Circulating microRNA Profiles during the Bovine Oestrous Cycle

Up to 50% of ovulations go undetected in modern dairy herds due to attenuated oestrus behavior and a lack of high-accuracy methods for detection of fertile oestrus. This significantly reduces overall herd productivity and constitutes a high economic burden to the dairy industry. MicroRNAs (miRNAs) are ubiquitous regulators of gene expression during both health and disease and they have been shown to regulate different reproductive processes. Extracellular miRNAs are stable and can provide useful biomarkers of tissue function; changes in circulating miRNA profiles have been reported during menstrual cycles. This study sought to establish the potential of circulating miRNAs as biomarkers of oestrus in cattle. We collected plasma samples from 8 Holstein-Friesian heifers on days Days 0, 8 and 16 of an oestrous cycle and analysed small RNA populations on each Day using two independent high-throughput approaches, namely, Illumina sequencing (n = 24 samples) and Qiagen PCR arrays (n = 9 sample pools, 3-4 samples / pool). Subsequently, we used RT-qPCR (n = 24 samples) to validate the results of high-throughput analyses, as well as to establish the expression profiles of additional miRNAs previously reported to be differentially expressed during reproductive cycles. Overall, we identified four miRNAs (let-7f, miR-125b, miR-145 and miR-99a-5p), the plasma levels of which distinctly increased (up to 2.2-fold, P < 0.05) during oestrus (Day 0) relative to other stages of the cycle (Days 8 and 16). Moreover, we identified several hundred different isomiRs and established their relative abundance in bovine plasma. In summary, our results reveal the dynamic nature of plasma miRNAs during the oestrous cycle and provide evidence of the feasibility of using circulating miRNAs as biomarkers of reproductive function in livestock in the future

Circulating miRNA signatures of early pregnancy in cattle

BACKGROUND: Low fertility remains a leading cause of poor productivity in dairy cattle. In this context, there is significant interest in developing novel tools for accurate early diagnosis of pregnancy. MicroRNAs (miRNAs) are short RNA molecules which are critically involved in regulating gene expression during both health and disease. MiRNAs have been shown to regulate ovarian function, uterine receptivity, embryonic development and placental function. Circulating miRNAs can provide useful biomarkers of tissue function and disease; importantly, differential miRNA profiles have been linked to pregnancy and preeclampsia in humans. This study sought to establish the potential of circulating miRNAs as biomarkers of early pregnancy in cattle. RESULTS: We applied Illumina small-RNA sequencing to profile miRNAs in plasma samples collected from eight non-pregnant heifers on Days 0, 8 and 16 of the oestrous cycle and 11 heifers on Days 16 and 24 of pregnancy. We sequenced a total of 46 samples and generated 9.2 million miRNA reads per sample. There were no differences in miRNA read abundance between any of the pregnant and non-pregnant time-points (FDR > 0.1). As a complementary approach, we analysed sample pools (3–4 samples/pool) corresponding to Days 0, 8 and 16 of the oestrous cycle and Day 24 of pregnancy (n = 3 pools/group) using Qiagen PCR arrays. A total of 16 miRNAs were differentially expressed (FDR < 0.1) in plasma between pregnant and non-pregnant animals. RT-qPCR validation using the same plasma samples confirmed that miR-26a was differentially upregulated on Day 16 pregnant relative to non-pregnant heifers (1.7-fold; P = 0.043), whereas miR-1249 tended to be upregulated in Day 16 pregnant heifers (1.6-fold; P = 0.081). Further validation in an independent group of heifers confirmed an increase in plasma miR-26a levels during early pregnancy, which was significant only on Day 24 (2.0-fold; P = 0.027). CONCLUSIONS: Through genome-wide analyses we have successfully profiled plasma miRNA populations associated with early pregnancy in cattle. We have identified miR-26a as a potential circulating biomarker of early pregnancy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2529-1) contains supplementary material, which is available to authorized users

LPS-induced expression of CD14 in the TRIF pathway is epigenetically regulated by sulforaphane in porcine pulmonary alveolar macrophages

Pulmonary alveolar macrophages (AMs) are important in defense against bacterial lung inflammation. Cluster of differentiation 14 (CD14) is involved in recognizing bacterial lipopolysaccharide (LPS) through MyD88-dependent and TRIF pathways of innate immunity. Sulforaphane (SFN) shows anti-inflammatory activity and suppresses DNA methylation. To identify CD14 epigenetic changes by SFN in the LPS-induced TRIF pathway, an AMs model was investigated in vitro. CD14 gene expression was induced by 5 µg/ml LPS at the time point of 12 h and suppressed by 5 µM SFN. After 12 h of LPS stimulation, gene expression was significantly up-regulated, including TRIF, TRAF6, NF-κB, TRAF3, IRF7, TNF-α, IL-1β, IL-6, and IFN-β. LPS-induced TRAM, TRIF, RIPK1, TRAF3, TNF-α, IL-1β and IFN-β were suppressed by 5 µM SFN. Similarly, DNMT3a expression was increased by LPS but significantly down-regulated by 5 µM SFN. It showed positive correlation of CD14 gene body methylation with in LPS-stimulated AMs, and this methylation status was inhibited by SFN. This study suggests that SFN suppresses CD14 activation in bacterial inflammation through epigenetic regulation of CD14 gene body methylation associated with DNMT3a. The results provide insights into SFN-mediated epigenetic down-regulation of CD14 in LPS-induced TRIF pathway inflammation and may lead to new methods for controlling LPS-induced inflammation in pigs

Expression of microRNA and microRNA processing machinery genes during early quail (Coturnix japonica) embryo development

Wondim, Dr. Dessie Salilew/0000-0002-8756-0865WOS: 000319314800023PubMed: 23436530MicroRNA (miRNA) are small regulatory RNA molecules that are implicated in regulating and controlling a wide range of physiological processes including cell division, differentiation, migration, apoptosis, morphogenesis, and organogenesis. The aim of this study was to determine the expression pattern of 32 miRNA and 18 miRNA processing machinery genes during somite formation in quail embryos. The embryos were collected at stages HH (Hamburger and Hamilton) 4, 6, and 9 of embryo development (19, 24, and 30 h of incubation, respectively). Total RNA including miRNA was isolated from 4 groups of embryos (each group consisting of 6 to 8 embryos) were collected at each of the 3 stages (19, 24, and 30 h). The expression pattern of candidate miRNA and miRNA processing machinery genes was performed using quantitative real-time PCR. The results demonstrated that 7 miRNA (let-7a, mir-122, mir-125b, mir-10b, P < 0.01; let-7b, mir-26a, and mir-126, P < 0.05) were differentially expressed during early quail embryo development. In addition, the expression profile of 18 miRNA processing machinery genes was not significantly increased at 30 h of incubation compared with both 19 and 24 h. Our results suggest that machinery genes for miRNA biogenetic pathways are functional, and hence, miRNA may be involved in the regulation of early quail development. These 7 differentially expressed miRNA are suggested to play critical roles in quail embryo somite formation

Endometrial receptivity in lactating dairy cows

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SABRE-hanke; Kohdun limakalvon geenitoiminta hyvän alkiovastaanottajan tunnistamisessa

Alkionsiirtoja hyödyntävät paitsi jalostusohjelmat myös maitotilat. Alkiohuuhteluiden avulla hyvistä tuotantoeläimistä voidaan saada useita jälkeläisiä vuodessa karjan muiden eläinten toimiessa alkioiden vastaanottajina, mikä mahdollistaa karjan nopean eläinaineksen parantamisen. Ennen alkionsiirtoa vastaanottajan keltarauhanen tarkastetaan manuaalisesti. Keltarauhanen kehittyy ovulaation seurauksena ja sen tuottama progesteroni-hormoni vaikuttaa kohdun limakalvon toimintaan tehden sen alkion kiinnittymiselle suotuisaksi. Nykyisin keskimäärin joka toinen tuorealkionsiirto johtaa tiinehtymiseen. Tämän tutkimuksen tarkoituksena oli selvittää alkionsiirrosta tiinehtyneiden/poikineiden ja tyhjäksi jääneiden vastaanottajien kohdun limakalvon toimintaa. Tavoitteena oli löytää tiinehtyvän eläimen kohdun limakalvon toiminnalle ominaisia aineenvaihduntareittejä ja mahdollisesti kandidaattigeenejä diagnostiikan työkaluiksi. Tutkimus tehtiin maidontuotannossa olevilla lehmillä. Poikimisen jälkeen alkiovastaanottajien kohdun limakalvosta otettiin näytepalat ensimmäisen näkyvän kiimankierron päivinä 0 (siemennyskiima), 7 ja 14. Näytepalat pakastettiin (-80 °C). Seuraavan kiimakierron päivänä 7 samaan vastaanottajaan siirrettiin alkio ja alkion kiinnittyminen tarkistettiin kuukauden kuluttua siirrosta. Poikimisten jälkeen pakastetut kohtunäytteet jaettiin poikineiden ja tyhjiksi jääneiden ryhmiin, ja näytteiden geenitoimintaa verrattiin mikrosirutekniikalla, joka mahdollistaa kymmenien tuhansien geenituotteiden samanaikaisen analysoinnin. Vastaanottajista kerättiin lisäksi tuotos- ja hedelmällisyystietoja. Mikrosirututkimuksen perusteella erot poikineiden ja tyhjiksi jääneiden lehmien välillä keskittyivät kiimankierron alkupuoliskolle, jolloin eroja löytyi 64 yksittäisen geenin osalta. Aineenvaihduntareittien osalta kohdun limakalvon toiminnassa todettiin ryhmien välillä eroja mm. rasvahappojen aineenvaihdunnassa ja immuunivasteissa. Tuotos- ja hedelmällisyysominaisuuksien osalta poikineet ja tyhjiksi jääneet alkiovastaanottajat poikkesivat toisistaan kokonaisjalostusarvojen ja rasvatuotos-indeksien osalta. Tämä tutkimus on osa EU:n rahoittamaa SABRE-projektia (2006–2010)