Increasing evidence indicates that paternal diet can result in metabolic changes in offspring, but the definite mechanism remains unclear in birds. Here, we fed breeder cocks five different diets containing 0, 0.25, 1.25, 2.50 and 5.00 mg kg −1 folate throughout life. Paternal folate supplementation (FS) was beneficial to the growth and organ development of broiler offspring. Most importantly, the lipid and glucose metabolism of breeder cocks and broiler offspring were affected by paternal FS, according to biochemical and metabolomic analyses. We further employed global analyses of hepatic and spermatozoal messenger RNA (mRNA), long non-coding RNA (lncRNA) and micro RNA (miRNA). Some key genes involved in the glycolysis or gluconeogenesis pathway and the PPAR signalling pathway, including PEPCK , ANGPTL4 and THRSP , were regulated by differentially expressed hepatic and spermatozoal miRNAs and lncRNAs in breeder cocks and broiler offspring. Moreover, the expression of ANGPTL4 could also be regulated by differentially expressed miRNAs and lncRNAs in spermatozoa via competitive endogenous RNA (ceRNA) mechanisms. Overall, this model suggests that paternal folate could transgenerationally regulate lipid and glucose metabolism in broiler offspring and the epigenetic transmission may involve altered spermatozoal miRNAs and lncRNAs.

Astragalus Polysaccharide (APS) is an important feed additive due to its immunomodulatory functions. Previous studies have proven that miRNAs play important roles in posttranscriptional gene regulation. Our goals were to identify differentially expressed miRNAs in testes in responses to APS dietary supplements and to find the effects of APS on breeder cock testes. We measured several enzymatic activities in testes and sperm samples and further generated miRNA expression profiles of testes from breeder cocks fed with control diets and extra APS. As a result, we found APS could increase testicular functional activities of marker enzymes. Meanwhile, there were 16 up-regulated and 17 down-regulated miRNAs in APS group, compared with the control group meeting the criteria of P-values < 0.05. Meanwhile, twelve differentially expressed miRNAs were validated by Mir-XTM miRNA RT-qPCR. Further GO and KEGG analyses of target genes for differentially expressed miRNAs revealed that some miRNAs may be involved in testicular nutrient metabolisms and NK cell mediated cytotoxicity pathway. Moreover, the effect of dietary APS supplements on NK cell mediated cytotoxicity pathway was also validated by RT-qPCR. Our results provided a novel insight into the effect of dietary APS supplements on testicular miRNA expression profiles and enzymatic changes of breeder cocks.

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The insulin-like growth factors ( IGFS: ) are synthesized in tissues and play an important role in embryonic development of avian via autocrine/paracrine mechanisms. In the study, mRNA expression of IGFs were detected by real-time PCR in the muscle and liver from d 10 to 20 of chick embryo ( E10: to E20: ). Methylation of IGF1 promoter in the muscle was analyzed by bisulfite sequencing PCR as well as IGF2 promoter in the liver. These results showed that there was obviously IGF1 expression in liver at E19 and E20. The higher IGF1 expression in muscle was found during E15 to E18 with the peak on E17, and then declined. Correspondingly, the lowest methylation level of IGF1 promoter was detectable on the same embryonic d 17. Expression of IGF2 in muscle increased gradually during embryonic growth and showed higher level in the later stages (E17 to E20) when IGF1 expression began to decrease. IGF2 expression in liver reached the first peak on E14, then declined but gradually elevated from E17. IGF2 promoter methylation in liver showed gradual decline on d 12, 15, 17 and 19 of incubation, meanwhile IGF2 expression of liver increased gradually. These results suggested that IGF1 and IGF2 might separately be more important for muscle and liver growth in chick embryonic development. Variation of IGFs expression during the incubation might be concerned with the methylation of gene promoter. The profile of IGFs expression in chick embryonic tissues may be meaningful for understanding organ growth and embryonic development in chick.