Dynamic intrauterine crosstalk promotes porcine embryo implantation during early pregnancy.

Dynamic crosstalk between the embryo and mother is crucial during implantation. Here, we comprehensively profile the single-cell transcriptome of pig peri-implantation embryos and corresponding maternal endometrium, identifying 4 different lineages in embryos and 13 cell types in the endometrium. Cell-specific gene expression characterizes 4 distinct trophectoderm subpopulations, showing development from undifferentiated trophectoderm to polar and mural trophectoderm. Dynamic expression of genes in different types of endometrial cells illustrates their molecular response to embryos during implantation. Then, we developed a novel tool, ExtraCellTalk, generating an overall dynamic map of maternal-foetal crosstalk using uterine luminal proteins as bridges. Through cross-species comparisons, we identified a conserved RBP4/STRA6 pathway in which embryonic-derived RBP4 could target the STRA6 receptor on stromal cells to regulate the interaction with other endometrial cells. These results provide insight into the maternal-foetal crosstalk during embryo implantation and represent a valuable resource for further studies to improve embryo implantation.

Treatment of Donor Cells with Oxidative Phosphorylation Inhibitor CPI Enhances Porcine Cloned Embryo Development.

Somatic cell nuclear transfer (SCNT) technology holds great promise for livestock industry, life science and human biomedicine. However, the development and application of this technology is limited by the low developmental potential of SCNT embryos. The developmental competence of cloned embryos is influenced by the energy metabolic status of donor cells. The purpose of this study was to investigate the effects of CPI, an oxidative phosphorylation inhibitor, on the energy metabolism pathways of pig fibroblasts and the development of subsequent SCNT embryos. The results showed that treatment of porcine fibroblasts with CPI changed the cellular energy metabolic pathways from oxidative phosphorylation to glycolysis and enhanced the developmental ability of subsequent SCNT embryos. The present study establishes a simple, new way to improve pig cloning efficiency, helping to promote the development and application of pig SCNT technology.

Calculating Volume of Pig Point Cloud Based on Improved Poisson Reconstruction.

Pig point cloud data can be used to digitally reconstruct surface features, calculate pig body volume and estimate pig body weight. Volume, as a pig novel phenotype feature, has the following functions: (a) It can be used to estimate livestock weight based on its high correlation with body weight. (b) The volume proportion of various body parts (such as head, legs, etc.) can be obtained through point cloud segmentation, and the new phenotype information can be utilized for breeding pigs with smaller head volumes and stouter legs. However, as the pig point cloud has an irregular shape and may be partially missing, it is difficult to form a closed loop surface for volume calculation. Considering the better water tightness of Poisson reconstruction, this article adopts an improved Poisson reconstruction algorithm to reconstruct pig body point clouds, making the reconstruction results smoother, more continuous, and more complete. In the present study, standard shape point clouds, a known-volume Stanford rabbit standard model, a measured volume piglet model, and 479 sets of pig point cloud data with known body weight were adopted to confirm the accuracy and reliability of the improved Poisson reconstruction and volume calculation algorithm. Among them, the relative error was 4% in the piglet model volume result. The average absolute error was 2.664 kg in the weight estimation obtained from pig volume by collecting pig point clouds, and the average relative error was 2.478%. Concurrently, it was determined that the correlation coefficient between pig body volume and pig body weight was 0.95.

Supplementation of SkQ1 Increases Mouse In Vitro Oocyte Maturation and Subsequent Embryonic Development by Reducing Oxidative Stress.

In vitro oocyte maturation (IVM) technology is important for assisted animal and human reproduction. However, the maturation rates and developmental potential of in vitro-matured oocytes are usually lower than those of in vivo-matured oocytes. Oxidative stress is a main factor that causes the lower maturation rates and quality of in vitro-matured oocytes. The purpose of this study was to investigate the effects of treatment with SkQ1, a mitochondria-targeted antioxidant, on mouse IVM and subsequent embryonic development. The results demonstrated that the supplementation of SkQ1 during IVM improves the maturation rates of mouse oocytes and the subsequent developmental competence of in vitro-fertilized embryos. The addition of SkQ1 to the IVM medium also decreased oxidative stress and apoptosis, and increased mitochondrial membrane potential in matured mouse oocytes. This study provides a new method through which to enhance the maturation rates and the quality of in vitro-matured mouse oocytes, thus promoting the application and development of assisted animal and human reproductive technology.

A deep learning-based approach for fully automated segmentation and quantitative analysis of muscle fibers in pig skeletal muscle.

Muscle fiber properties exert a significant influence on pork quality, with cross-sectional area (CSA) being a crucial parameter closely associated with various meat quality indicators, such as shear force. Effectively identifying and segmenting muscle fibers in a robust manner constitutes a vital initial step in determining CSA. This step is highly intricate and time-consuming, necessitating an accurate and automated analytical approach. One limitation of existing methods is their tendency to perform well on high signal-to-noise ratio images of intact, healthy muscle fibers but their lack of validation on more complex image datasets featuring significant morphological changes, such as the presence of ice crystals. In this study, we undertake the fully automatic segmentation of muscle fiber microscopic images stained with myosin adenosine triphosphate (mATPase) activity using a deep learning architecture known as SOLOv2. Our objective is to efficiently derive accurate measurements of muscle fiber size and distribution. Tests conducted on actual images demonstrate that our method adeptly handles the intricate task of muscle fiber segmentation, yielding quantitative results amenable to statistical analysis and displaying reliability comparable to manual analysis.

Structural and functional insights into yeast Tbf1 as an atypical telomeric repeat-binding factor.

Telomeric repeat-binding factor 1 (Tbf1) has a similar architecture as the TRF family of telomeric proteins and plays important roles in both telomere homeostasis and ribosome regulation. However, the molecular basis of why Tbf1 has such different functions compared to other TRFs remains unclear. Here, we present the crystal structures of the TRF homology (TRFH) and Myb-L domains from Schizosaccharomyces pombe Tbf1 (spTbf1). TRFH-mediated homodimerization is essential for spTbf1 stability. Importantly, spTbf1TRFH lacks the conserved docking motif for interactions with telomeric proteins, explaining why spTbf1 does not participate in the assembly of the shelterin complex. Finally, structural and biochemical analyses demonstrate that TRFH and Myb-L domains as well as the loop region of spTbf1 coordinate to recognize S. pombe telomeric double-stranded DNA. Overall, our findings provide structural and functional insights into how fungi Tbf1 acts as an atypical telomeric repeat-binding factor, which helps to understand the evolution of TRFH-containing telomeric proteins.

Protein Dynamic Landscape during Mouse Mammary Gland Development from Virgin to Pregnant, Lactating, and Involuting Stages.

The mammary gland undergoes significant physiological changes as it undergoes a transition from virgin to pregnancy, lactation, and involution. However, the dynamic role of proteins in regulating these processes during mouse mammary gland development has not been thoroughly explored. In this study, we collected mouse mammary gland tissues from mature virgins aged 8-10 weeks (V), day 16 of pregnancy (P16d), day 12 of lactation (L12d), day 1 of forced weaning (FW 1d), and day 3 of forced weaning (FW 3d) stages for analysis using DIA-based quantitative proteomics technology. A total of 3,312 proteins were identified, of which 843 were DAPs that were categorized into nine clusters based on their abundance changes across developmental stages. Notably, DAPs in cluster 2, which peaked at the L12d stage, were primarily associated with mammary gland development and lactation. The protein-protein interaction network revealed that the epidermal growth factor (EGF) was central to this cluster. Our study provides a comprehensive overview of the mouse mammary gland development proteome and identifies some important proteins, such as EGF, Janus kinase 1 (JAK1), and signal transducer and activator of transcription 6 (STAT6) that may serve as potential targets for future research to provide guidelines for a deeper understanding of the developmental biology of mammary glands.

Porcine Granulosa-Cell-Derived Exosomes Enhance Oocyte Development: An In Vitro Study.

Recent studies have established that exosomes (EXs) derived from follicular fluid (FF) can promote oocyte development. However, the specific sources of these EXs and their regulatory mechanisms remain elusive. It is universally acknowledged that oocyte development requires signal communication between granulosa cells (GCs) and oocytes. However, the role of GC-secreted EXs and their functions are poorly understood. This study aimed to investigate the role of porcine granulosa-cell-derived exosomes (GC-EXs) in oocyte development. In this study, we constructed an in vitro model of porcine GCs and collected and identified GC-EXs. We confirmed that porcine GCs can secrete EXs and investigated the role of GC-EXs in regulating oocyte development by supplementing them to cumulus-oocyte complexes (COCs) cultured in vitro. Specifically, GC-EXs increase the cumulus expansion index (CEI), promote the expansion of the cumulus, alleviate reactive oxygen species (ROS), and increase mitochondrial membrane potential (MMP), resulting in improved oocyte development. Additionally, we conducted small RNA sequencing of GC-EXs and hypothesized that miR-148a-3p, the highest-expressed microRNA (miRNA), may be the key miRNA. Our study determined that transfection of miR-148a-3p mimics exerts effects comparable to the addition of EXs. Meanwhile, bioinformatics prediction, dual luciferase reporter gene assay, and RT-qPCR identified DOCK6 as the target gene of miR-148a-3p. In summary, our results demonstrated that GC-EXs may improve oocyte antioxidant capacity and promote oocyte development through miR-148a-3p by targeting DOCK6.

RASGRP1 targeted by H3K27me3 regulates myoblast proliferation and differentiation in mice and pigs.

Skeletal muscle is not only the largest organ in the body that is responsible for locomotion and exercise but also crucial for maintaining the body's energy metabolism and endocrine secretion. The trimethylation of histone H3 lysine 27 (H3K27me3) is one of the most important histone modifications that participates in muscle development regulation by repressing the transcription of genes. Previous studies indicate that the RASGRP1 gene is regulated by H3K27me3 in embryonic muscle development in pigs, but its function and regulatory role in myogenesis are still unclear. In this study, we verify the crucial role of H3K27me3 in RASGRP1 regulation. The gain/loss function of RASGRP1 in myogenesis regulation is performed using mouse myoblast C2C12 cells and primarily isolated porcine skeletal muscle satellite cells (PSCs). The results of qPCR, western blot analysis, EdU staining, CCK-8 assay and immunofluorescence staining show that overexpression of RASGRP1 promotes cell proliferation and differentiation in both skeletal muscle cell models, while knockdown of RASGRP1 leads to the opposite results. These findings indicate that RASGRP1 plays an important regulatory role in myogenesis in both mice and pigs.

Genomic prediction based on preselected single-nucleotide polymorphisms from genome-wide association study and imputed whole-genome sequence data annotation for growth traits in Duroc pigs.

The use of whole-genome sequence (WGS) data is expected to improve genomic prediction (GP) power of complex traits because it may contain mutations that in strong linkage disequilibrium pattern with causal mutations. However, a few previous studies have shown no or small improvement in prediction accuracy using WGS data. Incorporating prior biological information into GP seems to be an attractive strategy that might improve prediction accuracy. In this study, a total of 6334 pigs were genotyped using 50K chips and subsequently imputed to the WGS level. This cohort includes two prior discovery populations that comprise 294 Landrace pigs and 186 Duroc pigs, as well as two validation populations that consist of 3770 American Duroc pigs and 2084 Canadian Duroc pigs. Then we used annotation information and genome-wide association study (GWAS) from the WGS data to make GP for six growth traits in two Duroc pig populations. Based on variant annotation, we partitioned different genomic classes, such as intron, intergenic, and untranslated regions, for imputed WGS data. Based on GWAS results of WGS data, we obtained trait-associated single-nucleotide polymorphisms (SNPs). We then applied the genomic feature best linear unbiased prediction (GFBLUP) and genomic best linear unbiased prediction (GBLUP) models to estimate the genomic estimated breeding values for growth traits with these different variant panels, including six genomic classes and trait-associated SNPs. Compared with 50K chip data, GBLUP with imputed WGS data had no increase in prediction accuracy. Using only annotations resulted in no increase in prediction accuracy compared to GBLUP with 50K, but adding annotation information into the GFBLUP model with imputed WGS data could improve the prediction accuracy with increases of 0.00%-2.82%. In conclusion, a GFBLUP model that incorporated prior biological information might increase the advantage of using imputed WGS data for GP.

Gender Control of Mouse Embryos by Activation of TLR7/8 on X Sperm via Ligands dsRNA-40 and dsRNA-DR.

Gender control technologies are promising for enhancing the production efficiency of the farm animal industry, and preventing sex-linked hereditary diseases in humans. It has been shown that the X sperm of mammalian animals specifically expresses X-chromosome-derived toll-like receptor 7/8 (TLR7/8), and the activation of TLR7/8 on the X sperm by their agonist, R848, can separate X and Y sperm via the specific inhibition of X sperm motility. The use of R848-preselected sperm for fertilization resulted in sex-ratio-skewed embryos or offspring. In this study, we aimed to investigate whether two other TLR7/8 ligands, double-stranded RNA-40 (dsRNA-40) and double-stranded RNA-DR (dsRNA-DR), are also effective in the separation of mouse X and Y sperm and the subsequent generation of gender-ratio-skewed in vitro fertilization (IVF) embryos. Our results indicated that cholesterol modification significantly enhances the transfection of dsRNA-40 and dsRNA-DR into sperm cells. dsRNA-40 and dsRNA-DR incubation with mouse sperm could separate X and Y sperm by the specific suppression of X sperm motility by decreasing its ATP level and mitochondrial activity. The use of a dsRNA-40- or dsRNA-DR-preselected upper layer of sperm, which predominantly contains high-motility Y sperm, for IVF caused a male-biased sex ratio shift in resulting embryos (with 65.90-74.93% of embryos being male). This study develops a simple new method for the efficient separation of mammalian X and Y sperm, enabling the selective production of male or female progenies.

Protein Dynamic Landscape of Pig Embryos during Peri-Implantation Development.

Properly developed embryos are critical for successful embryo implantation. The dynamic landscape of proteins as executors of biological processes in pig peri-implantation embryos has not been reported so far. In this study, we collected pig embryos from days 9, 12, and 15 of pregnancy during the peri-implantation stage for a PASEF-based quantitative proteomic analysis. In total, approximately 8000 proteins were identified. These proteins were classified as stage-exclusive proteins and stage-specific proteins, respectively, based on their presence and dynamic abundance changes at each stage. Functional analysis showed that their roles are consistent with the physiological processes of corresponding stages, such as the biosynthesis of amino acids and peptides at P09, the regulation of actin cytoskeletal organization and complement activation at P12, and the vesicular transport at P15. Correlation analysis between mRNAs and proteins showed a general positive correlation between pig peri-implantation embryonic mRNAs and proteins. Cross-species comparisons with human early embryos identified some conserved proteins that may be important in regulating embryonic development, such as STAT3, AP2A1, and PFAS. Our study provides a comprehensive overview of the pig embryo proteome during implantation, fills gaps in relevant developmental studies, and identifies some important proteins that may serve as potential targets for future research.

DIA-based quantitative proteomic analysis of porcine endometrium in the peri-implantation phase.

The 12th day of gestation is a critical period for embryo loss and the beginning of imminent implantation in sows. Data independent acquisition (DIA) technology is one of the high-throughput, high-resolution and reproducible proteomics technologies for large-scale digital qualitative and quantitative research. The aim of this study was to identify and characterize the protein abundance landscape of Yorkshire pig endometrium on the 12th day of pregnancy (P12) and estrous cycle (C12) using DIA proteomics. A total of 1251 differentially abundant proteins (DAPs) were identified, of which 882 were up-regulated and 369 were down-regulated at P12. Functional enrichment analysis showed that the identified proteins were related to metabolism, biosynthesis and signaling pathways. Three proteins were selected for Western blot (WB) validation and the results were consistent with the DIA data. Further combined with transcriptome data, fibrinogen like 2 (FGL2) and S100 calcium binding protein A8 (S100A8) were verified to be highly abundant in the P12 endometrial epithelium. In summary, there were significantly different abundance of proteome profiles in C12 and P12 endometrium, suggesting that DAPs are associated with changes in endometrial receptivity, which laid the foundation for further research on related regulatory mechanisms. SIGNIFICANCE: The 12th day of gestation is an important point in the peri-implantation period of pigs, when the endometrium presents a receptive state under the stimulation of estrogen. DIA proteomics technology is an emerging protein identification technology in recent years, which can obtain protein information through comprehensive and unbiased scanning. In this study, DIA technology was used to characterize endometrial proteins in pigs during the peri-implantation period. The results showed that higher protein abundance was detected using the DIA technique, and some of these DAPs may be involved in regulating embryo implantation. This study will help to better reveal the related proteins involved in embryo implantation, and lay a foundation for further research on the mechanism of endometrial regulation of embryo implantation. SIGNIFICANCE OF THE STUDY: The 12th day of gestation is an important point in the peri-implantation period of pigs, when the endometrium presents a receptive state under the stimulation of estrogen. DIA proteomics technology is an emerging protein identification technology in recent years, which can obtain protein information through comprehensive and unbiased scanning. In this study, DIA technology was used to characterize endometrial proteins in pigs during the peri-implantation period. The results showed that higher protein abundance was detected using the DIA technique, and some of these DAPs may be involved in regulating embryo implantation. This study will help to better reveal the related proteins involved in embryo implantation, and lay a foundation for further research on the mechanism of endometrial regulation of embryo implantation.

Genome-wide association studies for loin muscle area, loin muscle depth and backfat thickness in DLY pigs.

This study aimed at identifying genes associated with loin muscle area (LMA), loin muscle depth (LMD) and backfat thickness (BFT). We performed single-trait and multi-trait genome-wide association studies (GWASs) after genotyping 685 Duroc × (Landrace × Yorkshire) (DLY) pigs using the Geneseek Porcine 50K SNP chip. In the single-trait GWASs, we identified two, eight and two significant SNPs associated with LMA, LMD and BFT, respectively, and searched genes within the 1 Mb region near the significant SNPs with relevant functions as candidate genes. Consequently, we identified one (DOCK5), three (PID1, PITX2, ELOVL6) and three (CCR1, PARP14, CASR) promising candidate genes for LMA, LMD and BFT, respectively. Moreover, the multi-trait GWAS identified four significant SNPs associated with the three traits. In conclusion, the GWAS analysis of LMA, LMD and BFT in a DLY pig population identified several associated SNPs and candidate genes, further deepening our understanding of the genetic basis of these traits, and they may be useful for marker-assisted selection to improve the three traits in DLY pigs.

Increased Accuracy of Genomic Prediction Using Preselected SNPs from GWAS with Imputed Whole-Genome Sequence Data in Pigs.

Enhancing the accuracy of genomic prediction is a key goal in genomic selection (GS) research. Integrating prior biological information into GS methods using appropriate models can improve prediction accuracy for complex traits. Genome-wide association study (GWAS) is widely utilized to identify potential candidate loci associated with complex traits in livestock and poultry, offering essential genomic insights. In this study, a GWAS was conducted on 685 Duroc × Landrace × Yorkshire (DLY) pigs to extract significant single-nucleotide polymorphisms (SNPs) as genomic features. We compared two GS models, genomic best linear unbiased prediction (GBLUP) and genomic feature BLUP (GFBLUP), by using imputed whole-genome sequencing (WGS) data on 651 Yorkshire pigs. The results revealed that the GBLUP model achieved prediction accuracies of 0.499 for backfat thickness (BFT) and 0.423 for loin muscle area (LMA). By applying the GFBLUP model with GWAS-based SNP preselection, the average prediction accuracies for BFT and LMA traits reached 0.491 and 0.440, respectively. Specifically, the GFBLUP model displayed a 4.8% enhancement in predicting LMA compared to the GBLUP model. These findings suggest that, in certain scenarios, the GFBLUP model may offer superior genomic prediction accuracy when compared to the GBLUP model, underscoring the potential value of incorporating genomic features to refine GS models.

Modulation of cell cycle increases CRISPR-mediated homology-directed DNA repair.

BACKGROUND: Gene knock-in (KI) in animal cells via homology-directed repair (HDR) is an inefficient process, requiring a laborious work for screening from few modified cells. HDR tends to occur in the S and G2/M phases of cell cycle; therefore, strategies that enhance the proportion of cells in these specific phases could improve HDR efficiency. RESULTS: We used various types of cell cycle inhibitors to synchronize the cell cycle in S and G2/M phases in order to investigate their effect on regulating CRISPR/Cas9-mediated HDR. Our results indicated that the four small molecules-docetaxel, irinotecan, nocodazole and mitomycin C-promoted CRISPR/Cas9-mediated KI with different homologous donor types in various animal cells. Moreover, the small molecule inhibitors enhanced KI in animal embryos. Molecular analysis identified common signal pathways activated during crosstalk between cell cycle and DNA repair. Synchronization of the cell cycle in the S and G2/M phases results in CDK1/CCNB1 protein accumulation, which can initiate the HDR process by activating HDR factors to facilitate effective end resection of CRISPR-cleaved double-strand breaks. We have demonstrated that augmenting protein levels of factors associated with the cell cycle via overexpression can facilitate KI in animal cells, consistent with the effect of small molecules. CONCLUSION: Small molecules that induce cell cycle synchronization in S and G2/M phases promote CRISPR/Cas9-mediated HDR efficiency in animal cells and embryos. Our research reveals the common molecular mechanisms that bridge cell cycle progression and HDR activity, which will inform further work to use HDR as an effective tool for preparing genetically modified animals or for gene therapy.

Composition, function, and timing: exploring the early-life gut microbiota in piglets for probiotic interventions.

BACKGROUND: The establishment of a robust gut microbiota in piglets during their early developmental stage holds the potential for long-term advantageous effects. However, the optimal timeframe for introducing probiotics to achieve this outcome remains uncertain. RESULTS: In the context of this investigation, we conducted a longitudinal assessment of the fecal microbiota of 63 piglets at three distinct pre-weaning time points. Simultaneously, we gathered vaginal and fecal samples from 23 sows. Employing 16S rRNA gene and metagenomic sequencing methodologies, we conducted a comprehensive analysis of the fluctuation patterns in microbial composition, functional capacity, interaction networks, and colonization resistance within the gut microbiota of piglets. As the piglets progressed in age, discernible modifications in intestinal microbial diversity, composition, and function were observed. A source-tracking analysis unveiled the pivotal role of fecal and vaginal microbiota derived from sows in populating the gut microbiota of neonatal piglets. By D21, the microbial interaction network displayed a more concise and efficient configuration, accompanied by enhanced colonization resistance relative to the other two time points. Moreover, we identified three strains of Ruminococcus sp. at D10 as potential candidates for improving piglets' weight gain during the weaning phase. CONCLUSIONS: The findings of this study propose that D10 represents the most opportune juncture for the introduction of external probiotic interventions during the early stages of piglet development. This investigation augments our comprehension of the microbiota dynamics in early-life of piglets and offers valuable insights for guiding forthcoming probiotic interventions.

Genome-wide association analysis unveils candidate genes and loci associated with aplasia cutis congenita in pigs.

BACKGROUND: Aplasia cutis congenita (ACC) is a rare genetic disorder characterized by the localized or widespread absence of skin in humans and animals. Individuals with ACC may experience developmental abnormalities in the skeletal and muscular systems, as well as potential complications. Localized and isolated cases of ACC can be treated through surgical and medical interventions, while extensive cases of ACC may result in neonatal mortality. The presence of ACC in pigs has implications for animal welfare. It contributes to an elevated mortality rate among piglets at birth, leading to substantial economic losses in the pig farming industry. In order to elucidate candidate genetic loci associated with ACC, we performed a Genome-Wide Association Study analysis on 216 Duroc pigs. The primary goal of this study was to identify candidate genes that associated with ACC. RESULTS: This study identified nine significant SNPs associated with ACC. Further analysis revealed the presence of two quantitative trait loci, 483 kb (5:18,196,971-18,680,098) on SSC 5 and 159 kb (13:20,713,440-207294431 bp) on SSC13. By annotating candidate genes within a 1 Mb region surrounding the significant SNPs, a total of 11 candidate genes were identified on SSC5 and SSC13, including KRT71, KRT1, KRT4, ITGB7, CSAD, RARG, SP7, PFKL, TRPM2, SUMO3, and TSPEAR. CONCLUSIONS: The results of this study further elucidate the potential mechanisms underlying and genetic architecture of ACC and identify reliable candidate genes. These results lay the foundation for treating and understanding ACC in humans.

Identification of Candidate Genes for Economically Important Carcass Cutting in Commercial Pigs through GWAS.

During the process of pork production, the carcasses of pigs are divided and sold, which provides better economic benefits and market competitiveness for pork production than selling the carcass as a whole. Due to the significant cost of post-slaughter phenotypic measurement, the genetic architecture of tenderloin weight (TLNW) and rib weight (RIBW)-important components of pig carcass economic value-remain unknown. In this study, we conducted genome-wide association studies (GWAS) for TLNW and RIBW traits in a population of 431 Duroc × Landrace × Yorkshire (DLY) pigs. In our study, the most significant single nucleotide polymorphism (SNP) associated with TLNW was identified as ASGA0085853 (3.28 Mb) on Sus scrofa chromosome 12 (SSC12), while for RIBW, it was Affx-1115046258 (172.45 Mb) on SSC13. Through haplotype block analysis, we discovered a novel quantitative trait locus (QTL) associated with TLNW, spanning a 5 kb region on SSC12, and a novel RIBW-associated QTL spanning 1.42 Mb on SSC13. Furthermore, we hypothesized that three candidate genes, TIMP2 and EML1, and SMN1, are associated with TLNW and RIBW, respectively. Our research not only addresses the knowledge gap regarding TLNW, but also serves as a valuable reference for studying RIBW. The identified SNP loci strongly associated with TLNW and RIBW may prove useful for marker-assisted selection in pig breeding programs.

Progesterone and Androstenedione Are Important Follicular Fluid Factors Regulating Porcine Oocyte Maturation Quality.

Oocytes matured in vitro are useful for assisted human and farm animal reproduction. However, the quality of in vitro matured oocytes is usually lower than that of in vivo matured oocytes, possibly due to the absence of some important signal regulators in vitro. In this study, untargeted metabolomics was used to detect the changes in the metabolites in the follicular fluid (FF) during in vivo pig oocyte maturation and in the culture medium during in vitro maturation. Our results showed that the total metabolite changing profile of the in vivo FF was different from that of the in vitro maturation medium, but the levels of 23 differentially expressed metabolites (DEMs) changed by following the same trend during both in vivo and in vitro pig oocyte maturation. These 23 metabolites may be important regulators of porcine oocyte maturation. We found that progesterone and androstenedione, two factors in the ovarian steroidogenesis pathway enriched from the DEMs, were upregulated in the FF during in vivo pig oocyte maturation. The levels of these two factors were 31 and 20 fold, respectively, and they were higher in the FF than in the culture medium at the oocyte mature stage. The supplementation of progesterone and androstenedione during in vitro maturation significantly improved the pig oocyte maturation rate and subsequent embryo developmental competence. Our finding suggests that a metabolic abnormality during in vitro pig oocyte maturation affects the quality of the matured oocytes. This study identified some important metabolites that regulate oocyte maturation and their developmental potential, which will be helpful to improve assisted animal and human reproduction.