Bos grunniens is classified as a bovid in the class Artiodactyla, Mammalia, Chordata. They are mainly found in the Qinghai-Tibetan Plateau, where they naturally graze on alpine pastures [1]. Like other ruminants, the yak has four stomach chambers, including the reticulum, rumen, omasum and abomasum [2]. Microbial colonization and fermentation largely occur in the rumen. The rumen also provides a suitable anaerobic environment for the growth and multiplication of micro-organisms [3]. Bacteria, methanogenic bacteria, fungi and protozoa are the main microorganisms in the yak rumen. These microorganisms interact with each other to enhance nutrient digestion [30]. [34] isolated anaerobic fungi from sheep rumen contents and showed that rumen anaerobic fungi can produce cell wall degrading enzymes, such as cellulase, hemicellulase, pectinase and esterase [4]. The rate of dry matter degradation by rumen fungi in the absence of bacteria is approximately 62% [5]. Rumen fungi utilise all plant polysaccharides and soluble monosaccharides to produce volatile fatty acids, hydrogen and intermediate metabolites [6].

The anaerobic environment in the rumen limits the culture of rumen microorganisms in vitro. Therefore, omics technology has provided a useful tool to reveal the function of rumen microorganisms. For example, transcriptome analysis showed that mRNAs differentially expressed in yak rumen fluid are related to various mechanisms, such as HIF-1α, insulin and PI3K-Akt signalling pathway, nucleotide excision and repair, cell cycle, apoptosis and fatty acid metabolism. This suggests that the rumen regulates the nutritional and physiological metabolism of the host [7]. However, an mRNA-based study cannot comprehensively assess the structure and function of the yak rumen microbial community. This is because post-transcriptional translation and post-translational modification can weaken the association between mRNA and protein [8]. Proteomes can reveal the function of rumen microbes as proteins are directly involved in biological metabolism [28]. Proteomic analysis of rumen fluid from dairy cows at different feeding times identified 658 proteins in 19 rumen fluid samples. The different feeding times affected 68 differential proteins. Furthermore, the differential protein pathway shifted from transformation-centered biosynthesis to a broader DNA drive with increasing sampling time intervals [31]. The results of the co-culture solution of anaerobic rumen fungi from dairy cows and methanogens showed that the diversity of anaerobic fungi decreased with increasing sampling interval time. Anaerobic fungal species passing at the 5 d and 7 d intervals were significantly different from those passing at the 3 d interval [9].

Although studies have reported the effect of passage culture at different time intervals on the type of rumen anaerobic fungal flora, it remains unknown whether passage at different time intervals affects the proteomes of rumen anaerobic fungi and their functions. This study aimed to: 1) obtain a pure culture of yak rumen anaerobic fungi subjected to passage culture at different time intervals (3 d, 5 d, and 7 d) for seven times using consecutive batch culture; 2) analyze the proteins of anaerobic fungi subjected to the passage at different time intervals using the TMT-based quantitative proteomics; 3) count the differential proteins (DPs) and annotate their functions using bioinformatics.