Marine red yeast supplementation improves laying performance by regulating small intestinal homeostasis in aging chickens.

Recent studies have shown that age-related aging evolution is accompanied by imbalances in intestinal homeostasis. Marine red yeast (MRY) is a functional probiotic that has been shown to have antioxidant, immune and other properties. Therefore, we chose 900 healthy Hy-Line Brown hens at 433 d old as the research subjects and evaluated the correlation between intestinal health, laying performance, and egg quality in aged hens through the supplementation of MRY. These laying hens were assigned into 5 groups and received diet supplementation with 0%, 0.5%, 1.0%, 1.5%, and 2% MRY for 12 weeks. The results showed that MRY supplementation increased egg production rate, average egg weight, and egg quality, and decreased feed conversion ratio and daily feed intake (P < 0.05). The MRY supplement improved antioxidant indicators such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), stimulated villus height, and increased the villus height to crypt depth ratio (V/C ratio) in the intestine (P < 0.05). It also regulated the expression of intestinal inflammatory factors (transforming growth factor-ß [TGF-ß], interleukin [IL]-1ß, IL-8, tumor necrosis factor-α [TNF-α]) while increasing serum immunoglobulin G (IgG) levels (P < 0.05). Furthermore, MRY supplementation upregulated the mRNA expression of tight junction proteins (occludin and zonula occludens-1 [ZO-1]), anti-apoptotic gene (Bcl-2), and autophagy-related proteins (beclin-1 and light chain 3I [LC3I]) in the intestine (P < 0.05). The MRY supplement also led to an increase in the concentration of short-chain fatty acids in the cecum, and the relative abundance of the phylum Bacteroidetes, and genera Bacteroides and Rikenellaceae_RC9_gut_group. The LEfSe analysis revealed an enrichment of Sutterella and Akkermansia muciniphila. In conclusion, the results of this experiment indicated that the additional supplementation of MRY can improve the production performance of laying hens and may contribute to the restoration and balance of intestinal homeostasis, which supports the application potential of MRY as a green and efficient feed additive for improving the laying performance in chickens.

The role of AdipoQ on proliferation, apoptosis, and hormone Secretion in chicken primary adenohypophysis cells.

Adiponectin (AdipoQ), an adipokine secreted by adipocytes, has been reported to exist widely in various cell types and tissues, including the adenohypophysis of chickens. However, the molecular mechanism by which AdipoQ regulates the function of chicken adenohypophysis remains elusive. In this study, we investigated the effects of AdipoQ on proliferation, apoptosis, secretion of related hormones (FSH, LH, TSH, GH, PRL and ACTH) and expression of related genes (FSHß, LHß, GnRHR, TSHß, GH, PRL and ACTH) in primary adenohypophysis cells of chickens by using real-time fluorescent quantitative PCR (RT-qPCR), cell counting kit-8 (CCK-8), flow cytometry, enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) assays. Our results showed that AdipoQ promoted the proliferation of chicken primary adenohypophysis cells, up-regulated the mRNA expression of proliferation-related genes CDK1, PCNA, CCND1 and P21 (P < 0.05), as well as the increased protein expression of CDK1 and PCNA (P < 0.05). Furthermore, AdipoQ inhibited apoptosis of chicken primary adenohypophysis cells, resulting in down-regulation of pro-apoptotic genes Caspase3, Fas, and FasL mRNA expression, and decreased Caspase3 protein expression (P < 0.05). Moreover, there was an up-regulation of anti-apoptotic gene Bcl2 mRNA and protein expression (P < 0.05). Additionally, AdipoQ suppressed the secretion of FSH, LH, TSH, GH, PRL, and ACTH (P < 0.05), as well as the mRNA expression levels of related genes (P < 0.05). Treatment with AdipoRon (a synthetic substitute for AdipoQ) and co-treatment with RNA interference targeting AdipoQ receptors 1/2 (AdipoR1/2) had no effect on the secretion of FSH, LH, TSH, GH, PRL, and ACTH, as well as the mRNA expression levels of the related genes. This suggests that AdipoQ's regulation of hormone secretion and related gene expression is mediated by the AdipoR1/2 signaling axis. Importantly, we further demonstrated that the mechanism of AdipoQ on FSH, LH, TSH and GH secretion is realized through AMPK signaling pathway. In conclusion, we have revealed, for the first time the molecular mechanism by which AdipoQ regulates hormone secretion in chicken primary adenohypophysis cells.

Estrogen promotes gonadotropin-releasing hormone expression by regulating tachykinin 3 and prodynorphin systems in chicken.

The "KNDy neurons" located in the hypothalamic arcuate nucleus (ARC) of mammals are known to co-express kisspeptin, neurokinin B (NKB), and dynorphin (DYN), and have been identified as key mediators of the feedback regulation of steroid hormones on gonadotropin-releasing hormone (GnRH). However, in birds, the genes encoding kisspeptin and its receptor GPR54 are genomic lost, leaving unclear mechanisms for feedback regulation of GnRH by steroid hormones. Here, the genes tachykinin 3 (TAC3) and prodynorphin (PDYN) encoding chicken NKB and DYN neuropeptides were successfully cloned. Temporal expression profiling indicated that TAC3, PDYN and their receptor genes (TACR3, OPRK1) were mainly expressed in the hypothalamus, with significantly higher expression at 30W than at 15W. Furthermore, overexpression or interference of TAC3 and PDYN can regulate the GnRH mRNA expression. In addition, in vivo and in vitro assays showed that estrogen (E2) could promote the mRNA expression of TAC3, PDYN, and GnRH, as well as the secretion of GnRH/LH. Mechanistically, E2 could dimerize the nuclear estrogen receptor 1 (ESR1) to regulate the expression of TAC3 and PDYN, which promoted the mRNA and protein expression of GnRH gene as well as the secretion of GnRH. In conclusion, these results revealed that E2 could regulate the GnRH expression through TAC3 and PDYN systems, providing novel insights for reproductive regulation in chickens.

Evaluation of stimbiotic on growth performance and intestinal development of broilers fed corn- or wheat-based diets.

In the antibiotics-free era, stimbiotic (STB) has been suggested as a new alternative of antibiotic growth promoters to modulate intestinal health via stimulating dietary fiber utilization in poultry production. The aim of this study was to evaluate the effects of STB supplementation in corn- or wheat-basal diet on growth performance, intestinal development, and function of broilers. A total of 512 one-day-old Arbor Acres(AA)broilers were randomly allocated 4 treatments, including corn group (CG), corn + 100 g/t STB (CG + STB), wheat group (WG), wheat + 100 g/t STB (WG + STB). The broilers were weighed at the days of 14, 28, and 42, of which 8 repetitions per treatment were randomly selected to determine the intestinal morphology, intestinal barrier, and cecal microbiota and metabolites. Our data showed that STB increased (P < 0.05) feed intake, body weight and reduced FCR for the overall period (0-42 d). At 28 d of age, significant increases in villus height and the villus height-to-crypt depth ratio (V/C) were found in the STB supplementation groups (P < 0.05). Addition of STB significantly increased intestinal mucosal DAO and AMPK enzyme activity and the gene expression of OCLN, CLDN1, ZO1, MUC2, SGLT1, PEPT1, FABP2, Ghrelin, and GCG in jejunum (P < 0.05), and significantly decreased the expression of the PYY gene. In addition, STB increased the relative abundance of beneficial bacteria, such as Akkermansia, Bifidobacterium, and Oscillospirales (P < 0.05). A significant increase in cecal short-chain fatty acid (SCFAs) concentration was also observed in the STB supplementation groups. At the cellular level, STB cannot directly increase the expression of small intestinal epithelial cells, and may indirectly improve intestinal barrier function by increasing the level of sodium butyrate. Overall, these results indicated that STB supplementation could improve the growth performance, intestinal development and barrier functions, and fiber fermentation in cecum of broiler chickens.

Adiponectin inhibits GnRH secretion via activating AMPK and PI3K signaling pathways in chicken hypothalamic neuron cells.

It has been reported that adiponectin (AdipoQ), an adipokine secreted by white adipose tissue, plays an important role in the control of animal reproduction in addition to its function in energy homeostasis by binding to its receptors AdipoR1/2. However, the molecular mechanisms of AdipoQ in the regulation of animal reproduction remain elusive. In this study, we investigated the effects of AdipoQ on hypothalamic reproductive hormone (GnRH) secretion and reproduction-related receptor gene (estrogen receptor [ER] and progesterone receptor [PR]) expression in hypothalamic neuronal cells (HNCs) of chickens by using real-time fluorescent quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), Western blot (WB) and cell counting kit-8 (CCK-8) assays and found that overexpression of AdipoQ could increase the expression levels of AdipoR1/2 and reproduction-related receptor genes (P < 0.05) while decreasing the expression level of GnRH. In contrast, interference with AdipoQ mRNA showed the opposite results in HNCs. Furthermore, we demonstrated that AdipoQ exerts its functions through the AMPK and PI3K signaling pathways. Finally, our in vitro experiments found that AdipoRon (a synthetic substitute for AdipoQ) treatment and AdipoR1/2 RNAi interference co-treatment resulted in no effect on GnRH secretion, suggesting that the inhibition of GnRH secretion by AdipoQ is mediated by the AdipoR1/2 signaling axis. In summary, we uncovered, for the first time, the molecular mechanism of AdipoQ in the regulation of reproductive hormone secretion in hypothalamic neurons in chickens.

The adiponectin receptor agonist, AdipoRon, promotes reproductive hormone secretion and gonadal development via the hypothalamic-pituitary-gonadal axis in chickens.

Adiponectin is a key hormone secreted by fat tissues that has multiple biological functions, including regulating the energy balance and reproductive system by binding to its receptors AdipoR1 and AdipoR2. This study investigated the correlation between the levels of adiponectin and reproductive hormones in the hypothalamic-pituitary-ovarian (HPO) axis of laying hens at 4 different developmental stages (15, 20, 30, and 68 wk) and explored the effects of AdipoRon (an activator of adiponectin receptors) on the hypothalamic-pituitary-gonadal (HPG) axis and follicle and testicular Leydig cells in vitro and in vivo. The results demonstrated that the adiponectin level was significantly correlated with that of reproductive hormones in the HPO axis (e.g., GnRH, FSH, LH, and E2) in laying hens at 4 different ages. Moreover, AdipoRon could promote the expression of AdipoR1 and AdipoR2 and the secretion of reproductive hormones in the HPG axis, including GnRH, FSH, LH, P4, and T. AdipoRon could also upregulate the expression of genes related to follicular steroidogenesis (STAR, CYP19A1, CYP17A1, and CYP11A1), hepatic lipid synthesis (OVR, MTP), follicular lipid uptake (PPAR-g), and follicular angiogenesis (VEGFA1, VEGFA2, VEGFR1, ANGPT1, ANGPT2, TEK) in the oviposition period, and all of these findings were consistent with the results obtained from in vitro experiments after the transfection of small white follicles (SWFs) with AdipoRon. Furthermore, the results suggest that AdipoRon increases the diameter of testicular seminiferous tubules, the number of spermatogenic cells and sperm production in vivo and enhances the expression of AdipoR1, AdipoR2 and steroid hormones in vitro. Collectively, the findings suggest that AdipoRon could facilitate the expression and secretion of reproductive hormones in the HPG axis by activating its receptors and then improve the growth and development of follicles and testes in chickens.