Pigs In Stages Research Articles

Multi-omic characterization of allele-specific regulatory variation in hybrid pigs

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.

Long Non-Coding RNAs Differentially Expressed in Swine Fetuses.

Long non-coding RNAs (lncRNAs) are non-coding transcripts involved in various biological processes. The Y chromosome is known for determining the male sex in mammals. LncRNAs on the Y chromosome may play important regulatory roles. However, knowledge about their action mechanisms is still limited, especially during early fetal development. Therefore, we conducted this exploratory study aiming to identify, characterize, and investigate the differential expression of lncRNAs between male and female swine fetuses at 35 days of gestation. RNA-Seq libraries from 10 fetuses were prepared and sequenced using the Illumina platform. After sequencing, a data quality control was performed using Trimmomatic, alignment with HISAT2, and transcript assembly with StringTie. The differentially expressed lncRNAs were identified using the limma package of the R software (4.3.1). A total of 871 potentially novel lncRNAs were identified and characterized. Considering differential expression, eight lncRNAs were upregulated in male fetuses. One was mapped onto SSC12 and seven were located on the Y chromosome; among them, one lncRNA is potentially novel. These lncRNAs are involved in diverse functions, including the regulation of gene expression and the modulation of chromosomal structure. These discoveries enable future studies on lncRNAs in the fetal stage in pigs.

Single-Cell RNA Sequencing Reveals Differences in Chromatin Remodeling and Energy Metabolism among In Vivo-Developed, In Vitro-Fertilized, and Parthenogenetically Activated Embryos from the Oocyte to 8-Cell Stages in Pigs

In vitro-fertilized (IVF) and parthenogenetically activated (PA) embryos, key to genetic engineering, face more developmental challenges than in vivo-developed embryos (IVV). We analyzed single-cell RNA-seq data from the oocyte to eight-cell stages in IVV, IVF, and PA porcine embryos, focusing on developmental differences during early zygotic genome activation (ZGA), a vital stage for embryonic development. (1) Our findings reveal that in vitro embryos (IVF and PA) exhibit more similar developmental trajectories compared to IVV embryos, with PA embryos showing the least gene diversity at each stage. (2) Significant differences in maternal mRNA, particularly affecting mRNA splicing, energy metabolism, and chromatin remodeling, were observed. Key genes like SMARCB1 (in vivo) and SIRT1 (in vitro) played major roles, with HDAC1 (in vivo) and EZH2 (in vitro) likely central in their complexes. (3) Across different types of embryos, there was minimal overlap in gene upregulation during ZGA, with IVV embryos demonstrating more pronounced upregulation. During minor ZGA, global epigenetic modification patterns diverged and expanded further. Specifically, in IVV, genes, especially those linked to H4 acetylation and H2 ubiquitination, were more actively regulated compared to PA embryos, which showed an increase in H3 methylation. Additionally, both types displayed a distinction in DNA methylation. During major ZGA, IVV distinctively upregulated genes related to mitochondrial regulation, ATP synthesis, and oxidative phosphorylation. (4) Furthermore, disparities in mRNA degradation-related genes between in vivo and in vitro embryos were more pronounced during major ZGA. In IVV, there was significant maternal mRNA degradation. Maternal genes regulating phosphatase activity and cell junctions, highly expressed in both in vivo and in vitro embryos, were degraded in IVV in a timely manner but not in in vitro embryos. (5) Our analysis also highlighted a higher expression of many mitochondrially encoded genes in in vitro embryos, yet their nucleosome occupancy and the ATP8 expression were notably higher in IVV.

The Responses of Lactobacillus reuteri LR1 or Antibiotic on Intestinal Barrier Function and Microbiota in the Cecum of Pigs.

This study aimed to investigate responses of the Lactobacillus reuteri or an antibiotic on cecal microbiota and intestinal barrier function in different stages of pigs. A total of 144 weaned pigs (Duroc × Landrace × Yorkshire, 21 days of age) were randomly assigned to the control group (CON, fed with a basal diet), the antibiotic group (AO, fed with basal diet plus 100 mg/kg olaquindox and 75 mg/kg aureomycin), and the L. reuteri group (LR, fed with the basal diet + 5 × 1010 CFU/kg L. reuteri LR1) throughout the 164-d experiment. A total of 45 cecal content samples (5 samples per group) from different periods (14th, 42th, and 164th days) were collected for 16S rRNA gene amplification. The results revealed that although LR and AO did not change the diversity of cecal microbiota in pigs, the abundance of some bacteria at the genus level was changed with age. The proportion of Lactobacillus was increased by LR in early life, whereas it was decreased by AO compared with the control group. The relative abundance of Ruminococcaceae was increased along with age. In addition, the gas chromatography results showed that age, not AO or LR, has significant effects on the concentrations of SCFAs in the cecum of pigs (P < 0.05). However, the mRNA expression of tight junction proteins zonula occluden-1 (ZO-1) and occludin were increased by AO in the cecum of pigs on day 14, while LR increased the mRNA expression of intestinal barrier-related proteins ZO-1, occludin, mucin-1, mucin-2, PG1-5, and pBD2 in the cecum of pigs on days 14 and 164 (P < 0.05). In conclusion, LR and AO have different effects on the intestinal barrier function of the cecum, and neither LR nor AO damaged the intestinal barrier function of pig cecum. In addition, LR and AO have little effects on cecal microflora in different stages of the pigs. The microflora and their metabolite SCFAs were significantly changed along with age. These findings provide important information to understand the homeostasis of the cecum of pigs after antibiotic or probiotic treatment.

Alternative Splicing Dynamics of the Hypothalamus-Pituitary-Ovary Axis During Pubertal Transition in Gilts.

The timing of puberty in mammals marks the point at which reproduction becomes possible. Abnormalities in the timing of puberty may exert a series of negative effects on subsequent health outcomes. Alternative splicing (AS) has not only emerged as a significant factor in the transcription of genes but it is also reported to play a role in the timing of puberty. However, to date, the changes and dynamics of AS during the onset of puberty is extremely seldom explored. In the present study, we used gilts as a research model to investigated the dynamics of AS and differentially expressed AS (DEAS) events within the hypothalamus–pituitary–ovary (HPO) axis across pre-, in-, and post-puberty. We detected 3,390, 6,098, and 9,085 DEAS events in the hypothalamus, pituitary, and ovary when compared across pre-, in-, and post-pubertal stages, respectively. Within the entire HPO axis, we also identified 22,889, 22,857, and 21,055 DEAS events in the pre-, in-, and post-pubertal stages, respectively. Further analysis revealed that the differentially spliced genes (DSGs) associated with staged DEAS events were likely to be enriched in the oxytocin signaling pathway, thyroid hormone signaling pathway, GnRH signaling pathway, and oocyte meiosis signaling pathway. The DSGs associated with DEAS events across the entire HPO axis were enriched in endocytosis signaling pathway, the MAPK signaling pathway, and the Rap1 signaling pathway. Moreover. the ASs of TAC1, TACR3, CYP19A1, ESR1, ESRRA, and FSHR were likely to regulate the functions of the certain HPO tissues during the onset of puberty. Collectively, the AS dynamics and DEAS events were comprehensively profiled in hypothalamus, pituitary, and ovary across the pre-, in-, and post-pubertal stages in pigs. These findings may enhance our knowledge of how puberty is regulated by AS and shed new light on the molecular mechanisms underlying the timing of puberty in mammals.

Global Transcriptomic Analyses Reveal Genes Involved in Conceptus Development During the Implantation Stages in Pigs.

Prenatal mortality remains a significant concern to the pig farming industry around the world. Spontaneous fetal loss ranging from 20 to 45% by term occur after fertilization, with most of the loss happening during the implantation period. Since the factors regulating the high mortality rates of early conceptus during implantation phases are poorly understood, we sought to analyze the overall gene expression changes during this period, and identify the molecular mechanisms involved in conceptus development. This work employed Illumina’s next-generation sequencing (RNA-Seq) and quantitative real-time PCR to analyze differentially expressed genes (DEGs). Soft clustering was subsequently used for the cluster analysis of gene expression. We identified 8236 DEGs in porcine conceptus at day 9, 12, and 15 of pregnancy. Annotation analysis of these genes revealed rRNA processing (GO:0006364), cell adhesion (GO:1904874), and heart development (GO:0007507), as the most significantly enriched biological processes at day 9, 12, and 15 of pregnancy, respectively. In addition, we found various genes, such as T-complex 1, RuvB-like AAA ATPase 2, connective tissue growth factor, integrins, interferon gamma, SLA-1, chemokine ligand 9, PAG-2, transforming growth factor beta receptor 1, and Annexin A2, that play essential roles in conceptus morphological development and implantation in pigs. Furthermore, we investigated the function of PAG-2 in vitro and found that PAG-2 can inhibit trophoblast cell proliferation and migration. Our analysis provides a valuable resource for understanding the mechanisms of conceptus development and implantation in pigs.

SIN3A Regulates Porcine Early Embryonic Development by Modulating CCNB1 Expression.

SIN3A is the central scaffold protein of the SIN3/histone deacetylase (HDAC) transcriptional repressor complex. SIN3A participates in the mouse preimplantation development by fine-tuning HDAC1 expression. However, it remains unresolved if this functional significance of SIN3A was conserved in other mammals. Herein, RNA-seq results show a large amount of SIN3A mRNA is present in oocytes and early embryos prior to embryonic genome activation and a low amount thereafter, suggesting a maternal origin of SIN3A in pigs, cattle, mice, and humans. Interestingly, immunofluorescence data show that SIN3A protein level peaks at four-cell stage in pigs compared with morula stage in cattle. SIN3A depletion in early embryos causes a developmental arrest at two-cell stage in pigs but does not affect bovine early embryonic development. In contrast with mouse data, SIN3A depletion results in only a slight decrease and even no difference in HDAC1 expression in porcine and bovine early embryos, respectively. In addition, HDAC1 knockdown does not cause two-cell block but leads to a reduced blastocyst rate. By using unbiased RNA-seq approach, we found that Cyclin B1 (CCNB1) transcript level is dramatically reduced. Moreover, CCNB1 knockdown results in a similar phenotype as SIN3A depletion. Injection of exogenous CCNB1 mRNA into SIN3A-depleted embryos could partly rescue embryonic development to pass two-cell stage. In conclusion, our results indicate SIN3A plays an essential role in porcine early embryonic development, which probably involves the regulation of CCNB1 expression.

64 SIN3 transcription regulator family member A regulates porcine early embryonic development by modulating CCNB1 expression

SIN3 transcription regulator family member A (SIN3A) is the central scaffold protein of the SIN3/HDAC (histone deacetylase) transcriptional repressor complex. We previously found that SIN3A participates in the mouse pre-implantation development by finetuning HDAC1 expression. However, it remains unresolved whether this functional significance of SIN3A is conserved in other mammals. The objective of this work was thus to characterise the expression profiles and the functional role of SIN3A in pre-implantation development using non-rodent animal models. RNA sequencing results show that a large amount of SIN3A mRNA is present in oocytes and early embryos before embryonic genome activation and a low amount thereafter, suggesting a maternal origin of SIN3A in all species examined. Interestingly, immunofluorescence data show that SIN3A protein level peaks at the 4-cell stage in pigs compared with the morula stage in cattle, suggesting a differential role of SIN3A among species. To explore the function of SIN3A in early embryonic development, we used a short interfering (si)RNA-mediated knockdown approach in porcine parthenogenetic activated (PA) embryos. Immunocytochemical analysis showed that SIN3A levels were diminished ∼80% compared with nonspecific siRNA (NC) injected control (n=3). To monitor the developmental potential of embryo depleted of SIN3A, we injected SIN3A-siRNA into MII stage oocytes, followed by parthenogenetic activation, and percent cleavage and blastocyst formation were recorded. We found that SIN3A knockdown (KD) did not affect the cleavage rate (NC vs. KD, 83.63±3.63% vs. 80.08±4.66%, n=5), but significantly reduced blastocyst rate compared with the NC group (NC vs. KD, 36.64±4.28% vs. 6.33±3.12%, n=5). Specifically, SIN3A depletion in early embryos causes developmental arrest at 2-cell stage in pigs but does not affect early embryonic development in bovines. In contrast with mouse data, SIN3A depletion results in only a slight decrease and even no difference in HDAC1 expression in porcine and bovine early embryos, respectively. In addition, HDAC1 knockdown does not cause 2-cell block but leads to a reduced blastocyst rate, suggesting that the effect of SIN3A depletion on porcine early embryos is independent of HDAC1. RNA-Seq analysis was used to compare the global transcript content between NC and KD 2-cell embryos. A total of 23 genes (14 upregulated and 9 downregulated) had undergone significant changes. Interestingly, cyclin B1 (CCNB1) ranked second among downregulated genes. To test whether knockdown of CCNB1 would display a similar phenotype in porcine early embryos, we injected CCNB1-siRNA into pronuclear stage. CCNB1 KD resulted in a similar phenotype as SIN3A depletion. Injection of exogenous CCNB1 mRNA into SIN3A-depleted embryos could partly rescue embryonic development. In conclusion, our results indicate SIN3A plays an essential role in porcine early embryonic development, probably involving the regulation of CCNB1 expression. This work was funded by National Natural Science Foundation of China, the Anhui Provincial Natural Science Foundation and China Postdoctoral Science Foundation.

Pluripotency-associated genes reposition during early embryonic developmental stages in pigs.

We examined the allelic expression and positioning of two pluripotency‐associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4‐ and 8‐cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi‐allelically increased from 45% at the 4‐cell stage to 60% at the 8‐cell stage. Moreover, in 8‐cell embryos, SOX2 was expressed bi‐allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4‐ to 8‐cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4‐ or 8‐cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency‐associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.

PSII-24 Towards a better understanding of the gut microbiome functions in the swine production: extensive cultivation of the swine gut microbiome from different growth stages

Abstract Substantial progress has been made in the culture-omics of the human gut microbiota. However, little is known about the culture-omics of the swine gut microbiota, despite recent reports of their significant roles in swine health and production. To fill this knowledge gap in research, we tested 52 bacterial cultivation methods with different media and gas combinations. Fresh fecal samples (0.2g/sample) were collected from three pigs at the end of four growth stages: lactation, nursery, growing and finishing and were mixed with a stomacher in 20 mL saline. Aliquots of 50 uL microbial suspensions were then spread onto different media plates and incubated under aerobic and anaerobic conditions at 37C for up to 5 days. An additional aliquot of each sample was subjected to direct DNA extraction as a positive control. Bacterial colonies from each plate were collected and DNA was extracted from these samples using the Powersoil DNA isolation kit and sequenced with an Illumina Miseq sequencer targeting the V4 region of the 16S rRNA gene. Sequences were analyzed with the Deblur algorithm in the QIIME2 package. A total of 378, 482, 565, and 555 bacterial features were observed from microbial solutions at the end of lactation, nursery, growing and finishing. Our culturing methods recovered 415, 675, 808, and 823 features correspondingly, representing 45.2%, 54.8%, 53.3%, and 56.4% of total features observed in microbial solutions. The top ten most easily cultured genus were Escherichia, Streptococcus, Lactobacillus, Megasphaera, Acidaminococcus, Bacillus, Mitsuokella, Enterococcus and Prevotella. Non-parametric permutational multivariate analysis of variance shows that the main factors driving the swine culture-omics included medium, age and oxygen condition. This study identifies the cultivable bacteria from fecal samples collected at different growth stages of pigs and provides a guidance to cultivate potential beneficial or pathogenic bacteria of interests and validate their functions in swine production.

Temporal expression profiling of long noncoding RNA and mRNA in the peripheral blood during porcine development.

ObjectiveWe investigated the temporal expression profiles of long noncoding RNA (lncRNA) and mRNA in the peripheral blood of pigs during development and identified the lncRNAs that are related to the blood-based immune system.MethodsPeripheral blood samples were obtained from the pigs at 0, 7, 28, and 180 days and 2 years of age. RNA sequencing was performed to survey the lncRNA and mRNA transcriptomes in the samples. Short time-series expression miner (STEM) was used to show temporal expression patterns in the mRNAs and lncRNAs. Gene ontology and Kyoto encyclopedia of genes and genomes analyses were performed to assess the genes’ biological relevance. To predict the functions of the identified lncRNAs, we extracted mRNAs that were nearby loci and highly correlated with the lncRNAs.ResultsIn total of 5,946 lncRNA and 12,354 mRNA transcripts were identified among the samples. STEM showed that most lncRNAs and mRNAs had similar temporal expression patterns during development, indicating the expressional correlation and functional relatedness between them. The five stages were divided into two classes: the suckling period and the late developmental stage. Most genes were expressed at low level during the suckling period, but at higher level during the late stages. Expression of several T-cell-related genes increased continuously during the suckling period, indicating that these genes are crucial for establishing the adaptive immune system in piglets at this stage. Notably, lncRNA TCONS-00086451 may promote blood-based immune system development by upregulating nuclear factor of activated T-cells cytoplasmic 2 expression.ConclusionThis study provides a catalog of porcine peripheral blood-related lncRNAs and mRNAs and reveals the characteristics and temporal expression profiles of these lncRNAs and mRNAs during peripheral blood development from the newborn to adult stages in pigs.

Developmental atlas of the RNA editome in Sus scrofa skeletal muscle

Adenosine-to-inosine (A-to-I) RNA editing meditated by adenosine deaminases acting on RNA (ADARs) enzymes is a widespread post-transcriptional event in mammals. However, A-to-I editing in skeletal muscle remains poorly understood. By integrating strand-specific RNA-seq, whole genome bisulphite sequencing, and genome sequencing data, we comprehensively profiled the A-to-I editome in developing skeletal muscles across 27 prenatal and postnatal stages in pig, an important farm animal and biomedical model. We detected 198,892 A-to-I editing sites and found that they occurred more frequently at prenatal stages and showed low conservation among pig, human, and mouse. Both the editing level and frequency decreased during development and were positively correlated with ADAR enzymes expression. The hyper-edited genes were functionally related to the cell cycle and cell division. A co-editing module associated with myogenesis was identified. The developmentally differential editing sites were functionally enriched in genes associated with muscle development, their editing levels were highly correlated with expression of their host mRNAs, and they potentially influenced the gain/loss of miRNA binding sites. Finally, we developed a database to visualize the Sus scrofa RNA editome. Our study presents the first profile of the dynamic A-to-I editome in developing animal skeletal muscle and provides evidences that RNA editing is a vital regulator of myogenesis.

Transcriptome analysis to identify long non coding RNA (lncRNA) and characterize their functional role in back fat tissue of pig

Long non coding RNAs (lncRNA) have been previously found to be involved in important cellular activities like epigenetics, implantation, cell growth etc. in pigs. However, comprehensive analysis of lncRNA in back fat tissues at different developmental stages in pigs is still lacking. In this study we conducted transcriptome analysis in the back fat tissue of a F1 crossbred Korean Native Pig (KNP) × Yorkshire Pig to identify lncRNA. We investigated their role in 16 pigs at two different growth stages; stage 1 (10 weeks, n = 8) and stage 2 (26 weeks, n = 8). After quality assessment of sequencing reads, we got a total of 1,641,165 assembled transcripts out of eight paired end read from each stage. Among them, 6808 lncRNA transcripts were identified by filtering on the basis of multiple parameters like read length ≥ 200 nucleotides, exon numbers ≥2, FPKM ≥0.5, coding potential score < 0 etc. PFAM and RFAM were used to filter out all possible protein coding genes and housekeeping RNAs respectively. A total of 103 lncRNAs and 1057 mRNAs were found to be differentially expressed (DE) between the two stages (|log2FC| > 2, q < 0.05). We also identified 306 genes located around 100 kb upstream and 234 genes downstream around these DE lncRNA transcripts. The expression of top eleven DE lncRNAs (COL4A6, LY7S, MYH2, OXCT1, SMPDL3A, TMEM182, TTC36, RFOOOO4, RFOOO15, RFOOO45, CADM2) had been validating by qRT-PCR. Pathway and GO terms analysis showed that, positive regulation of biosynthetic process, Wnt signaling pathway, cellular protein modification process, and positive regulation of nitrogen compound were differentially enriched. Our results suggested that, KEGG pathways such as protein digestion and absorption, Arrhythmogenic right ventricular cardiomyopathy (ARVC) to be significantly enriched in both DE lncRNAs as well as DE mRNAs and involved in back fat tissues development. It also suggests that, identified lncRNAs are involved in regulation of important adipose tissues development pathways.

Comparative serum proteome analysis reveals potential early pregnancy-specific protein biomarkers in pigs

In this study, the comparative serum proteome profile of Day 5, 12 and 16 of gestation, representing three early embryonic events, namely formation, elongation and implantation of blastocysts, and non-pregnant control were explored by a label-free quantitation-based mass spectrometric approach to identify early pregnancy biomarkers in pigs. A total of 131 proteins were identified with respect to different groups, out of which 105 were found to be differentially expressed proteins (DEPs). Among the DEPs, 54 and 66 proteins were found to be up and downregulated respectively in early pregnancy groups (fold change >2) and the maximum number of upregulated proteins was observed in the Day 12 pregnancy stage. Functional classification and pathway analysis of the DEPs revealed involvement of most of the proteins in complement and coagulation cascades, metabolic processes and immune and inflammatory responses. Proteins such as glutathione peroxidise (GPX), pregnancy zone protein (PZP), thrombospondin-1 (THBS1), α-1-antitrypsin (AAT) and mannose-binding lectin C (MBLC) were differentially expressed during early pregnancy and actively involved in different pregnancy-related activities. To the best of our knowledge, this is the first report on comparative serum protein profiling of different early pregnancy stages in pigs and our results provide a set of proteins that can be used as potential biomarkers for early pregnancy diagnosis in pigs.

Expression analysis of multifunctional RNA-binding protein hnRNP K during development of mammalian testis.

Heterogeneous nuclear ribonucleoprotein K (hnRNP K), is a multifunctional protein that participates in a variety of regulatory processes of signal transduction and gene expression. To further characterize the significance of hnRNP K in different male germ cells, we investigated the expression profiles of hnRNP K at different developmental stages in pig and rat testes, and conducted a comparative analysis of expression patterns between these two species. In porcine testis development, both the mRNA and protein level of hnRNP K were down-regulated from 3 months to 8 months. However, the expression level of hnRNP K was abundant across the embryonic period in rats, and decreased gradually from 0 day post partum (dpp) to 14 dpp, then increased with the highest level presenting at 90 dpp. Immunolocalization analysis further confirmed the differential expression and localization of hnRNP K protein during testis development in pigs and rats. The results showed that hnRNP K was widely distributed in gonocytes, spermatogonia, sertoli cells and Leydig cells. The dynamic expression profile of hnRNP K may imply its crucial and potential roles in the development of the testis, which will provide a theoretical basis for the future study of molecular mechanism regulation of spermatogenesis.

Transmission of African swine fever virus from infected pigs by direct contact and aerosol routes

In 2014, African swine fever virus (ASFV) was introduced into the Baltic states and Poland. Since then, the disease has continued to spread within these regions, and recently, cases were reported in the Czech Republic and Romania. Currently, there is an increasing risk of ASFV introduction into Western Europe. Hence, there is an urgent need to assess current contingency plans. For this purpose, knowledge of modes-of-transmission and clinical outcome in pigs infected with new European ASFV strains is needed.In the present study, two experiments were conducted in pigs using an isolate of ASFV from Poland (designated here POL/2015/Podlaskie/Lindholm). In both studies, pigs were inoculated intranasally with the virus and contact pigs were exposed to the experimentally infected pigs, either directly (contact within and between pens) or by air.Pigs exposed to the virus by intranasal inoculation, by direct contact to infected animals and by aerosol developed acute disease characterized by viremia, fever and depression. Infectious virus was first detected in blood obtained from the inoculated pigs and then sequentially among the within-pen, between-pen and air-contact pigs. ASFV DNA and occasionally infectious virus was found in nasal-, oral-, and rectal swabs obtained from the pigs, and ASFV DNA was detected in air samples. No anti-ASFV antibodies were detected in sera.In conclusion, the study shows that the currently circulating strain of ASFV can be efficiently transmitted via direct contact and by aerosols. Also, the results provide quantitative transmission parameters and knowledge of infection stages in pigs infected with this ASFV.

A comparative proteomic analysis of blood serum for developmental stages in pigs.

This study aimed to differentiate genes at developmental stages of pigs from 0 to 150 days of age, to build up a protein database and to find candidate genetic markers for growth traits. The analysis of two-dimensional electrophoresis and matrix-assisted laser-desorption/ionization mass spectrometry separated 252 protein segments. After successfully blasting the peptide sequences, the analysis confirmed 37 differentially expressed proteins that increased from birth to 150days of age (type A), whereas the type B proteins presented the inverse pattern. The type C proteins included proteins that were expressed continuously throughout the developmental periods. A total of 319 primer sets for 33 genes were designed to find genetic variants using pooled DNA samples of Yorkshire pigs. Amplification products for all primer sets produced approximately 20000 clones that were sequenced, and 48 candidate SNP sites were finalized for genotyping. A total of 475 animals were used for high throughput genotyping analysis. Among these, phenotype data of all 475 animals were collected for average daily gain, backfat thickness and days to 90kg, whereas feed conversion data were collected for 300 animals and body measurement traits (starting weight, ending weight, body length, wither height and chest depth) were collected for 209 animals. Association analysis found significant statistical differences between the animals having genotypes of 13 SNPs (g.78935883C>T, g.147629986C>T, g.98266037T>C, g.214707340G>A, g.88350299C>T, g.17180956C>T, g.17181024C>T, g.2350283A>G, g.138361311C>T, g.44996379C>T, g.44996247A>C, g.107715245C>T, g.4149631C>T) for the various measured traits. The identified genetic polymorphisms, of which one was novel (g.214707340G>A), may serve as candidate molecular markers to change population means for the targeted growth traits.

Standardized total tract digestibility of phosphorus in flaxseed meal fed to growing and finishing pigs without or with phytase supplementation.

Two experiments were conducted to determine the apparent total tract digestibility (ATTD) and standardized total tract digestibility (STTD) of P in flaxseed meal (FM) and the effect of dietary microbial phytase on the digestibility of P in FM fed to growing and finishing pigs. In Exp. 1, eighteen growing barrows (26.6 ± 1.8 kg BW) were allotted to 1 of 3 experimental diets consisting of a diet containing 32% FM that was fed with or without phytase at 500 phytase units (FTU/kg and a P-free diet in a completely randomized design to give 6 replicates per diet. The experimental period lasted 12 d including first 7 d for adaptation and 5 d for total collection of feces. Pigs were fed their assigned diets at 4% of BW at the beginning of the experiment. The daily feed allowance was offered in 2 equal portions at 0800 and 1600 h. All experimental diets were provided in mash form. Results indicated that pigs fed the diets containing FM with dietary phytase had less ( < 0.05) fecal P concentration and daily P output than those fed the diets without phytase supplementation. Also, phytase supplementation increased ( < 0.05) the ATTD of P of the diets containing FM from 37.3% to 51.8% and STTD of P of the diets containing FM from 43.2% to 57.7%. The basal endogenous P losses (EPL) was calculated at 140 ± 11 mg/kg of DMI in growing pigs fed the P-free diet. In Exp. 2, eighteen finishing pigs (78.7 ± 2.4 kg BW) were randomly allotted to 1 of 3 dietary treatments. The experimental diets and procedures were similar to those described in Exp. 1. Similar to Exp. 1, pigs fed FM diets with phytase supplementation had less ( < 0.05) P concentration in feces than those fed diets without phytase supplementation. Also, daily P output was reduced ( = 0.08) when pigs were fed the FM diets with phytase compared to those fed the FM diets without phytase. The ATTD of P in FM diets was increased ( < 0.01) from 31.4% to 45.8%, whereas the STTD of P in FM diets was increased ( < 0.01) from 37.8% to 52.3% as a result of phytase supplementation. The basal EPL was calculated at 164 ± 19 mg/kg of DMI in finishing pigs fed the P-free diet. In conclusion, the ATTD and STTD of P in FM fed to growing pigs were 37.3% and 43.2%, respectively, whereas respective values for finishing pigs were 31.4%, and 37.8%, respectively. Also, dietary phytase supplementation improved both ATTD and STTD of P in FM for both stages of pigs by an average of 33%.

Silencing myotubularin related protein 7 enhances proliferation and early differentiation of C2C12 myoblast

Myotubularin related protein 7 (MTMR7) is a key member of the highly conserved myotubularin related proteins (MTMRs) family, which has phosphatase activity. MTMR7 was increased during myoblast differentiation and exhibited high expression level at primary fibers formation stages in pigs. This suggests that MTMR7 may be involved in myogenesis. In our study, we investigated the roles of MTMR7 on proliferation and differentiation of C2C12 myoblasts. Knocking down MTMR7 not only enhanced myoblast early differentiation via altering the expression of Myf5, but also promoted myoblast proliferation through increasing cyclinA2 expression. The improved proliferation capacity was related to the increased phosphorylation of AKT. Taken together, our research demonstrates that MTMR7 plays an important role in proliferation and early differentiation of C2C12 myoblast.

The effect of optimal space allowance on growth performance and physiological responses of pigs at different stages of growth

This study was conducted to determine the optimal space allowance for maximizing the growth performance of pigs at each of the following five growth stages (based on BW ranges): stage 1, 11 to 25 kg BW; stage 2, 25 to 45 kg BW; stage 3, 45 to 65 kg BW; stage 4, 65 to 85 kg BW; and stage 5, 85 to 110 kg BW. A total of 1590 crossbred (Landrace×Yorkshire×Duroc) pigs were assigned to one of four treatments at each growth stage, with three replicates each. Pen areas at each growth stage were 6, 11, 16, 19.5 and 20 m2 for stages 1 to 5, respectively. Space allowances for the four treatments at each growth stage were modified by varying the number of pigs per pen (22, 25, 28 and 31 pigs in T1, T2, T3 and T4, respectively). Blood samples were collected on the final day of each growth stage. The average daily gain (ADG) decreased significantly with decreased space allowances at all growth stages, except at stage 2. Average daily feed intake (ADFI) was not significantly affected by space allowances at stages 1 to 4; however, at stage 5, there was a linear effect of space allowance on ADFI. Thus, the feed conversion ratio showed results similar to those for ADG. Serum cortisol concentrations, indicating the level of stress response, increased as space allowances decreased. The highest serum cortisol concentrations were observed in T3 at stages 2 to 5. Serum tumor necrosis factor-α levels were significantly higher in association with a small space allowance than with at large space allowance at stages 2, 4 and 5. Serum interleukin-1β levels also increased in a significant linear manner at every growth stage in pigs reared at a low space allowance, except at stage 4 (P=0.068). This study found that limited space allowance decreases the growth performance of pigs and induces stress and inflammatory responses. We confirmed that no significant effect of space allowance on growth performance and serum cortisol concentrations are observed between T1 and T2 across all growth stages. We suggest that the optimal space allowances for pigs according to their BW are as follows: 0.24, 0.44, 0.64, 0.78 and 0.80 m2/pig for BWs of 11 to 25, 25 to 45, 45 to 65, 65 to 85 and 85 to 115 kg, respectively.