Polymorphism of M307 of the FUT1 Gene and Its Relationship with Some Immune Indexes in Sutai Pigs (Duroc????Meishan)

The alpha (1,2)fucosyltransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of the enterotoxigenic Escherichia coli (ETEC) F18 receptor. Polymorphisms were detected at the M307 position in FUT1 of a breeding base population of Sutai pigs and their correlations to immune parameters analyzed. After digestion by Hin6I, three genotypes were identified at M307, AA (frequency 0.235), AG (0.609), and GG (0.156), with significant deviation from Hardy-Weinberg equilibrium (P < 0.01). The hemoglobin and white blood cell count of the AA genotype pigs were significantly higher than those of AG and GG pigs (P < 0.05). The results indicated that AA pigs not only are resistant to edema disease and post-weaning diarrhea in piglets but also have relatively strong resistance to disease in general.     

The effect of mutation at M307 in <Emphasis Type="Italic">FUT1</Emphasis> gene on susceptibility of <Emphasis Type="Italic">Escherichia coli</Emphasis> F18 and gene expression in Sutai piglets

Escherichia coli F18 (ECF18) is a common porcine enteric pathogen. The pathogenicity of ECF18 bacteria depends on the existence of ECF18 receptor in the brush border membranes of piglet’s small intestinal mucosa. Alpha (1) fucosyltransferase gene (FUT1) has been identified as the candidate gene controlling the adhesion to ECF18 receptor. The genetic variations in the position of M307 nucleotide in open reading frame of FUT1 have been proposed as a marker for selecting resistant pigs. The piglets were divided into three groups, AA, AG and GG, according to the genotypes present at M307 of FUT1. Small intestinal epithelium cells of piglets with AA, AG and GG genotypes were selected to test the adhesion capability of the wild type E.coli expressing F18ab fimbriae, the recombinant E. coli expressing F18ac fimbriae or the recombinant E. coli secreting and surface-displaying the FedF subunit of F18ab fimbriae, respectively. Here, we examined the distribution and expression of porcine FUT1 mRNA in different tissues in Sutai pigs using real-time PCR. The results showed that piglets with AA genotype show resistance, whereas piglets with GG or AG genotypes are sensitive to the pathogenic E. coli F18 in Sutai piglets. FUT1 was expressed in all the tissues that were examined, and the gene’s expression was highest in the lungs. There was no significant difference in expression level among the three genotypes in the liver, lung, stomach and duodenum, where the gene expression was relatively high. The present analysis suggested that mutation at M307 in FUT1 gene determines susceptibility of small intestinal epithelium to E. coli F18 adhesion in Sutai piglet and the expression of FUT1 gene may be regulated by other factors or the mutation was likely to be in linkage disequilibrium with some cis-regulatory variants.     

Evaluation of M307 of <Emphasis Type="Italic">FUT1</Emphasis> gene as a genetic marker for disease resistance breeding of sutai pigs

Alpha (1,2) fucosyltransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of the receptor for ETEC F18. The genetic variations in the position of M307 nucleotide in open reading frame of FUT1 have been proposed as a marker for selecting ETEC F18 resistant pigs. The polymorphisms of M307 in FUT1 of breeding base group for ETEC F18 resistance of Sutai pigs (Duroc × Meishan) was detected and their correlations to some immune indexes, growth and development ability, carcass traits and meat quality were also analyzed, which aimed to investigate feasibility of further breeding for diseases resistance based on M307 of FUT1 for Sutai pigs. After digested by Hin6 I, M307 of FUT1 gene could be divided into three kinds of genotypes, AA, AG, and GG. The frequencies were 0.235, 0.609, and 0.156, respectively. The results indicated that Sutai pigs with the AA genotype in M307 of FUT1 gene not only have relatively strong general disease resistance ability in piglets, but also have higher growth and development ability and stable carcass traits and meat quality. It is entirely feasible to raise the new strains of Sutai pigs resistant to Escherichia coli F18 based on genetic marker of the M307 position in FUT1gene.     

Effects of natural resistance-associated macrophage protein 1 and toll-like receptor 2 gene polymorphisms on post-weaning piglet survivability

Predicting resistance or susceptibility to infectious diseases on the basis of the genotypes of animals regarding disease or immune related genes could be important in animal production. We evaluated the potential influence of the genetic polymorphisms of four immune related genes, porcine beta defensin 4, interferon-induced GTP-binding protein Mx1, natural resistance-associated macrophage protein 1 (Nramp1), and Toll-like receptor 2 (TLR2) on post-weaning piglet survivability in farms. We selected a single SNP that satisfied the criteria of non-synonymous substitutions and the minor allele frequency >0.1 from each gene, and performed PCR–RFLP. Distributions of allele and genotype frequencies of the SNPs were, in general, significantly different among the five breeds (Berkshire, Yorkshire, Duroc, Landrace, and Korean native pigs. Initially, 371 randomly collected Yorkshire × Landrace F1 piglets consisting of post-weaning survival (n = 185) and non-survival groups (n = 186) were genotyped for the selected SNPs. For the Nramp1 and TLR2 SNPs, genotype frequencies were significantly different between the two groups (P < 0.05). To confirm the results, additional animals that were collected in a different time-period were genotyped for the Nramp1 (n = 390) and TLR2 (n = 240) SNPs. The results using the combined samples (n = 761) were consistent with the initial analysis (P < 0.01), suggesting that the genetic polymorphisms of Nramp1 and TLR2 affect post-weaning piglet survivability. The relative risks, i.e. odds ratios, between the beneficial and non-beneficial genotypes to piglet survivability were 4.88 and 29.65 for the Nramp1 and TLR2 SNPs, respectively.     

Effects of FUT1 gene mutation on resistance to infectious disease

Alpha-(1,2)-fucosyltransferase (FUT1) gene has been identified as a candidate gene for regulating the expression of Escherichia coli F18 receptor gene (ECF18R) which promotes adherence of Enterotoxigenic (ETEC) and Verotoxigenic (VTEC) Escherichia coli (E. coli) via F18 fimbriae. In order to illustrate the polymorphisms of FUT1 and their effects on resistance to natural infection by Porcine Respiratory and Reproductive Symdrome Virus (PRRSV) and Haemophilus parasuis, the distributions of different genotypes and the relative risks of disease incidence in pigs were investigated. A total of 1,041 pigs representing three European breeds (Duroc, Landrace and LargeWhite), five Chinese local breeds (Wild pig, Small MeiShan, QinPing, JinHua, and JianLi) and three commercial populations (LargeWhite?×?JianLi, Duroc?×?Landrace?×?LargeWhite and Duroc?×?wild pig) were selected to analyze the genotype of the FUT1 gene by PCR-RFLP. Only the GG genotype associated with susceptibility to ECF18 bacteria was detected in Chinese local pig breeds and a population of LargeWhite?×?JianLi, while the AA genotype which confers resistance to ECF18 was detected in two European breeds (Duroc and LargeWhite), two populations of Duroc?×?wild pig and Duroc?×?Landrace?×?LargeWhite. Regarding relative risk of incidence, Duroc?×?Landrace?×?LargeWhite with genotypes GG or AG showed greater relative risk (OR?=?2.040, P?=?0.025; OR?=?1.750, P?=?0.081, respectively) than those with genotype AA during natural infection by both PRRSV and Haemophilus parasuis. It can be concluded that the mutation of FUT1 gene might play a role in pig infection by multi-pathogens, and that AA may be a favourable genotype for increasing the resistance to disease.     

猪a1-岩藻糖转移酶基因(FUT1)M857位点遗传变异分析

肠毒素大肠杆菌 (ETEC) F18是引起仔猪断奶后水肿和腹泻病的主要病原菌, a-1岩藻糖转移酶(FUT1)基因是ECEC F18侵染猪小肠的受体蛋白基因。利用PCR-RFLP方法检测了1个野猪以及20个中外家猪猪种(群)共696个个体在FUT1基因开放阅读框架的857核苷酸位点的遗传变异, 结果表明: 在所有猪种中, 均未检测到抗性的AA型纯合子, 在外来猪种杜洛克和约克夏、国内猪种临高猪和杂交猪种中检测到AG型杂合子, 外来猪种中的皮特兰、长白猪以及除临高猪外的所有国内猪种和野猪均表现为极端的单态分布, 只有易感的GG基因型。研究结果提示, 中国地方猪种不具备抵抗ETEC F18大肠杆菌的遗传基础, 与外来猪种确实存在差异, 这种差异可能与各自不同的起源有关, ETEC F18抗性基因可能起源于欧洲野猪; 并推测猪种的生长速度与ETEC F18大肠杆菌病的发生具有密切的关系。     

Variant in sulfonylurea receptor-1 gene is associated with high insulin concentrations in non-diabetic Mexican Americans: SUR-1 gene variant and hyperinsulinemia

The high-affinity sulfonylurea receptor (SUR1) gene regulates insulin secretion and may play a role in type 2 diabetes. A silent variant in exon 31 of SUR1 (AGG→AGA) was detected by single-strand conformational polymorphism and genotypes were determined for 396 Mexican American subjects (289 non-diabetic). The normal and mutant alleles were designated G and A, respectively. Among non-diabetics, those with the AA genotype had higher fasting insulin values than those with the AG and GG genotypes (113.4 pmol/l for AA vs 82.8 pmol/l for AG/GG, P=0.043). Similar results were observed for 2-h insulin (849.6 pmοl/l for AA vs 498.6 pmol/l for AG/GG, P=0.0003) and for the proinsulin to specific insulin ratio (0.068 for AA vs 0.056 for AG/GG, P=0.030). Specific insulin levels also differed significantly across the three genotypic classes (P=0.021). No differences in fasting glucose, body mass index, or waist circumference according to genotype were noted. Two-hour glucose was modestly higher in individuals with the AA genotype. Since we have previously reported linkage between SUR1 and hyperglycemia, the present association between a SUR1 variant and hyperinsulinemia in normal individuals from a high diabetes risk ethnic group raises the possibility of primary insulin hypersecretion as an antecedent of type 2 diabetes in at least some individuals from this population. Received: 3 April 1998 / Accepted: 5 June 1998     

FUT1基因多态性及其与产仔性状的关联性研究

采用PCR-RFLP技术6个中外品种共245头猪的FUT1基因进行了研究, 结果表明, Hin 6Ⅰ位点上, 大白猪、长白猪和杜洛克猪3个外来猪种均存在多态, 且以敏感型(GG型和AG型)居多; 山西黑猪、太原花猪和马身猪3个本地猪种的所有检测样品都表现为GG型。用方差分析方法分析FUT1基因型、品种和胎次与产仔性状之间的关系, 基因型和品种对猪的总产仔数的影响显著, 胎次对总产仔数影响不显著。而基因型、品种和胎次对产活仔数影响均不显著。     

猪a1-岩藻糖转移酶基因(FUT1)在26个中外猪种中的遗传变异研究

肠毒素大肠杆菌F18(ECF18)是引起仔猪断奶后水肿和腹泻病的主要病原菌,a1—岩藻糖转移酶基因(FUT1)是ECF18侵染猪小肠的受体蛋白候选基因。通过采用PCR—RFLP方法检测了5个西方商业猪种以及21个中国地方猪种(群)1458个个体在FUT1基因开放阅读框架的307核苷酸位点的G-A点突变(M307^G-A)遗传变异。结果表明：5个外来猪种以及中国地方猪种中的临高猪在该FUT1基因位点存在多态性,其他中国地方猪种均表现为极端的单态分布,只有易感的GG基因型,没有多态性。由此提示：1)如果猪FUT1 M307^G-A点突变是决定猪小肠上ECF18受体表达与否的关键因素,则绝大部分中国地方猪种均不具备抵抗ECF18的遗传基础,这除了表明ECF18抗性基因有可能起源于西方猪种外,同时也表明对中国地方猪种中在这个位点惟一存在多态性的海南临高猪的品种资源保存具有非常重要的意义。2)一般而言,在中国的养猪生产实践中,中国地方猪种的仔猪抗水肿与腹泻病能力普遍强于外来猪种,研究的结果提示有必要对中国地方猪种所具备的上述遗传抗性做更深入的研究,寻找、定位其相应的QTL或／和抗性基因。     

中外六个猪种FUT1基因多态性研究

目的对6个中外猪种共245头猪的FUT1基因进行了研究。方法采用PCR-RFLP技术。结果与结论Hin6I位点上,大白猪、长白猪、杜洛克猪3个外来猪种均存在多态,且以敏感型（GG型和AG型）居多;山西黑猪、太原花猪、马身猪3个本地猪种的所有检测样品都表现为GG型。     

Investigation Into the Humaneness of Slaughter Methods for Guinea Pigs (Cavia porcelus) in the Andean Region

Guinea pigs (Cavia porcelus) are an important source of nonhuman animal protein in the Andean region of South America. Specific guidelines regarding the welfare of guinea pigs before and during slaughter have yet to be developed. This study critically assessed the humaneness of 4 different stunning/slaughter methods for guinea pigs: cervical neck dislocation (n = 60), electrical head-only stunning (n = 83), carbon dioxide (CO₂) stunning (n = 21), and penetrating captive bolt (n = 10). Following cervical neck dislocation, 97% of guinea pigs had at least 1 behavioral or cranial/spinal response. Six percent of guinea pigs were classified as mis-stunned after electrical stunning, and 1% were classified as mis-stunned after captive bolt. Increased respiratory effort was observed during CO₂ stunning. Apart from this finding, there were no other obvious behavioral responses that could be associated with suffering. Of the methods assessed, captive bolt was deemed the most humane, effective, and practical method of stunning guinea pigs. Cervical neck dislocation should not be recommended as a slaughter method for guinea pigs.     

Polymorphisms of the Myostatin Gene and Its Relationship with Reproduction Traits in the Bian Chicken

Myostatin, or growth and differentiation factor 8, is a member of the transforming growth factor-β superfamily; it functions as a negative regulator of skeletal muscle development and growth in mammals. In this study, single nucleotide polymorphisms in the 5′ regulatory region and exon 1 of the myostatin gene were detected by PCR–SSCP in the Bian, Jinghai, Youxi, and Arbor Acre chickens, and the associations of the polymorphisms with reproduction traits were analyzed. Seven SNPs (A326G, C334G, C1346T, G1375A, A1473G, G1491A, and G2283A) were found in the myostatin gene. Association analysis showed that the G2283A were significantly associated with reproduction traits. Bian chickens of the GG genotype had a greater age at first egg than those of the GA and AA genotypes (P < 0.01). Correspondingly, Bian chickens of the GA and AA genotypes had larger egg number at 300 days than those of the GG genotype (P < 0.05 and P < 0.01, respectively). Bian chickens of the AA genotype had significantly higher body weight at 300 days than those of the GG genotype (P < 0.05). These results suggested that the myostatin gene may have certain effects on reproduction traits other than merely as a negative regulator of skeletal muscle development and growth in mammals previously reported.     

Effect of increasing the dietary tryptophan to lysine ratio on plasma levels of tryptophan,kynurenine and urea and on production traits in weaner pigs experimentally infected with an enterotoxigenic strain of Escherichia coli‡

This experiment examined if immune system stimulation of weaner pigs, initiated by inoculation an enterotoxigenic strain of Escherichia coli (ETEC), increased the requirement for dietary tryptophan (Trp), modulated the inflammatory response, altered plasma levels of Trp and its metabolite kynurenine (Kyn) and effected post-weaning diarrhoea. Individually housed pigs (n = 72) weaned at 21 d of age were allocated to one of six treatments (n = 12) according to a two by three factorial arrangement of (1) with or without ETEC infection and (2) three dietary ratios of standardised ileal digestible (SID) Trp to lysine (Lys) (SID Trp:Lys) of 0.16, 0.20 or 0.24, in a completely randomised block design. Pigs had ad libitum access to diets (per kg 14.13 MJ ME, 12.4 g SID Lys, 195 g crude protein) for 3 weeks after weaning. Pigs were infected with ETEC (O149:K98:K88) at 72, 96 and 120 h after weaning and then bled on day 3, 11 and 19. An increased dietary Trp:Lys ratio increased plasma Trp and Kyn (p < 0.001) without effect of infection. On day 3, pigs fed 0.24 SID Trp:Lys had lower levels of plasma urea than at 0.20 Trp:Lys (p = 0.047) and on day 11, plasma urea was lower at 0.20 than at 0.16 SID Trp:Lys (p = 0.007). Infection increased (p = 0.039) the diarrhoea index and deteriorated faecal consistency from day 4–10 (p < 0.05). Treatments did not affect haptoglobin and acid soluble glycoprotein levels or daily gain and feed intake. However, 0.24 SID Trp:Lys improved (p = 0.021) feed efficiency without an effect of infection. In conclusion, in the absence of dietary antibiotic growth promotants, increasing the dietary SID Trp:Lys ratio to 0.24 improved feed conversion ratio after weaning and increased plasma levels of Trp and Kyn regardless of infection with E. coli.     

Association analysis of the SNP (rs345476947) in the <Emphasis Type="Italic">FUT2</Emphasis> gene with the production and reproductive traits in pigs

The FUT2 gene was considered as an important candidate for pathogenic infections, while the potential associations between this gene and the production and reproductive traits of pigs have not been explored. In this study, we detected the genetic variants of porcine FUT2 gene and analyzed the associations of the polymorphisms with FUT2 mRNA expression and production and reproductive traits (age at 100 kg, backfat thickness at 100 kg, eye muscle thickness, the number of newborn piglets, the number of weaned piglets, and birth weight) in 100 Large White sows. One single nucleotide polymorphism (SNP) (rs345476947, C→T) in the intron of FUT2 and three genotypes (TT, CT and CC) were determined. Association analysis revealed significant associations between this SNP with the number of newborn piglets and weaned piglets. Furthermore, individuals with the TT genotype had significantly higher numbers of newborn piglets and weaned piglets than those with the CC genotype (P?<?0.05). Quantitative PCR analysis showed that FUT2 expression in individuals with CC genotype was significantly higher than those with TT and CT genotypes in the liver and lymph gland (P?<?0.05) and higher than that of CT in the spleen, kidney, and duodenum (P?<?0.05). These findings indicated that the TT genotype may be a favorable genotype for the reproductive traits of pigs. Our study revealed the genetic variants of the FUT2 gene and identified a promising candidate SNP (rs345476947) associated with the reproductive traits, which has the potential to be applied in selective breeding of pigs.     

An <Emphasis Type="Italic">Msp</Emphasis>I polymorphism in the inhibin alpha gene and its associations with superovulation traits in Chinese Holstein cows

To identify a predictor to forecast superovulation response on the basis of associations between superovulation performance and gene polymorphism, the PCR–RFLP method was applied to detect an A>G transition determining an MspI polymorphism at position 192 in the exon I of the bovine inhibin alpha (INHA) gene and evaluate its associations with superovulatory response in 118 Chinese Holstein cows treated for superovulation. Association analysis showed that cows with the GG genotype resulted in a significant increase in the number of ova (TNO) than AG and AA genotypes in the first (P = 0.023), second (P = 0.004) and third (P = 0.002) superovulation treatments and produced more transferable embryos (NTE) than that of AG and AA genotypes in the third (P = 0.045) superovulation treatment. Moreover, individuals with GG genotype produced more transferable embryos than AA (P < 0.05) genotype in the second superovulation treatment and all cows without superovulation response were mutations with genotypes of AA and AG. These results indicate that INHA gene can be used as a predictor for superovulation in Chinese Holstein cows, and imply that cows with AA genotype should be excluded for superovulation practices.     

Two α(1,2) fucosyltransferase genes on porcine Chromosome 6q11 are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18R) loci

The Escherichia coli F18 receptor locus (ECF18R) has been genetically mapped to the halothane linkage group on porcine Chromosome (Chr) 6. In an attempt to obtain candidate genes for this locus, we isolated 5 cosmids containing the α(1,2)fucosyltransferase genes FUT1, FUT2, and the pseudogene FUT2P from a porcine genomic library. Mapping by fluorescence in situ hybridization placed all these clones in band q11 of porcine Chr 6 (SSC6q11). Sequence analysis of the cosmids resulted in the characterization of an open reading frame (ORF), 1098 bp in length, that is 82.3% identical to the human FUT1 sequence; a second ORF, 1023 bp in length, 85% identical to the human FUT2 sequence; and a third FUT-like sequence thought to be a pseudogene. The FUT1 and FUT2 loci therefore seem to be the porcine equivalents of the human blood group H and Secretor loci. Direct sequencing of the two ORFs in swine being either susceptible or resistant to adhesion and colonization by F18 fimbriated Escherichia coli (ECF18) revealed two polymorphisms at bp 307 (M307) and bp 857 (M857) of the FUT1 ORF. Analysis of these mutations in 34 Swiss Landrace families with 221 progeny showed close linkage with the locus controlling resistance and susceptibility to E. coli F18 adhesion and colonization in the small intestine (ECF18R), and with the locus of the blood group inhibitor S. A high linkage disequilibrium of M307–ECF18R in Large White pigs makes the M307 mutation a good marker for marker-assisted selection of E. coli F18 adhesion-resistant animals in this breed. Whether the FUT1 or possibly the FUT2 gene products are involved in the synthesis of carbohydrate structures responsible for bacterial adhesion remains to be determined. Received: 17 February 1997 / Accepted: 30 May 1997     

Production of Cloned Wuzhishan Miniature Pigs and Application for Alloxan Toxicity Test

Wuzhishan miniature pig is one of the four most important pig breeds in China and has many major economic characteristics. Herein, we successfully used SCNT to clone Wuzhishan miniature pig. First, ear fibroblasts were isolated from a 2-year-old female Wuzhishan miniature piglet to be used as the donor cell. Second, good-quality COCs were selected from ovaries obtained from pigs at a local slaughterhouse and cultured. Mature eggs with the first polar body and ear fibroblasts were applied SCNT. Lastly, we in total produced 12 piglets with 7 piglets surviving to adults. Next, we used these pigs to test alloxan toxicity and to build T I D diabetes type. We know that diabetes mellitus is a chronic heterogeneous metabolic disease characterized by a high blood glucose level and abnormal insulin secretion. In this study, T I D (type I diabetes) was experimentally induced in cloned Wuzhishan miniature pigs with alloxan. In brief, an intravenous injection of alloxan (group B: 170 mg/kg, n = 3) was administered to pigs weighing between 27 and 39 kg. Sterile saline was administered to control pigs (n = 3). We determined the glycometabolism related index, performed an intravenous glucose tolerance test, and carried out immunohistochemistry experiments. There were no significant differences in body weight, blood glucose, and serum insulin in all groups, before treatment. The level of blood glucose was significantly higher (P < 0.05) in group B (12.18 ± 0.70 mmol/L) than in the control (2.93 ± 0.39 mmol/L). By contrast, the level of serum insulin was lower in group B (5.641 ± 0.573 μIU/mL) than in the control (7.578 ± 0.539 μIU/mL). Histological studies by hematoxylin and eosin (H&E) revealed a loss of β-cells in the pancreas from pigs treated with 170 mg/kg alloxan. Immunolocalization studies showed a decrease in insulin reactivity in this treatment group as well. To conclude, our model holds promise in future studies of diabetes drug testing and islet xenotransplantation.     

Piglet growth before and after weaning in relation to a qualitative estimate of solid (creep) feed intake during lactation: A pilot study*

Abstract

The experimental objectives were to verify whether a qualitative measure of creep feed consumption using a dye was related to performance, and associate this with teat order. Indigo carmine (5 g/kg) was added to a starter diet between days 12 and 31 (weaning) of lactation. On days 19, 23, 27 and 31, faeces from each piglet were assessed for colouration. Each piglet was categorized as a ‘good’, ‘moderate’ or ‘small/non’ eater of feed. There were no differences in pre-weaning growth rate between categories. Piglets classed as ‘good’ or ‘moderate’ eaters in lactation grew fastest (p = 0.009) in the first three days after weaning, but between days 4 and 7, the highest growth rate occurred in ‘moderate’ eaters. ‘Small/non' eaters grew slower (p < 0.01) between weaning and 28 days after weaning. Piglets drinking milk from anterior teats were heavier at weaning (p < 0.001) and for the first 14 days after weaning (p = 0.104) compared to piglets sucking posterior teats. Data from this study demonstrated that creep feed intake of piglets could qualitatively be assessed using indigo carmine, and that this categorization was related to performance in the immediate post-weaning period.