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Abstract Background Teat number is a vital reproductive trait in sows, crucial for providing immunity and nutrition to piglets during lactation. However, “missing heritability” in Single Nucleotide Polymorphism (SNP)-based Genome-Wide Association Studies (GWAS) has led to an increasing focus on structural variations in the genetic analysis of complex biological traits. Results In this study, we generated a comprehensive CNV map in a population of French Yorkshire pigs (n = 644) and identified 429 CNVRs. Notably, 44% (189 CNVRs) of these were detected for the first time. Subsequently, we conducted GWAS for teat number in the French Yorkshire pig population using both 80K chip and its imputed data, as well as a GWAS analysis based on CNV regions (CNVR). Interestingly, 80K chip GWAS identified two SNPs located on Sus scrofa chromosome 5 (SSC5) that were simultaneously associated with Total Teat Number (TTN), Left Teat Number (LTN), and Right Teat Number (RTN). The leading SNP (WU_10.2_5_76130558) explained 3.33%, 2.69%, and 2.67% of the phenotypic variance for TTN, LTN, and RTN, respectively. Moreover, through imputed GWAS, we successfully identified 30 genetic variants associated with TTN located within the 73.22 -73.30 Mb region on SSC5. The two SNPs identified in the 80K chip GWAS were also located in this region. In addition, CNVR-based GWAS revealed three significant CNVRs associated with TTN. Finally, through gene annotation, we pinpointed two candidate genes, TRIM66 and PRICKLE1, which are related to diverse processes such as breast cancer and abnormal vertebral development. Conclusions Our research provides an in-depth analysis of the complex genetic structure underlying teat number, contributing to the genetic enhancement of sows with improved reproductive performance and, ultimately, bolstering the economic benefits of swine production enterprises.

Abstract Hybrid mapping is a powerful approach to efficiently identify and characterize genes regulated through mechanisms in cis. In this study, using reciprocal crosses of the phenotypically divergent Duroc and Lulai pig breeds, we perform a comprehensive multi-omic characterization of regulatory variation across the brain, liver, muscle, and placenta through four developmental stages. We produce one of the largest multi-omic datasets in pigs to date, including 16 whole genome sequenced individuals, as well as 48 whole genome bisulfite sequencing, 168 ATAC-Seq and 168 RNA-Seq samples. We develop a read count-based method to reliably assess allele-specific methylation, chromatin accessibility, and RNA expression. We show that tissue specificity was much stronger than developmental stage specificity in all of DNA methylation, chromatin accessibility, and gene expression. We identify 573 genes showing allele specific expression, including those influenced by parent-of-origin as well as allele genotype effects. We integrate methylation, chromatin accessibility, and gene expression data to show that allele specific expression can be explained in great part by allele specific methylation and/or chromatin accessibility. This study provides a comprehensive characterization of regulatory variation across multiple tissues and developmental stages in pigs.

Abstract Proper development of the placenta, the transient support organ forms after embryo implantation, is essential for a successful pregnancy. However, the regulation of trophoblast invasion, which is most important during placentation, remains largely unknown. Here, rats, mice, and pigs are used as biomedical models, used scRNA‐seq to comparatively elucidate the regulatory mechanism of placental trophoblast invasion, and verified it using a human preeclampsia disease model combined with scStereo‐seq. A dual‐featured type of immune‐featured trophoblast (iTrophoblast) is unexpectedly discovered. Interestingly, iTrophoblast only exists in invasive placentas and regulates trophoblast invasion during placentation. In a normally developing placenta, iTrophoblast gradually transforms from an immature state into a functional mature state as it develops. Whereas in the developmentally abnormal preeclamptic placenta, disordered iTrophoblast transformation leads to the accumulation of immature iTrophoblasts, thereby disrupting trophoblast invasion and ultimately leading to the progression of preeclampsia.

(1) Background: Reproductive performance is crucial for the pork industry’s success. The Large White pig is central to this, yet the genetic factors influencing its reproductive traits are not well understood, highlighting the need for further research. (2) Methods: This study utilized Genome-Wide Association Studies to explore the genetic basis of reproductive traits in the Large White pig. We collected data from 2237 Large White sows across four breeding herds in southern China, focusing on eight reproductive traits. Statistical analyses included principal component analysis, linkage disequilibrium analysis, and univariate linear mixed models to identify significant single-nucleotide polymorphisms and candidate genes. (3) Results: Forty-five significantly related SNPs and 17 potential candidate genes associated with litter traits were identified. Individuals with the TT genotype at SNP rs341909772 showed an increase of 1.24 in the number of piglets born alive (NBA) and 1.25 in the number of healthy births (NHBs) compared with those with the CC genotype. (4) Conclusions: The SNPs and genes identified in this study offer insights into the genetics of reproductive traits in the Large White pig, potentially guiding the development of breeding strategies to improve litter size.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract Background Although the accumulation of whole-genome sequencing (WGS) data has accelerated the identification of mutations underlying complex traits, its impact on the accuracy of genomic predictions is limited. Reliable genotyping data and pre-selected beneficial loci can be used to improve prediction accuracy. Previously, we reported a low-coverage sequencing genotyping method that yielded 11.3 million highly accurate single-nucleotide polymorphisms (SNPs) in pigs. Here, we introduce a method termed selective linkage disequilibrium pruning (SLDP), which refines the set of SNPs that show a large gain during prediction of complex traits using whole-genome SNP data. Results We used the SLDP method to identify and select markers among millions of SNPs based on genome-wide association study (GWAS) prior information. We evaluated the performance of SLDP with respect to three real traits and six simulated traits with varying genetic architectures using two representative models (genomic best linear unbiased prediction and BayesR) on samples from 3579 Duroc boars. SLDP was determined by testing 180 combinations of two core parameters (GWAS P-value thresholds and linkage disequilibrium r2). The parameters for each trait were optimized in the training population by five fold cross-validation and then tested in the validation population. Similar to previous GWAS prior-based methods, the performance of SLDP was mainly affected by the genetic architecture of the traits analyzed. Specifically, SLDP performed better for traits controlled by major quantitative trait loci (QTL) or a small number of quantitative trait nucleotides (QTN). Compared with two commercial SNP chips, genotyping-by-sequencing data, and an unselected whole-genome SNP panel, the SLDP strategy led to significant improvements in prediction accuracy, which ranged from 0.84 to 3.22% for real traits controlled by major or moderate QTL and from 1.23 to 11.47% for simulated traits controlled by a small number of QTN. Conclusions The SLDP marker selection method can be incorporated into mainstream prediction models to yield accuracy improvements for traits with a relatively simple genetic architecture, however, it has no significant advantage for traits not controlled by major QTL. The main factors that affect its performance are the genetic architecture of traits and the reliability of GWAS prior information. Our findings can facilitate the application of WGS-based genomic selection.

Pig carcass traits are among the most economically significant characteristics and are crucial for genetic selection in breeding and enhancing the economic efficiency. Standardized and automated carcass phenotyping can greatly enhance the measurement efficiency and accuracy, thereby facilitating the selection and breeding of superior pig carcasses. In this study, we utilized phenotypic images and data from 3912 pigs to propose a deep learning-based approach for the automated determination of pig carcass phenotypic traits. Using the YOLOv8 algorithm, our carcass length determination model achieves an average accuracy of 99% on the test set. Additionally, our backfat segmentation model, YOLOV8n-seg, demonstrates robust segmentation performance, with a Mean IoU of 89.10. An analysis of the data distribution comparing manual and model-derived measurements revealed that differences in the carcass straight length are primarily concentrated between −2 cm and 4 cm, while differences in the carcass diagonal length are concentrated between −3 cm and 2 cm. To validate the method, we compared model measurements with manually obtained data, achieving coefficients of determination (R2) of 0.9164 for the carcass straight length, 0.9325 for the carcass diagonal length, and 0.7137 for the backfat thickness, indicating high reliability. Our findings provide valuable insights into automating carcass phenotype determination and grading in pig production.

Abstract Background One of the most critical periods for the loss of pig embryos is the 12th day of gestation when implantation begins. Recent studies have shown that non-coding RNAs (ncRNAs) play important regulatory roles during pregnancy. Circular RNAs (circRNAs) are a kind of ubiquitously expressed ncRNAs that can directly regulate the binding proteins or regulate the expression of target genes by adsorbing micro RNAs (miRNA). Results We used the Illumina Novaseq6,000 technology to analyze the circRNA expression profile in the endometrium of three Erhualian (EH12) and three Yorkshire (YK12) pigs on day 12 of gestation. Overall, a total of 22,108 circRNAs were identified. Of these, 4051 circRNAs were specific to EH12 and 5889 circRNAs were specific to YK12, indicating a high level of breed specificity. Further analysis showed that there were 641 significant differentially expressed circRNAs (SDEcircRNAs) in EH12 compared with YK12 (FDR

Objective Loin muscle area (LMA) is an important target trait of pig breeding. This study aimed to identify single nucleotide polymorphisms (SNPs) and genes associated with LMA in the Duroc×(Landrace×Yorkshire) crossbred pigs (DLY). Methods A genome-wide association study was performed using the Illumina 50K chip to map the genetic marker and genes associated with LMA in 511 DLY pigs (255 boars and 256 sows). Results After quality control, we detected 35,426 SNPs, including six SNPs significantly associated with LMA in pigs, with MARC0094338 and ASGA0072817 being the two key SNPs responsible for 1.77% and 2.48% of the phenotypic variance of LMA, respectively. Based on previous research, we determined two candidate genes (growth hormone receptor [GHR] and 3-oxoacid Co A-transferase 1 [OXCT1]) that are associated with fat deposition and muscle growth and found further additional genes (MYOCD, ARHGAP44, ELAC2, MAP2K4, FBXO4, FBLL1, RARS1, SLIT3, and RANK3) that are presumed to have an effect on LMA. Conclusion This study contributes to the identification of the mutation that underlies quantitative trait loci associated with LMA and to future pig breeding programs based on marker-assisted selection. Further studies are needed to elucidate the role of the identified candidate genes in the physiological processes involved in LMA regulation.

Abstract 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.

Duroc pigs are popular crossbred terminal sires, and accurate assessment of genetic parameters in the population can help to rationalize breeding programmes. The principle aim of this study were to evaluate the genetic parameters of production (birth weight, BW; age at 115 kg, AGE; feed conversion ratio, FCR) and body size (body length, BL; body height, BH; front cannon circumference, FCC) traits of Duroc pigs. The second objective was to analyze the fit of different genetic assessment models. The variance components and correlations of BW (28,348 records), AGE (28,335 records), FCR (11,135 records), BL (31,544 records), BH (21,862 records), and FCC (14,684 records) traits were calculated by using DMU and AIREMLF90 from BLUPF90 package. In the common environment model, the heritability of BW, AGE, FCR, BL, BH, and FCC traits were 0.17 ± 0.014, 0.30 ± 0.019, 0.28 ± 0.024, 0.16 ± 0.013, 0.14 ± 0.017, and 0.081 ± 0.016, with common litter effect values of 0.25, 0.20, 0.18, 0.23, 0.19, and 0.16, respectively. According to the results of the Akaike information criterion (AIC) calculations, models with smaller AIC values have a better fit. We found that the common environment model with litter effects as random effects for estimating genetic parameters had a better fit. In this Model, the estimated genetic correlations between AGE with BW, FCR, BL, BH, and FCC traits were −0.28 (0.040), 0.76 (0.038), −0.71 (0.036), −0.44 (0.060), and −0.60 (0.073), respectively, with phenotypic correlations of −0.17, 0.52, −0.22, −0.13 and −0.24, respectively. In our analysis of genetic trends for six traits in the Duroc population from 2012 to 2021, we observed significant genetic trends for AGE, BL, and BH. Particularly noteworthy is the rapid decline in the genetic trend for AGE, indicating an enhancement in the pig's growth rate through selective breeding. Therefore, we believe that some challenging-to-select traits can benefit from the genetic correlations between traits. By selecting easily measurable traits, they can gain from synergistic selection effects, leading to genetic progress. Conducting population genetic parameter analysis can assist us in devising breeding strategies.

Abstract Genetic mapping to identify genes and alleles associated with or causing economically important quantitative trait variation in livestock animals such as pigs is a major goal in animal genetic improvement. Despite recent advances in high-throughput genotyping technologies, the resolution of genetic mapping in pigs remains poor due in part to the low density of genotyped variant sites. In this study, we overcame this limitation by developing a reference haplotype panel for pigs based on 2259 whole genome-sequenced animals representing 44 pig breeds. We evaluated software combinations and breed composition to optimize the imputation procedure and achieved an average concordance rate in excess of 96%, a non-reference concordance rate of 88%, and an r 2 of 0.85. We demonstrated in two case studies that genotype imputation using this resource can dramatically improve the resolution of genetic mapping. A public web server has been developed to allow the pig genetics community to fully utilize this resource. We expect this resource to facilitate genetic mapping and accelerate genetic improvement in pigs.

Abstract Background Pork quality can directly affect customer purchase tendency and meat quality traits have become valuable in modern pork production. However, genetic improvement has been slow due to high phenotyping costs. In this study, whole genome sequence (WGS) data was used to evaluate the prediction accuracy of genomic best linear unbiased prediction (GBLUP) for meat quality in large-scale crossbred commercial pigs. Results We produced WGS data (18,695,907 SNPs and 2,106,902 INDELs exceed quality control) from 1,469 sequenced Duroc × (Landrace × Yorkshire) pigs and developed a reference panel for meat quality including meat color score, marbling score, L* (lightness), a* (redness), and b* (yellowness) of genomic prediction. The prediction accuracy was defined as the Pearson correlation coefficient between adjusted phenotypes and genomic estimated breeding values in the validation population. Using different marker density panels derived from WGS data, accuracy differed substantially among meat quality traits, varied from 0.08 to 0.47. Results showed that MultiBLUP outperform GBLUP and yielded accuracy increases ranging from 17.39% to 75%. We optimized the marker density and found medium- and high-density marker panels are beneficial for the estimation of heritability for meat quality. Moreover, we conducted genotype imputation from 50K chip to WGS level in the same population and found average concordance rate to exceed 95% and r 2 = 0.81. Conclusions Overall, estimation of heritability for meat quality traits can benefit from the use of WGS data. This study showed the superiority of using WGS data to genetically improve pork quality in genomic prediction.

Abstract Most pregnancy losses worldwide are caused by implantation failure for which there is a lack of effective therapeutics. Extracellular vesicles are considered potential endogenous nanomedicines because of their unique biological functions. However, the limited supply of ULF-EVs prevents their development and application in infertility diseases such as implantation failure. In this study, pigs were used as a human biomedical model, and ULF-EVs were isolated from the uterine luminal. We comprehensively characterized the proteins enriched in ULF-EVs and revealed their biological functions in promoting embryo implantation. By exogenously supplying ULF-EVs, we demonstrated that ULF-EVs improve embryo implantation, suggesting that ULF-EVs are a potential nanomaterial to treat implantation failure. Furthermore, we identified that MEP1B is important in improving embryo implantation by promoting trophoblast cell proliferation and migration. These results indicated that ULF-EVs can be a potential nanomaterial to improve embryo implantation.

We identified Wdr17 as a highly expressed gene in pachytene spermatocytes by transcriptomic analysis of mouse testis. Germ cell-deficient infertile mouse models had significantly reduced Wdr17 expression. We performed gene interference and overexpression in the mouse spermatocyte cell line GC-2spd(ts) and investigated how Wdr17 affects spermatocyte growth and development. Our results showed that Wdr17 suppression significantly decreased cell growth rate and increased cell apoptosis in GC-2spd(ts) cells. Wdr17 suppression also arrested the cell cycle at the G1 phase. On the contrary, Wdr17 overexpression significantly promoted cell proliferation and inhibited cell apoptosis in GC-2spd(ts) cells. More cells were enriched at the S stage with a concomitant reduction of cells at the G1 stage. Wdr17 promotes mouse spermatocyte proliferation by advancing cell cycle progression and inhibiting cell apoptosis, indicating its potential role in regulating spermatogenesis in the mouse.

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.

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.

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.

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.

Skeletal myogenesis is a highly ordered process finely regulated by various factors. Long non-coding RNAs play an important regulatory role in myogenesis via multiple mechanisms. In this study, we identified the lncRNA Gm10561, which was upregulated during myogenic differentiation and is highly expressed in skeletal muscle. Knockdown of Gm10561 inhibited the proliferation and differentiation of C2C12 myoblasts in vitro and muscle growth in vivo. Overexpression of Gm10561 promoted the proliferation and differentiation of both C2C12 myoblasts and porcine muscle satellite cells. Notably, lncRNA Gm10561 is localized in the cytoplasm and competitively bound to miR-432, which directly targets MEF2C and E2F3. It was confirmed that lncRNA Gm10561 regulates the proliferation and differentiation of myoblasts by acting as a sponge of miR-432 to modulate MEF2C and E2F3 expression. Thus, the lncRNA-Gm10561-miR-432-MEF2C/E2F3 axis plays an important role in myogenesis.

Abstract Background Different types of skeletal myofibers exhibit distinct physiological and metabolic properties that are associated with meat quality traits in livestock. Alternative splicing (AS) of pre-mRNA can generate multiple transcripts from an individual gene by differential selection of splice sites. N6-methyladenosine (m6A) is the most abundant modification in mRNAs, but its regulation for AS in different muscles remains unknown. Results We characterized AS events and m6A methylation pattern in pig oxidative and glycolytic muscles. A tota1 of 1294 differential AS events were identified, and differentially spliced genes were significantly enriched in processes related to different phenotypes between oxidative and glycolytic muscles. We constructed the regulatory network between splicing factors and corresponding differential AS events and identified NOVA1 and KHDRBS2 as key splicing factors. AS event was enriched in m6A-modified genes, and the methylation level was positively correlated with the number of AS events in genes. The dynamic change in m6A enrichment was associated with 115 differentially skipping exon (SE-DAS) events within 92 genes involving in various processes, including muscle contraction and myofibril assembly. We obtained 23.4% SE-DAS events (27/115) regulated by METTL3-meditaed m6A and experimentally validated the aberrant splicing of ZNF280D, PHE4DIP, and NEB. The inhibition of m6A methyltransferase METTL3 could induce the conversion of oxidative fiber to glycolytic fiber in PSCs. Conclusion Our study suggested that m6A modification could contribute to significant difference in phenotypes between oxidative and glycolytic muscles by mediating the regulation of AS. These findings would provide novel insights into mechanisms underlying muscle fiber conversion.

The quality of oocytes determines their development competence, which will be rapidly lost if the oocytes are not fertilized at the proper time after ovulation. SIRT1, one of the sirtuin family members, has been proven to protect the quality of oocytes during postovulatory oocyte aging. However, evidence of the effect of SIRT1 on the activity of organelles including the mitochondria, the endoplasmic reticulum (ER), the Golgi apparatus, and the lysosomes in postovulatory aging oocyte is lacking. In this study, we investigated the distribution and function of organelles in postovulatory aged oocytes and discovered abnormalities. Luteolin, which is a natural flavonoid contained in vegetables and fruits, is an activator of SIRT1. When the oocytes were treated with luteolin, the abnormal distribution of mitochondria, ER, and Golgi complex were restored during postovulatory oocyte aging. The ER stress protein GRP78 and the lysosome protein LAMP1 increased, while the mitochondrial membrane potential and the Golgi complex protein GOLPH3 decreased in aged oocytes, and these were restored by luteolin treatment. EX-527, an inhibitor of SIRT1, disrupted the luteolin-mediated normal distribution and function of mitochondria, ER, Golgi apparatus, and lysosomes. In conclusion, we demonstrate that luteolin regulates the distribution and function of mitochondria, ER, Golgi apparatus, and lysosomes during postovulatory oocyte aging by activating SIRT1.

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.

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.

The pig bone weight trait holds significant economic importance in southern China. To expedite the selection of the pig bone weight trait in pig breeding, we conducted molecular genetic research on these specific traits. These traits encompass the bone weight of the scapula (SW), front leg bone weight (including humerus and ulna) (FLBW), hind leg bone weight (including femur and tibia) (HLBW), and spine bone weight (SBW). Up until now, the genetic structure related to these traits has not been thoroughly explored, primarily due to challenges associated with obtaining the phenotype data. In this study, we utilized genome-wide association studies (GWAS) to discern single nucleotide polymorphisms (SNPs) and genes associated with four bone weight traits within a population comprising 571 Duroc × (Landrace × Yorkshire) hybrid pigs (DLY). In the analyses, we employed a mixed linear model, and for the correction of multiple tests, both the false discovery rate and Bonferroni methods were utilized. Following functional annotation, candidate genes were identified based on their proximity to the candidate sites and their association with the bone weight traits. This study represents the inaugural application of GWAS for the identification of SNPs associated with individual bone weight in DLY pigs. Our analysis unveiled 26 SNPs and identified 12 promising candidate genes (OPRM1, SLC44A5, WASHC4, NOPCHAP1, RHOT1, GLP1R, TGFB3, PLCB1, TLR4, KCNJ2, ABCA6, and ABCA9) associated with the four bone weight traits. Furthermore, our findings on the genetic mechanisms influencing pig bone weight offer valuable insights as a reference for the genetic enhancement of pig bone traits.

Abstract Background Carcass traits are important in pig breeding programs for improving pork production. Understanding the genetic variants underlies complex phenotypes can help explain trait variation in pigs. In this study, we integrated a weighted single-step genome-wide association study (wssGWAS) and copy number variation (CNV) analyses to map genetic variations and genes associated with loin muscle area (LMA), loin muscle depth (LMD) and lean meat percentage (LMP) in Duroc pigs. Results Firstly, we performed a genome-wide analysis for CNV detection using GeneSeek Porcine SNP50 Bead chip data of 3770 pigs. A total of 11,100 CNVs were detected, which were aggregated by overlapping 695 CNV regions (CNVRs). Next, we investigated CNVs of pigs from the same population by whole-genome resequencing. A genome-wide analysis of 21 pigs revealed 23,856 CNVRs that were further divided into three categories (851 gain, 22,279 loss, and 726 mixed), which covered 190.8 Mb (~ 8.42%) of the pig autosomal genome. Further, the identified CNVRs were used to determine an overall validation rate of 68.5% for the CNV detection accuracy of chip data. CNVR association analyses identified one CNVR associated with LMA, one with LMD and eight with LMP after applying stringent Bonferroni correction. The wssGWAS identified eight, six and five regions explaining more than 1% of the additive genetic variance for LMA, LMD and LMP, respectively. The CNVR analyses and wssGWAS identified five common regions, of which three regions were associated with LMA and two with LMP. Four genes (DOK7, ARAP1, ELMO2 and SLC13A3) were highlighted as promising candidates according to their function. Conclusions We determined an overall validation rate for the CNV detection accuracy of low-density chip data and constructed a genomic CNV map for Duroc pigs using resequencing, thereby proving a value genetic variation resource for pig genome research. Furthermore, our study utilized a composite genetic strategy for complex traits in pigs, which will contribute to the study for elucidating the genetic architecture that may be influenced and regulated by multiple forms of variations.

ABSTRACT Extensive research has explored the causes of embryo losses during early pregnancy by analyzing interaction mechanisms in sows’ uterus, ignoring the importance of the lower reproductive tract in pregnancy development regulation. Despite recent progress in understanding the diversity of vaginal microbes under different physiological states, the dynamic of sows’ vaginal microbiotas during pregnancy and the interaction between vaginal microbes and the host are poorly understood. Here, we performed a comprehensive analysis of sows’ vaginal microbial communities in early pregnancy coupled with overall patterns of vaginal mucosal epithelium gene expression. The vaginal microbiota was analyzed by 16s rRNA or metagenome sequencing, and the vaginal mucosal epithelium transcriptome was analyzed by RNA sequencing, followed by integration of the data layers. We found that the sows’ vaginal microbiotas in early pregnancy develop dynamically, and there is a homeostasis balance of Firmicutes and Proteobacteria. Subsequently, we identified two pregnancy-specific communities, which play diverse roles. The microbes in the vagina stimulate the epithelial cells, while vaginal epithelium changes its structure and functions in response to stimulation. These changes produce specific inflammation responses to promote pregnancy development. Our findings demonstrate the interaction between microbes and host in the sow vagina in early pregnancy to promote pregnancy development, meanwhile providing a reference data set for the study of targeted therapies of microbial homeostasis dysregulation in the female reproductive tract. IMPORTANCE This work sheds light on the dynamics of the sow vaginal microbiotas in early pregnancy and its roles in pregnancy development. Furthermore, this study provides insight into the functional mechanisms of reproductive tract microbes by outlining vaginal microbe-host interactions, which might identify new research and intervention targets for improving pregnancy development by modulating lower reproductive tract microbiota.

Abstract Background Embryo implantation in sows is an important event during pregnancy. During this process, blastocysts undergo dramatic morphologic changes, and the endometrium becomes receptive. Studies have shown that developmental changes associated with the crosstalk between peri-implantation embryos and embryo-uterine are driven by various biomolecules secreted by the endometrium and embryos. In sows, changes in the uterus are also reflected in circulating body fluids and urine. Metabolomics reveals the metabolic state of cells, tissues, and organisms. In this study, we collected urine samples from large white sows during the peri-implantation period. The levels of urinary metabolites at different periods were analyzed using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) analysis techniques. Results A total of 32 samples were collected from 8 sows during the estrus period and at each phase of early pregnancy (9, 12, and 15 days of gestation). A total of 530 metabolites were identified with high confidence in all samples. Compared with samples collected during the estrus phase, 269 differential metabolites were found in samples obtained during early pregnancy. Conclusions The identified metabolites included lipids and lipid-like molecules, organic acids and their derivatives, organic oxygen compounds, organoheterocyclic compounds, benzenoids, among others. Metabolites, such as choline and pregnanediol-3-glucuronide, play important roles in pregnancy in sows and other animals. These results reveal the metabolic changes in urine of sows during early pregnancy phase. The differential urinary metabolites can be used for assessing peri-implantation status in sows. Understanding these metabolic changes may promote the management of pregnant sows through various interventions such as provision of proper nutrition.

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.

Abstract Background Genomic selection using single nucleotide polymorphism (SNP) markers has been widely used for genetic improvement of livestock, but most current methods of genomic selection are based on SNP models. In this study, we investigated the prediction accuracies of haplotype models based on fixed chromosome distances and gene boundaries compared to those of SNP models for genomic prediction of phenotypic values. We also examined the reasons for the successes and failures of haplotype genomic prediction. Methods We analyzed a swine population of 3195 Duroc boars with records on eight traits: body judging score (BJS), teat number (TN), age (AGW), loin muscle area (LMA), loin muscle depth (LMD) and back fat thickness (BF) at 100 kg live weight, and average daily gain (ADG) and feed conversion rate (FCR) from 30 to100 kg live weight. Ten-fold validation was used to evaluate the prediction accuracy of each SNP model and each multi-allelic haplotype model based on 488,124 autosomal SNPs from low-coverage sequencing. Haplotype blocks were defined using fixed chromosome distances or gene boundaries. Results Compared to the best SNP model, the accuracy of predicting phenotypic values using a haplotype model was greater by 7.4% for BJS, 7.1% for AGW, 6.6% for ADG, 4.9% for FCR, 2.7% for LMA, 1.9% for LMD, 1.4% for BF, and 0.3% for TN. The use of gene-based haplotype blocks resulted in the best prediction accuracy for LMA, LMD, and TN. Compared to estimates of SNP additive heritability, estimates of haplotype epistasis heritability were strongly correlated with the increase in prediction accuracy by haplotype models. The increase in prediction accuracy was largest for BJS, AGW, ADG, and FCR, which also had the largest estimates of haplotype epistasis heritability, 24.4% for BJS, 14.3% for AGW, 14.5% for ADG, and 17.7% for FCR. SNP and haplotype heritability profiles across the genome identified several genes with large genetic contributions to phenotypes: NUDT3 for LMA, LMD and BF, VRTN for TN, COL5A2 for BJS, BSND for ADG, and CARTPT for FCR. Conclusions Haplotype prediction models improved the accuracy for genomic prediction of phenotypes in Duroc pigs. For some traits, the best prediction accuracy was obtained with haplotypes defined using gene regions, which provides evidence that functional genomic information can improve the accuracy of haplotype genomic prediction for certain traits.

Body conformation is the most direct production index, which can fully reflect pig growth status and is closely related to critical economic traits. In this study, we conducted a genome-wide association study (GWAS) on body conformation traits in a population of 1518 Duroc × (Landrace × Yorkshire) commercial pigs. These traits included body length (BL), body height (BH), chest circumference (CC), abdominal circumference (AC), and waist circumference (WC). Both the mixed linear model (MLM) and fixed and random model circulating probability unification (FarmCPU) approaches were employed for the analysis. Our findings revealed 60 significant single nucleotide polymorphisms (SNPs) associated with these body conformation traits in the crossbred pig population. Specifically, sixteen SNPs were significantly associated with BL, three SNPs with BH, thirteen SNPs with CC, twelve SNPs with AC, and sixteen SNPs with WC. Moreover, we identified several promising candidate genes located within the genomic regions associated with body conformation traits. These candidate genes include INTS10, KIRREL3, SOX21, BMP2, MAP4K3, SOD3, FAM160B1, ATL2, SPRED2, SEC16B, and RASAL2. Furthermore, our analysis revealed a novel significant quantitative trait locus (QTL) on SSC7 specifically associated with waist circumference, spanning an 84 kb interval. Overall, the identification of these significant SNPs and potential candidate genes in crossbred commercial pigs enhances our understanding of the genetic basis underlying body conformation traits. Additionally, these findings provide valuable genetic resources for pig breeding programs.

The quality of oocytes determines the development potential of an embryo and is dependent on their timely fertilization after ovulation. Postovulatory oocyte aging is an inevitable factor during some assisted reproduction technology procedures, which results in poor fertilization rates and impairs embryo development. We found that fisetin, a bioactive flavonol contained in fruits and vegetables, delayed postovulatory oocyte aging in mice. Fisetin improved the development of aged oocytes after fertilization and inhibited the Sirt1 reduction in aged oocytes. Fisetin increased the GSH level and Sod2 transcription level to inhibit ROS accumulation in aged oocytes. Meanwhile, fisetin attenuated aging-induced spindle abnormalities, mitochondrial dysfunction, and apoptosis. At the molecular level, fisetin decreased aging-induced aberrant expression of H3K9me3. In addition, fisetin increased the expression levels of the mitochondrial transcription factor Tfam and the mitochondrial genes Co2 and Atp8 by upregulating Sirt1 in aged oocytes. Finally, inhibition of Sirt1 reversed the anti-aging effects of fisetin. Taken together, fisetin delayed postovulatory oocyte aging by upregulating Sirt1.

The number of teats is a crucial reproductive trait with significant economic implications on maternal capacity and litter size. Consequently, improving this trait is essential to facilitate genetic selection for increased litter size. In this study, we performed a genome-wide association study (GWAS) of the number of teats in a three-way crossbred commercial Duroc × (Landrace × Yorkshire) (DLY) pig population comprising 1518 animals genotyped with the 50K BeadChip. Our analysis identified crucial quantitative trait loci (QTL) for the number of teats, containing the ABCD4 and VRTN genes on porcine chromosome 7. Our results establish SNP variants of ABCD4 and VRTN as new molecular markers for improving the number of teats in DLY pigs. Furthermore, the most significant noteworthy single nucleotide polymorphism (SNP) (7_97568284) was identified within the ABCD4 gene, exhibiting a significant association with the total teat number traits. This SNP accounted for a substantial proportion of the genetic variance, explaining 6.64% of the observed variation. These findings reveal a novel gene on SSC7 for the number of teats and provide a deeper understanding of the genetic mechanisms underlying reproductive traits.

Abstract Background Improving feed efficiency is economically and environmentally beneficial in the pig industry. A deeper understanding of feed efficiency is essential on many levels for its highly complex nature. The aim of this project is to explore the relationship between fecal metabolites and feed efficiency-related traits, thereby identifying metabolites that may assist in the screening of the feed efficiency of pigs. Results We performed fecal metabolomics analysis on 50 individuals selected from 225 Duroc x (Landrace x Yorkshire) (DLY) commercial pigs, 25 with an extremely high feed efficiency and 25 with an extremely low feed efficiency. A total of 6749 and 5644 m/z features were detected in positive and negative ionization modes by liquid chromatography-mass spectrometry (LC/MS). Regrettably, the PCA could not classify the the samples accurately. To improve the classification, OPLS-DA was introduced. However, the predictive ability of the OPLS-DA model did not perform well. Then, through weighted coexpression network analysis (WGCNA), we found that one module in each positive and negative mode was related to residual feed intake (RFI), and six and three metabolites were further identified. The nine metabolites were found to be involved in multiple metabolic pathways, including lipid metabolism (primary bile acid synthesis, linoleic acid metabolism), vitamin D, glucose metabolism, and others. Then, Lasso regression analysis was used to evaluate the importance of nine metabolites obtained by the annotation process. Conclusions Altogether, this study provides new insights for the subsequent evaluation of commercial pig feed efficiency through small molecule metabolites, but also provide a reference for the development of new feed additives.

The conserved shelterin complex caps chromosome ends to protect telomeres and regulate telomere replication. In fission yeast Schizosaccharomyces pombe, shelterin consists of telomeric single- and double-stranded DNA-binding modules Pot1-Tpz1 and Taz1-Rap1 connected by Poz1, and a specific component Ccq1. While individual structures of the two DNA-binding OB folds of Pot1 (Pot1OB1-GGTTAC and Pot1OB2-GGTTACGGT) are available, structural insight into recognition of telomeric repeats with spacers by the complete DNA-binding domain (Pot1DBD) remains an open question. Moreover, structural information about the Tpz1-Ccq1 interaction requires to be revealed for understanding how the specific component Ccq1 of S. pombe shelterin is recruited to telomeres to function as an interacting hub. Here, we report the crystal structures of Pot1DBD-single-stranded-DNA, Pot1372-555-Tpz1185-212 and Tpz1425-470-Ccq1123-439 complexes and propose an integrated model depicting the assembly mechanism of the shelterin complex at telomeres. The structure of Pot1DBD-DNA unveils how Pot1 recognizes S. pombe degenerate telomeric sequences. Our analyses of Tpz1-Ccq1 reveal structural basis for the essential role of the Tpz1-Ccq1 interaction in telomere recruitment of Ccq1 that is required for telomere maintenance and telomeric heterochromatin formation. Overall, our findings provide valuable structural information regarding interactions within fission yeast shelterin complex at 3' ss telomeric overhang.

Abstract Background In the process of pig breeding, the average daily gain (ADG), days to 100 kg (AGE), and backfat thickness (BFT) are directly related to growth rate and fatness. However, the genetic mechanisms involved are not well understood. Copy number variation (CNV), an important source of genetic diversity, can affect a variety of complex traits and diseases and has gradually been thrust into the limelight. In this study, we reported the genome-wide CNVs of Duroc pigs using SNP genotyping data from 6627 animals. We also performed a copy number variation region (CNVR)-based genome-wide association studies (GWAS) for growth and fatness traits in two Duroc populations. Results Our study identified 953 nonredundant CNVRs in U.S. and Canadian Duroc pigs, covering 246.89 Mb (~ 10.90%) of the pig autosomal genome. Of these, 802 CNVRs were in U.S. Duroc pigs with 499 CNVRs were in Canadian Duroc pigs, indicating 348 CNVRs were shared by the two populations. Experimentally, 77.8% of nine randomly selected CNVRs were validated through quantitative PCR (qPCR). We also identified 35 CNVRs with significant association with growth and fatness traits using CNVR-based GWAS. Ten of these CNVRs were associated with both ADG and AGE traits in U.S. Duroc pigs. Notably, four CNVRs showed significant associations with ADG, AGE, and BFT, indicating that these CNVRs may play a pleiotropic role in regulating pig growth and fat deposition. In Canadian Duroc pigs, nine CNVRs were significantly associated with both ADG and AGE traits. Further bioinformatic analysis identified a subset of potential candidate genes, including PDGFA, GPER1, PNPLA2 and BSCL2. Conclusions The present study provides a necessary supplement to the CNV map of the Duroc genome through large-scale population genotyping. In addition, the CNVR-based GWAS results provide a meaningful way to elucidate the genetic mechanisms underlying complex traits. The identified CNVRs can be used as molecular markers for genetic improvement in the molecular-guided breeding of modern commercial pigs.

Abstract Feed efficiency (FE) is an important economic indicator in pig production. Improving the FE of commercial pigs is an important strategy for minimizing pig production costs and providing sustainability to the pig industry. In this study, nontargeted LC–MS metabolomics was performed on the contents of the three intestine segments (ileum, cecum and colon) of high-FE and low-FE pigs to explore the effects of small-molecule metabolites in pig intestine on pig FE. A total of 225 Duroc × (Landrace × Yorkshire) pigs in the 30–100 kg stage were sorted based on FE, and 20 pigs with extreme phenotypes were selected, with 10 in each group. A total of 749 metabolites were identified, of which 15, 38 and 11 differed between high-FE and low-FE pigs in ileum, cecum and colon, respectively. These candidate biomarkers mainly comprised lipids and organic acids, which could partially explain the FE difference between the two groups. Among the identified differential metabolites, the lipids are mainly involved in combatting inflammation and oxidation in the ileum and cecum and in bile acid metabolism and vitamin D absorption in the cecum. A difference in organic acids was mainly observed in the hindgut, which is involved in the metabolism of amino acids and fatty acids. This comprehensive study provides new insight into the biochemical mechanisms associated with pig FE.

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.

Technologies that can preselect offspring gender hold great promise for improving farm animal productivity and preventing human sex-related hereditary diseases. The maternal Rlim allele is required for imprinted X-chromosome inactivation, which is essential for the normal development of female mouse embryos. In this study, we inactivated the maternal Rlim allele in embryos by crossing a male transgenic mouse line carrying an X-linked CMV-Cre transgene with a female line carrying a loxP-flanked Rlim gene. Knockout of the maternal Rlim gene in embryos resulted in a male-biased sex ratio skew in the offspring. However, it also reduced litter size, and this effect was not compensated for by superovulation in the mother mice. In addition, we showed that siRNA-mediated knockdown of Rlim in mouse embryos leads to the birth of male-only progenies. This study provides a new promising method for male-biased sex selection, which may help to improve the productivity in livestock and prevent sex-associated hereditary diseases in humans.

Abstract Background Average daily gain (ADG) and lean meat percentage (LMP) are the main production performance indicators of pigs. Nevertheless, the genetic architecture of ADG and LMP is still elusive. Here, we conducted genome-wide association studies (GWAS) and meta-analysis for ADG and LMP in 3770 American and 2090 Canadian Duroc pigs. Results In the American Duroc pigs, one novel pleiotropic quantitative trait locus (QTL) on Sus scrofa chromosome 1 (SSC1) was identified to be associated with ADG and LMP, which spans 2.53 Mb (from 159.66 to 162.19 Mb). In the Canadian Duroc pigs, two novel QTLs on SSC1 were detected for LMP, which were situated in 3.86 Mb (from 157.99 to 161.85 Mb) and 555 kb (from 37.63 to 38.19 Mb) regions. The meta-analysis identified ten and 20 additional SNPs for ADG and LMP, respectively. Finally, four genes (PHLPP1, STC1, DYRK1B, and PIK3C2A) were detected to be associated with ADG and/or LMP. Further bioinformatics analysis showed that the candidate genes for ADG are mainly involved in bone growth and development, whereas the candidate genes for LMP mainly participated in adipose tissue and muscle tissue growth and development. Conclusions We performed GWAS and meta-analysis for ADG and LMP based on a large sample size consisting of two Duroc pig populations. One pleiotropic QTL that shared a 2.19 Mb haplotype block from 159.66 to 161.85 Mb on SSC1 was found to affect ADG and LMP in the two Duroc pig populations. Furthermore, the combination of single-population and meta-analysis of GWAS improved the efficiency of detecting additional SNPs for the analyzed traits. Our results provide new insights into the genetic architecture of ADG and LMP traits in pigs. Moreover, some significant SNPs associated with ADG and/or LMP in this study may be useful for marker-assisted selection in pig breeding.

Runs of homozygosity (ROH) are widely used to estimate genomic inbreeding, which is linked to inbreeding depression on phenotypes. However, the adverse effects of specific homozygous regions on phenotypic characteristics are rarely studied in livestock. In this study, the 50 K SNP data of 3,770 S21 Duroc (American origin) and 2,096 S22 Duroc (Canadian origin) pigs were used to investigate the harmful ROH regions on five economic traits. The results showed that the two Duroc lines had different numbers and distributions of unfavorable ROHs, which may be related to the different selection directions and intensities between the two lines. A total of 114 and 58 ROH segments were found with significant adverse effects on the economic traits of S21 and S22 pigs, respectively. Serval pleiotropic ROHs were detected to reduce two or multiple phenotypic performances in two Duroc populations. Candidate genes in these shared regions were mainly related to growth, fertility, immunity, and fat deposition. We also observed that some ROH genotypes may cause opposite effects on different traits. This study not only enhances our understanding of the adverse effects of ROH on phenotypes, but also indicates that ROH information could be incorporated into breeding programs to estimate and control the detrimental effects of homozygous regions.

Runs of homozygosity (ROH) are widely used to investigate genetic diversity, demographic history, and positive selection signatures of livestock. Commercial breeds provide excellent materials to reveal the landscape of ROH shaped during the intense selection process. Here, we used the GeneSeek Porcine 50K single-nucleotide polymorphism (SNP) Chip data of 3,770 American Duroc (AD) and 2,096 Canadian Duroc (CD) pigs to analyze the genome-wide ROH. First, we showed that AD had a moderate genetic differentiation with CD pigs, and AD had more abundant genetic diversity and significantly lower level of inbreeding than CD pigs. In addition, sows had larger levels of homozygosity than boars in AD pigs. These differences may be caused by differences in the selective intensity. Next, ROH hotspots revealed that many candidate genes are putatively under selection for growth, sperm, and muscle development in two lines. Population-specific ROHs inferred that AD pigs may have a special selection for female reproduction, while CD pigs may have a special selection for immunity. Moreover, in the overlapping ROH hotspots of two Duroc populations, we observed a missense mutation (rs81216249) located in the growth and fat deposition-related supergene (ARSB-DMGDH-BHMT) region. The derived allele of this variant originated from European pigs and was nearly fixed in Duroc pigs. Further selective sweep and association analyses indicated that this supergene was subjected to strong selection and probably contributed to the improvement of body weight and length in Duroc pigs. These findings will enhance our understanding of ROH patterns in different Duroc lines and provide promising trait-related genes and a functional-altering marker that can be used for genetic improvement of pigs.

Abstract Background More teats are necessary for sows to nurse larger litters to provide immunity and nutrient for piglets prior to weaning. Previous studies have reported the strong effect of an insertion mutation in the Vertebrae Development Associated (VRTN) gene on Sus scrofa chromosome 7 (SSC7) that increased the number of thoracic vertebrae and teat number in pigs. We used genome-wide association studies (GWAS) to map genetic markers and genes associated with teat number in two Duroc pig populations with different genetic backgrounds. A single marker method and several multi-locus methods were utilized. A meta-analysis that combined the effects and P-values of 34,681 single nucleotide polymorphisms (SNPs) that were common in the results of single marker GWAS of American and Canadian Duroc pigs was conducted. We also performed association tests between the VRTN insertion and teat number in the same populations. Results A total of 97 SNPs were found to be associated with teat number. Among these, six, eight and seven SNPs were consistently detected with two, three and four multi-locus methods, respectively. Seven SNPs were concordantly identified between single marker and multi-locus methods. Moreover, 26 SNPs were newly found by multi-locus methods to be associated with teat number. Notably, we detected one consistent quantitative trait locus (QTL) on SSC7 for teat number using single-locus and meta-analysis of GWAS and the top SNP (rs692640845) explained 8.68% phenotypic variance of teat number in the Canadian Duroc pigs. The associations between the VRTN insertion and teat number in two Duroc pig populations were substantially weaker. Further analysis revealed that the effect of VRTN on teat number may be mediated by its LD with the true causal mutation. Conclusions Our study suggested that VRTN insertion may not be a strong or the only candidate causal mutation for the QTL on SSC7 for teat number in the analyzed Duroc pig populations. The combination of single-locus and multi-locus GWAS detected additional SNPs that were absent using only one model. The identified SNPs will be useful for the genetic improvement of teat number in pigs by assigning higher weights to associated SNPs in genomic selection.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is a precise genome manipulating tool that can produce targeted gene mutations in various cells and organisms. Although CRISPR/Cas9 can efficiently generate gene knockout, the gene knock-in (KI) efficiency mediated by homology-directed repair remains low, especially for large fragment integration. In this study, we established an efficient method for the CRISPR/Cas9-mediated integration of large transgene cassette, which carries salivary gland-expressed multiple digestion enzymes (≈ 20 kbp) in CEP112 locus in pig fetal fibroblasts (PFFs). Our results showed that using an optimal homology donor with a short and a long arm yielded the best CRISPR/Cas9-mediated KI efficiency in CEP112 locus, and the targeting efficiency in CEP112 locus was higher than in ROSA26 locus. The CEP112 KI cell lines were used as nuclear donors for somatic cell nuclear transfer to create genetically modified pigs. We found that KI pig (705) successfully expressed three microbial enzymes (β-glucanase, xylanase, and phytase) in salivary gland. This finding suggested that the CEP112 locus supports exogenous gene expression by a tissue-specific promoter. In summary, we successfully targeted CEP112 locus in pigs by using our optimal homology arm system and established a modified pig model for foreign digestion enzyme expression in the saliva.

NDH-1 is a key component of the cyclic-electron-transfer around photosystem I pathway, an antioxidant mechanism for efficient photosynthesis. Here, authors report a cryo-EM structure of the ferredoxin (Fd)-NDH-1L complex from the cyanobacterium Thermosynechococcus elongatus.

It has been proven that the random regression model has a great advantage over the repeatability model in longitudinal data analysis. At present, the random regression model has been used as a standard analysis method in longitudinal data analysis. The aim of this study was to estimate the variance components and heritability of semen traits over the reproductive lifetime of boars. The study data, including 124,941 records from 3,366 boars, were collected from seven boar AI centers in South China between 2010 and 2019. To evaluate alternative models, we compared different polynomial orders of fixed, additive, and permanent environment effects in total 216 models using Bayesian Information Criterions. The result indicated that the best model always has higher-order polynomials of permanent environment effect and lower-order polynomials of fixed effect and additive effect regression. In Landrace boars, the heritabilities ranged from 0.18 to 0.28, 0.06 to 0.43, 0.03 to 0.14, and 0.05 to 0.24 for semen volume, sperm motility, sperm concentration, and abnormal sperm percentage, respectively. In Large White boars, the heritabilities ranged from 0.20 to 0.26, 0.07 to 0.15, 0.10 to 0.23, and 0.06 to 0.34 for semen volume, sperm motility, sperm concentration, and abnormal sperm percentage, respectively.

Heterozygosity can effectively reflect the diverse models of population structure and demographic history. However, the genomic distribution of heterozygotes and the correlation between regions of heterozygosity (runs of heterozygosity, ROHet) and phenotypes are largely understudied in livestock. The objective of this study was to identify ROHet in the Duroc pig genome, and investigate the relationships between ROHet and eight important economic traits. Here, we genotyped 3,770 American Duroc (S21) and 2,096 Canadian Duroc (S22) pigs using 50 K single nucleotide polymorphism array to analyze heterozygosity. A total of 145,010 and 84,396 ROHets were characterized for S21 and S22 populations, respectively. ROHet segments were mostly enriched in 1–2 Mb length classification (75.48% in S21 and 72.25% in S22). The average genome length covered by ROHet was 66.53 ± 12.20 Mb in S21 and 73.32 ± 13.77 Mb in S22 pigs. Additionally, we detected 20 and 13 ROHet islands in S21 and S22 pigs. Genes in these genomic regions were mainly involved in the biological processes of immunity and reproduction. Finally, the genome-wide ROHet-phenotypes association analysis revealed that 130 ROHets of S21 and 84 ROHets of S22 were significantly associated with eight economic traits. Among the candidate genes in the significant ROHet regions, 16 genes related to growth, metabolism, and meat quality were considered as candidate genes for important economic traits of pigs. This work preliminarily explores the effect of heterozygosity-rich regions in the pig genome on production performance and provides new insights for subsequent research on pig genetic improvement.

Chest circumference (CC), abdominal circumference (AC), and waist circumference (WC) are regarded as important indicators for improving economic traits because they can reflect the growth and physiological status in pigs. However, the genetic architecture of CC, AC, and WC is still elusive. Here, we performed single-trait and multi-trait genome-wide association studies (GWASs) for CC, AC, and WC in 2,206 American origin Duroc (AOD) and 2,082 Canadian origin Duroc (COD) pigs. As a result, one novel quantitative trait locus (QTL) on Sus scrofa chromosome (SSC) one was associated with CC and AC in COD pigs, which spans 6.92 Mb (from 170.06 to 176.98 Mb). Moreover, multi-trait GWAS identified 21 significant SNPs associated with the three conformation traits, indicating the multi-trait GWAS is a powerful statistical approach that uncovers pleiotropic locus. Finally, the three candidate genes (ITGA11, TLE3, and GALC) were selected that may play a role in the conformation traits. Further bioinformatics analysis indicated that the candidate genes for the three conformation traits mainly participated in sphingolipid metabolism and lysosome pathways. For all we know, this study was the first GWAS for WC in pigs. In general, our findings further reveal the genetic architecture of CC, AC, and WC, which may offer a useful reference for improving the conformation traits in pigs.