Expression and genome polymorphism of ACSL1 gene in different pig breeds

Acyl coenzyme A long-chain 1 synthetase (ACSL1) plays a key role in animal fat synthesis and fatty acid β-oxidation. In order to research the function of the ACSL1 gene in pig, we analyzed the mRNA expression in liver, backfat and longissimus dorsi muscle by quantitative real-time PCR in Tibet pig (TP, n = 10), Diannan small ear pig (DSP, n = 10) and large white pig (LW, n = 10). The results showed that the mRNA expressions of the ACSL1 gene in liver and longissimus dorsi muscle of DSP and TP were significant higher than that of LW (P < 0.01). However, the expression in backfat of LW was significant higher than that of TP (P < 0.01) and DSP (P < 0.05). In addition, four SNPs located in 5' flanking region (T-1191C), exon 6(G173A), exon 14(C36T) and exon 17(T46C) were identified, and the allele frequencies of the four SNPs were significant different in indigenous and introduced pig breeds. The results indicated that the ACSL1 gene might be relative to the capacity of fat deposition and meat quality in pig breeds.     

猪脂肪组织中IGF2和IGFBP3基因表达的发育性变化及其品种差异

采用荧光定量PCR技术分析长白猪和太湖猪背部皮下脂肪组织中胰岛素样生长因子2和胰岛素样生长因子结合蛋白3基因在30、60、90、120和150日龄时表达水平的发育性变化。结果表明: (1)品种内日龄间比较, 长白猪IGF2 mRNA 在30日龄时的表达量极显著高于其他日龄(P<0.01), 之后逐渐下降, 至120日龄降到最低, 150日龄时又明显上升; 太湖猪IGF2 mRNA在30~60日龄的表达量较高, 90日龄降至最低, 120日龄迅速回升, 之后又有所下降。两品种IGFBP3 mRNA表达的发育性变化模式基本相同, 30日龄时表达量最高, 60日龄显著下降(P<0.05), 之后趋于平缓但略有波动。(2)品种间同日龄比较, 120日龄时太湖猪IGF2 mRNA的表达量极显著高于长白猪(P<0.01), 150日龄时太湖猪IGFBP3的表达量极显著低于长白猪(P<0.01), 其余日龄间两品种差异均不显著(P>0.05)。研究结果提示: 猪脂肪组织IGF2和IGFBP3基因表达存在明显的发育性变化和品种差异; IGF2基因表达水平的降低可能与脂肪细胞的增殖有关。     

Characterisation of androstenone metabolism in pig liver microsomes

Androstenone (5 alpha-androst-16-en-3-one) is a steroid pheromone produced in the testis. Excessive accumulation of androstenone together with skatole (3-methyl-indole) in the adipose tissue of some male pigs leads to "boar taint". In isolated pig hepatocytes androstenone represses the expression of cytochrome P450IIE1 (CYP2E1), the enzyme principally responsible for skatole metabolism. Androstenone can be metabolised in liver microsomes but the pathway has not been established. We have investigated androstenone metabolism in liver microsomes from two breeds of pigs exhibiting low and high levels of androstenone in adipose tissue-Large White (LW) and Meishan (M), respectively. Androstenone was reduced in isolated liver microsomes mainly to beta-androstenol using NADH as a co-factor. The rate of beta-androstenol formation in the presence of NADPH was very low. In microsomes from LW pigs the rate of beta-androstenol formation from androstenone was six times higher than in M pigs. 3beta-hydroxysteroid dehydrogenase (3beta-HSD) was investigated as a likely candidate for the enzyme catalysing androstenone reduction in pig liver. RT-PCR analysis showed that there was no sequence difference in the cDNA encoding 3beta-hydroxysteroid dehydrogenase from LW and M pigs. However, competitive RT-PCR analysis showed that the expression of 3beta-hydroxysteroid dehydrogenase mRNA was about 12 times higher in the case of LW compared to M pigs. It is concluded that the rate of androstenone metabolism in pig liver microsomes is determined by the level of expression of hepatic 3beta-hydroxysteroid dehydrogenase. The differential expression of this enzyme could be a factor affecting the rate of hepatic androstenone metabolism which in turn may influence the level of hepatic CYP2E1 expression and hence the rate of hepatic skatole metabolism.     

Expression profiling analysis for genes related to meat quality and carcass traits during postnatal development of backfat in two pig breeds

The competitive equilibrium of fatty acid biosynthesis and oxidation in vivo determines porcine sub-cutaneous fat thickness(SFT) and intramuscular fat(IMF) content.Obese and lean-type pig breeds show obvious differences in adipose deposition;however, the molecular mechanism underlying this phenotypic variation remains unclear.We used pathway-focused oligo microarray studies to examine the expression changes of 140 genes associated with meat quality and carcass traits in backfat at five growth stages(1―5 months) of Landrace(a leaner, Western breed) and Taihu pigs(a fatty, indigenous, Chinese breed).Variance analysis(ANOVA) revealed that differences in the expression of 25 genes in Landrace pigs were significant(FDR adjusted permutation, P<0.05) among 5 growth stages.Gene class test(GCT) indicated that a gene-group was very significant between 2 pig breeds across 5 growth stages(PErmineJ<0.01), which consisted of 23 genes encoding enzymes and regulatory proteins associ-ated with lipid and steroid metabolism.These findings suggest that the distinct differences in fat deposition ability between Landrace and Taihu pigs may closely correlate with the expression changes of these genes.Clustering analysis revealed a very high level of significance(FDR adjusted, P<0.01) for 2 gene expression patterns in Landrace pigs and a high level of significance(FDR adjusted, P<0.05) for 2 gene expression patterns in Taihu pigs.Also, expression patterns of genes were more diversified in Taihu pigs than those in Landrace pigs, which suggests that the regulatory mechanism of micro-effect polygenes in adipocytes may be more complex in Taihu pigs than in Landrace pigs.Based on a dy-namic Bayesian network(DBN) model, gene regulatory networks(GRNs) were reconstructed from time-series data for each pig breed.These two GRNs initially revealed the distinct differences in physiological and biochemical aspects of adipose metabolism between the two pig breeds;from these results, some potential key genes could be identified.Quantitative, real-time RT-PCR(QRT-PCR) was used to verify the microarray data for five modulated genes, and a good correlation between the two measures of expression was observed for both 2 pig breeds at different growth stages(R=0.874±0.071).These results highlight some possible candidate genes for porcine fat characteristics and provide some data on which to base further study of the molecular basis of adipose metabolism.     

Expression profiling analysis for genes related to meat quality and carcass traits during postnatal development of backfat in two pig breeds

The competitive equilibrium of fatty acid biosynthesis and oxidation in vivo determines porcine subcutaneous fat thickness (SFT) and intramuscular fat (IMF) content. Obese and lean-type pig breeds show obvious differences in adipose deposition; however, the molecular mechanism underlying this phenotypic variation remains unclear. We used pathway-focused oligo microarray studies to examine the expression changes of 140 genes associated with meat quality and carcass traits in backfat at five growth stages (1–5 months) of Landrace (a leaner, Western breed) and Taihu pigs (a fatty, indigenous, Chinese breed). Variance analysis (ANOVA) revealed that differences in the expression of 25 genes in Landrace pigs were significant (FDR adjusted permutation, P<0.05) among 5 growth stages. Gene class test (GCT) indicated that a gene-group was very significant between 2 pig breeds across 5 growth stages (P _ErmineJ<0.01), which consisted of 23 genes encoding enzymes and regulatory proteins associated with lipid and steroid metabolism. These findings suggest that the distinct differences in fat deposition ability between Landrace and Taihu pigs may closely correlate with the expression changes of these genes. Clustering analysis revealed a very high level of significance (FDR adjusted, P<0.01) for 2 gene expression patterns in Landrace pigs and a high level of significance (FDR adjusted, P<0.05) for 2 gene expression patterns in Taihu pigs. Also, expression patterns of genes were more diversified in Taihu pigs than those in Landrace pigs, which suggests that the regulatory mechanism of micro-effect polygenes in adipocytes may be more complex in Taihu pigs than in Landrace pigs. Based on a dynamic Bayesian network (DBN) model, gene regulatory networks (GRNs) were reconstructed from time-series data for each pig breed. These two GRNs initially revealed the distinct differences in physiological and biochemical aspects of adipose metabolism between the two pig breeds; from these results, some potential key genes could be identified. Quantitative, real-time RT-PCR (QRT-PCR) was used to verify the microarray data for five modulated genes, and a good correlation between the two measures of expression was observed for both 2 pig breeds at different growth stages (R=0.874±0.071). These results highlight some possible candidate genes for porcine fat characteristics and provide some data on which to base further study of the molecular basis of adipose metabolism.     

Single nucleotide polymorphism scanning and expression of the FRZB gene in pig populations

Secreted frizzled-related protein 3 (sFRP3), encoded by the gene FRZB, is a member of the sFRP family with important roles in inhibition of the Wnt signalling pathway through competitive binding of the Wnt receptor. Here, we investigated pig FRZB as a candidate gene for growth traits and identified three polymorphic sites, an insertion (A-532B) and two SNPs (G636A and C650T) in its 5′-UTR. The genotype distributions of G636A and C650T were significantly different among mini-type indigenous (Diannan Small-ear and Tibetan), normal indigenous (Laiwu and Huai), and introduced (Large Yorkshire and Landrace) breeds. In semi-quantitative PCR expression analysis, expression of FRZB mRNA was abundant in tissues of hypophysis, longissimus dorsi muscle, and adipose tissues, and low in the heart, hypothalamus, and brain. Quantitative determination of mRNA level and protein expression analysis were corresponding. The results demonstrated that FRZB gene expression in longissimus dorsi muscle and liver tissue was significantly higher in Diannan Small-ear and Tibetan pigs than in the Large Yorkshire breed (P < 0.05); however, in back fat tissue, the expression was significantly higher in Diannan Small-ear pig than in Tibetan or Large Yorkshire breeds (P < 0.05). Given the known growth and fat characteristics of the breeds, these results indicate that FRZB expression has a negative association with muscle growth and a positive association with fat deposition. In conclusion, FRZB may be a major candidate gene for growth traits in pigs.     

猪脂联素基因启动子区甲基化与其mRNA表达分析

脂联素(Adiponectin)是至今发现的唯一与肥胖呈负相关的脂肪细胞特异性蛋白, 是调控生物体的能量稳态、葡萄糖代谢和脂肪代谢的脂源性细胞因子之一。生物信息学分析发现, adiponectin基因启动子区-1500~-1350 bp 是CpG位点的富集区域。为了进一步研究脂联素基因的表达调控研究, 文章从表观遗传学角度出发, 通过Real-time PCR与甲基化特异性PCR(Methylation special PCR, MSP)的方法对脂联素基因的表达及其启动子区的甲基化状况进行了分析。结果表明, 在adiponectin基因启动子区CG富集的区域(-1500~-1350 bp)中, 90日龄长白猪大多去甲基化(83%), 90日龄蓝塘猪部分去甲基化(33%), 成年蓝塘猪全是高度甲基化(100%), 成年长白猪部分去甲基化(33%)。甲基化与去甲基化过程主要发生在某些特定CpG位点。脂联素基因在猪肌肉组织中以高度甲基化状态为主, 这一结果与该基因在肌肉组织中表达量相吻合。以上结果提示, 随个体发育, 脂联素基因的甲基化状态随基因表达的波动呈现动态的过程, 表现出与基因表达量波动基本一致的波动趋势。     

Evaluating environmental impacts of contrasting pig farming systems with life cycle assessment

Environmental impacts of 15 European pig farming systems were evaluated in the European Union Q-PorkChains project using life cycle assessment. One conventional and two non-conventional systems were evaluated from each of the five countries: Denmark, The Netherlands, Spain, France and Germany. The data needed for calculations were obtained from surveys of 5 to 10 farms from each system. The systems studied were categorised into conventional (C), adapted conventional (AC), traditional (T) and organic (O). Compared with C systems, AC systems differed little, with only minor changes to improve meat quality, animal welfare or environmental impacts, depending on the system. The difference was much larger for T systems, using very fat, slow-growing traditional breeds and generally outdoor raising of fattening pigs. Environmental impacts were calculated at the farm gate and expressed per kg of pig live weight and per ha of land used. For C systems, impacts per kg LW for climate change, acidification, eutrophication, energy use and land occupation were 2.3 kg CO₂-eq, 44.0 g SO₂-eq, 18.5 g PO₄-eq, 16.2 MJ and 4.1 m², respectively. Compared with C, differences in corresponding mean values were +13%, +5%, 0%, +2% and +16% higher for AC; +54%, +79%, +23%, +50% and +156% for T, and +4%, −16%, +29%, +11% and +121% for O. Conversely, when expressed per ha of land use, mean impacts were 10% to 60% lower for T and O systems, depending on the impact category. This was mainly because of higher land occupation per kg of pig produced, owing to feed production and the outdoor raising of sows and/or fattening pigs. The use of straw bedding tended to increase climate change impact per kg LW. The use of traditional local breeds, with reduced productivity and feed efficiency, resulted in higher impacts per kg LW for all impact categories. T systems with extensive outdoor raising of pigs resulted in markedly lower impact per ha of land used. Eutrophication potential per ha was substantially lower for O systems. Conventional systems had lower global impacts (global warming, energy use, land use), expressed per kg LW, whereas differentiated systems had lower local impacts (eutrophication, acidification), expressed per ha of land use.     

Genetic Diversity of Chinese Native Pigs Inferred from Protein Electrophoresis

We examined protein polymorphism of 20 nativepig breeds in China and 3 introduced pig breeds. Thirtyloci have been investigated, among which six loci werefound to be polymorphic. Especially, the polymorphismof malate dehydrogenase (MDH), adenylate kinase(AK), and two new alleles of adenosine deaminase (ADA)had not been reported in domestic pigs and wild pigs.The percentage of polymorphic loci (P), the meanheterozygosity (H), and the mean number ofalleles (A) are 0.200, 0.065, and 1.300, respectively.The degree of genetic variability of Chinese pigs as awhole was higher than that of goats, lower than thatof cattle and horses, and similar to that ofsheep. Using the gene frequencies of the 30 loci, Nei'sgenetic distance among the 20 native breeds in China and3 introduced pig breeds was calculated by theformula of Nei. The program NEIGHBOR in PHYLIP3.5c was chosen to construct an UPGMA tree and a NJtree. Our results show that, of the total geneticvariation found in the native pig breeds in China, 31%(0.31) is ascribable to genetic differencesamong breeds. About 69% of the total genetic variationis found within breeds. Most breeds are in linkagedisequilibrium. The patterns of genetic similaritiesbetween the Chinese native pig breeds were notin agreement with the proposed pig type classification.     

Single nucleotide polymorphism analysis of exons 3 and 4 of IGF-1 gene in pigs

The IGF-1 gene has been implicated as a candidate gene for the regulation of pig growth traits. We analyzed exons 3 and 4 of IGF-1 gene polymorphisms of the Banna mini-pig (28), the Tibetan mini-pig (30), the Junmu pig (55), and L. Yorkshire species (50) using PCR-SSCP. Three genotypes in exon 3 and 6 genotypes in exon 4 were observed, among which, one single nucleotide polymorphism, G201A, on exon 3 and two single nucleotide polymorphisms, A440G and T455C, on exon 4 were found. Statistical analysis of genotype frequencies revealed that the A allele was dominant in the large pig at the G201A locus (PIC = 0.20-0.34), and the AT alleles were dominant in the large pig at the A440G and T455C loci (PIC = 0.30-0.60). The genotype distribution between the various groups was significantly different (P< 0.01), with the highest heterozygosity seen in Junmu pigs at 0.223 and the lowest seen in L. Yorkshire at 0.098. The genetic distance of the Junmu pig from the L. Yorkshire is the smallest, the distance from the Tibetan miniature pigs is larger, and the distance from the Banna mini-pig is the largest. The IGF-1 gene polymorphism and heterozygosity results from various pig breeds indicate that IGF-1 is substantially polymorphic with significant difference of the polymorphic distribution and expression levels among various pig breeds. This information provides a theoretical basis for the genetic background of miniature pigs but also provides means to breed improved pig varieties.     

Differences in adiponectin protein expression: effect of fat depots and type 2 diabetic status.

Adiponectin is an adipocyte-derived hormone associated with insulin sensitivity and atherosclerotic risk. As central rather than gluteofemoral fat is known to increase the risk of type 2 diabetes and cardiovascular disease, we investigated the mRNA and protein expression of adiponectin in human adipose tissue depots. RNA was extracted from 46 human adipose tissue samples from non-diabetic subjects aged 44.33 +/- 12.4 with a BMI of 28.3 +/- 6.0 (mean +/- SD). The samples were as follows: 21 abdominal subcutaneous, 13 omentum, 6 thigh; samples were also taken from diabetic subjects aged 66.6 +/- 7.5 with BMI 28.9 +/- 3.17; samples were: 6 abdominal subcutaneous; 3 thigh. Quantitative PCR and Western analysis was used to determine adiponectin content. Protein content studies determined that when compared with non-diabetic abdominal subcutaneous adipose tissue (Abd Sc AT) (values expressed as percentage relative to Abd Sc AT -100 %). Adiponectin protein content was significantly lower in non-diabetic omental AT (25 +/- 1.6 %; p < 0.0001, n = 6) and in Abd Sc AT from diabetic subjects (36 +/- 1.5 %; p < 0.0001, n = 4). In contrast, gluteal fat maintained high adiponectin protein content from non-diabetic patients compared with diabetic patients. An increase in BMI was associated with lower adiponectin protein content in obese ND Abd Sc AT (25 +/- 0.4 %; p < 0.0001). These findings were in agreement with the mRNA expression data. In summary, this study indicates that adiponectin protein content in non-diabetic subjects remains high in abdominal subcutaneous fat, including gluteal fat, explaining the high serum adiponectin levels in these subjects. Omental fat, however, expresses little adiponectin. Furthermore, abdominal and gluteal subcutaneous fat appears to express significantly less adiponectin once diabetic status is reached. In conclusion, the adipose tissue depot-specific expression of adiponectin may influence the pattern of serum adiponectin concentrations and subsequent disease risk.     

Characterization of mineralocorticoid and glucocorticoid receptors in pigs: comparison of Meishan and Large White breeds.

Corticosteroids receptors were characterized and compared in central and peripheral tissues of two pig breeds, the Meishan (MS) and the Large White (LW) pigs, that display differences in the basal activity and stress-induced reactivity of the hypothalamic-pituitary-adrenal (HPA) axis. In vitro kinetic experiments on kidney and liver cytosols from adrenalectomized pigs allowed to identify two distinct corticosteroid receptors referred to as mineralocorticoid (MR) and glucocorticoid (GR) receptors. The binding specificities were determined for kidney and hippocampal MR and for liver and hippocampal GR. In hippocampus and peripheral tissues, cortisol showed a greater affinity for MR than for GR. As already described in the dog, mouse and human, dexamethasone and progesterone display a moderate affinity for MR. Putative differences in corticosteroid receptors binding capacities and affinities were investigated by saturation binding studies in specific regions implicated in the regulation of HPA axis (hippocampus and pituitary). The MS pigs evidenced higher densities of hippocampal MR, while LW pigs had higher densities of pituitary GR. Thus, this study suggests that a difference in the MR/GR balance in hippocampus and pituitary could be implicated in the different HPA activity between MS and LW pigs.     

Evolution of the fatty acid profile of subcutaneous back-fat adipose tissue in growing Iberian and Landrace × Large White pigs

The lipid content and fatty acid (FA) profile in pig tissues are strongly influenced by genotype and nutrient supply, with implications in meat quality. The de novo lipid synthesis and pattern of FA unsaturation could be an important cause of variation in the overall efficiency of energy utilization among breeds. To test the effects of pig genotype and CP supply on the evolution of back-fat tissue FA profile throughout the growing and finishing stages, 32 Iberian (IB) and Landrace × Large White (LR × LW) barrows were offered one of two diets differing in CP content (13% or 17% as fed). A pair-fed procedure (0.8 × ad libitum intake of IB pigs) was used. Subcutaneous fat samples were taken at the dorso-lumbar region at ∼38, 50, 65, 90 and 115 kg BW. Higher proportions of total monounsaturated FA (MUFA; P < 0.01) and lower proportions of total saturated FA (SFA; P < 0.01 to 0.05) were found in the outer back-fat layer of pigs both at 50 and 115 kg BW. Pig genotype affected the FA composition of both subcutaneous back-fat layers. The proportions of C18:0 and SFA in fat tissue were higher in IB than in LR × LW pigs from 38 to 65 kg BW, especially in the outer layer. In addition, MUFA contents were higher in IB pigs at 115 kg BW in both layers (+5% on average; P < 0.01). Increased proportions of C18:2 n-6 and polyunsaturated FA (PUFA) were found in LR × LW pigs, irrespective of the stage of growth and back-fat layer (P⩽0.02). At 50 kg BW, pigs receiving the high-protein diet presented the highest C18:2 n-6, C18:3 n-3, C20:5 n-3 and PUFA contents. A significant genotype × CP content interaction was observed for C18:3 n-3 because of the increased concentration of this FA in LR × LW pigs when offered the 17% CP diet (P < 0.05). Higher C16:0 and SFA contents (+5%; P = 0.03) were found in pigs offered the 13% CP diet and slaughtered at 115 kg BW. There was a genotype × CP interaction for MUFA concentration because of the higher MUFA content observed in IB pigs offered the highest protein content diet (P = 0.03). Our results suggest that genetic variation in de novo lipid synthesis and pattern of FA unsaturation might contribute to explain differences in back-fat FA profile of IB and LR × LW pigs under identical nutritional management. They could be also relevant to explain the low efficiency of nutrient and energy utilization in the IB pig.     

Lean and obese pig breeds exhibit differences in prenatal gene expression profiles of muscle development

Muscle development in domesticated animals is important for meat production. Furthermore, intramuscular fat content is an important trait of meat intended for consumption. Here, we examined differences in the expression of factors related to myogenesis, adipogenesis and skeletal muscle growth during fetal muscle development of lean (Yorkshire) and obese (Chenghua) pig breeds. At prenatal days 50 (d50) and 90 (d90), muscles and sera were collected from pig fetuses. Histology revealed larger diameters and numbers of myofibers in Chenghua pig fetuses than those in Yorkshire pig fetuses at d50 and d90. Yorkshire fetuses had higher serum concentrations of myostatin (d90), a negative regulator for muscle development, and higher mRNA expression of the growth hormone receptor Ghr (d90), myogenic MyoG (d90) and adipogenic LPL (d50). By contrast, Chenghua fetuses exhibited higher serum concentration of growth hormone (d90), and higher mRNA expression of myogenic MyoD (d90) as well as adipogenic PPARG and FABP4 (d50). Our results revealed distinct expression patterns in the two pig breeds at each developmental stage before birth. Compared with Chenghua pigs, development and maturation of fetal skeletal muscles may occur earlier in Yorkshire pigs, but the negative regulatory effects of myostatin may suppress muscle development at the later stage.