Study On The Effect Of The Phytogenic Feed Additives On Chicken Reproduction Performance,Antioxidant Capacity And Functional Role Of Quercetin On Follicle Development

With globalization,the growing demand for livestock products will continue in the future,and with feed resources becoming increasingly scarce,developing new feeds and improving the quality of livestock products are important research objectives for the livestock industry today.Moringa oleifera and mulberry leaves are rich in nutrients and can be used to replace some of the ingredients in livestock diets to improve livestock performance and the quality of livestock products.However,there are few reports on the feeding effects of mixing Moringa oleifera and Mulberry leaves in poultry feed.In this study,we added different level of Moringa oleifera leaf powder and different proportions of Moringa oleifera leaf powder and Mulberry leaf powder to the basic diet formulation at post-peak laying period in local hens to study their effects on feed utilization,production performance and physiological and biochemical indexes of chickens,and to provide a reference basis for the diversified development and utilization of phytogenic feed.Moringa oleifera and mulberry leaves are rich in flavonoids,which have the effect of improving the reproductive performance of poultry.Quercetin is one of the important flavonoids,but its effect on the regulation of chicken reproductive performance has rarely been reported.In this study,we investigated the effects of quercetin on the functional and transcriptional levels of cultured follicular granulosa cells in vitro and analyzed the mechanisms of its effects on the reproductive performance of chickens.Research 1: Effect of Moringa oleifera leaf powder on chicken performance,egg quality,lipid metabolism and antioxidant functionThe control group was fed the basal diet and the treatment group was supplemented with 2.5%,5%,7.5% and 10% Moringa oleifera Leaf Powder(MOLP).During the 6-week trial period,production performance was measured,mainly in terms of daily egg production,feed consumption and mortality rate.Thirty eggs(6 eggs per replicate)were collected from each group at the end of the 2nd,4th and 6th week of the trial to determine egg quality.Blood was collected at the end of week 6 of the trial period for antioxidant levels,lipid,and hormone levels.The chickens were slaughtered to determine internal organ index and the weight of ovaries and the number and weight of different follicles.Liver and ovarian tissues were taken for relative quantitative gene expression assays.The results showed that supplementation of Moringa oleifera leaf powder significantly reduced the feed intake and feed conversion ratio of the hens(P<0.05),while the laying rate of the 2.5% MOLP group was not significantly affected(P>0.05).There was no significant difference in heart index,liver index or kidney index(P>0.05);follicle number and weight increased significantly in the 2.5% MOLP group(P<0.05);serum triglyceride and HDL levels decreased significantly in the 2.5% MOLP group(P<0.05)and serum glucose levels decreased significantly in the MOLP group(P<0.05);serum superoxide dismutase(SOD)levels decreased significantly in the 2.5% MOLP group(P<0.05).(SOD)levels were significantly higher in the 2.5% MOLP group(P<0.05);SOD1 m RNA expression levels were significantly higher in the 2.5% and 7.5% MOLP groups than in the control group(P<0.05);cytochrome P450 family 19 subfamily A member 1(Cyp19a1)and steroidogenic acute regulatory protein(St AR)expression levels were significantly higher(P<0.05).In conclusion,the addition of 2.5% Moringa oleifera leaf powder to the diet had no adverse effects on chicken health,but improved yolk color,increased the antioxidant capacity,reduced feed conversion and abdominal fat index,increased follicle weight and number,and increased the expression of genes related to steroid synthesis.Research 2: Effect of different ratio of Moringa oleifera leaf powder and Mulberry leaf powder on chicken performance,egg quality,lipid metabolism and antioxidant functionThe subjects were randomly divided into 3 groups with 5 replicates of 14 chickens each.The control group was feed the basel diet.The treatment groups were supplemented with 2.5% Moringa oleifera leaf powder + 2.5% Mulberry leaf powder(MLP)and 5%Moringa oleifera leaf powder + 2.5% Mulberry leaf powder in the basal diet respectively.The treatment time,sample collection and statistical analysis were as same as in Trial 1.Results showed that the laying rate of supplementated of 5% MOLP + 2.5% MLP significantly reduced(P<0.05),but the feed conversion ratio was highly significantly reduced after the addition of Moringa oleifera leaf powder and Mulberry leaf powder compared to that in control group(P<0.001),and the number of clutches was significantly higher than that in the control group(P<0.05);egg yolk color of supplementated of MOLP and MLP significantly increased(P<0.05);Moringa oleifera leaf powder and Mulberry leaf powder had a significant effect(P<0.05)on reducing abdominal lipid index.In terms of antioxidant indicators and hormone levels,there were no significant differences in serum superoxide dismutase(SOD),malondialdehyde(MDA)and total antioxidant capacity(T-AOC)between the groups(P>0.05),and no significant differences in serum estrogen and progesterone levels(P>0.05).RT-q PCR analysis showed that CAT,Nrf2,SOD1 and ACC were not significantly different between the groups(P>0.05),and MOLP and MLP significantly reduced the expression level of apolipoprotein-b(APo B)(P<0.05)and significantly increased the expression level of SOD2 m RNA.The expression levels of ESR1 and Cyp19a1 in the 2.5% MOLP and 2.5% MLP were significantly upregulated compared to the other groups(P<0.05).In conclusion,the addition of 2.5% Moringa oleifera leaf powder and 2.5% Mulberry leaf powder to the diet had no significant effect on egg production performance,egg weight and most egg quality indicators,and was able to improve egg yolk color.Research 3: Regulation effect of quercetin on follicular granulosa cells development in chickenIn vitro isolation of SYFG(4-8 mm in diameter)and hierarchical follicles granulosa cells(>8 mm in diameter)was carried out to examine the effects on cell proliferation,hormone synthesis and transcriptome levels.The effects of quercetin on the granulosa cells from hierarchical follicles showed that: cell viability was significantly increased when quercetin treatment at 24 h(P < 0.05),progesterone secretion levels significantly increased(P < 0.05).Quercetin treatment had no significant effect on the expression of granulosa cell P450 scc,3β-HSD and St AR m RNA(P > 0.05).The 1000 ng/m L quercetin group significantly increased PCNA m RNA expression levels(P < 0.05).There was no significant effect on cell proliferation(P>0.05)after 24 h treatment with various concentrations of quercetin in SYFG,and the level of progesterone secretion in granulosa cells was significantly increased(P<0.05).The results of transcriptional regulatory group analysis showed that there were 665 differentially significantly expressed genes after quercetin treatment of SYFG,of which 402up-regulated and 263 down-regulated,mostly enriched in the Focal adhesion,TGF-βsignaling pathway,Gn RH signaling pathway.Granulosa cells may induce follicular development and selection by regulating the expression levels of genes related to receptors and steroid synthesis.