As an important ruminant in the Qinghai-Tibet Plateau, Tibetan sheep graze naturally throughout the year, and mainly obtain nutrients by eating natural forages. In ruminants, > 75% of SCFAs produced by rumen fermentation are absorbed via the rumen epithelium as the main host energy source . We found that the total SCFA, propionate and butyrate were higher in Aug and Oct, and the propionate and butyrate were significantly higher than in other months; NH3-N levels were significantly higher in Jun and Augr than in other months. Forages in the green grass stage have high protein and carbohydrate content , and Tibetan sheep can produce high NH3-N levels after eating them by increasing the level of the main rumen community in the rumen . In this study, in Aug, the Qinghai-Tibet Plateau was in the green grass stage, with rich pasture species and high nutrient levels. Therefore, In June, NH3-N mainly providing energy for the host, and in August and October, the total SCFA concentrations providing energy for the host. Regarding energy utilization, the A:P ratio is inversely proportional to the energy utilization efficiency of forage , indicating that the energy utilization efficiency of Tibetan sheep is lowest in Dec and highest in Apr. During the green grass stage, Tibetan sheep rapidly eat forages with sufficient nutrient levels within a small range , so metabolism decreases and more energy is stored by the body. In contrast, in the withered grass stage, Tibetan sheep need to travel across a larger area to search for forages, which increases metabolism and thereby decreases energy storage. In addition, ACX and CL activities in the rumen gradually increased from Dec, peaking in Jun and Apr, respectively. In the withered grass stage in plateau grasslands (from early October to early Apr), the cellulose proportion in the forages is higher than that of green grass. The increased ACX and CL activities allow more high-cellulose-containing substrates to be degraded into glucose that can be directly used by the host, in order to provide energy for the host to adapt to the reduced energy supply during this stage.
The rumen microbes of Tibetan sheep and their hosts have evolved a relatively stable microbiota structure during long-term co-evolution in order to adapt to the harsh environment of the Qinghai-Tibet Plateau. Among the many factors affecting the rumen microbiota structure, diet is key . In this study, there were differences in the microbial abundances and diversity in the rumen of Tibetan sheep in different months. In particular, the microbial diversity was significantly lower in Dec than in other months, and the microbial abundance was significantly higher in Aug than in other months. The analyses of KEGG gene families and COG functional genes of microorganisms found that > 57.4% of the genes that were differentially expressed among different months were related to the METABOLISM pathway. Among them, the Carbohydrate metabolism pathway had the most significant impact regarding the differences between months, followed by the Amino acid and Energy metabolism pathways. This reflects the fact that rumen microbes and forage supply affect the METABOLISM pathway (mainly the Carbohydrate, Amino acid, and Energy metabolism pathways), which can affect host gene expression.
At the phylum level, both Bacteroidetes and Firmicutes were dominant (> 83.3%) in each month, and the abundance of Bacteroidetes was higher than that of Firmicutes. This is consistent with the results reported by Liu et al . However, Kim et al.  found that the mean abundances of Bacteroides and Firmicutes in the rumens of low-altitude livestock were only 31% and 56%, respectively. Cunha et al.  also found that the mean abundances of Bacteroides and Firmicutes in goats in semi-arid regions of Brazil were only about 37.9% and 56.3%, respectively. In the current study, there were no significant differences in the abundance of Bacteroidetes in different months, and it only increased in Dec. Members of this phylum can effectively decompose protein and carbohydrates in feed into SCFAs, providing energy for the host , and they also promote rumen growth and increase its volume . The abundance of Firmicutes was significantly higher in Jun than in Feb and Dec. Firmicutes can help cells absorb glucose . The increase in the abundance of Bacteroidetes in Dec indicates that it plays a more important role than Firmicutes in the high-altitude adaptation of the host during the period of nutrient deficiency. The abundances of Bacteroidetes and Firmicutes in the rumen of Tibetan sheep were maintained at stable levels during certain periods (Aug to Oct) of the year, which is of great significance to the stability of the rumen internal environment. The increase in the abundance of Bacteroidetes in Dec may have promoted rumen development and forage decomposition, thereby providing a certain amount of energy for the host. The significant increase in the abundance of Firmicutes in Jun may have helped the host to obtain more energy.
At the genus level, Prevotella_1 and Rikenellaceae_RC9_gut_group were dominant. Prevotella_1 plays an important role in the degradation and utilization of plant non-cellulosic polysaccharides, including starch, xylan, and protein . Reduced protein and starch levels decrease the abundance of Prevotella_1 . In this study, the abundance of Prevotella_1 was highest in Jun and lowest in Dec, which may be due to the lower protein content of Dec hay stage forages. Rikenellaceae_RC9 is closely related to members of the Alipites family , and it may play a role in the degradation of plant-derived polysaccharides . In this study, Rikenellaceae_RC9_gut_group had the highest abundance in Dec, which may have increased high-cellulose forage degradation, thereby providing energy for the host.
In addition, many cellulolytic bacterial genera  were found in this study, such as Ruminococcus_2, Fibrobacter, Butyrivibrio_2, Treponema_2, and Pseudobutyrivibrio. Cellulolytic bacteria can degrade cellulose in the rumen and play key roles in SCFA production . Microorganisms have a rich library of protein-coding genes that can encode various enzymes related to metabolism . Changes in the abundances of microbes lead to changes in enzymes, which alter the fermentation in the rumen . ACX and CL activities dropped to its lowest level in Aug, ACX activity was significantly lower in Aug than in other months, while CL activity remained at a stable level (from Apr to Dec), which indicates that the digestion and utilization of cellulose in forage grass by ruminal microorganisms remained relatively stable. It also implies that after a long period of co-evolution, a relatively stable cellulolytic community exists in the rumen of Tibetan sheep. We speculate that the microorganisms encoding cellulase proteins are an important part of the “core microbiota”  in the rumen of Tibetan sheep, and cellulolytic bacteria play an important role in the plateau adaptation mechanism of Tibetan sheep.
Studies have shown that high abundances of Prevotella and low abundances of Methanobacter in the rumen of Tibetan sheep promote forage fermentation to produce high SCFA concentrations and reduce methane production to avoid energy loss . In this study, the total SCFA, propionate, and butyrate were higher in Aug to Oct, and SGLT1 expression in the rumen epithelium was also significantly increased. Butyrate is the main source of ruminant metabolic energy [38,39], and both propionate and butyrate can promote the development of rumen papilla . Prevotella_1 was significantly positively correlated with butyrate level (P < 0.05); Ruminococcaceae_NK4A214_group and Succiniclasticum were highly significantly positively correlated with SGLT1 expression (P < 0.01); Ruminococcaceae_NK4A214_group and Succiniclasticum were significantly positively correlated with NH3-N level; and Ruminococcus_1 and [Eubacterium]_coprostanoligenes_group were highly significantly negatively correlated with SGLT1 expression (P < 0.01), but highly significantly positively correlated with ACX and CL activities. This is because the Jun forage quantity and quality are higher than those of the withered grass and ACX activity, the NH3-N and total SCFA levels produced by rumen microbial fermentation, and glucose production are higher. The high SGLT1 expression in the rumen epithelium helps to transport D-glucose to the blood faster , reduce the glucose concentration in the rumen, and improve or prevent rumen acidosis . It also increases the host energy utilization. Rumen microbes have adapted to highly nutritional forages, so the various cellulose-degrading bacteria in the rumen of Tibetan sheep reach a stable level by Aug, and CL activity remains at a stable level between Apr and Dec. Therefore, Aug is most conducive to the rumen development and body growth of Tibetan sheep.
The abundance of Rikenellaceae_RC9_gut_group was highest in Dec and lowest in Aug. Rikenellaceae_RC9_gut_group was also a biomarker for Dec samples. Furthermore, random forest analysis showed that Rikenellaceae_RC9_gut_group played important roles in rumen microbial composition. The acetate concentration increased significantly in Oct and Dec, and the propionate and total SCFA concentrations remained at high levels in Oct and Dec. The expression of CLAUDIN4 and ZO1 (which encode rumen epithelial barrier-related proteins that form a physical barrier around the cells to prevent the free passage of small molecules ) decreased significantly in Dec, that is, the permeability of the rumen epithelial barrier increased. Succiniclasticum was significantly positively correlated with CLAUDIN4 and ZO1 expression; Rikenellaceae_RC9_gut_group and Fretibacterium were significantly negatively correlated with CLAUDIN4 and ZO1 expression, and significantly positively correlated with acetate concentration.
Further study of the OTU that was found to be unique to the Dec samples (OTU270), as shown in the Venn diagram (Fig. 2A), showed that the species belongs to the phylum Synergistetes, which mainly colonizes anaerobic environments  and can ferment amino acids and carry out glycolysis [44,45]. The forage cellulose ratio was slightly higher in Oct than in Aug, and ACX activity was also higher, so the cellulolytic bacteria still produced higher SCFA concentrations (such as a significant increase in acetic acid concentration). Additionally, Synergistetes thoroughly and effectively decomposes forage via amino acid fermentation and glycolysis, thereby providing the host with as much energy as possible. Synergistetes has been reported to be a biomarker of periodontitis in the human oral cavity . Therefore, we speculate that Synergistetes can increase the energy utilization of the host during the period of nutrient deficiency. In this situation, it is beneficial to the host. In Dec, when the quality of forage is further reduced, the host increases the abundance of Synergistetes to provide itself with more energy. At this time, CLAUDIN4 and ZO1 expression also reached their lowest levels, the permeability of the rumen epithelium increased, more small molecules (mainly nutrient molecules) in the rumen easily pass through, and the host obtained more energy. However, when the abundance of Synergistetes increases to a certain level, the rumen epithelium of Tibetan sheep becomes inflamed, the barrier protein structure is gradually destroyed, and the rumen epithelial function decreases. To avoid long-term inflammation, after entering the rejuvenation stage (Apr), the host regulates the microbiota structure by actively regulating rumen epithelial genes, which significantly increases CLAUDIN4 and ZO1 expression, inhibits the passage of harmful substances, and returns the CLAUDIN4 and ZO1 expression to normal levels by Aug. This is a typical self-regulatory protection mechanism in Tibetan sheep in alpine regions with insufficient pasture. To prevent the decline in rumen epithelial function of Tibetan sheep observed in Dec, Feb, and Apr due to insufficient forage and the corresponding decrease in production performance, supplementary feeding should be carried out between Dec and Apr of the following year.
In addition, compared to the Dec, Feb, and Apr samples, the Jun samples a significant increase in the number of differential species and the number of biomarkersthe (P < 0.05), and a significant change in microbiota structure (the diversity and abundance of rumen microbes). Thus, compared to other months, the rumen microbial composition and structure of the Jun samples underwent substantial adjustments. Based on the KEGG and COG analyses, the METABOLIC pathway also underwent a certain degree of change in Apr to Aug. Therefore, we believe that Jun is the most critical transition stage for Tibetan sheep in the Qinghai-Tibet Plateau, which is of great significance to their growth and development.