Integrative network analysis revealed the molecular function of folic acid on immunological enhancement in a sheep model.

We previously observed the beneficial role of folic acid supplemented from maternal or offspring diet on lamb growth performance and immunity. Twenty-four Hu lambs from four groups (mother received folic acid or not, offspring received folic acid or not) were used in the current study, which was conducted consecutively to elucidate the molecular regulatory mechanisms of folic acid in lambs by analyzing blood metabolome, liver transcriptome, and muscle transcriptome. Serum metabolomics analysis showed that L-homocitrulline, hyodeoxycholic acid, 9-Hpode, palmitaldehyde, N-oleoyl glycine, hexadecanedioic acid, xylose, 1,7-dimethylxanthine, nicotinamide, acetyl-N-formyl-5-methoxykynurenamine, N6-succinyl adenosine, 11-cis-retinol, 18-hydroxycorticosterone, and 2-acetylfuran were down-regulated and methylisobutyrate was up-regulated by the feeding of folic acid from maternal and/or offspring diets. Meanwhile, folic acid increased the abundances of S100A12 and IRF6 but decreased TMEM25 in the liver. In the muscle, RBBP9, CALCR, PPP1R3D, UCP3, FBXL4, CMBL, and MTFR2 were up-regulated, CYP26B1 and MYH9 were down-regulated by the feeding of folic acid. The pathways of bile secretion, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, and herpes simplex virus 1 infection were changed by folic acid in blood, liver, or muscle. Further integrated analysis revealed potential interactions among the liver, blood, and muscle, and the circulating metabolites, hub gene, and pathways, which might be the predominant acting targets of folic acid in animals. These findings provide fundamental information on the beneficial function of folic acid no matter from maternal or offspring, in regulating animal lipid metabolism and immune enhancement, providing a theoretical basis for the use of folic acid from the view of animal health care.

Folic acid supplementation during pregnancy modulates hepatic methyl metabolism and genes expression profile of neonatal lambs of different litter sizes.

Maternal folic acid (FA) plays an important role in the fetus development, but it is unknown the response of hepatic metabolism in the offspring from different litter sizes to maternal FA supplementation. In the present study, this was done by feeding the ewes with 0, 16 and 32 mg/(kg·DM) FA supplemented diet during pregnancy and analysing the hepatic one-carbon metabolism-related indices and gene expression in the neonatal lambs of different litter sizes (twins, TW; triplets, TR). Regardless of litter sizes, the concentrations of folate, methionine, S-adenosylmethionine and DNA methyltransferase increased significantly, but homocysteine and S-adenosylhomocysteine decreased in the liver of newborn lambs from ewes whose diet was supplemented with FA. In TW, maternal FA status has little effect on hepatic genes expression profile of newborn lambs, and no significant enriched pathway was found. However, DEG involved in cell proliferation such as CCNA2, CCNB2, CCNE2, CDK1 and BUB1 were significantly enriched when the ewes were supplemented with FA in TR groups. In addition, nucleotide synthesis-related genes such as POLD1, POLD2, MCM4 and MCM5 were enriched markedly in DNA replication and pyrimidine metabolism pathways in triplets when a higher FA ingestion [32 mg/(kg·DM)] was implemented in ewes. This finding demonstrated that the hepatic methyl metabolism in TW and TR newborn lambs was regulated by maternal FA status. The hepatic cell proliferation and nucleotide metabolism related genes in TR were more susceptible to maternal dietary FA supplementation during pregnancy.

Maternal Folic Acid Supplementation Differently Affects the Small Intestinal Phenotype and Gene Expression of Newborn Lambs from Differing Litter Sizes.

The purpose of this study was to investigate the effect of maternal dietary folic acid (FA) supplementation during gestation on small intestinal development of newborn lambs of different litter sizes, focusing on the intestinal morphology and development-, apoptosis- and digestion-related genes expression. One hundred and twenty Hu ewes (Ovis aries) were inseminated and randomly allotted to three groups. One group received a control diet [without FA supplementation, control (CON)] and the other two groups received control diets supplemented with different amount of FA [16 or 32 mg FA per kg dry matter (DM), i.e., F16 and F32] during pregnancy. After lambing, according to the dietary FA levels and litter size (twins, TW; triplets, TR), the neonatal lambs were divided into 6 (TW-CON, TW-F16, TW-F32, TR-CON, TR-F16, TR-F32) treatment groups. The results showed that the ratio of small intestinal weight to live body weight and the thickness of the intestinal muscle layer in the offspring was enhanced significantly with increasing maternal FA supplementation (p < 0.05). Meanwhile, the expression levels of insulin-like growth factor I (IGF-I), B-cell lymphoma-2 (BCL-2) and sodium/glucose co-transporter-1 (SGLT1) in the small intestines of the newborn lambs were increased, while the opposite was true for Bcl2-associated × (BAX) in response to FA supplementation (p < 0.05). Moreover, the small intestinal weights of twins were significantly higher than those of triplets (p < 0.01), and the expression levels of IGF-I (p < 0.05), sucrase-isomaltase (SI) (p < 0.05) and solute carrier family 2 member 5 (SLC2A5) (p < 0.01) were significantly lower than those in triplets. These findings suggest that maternal FA supplementation could improve the offspring's small intestinal phenotype and the expression of development-, apoptosis- and digestion-related genes, so it could promote the small intestinal development of newborn lambs. Furthermore, the small intestine phenotypic development of twins was generally better than that of triplets, while the expression levels of the above genes of twins were lower than those of triplets.

Effect of Dietary Folic Acid Supplementation during Pregnancy on Blood Characteristics and Milk Composition of Ewes.

The objective of the present study was to investigate the dynamic change of serum parameters and milk composition by dietary FA supplementation with ewes with different litter size from mating to lambing. The ewes were divided into six treatments (TW-CON, TW-F16, TW-F32, TR-CON, TR-F16, TR-F32) according to dietary FA levels (control, CON; 16 or 32 mg·kg-1 rumen-protect-FA supplementation, F16 and F32) and litter size (twin born, TW; and triplet born, TR). In serum, the concentration of folate increased linearly with dietary FA supplementation (P < 0.05), regardless of the litter size, they showed a quadratic response to gestation progression (P < 0.05). With dietary FA addition, IGFI-I levels significant increased from late gestation to after lambing (P < 0.05), and linearly increased immunoglobulin during the perinatal period (P < 0.05). In colostrum and milk at d 15, the content of folate, lactoferrin, and IgG were affected positively by FA supplementation (P < 0.05). IgG was higher in the TW group than TR in colostrum (P < 0.05), and lactoferrin in TW was lower than TR in milk of d 15 (P < 0.05). FA supplementation increased protein content in colostrum (P < 0.05), while it had no effect on the fat, lactose, and BUN of colostrum and milk of d 15 (P > 0.05). These results suggest that FA supplementation during gestation could regulate maternal blood metabolism and contribute to milk immune composition.

Maternal folic acid supplementation modulates the growth performance, muscle development and immunity of Hu sheep offspring of different litter size.

It is generally accepted that the phenotype and gene expression pattern of the offspring can be altered by maternal folic acid (FA) supplementation during the gestation period. The aims of this study were to investigate the effects of maternal FA supplementation on the growth performance, muscle development and immunity of newborn lambs of different litter size. According to litter size (twins, TW; triplets, TR) and maternal dietary FA supplementation levels (control, C; 16 or 32 mg·kg-1 FA supplementation, F16 and F32), neonatal lambs were randomly divided into six groups (TW-C, TW-F16, TW-F32, TR-C, TR-F16 and TR-F32). After farrowing, the birth weight in TW was higher than that in the TR group, and increased with FA supplementation of their mothers (P<.05). Folate, IGF-I, IgM and IgA concentrations of newborn lambs showed a litter size and FA supplementation interaction (P<.05). FA supplementation also increased diameter, area, perimeter and DNA content of the longissimus dorsi muscle of the lambs (P<.05) regardless of the litter size. Transcriptome analysis of the longissimus dorsi muscle revealed differentially expressed genes with dietary FA supplementation enriched in immunity- and cell development-related genes. Furthermore, FA supplementation upregulated the expression of myogenesis-related genes, while downregulated those involved in the inhibition of muscle development. In addition, immunity-related genes in the neonatal lambs showed lower expression levels in response to maternal dietary FA supplementation. Overall, maternal FA supplementation during gestation could increase the offspring's birth weight and modulate its muscle development and immunity.