Determination of microbiological characteristics in the digestive tract of different ruminant species

Abstract Holstein dairy cows, Chinese Luxi Yellow cattle, Chinese Laoshan dairy goats, and Chinese Bohai Black cattle were selected for the study. The 16S rDNA sequencing technique was used to analyze the microflora in the digestive tract. The rumen flora in high milk‐yield Holstein dairy cows showed significantly higher proportions of Treponema, Butyrivibrio, Coprococcus, Shuttleworthia, Lachnospira, and Selenomonas, compared with the rumen flora in Chinese Bohai Black cattle and Chinese Luxi Yellow cattle (p < 0.05). In addition, the abundances of Succiniclasticum, Ruminococcus, and Fibrobacter in the rumen fluid of high‐yield dairy cows were significantly higher than those in rumen flora of dairy goats. Compared with ruminal flora in Chinese Luxi Yellow cattle, the rumen flora in high‐yield dairy cattle showed significantly higher Prevotella. Compared with the rumen flora in Chinese Laoshan dairy goats, Chinese Bohai Black cattle, and Chinese Luxi Yellow cattle, the flora in high‐yielding dairy cows showed significantly lower proportions of CF231, 02d06, Oscillospira, RFN20, Desulfovibrio, Methanobrevibacter, and SHD‐231. In addition, compared with the rumen flora in dairy goats, the rumen flora in high‐yielding dairy cattle displayed significantly lower proportion of Enterococcus. Compared with the rumen flora in Chinese Bohai Black cattle, the flora in high‐yielding dairy cattle exhibited significantly lower Ruminococcus, YRC22, Pseudobutyrivibrio, L7A_E11, BF311, p‐75‐a5, and Dehalobacterium. Compared with the rumen flora in Chinese Luxi Yellow cattle, the flora in the high‐yield dairy cows also displayed significantly lower proportions of Ruminococcus, YRC22, BF311, Paludibacter, and Dehalobacterium.

It is resistant to coarse feed and disease, and it has good adaptability and genetic stability (Liu, 2015).
The Luxi cattle breed is a fine variety of meat livestock unique to Shandong Province. They have a strong ability to digest hay, straw, and other rough feed. Cellulose is composed of 36 single chains forming a highly crystalline microfibril structure with intrachain and interchain hydrogen bonds, van der Waals force, and hydrophobic force. The rumen microbial system of Luxi cattle can efficiently degrade plant cellulose substrates. It is speculated that this efficient mechanism may primarily depend on the various glycoside hydrolases contained in the rumen of the cattle, which are produced by a variety of symbiotic microorganisms in the rumen.
Ruminococcus produces fibrous bodies with a large molecular weight and a variety of glycoside hydrolase activities. In addition, we speculate that Ruminococcus may gradually degrade the natural plant cellulose substrate by rolling a large number of fibrous bodies on the surface to destroy the highly crystalline cellulose chains (Wang, 2011). Ruminococcus-fibrous body complexes may be necessary to degrade crystalline cellulose. Fibrous bodies that are free from Ruminococcus cells will lose most of their ability to degrade natural plant cellulose (Wang, 2011).
Chinese Laoshan dairy goats, Bohai Black, and Luxi cattle can undergo long-term adaptation to rough forage and a rough environment. Their digestive tract may have unique or dominant microorganisms that improve the utilization of poor-quality roughage. Therefore, this study is aimed at determining differences and similarities of ruminal microbial composition among high-performance Holstein dairy cows, Chinese Laoshan dairy goats, Chinese Bohai Black cattle, and Luxi cattle; it attempts to determine the typical characteristics of various types of ruminant intestinal flora.
It is hoped that these microorganisms in ruminants that can tolerate poor roughage can be used in the dairy cattle industry to improve the effectiveness of dairy cows using local poor-quality feed resources and to improve lactation performance for unit cost reduction. This study will provide a vital reference for screening, purification, and identification of bacteria that impart tolerance to roughage and to further researches into microbial nutrition control.

| Animals
The study included six healthy high-yield (milk production more than 30 kg) and six low-yield (milk production <20 kg) Holstein dairy

| Sample collection
Representative rumen fluid samples were obtained from all experimental animals via the animals' mouth with the oro-ruminal sampling device within two hours prior to morning feeding. The collected rumen fluid was dispensed into three 50-ml sterile centrifuge tubes.
All samples were immediately flash-frozen in liquid nitrogen and stored at −80°C for future testing.

| Experimental procedures for 16S rDNA sequencing
The samples were slowly thawed at 4°C. Total DNA was extracted from the rumen fluid samples using the Stool DNA Isolation kit (Tiangen, Beijing, China). DNA samples were quantified using a NanoDrop spectrophotometer (Nyxor Biotech, Paris, France) and then transferred to BGI Genomics for V4 region of the 16S rDNA gene sequencing with PE250 Miseq. The PCR primer used for 16S rDNA amplicon libraries was 515F-806R.

| Bioinformatics analysis for 16S rDNA sequencing
The raw data were filtered to eliminate the adapter pollution and low-quality reads to obtain clean sequences (Douglas et al., 2014).
Sequence reads with an average quality of under 20 over a 30-bp sliding window as per the phred algorithm were truncated, and trimmed reads having less than 75% of their original length, as well as its paired read, were removed. Then, paired-end reads with overlap were merged into tags using FLASH (Magoc & Salzberg, 2011; Fast Length Adjustment of Short reads, v1.2.11). Tags were clustered to OTU at 97% sequence similarity by scripts of software USEARCH (v7.0.1090;Edgar, 2013 VennDiagram and package "ade4" of software R (v3.0.3) were used separately in Venn diagram and OTU PCA analysis. The tag numbers of each taxonomic rank (phylum, class, order, family, genus, and species) or OTU in different samples were summarized in a profiling table. The species with abundances of less than 0.5% were classified into "others" in other ranks for all samples.
The Wilcoxon rank-sum test was used for comparison of two groups using the alpha-diversity indices. Beta-diversity analysis was done by software QIIME (v1.80; Caporaso et al., 2010). Principal coordinate analysis (PCoA) was used to exhibit the differences between the samples according to the matrix of beta-diversity distance.
Metastats (https://metastats.cbcb.umd.edu/) via a T test on the species abundance data between groups obtains p-values and corrects the p-values to obtain q-values. Finally, based on the p-value (or q-value), the species that cause differences in the composition of the two groups were screened, p ≥ 0.05.

| Sequence stitching
Paired-end reads were stitched into contigs based on their overlap.
A total of 2,733,352 contigs were obtained, an average of 94,253 contigs per sample. The SD value was 30,677. The contigs had an average length of 252 bp and an SD value of 0 bp.

| OTU statistics
After the stitched Tags are optimized, they were clustered at 97% similarity for operational taxonomic units (OTUs) to classify species.
The abundance information of each sample in each OTU was counted, and the abundance of OTU preliminarily indicated the species richness of the sample. A total of 4,314 OTUs were found in 29 samples.

| OTU Venn chart analysis
At a similarity level of 97%, the OTU number in each sample was obtained. The Venn chart ( Figure 1) depicts the number of OTUs unique to each sample or shared among multisamples and intuitively displayed OTU overlap among samples. There were 2,300 OTUs in total found in the rumen fluid of the high milk-yield Holstein dairy F I G U R E 1 Shared OTU across different samples or groups cows, 2,320 OTUs in the rumen fluid of the low-yield Holstein dairy cows, 2,731 OTUs identified from the rumen fluid of dairy goat, 2,850 OTUs from Bohai Black, and 2,807 OTUs from Luxi Yellow.

| PCA of OTUs
As shown in Figure 2, the Chinese Bohai Black cattle group was very close to the Chinese Luxi Yellow cattle group in terms of PCA results in the graph. These two groups contained similar OTUs, whereas the other two groups of samples distantly scattered; this indicated that the OTU compositions among the other groups were quite different.
A total of 113 genera were found in rumen fluid of dairy goats; among these, the genera that contributed than 0.5% included  Figure 5 shows the corresponding rarefaction curves of the observed species index. It was found that the rarefaction curves of all samples eventually plateaued, indicating that the sequencing depth was sufficient and there was substantial coverage of all the species in the sample.

| Single sample diversity analysis
The observed species index, the chao index, and the ACE index reflect the richness of the community in the sample. The Shannon index and the simpson index reflect the diversity of the community and are affected by species richness and species uniformity in the sample community. Figure 6 shows boxplots of microflora in the rumen fluid of high-yielding dairy cows, low-yielding dairy cows, dairy goats, Bohai Black cattle, and Luxi cattle. There were significant differences in species richness and evenness. The diversity of rumen bacteria in high-yield dairy cows was significantly lower than that in low-yielding dairy cows, dairy goats, Bohai Black cattle, and Luxi cattle.

| Comparison of diversity between samples
The results of PCoA are shown in Figure 7. The PC1 axis explained 50.83% of the variability, the PC2 axis explained 26.13% variance, and the PC3 axis explained 7.93% variability. These results clearly showed distinctions among the microbial compositions in the rumen fluid of high-yield dairy cows, low-yield dairy cows, dairy goats, Bohai Black cattle, and Luxi cattle.

F I G U R E 4 Genus-level taxonomic composition distribution in samples
F I G U R E 5 Sample-based rarefaction curve of observed species indices F I G U R E 6 Boxplot of alpha-diversity indices among groups F I G U R E 7 Weighted UniFrac PCoA results for all samples. High-yield dairy cows, low-yield dairy cows, milk goats, Bohai Black, and Luxi cattle were, respectively, represented by red triangles, blue triangles, orange triangles, green dots, and purple squares

Phylum level
A total of 0.06% bacteria in the rumen fluid of high-yield cows remained unidentified at the phylum level. The percentage of unidentified bacteria was 0.11% for samples from the low-yield dairy cows, 0.13% for samples from Chinese Laoshan dairy goats, 0.28% for samples from Chinese Bohai Black cattle, and 0.27% for samples from Luxi cattle (Table 1).
It can be seen from Table 2  It can be seen from Table 3  Anaeroplasma Selenomonas, and Ruminobacter (p < 0.05; Figure 8).
As shown in Figure  As shown in Figure 10, compared with the rumen fluid of Luxi cattle, that of high-yield dairy cattle contained a higher proportions TA B L E 1 Significant differences in the flora of rumen fluids of high-production dairy cows and low production dairy cows at the phylum level

| D ISCUSS I ON
The high milk-yield Holstein dairy cows were significantly enriched for the genera Butyrivibrio, Lachnospira, and Dialister when compared with the low milk-yield Holstein dairy cows. The genera Butyrivibrio and Lachnospira both belong to the Family Lachnospiraceae. In the rumen, some special strains of Butyrivibrio fibrisolvens completely and quickly degrade cellulose. Lachnospira sp. are mostly involved with pectin degradation (Cotta & Forster, 2006). Lima et al. (2015) revealed a positive correlation between Butyrivibrio abundance and milk yield. Jami, White, and Mizrahi (2014) showed a positive correlation between Dialister and milk yield. Ruminococcus, Coprococcus, and Succiniclasticum were suggested to have a negative impact on milk production (Jami et al., 2014), which was consistent with our results. TA B L E 4 Significant differences in rumen flora between high-production dairy cows and Luxi cattle at the level of phylum F I G U R E 8 Significant difference in rumen flora between high-production dairy cows and milk goat at the genus level F I G U R E 9 Significant differences in rumen fluid flora between high-yield dairy cows and Chinese Bohai Black cattle at the genus level to improve fiber digestion. Because these bacterial genera exist in the rumen fluid of Chinese Luxi Yellow cattle, Chinese Laoshan dairy goats, and Chinese Bohai Black cattle but not in the rumen fluid of low milk-yield dairy cows, we believe that these genera can promote the use of roughage in the rumen without reducing milk production of the high milk-yield Holstein dairy cows. Oscillospira is an anaerobic bacterium that is difficult to culture and is commonly found in the gut of herbivores (Mackie et al., 2003). The In some herbivores, such as Venezuelan sheep (2008)

| CON CLUS ION
There were significant differences in ruminal flora between the high-yield dairy cows and Chinese Laoshan dairy goats, Chinese Bohai Black cattle, and Chinese Luxi Yellow cattle, and the differences were mainly reflected in the relative contents of certain taxa.

ACK N OWLED G EM ENTS
The study was financially supported by the earmarked fund for F I G U R E 1 0 Significant differences in rumen flora between high-production dairy cows and Luxi Yellow cattle at the genus level