Anti-Heat Stress Effects And Molecular Mechanisms Of Dietary Manganese And Zinc In Broilers

Series of five trials on chronic heat stress in broilers breeders, embryonic development during hatching and chronic heat stress in offspring broilers were conducted to investigate the anti-heat stress effect and possible mechanisms of dietary manganese (Mn) or zinc (Zn) in broiler breeders, offspring embryos and broilers from the aspects of reproductive performance, antioxidant ability and epigenetics.Firstly, experiment 1 was conducted to investigate the effects of environmental temperature and dietary manganese (Mn) on egg production performance, egg quality, and some plasma biochemical traits of broiler breeders. A completely randomized factorial design involved 2 environmental temperatures (normal,21±1℃ and high,32±1℃)×3 dietary Mn treatments [a Mn-unsupplemented basal diet (CON), or a basal diet supplemented with 120 mg Mn/kg diet as inorganic Mn sulfate (iMn) or organic Mn (oMn). There were 6 treatments with 6 replicates (4 birds per replicate). High temperature decreased (P< 0.05) egg weight, laying rate, egg yield, feed intake, egg:feed ratio, eggshell strength and thickness, plasma triiodothyronine (T3) level and alkaline phosphatase (ALP) activity, while it increased (P< 0.02) rectal temperature, plasma malondialdehyde (MDA) level, and activities of lactic dehydrogenase (LDH) and creatine kinase (CK). Broiler breeders fed the diets supplemented with Mn regardless of source had greater (P< 0.05) eggshell strength, and lower (P<= 0.05) plasma T3 level and protein carbonyl content than those fed the control diet. Under normal temperature, dietary Mn did not affect the above four parameters, however, under high temperature, broiler breeders fed the diet supplemented with the organic Mn showed greater (P< 0.03) improvements in these four parameters than those fed the control diet. The results indicated that high temperature significantly impaired egg production performance and eggshell quality, and induced lipid peroxidation and tissue damage, while dietary supplementation of either organic or inorganic Mn improved eggshell strength and thermotolerance and reduced protein oxidation, and the organic Mn could alleviate the negative effect of high temperature on egg production performance of broiler breeders at the period of 32-45 wk of age.Secondly, we further investigated the effect of dietary Mn on antioxidant status and expressions of heat shock proteins/factors in tissues of laying broiler breeders subjected to heat challenge based on experiment 1. High temperature decreased (P<0.01) Mn content in the liver and heart, and increase (P <0.05) HSFs 1,3 mRNA as well as HSP70 mRNA and protein expressions in the liver, heart and muscle. An increased manganese superoxide dismutase (MnSOD) activity (P<0.05) and a slight increase of MDA level were only detected in breast muscle. Breast muscle is more susceptible to heat stress due to the induction of lipid peroxidation, as well as up-regulation of HSP90 expression. Broiler breeders fed either iMn or oMn had higher (P<0.01) tissue Mn content, heart MnSOD and CuZnSOD activitiesand breast muscle MnSOD protein, and lower breast muscle HSP70 mRNA and protein levels than those fed CON. The bone Mn content in oMn was higher than that in iMn (P<0.03). No interaction between environmental temperature and dietary Mn affect the aboved indcies (P>0.05). The results indicated that dietary supplementation with Mn in either source may enhance heart antioxidant ability and inhibit the expression of HSP70 in breast muscle. Finally, the organic Mn appears to be more available than inorganic Mn for bone in laying broiler breeders regardless of environmental temperatures.Thirdly, we further investigated whether maternal dietary Mn supplementation could protect offspring chick embryos against maternal hyperthermia-induced negative effects. Maternal hyperthermia decreased (P< 0.002) fertility and hatchability and increased embryonic mortality as well as increase (P < 0.05) mRNA expressions of HSP90, HSP70, HSP35, HSP25, COX2, CDK6 and BAX genes with a serious oxidative damage and apoptosis. Moreover, maternal hyperthermia altered the epigenetic status with the global DNA hypomethylation and AcH3K9 hyperacetyiation as well as the DNA hypermethylation of HSP70 promoter in embryonic heart. Maternal dietary Mn supplementation increased (P< 0.05) Mn contents in yolk and tissues, and subsequently heart MnSOD mRNA expression and activity, potentially by alterations of DNA methylation (lower) and histone AcH3K9 acetylation (higher) of MnSOD promoter. The interaction between maternal environmental temperature and maternal dietary Mn affected (P< 0.05) hatchability and heart BCL2 mRNA and protein expressions. Under NT, heart BCL2 mRNA and protein expressions were not affected (P> 0.28) by maternal dietary Mn, while under HT, heart BCL2 mRNA and protein expressions from either iMn or oMn were higher (P<0.05) than that from CON. Finally, maternal dietary supplementation of Mn, especially the organic Mn, could alleviate mortality and apoptosis of offspring chick embryos induced by maternal hyperthermia via enhancing MnSOD as well as BCL2 expressions.Fourthly, we further investigated effects of maternal heat stress and dietary Mn supplementation on offspring broilers under normal and high environmental temperatures. Therefore, offspring broilers from 6 maternal treatments at 29 d of age were allotted to 2 rearing temperatures (22 vs 33℃) with sex. In total there was 12 treatments with 6 replicates having 4 birds each (male:female=1:1) in during progeny stage. Maternal hyperthermia increased (P< 0.05) progeny BW, ADG and ADFI at 22-28 d of age, while compared to maternal Mn treatment, maternal CON had greater (P< 0.05) ADFI and plasma UA and T4 contents. The above results indicated that maternal heat stress and dietary Mn deficiency leaded to a compensatory growth and greater metabolism in offspring broilers during early growth stage. Notably, the addition of Mn improved meat quality progeny at 42 d of age subjected to heat stress (P<0.05). The interaction between maternal environmental temperature and maternal dietary Mn affected (P<0.05) gga-miR-34c-5p and gga-miR-1551-5p in embryonic heart by high-throughput miRNA sequencing and RT-PCR verification. And gga-miR-34c-5p and gga-miR-1551-5p might regulate the target gene MAP3K14 and BCL2 mRNA expressions, respectively, in the heart of chick embryo by the prediction and verification of target gene using dual luciferase reporter system. Compared with maternal normal temperature, maternal high temperature decreased gga-miR-34c-5p and gga-miR-1551-5p expressions and subsequently increased MAP3K14 and NF-κB mRNA and BCL2 mRNA and protein expressions in CON; however, maternal temperature treatments had no effect on gga-miR-34c-5p and gga-miR-1551-5p as well as their target gene expressions in iMn and oMn. Therefore, maternal dietary Mn had anti-heat stress effect on embryo and progeny involved in activation of NF-κB and antiapoptosis by downregulation of miRNA and upregulation of the target genes.Finally, we investigated maternal dietary Zn supplementation could protect offspring embryos and chick broilers against maternal hyperthermia-induced negative effects following the research approaches of Mn anti-heat stress experiment. During embryonic stage, a completely randomized design (n= 6) with 2 maternal environmental temperatures [normal 21±1℃ vs high 32±1℃]×3 maternal dietary supplemental Zn levels [Zn-unsupplemented control diet (CON), the control diet+110 mg of Zn/kg of diet as either inorganic ZnSO4 (iZn) or organic Zn with a moderate chelation strength (oZn)] was used. During progeny stage, offspring broilers from 6 maternal treatments at 29 d of age were allotted to 2 rearing temperatures (22 vs 33℃) with sex. In total there was 12 treatments with 6 replicates having 4 birds each (male:female=1:1) in during progeny stage. Maternal hyperthermia (P <=0.05) decreased fertility, hatchability, chick hatch weight, progeny BW, ADG and ADFI at 1-21, 22-28 and 29-42 d of age. Especially, heat stress was more susceptible to increase breast muscle drip loss and disturb the balance of antioxidant system in pancreas of progeny at 42 d of age from maternal heat-stressed breeders. Maternal dietary Zn supplementation with either iZn or oZn increased (P< 0.05) healthy chick ratio, Zn contents in yolk and liver, liver MT4 and MT1 mRNA, as well as progeny BW, ADFI and livability at 1-21 d of age. Notably, the addition of Zn alleviated the negative effect of maternal heat stress on growth performance of offspring broilers during the starter period and improved meat quality progeny at 42 d of age subjected to heat stress. Finally, maternal dietary supplementation of Zn improved hatchability, hatched chick quality and antioxidant ability. Maternal dietary supplementation of Zn also increased altered embryonic liver epigenetic patterns as well as the promoter histone AcH3K9 hyperacetylation related to the greater MT4 mRNA expression.In conclusion, dietary supplementation of Mn, especially the organic Mn, could alleviate the negative effect of high temperature on egg production performance of broiler breeders via enhancing antioxidant ability and reducing oxidative damage.Also, maternal dietary supplementation of Mn, especially the organic Mn, could alleviate mortality and apoptosis of offspring chick embryos and deline in meat quality induced by maternal hyperthermia via enhancing the abilities of antioxidant and antiapoptosis. In addition, maternal dietary supplementation of Zn alleviated the negative effect of maternal heat stress on growth performance of offspring broilers during the starter period and improved meat quality progeny at 42 d of age subjected to heat stress. Furthermore, maternal heat stress and maternal dietary Mn and Zn supplementation affected epigenetic status and the regulation of the inprint gene expressions, including alterations of DNA methylation, histone AcH3K9 acetylation and miRNA expression. And the addition of Mn and Zn in maternal diets altered embryonic tissue DNA methylation and histone AcH3K9 acetylation patterns related to the greater heart MnSOD and liver MT4 mRNA expression. The above new research achievements from the aspects of reproductive performance, antioxidant ability and epigenetics also provide scientific evidence to decrease the negative effects of global warming by supplementing Mn or Zn in diets.