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ABSTRACT: Photoperiod is an important environmental factor that influences seasonal reproduction behavior in birds. Birds translate photoperiodic information into neuroendocrine signals through deep brain photoreceptors (DBPs). OPN5 has been considered candidate DBPs involved in regulating seasonal reproduction in birds. We found that OPN5 could mediate light to regulate the follicle development in ducks. In this study, we further verified the effect of OPN5 on follicular development in Shan Partridge ducks by immunizing against the extracellular domain (ECD) of OPN5. We investigated the specific regulatory mechanism of photoperiod mediated by OPN5 on the reproductive activity of ducks. The trial randomly divided 120 Shan Partridge ducks into 3 groups with different treatments: the immunization of OPN5 group was done at d0, d15, d30, and d40 with 1 mL of vaccine containing OPN5 protein (thus containing 1, 1, 0.5, and 0.5 mg of OPN5-KLH protein), and the control group (CS and CL groups) was injected at the same time with the same dose of OPN5-uncontained blank vaccine. The group of CS (900 lux), OPN5 (600 lux), and CL (600 lux) lasted for 40 d in 12 L:12 D photoperiods, respectively. Then, the groups of CS, OPN5, and CL subsequently received 12 L:12 D, 12 L:12 D, and 17 L:7 D light treatments for 33 d, respectively. The ducks were caged in 3 constant rooms with the same feeding conditions for each group, free water, and limited feeding (150 g per duck each day). Duck serum and tissue samples were collected at d 40, d 62, and d 73 (n = 12). It was found that before prolonged light, the group of immunization (group OPN5) and the group of strong light intensity (group CS) were higher than the group of CL in egg production. Subsequent to prolonged light, the group CL in egg production rose about the same as the group immunization, while the strong light group (group CS) was lower. Group OPN5 increased the ovarian index of ducks, and both the immunization of group OPN5 and group CL (extended light) increased the thickness of the granular layer and promoted the secretion of E2, P4, LH, and PRL hormones. Compared with group CS, group CL and OPN5 increased the mRNA level and protein expression of OPN5 in the hypothalamus on d 62 and d 73 (P < 0.05). The gene or protein expression patterns of GnRH, TRH, TSHβ, DIO2, THRβ, VIP, and PRL were positively correlated with OPN5, whereas the gene expression patterns of GnIH and DIO3 were negatively correlated with OPN5. The results showed that immunization against OPN5 could activate the corresponding transmembrane receptors to promote the expression of OPN5, up-regulate the expression of TSHβ and DIO2, and then regulate the HPG axis-related genes to facilitate the follicular development of Shan Partridge ducks. In addition, in this experiment, prolonging the photoperiod or enhancing the light intensity could also enhance follicle development, but the effect was not as significant as immunizing against OPN5. Our results will offer beneficial data and more supportive shreds of evidence in favor of elucidating the role of OPN5 in relation to photoperiods and reproduction.

ABSTRACT: The mitochondrial quality control system is crucial in maintaining cellular homeostasis during environmental stress. Granulosa cells are the main cells secreting steroid hormones, and mitochondria are the key organelles for steroid hormone synthesis. The impact of the mitochondrial quality control system on granulosa cells’ steroid hormone synthesis and survival under heat stress is still unclear. Here, we showed that acute heat stress induces mitochondrial damage and significantly increases the number of mitophagy-like vesicles in the cytoplasm of duck ovary granulosa cells at the ultra-structural level. Meanwhile, we also found heat stress significantly increased mitochondrial fission and mitophagy-related protein expression levels both in vivo and in vitro. Furthermore, by confocal fluorescence analysis, we discovered that LC3 was distributed spot-like manner near the nucleus in the heat treatment group, and the LC3 spots and lysosomes were colocalized with Mito-Tracker in the heat treatment group. We further detected the mitophagy-related protein in the cytoplasm and mitochondria, respectively. Results showed that the PINK1 protein was significantly increased both in cytoplasm and mitochondria, while the LC3-Ⅱ/LC3-Ⅰ ratio increase only occurred in mitochondrial. In addition, the autophagy protein induced by acute heat treatment was effectively inhibited by the mitophagy inhibitor CysA. Finally, we demonstrated that the alteration of cellular mitophagy by siRNA interference with Drp1 and PINK1 inhibited the steroid synthesis of granulosa cells and increased cell apoptosis. Study provides strong evidence that the Drp1 regulated PINK1-dependent mitophagy pathway protects follicular granulosa cells from acute heat stress-induced injury.

ABSTRACT: This study sought to understand the regulation mechanism of OPN5 through the TSH-DIO2/DIO3 pathway mediated photoperiod on the breeding activity of short-day breeding birds. In this study, the reproductive activity of Magang goose was regulated by artificial light, and the reproductive activity of the ganders were determined according to the daily laying rate of female geese. The testicular development and the serum reproductive hormone concentrations of ganders were measured during the reproductive period (d 0), the reproductive degeneration period (d 13 and 27) and the resting period (d 45). The mRNA and protein expression patterns of OPN5, the HPG axis reproductive genes, and TSH-DIO2/DIO3 pathway related genes were examined. Results showed that the laying rate of geese and the gonadal indices (GSI) decreased gradually after the photoperiod increased. Histological observation found that the spermatogenic function of the testis was normal on d 0 and 13, while degeneration occurred by d 27 and 45. Serum testosterone, FSH, and LH concentration showed a slight increase on d 13, followed by a sharp decrease on d 27 and 45 (P < 0.01), while PRL concentrations were low on d 0 and 13, and increased rapidly on d 27 and 45 (P < 0.01).The expression pattern of GnRH, FSH, LH, and THRβ mRNA were similar, with high levels on d 0 and 13 and a decreasing trend on d 27 and 45 (P < 0.05 or P < 0.01); and GnRHR mRNA levels were higher on d 13 (P < 0.05), but then had decreased by d 27 and 45 (P < 0.01). The expression pattern of GnIH and GnIHR was similar, which was opposite to that of GnRHR. VIP, PRL, and PRLR increased gradually and peaked on d 45 (P < 0.01). The expression trend of TRH, TSHβ, and DIO2 was similar to that of GnRHR, and the expression abundance increased on d 13, and then decreased on d 27 and 45. GnRH protein expression was significantly higher than during the other 3 periods (P < 0.01) while the GnIH protein levels were extremely low on d 0, had gradually increased by d 13, and significantly increased by d 27 and 45 (P < 0.01). The protein expression trends of THR and DIO2 were similar to that of GNIH. DIO3 protein expression was low on d 0 and 13, and increased by d 27 and 45. These results suggest that when the photoperiod increased, the hypothalamus OPN5 gene and protein were upregulated and the pituitary TSHβ, TSHR, and hypothalamus THRβ, TRH, and DIO2 were downregulated, and thus the reproductive activity of geese was inhibited.

Abstract In quantitative PCR research, appropriate reference genes are key to determining accurate mRNA expression levels. In order to screen the reference genes suitable for detecting gene expression in tissues of the reproductive axis, a total of 420 (males and females = 1:5) 3-year-old Magang geese were selected and subjected to light treatment. The hypothalamus, pituitary and testicular tissues were subsequently collected at different stages. Ten genes including HPRT1, GAPDH, ACTB, LDHA, SDHA, B2M, TUBB4, TFRC, RPS2 and RPL4 were selected as candidate reference genes. The expression of these genes in goose reproductive axis tissues was detected by real-time fluorescent quantitative PCR. The ΔCT, geNorm, NormFinder and BestKeeper algorithms were applied to sort gene expression according to stability. The results showed that ACTB and TUBB4 were the most suitable reference genes for the hypothalamic tissue of Magang goose in the three breeding stages; HPRT1 and RPL4 for pituitary tissue; and HPRT1 and LDHA for testicular tissue. For all three reproductive axis tissues, ACTB was the most suitable reference gene, whereas the least stable reference gene was GAPDH. Altogether, these results can provide references for tissue expression studies in geese under light treatment.

ABSTRACT: 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.

ABSTRACT: Resveratrol, a natural antioxidant, anti-inflammatory plant extract, was found to have a protective effect in poultry subjected to heat stress. In this study, we strove to characterize resveratrol on intestinal of duck exposed to acute heat stress and investigate the underlying mechanism. A total of 120 Shan-ma ducks (60 days old) were randomly divided into 2 groups. The control group was fed a basal diet, and the resveratrol group was fed a basal diet supplemented with 400 mg/kg resveratrol. Animals in 2 groups were kept at a temperature of 24°C ± 2°C for 15 d. Then, animals of both groups were placed in an artificial climate room at 39°C. Twelve ducks of each group were sacrificed for sampling at 0, 30, and 60 min, respectively. Results indicated that resveratrol increased the ratio of villus height to crypt depth, increased the number of goblet cells, and reduced the histopathological damage of jejunum caused by acute heat stress. Furthermore, the gene expression of heat shock proteins (HSP60, HSP70, and HSP90) and tight junction proteins (CLDN1 and OCLN) was significantly increased in the resveratrol group compared to that in the control groups. Simultaneously, resveratrol significantly activated the SIRT1-NRF1/NRF2 signaling pathways, improved ATP level of jejunum, and increased SOD and CAT antioxidant enzymes activities. In addition, we found that the NF-κB/NLRP3 inflammasome signaling pathways were repressed under acute heat stress. Meanwhile, supplement resveratrol further inhibited the NLRP3 inflammasome pathway, decreased protein level of NLRP3 and caspase1 p20, reduced the secretion of IL-1β. Taken together, our results indicate that resveratrol against the oxidative damage and inflammation injury in duck jejunum induced by heat stress via active SIRT1 signaling pathways.

Granulosa cells (GCs) play an important role in the development of follicles. In this study, we investigate the impact of heat stress at 41°C and 43°C on duck GCs' proliferation and steroids secretion. And, the transcriptomic responses to heat treatment were examined using RNA-sequencing analysis. Digital gene expression profiling was used to screen and identify differentially expressed genes (fold change = 2 and Q value < 0.05). Further, the differential expression genes (DEGs) were classified into GO categories and KEGG pathways. The results show that duck GCs blocked in the G1 phase were increased on exposure to heat stress. Meanwhile, the expression of proliferative genes, which were essential for the transition from G1 to S phase, was inhibited. At the same time, heat stress inhibited the estradiol synthesis of GCs by decreasing CYP11A1 and CYP19A1 gene expression. A total of 241 DEGs including 181 upregulated and 60 downregulated ones were identified. Transcriptome result shows that heat shock protein and CXC chemokines gene were significantly activated during heat stress. While collagenases (MMP1 and MMP13) and strome lysins (MMP3) were downregulated. And, the hedgehog signaling pathway may be a prosurvival adaptive response under heat stress. These results offer a basis for better understanding the molecular mechanism underlying lay-eggs-less in ducks under heat stress. [ABSTRACT FROM AUTHOR]