关键词: RNA-seq, 育成期, 代谢能摄入量, 雌激素, 卵巢, 蛋鸡

Abstract:

This experiment was conducted to evaluate the effects of metabolizable energy (ME) intake during rearing on the growth and development of reproductive organ, hormone level and gene expression in ovary of laying hens. A total of 720 6-week-old Hyline-Brown laying hens were allocated equally to three groups with six replicates of 40 hens each, and were fed one of three diets that were nutritionally equal with the exception of ME levels. From 6 to 17 weeks of age, the crude protein (CP) levels in diet were 17.5% (6-12 weeks) and 15.5% (12-17 weeks), respectively, and the ME levels in diet were 12.34, 11.11 (12.34×90%) and 9.87(12.34×80%) MJ ·kg^-1 (control group, also called ad libitum feeding group, 90% energy-restricted feeding group, and 80% energy-restricted feeding group), respectively. The hens in control group were fed ad libitum, and the daily amount of feed in experimental groups was restricted to the absolute quantity of the diet consumed by hens in control group. The results showed as follows: 1) Body weight, body slope length and shank circumference of hens at 17 weeks of age decreased linearly (P < 0.001) with increasing levels of energy restriction. 2) The serum UN and GLU content decreased linearly (P=0.040 and 0.044, respectively), while the plasma oestradiol concentrations increased linearly (P=0.026). 3)The ovary stroma of hens in the ad libitum feeding group (ALF17W) and 80% energy-restricted feeding group (ERF17W) with significant differences (P < 0.05) in plasma oestradiol concentrations were used to screen the novel mRNA implemented by the RNA-seq. The proportion of pure reads matching to chicken reference genome was more than 94.61%, and the content of Q20 and Q30 was more than 97.38% and 92.87%, respectively. A total of 1 299 differential genes were screened in ALF17W and ERF17W, of which 961 were down-regulated and 338 were up-regulated in ERF17W. The GO functional enrichment analysis found that differentially expressed mRNAs were involved in 48 GO terms such as cell proliferation, development, and reproduction. The KEGG pathway were significantly enriched in 25 pathways, among these pathways, the cAMP signaling pathway, estrogen signaling pathway, steroid hormone biosynthesis and ovarian steroidogenesis pathway were related to energy metabolism or reproduction. The screened cAMP response element-binding protein (CREB) and steroid-producing acute regulatory protein (StAR) are enriched in the cAMP signaling pathway, and progesterone receptor (PGR), metabotropic glutamate receptor 1 (GRM1) and cytoskeletal keratin (KRT18) are enriched in the estrogen signaling pathway, they may be the potential target genes of ME intake regulating estrogen production in laying hens. The qRT-PCR results showed that the expression trends of 10 randomly selected differentially expressed genes were consistent with the RNA-Seq results. The above results showed that the body weight, serum UN and GLU decreased linearly with the decrease of ME intake during the rearing period, but plasma oestradiol level increased linearly. It is suggested that ME intake during the rearing period may regulate the expression of StAR, CREB1, KRT18, PGR and GRM1 genes in ovarian tissues, and act on the cAMP and estrogen signaling pathways to regulate the production of estrogen in laying hens.

Key words: RNA-seq, rearing period, ME intake, estrogen, ovary, laying hens