The effect of a strict breeding strategy on overall growth and the prevalence of inherited disorders in the double-muscled Belgian Blue beef breed

Muscular conformation is the main breeding goal in the double-muscled (I)M) Belgian Blue beef breed (BBB). In recent years the muscularity has improved enormously, though at the same time the growth rate has decreased and the prevalence of a number of inherited disorders has increased. Professionals working in the cattle breeding industry are encouraging the development of breeding strategies that will increase overall growth and decrease the amount of inherited disorders. One such breeding strategy that was tested in the field neither reduced the prevalence of inherited disorders nor improved overall growth. It can be concluded that breeding strategies should be based more on the relevant genetic values than on the phenotypic values of the parental generation

Meat quality in the double-muscled Belgian Blue Beef breed

The double-muscled Belgian Blue Beef breed (DM-BBB), being homozygous for the muscular hypertrophy (mh) allele, has meat quality attributes that differ from 'normal' beef cattle. These differences are due to this mutation in the myostatin locus. One of these differences is more tender meat, although this characteristic is in fact not observed in all individual animals. Most animals with this mutation have more tender meat, but some DM-BBB animals, especially those with an extremely muscular conformation, seem to have less tender meat. This is perhaps due to minor genes that influence muscular conformation and are at a higher frequency in these extremely muscular animals. Other genes or mutations also influence meat quality properties. Some are definitely present in DM-BBB; the presence of others is as yet unknown. Meat quality can be improved not only genetically, but also by improving feeding, handling and transportation. In current DM-BBB selection, meat quality is not taken into account. If it is to be considered, then selection should focus on the control of extreme meat quality disorders. Environmental factors may have a greater impact on the overall acceptance of DM-BBB meat

Quantification of Fibronectin 1 (FN1) splice variants, including two novel ones, and analysis of integrins as candidate FN1 receptors in bovine preimplantation embryos

<p>Abstract</p> <p>Background</p> <p>Fibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development.</p> <p>Results</p> <p>RT-qPCR quantification of the FN1 splice isoforms in oocytes, embryos, cumulus cells and adult tissue samples revealed a large variation in overall FN1 expression and in splice variant expression. Moreover, two new FN1 transcript variants were identified, the first one expressed in bovine preimplantation embryos and the second one expressed in cumulus cells.</p> <p>In the search for candidate receptors for the new embryo specific FN1 isoform, RNA expression analysis identified 5 α integrin subunits (ITGA2B, ITGA3, ITGA5, ITGA8, ITGAV) and 2 β integrin subunits (ITGB1 and ITGB3) with a similar or overlapping RNA expression pattern as compared to FN1. But double immunofluorescent stainings could not confirm complete co-localisation between FN1 and one out of 3 selected integrins alpha subunits (ITGA3, ITGA5, ITGAV).</p> <p>Conclusion</p> <p>The existence of a new FN1 transcript variant, specifically expressed in morulae and blastocysts strengthens the idea that FN1 is involved in the process of compaction and blastocyst formation. Analysis of the integrin expression could not identify the binding partner for the embryo specific FN1 transcript variant making further steps necessary for the identification of the FN1 receptor and the downstream effects of FN1-receptor binding.</p

Presence of the ABCB1 (MDR1) deletion mutation causing ivermectin hypersensitivity in certain dog breeds in Belgium

Hypersensitivity to ivermectin and certain other drugs in Collies and related breeds is caused by a 4-base pair deletion mutation in the ABCB1 gene, better known as the MDR1 gene, encoding P-glycoprotein. There is no information available, however, regarding the presence of this mutation in dogs in Belgium. In this study, the ABCB1 genotype was assessed in 92 dogs of breeds suspected to possess the deletion mutation. The results indicated that the mutation was present in the Australian Shepherd, Collie, Shetland Sheepdog and Swiss White Shepherd, but was not detected in the Bearded Collies, Border Collies and German Shepherds of this study, which is in accordance with the findings in similar breed populations of other countries. In Belgium it is therefore important to take the ABCB1 genotype of the breeds involved into account, in order to use drugs in a safe and efficient manner and to improve the selection procedure in dog breeding

Characterization of the genomic region containing the Shadow of Prion Protein (SPRN) gene in sheep

<p>Abstract</p> <p>Background</p> <p>TSEs are a group of fatal neurodegenerative diseases occurring in man and animals. They are caused by prions, alternatively folded forms of the endogenous prion protein, encoded by <it>PRNP</it>. Since differences in the sequence of <it>PRNP </it>can not explain all variation in TSE susceptibility, there is growing interest in other genes that might have an influence on this susceptibility. One of these genes is <it>SPRN</it>, a gene coding for a protein showing remarkable similarities with the prion protein. Until now, <it>SPRN </it>has not been described in sheep, a highly relevant species in prion matters.</p> <p>Results</p> <p>In order to characterize the genomic region containing <it>SPRN </it>in sheep, a BAC mini-contig was built, covering approximately 200,000 bp and containing the genes <it>ECHS1</it>, <it>PAOX</it>, <it>MTG1</it>, <it>SPRN</it>, <it>LOC619207, CYP2E1 </it>and at least partially <it>SYCE1</it>. FISH mapping of the two most exterior BAC clones of the contig positioned this contig on Oari22q24. A fragment of 4,544 bp was also sequenced, covering the entire <it>SPRN </it>gene and 1206 bp of the promoter region. In addition, the transcription profile of <it>SPRN </it>in 21 tissues was determined by RT-PCR, showing high levels in cerebrum and cerebellum, and low levels in testis, lymph node, jejunum, ileum, colon and rectum.</p> <p>Conclusion</p> <p>Annotation of a mini-contig including <it>SPRN </it>suggests conserved linkage between Oari22q24 and Hsap10q26. The ovine <it>SPRN </it>sequence, described for the first time, shows a high level of homology with the bovine, and to a lesser extent with the human <it>SPRN </it>sequence. In addition, transcription profiling in sheep reveals main expression of <it>SPRN </it>in brain tissue, as in rat, cow, man and mouse.</p

Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A

BACKGROUND: An essential part of using real-time RT-PCR is that expression results have to be normalized before any conclusions can be drawn. This can be done by using one or multiple, validated reference genes, depending on the desired accuracy of the results. In the pig however, very little information is available on the expression stability of reference genes. The aim of this study was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in porcine backfat and longissimus dorsi muscle, both representing an economically important part of a pig's carcass. Because of its multiple functions in fat metabolism and muscle fibre type composition, peroxisome proliferative activated receptor γ coactivator 1α (PPARGC1A) is a very interesting candidate gene for meat quality, and was an ideal gene to evaluate our developed set of reference genes for normalization of mRNA expression data of both tissue types. RESULTS: The mRNA expression stability of 10 reference genes was determined. The expression of RPL13A and SDHA appeared to be highly unstable. After normalization to the geometric mean of the three most stably expressed reference genes (ACTB, TBP and TOP2B), the results not only showed that the mRNA expression of PPARGC1A was significantly higher in each of the longissimus dorsi muscle samples than in backfat (P < 0.05), but also that the expression was significantly higher in the most cranial of the three muscle samples (P < 0.05). CONCLUSION: This study provides a new set of reference genes (ACTB, TBP and TOP2B) suitable for normalization of real-time RT-PCR data of backfat and longissimus dorsi muscle in the pig. The obtained PPARGC1A expression results, after application of this set of reference genes, are a first step in unravelling the PPARGC1A expression pattern in the pig and provide a basis for possible selection towards improved meat quality while maintaining a lean carcass

Comparative analysis of a BAC contig of porcine chromosome 13q31-q32 and human chromosome 3q21-q22

BACKGROUND: The gene(s) encoding the ETEC F4ab/ac receptors, involved in neonatal diarrhoea in pigs (a disease not yet described in humans), is located close to the TF locus on Sscr13. In order to reveal and characterize possible candidate genes encoding these receptors, a porcine physical map of the TF region is indispensable. RESULTS: A contig of 33 BAC clones, covering approximately 1.35 Mb surrounding the TF locus on Sscr13q31-q32, was built by chromosome walking. A total of 22,552 bp from the BAC contig were sequenced and compared with database sequences to identify genes, ESTs and repeat sequences, and to anchor the contig to the syntenic region of the human genome sequence (Hsap3q21-q22). The contig was further annotated based on this human/porcine comparative map, and was also anchored to the Sanger porcine framework map and the integrated map of Sscr13 by RH mapping. CONCLUSION: The annotated contig, containing 10 genes and 2 ESTs, showed a complete conservation of linkage (gene order and orientation) with the human genome sequence, based on 46 anchor points. This underlines the importance of the human/porcine comparative map for the identification of porcine genes associated with genetic defects and economically important traits, and for assembly of the porcine genome sequence

Identification and expression analysis of genes associated with bovine blastocyst formation

<p>Abstract</p> <p>Background</p> <p>Normal preimplantation embryo development encompasses a series of events including first cleavage division, activation of the embryonic genome, compaction and blastocyst formation.</p> <p>First lineage differentiation starts at the blastocyst stage with the formation of the trophectoderm and the inner cell mass. The main objective of this study was the detection, identification and expression analysis of genes associated with blastocyst formation in order to help us better understand this process. This information could lead to improvements of <it>in vitro </it>embryo production procedures.</p> <p>Results</p> <p>A subtractive cDNA library was constructed enriched for transcripts preferentially expressed at the blastocyst stage compared to the 2-cell and 8-cell stage. Sequence information was obtained for 65 randomly selected clones. The RNA expression levels of 12 candidate genes were determined throughout 3 stages of preimplantation embryo development (2-cell, 8-cell and blastocyst) and compared with the RNA expression levels of <it>in vivo </it>"golden standard" embryos using real-time PCR. The RNA expression profiles of 9 (75%) transcripts (<it>KRT18</it>, <it>FN1</it>, <it>MYL6</it>, <it>ATP1B3</it>, <it>FTH1</it>, <it>HINT1</it>, <it>SLC25A5</it>, <it>ATP6V0B</it>, <it>RPL10</it>) were in agreement with the subtractive cDNA cloning approach, whereas for the remaining 3 (25%) (<it>ACTN1</it>, <it>COPE</it>, <it>EEF1A1</it>) the RNA expression level was equal or even higher at the earlier developmental stages compared to the blastocyst stage. Moreover, significant differences in RNA expression levels were observed between <it>in vitro </it>and <it>in vivo </it>produced embryos. By immunofluorescent labelling, the protein expression of KRT18, FN1 and MYL6 was determined throughout bovine preimplantation embryo development and showed the same pattern as the RNA expression analyses.</p> <p>Conclusion</p> <p>By subtractive cDNA cloning, candidate genes involved in blastocyst formation were identified. For several candidate genes, important differences in gene expression were observed between <it>in vivo </it>and <it>in vitro </it>produced embryos, reflecting the influence of the <it>in vitro </it>culture system on the embryonic gene expression. Both RNA and protein expression analysis demonstrated that <it>KRT18</it>, <it>FN1 </it>and <it>MYL6 </it>are differentially expressed during preimplantation embryo development and those genes can be considered as markers for bovine blastocyst formation.</p

Regulatory microRNA network identification in bovine blastocyst development

Mammalian blastocyst formation is characterized by two lineage segregations resulting in the formation of the trophectoderm, the hypoblast, and the epiblast cell lineages. Cell fate determination during these early lineage segregations is associated with changes in the expression of specific transcription factors. In addition to the transcription factor-based control, it has become clear that also microRNAs (miRNAs) play an important role in the post-transcriptional regulation of pluripotency and differentiation. To elucidate the role of miRNAs in early lineage segregation, we compared the miRNA expression in early bovine blastocysts with the more advanced stage of hatched blastocysts. Reverse transcription-quantitative PCR-based miRNA expression profiling revealed eight upregulated miRNAs (miR-127, miR-130a, miR-155, miR-196a, miR-203, miR-28, miR-29c, and miR-376a) and four downregulated miRNAs (miR-135a, miR-218, miR-335, and miR-449b) in hatched blastocysts. Through an integrative analysis of matching miRNA and mRNA expression data, candidate miRNA-mRNA interaction pairs were prioritized for validation. Using an in vitro luciferase reporter assay, we confirmed a direct interaction between miR-218 and CDH2, miR-218 and NANOG, and miR-449b and NOTCH1. By interfering with the FGF signaling pathway, we found functional evidence that miR-218, mainly expressed in the inner cell mass, regulates the NANOG expression in the bovine blastocyst in response to FGF signaling. The results of this study expand our knowledge about the miRNA signature of the bovine blastocyst and of the interactions between miRNAs and cell fate regulating transcription factors

Characterization of the ovine ribosomal protein SA gene and its pseudogenes

Background: The ribosomal protein SA (RPSA), previously named 37-kDa laminin receptor precursor/67-kDa laminin receptor (LRP/LR) is a multifunctional protein that plays a role in a number of pathological processes, such as cancer and prion diseases. In all investigated species, RPSA is a member of a multicopy gene family consisting of one full length functional gene and several pseudogenes. Therefore, for studies on RPSA related pathways/pathologies, it is important to characterize the whole family and to address the possible function of the other RPSA family members. The present work aims at deciphering the RPSA family in sheep. Results: In addition to the full length functional ovine RPSA gene, 11 other members of this multicopy gene family, all processed pseudogenes, were identified. Comparison between the RPSA transcript and these pseudogenes shows a large variety in sequence identities ranging from 99% to 74%. Only one of the 11 pseudogenes, i.e. RPSAP7, shares the same open reading frame (ORF) of 295 amino acids with the RPSA gene, differing in only one amino acid. All members of the RPSA family were annotated by comparative mapping and fluorescence in situ hybridization (FISH) localization. Transcription was investigated in the cerebrum, cerebellum, spleen, muscle, lymph node, duodenum and blood, and transcripts were detected for 6 of the 11 pseudogenes in some of these tissues. Conclusions: In the present work we have characterized the ovine RPSA family. Our results have revealed the existence of 11 ovine RPSA pseudogenes and provide new data on their structure and sequence. Such information will facilitate molecular studies of the functional RPSA gene taking into account the existence of these pseudogenes in the design of experiments. It remains to be investigated if the transcribed members are functional as regulatory non-coding RNA or as functional proteins