Aohan Fine-wool Sheep Research Articles

The role of miR-369-3p in proliferation and differentiation of preadipocytes in Aohan fine-wool sheep.

MicroRNAs (miRNAs) are a large class of non-coding RNAs that play important roles in the proliferation and differentiation of adipocytes. Our previous sequencing analysis revealed higher expression of miR-369-3p in the longissimus muscle of 2-month-old Aohan fine-wool sheep (AFWS) compared to 12-month-old sheep (), suggesting that miR-369-3p may regulate fat deposition in AFWS. To test this, miR-369-3p mimics, inhibitors, and negative controls (NCs) were constructed and transfected into AFWS preadipocytes. After transfection with miR-369-3p mimics, we found a decrease () in the expression of genes and proteins related to cell proliferation and differentiation, detected by RT-qPCR (quantitative reverse transcription PCR) and western blot analyses. Moreover, EdU (5-ethynyl-2-deoxyuridine) detection and Oil Red O staining showed a decrease () in cell proliferation and lipid accumulation, respectively. The opposite trends () were obtained after transfection with miR-369-3p inhibitors. In conclusion, the results showed that miR-369-3p can inhibit the proliferation and differentiation of AFWS preadipocytes, providing a theoretical basis to further explore the molecular mechanism of fat deposition in sheep and other domestic animals.

Characteristics of Bacterial Microbiota in Different Intestinal Segments of Aohan Fine-Wool Sheep.

The microbial community performs vital functions in the intestinal system of animals. Modulation of the gut microbiota structure can indirectly or directly affect gut health and host metabolism. Aohan fine-wool sheep grow in semi-desert grasslands in China and show excellent stress tolerance. In this study, we amplified 16S rRNA gene to investigate the dynamic distribution and adaptability of the gut microbiome in the duodenum, jejunum, ileum, cecum, colon, and rectum of seven Aohan fine-wool sheep at 12 months. The results showed that the microbial composition and diversity of the ileum and the large intestine (collectively termed the hindgut) were close together, and the genetic distance and functional projections between them were similar. Meanwhile, the diversity index results revealed that the bacterial richness and diversity of the hindgut were significantly higher than those of the foregut. We found that from the foregut to the hindgut, the dominant bacteria changed from Proteobacteria to Bacteroidetes. In LEfSe analysis, Succiniclasticum was found to be significantly abundant bacteria in the foregut and was involved in succinic acid metabolism. Ruminococcaceae and Caldicoprobacteraceae were significantly abundant in hindgut, which can degrade cellulose polysaccharides in the large intestine and produce beneficial metabolites. Moreover, Coriobacteriaceae and Eggthellaceae are involved in flavonoid metabolism and polyphenol production. Interestingly, these unique bacteria have not been reported in Mongolian sheep or other sheep breeds. Collectively, the gut microbiota of Aohan fine-wool sheep is one of the keys to adapting to the semi-desert grassland environment. Our results provide new insights into the role of gut microbiota in improving stress tolerance and gut health in sheep.

Expression and distribution of EPHA4 and EphrinA3 in Aohan fine-wool sheep skin.

The objective of this study was to identify the expression and distribution of EPHA4 and Ephrin A3 genes in the development and morphogenesis of hair follicles in fine-wool sheep. The results could lay a theoretical basis for understanding the molecular mechanism that regulates hair follicle development. The skin of Aohan fine-wool sheep at different developmental stages (embryonic day 90, E90d, and 120, E120d, and postnatal day 1, B1d, and 30, B30d) were selected. Real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry were used to study the levels of mRNA and proteins, respectively. The RT-qPCR results showed that the mRNA expression level of EPHA4 at B1d was significantly lower than at E120d (). The expression of Ephrin A3 at E120d was significantly higher than that at E90d and B1d (). Immunohistochemical detection results showed that the level and localisation of EPHA4 and Ephrin A3 proteins had spatial and temporal specificity. EPHA4 expression in dermal papilla cells might be important for inducing Aohan fine-hair follicle regeneration and for controlling the properties of the hair. Ephrin A3 might play an important role in the redifferentiation of secondary hair follicles and might also be involved in the inhibition of apoptosis-related gene expression in hair follicles. The Ephrin A3 signalling pathway might accelerate the growth of fine-hair follicles and increase the density of hair follicles.

Identification and Characterization of Circular RNAs in Association With the Deposition of Intramuscular Fat in Aohan Fine-Wool Sheep.

Aohan fine-wool sheep (AFWS) is a high-quality fine-wool sheep breed that supplies wool and meat. Research is needed on the molecular mechanism behind intramuscular fat (IMF) deposition that greatly improves mutton quality. The widely expressed non-coding RNA is physiologically used in roles such as competitive endogenous RNA (ceRNA) that includes circular RNAs (circRNAs). Although circRNAs were studied in many fields, little research was devoted to IMF in sheep. We used the longissimus dorsi muscle of 2 and 12-month-old AWFS as research material to identify circRNAs related to IMF deposition in these sheep by RNA-seq screening for differentially expressed circRNAs in the two age groups. A total of 11,565 candidate circRNAs were identified, of which the 104 differentially expressed circRNAs in the two age groups were analyzed. Enrichment analysis was performed using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. The enriched pathways included lipid transport (GO:0006869), negative regulation of canonical Wnt signaling pathway (GO:0090090), fat digestion and absorption (ko04975), and sphingolipid metabolism (ko00600). The differentially expressed circRNAs included ciRNA455, circRNA9086, circRNA7445, circRNA4557, and others. The source genes involved in these pathways might regulate IMF deposition. We used the TargetScan and miRanda software for interaction analysis, and a network diagram of circRNA-miRNA interactions was created. CircRNA455-miR-127, circRNA455-miR-29a, circRNA455-miR-103, circRNA4557-mir149-5p, and circRNA2440-mir-23a might be involved in the IMF deposition process. The targeting relationship of circRNA4557-miR-149-5p was verified by a dual-luciferase reporter assay. The RT-qPCR results of seven randomly selected circRNAs were consistent with the sequencing results. This study provides additional information on circRNA regulation of IMF deposition in AFWS and is a useful resource for future research on this sheep breed.

Whole-genome re-sequencing association study on yearling wool traits in Chinese fine-wool sheep

To investigate single nucleotide polymorphism (SNP) loci associated with yearling wool traits of fine-wool sheep for optimizing marker-assisted selection and dissection of the genetic architecture of wool traits, we conducted a genome-wide association study (GWAS) based on the fixed and random model circulating probability unification (FarmCPU) for yearling staple length (YSL), yearling mean fiber diameter (YFD), yearling greasy fleece weight (YGFW), and yearling clean fleece rate (YCFR) by using the whole-genome re-sequenced data (totaling 577 sheep) from the following four fine-wool sheep breeds in China: Alpine Merino sheep (AMS), Chinese Merino sheep (CMS), Qinghai fine-wool sheep (QHS), and Aohan fine-wool sheep (AHS). A total of 16 SNPs were detected above the genome-wise significant threshold (P = 5.45E-09), and 79 SNPs were located above the suggestive significance threshold (P = 5.00E-07) from the GWAS results. For YFD and YGFW traits, 7 and 9 SNPs reached the genome-wise significance thresholds, whereas 10 and 12 SNPs reached the suggestive significance threshold, respectively. For YSL and YCFR traits, none of the SNPs reached the genome-wise significance thresholds, whereas 57 SNPs exceeded the suggestive significance threshold. We recorded 14 genes located at the region of ±50-kb near the genome-wise significant SNPs and 59 genes located at the region of ±50-kb near the suggestive significant SNPs. Meanwhile, we used the Average Information Restricted Maximum likelihood algorithm (AI-REML) in the “HIBLUP” package to estimate the heritability and variance components of the four desired yearling wool traits. The estimated heritability values (h2) of YSL, YFD, YGFW, and YCFR were 0.6208, 0.7460, 0.6758, and 0.5559, respectively. We noted that the genetic parameters in this study can be used for fine-wool sheep breeding. The newly detected significant SNPs and the newly identified candidate genes in this study would enhance our understanding of yearling wool formation, and significant SNPs can be applied to genome selection in fine-wool sheep breeding.

Identification of miRNA in Sheep Intramuscular Fat and the Role of miR-193a-5p in Proliferation and Differentiation of 3T3-L1.

Intramuscular fat (IMF) is one of the most critical parameters affecting meat quality and mainly affected by genetic factors. MicroRNA as an important regulatory factor, which is still a lack of research in the development of sheep IMF deposition. We used RNA sequencing (RNA-seq) and cell-level validation to explore the role of miRNA in IMF deposition. As for this purpose, longissimus thoracis et lumborum (LTL) samples of 2 month-old (Mth-2) and 12 months-old (Mth-12) Aohan fine-wool sheep (AFWS) were used to identified miRNAs expression. We found 59 differentially expressed miRNAs (DE-miRNA) between these age groups and predicted their 1,796 target genes. KEGG functional enrichment analysis revealed eight pathways involved in lipid metabolism-related processes, including fatty acid elongation and the AMPK signaling pathway. A highly expressed DE-miRNA, miR-193a-5p, was found to serve a function in 3T3-L1 preadipocyte differentiation. Luciferase assay demonstrated that miR-193a-5p directly binds to the 3′-UTR region of ACAA2. By constructing mimics and inhibitor vector transfecting into 3T3-L1 cells to explore the effect of miR-193a-5p on cell proliferation and differentiation, we demonstrated that overexpression of miR-193a-5p inhibited 3T3-L1 preadipocyte proliferation, as evidenced by decreased mRNA and protein expression of CDK4 and CyclinB. CCK-8 assay showed that miR-193a-5p significantly inhibited cell proliferation. Similarly, the overexpression of miR-193a-5p inhibited 3T3-L1 preadipocyte differentiation and adipocyte-specific molecular markers’ expression, leading to a decrease in PPARγ and C/EBPα and ACAA2. Inhibition of miR-193a-5p had the opposite effects. Our study lists the miRNAs associated with intramuscular lipid deposition in sheep and their potential targets, striving to improve sheep meat quality.

Genome-wide association studies detects candidate genes for wool traits by re-sequencing in Chinese fine-wool sheep

BackgroundThe quality and yield of wool determine the economic value of the fine-wool sheep. Therefore, discovering markers or genes relevant to wool traits is the cornerstone for the breeding of fine-wool sheep. In this study, we used the Illumina HiSeq X Ten platform to re-sequence 460 sheep belonging to four different fine-wool sheep breeds, namely, Alpine Merino sheep (AMS), Chinese Merino sheep (CMS), Aohan fine-wool sheep (AHS) and Qinghai fine-wool sheep (QHS). Eight wool traits, including fiber diameter (FD), fiber diameter coefficient of variance (FDCV), fiber diameter standard deviation (FDSD), staple length (SL), greasy fleece weight (GFW), clean wool rate (CWR), staple strength (SS) and staple elongation (SE) were examined. A genome-wide association study (GWAS) was performed to detect the candidate genes for the eight wool traits.ResultsA total of 8.222 Tb of raw data was generated, with an average of approximately 8.59X sequencing depth. After quality control, 12,561,225 SNPs were available for analysis. And a total of 57 genome-wide significant SNPs and 30 candidate genes were detected for the desired wool traits. Among them, 7 SNPs and 6 genes are related to wool fineness indicators (FD, FDCV and FDSD), 10 SNPs and 7 genes are related to staple length, 13 SNPs and 7 genes are related to wool production indicators (GFW and CWR), 27 SNPs and 10 genes associated with staple elongation. Among these candidate genes, UBE2E3 and RHPN2 associated with fiber diameter, were found to play an important role in keratinocyte differentiation and cell proliferation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results, revealed that multitude significant pathways are related to keratin and cell proliferation and differentiation, such as positive regulation of canonical Wnt signaling pathway (GO:0090263).ConclusionThis is the first GWAS on the wool traits by using re-sequencing data in Chinese fine-wool sheep. The newly detected significant SNPs in this study can be used in genome-selective breeding for the fine-wool sheep. And the new candidate genes would provide a good theoretical basis for the fine-wool sheep breeding.

Identification and co-expression analysis of long noncoding RNAs and mRNAs involved in the deposition of intramuscular fat in Aohan fine-wool sheep

BackgroundIntramuscular fat (IMF) content has become one of the most important indicators for measuring meat quality, and levels of IMF are affected by various genes. Long non-coding RNAs (lncRNAs) are widely expressed non-coding RNAs that play an important regulatory role in a variety of biological processes; however, research on the lncRNAs involved in sheep IMF deposition is still in its infancy. Aohan fine-wool sheep (AFWS), one of China’s most important meat-hair, dual-purpose sheep breed, provides a great model for studying the role of lncRNAs in the regulation of IMF deposition. We identified lncRNAs by RNA sequencing in Longissimus thoracis et lumborum (LTL) samples of sheep at two ages: 2 months (Mth-2) and 12 months (Mth-12).ResultsWe identified a total of 26,247 genes and 6935 novel lncRNAs in LTL samples of sheep. Among these, 199 mRNAs and 61 lncRNAs were differentially expressed. We then compared the structural characteristics of lncRNAs and mRNAs. We obtained target genes of differentially expressed lncRNAs (DELs) and performed enrichment analyses using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). We found that target mRNAs were enriched in metabolic processes and developmental pathways. One pathway was significantly enriched, namely tight junction. Based on the analysis of critical target genes, we obtained seven candidate lncRNAs that potentially regulated lipid deposition and constructed a lncRNA-mRNA co-expression network that included MSTRG.4051.3-FZD4, MSTRG.16157.3-ULK1, MSTRG.21053.3-PAQR3, MSTRG.19941.2-TPI1, MSTRG.12864.1-FHL1, MSTRG.2469.2-EXOC6 and MSTRG.21381.1-NCOA1. We speculated that these candidate lncRNAs might play a role by regulating the expression of target genes. We randomly selected five mRNAs and five lncRNAs to verify the accuracy of the sequencing data by qRT-PCR.ConclusionsOur study identified the differentially expressed mRNAs and lncRNAs during intramuscular lipid deposition in Aohan fine-wool sheep. The work may widen the knowledge about the annotation of the sheep genome and provide a working basis for investigating intramuscular fat deposition in sheep.

Transcriptomic Analysis Reveals the Involvement of lncRNA-miRNA-mRNA Networks in Hair Follicle Induction in Aohan Fine Wool Sheep Skin.

Long non-coding RNAs (lncRNA) and microRNAs (miRNA) are new found classes of non-coding RNAs (ncRNAs) that are not translated into proteins but regulate various cellular and biological processes. In this study, we conducted a transcriptomic analysis of ncRNA and mRNA expression in Aohan fine wool sheep (AFWS) at different growth stages (embryonic day 90, embryonic day 120, and the day of birth), and explored their relationship with wool follicle growth. In total, 461 lncRNAs, 106 miRNAs, and 1,009 mRNAs were found to be differentially expressed during the three stages of wool follicle development. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to clarify the roles of the differentially expressed lncRNA, miRNA, and mRNA in the different stages of wool follicle development. Quantitative real-time PCR (qRT-PCR) was used to validate the results of RNA-seq analysis. lncRNA (MSTRG.223165) was found to act as a competing endogenous RNA (ceRNA) and may participate in wool follicle development by acting as an miR-21 sponge. Network prediction implicated the MSTRG.223165-miR-21-SOX6 axis in the wool follicle development. The targeting relationships of miR-21 with SOX6 and MSTRG.223165 were validated in dual-luciferase assays. This is the first report indicating the association of the lncRNA–miRNA–mRNA network with wool follicle development in AFWS. This study provides new insights into the regulation of the wool follicle growth and represents a solid foundation for wool sheep breeding programs.

Molecular cloning and characterization of the β-catenin gene from fine-wool sheep.

β-Catenin is an evolutionarily conserved molecule that functions as a crucial effector in both cell-to-cell adhesion and Wnt signaling. To gain a better understanding of its role in the development of hair follicles, we cloned the cDNA sequence of the β-catenin gene from the skin of Aohan fine-wool sheep and performed a variety of bioinformatics analyses. We obtained the full-length sequence, which was 4573-bp long and contained a 2346-bp open reading frame encoding a protein of 781 amino acids. The protein had a predicted molecular weight of 85.4 kDa and a theoretical isoelectric point of 5.57. Domain architecture analysis of the β-catenin protein revealed an armadillo repeat region, which is a common feature of β-catenin in other species. The ovine β-catenin gene shares 97.91%, 94.25%, 94.59%, 83.89%, and 89.39% sequence identity with its homologs in Bos taurus, Homo sapiens, Sus scrofa, Gallus gallus, and Mus musculus, respectively, while the amino acid sequence is more than 99% identical with each of these species. The expression of β-catenin mRNA was detected in the heart, liver, spleen, lung, kidney, skin, muscle, and adipose tissue. Expression levels were maximal in the lung and minimal in the muscle, and the difference in expression in these tissues was significant (P<0.01). Western blot analysis revealed the presence of the β-catenin protein in all tissues examined; expression was lowest in the skin and adipose tissues.

Effects of dietary vitamin E on muscle vitamin E and fatty acid content in Aohan fine-wool sheep.

BackgroundIncreasing the polyunsaturated fatty acid (PUFA) content and decreasing the saturated fatty acid (SFA) content of mutton can help to improve its nutritional value for consumers. Several laboratories have evaluated the effects of vitamin E on the fatty acid (FA) composition of muscle in sheep. However, little information is available on wool sheep, even though wool sheep breeds are an important source of mutton, especially in northern China where sheep are extensively farmed. The present study was designed to address the effects of vitamin E on muscle FA composition in male Aohan fine-wool sheep.MethodsForty-two male Aohan fine-wool lambs (5 mo old) with similar initial body weight were randomly divided into seven groups and fed diets supplemented with 0 (control group), 20, 100, 200, 1,000, 2,000, or 2,400 IU/sheep/d vitamin E for 12 mo. Three lambs from each group were slaughtered to measure vitamin E and FA content in the longissimus lumborum (LL) and gluteus medius (GM) muscles.ResultsVitamin E concentrations in the LL and GM increased significantly after 12 mo of vitamin E supplementation (P < 0.05). However, this increase did not occur in a dose-dependent manner because the muscle vitamin E concentration was highest in the 200 IU/sheep/d group. Dietary vitamin E supplementation also caused a significant reduction in SFA content and an increase in monounsaturated FA (MUFA) content in the LL and GM (P < 0.05). All six doses of vitamin E significantly increased cis9 trans11-conjugated linoleic acid (c9t11-CLA) content in the LL compared with the control group (P < 0.05).ConclusionsDietary supplementation with vitamin E increased muscle vitamin E content and improved the nutritional value of mutton by decreasing SFA content and increasing MUFA and c9t11-CLA contents in Aohan fine-wool sheep. These effects were greatest in sheep fed a diet containing 200 IU/sheep/d vitamin E.

Effect of Vitamin E supplementation on the enzymatic activity of selected markers in Aohan fine-wool sheep testis

The aim of this study was to evaluate the effects of dietary Vitamin E supplementation on the testicular ‘marker’ enzyme activity and Vitamin E content in Aohan fine-wool sheep. Thirty male Aohan fine-wool sheep (5 months of age) with similar body weight were selected from the Aohan fine-wool sheep-breeding farm of Inner Mongolia Autonomous Region, China. The sheep were randomly divided into five groups and supplemented with 0, 20, 200, 1000 or 2400 IU sheep −1 d −1 Vitamin E for 12 months. Three sheep in each group were slaughtered at 17 months to collect a testis sample for testicular marker enzyme analysis. The results showed that, compared to Control, supplementing the diet with Vitamin E at 200 IU sheep −1 d −1 significantly increased the content of Vitamin E in testis and improved the activity of testicular mitochondrial ATPase ( P < 0.01), lactate dehydrogenase (LDH) ( P < 0.01), sorbitol dehydrogenase (SDH) ( P < 0.01), and alkaline phosphatase (ALP) ( P < 0.05). The present study demonstrated that supplementing Vitamin E can have a positive role in improving testicular marker enzyme activity and that the optimum range of dose appeared to be 100–200 IU sheep −1 d −1.

Effect of Vitamin E supplementation on semen quality and the testicular cell membranal and mitochondrial antioxidant abilities in Aohan fine-wool sheep

Thirty male Aohan fine-wool sheep (5 months of age) with similar body weight were procured from the Aohan fine-wool sheep breeding farm of Inner Mongolia Autonomous Region, China. The sheep were divided randomly into five groups, which were labeled as Group 1, 2, 3, 4 and 5 and supplemented respectively with 0, 20, 200, 1000 or 2400 IU sheep −1 d −1 Vitamin E, for 12 months, respectively. Three Aohan fine-wool sheep in each group were selected randomly for semen collection at the age of 16 months, then slaughtered at 17 months to collect the testis sample for testicular cell membranal and mitochondrial antioxidant abilities analysis. The results showed that supplementing Vitamin E at the concentration of 200 IU sheep −1 d −1 in diets may have a positive effect in increasing semen quality and quantity ( P < 0.05), significantly reduce malondialdehyde (MDA) level and improve the activities of superoxide dismutase (SOD)and glutathione peroxidase (GSH-PX) in testicular cell membrane and mitochondria ( P < 0.05). In conclusion, the present study demonstrated that that supplementing Vitamin E can have a positive role in improving semen quality via protecting testicular cell membrane and mitochondria from antioxidant abilities. However, the optimal level of Vitamin supplement has still to be determined.