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Article

Comparative Analysis of Rumen Bacterial Profiles and Functions during Adaption to Different Phenology (Regreen vs. Grassy) in Alpine Merino Sheep with Two Growing Stages on an Alpine Meadow

by
Xiang Gao
and
Hucheng Wang
*
State Key Laboratory of Herbage Improvement and Grassland Agro-Ecosystems, Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs, Engineering Research Center of Grassland Industry, Ministry of Education, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(1), 16; https://doi.org/10.3390/fermentation9010016
Submission received: 20 November 2022 / Revised: 16 December 2022 / Accepted: 21 December 2022 / Published: 24 December 2022
(This article belongs to the Special Issue Rumen Fermentation)
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Abstract

:
Phenological periods can affect the growth of forage, the single food source for grazing animals, and its nutrition and the stage of growth of the animals can affect the rumen microbiota. The aim of this study was to evaluate the effects of phenological periods (regreen vs. grassy) and growing stages (hoggets (1.5 years old) vs. rams (3 years old)) on rumen bacteria communities and functions in Alpine Merion sheep. The result showed that the Alpha diversity indices of ACE (p < 0.001), Chao (p < 0.001), and Shannon (p < 0.001) were higher in the regreen stage. At the phylum level, the abundances of Bacteroidetes (p = 0.003) and Firmicutes (p = 0.017) shifted with plant phenology. The abundance of fiber-degrading bacteria altered at the genus level (p < 0.05). Genes related to fatty acid degradation and metabolism increased in both the growing stage sheep (p < 0.05). In the grassy stage, the abundance of vitamin B6 metabolism (p = 0.046) was increased in hoggets. In summary, this study showed that the phenological stage had a significant effect on the rumen bacterial compartment and functions in two growing stages, while the growing stage only tended to change rumen bacterial diversity.

1. Introduction

Rumen microorganisms use dietary nutrition to produce volatile fatty acids (VFA) to provide energy for the host. Previous studies have shown that the population and function of microbial community are related to diet composition [1,2,3], growing stage [4], feeding strategy [5,6], breeds [7], and environment [8,9]. The Alpine Merino sheep is a new breed [10], which has great significance to the production system in Western China. The animals provide mutton, fabrics, and hides for the local population [11]. The sheep are in a state of natural grazing all year round, and they consume natural herbage as their predominant source of nutrients. Plant phenology is cyclical, involving regreen, grassy, and withering stages [12]. Due to the high altitude of the alpine meadow, its low levels of oxygen, and its cold climate, herbage turns green in April, grassy green from July to August, and withers in September. The forage had more quantity, with higher levels of protein and carbohydrate in the grassy stage than it did in the regreen and withering stage [13]. In this feeding mode, in response to the changes of forage quantity and quality in different phenological periods, rumen bacterial flora, as the largest proportion of rumen microorganisms, are bound to cause some adaptive changes. Ref. [14] reported that, in the regreen period, both the composition and function of rumen microbiota on Tibetan sheep had obvious disadvantages. Fan et al. also confirmed that Tibetan sheep presented low values of forage degradation and fermentation indicators in the cold season, while the microbial diversity index was increased [15]. In addition, animals have different digestive and utilization abilities of substrates at different growing stages, which can also affect the composition of gastrointestinal microflora. This point has been confirmed by previous studies in humans [16,17], goats [18,19], Tibetan sheep [20], and cows [21]. The signs of rumen maturation include an increase in volume, weight, and the length of intraruminal papillae, and rumen epithelium begins to keratinize, which is also partially associated with changes in the rumen microbiome [22].
Our earlier studies have shown divergent effects of the two plant phenology periods on ruminal digestibility and fermentation characteristics in hoggets [23] and rams [24] Alpine Merino sheep. There are great differences in forage nutrients between the two phenological periods, and the Ash (8.67%) and crude protein (12.74%) contents in the grassy stage were higher than in the regreen stage. The digestibility of crude protein, neutral detergent fiber, and acid detergent fiber were higher in the regreen stage than the grassy stage in both hoggets and rams, while the concentration of butyrate and branched-chain volatile fatty acids was lower in the regreen stage. We hypothesized that the phenological stage had a significant effect on rumen bacterial compartment and predicted function in two growing stages of Alpine Merino sheep as a consequence of the differences in nutritional composition and quantity, while the growing stage only tended to change rumen bacterial diversity in hoggets and rams. In this study, we aimed to investigate changes in rumen bacterial community composition and function in hoggets and rams of Alpine Merrion sheep under different plant phenology, and to explore their relationship with previously published feed nutrient composition. This work included 16S rRNA sequencing to characterize the composition of the rumen bacterial flora of sheep.

2. Materials and Methods

2.1. Experimental Site, Design, and Sampling Date

The experiment was carried out in the Sheep Breeding Technology Promotion Station of Gansu province from June 2018 to September 2018. The experimental site (37°48′ N, 101°45′ E, altitude range of 2600 to 4000 m) belongs to the typical place of the semi-arid, with an annual average temperature range of 0.6 to 3.8 °C, and the herbage growing period is about 150 days, with an average annual rainfall is about 361.6 mm. Generally, herbage begins to sprout in April and starts to wither in September. The main grassland type is the alpine meadow, and the dominant species are Kobresia, Elymus dahuricus, and Stipa. Associated species are mainly members of the Compositae, Ranunculaceae, and Gramineae.
During the experiment, six rams (R; 3-year-old) with similar body weight (104.94 ± 5.15 kg) and six hoggets (H; 1.5-year-old) with similar body weight (71.33 ± 1.72 kg) of Alpine Merino male sheep were selected from the same herd. During the forage regreen (R) and grassy (G) stage, animals graze on alpine meadows and samples are collected at the end of each phenological period. A total of 12 sheep had no supplementary feeding and free access to water during the experimental stage.

2.2. Rumen Fluid Collection

Samples were collected in regreen (July 2018) and grassy stage (September 2018), respectively. The rumen fluid was collected using the method described by Bainbridge et al. [25]. An esophageal tube with a vacuum pump was inserted through the mouth into the rumen for sampling. Each sample was obtained through two sequential collections: firstly, rumen fluid (approximately 100 mL) was collected and discarded to avoid sample contamination with saliva. After that, rumen fluids were collected and filtered through four layers of cheesecloth in each phenological period before the morning grazing. The rumen fluid samples were then transported to the laboratory and stored at −80 °C for bacteria DNA extraction.

2.3. DNA Extraction, PCR, and Sequencing

The DNA of rumen bacteria was extracted using a DNA extraction kit (EZNA DNA Stool Mini, Sigma-Aldrich Co., St. Louis, MI, USA), following the manufacturer’s instructions. The concentration and purity of DNA were measured using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The V3–V4 16S rRNA gene was amplified using primers 5′-ACTCCTACGGGAGGCAGCA-3′ and 5′-GGACTACAVGGGTGTCTAAT-3′. A 10-μL amplification system was used to determine the quality of the DNA. Each sample contained 0.5 μL full-type gold enzyme, 2 μL stock solution, 1 μL of each primer, 2 μL DNA template, and 3.5 μL ddH2O. The amplification conditions were as follows: 95 °C for 2 min, 95 °C for 20 s, 55 °C for 15 s, 30 cycles at 72 °C for 5 min, and a final cycle at 72 °C for 10 min. The PCR products were electrophoresed on a 1.8% (w/v) agarose gel. This band was separated and purified using a gel extraction kit (Takara, Biomedical Technology Co. Ltd., Beijing, China). Amplicon library sequencing was performed using a PacBio MiSeq platform (Beijing Biomarker Technologies Co., Ltd., Beijing, China).
Based on the raw data returned by the Illumina HiSeq sequencing platform, double-ended splicing (FLASH v 1.2.7), filtering (Trimmomatic v 0.33), and removal of chimeras (UCHIME v 4.2. 2) were used to obtain optimized sequences. QIIME (v 1.9.0) software was utilized to cluster tags at a similarity level of 97%, obtain operational taxonomic units (OTUs), and annotate the OTUs based on the Silva (bacteria) taxonomy database. The Alpha indexes (ACE, Chao, Shannon, and Simpson) of each sample were statistically calculated by using Mothur (version v 1.3.0). The PICRUSt2 (v 2.5.0) software was used to perform functional annotation and prediction of OTUs using the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional database. Mantel analysis was performed using R packages (Version 4.1.2) vegan and ggplot2.

2.4. Statistical Analysis

Alpha-diversity indices and rumen bacterial abundances were analyzed using the general linear model (GLM) and Duncan’s multiple test by SPSS software Version 19.0 (IBM, Armonk, NY, USA) to test the effect of the plant phenology and growing stage. The statistical significance level was p < 0.05, and the trend was defined when 0.05 < p < 0.1.

3. Results

3.1. Rumen Bacterial Profiles

Across all the samples, a total of 1,919,100 pairs of reads were obtained in this study, and 1,554,950 clean tags were generated after tiling and filtering of double-ended reads. At least 63,155 (average 64,790) clean tags were generated for each sample, with an average sequence length of 419 bp. An analysis of similarity (based on Bray–Curtis distance) showed a difference in the regreen (R = 0.309, p = 0.006) and grassy (R = 0.331, p = 0.007) stage between hoggets and rams or in hoggets (R = 0.715, p = 0.002) and rams (R = 693, p = 0.003) between the regreen and grassy stage (Figure 1). We found that the alpha-diversity indices of number of operational taxonomic units (p = 0.022), ACE (p = 0.013) and Chao (p = 0.015) were affected by the interaction between phenology and growing stage. In the grassy stage, the alpha-diversity index showed a trend towards lower ACE (p = 0.066) and Chao (p = 0.074) in hoggets than in rams, while hoggets had a tendency to have a higher ACE (p < 0.001) and Chao (p < 0.001) in the regreen stage than in the grassy stage. The other alpha-diversity (Simpson and Shannon) had no significant difference between hoggets and rams, while the Simpson (p = 0.010) and Shannon (p < 0.001) were higher in the regreen stage than the grassy stage (Table 1).

3.2. Composition of Rumen Bacterial Structure

Bacteroidetes and Firmicutes were the most abundant phyla in the rumen fluid, and they represented 50.57% and 42.95% of the bacterial community, respectively (Table 2). The abundance of three phyla were altered by the plant phenology including Bacteroidetes (p = 0.003), Firmicutes (p = 0.017), and Saccharibacteria (p < 0.001). One of the taxa was affected by the growing stage. The abundance of Saccharibacteria was higher in rams compared to the hoggets in the regreen and grassy stage (p < 0.001). The phenology × growing stage interaction had a tendency to affect the abundance of Bacteroidetes (p = 0.068) and [Eubacterium]_coprostanoligenes_group (p = 0.041). At the genus level (Table 3), Prevotella-1 (19.09%) was the predominant genera in all samples. Of the 10 genera that changed significantly, of these, the relative abundances of Succiniclasticum (p = 0.019), Christensenellaceae_R-7_group (p = 0.004), Ruminococcus_1 (p < 0.001), Ruminococcaceae_UCG-014 (p = 0.004), Ruminococcaceae_UCG-010 (p < 0.001), Saccharofermentans (p < 0.001), Lachnospiraceae_AC2044_group (p = 0.002), [Eubacterium]_coprostanoligenes_group (p < 0.001), and Candidatus_Saccharimonas (p < 0.001) were higher in the regreen stage, while the abundance of Selenomonas_1 (p < 0.001) was higher in the grassy stage. One of the taxa was affected by the growing stage. The abundance of Candidatus_Saccharimonas (p < 0.001) in rams was higher than hoggets between in the regreen and grassy stage. There were significant differences between plant phenology and growing stage. In the regreen stage, the ratio of F/B (Firmicutes/Bacteroides) in rams was higher than in other groups (Figure 2).

3.3. Forage Nutrition Determined Rumen Bacterial Diversity

In order to explore the factors that affected rumen bacteria in different phenological periods and growing stages, we analyzed the nutrition matrix distance and number of operational taxonomic unit matrix distance using a Mantel test, as shown in Table 3. The content of Ash (r = 0.619, p = 0.003) and CP (r = 0.566, p = 0.002) were significantly correlated with rumen bacterial community structure in hoggets. For the rams, the content of Ash (r = 0.552, p = 0.002) and CP (r = 0.520, p = 0.004) were significantly correlated with rumen bacterial community structure. The data of forage nutrients are shown in Supplementary Table S1.

3.4. Function of Rumen Bacteria

A total of 468 KEGG gene families were identified in the 16S rRNA gene sequencing data using reconstruction of unobserved states (PICRUSt) analysis to predict gene function. Among these predictions, 50 and 19 KEGG gene families showed significant differences between regreen and grassy stage in hoggets and rams, respectively (Figure 3A,B). The pathways related to phenylalanine, fatty acid, and propanoate metabolism, and fatty acid degradation was enriched in the regreen stage than the grassy stage, while vitamin B6 was higher in the grassy stage in hoggets. In rams, the higher relative abundance of carbon and fatty acid metabolism were detected in the regreen stage compared to the grassy stage, while the abundance of amino sugar and nucleotide sugar was lower in the regreen stage. However, there was no difference in rumen bacteria at genes abundance between hoggets and rams (p > 0.05). All details are shown in Supplementary Table S2 and Supplementary Table S3, respectively.

4. Discussion

4.1. Effects of the Plant Phenology on Rumen Bacterial Profiles and Functions in Hoggets and Rams Alpine Merino Sheep

In the alpine meadow, the plant phenology period can be divided into three stages: withering, regreen, and grassy stage [12]. The plant phenology period might have affected the quantity and quality of forage, and then affected the performance of animals. Grazing animals rely on grass and other plants to survive and maintain their growing requirement. In this process, microorganisms are known as the “second genome” [26], and this may play an important role in the adaptive mechanisms [27]. Due to the high aboveground biomass and rich nutrients, the grassy stage plays a crucial role for the rapid weight gain of grazing cattle and sheep. Evidence from previous research on Tibetan sheep grazing in Qinghai-Tibetan pasture showed that, in the grassy stage, sheep consumed more diverse forage with high nutrition, whereas in the regreen stage, as the biomass of forage decreased, high nutrition forage could not meet the growing requirements. The animal had to eat forage with poor palatability and nutritional composition [28]. The diversity of diet leads to the increase of rumen bacterial diversity, and our results were in agreement with Liu et al. [14], who reported that, due to the differences of feeding niches and forage nutrition structure, the rumen bacterial diversity in the regreen stage was higher than the grassy stage. Analysis of similarity showed a difference in bacteria profiles among groups. This result suggests that specific bacteria might change with the plant phenology and growing stage.
Rumen bacteria are composed of variable bacteria and core bacteria, with Firmicutes, Bacteroidetes, and Proteobacteria being the dominant phyla and constitute the core microbiota [29]. In the current study, the rumen bacteria community in sheep was dominated by Bacteroidetes, followed by Firmicutes, which is consistent with previous studies on Tibetan sheep [14,15]. The main function of members of the phylum Bacteroides is participating in the degradation of nutrients, such as carbohydrates and proteins [30,31,32]. Firmicutes carry many genes encoding enzymes related to energy metabolism, which can help the host to digest and absorb nutrients [33]. The ratio of F/B (Firmicutes/Bacteroides) is related to absorbing energy substances and maintaining the metabolic balance [30,34,35]. In the current study, the F/B ratio of hoggets in the regreen stage was higher than other groups, which may be due to a lack of energy caused by nutrient deficiencies in the regreen stage. At the genus level, Prevotella_1 was the most abundant in the rumen, and no difference was found in the abundance of Prevotella_1 among treatments. In accordance with the literature [36], the Succiniclasticum plays an important role in rumen lignocellulose fermentation. The higher abundance of Succiniclasticum in the regeneration stage in our study is inconsistent with previous studies in Tibetan sheep [14] and may be due to the higher fiber content of the forage in our study area. Similarly, in the present study, the abundance of Christensenellaceae_R-7_group, Ruminococcus_1, Ruminococcaceae_UCG-014, and Ruminococcaceae_UCG-010 was lower in the grassy stage. In the regreen and withering stages, to obtain enough foods, the animals had to seek a wider range of forage with poor nutrition [14]. To test this hypothesis, we analyzed the correlation between forage nutrition and bacteria. We found that CP and Ash significantly affected the rumen bacterial community.
PICRUSt was used to predict the gene function of the rumen bacteria and revealed that there were significant differences in the gene function of bacteria between the regreen and grassy stage in hoggets and rams, respectively. KEGG gene family predictions showed that there were significant increases in functions related to vitamin B6 metabolism in the regreen stage compared with the grassy stage in hoggets. Previous studies reported that vitamin B6 as a co-factor for more than 150 enzymes has been reported to be involved in amino acid, glucose, and lipid metabolism [37], and the upregulated levels of vitamin B6 and its metabolites may indicate increased nutrient digestibility [38]. An increased abundance of amino acid sugars and nucleotide sugar pathways was found in a study of saponin on rumen microbes in young cattle [39] because the effect of treatment on rumen metabolism involves carbon metabolism, amino acid metabolism, and nucleotide metabolism. These results are consistent with our observations on rams. In addition, the abundance of fatty acid metabolism and degradation was higher in the regreen stage than the grassy stage. This result is consistent with Dervishi et al. [40], who reported that genes involved in fatty acid synthesis and metabolism were downregulated in the grazing group by comparing the effects of different feeding systems (indoor or grazing) on gene expression in sheep.

4.2. Rumen Bacterial Profiles and Functions in Hoggets and Rams Alpine Merino Sheep

In the current study, pronounced differences in the bacteria composition among different growing stages were evident, in line with previous studies [19,21,41]. Most of the alpha diversity indices (except Simpson and Shannon index) tended to change with the growing stage. These three alpha diversity indices increased with the growing stage in the grassy stage, while decreasing with growing stage in the regreen stage. This finding was inconsistent with the previous studies [19,42], in which the alpha diversity significantly increased with the growing stage in goats. Possible reasons for this are the species-specific differences and the different age stages. This study also detected growing stage-related changes in the population structure at the phyla level. The abundance of Saccharibacteria was higher in rams than hoggets. A similar compositional change was reported in a study of gastrointestinal microbes in goats fed diets with high calcium digestibility [42].
When the microbial function does not change, it may mean that the host has higher adaptability. The microbial function is flexible and change under the influence of diet, environment, and other factors [43]. By KEGG analysis, the results of the current study indicated that hoggets have more differential genes than rams. To sum up, the rumen flora of rams fluctuated less than hoggets, and the internal environment was relatively stable. Compared to rams, the hoggets have some special significant pathways, such as phenylethylamine, which takes part in phenylalanine metabolism and was more enriched in the regreen stage [20]. Phenylethylamine is one of the essential amino acids for animals. Most of them can participate in the synthesis of neurotransmitters and hormones in the form of tyrosine, which are involved in glucose metabolism and fat metabolism. These results demonstrated that the nutritional requirement of essential amino acids was different for the rams and hoggets.

5. Conclusions

In this study, the composition and function of rumen bacteria in hoggets and rams Alpine Merino sheep under different phenology periods were compared. Our results indicated that the nutritional quality of herbage, especially the CP and Ash content, was the key factor in determining the composition and function of the rumen bacteria. Further analysis revealed that rumen bacteria of grazing Alpine Merino sheep have varying adaptive mechanisms in different phenological periods and growing stages. The alpha diversity indices of rumen bacteria were higher in the regreen stage than the grassy stage. In the regreen stage, the greater abundance of Bacteroidetes and Firmicutes promoted forage conversion and utilization, and the members of cellulolytic bacteria were enriched, potentially increasing plant biomass decomposition and improving the growth of sheep. Compared with rams, hoggets had a higher abundance of metabolic pathways in the regreen stage, which could enable the animals to make maximum use of forage. This study will help to make more efficient use of grassland resources by grazing animals. In addition, further research should be conducted on the effects of phenological and age stages on fungi and protozoa.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation9010016/s1, Table S1: Common forage nutrient composition in different phonological periods (%, DM basis); Table S2: The relative abundance of Kyoto Encyclopedia of Genes and Genomes (KEGGs) genes in rumen microbiome of hoggets (H) of grazing Alpine Merino sheep under different phonological periods; Table S3: The relative abundance of Kyoto Encyclopedia of Genes and Genomes (KEGGs) genes in rumen microbiome of rams (R) of grazing Alpine Merino sheep under different phonological periods.

Author Contributions

X.G. wrote the original manuscript. X.G. conducted animal experiments and sample collection, and analyzed the data. H.W. designed the study and reviewed the draft of the manuscript and provided laboratory facilities for analyzing and financial support. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Program for Key Research and Development plan of Gansu Province (20YE3NA006) and the Second Tibetan Plateau Scientific Expedition and Research (2019QZKK0302).

Institutional Review Board Statement

All trial procedures strictly followed the rules and regulations of the Experimental Field Management protocols (File No: 2010-1 and 2010-2) of Lanzhou University and were approved by the Animal Ethics Committee of the University. Written informed consent was obtained from the owners for the participation of their animals in this study.

Informed Consent Statement

Not applicable.

Data Availability Statement

The 16S rRNA sequence data in the present study have been submitted to the NCBI Sequence Read Archive database (SRA; http://www.ncbi.nlm.nih.gov/Traces/sra/, accessed on 20 December 2022) under accession no. PRJNA816442.

Acknowledgments

We would like to extend our sincere gratitude to Shumiao Zhang, Hongwei Zhang, Zhiwei Zhao, Chen Wang, Zhiyuan Ma, and Chao Peng for their assistance in sample collection. We would also like to thank the Gansu Province Sheep Breeding Technology Extension Station for providing the experiment site and the relevant personnel for their help and care.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 09 00016 g001 550
Figure 1. Analysis of similarity of rumen bacteria diversity based on Bray–Curtis distance. HR, hoggets of grazing Alpine Merino sheep in the regreen stage; RR, rams of grazing Alpine Merino sheep in the regreen stage, HG, hoggets of grazing Alpine Merino sheep in the grassy stage; RG, rams of grazing Alpine Merino sheep in the grassy stage.
Figure 1. Analysis of similarity of rumen bacteria diversity based on Bray–Curtis distance. HR, hoggets of grazing Alpine Merino sheep in the regreen stage; RR, rams of grazing Alpine Merino sheep in the regreen stage, HG, hoggets of grazing Alpine Merino sheep in the grassy stage; RG, rams of grazing Alpine Merino sheep in the grassy stage.
Fermentation 09 00016 g001
Fermentation 09 00016 g002 550
Figure 2. Fold change (Firmicutes/Bacteroides) in the relative abundances of the rumen bacteria of hoggets (H) and rams (R) of grazing Alpine Merino sheep under different plant phenology. HR, hoggets of grazing Alpine Merino sheep in the regreen stage; RR, rams of grazing Alpine Merino sheep in the regreen stage, HG, hoggets of grazing Alpine Merino sheep in the grassy stage; RG, rams of grazing Alpine Merino sheep in the grassy stage.
Figure 2. Fold change (Firmicutes/Bacteroides) in the relative abundances of the rumen bacteria of hoggets (H) and rams (R) of grazing Alpine Merino sheep under different plant phenology. HR, hoggets of grazing Alpine Merino sheep in the regreen stage; RR, rams of grazing Alpine Merino sheep in the regreen stage, HG, hoggets of grazing Alpine Merino sheep in the grassy stage; RG, rams of grazing Alpine Merino sheep in the grassy stage.
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Fermentation 09 00016 g003 550
Figure 3. Functional profiles of microbial communities of hoggets (H) and rams (R) of grazing Alpine Merino sheep under different plant phenology. The extended error bars show significantly different KEGG pathways between the fractions. (A) relative abundance of metabolic pathways in hoggets (H); (B) relative abundances of pathways in rams (R).
Figure 3. Functional profiles of microbial communities of hoggets (H) and rams (R) of grazing Alpine Merino sheep under different plant phenology. The extended error bars show significantly different KEGG pathways between the fractions. (A) relative abundance of metabolic pathways in hoggets (H); (B) relative abundances of pathways in rams (R).
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Table
Table 1. Alpha-diversity indices of rumen microbiome of hoggets (H) and rams (R) of grazing Alpine Merino sheep under different phonological periods.
Table 1. Alpha-diversity indices of rumen microbiome of hoggets (H) and rams (R) of grazing Alpine Merino sheep under different phonological periods.
Regreen StageGrassy Stage p-Value 2
ItemHRHRSEM 1PGP × G
OTUs no. 31057.2 a1034.8 a828.3 b965.8 a16.100<0.0010.0890.022
ACE 41093.9 a1070.5 a873.2 b1016.4 a15.364<0.0010.0660.013
Chao 41105.4 a1081.2 a883.7 b1024.8 a15.512<0.0010.0740.015
Simpson 50.008 ab0.007 b0.012 a0.011 ab0.0010.0100.4660.960
Shannon 55.84 a5.86 a5.41 b5.57 b0.039<0.0010.3050.375
OTU, operational taxonomic unit; ACE, abundance-based coverage estimator; 1 SEM, standard error of means; 2 P, plant phenology; G, growing stage; P × G, interaction between plant phenology and growing stage; 3 OTUs no., Number of operational taxonomic units; 4 ACE and Chao species richness estimators; 5 Simpson and Shannon diversity index.
Table
Table 2. The relative abundance of bacterial taxa (relative abundance > 1% at least in one group) in rumen microbiome of hoggets (H) and rams (R) of grazing Alpine Merino sheep under different phonological periods.
Table 2. The relative abundance of bacterial taxa (relative abundance > 1% at least in one group) in rumen microbiome of hoggets (H) and rams (R) of grazing Alpine Merino sheep under different phonological periods.
Regreen StageGrassy Stage p-Value 2
Bacterial Taxa, %HRHRSEM 1PGP × G
Bacteroidetes44.55 b49.72 ab55.38 a52.61 a1.0300.0030.5660.068
  Prevotella_116.0419.4521.5619.320.9560.1740.7640.155
  Rikenellaceae_RC9_gut_group8.0210.469.449.930.6250.7250.2560.444
  Prevotellaceae_UCG-0011.671.591.591.160.1480.4050.4050.553
  Prevotellaceae_UCG-0030.861.361.541.400.1120.1260.4260.173
Firmicutes49.62 a42.43 b40.21 b39.53 b1.1850.0170.1120.185
  Christensenellaceae_R-7_group5.96 a4.37 ab3.14 b2.99 b0.3170.0040.1850.271
  Succiniclasticum5.73 a4.89 ab3.92 ab3.05 b0.3580.0190.2440.987
  Ruminococcaceae_NK4A214_group4.333.133.013.530.3310.4970.6130.210
  Ruminococcus_12.45 a2.47 a1.26 b0.94 b0.122<0.0010.5460.507
  Butyrivibrio_22.301.692.352.440.1560.2120.4230.274
  Saccharofermentans2.10 a1.98 a0.71 b0.77 b0.089<0.0010.8500.629
  Ruminococcaceae_UCG-0141.76 ab2.45 a0.92 b1.33 b0.1510.0040.0860.650
  Pseudobutyrivibrio1.721.351.672.220.1410.1610.7480.123
  Selenomonas_11.72 b0.60 b3.51 a3.22 a0.237<0.0010.1510.388
  Lachnospiraceae_AC2044_group1.71 a1.65 a0.83 b1.15 ab0.1000.0020.5220.343
  Ruminococcaceae_UCG-0101.58 a1.86 a1.15 b1.11 b0.056<0.0010.2850.166
  [Eubacterium]_coprostanoligenes_group1.31 b1.73 a1.03 bc0.88 c0.066<0.0010.3020.041
  Roseburia0.910.571.511.320.1390.0250.3640.785
Proteobacteria0.710.801.051.550.2200.2270.5160.646
  Candidatus_Saccharimonas1.16 b1.92 a0.29 c1.08 b0.070<0.001<0.0010.913
SR11.262.070.951.170.2350.2140.2870.533
Saccharibacteria1.16 b1.92 a0.29 c1.08 b0.070<0.001<0.0010.913
1 SEM, standard error of means; 2 P, plant phenology; G, growing stage; P × G, interaction between plant phenology and growing stage.
Table
Table 3. Mantel test revealed the correlation between forage nutrition and rumen bacteria (OTU level) of hoggets (H) and rams (R) of grazing Alpine Merino sheep.
Table 3. Mantel test revealed the correlation between forage nutrition and rumen bacteria (OTU level) of hoggets (H) and rams (R) of grazing Alpine Merino sheep.
HoggetRam
Forage Nutrition 1r2pr2p
Ash0.6190.0030.5520.002
CP0.5660.0020.5200.004
NDF0.1470.1450.2020.080
ADF0.1630.1450.0710.314
1 CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; 2 r, Mantel test r statistic.
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Gao, X.; Wang, H. Comparative Analysis of Rumen Bacterial Profiles and Functions during Adaption to Different Phenology (Regreen vs. Grassy) in Alpine Merino Sheep with Two Growing Stages on an Alpine Meadow. Fermentation 2023, 9, 16. https://doi.org/10.3390/fermentation9010016

AMA Style

Gao X, Wang H. Comparative Analysis of Rumen Bacterial Profiles and Functions during Adaption to Different Phenology (Regreen vs. Grassy) in Alpine Merino Sheep with Two Growing Stages on an Alpine Meadow. Fermentation. 2023; 9(1):16. https://doi.org/10.3390/fermentation9010016

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Gao, Xiang, and Hucheng Wang. 2023. "Comparative Analysis of Rumen Bacterial Profiles and Functions during Adaption to Different Phenology (Regreen vs. Grassy) in Alpine Merino Sheep with Two Growing Stages on an Alpine Meadow" Fermentation 9, no. 1: 16. https://doi.org/10.3390/fermentation9010016

APA Style

Gao, X., & Wang, H. (2023). Comparative Analysis of Rumen Bacterial Profiles and Functions during Adaption to Different Phenology (Regreen vs. Grassy) in Alpine Merino Sheep with Two Growing Stages on an Alpine Meadow. Fermentation, 9(1), 16. https://doi.org/10.3390/fermentation9010016

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