Study On The Dietary Folic Acid Requirement Of Juvenile Largemouth Bass(Micropterus Salmoides)

Largemouth bass(Micropterus salmoides)is currently a major species in freshwater aquaculture in China.With the rapid development of its aquaculture,the development of specialised compound feeds in term of the precise nutritional requirements of has become increasedly important.As one of the B-soluble vitamins essential for growth maintenance in aquatic animals,folic acid is involved in the metabolism of substances in the body as a coenzyme tetrahydrofolate,and provides methyl for methylation as a one-carbon unit donor to amino acid metabolism.In order to investigate the optimum requirement of folic acid in practical diets for juvenile largemouth bass and its mechanism of action,based on traditional feeding experiments,the optimal dietary folic acid requirement for juvenile largemouth bass was determined by analysing the effects of graded levels of folic acid on growth performance,body composition,haematological parameters,nutritional metabolism and antioxidant immunity,combined with regression analysis of growth performance and addition gradients.The response and mechanism of action of the dietary folic acid in juvenile Largemouth bass was investigated by integrated transcriptional and translational analysis.1.Effect of dietary level of folic acid on growth performance,blood biochemistry,antioxidant capacity and immunity of juvenile largemouth bassTo obtain the optimal requirement of dietary folic acid(FA)for juvenile largemouth bass,five experimental diets were prepared with 0,0.5,1.5,4.5 and 13.5 mg/kg of supplemental FA in the basal practical diet(measured FA value < 0.18 mg/kg in the basal diet)and fed to juvenile largemouth bass with an initial body weight of(14.49± 0.06)g for 7 weeks.The results showed that the weight gain rate(WGR)and specific growth rate(SGR)of the experimental fish in each FA supplementation groups were higher than those in the control group to varying degree and reached maximum values at 1.5 mg/kg in the experimental group,which were significantly higher than those in the FA-free group(P < 0.05);the increase in the addition of FA to the diet significantly reduced the visceral to body ratio(VSI)(P < 0.05),liver to body ratio and relative intestine weight.The addition of no less than 0.5 mg/kg FA to the feed significantly increased the whole fish protein content(P < 0.05).The hemoglobin content and haematocrit of whole blood cells were significantly increased in the fish group supplemented with 0.5-1.5 mg/kg FA(P < 0.05),and the addition of 0.5 mg/kg FA significantly increased the number of red blood cells(P < 0.05).Plasma total protein(TP)and albumin(ALB)levels increased and then decreased with the addition of FA,reaching a maximum at 1.5 mg/kg supplementation,and differed significantly from the FA-free group(P < 0.05).The addition of 0.5mg/kg and more of FA significantly decreased plasma glutamic-pyruvic transaminase(GPT)activity and improved liver GPT activity(P < 0.05),the addition of0.5mg/kg and more of FA significantly decreased serum malonaldehyde activity(P <0.05),0.5mg/kg of FA significantly increased total hepatic antioxidant capacity(P <0.05),1.5mg/kg of FA significantly increased hepatic catalase and head kidney lysozyme activity(P < 0.05).The addition of 0.5mg/kg and above of FA significantly reduced liver glycogen content(P < 0.05).In conclusion,the addition of FA to the diet significantly improved the growth performance,haematopoiesis,metabolism of protein and carbohydrate,antioxidant capacity of the liver,and protected liver health of juvenile largemouth bass(P < 0.05).The regression analysis based on growth performance showed that the optimal amount of FA in the feed of juvenile largemouth bass was 1.42-1.46 mg/kg.2.Effects of folic acid on digestibility,intestinal antioxidant capacity and fillet quality of largemouth bassThe samples from the end of the feeding experiment in Chapter1 were analyzed to evaluate the effects on digestibility,intestinal antioxidant capacity and muscle quality were analyzed.The results showed that pepsin was significantly higher in the 13.5 mg/kg folic acid supplemented group than in the FA free group(P < 0.05),and gastric lipase was significantly higher in the FA0.5 and FA13.5 groups than in the FA0 group(P <0.05);pyloric blind sac pepsin was significantly higher in the folic acid supplemented groups than in the folic acid free group(P < 0.05),and pyloric blind sac lipase increased and then decreased with the addition of folic acid in the FA0.5 and FA13.5 groups(P <0.05).The amylase activity of all digestive sites decreased with the addition of FA.Midgut sodium-potassium ATPase activity increased and then decreased with the addition of FA,reaching a maximum in the FA1.5 group(P < 0.05).The total antioxidant capacity showed a trend of increasing followed by decreasing with the addition of FA and reached the maximum value in the FA4.5 group(P < 0.05),while there was no significant difference on peroxidase among all groups(P < 0.05).The histidine,lysine and total essential amino acid contents were significantly higher in the groups supplemented with more than 1.5 mg/kg of FA than in the group without FA(P < 0.05).The addition of 13.5 mg/kg FA significantly reduced the total saturated fatty acid content(P < 0.05),and the total monounsaturated fatty acid and polyunsaturated fatty acid contents were significantly higher in the FA1.5 group than in the other groups(P < 0.05),with consistent results for all fatty acids.The study showed that the addition of FA to the diet could improve the protein and fat utilization of largemouth bass,and the addition of4.5 mg/kg of FA could maximize the intestinal antioxidant capacity,and the addition of4.5 mg/kg of FA could optimize the muscle composition of largemouth bass.3.Analysis of hepatic transcriptional mechanisms in response to dietary folic acid in juvenile largemouth bassIn order to investigate the physiological regulation mechanism of FA in feed for the growth of largemouth bass,therefore,in this chapter,the liver of the FA-free group(FA0)and the best growth group(FA1.5)were selected to be studied at the transcriptional level based on the growth performance results in Chapter 1.The transcriptomic results showed that the total number of differential Unigene detected was 638(P < 0.05),with 266 upregulated Unigene and 372 down-regulated Unigene,using FA0 as the control group and FA1.5 as the experimental group.The results of GO enrichment analysis showed that a total of 627 enrichment entries were obtained,and a total of 437 significant enrichment entries were screened(P-adj < 0.05),the most enriched differential genes were related to cellular components,and the most significant differential entries were enriched for immune response,DNA synthesis,antigen processing and presentation.After screening by KEGG significant enrichment pathways(P-adj < 0.05),a total of 6 pathways were found to be significantly enriched,mainly in digestion,endocrine system,translation and cardiovascular disease,with the most differentially expressed genes enriched in the protein digestion and absorption pathway.Seven differentially expressed genes were selected for validation and the results of RT-PCR validation were consistent with the trend of FPKM values obtained from differential gene transcriptome sequencing.All the results preliminarily elucidated the biological processes,metabolic pathways or signaling pathways involved in the liver tissue genes of largemouth bass when folic acid was added to the diet.It is possible that FA addition activated PI3K-Akt and MAPK signaling pathways to further activate downstream effectors and promote protein synthesis.4.Response of juvenile largemouth bass to dietary folic acid analyzed by joint analysis of the liver proteomics and transcriptome To further investigate the mechanism of FA in feed for the regulation of largemouth bass,the experimental group was selected as in Chapter 3 and studied at the translational level.Proteomics results showed that a total of 183 significantly differentially expressed proteins(P < 0.05)were detected,with 31 differentially expressed proteins significantly up-regulated and 152 differentially expressed proteins significantly down-regulated,using FA0 as the control group and FA1.5 as the experimental group.According to the GO functional annotation results,37,987 proteins were annotated to the GO library,of which 151 total differential proteins were annotated to the GO library.The largest number of entries enriched to the top 30 entries was for biological processes with 17 significant enrichments,followed by molecular functions with 9 entries and finally cellular components,with the largest number of genes enriched to extracellular regions,followed by cell adhesion.The entries enriched for differential proteins were UV protection,regulation of collagen metabolic processes,elastic fibre composition,cellular response to UVB,supramolecular fibre organisation,and structural molecular activity.According to the KEGG annotation results,a total of 15404 proteins were annotated in which 54 differential proteins were included,and a total of 104 pathways were enriched,containing25 up-regulated ones and 91 down-regulated ones.Compared to the control group,the two differential proteins enriched to the lipid metabolism pathway were significantly upregulated in the experimental group,and the differential proteins enriched to 13 pathways were significantly down-regulated,metabolic and organic systems were mainly involved.The sequence alignment of differential genes and differential proteins revealed only one set of genes and proteins with high similarity,but the differential genes and differential proteins were expressed in opposite trends.3 GO entries were enriched for serine endopeptidase activity,extracellular region and metal ion binding,and only two metabolic pathways were enriched for KEGG,and one pathway was enriched for pancreatic secretion-related pathway after integration.FA may regulate cela expression in pancreatic secretion and thus the pathways of protein digestion and absorption.