Ruminant animals lack the carbohydrate-active enzyme encoding genes, so feed (carbohydrate) metabolism is completely dependent on the microorganisms residing in their rumen (Kameshwar and Qin 2018). Current research on rumen fungi has focused on anaerobic rumen fungi. Anaerobic rumen fungi play a very important role in the digestion and metabolism of carbohydrates in the rumen (Gruninger et al. 2018; Kameshwar and Qin 2018). Anaerobic rumen fungi can secrete large amounts of cellulolytic enzymes. Their hyphae can destroy the cell wall structure of plant feed owing to the combination of enzymes and degradable cellulose and this improves the degradation and utilization rates of plant feed (Lee et al. 2000; Gruninger et al. 2018; Kameshwar et al. 2018). Currently, the rumen AF (anaerobic fungi) are classified into phylum Neocallimastigomycota (Gruninger et al. 2014) and Neocallimasticaceae (Hibbett et al. 2007). Neocallimasticaceae was divided into eleven genera, containing a large number of monocentric rumen AF:
Little is known about rumen AF and most of them were identified by microscopic conventional cultivation techniques, which provides important information for rumen AF (Breton et al. 1990; Ho and Barr 1995). The limitations of these methods are mainly due to the strict growing requirements and the low survival rate but molecular biology techniques can overcome these problems (Pryce et al. 2006). The fungal ribosomal RNA gene includes a gene encoding 28S ribosomal DNA, 18S ribosomal DNA, and 5.8S ribosomal DNA, which in the internal transcribed spacer Region1 evolves rapidly (Sirohi et al. 2013; Elekwachi et al. 2017). The last-mentioned gene has the interspecies specificity and intraspecies conservation, and the length of the sequence is moderate enough to get sufficient information (Pryce et al. 2006; Campa et al. 2008; Bellemain et al. 2010). ITS sequencing is used to study the diversity of the community of rumen AF and provide genetic information for the classification and identification of fungi (Liggenstoffer et al. 2010; Koljalg et al. 2013).
The free-range gayals are mainly distributed in the Nujiang River and Dulong River areas of Yunnan Province, China. Yaks live exclusively on the Qinghai-Tibetan Plateau, China (An et al. 2005) and are well adapted to harsh environmental conditions. Yunnan yellow cattle and Tibetan yellow cattle are common, wide-ranging cattle. Yunnan yellow cattle live in the same region as gayals and Tibetan yellow cattle in the same region as yaks (Deng et al. 2007; Leng et al. 2012). Rumen bacteria in gayals, Yunnan yellow cattle and yak have been already studied (Deng et al. 2007), but there is no research on their rumen fungi. Rumen anaerobic fungi are the first microorganisms attached to fibers during rumen microbial degradation (Bauchop 1979) and play an important role in the degradation process (Dagar et al. 2015b). Anaerobic fungi degrade lignocellulose using a large portfolio of Carbohydrate-Active enZymes (CAZymes) and penetrating hyphae that physically disrupt the ultrastructure of the plant cell wall; such action may help to increase the surface area for bacterial colonization and further enzymatic digestion (Lee et al. 2000; Gruninger et al. 2018; Kameshwar et al. 2018). Rumen anaerobic fungi have great application potential in industrial production. The AF cellulose degradation ability shows that it can increase biogas production in co-culture with methanogens (Cheng 2018). Studies have also shown that rumen AF can reduce animal energy loss by reducing CH4 (greenhouse gas) production during digestion, and it can also be used to improve the straw lignocellulosic structure in biofuels and biochemical production (Andrea et al. 2018; Oliver and Schilling 2018). These four cattle breeds are very important cattle species in China, and there are very few studies on their rumen fungi. Therefore, this paper conducted a comprehensive analysis of rumen fungi from four breeds of cattle to help us understand their rumen fungi function.
Diversity index of anaerobic fungal communities among the cattle breeds.
Cattle breeds | Feed | Reads | 0.97 (level) | ||||
---|---|---|---|---|---|---|---|
OTU0.05 | Ace | Chao | Shannon | Simpson | |||
D | weeds | 64818 | 255 | a272 (b263, c288) | a263 (b258, c276) | a2.17 (b2.15, c2.18) | a0.3102 (b0.3063, c0.3142) |
H | feed | 57567 | 166 | a193 (b180, c220) | a191 (b176, c225) | a2.78 (b2.76, c2.79) | a0.1352 (b0.1332, c0.1372) |
M | weeds | 98550 | 463 | a487 (b476, c505) | a477 (b470, c494) | a2.82 (b2.80, c2.83) | a0.1622 (b0.1606, c0.1639) |
ZH | weeds | 64694 | 441 | a461 (b452, c477) | a451 (b445, c466) | a1.74 (b1.72, c1.76) | a0.4905 (b0.4858, c 0.4953) |
Weeds: bamboo or other wild grass; feed: rice bran and corn
a – average; b – minimum number; c – maximum number; D – gayals; H – Yunnan yellow cattle; M – yaks; ZH – Tibetan yellow cattle
Cattle diet and nutrient levels.
Feeds | Dietary nutrients (%) | ||||
---|---|---|---|---|---|
DM | CP | EE | NDF | ADF | |
Bamboo diet | 48.10 ± 9.85 | 13.06 ± 1.20 | 3.08 ± 0.69 | 72.13 ± 1.54 | 42.76 ± 3.02 |
Wild grass | 70.24 ± 0.56 | 1.44 ± 0.10 | 0.04 ± 0.02 | 30.12 ± 0.17 | 18.83 ± 0.10 |
Rice bran | 87.03 ± 0.22 | 12.82 ± 0.16 | 16.53 ± 0.18 | 22.91 ± 0.21 | 13.44 ± 0.52 |
Corn | 86.15 ± 0.14 | 8.38 ± 0.13 | 3.01 ± 0.24 | 8.59 ± 0.62 | 3.27 ± 0.54 |
DM – Dry matter; CP – Crude protein; EE – Ether extract; NDF – Neutral detergent fiber; ADF – Acid detergent fiber
A total of 622 176 original sequences were obtained from four cattle breeds by Illumina sequencing of which 297 745 sequences remained after quality control measures, and 285 092 sequences were used for phylogenetic analyses. The average length of trim sequences used for analyses was 317.6 bp. The average number of sequences from the different cattle breeds is shown in Table I. Rarefaction curve analysis (Fig. 1B) indicated that sequences collected in this study comprised the majority of rumen fungi sequences from the four cattle breeds. The taxonomic analysis was reflected in the cluster analysis of OTUs, with 904 OTUs from the four cattle breeds: 255 OTUs from gayals, 166 from Yunnan yellow cattle, 463 from yaks, and 441 from Tibetan yellow cattle (Table I). The four cattle breeds had unique OTUs of rumen AF and shared OTUs across rumen AF (Fig. 1A).
Venn diagram, rarefaction index, and abundance distribution curves for D, H, M and ZH based on OTUs from cattle breeds, which had ≥ 97% similarity.
D – gayals; H – Yunnan yellow cattle; M – yaks; ZH – Tibetan yellow cattle. Red line represents D. Blue line represents ZH.
Green line represents M. Yellow line represents H.
With increasing sequencing depth, the number of OTUs was unchanged (Fig. 1B). A rank-abundance distribution curve was constructed to reflect the abundance and uniformity of rumen fungi. The width of the curve indicated the highest abundance of rumen fungi for yaks and the least abundance for Yunnan yellow cattle; the abundance of Tibetan yellow cattle was close to yaks (Fig. 1C). According to the sequence abundance of the top 50 OTUs, the relative abundance of rumen fungi was similar. However, abundance differed among the different cattle breeds with increasing OTU ranking (Fig. 1C). The uniformity of rumen fungi was similar when the relative abundance was under 0.01, indicating that yaks, Tibetan yellow cattle, and gayals had a similar abundance and uniformity of fungi (Fig. 1C).
Composition of rumen fungi genera. Others represent the abundance of rumen fungi lower than 1%.
D – gayals; H – Yunnan yellow cattle; M – yaks; ZH – Tibetan yellow cattle.
Phylogenetic tree. The tree was based on taxonomic information such as the abundance of all genera and the genera of corresponding OTUs based on taxonomic information from the NCBI database to reflect the diversity and community of rumen fungi of D, H, M, and ZH.
D – gayals; H – Yunnan yellow cattle; M – yaks; ZH – Tibetan yellow cattle.
Fungal classification and the percentage statistics.
Total sequence | Phylum | Sequences | Percents | Dominant genus |
---|---|---|---|---|
285092 | Neocallimastigomycota | 63 535 | 22.28% | |
Basidiomycota | 6 030 | 2.11% | ||
Ascomycota | 2 740 | 0.96% |
The hierarchical clustering heatmap analysis was performed at the class level based on the top 95 most abundant communities across the four cattle breeds (Fig. 4). Results were separated into five clusters. The abundance of
Heatmap formed using the Bray-Curtis algorithm and the complete linkage method. The heatmap-plot describes the relative percentage of each fungal class within each cattle breed. Relative values for the fungal class are indicated by color intensity.
D – gayals; H – Yunnan yellow cattle; M – yaks; ZH – Tibetan yellow cattle.
The previous studies have shown that Illumina sequencing has a higher capacity to explore rumen bacteria diversity than culture-dependent methods (Peng et al. 2015). PCR amplification of universal primers for conserved regions within the rRNA genes, followed by DNA sequencing of the internal transcribed spacer (ITS) is widely used in fungal identification studies (Pryce et al. 2006). Primers using ITS1 can avoid bias in PCR amplification and reliably study the fungal abundance and species richness (Bellemain et al. 2010). This study used the second-generation sequencing technology to investigate the structure and diversity of rumen fungi communities in four cattle breeds. The results provide new information about rumen fungi communities. The analysis showed that the dominant rumen fungi clusters, distribution, and abundance present major differences among the cattle breeds, location, and feeds.
Free-range ruminants that use grass as food may require more anaerobic fungal cellulase to aid digestion than domesticated ruminants. When compared with Yunnan yellow cattle, gayals, yak, and Tibetan yellow cattle have abundant rumen fungi sequences (Table I) and more unique OTUs (Fig. 1A). Analysis of ACE, Chao, and Simpson indexes showed that gayals, yaks and Tibetan yellow cattle had higher indexes than Yunnan yellow cattle, but the Shannon index was smaller than for Yunnan yellow cattle (Table I). These results suggest that free-range gayals and Tibetan cattle can have higher rumen fungi diversity than domesticated Yunnan cattle. Unclassified sequences were 90.63% for gayals, 98.52% for yaks, 97.79% for Tibetan yellow cattle, and 27.01% for Yunnan yellow cattle (Fig. 2), which was consistent with the heatmap analysis (Fig. 4). These results showed that the class levels could be divided into five clusters based on the top 95 genera. Many unidentified genera were distributed in the third cluster and were dominant in gayals, yaks, and Tibetan yellow cattle. These results indicated that free-range ruminants were more likely to have unknown yet fungi.
Animal species and location may be the important factors influencing the distribution and abundance of dominant rumen fungi clusters. Analysis of phylogenetic trees detected three dominant phyla rumen fungi: Ascomycota, Basidiomycota, and Neocallimastigomycota in the four cattle breeds in this study (Fig. 3), similar to the results of Zhang and Han studies (Zhang et al. 2017; Han et al. 2019). But the abundance of Neocallimastigomycota is superior to Ascomycota and Basidiomycota in this study, contrary to the results of the study on the cashmere goats (Han et al. 2019). However, Neocallimastigomycota predominates in the rumen, which is similar to the results on most ruminant rumen anaerobic fungi (Youssef et al. 2013; Wei et al. 2016; Rabee et al. 2018). A previous study showed that the genera of Ascomycota and Basidiomycota efficiently produce beta-glucanase (Mintz-Cole et al. 2013), possibly promoting the digestibility of feed, even though their abundance is low.
Different cattle breeds have different dominant rumen fungi clusters (Fig. 4) and the abundance of rumen fungi (Table I).
Diet can also be an important factor. Han and coworkers research has shown that the concentrated feed has an important impact on the anaerobic fungal population of cashmere goats AF (Han et al. 2019). AF secrete a range of cell wall degrading enzymes such as free enzymes and cellulase multienzyme complexes (Cheng et al. 2018), which are effective degradation products of plant biomass (Haitjema et al. 2014). Screening of all rumen microbial CAZyme transcripts indicated that the AF of Neocallimastigaceae produced the largest share of cellulase transcripts (Söllinger et al. 2018). The studies have shown that
This study found a large difference in rumen fungi abundance among four cattle breeds. Rumen fungi diversity and composition were mainly related to diet, and the use of its components depends on enzyme activity and quantity produced by these fungi. To better understand the relationship between fungal composition and function and the ruminant growth, as well as to extract cellulases from rumen fungi, the metagenomic and metatranscriptomic analyses should be used in future studies.