Genomic evidence for human-mediated introgressive hybridization and selection in the developed breed.

BACKGROUND: The pig (Sus Scrofa) is one of the oldest domesticated livestock species that has undergone extensive improvement through modern breeding. European breeds have advantages in lean meat development and highly-productive body type, whereas Asian breeds possess extraordinary fat deposition and reproductive performance. Consequently, Eurasian breeds have been extensively used to develop modern commercial breeds for fast-growing and high prolificacy. However, limited by the sequencing technology, the genome architecture of some nascent developed breeds and the human-mediated impact on their genomes are still unknown. RESULTS: Through whole-genome analysis of 178 individuals from an Asian locally developed pig breed, Beijing Black pig, and its two ancestors from two different continents, we found the pervasive inconsistent gene trees and species trees across the genome of Beijing Black pig, which suggests its introgressive hybrid origin. Interestingly, we discovered that this developed breed has more genetic relationships with European pigs and an unexpected introgression from Asian pigs to this breed, which indicated that human-mediated introgression could form the porcine genome architecture in a completely different type compared to native introgression. We identified 554 genomic regions occupied 63.30 Mb with signals of introgression from the Asian ancestry to Beijing Black pig, and the genes in these regions enriched in pathways associated with meat quality, fertility, and disease-resistant. Additionally, a proportion of 7.77% of genomic regions were recognized as regions that have been under selection. Moreover, combined with the results of a genome-wide association study for meat quality traits in the 1537 Beijing Black pig population, two important candidate genes related to meat quality traits were identified. DNAJC6 is related to intramuscular fat content and fat deposition, and RUFY4 is related to meat pH and tenderness. CONCLUSIONS: Our research provides insight for analyzing the origins of nascent developed breeds and genome-wide selection remaining in the developed breeds mediated by humans during modern breeding.

Genetic introgression from commercial European pigs to the indigenous Chinese Lijiang breed and associated changes in phenotypes.

BACKGROUND: Gene flow is crucial for enhancing economic traits of livestock. In China, breeders have used hybridization strategies for decades to improve livestock performance. Here, we performed whole-genome sequencing of a native Chinese Lijiang pig (LJP) breed. By integrating previously published data, we explored the genetic structure and introgression of genetic components from commercial European pigs (EP) into the LJP, and examined the impact of this introgression on phenotypic traits. RESULTS: Our analysis revealed significant introgression of EP breeds into the LJP and other domestic pig breeds in China. Using a haplotype-based approach, we quantified introgression levels and compared EP to LJP and other Chinese domestic pigs. The results show that EP introgression is widely prevalent in Chinese domestic pigs, although there are significant differences between breeds. We propose that LJP could potentially act as a mediator for the transmission of EP haplotypes. We also examined the correlation between EP introgression and the number of thoracic vertebrae in LJP and identified VRTN and STUM as candidate genes for this trait. CONCLUSIONS: Our study provides evidence of introgressed European haplotypes in the LJP breed and describes the potential role of EP introgression on phenotypic changes of this indigenous breed.

Unveiling the Genetic Mechanism of Meat Color in Pigs through GWAS, Multi-Tissue, and Single-Cell Transcriptome Signatures Exploration.

Meat color traits directly influence consumer acceptability and purchasing decisions. Nevertheless, there is a paucity of comprehensive investigation into the genetic mechanisms underlying meat color traits in pigs. Utilizing genome-wide association studies (GWAS) on five meat color traits and the detection of selection signatures in pig breeds exhibiting distinct meat color characteristics, we identified a promising candidate SNP, 6_69103754, exhibiting varying allele frequencies among pigs with different meat color characteristics. This SNP has the potential to affect the redness and chroma index values of pork. Moreover, transcriptome-wide association studies (TWAS) analysis revealed the expression of candidate genes associated with meat color traits in specific tissues. Notably, the largest number of candidate genes were observed from transcripts derived from adipose, liver, lung, spleen tissues, and macrophage cell type, indicating their crucial role in meat color development. Several shared genes associated with redness, yellowness, and chroma indices traits were identified, including RINL in adipose tissue, ENSSSCG00000034844 and ITIH1 in liver tissue, TPX2 and MFAP2 in lung tissue, and ZBTB17, FAM131C, KIFC3, NTPCR, and ENGSSSCG00000045605 in spleen tissue. Furthermore, single-cell enrichment analysis revealed a significant association between the immune system and meat color. This finding underscores the significance of the immune system associated with meat color. Overall, our study provides a comprehensive analysis of the genetic mechanisms underlying meat color traits, offering valuable insights for future breeding efforts aimed at improving meat quality.

Comparative transcriptome analysis of longissimus dorsi muscle reveal potential genes affecting meat trait in Chinese indigenous Xiang pig.

In this study, we compared the transcriptome of longissimus dorsi muscle between Guizhou Xiang pigs (XP) and Western commercial Large White pigs (LW), which show diffirent meat quality between them. In terms of meat quality traits, the pH 45 min, color score, backfat thickness, and intramuscular fat (IMF) content were higher in Xiang pigs than in Large White pigs (P < 0.01), while the drip loss, lean meat percentage, shear force, and longissimus dorsi muscle area of Xiang pigs were lower than that of Large White pigs (P < 0.01). Nutrients such as monounsaturated fatty acid (MUFA), total amino acids (TAA), delicious amino acids (DAA) and essential amino acids (EAA) in Xiang pigs were higher than that in Large White pigs, and the proportion of polyunsaturated fatty acid (PUFA) of Xiang pigs was significantly lower than Large White pigs (P < 0.01). Transcriptome analysis identified 163 up-regulated genes and 88 genes down-regulated in Xiang pigs longissimus dorsi muscle. Combined with the correlation analysis and quantitative trait locis (QTLs) affecting meat quality, a total of 227 DEGs were screened to be significantly associated with meat quality values. Enrichment analysis indicated that numerous members of genes were gathered in muscle development, adipogenesis, amino acid metabolism, fatty acid metabolism and synthesis. Of those, 29 genes were identified to be hub genes that might be related with the meat quality of Xiang pig, such as MYOD1, ACTB, ASNS, FOXO1, ARG2, SLC2A4, PLIN2, and SCD. Thus, we screened and identified the potential functional genes for the formation of meat quality in Xiang pigs, which provides a corresponding theoretical basis for the study of the molecular regulatory mechanism of pork quality and the improvement of pork quality.

Estimating genetic parameters of digital behavior traits and their relationship with production traits in purebred pigs.

BACKGROUND: With the introduction of digital phenotyping and high-throughput data, traits that were previously difficult or impossible to measure directly have become easily accessible, offering the opportunity to enhance the efficiency and rate of genetic gain in animal production. It is of interest to assess how behavioral traits are indirectly related to the production traits during the performance testing period. The aim of this study was to assess the quality of behavior data extracted from day-wise video recordings and estimate the genetic parameters of behavior traits and their phenotypic and genetic correlations with production traits in pigs. Behavior was recorded for 70 days after on-test at about 10 weeks of age and ended at off-test for 2008 female purebred pigs, totaling 119,812 day-wise records. Behavior traits included time spent eating, drinking, laterally lying, sternally lying, sitting, standing, and meters of distance traveled. A quality control procedure was created for algorithm training and adjustment, standardizing recording hours, removing culled animals, and filtering unrealistic records. RESULTS: Production traits included average daily gain (ADG), back fat thickness (BF), and loin depth (LD). Single-trait linear models were used to estimate heritabilities of the behavior traits and two-trait linear models were used to estimate genetic correlations between behavior and production traits. The results indicated that all behavior traits are heritable, with heritability estimates ranging from 0.19 to 0.57, and showed low-to-moderate phenotypic and genetic correlations with production traits. Two-trait linear models were also used to compare traits at different intervals of the recording period. To analyze the redundancies in behavior data during the recording period, the averages of various recording time intervals for the behavior and production traits were compared. Overall, the average of the 55- to 68-day recording interval had the strongest phenotypic and genetic correlation estimates with the production traits. CONCLUSIONS: Digital phenotyping is a new and low-cost method to record behavior phenotypes, but thorough data cleaning procedures are needed. Evaluating behavioral traits at different time intervals offers a deeper insight into their changes throughout the growth periods and their relationship with production traits, which may be recorded at a less frequent basis.

Constructing eRNA-mediated gene regulatory networks to explore the genetic basis of muscle and fat-relevant traits in pigs.

BACKGROUND: Enhancer RNAs (eRNAs) play a crucial role in transcriptional regulation. While significant progress has been made in understanding epigenetic regulation mediated by eRNAs, research on the construction of eRNA-mediated gene regulatory networks (eGRN) and the identification of critical network components that influence complex traits is lacking. RESULTS: Here, employing the pig as a model, we conducted a comprehensive study using H3K27ac histone ChIP-seq and RNA-seq data to construct eRNA expression profiles from multiple tissues of two distinct pig breeds, namely Enshi Black (ES) and Duroc. In addition to revealing the regulatory landscape of eRNAs at the tissue level, we developed an innovative network construction and refinement method by integrating RNA-seq, ChIP-seq, genome-wide association study (GWAS) signals and enhancer-modulating effects of single nucleotide polymorphisms (SNPs) measured by self-transcribing active regulatory region sequencing (STARR-seq) experiments. Using this approach, we unraveled eGRN that significantly influence the growth and development of muscle and fat tissues, and identified several novel genes that affect adipocyte differentiation in a cell line model. CONCLUSIONS: Our work not only provides novel insights into the genetic basis of economic pig traits, but also offers a generalizable approach to elucidate the eRNA-mediated transcriptional regulation underlying a wide spectrum of complex traits for diverse organisms.

Circular RNAs Mediate the Effects of Dietary Tryptophan on the Transformation of Muscle Fiber Types in Weaned Piglets.

The nutritional composition of the diet significantly impacts the overall growth and development of weaned piglets. The current study aimed to explore the effects and underlying mechanisms of dietary tryptophan consumption on muscle fiber type transformation during the weaning period. Thirty weaned piglets with an average body weight of 6.12 ± 0.16 kg were randomly divided into control (CON, 0.14% Trp diet) and high Trp (HT, 0.35% Trp) groups and maintained on the respective diet for 28 days. The HT group of weaned piglets exhibited highly significant improvements in growth performance and an increased proportion of fast muscle fibers. Transcriptome sequencing revealed the potential contribution of differentially expressed circular RNAs toward the transformation of myofiber types in piglets and toward the regulation of expression of related genes by targeting the microRNAs, miR-34c and miR-182, to further regulate myofiber transformation. In addition, 145 DE circRNAs were identified as potentially protein-encoding, with the encoded proteins associated with a myofiber type transformation. In conclusion, the current study greatly advances and refines our current understanding of the regulatory networks associated with piglet muscle development and myofiber type transformation and also contributes to the optimization of piglet diet formulation.

Comparison of ICSI, IVF, and in vivo derived embryos to produce CRISPR-Cas9 gene-edited pigs for xenotransplantation.

Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and ß4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo-derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and ß4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo-derived embryos showing efficient multiple gene editing for GGTA1 and GHR.

Transcriptome Analysis of miRNA and mRNA in Porcine Skeletal Muscle following Glaesserella parasuis Challenge.

Glaesserella parasuis (G. parasuis) causes systemic infection in pigs, but its effects on skeletal muscle and underlying mechanisms are poorly understood. We investigated G. parasuis infection in colostrum-deprived piglets, observing decreased daily weight gain and upregulation of inflammatory factors in skeletal muscle. Muscle fiber area and diameter were significantly reduced in the treated group (n = 3) compared to the control group (n = 3), accompanied by increased expression of FOXO1, FBXO32, TRIM63, CTSL, and BNIP3. Based on mRNA and microRNA (miRNA) sequencing, we identified 1642 differentially expressed (DE) mRNAs and 19 known DE miRNAs in skeletal muscle tissues between the two groups. We predicted target genes with opposite expression patterns to the 19 miRNAs and found significant enrichment and activation of the FoxO signaling pathway. We found that the upregulated core effectors FOXO1 and FOXO4 were targeted by downregulated ssc-miR-486, ssc-miR-370, ssc-miR-615, and ssc-miR-224. Further investigation showed that their downstream upregulated genes involved in protein degradation were also targeted by the downregulated ssc-miR-370, ssc-miR-615, ssc-miR-194a-5p, and ssc-miR-194b-5p. These findings suggest that G. parasuis infection causes skeletal muscle atrophy in piglets through accelerated protein degradation mediated by the "miRNAs-FOXO1/4" axis, while further research is necessary to validate the regulatory relationships. Our results provide new insights into the understanding of systemic inflammation growth mechanisms caused by G. parasuis and the role of miRNAs in bacterial infection pathogenesis.

Genome-Wide Association Analysis of Semen Characteristics in Piétrain Boars.

Since artificial insemination is common practice in pig breeding, the quality and persistence of the semen are decisive for the usability of individual boars. In the current study, genome-wide association analyses were performed to investigate the genetic variability underlying phenotypic variations in semen characteristics. These traits comprise sperm morphology and sperm motility under different temporal and thermal storage conditions, in addition to standard semen quality parameters. Two consecutive samples of the fourth and fifth ejaculates from the same boar were comprehensively analyzed in a genotyped Piétrain boar population. A total of 13 genomic regions on different chromosomes were identified that contain single-nucleotide polymorphisms significantly associated with these traits. Subsequent analysis of the genomic regions revealed candidate genes described to be involved in spermatogenesis, such as FOXL3, GPER1, PDGFA, PRKAR1B, SNRK, SUN1, and TSPO, and sperm motility, including ARRDC4, CEP78, DNAAF5, and GPER1. Some of these genes were also associated with male fertility or infertility in mammals (e.g., CEP78, GPER1). The analyses based on these laboriously determined and valuable phenotypes contribute to a better understanding of the genetic background of male fertility traits in pigs and could prospectively contribute to the improvement of sperm quality through breeding approaches.

Omics-based construction of regulatory variants can be applied to help decipher pig liver-related traits.

Genetic variants can influence complex traits by altering gene expression through changes to regulatory elements. However, the genetic variants that affect the activity of regulatory elements in pigs are largely unknown, and the extent to which these variants influence gene expression and contribute to the understanding of complex phenotypes remains unclear. Here, we annotate 90,991 high-quality regulatory elements using acetylation of histone H3 on lysine 27 (H3K27ac) ChIP-seq of 292 pig livers. Combined with genome resequencing and RNA-seq data, we identify 28,425 H3K27ac quantitative trait loci (acQTLs) and 12,250 expression quantitative trait loci (eQTLs). Through the allelic imbalance analysis, we validate two causative acQTL variants in independent datasets. We observe substantial sharing of genetic controls between gene expression and H3K27ac, particularly within promoters. We infer that 46% of H3K27ac exhibit a concomitant rather than causative relationship with gene expression. By integrating GWAS, eQTLs, acQTLs, and transcription factor binding prediction, we further demonstrate their application, through metabolites dulcitol, phosphatidylcholine (PC) (16:0/16:0) and published phenotypes, in identifying likely causal variants and genes, and discovering sub-threshold GWAS loci. We provide insight into the relationship between regulatory elements and gene expression, and the genetic foundation for dissecting the molecular mechanism of phenotypes.

Haplotype-resolved 3D chromatin architecture of the hybrid pig.

In diploid mammals, allele-specific three-dimensional (3D) genome architecture may lead to imbalanced gene expression. Through ultradeep in situ Hi-C sequencing of three representative somatic tissues (liver, skeletal muscle, and brain) from hybrid pigs generated by reciprocal crosses of phenotypically and physiologically divergent Berkshire and Tibetan pigs, we uncover extensive chromatin reorganization between homologous chromosomes across multiple scales. Haplotype-based interrogation of multi-omic data revealed the tissue dependence of 3D chromatin conformation, suggesting that parent-of-origin-specific conformation may drive gene imprinting. We quantify the effects of genetic variations and histone modifications on allelic differences of long-range promoter-enhancer contacts, which likely contribute to the phenotypic differences between the parental pig breeds. We also observe the fine structure of somatically paired homologous chromosomes in the pig genome, which has a functional implication genome-wide. This work illustrates how allele-specific chromatin architecture facilitates concomitant shifts in allele-biased gene expression, as well as the possible consequential phenotypic changes in mammals.

Impact of media supplements FGF2, LIF and IGF1 on the genome activity of porcine embryos produced in vitro.

In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.

Transcript abundance of hepatic drug-metabolizing enzymes in two dog breeds compared with 14 species including humans.

Drug-metabolizing enzymes are important in drug development and therapy, but have not been fully identified and characterized in many species, lines, and breeds. Liver transcriptomic data were analyzed for phase I cytochromes P450, flavin-containing monooxygenases, and carboxylesterases and phase II UDP-glucuronosyltransferases, sulfotransferases, and glutathione S-transferases. Comparisons with a variety of species (humans, rhesus macaques, African green monkeys, baboons, common marmosets, cattle, sheep, pigs, cats, dogs, rabbits, tree shrews, rats, mice, and chickens) revealed both general similarities and differences in the transcript abundances of drug-metabolizing enzymes. Similarly, Beagle and Shiba dogs were examined by next-generation sequencing (RNA-seq). Consequently, no substantial differences in transcript abundance were noted in different breeds of pigs and dogs and in different lines of mice and rats. Therefore, the expression profiles of hepatic drug-metabolizing enzyme transcripts appear to be similar in Shiba and Beagle dogs and pig breeds and the rat and mouse lines analyzed, although some differences were found in other species.

Identification and characterization of unknown disturbances in a structured population using high-throughput phenotyping data and measurement of robustness: application to growing pigs.

Improving the robustness of animals has become a priority in breeding due to climate change, new societal demands, and the agroecological transition. Components of animal robustness can be extracted from the analysis of the adaptive response of an animal to disturbance using longitudinal data. Nonetheless, this response is a function of animal robustness as well as of disturbance characteristics (intensity and duration). To correctly assess an animal's robustness potential, it is therefore useful to know the characteristics of the disturbances it faces. The UpDown method, which detects and characterizes unknown disturbances at different levels of organization of the population (e.g., individual, pen, and batch disturbances), has been proposed for this purpose. Furthermore, using the outputs of the method, it is possible to extract proxies of the robustness of animals. In this context, the objective of the study was to evaluate the performances of the UpDown method to detect and characterize disturbances and quantify the robustness of animals in a genetic framework using different sets of simulations, and to apply this method to real pig longitudinal data recorded during the fattening period (body weight, cumulative feed intake, and feeding rate). Based on the simulations, the specificity of the UpDown method was high (>0.95). Its sensitivity increased with the level of organization exposed (from 0.23 to 0.32 for individual disturbances, from 0.45 to 0.59 for pen disturbances, and from 0.77 to 0.88 for batch disturbances). The UpDown method also showed a good ability to characterize detected disturbances. The average time interval between the estimated and true start date or duration of the disturbance was lower than 3 d. The correlation between the true and estimated intensity of the disturbance increased with the hierarchical level of organization (on average, 0.41, 0.78, and 0.83 for individual, pen, and batch disturbance, respectively). The accuracy of the estimated breeding values of the proxies for robustness extracted from the analysis of individual trajectories over time were moderate (lower than 0.33). Applied to real data, the UpDown method detected different disturbances depending on the phenotype analyzed. The heritability of the proxies of robustness were low to moderate (ranging from 0.11 to 0.20).

Improving the response of animals to environmental disturbances in terms of robustness is a key element to face the new breeding constraints related to climate change and the agroecological transition. Characterizing the disturbances that an animal experiences is a necessary first step to correctly evaluate its robustness. We propose a new method to do so based on the analysis of high-throughput phenotyping data. Using simulated data, we demonstrate that this method is effective for detecting and characterizing unknown disturbances and is thus helpful in correctly evaluating an animal's robustness. Applied to real growing pig data, it allowed us to obtain new measurements of robustness and to estimate their heritability in order to consider including these new traits for selection.

Identification and characterization of structural variants related to meat quality in pigs using chromosome-level genome assemblies.

BACKGROUND: Many studies have been performed to identify various genomic loci and genes associated with the meat quality in pigs. However, the full genetic architecture of the trait still remains unclear in part because of the lack of accurate identification of related structural variations (SVs) which resulted from the shortage of target breeds, the limitations of sequencing data, and the incompleteness of genome assemblies. The recent generation of a new pig breed with superior meat quality, called Nanchukmacdon, and its chromosome-level genome assembly (the NCMD assembly) has provided new opportunities. RESULTS: By applying assembly-based SV calling approaches to various genome assemblies of pigs including Nanchukmacdon, the impact of SVs on meat quality was investigated. Especially, by checking the commonality of SVs with other pig breeds, a total of 13,819 Nanchukmacdon-specific SVs (NSVs) were identified, which have a potential effect on the unique meat quality of Nanchukmacdon. The regulatory potentials of NSVs for the expression of nearby genes were further examined using transcriptome- and epigenome-based analyses in different tissues. CONCLUSIONS: Whole-genome comparisons based on chromosome-level genome assemblies have led to the discovery of SVs affecting meat quality in pigs, and their regulatory potentials were analyzed. The identified NSVs will provide new insights regarding genetic architectures underlying the meat quality in pigs. Finally, this study confirms the utility of chromosome-level genome assemblies and multi-omics analysis to enhance the understanding of unique phenotypes.

Population genetic analysis based on the polymorphisms mediated by transposons in the genomes of pig.

Transposable elements (TEs) mobility is capable of generating a large number of structural variants (SVs), which can have considerable potential as molecular markers for genetic analysis and molecular breeding in livestock. Our results showed that the pig genome contains mainly TE-SVs generated by short interspersed nuclear elements (51,873/76.49%), followed by long interspersed nuclear elements (11,131/16.41%), and more than 84% of the common TE-SVs (Minor allele frequency, MAF > 0.10) were validated to be polymorphic. Subsequently, we utilized the identified TE-SVs to gain insights into the population structure, resulting in clear differentiation among the three pig groups and facilitating the identification of relationships within Chinese local pig breeds. In addition, we investigated the frequencies of TEs in the gene coding regions of different pig groups and annotated the respective TE types, related genes, and functional pathways. Through genome-wide comparisons of Large White pigs and Chinese local pigs utilizing the Beijing Black pigs, we identified TE-mediated SVs associated with quantitative trait loci and observed that they were mainly involved in carcass traits and meat quality traits. Lastly, we present the first documented evidence of TE transduction in the pig genome.

Target silencing of porcine SPAG6 and PPP1CC by shRNA attenuated sperm motility.

The quality of sperm significantly influences the reproductive efficiency of pig herds. High-quality sperm is necessary for efficient fertilization and to maximize the litter numbers in commercial pig farming. However, the understanding of genes regulating porcine sperm motility and viability is limited. In this study, we validated porcine sperm/Sertoli-specific promoters through the luciferase reporter system and identified vital genes for sperm quality via loss-of-function means. Further, the shRNAs driven by the ACE and SP-10 promoters were used to knockdown the SPAG6 and PPP1CC genes which were provisionally important for sperm quality. We assessed the effects of SPAG6 and PPP1CC knockdown on sperm motility by using the sperm quality analyzer and flow cytometry. The results showed that the ACE promoter is active in both porcine Sertoli cells and sperms, whereas the SP-10 promoter is operating exclusively in sperm cells. Targeted interference with SPAG6 and PPP1CC expression in sperm cells decreases the motility and increases apoptosis rates in porcine sperms. These findings not only offer new genetic tools for targeting male germ cells but also highlight the crucial roles of SPAG6 and PPP1CC in porcine sperm function.

Integrated analysis of lncRNA, miRNA and mRNA expression profiles reveals regulatory pathways associated with pig testis function.

Long noncoding RNA (lncRNA) and microRNA (miRNA) are known to play pivotal roles in mammalian testicular function and spermatogenesis. However, their impact on porcine male reproduction has yet to be well unraveled. Here, we sequenced and identified lncRNA and miRNA expressed in the testes of Chinese indigenous Banna mini-pig inbred line (BMI) and introduced Western Duroc (DU) and Large White (LW) pigs. By pairwise comparison (BMI vs DU, BMI vs LW, and DU vs LW), we found the gene expression differences in the testes between Chinese local pigs and introduced Western commercial breeds were more striking than those between introduced commercial breeds. Furthermore, we found 1622 co-differentially expressed genes (co-DEGs), 122 co-differentially expressed lncRNAs (co-DELs), 39 co-differentially expressed miRNAs (co-DEMs) in BMI vs introduced commercial breeds (DU and LW). Functional analysis revealed that these co-DEGs and co-DELs/co-DEMs target genes were enriched in male sexual function pathways, including MAPK, AMPK, TGF-ß/Smad, Hippo, NF-kappa B, and PI3K/Akt signaling pathways. Additionally, we established 10,536 lncRNA-mRNA, 11,248 miRNA-mRNA pairs, and 62 ceRNA (lncRNA-miRNA-mRNA) networks. The ssc-miR-1343 had the most interactive factors in the ceRNA network, including 20 mRNAs and 3 lncRNAs, consisting of 56 ceRNA pairs. These factors played extremely important roles in the regulation of testis function as key nodes in the interactive regulatory network. Our results provide insight into the functional roles of lncRNAs and miRNAs in porcine testis and offer a valuable resource for understanding the differences between Chinese indigenous and introduced Western pigs.