Creatine is phosphorylated to produce phosphocreatine, which can provide energy for cell activities. Creatine produced inside the body of animals can only meet 50–70% of the body’s needs, and the remaining amount should be supplemented by exogenous creatine (Ostojic, 2017). Creatine has a high production cost, poor stability, and a low utilization rate of additional creatine in animals (Chamruspollert et al., 2002). Guanidinoacetic acid (GAA), the direct precursor of creatine biosynthesis in animals, has stable chemical properties and high biological value and is suitable for use as a feed additive (Baker, 2009). Currently, GAA, as a nutritional feed additive, has been approved for use in animals by the European Food Safety Authority (Poaa, 2016) and the Chinese Ministry of Agriculture (MOA Ministry of Agriculture (PRC), 2014). Supplementation of GAA in poultry diets was proven to enhance the feed conversion rate and muscle quality (Lemme et al., 2007; Khalil et al., 2021), as well as liver antioxidant capacity (Zhao et al., 2021) and muscle energy metabolism (Michiels et al., 2012). In addition, GAA supplementation can significantly increase the average daily feed intake, feed conversion and lean meat percentage of pigs (Jayaraman et al., 2018) and improve carcass traits and meat quality by reducing the mandibular fat index and changing muscle fiber characteristics (Zhu et al., 2020). Liu et al. (2021) reported that dietary supplementation with 0.6 g/kg GAA significantly increased the body weight and average daily gain of Angus bulls.

In fact, the function of the rumen is very important for nutrition. Rumen microbes can degrade protein in feed to provide amino acids as a nitrogen source for the body (Sari et al., 2022) and digest carbohydrates to produce VFAs as an energy source (Wilk et al., 2021). Research on GAA has mainly focused on animal growth, meat quality, and antioxidant properties of poultry and pigs (Lemme et al., 2007; Jayaraman et al., 2018; Khalil et al., 2021; Zhao et al., 2021). However, only a few studies have investigated the effect of adding GAA on ruminants, and the effect of GAA on rumen fermentation and rumen microflora is unclear. Therefore, an in vitro experiment was carried out to study the effects of different levels of GAA on rumen gas production, gas composition and fermentation parameters, as well as the structure and gene function of rumen microflora through 16S rRNA high-throughput sequencing technology, to provide a reference for the use of GAA in ruminant feed.

The GAA used in the experiment was provided by Beijing Gendone Biotechnology Co., LTD. GAA is a white powder with 98% purity. All procedures in this study were approved by the Laboratory Animal Ethics Committee of Inner Mongolia Minzu University (protocol number: 2018090516001).

In vitro rumen-simulated fermentation techniques are commonly used to evaluate the extent of rumen microbial fermentation and the utilization of diets (Yejun et al., 2019; Grubješić et al., 2020; Choi et al., 2021). Rumen microorganisms ferment carbohydrates to produce rumen gasses (H₂, CH₄, CO₂, etc.) and VFAs (Ju et al., 2023). Under the action of some microorganisms, these gasses also have a certain mutual transformation (Lv et al., 2022). Rumen gas production and gas composition reflect not only the digestion process of feed in the rumen but also the changes in rumen microorganisms and are usually affected by fermentation substrates and additives. It is obvious that substrate composition affects rumen fermentation kinetics. It has been shown that supplementing 0.4% GAA to a fermentation substrate with a ratio of concentrate to forage (C/F) of 7:3 can increase the total gas production after 24 h of fermentation (Ren et al., 2023), and a C/F of 5:5 can also promote nutrient digestion and rumen fermentation in Angus bulls (Li et al., 2020). Therefore, it is worth discussing the effect of GAA when the ratio of concentrate to forage is further reduced. In this experiment (with C/F 4:6), there was no significant difference in GP_{48 h} between the GAA groups and the CON group, but the gas production in the GAA groups was numerically lower (p > 0.05). In addition, the gas production rate is also an important indicator of rumen fermentation kinetics (Wu et al., 2022). In this experiment, the theoretical maximum gas production (B in Table 2) and gas production rate (C in Table 2) predicted by the model showed no significant differences between groups, and the gas proportions of H₂, CH₄, and CO₂ did not change. Since rumen gas was mainly produced from carbohydrates in the diet, it is considered that GAA barely influenced carbohydrate digestion of the substrate with this C/F (4:6).

A relatively stable rumen environment is important for the colonization and function of rumen microorganisms; in general, the indicators examined include rumen pH, NH₃-N and VFAs (Ma et al., 2022). Rumen pH is particularly important (Ma, 2021) because it is also the result of the combined effect of rumen VFA and NH₃-N. The rumen pH fluctuates from 5.5 to 7.5, which is conducive to rumen microorganisms and to the normal function of ruminants (Calsamiglia et al., 2008). In this experiment, the pH of the rumen in each group fluctuated within the normal range of 6.09–6.13, indicating that GAA supplementation did not disrupt the balance of volatile acids and ammonia nitrogen and maintained the acid–base environment, which was consistent with the results of Wales et al. (2004).

The NH₃-N concentration in the rumen is affected by the substrate’s degradable protein content and the activity of rumen microorganisms (Zain et al., 2023). The optimal NH₃-N concentration for rumen microorganism growth is generally within the range of 20–50 mg/dL under in vitro conditions (Satter and Slyter, 1974). The results of this experiment showed that the NH₃-N concentrations of all groups were similar and fluctuated within the normal range from 31.99 to 35.31 mg/dL, which means that microbial nitrogen sources were adequately supplied. Since GAA contains 35.86% nitrogen and does not affect the rumen ammonia nitrogen content but increases MCP, it is speculated that it promotes the conversion of NH₃-N into MCP. The same result was reported in lambs (Ren et al., 2023). However, it may depend on more factors than the amount added. A decreased NH₃-N concentration was also observed (Li et al., 2020; Liu et al., 2021). Supplementation with GAA (from 300 mg to 900 mg/kg) even affected the growth performance and nutrient digestion of lambs (Liu et al., 2021).

Carbohydrates are fermented in the rumen and produce VFAs, which can meet 70–80% of ruminant energy needs (Rey et al., 2014); therefore, carbohydrates are an important source of energy for ruminants. In addition, acetate and butyrate are important substances for milk fat synthesis, while propionate is an important precursor for glucose synthesis in gluconeogenesis, which is also mainly used for synthesizing body fat and lactose (Li et al., 2023). The proportion of VFA depends on the rumen fermentation pattern, especially C/F. More acetic acid and less propionic acid are produced when C/F is low. To distinguish rumen fermentation patterns, when A/P is greater than 3, it is considered an acetate-type fermentation. Otherwise, it is a propionate-type fermentation (Gong, 2020). The lower C/F in this experiment was achieved by acetate-type fermentation, but the concentration of propionate significantly increased with GAA supplementation. Therefore, the A/P showed a decreasing trend due to the increase in propionate. This result was consistent with the report of Liu et al. (2023). This meant that GAA supplementation has a regulatory effect on the rumen fermentation pattern to perform propionate-type fermentation. In fact, propionate-type fermentation has important physiological significance in finishing cattle, such as the lowest heat consumption, less methane production and the highest energy conversion efficiency. This means that beef cattle can obtain more energy for weight gain (Xie et al., 2021). The effect of GAA on the rumen fermentation pattern may be attributed to rumen microorganisms. The results of 16S in this experiment indicated that GAA supplementation increased the abundance of Prevotella and Prevotellaceae_UCG-001, which are involved in propionate metabolism in the rumen, and thus led to an increase in the concentration of propionate.

Branched chain amino acids (valine, leucine, and proline) undergo oxidative deamination or decarboxylation by rumen microorganisms (Ren and Zhao, 2008), which produce corresponding isomeric acids (butyrate, isobutyrate, isovalerate, and valerate) (Zhang et al., 2022). Among them, isovalerate is associated with fiber degradation and was reported to increase acetate, butyrate and TVFA in the rumen of Simmental cattle and reduce propionate, which leads to acetate-type fermentation (Liu et al., 2007). In this experiment, propionate significantly increased and isovalerate showed a significant downwards trend with increasing GAA addition. This means that isovaleric acid-related fiber degradation is weakened and propionic acid-related nonfiber carbohydrate degradation is enhanced. This was consistent with previous reports. However, GAA supplementation did not affect acetate and butyrate concentrations.

The rumen is a complex anaerobic fermentation system composed of various microorganisms with a high degree of microbial diversity (Mizrahi and Jami, 2018). These microorganisms are important in maintaining the stability of the rumen environment, increasing the nutrient digestion rate and maintaining animal productivity (Lin et al., 2018). Alpha diversity reflects the diversity within a specific ecosystem, the Shannon index is used to reflect microbial diversity, and the Chao index is an indicator of richness (Wei et al., 2017). Shabat et al. (2016) found that the diversity of rumen microorganisms decreased while the concentration of propionate increased. In this study, the same results were found. As the level of GAA addition increased, the Shannon index decreased, but the Chao index did not show significant changes. This suggested that supplementation with different GAA levels may partially affect the diversity of the rumen microbiota.

Firmicutes degrade dietary fiber to produce acetate and butyrate, while Bacteroidota mainly produces propionate through nonfibrous substance degradation. In this study, Firmicutes and Bacteroidota were also the dominant microbiota in the rumen, which was consistent with the results of other studies (Lee et al., 2011; Yang et al., 2020). However, the relative abundance of Firmicutes showed a trend of first increasing and then decreasing with GAA addition, while Bacteroidota showed a significant decrease at first and then increasing. The most obvious changes were observed within the GAA02 and GAA08 groups, and between them, they showed almost opposite results. This meant that the result may depend on the GAA level, and the 0.8% level may be a critical value. When the amount of GAA supplemented was 0.8%, Bacteroidota significantly increased, and the results of rumen volatile acids also indicated a significant increase in propionate concentration. This indicated that low levels of GAA may be beneficial for Firmicutes survival, while high levels of GAA are more suitable for Bacteroidota to degrade nonfibrous substances and produce more propionate.

Further research and analysis of the differences in genus-level communities revealed that Rikenellaceae_RC9_gut_group is the dominant genus in the rumen, which is the same as the reports of Liu et al. (2022) and Ahmad et al. (2022). Cox et al. (2014) found that Rikenellaceae_RC9_gut_group is a beneficial bacterium with intestinal protective functions that is mainly involved in the degradation of plant-based polysaccharides in the body, and its relative abundance is directly proportional to dietary fiber content (Fan et al., 2020). This indicates that GAA supplementation does not negatively impact dietary fiber degradation that can be used by beneficial dominant bacteria in the rumen. In this experiment, the addition of 0.6% GAA significantly increased norank_f__F082 and Papillibacter. The norank_f__F082 belongs to Bacteroides and mainly participates in nonstructural carbohydrate degradation. Papillibacter has been found to exist in the rumen in studies (Ma et al., 2020; Zhang et al., 2022), but the functions of norank_f__F082 and Papillibacter have not been reported. The relative abundance of Prevotella and Prevotellaceae_UCG-001 was significantly increased when 0.8% GAA was supplemented. Prevotella and Prevotellaceae_UCG-001 belong to the phylum Bacteroidei, among which Prevotella has a strong ability to degrade nonstructural carbohydrates and proteins and can ferment sugars through the acrylic and succinic acid pathways to produce propionate (Flinth, 2008). By examining the correlation between rumen fermentation parameters and microbial communities, propionate concentration was positively correlated with the relative abundance of Prevotellaceae_UCG-001. The increase in the relative abundance of Prevotella often leads to an increase in propionate content (Poudel et al., 2019). This also explains the increasing rumen propionate concentration with increasing GAA addition levels from the perspective of the microbial genus level.

The rumen microbiota is a complex ecosystem closely related to the biology of the host, and species interactions are crucial for the stability of the community in healthy symbiotic microbiota (Clemente et al., 2012; Faust et al., 2012). A network diagram was drawn based on the correlation between species to reflect the interactions between species in the sample. Ramayo-Caldas studied the relationship between gut microbes in 518 pigs (60 days of age) through correlation network analysis and found that genus-level correlation network analysis revealed significant interactions between different genera of pig gut flora (Ramayo-Caldas et al., 2016). Prevotella and Prevotellaceae_UCG-001 showed a positive correlation, while the two were negatively correlated with other bacterial genera. This was consistent with the results of the genus-level relative abundance analysis above, where the addition of 0.8% GAA significantly increased the relative abundance of Prevotella and Prevotellaceae_UCG-001 while decreasing the relative abundance of NK4A214_group and Christensenellaceae_R-7_group. Through correlation network analysis, it was found that NK4A214_group, Prevotella, Prevotellaceae_UCG-001 and Christensenellaceae_R-7_group are important genera in the rumen microbiota, which may partially affect the stability of the rumen microbiota.

Rumen microorganisms participate in body metabolism and provide nutrients to the host by decomposing dietary nutrients, and the abundance and function of microflora are closely related to the host (Vasta et al., 2019). Further analysis showed downregulated expression of flagellar assembly, bacterial chemotaxis and plant–pathogen interaction in the GAA08 group. Flagellar assembly and bacterial chemotaxis are important in the bacterial adhesion process (Wang, 2019). Zhang et al. (2022) showed that plant–pathogen interactions mainly involve pathogenic microorganisms plundering nutrients from host cells for survival and reproduction, while host plants use various defense strategies to inhibit pathogen growth. This indicates that high GAA levels reduce the cell motility ability of bacteria and the plant–pathogen interaction. In addition, Hamana et al. (2012) showed that replication-and repair-related genes may help the body restore the molecular structure of genes and reduce the damage caused by mismatching biological molecules. In this experiment, the expression of mismatch repair and nucleotide excision repair in the GAA08 group was downregulated, indicating that supplementing high levels of GAA may reduce the body’s ability to replicate and repair biomolecules. The relative abundance of bile secretion and protein digestion and absorption in the GAA08 group was significantly higher than that in the other groups. Based on the above results of MCP concentration and relative abundance of bacteria, the addition of 0.8% GAA promotes nutrient utilization, such as lipids and proteins, by rumen microorganisms, leading to an increase in these genes.

The results showed that when GAA was supplemented, the concentrations of MCP and propionate in the rumen could be significantly increased, but isovalerate was decreased. In addition, the relative abundances of Bacteroidota, Prevotella and Prevotellaceae_UCG-001 could be significantly enhanced. The metabolic pathways related to bile secretion, protein digestion and absorption were increased. In conclusion, supplementation with 0.8% GAA can enhance in vitro rumen fermentation parameters and increase the relative abundance of bacteria related to nonfiber substance degradation, which would enable beef cattle to obtain more energy.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI - PRJNA1011887.

The animal studies were approved by Laboratory Animal Ethics Committee of Inner Mongolia Minzu University. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

CD: Conceptualization, Data curation, Formal analysis, Writing – original draft. MW: Funding acquisition, Project administration, Resources, Writing – review & editing. JJ: Conceptualization, Writing – review & editing. LD: Methodology, Writing – review & editing. RZ: Investigation, Writing – review & editing. MX: Investigation, Writing – review & editing. YZ: Visualization, Writing – review & editing. HB: Visualization, Writing – review & editing. MB: Supervision, Writing – review & editing.