Integrative methylome and transcriptome analysis of porcine abdominal fat indicates changes in fat metabolism and immune responses during different development.

Fat is involved in synthesizing fatty acids (FAs), FA circulation, and lipid metabolism. Various genetic studies have been conducted on porcine fat but understanding the growth and specific adipose tissue is insufficient. The purpose of this study is to investigate the epigenetic difference in abdominal fat according to the growth of porcine. The samples were collected from the porcine abdominal fat of different developmental stages (10 and 26 weeks of age). Then, the samples were sequenced using MBD-seq and RNA-seq for profiling DNA methylation and RNA expression. In 26 weeks of age pigs, differentially methylated genes (DMGs) and differentially expressed genes (DEGs) were identified as 2,251 and 5,768, compared with 10 weeks of age pigs, respectively. Gene functional analysis was performed using GO and KEGG databases. In functional analysis results of DMGs and DEGs, immune responses such as chemokine signaling pathways, B cell receptor signaling pathways, and lipid metabolism terms such as PPAR signaling pathways and fatty acid degradation were identified. It is thought that there is an influence between DNA methylation and gene expression through changes in genes with similar functions. The effects of DNA methylation on gene expression were investigated using cis-regulation and trans-regulation analysis to integrate and interpret different molecular layers. In the cis-regulation analysis using 629 overlapping genes between DEGs and DMGs, immune response functions were identified, while in trans-regulation analysis through the TF-target gene network, the co-expression network of lipid metabolism-related functions was distinguished. Our research provides an understanding of the underlying mechanisms for epigenetic regulation in porcine abdominal fat with aging.

Fat is involved in the synthesis of new fatty acids (FAs), FA circulation, and lipid metabolism. Various genetic studies have been conducted on porcine fat but understanding the growth and specific adipose tissue is insufficient. The purpose of this study is to investigate the epigenetic difference in abdominal fat according to the growth of porcine. Modifications in DNA methylation and expression values were confirmed epigenetically with growth. Changed genes in each DNA and RNA showed identical trends in the function of immune response and lipid metabolism. The effects of DNA methylation on gene expression were investigated using cis-regulation (functional enrichment analysis of overlapping genes) and trans-regulation (transcription factor and target gene networking) analysis to integrate and interpret different molecular layers. Our research provides an understanding of the underlying mechanisms for epigenetic regulation in porcine abdominal fat with aging.

Analysis of 16S rRNA gene sequencing data for the taxonomic characterization of the vaginal and the fecal microbial communities in Hanwoo.

OBJECTIVE: The study of Hanwoo (Korean native cattle) has mainly been focused on meat quality and productivity. Recently the field of microbiome research has increased dramatically. However, the information on the microbiome in Hanwoo is still insufficient, especially relationship between vagina and feces. Therefore, the purpose of this study is to examine the microbial community characteristics by analyzing the 16S rRNA sequencing data of Hanwoo vagina and feces, as well as to confirm the difference and correlation between vaginal and fecal microorganisms. As a result, the goal is to investigate if fecal microbiome can be used to predict vaginal microbiome. METHODS: A total of 31 clinically healthy Hanwoo that delivered healthy calves more than once in Cheongju, South Korea were enrolled in this study. During the breeding season, we collected vaginal and fecal samples and sequenced the microbial 16S rRNA genes V3-V4 hypervariable regions from microbial DNA of samples. RESULTS: The results revealed that the phylum-level microorganisms with the largest relative distribution were Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria in the vagina, and Firmicutes, Bacteroidetes, and Spirochaetes in the feces, respectively. In the analysis of alpha, beta diversity, and effect size measurements (LefSe), the results showed significant differences between the vaginal and fecal samples. We also identified the function of these differentially abundant microorganisms by functional annotation analyses. But there is no significant correlation between vaginal and fecal microbiome. CONCLUSION: There is a significant difference between vaginal and fecal microbiome, but no significant correlation. Therefore, it is difficult to interrelate vaginal microbiome as fecal microbiome in Hanwoo. In a further study, it will be necessary to identify the genetic relationship of the entire microorganism between vagina and feces through the whole metagenome sequencing analysis and meta-transcriptome analysis to figure out their relationship.

Insight into the potential candidate genes and signaling pathways involved in lymphoma disease in dogs using a comprehensive whole blood transcriptome analysis.

Lymphoma is one of the most prevalent hematological cancers, accounting for 15-20 % of new cancer diagnoses in dogs. Therefore, this study aims to explore the important genes and pathways involved in canine lymphoma progression and understand the underlying molecular mechanisms using RNA sequencing. In this study, RNAs acquired from seven pairs of lymphoma and non-lymphoma blood samples were sequenced from different breeds of dogs. Sequencing reads were preprocessed, aligned with the reference genome, assembled and expressions were estimated through bioinformatics approaches. At a false discovery rate (FDR) < 0.05 and fold change (FC) ≥ 1.5, a total of 625 differentially expressed genes (DEGs) were identified between lymphoma and non-lymphoma samples, including 347 up-regulated DEGs such as SLC38A11, SCN3A, ZIC5 etc. and 278 down-regulated DEGs such as LOC475937, CSMD1, KRT14 etc. GO enrichment analysis showed that these DEGs were highly enriched for molecular function of ATP binding and calcium ion binding, cellular process of focal adhesion, and biological process of immune response, and defense response to virus. Similarly, KEGG pathways analysis revealed 11 significantly enriched pathways such as ECM-receptor interaction, cell cycle, PI3K-Akt signaling pathway, ABC transporters etc. In the protein-protein interaction (PPI) network, CDK1 was found to be a top hub gene with highest degree of connectivity. Three modules selected from the PPI network showed that canine lymphoma was highly associated with cell cycle, ECM-receptor interaction, hypertrophic cardiomyopathy, dilated cardiomyopathy and RIG-I-like receptor signaling pathway. Overall, our findings highlighted new candidate therapeutic targets for further testing in canine lymphoma and facilitate the understanding of molecular mechanism of lymphoma's progression in dogs.

Comprehensive Transcriptomic Comparison between Porcine CD8- and CD8+ Gamma Delta T Cells Revealed Distinct Immune Phenotype.

We aimed to comprehensively understand the functional mechanisms of immunity, especially of the CD8+/- subsets of gamma delta (γδ) T cells, using an RNA-sequencing analysis. Herein, γδ T cells were obtained from bronchial lymph node tissues of 38-day-old (after weaning 10-day: D10) and 56-day-old (after weaning 28-day: D28) weaned pigs and sorted into CD8+ and CD8- groups. Differentially expressed genes (DEGs) were identified based on the CD8 groups at D10 and D28 time points. We confirmed 1699 DEGs between D10 CD8+ versus D10 CD8- groups and 1784 DEGs between D28 CD8+ versus D28 CD8- groups; 646 upregulated and 561 downregulated DEGs were common. The common upregulated DEGs were enriched in the cytokine-cytokine receptor interaction and T cell receptor (TCR) signaling pathway, and the common downregulated DEGs were enriched in the B cell receptor signaling pathway. Further, chemokine-related genes, interferon gamma, and CD40 ligand were involved in the cytokine-cytokine receptor interaction and TCR signaling pathway, which are associated with inter-regulation in immunity. We expect our results to form the basic data required for understanding the mechanisms of γδ T cells in pigs; however, further studies are required in order to reveal the dynamic changes in γδ T cells under pathogenic infections, such as those by viruses.

Return to Play After Open Calcaneoplasty for Insertional Achilles Tendinopathy With Haglund Deformity in Competitive Professional Athletes.

BACKGROUND: Surgical treatment of insertional Achilles tendinopathy with Haglund deformity in highly competitive professional athletes has not been previously investigated. PURPOSE: To assess clinical outcomes, including return to play (RTP), after surgical treatment of insertional Achilles tendinopathy in professional athletes. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: This retrospective study included 20 professional athletes who were surgically treated for insertional Achilles tendinopathy and had at least 2 years of follow-up. An open longitudinal lateral approach was used for the operation, without violation of the Achilles tendon. Outcome evaluation included American Orthopaedic Foot and Ankle Society (AOFAS) hindfoot score, subjective patient satisfaction, mean time of return to competition (RTC), and rate of RTP. We defined RTC as return to an official match for at least 1 minute and RTP rate as the percentage of patients who were able to participate in at least 2 full seasons. A subgroup analysis was performed to compare the RTP and no-RTP groups. RESULTS: The AOFAS score improved significantly from preoperatively to the final follow-up (from 65.1 to 88.4; P < .001), and 75% of the patients reported good to excellent satisfaction. The mean time of RTC was 7.45 months (range, 4-18 months), and the rate of RTP was 60%. The RTP group had a significantly lower mean body mass index than did the no-RTP group (22.03 vs 23.86, respectively; P = .005) and faster mean RTC (5.0 vs 11.1 months, respectively; P < .001). CONCLUSION: Open calcaneoplasty for surgical treatment of insertional Achilles tendinopathy with Haglund deformity in highly competitive professional athletes should be approached cautiously. Our patients had a 7.5-month recovery period before return to their first official match, and only 60% of the patients returned to their sports activity and participated in at least 2 full seasons. Lower body mass index and a faster RTC after surgery were related to longer functional maintenance.

Transcriptome analysis of pig macrophages expressing porcine reproductive and respiratory syndrome virus non-structural protein 1.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a causative pathogen of PRRS, one of the most economically disastrous swine diseases. Non-structural protein 1 (NSP1) of PRRSV consists of NSP1α and NSP1ß which exhibit papain like cysteine protease activity. Recent evidence demonstrates that PRRSV NSP1 may be participated in modulating host immunity, but very few host proteins were discovered as targets for NSP1. In this study, we used RNA-seq to investigate the functional role of PRRSV NSP1 in porcine alveolar macrophages, 3D4/31 cells. Compared to empty vector (mock) transfectant, NSP1, NSP1α, and NSP1ß expressing 3D4/31 cells displayed a total of 60 genes, 63 genes, and 80 genes as differentially expressed genes (DEGs), respectively. Most of DEGs are involved in early inflammatory responses including interleukin (IL)-17 signaling pathway, chemokine signaling pathway, tumor necrosis factor (TNF)-α signaling pathway, and cell adhesion molecules. Interestingly, PRRSV NSP1 expression in 3D4/31 cells decreased mRNA transcripts of Fosb and Gdf15 known to be involved in host cell signaling or host cell protection during inflammation. Therefore, PRRSV NSP1 might block the signaling involved in host immune surveillance. Further study is required to define the mechanism on how PRRSV NSP1 protein represses mRNA transcripts of specific host genes.

Identification of Monoallelically Expressed Genes Associated with Economic Traits in Hanwoo (Korean Native Cattle).

Hanwoo, an indigenous Korean cattle breed, has been genetically improved by selecting superior sires called Korean-proven bulls. However, cows still contribute half of the genetic stock of their offspring, and allelic-specific expressed genes have potential, as selective targets of cows, to enhance genetic gain. The aim of this study is to identify genes that have MAEs based on both the genome and transcriptome and to estimate their effects on breeding values (BVs) for economically important traits in Hanwoo. We generated resequencing data for the parents and RNA-sequencing data for the muscle, fat, and brain tissues of the offspring. A total of 3801 heterozygous single nucleotide polymorphisms (SNPs) in offspring were identified and they were located in 1569 genes. Only 14 genes showed MAE (seven expressing maternal alleles and seven expressing paternal alleles). Tissue-specific MAE was observed, and LANCL1 showed maternal allele expression across all tissues. MAE genes were enriched for the biological process of cell death and angiogenesis, which included ACKR3 and PDCL3 genes, whose SNPs were significantly associated with BVs of lean meat production-related traits, such as weight at 12 months of age, carcass weight, and loin eye area. In the current study, monoallelically expressed genes were identified in various adult tissues and these genes were associated with genetic capacity in Hanwoo.

Lesion Size May Predict Return to Play in Young Elite Athletes Undergoing Microfracture for Osteochondral Lesions of the Talus.

PURPOSE: To evaluate the clinical and sports-related outcomes of arthroscopic microfracture (MFx) for osteochondral lesion of the talus (OLT) in elite athletes. METHODS: The athletes who underwent arthroscopic MFx for OLTs at our institution between January 2011 and September 2015 with minimum 2 years of follow-up were reviewed. The Foot and Ankle Outcome Score, American Orthopaedic Foot & Ankle Society, and visual analog scale pain score, time and rate of "return-to-competition" (RTC, return to an official match for at least 1 minute after treatment), and rate of "return-to-play" (RTP, participation in at least 2 entire seasons after treatment) were used to evaluate the outcomes. We compared athletes who were able to RTP with those who were not. RESULTS: In total, 41 patients (mean age 19.34 ± 3.76 years) were included. The mean follow-up was 54.9 ± 13.72 months. In total, 36 patients had medial lesions, and 5 patients had lateral lesions. All subscales of preoperative Foot and Ankle Outcome Score were significantly improved at the final follow-up. The mean preoperative American Orthopaedic Foot & Ankle Society score of 74.46 ± 8.10 improved to 91.62 ± 2.99 (P < .001) at the final follow-up. The mean preoperative visual analog scale pain score of 5.44 ± 1.57 improved to 2.66 ± 1.04 (P < .001). All patients achieved RTC (100%) at mean time of 5.45 ± 3.18 months, and 74.4% of patients were able to RTP. The RTP-group showed significantly smaller lesions compared to the No-RTP group (71.52 ± 43.29 vs 107.00 ± 45.28 mm2, P = .009). The cut-off OLT size for predicting RTP was 84.0 mm2, with a sensitivity of 90.0% and specificity of 75.9%. CONCLUSIONS: All athletes were able to RTC at average of 5.45 months after MFx for OLTs with minimal subchondral involvement, and 74.4% were able to RTP. The only prognostic variable for RTP was lesion size, and its predictive cut-off was 84.0 mm2. LEVEL OF EVIDENCE: IV, Case series.

Author Correction: Comprehensive genome and transcriptome analyses reveal genetic relationship, selection signature, and transcriptome landscape of small-sized Korean native Jeju horse.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Integrated time-serial transcriptome networks reveal common innate and tissue-specific adaptive immune responses to PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection is the most important viral disease causing severe economic losses in the swine industry. However, mechanisms underlying gene expression control in immunity-responsible tissues at different time points during PRRSV infection are poorly understood. We constructed an integrated gene co-expression network and identified tissue- and time-dependent biological mechanisms of PRRSV infection through bioinformatics analysis using three tissues (lungs, bronchial lymph nodes [BLNs], and tonsils) via RNA-Seq. Three groups with specific expression patterns (i.e., the 3-dpi, lung, and BLN groups) were discovered. The 3 dpi-specific group showed antiviral and innate-immune signalling similar to the case for influenza A infection. Moreover, we observed adaptive immune responses in the lung-specific group based on various cytokines, while the BLN-specific group showed down-regulated AMPK signalling related to viral replication. Our study may provide comprehensive insights into PRRSV infection, as well as useful information for vaccine development.

Comparison of prediction accuracy for genomic estimated breeding value using the reference pig population of single-breed and admixed-breed.

This study was performed to increase the accuracy of genomic estimated breeding value (GEBV) predictions for domestic pigs using single-breed and admixed reference populations (single-breed of Berkshire pigs [BS] with cross breed of Korean native pigs and Landrace pigs [CB]). The principal component analysis (PCA), linkage disequilibrium (LD), and genome-wide association study (GWAS) were performed to analyze the population structure prior to genomic prediction. Reference and test population data sets were randomly sampled 10 times each and precision accuracy was analyzed according to the size of the reference population (100, 200, 300, or 400 animals). For the BS population, prediction accuracy was higher for all economically important traits with larger reference population size. Prediction accuracy was ranged from -0.05 to 0.003, for all traits except carcass weight (CWT), when CB was used as the reference population and BS as the test. The accuracy of CB for backfat thickness (BF) and shear force (SF) using admixed population as reference increased with reference population size, while the results for CWT and muscle pH at 24 hours after slaughter (pH) were equivocal with respect to the relationship between accuracy and reference population size, although overall accuracy was similar to that using the BS as the reference.

Population analysis of the Korean native duck using whole-genome sequencing data.

BACKGROUND: Advances in next-generation sequencing technologies have provided an opportunity to perform population-level comparative genomic analysis to discover unique genomic characteristics of domesticated animals. Duck is one of the most popular domesticated waterfowls, which is economically important as a source of meat, eggs, and feathers. The objective of this study is to perform population and functional analyses of Korean native duck, which has a distinct meat flavor and texture phenotype, using whole-genome sequencing data. To study the distinct genomic features of Korean native duck, we conducted population-level genomic analysis of 20 Korean native ducks together with 15 other duck breeds. RESULTS: A total of 15.56 million single nucleotide polymorphisms were detected in Korean native duck. Based on the unique existence of non-synonymous single nucleotide polymorphisms in Korean native duck, a total of 103 genes related to the unique genomic characteristics of Korean native duck were identified in comparison with 15 other duck breeds, and their functions were investigated. The nucleotide diversity and population structures among the used duck breeds were then compared, and their phylogenetic relationship was analyzed. Finally, highly differentiated genomic regions among Korean native duck and other duck breeds were identified, and functions of genes in those regions were examined. CONCLUSIONS: This is the first study to compare the population of Korean native duck with those of other duck breeds by using whole-genome sequencing data. Our findings can be used to expand our knowledge of genomic characteristics of Korean native duck, and broaden our understanding of duck breeds.

Conservative treatment of refractures after modified tension band wiring of fifth metatarsal base stress fractures in athletes.

OBJECTIVE: Recurrent fifth metatarsal base stress fractures (MT5-BSF) in athletes present a challenging problem. The aim of this study was to evaluate the result of conservative treatment for the refracture of MT5-BSF after modified tension band wiring (MTBW). MATERIALS AND METHODS: The outcomes of 15 elite athletes undergoing conservative treatment for refracture of MT5-BSF after MTBW were retrospectively reviewed. They were instructed to avoid weight-bearing with short leg cast for 6 weeks. After that, they started partial weight with a postop shoe. Stepwise exercise followed bone union by radiographs. RESULTS: Thirteen cases (86.6%) had a complete bone union after a mean of 18.9 ± 8.6 weeks. Twelve cases (80%) returned to their previous activity level and maintained for at least two consecutive seasons. CONCLUSION: Eighty percent of all athletic patients with the conservative treatment for refractures with healed MT5-BSF after MTBW on the plantar-lateral side could maintain and return to their previous sports activity for at least 2 years.

Operative Results of Plantar Plating for Fifth Metatarsal Stress Fracture.

BACKGROUND: The treatment of fifth metatarsal stress fractures can be challenging. Various operative fixation methods have been reported for fracture management. Among them, intramedullary screw fixation has become increasingly popular. However, recent reports have described failures after screw fixation in athletes. The aim of this study was to determine the rates of clinical and radiographic healing, time to return to sport, and complications of elite athletes with proximal fifth metatarsal fractures treated with plantar plating. METHODS: Thirty-eight athletes with fifth metatarsal stress fractures treated using a plantar plating technique in 3 hospitals from 2013 to 2018 were evaluated retrospectively. Demographic data, radiographic evaluation, and the time until union and return to sports activities were collected and analyzed. A total of 38 patients underwent the plantar plating for a fifth metatarsal stress fracture with a mean follow-up of 23 (range, 12-49) months. RESULTS: The mean time to the radiologic union, as determined by plain radiography, was 9.3 (range, 8-16) weeks. Although there were no nonunions or delayed unions during follow-up, 4 refractures developed (10.5%). All but 1 patient were able to return to their previous levels of sporting activity at 22.2 ± 4.5 (range, 12-40) weeks. CONCLUSION: With a minimum of 1-year follow-up, the described plantar plating technique could be an alternative method for the operative treatment of fifth metatarsal stress fractures without nonunion problems. LEVEL OF EVIDENCE: Level IV, case series.

Serial gene co-expression network approach to mine biological meanings from integrated transcriptomes of the porcine endometrium during estrous cycle.

The estrous cycle is a complex process regulated by several hormones. To understand the dynamic changes in gene expression that takes place in the swine endometrium during the estrous cycle relative to the day of estrus onset, we performed RNA-sequencing analysis on days 0, 3, 6, 9, 12, 15, and 18, resulting in the identification of 4495 differentially expressed genes (DEGs; Q ≤ 0.05 and |log2FC| ≥ 1) at various phases in the estrous cycle. These DEGs were integrated into multiple gene co-expression networks based on different fold changes and correlation coefficient (R2) thresholds and a suitable network, which included 899 genes (|log2FC| ≥ 2 and R2 ≥ 0.99), was identified for downstream analyses based on the biological relevance of the Gene Ontology (GO) terms enriched. The genes in this network were partitioned into 6 clusters based on the expression pattern. Several GO terms including cell cycle, apoptosis, hormone signaling, and lipid biosynthetic process were found to be enriched. Furthermore, we found 15 significant KEGG pathways, including cell adhesion molecules, cytokine-cytokine receptor signaling, steroid biosynthesis, and estrogen signaling pathways. We identified several genes and GO terms to be stage-specific. Moreover, the identified genes and pathways extend our understanding of porcine endometrial regulation during estrous cycle and will serve as a good resource for future studies.

Erratum to: Comparison of prediction accuracy for genomic estimated breeding value using the reference pig population of single-breed and admixed-breed.

[This corrects the article DOI: 10.5187/jast.2020.62.4.438.].

Comprehensive genome and transcriptome analyses reveal genetic relationship, selection signature, and transcriptome landscape of small-sized Korean native Jeju horse.

The Jeju horse, indigenous to the Jeju Island in Korea may have originated from Mongolian horses. Adaptations to the local harsh environment have conferred Jeju horse with unique traits such as small-sized body, stocky head, and shorter limbs. These characteristics have not been studied previously at the genomic level. Therefore, we sequenced and compared the genome of 41 horses belonging to 6 breeds. We identified numerous breed-specific non-synonymous SNPs and loss-of-function mutants. Demographic and admixture analyses showed that, though Jeju horse is genetically the closest to the Mongolian breeds, its genetic ancestry is independent of that of the Mongolian breeds. Genome wide selection signature analysis revealed that genes such as LCORL, MSTN, HMGA2, ZFAT, LASP1, PDK4, and ACTN2, were positively selected in the Jeju horse. RNAseq analysis showed that several of these genes were also differentially expressed in Jeju horse compared to Thoroughbred horse. Comparative muscle fiber analysis showed that, the type I muscle fibre content was substantially higher in Jeju horse compared to Thoroughbred horse. Our results provide insights about the selection of complex phenotypic traits in the small-sized Jeju horse and the novel SNPs identified will aid in designing high-density SNP chip for studying other native horse breeds.

Genome-Wide Analysis of Allele-Specific Expression Patterns in Seventeen Tissues of Korean Cattle (Hanwoo).

The functional hemizygosity could be caused by the MAE of a given gene and it can be one of the sources to affect the phenotypic variation in cattle. We aimed to identify MAE genes across the transcriptome in Korean cattle (Hanwoo). For three Hanwoo family trios, the transcriptome data of 17 tissues were generated in three offspring. Sixty-two MAE genes had a monoallelic expression in at least one tissue. Comparing genotypes among each family trio, the preferred alleles of 18 genes were identified (maternal expression, n = 9; paternal expression, n = 9). The MAE genes are involved in gene regulation, metabolic processes, and immune responses, and in particular, six genes encode transcription factors (FOXD2, FOXM1, HTATSF1, SCRT1, NKX6-2, and UBN1) with tissue-specific expression. In this study, we report genome-wide MAE genes in seventeen tissues of adult cattle. These results could help to elucidate epigenetic effects on phenotypic variation in Hanwoo.

Comparison of Bacterial Populations in the Ceca of Swine at Two Different Stages and their Functional Annotations.

The microbial composition in the cecum of pig influences host health, immunity, nutrient digestion, and feeding requirements significantly. Advancements in metagenome sequencing technologies such as 16S rRNAs have made it possible to explore cecum microbial population. In this study, we performed a comparative analysis of cecum microbiota of crossbred Korean native pigs at two different growth stages (stage L = 10 weeks, and stage LD = 26 weeks) using 16S rRNA sequencing technology. Our results revealed remarkable differences in microbial composition, α and ß diversity, and differential abundance between the two stages. Phylum composition analysis with respect to SILVA132 database showed Firmicutes to be present at 51.87% and 48.76% in stages L and LD, respectively. Similarly, Bacteroidetes were present at 37.28% and 45.98% in L and LD, respectively. The genera Prevotella, Anaerovibrio, Succinivibrio, Megasphaera were differentially enriched in stage L, whereas Clostridium, Terrisporobacter, Rikenellaceae were enriched in stage LD. Functional annotation of microbiome by level-three KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed that glycine, serine, threonine, valine, leucine, isoleucine arginine, proline, and tryptophan metabolism were differentially enriched in stage L, whereas alanine, aspartate, glutamate, cysteine, methionine, phenylalanine, tyrosine, and tryptophan biosynthesis metabolism were differentially enriched in stage LD. Through machine-learning approaches such as LEfSe (linear discriminant analysis effect size), random forest, and Pearson's correlation, we found pathways such as amino acid metabolism, transport systems, and genetic regulation of metabolism are commonly enriched in both stages. Our findings suggest that the bacterial compositions in cecum content of pigs are heavily involved in their nutrient digestion process. This study may help to meet the demand of human food and can play significant roles in medicinal application.