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Abstract Background 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. Methods 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. Results 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. Conclusion 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. Video Abstract

With the increase of cadmium content in the environment, the losses caused by cadmium-induced intestinal diseases to animal husbandry are increasing year by year. However, most of the on-going research activities focus on zoonotic diseases rather than exploring the mechanisms of animal disease occurrence from an anthropogenic environmental perspective. In this study, stressed Hu sheep under cadmium environmental exposure were selected to explore the mechanism of inflammatory bowel disease development. 16 s, untargeted metabolomics and transcriptomic multiomics were used to analyze the changes of their intestinal tract and intestinal contents. The results showed that the beneficial microorganisms (s_Ruminococcus_sp) in the Cd group were significantly decreased and the potentially harmful microorganisms were significantly enriched, and the changes of these microorganisms affected the changes of metabolites (caprylic acid) to a certain extent, resulting in a decrease in fatty acids in the intestine. Due to the combined effect of cadmium ion and fatty acid reduction, the PPAR signaling pathway was inhibited, and the fatty acid transport and binding were further reduced, causing very serious damage to the intestine. We revealed for the first time the mechanism of intestinal injury in Hu sheeps under cadmium environmental exposure and provided new prevention and treatment methods of intestinal diseases under the environmental exposure to trace metals.

The emergence and development of induced pluripotent stem cells (iPSCs) provides an approach to understand the regulatory mechanisms of cell pluripotency and demonstrates the great potential of iPSCs in disease modeling. Acute myelitis defines a group of inflammatory diseases that cause acute nerve damage in the spinal cord; however, its pathophysiology remains to be elusive. In this study, we derived skin fibroblasts from a patient with acute myelitis (P-HAF) and then reprogrammed P-HAF cells to iPSCs using eight exogenous factors (namely, OCT4, SOX2, c-MYC, KLF4, NANOG, LIN28, RARG, and LRH1). We performed transcriptomic analysis of the P-HAF and compared the biological characteristics of the iPSCs derived from the patient (P-iPSCs) with those derived from normal individuals in terms of pluripotency, transcriptomic characteristics, and differentiation ability toward the ectoderm. Compared to the control iPSCs, the P-iPSCs displayed similar features of pluripotency and comparable capability of ectoderm differentiation in the specified culture. However, when tested in the common medium, the P-iPSCs showed attenuated potential for ectoderm differentiation. The transcriptomic analysis revealed that pathways enriched in P-iPSCs included those involved in Wnt signaling. To this end, we treated iPSCs and P-iPSCs with the Wnt signaling pathway inhibitor IWR1 during the differentiation process and found that the expression of the ectoderm marker Sox1 was increased significantly in P-iPSCs. This study provides a novel approach to investigating the pathogenesis of acute myelitis.

The incomplete silencing of exogenous transcription factors (TFs) and the lack of endogenous counterpart activation hampers the application of porcine induced pluripotent stem cells (piPSCs). We used porcine, bovine and murine TFs to reprogram porcine fetal fibroblasts. Porcine TFs-derived piPSCs (ppiPSCs) showed the highest levels of endogenous pluripotency markers activation, were able to differentiate into three germ layers and primordial germ cell-like cells (PGCLCs) and integrated into neural ectoderm of E7.5 mouse embryos in vitro. The bovine TFs derived piPSCs (bpiPSCs) expressed endogenous pluripotency markers higher than murine TFs derived piPSCs (mpiPSCs), but both had limited differentiation ability in vitro and depended on continuous expression of exogenous TFs for the maintenance. RNA sequencing confirmed ppiPSCs had distinct global transcriptional profiling, upregulated Hippo, PI3K-Akt, MAPK and relevant pluripotency signaling pathways as porcine blastocyst inner cell mass and expressed PGC early related genes. In addition, a positive and a negative correlation between exogenous and endogenous TFs’ expression level were observed in ppiPSCs and bpiPSCs lines, respectively. The TFs’ protein structures in pig were more similar to cattle than to mouse. In conclusion, the species affinity of the exogenous TFs is a key element, and the own species origin of TFs is optimal for iPSCs generation and application.

Objective Owing to the lack of a breeding index for efficient and quick fertility evaluations of Holstein bulls when using traditional or genome-wide detection methods, this study aimed to determine whether in vitro fertilization (IVF) could be used as an indicator of conception rate of artificial insemination (AI). Methods Conventional and sexed frozen semen from nine bulls were used for IVF and AI. Results The IVF and AI conception rates of each bull were confirmed to be positively correlated between the conventional frozen and sexed frozen semen. The correlation coefficient R values of nine bulls between IVF and AI methods were 0.73 and 0.97 for the conventional frozen and sexed frozen semen, respectively. The average conception rate of three bulls undergoing AI was 69.5% and 64.2%, 61.8% and 58.8%, and 48.2% and 46.2% in first-, second-, and third-born cows when conventional frozen and sexed frozen semen were used, respectively, which showed a positive correlation with the fertilization rate in the same parity. We propose an evaluation standard to assess the fertilization ability of bulls based on their IVF test results, which is categorized into three grades: grade one, normal fertility bull with an AI conception rate of 40%±5% and IVF rate of 45% to 60%; grade two, higher fertility bull with an AI conception rate of 50%±5% and IVF rate of 61% to 80%; and grade three, highest fertility bull with an AI conception rate of 60%±5% and IVF rate of >80%. Conclusion These findings reveal that IVF results can be used as a breeding index for bulls to evaluate their AI conception ability, which may shorten the time required to select bulls for breeding.

Abstract Background It is important to resolve the evolutionary history of species genomes as it has affected both genome organization and chromosomal architecture. The rapid innovation in sequencing technologies and the improvement in assembly algorithms have enabled the creation of highly contiguous genomes. DNA Zoo, a global organization dedicated to animal conservation, offers more than 150 chromosome-length genome assemblies. This database has great potential in the comparative genomics field. Results Using the donkey (Equus asinus asinus, EAS) genome provided by DNA Zoo as an example, the scaffold N50 length and Benchmarking Universal Single-Copy Ortholog score reached 95.5 Mb and 91.6%, respectively. We identified the cytogenetic nomenclature, corrected the direction of the chromosome-length sequence of the donkey genome, analyzed the genome-wide chromosomal rearrangements between the donkey and horse, and illustrated the evolution of the donkey chromosome 1 and horse chromosome 5 in perissodactyls. Conclusions The donkey genome provided by DNA Zoo has relatively good continuity and integrity. Sequence-based comparative genomic analyses are useful for chromosome evolution research. Several previously published chromosome painting results can be used to identify the cytogenetic nomenclature and correct the direction of the chromosome-length sequence of new assemblies. Compared with the horse genome, the donkey chromosomes 1, 4, 20, and X have several obvious inversions, consistent with the results of previous studies. A 4.8 Mb inverted structure was first discovered in the donkey chromosome 25 and plains zebra chromosome 11. We speculate that the inverted structure and the tandem fusion of horse chromosome 31 and 4 are common features of non-caballine equids, which supports the correctness of the existing Equus phylogeny to an extent.

Cell types have been established during organogenesis based on early mouse embryos. However, our understanding of cell types and molecular mechanisms in the early embryo development of Mongolian sheep has been hampered. This study presents the first comprehensive single-cell transcriptomic characterization at E16 in Ujumqin sheep and Hulunbuir short-tailed sheep. Thirteen major cell types were identified at E16 in Ujumqin sheep, and eight major cell types were identified at E16 in Hulunbuir short-tailed sheep. Function enrichment analysis showed that several pathways were significantly enriched in the TGF-beta signaling pathway, the Hippo signaling pathway, the platelet activation pathway, the riboflavin metabolism pathway, the Wnt signaling pathway, regulation of the actin cytoskeleton, and the insulin signaling pathway in the notochord cluster. Glutathione metabolism, glyoxylate, and dicarboxylate metabolism, the citrate cycle, thyroid hormone synthesis, pyruvate metabolism, cysteine and methionine metabolism, thermogenesis, and the VEGF signaling pathway were significantly enriched in the spinal cord cluster. Steroid biosynthesis, riboflavin metabolism, the cell cycle, the Hippo signaling pathway, the Hedgehog signaling pathway, the FoxO signaling pathway, the JAK-STAT signaling pathway, and the Wnt signaling pathway were significantly enriched in the paraxial mesoderm cluster. The notochord cluster, spinal cord cluster, and paraxial mesoderm cluster were found to be highly associated with tail development. Pseudo-time analysis demonstrated that the mesenchyme can translate to the notochord in Ujumqin sheep. Molecular assays revealed that the Hippo signaling pathway was enriched in Ujumqin sheep. This comprehensive single-cell map revealed previously unrecognized signaling pathways that will further our understanding of the mechanism of short-tailed sheep formation.

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 (FecGA) 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.

Abstract Background Pyroptosis is a recently identified pathway of caspase-mediated cell death in response to microbes, lipopolysaccharide, or chemotherapy in certain types of cells. However, the mechanism of how pyroptosis is regulated is not well-established. Methods Herein, the intracellular bacteria were detected by staining and laser confocal microscopy and TEM. Live/dead cell imaging assay was used to examine macrophage death. Western blot and immunohistochemical staining were used to examine the protein changes. IFA was used to identify typical budding vesicles of pyroptosis and the STAT3 nuclear localization. SEM was used to observe the morphological characteristics of pyroptosis. ELISA was used to detect the level of inflammatory cytokines. Pyroptosis was filmed in macrophages by LSCM. Results S. aureus was internalized by human macrophages. Intracellular S. aureus induced macrophage death. S. aureus invasion increased the expression of NLRP3, Caspase1 (Casp-1 p20) and the accumulation of GSDMD-NT, GSDMD-NT pore structures, and the release of IL-1β and IL-18 in macrophages. Macrophages pyroptosis induced by S. aureus can be abrogated by blockage of S. aureus phagocytosis. The pyroptosic effect by S. aureus infection was promoted by either rapamycin or Stattic, a specific inhibitor for mTORC1 or STAT3. Inhibition of mTORC1 or STAT3 induced pyroptosis. mTORC1 regulated the pyroptosic gene expression through governing the nuclear localization of STAT3. mTORC1/STAT3 axis may play a regulatory role in pyroptosis within macrophages. Conclusions S. aureus infection induces human macrophage pyroptosis, inhibition of mTORC1/STAT3 axis facilitates S. aureus-induced pyroptosis. mTORC1 and STAT3 are associated with pyroptosis. Our findings demonstrate a regulatory function of the mTORC1/STAT3 axis in macrophage pyroptosis, constituting a novel mechanism by which pyroptosis is regulated in macrophages. Video Abstract Macrophages were infected with S. aureus for 3 h (MOI 25:1), and pyroptosis was filmed in macrophages by laser confocal microscopy. A representative field was recorded. Arrow indicates lysing dead cell.

Summary: Inhibitors of Mek1/2 and Gsk3β, known as 2i, and, together with leukemia inhibitory factor, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency (2i/L-ESCs). However, recent reports show that prolonged Mek1/2 suppression impairs developmental potential of ESCs, and is rescued by serum (S/L-ESCs). Here, we show that culturing ESCs in Activin A and BMP4, and in the absence of MEK1/2 inhibitor (ABC/L medium), establishes advanced stem cells derived from ESCs (esASCs). We demonstrate that esASCs contributed to germline lineages, full-term chimeras and generated esASC-derived mice by tetraploid complementation. We show that, in contrast to 2i/L-ESCs, esASCs display distinct molecular signatures and a stable hypermethylated epigenome, which is reversible and similar to serum-cultured ESCs. Importantly, we also derived novel ASCs (blASCs) from blastocysts in ABC/L medium. Our results provide insights into the derivation of novel ESCs with DNA hypermethylation from blastocysts in chemically defined medium. : In this article, Bao and colleagues show that ASCs cultured in Activin A and BMP4, and in the absence of MEK1/2 inhibitor, possess reversible epigenetic changes that extend their developmental potency, distinct transcriptome pattern, and a stable hypermethylated epigenome. Importantly, they also derived novel hypermethylated ASCs from blastocysts in ABC/L medium. Keywords: mouse, embryonic stem cells, epigenetics, hypermethylation, development, chemically defined, genomic imprinting, blastocyst, pluripotency, implantation

Intramuscular fat content and marbling affecting meat quality are important economic traits in beef cattle. CDC10 (cell division cycle 10 or Septin 7), a member of the septin family involved in cellular proliferation, was considered as a functional and positional candidate gene for beef marbling. In a previous study, we revealed that the expression levels of CDC10 were also positively correlated with marbling scores in Japanese Black cattle. However, the regulatory mechanism of the CDC10 gene on IMF deposition in cattle remains unclear. In the present study, flow cytometry, EdU proliferation assays, and Oil Red O staining results showed that overexpression of CDC10 could promote the differentiation of bovine intramuscular preadipocyte (BIMP) and 3T3-L1 cells, whereas knockdown of CDC10 resulted in the opposite consequences. Furthermore, quantitative PCR and Western blotting results showed that overexpression of CDC10 could promote the expression levels of adipogenic marker genes PPARγ and C/EBPα at both mRNA and protein levels in BIMP and 3T3-L1 cells, whereas knockdown of CDC10 resulted in the opposite consequences. Our results provide new insights into the regulatory roles of CDC10 in adipocytes in animals.

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.

Pluripotent stem cells (PSCs) have the potential to differentiate to all cell types of an adult individual and are useful for studying mammalian development. Establishing induced pluripotent stem cells (iPSCs) capable of expressing pluripotent genes and differentiating to three germ layers will not only help to explain the mechanisms underlying somatic reprogramming but also lay the foundation for the establishment of sheep embryonic stem cells (ESCs) in vitro. In this study, sheep somatic cells were reprogrammed in vitro into sheep iPSCs with stable morphology, pluripotent marker expression, and differentiation ability, delivered by piggyBac transposon system with eight doxycycline (DOX)-inducible exogenous reprogramming factors: bovine OCT4, SOX2, KLF4, cMYC, porcine NANOG, human LIN28, SV40 large T antigen, and human TERT. Sheep iPSCs exhibited a chimeric contribution to the early blastocysts of sheep and mice and E6.5 mouse embryos in vitro. A transcriptome analysis revealed the pluripotent characteristics of somatic reprogramming and insights into sheep iPSCs. This study provides an ideal experimental material for further study of the construction of totipotent ESCs in sheep.

In mice, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are established from pre- and post-implantation embryos and represent the naive and primed state, respectively. Herein we used mouse leukemia inhibitory factor (LIF), which supports ESCs self-renewal and Activin A (Act A), which is the main factor in maintaining EpiSCs in post-implantation epiblast cultures, to derive a primed stem cell line named ALSCs. Like EpiSCs, ALSCs express key pluripotent genes Oct4, Sox2, and Nanog; one X chromosome was inactivated; and the cells failed to contribute to chimera formation in vivo. Notably, compared to EpiSCs, ALSCs efficiently reversed to ESCs (rESCs) on activation of Wnt signaling. Moreover, we also discovered that culturing EpiSCs in AL medium for several passages favored Wnt signaling-driven naive pluripotency. Our results show that ALSCs is a primed state stem cell and represents a simple model to study the control of pluripotency fate and conversion from the primed to the naive state.

Dosage compensation is a mechanism first proposed by Susumu Ohno, whereby X inactivation balances X gene output between males (XY) and females (XX), while X upregulation balances X genes with autosomal gene output. These mechanisms have been actively studied in Drosophila and mice, but research regarding them lags behind in domestic species. It is unclear how the X chromosome is regulated in the sheep male germline. To address this, using single-cell RNA sequencing, we analyzed testes in three important developmental stages of sheep. We observed that the total RNA per cell from X and autosomes peaked in SSCs and spermatogonia and was then reduced in early spermatocytes. Furthermore, we counted the detected reads per gene in each cell type for X and autosomes. In cells experiencing dose compensation, close proximity to MSL (male-specific lethal), which is regulated the active X chromosome and was observed. Our results suggest that there is no dose compensation in the pre-meiotic germ cells of sheep testes and, in addition, MSL1 and MSL2 are expressed in early germ cells and involved in regulating mammalian X-chromosome inactivation and activation.

Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine and metabolic disorder in women, which is characterized by androgen excess, ovulation dysfunction, and polycystic ovary. Although the etiology of PCOS is largely unknown, many studies suggest that aberrant DNA methylation is an important contributing factor for its pathological changes. In this study, we investigated DNA methylation characteristics and their impact on gene expression in granulosa cells obtained from PCOS patients. Transcriptome analysis found that differentially expressed genes were mainly enriched in pathways of insulin resistance, fat cell differentiation, and steroid metabolism in PCOS. Overall DNA methylation level in granulosa cells was reduced in PCOS, and the first introns were found to be the major genomic regions that were hypomethylated in PCOS. Integrated analysis of transcriptome, DNA methylation, and miRNAs in ovarian granulosa cells revealed a DNA methylation and miRNA coregulated network and identified key candidate genes for pathogenesis of PCOS, including BMP4, ETS1, and IRS1. Our study shed more light on epigenetic mechanism of PCOS and provided valuable reference for its diagnosis and treatment.

3-hydroxybutyrate dehydrogenase-2 (Bdh2), a short-chain dehydrogenase, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore, playing a key role in iron homeostasis, energy metabolism and apoptosis. However, the function of Bdh2 in embryonic stem cells (ESCs) remains unknown. To gain insights into the role of Bdh2 on pluripotency and cell fate decisions of mouse ESCs, we generated Bdh2 homozygous knockout lines for both mouse advanced embryonic stem cell (ASC) and ESC using CRISPR/Cas9 genome editing technology. Bdh2 deficiency in both ASCs and ESCs had no effect on expression of core pluripotent transcription factors and alkaline phosphatase activity, suggesting dispensability of Bdh2 for self-renewal and pluripotency of ESCs. Interestingly, cells with Bdh2 deficiency exhibited potency of endoderm differentiation in vitro; with upregulated endoderm associated genes revealed by RNA-seq and RT-qPCR. We further demonstrate that Bdh2 loss inhibited expression of multiple methyltransferases (DNMTs) at both RNA and protein level, suggesting that Bdh2 may be essentially required to maintain DNA methylation in ASCs and ESCs. Overall, this study provides valuable data and resources for understanding how Bdh2 regulate earliest cell fate decision and DNA methylation in ASCs/ESCs.

In vitro maturation (IVM) of sheep oocytes and early embryonic development are of great scientific importance for the study of reproductive development in sheep. Ghrelin is an important hormone that regulates the secretion of the growth hormone (GH). In this study, different gradients of ghrelin (0, 100, 200, and 300 ng/mL) were added to the IVM system of sheep oocytes to observe their cell morphology, and Hosesth 33342 staining was used to determine the time taken for oocytes to reach different developmental stages. We found 200 ng/mL ghrelin to be the optimal concentration. The RNA-seq analysis showed that many signaling pathways were significantly altered by ghrelin. Cell cycle, Wnt, and oxidative phosphorylation were activated; the P53 was inhibited. These pathways together regulate the maturation of oocytes and early embryonic development in vitro. The effects of the addition of ghrelin were verified by the expression of GLUT1 in early embryonic development. The results suggest that adding ghrelin shortens the duration of the IVM of sheep oocytes and hinders early embryonic development. This study provides new insights into the effects of exogenous ghrelin on sheep oocyte maturation and early embryonic development in vitro.

The short-tailed phenotype has long been considered one of the best traits for population genetic improvement in sheep breeding. In short-tailed sheep, not only is tail fat eliminated but also the pubic area is exposed due to the lack of a tail covering, giving them an advantage in reproduction. Recent studies have shown that two linked mutations in sheep TBXT at nucleotides 333 and 334 are associated with the short-tailed phenotype. In the population of short-tailed sheep, several heterozygous mutants of this gene are found. In our research, we used high-resolution melting (HRM) to identify homozygous and heterozygous genotypes in a flock of short-tailed sheep and compared the results with those of Sanger sequencing, which were identical. This demonstrates that our established HRM method, a rapid and inexpensive genotyping method, can be used to identify homozygous and heterozygous individuals in short-tailed sheep flocks.

The differences in small noncoding RNAs (sncRNAs), including miRNAs, piRNAs, and tRNA-derived fragments (tsRNAs), between X and Y sperm of mammals remain unclear. Here, we employed high-throughput sequencing to systematically compare the sncRNA profiles of X and Y sperm from bulls (n = 3), which may have a wider implication for the whole mammalian class. For the comparison of miRNA profiles, we found that the abundance of bta-miR-652 and bta-miR-378 were significantly higher in X sperm, while nine miRNAs, including bta-miR-204 and bta-miR-3432a, had greater abundance in Y sperm (p

Although cryopreservation of bull semen is widely used commercially in the livestock breeding industry, cryopreservation results in low fertility of bull sperm. As an important regulatory factor, the alteration of small non-coding RNA (sncRNA) profile during cryopreservation of bull sperm is not yet completely known. In the present study, we sequenced sncRNAs of frozen and fresh sperm to study the link of alteration of the sncRNA profiles (particularly in miRNAs and mRNA fragments) with low sperm fertility caused by cryopreservation. We identified 55 miRNAs and 526 mRNA fragments differentially expressed (DE) between frozen and fresh sperm. Subsequently, the functional analysis revealed that targeted genes of DE miRNAs in sperm had roles in the fertilization, ATP, and apoptosis. Instead, targeted genes of DE miRNAs in cow metaphase II oocyte were significantly enriched in the MAPK signaling pathway, autophagy-animal pathway, and mitophagy-animal pathway. Additionally, biological processes of DNA repair, spermatid development, response to temperature stimulus, and cellular response to DNA damage stimulus were enriched by mRNA fragments. In conclusion, we found that DE miRNAs or DE mRNA fragments in cryopreservation may influence the fertility of sperm, these findings will provide the reference to improve the cryopreservation technology of bull semen.

Most hinnies (female donkey×male horse) and mules (female horse×male donkey) are sterile with few reports of equine fertile hybrids. The main cause of this sterility is thought to be a meiotic block to spermatogenesis and oogenesis. This study compared the developmental features of the testes and a histological analyses of spermatogenesis in a male hinny with those of a normal, fertile stallion and Jack donkey. Hinny testes showed a thicker tunica albuginea, fewer blood vessels and more connective tissue in the testis parenchyma than those of the stallion and Jack donkey. Although the mean number of seminiferous tubules was significantly higher in stallion and hinny than Jack donkey (p

Blastocyst injection and morula aggregation are commonly used to evaluate stem cell pluripotency based on chimeric contribution of the stem cells. To assess the protocols for generating chimeras from stem cells, 8-cell mouse embryos were either injected or cocultured with mouse embryonic stem cells and induced pluripotent stem cells, respectively. Although a significantly higher chimera rate resulted from blastocyst injection, the highest germline contribution resulted from injection of 8-cell embryos with embryonic stem cells. The fully agouti colored chimeras were generated from both injection and coculture of 8-cell embryos with embryonic stem cells. Additionally, microsatellite DNA screening showed that the fully agouti colored chimeras were fully embryonic stem cell derived mice. Unlike embryonic stem cells, the mouse chimeras were only generated from injection of 8-cell embryos with induced pluripotent stem cells and none of these showed germline transmission. The results indicated that injection of 8-cell embryos is the most efficient method for assessing stem cell pluripotency and generating induced pluripotent stem cell chimeras, embryonic stem cell chimeras with germline transmission, and fully mouse embryonic stem cell derived mice.

Early embryonic development determines fetal and adult development. However, little is known about the specific development of the early Mongolian sheep embryo. We aim to analyze the differentially expressed genes (DEGs) between 16-day embryos of two breeds of Mongolian sheep, namely, Hulunbuir short-tailed sheep and Ujumqin sheep, by mRNA sequencing. A total of 2152 DEGs were identified. Significantly DEGs include WNT5A, WNT2, AXIN2, BMP5, TGFB2, and SMAD9, which are involved in multiple signaling pathways, and genes involved in digestion and absorption and cellular differentiation, such as DPP4, ACE2, PLPP1, CD22, CD44, TEC, and JAK1. In the pathway analysis, the development of Hulunbuir short-tailed sheep 16-day embryo H(E16) highlighted pathways primarily associated with cellular interactions and organogenesis. These pathways are critical for transcriptional control during early embryonic development, especially organ formation. Genes that sophisticate organs and body mechanisms, and genes associated with protein and fat digestion, hematopoietic stem cell lineage and osteoblast differentiation, are enriched during the development of the Ujumqin sheep 16-day embryo U(E16). Our work provides important complementary reference data for the study of early embryonic development in sheep. [ABSTRACT FROM AUTHOR]