Shuji Sato

Identification of a second gene associated with variation in vertebral number in domestic pigs

BackgroundThe number of vertebrae in pigs varies and is associated with body size. Wild boars have 19 vertebrae, but European commercial breeds for pork production have 20 to 23 vertebrae. We previously identified two quantitative trait loci (QTLs) for number of vertebrae on Sus scrofa chromosomes (SSC) 1 and 7, and reported that an orphan nuclear receptor, NR6A1, was located at the QTL on SSC1. At the NR6A1 locus, wild boars and Asian local breed pigs had the wild-type allele and European commercial-breed pigs had an allele associated with increased numbers of vertebrae (number-increase allele).ResultsHere, we performed a map-based study to define the other QTL, on SSC7, for which we detected genetic diversity in European commercial breeds. Haplotype analysis with microsatellite markers revealed a 41-kb conserved region within all the number-increase alleles in the present study. We also developed single nucleotide polymorphisms (SNPs) in the 450-kb region around the QTL and used them for a linkage disequilibrium analysis and an association study in 199 independent animals. Three haplotype blocks were detected, and SNPs in the 41-kb region presented the highest associations with the number of vertebrae. This region encodes an uncharacterized hypothetical protein that is not a member of any other known gene family. Orthologs appear to exist not only in mammals but also birds and fish. This gene, which we have named vertnin (VRTN) is a candidate for the gene associated with variation in vertebral number. In pigs, the number-increase allele was expressed more abundantly than the wild-type allele in embryos. Among candidate polymorphisms, there is an insertion of a SINE element (PRE1) into the intron of the Q allele as well as the SNPs in the promoter region.ConclusionsGenetic diversity of VRTN is the suspected cause of the heterogeneity of the number of vertebrae in commercial-breed pigs, so the polymorphism information should be directly useful for assessing the genetic ability of individual animals. The number-increase allele of swine VRTN was suggested to add an additional thoracic segment to the animal. Functional analysis of VRTN may provide novel findings in the areas of developmental biology.

Transdermal administration of salmon calcitonin by pulse depolarization-iontophoresis in rats

Using the pulse depolarization-iontophoresis (PDP-IP) system, salmon calcitonin (sCT), a drug for the treatment of osteoporosis, was transdermaly administered in rats. While absorption of sCT was not observed after passive transdermal administration, the serum sCT concentration was confirmed at a dose of 0.2-4 microg when the PDP-IP system was employed. The results indicated that PDP-IP could enhance transdermal absorption of peptide drugs. Also noted was the increased amount of absorption of sCT along with an increase in the dose. We investigated the influence of electrical parameters (current, frequency) in PDP-IP on the transdermal absorption of sCT. An optimal current for drug absorption was found within the range of transported current (0.1-1.0 mA) employed for PDP-IP. In comparison with the results obtained at 0.1 mA, the drug absorption increased, along with an increase in transported current, when the current was set at 0.5 mA, while the drug absorption decreased at 1.0 mA in comparison. The decrease in drug absorption was assumed to be attributable to the structural destruction of skin by application of excessive current. There was no change in skin resistance attributable to the frequency; nor was there any influence of the frequency on the amount of drug absorption.

Genome-wide mapping and identification of new quantitative trait loci affecting meat production, meat quality, and carcass traits within a Duroc purebred population

Most QTL detection studies in pigs have been carried out in experimental F(2) populations. However, segregation of a QTL must be confirmed within a purebred population for successful implementation of marker-assisted selection. Previously, QTL for meat quality and carcass traits were detected on SSC 7 in a Duroc purebred population. The objectives of the present study were to carry out a whole-genome QTL analysis (except for SSC 7) for meat production, meat quality, and carcass traits and to confirm the presence of segregating QTL in a Duroc purebred population. One thousand and four Duroc pigs were studied from base to seventh generation; the pigs comprised 1 closed population of a complex multigenerational pedigree such that all individuals were related. The pigs were evaluated for 6 growth traits, 7 body size traits, 8 carcass traits, 2 physiological traits, and 11 meat quality traits, and the number of pigs with phenotypes ranged from 421 to 953. A total of 119 markers were genotyped and then used for QTL analysis. We utilized a pedigree-based, multipoint variance components approach to test for linkage between QTL and the phenotypic values using a maximum likelihood method; the logarithm of odds score and QTL genotypic heritability were estimated. A total of 42 QTL with suggestive linkages and 3 QTL with significant linkages for 26 traits were detected. These included selection traits such as daily BW gain, backfat thickness, loin eye muscle area, and intramuscular fat content as well as correlated traits such as body size and meat quality traits. The present study disclosed QTL affecting growth, body size, and carcass, physiological, and meat quality traits in a Duroc purebred population.

Genome-wide mapping for fatty acid composition and melting point of fat in a purebred Duroc pig population

The fatty acid composition and melting point of fatty tissue are among the most important economic traits in pig breeding because of their influence on the eating quality of meat. Identifying the quantitative trait locus (QTL) of these traits may help reveal the genetic structure of fatty acid composition and the melting point of fatty tissue and improve meat-quality traits by marker-assisted selection. We conducted whole-genome QTL analysis for fatty acid composition and melting point of inner and outer subcutaneous fat and inter- and intramuscular fat in a purebred Duroc population. A total of 129 markers were genotyped and used for QTL analysis. For fatty acid compositions of inner and outer subcutaneous fat, three significant QTL and 17 suggestive QTL were detected on SSC2, 4, 6, 8, 9, 10, 11, 12, 14 and 18. For the melting point of inner and outer subcutaneous fat, two significant QTL were detected on the same region of SSC14. For fatty acid compositions of inter- and intramuscular fat, five significant QTL and 13 suggestive QTL were detected on SSC2, 4, 6, 8, 9, 10, 14 and 15. On SSC14, significant QTL for C18:0 and C18:1 of outer subcutaneous fat and intramuscular fat, and melting point of subcutaneous fat, which had high likelihood of odds (LOD) scores (2.67-5.78), were detected in the same region. This study determined QTL affecting fatty acid composition and melting point of different fat tissues in purebred Duroc pigs.

Quantitative trait loci analysis on Sus scrofa chromosome 7 for meat production, meat quality, and carcass traits within a Duroc purebred population

Many QTL analyses related to meat production and meat quality traits have been carried out using an F(2) resource population produced by crossing 2 genetically different breeds. This experiment was intended to investigate whether these QTL were segregating in a purebred Duroc population that had been selected for meat production and meat quality traits during 7 generations. Sus scrofa chromosome 7, for which significant QTL of intramuscular fat and many other traits have already been reported, was studied. The polymorphism of 10 microsatellite markers that were arranged at about 20-cM intervals was investigated on 1,004 pigs. In the selected population, 954 progeny were produced from mating of 99 sires and 286 dams. The QTL analysis for a full-sib family population was examined with the multigeneration pedigree structure of the population. Variance component analysis was used to detect QTL in this population and was examined for the multigeneration pedigree population. In this study, multigenerational pedigree estimated identical by descent coefficients among sibs were produced using Markov chain Monte Carlo methods. The maximum likelihood of odds score was found at the 70-cM position for the LM area, at the 0-cM position for the pork color standard, and at the 120-cM position for the number of thoracic vertebra, but no significant QTL for intramuscular fat were detected on SSC 7. These results indicate that QTL analysis via a variance component method within a purebred population was effective to determine that QTL were segregating in a population of purebred Durocs.

Fine mapping of porcine SSC14 QTL and SCD gene effects on fatty acid composition and melting point of fat in a Duroc purebred population.

The stearoyl-CoA desaturase (delta-9-desaturase; SCD) gene is a candidate gene for fatty acid composition. It is located on pig SSC14 in a region where quantitative trait loci (QTL) for fatty acid composition were previously detected in a Duroc purebred population. The objective of the present study was to fine map the QTL, to identify polymorphisms of the pig SCD gene and to examine the effects of SCD polymorphisms on fatty acid composition and melting point of fat in the population. The pigs were examined for fatty acid composition and melting point of inner and outer subcutaneous fat and inter- and intramuscular fat; the number of pigs examined was 479-521. Two SNPs (g.-353C>T and g.-233T>C) were identified in the promoter region of the SCD gene and were completely linked in the pigs from the base generation. In all pigs, 19 microsatellite markers and SCD haplotypes were then genotyped. Different statistical models were applied to evaluate the effects of QTL and the possible causality of the SCD gene variants with respect to the QTL. The results show that all significant QTL for C14:0, C18:0, C18:1 and melting point of fat were detected in the same region, located near the SCD gene. The results also show a significant association between SCD haplotypes and fatty acid composition and fat melting point in this population. These results indicate that the haplotype of the SCD gene has a strong effect on fatty acid composition and melting point of fat.

Effects of porcine leptin receptor gene polymorphisms on backfat thickness, fat area ratios by image analysis, and serum leptin concentrations in a Duroc purebred population

The leptin receptor (LEPR) gene is considered a candidate gene for fatness traits. It is located on SSC 6 in a region in which quantitative trait loci (QTLs) for backfat thickness (BF), fat area ratios, and serum leptin concentration (LEPC) have previously been detected in a Duroc purebred population. The objectives of the present study were to identify porcine LEPR polymorphisms and examine the effects of LEPR polymorphisms on fatness traits in this same population. The Duroc pigs (226 to 953 pigs) were evaluated for BF, fat area ratios using image analysis, and LEPC. A total of seven single nucleotide polymorphisms (SNPs) in the full-length LEPR coding region were identified in pigs from the base population. Four non-synonymous SNPs of the LEPR gene and 15 microsatellite markers on SSC 6 were then genotyped in all pigs. During candidate gene analysis, we detected significant effects of the non-synonymous SNP c.2002C>T in exon 14 on all traits. In fine mapping analysis, significant QTLs for BF, fat area ratios, and LEPC were detected near the LEPR gene in the same region. These results indicated that the c.2002C>T SNP of LEPR has a strong effect on BF, fat area ratios and LEPC.

Quantitative trait loci for leg weakness traits in a Landrace purebred population

Leg weakness in pigs is a serious problem in the pig industry. We performed a whole genome quantitative trait locus (QTL) analysis to find QTLs affecting leg weakness traits in the Landrace population. Half-sib progeny (n = 522) with five sires were measured for leg weakness traits. Whole genome QTL mapping was performed using a half-sib regression-based method using 190 microsatellite markers. No experiment-wide significant QTLs affecting leg weakness traits were detected. However, at the 5% chromosome-wide level, QTLs affecting leg weakness traits were detected on chromosomes 1, 3, 10 and 11 with QTL effects ranging from 0.07 to 0.11 of the phenotypic variance. At the 1% chromosome-wide level, QTLs affecting rear feet score and total leg score were detected on chromosomes 2 and 3 with QTL effects of 0.11 and 0.13 of the phenotypic variance, respectively. On chromosome 3 and 10, some QTLs found in this study were located at nearby positions. The present study is one of the first reports of QTLs affecting fitness related traits such as leg weakness traits, that segregate within the Landrace population. The study also provides useful information for studying QTLs in purebred populations.

Preparation and evaluation of biodegradable thermo-responsive microspheres

Abstract In order to develop thermoresponsive microspheres containing antitumor agents, which are expected to embolize the artery or arteriole feeding the tumor region and to release the antitumor drugs pulsatively by the heat in the hyperthermia treatment, biodegradable microspheres consisting of a liquid crystalline and poly( d,l -lactic acid) were prepared by a solvent evaporation method. In vitro studies on the release of model drugs, indomethacin (IND) or aclarubicin (ACR), from the microspheres were performed by changing the temperature between 37 and 43°C (the latter is usually applied in the hyperthermia therapy). Although the differences in ACR release between 37 and 43° C from the microspheres consisting of high-molecular-weight (Mv 64 000) poly ( d,l -lactic acid) (HPLA) as a polymer component were observed, the pulsatile release of ACR from the microspheres was not achieved. The effect of an addition of low-molecular-weight (Mn 2600) poly ( d,l -lactic acid) (LPLA) as a second polymer component to the microspheres on the pulsatile drug release was evaluated using several microspheres containing IND. It was clarified that the composition of HPLA, LPLA and the liquid crystalline had an important role in the pulsatile release of IND. The pulsatile release of ACR could be achieved with the microspheres which consisted of HPLA, LPLA and polyoxyethylene glyceryl tristearate (PGTS) (5/5/2).

Application of a thermo-responsive membrane to the transdermal delivery of non-steroidal anti-inflammatory drugs and antipyretic drugs

Abstract A thermo-responsive membrane was incorporated into a transdermal therapeutic system (TTS) which responds to thermal stimuli associated with skin temperature changes. The thermo-responsive membrane was prepared by pouring monooxyethylene trimethylolpropane tristearate (MTTS) between two porous polypropylene films. The TTS with this thermoresponsive membrane as a rate controlling membrane released non-steroidal anti-inflammatory drugs (indomethacin, ketoprofen) and antipyretic drugs (acetoaminophen, ethenzamide) in response to temperature changes between 32 and 38°C in the in vitro experiments. It was then shown that the temperature-activated TTS was operative in vivo in a rabbit model .