Effects Of Dietary Zinc Nutrition Of The Broiler Breeders On The Immunity Function Of Their Offsprings And The Related Molecular Mechanism

Five experiments were conducted to study the effects and mechanism of dietary zinc nutrition of the broiler breeders on the immunity of their offsprings.Exp.l was conducted to analyze the epigenetic patterns of developing embryos and posthatched chicks. Embryos at embryonic day5(E5), E8, E11, E14, E17, and E20and newly hatched chicks on day of life1(D1), D7, D14, D21were collected. The levels of global DNA methylation and histone H3at lysine9residue (H3K9) modifications were measured in samples of liver, jejunum, and breast skeletal muscles by Western blotting and immunofluorescence staining. According to our data, levels of DNA methylation and H3K9acetylation decreased in liver over time, while the pattern was N-shaped in jejunal tissue and W-shaped in pectoral muscles. Moreover, dimethylation, trimethylation, and acetylation of H3K9were expressed in a time-and tissue-dependent manner. After birth, epigenetic marks were relatively stable. These results indicate that spatiotemporal specific epigenetic alterations could be critical for the late development of chick embryos and neonates.Exp.2was carried out to investigate the effects of maternal Zn nutrition in avian animals on the immunity of their progeny. Seven hundred and twenty Ross308broiler breeders were fed with zinc-deficient diet for2weeks and then allocated randomly into five groups fed basal diets supplemented with Zn from methionine hydroxy analog chelated Zn (MHA-Zn) or ZnSO4at level of0,50or300mg/kg, respectively. After8weeks, fertilized eggs were collected and incubated. Newly-hatched chicks from each treatment were fed with low (20mg/kg) or normal (70mg/kg) Zn diet. Antibody levels and B cell proliferation in bursas were assayed by ELISA and Western blot, respectively. Maternal Zn deficiency significantly reduced maternal antibody (IgY and IgA) levels in the yolk and the newborn serum, and resulted in poorer immune responses to Newcastle disease virus (NDV) and infectious bursal disease virus (IBDVV) vaccines, and lower protein expression of PCNA in bursas of the offspring at age of day14. These carry-over effects partially persisted to the age of day35. However, adequate or excessive Zn supplementation into the diets of hens increased maternal non-specific (IgY and IgA) and specific (anti-NDV and anti-IBDVV) antibody production. Supplemental high dose of organic Zn presented better effects on the antibody production in the offspring circulation when compared with supplementation of normal dose of Zn in the maternal diet. Maternal organic Zn exposure resulted in significantly improved intestinal morphological characteristics, increased mucin2(MUC2) abundance, and secretory IgA (slgA) production in jejunums of progeny. Maternal and offspring Zn supplementation partially alleviated Zn-deficiency-induced inflammatory response, accompanied by repression of NF-κB signaling. Compared with the offsprings fed with adequate Zn diet, the progeny birds fed with low Zn diet had lower relative weights of lymphoid organs, ratio of CD4+/CD8+, antibody titers against IBDV and NDV antigens. Additionally, we observed DNA hypomethylation and histone H3at lysine9(H3K9) hyperacetylation at the A20promoter region and subsequent activated A20expression in Zn-supplemented hens compared with control. Notably, maternal dietary organic Zn exposure exhibited greater attenuation of gut impairment, along with increased MUC2expression and sIgA level, and decreased the abundance of TNF-a and A20relative to the inorganic-Zn group. Furthermore, enhanced acetylated H3K9and A20transcription at day14was found in the offspring adequate dietary Zn group. Thus, supplementation of MHA-Zn to broiler breeder’s diet at50-300mg/kg had long-term beneficial effects on the immunity of the offspring, and A20may be a novel inflammatory-suppressed factor of chick gut that is persistently promoted by dietary Zn supplementation via epigenetic modifications at A20promoter.Exp.3was conducted to analyze the effect of maternal Zn nutrition on LPS-induced inflammation in their offsprings. Female broiler chicks from the breeders fed with0,50or300mg/kg of Zn in the form of either MHA-Zn or ZnSO4, were selected and allocated into8groups, and fed with normal Zn diet containing94.46mg/kg of Zn. The progeny chicks on day15were subjeced to LPS or saline injection every other day for total of8days. The resluts indicated that LPS chanllenge led to lower body weight, body weight gain, feed intake, antobody levels of serum IgA and IgG after LPS injection for8or24h. Moreover, LPS stress increased relative spleen weight, and up-regulated gene expression of IL-8, IL-1β, TNF-α, NF-κB p65and A20in the jejunum and spleen tissues. However, organic Zn supplementation in the breeder diet increased the production of serum IgA and IgG, and alleviated LPS-induced transcription of NF-κB p65and pro-inflammatory cytokines in the jejunal and splenic samples. Therefore, dietary organic zinc addition at300mg/kg in broiler breeders could decrease the inflammation in their offspring via inactivation of NF-κB pathway.Exp.4was performed to analyze the effects of maternal Zn nutrition on the development of lymphoid organs and gut barrier function of their progeny broilers. Three groups of broiler breeders were subjected to dietary Zn deficiency, supplementation with methionine hydroxy analog chelated Zn (ZnMHA) or ZnSO4treatments for7weeks, respectively. Breeding eggs were collected during the last four days and incubated, and offspring birds were fed with low Zn (52.96mg/kg) diet. Zn supplementation increased the proliferation and subsets of T and B cells in the peripheral blood of hens. Dietary ZnMHA addition into the diet for the breeders resulted in more maternally-derived antibody transferred to progeny circulation, enhanced proliferative response and inhibited apoptosis of bursal lymphocytes in the progeny chicks, and up-regulated protein expression of PCNA and down-regulated expression of Caspase-3in bursal tissues. Improved proliferation of T cells, percentages of CD3+and CD4+T cells and PCNA expression in the spleens, and the suppressed LPS-induced activation of NF-κB pathway and subsequent inflammatory response were observed in the treatment with maternal ZnMHA supplementation. The transcription of A20gene was downregulated in the Zn-deficient birds. In addition, maternal Zn deficiency had lower ratio of villus height to crypt depth and protein expression of PCNA in the jejunums of offspring on day12. LPS chanllenge resulted in a decrease of body.weight, increase of serum DAO and HRP after administration for45and120min, up-regulation of genes IL-8, IL-1β, TNF-α, TLR-4and NF-κB p65, down-regulation of gene A20and protein IκBα, disorder of both distribution and protein expression of E-Cadherin in the jejunums. Supplementation of 100mg/kg inorganic or organic Zn into the breeder diet showed significant improvements of gut barrier and immune function before or after LPS chanllenge in the progeny chicks. Maternal organic Zn addition had better effect on intestinal barrier function than that in inorganic Zn group. There was no obvious difference of intestinal immunity between inorgnaic and organic Zn treatments. The results indicate that maternal dietary organic-Zn supplementation of100mg/kg through the breeders’ diet could enhance the immunity of the post-hatch offspring via enhancing the development of immune organs, improving humoral and cellular immunity, and increasing intestinal barrier function.Exp.5was conducted to disclose the role of A20in Zn alleviating LPS-induced inflammatory response in primary intestinal epithelial cells of chicken embryos. The results indicated that addition of25μM Zn could decrease the production of pro-inflammatory cytokines under LPS stress. Protein expression of A20was significantly decreased; gene expression and protein secretion of IL-1β, IL-8and TNF-α were increased; LPS-induced up-regulated transcription of IL-8, IL-1βand increased secretion of the IL-1β, TNF-α were enhanced after culturing with A20-siRNA lentivirus for72h in primary intestinal epithelial cells. Additionally, Zn was beneficial to the protein expression of A20and NF-κB p65, but inhibited protein expression of phospho (P)-NF-κB p65. However, no effects of Zn on the down-regulated expression of proteins A20, NF-κB p65and up-regulated expression of cytoplasmic proteins P-NF-κB p65, P-IκBα, and nuclear protein P-NF-κB p65under A20gene silence were observed. Therefore, A20-NF-κB pathway plays an important role in the alleviation of LPS-induced inflammation by Zn.In conclusion, dietary supplementation of300mg/kg ZnMHA into broiler breeders increased mRNA expression of Zn finger protein A20in the jejunums via affecting epigentic modifications in the promoter of A20gene, inhibited inflammation induced by LPS and enhanced intestinal mucosal immunity in the progeny chicks. Compared with inorganic Zn treatment, maternal supplementation of100mg/kg organic Zn had no better effects on the immunity of offspring chicks, but enhanced gut barrier function. Zn could up-regulate protein expression of A20, which inhibited the activation of NF-κB pathway and the production of pro-inflammatory cytokines. Therefore, the up-regulation of A20is a crucial factor for enhancing intestinal mucosal immunity in the offspring birds from broiler breeders fed with organic Zn.