Bioinformatics in horses involves the application of computational tools and techniques to analyze and interpret biological data related to equine species. This interdisciplinary field integrates biology, computer science, and information technology to study genetic, genomic, and proteomic information in horses. Bioinformatics can be used to investigate genetic variations, understand disease mechanisms, and assist in the development of targeted therapies and breeding programs. Key areas of focus include genome sequencing, gene expression analysis, and the identification of genetic markers associated with specific traits or conditions. This page compiles peer-reviewed research studies and scholarly articles that explore the application and impact of bioinformatics on equine genetics, health, and breeding.
Paulis M, Moralli D, Bensi M, De Carli L, Raimondi E.Tigger elements are human DNA transposons homologous to the pogo element of Drosophila melanogaster. They contain an open reading frame for a transposase very similar to the major mammalian centromere protein CENP-B. We found in the horse genome a DNA element ( Ecatig3) sharing 88% homology with human Tigger3. The presence of Tigger elements in the horse genome confirms previous data that date these elements before the divergence between Perissodactyla and Primates (80-90 Myr ago). Copy number evaluation indicates that the horse element is much more abundant than its human counterpart. Souther...
Chikuni K, Muroya S, Nakajima I.Nucleotide sequences which included the full coding region for three types of myosin heavy chain (MyHC) isoforms were determined from equine skeletal muscles. The deduced amino acid sequences were 1937, 1938, and 1935 residues for the MyHC-2a, -2x, and -slow, respectively. No MyHC-2b isoform was amplified from the equine muscle cDNA except for one pseudogene fragment. One nucleotide was inserted in the coding region of the equine pseudogene product, a minute amount of which was expressed in the skeletal muscle. The 596 bp sequence of the equine MyHC pseudogene was categorized into the MyHC-2b ...
Davis EG, Sang Y, Blecha F.beta-Defensins are cysteine-rich endogenously produced antimicrobial peptides that play an important role in innate immune defense. Although, previous investigations have identified beta-defensins in several mammalian species, no reports have identified equine beta-defensins. Using a strategy of database searching for expressed sequence tags (EST) we identified putative expression of equine beta-defensins in hepatic tissue. Based on this information, sequence specific primers were designed for the equine gene enabling the identification of the full-length cDNA sequence of equine beta-defensin-...
Poloumienko A.A growing body of evidence suggests the involvement of sex chromosome genes in mammalian development. We report the cloning and characterization of the complete coding regions of the bovine Y chromosome ZFY and X chromosome ZFX genes, and partial coding regions of porcine and equine ZFX and ZFY genes. Bovine ZFY and ZFX are highly similar to each other and to ZFX and ZFY from other species. While bovine and human ZFY proteins are both 801 amino acids long, bovine ZFX is 5 amino acids shorter than human ZFX. Like in humans, both bovine ZFY and ZFX contain 13 zinc finger motifs and belong to the...
Raudsepp T, Lee EJ, Kata SR, Brinkmeyer C, Mickelson JR, Skow LC, Womack JE, Chowdhary BP.Development of a dense map of the horse genome is key to efforts aimed at identifying genes controlling health, reproduction, and performance. We herein report a high-resolution gene map of the horse (Equus caballus) X chromosome (ECAX) generated by developing and typing 116 gene-specific and 12 short tandem repeat markers on the 5,000-rad horse x hamster whole-genome radiation hybrid panel and mapping 29 gene loci by fluorescence in situ hybridization. The human X chromosome sequence was used as a template to select genes at 1-Mb intervals to develop equine orthologs. Coupled with our previou...
Anastasiou A, Hall LD.Cramer-Rao theory and computer simulations were used to show that the errors involved in calculating the magnetization and relaxation parameters of a two-component system decrease with: (1) increasing SNR, (2) increasing number of echoes used in the fitting procedure, and (3) increasing ratio of the relaxation times of the two components, T(22)/T(21). Images of bi-compartmental phantoms of known T(2) values were acquired using an optimized imaging sequence, and an optimized fitting algorithm was used to calculate the T(2) values of the two components by fitting the resulting images to a bi-exp...
Chowdhary BP, Bailey E.Analysis of the horse genome is proceeding at a rapid pace. Within a short span of 6-7 years, approximately 1,500 markers have been mapped in horse, of which at least half are genes/ESTs. Health, performance and phenotypic characteristic are of major concern/interest to horse breeders and owners. Current efforts to analyze the equine genome are primarily aimed at developing critical resources (including an advanced gene map) that could readily be used in the near future to i) identify genes and mutations responsible for inherited equine diseases/disorders and to formulate approaches for accura...
Gustafson AL, Tallmadge RL, Ramlachan N, Miller D, Bird H, Antczak DF, Raudsepp T, Chowdhary BP, Skow LC.A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contain...
Frazer KA, Tao H, Osoegawa K, de Jong PJ, Chen X, Doherty MF, Cox DR.Cross-species DNA sequence comparison is a fundamental method for identifying biologically important elements, because functional sequences are evolutionarily conserved, wheres nonfunctional sequences drift. A recent genome-wide comparison of human and mouse DNA discovered over 200,000 conserved noncoding sequences with unknown function. Multispecies DNA comparison has been proposed as a method to prioritize these conserved noncoding sequences for functional analysis based on the hypothesis that elements present in many species are more likely to be functional than elements present in limited ...
Farber CR, Medrano JF.In this study the flanking sequences of 1534 horse microsatellites were used in a BLAST search to identify putative human-horse homologies. BLAST searches revealed 129 flanking sequences with significant blastn matches [alignment scores (S) > or = 60 and sum probability values (E) < or = 3.0E-6], also, 25 of these produced significant blastx matches. To provide a reference point in the human genome the flanking sequences with matches were subjected to a BLAT search of the University of California Santa Cruz (UCSC) human genome assembly (July 2003 freeze). Eighty-three of the flanking seq...
Lee EJ, Raudsepp T, Kata SR, Adelson D, Womack JE, Skow LC, Chowdhary BP.Comparative genomics has served as a backbone for the rapid development of gene maps in domesticated animals. The integration of this approach with radiation hybrid (RH) analysis provides one of the most direct ways to obtain physically ordered comparative maps across evolutionarily diverged species. We herein report the development of a detailed RH and comparative map for horse chromosome 17 (ECA17). With markers distributed at an average interval of every 1.4 Mb, the map is currently the most informative among the equine chromosomes. It comprises 75 markers (56 genes and 19 microsatellites),...
Wagner B, Greiser-Wilke I, Antczak DF.Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse ( Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5' end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding ...
Chen C, Montelaro RC.Synthesis of Gag-Pol polyproteins of retroviruses requires ribosomes to shift translational reading frame once or twice in a -1 direction to read through the stop codon in the gag reading frame. It is generally believed that a slippery sequence and a downstream RNA structure are required for the programmed -1 ribosomal frameshifting. However, the mechanism regulating the Gag-Pol frameshifting remains poorly understood. In this report, we have defined specific mRNA elements required for sufficient ribosomal frameshifting in equine anemia infectious virus (EIAV) by using full-length provirus rep...
Kriegshäuser G, Wutz G, Lea S, Stuart D, Skern T, Kuechler E.Mouse monoclonal antibodies (mAbs) were employed to select neutralization escape mutants of equine rhinitis A virus (ERAV). Amino acid changes in the ERAV mutants resulting in resistance to neutralization were identified in capsid protein VP1 at Lys-114, Pro-240 and Thr-241. Although the changes were located in different parts of the polypeptide chain, these mutants exhibited cross-resistance against all four mAbs employed, indicating that these residues contribute to a single immunogenic site. To explain this result, we constructed a model of the three-dimensional structure of the ERAV capsid...
Ladrón N, Fernández M, Agüero J, González Zörn B, Vázquez-Boland JA, Navas J.The actinomycete Rhodococcus equi is an important pathogen of horses and an emerging opportunistic pathogen of humans. Identification of R. equi by classical bacteriological techniques is sometimes difficult, and misclassification of an isolate is not uncommon. We report here on a specific PCR assay for the rapid and reliable identification of R. equi. It is based on the amplification of a fragment of the choE gene encoding cholesterol oxidase. The choE-based PCR was assessed by using a panel of strains comprising 132 isolates from different sources and of different geographical origins, all i...
Schwartz S, Elnitski L, Li M, Weirauch M, Riemer C, Smit A, Green ED, Hardison RC, Miller W.Analysis of multiple sequence alignments can generate important, testable hypotheses about the phylogenetic history and cellular function of genomic sequences. We describe the MultiPipMaker server, which aligns multiple, long genomic DNA sequences quickly and with good sensitivity (available at http://bio.cse.psu.edu/ since May 2001). Alignments are computed between a contiguous reference sequence and one or more secondary sequences, which can be finished or draft sequence. The outputs include a stacked set of percent identity plots, called a MultiPip, comparing the reference sequence with sub...
Dimsoski P.To describe the development and performance of the new horse genotyping kit. Methods: Highly discriminatory 17-Plex horse genotyping kit was designed by adding the fifth dye to the StockMarks kit for genotyping horses and taking advantage of the new instrument platforms. This was accomplished by using a new set of five fluorescent dyes developed by Applied Biosystems (DS-31), with four of the dyes used to label the forward amplification primers (6-FAM, VIC, NED, and PET) in each primer set. Results: The new equine kit contained five extra loci (ASB17, LEX3, HMS1, CA425, and ASB23) in addition ...
Wille KH, Schnorr B.Arterioles, precapillary sphincters, capillary endothelium, and pericytes probably regulate the blood flow in the intestinal microvascular bed similar to other regions of the body because of their equipment with contractile filaments. Only throttle veins with their arrangement of pools and their characteristics probably exert influence on the hemodynamic qualities of the blood flow in the intestinal mucosa.
Lannergård J, Frykberg L, Guss B.Streptococcus equi subspecies equi is an important horse pathogenic bacterium causing a serious disease called strangles. Using bioinformatics we identified a gene denoted cne (gene encoding collagen-binding protein from S. equi) coding for a novel potential virulence factor of this species called protein CNE. The protein is composed of 657 amino acids and has the typical features found in cell surface-anchored proteins in Gram-positive bacteria. CNE displays amino acid sequence similarities to the previously well-studied collagen-binding protein CNA from Staphylococcus aureus, a proven virule...
Guérin G, Bailey E, Bernoco D, Anderson I, Antczak DF, Bell K, Biros I, Bjørnstad G, Bowling AT, Brandon R, Caetano AR, Cholewinski G, Colling D....A low-density, male-based linkage map was constructed as one of the objectives of the International Equine Gene Mapping Workshop. Here we report the second generation map based on testing 503 half-sibling offspring from 13 sire families for 344 informative markers using the CRIMAP program. The multipoint linkage analysis localized 310 markers (90%) with 257 markers being linearly ordered. The map included 34 linkage groups representing all 31 autosomes and spanning 2262 cM with an average interval between loci of 10.1 cM. This map is a milestone in that it is the first map with linkage groups ...
McAleese SM, Miller HR.The high-affinity receptor for IgE is expressed on the surface of mast cells and basophils. It is a transmembrane protein with one alpha, one beta and two gamma subunits. The cDNA sequences for the alpha subunit have already been determined. We report here the cDNA sequences for the beta and gamma subunits. The cytoplasmic domains of these subunits are important for intracellular signalling and the deduced amino acid sequences show the expected immunoreceptor tyrosine-based activation motifs. The gamma subunit is highly conserved between species but more variation is seen with the beta subunit...
Chowdhary BP, Raudsepp T, Kata SR, Goh G, Millon LV, Allan V, Piumi F, Guérin G, Swinburne J, Binns M, Lear TL, Mickelson J, Murray J, Antczak DF....A first-generation radiation hybrid (RH) map of the equine (Equus caballus) genome was assembled using 92 horse x hamster hybrid cell lines and 730 equine markers. The map is the first comprehensive framework map of the horse that (1) incorporates type I as well as type II markers, (2) integrates synteny, cytogenetic, and meiotic maps into a consensus map, and (3) provides the most detailed genome-wide information to date on the organization and comparative status of the equine genome. The 730 loci (258 type I and 472 type II) included in the final map are clustered in 101 RH groups distribute...
Barrera R, Torres N, Freier JE, Navarro JC, García CZ, Salas R, Vasquez C, Weaver SC.The distribution of the sylvatic subtype ID Venezuelan equine encephalitis (VEE) viruses in the lowland tropical forests of western Venezuela was investigated using remote sensing and geographic information system technologies. Landsat 5 Thematic Mapper satellite imagery was used to study the reflectance patterns of VEE endemic foci and to identify other locations with similar reflectance patterns. Enzootic VEE virus variants isolated during this study are the closest genetic relatives of the epizootic viruses that emerged in western Venezuela during 1992-1993. VEE virus surveillance was condu...
Lenasi T, Rogelj I, Dovc P.Here we report the entire cDNA sequences for equine alphaS1-, beta- and kappa-casein. Based on interspecies comparison, nine exons were found in equine beta-casein and five in kappa-casein. In equine alphaS1-casein cDNA the exon 5 was missing, which resulted in the total of 18 exons instead of 19 theoretically possible exons in alphaS1-casein cDNA. Comparison of DNA sequences representing exon 5 in other species with corresponding equine genomic region confirmed the presence of cryptic exon in horse genomic DNA. Equine alphaS1-casein mRNA was present in three forms in the lactating mammary gla...
Mariat D, Taourit S, Guérin G.In horses, basic colours such as bay or chestnut may be partially diluted to buckskin and palomino, or extremely diluted to cream, a nearly white colour with pink skin and blue eyes. This dilution is expected to be controlled by one gene and we used both candidate gene and positional cloning strategies to identify the "cream mutation". A horse panel including reference colours was established and typed for different markers within or in the neighbourhood of two candidate genes. Our data suggest that the causal mutation, a G to A transition, is localised in exon 2 of the MATP gene leading to an...
Sato F, Hasegawa T, Katayama Y, Ishida N.Complementary DNA (cDNA) encoding equine dopamine beta-hydroxylase (DBH) was amplified with a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method, and their nucleotide sequences (Accession No. AB029430: the DDBJ nucleotide sequence database) was determined. A total of 3842 bp cDNA sequence was consisted with 5 bp of 5' flanking untranslated sequence, 1833 bp of open reading frame encoding 610 amino acids, and 2004 bp of 3' flanking untranslated sequence. The deduced amino acid sequence of equine DBH was very similar to the ...
Tayyab S, Khan NJ, Khan MA, Kumar Y.Bilirubin (BR) binding properties of serum albumins from different mammalian species viz. human (HSA), equine (ESA), dog (DSA) and guinea pig (GPSA) were studied by absorption, fluorescence and CD spectroscopy. Whereas, a complex of BR with ESA produced maximum change, GPSA-BR complex showed weaker interaction as reflected from absorption and fluorescence spectroscopic data. Conformational analysis of these albumins by near- and far-UV CD spectra suggested similar structural characteristics (both secondary and tertiary structures) for ESA and HSA, whereas, DSA and GPSA had lower amounts of sec...
Ward RJ, Ramsey M, Dickson DP, Hunt C, Douglas T, Mann S, Aquad F, Peters TJ, Crichton RR.The biochemical and biophysical properties of isolated haemosiderins have been compared to that of another iron-containing protein, termed prehaemosiderin, which sediments through chaotropic potassium iodide only after 20 h of ultracentrifugation, in contrast to that of haemosiderin which is recovered after 2 h of ultracentrifugation. The iron/protein ratio and iron/phosphate ratio were less that that of the corresponding haemosiderin, while the elemental composition was also reduced in many of the prehaemosiderin samples. Mossbauer spectroscopy and electron diffraction identified the predomin...
Tikhonov VN, Cothran EG, Kniazev SP.This study was the first to analyze the polymorphic characteristics of a wide range of biochemical markers in aboriginal Yakut horses. A total of 124 alleles, including 48 alleles of seven blood-group loci and 76 alleles of ten loci for enzymes and other proteins, were studied. For these polymorphic systems, a computer analysis of the genetic distances between 85 horse breeds of different origin from all parts of the world was performed. The low level of hereditary variation in the Yakut horses confirmed that this breed is old and has long been an isolated population. Phylogenetic analysis dem...
Gim JA, Kim HS.MicroRNAs (miRNAs) are single-stranded, small RNAs (21-23 nucleotides) that function in gene silencing and translational inhibition via the RNA interference mechanism. Most miRNAs originate from host genomic regions, such as intergenic regions, introns, exons, and transposable elements (TEs). Here, we focused on the palindromic structure of medium reiteration frequencies (MERs), which are similar to precursor miRNAs. Five MER consensus sequences (MER5A1, MER53, MER81, MER91C, and MER117) were matched with paralogous transcripts predicted to be precursor miRNAs in the horse genome (equCab2) and...
Bramucci M, Miano A, Quassinti L, Maccari E, Murri O, Amici D.This study was conducted to determine the presence of the angiotensin converting enzyme in commercial sera used in cell culture medium. The aim of the research was to bring the presence of proteinases (angiotensin converting enzyme) to cell culture users' knowledge and to give some data for solving problems about the development of peptides as useful drugs. The enzymes, purified from foetal bovine, adult bovine, foetal equine, adult equine, and human sera, showed molecular weights of about 170 kDa. Captopril and lisinopril inhibited enzyme activities at nanomolar concentrations. The enzymes we...
Harnal VK, Wildt DE, Bird DM, Monfort SL, Ballou JD.Genome resource banks (GRBs) and assisted reproductive techniques are increasingly recognized as useful tools for the management and conservation of biodiversity, including endangered species. Cryotechnology permits long-term storage of valuable genetic material. Although, the actual application to endangered species management requires technical knowledge about sperm freezing and thawing, a systematic understanding of the quantitative impacts of various germ plasm storage and use scenarios is also mandatory. In this study, various GRB strategies were analyzed using the historical data from th...
McIntyre JC, Hundley P, Behnke WD.Fluorescence techniques have been employed to study the interaction of porcine and equine colipase with pure taurodeoxycholate and mixed micelles. Nitrotyrosine-55 of porcine colipase is obtained by modification with tetranitromethane (low excess, in the presence of taurodeoxycholate) of the protein followed by gel filtration and ion-exchange chromatography. Verification of the residue modified was obtained by h.p.l.c. peptide purification and sequence analysis. Reduction and quantitative reaction with dansyl chloride yields a fluorescent derivative that is twice as active in conjunction with ...
Coleman SJ, Gong G, Gaile DP, Chowdhary BP, Bailey E, Liu L, MacLeod JN.Loci for 9322 equine expressed sequence tags (ESTs) were predicted using the Comparative Mapping by Annotation and Sequence Similarity (Compass) strategy in order to evaluate the programme's ability to make accurate locus predictions in species with comparative gene maps. Using human genome sequence information from Build 35 (May 2004) and published marker information from the radiation hybrid (RH) maps for equine chromosomes (ECA) 17 and X, 162 ESTs were predicted to locations on ECA17 and 328 ESTs to locations on ECAX by selection of the 'top blast hit'. The locations of 30 ESTs were assesse...
Palomino Lago E, Baird A, Blott SC, McPhail RE, Ross AC, Durward-Akhurst SA, Guest DJ.Fractures caused by bone overloading are a leading cause of euthanasia in Thoroughbred racehorses. The risk of fatal fracture has been shown to be influenced by both environmental and genetic factors but, to date, no specific genetic mechanisms underpinning fractures have been identified. In this study, we utilised a genome-wide polygenic risk score to establish an in vitro cell system to study bone gene regulation in horses at high and low genetic risk of fracture. Candidate gene expression analysis revealed differential expression of and genes in osteoblasts derived from high- and low-risk...
Semik-Gurgul E, Pawlina-Tyszko K, Gurgul A, Szmatoła T, Rybińska J, Ząbek T.DNA methylation and microRNA (miRNA) expression are epigenetic mechanisms essential for regulating tissue-specific gene expression and metabolic processes. However, high-resolution transcriptome, methylome, or miRNAome data is only available for a few model organisms and selected tissues. Up to date, only a few studies have reported on gene expression, DNA methylation, or miRNA expression in adult equine tissues at the genome-wide level. In the present study, we used RNA-Seq, miRNA-seq, and reduced representation bisulfite sequencing (RRBS) data from the heart, lung, and liver tissues of healt...
Ma L, Li J, He J, Jiang T, Hao Y, Bu Y.Cylicostephanus longibursatus is a common parasite in equine animals. Hosts infected by these nematodes might face disease or death. This study utilized next-generation sequencing technology to sequence the complete mitochondrial genome (mt genome) of C. longibursatus. Through bioinformatics techniques, the genomic base composition, codon usage, tRNA secondary structures, evolutionary relationships, and taxonomic status were analyzed. The results revealed that the mitochondrial genome of C. longibursatus is a double-stranded, 13,807-bp closed circular molecule with an AT content of 76.0%, indi...
Ahlawat S, Sharma U, Niranjan SK, Chhabra P, Arora R, Sharma R, Singh KV, Vijh RK, Mehta SC.This study explored the maternal genetic diversity of six indigenous Indian horse and pony breeds (Bhutia, Kathiawari, Manipuri, Marwari, Spiti, and Zanskari) using comprehensive mitochondrial genome (mitogenome) analysis. Blood samples from 53 horses across diverse agro-climatic zones of India were analyzed, revealing 36 distinct haplotypes, with a haplotype diversity of 0.889 and nucleotide diversity of 0.00347. These indices suggest significant maternal genetic diversity in Indian equines. A median-joining (MJ) network, based on the hypervariable region of the D-loop along with sequences of...
Ding W, Gong W, Bou T, Shi L, Lin Y, Wu H, Dugarjaviin M, Bai D.Muscle fibers, as the fundamental units of muscle tissue, play a crucial role in determining skeletal muscle function through their growth, development, and composition. To investigate changes in muscle fiber types and their regulatory mechanisms in Mongolian horses (MG), Xilingol horses (XL), and Grassland-Thoroughbreds (CY), we conducted histological and bioinformatic analyses on the gluteus medius muscle of these three horse breeds. Immunofluorescence analysis revealed that Grassland-Thoroughbreds had the highest proportion of fast-twitch muscle fibers at 78.63%, while Mongolian horses had ...
Luciani M, Armillotta G, Di Febo T, Krasteva I, Ulisse S, Di Pancrazio C, Laguardia C, Perletta F, Serroni A, Maggetti M, Testa L, Sacchini F....The significance of as the causative agent of dourine cannot be understated, especially given its high mortality rate among equids. International movement of equids should be subject to thorough health checks and screenings to ensure that animals are not infected with . This involves the implementation of quarantine protocols, testing procedures, and the issuance of health certificates to certify the health status of the animals. Three proteins, the peptidyl-prolyl cis-trans isomerase (A0A1G4I8N3), the GrpE protein homolog (A0A1G4I464) and the transport protein particle (TRAPP) component, put...
Yang P, Bi Q, Li Y, Liao J, Ding Y, Huang D, Luo X, Huang Y, Yao C, Zhang J, Wei W, Li Z, Meng J, Guo D.In this study, a novel pseudo-targeted peptidomics strategy, integrating the transition list generated by an in-house software (Pep-MRMer) and the retention time transfer by high-abundance ion-based retention time calibration (HAI-RT-cal), was developed to screen marker peptides of gelatins from five closely related animal species, including porcine, bovine, horse, mule, and donkey. Five marker peptides were screened from the molecular phenotypic differences of type I collagen. Furthermore, a simple and robust 10 min multiple reaction monitoring (MRM) method was established and performed well ...
Groesel M, Gfoehler M, Peham C.In this paper, an alternative method is presented to convert computed tomography (CT)-scans into 3D biomodels. The CT-data of an equine spine was converted into TIF format to work with it in a 2D CAD program. Then the bony structure has been marked manually with closed splines and saved as IGS files for the next procedure with 3D CAD software to create virtual biomodels of every single bone. Therefore, the different layers of the CT-scans were positioned in correct distance and then a closed surface was created to cover all spline-curves. Finally, the cover was filled up with material to creat...