| Egg production performance is not only influenced by the nutritional level of the diet during the egg-laying period,but also related to the development of the laying hens during the breeding period.High-energy diets promote egg growth but may increase oxidative stress in the body.The source and level of energy in the diet during the breeding period have a great influence on the production performance and oxidative stress of laying hens.Tea polyphenols,mainly composed of EGCG,play an important role in mitigating oxidative damage in the body.It is of great theoretical and practical importance to study the effects of high-energy diets on the performance of laying hens during the breeding period and the modulating effect of EGCG on oxidative stress.In this paper,we systematically investigated the effects of tea polyphenols on the antioxidant capacity of laying hens and the effects of EGCG on growth,body health and subsequent egg production performance of laying hens at different energy sources and levels in the breeding ration through four experiments,and explored the regulatory effect of EGCG on oleic acid-induced hepatocyte steatosis through in vitro hepatocyte culture tests to analyze the molecular mechanism of the effects of breeding high energy ration on egg production performance of laying hens.The effect of EGCG on oleic acid-induced hepatocyte steatosis was investigated through an ex vivo hepatocyte culture test.The main findings are as follows.Experiment 1.Effects of different tea polyphenol products on production performance and antioxidant capacity of laying hensIn this experiment,840 Lohmann laying hens at 253 days of age were used in a single-factor test design,divided into 7 treatments with 6 replicating groups for each treatment.Each replicate has 20 hens.The control group was fed with the basal diet,while the remaining 6 groups were fed with 6 tea polyphenol products at 1266.7 mg/kg.The 6 tea polyphenol products have different contents of EGCG and caffeine,of which product 2 had the highest EGCG content.The trial period was 12 weeks.The results are shown in the followings.1.For production performance,different tea polyphenol products significantly reduced the feed-to-egg ratio of 1-4 wk(P < 0.05),and all products except T2 and T6 reduced the feed intake of 1-4 wk(P < 0.05).In addition,T2 and T4 significantly increased the 5-8 wk dirty egg rate and decreased the qualified egg rate(P < 0.05).2.Regarding egg quality,T6 and T7 significantly reduced eggshell strength and eggshell weight at week 1(P < 0.05);the rest of the groups excluding T4 and T6 significantly increased eggshell thickness(P < 0.05);T6 significantly reduced eggshell strength at week 4(P < 0.05);T6 and T7 significantly reduced eggshell strength,thickness and eggshell weight at week 8(P < 0.05);T3 and T4 significantly reduced T3 and T4 significantly reduced egg white height and Hastelloy units at week 8(P < 0.05);T2significantly increased shell thickness at week 12(P < 0.05);T3,T5 and T7 significantly reduced shell color brightness values at week 12(P < 0.05)and significantly increased shell redness and yellowness values(P < 0.05).The egg storage test showed that at the end of the 10-day test,the tea polyphenol-added groups all significantly reduced the shell thickness at 10 days of storage(P < 0.05),with T2 showing a modest decrease;T3,T5,and T7 significantly reduced the shell strength of eggs stored for 20 days,T4 and T7 reduced the shell thickness,and T5 and T7 reduced the egg weight(P < 0.05).3.Regarding antioxidant capacity,different tea polyphenol add-ons significantly increased total serum antioxidant capacity(T-AOC)viability and significantly reduced malondialdehyde(MDA)content(P < 0.05),with T2 and T3 having the best effect.The results of this experiment showed that the effects of different tea polyphenol products on the production performance and egg quality of laying hens differ,but all of them significantly improved the antioxidant capacity of laying hens,and the tea polyphenol products with higher EGCG content had a better effect on the antioxidant capacity and had a protective effect on the health of laying hens.Experiment 2.Effects and mechanisms of energy sources and EGCG of high-energy diets on growth and subsequent performance of egg production of breeding hensIn this experiment,810 young Lohmann laying hens of similar weight at 56 days of age were used in a 2 × 3 factorial design,including 6 treatments with 3 diets,namely normal energy level(11.02 MJ/kcal for basal diet),two high energy diets(2 energy sources:soybean oil and flax oil,ME 13.57 MJ/kcal),2 EGCG levels(0,400 mg/kg),9 replicates for each treatment and 15 replicates for each treatment(single cage feeding,15 cages as one replicate).The control group was fed with the basal diet and the high-energy diet was supplemented with soybean oil or flax oil at an appropriate dose in the basal diet according to the level of energy increase.The test period was from 9 to 18 weeks of age and the subsequent egg-laying period was from 19 to 44 weeks of age.The results showed that:1.For growth performance,the age at start of production was 3 days earlier in the soybean oil group and 7 days earlier in the linseed oil group compared to the control group,and both energy sources significantly reduced feed intake by 17-18 wk(P < 0.01).2.As for blood indicators,both energy sources significantly reduced the levels of serum total cholesterol(TC)and very low-density lipoprotein cholesterol(VLDL-C)(P <0.05)and significantly increased MDA levels(P < 0.05)in 18-week-old hens week.Flax oil significantly reduced serum glutathione peroxidase(GSH-PX)activity(P < 0.05);additional EGCG significantly reduced serum MDA level(P < 0.05)and increased serum total antioxidant capacity(T-AOC)and superoxide dismutase(SOD)activity(P < 0.05).3.Regarding liver function,both energy sources significantly increased levels of liver fat content and triglyceride in 18-week-old hens(P < 0.05),but liver fat content was higher in the flax oil group(P < 0.05)and EGCG significantly decreased liver fat content and triglyceride levels(P < 0.05);both energy sources significantly increased the m RNA expression of liver synthesis-related genes ACACA and FASN(P < 0.05)and significantly decreased the m RNA expression of the liver transport-related genes Apo VLDL-II and CPT1(P < 0.05);EGCG significantly decreased the m RNA expression of ACACA and FASN(P < 0.05)and increased the m RNA expression of Apo VLDL-II(P < 0.05).4.For ovarian function,both energy sources significantly reduced the levels of serum estradiol(E2)and progesterone(PROG)(P < 0.05),and EGCG significantly increased the levels of E2 and follicle stimulating hormone(FSH)(P < 0.05);significantly increased ovarian weight in 18-week-old hens(P < 0.05);the dietary energy source also significantly reduced SOD and GPX gene m RNA expression(The dietary energy source also significantly reduced the expression level of SOD and GPX(P < 0.05)and significantly down-regulated the expression level of Nrf2 and HO-1(P < 0.05);EGCG significantly increased the expression level of SOD,GPX,Nrf2 and HO-1 gene(P < 0.05).5.For egg production performance,both energy sources significantly reduced the egg production rate at 21-44 wk(P < 0.05)and reduced the average feed intake comparing to the control group;the soybean oil group significantly increased the feed-to-egg ratio at25-28 wk,29-32 wk and 41-44 wk(P < 0.05);EGCG significantly reduced the feed-to-egg ratio at 21-24 wk(P < 0.05),but EGCG significantly reduced 21-24 wk feed-to-egg ratio(P < 0.05),while also significantly increased 41-44 wk feed-to-egg ratio(P < 0.05).In this experiment,it was found that the high energy diet started the laying age of laying hens earlier,but through reducing liver lipid metabolism and reproductive hormone levels,thus reducing follicular development of fertile hens,lowering the antioxidant capacity of the body and ovaries,and producing oxidative stress;the high energy diet group had poor follicular development quality and lower egg production rate during egg laying,and the effect was worse in the linseed oil group;the addition of EGCG significantly improved the antioxidant capacity of the body and had a tendency to improve egg production performance.Experiment 3.Effects and mechanisms of high-energy diets and EGCG on growth and subsequent egg production performance of fertile hensIn this experiment,567 young Lohmann laying hens of similar weight at 78 days of age were used in a 2 × 3 + 1 factorial design,including 7 treatments with 3 diets of 9replicates each: basal diet 11.40 MJ/kcal,high energy diet 13.22 MJ/kcal(HE,other nutrient levels were kept the same as normal energy levels),high nutrition diet(HN,energy levels were the same as HE,keeping the diets(other nutrient levels and energy ratios were the same as the basal diet);2 EGCG levels(0,200 mg/kg),all 6 treatments were fed with ad libitum;in addition,a basal diet ration feeding treatment(CONR)was added.The test period was 12-17 weeks of age.The results showed that:1.For growth performance,the CONR group significantly reduced the body weight of laying hens at 14,15,16 and 17 wk,comparing to the control group(P < 0.05).The high energy level significantly reduced the mean daily feeding intake of laying hens at 13,14 and 18 wk(P < 0.01).EGCG significantly reduced feeding intake of laying hens at 16wk(P = 0.04)and CONR significantly reduced ADFI(except 13 wk and 17 wk)comparing to the control group(P < 0.05);the high energy level significantly increased abdominal fat weight(P = 0.02)and abdominal fat index(P = 0.03).2.For blood indices,high energy diets significantly reduced the level of serum VLDL-C in 17-week-old hens(P < 0.01),while EGCG significantly increased the level of serum VLDL-C(P < 0.01),it also significantly increased serum MDA levels(P < 0.01)and CAT activity(P < 0.01)and significantly decreased serum SOD activity(P < 0.01);EGCG significantly increased serum T-AOC,SOD and GSH-PX activities(P < 0.01),but significantly decreased serum MDA content and CAT activity(P < 0.01).3.For liver function,high energy level significantly decreased hepatic T-AOC activity in 17-week-old hens(P < 0.05)but significantly increased hepatic MDA content(P< 0.01).HE group was significantly higher in the high nutrition group than in the remaining two groups;EGCG significantly increased hepatic T-AOC,SOD and GSH-PX activities(P < 0.01)and significantly decreased hepatic MDA content and CAT activity(P< 0.01).High energy levels diet? significantly upregulated the expression of ACACA and FASN in the liver(P < 0.01),and addition of EGCG significantly downregulated m RNA expression of these 2 genes(P < 0.01),but significantly increased m RNA expression of lipid transporter Apo VLDL-II and CPT1(P < 0.01).4.In terms of ovarian function,high energy levels significantly reduced serum PROG,luteinizing hormone(LH)and leptin(LEP)concentrations in 17-week-old hens(P< 0.05)and EGCG significantly increased serum E2,PROG,IGF-1 and AMH concentrations(P < 0.05);high energy levels diet?? significantly reduced ovarian T-AOC activity(P = 0.01)and significantly increased MDA levels(P = 0.03),EGCG significantly increased ovarian T-AOC,SOD(P < 0.01)and GSH-PX activity(P = 0.01)and significantly decreased MDA content and CAT activity(P < 0.01);high energy levels significantly down-regulated NQO1,Nrf2 and SOD gene m RNA expression(P < 0.01),EGCG significantly increased GPx,The m RNA expression of GPx,NQO1,SOD and Nrf2 were significantly increased by EGCG(P < 0.01).5.For egg production performance,dietary energy and EGCG had no significant effect(P > 0.05)on the 18-41 wk egg production rate of hens,but it was observed that the HE groups consistently had higher egg production rate than the rest of the groups.The effect on egg quality was found to be significantly lower in the CONR group compared to the control group in terms of HN units(P = 0.02),with a tendency to reduce protein height and improve yolk color(P = 0.05).Ovarian morphology was observed in the HN group with a lower number of pregraded follicles(large yellow,small yellow and large white follicles)than the rest of the group and with poor follicular development.In this experiment,it was found that free-feeding high-energy diets reduced liver lipid metabolism and reproductive hormone levels,thereby promoting follicular development in fertile hens and improving the quality of follicular development after laying,thereby increasing egg production;however,high-energy diets,especially those with high nutrient concentrations(HN),significantly reduced the antioxidant capacity of the serum,liver and ovaries of hens;the addition of EGCG significantly improved the antioxidant capacity of the body.Experiment 4.The regulatory mechanism of oleic acid-induced hepatocyte steatosis in chickens by EGCGIn this experiment,liver tissues were collected from 25-week-old laying hens,and liver cells were isolated for in vitro culture.The hepatocytes were treated with 0.6 m M oleic acid(OA)and 1 u M EGCG.The test treatments were: NC,EGCG,OA,OA+EGCG.the results were shown as followings:1.EGCG significantly reduced OA-induced TG and TC content in hepatocytes(P <0.05),reduced intracellular lipid droplet accumulation,and significantly increased intracellular fatty acid β-oxidation in liver(P < 0.05).2.OA intervention significantly decreased cellular antioxidant enzyme activities and expression of antioxidant-related genes.EGCG treatment increased antioxidant enzyme activity and expression of related genes(P < 0.05),and EGCG significantly alleviated OA-induced increases in hepatocyte reactive oxygen species(ROS)and mitochondrial membrane potential levels(P < 0.05).3.OA intervention significantly increased cellular inflammation-related gene m RNA expression(P < 0.05),and EGCG add-ons decreased cellular inflammation-related gene m RNA expression,and cellular IL-6 protein expression level was significantly increased after OA induction(P < 0.05).EGCG showed a rising trend(P < 0.05).EGCG significantly reduced the levels of OA-induced IL-6 and IL-1β(P < 0.05).4.EGCG significantly reduced the m RNA expression of DNA damage-related geneγ-H2AX(P < 0.05).OA increased the expression abundance of γ-H2 AX protein(P < 0.05),while EGCG treatment decreased the expression of γ-H2 AX protein(P < 0.05).Immunofluorescence assay further verified that EGCG significantly increased the expression of DNA repair pathway-related genes(P < 0.05).5.OA intervention significantly increased the expression of autophagy and apoptosis-related genes and proteins(P < 0.05),and EGCG significantly decreased the expression of autophagy and apoptosis-related genes(P < 0.05).6.A total of 56,757 genes were detected by transcriptome sequencing of different treatment groups,and the results of Wayne analysis showed that 33688 genes were expressed simultaneously within the four groups.350 genes were down-regulated in the OA group,and the downward trend of these genes was reversed EGCG add-ons;355 genes were increased in the OA group,but the expression of genes in this group was reduced by adding EGCG.KEGG enrichment analysis of the differentially expressed genes from both groups revealed that both could be enriched to MAPK pathway.OA intervention significantly increased the phosphorylation level of p38,a key protein of MAPK pathway,but extra EGCG significantly inhibited the rising trend of OA-induced p-p38,indicating that EGCG could activate MAPK pathway by inhibiting the phosphorylation level of p38.In this experiment,we found that exogenous sources of oleic acid significantly reduced the antioxidant stress capacity of hepatocytes cultured in vitro,increased the level of autophagy and led to apoptosis,and EGCG could alleviate hepatocyte damage through MAPK signaling pathway.In conclusion,the results of this study showed that tea polyphenols could improve antioxidant capacity,and the products rich in EGCG and low in caffeine had the most obvious effect;the quantitative feeding of soybean oil or flax oil high-energy rations during the breeding period improved the body weight of breeding hens,started laying earlier,and increased liver fat content,but significantly reduced liver fat transfer capacity and antioxidant capacity,which affected the health of laying hens and reduced egg production rate after subsequent laying,with no difference between the two There was no difference in the degree of effect between the two sources;the addition of EGCG alleviated the adverse effects of the high-energy diet.The addition of EGCG alleviated the adverse effects of the high-energy diet.The free-feeding basal diet increased the body weight of laying hens during the breeding period but did not affect the start of laying;the free-feeding high-energy diet significantly reduced the feed intake of breeding hens,significantly increased the body weight of breeding hens and started laying earlier;by affecting liver lipid metabolism and reproductive hormone levels,it promoted the follicular development of breeding hens and started laying earlier,and improved the quality of follicular development after laying,thus increasing the egg production rate;however,the high-energy diet,especially the high-nutrient concentration diet(HN),significantly reduced the egg production rate after laying.The addition of EGCG significantly improved the antioxidant capacity of hepatocytes through the MAPK signaling pathway,and regulated autophagy and apoptosis of hepatocytes caused by high energy and alleviated hepatocyte steatosis.The results of this study can provide a theoretical basis for analyzing the regulatory mechanism of high energy levels affecting egg production performance and oxidative stress in laying hens. |
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