Lambskin of the Hu sheep exhibits high economic value due to its water-wave pattern. Wool curvature is the key factor of the pattern types and quality of lambskin, and it is formed by the interaction between dermal papilla cells and hair matrix cells in the hair follicle, which is regulated by various genes and signaling pathways. Herein, three full-sibling pairs of two-day-old healthy lambs (n = 6) were divided into a straight wool group (ST) and small waves group (SM) with three repetitions. RNA-seq was applied to determine the expression profile of mRNAs and lncRNAs in Hu sheep hair follicles. 25 differentially expressed mRNAs and 75 differentially expressed lncRNAs were found between SM and ST. FGF12, ATP1B4, and TCONS_00279168 were probably associated with hair follicle development. Then, Gene Ontology (GO) and KEGG enrichment analysis were implemented for the functional annotation of target genes of differentially expressed lncRNAs. The results showed that many genes, such as FGF12 and ATP1B4, were found enriched in PI3K-Akt signaling, MAPK signaling, and Ras signaling pathway associated with hair follicle growth and development. In addition, the interaction network of differentially expressed lncRNAs and mRNAs showed that a total of 6 differentially expressed lncRNAs were associated with 12 differentially expressed mRNAs, which may be as candidate mRNAs and lncRNAs. TCONS_00279168 may target ATP1B4 and FGF12 to regulate MAPK, PI3K-Akt, Ras signaling pathways involved in the sheep hair follicle development process. These results will provide the basis for exploring hair follicle development.

Hair follicle development is closely associated with wool curvature. Current studies reveal the crucial role of microRNAs (miRNAs) in hair follicle growth and development. However, few studies are known regarding their role in wool curvature. To reveal the potential roles of miRNAs in Hu sheep lambskin with different patterns, a total of 37 differentially expressed (DE) miRNAs were identified in hair follicles between small waves (SM) and straight wool (ST) groups using RNA-seq. Through functional enrichment and miRNA-mRNA co-expression analysis, some key miRNAs (oar-miR-143, oar-miR-200b, oar-miR-10a, oar-miR-181a, oar-miR-10b, oar-miR-125b, etc.) and miRNA-mRNA pairs (miR-125b target CD34, miR-181a target FGF12, LMO3, miR-200b target ZNF536, etc.) were identified. Though direct or indirect ways affecting hair follicle development, these miRNAs and mRNAs may have possible effects on wool curvature, and this study thus provides valuable insight on potential pattern formation.

It has long been recognized that enterotoxigenic Escherichia coli (ETEC) is the major pathogen responsible for vomiting and diarrhea. E. coli F17, a main subtype of ETEC, is characterized by high morbidity and mortality in young livestock. However, the transcriptomic basis underlying E. coli F17 infection has not been fully understood. In this study, RNA sequencing was performed to explore the expression profiles of circRNAs and miRNAs in the jejunum of E. coli F17-antagonism (AN) and -sensitive (SE) lambs. A total of 16,534 circRNAs and 271 miRNAs (125 novel miRNAs and 146 annotated miRNAs) were screened, and 214 differentially expressed (DE) circRNAs and 53 DE miRNAs were detected between the AN and SE lambs (i.e., novel_circ_0025840, novel_circ_0022779, novel_miR_107, miR-10b). Functional enrichment analyses showed that source genes of DE circRNAs were mainly involved in metabolic-related pathways, while target genes of DE miRNAs were mainly enriched in the immune response pathways. Then, a two-step machine learning approach combining Random Forest (RF) and XGBoost (candidates were first selected by RF and further assessed by XGBoost) was performed, which identified 44 circRNAs and 39 miRNAs as potential biomarkers (i.e., novel_circ_0000180, novel_circ_0000365, novel_miR_192, oar-miR-496-3p) for E. coli infection. Furthermore, circRNA-related and lncRNA-related ceRNA networks were constructed, containing 46 circRNA-miRNA-mRNA competing triplets and 630 lncRNA-miRNA-mRNA competing triplets, respectively. By conducting a serious of bioinformatic analyses, our results revealed important circRNAs and miRNAs that could be potentially developed as candidate biomarkers for intestinal inflammatory response against E. coli F17 infection; our study can provide novel insights into the underlying mechanisms of intestinal immunity.

Diarrhea is one of the most commonly reported diseases in young farm animals. Escherichia coli (E. coli) F17 is one of the major pathogenic bacteria responsible for diarrhea. However, the pathogenicity of diarrhea in lambs involving E. coli F17 strains and how E. coli F17 infection modifies lambs’ intestinal microbiota are largely unknown. To evaluate diarrhea in newborn lambs with an infection of E. coli F17, 50 lambs were selected for challenge experiments and divided into four groups, namely, a high-dose challenge group, low-dose challenge group, positive control group, and negative control group. The E. coli F17 challenge experiments caused diarrhea and increased mortality in the experimental lamb population, with a higher prevalence (90%), mortality (35%), and rapid onset time (4–12 h) being observed in the high-dose challenge group than the results observed in the low-dose challenge group (75%, 10%, 6–24 h, respectively). After the challenge experiment, healthy lambs in the high-dose challenge group and severely diarrheic lamb in the low-dose challenge group were identified as lambs sensitive/resistant to E. coli F17 (E. coli F17 -resistant/-sensitive candidate, AN/SE) according to the histopathological detection. Results of intestinal contents bacteria plate counting revealed that the number of bacteria in the intestinal contents of SE lambs was 10^2~3-fold greater than that of the AN lambs, especially in the jejunum. Then, 16S rRNA sequencing was conducted to profile the intestinal microbiota using the jejunal contents, and the results showed that SE lambs had higher Lactococcus and a lower Bacteroidetes:Firmicutes ratio and intestinal microbiota diversity in the jejunum than AN lambs. Notably, high abundance of Megasphaera elsdenii was revealed in AN lambs, which indicated that Megasphaera elsdenii may serve as a potential probiotic for E. coli F17 infection. Our study provides an alternative challenge model for the identification of E. coli F17-sensitive/-resistant lambs and contributes to the basic understandings of intestinal microbiota in lambs with different susceptibilities to E. coli F17.

Previously, NCAPG was identified as a candidate gene associated with sheep growth traits. This study aimed to investigate the direct role of NCAPG in regulating myogenesis in embryonic myoblast cells and to investigate the association between single-nucleotide polymorphisms (SNPs) in its promoter region and sheep growth traits. The function of NCAPG in myoblast proliferation and differentiation was detected after small interfering RNAs (siRNAs) knocked down the expression of NCAPG. Cell proliferation was detected using CCK-8 assay, EdU proliferation assay, and flow cytometry cell cycle analysis. Cell differentiation was detected via cell immunofluorescence and the quantification of myogenic regulatory factors (MRFs). SNPs in the promoter region were detected using Sanger sequencing and genotyped using the improved multiplex ligation detection reaction (iMLDR^®) technique. As a result, a notable decrease (p < 0.01) in the percentage of EdU-positive cells in the siRNA-694-treated group was observed. A significant decrease (p < 0.01) in cell viability after treatment with siRNA-694 for 48 h and 72 h was detected using the CCK-8 method. The quantity of S-phase cells in the siRNA-694 treatment group was significantly decreased (p < 0.01). After interfering with NCAPG in myoblasts during induced differentiation, the relative expression levels of MRFs were markedly (p < 0.05 or p < 0.01) reduced compared with the control group on days 5–7. The myoblast differentiation in the siRNA-694 treatment group was obviously suppressed compared with the control group. SNP1, SNP2, SNP3, and SNP4 were significantly (p < 0.05) associated with all traits except body weight measured at birth and one month of age. SNP5 was significantly (p < 0.05) associated with body weight, body height, and body length in six-month-old sheep. In conclusion, interfering with NCAPG can inhibit the proliferation and differentiation of ovine embryonic myoblasts. SNPs in its promoter region can serve as potential useful markers for selecting sheep growth traits.

Escherichia coli (E. coli) F17 is one of the most common pathogens causing diarrhea in farm livestock. In the previous study, we accessed the transcriptomic and microbiomic profile of E. coli F17-antagonism (AN) and -sensitive (SE) lambs; however, the biological mechanism underlying E. coli F17 infection has not been fully elucidated. Therefore, the present study first analyzed the metabolite data obtained with UHPLC-MS/MS. A total of 1957 metabolites were profiled in the present study, and 11 differential metabolites were identified between E. coli F17 AN and SE lambs (i.e., FAHFAs and propionylcarnitine). Functional enrichment analyses showed that most of the identified metabolites were related to the lipid metabolism. Then, we presented a machine-learning approach (Random Forest) to integrate the microbiome, metabolome and transcriptome data, which identified subsets of potential biomarkers for E. coli F17 infection (i.e., GlcADG 18:0-18:2, ethylmalonic acid and FBLIM1); furthermore, the PCCs were calculated and the interaction network was constructed to gain insight into the crosstalk between the genes, metabolites and bacteria in E. coli F17 AN/SE lambs. By combing classic statistical approaches and a machine-learning approach, our results revealed subsets of metabolites, genes and bacteria that could be potentially developed as candidate biomarkers for E. coli F17 infection in lambs.