1. Background
The rumen, the forestomach of ruminants, harbors bacteria, archaea, fungi, and ciliate protozoa [
1]. An indispensable function of these microorganisms is to break down plant polymers into volatile fatty acids through various fermentation pathways to be absorbed and used by host animals [
1,
2]. The composition and function of rumen microbial communities are not only affecting growth and milk production but are also related to the host health and nutrient utilization in dairy cattle [
3,
4]. The unique metabolites (e.g., saturated fatty acids, organic acids, amine, or polysaccharide) produced by rumen microbes may play a vital role in ruminant physiology [
5]. Jewell et al. [
6] reported that some rumen bacteria are associated with milk production efficiency and ketosis.
Many factors, including diet, species, age, and geographic location, can affect the composition and function of rumen microbiota [
6,
7]. Furthermore, the acidosis, subacidosis, and metabolic dysfunction of the rumen can change the composition and function of rumen microbiota [
8,
9,
10]. Given the importance of rumen microbial communities on ruminant health and productivity, many researchers have attempted to manipulate rumen microbiota through various strategies, such as using chemical agents and enzymes as feed additives or probiotic supplements. However, modification of rumen microbial compositions in adult ruminants appears difficult. Changes in rumen microbiota from exogenous factors can be rapidly restored by eliminating the influential factors [
11], but little is known of the ecological and physiological roles of predominant core bacteria in the rumen microbial ecosystem.
In addition, the lactation cycle is split into lactation and dry periods. Most studies have focused on investigating rumen microbiota during lactation; for instance, studies have assessed how rumen microbiota respond to exogenous butyrate [
12] and the composition of rumen microbiota [
13,
14]. The dry period is a crucial rest period for cows in which new hormonal stimulation is gained for subsequent lactation. The precise interaction between a rumen microbial community and the host animal should be investigated for the whole lactation cycle to investigate the possibility of maintaining the health and productivity of ruminants by manipulating rumen microbial composition. Nevertheless, limited information has been reported concerning rumen microbiomes and their functions during dry periods.
Thus, in the present study, the composition of rumen microbiomes from three dairy farms in the northern, middle, and southern regions of Taiwan, respectively, were investigated. We identified core bacteria that contribute to various physiological roles during rumen fermentation in dry cows. The exogenous factors affecting rumen microbiomes were also studied. Fundamental knowledge of rumen core microbiomes and their relationship to physiological functionality in rumen microbial ecosystems during dry periods can provide insight into potential manipulation of rumen microbiota to enhance dairy performance, such as milk production and cow health.
4. Discussion
Our findings revealed that ruminal fluid in dry dairy cows across regional farms had similar core rumen microbiota but in different proportions. Although bacterial enumeration is difficult to extrapolate from sequencing data, proportional changes within the core microbiota species may be crucial and merit investigation. Some core bacterial taxa identified during the dry period were consistent with those identified in lactating dairy cows [
14,
32] and beef cattle [
33]. The genera
Prevotella,
Ruminococcus, and
Butyrivibrio, which are the most abundant in rumens of lactating dairy cows [
14], were the predominant genera in rumens during the dry period.
Prevotella, from Bacteroidetes, occupies the ecological niche of second line degrader and possesses oligosaccharolytic and xylanolytic activity to produce substantial amounts of succinate and acetate [
34]. The genus
Ruminococcus, which breaks down fibrous plant material to generate acetate, formate, succinate, and other short-chain fatty acids [
1], was identified as the second most predominant (8.42%) core taxon in the present study.
Butyrivibrio, in the class Clostridia, is involved in various ruminal functions, including fiber degradation, protein degradation, lipid biohydrogenation [
35], and microbial inhibitor production [
36,
37]. Mrázek et al. [
38] reported that high-fiber intake essentially increases
Butyrivibrio in rumens, whereas high-energy food additives suppress it. This genus also significantly differed among somatic cell count groups [
39]. Our study was paralleled with previous findings in
Butyrivibrio. Farm B, with a higher fiber intake than the other two farms, showed more abundance in
Butyrivibrio (Farm A: 1.5 ± 0.2%, Farm B: 2.7 ± 1.3%, Farm C: 2.0 ± 0.6%). Although ruminal fluid in dry dairy cows across regional farms possessed similar core rumen microbiota, variation in rumen microbiota composition could effectively separate each farm by PCA plot and unweighted UniFrac.
Prevotella,
Methanobrevibacter,
Pseudobutyrivibrio,
Ruminococcus,
Bacteroides, and
Streptococcus were major contributors in this respect. Variations in identified core rumen microbiomes in dry dairy cows may be attributed to differences in dietary conditions (forage-to-concentrate ratio), geographical location, and management regime. Indugu et al. [
40] indicated that differences in microbial communities between farms are greater than within farms, which is similar to our findings.
Factors affecting specific bacterial genera must be evaluated. We identified key microorganisms associated with each farm using LEfSe. The results agreed with our PCA findings. Through additional Spearman correlations, the genus
Prevotella, a biomarker in Farm A, was positively correlated with milk yield in the previous lactating period. Studies have indicated that ruminal
Prevotella, which can convert sugars, amino acids, and peptides into energy [
41,
42], was significantly higher in high milk yielding cows as compared to the low milk yielding cows [
40]; this association has been shown for both high- and low-milk yielding cows [
6]. Moreover, the relative abundance of short-chain fatty acid (SCFA)-producing genera, including
Prevotella and other genera (
Bacteroides,
Oscillibacter,
Clostridium,
Succinivibrio, and
Phascolarctobacterium), among the identified core ruminal microorganisms in Farm A comprised more than 25% of the total sequences in our dataset, which was higher than that of the other two farms. SCFAs serves as an important energy source for epithelial cells in ruminants [
43] and are significant in maintaining colonic health in both humans and animals [
44].Sufficient energy is required to maintain cow health and support a high milk yield because gut SCFAs are precursors to milk fats [
45]. The relative abundance of SCFA-producing genera may partially explain the higher milk yield of Farm A in the previous lactating period. The results revealed that the abundance of
Prevotella with SCFA-producing microorganisms in dry dairy cows may indicate milk yield during the previous lactating period.
Pseudobutyrivibrio, the specific biomarker from Farm B, was positively correlated with fiber and negatively correlated with milk yield.
Pseudobutyrivibrio, a Gram-negative, anaerobic, and non-spore-forming bacterial genus from the Lachnospiraceae family, was reported to have a functional role in the digestion of hemicellulose [
46]. The dry cows from Farm B were fed 80% from pastures, and cows from Farms A and C were fed 70% and 60% from pastures, respectively. Therefore, the dry cows from Farm B had a higher relative abundance of
Pseudobutyrivibrio.
In Farm C, the specific biomarkers were the genera
Methanobrevibacter,
Ruminococcus,
Bacteroides, and
Streptococcus, which were all negatively related to fiber in the present study.
Methanobrevibacter is in the Methanobacteriaceae family. Certain
Methanobrevibacter groups of
Methanobrevibacter species, including
M. smithii,
M. gottschalkii,
M. millerae, and
M. thaueri, were correlated with individuals with higher CH
4 production [
47,
48], with no effect on fiber digestion or milk production [
49].
Ruminococcus, a major fiber and cellulose degrader in the rumen of ruminants [
50], was negatively correlated with fiber and milk yield in the present study. Jami et al. [
13] reported that
Ruminococcus was negatively correlated with milk production, whereas
Streptococcus was positively correlated with starchy diets. In a study,
Bacteroides was found to be significantly reduced in abundance in rumen fluids because of some diseases [
51]. The feeding of dairy cows with probiotics in Farm C may have resulted in significant increases in rumen fermentative bacteria (including
Bacteroides and
Ruminococcus), which corresponds with a previous study [
52]. In addition, introducing other microorganisms, such as probiotics, can modulate gut SCFAs by changing the metabolism of certain intestinal microflora during ruminal fermentation [
53]. In the present study, only Farm C provided probiotics to dry dairy cows, which did not reveal more SCFA-producing microorganisms. Weimer [
11] reported that rumen microbiomes exhibit remarkable specificity and resilience within hosts. Most attempts at introducing probiotics to rumens have resulted in only a temporary change after days or a few weeks, suggesting high host-specificity of rumen microbiome composition once established [
54].
The similarity of core bacteria was associated with the major metabolic pathways, and functional pathway analysis of rumen bacterial communities unsurprisingly revealed that amino acid and carbohydrate metabolisms were the major pathways. Moreover, the consistent presence of core taxa in the rumen indicated the vital functions of rumen ecological niches in dairy cows [
14]. A metaproteomics study agreed with our findings regarding relative bacterial abundance, reporting that Bacteriodete activity dominated the metaproteome most abundantly with the Prevotellaecae family [
55].
Prevotella are typically related to microbial proteolytic activity in the rumen [
56] and early colonization associated with fiber degradation [
57]. Degradation of fiber through fibrolytic bacteria activity is crucial for rumen microbiota to gain energy [
58]. Once thought to be abundant in the rumen,
Ruminococcus, the second most dominant genus in rumen bacterial communities, contributes to the degradation of plant polymers, which suggests that this genus plays a key role in carbohydrate metabolism. However, in the present study, we were unable to clarify which factor (regional difference or diet composition) was more effective on the change of rumen microbiota in dry dairy cows. Further studies should be conducted in various regional farms with the same diet composition. On the other hand, the present study revealed that several genera were the biomarkers in each farm with a different diet composition. How the diet composition affects the metabolic interaction of those microorganisms in dry cows remains unclear. The metabolomics of dry cows will be investigated in our future study as well.