The motility of the rumen plays a vital role in ruminant nutrient degradation, and could be featured by cyclic movements basically including the primary contraction and the secondary contraction (Ruckebusch, 1988; Beauchemin, 2018). The primary contraction basically facilitates the mix of rumen ingesta and microbial fermentation, as well as the transfer of rumen digesta into the omasum, whereas the secondary contraction usually leads to the eructation of ruminants (Imran et al., 2011; Arai et al., 2019). It was observed by the transabdominal ultrasonography on cows that, the average time interval between the two successive peaks of ruminal contractions was 48.6 ± 1.53 s, without the differentiation between the primary and secondary ruminal contractions (Imran et al., 2011). Through the ultrasonographic examinations performed on dairy cows, Braun and Schweizer (2015) found that the duration of a complete cycle of the primary contraction was approximately 45 s. As significant variables of rumen motility, the duration and interval of ruminal contractions could be bound up with the production performance, health, and welfare of ruminants (Braun and Schweizer, 2015; Arai et al., 2019). However, until now, reports on the exact time of the contraction duration and interval in different ruminants, and their impacts on the rumen fermentation and ruminal microbiota are rather limited.

With the constant inflows and outflows, the rumen simulation technique (RUSITEC) system could serve as a relatively more effective tool in assessing rumen fermentation in vitro in comparison with the traditional method of batch culture (Deitmers et al., 2022). So far, two major types of the RUSITEC system have been separately invented and are commonly applied: the semi-continuous culture system and the continuous culture system (Hoover et al., 1976; Czerkawski and Breckenridge, 1977). These artificial rumen systems vary from simple vessel sorts with different volumes to complex continuous-flow types with diverse stirrer, and various in- and outflow systems (Deitmers et al., 2022). In our previous investigation on the impacts of rotation speed on rumen fermentation and ruminal microorganisms, a novel dual-flow continuous RUSITEC system was developed and adopted with detailed descriptions (Adebayo Arowolo et al., 2022). This dual-flow continuous RUSITEC system can also be used to examine our hypothesis that the contraction duration and interval could affect the rumen microbial fermentation, through the simulation of different contraction durations and intervals by setting different stirring times and intervals for the stirrer and the elimination of the interferences from other irrelevant factors.

Therefore, the aim of the current study was to evaluate the influences of stirring time on nutrient degradation, fermentation characteristics, and bacterial microflora in a RUSITEC system. We assume that this study could not only gain more insights into the biofunction of rumen motility pattern, but also help to improve the validity of RUSITEC.

The ruminal motility, which primarily consists of the primary and secondary contractions, has been proved to facilitate the microbial colonization on feed particles and thence enhance the rumen fermentation (Ruckebusch, 1988; Beauchemin, 2018). The reticulorumen contractions are crucial for the digesta mixing and movement, as well as the gas eructation (Imran et al., 2011; Arai et al., 2019). It has been detected that the strength, frequency, and duration of ruminal contractions generally grow around feeding (Egert et al., 2014; Arai et al., 2019). Conversely, declines in the rumen contractions could be a clinical sign of reticuloruminal stasis, which is usually related to metabolic disease such as ruminal acidosis, tympany, and hypocalcaemia (Arai et al., 2019). In the present study, the conversion from constant stirring to intermittent stirring significantly improved the ruminal TVFA concentration, nutrient disappearance rates, and total gas production, indicating that proper reduction of the stirring time might help to prolong the retention time during which the feed particles were degraded by the ruminal microbes in the fermentation vessels, and thereby promote the RUSITEC fermentation (Dufreneix et al., 2019; Adebayo Arowolo et al., 2022).

The NH₃-N is exploited by the ruminal microbes in synthesizing microbial protein, and the simultaneous supply of nitrogen and energy is essential for the NH₃-N utilization efficiency and MCP production of the ruminal microorganisms (Nocek and Russell, 1988; Bharanidharan et al., 2018; Lu et al., 2019). It has been reported that the rumen motility could help the microbes to colonize the substrate and hence contribute to the synchronization of nitrogen and energy release (Beauchemin, 2018; Adebayo Arowolo et al., 2022). In this trial, the MCP level rose significantly with the reducing stirring time, which was in line with the significant decrement in the ruminal NH₃-N concentration. The improved MCP synthesis observed in this study further supported our previous assumption that appropriate motility could promote the acquisition of NH₃-N by the ruminal microorganisms for MCP production (Adebayo Arowolo et al., 2022).

Our precedent investigation found that elevating the stirring speed from 5 rpm to 25 rpm quadratically increased the copy number of protozoa in the rumen solid (Adebayo Arowolo et al., 2022). In the current experiment, the highest protozoa count in the rumen fluid was exhibited in the IS2 treatment, while the protozoa number in IS2 was also higher than that in the CS group. This phenomenon could be an explanation for the significant increments of CH₄ yield in response to the increasing interval time between stirring, as a major portion of methanogens are attached to ruminal protozoa and considered as one of the most active contributor for methanogenesis (Belanche et al., 2014; Wang et al., 2017b). It could be thus inferred that, compared to the continuous stir, reducing the stirring time and increasing the interval properly would also benefit the proliferation of rumen protozoal populations.

In the rumen liquid obtained from the RUSITEC system of this trial, the bacterial community at the phylum level was successively dominated by Proteobacteria, Bacteroidota, and Firmicutes, which further confirmed the predominance of these three bacterial phyla in the ruminal microbiome reported in precedent investigations including our prior studies (Rey et al., 2014; Wang et al., 2017a, 2020, 2021, 2022; Zhong et al., 2018). Besides, the genera Rikenellaceae_RC9_gut_group, Succinivibrionaceae_UCG_002, and Ruminobacter, and the species Ruminobacter amylophilus were, respectively, identified as the prominent ruminal bacterial taxa in this experiment, and this result was also supported by the findings of previous researches (Cui et al., 2021; Daghio et al., 2021; Wu et al., 2021).

In this study, the greatest indices of Ace and Chao 1 were both marked in IS2, implying that the intermittent stirring raised the richness of the bacterial populations when compared with the constant stirring (Granja-Salcedo et al., 2017). Since the diversity and richness of the rumen microflora are considered as the fundamental factor which could affect the rumen function (Weimer, 2015; Xie et al., 2018), the elevated richness of the bacterial microbiome might lead to the above-mentioned enhancement of rumen fermentation metrics. Further distinctions of the bacterial community within different treatments were shown in the PCoA plots based on both unweighted and weighted Unifrac matrix, which suggested the influences of stirring time on the structure of rumen bacterial microbiota.

The genus Treponema is a bacterial taxon that commonly detected in the rumen as a major participator involving the lignocellulose degradation (Bekele et al., 2011; Xie et al., 2018), while the Lactobacillaceae is a lactate-producing bacterial family precedently found in the bovine rumen (Wu et al., 2019; Baek et al., 2020). The prevalence of these two bacterial taxa in the CS treatment might indicate their preference for the continuous stirring condition, and requires further studies to be verified. Moreover, Li and Guan (2017) reported that the abundance of Lactobacillaceae tended to be higher in the rumen of cattle with low feed efficiency, when compared to cattle possessing high feed efficiency. It could be thereby assumed that the significant enrichment of Lactobacillaceae in the CS could be correlated with the lower nutrient disappearance rates than those in the IS1 and IS2. Agathobacter ruminis was previously isolated from the rumen of grazing sheep and cows, and it had been identified as an obligatory anaerobic and saccharolytic genus with the capacity to produce acetate and butyrate (Rosero et al., 2016). Therefore, the enrichment of Agathobacter ruminis might partly contribute to the boost in ruminal TVFA in IS2. Very little is known about the Bacteroidales_bacterium_Bact_15 and uncultured_rumen_bacterium that were enriched in IS2. It could be presumed that these two genera might favor the environment with reduced stirring time, and more studies are necessitated to disclose their role in rumen fermentation during the frequency change.

It was noteworthy in this study that, according to the Tax4Fun analysis, the KEGG pathways of carbohydrate metabolism and digestive system were both enriched in IS2 compared with IS1. Nevertheless, no difference was observed in the abundances of metabolic pathways between IS2 and CS, despite those significant differences in fermentation parameters and nutrient disappearance rates between these two treatments. This disparity could stem from the distinctions between the bacterial populations in rumen fluid and rumen solid, differences between the metagenomics analysis and actual metabolic activities of the rumen microflora, and the limitations of Tax4Fun (Wang et al., 2017a, 2020, 2021).

In conclusion, the highest TVFA, MCP, protozoa count, nutrient disappearance rates, gas production, and bacterial richness indices of Ace and Chao 1 were all presented in the IS2 treatment of this trial. In addition, the relative abundances of Agathobacter ruminis and another two less known bacterial genera were also enriched by raising the interval time from 0 to 3 min. The current investigation not only supported the theory that the proper cyclic contractions could be beneficial to the ruminal microbial fermentation, but also provide the improvements of RUSITEC system with more references. Further studies are required to explore the optimum stirring time and interval for the enhancement of RUSITEC fermentation, as well as to elucidate the roles and relevant mechanisms for those altered bacteria during the frequency change.

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 at: https://www.ncbi.nlm.nih.gov/, PRJNA877046.

All animal procedures in the present study were reviewed and approved by the Animal Care Committee (approval number: 20211003), College of Animal Science and Technology, Hunan Agricultural University, Changsha, China.

ZW, QL, and WS designed the research. ZW, QL, XL, WS, FW, JH, ST, and ZT conducted the research. ZW and QL analyzed the data. ZW wrote the manuscript. All authors contributed to this study and read and approved the final manuscript.

The present work was supported by the Hunan Herbivores Industry Technological System, Science and Technology Department of Xinjiang Uygur Autonomous Region (Grant No. 2022A02001-1), Hunan Provincial Science and Technology Department (Grant No. 2021RC4060), Hunan Provincial Natural Science Foundation (Grant No. 2021JJ30011), and National Natural Science Foundation of China (Grant No. 32172758).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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