Comprehensive RNA-Seq profiling to evaluate lactating sheep mammary gland transcriptome.
ABSTRACT: RNA-Seq enables the generation of extensive transcriptome information providing the capability to characterize transcripts (including alternative isoforms and polymorphism), to quantify expression and to identify differential regulation in a single experiment. Our aim in this study was to take advantage of using RNA-Seq high-throughput technology to provide a comprehensive transcriptome profiling of the sheep lactating mammary gland. Eight ewes of two dairy sheep breeds with differences in milk production traits were used in this experiment (four Churra and four Assaf ewes). Milk samples from these animals were collected on days 10, 50, 120 and 150 after lambing to cover the various physiological stages of the mammary gland across the complete lactation. RNA samples were extracted from milk somatic cells. The RNA-Seq dataset was generated using an Illumina HiSeq 2000 sequencer. The information reported here will be useful to understand the biology of lactation in sheep, providing also an opportunity to characterize their different patterns on milk production aptitude.
Project description:This study presents a dynamic characterization of the sheep milk transcriptome aiming at achieving a better understanding of the sheep lactating mammary gland. Transcriptome sequencing (RNA-seq) was performed on total RNA extracted from milk somatic cells from ewes on days 10, 50, 120 and 150 after lambing. The experiment was performed in Spanish Churra and Assaf breeds, which differ in their milk production traits. Nearly 67% of the annotated genes in the reference genome (Oar_v3.1) were expressed in ovine milk somatic cells. For the two breeds and across the four lactation stages studied, the most highly expressed genes encoded caseins and whey proteins. We detected 573 differentially expressed genes (DEGs) across lactation points, with the largest differences being found, between day 10 and day 150. Upregulated GO terms at late lactation stages were linked mainly to developmental processes linked to extracellular matrix remodeling. A total of 256 annotated DEGs were detected in the Assaf and Churra comparison. Some genes selectively upregulated in the Churra breed grouped under the endopeptidase and channel activity GO terms. These genes could be related to the higher cheese yield of this breed. Overall, this study provides the first integrated overview on sheep milk gene expression.
Project description:This study presents a dynamic characterization of the sheep milk transcriptome aiming at achieving a better understanding of the sheep lactating mammary gland. Transcriptome sequencing (RNA-seq) was performed on total RNA extracted from milk somatic cells from ewes on days 10, 50, 120 and 150 after lambing. The experiment was performed in Spanish Churra and Assaf breeds, which differ in their milk production traits. Nearly 67% of the annotated genes in the reference genome (Oar_v3.1) were expressed in ovine milk somatic cells. For the two breeds and across the four lactation stages studied, the most highly expressed genes encoded caseins and whey proteins. We detected differentially expressed genes (DEGs) across lactation points, with the largest differences being found, between day 10 and day 150. Upregulated GO terms at late lactation stages were linked mainly to developmental processes linked to extracellular matrix remodeling. A total of 256 annotated DEGs were detected in the Assaf and Churra comparison. Some genes selectively upregulated in the Churra breed grouped under the endopeptidase and channel activity GO terms. These genes could be related to the higher cheese yield of this breed. Overall, this study provides the first integrated overview on sheep milk gene expression. Overall design: A total of eight healthy sheep were selected to be included in the experiment, four Assaf and four Churra ewes. 32 Milk Somatic Cells (MSCs) samples were collected on days 10, 50, 120 and 150 after lambing. In each time point 4 biological replicates from each breed were collected unless on day 120 that only three biological replicates from each breed were sequenced.
Project description:Small-Tailed Han (STH) sheep are known for their high fecundity, but the survival of lambs is compromised and influences the commercial return from farming these sheep, with this being attributed in part to starvation from insufficient milk production by the ewes. In this study, the transcriptome profiles of the mammary gland of lactating and non-lactating STH ewes were investigated using paired-end RNA sequencing (RNA-Seq). An average of 14,447 genes were found to be expressed at peak-lactation in the STH sheep, while 15,146 genes were expressed in non-lactating ewes. A total of 4,003 differentially expressed genes (DEGs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the DEGs were associated with a wide range of cellular components, biological processes and metabolic pathways, including binding activities, signaling pathways, cellular structures, and immune responses. The most highly expressed genes at peak-lactation included CSN2, LGB, LALBA, CSN1S1, CSN1S2, and CSN3, and the 10 most highly expressed genes accounted for 61.37% of the total Reads Per Kilobase of transcript, per Million mapped reads (RPKM). The most highly expressed genes in the mammary gland of non-lactating ewes included IgG, THYMB4X, EEF1A1, IgA, and APOE, and the 10 most highly expressed genes accounted for only 12.97% of the total gene RPKM values. This suggests that the sheep mammary gland undergoes a substantial development in milk protein synthesis infrastructure and promotion of protein transportation during lactation.
Project description:Long non-coding RNAs (lncRNAs) are a kind of non-coding RNA with >200 nucleotides in length. Some lncRNAs have been proven to have clear regulatory functions in many biological processes of mammals. However, there have been no reports on the roles of lncRNAs in ovine mammary gland tissues. In the study, the expression profiles of lncRNAs were studied using RNA-Seq in mammary gland tissues from lactating Small-Tailed Han (STH) ewes and Gansu Alpine Merino (GAM) ewes with different milk yield and ingredients. A total of 1894 lncRNAs were found to be expressed. Compared with the GAM ewes, the expression levels of 31 lncRNAs were significantly up-regulated in the mammary gland tissues of STH ewes, while 37 lncRNAs were remarkably down-regulated. Gene Ontogeny (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the target genes of differentially expressed lncRNAs were enriched in the development and proliferation of mammary epithelial cells, morphogenesis of mammary gland, ErbB signaling pathway, and Wnt signaling pathway. Some miRNA sponges of differentially expressed lncRNAs, reported to be associated with lactation and mammary gland morphogenesis, were found in a lncRNA-miRNA network. This study reveals comprehensive lncRNAs expression profiles in ovine mammary gland tissues, thereby providing a further understanding of the functions of lncRNAs in the lactation and mammary gland development of sheep.
Project description:The identification of genetic variation underlying desired phenotypes is one of the main challenges of current livestock genetic research. High-throughput transcriptome sequencing (RNA-Seq) offers new opportunities for the detection of transcriptome variants (SNPs and short indels) in different tissues and species. In this study, we used RNA-Seq on Milk Sheep Somatic Cells (MSCs) with the goal of characterizing the genetic variation within the coding regions of the milk transcriptome in Churra and Assaf sheep, two common dairy sheep breeds farmed in Spain.A total of 216,637 variants were detected in the MSCs transcriptome of the eight ewes analyzed. Among them, a total of 57,795 variants were detected in the regions harboring Quantitative Trait Loci (QTL) for milk yield, protein percentage and fat percentage, of which 21.44% were novel variants. Among the total variants detected, 561 (2.52%) and 1,649 (7.42%) were predicted to produce high or moderate impact changes in the corresponding transcriptional unit, respectively. In the functional enrichment analysis of the genes positioned within selected QTL regions harboring novel relevant functional variants (high and moderate impact), the KEGG pathway with the highest enrichment was "protein processing in endoplasmic reticulum". Additionally, a total of 504 and 1,063 variants were identified in the genes encoding principal milk proteins and molecules involved in the lipid metabolism, respectively. Of these variants, 20 mutations were found to have putative relevant effects on the encoded proteins.We present herein the first transcriptomic approach aimed at identifying genetic variants of the genes expressed in the lactating mammary gland of sheep. Through the transcriptome analysis of variability within regions harboring QTL for milk yield, protein percentage and fat percentage, we have found several pathways and genes that harbor mutations that could affect dairy production traits. Moreover, remarkable variants were also found in candidate genes coding for major milk proteins and proteins related to milk fat metabolism. Several of the SNPs found in this study could be included as suitable markers in genotyping platforms or custom SNP arrays to perform association analyses in commercial populations and apply genomic selection protocols in the dairy production industry.
Project description:The aim of this study was to examine the expression patterns of SREBP-1 gene in milk somatic cells and its association with milk fat yield during early lactation in Sarda breed sheep. A sample of 20 Sarda ewes, aged between 4 and 5 years, in their third to fourth lactation were chosen. From each ewe 28 days after lambing milk yield was measured, and a 160 ml milk sample for the RNA extraction and to test somatic cells count and lactose, fat and protein contents were collected. From the obtained RNA, total cDNA was synthesized and the quantitative PCR was performed. The fat, proteins and lactose content showed many differences among the animals, but these variations were no correlated with the milk yield. The SREBP-1 gene expression resulted higher in the high milk fat producing ewes. The correlation analysis showed that the SREBP-1 expression level is directly related to the amount of milk fat (g/die) (P < 0.001), while the total RNA obtained from each sample was found to be related to the somatic cells number (P < 0.001). Instead the expression of this gene showed no relations with the concentration of fat in milk. Our data highlight that in sheep SREBP-1 gene is expressed in the mammary gland during early lactation. Moreover, the positive relationship between SREBP-1 gene expression and the milk fat yield suggests that SREBP-1 gene is required for the lipid synthesis in the sheep mammary gland.
Project description:The mammary gland is a crucial tissue for milk synthesis and plays a critical role in the feeding and growth of mammalian offspring. The aim of this study was to use RNA-sequencing (RNA-Seq) technology to provide a transcriptome profile of the ovine mammary gland at the peak of lactation. Small-Tailed Han (STH) sheep (n = 9) and Gansu Alpine Merino (GAM) sheep (n = 9), breeds with phenotypic differences in milk production traits, were selected for the RNA-Seq analysis. This revealed 74 genes that were more highly expressed in the STHs than in the GAMs. Similarly, 143 genes that were expressed at lower levels in the STHs than in the GAMs, were identified. Gene ontogeny (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that these differentially expressed genes (DEGs) were associated with binding and catalytic activities, hematopoietic cell lineages, oxytocin signaling pathway and neuroactive ligand-receptor interaction. This is the first study of the transcriptome profile of the ovine mammary gland in these Chinese breeds at peak lactation. The results provide for a better understanding of the genetic mechanisms involved in ovine lactation.
Project description:Different studies have shown that polymorphisms in the sequence of genes coding for the milk proteins and milk fatty acids are associated with milk composition traits as well as with cheese-making traits. However, the lack of coincident results across sheep populations has prevented the use of this information in sheep breeding programs. The main objective of this study was to exploit the information derived from a total of 175 whole genome resequencing (WGR) datasets from 43 domestic sheep breeds and three wild sheep to evaluate the genetic diversity of 24 candidate genes for milk composition and identify genetic variants with a potential phenotypic effect. The functional annotation of the identified variants highlighted five single nucleotide polymorphisms (SNPs) predicted to have a high impact on the protein function and 42 missense SNPs with a putative deleterious effect. When comparing the allelic frequencies at these 47 polymorphisms with relevant functional effects between the genomes of Assaf and Churra sheep breeds, two missense deleterious variants were identified as potential markers associated to the milk composition differences found between the Churra and Assaf: XDH:92215727C>T and LALBA:137390760T>C. Future research is required to confirm the effect of the potential functionally relevant variants identified in the present study on milk composition and cheese-making traits.
Project description:The mammary gland is a dynamic organ that undergoes dramatic physiological adaptations during the transition from late pregnancy to lactation. Investigation of the molecular basis of mammary development and function will provide fundamental insights into tissue remodelling as well as a better understanding of milk production and mammary disease. This is important to livestock production systems and human health. Here we use RNA-seq to identify differences in gene expression in the ovine mammary gland between late pregnancy and lactation.Between late pregnancy (135 days of gestation ± 2.4 SD) and lactation (15 days post partum ± 1.27 SD) 13 % of genes in the sheep genome were differentially expressed in the ovine mammary gland. In late pregnancy, cell proliferation, beta-oxidation of fatty acids and translation were identified as key biological processes. During lactation, high levels of milk fat synthesis were mirrored by enrichment of genes associated with fatty acid biosynthesis, transport and lipogenesis. Protein processing in the endoplasmic reticulum was enriched during lactation, likely in support of active milk protein synthesis. Hormone and growth factor signalling and activation of signal transduction pathways, including the JAK-STAT and PPAR pathways, were also differently regulated, indicating key roles for these pathways in functional development of the ovine mammary gland. Changes in the expression of epigenetic regulators, particularly chromatin remodellers, indicate a possible role in coordinating the large-scale transcriptional changes that appear to be required to switch mammary processes from growth and development during late pregnancy to synthesis and secretion of milk during lactation.Coordinated transcriptional regulation of large numbers of genes is required to switch between mammary tissue establishment during late pregnancy, and activation and maintenance of milk production during lactation. Our findings indicate the remarkable plasticity of the mammary gland, and the coordinated regulation of multiple genes and pathways to begin milk production. Genes and pathways identified by the present study may be important for managing milk production and mammary development, and may inform studies of diseases affecting the mammary gland.
Project description:These trials were carried out to determine firstly the effect of diet and type of pregnancy on the transcriptional expression of genes involved in angiogenesis and cell turnover/lactogenesis inside the sheep mammary gland from late gestation to late lactation. Eighteen Ile de France sheep, 8 twin- and 10 single-bearing ewes were alloted into two groups according to their diet, either based on ad libitum naturalized pasture or red clover hay plus lupine from day -45 pre-partum until day +60 post-partum. Samples from diets and mammary glands were collected at day -10 pre partum (time 1), day +30 (time 2) and day +60 post-partum (time 3) and analyzed by qRT-PCR. Additionally, samples from longissimus dorsi muscle were taken from lambs twice, at weaning and 45 days later, to determine the effect of the maternal treatment with regard to diet and type of pregnancy, on the mRNA expression of genes involved in lipid metabolism. The data was processed using the lme4 package for R, and SPSS Statistics 23.0 for Windows®. The results showed that the group of twin-bearing ewes fed red clover showed a higher expression of genes involved in angiogenesis before lambing and in cell turnover/lactogenesis during late lactation, explained by a lamb survival mechanism to delay apoptosis as a way to keep a secretory cells population and boosted by the diet quality, assuring a longer milk production potential during late lactation. Regarding lambs, apparently the maternal diet would influence the transcriptional expression of lipogenic enzymes in the longissimus dorsi muscle after weaning, but further studies are necessary to validate these results. In summary, Twin-bearing ewes fed red clover performed best at increasing the expression of genes associated with angiogenesis and cell turnover/lactogenesis in the mammary gland.