The Effect of BSCL2 Gene on Fat Deposition Traits in Pigs.

BSCL2 encodes seipin, a transmembrane endoplasmic reticulum protein associated with lipodystrophy and severe metabolic complications, including diabetes and hepatic steatosis. In pigs, BSCL2 expression increases during adipocyte differentiation. In the present study, we identified significant gene variants associated with fat deposition (FD)-related processes based on subcutaneous fat tissue RNA-seq data. In the association study, to prove our hypothesis, three Polish pig breeds were included: Zlotnicka White (ZW, n = 72), Polish Landrace (PL, n = 201), and Polish Large White (PLW, n = 169). Based on variant calling analysis and χ2 tests, BSCL2 mutations showing significantly different genotype/allele distribution between high- and low-fat pigs were selected for a comprehensive association study. Four interesting BSCL2 variants (rs346079334, rs341493267, rs330154033, and rs81333153) belonging to downstream and missense mutations were investigated. Our study showed a significant decrease in minor allele frequency for two BSCL2 variants (rs346079334 and rs341493267) in PL pigs in 2020-2021. In ZW, BSCL2 mutations significantly affected loin and ham fats, meat redness, and growth performance traits, such as feed conversion and daily feed intake. Similar observations were noted for PLW and PL, where BSCL2 mutations influenced fat depositions and meat traits, such as loin eye area, loin mass and fat, carcass yield, and growth performance traits. Based on the observation in pigs, our study supports the theory that BSCL2 expressed in subcutaneous fat is involved in the FD process.

Effect of different doses of cholecalciferol and calcidiol on meat quality parameters and skeletal muscle transcriptome profiles in swine.

Skeletal muscle tissue is one of the potential targets for vitamin D actions. There are indications that vitamin D supplementation to swine has a positive effect on meat quality. However, these issues need further study, especially in terms of response to the use of different forms of vitamin D. We carried out a multi-purpose study to compare the effects of cholecalciferol and calcidiol on meat quality and muscle tissue transcriptome. Meat quality assessment and gene expression analysis were performed on longissimus dorsi samples collected from swine fed grower/finisher diets containing 2000 IU cholecalciferol/1500 IU cholecalciferol per kg (n = 8), 3000 IU cholecalciferol/2500 IU cholecalciferol per kg (n = 10), 2000 IU cholecalciferol +1000 IU calcidiol/1500 IU cholecaliferol +1000 IU calcidiol per kg (n = 8), and 2000 IU calcidiol/1500 IU calcidiol per kg (n = 8). The results suggest that increasing the dose of cholecalciferol and using calcidiol in the diet of finishers may improve meat texture parameters (shear force P = 0,014, toughness P = 0,048, cohesiveness P = 0,017, resilience P = 0,002). Shear force (68.46 N-51.42 N) and toughness (145.85 N-114.52 N) decreased the most under the effect of increasing cholecalciferol dosage. In turn, cohesiveness (0.60 N-0.65 N) and resilience (0.23 N-0.28 N) increased most strongly under the use of cholecalciferol+calcidiol. Moreover, the results indicate no significant effect of increasing cholecalciferol dose and use calcidiol in the swine diet on muscle tissue transcriptome.

Variations in Fibrinogen-like 1 (FGL1) Gene Locus as a Genetic Marker Related to Fat Deposition Based on Pig Model and Liver RNA-Seq Data.

The goal of this study was to evaluate the effects of mutations in the FGL1 gene associated with pig productive traits to enrich the genetic marker pool for further selection and to support the studies on FGL1 in the context of the fat deposition (FD) process. The variant calling and χ2 analyses of liver RNA-seq data were used to indicate genetic markers. FGL1 mutations were genotyped in the Zlotnicka White (n = 72), Polish Large White (n = 208), Duroc (n = 72), Polish Landrace (PL) (n = 292), and Pulawska (n = 178) pig breeds. An association study was performed using a general linear model (GLM) implemented in SAS® software. More than 50 crucial mutations were identified in the FGL1 gene. The association study showed a significant effect of the FGL1 on intramuscular fat (IMF), loin eye area, backfat thickness at the lumbar, ham mass (p = 0.0374), meat percentage (p = 0.0205), and loin fat (p = 0.0003). Alternate homozygotes and heterozygotes were found in the PL and Duroc, confirming the selective potential for these populations. Our study supports the theory that liver FGL1 is involved in the FD process. Moreover, since fat is the major determinant of flavor development in meat, the FGL1 rs340465447_A allele can be used as a target in pig selection focused on elevated fat levels.

The Effect of Replacement of Soybean Meal with Corn Dried Distillers Grains with Solubles (cDDGS) and Differentiation of Dietary Fat Sources on Pig Meat Quality and Fatty Acid Profile.

The aim of the study was to investigate mixtures comprising corn distillers dried grain with solubles as a partial replacer for soybean meal (SBM) and different dietary fat sources, in order to determine their effect on the meat quality and fatty acid profile. Thirty-two crossbred fatteners were divided into four groups: I-SBM + rapeseed oil, II-cDDGS + rapeseed oil, III-cDDGS + beef tallow, IV-cDDGS + coconut oil. The experiment took place from 60 to 118 kg. At the end of fattening, all pigs were slaughtered and samples of meat (musculuslongissimus lumborum) were taken. The fatty acid profile, texture, and quality traits were analyzed. Corn DDGS affected drip loss. Beef tallow and coconut oil improved water holding capacity and drip loss and increased fat content, compared to the control group. The dietary fat type affected the fatty acid composition, iodine value, and consequently some quality traits of meat. However, these relationships varied. Fat content in the meat was inversely correlated with shear force and texture parameters, but positively with tenderness and juiciness. The fatty acid profile significantly influenced cohesiveness, chewiness, resilience and sensory traits, which were the most beneficial in meat with higher fat content and higher fat saturation index.

Identification of Molecular Mechanisms Related to Pig Fatness at the Transcriptome and miRNAome Levels.

Fat deposition and growth rate are closely related to pork quality and fattening efficiency. The next-generation sequencing (NGS) approach for transcriptome and miRNAome massive parallel sequencing of adipocyte tissue was applied to search for a molecular network related to fat deposition in pigs. Pigs were represented by three breeds (Large White, Pietrain, and Hampshire) that varied in fat content within each breed. The obtained results allowed for the detection of significant enrichment of Gene Ontology (GO) terms and pathways associated directly and indirectly with fat deposition via regulation of fatty acid metabolism, fat cell differentiation, inflammatory response, and extracellular matrix (ECM) organization and disassembly. Moreover, the results showed that adipocyte tissue content strongly affected the expression of leptin and other genes related to a response to excessive feed intake. The findings indicated that modification of genes and miRNAs involved in ECM rearrangements can be essential during fat tissue growth and development in pigs. The identified molecular network within genes and miRNAs that were deregulated depending on the subcutaneous fat level are proposed as candidate factors determining adipogenesis, fatness, and selected fattening characteristics in pigs.

Evaluation of SCD, ACACA and FASN Mutations: Effects on Pork Quality and Other Production Traits in Pigs Selected Based on RNA-Seq Results.

In recent years, pig producers have struggled with the problem of low intramuscular fat levels in pork, which impacts palatability and ultimately meat quality. Reduced levels of intramuscular fat are likely the result of breeding objectives aimed at increasing lean meat content. In this study, three mutations within candidate genes for fat content (SCD, ACACA, and FASN) were selected, based on RNA-seq results and the relationship between polymorphisms in genes related to lipid metabolism, fattening and slaughter characteristics, as well as pork quality, including IMF level, were evaluated to identify selection markers. Moreover, their impact on gene expression was also examined. The PCR-RFLP (polymerase cha- in reaction - restriction fragments length) method was used to establish genotypes and effect sizes of potential genetic markers were estimated using a GLM model. It was identified that a FASN missense variant was positively associated with the expression level of this gene, which suggested its linkage with a mutation having a regulatory function. The association study indicated that the FASN missense variant may play a role in the determination of feed conversion and meat colour. In turn, a mutation in the ACACA gene showed a relationship with IMF content in the Pulawska breed where the differences reached as much as 20%. We suggest considering all three mutations in further studies based on different pig populations due to the crucial role of SCD, ACACA, and FASN genes in lipid metabolism.

The Pituitary Transcriptional Response Related to Feed Conversion in Pigs.

Over the decades, pig breeding objectives have focused on improving the meat content in the carcass without taking into consideration the more effective fattening indicators that affect feed conversion. At present, pig growth traits associated particularly with animal feeding have become crucial due to their economic significance. This is especially evident in countries where pigs are maintained on large farms. The present study indicates that pituitary differentially expressed genes (DEGs) are activated in response to variable feed conversion (FC) in pigs. The experiment included two native Polish breeds: Pulawska and Zlotnicka White (ZW). The whole pituitary transcriptome was sequenced using next-generation sequencing (NGS) technology. The RNA-seq method identified over 500 and 300 DEGs in the pituitaries of the ZW and the Pulawska pig populations, respectively, that were associated with hormonal regulation, notch signaling, and Wnt pathways. Lower FC in the ZW pigs favoured increased fat content in the body and significantly higher prolactin expression. The obtained results indicate that low FC values in pigs are related to slower growth or increased fat content, which suggests various pituitary responses. Therefore, the identified candidate genes were not directly associated with feed conversion values but with other factors. However, the present study delivers new insights into pituitary regulation in pigs.

Association of Ghrelin Gene Polymorphisms with Fattening Traits and Feed Intake in Pig: A Preliminary Study.

Numerous studies have been conducted to explain the biological functions and mechanism of ghrelin (GHRL) action in animals. However, the exact role of ghrelin in the regulation of growth and development in pigs is still unclear. The ghrelin gene is considered to be a good candidate marker for the identification of economically important traits in pig production such as feed intake, growth or carcass quality. The objectives of the present study were to investigate the regulatory regions and coding sequence of the porcine GHRL gene and determine the effect of polymorphisms at the ghrelin gene locus on selected fattening traits. Data were obtained from 346 gilts (pure breeds: Landrace, 188; Duroc, 74; Pietrain, 84). The PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method was used to detect polymorphisms within GHRL. Three polymorphisms were found, one in the promoter region (c.-93A>G) and two in the 3'UTR sequence (g.4428T>C; g.4486C>T). A significant (p ≤ 0.01) additive effect on daily gain (negative) and age at slaughter (positive) was obtained at the locus c.-93A>G. However, the most promising mutation was at the locus g.4486C > T, which is associated with total feed intake. Overall, the described GHRL polymorphisms may be useful as molecular markers in pig selection but future studies are required.

Functional Analysis of Genes Involved in Glycerolipids Biosynthesis (GPAT1 and GPAT2) in Pigs.

Glycerol-3-phosphate acyltransferase (GPAT) enzymes catalyze the first step in triacylglycerol (TAG) synthesis. Genes that belong to the GPAT family are potential genetic markers for intramuscular fat content (IMF) content and thus meat quality. The objective of this study was to analyze the expression of GPAT1 and GPAT2 genes in musculus longissimus lumborum, liver and subcutaneous fat of various breeds of pigs. Furthermore, correlations between the genes' expression abundance and utility traits, meat quality and meat texture parameters of pork were determined. The results obtained showed significant differences in the mRNA level of GPAT1 between analyzed tissues and breeds. The highest expression of GPAT1 gene was observed in liver tissue (p ≤ 0.01). Furthermore, significantly higher GPAT1 transcript level in the m. longissimus lumborum was observed for duroc in comparison to other analyzed breeds (p ≤ 0.05). Expression of the GPAT2 gene was shown only in the liver tissues, however statistically significant differences between the analyzed breeds were not observed. Correlation analysis confirmed the highest association between GPAT2 gene expression level in liver and cohesiveness and resilience traits of m. longissimus lumborum (p ≤ 0.01).

The effect of QTL-rich region polymorphisms identified by targeted DNA-seq on pig production traits.

The aim of the present study was to analyse the effect of PLCD4, PECR, FN1 and PNKD mutations on pig productive traits and tested the usefulness of targeted enrichment DNA sequencing method as tool for preselection of genetic markers. The potential genetic markers for pig productive traits were identified by using targeted enrichment DNA sequencing of chromosome 15 region that is QTL-rich. The selected mutations were genotyped by using HRM, Sanger sequencing and PCR-ACRS methods. The association study was performed by using GLM model in SAS program and included over 500 pigs of 5 populations maintained in Poland. The variation (C/T) of PLCD4 gene affected feed conversion, intramuscular fat and water exudation. The PNKD mutations were associated with texture parameters measured after cooking. In turn, the variation rs792423408 (C/T) in the FN1 gene influenced toughness measured in semimembranosus muscle and growth traits that was observed particularly in Duroc breed. Summarizing, the investigated gene variants delivered valuable information that could be used during developing SNP microarray for genomic estimated breeding value procedure in pigs. Moreover, the study showed that the TEDNA-seq method could be used to preselect the molecular markers associated with pig traits. However, the further association study that included large number animal populations is necessary.

A comprehensive transcriptome analysis of skeletal muscles in two Polish pig breeds differing in fat and meat quality traits.

Pork is the most popular meat in the world. Unfortunately, the selection pressure focused on high meat content led to a reduction in pork quality. The present study used RNA-seq technology to identify metabolic process genes related to pork quality traits and fat deposition. Differentially expressed genes (DEGs) were identified between pigs of Pulawska and Polish Landrace breeds for two the most important muscles (semimembranosus and longissimus dorsi). A total of 71 significant DEGs were reported: 15 for longissimus dorsi and 56 for semimembranosus muscles. The genes overexpressed in Pulawska pigs were involved in lipid metabolism (APOD, LXRA, LIPE, AP2B1, ENSSSCG00000028753 and OAS2) and proteolysis (CST6, CTSD, ISG15 and UCHL1). In Polish Landrace pigs, genes playing a role in biological adhesion (KIT, VCAN, HES1, SFRP2, CDH11, SSX2IP and PCDH17), actin cytoskeletal organisation (FRMD6, LIMK1, KIF23 and CNN1) and calcium ion binding (PVALB, CIB2, PCDH17, VCAN and CDH11) were transcriptionally more active. The present study allows for better understanding of the physiological processes associated with lipid metabolism and muscle fiber organization. This information could be helpful in further research aiming to estimate the genetic markers.

Screening for candidate genes related with histological microstructure, meat quality and carcass characteristic in pig based on RNA-seq data.

OBJECTIVE: The aim of the present study was to identify genetic variants based on RNA-seq data, obtained via transcriptome sequencing of muscle tissue of pigs differing in muscle histological structure, and to verify the variants' effect on histological microstructure and production traits in a larger pig population. METHODS: RNA-seq data was used to identify the panel of single nucleotide polymorphisms (SNPs) significantly related with percentage and diameter of each fiber type (I, IIA, IIB). Detected polymorphisms were mapped to quantitative trait loci (QTLs) regions. Next, the association study was performed on 944 animals representing five breeds (Landrace, Large White, Pietrain, Duroc, and native Pulawska breed) in order to evaluate the relationship of selected SNPs and histological characteristics, meat quality and carcasses traits. RESULTS: Mapping of detected genetic variants to QTL regions showed that chromosome 14 was the most overrepresented with the identification of four QTLs related to percentage of fiber types I and IIA. The association study performed on a 293 longissimus muscle samples confirmed a significant positive effect of transforming acidic coiled-coil-containing protein 2 (TACC2) polymorphisms on fiber diameter, while SNP within forkhead box O1 (FOXO1) locus was associated with decrease of diameter of fiber types IIA and IIB. Moreover, subsequent general linear model analysis showed significant relationship of FOXO1, delta 4-desaturase, sphingolipid 1 (DEGS1), and troponin T2 (TNNT2) genes with loin 'eye' area, FOXO1 with loin weight, as well as FOXO1 and TACC2 with lean meat percentage. Furthermore, the intramuscular fat content was positively associated (p<0.01) with occurrence of polymorphisms within DEGS1, TNNT2 genes and negatively with occurrence of TACC2 polymorphism. CONCLUSION: This study's results indicate that the SNP calling analysis based on RNA-seq data can be used to search candidate genes and establish the genetic basis of phenotypic traits. The presented results can be used for future studies evaluating the use of selected SNPs as genetic markers related to muscle histological profile and production traits in pig breeding.

Deep sequencing of a QTL-rich region spanning 128-136Mbp of pig chromosome 15.

The present study shows the characterization of the chromosome 15 (SSC15) region that is highly rich in quantitative traits loci (QTLs) associated with pork quality, growth performance, fat and meat carcass contents. The analytic method that was utilized included targeted enrichment DNA sequencing and RNA hybridisation probes. The research included two pig breeds (Pulawska and Polish Landrace) that are significantly different in terms of carcass and meat quality features. Filtered sequences were aligned to the Sscrofa10.2 assembly genome with the STAR aligner and GATK HaplotypeCaller was used for identified gene variants in SSC15 region. In Pulawska pigs, which were characterized by high meat quality, mutations were predominantly observed in non-coding regions such as introns and intergenic regions. The highest over 50% frequencies of alternate alleles were identified in the introns of TNS1, VIL1 and USP37 genes. In the upstream gene regions of the Polish Landrace pigs, were observed more mutations than in the upstream gene regions of Pulawska. The present study showed interesting gene variant panel that could be analyzed in the further association studies in order to understand the impact on important productive pig traits.

Analysis of the associations between polymorphisms in GNAS complex locus and growth, carcass and meat quality traits in pigs.

Imprinted genes are interesting candidates for marker assisted selection in farm animals. One of them-GNAS complex locus is engaged in obesity pathogenesis in humans and mice. In our study, we identified new polymorphism in porcine GNAS gene (variable number of CT repeats, accession number: rs196952953) and found that this polymorphism is in linkage disequilibrum with GNAS AM490165:g.324C>T. Statistical analysis (GLM procedure), performed on 552 animals (Large White n = 258 and Landrace n = 269), revealed that deduced haplotypes and GNAS AM490165:g.324C>T are associated with growth performance and a few carcass traits, but not with feed intake. We observed significant additive effects of GNAS AM490165:g.324C>T genotype and haplotype 2 (C/278 bp) on test daily gain (TDG), average daily gain (ADG), number of days on test, age of the slaughter (P < 0.01) and FCR ratio (P < 0.05). Animals with two copies of C/278 haplotype had significantly higher: TDG, ADG, lower feed:gain ratio and faster reached the weight of 100 kg. When carcass traits were considered, significant associations between GNAS AM490165:g.324C>T polymorphism, haplotype 2 (C/278) and weight of ham with and without backfat and skin (WH) (WH2), length of the carcass, height and the width of the loin, meat percentage, weight of the main cuts were identified. The significant dominance effects of GNAS AM490165:g.324C>T polymorphism and haplotype 2 on WH and WH2 were observed (P < 0.05). When the two breeds were analyzed separately significant associations were observed for most of the traits in Landrace while in Large White the same trends were present but the differences were mostly not significant. Among meat quality traits we found significant association between haplotype and IMF content in Landrace (P < 0.03). Our results show for the first time that GNAS complex locus may modulate economically important traits in pigs.

Effect of EGF, AREG and LIF genes polymorphisms on reproductive traits in pigs.

The aim of our study was to determine the associations between mutations in the EGF, AREG, LIF genes and the reproductive traits in pigs. We examined sows of two Polish breeds, which are used as a dam line in Polish breeding: Large White and Landrace. The reproductive traits investigated were: number of piglets born alive (NBA), number of alive piglets on 21st day (N21), age of sows at first farrowing (AFF) and interval between two successive litters (IBTSL). Polymorphisms in AREG and LIF (C4950G) genes were detected by using PCR-RFLP methods, with restriction enzymes StyI and DraIII, respectively, in EGF gene by using PCR method, while C6988T in LIF gene by PCR-SSCP methods. The statistically significant differences were found at the locus EGF: sows with BB genotype showed higher number of piglets born alive (NBA) and higher number of alive piglets on 21st day (N21) (P<0.01 and P<0.05) when compared to the other genotypes. At the LIF1 locus sows of AA genotype had higher number of NBA (P<0.01) when compared to BB genotype, and at the LIF3 sows of BB had better results for this trait when compared to AA and AB genotype (P<0.01 and P<0.05). In our study, we obtained also statistically significant results for association between AREG gene and reproductive traits. In parities 2 to 4, statistically significant differences were observed between sows of A1A1 and A1A2 genotype of the AREG gene for NBA (P<0.05) and between AA and BB genotypes of the LIF1 gene for NBA and N21 (P<0.01 and P<0.05).