The Nutri-epigenetic Mechanism Of Immune Function Regulated By Folic Acid In Broilers During Incubation Period

Folic acid is a water-soluable vitamin B, which participates in many physiologicalprocesses: involved in metabolism of genetic material and proteins, influencing of animalbreeding, promoting animal growth and improving animal immunity and so on. Thecirculation speed is fast for folic acid in the body and its low deposition rate leads to folic aciddeficiency. Embryonic period is the key of larval form, the adequate supply of nutrients canensure the normal embryonic development and subsequent health. For mammalian, theyobtain all kinds of nutrients from female parent during embryonic period, but the process ofembryonic development for chickens is away from their mothers. Therefore, folic acid duringincubation might be important for embryonic development, broiler performance and immunity.In this study, we injected different levels of folic acid at different embryonic ages toinvestigate the broiler performance and immune function. The epigenetic mechanism ofimmune effector molecules expression was investigated by using the methods ofMnase-qPCR and Chip-qPCR, which will provide a scientific data for nutri-epigeneticmechanism of immune function regulated by early nutrients in broilers.Trial1Effects of injecting different levels of folic acid at different embryonic agesduring incubation period on growth performance and liver antioxidant capacityof broilersThis experiment was conducted to explore the growth performance and immunefunction of broilers regulated by the suitable dose of folic acid and the proper time throughinjecting different levels of folic acid at3,7and11embryonic ages. A total of342(Sasso)hatching eggs were collected, using33factorial experiment design. Experimental factorsconsist of embryonic age and folic acid level. The former includes3,7and11embryonic ages;the latter contains saline group (injected0.1mL saline per egg),45g folic acid group(injected0.1mL saline with45g folic acid per egg) and90g folic acid group (injected0.1mL saline with90g folic acid per egg). Each group was allocated to4replicates with6broilers per replicate after incubation. Incubation period was21d, breeding period was42d.The results showed as follows:1) injecting different levels of folic acid at different embryonic ages significantly affected the hatchability of eggs (P <0.01), injecting different levels offolic acid at3and11embryonic ages had no significant effects on birth weight and liverindex of1-day-old broilers (P>0.05), had significant effects on spleen index and the index ofbursa of Fabricus of1-day-old broilers (P <0.05);had no significant effects on body weight,the index of bursa of Fabricus and liver index of broilers aged21d (P>0.05),but both ofembryonic age and folic acid level had an increasing trend in the body weight of broilers aged21d. Injecting different levels of folic acid at3and11embryonic ages had significant effectson spleen index of broilers aged21days, had no significant effects on spleen index, liverindex and the index of bursa of Fabricus of broilers aged42d (P>0.05),had significanteffects on body weight of broilers aged42d, and compared with3embryonic age,11embryonic age significantly increased body weight of broilers aged42d, and the body weightof broilers aged42d had an increasing trend with an increased level of folic acid.2) injectingdifferent levels of folic acid at3and11embryonic ages had no significant effects on theplasma albumin content of broilers aged1day (P>0.05), had significant effects on plasmatotal protein and albumin content (P <0.05), and compared with3embryonic age, plasmatotal protein content significantly increased at11embryonic ages, and there had an interactionbetween embryonic age and folic acid. Compared with3embryonic age, plasma globulincontent significantly increased at11embryonic age (P <0.05), and compared with3embryonic age, plasma globulin content significantly increased at11embryonic ages injectingfolic acid (P <0.05), plasma globulin content had an increasing trend with an increased levelof folic acid and there had no interaction between embryonic age and folic acid.3) injectingdifferent levels of folic acid at3and11embryonic ages had no significant effects on liverantioxidant capacity (P>0.05), the activity of glutathione peroxidase (GSH-Px) had anincreasing trend with an increased level of folic acid, meanwhile the activity of GSH-Px hadan increasing trend at11embryonic ages compared with3embryonic age injecting folic acid.These results suggested that injecting folic acid at11embryonic ages during incubation canimprove growth performance, plasma protein content and liver antioxidant capacity ofbroilers.Trial2Effects of injecting different levels of folic acid at11embryonic ages on growthperformance and the mRNA levels of immune effector molecules of broilersThis experiment was aimed to investigate the effects of injecting folic acid at11embryonic ages on growth performance, the mRNA level of folic acid metabolism genes andimmune effector molecules of broilers by measuring plasma lysozyme, IgG and IgM content,liver folic acid content, the mRNA levels of MTHFR and MTRR in liver, the mRNA levels of IL-2, IL-4and IL-6in spleen. A total of342(Cobb) hatching eggs were randomly subjected toone of the following4groups: saline group (injected0.1mL saline per egg),50g folic acidgroup (injected0.1mL saline with50g folic acid per egg),100g folic acid group (injected0.1mL saline with100g folic acid per egg) and150g folic acid group (injected0.1mLsaline with150g folic acid per egg), using single factor experiment design. Each group wasallocated to4replicates with8broilers per replicate after incubation. Incubation period was21d, breeding period was42d. The results showed as follows:1) injecting different levels offolic acid at11embryonic ages had significantly effects on the hatchability of eggs (P <0.05).There had extremely significant difference in the average daily gain (ADG) of broilersaged1to21d among all groups (P <0.008),the average daily feed intake (ADFI) of broilershad an increasing trend with an increased level of folic acid, while feed/gain (F/G) of broilershad a decreased trend with an increased level of folic acid. There had extremely significantdifference in feed/gain (F/G) of broilers aged21-42d among all groups (P=0.001), ADG andADFI of broilers ages21to42d had no significant difference among all groups (P>0.05).The ADFI and F/G of broilers aged0-42days had significant difference among allgroups (P <0.05), but ADG had no significant difference among all groups (P>0.05). Heartindex and liver index of broilers aged1day had no significant difference among all groups(P>0.05).2) injecting different levels of folic acid at11embryonic ages had significanteffects on plasma lysozyme activity, IgG and IgM content of broilers at different time.3)Liverfolic acid content of broilers aged1,21and42d had significant difference among all groups(P <0.05),the relative expression of MTHFR of broilers aged21d in liver had no significantdifference among all groups (P>0.05),the relative expression of MTHFR of broilers aged1and42d in liver had significant difference among all groups (P <0.05),the relative expressionof MTRR in liver at1and42d had no significant difference among all groups(P>0.05), butthe relative expression of MTRR in liver at1day had an increasing trend with an increasedlevel of folic acid. The MTRR relative expression at21d of broilers had significant differenceamong all groups (P<0.05).4)injecting different levels of folic acid at11embryonic ages hadno effects on IL-2relative expression of broilers aged1and21d in spleen (P>0.05),but therelative expression of IL-2had an increasing trend with an increased level of folic acid; hadextremely impact on the relative expression of IL-4and IL-6of broilers aged1,21and42d(P <0.01).These results suggested that injecting150g folic acid at11embryonic agessignificantly improved growth performance and immune function of broilers.Trial3Explore the nutri-epigenetic mechanism of immune effector molecularsregulated by folic acid during incubation This experiment was carried out to assess whether the nutri-epigenetic mechanism ofimmune function of broilers was associated with folic acid during incubation by measuringstructural tightness and CHIP analysis of IL-2, IL-4and IL-6in promoter region.1)Thetightness of IL-2of broilers aged21and42d in promoter region had significant differenceamong all groups(P=0.034; P=0.008), IL-4and IL-6structural tightness of broilers aged21and42d in promoter region had no significant difference among all groups (P>0.05).2)injecting150g folic acid at11embryonic ages had no significant effects on H3K4methylation of IL-2in spleen of broilers aged21d (P>0.05),but had significant effects onH3K4methylation of IL-2of broilers aged42d (P=0.012),the H3K4methylation of IL-4inspleen of broilers aged21and42d had significant difference among all groups (P <0.01), theH3K9methylation of IL-6in spleen of broilers aged21and42d had significant differenceamong all groups (P <0.05).These data suggested that injecting150g folic acid at11embryonic ages had significant effects on structural tightness and histone modifications ofIL-2, IL-4and IL-6. Both of structural tightness and H3K4methylation in promoter regionregulate the relative expression of IL-2, the relative expression of IL-4regulated by H3K4methylation in promoter region, while relative expression of IL-6regulated by H3K9methylation in promoter region.