| The continuous breeding selection,nutrient optimization and intensive farming shortened production cycle of broilers,however the resulting high-intensity metabolism leads to immune dysfunction.Folic acid is an important metabolic regulator of life metabolism with many researches on trans-generation regulation.In the present study,we made different levels of folic acid supplementation to the breeding cocks.We attempted to study the effects of folic acid on the growth performanceand antioxidant activityof the cocks and their offspring.We also investigated the potential trans-generation mechanisms of the folic acid on the sperm piRNAs by using high-throughput sequencing technology,which we wish to provide theoretical evidence to the trans-generational regulation mechanisms.Experiment 1 Effects of paternal folic acid on the production performance and organ development of breeding cocks and F1 generationsInexperiment,200 one-day-old healthy Arbor Acres broiler cocks were randomly divided into 5 treatments with 5 repeats per treatment and 8 males in each repeat.The broilers were fed a corn and soybean diet with 5 FA levels(0,0.25,1.25,2.5,and 5 mg/kg).The cocks fed with 2.5mg/kg folic acid supplementation in diet had the highest body weight at 36 weeks,with the 1.25mg/kg goups were the lowest(P < 0.05).For the 21 d F1 generations,folic acid supplementation in diet had no effects on the body weight,feed intake and average daily gain(P >0.05).Feed meat ratio was the highest when fed with 0.25mg/kg folic acid dietary supplementation during the first 21days(P < 0.05).For the bursa of fabricius index of the breeding cocks,5mg/kg supplementation group was the highest and the control group was the lowest(P < 0.05).For the F1 one-day-old generation,folic acid had no effects on bursa of fabricius index(P >0.05).For the F1 21-day-old generation,the bursa of fabricius index was the lowest when cocks were fed with 0.25mg/kg folic acid and was the highest with 5mg/kg folic acid supplementation and the control group(P < 0.05).For the breeding cocks,the duodenal villi height was the highest in the 5 mg / kg treatment group(P <0.05).For the F1 21-day-old generation,1.25 mg/kg group reduced the duodenum villus height and the ratio of duodenum villus height to the crypt depth(P <0.05).This study indicates that 5mg/kg folic acid dietary supplementation promotes the development of the villus of the duodenum,delays the degeneration of the bursa of fabricius.Moreover,it may have potential transgenerational regulation effect on the development of the bursa of fabricius of the F1 generations,and reduces feed meat ratio.Experiment2Effects of breeding cock dietary folic acid supplementation on the antioxidant activity and immune function of cocks and offspring broilersIn the present study,we collected serum,liver and spleen of 40-week breeding cocks and 1-day and 21-day F1 generations to detect antioxidant capacity and expression levels of spleen immune factors.The results showed that when dietary was supplemented with 5 mg/kg folic acid,the rooster got the highest serum total antioxidant capacity(T-AOC)(P < 0.05).For the 1d and 21 d F1 generations,the broilers fed with 1.25,2.5,5 mg/kg folic acidsupplemented dietary had higher serum T-AOC(P < 0.05).However,there were no differences on liver T-AOC of the breeding cocks and F1 generations(P > 0.05).When treated with 2.5 mg/kg folic acid,the 21 d broiler showed higher serum superoxide dismutase(SOD)than other treatments(P < 0.05),however there were no differences about this index in cocks' serum and liver,as well as 1d broiler's serum and 21-day broiler's liver(P > 0.05).For the breeding cock,0 and 0.25 mg/kg folic acid supply improved liver SOD level compared with 1.25 mg/kg and 5 mg/kg supply(P < 0.05).In addition,2.5 and 5 mg/kg folic acid supplementation reduced the roosters' serum MDA contents but improved liver MDA contents(P < 0.05).No significant impacts were found on the 1d and 21 d broiler serum and liver(P > 0.05).For the breeding cocks and F1 generations,there were no significant differences on serum and liver GSH-Px level(P > 0.05).For the immune factor expression levels of the spleen as well as the apoptotic factor expression levels of the thymus and bursa of Fabricius,there were no significant differences on the breeding cocks and their F1 generations(P > 0.05).The same results were also shown in spleen DNA 5-mc content(P > 0.05).Collectively,these results indicate that 5 mg/kg folic acid dietary supplementation reduces the breeding cocks' serum MDA levels and improves serum T-AOC level of the breeding cocks and F1 generations.Experiment3Effects of adding different levels of folic acid to the diet of cocks on the semen piRNA expressionTo further investigate the potential trans-generational mechanisms of parental folic acid on their generations,we collected the semen samples of the 32-weeks rooster for highthroughput sequencing analysis.The results showed that a total of 2510 piRNAs were found in the cock semen samples,of which 473 were already known and 2037 were novel.Differential expression analysis showed that 1630 piRNAs were differentially expressed in the cock semen due to folic acid supplementation.Comparing with the control group,we found 594,589,972 and 439 differentially expressed pi RNAs in the four groups with different folic acid supplementation levels respectively.To predict that the biological processes and functions of the target genes,GO and KEGG analysis were performed.GO analysis showed that many target genes were involved in cell formation and development processes.KEGG analysis showed that the predicted target genes of the piRNAs were enriched in 14 KEGG pathways.These potential target genes were mainly enriched in glycolipid metabolism related pathways,indicating that folic acid could trans-generationally regulate glycolipid metabolism processes.In conclusion,our study indicate that 5 mg/kg dietary folic acid supplementation promotes the development of the villus of the duodenum,delays the degeneration of the bursa of fabricius and improves serum T-AOC level.Moreover,5 mg/kg dietary folic acid supplementation also improves development of the bursa of fabricius and duodenum villus of 21-day F1 generation,as well as reduces feed meat ratio. |
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