile secretion in the digestive tract (Lee et al., 2021). The peristalsis and digestive enzyme secretion in the gastrointestinal tract are enhanced, promoting feed digestion and nutrient absorption (Ma J. et al., 2020). Previous research has shown that adding essence and spices to ruminant diets can enhance taste and smell, increasing food intake (Hussein et al., 2018). Furthermore, supplementation of sweeteners in the diet has been shown to increase the average daily gain of cattle (McMeniman et al., 2006; Ponce et al., 2014). Sweeteners contribute to animal growth, development, production performance, and feed utilization efficiency.

Steviol glycosides (STE) is an emerging natural sweetener derived from the herbal plant stevia (Chatsudthipong and Muanprasat, 2009; Lemus-Mondaca et al., 2012). It is a zero-calorie sweetener 470 times sweeter than sucrose (Salehi et al., 2019). STE has been discovered to possess immune regulatory activities and positive effects regarding anti-inflammatory, antioxidant, and antibacterial properties and maintaining blood lipid levels (Pariwat et al., 2008; Casas-Grajales et al., 2019; Wei et al., 2021). Moreover, scientific studies have demonstrated that STE also exhibits antibacterial properties in monogastric animals such as chickens and rats (Lopez-Carbon et al., 2019; Alavala et al., 2020; Jiang et al., 2021). As the biological activity of STE has been discovered, there is growing interest in their application in animal production. STE has demonstrated potential effects as a bioactive substance in goat digestion rate and rumen fermentation in animal experiment (Xu et al., 2021). Additionally, dietary STE supplementation significantly increased Bifidobacteria and decreased Escherichia coli of the cecal digesta in broilers (Wu et al., 2019). Recent research has investigated that the total or partial replacement of sugar with STE influences the general state of health, especially the human microbiota’s response. This study demonstrated the modulatory role of STE on the human microbiota and on the fermentation processes that determine the essential SCFA synthesis in maintaining homeostasis (Gatea et al., 2021). These results indicate that STE may play an important role in regulating the rumen microbial communities. To our knowledge, no studies have reported the impact of STE on rumen microbial communities in calves.

Calves are crucial to the success and sustainability of dairy farms as they are the backup force and the future hope for high milk production. Proper management of calves feeding is essential for the profitability of the dairy industry. The rumen is a vital organ in ruminants due to its connection with nutrient metabolism, uptake, and transport (Nishihara et al., 2019). During this process, the development of the rumen in calves is extremely important for their growth and development, and appropriate feed intake plays a crucial role in its complete development. Reducing production costs and accelerating rumen development in calves due to early weaning is currently a common feeding method for calves (Ma J. et al., 2020). However, early weaning will lead to weaning stress. During this stage of development, the calves’ immune organs and digestive system are still developing, making them more susceptible to gastrointestinal issues and threatening their health (Browne et al., 2021). The microbiota is involved in regulating the development and health of the gastrointestinal tract. Its importance lies in providing essential metabolites, maintaining metabolic function, supporting immune system development, and defending against pathogens, which are crucial for overall host well-being (Guo et al., 2020). In recent years, an increasing emphasis has been on investigating the relationship between host performance and microbial composition (Cunha et al., 2019; Sofyan et al., 2019), the relationships between gastrointestinal flora and milk production in dairy cows and calf diarrhea (Sofyan et al., 2019; Ma T. et al., 2020). Some scholars have demonstrated that dietary STE supplementation affects the gut microbiota of chickens (Wu et al., 2019; Jiang et al., 2021). Additionally, previous studies have indicated that early intervention of starter feed contributes to the prompt colonization of rumen microflora, leading to high levels of volatile fatty acids (VFA) in the rumen, which may stimulate the early development of rumen epithelium tissue (Lin et al., 2019). It is unclear whether dietary STE affects calf rumen development, fermentation parameters, and microbiota. Therefore, this study aimed to determine the effects of dietary supplementation with STE on rumen development and microbiota in weaned calves.

The experiment was conducted at the experimental farm of Yangzhou University (Yangzhou, China), and the Yangzhou University Institution Animal Care and Use Committee (IACUC) approved all procedures involving animals (SYXK (Su) 2021-0026).

The rumen is the first stomach of ruminants, and the level of rumen development directly affects the subsequent production performance of ruminants. With the introduction of pellets at weaning and the subsequent establishment of the rumen microflora, the rumen not only develops physically but also begins to perform metabolic functions. The physical development of the rumen encompasses two key aspects. Firstly, the growth of rumen weight and capacity, and secondly, the development of rumen papillae (Liang et al., 2019). Rumen quality is stimulated by the quantity and physical structure of the ration consumed by the animal (Zitnan et al., 1998). Increased early intake of solid feed pellets stimulates increased rumen mass and growth of rumen teats. In our study, STE supplementation significantly increased rumen weight. It has been reported that the increase in rumen weight is due to the physical stimulation of solid feed, and the growth and keratinization of rumen papillae are due to chemical stimulation by fermentation products such as VFA (Baldwin and Connor, 2017).

Rumen development parameters (such as nipple length and width) are fundamental indicators of feed digestibility, rumen development, and feed efficiency (Na and Guan, 2022). The length and surface area of the ventral rumen papilla of calves supplemented with STE were increased, indicating that the calves in the STE group may have a larger surface area for absorbing nutrients in the ventral rumen. Previously, a study reported greater papillae development in the rumen’s ventral sac (O’Shea et al., 2016). Researchers suggested that this specific development allows for enhanced absorption of VFA compared to the dorsal sac due to increased contact between the ruminal fluid and the ventral wall. Furthermore, another study demonstrated that butyrate concentration is the most effective activator for promoting rumen papillae growth (Shen et al., 2004; Gorka et al., 2011; Kato et al., 2011). It has been shown that solid feed intake during weaning reduces the gene expressions of Insulin-like growth factor binding proteins 2 (IGFBP 2), IGFBP3, and IGFBP6, causing rumen epithelial cell proliferation in weaned calves to stimulate the growth of rumen papillae (Nishihara et al., 2019). Whether the addition of STE works through this pathway needs further proof. We hypothesized that STE induced calves to consume many pellets after weaning, which enhanced rumen development, increased the weight of the forestomach, stimulated nipple growth, promoted muscle development, and enhanced absorption capacity.

In order to promote the normal development of the rumen epithelium, the rumen must have a normal microbial flora to establish viable fermentation, and VFA in the rumen can promote the normal development of the papilla (Bannink et al., 2016). Therefore, we performed an analysis of rumen fermentation parameters. In this study, rumen pH was not significantly altered and remained within the normal range, supplementing with STE, indicating normal rumen fermentation function and no acidosis in calves. However, the trend of a decrease in pH after supplementing STE may be attributed to an increased intake of calves’ pellets, resulting in higher VFA production. As reported, when the microbial production rate of VFA surpasses the rate at which the rumen epithelium absorbs VFA, it leads to an accumulation of VFA in the rumen, ultimately resulting in a decrease in rumen pH value (Zhang et al., 2018).

As the main fermentation product of rumen microorganisms, a high level of proton release accompanies VFA production, and the absorption of VFA in the rumen epithelium is closely linked to the transport of protons or anions to adjust the pH in the rumen, which can directly affect the activity of microorganisms (Gabel et al., 2002). Bicarbonate-dependent transporters on the apical and basolateral membranes facilitate the exchange of free VFA with bicarbonate (HCO₃⁻), transferring HCO₃⁻ into the rumen lumen to increase or maintain luminal pH (Dengler et al., 2014). Undissociated VFA passively diffuses through the epithelial cell membrane, and the released protons are exported to the extracellular space via the Na/H exchanger, regulating the intracellular pH (Zhang et al., 2020). Supplementing 1.0% stevia stalk has been reported to more effectively enhance microbial fermentation activity in the rumen and promote the digestion and absorption of fiber (Zhang et al., 2023). The levels of propionate, butyrate, and TVFA in the STE supplementation group increased significantly in our experiment. Acetate and propionate, absorbed by the rumen epithelium, serve as energy sources or precursors for FA synthesis and gluconeogenesis. On the other hand, around 95% of the absorbed butyrate is metabolized within the rumen epithelium to acetoacetate and D-3-hydroxybutyrate, which act as energy sources for ruminants (Gabel et al., 2002). After weaning, VFA such as butyrate are used as the primary energy source and ketogenic precursor of rumen epithelial cells (Baldwin and Jesse, 1992).

STE has been suggested to stimulate rumen development in calves, possibly due to enhanced energy production. In the rumen, VFA production is related to the total amount of organic matter fermented by rumen microorganisms (Ungerfeld, 2020), while the balance between production and absorption regulates rumen VFA concentrations (Islam et al., 2021). VFA production in the developing rumen begins with the ingestion of solid feed, which provides the substrate for anaerobic fermentation (Kim et al., 2016). Steady increases in solids intake corresponded to increases in rumen VFA concentrations, reaching adult-like concentrations at a very young age (Cristobal-Carballo et al., 2021). This also suggested that the effect of STE on rumen VFA may also be due to the calves’ increased intake of pellets. Artificial sweeteners increased the concentration of propionic acid in mouse feces, and enhanced glycan degradation pathways were observed through metagenomic sequencing, in which glycans are fermented to form various compounds, including VFA, further suggesting a potential bacterium for sweeteners metabolism (Suez et al., 2014). This may explain the apparent variation in VFA observed in the STE group in this experiment. Since the metabolic pathways used to synthesize VFA are encoded in the genome of the rumen-content microbiota or the rumen epithelial microbiota, further research is needed to confirm the influence of STE on microbiota function.

The calves supplied with STE had higher valerate concentration in their rumen digesta than the CON group. Valerate in rumen digesta was a product of rumen fermentation, synthesized by propionyl-CoA and acetyl-CoA (Van Soest et al., 1991). In the presence of isobutyrate and isovalerate, valerate fulfills the necessary straight carbon chain requirement for cellulolytic rumen bacteria such as Bacteroides succinogenes (Bryant and Doetsch, 1954; Zimmermann et al., 2021). Interestingly, rats ingested stevia (rebaudioside A) showed elevated concentrations of cecal valerate and a strong positive correlation between valeric acid concentration and body weight was observed (Nettleton et al., 2019). Appropriate rumen ammonia-N concentration is a prerequisite for rumen microbial protein synthesis, which is conducive to the growth and reproduction of cellulolytic bacteria. Previous research found that the optimal concentration of ammonia-N for microbial protein synthesis in the rumen should be greater than 5 mg/100 mL (Karimizadeh et al., 2017). The concentration of ammonia-N in this experiment was at a normal level, indicating normal fermentation of rumen microorganisms. However, Zhang et al. (2023) found that the concentration of ammonia-N increased for 48 h, which is inconsistent with the results of this study. This observation may be attributed to two main factors: in vitro fermentation and feed type.

The growth and development of calves are primarily related to the rumen microbial community (Malmuthuge and Guan, 2017). Gastrointestinal microbial diversity is crucial in maintaining a stable microbial community. The alpha diversity analysis indicated that the difference between the two groups was non-significant. Research on beef cattle has indicated that rumens with higher microbial diversity can ferment a broader range of substrates, resulting in lower feed conversion rates. Conversely, rumen with less diversity, known as “simple rumen,” may generate more specific products that the host can efficiently absorb and utilize (Li et al., 2019). Therefore, the role of rumen microorganisms may depend on their heritability or function rather than their richness and diversity.

The increased abundance of Firmicutes is associated with higher ADG in animals and is vital in influencing feed efficiency (Min et al., 2019). There is limited research on the effects of STE on the microbiome of livestock and poultry, especially about the ruminants we describe. The abundance of Firmicutes exhibited a significant increase in the rumen of the STE group, and studies have shown that they can degrade fiber and cellulose (Michalak et al., 2021). Vamanu et al. (2019) used an in vitro simulated intestinal method and found that sodium saccharin significantly reduced the abundance of Firmicutes, mainly due to the increased abundance of E. coli caused by sodium saccharin, which lowered intestinal pH and disrupted the original microbial community. This may be due to the different metabolic characteristics of artificial sweeteners and natural sweeteners in animal bodies and differences in the pathways affecting microorganisms. It has been reported that STE positively affected the intestinal flora of broiler chickens, increasing the concentration of Streptococcus and reducing the concentration of E. coli in the cecum (Wu et al., 2019). The residue of stevia has been shown to have a significant inhibitory effect on the growth of harmful bacteria while promoting the proliferation of beneficial bacteria (Xiao et al., 2022). However, we did not find that supplementing with STE in the diet significantly inhibited the abundance of harmful bacteria such as Escherichia coli in the rumen.

In our study, the relative abundance of Succiniclasticum, Lachnospiraceae_NK3A20_group, and Olsenella was raised in the rumen of calves in the STE group. Succiniclasticum predominantly relies on starch and various other carbohydrates as their primary source of fermentation substrates. They metabolize these substrates through a series of biochemical reactions, yielding propionate. Moreover, Succiniclasticum can ferment fiber and cellulose (An et al., 2005). Ma et al. (2022) found that Succiniclassicum was significantly enriched in the foregut of Aohan fine-wooled sheep and was involved in the metabolism of succinate in LEfSe analysis. At the same time, He et al. (2021) observed that Succinniclassicum can utilize succinate to produce propionate, which is beneficial for intestinal health. Succiniclasticum significantly increased in the STE group, indicating that carbohydrates in the substrate are beneficial for the reproduction of succinate solubilizing bacteria (Wang et al., 2022). Lachnospiraceae_NK3A20_group is an important constituent of the gastrointestinal microbiota in ruminants, characterized by cellulose decomposition activity and starch hydrolysis, which can produce acetate and formate and is closely associated with butyrate production (Russell and Rychlik, 2001). Lachnospiraceae_NK3A20_group was positively correlated with the levels of TVFA, propionate, butyrate, isobutyrate, and isovalerate, whereas negatively correlated with pH, acetate concentration, acetate-to-propionate ratio, and xylanase activity (Liu et al., 2022). This is consistent with this study’s positive correlation between Lachnospiraceae_NK3A20_group and propionate concentration.

Lachnospiraceae can degrade fibrous substances in the intestine to generate VFA, lower pH values, prevent harmful microbial reproduction, and maintain intestinal microbiota homeostasis. A notable finding was that the abundance of Lachnospiraceae_NK3A20_group was significantly higher in the rumen of calves in the STE group. This increase in abundance is believed to promote rumen development through enhanced production of butyrate, which directly impacts the development of the rumen. As the abundance of Lachnospiraceae_NK3A20_group increased, calves’ rumen utilization rate increased significantly, enabling calves to obtain more energy while promoting rumen growth and development. Zhao (2023) found that artificial sweetener neotame significantly increased the abundance of Lachnospiraceae NK3A20_group in the rumen. This phenomenon can be the reason for the increase of the rumen butyrate. This is consistent with our results.

Olsenellae is the primary bacterium responsible for choline degradation in healthy and acidotic bovine rumen. Petri et al. (2013) reported that Olsenella dominates during subacute rumen acidosis in beef cattle. There is a significant negative correlation between Olsenella and propionate concentration in the rumen of Holstein cows (Guo et al., 2023). On the contrary, it was shown that the abundance of Olsenella in the rumen of yaks fed with high-concentrate diets was significantly lower than that of yaks fed with low-concentrate diets (Khafipour et al., 2009). The relative abundance of Olsenella in the rumen of fattened cattle was negatively correlated with the methane phenotype, positively correlated with the percentage of propionate, and negatively correlated with the acetate-to-propionate ratio (Smith et al., 2022). This may prove that the relative abundance of Olsenella increased by supplementing STE, which may also reduce methane production and positively correlate with VFA production in the rumen.

Interestingly, Olsenella is a lactobacillus that ferments carbohydrates into lactate (Khafipour et al., 2009). It has been found in the gastrointestinal tract of humans and animals to ferment starch and glycogen substrates, producing lactate, acetate, and formate (Goker et al., 2010). However, the abundance of Acinetobacter in the rumen was significantly lower in the STE-supplemented rumen in this experiment. Acinetobacter is a Gram-negative aerobic bacterium. Most Acinetobacter infections occur primarily in immunocompromised hosts. Usually, Acinetobacter infections are characterized by localized suppurative inflammation; in severe cases, meningitis and sepsis may occur (Chebotar et al., 2014). Meanwhile, Acinetobacter was shown to have a higher relative abundance in the rumen epithelium of goats in the high-concentrate diet group compared to the low-concentrate group (Shen et al., 2019). So far, most known Acinetobacter genus members exhibit pathogenic and commensal lifestyles in the gastrointestinal tract (Shen et al., 2019). Therefore, STE supplementation may have a positive effect on rumen health.

In addition, valerate level was significantly negatively correlated with the abundance of rumen bacterial genera Olsenella (Li et al., 2022), which is inconsistent with the results of this experiment. Isobutyrate and isovalerate levels are significantly correlated with the abundance of rumen bacterial genera Rikenellaceae_RC9_gut_group (Li et al., 2022), which is consistent with the results of this experiment. Interestingly, we found that the abundance of Succiniclassicum increased after supplementing with STE and was positively correlated with the propionate concentration. More importantly, Succinivibrionaceae_UCG-001 and Rikenellaceae_RC9_gut_group were positively correlated with rumen weight (Li et al., 2022). This may prove our results that STE promotes rumen weight, which is related to the microorganisms in the rumen, and the good fermentation of rumen microorganisms produces metabolites that promote rumen development. Thus, changes in rumen microbial abundance correlate with observed phenotypic changes in fermentation parameters, and further studies are needed to examine, alter, and optimize microbial populations to improve rumen homeostasis and promote rumen development. We speculate that the appropriate STE concentration can promote the rumen’s microbial fermentation activity and improve the rumen digestive function of calves, thereby improving feed digestibility, promoting rumen development, and maintaining body health.

In summary, we found that the rumen weight, papilla length, and papilla surface area of calves supplemented with STE significantly increased compared to the CON group. In addition, calves fed STE displayed increased propionate, butyrate, and TVFA levels in the rumen. STE treatment impacted the rumen microbiota composition in weaned calves. The relative abundance of Firmicutes and Actinobacteriota in the rumen of calves fed with STE significantly increased, while those of Proteobacteria and Spirochaetota significantly decreased at the phylum level. At the genus level, Succiniclasticum, Lachnospiraceae_NK3A20_group, and Olsenella significantly increased, while the relative abundance of Acinetobacter significantly reduced. This also indicates that it may be possible to enhance fermentation efficiency by modifying the composition of bacterial communities to promote rumen development in weaned calves. Therefore, STE may be a beneficial feed additive to enhance weaned calves’ rumen development.

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA016842) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

The animal study was approved by the experiment was conducted at the experimental farm of Yangzhou University (Yangzhou, China), and the Yangzhou University Institution Animal Care and Use Committee (IACUC) approved all procedures involving animals (SYXK (Su) 2021-0026). The study was conducted in accordance with the local legislation and institutional requirements.

KW: Conceptualization, Data curation, Formal analysis, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing. MJ: Methodology, Validation, Writing – review & editing. YC: Investigation, Visualization, Writing – review & editing. YH: Investigation, Methodology, Visualization, Writing – review & editing. ZC: Validation, Writing – review & editing. OD: Writing – review & editing. SJ: Validation, Writing – review & editing. LZ: Resources, Writing – review & editing. YL: Resources, Writing – review & editing. GZ: Funding acquisition, Project administration, Writing – review & editing. ML: Funding acquisition, Project administration, Writing – review & editing.