Search

The present study was designed to investigate the effect of dietary fish oil supplementation on piglet T helper cells (Th) polarization in relation to its impact on piglet serum interferon γ (IFN-γ) and interleukin 10 (IL-10) concentrations and splenic expression of Th1/Th2 characteristic genes. The diets of 18 gestating sows were supplemented with 7% lard (C) (n = 10) or 7% fish oil (T) (n = 8) from 10 d before parturition to weaning. At weaning, a split plot experiment was designed, 56 piglets, 28 each from sows fed with fish oil diet or lard diet, were divided into four groups of 7 replicates (one female and one castrated male per replicate) based on both sow diet during lactation and post-weaning piglet diet (C had 7% lard and T had 7% fish oil): CC, CT, TC, TT, and were fed the 7% fish oil or lard diet from day 35 to day 70. Serum concentrations of IFN-γ and IL-10, and Th1/Th2 related genes expression levels in spleen were measured and analyzed. The results showed that piglets fed with fish oil diet during post-weaning tended to have higher serum IFN-γ/IL-10 ratio (P = 0.09) than lard diet fed piglets. Lactation fish oil feeding increased splenic IL-12b, IL-12 receptor β2 (IL-12Rβ2), IL-2 and IFN-γ genes expression (P

The aim of the study was to investigate the effect of duration of feeding linseed (rich in n -3 PUFA) on peroxisome proliferator-activated receptor γ (PPARγ) and tumor necrosis factor (TNF-α) gene expression, and muscle mass of growing–finishing barrows. Two isoenergetic and isonitrogenous diets were formulated, and one of which was the basal diet and another one was the linseed diet including linseed at the level of 10%. Twenty-four Landrace × Yorkshire barrows weighing 35 ± 3.7 kg were randomly assigned to four treatments with six individuals per treatment. Pigs in treatment 1 (T1) fed the control diet throughout the experimental period, while pigs in T2, T3 and T4 fed the control diet except for 30, 60, and 90 d prior to slaughter when the linseed diet were fed. The experiment was conducted for 90 days. The longissimus muscle mass and each muscle mass in the hind leg were weighted. PPARγ and TNF-α mRNA expression levels in muscle, spleen and adipose tissue, and plasma concentrations of TNF-α data were measured and analyzed. The results showed that the longissimus muscle mass, quadriceps femoris muscle mass and semitendinosus muscle mass increased linearly ( P longissimus muscle and spleen increased ( P P = 0.095). Duration of linseed addition linearly decreased ( P longissimus dorsi muscle , adipose and spleen, and serum concentration of TNF-α as well. The expression levels of PPARγ negatively correlated with the expression of TNF-α in muscle ( R 2 = 0.70, P R 2 R 2 = 0.77, P R 2 R 2 = 0.59, P R 2 R 2 = 0.52, P R 2 R 2 = 0.80, P R 2 R 2 = 0.87, P longissimus dorsi muscle mass. These data suggested that duration of feeding linseed diet lead to a linear decrease of TNF-α gene expression, which may increase the muscle mass in growing–finishing barrows, at least in part, through a PPARγ-dependent mechanism.

ATP-citrate lyase (ACL), one of the lipogenic enzymes, catalyses the formation of acetyl-coenzyme A (CoA) involved in the synthesis of fatty acid and cholesterol. In pig, very little is known about the ACL gene. In this work, the mRNA differential display technique was used to analyse the differences in gene expression between Meishan and Large White pigs and the F1 hybrids of both direct and reciprocal crosses. Our results show that among the differentially expressed genes ACL is up-regulated in the backfat of the F1 hybrids. After cloning and analysing the fulllength cDNA and the 870 bp 5'-flanking sequence of the porcine ACL gene, a C/T mutation at position -97 bp upstream of the transcription site was detected. Luciferase activity detection showed that this mutation changed the transcriptional activity. In F1 hybrids, the heterozygous genotype CT was more frequent than the homozygous genotypes CC and TT. Real-time PCR analysis showed that in Meishan pigs, ACL mRNA expression was more abundant in individuals with genotype CT than in those with genotype CC or TT or in Large White pigs. These results indicate that the C/T mutation affects ACL mRNA expression, probably via the activator protein 2.

AMPD1 is the muscle-specific form of the AMPD multigene families in mammals and plays an important role in the purine nucleotide cycle and energy metabolism in skeletal muscle. In this study, we cloned and characterized AMPD1 from Sus scrofa muscle. The promoter of porcine AMPD1 contained several putative muscle-specific transcription factor binding sites (E box, myogenin, MEF2, Spl-CTF/NF-l), one RORalpha2 binding motif and NF-kappaB site. The deduced amino acid sequence of porcine AMPD1 contains an AMP deaminase signature sequence (SLSTDDP). RT-PCR analyses showed that AMPD1 was expressed specifically in skeletal muscle. Expression of AMPD1 was up-regulated during the muscle development and was higher in Yorkshire than in Meishan pigs. AMPD1 gene was expressed at higher levels in longissimus dorsi and bicepsfemoris muscles compared with soleus and masseter muscles in both Yorkshire and Meishan pigs. Moreover, we found that a single nucleotide polymorphism (SNP, T/C(426)) in exon12 of the AMPD1 gene was significantly associated with loin muscle area trait (p

In this experiment, DNA samples from parental lines Large White, Landrace, and Meishan pigs, and their hybrids Large White × Landrace, Landrace × Large White, Large White × Meishan, and Meishan × Large White pigs were used for the determination of DNA methylation content in both blood and muscle tissue. The differences about DNA methylation content between parental lines and their hybrids were analyzed. These will offer theoretical support from molecular level for heterosis. High performance liquid chromatography (HPLC) was firstly used to detect DNA methylation content. The average DNA methylation content in 163 DNA samples of muscle tissue was 16.92%, whereas, the average DNA methylation content in 182 samples of blood was 6.49%, the difference between which was especially prominent (P 0.05); and the differences between reciprocal cross hybrids in both hybrid systems were not significant (P > 0.05), but between different hybrid systems, the hybrids had a significant difference (P < 0.05). The average methylation content in muscle samples was higher than that in blood samples, and the methylation in different tissues was different.

Smad proteins are principal intracellular signaling mediators of transforming growth factor beta (TGF-beta) that regulate a wide range of biological processes. However, the identities of Smad partners mediating TGF-beta signaling are not fully understood. We firstly examined the expression of Smad2 and Smad3 induced by TGF-beta 1 in normal NIH/3T3 cells. The expression of Smad2 and Smad3 was assessed by RT-PCR and Western blotting. The results showed that the expression of Smad2 was increased after treatment with TGF-betaI, but Smad3 was more sensitive to TGF-betaI than Smad2. RNA interference (RNAi) provides a new approach for elucidation of gene function. Use of hairpin siRNA expression vectors for RNAi has provided a rapid and versatile method for assessing gene function in mammalian cells. Here, we have constructed Smad2 and Smad3 hairpin siRNA expression plasmids, and then transfected them into mouse NIH/3T3 cells. Endogenous Smad2 and Smad3 proteins decreased significantly at 48 h after transfection. We found the expression of Smad3 in Smad2-depleted cells was increased, however, the expression of Smad2 in Smad3-depleted cells was not changed. Consistently, the expression of Smad4 mRNA was also attenuated in Smad3-depleted cells. From these data, we suggest that Smad3, but not Smad2, may play a key role in TGF-beta signaling.

A number of epithelial lineages have been derived from mouse embryonic stem cells during the past decades, but the long lasting culture has never been reported. In this paper, we report when mouse embryonic stem cells were dispersed into small clumps containing approximately 50 to 100 cells and grown on mitotically inactivated mouse embryonic fibroblast feeder layers for up to 10 d to form epithelial-like colonies. Through subsequent cultivation without mouse embryonic fibroblast feeder layers, a serially subcultured keratinocyte-like cell lineage was established under these conditions. Pan cytokeratin, cytokeratin 14, and cytokeratin 18 were observed in these proliferating cells using immunocytochemistry and flow cytometry. E-cadherin, Involucrin, and keratin mRNAs were determined by a semi-quantitative and a quantitative real time reverse transcription-polymerase chain reaction (RT-PCR). These results confirmed the establishment of a keratinocyte-like cell lineage derived from mouse embryonic stem cells. In this paper also, we describe a method by which mouse embryonic stem cells can be differentiated into cells with some characteristics of epidermal keratinocytes and kept these cells in long-term culture. Potential applications of this method are the in vitro differentiation of cells of interest from embryonic stem (ES) cells of mice during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments or better understanding the mechanisms for epithelial differentiation of embryonic stem cells.

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation, and remodelling of the cell cytoskeleton by their interaction with some structural proteins, kinases, transcriptional regulators, etc. The presence of LIM domains in LMCD1 gene implies it may be involved in skeletal muscle protein-protein interactions. This study was to investigate polymorphisms of LIM and cysteine-rich domain 1 ( LMCD1 ) gene and its effect on meat quality and carcass traits in pig. The polymorphism (G294A) in exon 3 region of porcine LMCD1 gene, which is synonymous mutation, was genotyped in the population of 178 F 2 pigs of a Large White x Meishan resource family. Statistical results indicated the distribution of allele G (with a A → G mutation) and A (without mutation). Analysis of variance showed that the polymorphism of LMCD1 gene was associated with variation in several carcass traits of interest for pig breeding. Some carcass traits and meat quality traits are close to significance by association. An analysis of more animals is necessary to analyze the polymorphisms in exon 3 of porcine LMCD1 gene if it was selected as a marker for the pig carcass traits.

The glutathione S-transferase mu 2 gene (GSTM2) encodes a GST functioning in the elimination of electrophilic compounds and the regulation of cell growth. In this study, the sequence of porcine GSTM2 gene that contains the complete sequence encoding a protein of 218 amino acids was cloned. The deduced amino acid sequence shared 76%, 78% and 76% identity with that of human, mouse and rat, respectively. mRNA expression analysis showed that the porcine GSTM2 gene was expressed at a high level in liver and testis, at a medium level in longissimus dorsi muscle, adipose tissue, spleen and lung, at a low level in kidney, and at a very low level in heart and embryo. A nonsense mutation (CGA→TGA) resulted from C27T substitution in the fifth exon to produce a premature translation termination codon was identified, and it was discovered that nonsense-mediated mRNA decay might have an effect on the regulation of porcine GSTM2 gene expression. This polymorphism was analyzed in Large White, Landrace, Meishan and Qingping pig populations using the Taq I-polymerase chain reaction-restriction fragment length polymorphism method. The result showed that allele C had a higher frequency than allele T in each population.

To investigate the molecular basis of porcine heterosis, suppression subtractive hybridization (SSH) was performed to detect the differences in gene expression between porcine longissirnus dorsi of Meishan × Large White (MS × LW) F, hybrids and their parents Meishan pigs. An expression sequence tag (EST) differentially expressed was found, designated as ML556, which was homologous to a hypothetical protein HSPC117, from human hematopoietic stedprogenitor cells (HSPCs), and the full-length cDNA of porcine HSPC117 was cloned using rapid amplification of cDNA ends (RACE) method. Translation of the mRNA transcript revealed an open reading frame (ORF) of 505 amino acid residues encoding a peroxisomal targeting signal (PTS) with theoretical molecular weight of 55 kDa. Alignment analysis revealed that the deduced protein sequence exhibit 98, 98, 98, 97, and 97% identity with that of cattle, human, dog, rat, and mouse, respectively. The tissue expression analysis indicated that the porcine HSPC117 gene is highly expressed in muscle, spleen, lung, kidney, uterus, ovary and testis, moderately expressed in fat, heart, and liver, and not expressed in stomach and small intestine. The possible role of porcine HSPC117 and its relationship with porcine heterosis were discussed.

Quantitative trait loci (QTL) were detected for 8 internal organ traits, 3 carcass length traits, and teat number trait in 214 pigs in a resource population that included 180 F(2) individuals. A total of 39 microsatellite markers were examined on SSC4, SSC6, SSC7, SSC8, and SSC13. The genetic traits included heart weight (HW), lung weight (LW), liver and gallbladder weight (LGW), spleen weight (SPW), stomach weight (STW), small intestine weight (SIW), large intestine weight (LIW), kidney weight (KW), carcass length to the first cervical vertebra (CL1), carcass length to the first thoracic vertebra (CL2), rib numbers (RNS), and teat numbers (TNS). Results indicated that, 3 highly significant QTL (P

Phenotypic information about several pig meat quality traits on 334 Large White × Meishan F2 pigs was collected. Effects of the association of the FokI variants in the seventh intron of the skeletal muscle glycogen synthase (GYS1) gene and the PstI variants in the ninth intron of the palmitoyl acyl-CoA oxidase 1 (ACOX1) gene on the meat quality traits were examined on all pigs. The FokI variants of the GYS1 gene showed significant effects on pH of m. semipinalis capitis (P < 0.05). Linkage analysis indicated that the peak of the quantitative trait loci (QTL) curve was located around this marker for pH, but it did not reach significance (P > 0.05). The results may be due to several reasons such as linkage disequilibrium to the causal mutations, the limited number of animals or balance of another QTL or marker with negative effects. Significant effects of PstI variants of ACOX1 gene were also found on meat colour value and meat marbling score of both m. longissimus dorsi and m. biceps femoris (P < 0.05). Dominant effects for the affected traits at those two loci were significant except for meat marbling score of m. biceps femoris (P < 0.05). The results of this study give us some evidence for the potential of those dominant markers used in the marker-assisted selection of crossbreeding of the Large White pig sire lines and Meishan-derived synthetic dam lines.

Skeletal muscle genes are potential candidates for production and meat quality. Screening a subtracted cDNA library constructed with mRNA obtained from longissimus dorsi muscles of F1 hybrids Landrace × Yorkshire and their female parents Yorkshire, we isolated two partial sequences coding for the H3-K4-specific methyltransferase ( KIAA1717 ) and skeletal muscle myosin regulatory light chain ( HUMMLC2B ) genes. Database search revealed KIAA1717 and HUMMLC2B encoded proteins with SET domain and EF-hand calcium binding motif, respectively. In the present work we identified their partial polymorphisms and two SNPs, one (C1354T) at the 3′ untranslated region (UTR) of KIAA1717 and one (A345G) at the SINE (PRE-1) element of HUMMLC2B , both created/disrupted a restriction site for endonuclease Msp I. The selected pigs were genotyped at the KIAA1717 C1354T and HUMMLC2B A345G sites by means of a PCR-RFLP protocol. Significant associations were observed for the KIAA1717 C1354T polymorphic site with meat marbling ( longissimus doris ( p biceps femoris ( p p HUMMLC2B A345G were significantly associated with meat pH ( longissimus doris ( p biceps femoris ( p p p longissimus doris ( p biceps femoris ( p

Significant tissue destruction, repair, and remodeling are involved in parturition, which involves fetal membrane rupture, cervical ripening, and uterine contraction and its subsequent involution. Extracellular matrix degradation and remodeling by proteolytic enzymes, such as matrix metalloproteinases (MMPs), are required for the final steps of parturition. MMPs participate in physiological degradation and remodeling through their proteolytic activities on specific substrates, and are balanced by the action of their inhibitors. Disruption to this balance can result in pathological stress that ends with preterm or post-term birth or pre-eclampsia. In this review, we examine the roles and regulation of the MMP system in physiological and pathological labor, and propose a model that illustrates the mechanisms by which the MMP system contributes to these processes.

MicroRNAs (miRNAs) are small ∼22 nucleotide regulatory RNAs that regulate the stability and translation of cognate mRNAs. MiRNAs participate in the regulation of adipogenesis, and identification of the full repertoire of miRNAs expressed in adipose tisse is likely to improve our understanding of adipose tissue growth and development significantly. In the present study, miR-215-5p was found to inhibit adipocyte differentiation of 3T3-L1 cells. Moreover, fibronectin type III domain containing 3B (FNDC3B) and catenin, beta interacting protein 1 (CTNNBIP1) were found to be direct targets of miR-215-5p. Further studies in mouse 3T3-L1 cell-line suggests that miR-215-5p is a negative regulator of adipocyte differentiation through post-transcriptional regulation of FNDC3B and CTNNBIP1 during early adipogenesis.

The aim of the present study was to evaluate the effects of dietary n-6:n-3 fatty acid (FA) ratio and vitamin E on the semen quality, FA composition and antioxidant status of boars. Forty-eight Landrace boars were randomly distributed in a 3×2 factorial design with three n-6:n-3 FA ratios (14.4, 6.6 and 2.2) by the inclusion of three oil sources (soybean, fish/soybean, fish) and two vitamin E levels (200 and 400mg/kg). During the 8 weeks of treatment, semen parameters were evaluated. Serum, sperm and seminal plasma samples were taken at 0 and 8 weeks to monitor the FA composition and antioxidant status. Results showed that the 6.6 and 2.2 dietary ratios very effectively increased docosahexaenoic acid (DHA) and n-3 polyunsaturated fatty acid (PUFA) and decreased docosapentaenoic acid (DPA) and n-6:n-3 ratio in spermatozoa. The 6.6 dietary ratio contributed to a greater progressive sperm motility (P

Background To investigate the effect of feeding a linseed-enriched diet to growing-finishing pigs on gene expression in skeletal muscle, pigs were fed with a linseed-enriched diet for 0, 30, 60 and 90 d. Transcriptional profiles of longissimus dorsi muscle were measured using Affymetrix Genechip. Results Results showed that 264 genes were identified as differentially expressed genes (DEGs). The strongest transcriptional response was clearly observed at 30 d. DEGs were assigned to several main functional terms, including transcription, apoptosis, intracellular receptor-mediated signaling, muscle organ development, fatty acid metabolic process, cell motion, regulation of glucose metabolic process, spermatogenesis and regulation of myeloid cell differentiation. We also found that transcriptional changs of several transcription cofactors might contribute to n-3 PUFAs regulated gene expression. In addition, the increased expression of IGF-1, insulin signaling pathway and the metabolism of amino acids might involve in the muscle growth induced by feeding a linseed-enriched diet. The results also provide the new evidence that the expression changes of PTPN1, HK2 and PGC-1α might contribute to the regulation of insulin sensitivity by n-3 PUFAs. Conclusions Our finding provided correlative evidence that feeding the linseed enriched diet affact expression of genes involved in insulin signaling pathway and the metabolism of amino acids.

Labour onset is a very complex physiological process and its mechanism is poorly understood. Here, we obtained the mRNA and miRNA expression profiles from the placentas of four groups of sows: Qingping sows 112 days after insemination with signs of labour onset (QS), Qingping sows 114 days after insemination with signs of labour onset (QL), Large White sows 114 days after insemination with signs of labour onset (LL) and Large White sows 112 days after insemination without signs of labour onset (LN). A set of differentially expressed genes, including 2164 mRNAs and 39 miRNAs, were found. A DAVID analysis of these differentially expressed genes revealed their critical roles in response to hormone stimulus, immune response. Cytoscape Network analysis of the functional genes found node mRNAs and that the regulatory network between the node mRNAs and miRNAs was established. A comparison of the sequencing data from the shorter gestation period (QS) and the normal gestation period (QL) indicated that these genes were responsible for the quicker and more sensitive reaction to the regulation of labour onset. This research not only detected the key factors that were involved in labour onset but also provided useful information for the research of gynaecological diseases.

Fifty-four multiparous large white sows were used to determine the effects of supplementing oregano essential oil (OEO) to the gestation and lactation diets on oxidative stress status, lactation feed intake, and their piglet performance. Two groups were fed diets with (OEO;n=28) or without (Control;n=26) supplemental 15 mg/kg OEO during gestation and lactation. The serum levels of reactive oxygen species (ROS) (P<0.05), 8-hydroxy-deoxyguanosine (8-OHdG) (P<0.05), and thiobarbituric acid reactive substances (TBARS) (P<0.05) were higher during gestation (days 90 and 109) and lactation (days 1 and 3) than in early gestation (day 10). Compared with the control group, the OEO diet significantly reduced sows’ serum concentrations of 8-OHdG (P<0.05) and TBARS (P<0.01) on day 1 of lactation. The OEO diet increased the sows’ counts of faecallactobacillus(P<0.001) while reducingEscherichia coli(P<0.001) andEnterococcus(P<0.001). In the third week of lactation the treatment tended to increase sow’s feed intake (P=0.07), which resulted in higher average daily gain (P<0.01) of piglets. Our results demonstrated that there is an increased systemic oxidative stress during late gestation and early lactation of sows. The OEO supplementation to sows’ diet improved performance of their piglets, which may be attributed to the reduced oxidative stress.

It has been proved that terminally differentiated mature adipocytes possess abilities to dedifferentiate into fibroblast-like progeny cells with self-renewal and multiple differentiation, termed dedifferentiated fat (DFAT) cells. However, the biological properties of DFAT cells during long-term culturein vitrohave not been elucidated. Here, we obtained fibroblast-like morphology of porcine DFAT cells by ceiling culture. During the dedifferentiation process, round mature adipocytes with single large lipid droplets changed into spindle-shaped cells accompanied by the adipogenic markersPPARγ,aP2,LPL, andAdiponectinsignificant downregulation. Flow cytometric analysis showed that porcine DFAT cells displayed similar cell-surface antigen profile to mesenchymal stem cells (MSCs). Furthermore, different passages of porcine DFAT cells during long-term culturein vitroretained high levels of cell viabilities (>97%), efficient proliferative capacity including population doubling time ranged from 20 h to 22 h and population doubling reached47.40±1.64by 58 days of culture. In addition, porcine DFAT cells maintained the multiple differentiation capabilities into adipocytes, osteoblasts, and skeletal myocytes and displayed normal chromosomal karyotypes for prolonged passaging. Therefore, porcine DFAT cells may be a novel model of stem cells for studying the functions of gene in the different biological events.

PPARγ and Wnt signaling are central positive and negative regulators of adipogenesis, respectively. Here we identified that, eicosapentaenoic acid (EPA) could effectively induce the transdifferentiation of myoblasts into adipocytes through modulation of both PPARγ expression and Wnt signaling. During the early stage of transdifferentiation, EPA activates PPARδ and PPARγ1, which in turn targets β-catenin to degradation and down-regulates Wnt/β-catenin signaling, such that the myogenic fate of myoblasts could be switched to adipogenesis. In addition, EPA up-regulates the expression of PPARγ1 by activating RXRα, then PPARγ1 binds to the functional peroxisome proliferator responsive element (PPRE) in the promoter of adipocyte-specific PPARγ2 to continuously activate the expression of PPARγ2 throughout the transdifferentiation process. Our data indicated that EPA acts as a dual-function stimulator of adipogenesis that both inhibits Wnt signaling and induces PPARγ2 expression to facilitate the transdifferentiation program, and the transcriptional activation of PPARγ2 by PPARγ1 is not only the key factor for the transdifferentiation of myoblasts to adipocytes, but also the crucial evidence for successful transdifferentiation. The present findings provided insight for the first time as to how EPA induces the transdifferentiation of myoblasts to adipocytes, but also provide new clues for strategies to prevent and treat some metabolic diseases.

In order to detect the molecular basis of heterosis in pigs, suppression subtractive hybridization was carried out to investigate the difference in gene expression in the Longissimus dorsi muscle tissues between Meishan×Yorkshire F1 crossbreeds and their parents, Meishan pigs. The swine myosin regulatory light chain 2 (MRLC2) gene differentially expressed between the crossbreeds and the purebreds was isolated and identified using semi-quantitative reverse transcriptase polymerase chain reaction and its complete cDNA sequence was obtained using the rapid amplification of cDNA ends method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis reveals that the open reading frame of this gene encodes a protein of 172 amino acids containing the putative conserved domain of the EF-hand superfamily. This predicted amino acid sequence of porcine MRLC2 protein exhibits 99%, 98%, 98%, 98% and 97% identity with that of cattle, human, dog, rat and mouse, respectively. The homology analysis revealed that the MRLC2 protein was very much conserved in evolution. The tissue expression analysis indicated that the swine MRLC2 gene is highly expressed in muscle, fat, heart, liver, spleen, lung, kidney, stomach, small intestine, ovary and testis, but not expressed in pancreas.

To investigate the correlation of individual heterozygosity and heterosis of three traits in crossbred F1 pig populations, the F1 populations were built by random mating Yorkshire × Meishan (YM, n = 82), and its reciprocal (MY, n = 47) and two straightbred populations (Yorkshire = 34, Meishan = 55) were used as control groups. The heterosis of birth weight (BWT), average daily gain (ADG), and feed conversion ratio (FCR) were acquired as well. In the research, the significant marker loci for the heterosis of the three traits were observed by one-way ANOVA (P

Hexokinase Ⅱ has been demonstrated to play the role of a key enzyme member in the glycolysis reaction. It catalyzes the conversion of glucose to glucose-6-phosphate, thus committing glucose to the glycolytic pathway. In this paper, the partial exons and introns 10, 11, 13 and 14 of the porcine HK2 gene were cloned and sequenced by comparative genomics. Comparative sequencing of three pig breeds revealed ten putative single-nucleotide polymorphisms (SNPs), one of which in intron 10 with differing bases (G981 A) is within the restriction site for enzyme Msp Ⅰ. Distribution of Msp Ⅰ-RFLP genotype and allele frequencies among different pig breeds were studied. By association analysis between Msp ⅠPCRRFLP polymorphism (AA, AB, BB genotypes of HK2 gene intron 10) and some meat quality and carcass traits in F2 group, which was constructed by our laboratory, a significant difference of pig average backfat at rump was found between AB and BB genotypes (P＜005) in F2 group. In addition, the pattern of expression of HK2 in a variety of tissues in pig was also determined using semi-quantitative RT-PCR. The expression of HK2 mRNA was detected only in pig skeletal muscle.

A 2 × 2 factorial experiment (10 boars per treatment) was conducted for 16 weeks to evaluate the effects of the dietary n-6 : n-3 ratio (14 : 1 vs 6 : 1) and vitamin E (200 vs 400 mg kg–1) on boar sperm morphology and oxidative stress. Sperm mitochondrial membrane potential (MMP), reactive oxygen species (ROS), DNA damage (8-hydroxydeoxyguanosine; 8-OHdG), seminal lipoperoxidation (malondialdehyde; MDA) and antioxidant capacity in the serum, spermatozoa and seminal plasma were assessed as indicators of oxidative stress. Sperm production was similar among groups but increased (P

Myozenin-1 (Myoz1) gene-encoded calsarcin-2 protein was expressed exclusively in fast-twitch muscles. Peroxisome proliferator-activated receptor γ2 (PPAR-γ2) is a key regulator of adipocyte differentiation, fatty acid uptake and storage in mammals. In this study, transgenic (TG) mice were generated by injecting linearized DNA that contained mouse creatine kinase M-type enhancer, Myoz1 core promoter, swine PPAR-γ2 (sPPAR-γ2) and SV40 polyadenylation sequences into pronuclei of fertilized FVB/NJ mouse embryos using microinjection technology. Then, the TG mice were used to identify whether swine Myoz1 (sMyoz1) promoter could upregulate sPPAR-γ2 expression in skeletal muscle in a TG mouse model. The results showed that the sMyoz1 promoter indeed upregulated sPPAR-γ2 expression on both the RNA and protein levels. The target genes of PPAR-γ in fat formation pathways, such as fatty acid-binding protein 4 (FABP4) and lipoprotein lipase (LPL), were also overexpressed on the RNA level. Meanwhile, the level of skeletal muscle triacylglycerol in TG mice was increased (P < 0.05), and the result of Oil Red-O staining in the skeletal muscle sections also showed that the number of lipid droplets was significantly increased in TG mice compared to wild-type mice, which might improve the intramuscular fat (IMF) content. For pork, the quality was mostly influenced by the IMF; the identification of swine muscle-specific promoter, sMyoz1, could further serve to develop transgenic pigs with higher intramuscular fat contents and improve pork quality.

This study was conducted to investigate the effects of different amounts of konjac flour (KF) inclusion in the gestation diet on the physio-chemical properties of diets, postprandial satiety in pregnant sows, lactation feed intake of sows and piglet performance during two successive reproductive cycles. Multiparous Landrace sows (n=140) were assigned randomly to one of four experimental diets, and four gestation diets were formulated to contain varying amounts of KF at 0%, 0.6%, 1.2% or 2.2%, respectively. The water binding capacity (WBC) (P

Obesity is a serious health problem worldwide associated with an increased risk of life-threatening diseases such as type 2 diabetes, atherosclerosis, and certain types of cancer. Understanding the molecular basis of adipogenesis and fat cell development in obesity is essential to identify new biomarkers and therapeutic targets for the development of anti-obesity drugs. Recent computational and experimental studies have shown that microRNAs (miRNAs) appear to play regulatory roles in many biological processes associated with obesity, including adipocyte differentiation and lipid metabolism. In addition, many miRNAs are dysregulated in metabolic tissues from obese animals and humans, which potentially contributes to the pathogenesis of obesity-associated complications. The discovery of circulating miRNAs has highlighted their potential as both endocrine signaling molecules and disease markers. The potential of miRNA based therapeutics targeting obesity is highlighted as well as recommendations for future research which could lead to a breakthrough in the treatment of obesity.

Adipogenesis is a complex process strictly regulated by a well-established cascade that has been thoroughly studied in the last two decades. This process is governed by complex regulatory networks that involve the activation/inhibition of multiple functional genes, and is controlled by histone-modifying enzymes. Among such modification enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the transcriptional regulation and post-translational modification of protein acetylation. HATs and HDACs have been shown to respond to signals that regulate cell differentiation, participate in the regulation of protein acetylation, mediate transcription and post-translation modifications, and directly acetylate/deacetylate various transcription factors and regulatory proteins. In this paper, we review the role of HATs and HDACs in white and brown adipocyte differentiation and adipogenesis, to expand our knowledge on fat formation and adipose tissue biology.

The study was conducted to investigate whether dietary fish oil could influence growth of piglets via regulating the expression of proinflammatory cytokines. A split-plot experimental design was used with sow diet effect in the main plots and differing piglet diet effect in the subplot. The results showed that suckling piglets from fish oil fed dams grew rapidly (P<0.05) than control. It was also observed that these piglets had higher ADG, feed intake, and final body weight (P<0.05) during postweaning than those piglets from lard fed dams. Furthermore, there was a significant decrease (P<0.01) in the expression of interleukin 6 and tumor necrosis factor-αinlongissimus dorsimuscle. In contrast, there was a tendency (P<0.10) towards lower ADG and higher feed : gain in weaned piglets receiving fish oil compared with those receiving lard. Meanwhile, splenic proinflammatory cytokines expression was increased (P<0.01) in piglets receiving fish oil during postweaning period. The results suggested that 7% fish oil addition to sows' diets alleviated inflammatory response via decreasing the proinflammatory cytokines expression in skeletal muscle and accelerated piglet growth. However, 7% fish oil addition to weaned piglets' diets might decrease piglet growth via increasing splenic proinflammatory cytokines expression.

Background Recent studies have shown that copy number variation (CNV) in mammalian genomes contributes to phenotypic diversity, including health and disease status. In domestic pigs, CNV has been catalogued by several reports, but the extent of CNV and the phenotypic effects are far from clear. The goal of this study was to identify CNV regions (CNVRs) in pigs based on array comparative genome hybridization (aCGH). Results Here a custom-made tiling oligo-nucleotide array was used with a median probe spacing of 2506 bp for screening 12 pigs including 3 Chinese native pigs (one Chinese Erhualian, one Tongcheng and one Yangxin pig), 5 European pigs (one Large White, one Pietrain, one White Duroc and two Landrace pigs), 2 synthetic pigs (Chinese new line DIV pigs) and 2 crossbred pigs (Landrace × DIV pigs) with a Duroc pig as the reference. Two hundred and fifty-nine CNVRs across chromosomes 1–18 and X were identified, with an average size of 65.07 kb and a median size of 98.74 kb, covering 16.85 Mb or 0.74% of the whole genome. Concerning copy number status, 93 (35.91%) CNVRs were called as gains, 140 (54.05%) were called as losses and the remaining 26 (10.04%) were called as both gains and losses. Of all detected CNVRs, 171 (66.02%) and 34 (13.13%) CNVRs directly overlapped with Sus scrofa duplicated sequences and pig QTLs, respectively. The CNVRs encompassed 372 full length Ensembl transcripts. Two CNVRs identified by aCGH were validated using real-time quantitative PCR (qPCR). Conclusions Using 720 K array CGH (aCGH) we described a map of porcine CNVs which facilitated the identification of structural variations for important phenotypes and the assessment of the genetic diversity of pigs.

The domestic pig (Sus scrofa), an important species in animal production industry, is a right model for studying adipogenesis and fat deposition. In order to expand the repertoire of porcine miRNAs and further explore potential regulatory miRNAs which have influence on adipogenesis, high-throughput Solexa sequencing approach was adopted to identify miRNAs in backfat of Large White (lean type pig) and Meishan pigs (Chinese indigenous fatty pig). We identified 215 unique miRNAs comprising 75 known pre-miRNAs, of which 49 miRNA*s were first identified in our study, 73 miRNAs were overlapped in both libraries, and 140 were novelly predicted miRNAs, and 215 unique miRNAs were collectively corresponding to 235 independent genomic loci. Furthermore, we analyzed the sequence variations, seed edits and phylogenetic development of the miRNAs. 17 miRNAs were widely conserved from vertebrates to invertebrates, suggesting that these miRNAs may serve as potential evolutional biomarkers. 9 conserved miRNAs with significantly differential expressions were determined. The expression of miR-215, miR-135, miR-224 and miR-146b was higher in Large White pigs, opposite to the patterns shown by miR-1a, miR-133a, miR-122, miR-204 and miR-183. Almost all novel miRNAs could be considered pig-specific except ssc-miR-1343, miR-2320, miR-2326, miR-2411 and miR-2483 which had homologs in Bos taurus, among which ssc-miR-1343, miR-2320, miR-2411 and miR-2483 were validated in backfat tissue by stem-loop qPCR. Our results displayed a high level of concordance between the qPCR and Solexa sequencing method in 9 of 10 miRNAs comparisons except for miR-1a. Moreover, we found 2 miRNAs, miR-135 and miR-183, may exert impacts on porcine backfat development through WNT signaling pathway. In conclusion, our research develops porcine miRNAs and should be beneficial to study the adipogenesis and fat deposition of different pig breeds based on miRNAs.

Piwi- interacting RNAs (piRNAs), a new class of small RNAs discovered from mammalian testes, are involved in transcriptional silencing of retrotransposons and other genetic elements in germ line cells. In order to identify a full transcriptome set of piRNAs expressed in the sexually mature porcine testes, small RNA fractions were extracted and were subjected to a Solexa deep sequencing. We cloned 6,913,561 clean reads of Sus Scrofa small RNAs (18–30 nt) and performed functional characterization. Sus Scrofa small RNAs showed a bimodal length distribution with two peaks at 21 nt and 29 nt. Then from 938,328 deep-sequenced small RNAs (26–30 nt), 375,195 piRNAs were identified by a k-mer scheme and 326 piRNAs were identified by homology searches. All piRNAs predicted by the k-mer scheme were then mapped to swine genome by Short Oligonucleotide Analysis Package (SOAP), and 81.61% of all uniquely mapping piRNAs (197,673) were located to 1124 defined genomic regions (5.85 Mb). Within these regions, 536 and 501 piRNA clusters generally distributed across only minus or plus genomic strand, 48 piRNA clusters distributed on two strands but in a divergent manner, and 39 piRNA clusters distributed on two strands in an overlapping manner. Furthermore, expression pattern of 7 piRNAs identified by homology searches showed 5 piRNAs displayed a ubiquitous expression pattern, although 2 piRNAs were specifically expressed in the testes. Overall, our results provide new information of porcine piRNAs and their specific expression pattern in porcine testes suggests that piRNAs have a role in regulating spermatogenesis.

Myostation (MSTN), which is primarily expressed in muscle, plays an important role in myogenic and adipogenic cells. However, there is little information about whether MSTN displays different roles between adipose-derived stem cells (ADSCs) and muscle satellite cells (MSCs). The two kinds of cells can both exist in the muscle and differentiate into adiposities. In this research, we isolated ADSCs and MSCs from porcine fat tissues and semitendinosus muscle, respectively, to investigate the effect of MSTN on the adipogenesis of those cells. ADSCs and MSCs were treated with recombinant human MSTN during the induction of adipogenesis or before the induction of differentiation. Then, we evaluated adipogenesis by Oil Red O staining and assessed the expression patterns of adipocyte-specific fatty acid binding protein (aP2) and peroxisome proliferator-activated receptor (PPAR) γ using real-time polymerase chain reaction methods. Our results indicated that the treatment with MSTN before or during the induction of differentiation in MSCs could both inhibit the adipogenesis. However, the treatment with MSTN only during the induction of differentiation in ADSCs could suppress the adipogenesis. Those results showed that MSTN had different roles in the adipogenesis of ADSCs and MSCs. It can shed new light on the origin of adipocyte located in muscle.

Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) has been identified as 5′-inositol phosphatase that hydrolyzes PI(3,4,5)P3 to PI(3,4)P2, which negatively regulates insulin-induced Akt signaling in skeletal muscle. In this study, we obtained a 3,795-bp mRNA sequence of porcine SHIP2 that included the full coding region for a protein of 1,264 amino acids. With the use of comparative mapping, we mapped this gene to SSC9 p23–24, where many QTLs affect average backfat thickness, average daily weight gain (birth-10 weeks), adipocyte number, belly fat area, and mid-back fat traits. As a candidate gene for carcass traits, a novel single nucleotide polymorphism in intron 21 (A > G) was detected by PCR-RFLP. The results showed that the AA genotype had higher skin percentage, shoulder fat thickness, and m. longissimus dorsi width, but lower m. longissimus dorsi height compared with the genotype GG (P

Background The Chinese Taihu is one of the most prolific pig breeds in the world, which farrows at least five more piglets per litter than Western pig breeds partly due to a greater ovulation rate. Variation of ovulation rate maybe associated with the differences in the transcriptome of Chinese Taihu and Large White ovaries. In order to understand the molecular basis of the greater ovulation rate of Chinese Taihu sows, expression profiling experiments were conducted to identify differentially expressed genes in ovarian follicles at the preovulatory stage of a PMSG-hCG stimulated estrous cycle from 3 Chinese Taihu and 3 Large White cycling sows by using the Affymetrix Porcine Genechip™. Results One hundred and thirty-three differentially expressed genes were identified between Chinese Taihu and Large White sows by using Affymetrix porcine GeneChip (p ≤ 0.05, Fold change ≥ 2 or ≤ 0.5). Gene Ontology (GO) analysis revealed that these genes belonged to the class of genes that participated in regulation of cellular process, regulation of biological process, biological regulation, developmental process, cell communication and signal transduction and so on. Significant differential expression of 6 genes including WNT10B and DKK2 in the WNT signaling pathway was detected. Real-time RT-PCR confirmed the expression pattern in seven of eight selected genes. A search of chromosomal location revealed that 92 differentially expressed transcripts located to the intervals of quantitative trait loci (QTLs) for reproduction traits. Furthermore, SNPs of two differentially expressed genes- BAX and BMPR1B were showed to be associated with litter size traits in Large White pigs and Chinese DIV line pigs (p ≤ 0.1 or p ≤ 0.05). Conclusions Our study detected many genes that showed differential expression between ovary follicles of two divergent breeds of pigs. Genes involved with regulation of cellular process, regulation of biological process, in addition to several genes not previously associated with ovarian physiology or with unknown function, were differentially expressed between two breeds. The suggestive or significant associations of BAX and BMPR1B gene with litter size indicated these genetic markers had the potentials to be used in pig industry after further validation of their genetic effects. Taken together, this study reveals many potential avenues of investigation for seeking new insights into ovarian physiology and the genetic control of reproduction.

Embryonic stem (ES) cells have been investigated in many animal models of severe injury and degenerative disease. However, few studies have examined the ability of ES cells to improve functional outcome following mammary gland injury. This study investigates the feasibility of implanting mouse ES cells labeled with enhanced green fluorescence protein in the developing mammary glands in order to acquire lineage-committed cells in mammary (mammary gland epithelial cell or luminal cell). Cells implanted in high numbers (5 × 10(6) cells per mammary gland) survived in the majority of the mice and nearly 38.4% of the surviving cells were CK18(+) at 15th week following the transplantation. Our results may provide a technique instrument on advanced therapy of breast diseases and the mammary regeneration after breast ablated partly.

Bone morphogenetic protein 5 (BMP5) gene was suggested to be a potential positional candidate gene for fatness trait QTLs on SSC7. Here by comparative sequencing of BMP5 gene in Meishan (MS) and Large White (LW) pigs, one SNP *131CT in 3' un-translated region was detected. Association analysis results in 322 LW×MS F(2) pigs showed that CC pigs had thinner back fatness and heavier ham than TC (P0.05 or P0.1), and had a higher fat percentage and a lower lean meat percentage (P0.1) than TT. Moreover, this C/T transition was predicted to alter BMP5 interaction with let-7c and miR-184 by using RNA22 and RNAhybrid. The negative expression of BMP5 gene with let-7c and miR-184 detected from miRNAs overexpression in swine fibroblast, indicating these 2 miRNAs might participate in the translational inhibition of BMP5 gene. Overall, SNP *131CT might be a good marker for fatness traits.

Previous evidence shows that the extensive catabolism of dietary essential amino acids (AA) by the intestine results in decreased availability of these AA for protein synthesis in extraintestinal tissues. This raises the possibility that extraintestinal availability of AA may be improved by supplying the animal with an AA source more of which can bypass the intestine. To test this hypothesis, six barrows (35-day-old, 8.6 +/- 1.4 kg), implanted with arterial, portal, and mesenteric catheters, were fed a DL-methionine (DL-MET) or DL-2-hydroxy-4-methylthiobutyrate (DL-HMTB) diet once hourly and infused intramesenterically with 1% p-amino hippurate. Although the directly available L-MET in DL-MET diet was about 1.2-fold that in DL-HMTB diet, the net portal appearance of L-MET was not different between the two diets. Compared with the low mRNA abundance and low activity of D-2-hydroxy acid dehydrogenase (D-HADH) and l-2-hydroxy acid oxidase (L-HAOX) in the intestine, the high mRNA abundance and high activity of D-AA oxidase (D-AAOX) indicated that the intestine had a relatively higher capacity of D-MET utilization than of dl-HMTB utilization to L-MET synthesis and its subsequent metabolism. However, in contrast to the much lower D-AAOX activity (nmol/g tissue) in the stomach than in the liver and kidney, both d-HADH and L-HAOX activity in the stomach was comparable with those in the liver and/or kidney, indicating the substantial capacity of the stomach to convert DL-HMTB to L-MET. Collectively, the difference in distribution of activity and mRNA abundance of D-AAOX, D-HADH, and L-HAOX in the piglets may offer a biological basis for the similar portal appearance of L-MET between DL-MET and DL-HMTB diets, and thus may provide new important insights into nutritional efficiency of different L-MET sources.

Sarcoendoplasmic reticulum Ca2+-ATPase 1 (SERCA1) as a Ca2+ release channel plays a key role in the relaxation of skeletal muscle through pumping cytosolic Ca2+ into the SR (sarcoplasmic reticulum). In this study, a novel single nucleotide polymorphism (SNP) in exon 8 (C > T) was detected by tetra-primer ARMS-PCR and the tissue expression pattern of SERCA1 was analyzed in eleven tissues. A model of primary skeletal muscle cells in vitro exposed to dexamethasone (DEX, a synthetic corticosteroid) was also employed to determine whether stress hormones cause an increase in intracellular Ca2+ concentration that is associated with alteration in SERCA1 and in turn subsequently affect meat quality. The results showed that the CC genotype has lower content intramuscular fat and higher water than pig carrying the genotype CT and CC. In addition, the additive effects were both significantly (P < 0.05) and allele T seemed to be associate with increase in intramuscular fat, while decrease in water content. Accompanied with previous studies, the high abundance of porcine SERCA1 was found in skeletal muscle tissue. DEX markedly down-regulated the expression of SERCA1, leading to Ca2+ overload. Furthermore, the imbalance of Ca2+ homeostasis up-regulated the transcription level of Calpain1. Taken together, we demonstrated a novel mechanism that the changes in expression of SERCA1 potential disturb the normal Ca2+ channel as well as the balance of Ca2+ homeostasis and which in turn finally activated Ca2+-dependent proteases such as Calpain1 which could affect meat quality.

An effective cell lysis method for extraction of bacterial genomic DNA from compost was developed in this study. Enzymatic disruption method, physical-chemical combination method, and commercial kit method were used to extract DNA from compost samples and were compared by analyzing DNA yield and efficient cell lysis. The results showed that all the three methods can be used to extract high-quality DNA from compost, but the enzymatic method had better cell lysis efficiency and DNA yields than others without the use of special equipment and expensive spending. Comparison of different methods for lysing gram-positive bacteria Bacillus subtilis indicated that the enzymatic cell lysis is superior for destroying the gram-positive cell wall. Spin-bind DNA column was used for DNA purification, and the purity of the purified sample was checked by polymerase chain reaction to amplify a region of the 16S rRNA. Results indicated that the part of 16S rRNA were amplified from all the purified DNA samples, and all the amplification products could be digested by the restriction enzyme HhaI.

The lecithin cholesterol acyltransferase gene (LCAT) plays an important role in lipoprotein metabolism, especially in the process termed ‘reverse cholesterol transport’. In this study, we obtained the 1,434 bp mRNA sequence of porcine LCAT including the full coding region and encoding a protein of 472 amino acids. The sequence was deposited into the GenBank under the accession no. EU717835. The genomic sequence of this gene which contains six exons and five introns, is 3,712 bp in length (GQ379050). Bioinformatic analysis of the 5′ regulatory region has revealed that some transcription factor Sp1, AP-1, AP-2 and NF-kappaB were represented in this region. Tissue expression analysis showed that the porcine LCAT gene is ubiquitously expressed in all examined tissues. Phylogenetic tree was constructed by aligning the amino acid sequences of different species. Moreover, we found a single nucleotide polymorphism (SNP, C/G266) in intron 1 of the LCAT gene and association analysis showed that it was significantly associated with ratio of lean to fat (P

To determine whether portal plasma flow (PPF) and net portal appearance of amino acids (AA) could be affected by 2-hydroxy-4-methylthiobutyrate (HMB), six barrows (35-day-old, 8.6+/-1.4 kg), implanted with arterial, portal and mesenteric catheters, were fed a DL-methionine (as the control) or HMB-supplemented diet once hourly and infused intramesenterically with 1% p-amino hippurate. PPF was numerically 9% higher (P=0.09) in HMB-fed pigs than in controls over a 4-6 h period. Compared with controls, pigs fed the HMB diet had increased (P

The ring finger protein 4 gene (RNF4), which might play a role in fetal germ cell development as well as in oocyte and granulosa cell maturation, was one of the potential candidate genes for reproductive traits. In the present work, we isolated the complete coding sequence of porcine RNF4 gene, identified a single nucleotide polymorphism (SNP: T/C) in intron5, and developed a PCR-SacII-RFLP genotyping assay. Association of this SNP with reproductive traits was assessed in three populations with diverse genetic backgrounds. One was Chinese Qingping sows. Another was consisted of crossbred sows derived from Landrace, Large White, Chinese Tongcheng and/or Chinese Meishan (Line DIV). The third is Large White x Meishan (LW x M) F(2) slaughtered population. Statistical analysis demonstrated that, in the first parity, the difference between RNF4 genotypes and reproductive traits of both Qingping and Line DIV sows was not significant. In the second and subsequent litters, CC animals in Qingping population had more piglets born (+1.74 piglets) and piglets born alive (+2.02 piglets) than sows with the TT genotype (P

The glutathione S-transferase mu 2 gene (GSTM2) encodes a GST functioning in the elimination of electrophilic compounds and the regulation of cell growth. In this study, the sequence of porcine GSTM2 gene that contains the complete sequence encoding a protein of 218 amino acids was cloned. The deduced amino acid sequence shared 76%, 78% and 76% identity with that of human, mouse and rat, respectively. mRNA expression analysis showed that the porcine GSTM2 gene was expressed at a high level in liver and testis, at a medium level in longissimus dorsi muscle, adipose tissue, spleen and lung, at a low level in kidney, and at a very low level in heart and embryo. A nonsense mutation (CGA--TGA) resulted from C27T substitution in the fifth exon to produce a premature translation termination codon was identified, and it was discovered that nonsense-mediated mRNA decay might have an effect on the regulation of porcine GSTM2 gene expression. This polymorphism was analyzed in Large White, Landrace, Meishan and Qingping pig populations using the Taq I-polymerase chain reaction-restriction fragment length polymorphism method. The result showed that allele C had a higher frequency than allele T in each population.

The use of markers and linkage map construction are important for QTL mapping in pigs. In this article, the genetic characteristics were studied and the linkage map was constructed in a pig resource population including 214 individuals by typing 39 microsatellite marker loci on Sus scrofa chromosomes, SSC4, SSC6, SSC7, SSC8, and SSC13. Results indicated that the average allele number, the average observed heterozygosity (H o ), and the average polymorphism information content ( PIC ) in F 1 and F 2 population were 3.2, 0.528, 0.463 and 3.2, 0.496, 0.447, respectively. In the pig resource population, the average informative meiosis (IM) was 217.4 (44-316), and the average linkage map length between the two sexes on the five chromosomes were 172.3 cM (SSC4), 168.7 cM (SSC6), 191.7 cM (SSC7), 197.3 cM (SSC8), and 178.3 cM (SSC13). The orders of microsatellite marker loci in the linkage maps were identical to, but the length was greater than, those of USDA-MARC reference map. The results of this research showed the genetic relationship and genetic characteristics of the microsatellite markers in the pig resource family population, and the linkage map could be used to for QTL mapping in the subsequent study.

LIM domain proteins are important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton by their interaction with various structural proteins, kinases and transcriptional regulators. Using molecular biology combined with in silico cloning, we have cloned the complete coding sequence of pig LIM and the cysteine-rich domain 1 gene (LMCD1) which encodes a 363 amino acid protein. The estimated molecular weight of the LMCD1 protein is 40,788 Da with a pI of 8.39. It was found to be highly expressed in both skeletal muscle and cardiac muscle. Alignment analysis revealed that the deduced protein sequence shares 86%, 91% and 93% homology with that of its human, mouse and rat counterparts, respectively. The LMCD1 protein was predicted by bioinformatics software to contain a novel cysteine-rich domain in the N-terminal region, two LIM domains in the C-terminal region, nine potential protein kinase C phosphorylation sites, seven casein kinase II phosphorylation sites, a tyrosine kinase phosphorylation site, seven N-glycosylation and N-myristoylation sites and a single potential N-glycosylation site, which is similar to the protein's human counterpart. Phylogenetic tree was constructed by aligning the amino acid sequences of the LIM domain from different species. In addition, four base mutations were detected by comparing the sequences of Large White pigs with those of Chinese Meishan pigs. The G294A mutation site was confirmed by polymerase chain reaction-single-strand conformation polymorphism analysis. Its allele frequencies were studied in five pig breeds.