L-Carnitine sustainably affects bioenergetic profile of bovine blastocysts and transcriptome profile of elongation-stage embryos.

In brief: In the present study the sustainable effect of L-carnitine during the culture period on the post-transfer development was investigated. Taken together, we uncovered direct effects of L-carnitine on the bioenergetic profile of day 7 blastocysts along with sustainable effects on mtDNA copy numbers and transcriptome profile of bovine day 14 embryos. Abstract: L-Carnitine (LC) is known to play key roles in lipid metabolism and antioxidative activity, implicating enhanced cryotolerance of bovine blastocysts. However, sustainability of LC supplementation during culture period on preimplantation development beyond the blastocyst stage has not been investigated so far. Therefore, all embryos were cultured under fatty acid-free conditions, one group with LC (LC embryos) and the control group without LC (control) supplementation. Transfer to recipients was conducted on day 6. Elongation-stage embryos were recovered on day 14; metrics of embryo recollection, developmental rates as regards early elongation-stage as well as mean embryo length did not differ between the groups. Gene expression analyses via NGS revealed 341 genes to be differentially regulated between elongation-stage embryos derived from LC supplementation compared to controls. These played mainly a role in molecular functions and biological processes like oxidoreductase activity, ATP-dependent activity, cellular stress, and respiration. Pathways like oxidative phosphorylation and thermogenesis, extracellular matrix receptor signaling, PI3K-Akt, and focal adhesion were affected by differentially regulated genes. Moreover, all DEGs located on the mitochondria were significantly downregulated in LC embryos, being in line with lower mitochondrial copy number and mtDNA integrity compared to the control group. Finally, we uncovered alterations of the bioenergetic profile on day 7 as a consequence of LC supplementation for the first time, revealing significantly higher oxygen consumption rates, ATP linked respiration and spare capacity for LC embryos. In summary, we uncovered direct effects of LC supplementation during the culture period on the bioenergetic profile along with sustainable effects on mtDNA copy numbers and transcriptome profile of bovine day 14 embryos.

Multivariate genome-wide associations for immune traits in two maternal pig lines.

BACKGROUND: Immune traits are considered to serve as potential biomarkers for pig's health. Medium to high heritabilities have been observed for some of the immune traits suggesting genetic variability of these phenotypes. Consideration of previously established genetic correlations between immune traits can be used to identify pleiotropic genetic markers. Therefore, genome-wide association study (GWAS) approaches are required to explore the joint genetic foundation for health biomarkers. Usually, GWAS explores phenotypes in a univariate (uv), trait-by-trait manner. Besides two uv GWAS methods, four multivariate (mv) GWAS approaches were applied on combinations out of 22 immune traits for Landrace (LR) and Large White (LW) pig lines. RESULTS: In total 433 (LR: 351, LW: 82) associations were identified with the uv approach implemented in PLINK and a Bayesian linear regression uv approach (BIMBAM) software. Single Nucleotide Polymorphisms (SNPs) that were identified with both uv approaches (n = 32) were mostly associated with immune traits such as haptoglobin, red blood cell characteristics and cytokines, and were located in protein-coding genes. Mv GWAS approaches detected 647 associations for different mv immune trait combinations which were summarized to 133 Quantitative Trait Loci (QTL). SNPs for different trait combinations (n = 66) were detected with more than one mv method. Most of these SNPs are associated with red blood cell related immune trait combinations. Functional annotation of these QTL revealed 453 immune-relevant protein-coding genes. With uv methods shared markers were not observed between the breeds, whereas mv approaches were able to detect two conjoint SNPs for LR and LW. Due to unmapped positions for these markers, their functional annotation was not clarified. CONCLUSIONS: This study evaluated the joint genetic background of immune traits in LR and LW piglets through the application of various uv and mv GWAS approaches. In comparison to uv methods, mv methodologies identified more significant associations, which might reflect the pleiotropic background of the immune system more accurately. In genetic research of complex traits, the SNP effects are generally small. Furthermore, one genetic variant can affect several correlated immune traits at the same time, termed pleiotropy. As mv GWAS methods consider strong dependencies among traits, the power to detect SNPs can be boosted. Both methods revealed immune-relevant potential candidate genes. Our results indicate that one single test is not able to detect all the different types of genetic effects in the most powerful manner and therefore, the methods should be applied complementary.

Comparison of the choice of animals for re-sequencing in two maternal pig lines.

Next-generation sequencing is a promising approach for the detection of causal variants within previously identified quantitative trait loci. Because of the costs of re-sequencing experiments, this application is currently mainly restricted to subsets of animals from already genotyped populations. Imputation from a lower to a higher marker density could represent a useful complementary approach. An analysis of the literature shows that several strategies are available to select animals for re-sequencing. This study demonstrates an animal selection workflow under practical conditions. Our approach considers different data sources and limited resources such as budget and availability of sampling material. The workflow combines previously described approaches and makes use of genotype and pedigree information from a Landrace and Large White population. Genotypes were phased and haplotypes were accurately estimated with AlphaPhase. Then, AlphaSeqOpt was used to optimize selection of animals for re-sequencing, reflecting the existing diversity of haplotypes. AlphaSeqOpt and ENDOG were used to select individuals based on pedigree information and by taking into account key animals that represent the genetic diversity of the populations. After the best selection criteria were determined, a subset of 57 animals was selected for subsequent re-sequencing. In order to evaluate and assess the advantage of this procedure, imputation accuracy was assessed by setting a set of single nucleotide polymorphism (SNP) chip genotypes to missing. Accuracy values were compared to those of alternative selection scenarios and the results showed the clear benefits of a targeted selection within this practical-driven approach. Especially imputation of low-frequency markers benefits from the combined approach described here. Accuracy was increased by up to 12% compared to a randomized or exclusively haplotype-based selection of sequencing candidates.

Genetic parameters of immune traits for Landrace and Large White pig breeds.

Improving the immunocompetence towards pathogens represents a desirable objective of breeding strategies to increase resilience. However, the immune system is complex and the genetic foundation of the underlying components is not yet clarified. In the present study, we focused on 22 blood parameters of 1,144 Landrace (LR) and Large White (LW) piglets at the age of 6-7 weeks. The immune profiles covered immune cells, red blood cell characteristics and cytokines. Genetic parameters based on pedigree information along with possible environmental effects were estimated. Litter effects play an important role in the expression of immune parameters of their young progenies. Hence, litter impacts on the piglet's immune profile including the immune parameters of the dam itself were investigated by different models. To incorporate the complexity of the immune network, the data were further investigated with a principal component analysis. Immune traits showed low to high breed-specific heritabilities (h2 ). Strong positive rg were estimated among red blood cell characteristics (0.77-0.99) and among cytokines (0.48-0.99). Neutrophils and lymphocytes illustrated a high negative rg (-0.96 to -0.98). The litter impact on piglet's immunity was examined and strengthened already observed breed differences. In LR, h2 (0.22-0.15) and litter effect (c2 ) (0.52-0.44) for IFN-γ decreased after statistical consideration of maternal impact. In LW, a decrease in h2 (0.32-0.18) for IFN-γ and an increase in c2 (0.54-0.56) were observed. Here, sufficient correlations were detected within various immune traits and functional biological networks of principal components. Most immune traits are heritable and are promising to cover global breed-specific immunocompetence in pigs. The analysis of immune traits has to be extended in order to find an optimal range and to characterize relationships between immunity and performance to gain an improved immune system without accidental losses in productivity.

Oregano essential oil showed limited effects on pigs' carcass quality and haematology whereas a transcriptome analysis revealed significant modulations in the jejunum and the ileum.

Pig production depends on a health and performance balance. An approach to improve intestinal health is the oregano essential oil (OEO) supplementation within a conventional diet. Intestinal integrity regulating effects, for example gene expression, of some feed ingredients are important key factors for that balance. We hypothesized that OEO affects the expression of genes associated with pigs' intestinal integrity. In four trials, a total of 86 pigs have been used. From weaning, the 'treated' group (n = 42) was additionally fed an oregano flavour additive [1500 mg/kg (7.5% pure OEO)] within the basal diet. The 'control' group (n = 44) was kept under identical environmental conditions, except the OEO. At age of 6 months, pigs were slaughtered with an average weight of 111.1 ± 10.9 kg. In addition to automatically generated 'Fat-o-Meter' (AutoFOM) data, carcass quality factors have been measured manually. Valuable cuts of meat, such as ham and belly, were significantly reduced in the OEO group. Effects of OEO on pigs' haematologic parameters were very limited. For transcriptome analysis, the most interesting microarray expression results have been listed in a table (topTable). Selected genes were technically validated by qPCR. As a result, few significant differences in animal development and meat quality have been found between the OEO treated and the control group. Depending on OEO supplementation, we found 93 differently regulated genes in the jejunal tissue (70 up, 23 down) and 60 in the ileal tissue (48 up, 12 down). Just three genes (GRIN3B [glutamate ionotropic receptor NMDA type subunit 3B], TJP1/ZO-1 [tight junction protein ZO-1] and one uncharacterized gene) were affected by OEO both in jejunum and ileum. qPCR validation revealed AKT serine/threonine kinase 3 (AKT3), Interferon (IFN) -ε, -ω, tight junction protein (TJP1)/ZO-1 (ZO-1) to be upregulated in the jejunum and C-C motif chemokine ligand 21 (CCL21) was upregulated in the ileum of pigs that were supplemented with OEO. OEO supplementation had limited effects on pigs' performance traits. However, we were able to demonstrate that OEO alters the expression of genes associated with adaptive immune response in pigs' small intestine. These findings help to explain OEOs' beneficial impact on pigs' intestinal integrity.

Genome-wide associations for immune traits in two maternal pig lines.

BACKGROUND: In recent years, animal welfare and health has become more and more important in pig breeding. So far, numerous parameters have been considered as important biomarkers, especially in the immune reaction and inflammation. Previous studies have shown moderate to high heritabilities in most of these traits. However, the genetic background of health and robustness of pigs needs to be extensively clarified. The objective of this study was to identify genomic regions with a biological relevance for the immunocompetence of piglets. Genome-wide Association Studies (GWAS) in 535 Landrace (LR) and 461 Large White (LW) piglets were performed, investigating 20 immune relevant traits. Besides the health indicators of the complete and differential blood count, eight different cytokines and haptoglobin were recorded in all piglets and their biological dams to capture mediating processes and acute phase reactions. Additionally, all animals were genotyped using the Illumina PorcineSNP60v2 BeadChip. RESULTS: In summary, GWAS detected 25 genome-wide and 452 chromosome-wide significant SNPs associated with 17 immune relevant traits in the two maternal pig lines LR and LW. Only small differences were observed considering the maternal immune records as covariate within the statistical model. Furthermore, the study identified across- and within-breed differences as well as relevant candidate genes. In LR more significant associations and related candidate genes were detected, compared with LW. The results detected in LR and LW are partly in accordance with previously identified quantitative trait loci (QTL) regions. In addition, promising novel genomic regions were identified which might be of interest for further detailed analysis. Especially putative pleiotropic regions on SSC5, SSC12, SSC15, SSC16 and SSC17 are of major interest with regard to the interacting structure of the immune system. The comparison with already identified QTL gives indications on interactions with traits affecting piglet survival and also production traits. CONCLUSION: In conclusion, results suggest a polygenic and breed-specific background of immune relevant traits. The current study provides knowledge about regions with biological relevance for health and immune traits. Identified markers and putative pleiotropic regions provide first indications in the context of balancing a breeding-based modification of the porcine immune system.

Genomic background and genetic relationships between boar taint and fertility traits in German Landrace and Large White.

BACKGROUND: Due to ethical reasons, surgical castration of young male piglets in their first week of life without anesthesia will be banned in Germany from 2021. Breeding against boar taint is already implemented in sire breeds of breeding organizations but in recent years a low demand made this trait economically less important. The objective of this study was to estimate heritabilities and genetic relationships between boar taint compounds androstenone and skatole and maternal/paternal reproduction traits in 4'924 Landrace (LR) and 4'299 Large White (LW) animals from nucleus populations. Additionally, genome wide association analysis (GWAS) was performed per trait and breed to detect SNP marker with possible pleiotropic effects that are associated with boar taint and fertility. RESULTS: Estimated heritabilities (h2) were 0.48 (±0.08) for LR (0.39 ± 0.07 for LW) for androstenone and 0.52 (±0.08) for LR (0.32 ± 0.07 for LW) for skatole. Heritabilities for reproduction did not differ between breeds except age at first insemination (LR: h2 = 0.27 (±0.05), LW: h2 = 0.34 (±0.05)). Estimates of genetic correlation (rg) between boar taint and fertility were different in LR and LW breeds. In LR an unfavorable rg of 0.31 (±0.15) was observed between androstenone and number of piglets born alive, whereas this rg in LW (- 0.15 (±0.16)) had an opposite sign. A similar breed-specific difference is observed between skatole and sperm count. Within LR, the rg of 0.08 (±0.13) indicates no relationship between the traits, whereas the rg of - 0.37 (±0.14) in LW points to an unfavorable relationship. In LR GWAS identified QTL regions on SSC5 (21.1-22.3 Mb) for androstenone and on SSC6 (5.5-7.5 Mb) and SSC14 (141.1-141.6 Mb) for skatole. For LW, one marker was found on SSC17 at 48.1 Mb for androstenone and one QTL on SSC14 between 140.5 Mb and 141.6 Mb for skatole. CONCLUSION: Knowledge about such genetic correlations could help to balance conventional breeding programs with boar taint in maternal breeds. QTL regions with unfavorable pleiotropic effects on boar taint and fertility could have deleterious consequences in genomic selection programs. Constraining the weighting of these QTL in the genomic selection formulae may be a useful strategy to avoid physiological imbalances.

MicroRNAs: tiny molecules with a significant role in mammalian follicular and oocyte development.

The genetic regulation of female fertility (follicular development, oocyte maturation and early preimplantation embryo development) involves the spatio-temporal regulation of those genes that play key roles in various stages of the female reproductive axis. MicroRNAs (miRNAs), a class of small non-coding RNAs, are known to regulate the expression of a large proportion of such genes. In recent decades, multiple studies have aimed to determine the roles of these non-coding RNAs in mammalian follicular development, oocyte growth and embryo development. These studies have applied a variety of approaches, including conditional knockout of miRNA biogenesis genes, high-throughput sequencing technologies for pattern recognition in miRNA expression and loss- and gain-of-function of miRNAs in various animal models. In addition to the cellular miRNAs, a large variety of RNAs are found in circulation, being coupled with extracellular vesicles, proteins and lipids. Because of their potential as diagnostic markers for abnormal physiologies, there is increasing interest in the identification of extracellular miRNAs in various biological fluids and spent in vitro culture media. This review focuses on studies addressing the expression and potential role of cellular and extracellular miRNAs in mammalian follicular cell physiology and subsequent ovarian functionality and oocyte maturation.

Deciphering transcriptome profiles of peripheral blood mononuclear cells in response to PRRSV vaccination in pigs.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important viral diseases affecting swine industry worldwide. Despite routine farm vaccination, effective control strategies for PRRS remained elusive which underscores the need for in-depth studies to gain insight into the host immune response to vaccines. The current study aimed to investigate transcriptional responses to PRRS Virus (PRRSV) vaccine in the peripheral blood mononuclear cells (PBMCs) within 3 days following vaccination in German Landrace pigs. RESULTS: Transcriptome profiling of PBMCs from PRRSV vaccinated and age-matched unvaccinated pigs at right before (0 h), and at 6, 24 and 72 h after PRRSV vaccination was performed using the Affymetrix gene chip porcine gene 1.0 st array. Comparison of PBMCs transcriptome profiles between vaccinated and unvaccinated pigs revealed a distinct host innate immune transcriptional response to PRRSV vaccine. There was a significant temporal variation in transcriptional responses of PRRSV vaccine in PBMCs accounting 542, 2,263 and 357 differentially expressed genes (DEGs) at 6, 24 and 72 h post vaccination, respectively compared to the time point before vaccination (controls). Gene ontology analysis revealed the involvement of these DEGs in various biological process including innate immune response, signal transduction, positive regulation of MAP kinase activity, TRIF-dependent toll-like receptor signaling pathway, T cell differentiation and apoptosis. Immune response specific pathways such as cytokine-cytokine receptor interaction, chemokine signaling pathway, signal transduction, JAK-STAT pathway and regulation, TRAF6 mediated induction of NF-kB and MAPK, the NLRP3 inflammasome, endocytosis and interferon signaling were under regulation during the early stage of PRRSV vaccination. Network enrichment analysis revealed APP, TRAF6, PIN1, FOS, CTNNB1, TNFAIP3, TIP1, CDKN1, SIRT1, ESR1 and HDAC5 as the highly interconnected hubs of the functional network of PRRSV vaccine induced transcriptome changes in PBMCs. CONCLUSIONS: This study showed that a massive gene expression change occurred in PBMCs following PRRSV vaccination in German Landrace pigs. Within first 3 days of vaccine exposure, the highest transcript abundance was observed at 24 h after vaccination compared to that of control. Results of this study suggest that APP, TRAF6, PIN1, FOS, CDKN1A and TNFAIP3 could be considered as potential candidate genes for PRRSV vaccine responsiveness.

Integrative Analysis of Metabolomic, Proteomic and Genomic Data to Reveal Functional Pathways and Candidate Genes for Drip Loss in Pigs.

The aim of this study was to integrate multi omics data to characterize underlying functional pathways and candidate genes for drip loss in pigs. The consideration of different omics levels allows elucidating the black box of phenotype expression. Metabolite and protein profiling was applied in Musculus longissimus dorsi samples of 97 Duroc × Pietrain pigs. In total, 126 and 35 annotated metabolites and proteins were quantified, respectively. In addition, all animals were genotyped with the porcine 60 k Illumina beadchip. An enrichment analysis resulted in 10 pathways, amongst others, sphingolipid metabolism and glycolysis/gluconeogenesis, with significant influence on drip loss. Drip loss and 22 metabolic components were analyzed as intermediate phenotypes within a genome-wide association study (GWAS). We detected significantly associated genetic markers and candidate genes for drip loss and for most of the metabolic components. On chromosome 18, a region with promising candidate genes was identified based on SNPs associated with drip loss, the protein "phosphoglycerate mutase 2" and the metabolite glycine. We hypothesize that association studies based on intermediate phenotypes are able to provide comprehensive insights in the genetic variation of genes directly involved in the metabolism of performance traits. In this way, the analyses contribute to identify reliable candidate genes.

Genome-wide association analyses for boar taint components and testicular traits revealed regions having pleiotropic effects.

BACKGROUND: The aim of this study was to perform a genome-wide association analyses (GWAS) for androstenone, skatole and indole in different Pietrain sire lines and compare the results with previous findings in purebred populations. Furthermore, the genetic relationship of androstenone and skatole were investigated with respect to pleiotropy. In order to characterize the performance of intact boars, crossbred progenies of 136 Pietrain boars mated to crossbred sows from three different breeding companies were tested on four test stations. A total of 598 boars were performance tested according to the rules of stationary performance testing in Germany. Beside common fattening and carcass composition traits, the concentrations of the boar taint components and testicular size parameters were recorded. All boars were genotyped with the PorcineSNP60 Illumina BeadChip. The GWAS were performed using the whole data set as well as in sub groups according to the line of origin. Besides an univariate GWAS approach, principal component (PC) techniques were applied to identify common expression pattern affecting the biosynthesis and the metabolism of androstenone. RESULTS: In total, 33 SNPs were significantly associated with at least one of the boar taint components. Only one SNP was identified being significant in both subgroups. The analyses of the testes size parameters revealed 31 significant associations. The numbers of significant SNPs within the genetic groups evidenced the strong population specific effects. A multivariate approach using PC revealed 33 significant associations for five different PC. CONCLUSIONS: Based on Pietrain sired cross bred boars, the mayor objective of our study was to identify QTL for boar taint components and to detect pleiotropy among boar taint and testes traits. The high number of identified QTL revealed that boar taint traits are influenced by a large number of loci. Analyzing pleiotropy allowed identifying a QTL affecting androstenone and the gonasomatic index. In this region, QTL for ovulation rate and age at puberty of sows have been described in literature. This supports the physiological findings that the androstenone level of boars and reproduction performance of sows might be linked by an antagonistic relationship.

Identification of gene co-expression clusters in liver tissues from multiple porcine populations with high and low backfat androstenone phenotype.

BACKGROUND: Boar taint is principally caused by accumulation of androstenone and skatole in adipose tissues. Studies have shown high heritability estimates for androstenone whereas skatole production is mainly dependent on nutritional factors. Androstenone is a lipophilic steroid mainly metabolized in liver. Majority of the studies on hepatic androstenone metabolism focus only on a single breed and very few studies account for population similarities/differences in gene expression patterns. In this work, we concentrated on population similarities in gene expression to identify the common genes involved in hepatic androstenone metabolism of multiple pig populations. Based on androstenone measurements, publicly available gene expression datasets from three porcine populations were compiled into either low or high androstenone dataset. Gene expression correlation coefficients from these datasets were converted to rank ratios and joint probabilities of these rank ratios were used to generate dataset specific co-expression clusters. Finally, these networks were clustered using a graph clustering technique. RESULTS: Cluster analysis identified a number of statistically significant co-expression clusters in the dataset. Further enrichment analysis of these clusters showed that one of the clusters from low androstenone dataset was highly enriched for xenobiotic, drug, cholesterol and lipid metabolism and cytochrome P450 associated metabolism of drugs and xenobiotics. Literature references revealed that a number of genes in this cluster were involved in phase I and phase II metabolism. Physical and functional similarity assessment showed that the members of this cluster were dispersed across multiple clusters in high androstenone dataset, possibly indicating a weak co-expression of these genes in high androstenone dataset. CONCLUSIONS: Based on these results we hypothesize that majority of the genes in this cluster forms a signature co-expression cluster in low androstenone dataset in our experiment and that majority of the members of this cluster might be responsible for hepatic androstenone metabolism across all the three populations used in our study. We propose these results as a background work towards understanding breed similarities in hepatic androstenone metabolism. Additional large scale experiments using data from multiple porcine breeds are necessary to validate these findings.

Sexual dimorphic miRNA-mediated response of bovine elongated embryos to the maternal microenvironment.

A skewed male-to-female ratio in cattle is believed to be due to the biased embryo losses during pregnancy. The changes in biochemical secretion such as miRNAs by the embryo due to altered maternal environment could cause a sex biased selective implantation resulting in a skewed male to female ratio at birth. Nevertheless, it is still not clear whether the male and female embryos could modify their miRNA expression patterns differently in response to altered physiological developmental conditions. Therefore, this study was focused on identifying sex specific miRNA expression patterns induced in the embryo during the elongation period in response to the maternal environment. For this, in vitro produced day female and male embryos were transferred to Holsteins Frisian cows and heifers. The elongated female and male embryos were then recovered at day 13 of the gestation period. Total RNA including the miRNAs was isolated from each group of elongated embryo samples were subjected to the next generation miRNA sequencing. Sequence alignment, identification and quantification of miRNAs were done using the miRDeep2 software package and differential miRNA expression analyses were performed using the edgeR bioconductor package. The recovery rate of viable elongating embryos at day 13 of the gestation period was 26.6%. In cows, 2.8 more viable elongating male embryos were recovered than female embryos, while in heifers the sex ratio of the recovered elongating embryos was close to one (1.05). The miRNA analysis showed that 254 miRNAs were detected in both male and female elongated embryos developed either in cows or heifers, of which 14 miRNAs including bta-miR-10b, bta-miR-148a, bta-miR-26a, and bta-miR-30d were highly expressed. Moreover, the expression level of 32 miRNAs including bta-let-7c, bta-let-7b, bta-let-7g, bta-let-7d and bta-let-7e was significantly different between the male and female embryos developed in cows, but the expression level of only 4 miRNAs (bta-miR-10, bta-mR-100, bta-miR-155 and bta-miR-6119-5p) was different between the male and female embryos that were developed in heifers. Furthermore, 19 miRNAs including those involved in cellular energy homeostasis pathways were differentially expressed between the male embryos developed in cows and heifers, but no significantly differentially expressed miRNAs were detected between the female embryos of cows and heifers. Thus, this study revealed that the sex ratio skewed towards males in embryos developed in cows was accompanied by increased embryonic sexual dimorphic miRNA expression divergence in embryos developed in cows compared to those developed in heifers. Moreover, male embryos are more sensitive to respond to the maternal reproductive microenvironment by modulating their miRNA expression.

A genetical genomics approach reveals new candidates and confirms known candidate genes for drip loss in a porcine resource population.

In this study lean meat water-holding capacity (WHC) of a Duroc × Pietrain (DuPi) resource population with corresponding genotypes and transcriptomes was investigated using genetical genomics. WHC was characterized by drip loss measured in M. longissimus dorsi. The 60K Illumina SNP chips identified genotypes of 169 F2 DuPi animals. Whole-genome transcriptomes of muscle samples were available for 132 F2 animals using the Affymetrix 24K GeneChip® Porcine Genome Array. Performing genome-wide association studies of transcriptional profiles, which are correlated with phenotypes, allows elucidation of cis- and trans-regulation. Expression levels of 1,228 genes were significantly correlated with drip loss and were further analyzed for enrichment of functional annotation groups as defined by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. A hypergeometric gene set enrichment test was performed and revealed glycolysis/glyconeogenesis, pentose phosphate pathway, and pyruvate metabolism as the most promising pathways. For 267 selected transcripts, expression quantitative trait loci (eQTL) analysis was performed and revealed a total of 1,541 significant associations. Because of positional accordance of the gene underlying transcript and the eQTL location, it was possible to identify eight eQTL that can be assumed to be cis-regulated. Comparing the results of gene set enrichment and the eQTL detection tests, molecular networks and potential candidate genes, which seemed to play key roles in the expression of WHC, were detected. The α-1-microglobulin/bikunin precursor (AMBP) gene was assumed to be cis-regulated and was part of the glycolysis pathway. This approach supports the identification of trait-associated SNPs and the further biological understanding of complex traits.

The mitochondrial respiration signature of the bovine blastocyst reflects both environmental conditions of development as well as embryo quality.

The major limitation of the widespread use of IVP derived embryos is their consistent deficiencies in vitality when compared with their ex vivo derived counterparts. Although embryo metabolism is considered a useful metric of embryo quality, research connecting mitochondrial function with the developmental capacity of embryos is still lacking. Therefore, the aim of the present study was to analyse bovine embryo respiration signatures in relation to developmental capacity. This was achieved by taking advantage of two generally accepted metrics for developmental capacity: (I) environmental conditions during development (vivo vs. vitro) and (II) developmental kinetics (day 7 vs. day 8 blastocysts). Our study showed that the developmental environment affected total embryo oxygen consumption while different morphokinetics illustrating the embryo qualities correlate with maximal mitochondrial respiration, mitochondrial spare capacity, ATP-linked respiration as well as efficiency of ATP generation. This respiration fingerprint for high embryo quality is reflected by relatively lower lipid contents and relatively higher ROS contents. In summary, the results of the present study extend the existing knowledge on the relationship between bovine embryo quality and the signature of mitochondrial respiration by considering contrasting developmental environments as well as different embryo morphokinetics.

The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events.

At the embryonic level, CRISPR technologies have been used to edit genomes reliably and efficiently in various mammalian models, with Ribonucleoprotein (RNP) electroporation potentially representing a superior delivery method into mammalian zygotes. However, detailed insights of the interactions between varying technical settings as well as the time point of electroporation in a bovine zygote's cell cycle on developmental metrics and the frequency and type of editing events are largely unknown. The present study uncovers that increasing pulse lengths result in higher Full Edit rates, with Mosaicism in Full-Edit embryos being significantly affected by adjusting RNP-electroporation relative to zygote cell cycle. A considerable proportion of Full Edit embryos demonstrated loss-of-heterozygosity after RNP-electroporation prior to S-phase. Some of these loss-of-heterozygosity events are a consequence of chromosomal disruptions along large sections of the target chromosomes making it necessary to check for their presence prior use of this technique in animal breeding. One out of 2 of these loss-of-heterozygosity events, however, was not associated with loss of an entire chromosome or chromosomal sections. Whether analysed loss-of-heterozygosity in these cases, however, was a false negative result due to loss of PCR primer sequences after INDEL formation at the target side or indeed due to interhomolog recombination needs to be clarified in follow up studies since the latter would for sure offer attractive options for future breeding schedules.

Quantitative trait loci analysis for leg weakness-related traits in a Duroc × Pietrain crossbred population.

BACKGROUND: Leg weakness issues are a great concern for the pig breeding industry, especially with regard to animal welfare. Traits associated with leg weakness are partly influenced by the genetic background of the animals but the genetic basis of these traits is not yet fully understood. The aim of this study was to identify quantitative trait loci (QTL) affecting leg weakness in pigs. METHODS: Three hundred and ten F2 pigs from a Duroc × Pietrain resource population were genotyped using 82 genetic markers. Front and rear legs and feet scores were based on the standard scoring system. Osteochondrosis lesions were examined histologically at the head and the condylus medialis of the left femur and humerus. Bone mineral density, bone mineral content and bone mineral area were measured in the whole ulna and radius bones using dual energy X-ray absorptiometry. A line-cross model was applied to determine QTL regions associated with leg weakness using the QTL Express software. RESULTS: Eleven QTL affecting leg weakness were identified on eight autosomes. All QTL reached the 5% chromosome-wide significance level. Three QTL were associated with osteochondrosis on the humerus end, two with the fore feet score and two with the rear leg score. QTL on SSC2 and SSC3 influencing bone mineral content and bone mineral density, respectively, reached the 5% genome-wide significance level. CONCLUSIONS: Our results confirm previous studies and provide information on new QTL associated with leg weakness in pigs. These results contribute towards a better understanding of the genetic background of leg weakness in pigs.

Endocrine Fertility Parameters-Genomic Background and their Genetic Relationship to Boar Taint in German Landrace and Large White.

The surgical castration of young male piglets without anesthesia is no longer allowed in Germany from 2021. One alternative is breeding against boar taint, but shared synthesis pathways of androstenone (AND) and several endocrine fertility parameters (EFP) indicate a risk of decreasing fertility. The objective of this study was to investigate the genetic background between AND, skatole (SKA), and six EFP in purebred Landrace (LR) and Large White (LW) populations. The animals were clustered according to their genetic relatedness because of their different origins. Estimated heritabilities (h2) of AND and SKA ranged between 0.52 and 0.34 in LR and LW. For EFP, h2 differed between the breeds except for follicle-stimulating hormone (FSH) (h2: 0.28-0.37). Both of the breeds showed unfavorable relationships between AND and testosterone, 17-ß estradiol, and FSH. The genetic relationships (rg) between SKA and EFP differed between the breeds. A genome-wide association analysis revealed 48 significant associations and confirmed a region for SKA on S us S crofa chromosome (SSC) 14. For EFP, the results differed between the clusters. In conclusion, rg partly confirmed physiologically expected antagonisms between AND and EFP. Particular attention should be spent on fertility traits that are based on EFP when breeding against boar taint to balance the genetic progress in both of the trait complexes.

Mapping of quantitative trait loci for mycoplasma and tetanus antibodies and interferon-gamma in a porcine F(2) Duroc x Pietrain resource population.

The aim of the present study was to detect quantitative trait loci (QTL) for innate and adaptive immunity in pigs. For this purpose, a Duroc x Pietrain F(2) resource population (DUPI) with 319 offspring was used to map QTL for the immune traits blood antibodies and interferon-gamma using 122 microsatellites covering all autosomes. Antibodies response to Mycoplasma hyopneumoniae and tetanus toxoid vaccine and the interferon-gamma (IFNG) serum concentration were measured at three different time points and were used as phenotypes. The differences of antibodies and interferon concentration between different time points were also used for the linkage mapping. Line-cross and imprinting QTL analysis, including two-QTL, were performed using QTL Express. A total of 30 QTL (12, 6, and 12 for mycoplasma, tetanus antibody, and IFNG, respectively) were identified at the 5% chromosome-wide-level significant, of which 28 were detected by line-cross and 2 by imprinting model. In addition, two QTL were identified on chromosome 5 using the two-QTL approach where both loci were in repulsion phase. Most QTL were detected on pig chromosomes 2, 5, 11, and 18. Antibodies were increased over time and immune traits were found to be affected by sex, litter size, parity, and month of birth. The results demonstrated that antibody and IFNG concentration are influenced by multiple chromosomal areas. The flanking markers of the QTL identified for IFNG on SSC5 did incorporate the position of the porcine IFNG gene. The detected QTL will allow further research in these QTL regions for candidate genes and their utilization in selection to improve the immune response and disease resistance in pig.

Epistatic QTL pairs associated with meat quality and carcass composition traits in a porcine Duroc × Pietrain population.

BACKGROUND: Quantitative trait loci (QTL) analyses in pig have revealed numerous individual QTL affecting growth, carcass composition, reproduction and meat quality, indicating a complex genetic architecture. In general, statistical QTL models consider only additive and dominance effects and identification of epistatic effects in livestock is not yet widespread. The aim of this study was to identify and characterize epistatic effects between common and novel QTL regions for carcass composition and meat quality traits in pig. METHODS: Five hundred and eighty five F2 pigs from a Duroc × Pietrain resource population were genotyped using 131 genetic markers (microsatellites and SNP) spread over the 18 pig autosomes. Phenotypic information for 26 carcass composition and meat quality traits was available for all F2 animals. Linkage analysis was performed in a two-step procedure using a maximum likelihood approach implemented in the QxPak program. RESULTS: A number of interacting QTL was observed for different traits, leading to the identification of a variety of networks among chromosomal regions throughout the porcine genome. We distinguished 17 epistatic QTL pairs for carcass composition and 39 for meat quality traits. These interacting QTL pairs explained up to 8% of the phenotypic variance. CONCLUSIONS: Our findings demonstrate the significance of epistasis in pigs. We have revealed evidence for epistatic relationships between different chromosomal regions, confirmed known QTL loci and connected regions reported in other studies. Considering interactions between loci allowed us to identify several novel QTL and trait-specific relationships of loci within and across chromosomes.