Multi-omics analysis reveals substantial linkages between the oral-gut microbiomes and inflamm-aging molecules in elderly pigs.

Introduction: The accelerated aging of the global population has emerged as a critical public health concern, with increasing recognition of the influential role played by the microbiome in shaping host well-being. Nonetheless, there remains a dearth of understanding regarding the functional alterations occurring within the microbiota and their intricate interactions with metabolic pathways across various stages of aging. Methods: This study employed a comprehensive metagenomic analysis encompassing saliva and stool samples obtained from 45 pigs representing three distinct age groups, alongside serum metabolomics and lipidomics profiling. Results: Our findings unveiled discernible modifications in the gut and oral microbiomes, serum metabolome, and lipidome at each age stage. Specifically, we identified 87 microbial species in stool samples and 68 in saliva samples that demonstrated significant age-related changes. Notably, 13 species in stool, including Clostridiales bacterium, Lactobacillus johnsonii, and Oscillibacter spp., exhibited age-dependent alterations, while 15 salivary species, such as Corynebacterium xerosis, Staphylococcus sciuri, and Prevotella intermedia, displayed an increase with senescence, accompanied by a notable enrichment of pathogenic organisms. Concomitant with these gut-oral microbiota changes were functional modifications observed in pathways such as cell growth and death (necroptosis), bacterial infection disease, and aging (longevity regulating pathway) throughout the aging process. Moreover, our metabolomics and lipidomics analyses unveiled the accumulation of inflammatory metabolites or the depletion of beneficial metabolites and lipids as aging progressed. Furthermore, we unraveled a complex interplay linking the oral-gut microbiota with serum metabolites and lipids. Discussion: Collectively, our findings illuminate novel insights into the potential contributions of the oral-gut microbiome and systemic circulating metabolites and lipids to host lifespan and healthy aging.

Efficient Simultaneous Introduction of Premature Stop Codons in Three Tumor Suppressor Genes in PFFs via a Cytosine Base Editor.

Base editing is an efficient and precise gene-editing technique, by which a single base can be changed without introducing double-strand breaks, and it is currently widely used in studies of various species. In this study, we used hA3A-BE3-Y130F to simultaneously introduce premature stop codons (TAG, TGA, and TAA) into three tumor suppressor genes, TP53, PTEN, and APC, in large white porcine fetal fibroblasts (PFFs). Among the isolated 290 single-cell colonies, 232 (80%) had premature stop codons in all the three genes. C−to−T conversion was found in 98.6%, 92.8%, and 87.2% of these cell colonies for TP53, PTEN, and APC, respectively. High frequencies of bystander C−to−T edits were observed within the editing window (positions 3−8), and there were nine (3.01%) clones with the designed simultaneous three-gene C−to−T conversion without bystander conversion. C−to−T conversion outside the editing window was found in 9.0%, 14.1%, and 26.2% of the 290 cell colonies for TP53, PTEN, and APC, respectively. Low-frequency C−to−G or C−to−A transversion occurred in APC. The mRNA levels of the three genes showed significant declines in triple-gene-mutant (Tri-Mut) cells as expected. No PTEN and a significantly lower (p < 0.05) APC protein expression were detected in Tri-Mut cells. Interestingly, the premature stop codon introduced into the TP53 gene did not eliminate the expression of its full-length protein in the Tri-Mut cells, suggesting that stop codon read-through occurred. Tri-Mut cells showed a significantly higher (p < 0.05) proliferation rate than WT cells. Furthermore, we identified 1418 differentially expressed genes (DEGs) between the Tri-Mut and WT groups, which were mainly involved in functions such as tumor progression, cell cycle, and DNA repair. This study indicates that hA3A-BE3-Y130F can be a powerful tool to create diverse knockout cell models without double-strand breaks (DSBs), with further possibilities to produce porcine models with various purposes.

BMPR-IB gene disruption causes severe limb deformities in pigs.

In an attempt to generate g.A746G substitution in the BMPR-IB gene, we unexpectedly obtained BMPR-IB homozygous knockout piglets ( BMPR-IB -/-) and heterogeneous knockout piglets with one copy of the A746G mutation ( BMPR-IB -/746G) via CRISPR/Cas9 editing. Polymerase chain reaction (PCR) and sequencing revealed complex genomic rearrangements in the target region. All BMPR-IB-disrupted piglets showed an inability to stand and walk normally. Both BMPR-IB -/- and BMPR-IB -/746G piglets exhibited severe skeletal dysplasia characterized by distorted and truncated forearms (ulna, radius) and disordered carpal, metacarpal, and phalangeal bones in the forelimbs. The piglets displayed more severe deformities in the hindlimbs by visual inspection, including fibular hemimelia, enlarged tarsal bone, and disordered toe joint bones. Limb deformities were more profound in BMPR-IB -/- piglets than in the BMPR-IB -/746G piglets. Proteomic analysis identified 139 differentially expressed proteins (DEPs) in the hindlimb fibula of BMPR -IB -/746G piglets compared to the wild-type (WT) controls. Most DEPs are involved in skeletal or embryonic development and/or the TGF-ß pathway and tumor progression. Gene Ontology (GO) and protein domain enrichment analysis suggested alterations in these processes. Of the top 50 DEPs, a large proportion, e.g., C1QA, MYO1H, SRSF1, P3H1, GJA1, TCOF1, RBM10, SPP2, MMP13, and PHAX, were significantly associated with skeletal development. Our study provides novel findings on the role of BMPR-IB in mammalian limb development.

Integrated analysis of miRNA and mRNA expression profiles in p53-edited PFF cells.

p53 is the most frequently mutated gene in human cancers, with over half of all tumors harboring mutation at this locus. R248 and R249 (corresponding to porcine R241 and R242), are among the hotspot mutations frequently mutated in liver, lung, breast, and some other cancers. In this study, p53 gene was knocked out or point-edited (R241 and R242 were converted to 241W and 242S) in porcine fetal fibroblast (PFF) cells via CRISPR-Cas9 technique. High throughput sequencing of miRNA and mRNA uncovered a total of 225 differentially expressed miRNAs (DEMs) and 738 differentially expressed genes (DEGs) in the p53 knockout (p53-KO) cells, and a total of 211 DEMs and 722 DEGs in the point-modified (p53-241W242S) cells. Totally 28 annotated DEMs were found to overlap between p53-KO/p53-WT and p53-241W242S/p53-WT miRNAs datasets, of which miR-34 c, miR-218, miR-205, miR-105-1, miR-105-2, miR-206, miR-224 and miR-429 play important roles in p53 regulatory network. Among the top 10 DEGs in p53-KO and p53-241W242S cells, most genes were reported to be involved in tumors, cell proliferation or cell migration. p53-KO and p53-241W242S cells showed a significantly higher (P < 0.01) proliferation rate compared with p53-WT cells. In conclusion, genetic modifications of p53 gene significantly affect the expression levels of a large number of genes and miRNAs in the PFF cells. The p53-edited PFF cells could be used as non-tumor cell models for investigating the p53 signaling network, and as donor cells for somatic nuclear transfer, with the aim to develop porcine models with the corresponding p53 mutations.Abbreviations: CRISPR-Cas9: Clustered regularly interspaced short palindromic repeats-associated protein 9; PFF: porcine fetal fibroblasts; SCNT: somatic cell nuclear transfer; RNA sequencing: small RNA sequencing and mRNA sequencing; DEGs: differentially expressed mRNAs; DEMs: differentially expressed miRNAs.

Semen quality improvement in boars fed with supplemental wolfberry (Lycium barbarum).

Wolfberry is well known for its health benefits in Asian countries. This study consisted of two experiments. In Experiment 1, nine boars were provided 40 g dried wolfberry per 100 kg body weight per day in addition to regular feed for 160 days (divided into 40 days phases: I, II, III, and IV) under step-down air temperature conditions. Controls (n = 9) were fed regular feed only. Significant (p < .05 or p < .01) or slight improvements in sperm progressive motility, total abnormality rate, sperm concentration, and total sperm per ejaculate were observed in the wolfberry group during phases II and III. No differences were observed in semen volume. After combining the data from phases II ~ IV, significant improvements were detected in all aforementioned traits (p < .05 or p < .01), except semen volume. In Experiment 2, the wolfberry group (n = 5) was fed wolfberry for 90 days and exhibited significantly reduced head, tail, and total abnormality rates (p < .05 or p < .01) in both fresh semen and semen stored for 72 hr at 17°C compared to the control group (n = 5). SOD activity also significantly increased in this group of boars. Collectively, the findings of this study suggest that wolfberry has a positive effect on boar semen quality.

Unravelling the genetic loci for growth and carcass traits in Chinese Bamaxiang pigs based on a 1.4 million SNP array.

Bamaxiang pig is from Guangxi province in China, characterized by its small body size and two-end black coat colour. It is an important indigenous breed for local pork market and excellent animal model for biomedical research. In this study, we performed genomewide association studies (GWAS) on 43 growth and carcass traits in 315 purebred Bamaxiang pigs based on a 1.4 million SNP array. We observed considerable phenotypic variability in the growth and carcass traits in the Bamaxiang pigs. The corresponding SNP based heritability varied greatly across the 43 traits and ranged from 9.0% to 88%. Through a conditional GWAS, we identified 53 significant associations for 35 traits at p value threshold of 10-6 . Among which, 26 associations on chromosome 3, 7, 14 and X passed a genomewide significance threshold of 5 × 10-8 . The most remarkable loci were at around 30.6 Mb on chromosome 7, which had growth stage-dependent effects on body lengths and cannon circumferences and showed large effects on multiple carcass traits. We discussed HMGA1 NUDT3, EIF2AK1, TMEM132C and AFF2 that near the lead SNP of significant loci as plausible candidate genes for corresponding traits. We also showed that including phenotypic covariate in GWAS can help to reveal additional significant loci for the target traits. The results provide insight into the genetic architecture of growth and carcass traits in Bamaxiang pigs.

The increased expression of follicle-stimulating hormone leads to a decrease of fecundity in transgenic Large White female pigs.

Follicle-stimulating hormone (FSH) is a pituitary gonadotropin regulating reproduction in mammals. Overexpression of the exogenous FSHα/ß genes from Chinese Erhualian pigs improved female fecundity of transgenic (TG) mice and male spermatogenesis ability of Large White TG boars. Here, we investigated the impact of the exogenous FSHα/ß genes on female reproductive performance of Large White TG pigs. First, we identified the integration site of the exogenous FSHα/ß genes at 140,646,456 bp on chromosome 9 in these TG pigs using whole-genome sequencing. Then, we showed that TG gilts had higher levels of serum FSH and FSHß protein in pituitary while had a potentially lower number of born piglets than their wild-type half sibs. TG gilts grew healthily and normally without significant difference in growth and health parameters as compared to WT gilts. The expression levels of FSHR, LHR, ESR1 and ESR2 were significantly lower in TG gilts than in WT gilts at the age of 300 days. Taken together, we proposed that the overexpressed FSHα/ß transgenes could cause deteriorate fecundity via disturbing the normal expression of the endogenous reproduction-related genes in female pigs. Our findings provide insight into the effect of overexpression of FSHα/ß on female reproduction performance in pigs.

BAC mediated transgenic Large White boars with FSHα/ß genes from Chinese Erhualian pigs.

Follicle-stimulating hormone (FSH) is a critical hormone regulating reproduction in mammals. Transgenic mice show that overexpression of FSH can improve female fecundity. Using a bacterial artificial chromosome (BAC) system and somatic cell nuclear transfer, we herein generated 67 Large White transgenic (TG) boars harboring FSHα/ß genes from Chinese Erhualian pigs, the most prolific breed in the world. We selected two F0 TG boars for further breeding and conducted molecular characterization and biosafety assessment for F1 boars. We showed that 8-9 copies of exogenous FSHα and 5-6 copies of exogenous FSHß were integrated into the genome of transgenic pigs. The inheritance of exogenous genes conforms to the Mendel's law of segregation. TG boars had higher levels of serum FSH, FSHα mRNA in multiple tissues, FSHß protein in pituitary and more germ cells per seminiferous tubule compared with their wild-type half sibs without any reproductive defects. Analysis of growth curve, hematological and biochemical parameters and histopathology illustrated that TG boars grew healthily and normally. By applying 16S rRNA gene sequencing, we demonstrated that exogenous genes had no impact on the bacterial community structures of pig guts. Moreover, foreign gene drift did not occur as verified by horizontal gene transfer. Our findings indicate that overexpression of FSH could improve spermatogenesis ability of boars. This work provides insight into the effect of FSHα/ß genes on male reproductive performance on pigs by a BAC-mediated transgenic approach.