Maternal Hypermethylated Genes Contribute to Intrauterine Growth Retardation of Piglets in Rongchang Pigs.

The placenta is a crucial determinant of fetal survival, growth, and development. Deficiency in placental development directly causes intrauterine growth retardation (IUGR). IUGR can lead to fetal growth restriction and an increase in the mortality rate. The genetic mechanisms underlying IUGR development, however, remain unclear. In the present study, we integrated whole-genome DNA methylation and transcriptomic analyses to determine distinct gene expression patterns in various placental tissues to identify pivotal genes that are implicated with IUGR development. By performing RNA-sequencing analysis, 1487 differentially expressed genes (DEGs), with 737 upregulated and 750 downregulated genes, were identified in IUGR pigs (H_IUGR) compared with that in normal birth weight pigs (N_IUGR) (p < 0.05); furthermore, 77 miRNAs, 1331 lncRNAs, and 61 circRNAs were differentially expressed. The protein-protein interaction network analysis revealed that among these DEGs, the genes GNGT1, ANXA1, and CDC20 related to cellular developmental processes and blood vessel development were the key genes associated with the development of IUGR. A total of 495,870 differentially methylated regions were identified between the N_IUGR and H_IUGR groups, which included 25,053 differentially methylated genes (DMEs); moreover, the overall methylation level was higher in the H_IUGR group than in the N_IUGR group. Combined analysis showed an inverse correlation between methylation levels and gene expression. A total of 1375 genes involved in developmental processes, tissue development, and immune system regulation exhibited methylation differences in gene expression levels in the promoter regions and gene ontology regions. Five genes, namely, ANXA1, ADM, NRP2, SHH, and SMAD1, with high methylation levels were identified as potential contributors to IUGR development. These findings provide valuable insights that DNA methylation plays a crucial role in the epigenetic regulation of gene expression and mammalian development and that DNA-hypermethylated genes contribute to IUGR development in Rongchang pigs.

Lactobacillus sp. participated in the adaptation of Rongchang piglets to cold stress.

Rongchang piglets were easily induced to cold stress and diarrhoea in the winter when raised in an open hog house. However, they also gradually recovered under mid-cold stress. Other studies have suggested gut microbiome might be involved in the host energy metabolism to relieve stress. To study how to adapt Rongchang piglets to cold stress by gut microbiome, thirty Rongchang piglets were randomly divided into a mild cold stress group and a control group for 30 consecutive days. The findings revealed that the piglets had low growth performance and a high diarrhoea rate and mortality rate during the first half of the cold treatment, but subsequently stabilised. The level of cortisol (COR) also displayed a similar trend. In the mild cold stress group, the relative abundance of Muribaculaceae significantly increased on day 15, and the predominant bacterial on day 30 was Lactobacillus sp. Our results indicated that the Rongchang piglet's production performance and health were impaired at the start of the mild cold stress. However, as time passed, the body could progressively adapt to the low temperature, and Lactobacillus sp. participated in this process. This study provides new insight into how to alleviate health damage caused by cold stress.

Deficient of LRRC8A attenuates hypoxia-induced necrosis in 3T3-L1 cells.

Under acute hypoxia, multiple ion channels on the cell membrane are activated, causing cell swelling and eventually necrosis. LRRC8A is an indispensable protein of the volume-regulated anion channel (VRAC), which participates in swelling and the acceleration of cell necrosis. In this study, we revealed a dynamic change in the expression level of the LRRC8 family during hypoxia in 3T3-L1 cells. The disruption of LRRC8A in 3T3-L1 cells was also associated with a significant anti-necrotic phenotype upon hypoxia accompanied by the reduced expression of necrosis-related genes. In vivo, differential expression of LRRC8 family members was also identified between high-altitude pigs and their low-altitude relatives. Taken these findings together, this study demonstrates the involvement of LRRC8A in hypoxia-induced cell necrosis.

Spontaneous single nucleotide polymorphism in porcine microRNA-378 seed region leads to functional alteration.

Sequence variation in a microRNA (miRNA) seed region can influence its biogenesis and effects on target mRNAs; however, in mammals, few seed region mutations leading to functional alterations have been reported to date. Here, we report the identification of a single nucleotide polymorphism (SNP) with functional consequence located in the seed region of porcine miR-378. In vitro analysis of this rs331295049 A17G SNP showed significantly up-regulated expression of the mature miR-378 (miR-378/G). In silico target prediction indicated that the SNP would modulate secondary structure and result in functional loss affecting >85% of the known target genes of the wild-type miR-378 (miR-378/A), and functional gain affecting >700 new target genes, and dual-luciferase reporter assay verified this result. This report of a SNP in the seed region of miR-378 leads to functional alteration and indicates the potential for substantive functional consequences to the molecular physiology of a mammalian organism.

A de novo silencer causes elimination of MITF-M expression and profound hearing loss in pigs.

BACKGROUND: Genesis of novel gene regulatory modules is largely responsible for morphological and functional evolution. De novo generation of novel cis-regulatory elements (CREs) is much rarer than genomic events that alter existing CREs such as transposition, promoter switching or co-option. Only one case of de novo generation has been reported to date, in fish and without involvement of phenotype alteration. Yet, this event likely occurs in other animals and helps drive genetic/phenotypic variation. RESULTS: Using a porcine model of spontaneous hearing loss not previously characterized we performed gene mapping and mutation screening to determine the genetic foundation of the phenotype. We identified a mutation in the non-regulatory region of the melanocyte-specific promoter of microphthalmia-associated transcription factor (MITF) gene that generated a novel silencer. The consequent elimination of expression of the MITF-M isoform led to early degeneration of the intermediate cells of the cochlear stria vascularis and profound hearing loss, as well as depigmentation, all of which resemble the typical phenotype of Waardenburg syndrome in humans. The mutation exclusively affected MITF-M and no other isoforms. The essential function of Mitf-m in hearing development was further validated using a knock-out mouse model. CONCLUSIONS: Elimination of the MITF-M isoform alone is sufficient to cause deafness and depigmentation. To our knowledge, this study provides the first evidence of a de novo CRE in mammals that produces a systemic functional effect.

Multiomics Reveals the Microbiota and Metabolites Associated with Sperm Quality in Rongchang Boars.

In this study, we investigated the correlation between the composition and function of the gut microbiota and the semen quality of Rongchang boars. Significant differences in gut microbial composition between boars with high (group H) and low (group L) semen utilization rates were identified through 16S rRNA gene sequencing, with 18 differential microbes observed at the genus level. Boars with lower semen utilization rates exhibited a higher relative abundance of Treponema, suggesting its potential role in reducing semen quality. Conversely, boars with higher semen utilization rates showed increased relative abundances of Terrisporobacter, Turicibacter, Stenotrophomonas, Clostridium sensu stricto 3, and Bifidobacterium, with Stenotrophomonas and Clostridium sensu stricto 3 showing a significant positive correlation with semen utilization rates. The metabolomic analyses revealed higher levels of gluconolactone, D-ribose, and 4-pyridoxic acid in the H group, with 4 pyridoxic acid and D-ribose showing a significant positive correlation with Terrisporobacter and Clostridium sensu stricto 3, respectively. In contrast, the L group showed elevated levels of D-erythrose-4-phosphate, which correlated negatively with Bifidobacterium and Clostridium sensu stricto 3. These differential metabolites were enriched in the pentose phosphate pathway, vitamin B6 metabolism, and antifolate resistance, potentially influencing semen quality. These findings provide new insights into the complex interplay between the gut microbiota and boar reproductive health and may offer important information for the discovery of disease biomarkers and reproductive health management.

Chromatin accessibility landscape of stromal subpopulations reveals distinct metabolic and inflammatory features of porcine subcutaneous and visceral adipose tissue.

Background: Fat accumulation in visceral adipose tissue (VAT) confers increased risk for metabolic disorders of obesity, whereas accumulation of subcutaneous adipose tissue (SAT) is associated with lower risk and may be protective. Previous studies have shed light on the gene expression profile differences between SAT and VAT; however, the chromatin accessibility landscape differences and how the cis-regulatory elements govern gene expression changes between SAT and VAT are unknown. Methods: Pig were used to characterize the differences in chromatin accessibility between the two adipose depots-derived stromal vascular fractions (SVFs) using DNase-sequencing (DNase-seq). Using integrated data from DNase-seq, H3K27ac ChIP-sequencing (ChIP-seq), and RNA-sequencing (RNA-seq), we investigated how the regulatory locus complexity regulated gene expression changes between SAT and VAT and the possible impact that these changes may have on the different biological functions of these two adipose depots. Results: SVFs form SAT and VAT (S-SVF and V-SVF) have differential chromatin accessibility landscapes. The differential DNase I hypersensitive site (DHS)-associated genes, which indicate dynamic chromatin accessibility, were mainly involved in metabolic processes and inflammatory responses. Additionally, the Krüppel-like factor family of transcription factors were enriched in the differential DHSs. Furthermore, the chromatin accessibility data were highly associated with differential gene expression as indicated using H3K27ac ChIP-seq and RNA-seq data, supporting the validity of the differential gene expression determined using DNase-seq. Moreover, by combining epigenetic and transcriptomic data, we identified two candidate genes, NR1D1 and CRYM, could be crucial to regulate distinct metabolic and inflammatory characteristics between SAT and VAT. Together, these results uncovered differences in the transcription regulatory network and enriched the mechanistic understanding of the different biological functions between SAT and VAT.

miR-152 targets pyruvate kinase to regulate the glycolytic activity of pig skeletal muscles and affects pork quality.

As a type of non-coding RNA, microRNAs are widely involved in the biological processes of animals. In the present study, the expression of miR-152 in glycolytic muscle fibers (Longissimus thoracis, LT) was lower than that of oxidative muscle fibers (Psoas major, PM). Using dual luciferase assay, miR-152 was shown to target muscle pyruvate kinase (PKM) to perform biological functions. Moreover, overexpression of miR-152 in primary porcine cells inhibited PKM gene expression and reduced lactic acid production in cells, whereas inhibition of miR-152 expression promoted PKM gene expression and increased lactic acid production. Correlation analysis showed that the expression of miR-152 was significantly positively correlated with the ultimate pH of LT after slaughter, while the expression of the PKM gene was significantly negatively correlated with the final pH of LT. In vivo and in vitro experiments discussed herein suggest that miR-152 may affect muscle pH by targeting the expression of the PKM gene. Our findings enrich the understanding of the genetic regulatory network that influences pork quality.

Distinct Gut Microbiome Induced by Different Feeding Regimes in Weaned Piglets.

It is well accepted that the gut microbiota of breast-fed (BF) and formula-fed (FF) infants are significantly different. However, there is still a limited number of studies comparing the gut microbiota of BF and FF piglets, despite increasing numbers of FF piglets in the modern pig industry. The present study identified the differences in gut microbiota composition between BF- and FF-weaned Rongchang piglets at 30 days old, using pair-end sequencing on the Illumina HiSeq 2500 platform. The BF piglets had lower microbiota diversities than FF piglets (p < 0.05), and the community structures were well clustered as a result of each feeding pattern. Firmicutes and Bacteroidetes represented the most dominant phyla, and Ruminococcus, Prevotella, and Gemmiger were prominent genera in all piglets. Ruminococcus, Prevotella, Oscillospira, Eubacterium, Gemmiger, Dorea, and Lactobacillus populations were significantly higher, while Treponema and Coprococcus were significantly lower in BF piglets compared to FF piglets (p < 0.05). The metabolism pathways in the BF piglets were significantly different from FF piglets, which included carbohydrate and amino acid metabolism (p < 0.05). In addition, the top 10 abundance of microbiota were more or less significantly associated with the two phenotypes (p < 0.05). Collectively, these findings provide probable explanations for the importance of BF in neonates and support a theoretical basis for feeding regimes in indigenous Chinese piglets.

ssc-miR-451 Regulates Porcine Primary Adipocyte Differentiation by Targeting ACACA.

miRNA is a small non-coding RNA, which plays an important role in diverse biological processes. In the present study, we explore the effect of ssc-miR-451 on porcine adipose development and meat quality. We observed that ssc-miR-451 was downregulated during porcine primary adipocyte differentiation. Overexpression of ssc-miR-451 inhibited adipogenic differentiation, while inhibition of ssc-miR-451 promoted adipogenic differentiation. The dual luciferase reporter system indicated Acetyl-CoA carboxylase alpha (ACACA) as a target gene of ssc-miR-451. Correlation analysis negatively correlated miR-451 expression with intramuscular fat content (IMF) and positively correlated ACACA expression with IMF. Further analysis of fatty acid composition revealed that pigs with high expression of ssc-miR-451 had higher monounsaturated fatty acid (MUFA) and lower polyunsaturated fatty acid (PUFA). Taken together, our study suggests that ssc-miR-451 regulates lipid deposition and fatty acid composition by targeting ACACA, and ssc-miR-451 may serve as a potential genetic marker to improve pork quality.

MiR-1246 is upregulated and regulates lung cell apoptosis during heat stress in feedlot cattle.

Globally, heat stress seriously threatens productivity of cattle. The objective of this study was to identify novel miRNAs that regulated heat stress in feedlot cattle. Experiment was conducted under heat stress and normal conditions. With profiling miRNAs of each feedlot cattle, our results showed the level of miR-1246 was significantly increased in these heat-stressed cattle (P < 0.05). Furthermore, by using bioinformatics analysis and luciferase reporter assays combined with qPCR and western blot, we found miR-1246 negatively regulated poly (C) binding protein 2 (PCBP2) and cAMP response element binding protein-like 2 (CREBL2) mRNA and protein levels through binding to the 3'-UTR region (P < 0.05); further, it inhibited heat-induced apoptosis in lung cells. Finally, our results suggested that miR-1246 plays an important role in heat stress and it has the potential to be a novel modulation factor for heat stress in feedlot cattle.

A mutation in porcine pre-miR-15b alters the biogenesis of MiR-15b\16-1 cluster and strand selection of MiR-15b.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in translational regulation of the messenger RNA molecules. Sequence variations in the genes encoding miRNAs could influence their biogenesis and function. MiR-15b plays an important role in cellular proliferation, apoptosis and the cell cycle. Here, we report the identification of a C58T mutation in porcine pre-miR-15b. Through in vitro and in vivo experiments, we determined that this mutation blocks the transition from pri-miRNA to pre-miRNA, alters the strand selection between miR-15b-5p and miR-15b-3p, and obstructs biogenesis of the downstream miR-16-1. These results serve to highlight the importance of miRNA mutations and their impacts on miRNA biogenesis.