MAPK, AKT/FoxO3a and mTOR pathways are involved in cadmium regulating the cell cycle, proliferation and apoptosis of chicken follicular granulosa cells.

The occurrence of cadmium (Cd) in feed is a major problem in animal health and production. Studies have confirmed that Cd depresses egg production of laying hens, which is closely related to follicular atresia. This study aimed to assess the toxic impacts of Cd on the ovarian tissue, and to examine the mechanism of Cd-induced granulosa cell proliferation and apoptosis. Results from the nitric oxide (NO) and malondialdehyde (MDA) content, total superoxide dismutase (T-SOD), glutathione peroxide (GSH-Px), total nitric oxide synthase (T-NOS) and adenosine triphosphatase (ATPase) activities, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and hematoxylin-eosin (H & E) staining indicated that excess Cd induced oxidative stress, granulosa cell apoptosis and follicular atresia in the layer ovary. Low-dose Cd exposure (1 µM) induced the granulosa cell proliferation, upregulated the mRNA levels of RSK1 and RHEB, activated FoxO3a, AKT, ERK1/2, mTOR and p70S6K1 phosphorylation, and promoted cell cycle progression from phase G1 to S. However, high-dose Cd exposure (15 µM) induced reactive oxygen species (ROS) generation and cell apoptosis, upregulated the mRNA levels of the inflammatory factors, ASK1, JNK, p38 and TAK1, downregulated the expressions of RSK1 and RHEB genes, and inhibited the phosphorylation of ERK1/2, mTOR and p70S6K1 proteins, and the cell cycle progression. Rapamycin pre-treatment completely blocked the phosphorylation of mTOR and p70S6K1 proteins, and the cell cycle progression induced by 1 µM Cd, and accelerated 15 µM Cd-induced cell apoptosis and cell cycle arrest. The microRNA sequencing result showed that 15 µM Cd induced differential expression of microRNA genes, which may regulate AKT, ERK1/2 and mTOR signaling and cell cycle progression by regulating the activity of G proteins and cell cycle-related proteins. Conclusively, these results indicated that Cd can cause the ovarian damage and follicular atresia, and regulate cell cycle, cell proliferation or apoptosis of granulosa cells through MAPK, AKT/FoxO3a and mTOR pathways in laying hens.

Effect of Glycine Nano-Selenium Supplementation on Production Performance, Egg Quality, Serum Biochemistry, Oxidative Status, and the Intestinal Morphology and Absorption of Laying Hens.

The objective of this study was to investigate the feasibility of using glycine nano-selenium (NS-Gly) as a feed supplement and to evaluate its influence on production performance, egg quality, serum biochemistry, oxidative status, and the intestinal morphology and absorption of laying hens. A total of 864 hens at 40 weeks were randomly assigned into six groups including the basal diet (control, 0.13 mg Se/kg); basal diet + 0.30 mg Se/kg (Na2SeO3) diet; and basal diet + 0.15, 0.30, 0.45, and 0.60 mg Se/kg (NS-Gly) diet. After 8 weeks of Se supplementation, no difference was observed among the treatments on production performance and egg quality (P > 0.05). The levels of albumin (ALB) and alanine aminotransferase (GPT) were significantly influenced by dietary Se supplementation (P < 0.05). In the serum, the level of glutathione peroxide (GSH-Px) was significantly increased in the groups with the dietary NS-Gly supplementation (P < 0.05). The superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) levels in all groups of NS-Gly supplementation had a remarkable increase (P < 0.05). In the liver, GSH-Px was significantly increased in 0.45 and 0.60 mg/kg NS-Gly groups (P < 0.05). The activities of SOD and catalase (CAT) were significantly increased in the groups of 0.30 mg/kg NS-Gly diet (P < 0.05). The results of intestinal morphology showed that the crypt depth was affected by higher dose groups of NS-Gly diets in the duodenum, and the differences (P < 0.05) were obtained in villus height, the crypt depth, and the V/C in the jejunum. In the ileum, a significant increase (P < 0.05) of villus height was observed in 0.15 and 0.3 mg/kg Se-added groups. The V/C was the highest in the SS groups (P < 0.05). The mRNA levels of solute carrier family 3 member 1 (rBAT), solute carrier family 6 member 19 (B0AT1), and solute carrier family 15 member 1 (PepT1) increased at different degrees in the duodenum, especially in 0.15 and 0.60 mg/kg NS-Gly groups (P < 0.05). In the jejunum, the expression of B0AT1 was similar to that in the duodenum, and the expression of rBAT increased significantly in the 0.30 and 0.45 mg/kg NS-Gly groups (P < 0.05). The mRNA level of PepT1 increased significantly in the 0.30 mg/kg SS group. Conclusively, dietary NS-Gly supplementation could improve the antioxidant capacity, as well as the structure of small intestine in laying hens, although have no significant effects on the production performance and egg quality.

Dietary cadmium chloride impairs shell biomineralization by disrupting the metabolism of the eggshell gland in laying hens.

In this study, we identified cadmium (Cd) as a potential endocrine disruptor that impairs laying performance, egg quality, and eggshell deposition and induces oxidative stress and inflammation in the eggshell glands of laying hens. A total of 480 38-wk-old laying hens were randomly assigned into 5 groups that were fed a basal diet (control) or a basal diet supplemented with Cd (provided as CdCl2·2.5 H2O) at 7.5, 15, 30, and 60 mg Cd per kg feed for 9 wk. The results showed that, when compared with the control group, a low dose of dietary Cd (7.5 mg/kg) had positive effects on egg quality by improving albumen height, Haugh unit, yolk color, and shell thickness at the third or ninth week. However, with the increase in the dose and duration of Cd exposure, the laying performance, egg quality, and activities of eggshell gland antioxidant enzymes (catalase [CAT], glutathione peroxide [GSH-Px]), and ATPase (Na+/K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase) deteriorated, and the activity of total nitric oxide synthase (T-NOS) and the level of malondialdehyde (MDA) increased significantly (P < 0.05). The histopathology and real-time quantitative PCR results showed that Cd induced endometrial epithelial cell proliferation accompanied by upregulation of the mRNA levels of progesterone receptor (PgR) and epidermal growth factor receptor (EGFR), downregulation of the mRNA levels of estrogen receptor α (ERα) and interleukin 6 (IL6), and inflammation of the eggshell gland accompanied by significantly increased expression of complement C3 and pro-inflammatory cytokine tumor necrosis factor α (TNFα) (P < 0.05). In addition, the ultrastructure of the eggshell showed that dietary supplementation with 7.5 mg/kg Cd increased the palisade layer and total thickness of the shell, but with the increase in dietary Cd supplementation (30 and 60 mg/kg) the thickness of the palisade layer and mammillary layer decreased significantly (P < 0.05), and the outer surface of the eggshell became rougher. Correspondingly, the expression of calbindin 1 (CALB1), ovocalyxin-32 (OCX-32), ovocalyxin-36 (OCX-36), osteopontin (SPP1), and ovocledidin-17 (OC-17) decreased significantly (P < 0.05) with increasing dietary Cd supplementation. Conclusively, the present study demonstrates that dietary supplementation with Cd negatively affects laying performance, egg quality, and eggshell deposition by disturbing the metabolism of eggshell glands in laying hens but has a positive effect on egg quality at low doses.

Dietary Cadmium Chloride Supplementation Impairs Renal Function and Bone Metabolism of Laying Hens.

This study was conducted to evaluate the toxic effects of cadmium (Cd) on the kidney function and bone development in laying hens. A total of 480 Hy-line laying hens aged 38 weeks were randomly allocated into five treatments, each of which included six replicates of 16 birds. The concentrations of Cd in the diets of the five groups were 0.47, 7.58, 15.56, 30.55, and 60.67 mg/kg. Results showed that serum calcium (Ca) levels decreased significantly in the 60.67 mg Cd/kg diet group (p < 0.05). The activities of serum alkaline phosphatase (ALP) and bone ALP (BALP) decreased significantly in the 15.56, 30.55 and 60.67 mg Cd/kg diet groups (p < 0.05). The levels of parathyroid hormone (PTH) increased significantly in the 30.55 and 60.67 mg Cd/kg diet groups, and the estradiol (E2), 1,25-(OH)2-D3 and calcitonin (CT) decreased significantly with the increase of dietary Cd supplementation (p < 0.05). Histological results presented enlargements of renal tubules and tubular fibrosis in the kidney and decreased trabecular bone in the tibia. Tartrate-resistant acidic phosphatase (TRAP) staining results of tibia showed that osteoclast was significantly increased at the relatively high dose of dietary Cd (p < 0.05). In addition, the renal function indicators of blood urea nitrogen (BUN), urea acid (UA), and creatinine were significantly increased in Cd supplemented groups compared with the control group (p < 0.05). Low dose Cd exposure induced antioxidant defenses accompanying the increase in activities of catalase (CAT), glutathione peroxidase (GSH-Px), and the levels of glutathione (GSH) in renal tissue. At the same time, with the increased Cd levels, the activities of CAT, GSH-Px decreased significantly, and the level of malondialdehyde (MDA) increased significantly (p < 0.05). The activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase decreased significantly in the relatively high levels of dietary Cd (p < 0.05). These results suggest that Cd can damage renal function and induce disorders in bone metabolism of laying hens.

Prevention of Aflatoxin B1 Hepatoxicity by Dietary Selenium Is Associated with Inhibition of Cytochrome P450 Isozymes and Up-Regulation of 6 Selenoprotein Genes in Chick Liver.

BACKGROUND: The involvement of cytochrome P450 (CYP450) isozymes and the selenogenome in selenium-mediated protection against aflatoxin B1 (AFB1)-induced adverse effects in broilers remains unclear. OBJECTIVE: This study was designed first to determine whether selenium could reduce AFB1-induced hepatotoxic effects and then to determine whether these effects were due to changes in the CYP450 isozymes and selenogenome expression in the liver of chicks. METHODS: Male avian broilers (aged 120 d) were allocated to 4 groups with 5 replicates of 6 birds to be included in a 2-by-2 factorial trial in which the main factors included supplementation of AFB1 (<5 compared with 100 µg/kg) and selenium (0.2 compared with 0.5 mg/kg) in a corn/soybean-based diet for 4 wk. Serum biochemistry, hepatic histology, and mRNA and/or activities of hepatic antioxidant enzymes, CYP450 isozymes, and 26 selenoproteins were analyzed at week 2 and/or 4. RESULTS: Administration of AFB1 induced liver injury, decreasing (P < 0.05) total protein and albumin concentrations by 33.3-43.8% and increasing (P < 0.05) alanine aminotransferase and aspartate aminotransferase activities by 26.0-33.8% in serum, and induced hepatic necrosis and bile duct hyperplasia at week 2. AFB1 also decreased (P < 0.05) hepatic activities of glutathione peroxidase (GPX), thioredoxin reductase (TXNRD), and catalase, and the glutathione concentration by 13.1-59.9% and increased (P < 0.05) malondialdehyde, 8-hydroxydeoxyguanosine and exo-AFB1-8,9-epoxide (AFBO) DNA concentrations by 17.9-1200%. In addition, the mRNA and activity of enzymes responsible for the bioactivation of AFB1 into AFBO, which included CYP450 A1, 1A2, 2A6, and 3A4, were significantly induced (P < 0.05) by 29.2-271% in liver microsomes after 2-wk exposure to AFB1. These alterations induced by AFB1 were prevented by selenium supplementation. Dietary selenium supplementation increased (P < 0.05) mRNA and/or activities of 6 selenoprotein genes (Gpx3, Txnrd1, Txnrd2, Txnrd3, iodothyronine deiodinase 2, and selenoprotein N) in the liver of AFB1-treated groups at week 2. CONCLUSIONS: Dietary selenium protected chicks from AFB1-induced liver injury, potentially through the synergistic actions of inhibition of the pivotal CYP450 isozyme-mediated activation of AFB1 to toxic AFBO, and increased antioxidant capacities by upregulation of selenoprotein genes coding for antioxidant proteins.