The long-term selection for meat has led to the poor egg production efficiency in broiler. In this study, we analyzed the transcriptional levels of hypothalamus and ovary during the pre-laying (PP) and laying periods (LP) of broiler breeders. By combining these with the levels of reproductive hormones and ovarian metabolism, to reveal the neuroendocrine control mechanism of ovarian development. Results showed that during LP, the number of LYFs, SYFs and WFs, the thickness of the granular cell layer, and the serum LH, FSH, P4 and E2 levels were significantly increased (P < 0.05). A total of 1188 and 2481 differentially expressed genes (DEGs) were detected in hypothalamus and ovary, respectively. 1972 significantly differentially metabolites (DMs) were detected in ovary. In hypothalamus, the expression of neuroendocrine regulatory genes such as TRH, AVT, VIP, and NYB in the Neuroactive ligand-receptor interaction pathway regulated the LH and FSH secretion via the HPG axis. In ovary, the promotion of GCs proliferation may occur through the glycerophospholipid metabolism pathway, which increased the thickness of the GCs layer. This helped to receive gonadotropin signals and increased P4 and E2 secretion. Meanwhile, the decreased expression levels of ovarian development inhibitory factors in the TGF-beta signaling pathway, including BMP2, BMP4, BMP15 and AMHR2, and the increased expression levels of MMPs, including MMP9, MMP11 and MMP13, may regulate the synthesis of metabolites associated with steroid hormone secretion and ovarian development, such as E2, E2-3S, 7α-OH-DHEA, CHO and AD. These genes and metabolites may play an important role in HPG axis in regulating ovarian development.

OPN5 is one of the main deep brain photoreceptors (DBPs), converting photoperiodic information into neuroendocrine signals to regulate reproduction in birds. This study investigated the mechanism of OPN5-mediated photoperiodic regulation of reproduction by active immunization against OPN5. 96 female quail were divided into OPN5-immunized and control group under the same photoperiod: 16 L:8 D (d 1 to d 35), 8 L:16 D (d 36 to d 70) and 12 L:12 D (d 71 to d 126). OPN5-immunized group was conducted with OPN5 protein vaccination and control group was given a blank vaccine. Samples were collected on d 1, d 30, d 60, and d 126. Results showed switching photoperiod to 8 L:16 D decreased the laying rate, GSI%, numbers of YFs and WFs, serum levels of PRL, P4 and E2, and pituitary PRL and TSHβ protein expressions in both groups (P < 0.05). Whereas the OPN5-immunized group exhibited higher laying rates than the control group (P < 0.05). The control group showed reduced GnRHR and TSHβ gene expressions in the pituitary and increased GnIH and DIO3 transcript and/or protein abundance in the hypothalamus. (P < 0.05). The OPN5-immunized group had lower DIO3 expression at both mRNA and protein levels. (P < 0.05). Switching photoperiod from 8 L:16 D to 12 L:12 D increased the laying rates, GSI%, numbers of YFs and WFs, serum levels of PRL, and PRL protein expression in both groups (P < 0.05), and the responses were more pronounced in OPN5-immunized group (P < 0.05). In contrast to the control group, quail with OPN5-immunization had higher OPN5 and DIO2 transcript and/or protein levels but lower DIO3 expressions in the hypothalamus along the transition photoperiods (P < 0.05). The results revealed that OPN5 responds to photoperiod transition, and its activation mediates related signaling to up-regulate TSH-DIO2/DIO3 pathway and VIP-PRL secretion to prime quail reproductive functions.

1. Due to seasonal breeding, geese breeds from Southern China have low egg yield. The genetic makeup underlying performance of local breeds is largely unknown, and few studies have investigated this problem. This study integrated 21 newly generated and 50 publicly existing RNA-seq libraries, representing the hypothalamus, pituitary and testis, to identify candidate genes and importantly related pathways associated with seasonal breeding in male Lion-Head geese.2. In total, 19, 119 and 302 differentially expressed genes (DEGs) were detected in the hypothalamus, pituitary and testis, respectively, of male Lion-Head geese between non-breeding and breeding periods. These genes were significantly involved in the neuropeptide signalling pathway, gland development, neuroactive ligand-receptor interaction, JAK-STAT signalling pathway, cAMP signalling pathway, PI3K-Akt signalling pathway and Foxo signalling pathway.3. By integrating another 50 RNA-seq samples 4, 18 and 40 promising DEGs were confirmed in hypothalamus, pituitary and testis, respectively.4. HOX genes were identified as having important roles in the development of testis between non-breeding and breeding periods of male Lion-Head geese.

[目的]探究抗缪勒氏管激素(anti-Müllerian hormone,AMH)抑制山麻鸭卵泡发育的分子调控机制.[方法]选取40只29周龄产蛋山麻鸭,随机分为4组:AMH1、AMH2、AMH3和对照组,AMH1、AMH2和AMH3组给予2、20和200 μg/d AMH多肽;对照组给予同等体积的生理盐水,整个试验持续21 d;每天记录产蛋量,在试验末采集卵巢和卵泡进行计数,称重计算卵巢指数,测定血清促黄体生成素(luteinizing hormone,LH)、孕酮(progesterone,P4)和雌二醇(estradiol,E2)水平,检测等级卵泡(F6)、小黄卵泡(SYF)和大白卵泡(LWF)中卵泡刺激素受体(FSHR)、促黄体生成素受体(LHR)、类固醇生成急性调节蛋白(StAR)、3β-羟类固醇脱氢酶(3β-HSD)、雌激素合成酶(CYP19A1)、生长分化因子9(GDF9)、骨形态发生蛋白15(BMP15)基因表达量,以及F6和SYF中StAR、GDF9蛋白表达水平.[结果]与对照组相比,AMH2和AMH3组的产蛋率显著下降(P＜0.05),其中AMH3组对产蛋率的抑制作用最为明显;AMH1和AMH2组SYF和LWF数量显著上升(P＜0.05),而卵巢指数和LYF数量没有明显变化,血清LH、P4和E2水平呈剂量依赖性下降,AMH3组对生殖激素抑制作用较明显,其中AMH3组LH水平显著下降(P＜0.05).与对照组相比,在F6中,AMH2组GDF9基因和AMH1组BMP15基因表达水平显著上升(P＜0.05);在SYF中,除AMH2组GDF9基因外,各试验组FSHR基因表达水平显著下降,GDF9和BMP15基因表达水平显著上升(P＜0.05),AMH3组GDF9蛋白水平显著上升(P＜0.05);在LWF中AMH2和AMH3组的GDF9和BMP15基因表达水平显著上升(P＜0.05).给予外源性AMH对F6和LWF中CYP19A1、3β-HSD和StAR基因表达水平均无显著影响(P＞0.05),在SYF中,AMH1、AMH2和AMH3组中CYP19A1、3β-HSD和StAR基因表达水平均显著低于对照组(除AMH2组的CYP19A1基因外,P＜0.05);与对照组相比,给予外源性AMH对F6和SYF中StAR的蛋白水平均无显著影响(P＞0.05).[结论]200 μg/d外源性AMH可显著抑制卵泡发育,主要通过抑制LH、P4 和E2的分泌,上调卵泡中GDF9和BMP15表达水平,下调FSHR和LHR以及影响类固醇激素合成通路关键因子的表达来影响卵泡发育.

Photoperiod is a key environmental factor in regulating bird reproduction and induces neuroendocrine changes through the hypothalamic-pituitary-gonadal (HPG) axis. OPN5, as a deep-brain photoreceptor, transmits light signals to regulate follicular development through TSH-DIO2/DIO3. However, the mechanism among OPN5, TSH-DIO2/DIO3, and VIP/PRL in the HPG axis underlying the photoperiodic regulation of bird reproduction is unclear. In this study, 72 laying quails with 8-week-old were randomly divided into the long-day (LD) group [16 light (L): 8 dark (D)] and the short-day (SD) group (8 L:16 D), and then samples were collected on d 1, d 11, d 22, and d 36 of the experiment. The results showed that compared with the LD group, the SD group significantly inhibited follicular development (P < 0.05), decreased the P4, E2, LH, and PRL in serum (P < 0.05), downregulated the expression of GnRHR, VIP, PRL, OPN5, DIO2, and LHβ (P < 0.05), reduced the expression of GnRH and TSHβ (P > 0.05), and promoted DIO3, GnIH gene expression (P < 0.01). The short photoperiod downregulates OPN5, TSHβ, and DIO2 and upregulates DIO3 expression to regulate the GnRH/GnIH system. The downregulation of GnRHR and upregulation of GnIH resulted in a decrease in LH secretion, which withdrew the gonadotropic effects on ovarian follicles development. Slow down of follicular development and egg laying may also arise from lack of PRL potentiation to small follicle development under short days.

蛋鸭开产与卵巢发育有紧密联系,本实验旨在探索山麻鸭开产前后卵巢形态变化及基因表达的差异,鉴别参与启动开产的关键基因,为后期蛋鸭开产相关的分子遗传机制研究奠定基础.在山麻鸭开产前后的S1期(81日龄)和S2期(137日龄)分别选取3个个体的卵巢基质进行转录组测序,对转录组数据开展差异表达分析、功能富集分析及关键基因筛选,同时对关键基因进行荧光定量PCR(qRT-PCR)验证.在差异倍数log2转换的绝对值(|log2(FoldChange)|)大于1.5、矫正P值<0.05的筛选条件下鉴别出382个差异表达基因.基于382个差异表达基因的GO功能注释发现与卵泡生长和卵巢功能调节相关的通路:细胞外基质的组织、细胞外基质结构成分和含有胶原蛋白的细胞外基质相关途径;KEGG富集分析发现上述基因显著富集于ECM受体相互作用和PI3K-Akt信号通路.在涉及上述重要富集通路的基因中有9个基因交集:CD44、COL1A1、COL6A1、CREB3L1、ITGB2、THBS2、VWF、VEGFC、WNT5B,通过qRT-PCR验证了这9个基因在转录组数据差异表达方面的可靠性.在这9个基因中,COL1A1、COL6A1、ITGB2和VWF已在之前研究中报道与卵泡发育有关,而CD44、CREB3L1、THBS2、VEGFC、WNT5B基因为本研究新发现.本研究为后期山麻鸭开产相关性状的分子遗传机制研究及分子标记辅助选择奠定基础.

Photoperiod is an important environmental factor that influence seasonal reproduction behavior in bird and GnIH can play a function in this process through the reproductive axis, and some studies suggest that GnIH may have a direct role at the gonadal level. To investigate the expression of GnIH and its effects on follicle development and steroidogenesis in quail ovaries under different photoperiods, 72 healthy laying quails of 8-wk-old were randomly divided into long day (LD) group [16 light (L): 8 dark (D)] (n = 36) and short day (SD) group (8L:16D) (n = 36). Samples were collected from each group on d1, d11, d22, and d36 of the experiment. The result showed that short day treatment upregulated the level of GnIH in the gonads (P < 0.05), decreased the expression level of CYP19A1,3β-HSD, StAR, LHR, and FSHR and increased the expression level of AMH, AMHR2, GDF9, and BMP15 to inhibit follicle development and ovulation, thus affecting the egg production performance of quails. In vitro culture of quail granulosa cells and treatment with different concentrations of GnIH (0, 1, 10, and 100 ng/mL) for 24 h. Result showed that GnIH inhibited the levels of FSHR, LHR, and steroid synthesis pathways in granulosa cells, upregulated the levels of AMHR2, GDF9, and BMP15. The results suggest that the inhibition of follicle development and reduced egg production in quail by short day treatment is due to GnIH acting at the gonadal level, and GnIH affected the steroid synthesis by inhibiting gonadotropin receptors.

Stocking density critically affects the growth and subsequent performance of animals in modern poultry production. This study investigated the effects of stocking density on ovarian development, ovarian maturation, and the mRNA expression of key genes in the reproductive axis during the rearing period of Shan-ma ducks. The experiments involved 180 healthy 7-wk-old Shan-ma ducks and randomly divided into low stocking density (LSD; n = 30, density = 5 birds/m2), medium stocking density (MSD; n = 60, density = 10 birds/m2) and high stocking density groups (HSD; n = 90, density = 15 birds/m2), for rearing. After examining ovarian development and measuring hormone levels in the plasma and expression levels of key regulatory genes in the reproductive axis at 19 wk of rearing, analysis of the gonad index analysis, reflecting stocking density, uncovered statistically significant differences. The gonad index of the LSD group was significantly higher than those of the MSD and HSD groups (P < 0.01), while no significant difference was observed between the MSD and HSD groups. pre-ovulatory follicles (POFs) and small yellow follicles (SYFs) development was only apparent in the LSD group, with the large white follicles (LWFs) number of this group being significantly higher than that of the MSD group (P < 0.05). The blood levels of E2 (estradiol), P4 (progesterone), and T (testosterone) were significantly higher in the LSD group than in the MSD and HSD groups (P < 0.05 or 0.01). Also, the levels of both P4 and T were significantly higher in the MSD group than in the HSD group (P < 0.01). The gene expression levels of GnRHR, FSH, AMHR, and FSHR were significantly increased in the LSD group compared to the MSD and HSD groups (P < 0.05 or 0.01), while the expression levels of GnIHR and GDF9 were significantly decreased in the LSD and MSD groups compared to the HSD group (P < 0.05 or 0.01). Steroid biosynthesis pathway genes such as StAR, CYP11A1, 3β-HSD, CYP19A1, and BMP15 were significantly downregulated at greater stocking densities (P < 0.05 or 0.01). Likewise, the protein expression of StAR, 3β-HSD, and CYP19A1 was also significantly decreased (P < 0.05 or 0.01). These results demonstrate that both medium and high stocking densities suppressed the expression of the key reproduction-promoting factors, while the expression level of the key reproductive inhibitory factors was enhanced. Therefore, rates of ovarian development and maturation could be reduced by a high stocking density leading to a delay in reproduction performance during the rearing period of Shan-ma ducks.