A conjoint analysis of renal structure and omics characteristics reveal new insight to yak high-altitude hypoxia adaptation.

BACKGROUND: Yaks have unique adaptive mechanisms to the hypoxic environment, in which the kidney plays an important role. The aim of this study was to explore the histological changes of yak kidney at different altitudes and the metabolites and genes associated with adaptation to the hypoxic environment. METHODS: We analyzed the tissue structure and transcriptomic metabolomic data of yak kidney tissue at two altitudes, 2600 and 4400 m. We compared and identified the morphological adaptations of the kidney and the metabolites and genes associated with hypoxia adaptation in yaks. Changes in renal morphological adaptations, differential metabolites and genes were compared and identified, combining the two in a joint analysis. RESULTS: High-altitude yak kidneys showed significant adaptive changes: increased mitochondria, increased glomerular thylakoid area, and decreased localized ribosomes. Transcriptomics and metabolomics identified 69 DAMs (Differential metabolites) and 594 DEGs (differential genes). Functional enrichment analysis showed that the DAMs were associated with protein digestion and absorption, ABC transporter, and MTOR signaling pathway; the DEGs were significantly enriched in Cholesterol metabolism and P53 signaling pathway. The joint analysis indicated that metabolites such as lysine and arginine, as well as key genes such as ABCB5 and COL1A2, were particularly affected under hypoxic conditions, whereas changes in mitochondria in the tissue structure may be related to the expression of MFN1 and OPA1, and changes in glomerular thylakoid membranes are related to VEGFA and TGFB3. CONCLUSION: The kidney regulates metabolites and gene expression related to hormone synthesis, protein metabolism, and angiogenesis by adjusting the mitochondrial and glomerular thylakoid membrane structure to support the survival of yaks in high-altitude environments.

Circular RNA_015343 sponges microRNA-25 to regulate viability, proliferation, and milk fat synthesis of ovine mammary epithelial cells via INSIG1.

In our previous study, circ_015343 was found to inhibit the viability and proliferation of ovine mammary epithelial cells (OMECs) and the expression levels of milk fat synthesis marker genes, but the regulatory mechanism underlying the processes is still unclear. Accordingly in this study, the target relationships between circ_015343 with miR-25 and between miR-25 with insulin induced gene 1 (INSIG1) were verified, and the functions of miR-25 and INSIG1 were investigated in OMECs. The dual-luciferase reporter assay revealed that miR-25 mimic remarkably decreased the luciferase activity of circ_015343 in HEK293T cells cotransfected with a wild-type vector, while it did not change the activity of circ_015343 in HEK293T cells cotransfected with a mutant vector. These suggest that cic_015343 can adsorb and bind miR-25. The miR-25 increased the viability and proliferation of OMECs, and the content of triglycerides in OMECs. In addition, INSIG1 was found to be a target gene of miR-25 using a dual-luciferase reporter assay. Overexpression of INSIG1 decreased the viability, proliferation, and level of triglycerides of OMECs. In contrast, the inhibition of INSIG1 in expression had the opposite effect on activities and triglycerides of OMECs with overexpressed INSIG1. A rescue experiment revealed that circ_015343 alleviated the inhibitory effect of miR-25 on the mRNA and protein abundance of INSIG1. These results indicate that circ_015343 sponges miR-25 to inhibit the activities and content of triglycerides of OMECs by upregulating the expression of INSIG1 in OMECs. This study provided new insights for understanding the genetic molecular mechanism of lactation traits in sheep.

Mutations in the FOXO3 Gene and Their Effects on Meat Traits in Gannan Yaks.

The FOXO3 gene, a prominent member of the FOXO family, has been identified as a potential quantitative trait locus for muscle atrophy and lipid metabolism in livestock. It is also considered a promising candidate gene for meat quality traits such as Warner-Bratzler shear force (WBSF) and water holding capacity (WHC). The aim of this study was to identify sequence mutations in the FOXO3 gene of yaks and to analyze the association of genotypes and haplotypes with meat traits such as WBSF and WHC. Quantitative reverse-transcriptase PCR (RT-qPCR) was applied to determine the expression levels of FOXO3 in yak tissues, with the results revealing a high expression in the yak longissimus dorsi muscle. Exons of the FOXO3 gene were then sequenced in 572 yaks using hybrid pool sequencing. Five single nucleotide polymorphisms were identified. Additionally, four effective haplotypes and four combined haplotypes were constructed. Two mutations of the FOXO3 gene, namely C>G at exon g.636 and A>G at exon g.1296, were associated with cooked meat percentage (CMP) (p < 0.05) and WBSF (p < 0.05), respectively. Furthermore, the WBSF of the H2H3 haplotype combination was significantly lower than that of other combinations (p < 0.05). The findings of this study suggest that genetic variations in FOXO3 could be a promising biomarker for improving yak meat traits.

Molecular Characteristics of JAK2 and Its Effect on the Milk Fat and Casein Synthesis of Ovine Mammary Epithelial Cells.

In addition to its association with milk protein synthesis via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, JAK2 also affects milk fat synthesis. However, to date, there have been no reports on the effect of JAK2 on ovine mammary epithelial cells (OMECs), which directly determine milk yield and milk contents. In this study, the coding sequence (CDS) region of ovine JAK2 was cloned and identified and its tissue expression and localization in ovine mammary glands, as well as its effects on the viability, proliferation, and milk fat and casein levels of OMECs, were also investigated. The CDS region of ovine JAK2, 3399 bp in length, was cloned and its authenticity was validated by analyzing its sequence similarity with JAK2 sequences from other animal species using a phylogenetic tree. JAK2 was found to be expressed in six ovine tissues, with the highest expression being in the mammary gland. Over-expressed JAK2 and three groups of JAK2 interference sequences were successfully transfected into OMECs identified by immunofluorescence staining. When compared with the negative control (NC) group, the viability of OMECs was increased by 90.1% in the pcDNA3.1-JAK2 group. The over-expression of JAK2 also increased the number and ratio of EdU-labeled positive OMECs, as well as the expression levels of three cell proliferation marker genes. These findings show that JAK2 promotes the viability and proliferation of OMECs. Meanwhile, the triglyceride content in the over-expressed JAK2 group was 2.9-fold higher than the controls and the expression levels of four milk fat synthesis marker genes were also increased. These results indicate that JAK2 promotes milk fat synthesis. Over-expressed JAK2 significantly up-regulated the expression levels of casein alpha s2 (CSN1S2), casein beta (CSN2), and casein kappa (CSN3) but down-regulated casein alpha s1 (CSN1S1) expression. In contrast, small interfered JAK2 had the opposite effect to JAK2 over-expression on the viability, proliferation, and milk fat and milk protein synthesis of OMECs. In summary, these results demonstrate that JAK2 promotes the viability, proliferation, and milk fat synthesis of OMECs in addition to regulating casein expression in these cells. This study contributes to a better comprehension of the role of JAK2 in the lactation performance of sheep.

Integrative ATAC-seq and RNA-seq Analysis of the Longissimus Dorsi Muscle of Gannan Yak and Jeryak.

Jeryak is the F1 generation of the cross between Gannan yak and Jersey cattle, which has the advantages of fast growth and high adaptability. The growth and development of skeletal muscle is closely linked to meat production and the quality of meat. However, the molecular regulatory mechanisms of muscle growth differences between Gannan yak and Jeryak analyzed from the perspective of chromatin opening have not been reported. In this study, ATAC-seq was used to analyze the difference of chromatin openness in longissimus muscle of Gannan yak and Jeryak. It was found that chromatin accessibility was more enriched in Jeryak compared to Gannan yak, especially in the range of the transcription start site (TSS) ± 2 kb. GO and KEGG enrichment analysis indicate that differential peak-associated genes are involved in the negative regulation of muscle adaptation and the Hippo signaling pathway. Integration analysis of ATAC-seq and RNA-seq revealed overlapping genes were significantly enriched during skeletal muscle cell differentiation and muscle organ morphogenesis. At the same time, we screened FOXO1, ZBED6, CRY2 and CFL2 for possible involvement in skeletal muscle development, constructed a genes and transcription factors network map, and found that some transcription factors (TFs), including YY1, KLF4, KLF5 and Bach1, were involved in skeletal muscle development. Overall, we have gained a comprehensive understanding of the key factors that impact skeletal muscle development in various breeds of cattle, providing new insights for future analysis of the molecular regulatory mechanisms involved in muscle growth and development.

MicroRNA-148a Targets DNMT1 and PPARGC1A to Regulate the Viability, Proliferation, and Milk Fat Synthesis of Ovine Mammary Epithelial Cells.

In this study, the expression profiles of miR-148a were constructed in eight different ovine tissues, including mammary gland tissue, during six different developmental periods. The effect of miR-148a on the viability, proliferation, and milk fat synthesis of ovine mammary epithelial cells (OMECs) was investigated, and the target relationship of miR-148a with two predicted target genes was verified. The expression of miR-148a exhibited obvious tissue-specific and temporal-specific patterns. miR-148a was expressed in all eight ovine tissues investigated, with the highest expression level in mammary gland tissue (p < 0.05). Additionally, miR-148a was expressed in ovine mammary gland tissue during each of the six developmental periods studied, with its highest level at peak lactation (p < 0.05). The overexpression of miR-148a increased the viability of OMECs, the number and percentage of Edu-labeled positive OMECs, and the expression levels of two cell-proliferation marker genes. miR-148a also increased the percentage of OMECs in the S phase. In contrast, transfection with an miR-148a inhibitor produced the opposite effect compared to the miR-148a mimic. These results indicate that miR-148a promotes the viability and proliferation of OMECs in Small-tailed Han sheep. The miR-148a mimic increased the triglyceride content by 37.78% (p < 0.01) and the expression levels of three milk fat synthesis marker genes in OMECs. However, the miR-148a inhibitor reduced the triglyceride level by 87.11% (p < 0.01). These results suggest that miR-148a promotes milk fat synthesis in OMECs. The dual-luciferase reporter assay showed that miR-148a reduced the luciferase activities of DNA methyltransferase 1 (DNMT1) and peroxisome proliferator-activated receptor gamma coactivator 1-A (PPARGC1A) in wild-type vectors, suggesting that they are target genes of miR-148a. The expression of miR-148a was highly negatively correlated with PPARGC1A (r = -0.789, p < 0.001) in ovine mammary gland tissue, while it had a moderate negative correlation with DNMT1 (r = -0.515, p = 0.029). This is the first study to reveal the molecular mechanisms of miR-148a underlying the viability, proliferation, and milk fat synthesis of OMECs in sheep.

Unlocking the Transcriptional Control of NCAPG in Bovine Myoblasts: CREB1 and MYOD1 as Key Players.

Muscle formation directly determines meat production and quality. The non-SMC condensin I complex subunit G (NCAPG) is strongly linked to the growth features of domestic animals because it is essential in controlling muscle growth and development. This study aims to elucidate the tissue expression level of the bovine NCAPG gene, and determine the key transcription factors for regulating the bovine NCAPG gene. In this study, we observed that the bovine NCAPG gene exhibited high expression levels in longissimus dorsi and spleen tissues. Subsequently, we cloned and characterized the promoter region of the bovine NCAPG gene, consisting of a 2039 bp sequence, through constructing the deletion fragment double-luciferase reporter vector and site-directed mutation-identifying core promoter region with its key transcription factor binding site. In addition, the key transcription factors of the core promoter sequence of the bovine NCAPG gene were analyzed and predicted using online software. Furthermore, by integrating overexpression experiments and the electrophoretic mobility shift assay (EMSA), we have shown that cAMP response element binding protein 1 (CREB1) and myogenic differentiation 1 (MYOD1) bind to the core promoter region (-598/+87), activating transcription activity in the bovine NCAPG gene. In conclusion, these findings shed important light on the regulatory network mechanism that underlies the expression of the NCAPG gene throughout the development of the muscles in beef cattle.

Variation in Acetyl-CoA Carboxylase Beta Gene and Its Effect on Carcass and Meat Traits in Gannan Yaks.

Acetyl-CoA carboxylase beta (ACACB) is a functional candidate gene that impacts fat deposition. In the present study, we sequenced exon 37-intron 37, exon 46-intron 46, and intron 47 of yak ACACB using hybrid pool sequencing to search for variants and genotyped the gene in 593 Gannan yaks via Kompetitive allele-specific polymerase chain (KASP) reaction to determine the effect of ACACB variants on carcass and meat quality traits. Seven single nucleotide polymorphisms were detected in three regions. Eight effective haplotypes and ten diplotypes were constructed. Among them, a missense variation g.50421 A > G was identified in exon 37 of ACACB, resulting in an amino acid shift from serine to glycine. Correlation analysis revealed that this variation was associated with the cooking loss rate and yak carcass weight (p = 0.024 and 0.012, respectively). The presence of haplotypes H5 and H6 decreased Warner-Bratzler shear force (p = 0.049 and 0.006, respectively), whereas that of haplotypes H3 and H4 increased cooking loss rate and eye muscle area (p = 0.004 and 0.034, respectively). Moreover, the presence of haplotype H8 decreased the drip loss rate (p = 0.019). The presence of one and two copies of haplotypes H1 and H8 decreased the drip loss rate (p = 0.028 and 0.004, respectively). However, haplotype H1 did not decrease hot carcass weight (p = 0.011), whereas H3 increased the cooking loss rate (p = 0.007). The presence of one and two copies of haplotype H6 decreased Warner-Bratzler shear force (p = 0.014). The findings of the present study suggest that genetic variations in ACACB can be a preferable biomarker for improving yak meat quality.

Transcriptome Analysis of mRNA and lncRNA Related to Muscle Growth and Development in Gannan Yak and Jeryak.

The production performance of Jeryak, resulting from the F1 generation of the cross between Gannan yak and Jersey cattle, exhibits a significantly superior outcome compared with that of Gannan yak. Therefore, we used an RNA-seq approach to identify differentially expressed mRNAs (DEMs) and differentially expressed lncRNAs (DELs) influencing muscle growth and development in Gannan yaks and Jeryaks. A total of 304 differentially expressed lncRNAs and 1819 differentially expressed mRNAs were identified based on the screening criteria of |log 2 FC| > 1 and FDR < 0.05. Among these, 132 lncRNAs and 1081 mRNAs were found to be down-regulated, while 172 lncRNAs and 738 mRNAs were up-regulated. GO and KEGG analyses showed that the identified DELs and DEMs were enriched in the entries of pathways associated with muscle growth and development. On this basis, we constructed an lncRNA-mRNA interaction network. Interestingly, two candidate DELs (MSTRG.16260.9 and MSTRG.22127.1) had targeting relationships with 16 (MYC, IGFBP5, IGFBP2, MYH4, FGF6, etc.) genes related to muscle growth and development. These results could provide a basis for further studies on the roles of lncRNAs and mRNAs in muscle growth in Gannan yaks and Jeryak breeds.

Integration of ATAC-Seq and RNA-Seq Analysis to Identify Key Genes in the Longissimus Dorsi Muscle Development of the Tianzhu White Yak.

During the postnatal stages, skeletal muscle development undergoes a series of meticulously regulated alterations in gene expression. However, limited studies have employed chromatin accessibility to unravel the underlying molecular mechanisms governing muscle development in yak species. Therefore, we conducted an analysis of both gene expression levels and chromatin accessibility to comprehensively characterize the dynamic genome-wide chromatin accessibility during muscle growth and development in the Tianzhu white yak, thereby elucidating the features of accessible chromatin regions throughout this process. Initially, we compared the differences in chromatin accessibility between two groups and observed that calves exhibited higher levels of chromatin accessibility compared to adult cattle, particularly within ±2 kb of the transcription start site (TSS). In order to investigate the correlation between alterations in chromatin accessible regions and variations in gene expression levels, we employed a combination of ATAC-seq and RNA-seq techniques, leading to the identification of 18 central transcriptional factors (TFs) and 110 key genes with significant effects. Through further analysis, we successfully identified several TFs, including Sp1, YY1, MyoG, MEF2A and MEF2C, as well as a number of candidate genes (ANKRD2, ANKRD1, BTG2 and LMOD3) which may be closely associated with muscle growth and development. Moreover, we constructed an interactive network program encompassing hub TFs and key genes related to muscle growth and development. This innovative approach provided valuable insights into the molecular mechanism underlying skeletal muscle development in the postnatal stages of Tianzhu white yaks while also establishing a solid theoretical foundation for future research on yak muscle development.

Characterization of the promoter region of bovine ATP5B: roles of MyoD and GATA1 in the regulation of basal transcription.

Intramuscular fat (IMF) content is a key determinant of beef quality, making it a key topic of research interest. ATP5B serves as the catalytic component of the mitochondrial ATP synthase complex and plays essential roles in controlling fat contents and oxidative metabolism in bovine skeletal muscle. In this study, we determined that bovine ATP5B was highly expressed in longissimus thoracis. To elucidate the molecular mechanisms involved in bovine ATP5B regulation, we cloned and characterized the promoter region of ATP5B. Applying 5'-rapid amplification of cDNA end analysis (RACE), we identified two transcriptional start sites (TSSs) in its promoter region. Using a series of 5'-deletion promoter plasmids in luciferase reporter assay, we found that the proximal minimal promoter of ATP5B was located within the region -539/220 relative to the TSS. Site-directed mutation in combination with chromatin immunoprecipitation (ChIP) assays demonstrated that MyoD and GATA1 binding to the promoter region drives bovine ATP5B transcription. Taken together, these results provide new insight into the regulatory mechanisms of ATP5B transcription in mediating the IMF content of beef.

Interference with ACSL1 gene in bovine adipocytes: Transcriptome profiling of circRNA related to unsaturated fatty acid production.

Long-chain acyl-CoA synthetase 1 (ACSL1) is a member of the acyl-CoA synthetase family that plays a vital role in lipid metabolism. We have previously shown that the ACSL1 gene regulates the composition of unsaturated fatty acids (UFAs) in bovine skeletal muscle, which in turn regulates the fatty acid synthesis and the generation of lipid droplets. Here, we used RNA-Seq to screen circRNAs that regulated the expression of ACSL1 gene and other UFA synthesis-related genes by RNA interference and noninterference in bovine adipocytes. The results of KEGG pathway analysis showed that the parental genes of differentially expressed (DE)-circRNAs were primarily enriched in the adipocytokine signaling pathway. The prediction results showed that novel_circ_0004855, novel_circ_0001507, novel_circ_0001731, novel_circ_0005276, novel_circ_0002060, novel_circ_0005405 and novel_circ_0004254 regulated UFA synthesis-related genes by interacting with the related miRNAs. These results could help expand our knowledge of the molecular mechanisms of circRNAs in the regulation of UFA synthesis in bovine adipocytes.

Rumen Epithelial Development- and Metabolism-Related Genes Regulate Their Micromorphology and VFAs Mediating Plateau Adaptability at Different Ages in Tibetan Sheep.

The rumen is an important hallmark organ of ruminants and plays an important role in the metabolism and immune barrier of Tibetan sheep on the Plateau. However, there are few studies on rumen development and metabolism regulation in Tibetan sheep at different ages. Here, we comprehensively analyzed the immune function, fermentation function, rumen epithelial micromorphology and transcriptome profile of Tibetan sheep at different ages. The results showed that the concentration of IgG decreased and the concentration of IgM increased with age (p < 0.05), and the highest concentration of IgA was observed at 1.5 and 3.5 years of age. In terms of rumen fermentation characteristics, VFAs of 4-month-old lambs were the highest, followed by VFAs and NH3-N of Tibetan sheep at 3.5 years of age. Hematoxylin-eosin staining and transmission electron microscopy section examination of rumen epithelial tissue showed that the rumen papilla width increased with age (p < 0.001), the thickness of the stratum corneum decreased, the cells in the stratum corneum showed accelerated migration and the thickness of the rumen muscle layer increased (p < 0.001). Desmosomal junctions between the layers of rumen epithelium increased at 1.5 and 3.5 years old, forming a compact barrier structure, and the basal layer had more mitochondria involved in the regulation of energy metabolism. RNA-seq analysis revealed that a total of 1006 differentially expressed genes (DEGs) were identified at four ages. The DEGs of Tibetan sheep aged 4 months and 6 years were mainly enriched in the oxidation−reduction process and ISG15-protein conjugation pathway. The 1.5 and 3.5-year-olds were mainly enriched in skeletal muscle thin filament assembly, mesenchyme migration and the tight junction pathway. WGCNA showed that DEGs related to rumen microbiota metabolite VFAs and epithelial morphology were enriched in "Metabolism of xenobiotics by cytochrome P450, PPAR signaling pathway, Butanoate metabolism pathways" and participated in the regulation of rumen epithelial immune and fermentation metabolism functions of Tibetan sheep at different ages. This study systematically revealed the regulatory mechanism of rumen epithelial development and metabolism in the plateau adaptation of Tibetan sheep, providing a new approach for the study of plateau adaptation.

MicroRNA-200c Affects Milk Fat Synthesis by Targeting PANK3 in Ovine Mammary Epithelial Cells.

Milk fat is the foremost nutrient of milk and a vital indicator in evaluating milk quality. Accumulating evidence suggests that microRNAs (miRNAs) are involved in the synthesis of milk fat. The miR-200c is closely related to lipid metabolism, but little is known about its effect on the synthesis of milk fat in MECs of ewes. Herein, the effect of miR-200c on the proliferation of ovine mammary epithelial cells (MECs) and its target relationship with a predicted target gene were investigated. The regulatory effects of miR-200c on the expression of the target genes and the content of triglycerides in ovine MECs were further analyzed. The results revealed that the expression level of miR-200c was differentially expressed in both eight tissues selected during lactation and in mammary gland tissues at different physiological periods. Overexpression of miR-200c inhibited the viability and proliferation of ovine MECs, while inhibition of miR-200c increased cell viability and promoted the proliferation of ovine MECs. Target gene prediction results indicated that miR-200c would bind the 3'UTR region of pantothenate kinase 3 (PANK3). Overexpression of miR-200c reduced the luciferase activity of PANK3, while inhibition of miR-200c increased its luciferase activity. These findings illustrated that miR-200c could directly interact with the target site of the PANK3. It was further found that overexpression of miR-200c reduced the expression levels of PANK3 and, thus, accelerated the synthesis of triglycerides. In contrary, the inhibitor of miR-200c promoted the expression of PANK3 that, thus, inhibited the synthesis of triglycerides in ovine MECs. Together, these findings revealed that miR-200c promotes the triglycerides synthesis in ovine MECs via increasing the lipid synthesis related genes expression by targeting PANK3.

Sequence and haplotypes of ankyrin 1 gene (ANK1) and their association with carcass and meat quality traits in yak.

Ankyrin 1 (ANK1) gene has been demonstrated to be a functional candidate gene for meat quality that helps to constitute and maintain the structure of the cell skeleton. In this study, three contiguous ANK1 regions from yak were analyzed using polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP). As a result, nine single-nucleotide polymorphisms (SNPs) were identified, four of them in the coding region and three (c.179 C/A, c.250 G/C, and c.313 C/T) putatively resulting in amino acid changes (p. Ala 60 Glu, p. Asp 84 His, and p. Pro 105 Ser). Some SNPs in promoter region were located within or nearby the putative transcription factor binding sites, such as Sp1 and GATA, which might have an impact on the expression of the yak ANK1 gene. The presence of C1-D3 and C1-A3 were associated with an increased hot carcass weight (p = 0.0045) and a decreased drip loss rate (p = 0.0046). The presence of B1-B3, C1-A3 and C1-D3 had decreased Warner-Bratzler shear force (p = 0.0066, p = 0.0343 and p = 0.0004). The presence of one and two copies of B1-B3 and C1-A3 had decreased Warner-Bratzler shear force (p = 0.0005 and p = 0.0443), and C1-A3 had also decreased drip loss rate (p = 0.0164). These findings indicated that genetic variations of the ANK1 gene would be a preferable biomarker for the improvement of yak meat quality.

Effects of overexpression of ACSL1 gene on the synthesis of unsaturated fatty acids in adipocytes of bovine.

There exists a positive correlation between the unsaturated fatty acids (UFA) content in the bovine species and their taste and nutritional significance. Long-chain acyl-CoA synthetase 1 (ACSL1) is known to be involved in lipid synthesis as well as fatty acid transport and degradation. This gene has been identified as the key candidate gene for regulating lipid composition in the bovine skeletal muscle; however, its mechanism of action in regulating UFA synthesis in bovine adipocytes is unclear. In this study, we used a recombinant adenovirus vector (Ad-ACSL1) to overexpress the ACSL1 gene using Ad-NC (recombinant adenovirus of green fluorescent protein) as the control. Quantitative real-time PCR (qRT-PCR) was done to examine the gene expression associated with the synthesis of UFA, followed by the analysis of the fatty acid composition. Oil red O staining was done to examine the aggregation of lipid droplets. We found that ACSL1 overexpression was associated with an upregulated expression of PPARγ, FABP3, ACLY, SCD1, and FASN, and downregulated expression of CPT1A. Additionally, ACSL1 overexpression resulted in elevated saturated fatty acid content, especially C16:0 and C18:0, than the control group (Ad-NC cells) (p < 0.05). Furthermore, the overexpression of ACSL1 enhanced the proportion of eicosapentaenoic acid (EPA), decreased the proportion of C22:4, and significantly upregulated polyunsaturated fatty acid (PUFA) content. These results were supported by oil red O staining, which revealed an increase in the lipid droplets in bovine adipocytes after the overexpression of the ACSL1 gene. Thus, the results of this study indicated that ACSL1 positively regulated PUFA synthesis in bovine adipocytes.

Identification and screening of circular RNAs during adipogenic differentiation of ovine preadipocyte by RNA-seq.

Circular RNAs (circRNAs) are a class of non-coding RNAs that play important roles in preadipocyte differentiation and adipogenesis. However, little is known about genome-wide identification, expression profile, and function of circRNAs in sheep. To investigate the role of circRNAs during ovine adipogenic differentiation, the subcutaneous adipose tissue of Tibetan rams was collected in June 2022. Subsequently, the preadipocytes were immediately isolated from collected adipose tissue and then induced to begin differentiation. The adipocytes samples cultured on days 0, 2, and 8 of preadipocytes differentiation were used to perform RNA sequencing (RNA-seq) analysis to construct the expression profiles of circRNAs. Subsequently, the function of differentially expressed circRNAs was investigated by performing the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of their parent genes. Finally, a circRNAs-miRNAs-mRNAs network involved in adipogenic differentiation was been analyzed. As a result, a total of 6,449 candidate circRNAs were identified in ovine preadipocytes. Of these circRNAs identified, 63 candidate circRNAs were differentially expressed among the three differentiation stages and their parent genes were mainly enriched in acetyl-CoA metabolic process, positive regulation of lipid biosynthetic process, positive regulation of steroid biosynthetic process, and focal adhesion pathway (P < 0.05). Based on a circRNAs-miRNAs-mRNAs regulatory network constructed, circ_004977, circ_006132 and circ_003788 were found to function as competing endogenous RNAs (ceRNAs) to regulate ovine preadipocyte differentiation and lipid metabolism. The results provide an improved understanding of functions and molecular mechanisms of circRNAs underlying ovine adipogenesis in sheep.

The moderate fat deposition contributes to improve mutton quality, which is associated with the differentiation of preadipocytes. To investigate roles of circular RNAs (circRNAs) in preadipocyte differentiation, we identified circRNAs on days 0, 2, and 8 of preadipocytes differentiation and compared the expression profile of circRNAs at different adipogenic differentiation stages. A total of 6,449 candidate circRNAs were identified, among which 63 candidate circRNAs were differentially expressed among the three differentiation stages. The parent genes of differentially expressed circRNAs were enriched in several biological process and pathways related to lipid metabolism and synthesis. In addition, several circRNAs may regulate ovine preadipocyte differentiation by interacting with microRNAs (miRNAs). The results reveal the potential roles of circRNAs in adipogenic differentiation of sheep.

Oregano Essential Oil as a Natural Plant Additive Affects Growth Performance and Serum Antibody Levels by Regulating the Rumen Microbiota of Calves.

This experiment aimed to investigate whether supplementation of calves with different doses of oregano essential oil (OEO) could promote the development of the gastrointestinal tract and enhance the immune ability of calves by regulating the rumen microbiota. Twenty-four 70-day-old healthy and disease-free Holstein male calves were randomly divided into four groups, with the control group fed a basal diet, and the treatment group provided 4 g, 6 g, and 8 g of oregano essential oil per day in addition to the basal diet. After the 14-day pre-test, a 56-day formal test was conducted. At days 0 and 56 of the standard test period, calves were weighed, the average daily weight gain of calves during the test period was calculated, and serum samples were collected to measure the concentration of immunoglobulins (IgA, IgG, and IgM) in the serum; at day 56 of the formal test period, rumen fluid was collected from the calves, and 16SrRNA was sequenced to analyze changes in the rumen microbiota of the calves. The changes in the rumen microbiota of calves were analyzed by 16SrRNA sequencing. The results of the study showed that (1) OEO supplementation in calves significantly increased end weight and average daily gain (p < 0.05); (2) OEO supplementation in calves significantly increased serum concentrations of immunoglobulins IgA and IgM (p < 0.05); (3) OEO supplementation in calves significantly increased the abundance and diversity of rumen microbial organisms (p < 0.05); (4) OEO supplementation in calves significantly regulates the relative abundance of some species, and biomarkers with significant differences were screened by LEfSe analysis: g_Turicibacter, g_Romboutsia, f_Peptostreptococcaceae, f_Clostridiaceae, g_Clostridium_sensu_stricto_1, o_Clostridiales, g_unclassified_f_Synergistaceae, c_Coriobacteriia, o_Coriobacteriales, f_Atopobiaceae, g_Olsenella, p_Actinobacteriota, g_Defluviitaleaceae_UCG-011, f_Defluviitaleaceae, o_Corynebacteriales, g_Corynebacterium, f_Corynebacteriaceae, g_Shuttleworthia, f_Hungateiclostridiaceae, o_norank_c_Clostridia, g_Saccharofermentans, g_Streptococcus, f_Streptococcaceae, g_unclassified_o_Oscillospirales, and f_unclassified_o_Oscillospirales (p < 0.05, LDA ≥ 3); and (5) OEO supplementation in calves significantly enriched the metabolism of cofactors and vitamins pathway (p < 0.05). (6) Using Superman's correlation analysis, we screened unclassified_c_Clostridia, Shuttleworthia, and Christensenellaceae_R-7_group, three beneficial strains for calves. (7) Daily supplementation with 8g of OEO significantly affected rumen microbiota regulation in calves.

Spatiotemporal Expression and Haplotypes Identification of KRT84 Gene and Their Association with Wool Traits in Gansu Alpine Fine-Wool Sheep.

Keratin (K) is a major protein component of hair and is involved in hair growth and development. In this study, we analysed the expression, localization, and polymorphism of the K84 gene (KRT84) in Gansu Alpine Fine-wool sheep using immunofluorescence, RT-qPCR, and PARMS (penta-primer amplification refractory mutation system). Haplotypes of KRT84 were also constructed and their relationship with wool traits analysed. It was revealed that KRT84 was highly expressed in hair follicles, including the inner root sheath, outer root sheath, and hair medulla and at all six lamb ages investigated from 1 to 270 days of age. Three SNPs were detected in KRT84 exon 1, and they formed three haplotypes (named H1, H2, and H3) and six genotypes. Analyses revealed an association between haplotype combinations (diplotypes) and the mean fibre curvature, mean staple length, mean staple strength, mean fibre diameter, the coefficient of variation of fibre diameter, and comfort factor for these sheep. These results suggest that KRT84 is of importance in determining several key traits in Gansu Alpine Fine-wool sheep and that the gene could possibly be used as a genetic marker for wool trait selection in these sheep.

Proteome Analysis Related to Unsaturated Fatty Acid Synthesis by Interfering with Bovine Adipocyte ACSL1 Gene.

Unsaturated fatty acids (UFAs) in beef play a vital role in promoting human health. Long-chain fatty acyl-CoA synthase 1 (ACSL1) is a crucial gene for UFA synthesis in bovine adipocytes. To investigate the protein expression profile during UFA synthesis, we performed a proteomic analysis of bovine adipocytes by RNA interference and non-interference with ACSL1 using label-free techniques. A total of 3558 proteins were identified in both the NC and si-treated groups, of which 1428 were differentially expressed proteins (DEPs; fold change ≥ 1.2 or ≤ 0.83 and p-value < 0.05). The enrichment analysis of the DEPs revealed signaling pathways related to UFA synthesis or metabolism, including cAMP, oxytocin, fatty acid degradation, glycerol metabolism, insulin, and the regulation of lipolysis in adipocytes (p-value < 0.05). Furthermore, based on the enrichment analysis of the DEPs, we screened 50 DEPs that potentially influence the synthesis of UFAs and constructed an interaction network. Moreover, by integrating our previously published transcriptome data, this study established a regulatory network involving differentially expressed long non-coding RNAs (DELs), highlighting 21 DEPs and 13 DELs as key genes involved in UFA synthesis. These findings present potential candidate genes for further investigation into the molecular mechanisms underlying UFA synthesis in bovines, thereby offering insights to enhance the quality of beef and contribute to consumer health in future studies.