Significance: Bovine embryonic stem cells and pluripotent stem cells hold the potential to substantially advance biotechnology and agriculture. We report the establishment of bovine expanded potential stem cells (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer (SCNT). EPSCs have broader developmental potential to generate embryonic and extraembryonic cell lineages. bEPSCs express high levels of pluripotency genes, propagate robustly in single cell passaging, are genetically stable, and permit efficient precise gene editing. They differentiate in vitro and in chimeras to both the embryonic and extraembryonic cell lineages. Importantly, genetically modified bEPSCs can be used as donors in SCNT or cloning.

Prolificacy is a crucial characteristic of livestock, particularly for species such as sheep that have many births. The objectives of this study were as follows: (1) to investigate the genetic diversity of the 13 new and 7 known variants in the BMPRIB, GDF9, BMP15, LEPR, and B4GALNT2 genes in Ujimqin (UM), the F1 population of Dorper × Ujimqin crossbred (DPU), the F1 population of Suffolk × Ujimqin crossbred (SFKU), Sonid sheep (SN), Tan sheep (Tan), Hu sheep (Hu), and Small-tailed Han sheep (STH) sheep breeds/populations; (2) to perform an association analysis of the above 20 variants with litter size in 325 UM, 304 DPU, and 66 SFKU sheep populations; (3) to compare the frequencies of the litter-size-related alleles of these 20 variants among 8 sheep breeds/populations (the above seven sheep breeds + Mongolia sheep breed). With the use of the Sequenom MassARRAY^®SNP assay technology, these 20 mutations were genotyped. The association analysis results showed that the c.746A>G (FecB) mutation in BMPR1B was significantly associated with the litter size of UM and DPU, the c.994A>G (FecG^A) in GDF9 was significantly associated with the litter size of SFKU, and the c.31_33CTTinsdel (B1) in BMP15 was significantly associated with the litter size of UM. Our findings might provide valuable genetic markers for expanding sheep litter sizes.

The disease resistance phenotype is closely related to immunomodulatory function and immune tolerance and has far-reaching implications in animal husbandry and human health. Microbes play an important role in the initiation, prevention, and treatment of diseases, but the mechanisms of host–microbiota interactions in disease-resistant phenotypes are poorly understood. In this study, we hope to uncover and explain the role of microbes in intestinal diseases and their mechanisms of action to identify new potential treatments. First, we established the colitis model of DSS in two breeds of sheep and then collected the samples for multi-omics testing including metagenes, metabolome, and transcriptome. Next, we made the fecal bacteria liquid from the four groups of sheep feces collected from H-CON, H-DSS, E-CON, and E-DSS to transplant the fecal bacteria into mice. H-CON feces were transplanted into mice named HH group and H-DSS feces were transplanted into mice named HD group and Roseburia bacteria treatment named HDR groups. E-CON feces were transplanted into mice named EH group and E-DSS feces were transplanted into mice in the ED group and Roseburia bacteria treatment named EDR groups. After successful modeling, samples were taken for multi-omics testing. Finally, colitis mice in HD group and ED group were administrated with Roseburia bacteria, and the treatment effect was evaluated by H&E, PAS, immunohistochemistry, and other experimental methods. The difference in disease resistance of sheep to DSS-induced colitis disease is mainly due to the increase in the abundance of Roseburia bacteria and the increase of bile acid secretion in the intestinal tract of Hu sheep in addition to the accumulation of potentially harmful bacteria in the intestine when the disease occurs, which makes the disease resistance of Hu sheep stronger under the same disease conditions. However, the enrichment of harmful microorganisms in East Friesian sheep activated the TNFα signalling pathway, which aggravated the intestinal injury, and then the treatment of FMT mice by culturing Roseburia bacteria found that Roseburia bacteria had a good curative effect on colitis. Our study showed that in H-DSS-treated sheep, the intestinal barrier is stabilized with an increase in the abundance of beneficial microorganisms. Our data also suggest that Roseburia bacteria have a protective effect on the intestinal barrier of Hu sheep. Accumulating evidence suggests that host–microbiota interactions are associated with IBD disease progression.

Naive pluripotency can be maintained in medium with two inhibitors plus leukemia inhibitory factor (2i/LIF) supplementation, which primarily affects canonical WNT, FGF/ERK, and JAK/STAT3 signaling. However, whether one of these three supplements alone is sufficient to maintain naive self-renewal remains unclear. Here we show that LIF alone in medium is sufficient for adaptation of 2i/L-ESCs to embryonic stem cells (ESCs) in a hypermethylated state (L-ESCs). Global transcriptomic analysis shows that L-ESCs are close to 2i/L-ESCs and in a stable state between naive and primed pluripotency. Notably, our results demonstrate that DNA methyltransferases (DNMTs) play an important role in LIF-dependent mouse ESC adaptation and self-renewal. LIF-dependent ESC adaptation efficiency is significantly increased in serum treatment and reduced in Dnmt3a or Dnmt3l knockout ESCs. Importantly, unlike epiblast stem cells, L-ESCs contribute to somatic tissues and germ cells in chimeras. L-ESCs cultured under such simple conditions as in this study would provide a more conducive platform to clarify the molecular mechanism of ESCs in in vitro culture.

Many progresses have recently been achieved in animal somatic cell nuclear transfer (SCNT). However, embryos derived from SCNT rarely result in live births. Single‐cell RNA sequencing (scRNA‐seq) can be used to investigate the development details of SCNT embryos. Here, bovine fibroblasts and three factors bovine iPSCs (3F biPSCs) were used as donors for bovine nuclear transfer, and the single blastomere transcriptome was analysed by scRNA‐seq. Compared to in vitro fertilization (IVF) embryos, SCNT embryos exhibited many defects. Abnormally expressed genes were found at each stage of embryos, which enriched in metabolism, and epigenetic modification. The DEGs of the adjacent stage in SCNT embryos did not follow the temporal expression pattern similar to that of IVF embryos. Particularly, SCNT 8‐cell stage embryos showed failures in some gene activation, including ZSCAN4, and defects in protein association networks which cored as POLR2K, GRO1, and ANKRD1. Some important signalling pathways also showed incomplete activation at SCNT zygote to morula stage. Interestingly, 3F biPSCNT embryos exhibited more dysregulated genes than SCNT embryos at zygote and 2‐cell stage, including genes in KDM family. Pseudotime analysis of 3F biPSCNT embryos showed the different developmental fate from SCNT and IVF embryos. These findings suggested partial reprogrammed 3F biPS cells as donors for bovine nuclear transfer hindered the reprogramming of nuclear transfer embryos. Our studies revealed the abnormal gene expression and pathway activation of SCNT embryos, which could increase our understanding of the development of SCNT embryos and give hints to improve the efficiency of nuclear transfer.

Fatty acid (FA) composition has an important impact on the nutrition and flavor of meat, and on consumer health, and is receiving more attention in the sheep industry. This study aimed to evaluate the relationship between the expression levels of the CAST gene and the FA composition in the longissimus thoracis (LL) muscle, to identify novel variants of CAST, and to perform association analysis with the FA composition in grazing Sonid lambs. The correlation results showed that high expression levels of CAST are correlated with better FA compositions and classes in LL. For association studies, the results showed that c.1210C>T and c.1437G>A in LD-M, and c.2097C>T mutations are associated with some compositions and classes of FA in the LL of grazing Sonid sheep. Two missense c.646G>C (G216R) and c.1210C>T (R404C) mutations were predicted to influence the Calpain_inhib domains of CAST. Thus, the correlation results and associated mutations are expected to be genetic selection markers for the FA composition and meat quality of grazing Sonid lamb muscle and provide new insights into sheep meat quality traits influenced by the ovine CAST gene.