Identification And Function Analysis Of Differentially Expressed Mirna In Breeder Cock Testes Responsed To Astragalus Polysaccharide

As a critical process in broiler production, the breeder cock feeding process and its’ effect on breeder cock reproductive performance could influence the fertility rate and hatchability of eggs and the growth performance of commercial broilers. And the good condition of metabolism and immunity in testis could influence the reproductive performance and efficient utilization of breeder cocks. The major ingredient of Radix Astragali, Astragalus Polysaccharide(APS), is an important feed additive due to its immunomodulatory functions and nutritional metabolism regulation roles. Our results provided a novel insight into the role of miRNAs in response to the intake of APS, which will promote research and development of the relationship between APS and testicular function of breeder cocks.Experiment 1 Effects of dietary APS supplement on the expression of mi RNAs in testis of breeder cocksIn order to identify differentially expressed miRNAs in response to APS dietary supplements, we generated miRNA expression profiles of testis from breeder cocks fed with control diets and extra APS. Based on the single factor experimental design, a total of 64one-day-old Cobb500 breeder cocks were randomly assigned to two groups with four replicates per treatment and 8 birds per replicate, including two treatments: control and dietary APS supplements(10 g/kg) group. The chickens in control group were fed with corn soybean diet, and 10 g APS per kilogram fodder were added to the feed of APS group. At forty weeks of age, samples of each treatment were selected randomly for next generation sequencing analysis of miRNAs. As the result, a total of 1305 miRNAs were identified in control and APS group; 584 miRNAs were co-expressed in both libraries; 74 miRNAs were detected in control libraries and 79 were detected in the APS libraries. Moreover, compared with the control group meeting the criteria of P-value<0.05, we identified 33 differetially expressed miRNAs. There were 16 up-expressed mi RNAs and 17 down-expressed miRNAs in APS group. Based on Mir-XTM miRNA RT-qPCR, seven differentially expressed miRNAs were detected and the results suggested that the miRNA expression profiles data could represent actual miRNA expression levels.Experiment 2 Target genes prediction of differentially expressed miRNAs and functional analysesBased on the differentially expressed miRNAs we identified, target genes prediction of these 33 miRNAs and further GO and KEGG analysis of target genes for differentially expressed miRNAs were performed, the results revealed that some candidate miRNAs may be involved in testicular nutrient metabolisms, including nucleotide metabolic process(GO:0009117), regulation of proteolysis(GO: 0030162), propanoate metabolism(ko00640), fatty acid metabolism(ko00071) and phospholipid biosynthetic process(GO: 0008654), a carbon unit metabolisms including nicotinate and nicotinamide metabolism(ko00760), cysteine and methionine metabolism(ko00270), and other metabolic pathways(ko01100). These metabolic processes were correlated with development and function of testis. Furthermore, more researches are focusing on APS immunoregulation. In our studies, we discovered a pathway called natural killer cell mediated cytotoxicity, which is associated with testis innate immunity.In order to illuminate the function of miRNAs in regulating the process of NK cell mediated cytotoxicity, we made an analysis of miRNA-gene networks, and we found the gga-miR-16-5p plays an important role in the NK cell mediated cytotoxicity pathway.Subsequently, we chose miR-16-5p and the target genes of miR-16-5p as our further research object. Further validation of these four target genes expressions by RT-qPCR proved that the expressions of IFNGR2 and MAP2K1 decreased, and the expression of MAP2K2 increased.Therefore, further dual luciferase reporter experiments were performed by taking IFNGR2 and MAP2K1 as target genes, and the results confirmed that miR-16-5p repressed potential target genes(IFNGR2 and MAP2K1) expression by interacting with their 3’UTR. According to the validation of differentially expressed mi RNAs and target gene expressions of miR-16-5p both in vivo and in vitro, we believe that the APS could change miR-16-5p expression and its target gene(IFNGR2 and MAP2K1) expressions.In conclusion, our study showed that 10 g/kg APS supplement into the diet of breeder cocks could significantly affect the expression of testicular miRNAs, and the differently expressed miRNAs could regulate the testicular nutrient metabolism and immunoregulation process. And APS could decrease miR-16-5p expression and futher increase IFNGR2 and MAP2K1 expression.