Effects of dietary energy levels on rumen fermentation, microbiota, and gastrointestinal morphology in growing ewes

Abstract This study investigated whether dietary metabolizable energy (ME) could generate dynamical effects on rumen fermentation, gastrointestinal tract (GIT) morphology, and microbial composition of growing ewes. A total of twenty‐eight female Hu lambs were randomly allotted to two treatments with different dietary ME levels: 9.17 (FEA) and 10.41 MJ/kg (FEB). These lambs were further made ready for a 67‐day feeding trial. Results showed that the molar proportions of butyrate (p = .020), iso‐valerate (p = .028), and valerate (p = .005) were significantly higher in the FEB group than those in the FEA group. The results of the GIT morphologic properties showed that the villus height (VH) (p = .005) was significantly higher and crypt depth was significantly deeper (CD) (p = .005) in the duodenum and that the rumen papillary height (PH) was significantly higher (p = .020) in FEB group compared with the FEA group. High‐throughput sequencing results showed that 1826 operational taxonomic units (OTUs) were obtained and that the OTU number (p = .039), the ACE (p = .035), and Chao1 indices (p = .005) were lower in the FEB group. Moreover, 76 genera belonging to 21 phyla were detected in all samples; the relative abundance of Papillibacter (p = .036) and Flexilinea (p = .046) was significantly lower in the high energy group, whereas the relative abundance of unidentified Lachnospiraceae (p = .019), Acetitomaculum (p = .029), unidentified Veillonellaceae (p = .017), Anaerovibrio (p = .005), and Succinivibrio (p = .035) was significantly higher in the FEB group at the genus level. Furthermore, the relative abundance of genes and metabolic pathways were predicted by PICRUSt. The relative abundance of gene families related to carbohydrate metabolism was particularly higher (p = .027) in the FEB group. In summary, these results reveal that the dietary energy levels altered the composition and function of rumen microbiota and GIT morphology in growing female Hu sheep and provide a reference for optimizing diet formula and 10.41MJ/kg of ME level has been recommended in the growing period.


| INTRODUC TI ON
The Hu sheep are known for their high adaptability and reproductive ability in China. Prolific Hu sheep are considered a very good maternal resource in the current intensive and factory farming (Lv et al., 2020;. Diet is a critical determinant factor that influences the composition and function of the rumen microbiome, molar concentration, and proportion of volatile fatty acids (VFAs) and gastrointestinal morphology in ruminants (Carberry et al., 2012;Lee et al., 2012;Wang et al., 2012;da Silva et al., 2020).
With the transformation of ruminant farming patterns, the diets are witnessing a predictable change. The rumen micro-ecosystem and gastrointestinal development have altered accordingly, which ultimately lead to performance traits such as growth and fertility change (Morandfehr et al., 2007;Wang, et al., 2017b;. Bacteria is the dominant microorganism group in the rumen micro-ecosystem. Some of which are attached to feed particles can transform the plant ingredients into animal products (Han et al., 2015;Knoell et al., 2016;Pitta et al., 2016). Microbial activity is mostly limited by dietary nutrition, and energy and protein, particularly, are the two determinants (Clark, 1975;Clark & Davis, 1980). For instance, a high energy diet can stimulate the synthesis of microbial proteins by providing sufficient available energy for microbial growth (Bach et al., 2005;Owens et al., 2016). The rumen is the main site for carbohydrate digestion, in which feed ingredients are fermented by rumen microbes into VFAs and absorbed by the GIT (Cunha et al., 2011;McGovern et al., 2018).
Applying high energy diets to improve ruminant performance has been a popular strategy in intensive production, for example, by increasing the proportion of concentrate and using high grain (corn) diets. Fernando et al. (2010) find that feeding a high grain diet causes accelerated accumulation of the VFA and alters the composition and function of the rumen bacterial community. Further studies prove that long-term feeding of a high grain diet alters the ruminal fermentation and the rumen and ruminal epithelium-associated microbiome and cause rumen damage and metabolic disorders in cows, goats, and sheep (Hua et al., 2017;Saleem et al., 2012). Impaired rumen and intestinal epithelium can definitely affect the digestion and absorption of VFAs for the GIT. Therefore, keeping rumen fermentation normal and stable, the bacterial community and gastrointestinal morphology are crucial to guarantee the ruminant health and production capacity. Thus, we hypothesize that sheep growth rate and reproductivities may mostly depend on adequate nutrition, the appropriate dietary energy level may directly determine the production level of Hu sheep. However, the relationship between rumen microbial community, GIT development, and feed efficiency remains to be elucidated further in Hu sheep; Whether dietary energy levels have a dynamic effect on rumen fermentation, gastrointestinal morphology, and rumen bacteria in prebreeding ewes needs further investigation. The aim of this study was to systematically explore the effects of different dietary energy levels on the structural properties of the GIT and the rumen microbial diversity and predicted function in candidate ewes and recommend an appropriate dietary energy level in growing ewes.

| Ethical statements
The experimental procedures of this study were approved by the Animal Care Committee of Hunan Normal University with reference from the Administration of Affairs Concerning Experimental Animals.

| Animals, treatments, and sampling
Twenty-eight four-month-old female Hu lambs with the average initial body weight (IBW) of 18.43 ± 0.34 kg were chosen and randomly allotted to two dietary treatments: 9.17 MJ/kg (FEA) and 10.41MJ/kg (FEB) of ME. Experimental diets met the nutritional requirements (NY/T 816-2004 andNRC, 2007) for prebreeding ewes; The feed composition and nutritional ingredients are shown in Table 1. Feeding and management were implemented after referring to . Each group was fed in an individual pen with free feeding and automatic water supply. All experimental lambs were fed twice a day at 7:00 and 16:00 hr. The preliminary were predicted by PICRUSt. The relative abundance of gene families related to carbohydrate metabolism was particularly higher (p = .027) in the FEB group. In summary, these results reveal that the dietary energy levels altered the composition and function of rumen microbiota and GIT morphology in growing female Hu sheep and provide a reference for optimizing diet formula and 10.41MJ/kg of ME level has been recommended in the growing period.

K E Y W O R D S
Gastrointestinal tract, High-throughput sequencing, Rumen microbiota, Volatile fatty acid feeding period was 7 days, and the fixed trial period lasted 60 days.
Five lambs with body weight closest to the group's average body weight were selected for euthanasia after 12 hr of fasting from the two treatments according to veterinary police rules at the end of the experiment. The rumen was separated from each sheep, and approximately 100 ml of rumen content was collected and transferred into two 50 ml sterile plastic tubes and then immediately stored at −80°C. A 1 × 1 cm section of the rumen wall was cut, approximately 2 cm long intestinal tissues (duodenum, jejunum, and ileum) from the middle sections were isolated and flushed with ice-cold phosphatebuffered saline, and then all the samples were immediately fixed in 4% neutral formalin for morphometric analysis .

| Rumen fermentation parameters
The VFA concentration was determined by gas chromatography (Agilent 7890A, NYSE: A, Palo Alto, America) according to the method of Wang, et al. (2017). The processing of rumen fluid has been described in Supplementary Material S1 (Section S1.1).
Processed samples were automatically injected into an Agilent DB-FFAP gas-phase capillary column (30 m × 0.25 mm × 0.25 µm). The injector temperature was set at 250°C and the detector temperature at 280°C. The split ratio of all samples was set at 50:1. The column temperature was heated by programing from 60°C to 220°C with a rate of 20°C/min and followed by holding for a 5 min.

| Rumen and intestinal morphology
The rumen and intestinal morphology were analyzed by hematoxylin-eosin (HE) staining and optical microscopy. Paraffin sections of tissues were prepared by referring to the method described by Deng et al. (2020) and Wang et al. (2020aWang et al. ( , 2020b. Formalin-fixed rumen, duodenum, jejunum, and ileum samples were dehydrated and embedded in paraffin, and then, cross-sections of 5-μm thickness were cut and stained with HE. The morphological structure of villus height (VH), villus width (VW), crypt depth (CD), and papillary height (PH) was acquired by a microscope using an image processing and analysis system (Version 1, Leica Imaging Systems Ltd., Cambridge, UK).
At least ten well-oriented intact villi and their corresponding crypts were blindly measured by the Image-Pro Plus 6.0 software in each rumen and intestinal section of each female Hu lamb, the VH to CD ratio (VH/CD) was also calculated.
The Ion Plus Fragment Library Kit 48 rxns (Thermo Scientific) was used to construct the sequencing libraries, and the Ion S5 TM XL platform was used to sequence the library and generate 400-bp/600-bp single-end reads.

| Sequencing and bioinformatics analysis
The raw reads were cleaned by the Cutadapt quality control process (Martin, 2011), using the UCHIME algorithm (Edgar et al., 2011) to detect and remove the chimera sequences and finally obtain the clean reads. Sequence analysis was performed by the UPARSE software (UPARSE v7.0.1001) (Edgar, 2013) to cluster the operational taxonomic units (OTUs) with ≥ 97% similarity. Silva Database (Quast et al., 2013) was used to annotate taxonomic information

| Statistical analysis
The experimental data were analyzed using the one-way ANOVA in SPSS 18.0 software packages (SPSS, Chicago, IL, USA). The final results were presented with meaning values. Differences were considered to be tendency at 0.05 < p < .1 and statistically significant at p ≤ .05.

| Rumen fermentation parameters
The results of rumen fermentation parameters are shown in Table 2.
The molar proportion of butyrate (p = .020), iso-valerate (p = .028), and valerate (p = .005) was significantly higher in FEB group than that in FEA group.

| Rumen and intestinal morphology
The rumen and intestinal morphologic properties are shown in Table 3 and Figure 1. Compared with the FEA group, the VH (p = .005) and CD (p = .005) were significantly higher in the duodenum, and the VW and VH/CD had not significantly difference in the FEB group.
In the jejunum, the VH (p = .099) and VH/CD (p = .077) tended to decrease, whereas the CD and VW had no significant difference in the FEB group. In the ileum, there were no significant differences in VH, CD, VW, and VH/CD. The rumen PH was significantly higher in the FEB group than that in the FEA group (p = .020).

| Sequences across different diets
Sequencing analysis showed that a total of 1826 OTUs were ob-

| Composition of the rumen bacterial community
A total of 21 phyla were identified by taxonomic analysis, and there were other unclassified bacteria. The five phyla that were most abundant were Firmicutes, Bacteroidetes, Tenericutes, Proteobacteria, and Gracilibacteria ( Figure 2a and

TA B L E 4
Diversity indices of ruminal microflora in female Hu lambs fed different dietary energy levels accounting for more than 95% of the total abundance. Additionally, the relative abundance of Chloroflexi (p = .041) was considerably lower in the FEB group than that in the FEA group at the phyla level (Table S2).
A total of 76 bacterial genera were detected at the genus level.
The ten most abundant genera, which might relate to the most important bacteria affecting the rumen ecosystem, are elucidated in The relative abundance of Papillibacter (p = .036) and Flexilinea (p = .046) in the low energy group (FEA) was significantly higher than that in high energy group (FEB), while the relative abundance of unidentified Lachnospiraceae (p = .019), Acetitomaculum (p = .029), unidentified Veillonellaceae (p = .017), Anaerovibrio (p = .005), and Succinivibrio (p = .035) in the low energy group was significantly lower than that in the high energy group at the genus level ( Figure 3a and Table S2). Additionally, the relative abundance of bacterium_ VCB2013 (p = .027), Bacteroidales_bacterium_RM71 (p = .039), and Selenomonas ruminantium (p = .023) in the high energy group was significantly higher than that in the low energy group at the species level ( Figure 3b).

| Clustering dissimilarities of rumen microbes
The results of principal coordinate analysis (PCoA) showed that the ruminal bacterial communities accounted for 23.17% of the total variations and were obviously distinguished from groups FEA and FEB by PC1, and the bacterial communities between groups FEA and FEB were distinguished and represented 13.27% of the total variation by PC2 ( Figure 4). The results of the nonmetric multidimensional scaling (NMDS) analysis also revealed that the bacterial communities of the FEA group were separately clustered from the FEB group, and the stress of <0.001 indicated that the NMDS results could accurately reflect the degree of difference from all the samples ( Figure S1).

| Predicted metabolic pathways and functions of rumen microbiota
The metabolic function of the rumen microbiome was predicted by PICRUSt in the present study. The results of KEGG level 1 showed that "metabolism" was in the highest abundance with more than 47% of the total reads for each group ( Figure S2). At KEGG level 2, the 33 gene families of the most abundant (relative abundance > 0.10%) from all rumen samples are presented in Table S3. Genes belonging to membrane transport, amino acid metabolism, carbohydrate metabolism, replication and repair, translation, and energy metabolism had the most relative abundance in the two groups ( Figure 5a). Among these gene families, the genes associated with carbohydrate metabolism were dramatically higher (p = .027) in the FEB group, and the gene families of metabolism (p = .015), infectious diseases (p = .037), and nervous system (p = .004) were significantly lower in FEB group than those in the FEA group (Figure 5c). At KEGG level 3, the 35 most abundant pathways are shown in Table S3. Among these pathways, transporters, general function prediction only, DNA repair and recombination proteins, ABC transporters, and ribosome were highly represented (Figure 5b).
With an increase in the dietary energy levels, the relative abundance of eleven pathways showed significant variation between the two groups ( Figure 5d). The pathways related to glycolysis/glucone-

| DISCUSSION
The rumen is the most powerful digestive organ for degrading and converting plant materials to VFAs in ruminants. In this regard, possessing the complex microflora plays a crucial role in feed fermentation and energy metabolism, and more than 70% of the energy F I G U R E 3 Abundance maps of bacteria with significant differences in (a) genus and (b) species of female Hu lambs fed the low energy diet (FEA,9.17  is provided by VFAs to ensure host growth and reproduction performance (Flint et al., 2007). Previous studies have demonstrated that the VFA concentration and the proportion of acetate, propionate, and butyrate in the rumen were closely related to the feed type and nutrient level, high energy or concentrate-based diets tended to increase the VFAs concentration, especially propionate (Agle et al., 2010;Corley & Murphy, 2004;Keady et al., 2001;van Soest., 1994). In the present study, compared with the low energy group, a high energy level significantly increased the molar proportion of butyrate, iso-valerate, and valerate. These results were similar to that of previous studies , in which they found that the proportion of the butyrate, iso-valerate, and valerate is significantly higher in the high-concentrate group than those of the high-forage group.
Previous studies have found that feed conversion efficiency is influenced by dietary components and rumen environment in ruminants, and diet can mainly affect the composition of the rumen microbiome, while the key microbial species may specifically regulate feed efficiency. For instance, Methanobrevibacter smithii and Mitsuokella jalaludinii may improve ruminal fermentation and further influence feed efficiency (Ellison et al., 2017;Guan et al., 2008;Shabat et al., 2016). Therefore, we speculated that these results may be related to the rumen microbiota and the morphological characteristics of the GIT.
The GIT is the primary site of nutrients for digestion and absorption in ruminants. The GIT tissues are affected by ME intake and dietary energy density. Dietary energy levels can sufficiently promote GIT development and epithelial proliferation, for instance, the high concentrate diet significantly increased the length and width of ruminal papillae, and a decrease in dietary energy decreased the length and width of papillae (Cui et al., 2019;Steele et al., 2016;Wester et al., 1995). The particle size and composition of the diet greatly influenced the morphological structure of rumen papillae . Previous studies showed that a high energy diet can promote ruminal papillae proliferation in young goats (Shen et al., 2004). In the present study, a high energy diet significantly increased the papillae height compared to the low energy group. These results are consistent with that of previous studies (Kim et al., 2012).  (Mcleod et al., 2000;Wang et al., 2009). Compared with the low energy group, the VH and CD of duodenal increased significantly in the high energy group, while a tendentious decrease was observed in VH and VH/CD in the jejunum, thereby demonstrating that high energy diet may lead to a negative effect on the jejunum morphology of female Hu lambs.
Rumen microbial diversity affects the nutritional health and growth of ruminants and the host provides an appropriate anaerobic environment and fermentation substrates for rumen microorganisms to survive (Guan et al., 2008;Pokharel et al., 2018). In this study, the V4 region of rumen microbial 16S r DNA gene sequencing was sequence to investigate the relationship between rumen microbiota and dietary ME levels in female Hu sheep. Previous studies found that a high grain diet can significantly decrease the number of OTUs . We observed that the number of OUTs, Chao1 value, and ACE value were indeed significantly decreased in the diet of a high ME level. These results indicated that the relative abundance of rumen microbial communities in female Hu lambs is altered by different ME levels in the diets. Moreover, Liu et al. (2013) found ing that the rumen microbiota of sheep was also relatively stable at the phyla level. These results are consistent with that of previous studies on cattle (Plaizier et al., 2017;Wetzels et al., 2017). Such studies showed that the most dominant phyla were Firmicutes and Bacteroidetes in the rumen of ruminants, which were closely related to carbohydrate and protein metabolism (Hook et al., 2011;Yang, et al., 2013). In the present study too, the most dominant phyla in the rumen were Firmicutes and Bacteroidetes in female Hu sheep.
At the genus level, the dominant four genera were unidentified Ruminococcaceae, unidentified Lachnospiraceae, unidentified Bacteroidales, and Papillibacter, similar to previous studies (Wang, et al., 2017b;Wang et al., 2016). Notably, it is revealed in our study that the relative abundance of unidentified Lachnospiraceae was significantly higher, and unidentified Ruminococcaceae too had a higher tendency in the high ME group. Conversely, the relative abundance of Papillibacter was very significantly reduced in the high dietary energy level compared with the low dietary energy level.
These results indicated that the rumen bacterial dominant genera were dramatically influenced by the energy level of the diet and exhibited great differences among different breeds. The current study also identified the changes of the represented genera in the rumen between the different dietary ME levels. For instance, unidentified Ruminococcaceae comprised 5.23%-7.02% of the relative abundance, which was inconsistent with previous studies (Seddik et al., 2018;Wang, et al., 2017). Ruminococcaceae strains play a critical role in energy and lipid metabolism, and its relative abundance is negatively associated with vascular sclerosis (Menni et al., 2018).  (Lamendella et al., 2011). These results are similar to that of previous studies (Wang, et al., 2017b). As expected, the relative abundance of carbohydrate metabolism in the high energy group was significantly higher than that in the low energy group. At level 3, the relative abundance of transporters was the highest, followed by general function prediction only, DNA repair and recombination proteins, ATP-binding cassette (ABC) transporter, ribosome, and others. Furthermore, previous studies found that ABC transporters play an important role in the digestion and absorption of nutrients and that ribosomes are closely associated with protein synthesis (Gifford et al., 2013;Yan et al., 2016). The present study showed that the genes responsible for glycolysis/ gluconeogenesis, pentose phosphate pathway, glycerolipid metabolism, and biosynthesis of ansamycins were upregulated in high energy feeding which indicates an enhanced fermentation rate performed by the rumen microbiota, with a decrease in the general function prediction only, arginine and proline metabolism, drug metabolism-other enzymes, riboflavin metabolism, amino acid metabolism, novobiocin biosynthesis, and glutamatergic synapse-related genes. These results are inconsistent with that of previous research (Seddik et al., 2018). The current study implied that feeding a high energy diet by using corn as the main energy source and decreasing the roughage percentage alters the ruminal microbial composition and the inferred microbial functions.

| CON CLUS ION
The present study mainly studies rumen fermentation, gastrointestinal morphology, and the composition and function of rumen microbiota of growing ewes in Hu sheep with different energy feeding.
The results suggest that dietary energy levels have major effects on the molar proportion of VFA, morphological structure of GIT, rumen microbial diversity, and inferred metabolic functions. These conclusions provide a significant reference for targeting appropriate dietary energy levels in Hu sheep, recommending a high ME level of 10.41MJ/kg in production.

| ANIMAL WELFARE S TATEMENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes and feed legislation.

ACK N OWLED G M ENTS
We thank Dr. Yulong Yin and Dr. Xunping Jiang for their advice on experiment design. We aslo thank Mr. Jian guo Zhen and Mr. Chunhua Yang for their assistance on lambs feeding experiments. This work was supported by Hunan Province's Strategic and Emerging Industrial Projects (2018GK4035), Hunan Province's Changsha-

CO N FLI C T O F I NTE R E S T
The authors declare that they have no potential conflict of interest.