Bioactive Lignan Honokiol Alleviates Ovarian Oxidative Stress in Aging Laying Chickens by Regulating SIRT3/AMPK Pathway.

Aging is not only a key internal cause of age-related diseases in humans but also poses a threat to the productivity of farm animals with longer breeding cycles, such as laying chickens. Various measures were taken to prolong the laying period by reducing oxidative stress to improve poultry ovarian functions. Within the mitochondria, SIRT3, a member of the Sirtuin family, plays an important role in post-translational modifications and the regulation of protein activities involved in energy metabolism and oxidative response. This study aimed to investigate the alleviating effect of a bioactive lignan Honokiol (HKL) on oxidative stress in aging chicken ovaries in order to retard decline in egg production. The results showed that HKL treatment restored the abnormal balance between cell proliferation and apoptosis, and it enhanced the antioxidant capacity of the H2O2-induced small white follicles (SWFs) by activating the SIRT3/AMPK pathway. Moreover, HKL significantly increased total egg production, the number of yellow follicles, and the mRNA expression of yolk synthesis and deposition-related genes, serum estrogen, and antioxidant levels. These findings suggest that HKL holds promise in enhancing the egg productivity of aging laying chickens by promoting yolk deposition and reducing ovarian oxidative stress.

Nobiletin Ameliorates Aging of Chicken Ovarian Prehierarchical Follicles by Suppressing Oxidative Stress and Promoting Autophagy.

With the increase in the age of laying chickens, the aging of follicles is accelerated, and the reproductive ability is decreased. Increased oxidative stress and mitochondrial malfunction are indispensable causes of ovarian aging. In this study, the physiological condition of prehierarchical small white follicles (SWFs) was compared between D280 high-producing chickens and D580 aging chickens, and the effect of a plant-derived flavonoid nobiletin (Nob), a natural antioxidant, on senescence of SWFs granulosa cells (SWF-GCs) was investigated. The results showed that Nob treatment activated cell autophagy by activating the AMP-activated protein kinase (AMPK) and Sirtuin-1 (SIRT1) pathways in D-galactose (D-gal)-generated senescent SWF-GCs, restoring the expression of proliferation-related mRNAs and proteins. In addition, the expression of inflammation-related protein NF-κB was significantly enhanced in aging GCs that were induced by D-gal. Nob supplementation significantly increased the antioxidant capacity and decreased the expression of several genes associated with cell apoptosis. Furthermore, Nob promoted activation of PINK1 and Parkin pathways for mitophagy and alleviated mitochondrial edema. Either the AMPK inhibitor dorsomorphin (Compound C) or SIRT1 inhibitor selisistat (EX-527) attenuated the effect of Nob on mitophagy. The protective effect of Nob on natural aging, GC proliferation, and elimination of the beneficial impact on energy regulation of naturally aging ovaries was diminished by inhibition of Nob-mediated autophagy. These data suggest that Nob treatment increases the expression of mitophagy-related proteins (PINK1 and Parkin) via the AMPK/SIRT1 pathways to prevent ovarian aging in the laying chickens.

Krüppel-like factor 5 activates chick intestinal stem cell and promotes mucosal repair after impairment.

The mucosal renewal, which depends on the intestinal stem cell (ISC) activity, is the foundation of mucosal repairment. Importantly, activation of reserve ISCs (rISCs) plays a vital role in initiating mucosal repair after injury. However, the underlying regulatory mechanism of rISCs activation in chickens remains unclear. In this study, immediately after lipopolysaccharide (LPS) challenge, mitochondrial morphological destruction and dysfunction appeared in the crypt, accompanied by decreased epithelial secretion (decreased Muc2 mRNA abundance and LYSOZYME protein level). However, immediately after mucosal injury, the mucosal renewal accelerated, as indicated by the increased BrdU positive rate, proliferating cell nuclear antigen (PCNA) protein level and mRNA abundance of cell cycle markers (Ccnd1, Cdk2). Concerning the ISCs activity, during the early period of injury, there appeared a reduction of active ISCs (aISCs) marker Lgr5 mRNA and protein, and an increasing of rISCs marker Hopx mRNA and protein. Strikingly, upon LPS challenge, increased mRNA transcriptional level of Krüppel-like factor 5 (Klf5) was detected in the crypt. Moreover, under LPS treatment in organoids, the KLF5 inhibitor (ML264) would decrease the mRNA and protein levels of Stat5a and Hopx, the STAT5A inhibitor (AC-4-130) would suppress the Lgr5 mRNA and protein levels. Furthermore, the Dual-Luciferase Reporter assay confirmed that, KLF5 would bind to Hopx promoter and activate the rISCs, STAT5A would trigger Lgr5 promoter and activate the aISCs. Collectively, KLF5 was upregulated during the early period of injury, further activate the rISCs directly and activate aISCs via STAT5A indirectly, thus initiate mucosal repair after injury.

Protective effect of rutin on ferroptosis-induced oxidative stress in aging laying hens through Nrf2/HO-1 signaling.

Oxidative stress is a major cause of ovarian aging and follicular atresia. There is growing evidence that showed potential roles of rutin in antidiabetic, anti-inflammatory, antitumor, antibacterial and antioxidant, although it is yet unclear what the underlying mechanism is. Here, we looked into the potential effects of rutin on oxidative stress in the prehierarchical small white follicles (SWFs) from 580-day-old (D580) laying chickens. According to the findings, aging D580 layer ferroptosis was much higher than it was for laying hens during the peak period (280-day-old, D280). In both naturally aged and d-gal-induced chicken SWFs, rutin treatment concurrently boosted cell proliferation and prevented apoptosis. In addition, rutin inhibited the increased ferroptosis in aging hens. Meanwhile, rutin markedly suppressed the elevated ferroptosis and descending antioxidant capacity of D280-culture-SWFs from chicken elicited by d-gal. Rutin's activation of the Nrf2/HO-1 pathway hastened the SWFs' verbal battle with oxidative damage and reduced ferroptosis. Furthermore, activation of the ferroptosis signal increased the oxidative damage in SWFs. In conclusion, rutin alleviated oxidative stress that was induced by ferroptosis in aging chicken SWFs through Nrf2/HO-1 pathway. These findings point to a novel mechanism by which rutin protects SWFs from oxidative stress by suppressing ferroptosis, which is presumably a fresh approach to slowing ovarian aging in laying hens.

Lactobacillus salivarius Promotion of Intestinal Stem Cell Activity in Hens Is Associated with Succinate-Induced Mitochondrial Energy Metabolism.

Currently, the regulation of Lactobacillus on intestinal stem cells (ISCs) attracts broad attention, but their active ingredients and the underlying mechanism are worthy of further study. Previously, host intestinal commensal bacteria were verified to drive the differentiation of ISCs. In this study, the strong bacteriostatic activity of Lactobacillus salivarius and Lactobacillus agilis were illustrated, and the components (supernatant, precipitation) of L. salivarius or L. agilis were further demonstrated to decrease the differentiation of ISCs in vivo. Interestingly, antibiotics feeding decreased ISCs differentiation in vivo as well. However, the administration of L. salivarius supernatant following antibiotics feeding was shown to promote ISCs differentiation dramatically when compared with the antibiotics feeding group, indicating that some active ingredients existed in its supernatant to promote ISCs activity. Strikingly, in vitro, the treatment of L. salivarius supernatant was further confirmed to promote the intestinal organoids' size, budding, and LGR5 expression. Next, the metabolomics analysis of Lactobacilli' supernatants suggested that succinate might be a crucial metabolite to promote ISCs activity. Further, the succinate treatment in vitro (1000 µM) and in vivo (50 mM) was confirmed to enhance the expression of LGR5 and PCNA. SLC13A3 (a sodium/dicarboxylate cotransporter) was detected in the intestinal organoids and demonstrated to transport succinate into ISCs, as confirmed by the contact of FITC-succinate with ISCs nucleus. Subsequently, high mitochondrial membrane potential and reactive oxygen species levels appeared in the intestinal organoids upon succinate treatment. Collectively, the promotion of L. salivarius on ISCs activity is associated with succinate-induced mitochondrial energy metabolism. IMPORTANCE In our previous study, Lactobacillus salivarius and Lactobacillus agilis were demonstrated to regulate intestinal stem cell activity in hens, but their active ingredients and the underlying mechanism remain unclear. In this study, L. salivarius supernatant was shown to directly promote intestinal stem cell activity. Furthermore, the succinate (a critical metabolite of L. salivarius) was screened out to promote intestinal stem cell activity. Moreover, the succinate was confirmed to enter intestinal stem cells and induce high mitochondrial energy metabolism, finally promoting intestinal stem cell activity. These findings will advance uncovering the mechanism by which Lactobacillus regulate intestinal stem cell activity in chickens.

Follicle-Stimulating Hormone Alleviates Ovarian Aging by Modulating Mitophagy- and Glycophagy-Based Energy Metabolism in Hens.

As a predominant hormone in the reproductive axis, follicle-stimulating hormone (FSH) is known as the primary surviving factor for follicular growth. In this study, the alleviating effect of FSH on aging chicken granulosa cells (GCs) was investigated. Results showed that FSH activated mitophagy and relieved mitochondrial edema in D-gal-induced senescent GCs, which was evidenced by an increased number of mitophagosomes as well as increased mitochondria-light chain 3 (LC3) colocalization. Mitophagy activation was accompanied by the activation of the AMP-activated protein kinase (AMPK) signaling pathway. Furthermore, upregulated glycophagy was demonstrated by an increased interaction of starch-binding domain protein 1 (STBD1) with GABA type A receptor-associated protein-like 1 (GABARAPL1) in D-gal-induced senescent GCs. FSH treatment further promoted glycophagy, accompanied by PI3K/AKT activation. PI3K inhibitor LY294002 and AKT inhibitor GSK690693 attenuated the effect of FSH on glycophagy and glycolysis. The inhibition of FSH-mediated autophagy attenuated the protective effect of FSH on naturally aging GC proliferation and glycolysis. The simultaneous blockage of PI3K/AKT and AMPK signaling also abolished the positive effect of FSH on naturally senescent ovarian energy regulation. These data reveal that FSH prevents chicken ovarian aging by modulating glycophagy- and mitophagy-based energy metabolism through the PI3K/AKT and AMPK pathways.

Mechanistic elucidation of freezing-induced surface decomposition of aluminum oxyhydroxide adjuvant.

The freezing-induced aggregation of aluminum-based (Alum) adjuvants has been considered as the most important cause of reduced vaccine potency. However, the intrinsic properties that determine the functionality of Alum after freezing have not been elucidated. In this study, we used engineered aluminum oxyhydroxide nanoparticles (AlOOH NPs) and demonstrated that cryogenic freezing led to the mechanical pressure-mediated reduction of surface hydroxyl. The sugar-based surfactant, octyl glucoside (OG), was demonstrated to shield AlOOH NPs from the freezing-induced loss of hydroxyl content and the aggregation through the reduction of recrystallization-induced mechanical stress. As a result, the antigenic adsorption property of frozen AlOOH NPs could be effectively protected. When hepatitis B surface antigen (HBsAg) was adjuvanted with OG-protected frozen AlOOH NPs in mice, the loss of immunogenicity was inhibited. These findings provide insights into the freezing-induced surface decomposition of Alum and can be translated to design of protectants to improve the stability of vaccines.

Naringin prevents follicular atresia by inhibiting oxidative stress in the aging chicken.

Oxidative stress is an essential inducement in follicle atresia and ovarian aging, resulting in decline in female fecundity. As a natural and effective antioxidant, naringin was investigated to relieve chicken follicle atresia and ovarian aging. First, the cultured small white follicles (SWFs) from D280 hens were pretreated with 0.5 mM naringin for 24 h and then treated with H2O2 for 72 h to establish the oxidative stress model to evaluate the putative attenuating effects of naringin on follicle atresia. Meanwhile, SWFs of D580 hens were treated with naringin for 72 h to examine the attenuating effect on the physiological aging of SWFs. Finally, each hen was fed with naringin at a dose of 50 mg/kg every day to explore the effect of naringin on follicular development and laying performance in D580 hens. Results showed that naringin could rescue the antioxidant capacity decline by increasing the antioxidant-related indexes and expression of antioxidation-associated genes. It could also maintain the homeostasis of SWFs in both the H2O2-induced group and natural physiological aging group. In addition, naringin increased estrogen levels, capacity of antioxidants, and the laying performance in aged laying chickens. The thickness and strength of the eggshell were increased in the naringin-treated group as well. In conclusion, this study showed that naringin is capable of relieving SWFs atresia that was induced by oxidative stress and maintaining the laying performance of aging low-yielding hens by reducing oxidative stress.

Autophagy participates in germline cyst breakdown and follicular formation by modulating glycolysis switch via Akt signaling in newly-hatched chicken ovaries.

In females, the establishment of the primordial follicle pool is accompanied by a remarkable programmed oocyte loss for unclear reasons. In this study, the role of autophagy was investigated to serve as a protective mechanism for oocyte survival during chicken folliculogenesis. Inhibition of autophagy by 3-methyladenine (3-MA) led to a remarkable delay in germ cell cyst breakdown that resulted in fewer primordial follicles and retarded sequent follicular development either in vivo or in the ovarian organ culture. Furthermore, the glycolysis level was downregulated in ovaries treated with 3-MA, while Recilisib (a specific activator of Akt) reversed this inhibiting effect of 3-MA on primordial folliculogenesis. Collectively, these data indicate that autophagy functions to maintain germ cell cyst breakdown and primordial follicle assembly by regulating ovarian glycolysis involving Akt signaling in the ovaries of newly-hatched chickens.

Protective Effect of Follicle-Stimulating Hormone on DNA Damage of Chicken Follicular Granulosa Cells by Inhibiting CHK2/p53.

The increase in follicular atresia and the decrease in the fecundity of laying hens occur with the aging process. Therefore, the key measure for maintaining high laying performance is to alleviate follicular atresia in the aging poultry. Follicle-stimulating hormone (FSH), as an important pituitary hormone to promote follicle development and maturation, plays an important role in preventing reproductive aging in diverse animals. In this study, the physiological state of the prehierarchical small white follicles (SWFs) and atretic SWFs (ASWFs) were compared, followed by an exploration of the possible capacity of FSH to delay ASWFs' progression in the hens. The results showed that the DNA damage within follicles increased with aging, along with Golgi complex disintegration, cell cycle arrest, increased apoptosis and autophagy in the ASWFs. Subsequently, the ACNU-induced follicular atresia model was established to evaluate the enhancing capacity of FSH on increasing cell proliferation and attenuating apoptosis in ASWFs. FSH inhibited DNA damage and promoted DNA repair by regulating the CHK2/p53 pathway. Furthermore, FSH inhibited CHK2/p53, thus, suppressing the disintegration of the Golgi complex, cell cycle arrest, and increased autophagy in the atretic follicles. Moreover, these effects from FSH treatment in ACNU-induced granulosa cells were similar to the treatment by a DNA repair agent AV-153. These results indicate that FSH protects aging-resulted DNA damage in granulosa cells by inhibiting CHK2/p53 in chicken prehierarchical follicles.

Protective Effect of Grape Seed Proanthocyanidins on Oxidative Damage of Chicken Follicular Granulosa Cells by Inhibiting FoxO1-Mediated Autophagy.

A significant decrease in poultry egg production occurs due to ovarian aging and autophagy is one of the important factors of ovarian aging that is induced predominantly by oxidative stress. Increasing evidence showed potential roles of plant-derived grape seed proanthocyanidin (GSPs) in protecting ovarian granulosa cells (GCs) from oxidative damage, although the underlying mechanism is still unclear. Here we investigated the possible functions of autophagy involved in the preventive effect of GSPs on oxidative stress in the GCs of ovarian hierarchical follicles of laying chickens. The results showed that increased autophagy was observed in the aging hens (580-day-old, D580) compared with the peak-lay hens (D280). Treatment of GSPs significantly restored the elevated autophagy and decreased viability of cultured D280 chicken GCs that were elicited by hydrogen peroxide. GSPs also suppressed the increased autophagy in the natural aging hens. Similar to the effect of GSPs on GC viability, inhibition of autophagy also showed a protective effect on the decreased viability of GCs under oxidative damage. However, GSPs were not able to provide further protection in GCs that were pretreated with 3-methyladenine (an autophagy inhibitor). In addition to its promoting action on antioxidant capacity, treatment with GSPs increased survival of GCs from autophagy that was caused by oxidative stress through the FoxO1-related pathway. Inhibition of FoxO1 or activation of PI3K-Akt pathway by GSPs increased the confrontation of GCs to oxidative damage and decreased autophagy in GCs. In addition, activation of the SIRT1 signal inhibited the GCs autophagy that was caused by oxidative stress via GSPs-induced deacetylation of FoxO1. These results revealed a new mechanism of GSPs against oxidative stress of GCs via inhibiting FoxO1, which was probably a possible target for alleviating ovarian aging in laying poultry.

Leukemia inhibitory factor prevents chicken follicular atresia through PI3K/AKT and Stat3 signaling pathways.

Autophagy of granulosa cell (GC) may be a supplementary mechanism involved in follicular atresia through cooperating with apoptosis. Leukemia inhibitory factor (LIF) has been shown to promote follicular growth, through the underlying molecular mechanisms remain unclear. Rapamycin, an autophagy inducer, triggered the elevation of GC apoptosis within follicles, and then prevented follicular growth. However, combined treatment with LIF relieved the follicular regression caused by rapamycin, mainly resulting in alleviating the decline of GCs viability and cell autophagic apoptosis, and eventually, promoting follicle development. Further investigation revealed that LIF inhibited the GC autophagic apoptosis by activating PI3K/AKT and Stat3 pathways, reflecting an increase of BCL-2 expression but a decrease in BECN1. Additionally, blocking PI3K/AKT and Stat3 pathways resulted in the reduction of LIF protection against follicular atresia. These findings illustrated that LIF activated the PI3K/AKT and Stat3 signaling pathways to inhibit GC autophagic cell death, and further relieve chicken follicular atresia.

Transplantion of predominant Lactobacilli from native hens to commercial hens could indirectly regulate their ISC activity by improving intestinal microbiota.

In poultry, HyLine (HL) Hens are known for their excellent laying performance. However, ZhenNing (ZN) Hens, a native chicken breed in China, are known for their unique flavour. The intestinal mucosa, which is the main organ for nutrient absorption, could affect livestock product quality. In ZN Hens' intestinal mucosa, we found more villus wrinkles, larger villus circumference and higher amino acid transporters mRNA abundance compared with HL Hens. Among three laying periods of ZN Hens, in the intestinal lumen, Lactobacillus salivarius (L. sa.), Lactobacillus agilis (L. ag.) and Lactobacillus aviarius were the predominant species in the laying peak period. Furthermore, multiple-antibiotics feeding in ZN Hens and predominant Lactobacillus feeding in HL Hens suggested that these Lactobacilli could indeed increase villus wrinkles and improve intestinal absorption. In HL Hens, L. sa. + L. ag. treatment could promote organoids budding in vitro, and promote epithelial proliferation in vivo. Collectively, the unique intestinal mucosa morphology in ZN Hens was due to the high abundance of intestinal L. sa. and L. ag. Transplant these Lactobacilli to HL Hens could increase their intestinal probiotics abundance, fine adjust the intestinal stem cell function and promote the epithelial proliferation, in turn, increase villus winkles and mucosal absorption area.

Engineered Hydroxyapatite Nanoadjuvants with Controlled Shape and Aspect Ratios Reveal Their Immunomodulatory Potentials.

Hydroxyapatite (HAP) has been formulated as adjuvants in vaccines for human use. However, the optimal properties required for HAP nanoparticles to elicit adjuvanticity and the underlying immunopotentiation mechanisms have not been fully elucidated. Herein, a library of HAP nanorods and nanospheres was synthesized to explore the effect of the particle shape and aspect ratio on the immune responses in vitro and adjuvanticity in vivo. It was demonstrated that long aspect ratio HAP nanorods induced a higher degree of cell membrane depolarization and subsequent uptake, and the internalized particles elicited cathepsin B release and mitochondrial reactive oxygen species generation, which further led to pro-inflammatory responses. Furthermore, the physicochemical property-dependent immunostimulation capacities were correlated with their humoral responses in a murine hepatitis B surface antigen immunization model, with long aspect ratio HAP nanorods inducing higher antigen-specific antibody productions. Importantly, HAP nanorods significantly up-regulated the IFN-γ secretion and CD107α expression on CD8+ T cells in immunized mice. Further mechanistic studies demonstrated that HAP nanorods with defined properties exerted immunomodulatory effects by enhanced antigen persistence and immune cell recruitments. Our study provides a rational design strategy for engineered nanomaterial-based vaccine adjuvants.

Lactobacillus salivarius and Lactobacillus agilis feeding regulates intestinal stem cells activity by modulating crypt niche in hens.

Previously, we found that Lactobacillus salivarius, Lactobacillus agilis, and Lactobacillus aviarius were associated with excellent egg nutrition in native chicken. Next, the optimal Lactobacillus combination is worth studying. Here, a total of 120 HyLine hens (30 hens per group contained 3 replicate cells, 10 hens/cell) in the laying peak period were randomly divided into (1) control, (2) L. salivarius + L. agilis, (3) L. salivarius + L. aviarius, and (4) L. agilis + L. aviarius groups, fed with diet only or with corresponding Lactobacilli (108 colony-forming units/hen/day) for 30 days. As a result, L. salivarius + L. agilis feeding could (1) improve egg-laying rate, egg weight, and albumen's amino acid levels; (2) increase Lactobacillus abundance, decrease Escherichia coli abundance, upregulate the tryptophan metabolism pathway-related molecules, and downregulate the primary bile acid biosynthesis pathway-related molecules in intestinal contents; and (3) upregulate oxidative-phosphorylation pathway-related genes, reactive oxygen species levels, and mRNA abundance of Wnt3a, Dll1, Lgr5, CCDN1, and CDK2 in the crypt. Collectively, L. salivarius + L. agilis feeding in hens could improve intestinal microflora and metabolism profile, promote crypt's local energy metabolism and reactive oxygen species levels, and thus enhance Paneth cells and intestinal stem cells activity.Key points• Lactobacilli co-feeding could improve laying performance and egg nutrition.• Lactobacilli co-feeding could improve intestinal microflora and metabolism profile.• Lactobacilli co-feeding could enhance Paneth cells and intestinal stem cells activity.

LIF and bFGF enhanced chicken primordial follicle activation by Wnt/ß-catenin pathway.

The cytokines leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) are closely related to the development of primordial follicles. In this study, the functions and correlation of LIF and bFGF in the development of chicken primordial follicles were examined, along with the signaling pathways including protein kinase B (AKT), extracellular regulated protein kinase (ERK) and Wnt/ß-catenin signaling pathways. Ovarian tissues were collected from four-day-old chicks and incubated with LIF and bFGF alone or in combination for three days to observe the changes in follicular development. Results showed that there was a time-dependent correlation between the changes in expression of LIF/its receptor (LIFR) and the developmental process of primordial follicles. LIF and bFGF exerted a synergistic effect on the activation of primordial follicles. However, SC144 (an antagonist of LIFR) inhibited this stimulating action. The effect by LIF and bFGF were shown to operate at AKT and ERK signaling pathways to suppress cell apoptosis and promote proliferation (P < 0.05) via the Wnt/ß-catenin signaling (P < 0.05). In conclusion, local cytokines LIF and bFGF functioned to enhance the activation of chicken primordial follicles by increasing cell proliferation and decreasing apoptosis in the ovary involving AKT, ERK and Wnt/ß-catenin signaling.

TGF-ß1-induced collagen promotes chicken ovarian follicle development via an intercellular cooperative pattern.

Follicle development is a complex process under strict regulation of diverse hormones and cytokines including transforming growth factor ß (TGF-ß) superfamily members. TGF-ß is pivotal for the regulation of ovarian functions under physiological and pathological conditions. In this study, effect of TGF-ß1 on chicken follicle development was examined through investigating the accumulation and action of collagen, an indispensable member of the extracellular matrix (ECM) involved in this process. The granulosa cells (GCs) and theca cells (TCs) were separated from growing follicles of the laying chicken for treatment of TGF-ß1 and analysis of expression of ECM components and key proteins in intracellular signaling pathways. Results showed that collagen was mainly distributed in the follicular theca layer and was produced with the formation of the granulosa layer during ovarian development. Collagen accumulation increased with follicle growth and treatment of GCs with TGF-ß1 elicited an increased expression of collagen. After production from GCs, collagen was transferred to the neighboring TCs to promote cell proliferation and inhibit apoptosis. Treatment of collagen remarkably increased expression of p-ERK, mitogen-activated protein kinase (MAPK), and p-MAPK, but treatment with hydroxylase inhibitor (to break collagen structure) reversed these alterations. In conclusion, during follicle growth collagen was secreted by GCs under TGF-ß1 stimulation and was subsequently collaboratively transferred to neighboring TCs to increase cell proliferation and thus to promote follicle development via an intercellular cooperative pattern during development of chicken growing follicles.

Paneth cells mediated the response of intestinal stem cells at the early stage of intestinal inflammation in the chicken.

The rapid renewal and repair of the intestinal mucosa are based on intestinal stem cells (ISC), which are located at the crypt bottom. Paneth cells are an essential component in the crypt, which served as the niche for ISC development. However, in the chicken, how the function of Paneth cells changes during intestinal inflammation is unclear and is the key to understand the mechanism of mucosal repair. In the present study, 36 HyLine White chickens (7 d of age, n = 6) were randomly divided into 1 control and 5 lipopolysaccharide (LPS) injection groups. The chickens were injected (i.p.) with PBS in the control group, however, were injected (i.p.) with LPS (10 mg/kg BW) in the LPS injection groups, which would be sampled at 5 time points (1 h postinjection [hpi], 2 hpi, 4 hpi, 6 hpi, and 8 hpi). Results showed that tumor necrosis factor-α mRNA transcription in duodenal tissue increased gradually since 1 hpi, peaked at 4 hpi, and then reduced remarkably, indicating that 4 hpi of LPS was the early stage of intestinal inflammation. Meanwhile, the MUC2 expression in duodenal tissue was dramatically reduced since 1 hpi of LPS. The ISC marker, Lgr5 and Bmi1, in the duodenal crypt were reduced from 1 hpi to 4 hpi and elevated later. Accordingly, the hydroethidine staining showed that the reactive oxygen species level, which drives the differentiation of ISC, in the duodenal crypt reduced obviously at 1 hpi and recovered gradually since 4 hpi. The analysis of Paneth cells showed that many swollen mitochondria appeared in Paneth cells at 4 hpi of LPS. Meanwhile, the Lysozyme transcription in the duodenal crypt was substantially decreased since 1 hpi of LPS. However, the Wnt3a and Dll1 in duodenal crypt decreased at 1 hpi of LPS, then increased gradually. In conclusion, Paneth cells were impaired at the early stage of intestinal inflammation, then recovered rapidly. Thus, the ISC activity was reduced at first and recovery soon.

Metformin Prevents Follicular Atresia in Aging Laying Chickens through Activation of PI3K/AKT and Calcium Signaling Pathways.

Increased follicular atresia occurs with aging and results in reduced fecundity in laying chickens. Therefore, relieving follicular atresia of aging poultry is a crucial measure to maintain sustained high laying performance. As an antiaging agent, metformin was reported to play important roles in preventing aging in diverse animals. In this study, the physiological state of the prehierarchical follicles in the peak-laying hens (D280) and aged hens (D580) was compared, followed with exploration for the possible capacity of metformin in delaying atresia of the prehierarchical follicles in the aged D580 hens. Results showed that the capacity of yolk deposition within follicles declined with aging, and the point of endoplasmic reticulum- (ER-) mitochondrion contact decreased in the ultrastructure of the follicular cells. Meanwhile, the expression of apoptosis signaling genes was increased in the atretic small white follicles. Subsequently, the H2O2-induced follicular atresia model was established to evaluate the enhancing capacity of metformin on yolk deposition and inhibition of apoptosis in the atretic small white follicles. Metformin inhibited apoptosis through regulating cooperation of the mitochondrion-associated ER membranes and the insulin (PI3K/AKT) signaling pathway. Furthermore, metformin regulated calcium ion homeostasis to relieve ER-stress and inhibited release of mitochondrion apoptosis factors (BAD and caspase). Additionally, metformin activated PI3K/AKT that suppressed activation of BAD (downstream of the insulin signaling pathway) in the atretic follicles. Further, serum estrogen level and liver estrogen receptor-α expression were increased after dietary metformin supplementation in D580 hens. These results indicated that administration of dietary metformin activated the PI3K/AKT and calcium signaling pathway and enhanced yolk deposition to prevent chicken follicular atresia.

Enhancing effect of FSH on follicular development through yolk formation and deposition in the low-yield laying chickens.

Healthy and efficient development of ovarian follicles largely determines poultry laying performance. In low-yield laying chickens, retarded follicle progression resulted in decreased prehierarchical follicles. In this study the extenuating effect of follicle-stimulating hormone (FSH) on delayed follicular development was investigated in the low-yield chickens. Results showed that FSH administration in vivo accelerated development of prehierarchical follicles, with increased expression of steroidogenic enzymes and follicular angiogenesis through elevating plasma levels of 17ß-estradiol, progesterone, luteinizing hormone and the expression of vascular endothelial growth factor and its receptor as well as angiopoietins. Furthermore, treatment with FSH raised expression of lipid uptake and adipogenesis-related proteins and decreased tight junctions between granulosa cells. Meanwhile, the results of the in vivo studies were confirmed by the in vitro studies as FSH promoted development of the cultured prehierarchical follicles with increased angiogenesis, cell proliferation, steroid hormones synthesis and yolk deposition. These results indicated FSH enhanced follicular development in the low-yield laying chickens involving increased follicular angiogenesis.