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.

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.

Screening and identification of lncRNAs in preadipocyte differentiation in sheep.

Studies of preadipocyte differentiation and fat deposition in sheep have mainly focused on functional genes, and with no emphasis placed on the role that long non-coding RNAs (lncRNAs) may have on the activity of those genes. Here, the expression profile of lncRNAs in ovine preadipocyte differentiation was investigated and the differentially expressed lncRNAs were screened on day 0 (D0), day 2(D2) and day 8(D8) of ovine preadipocyte differentiation, with their target genes being predicted. The competing endogenous RNA (ceRNA) regulatory network was constructed by GO and KEGG enrichment analysis for functional annotation, and some differentially expressed lncRNAs were randomly selected to verify the RNA-Seq results by RT-qPCR. In the study, a total of 2517 novel lncRNAs and 3943 known lncRNAs were identified from ovine preadipocytes at the three stages of differentiation, with the highest proportion being intergenic lncRNAs. A total of 3455 lncRNAs were expressed at all three stages of preadipocyte differentiation, while 214, 226 and 228 lncRNAs were uniquely expressed at day 0, day 2 and day 8, respectively. By comparing the expression of the lncRNAs between the three stages of differentiation stages, a total of 405, 272 and 359 differentially expressed lncRNAs were found in D0-vs-D2, D0-vs-D8, and D2-vs-D8, respectively. Functional analysis revealed that the differentially expressed lncRNAs were enriched in signaling pathways related to ovine preadipocyte differentiation, such as mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide 3-kinase protein kinase B (PI3K-Akt) pathway, and the transforming growth factor beta (TGF-ß) pathway. In summary, lncRNAs from preadipocytes at different stages of differentiation in sheep were identified and screened using RNA-Seq technology, and the regulatory mechanisms of lncRNAs in preadipocyte differentiation and lipid deposition were explored. This study provides a theoretical reference for revealing the roles of lncRNAs in ovine preadipocyte differentiation and also offers a theoretical basis for further understanding the regulatory mechanisms of ovine preadipocyte differentiation.

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.

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.

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.

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.

Variation in the HSL Gene and Its Association with Carcass and Meat Quality Traits in Yak.

Hormone-sensitive lipase (HSL) is involved in the breakdown of triacylglycerols in adipose tissue, which influences muscle tenderness and juiciness by affecting the intramuscular fat content (IMF). This study analyzed the association between different genotypes and haplotypes of the yak HSL gene and carcass and meat quality traits. We used hybridization pool sequencing to detect exon 2, exon 8, and intron 3 variants of the yak HSL gene and genotyped 525 Gannan yaks via KASP to analyze the effects of the HSL gene variants on the carcass and meat quality traits in yaks. According to the results, the HSL gene is highly expressed in yak adipose tissue. Three single nucleotide polymorphisms (SNPs) were identified, with 2 of them located in the coding region and one in the intron region. Variants in the 2 coding regions resulted in amino acid changes. The population had 3 genotypes of GG, AG, and AA, and individuals with the AA genotype had lower WBSF values (p < 0.05). The H3H3 haplotype combinations could improve meat tenderness by reducing the WBSF values and the cooking loss rate (CLR) (p < 0.05). H1H1 haplotype combinations were associated with the increased drip loss rate (DLR) (p < 0.05). The presence of the H1 haplotype was associated the increased CLR in yaks, while that of the H2 haplotype was associated with the decreased DLR in yaks (p < 0.05). These results demonstrated that the HSL gene may influence the meat quality traits in yaks by affecting the IMF content in muscle tissues. Consequently, the HSL gene can possibly be used as a biomarker for improving the meat quality traits in yaks in the future.

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.

Comprehensive Analysis of miRNA and mRNA Expression Profiles during Muscle Development of the Longissimus Dorsi Muscle in Gannan Yaks and Jeryaks.

A hybrid offspring of Gannan yak and Jersey cattle, the Jeryak exhibits apparent hybrid advantages over the Gannan yak in terms of production performance and other factors. The small non-coding RNAs known as miRNAs post-transcriptionally exert a significant regulatory influence on gene expression. However, the regulatory mechanism of miRNA associated with muscle development in Jeryak remains elusive. To elucidate the regulatory role of miRNAs in orchestrating skeletal muscle development in Jeryak, we selected longissimus dorsi muscle tissues from Gannan yak and Jeryak for transcriptome sequencing analysis. A total of 230 (DE) miRNAs were identified in the longissimus dorsi muscle of Gannan yak and Jeryak. The functional enrichment analysis revealed a significant enrichment of target genes from differentially expressed (DE)miRNAs in signaling pathways associated with muscle growth, such as the Ras signaling pathway and the MAPK signaling pathway. The network of interactions between miRNA and mRNA suggest that some (DE)miRNAs, including miR-2478-z, miR-339-x, novel-m0036-3p, and novel-m0037-3p, played a pivotal role in facilitating muscle development. These findings help us to deepen our understanding of the hybrid dominance of Jeryaks and provide a theoretical basis for further research on the regulatory mechanisms of miRNAs associated with Jeryak muscle growth and development.

Long-stranded non-coding RNAs temporal-specific expression profiles reveal longissimus dorsi muscle development and intramuscular fat deposition in Tianzhu white yak.

The process of muscle development and intramuscular fat (IMF) deposition is quite complex and controlled by both mRNAs and ncRNAs. Long-stranded non-coding RNAs (LncRNAs) are involved in various biological processes in mammals while also playing a critical role in muscle development and fat deposition. In the present study, RNA-Seq was used to comprehensively study the expression of lncRNAs and mRNAs during muscle development and intramuscular fat deposition in postnatal Tianzhu white yaks at three stages, including 6 mo of age (calve, n = 6), 30 mo of age (young cattle, n = 6) and 54 mo of age (adult cattle, n = 6). The results indicated that a total of 2,101 lncRNAs and 20,855 mRNAs were screened across the three stages, of which the numbers of differential expression (DE) lncRNAs and DE mRNAs were 289 and 1,339, respectively, and DE lncRNAs were divided into eight different expression patterns based on expression trends. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that some DE mRNAs overlapped with target genes of lncRNAs, such as NEDD4L, SCN3B, AGT, HDAC4, DES, MYH14, KLF15 (muscle development), ACACB, PCK2, LIPE, PIK3R1, PNPLA2, and MGLL (intramuscular fat deposition). These DE mRNAs were significantly enriched in critical muscle development and IMF deposition-related pathways and GO terms, such as AMPK signaling pathway, PI3K-Akt signaling pathway, PPAR signaling pathway, etc. In addition, lncRNA-mRNA co-expression network analysis revealed that six lncRNAs (MSTRG.20152.2, MSTRG.20152.3, XR_001351700.1, MSTRG.8190.1, MSTRG.4827.1, and MSTRG.11486.1) may play a major role in Tianzhu white yak muscle development and lipidosis deposition. Therefore, this study enriches the database of yak lncRNAs and could help to further explore the functions and roles of lncRNAs in different stages of muscle development and intramuscular fat deposition in the Tianzhu white yak.

In-depth analysis of the molecular regulatory mechanisms involved in the development of yak skeletal muscle can aid in the identification of crucial functional genes that may enhance the quality of yak meat in the future. Our research is the first to investigate the expression of long-stranded non-coding RNAs (lncRNAs) during muscle development and intramuscular fat deposition in Tianzhu white yaks at different ages (6, 30, and 54 mo). We have discovered numerous novel lncRNAs and their associated target genes that are linked to muscle development and fat deposition, and have constructed relevant interaction network diagrams. This study is expected to promote a genetics-based approach to further enhance the Tianzhu white yak and other yak breeds.

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.

Identification and Characterization of Circular RNAs (circRNAs) Using RNA-Seq in Two Breeds of Cashmere Goats.

Circular RNA (circRNA) is a type of non-coding RNA generated from back-splicing the reactions of linear RNA. It plays an important role in various cellular and biological processes. However, there are few studies about the regulatory effect of circRNAs on cashmere fiber traits in cashmere goats. In this study, the expression profiles of circRNAs in skin tissue were compared between Liaoning cashmere (LC) goats and Ziwuling black (ZB) goats, with a significant difference in cashmere fiber yield, cashmere fiber diameter, and cashmere fiber color, using RNA-seq. A total of 11,613 circRNAs were expressed in the caprine skin tissue, and their type, chromosomal distribution, and length distribution were characterized. A total of 115 up-regulated circRNAs and 146 down-regulated circRNAs in LC goats were screened compared to ZB goats. The authenticity of 10 differentially expressed circRNAs was validated by detecting their expression levels and the head-to-tail splice junction using RT-PCR and DNA sequencing, respectively. The parent genes of differentially expressed circRNA were mainly enriched in some Gene Ontology (GO) terms and pathways related to cashmere fiber traits, such as the canonical Wnt signaling pathway, which is involved in the regulation of cell promotion, stem cell proliferation, Wnt signaling pathway regulation, epithelial morphogenesis, MAPK signaling pathway, and cell adhesion molecules pathway. Eight differentially expressed circRNAs were further selected to construct a circRNA-miRNA network, and some miRNAs that were previously reported as related to fiber traits were found in the network. This study provides a deep understanding of the roles of circRNAs in the regulation of cashmere fiber traits in cashmere goats and the involvement of differential splicing in phenotypic expression according to breed and region.

RNA-Seq Reveals the Roles of Long Non-Coding RNAs (lncRNAs) in Cashmere Fiber Production Performance of Cashmere Goats in China.

Long non-coding RNAs (lncRNAs) are a kind of non-coding RNA being >200 nucleotides in length, and they are found to participate in hair follicle growth and development and wool fiber traits regulation. However, there are limited studies reporting the role of lncRNAs in cashmere fiber production in cashmere goats. In this study, Liaoning cashmere (LC) goats (n = 6) and Ziwuling black (ZB) goats (n = 6) with remarkable divergences in cashmere yield, cashmere fiber diameter, and cashmere color were selected for the construction of expression profiles of lncRNAs in skin tissue using RNA sequencing (RNA-seq). According to our previous report about the expression profiles of mRNAs originated from the same skin tissue as those used in the study, the cis and trans target genes of differentially expressed lncRNAs between the two caprine breeds were screened, resulting in a lncRNA-mRNA network. A total of 129 lncRNAs were differentially expressed in caprine skin tissue samples between LC goats and ZB goats. The presence of 2 cis target genes and 48 trans target genes for the differentially expressed lncRNAs resulted in 2 lncRNA-cis target gene pairs and 93 lncRNA-trans target gene pairs. The target genes concentrated on signaling pathways that were related to fiber follicle development, cashmere fiber diameter, and cashmere fiber color, including PPAR signaling pathway, metabolic pathways, fatty acid metabolism, fatty acid biosynthesis, tyrosine metabolism, and melanogenesis. A lncRNA-mRNA network revealed 22 lncRNA-trans target gene pairs for seven differentially expressed lncRNAs selected, of which 13 trans target genes contributed to regulation of cashmere fiber diameter, while nine trans target genes were responsible for cashmere fiber color. This study brings a clear explanation about the influences of lncRNAs over cashmere fiber traits in cashmere goats.

Effects of Two Different Straw Pellets on Yak Growth Performance and Ruminal Microbiota during Cold Season.

The Tianzhu white yaks (Bos grunniens) live on the Qinghai-Tibet Plateau. During winter, a lack of resources and low nutritional levels seriously affect their growth performance. In this study, we aimed to explore the effect of supplementation straw pellets on the growth performance and ruminal microbiota of yaks. Overall, at 6 (6M, n = 24), 18 (18M, n = 26), 30 (30M, n = 20), 42 (42M, n = 24), and 54 (54M, n = 22) month old Tianzhu white yaks were selected (total n = 116) and divided into the mixed straw + grazing (MSG), corn straw + grazing (CSG), and the grazing control (G) groups according to age and gender. Their growth performance was measured as per different dietary treatments. The rumen microbial community structure and levels of VFAs were analyzed from the 6M, 30M, and 54M male yaks from each group. The supplementary diets led to an increase in the ADG, which was the highest in the MSG group. The MSG group exhibited the highest level of acetate and total VFAs (TVFAs) among the three groups (p < 0.05). In addition, the 16S rRNA sequencing results proved that the microbial composition was dominated by the members of Firmicutes and Bacteroidetes. Christensenellaceae R-7 group was significantly abundant in the CSG and MSG groups compared to the G group (p < 0.05). Principal coordinate analysis (PCoA) revealed that the bacterial community structure of rumen in the MSG and CSG groups was considerably different from that in the G group; 6M samples exhibited different rumen microbial diversity compared with the other samples. Correlation analysis revealed that Christensenellaceae_R-7_group was positively correlated with the levels of acetate, TVFAs, and ADG. These results demonstrated that mixed straw pellets improved the growth performance of yaks, increased the abundance of Christensenellaceae R-7_group involved in cellulose degradation in the rumen, and produced large amounts of VFAs, which were absorbed by yaks, thus increasing their ADG. This study provides new insights into the effects of straw pellet supplementation on the changes in the rumen microbiota and growth performance of yaks.

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.

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.

Characteristics and Expression of circ_003628 and Its Promoted Effect on Proliferation and Differentiation of Skeletal Muscle Satellite Cells in Goats.

In our previous a study, circ_003628 was one of the most highly expressed circular RNAs (circRNAs) in the Longissimus dorsi muscle of goats found by RNA-seq, suggesting that the circRNA may be important for caprine muscle growth and development. However, there have been no reports describing the molecular mechanisms by which circ_003628 regulates the activities of goat skeletal muscle satellite cells (SMSCs). In this study, reverse transcriptase-PCR (RT-PCR) and DNA sequencing were used to validate the authenticity of circ_003628, and its characteristics, expression profile and effect on goat SMSCs were also studied using real-time quantitative-PCR (RT-qPCR), EdU, CCK-8 and immunofluorescence assays. Circ_003628 is partially originated from 13 exons, 12 introns and 3'-untranslated regions (UTR) of caprine Myosin Heavy Chain 1 (MYH1), and 25 exons and 5' UTR of Myosin Heavy Chain 4 (MYH4), as well as intergenic sequences between the two genes. A total of 77.07% of circ_003628 were located in the nuclei of goat SMSCs, while 22.93% were expressed in the cytoplasm. The circRNAs were only expressed in triceps brachii, quadriceps femoris and longissimus dorsi muscle tissues in nine caprine tissues investigated, with the highest expression level in longissimus dorsi muscle. The expression level of circ_003628 gradually increased during differentiation periods of goat SMSCs and reached the maximum on day 6 after differentiation. The small interfering RNA of circ_003628 (named si-circ_003628) inhibited the viability and proliferation of goat SMSCs, and also decreased the expression of four cell proliferation marker genes: paired box 7 (Pax7), cyclin-dependent kinase 2 (CDK2), CDK4 and CyclinD1 in goat SMSCs. Transfection of si-circ_003628 significantly decreased the area of MyHC-labeled myotubes of goat SMSCs, as well as the expression levels of three differentiation marker genes: myosin heavy chain (MyHC), myogenin (MyoG), and myocyte enhancer factor 2C (MEF2C). These results suggest that circ_003628 promotes the viability, proliferation, and differentiation of goat SMSCs, and they also provide an improved understanding of the roles of circ_003628 in skeletal muscle growth and development in goats.

Transcriptional analysis of microRNAs related to unsaturated fatty acid synthesis by interfering bovine adipocyte ACSL1 gene.

Long-chain fatty acyl-CoA synthase 1 (ACSL1) plays a vital role in the synthesis and metabolism of fatty acids. The proportion of highly unsaturated fatty acids in beef not only affects the flavor and improves the meat's nutritional value. In this study, si-ACSL1 and NC-ACSL1 were transfected in bovine preadipocytes, respectively, collected cells were isolated on the fourth day of induction, and then RNA-Seq technology was used to screen miRNAs related to unsaturated fatty acid synthesis. A total of 1,075 miRNAs were characterized as differentially expressed miRNAs (DE-miRNAs), of which the expressions of 16 miRNAs were upregulated, and that of 12 were downregulated. Gene ontology analysis indicated that the target genes of DE-miRNAs were mainly involved in biological regulation and metabolic processes. Additionally, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis identified that the target genes of DE-miRNAs were mainly enriched in metabolic pathways, fatty acid metabolism, PI3K-Akt signaling pathway, glycerophospholipid metabolism, fatty acid elongation, and glucagon signaling pathway. Combined with the previous mRNA sequencing results, several key miRNA-mRNA targeting relationship pairs, i.e., novel-m0035-5p-ACSL1, novel-m0035-5p-ELOVL4, miR-9-X-ACSL1, bta-miR-677-ACSL1, miR-129-X-ELOVL4, and bta-miR-485-FADS2 were screened via the miRNA-mRNA interaction network. Thus, the results of this study provide a theoretical basis for further research on miRNA regulation of unsaturated fatty acid synthesis in bovine adipocytes.