Deep-Sequencing Identification of MicroRNA Biomarkers in Serum Exosomes for Early Pig Pregnancy.

Chen Zhou,Zhiqian Xu,Bin Hu,Linjun Hong,Fanming Meng,Gengyuan Cai,Zheng Xu,Sixiu Huang,Ting Gu,Qun Hu,Zhenfang Wu,Yanjuan He,Enqin Zheng

doi:10.3389/fgene.2020.00536

Abstract

Early pregnancy diagnosis in sows can significantly improve the efficiency of pig industry. Exosomes are membrane-covered nanovesicles that can transport microRNAs (miRNAs) and other molecular signals between cells. In other species, serum exosome-derived miRNAs can serve as good biomarkers of diseases and different physiological states, including pregnancy status. We hypothesized that circulating exosome-derived miRNAs might be used to differentiate the pregnancy status as early as several days after insemination in pigs. To test this hypothesis, we randomly assigned pigs for artificial insemination with fertile or dead semen (control group). Serum samples were obtained from pregnant pigs on days 9, 12, and 15 after insemination and from non-pregnant pigs on days 0, 9, 12, and 15 after insemination. Exosomes were isolated for RNA extraction. The exosomal RNA samples from pigs on day 9 of the estrus cycle and pregnancy were used for small-RNA sequencing. A total 321 miRNAs were identified in all samples. Twenty eight differentially abundant miRNAs were identified between the pregnant and control groups. miRNAs with | log2 (fold change)| > 2 from sequencing results were selected for validation by quantitative reverse-transcription-polymerase chain reaction (RT-qPCR) in larger samples. Finally two upregulated miRNAs (miR-92b-3p and miR-17-5p) in the pregnant groups (on days 9, 12, and 15 of pregnancy) were confirmed by RT-qPCR. In summary, we have successfully identified circulating exosomal miRNA profiles in the serum of pigs in early pregnancy. miR-92b-3p and miR-17-5p could be used as potential circulating biomarkers for early pregnancy diagnosis.

Highlights

Pregnancy diagnosis in sows is an important technique to improve the breeding efficiency of the swine industry
Biomarkers for Early Pig Pregnancy cycle and the increase in ventral abdomen during pregnancy combined with ultrasonic diagnosis
Analysis showed that more than 89.4% of the vesicles were 30– 150 nm in diameter (Figure 1B). This size range is similar to that detected by Transmission Electron Microscopy (TEM) and further confirms the identity of these vesicles as exosomes

Summary

Introduction

Pregnancy diagnosis in sows is an important technique to improve the breeding efficiency of the swine industry. Pregnancy status can be detected by concentrations of progesterone or estrogen in blood, urine, or feces (Resnis et al, 2000; Kauffold and Althouse, 2007; Rousian et al, 2009; Agnieszka, 2011). These methods can only detect the pregnancy status as early as days 25–30, and no currently available method diagnoses pregnancy before 25 days after fertilization. The specific circulating miRNAs detected in maternal plasma during pregnancy could be associated with pregnancy status. The detection of maternal blood circulating miRNAs shows potential to become a promising non-invasive prenatal diagnostic method in animals (Gu et al, 2019)

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Discussion

Conclusion