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Egg quality defects seriously reduce the quality grade and increase egg breakage in egg marketing activities. In this study, the effect of N-carbamylglutamate ( NCG ) on eggshell quality was investigated by evaluating calcium absorption and calcification in laying hens. A total of 30 newly hatched female Hy-Line chicks were randomly assigned to the control group (basal diet) and treatment group (basal diet supplemented with 1% NCG). At 25 wk, eggs from each group were obtained to assess egg quality parameters. Blood samples were collected for analysis of mineral, hormone, and amino acids levels at 16 h after laying egg. Uterine tissues were removed and fixed in 4% neutral paraformaldehyde solution or kept in liquid nitrogen for mineral determination, quantitative PCR, and Western blot. Results showed that the egg quality (eggshell thickness, strength and percentage, egg specific gravity, and eggshell effective thickness) was significantly increased while effective thickness of mastoid layer, width of mastoid gap, and mammillary knobs were significantly decreased by dietary NCG supplementation ( P < 0.05). The levels of minerals (Ca, P, Fe, Mg, Na, K) in eggshell, plasma, and uterus were remarkably elevated ( P < 0.05). Meanwhile, the concentrations of calcium metabolism–related hormones (17β-estradiol, parathyroid hormone, and calcitonin) were increased in the NCG group ( P < 0.05). Moreover, expression of calbindin 1, carbonic anhydrase 2, ovalbumin, ovotransferrin, ovocleidin-17, ovocleidin-116, and clusterin mRNAs, as well as calbindin 1 and ATP2A1 proteins in uterus, duodenum, and kidney, was all upregulated in hens fed with NCG ( P < 0.05). In addition, the number of blood vessels in the uterus, height of uterine mucosal folds, villus length in endometrium, and areas of uterine mucosal folds were significantly increased in the NCG group ( P < 0.05). In conclusion, dietary 1% NCG supplementation during 0 to 25 wk can improve eggshell quality through changes in endometrial morphology, expression of calcium metabolism–related genes, and secretion of related hormones to promote eggshell formation in the laying hens.

In the poultry, only less than 5% primordial follicles in the ovary can develop into the prehierarchical follicles (PHFs) leading to progressive development, ovulation, and egg formation. This low rate of recruitment indicates a huge potential for improvement of the laying performance. A great reduction in egg production is caused by aging with extensive follicular atresia. In this study, age-related changes in the laying performance and ovarian status were compared between the peak-lay (D280) and aged (D580) chickens. Subsequently, a cross coculture of PHFs and granulosa cells (GCs) from D280 or D580 hens was adopted to reveal the mechanism of declined follicle development. Results showed that persistent endoplasmic reticulum (ER) stress in GCs of the aged hens was accompanied with intensified apoptosis. Bone morphogenetic protein 4 (BMP4) secreted by GCs of PHFs in D280 hens was capable of relieving ER stress and improving follicular dominance for selection in D580 hens. During this action, BMP4 reduced free calreticulin (CALR, an ER marker) content and attenuated cell apoptosis in PHFs of D580 hens via the PERK-CHOP-BCL2/caspase3 or CALR-Ca2+-BCL2-caspase12 pathway. Furthermore, BMP4 prevented follicular atresia by promoting production of steroid hormones to improve survival of GCs in PHFs from the aged hens. In conclusion, intensified ER stress and apoptosis occurred in GCs of PHFs in aged chickens, while BMP4 secreted by GCs was capable of improving follicular viability by alleviating ER stress to promote follicular development.

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.

Follicle development is a complex process under strict regulation of diverse hormones and cytokines including transforming growth factor β (TGF‐β) superfamily members. TGF‐β is pivotal for the regulation of ovarian functions under physiological and pathological conditions. In this study, effect of TGF‐β1 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‐β1 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‐β1 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‐β1 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.

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