Proteomics analysis reveals promotion effect of 1α,25-dihydroxyvitamin D3 on mammary gland development and lactation of primiparous sows during gestation.

1α,25(OH)2VD3 is the most active form of vitamin D3 in animals, and it plays an important role in regulating mineral metabolism and reproduction. In this study, 140 crossbred gilts (Landrace × Yorkshire) were selected, randomly divided into four groups, and fed with a commercial diet supplemented with 0, 1, 2, and 4 µg/kg of 1α,25(OH)2VD3 in the form of 1α,25(OH)2VD3-glycosides. The mammary gland tissues were sampled from sows on day 114 of gestation. The production data of sows in each group were analyzed, and the colostrum quality was evaluated. Differentially abundant proteins (DAPs) in the mammary tissues were identified by tandem mass tag (TMT) technique and were verified by Western blot and parallel reaction monitoring (PRM). The results showed that 4 µg/kg 1α,25(OH)2VD3-glycosides significantly promoted the piglet birth weight, weaning weight, colostrum quality, and lactation ability of primiparous sows. The proteomics analysis showed that of the identified 53,118 peptides, 48,868 were unique peptides. A total of 5029 DAPs were identified, of which 4292 DAPs contained quantitative information. Our data indicated that 1α,25(OH)2VD3 was involved in the regulation of the mammary gland development and lactation in a dose-dependent manner through multiple pathways during gestation of primiparous sows. SIGNIFICANCE: The mammary gland is an important lactation organ of female mammals. Our research aims to reveal the effect of dietary supplementation with 1α,25(OH)2VD3 on mammary gland development and lactation of primiparous sow. This study identified potential signaling pathways and DAPs involved in regulating the mammary gland development and lactation in sows. Our findings provides theoretical basis for improving the fecundity of sows.

Proteomics analysis reveals the effect of 1α,25(OH)2VD3-glycosides on development of early testes in piglets.

1α,25(OH)2VD3 is the most active form of VD3 in animals. It plays an important role in regulating mineral metabolism but also in reproduction. Testes are the main reproductive organs of male mammals. Our research aims to reveal the effect of 1α,25(OH)2VD3-glycosides on development of early testes in piglets. 140 weaned 21-day old piglets were selected. The piglets were randomly divided into four groups and were fed a commercial diet supplemented with 0, 1, 2 and 4 µg/kg of 1α,25(OH)2VD3, provided as 1α,25(OH)2VD3-glycosides. Sixty days after the start of the experiment, at piglet age 82 days, testes were harvested. The morphology and histology of early testicular development were assessed. In addition, the proteomic TMT/iTRAQ labelling technique was used to analyse the protein profile of the testes in each group. Western blotting was applied to verify the target of differentially abundant proteins (DAPs). The analysis of morphology and histology of testes showed that a certain concentration of 1α,25(OH)2VD3-glycosides had a positive and significant effect on testicular development. And the results of proteomics analysis showed that of the identified 132,715 peptides, 122,755 were unique peptides. 7852 proteins, of which 6573 proteins contain quantitative information. Screening for DAPs focused on proteins closely related to the regulation of testicular development such as steroid hormone synthesis, steroid biosynthesis, peroxisome and fatty acid metabolism pathways. These results indicated that 1α,25(OH)2VD3 is involved in the regulation of early testicular development in piglets. At the same time, these findings provide valuable information for the proteins involved in the regulation of testicular development, and help to better understand the mechanisms of 1α,25(OH)2VD3 in regulating the development of piglets' testes.

Comparative analyses of longissimus muscle miRNAomes reveal microRNAs associated with differential regulation of muscle fiber development between Tongcheng and Yorkshire pigs.

Tongcheng (TC) and Yorkshire (YK) are two pig breeds with distinctive muscle morphology. Porcine microRNAome (miRNAome) of the longissimus muscle during five developmental stages (40, 55, 63, 70, and 90 days post coitum (dpc)) was explored by Solexa sequencing in the present study to find miRNAs involved in the different regulation of skeletal muscle development between the two breeds. A total of 320 known porcine miRNAs, 64 miRNAs corresponding to other mammals, and 224 potentially novel miRNAs were identified. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) suggested that the factor "pig breed" affected the miRNA expression profiles to a lesser extent than the factor "developmental stage". Fifty-seven miRNAs were differentially expressed (DE) between the neighbor developmental stages in TC and 45 such miRNAs were found in YK, 34 in common; there were more down-regulated stage-DE miRNAs than up-regulated. And a total of 23, 30, 12, 6, and 30 breed-DE miRNAs between TC and YK were identified at 40, 55, 63, 70, and 90 dpc, respectively, which were mainly involved in cellular protein modification process, protein transport, and metabolic process. As the only highly expressed breed-DE miRNA found in no less than four developmental stages, and also a stage-DE miRNA found both in TC and YK, miR-499-5p could bind the 3'-UTR of a myofibrillogenesis regulator, destrin/actin depolymerizing factor (DSTN), as validated in dual luciferase reporter assay. The results suggested that miR-499-5p possibly play a noteworthy role in the breed-distinctive porcine muscle fiber development associated with the regulation of DSTN.

Dynamic transcriptome profiles of skeletal muscle tissue across 11 developmental stages for both Tongcheng and Yorkshire pigs.

BACKGROUND: The growth and development of skeletal muscle directly impacts the quantity and quality of pork production. Chinese indigenous pig breeds and exotic species vary greatly in terms of muscle production and performance traits. We present transcriptome profiles of 110 skeletal muscle samples from Tongcheng (TC) and Yorkshire (YK) pigs at 11 developmental periods (30, 40, 55, 63, 70, 90, and 105 days of gestation, and 0, 1, 3, and 5 weeks of age) using digital gene expression on Solexa/Illumina's Genome Analyzer platform to investigate the differences in prenatal and postnatal skeletal muscle between the two breeds. RESULTS: Muscle morphological changes indicate the importance of primary fiber formation from 30 to 40 dpc (days post coitus), and secondary fiber formation from 55 to 70 dpc. We screened 4,331 differentially expressed genes in TC and 2,259 in YK (log2 ratio >1 and probability >0.7). Cluster analysis showed different gene expression patterns between TC and YK pigs. The transcripts were annotated in terms of Gene Ontology related to muscle development. We found that the genes CXCL10, EIF2B5, PSMA6, FBXO32, and LOC100622249 played vital roles in the muscle regulatory networks in the TC breed, whereas the genes SGCD, ENG, THBD, AQP4, and BTG2 played dominant roles in the YK breed. These genes showed breed-specific and development-dependent differential expression patterns. Furthermore, 984 genes were identified in myogenesis. A heat map showed that significantly enriched pathways (FDR <0.05) had stage-specific functional regulatory mechanisms. Finally, the differentially expressed genes from our sequencing results were confirmed by real-time quantitative polymerase chain reaction. CONCLUSIONS: This study detected many functional genes and showed differences in the molecular mechanisms of skeletal muscle development between TC and YK pigs. TC pigs showed slower muscle growth and more complicated genetic regulation than YK pigs. Many differentially expressed genes showed breed-specific expression patterns. Our data provide a better understanding of skeletal muscle developmental differences and valuable information for improving pork quality.

A 304 bp insertion/deletion mutation in promoter region induces the increase of porcine IDH3ß gene expression.

Obese and lean pig breeds show obvious differences in adipose metabolism/fat deposition; however, the molecular mechanism underlying phenotype variation remains unknown. In order to understand it, we analyzed the differences of gene expression in backfat between Meishan (a typical Chinese indigenous obese breed) and Large White (a lean Western breed) pigs. Here, we cloned porcine ß subunit of IDH3 (IDH3B) and 2447 bp 5'-flanking sequence of this gene, and determined the genomic structure. Porcine IDH3B contains three isoforms, IDH3B ( 1 ), IDH3B ( 2 ) and IDH3B ( 3 ). Real-time RT-PCR revealed that these three isoforms were prevalently up-regulated in backfat of western commercial pigs, Large White, Landrace and Duroc, compared with Chinese indigenous breeds, Meishan and Tongcheng pigs. A 304 bp insertion/deletion variant was found in the 5'-flanking region. Dual-luciferase reporter assays showed that in vitro the promoter of IDH3B gene with the insertion had higher luciferase activity as compared with the wild type. Three genotypes AA, AB and BB, due to this insertion, were detected, and the frequency of allele A was dominant in western commercial pigs, whereas allele B predominated in Chinese indigenous breeds. IDH3B mRNA expression in Meishan pigs was more abundant with genotype AA than with genotype AB or BB, as in Large White pigs. In addition, the polymorphism was detected in 317 pigs of a Large White × Meishan F2 resource population. Association analysis showed that pigs with genotype AA possessed higher backfat thickness at buttocks than those with genotype AB (P < 0.05) or BB. These data suggested that the 304 bp insertion mutation in promoter region increased the expression of porcine IDH3ß transcripts and this mutation might be a candidate marker for marker assistant selection in swine breeding.