Gary Evans

Relationships of myosin heavy chain fibre types to meat quality traits in traditional and modern pigs

Porcine skeletal muscle fibres were molecularly classified, using in situ hybridisation and immunocytochemistry, into four types, according to the isoform of myosin heavy chain (MyHC) that was present in each fibre (MyHC slow/I, MyHC 2a, MyHC 2x and MyHC 2b). The relationship between MyHC fibre types and meat quality traits between two phenotypically divergent muscles [longissimus dorsi (LD) and psoas], and between the same muscles of different breeds (traditional Berkshire and Tamworth, and modern Duroc-based and Large White-based) were examined. We found that the greater abundance of fast oxidative-glycolytic MyHC 2a and 2x fibres in the psoas was associated with superior meat quality traits, and that the greater presence of fast glycolytic MyHC 2b fibres in the LD could account for less favourable quality traits, both in terms of pH, drip loss, grain, colour, yield force and work done. Although significant correlations were found between specific fibre types and quality traits, within either the psoas or LD muscle of some breeds, no consistent correlation was found across both muscles and all breeds. This finding was in line with the view that a given fibre type could have considerable differences in phenotype between breeds, and between muscles. The observed inverse compositional and functional-meat quality relationship between MyHC 2b and 2x fibres, and MyHC 2b and 2a fibres could form a basis of fibre type manipulation to improve meat quality.

Development of a porcine skeletal muscle cDNA microarray: analysis of differential transcript expression in phenotypically distinct muscles.

BackgroundMicroarray profiling has the potential to illuminate the molecular processes that govern the phenotypic characteristics of porcine skeletal muscles, such as hypertrophy or atrophy, and the expression of specific fibre types. This information is not only important for understanding basic muscle biology but also provides underpinning knowledge for enhancing the efficiency of livestock production.ResultsWe report on the de novo development of a composite skeletal muscle cDNA microarray, comprising 5500 clones from two developmentally distinct cDNA libraries (longissimus dorsi of a 50-day porcine foetus and the gastrocnemius of a 3-day-old pig). Clones selected for the microarray assembly were of low to moderate abundance, as indicated by colony hybridisation. We profiled the differential expression of genes between the psoas (red muscle) and the longissimus dorsi (white muscle), by co-hybridisation of Cy3 and Cy5 labelled cDNA derived from these two muscles. Results from seven microarray slides (replicates) correctly identified genes that were expected to be differentially expressed, as well as a number of novel candidate regulatory genes. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray.ConclusionWe have developed a porcine skeletal muscle cDNA microarray and have identified a number of candidate genes that could be involved in muscle phenotype determination, including several members of the casein kinase 2 signalling pathway.

Association of HPA axis-related genetic variation with stress reactivity and aggressive behaviour in pigs

BackgroundStress, elicited for example by aggressive interactions, has negative effects on various biological functions including immune defence, reproduction, growth, and, in livestock, on product quality. Stress response and aggressiveness are mutually interrelated and show large interindividual variation, partly attributable to genetic factors. In the pig little is known about the molecular-genetic background of the variation in stress responsiveness and aggressiveness. To identify candidate genes we analyzed association of DNA markers in each of ten genes (CRH g.233C>T, CRHR1 c.*866_867insA, CRHBP c.51G>A, POMC c.293_298del, MC2R c.306T>G, NR3C1 c.*2122A>G, AVP c.207A>G, AVPR1B c.1084A>G, UCN g.1329T>C, CRHR2 c.*13T>C) related to the hypothalamic-pituitary-adrenocortical (HPA) axis, one of the main stress-response systems, with various stress- and aggression-related parameters at slaughter. These parameters were: physiological measures of the stress response (plasma concentrations of cortisol, creatine kinase, glucose, and lactate), adrenal weight (which is a parameter reflecting activity of the central branch of the HPA axis over time) and aggressive behaviour (measured by means of lesion scoring) in the context of psychosocial stress of mixing individuals with different aggressive temperament.ResultsThe SNP NR3C1 c.*2122A>G showed association with cortisol concentration (p = 0.024), adrenal weight (p = 0.003) and aggressive behaviour (front lesion score, p = 0.012; total lesion score p = 0.045). The SNP AVPR1B c.1084A>G showed a highly significant association with aggressive behaviour (middle lesion score, p = 0.007; total lesion score p = 0.003). The SNP UCN g.1329T>C showed association with adrenal weight (p = 0.019) and aggressive behaviour (front lesion score, p = 0.029). The SNP CRH g.233C>T showed a significant association with glucose concentration (p = 0.002), and the polymorphisms POMC c.293_298del and MC2R c.306T>G with adrenal weight (p = 0.027 and p < 0.0001 respectively).ConclusionsThe multiple and consistent associations shown by SNP in NR3C1 and AVPR1B provide convincing evidence for genuine effects of their DNA sequence variation on stress responsiveness and aggressive behaviour. Identification of the causal functional molecular polymorphisms would not only provide markers useful for pig breeding but also insight into the molecular bases of the stress response and aggressive behaviour in general.

Association between the porcine Escherichia coli F18 receptor genotype and phenotype and susceptibility to colonisation and postweaning diarrhoea caused by E. coli O138:F18

Porcine postweaning Escherichia coli enteritis is a cause of significant morbidity and mortality in pigs worldwide, and effective prevention remains an unsolved problem. This study examined the correlation between susceptibility of pigs to experimental infection with an E. coli F18 strain and the porcine intestinal F18 receptor genotypes. Thirty-one pigs classified as either belonging to the susceptible or the resistant genotype were inoculated with cultures of an E. coli O138:F18 isolated from a pig with postweaning diarrhoea. Susceptibility to colonisation and diarrhoea was assessed by clinical observations, faecal shedding of the challenge strain, histopathology and microscopic adhesion tests. Ten of 14 (71.4%) genetically susceptible pigs and one of 17 (5.9%) resistant pigs developed diarrhoea attributable to the challenge strain. There was no difference in susceptibility between homozygotic and heterozygotic susceptible pigs. Faecal shedding of the challenge strain correlated with the genetic receptor profile. Twenty pigs examined immunohistochemically revealed focal to extensive small intestinal mucosal colonisation by E. coli O138:F18 in nine of 10 susceptible and three of 10 resistant pigs. Results of in vitro adhesion assays performed with F18 cells on enterocyte preparations from 24 pigs, showed complete concordance with the F18 genotypes. In conclusion, this study showed a high correlation between the porcine intestinal F18 receptor genotypes and susceptibility to disease. However, pigs of the resistant F18 receptor genotype were not entirely protected against intestinal colonisation by E. coli F18.

Components of litter size in gilts with different prolactin receptor genotypes

Behavioral estrus and components of litter size at Day 35/36 of pregnancy were studied in gilts with prolactin receptor (PRLR) genotype AA (n=9), AB (n=25), and BB (n=22). This PRLR polymorphism (two alleles, A and B) has been associated with litter size, although it is not known whether the polymorphism itself causes differences in litter size or whether it is a marker for a closely linked causative gene. Estrus length in three successive estrous cycles was not affected by genotype, but estrous cycle length tended (P<0.1) to be longer for AA gilts compared to AB and BB gilts. AA gilts had a significantly (P<0.05) higher ovulation rate (21.5+/-0.9) than BB gilts (18.7+/-0.6), resulting in a numerically higher number of embryos at Day 35/36 (17.0+/-1.3, 15.6+/-0.8, and 13.7+/-0.9 for AA, AB, and BB gilts, respectively) which may lead to a subsequent difference in litter size. Ovulation rate of AB gilts (20.0+/-0.5) was intermediate. Genotype affected the total weight of the ovaries (P<0.05). Even after subtraction of the total weight of corpora lutea, ovarian weight in AA gilts was highest (16.6+/-1.0 g), in BB lowest (13.4+/-0.6g), and in AB gilts intermediate (15.0+/-0.6g; P<0.05). Unlike AB gilts, in AA and BB gilts uterine length was adapted to litter size, which led to longer (P<0.05) uteri for AA gilts (669+/-28 cm) compared to BB gilts (566+/-18 cm). Furthermore, embryos of AA gilts had heavier placentae (52.5+/-3.4 g) and larger implantation surface areas (309+/-19 cm(2)) than embryos of BB (42.0+/-2.3g, P<0.05; 256+/-12 cm(2), P<0.1) or AB (43.2+/-2.0 g, P<0.1; 257+/-11 cm(2), P<0.05) gilts. Results of this experiment show that the PRLR gene or a very closely linked gene affects porcine ovaries, uterus, and placenta in a way that might lead to differences in litter size. Since other genes and also environmental factors, however, might change the effect within the 112 days to parturition, it is preferable to state that the PRLR gene is a candidate gene for ovulation rate rather than for litter size.

Functional Implication of an Arg307Gly Substitution in Corticosteroid-Binding Globulin, a Candidate Gene for a Quantitative Trait Locus Associated With Cortisol Variability and Obesity in Pig

We previously reported that corticosteroid-binding globulin gene (Cbg) may be the causal gene of a quantitative trait locus associated with cortisol levels, fat deposition, and muscle content in a pig intercross. Sequence analysis of parental animals allowed us to identify four amino-acid substitutions. Here we have examined if any of these single amino acid substitutions could be responsible for the difference in CBG binding and affinity for cortisol between the parental breeds, using in vitro assays of Cbg variants after transfection of mammalian cells. Additionally, the Cbg coding region was analyzed in samples from a synthetic pig line to study association between polymorphism and CBG biochemical properties, carcass composition, and meat quality. Both in vitro transfection assays and the association studies suggest a role of the Arg307Gly mutation in increasing CBG capacity (by >70%) and decreasing CBG affinity for cortisol (by 30%). The Ile265Val substitution may also have an effect on decreasing CBG affinity for cortisol by 25%. The mutations Ser15Ile and Thr257Met do not seem to have an effect on CBG parameters. The Arg307Gly substitution was the only mutation associated with a parameter of meat quality and no mutation was linked to carcass composition.

SNPchiMp v.3: integrating and standardizing single nucleotide polymorphism data for livestock species

BackgroundIn recent years, the use of genomic information in livestock species for genetic improvement, association studies and many other fields has become routine. In order to accommodate different market requirements in terms of genotyping cost, manufacturers of single nucleotide polymorphism (SNP) arrays, private companies and international consortia have developed a large number of arrays with different content and different SNP density. The number of currently available SNP arrays differs among species: ranging from one for goats to more than ten for cattle, and the number of arrays available is increasing rapidly. However, there is limited or no effort to standardize and integrate array- specific (e.g. SNP IDs, allele coding) and species-specific (i.e. past and current assemblies) SNP information.ResultsHere we present SNPchiMp v.3, a solution to these issues for the six major livestock species (cow, pig, horse, sheep, goat and chicken). Original data was collected directly from SNP array producers and specific international genome consortia, and stored in a MySQL database. The database was then linked to an open-access web tool and to public databases. SNPchiMp v.3 ensures fast access to the database (retrieving within/across SNP array data) and the possibility of annotating SNP array data in a user-friendly fashion.ConclusionsThis platform allows easy integration and standardization, and it is aimed at both industry and research. It also enables users to easily link the information available from the array producer with data in public databases, without the need of additional bioinformatics tools or pipelines. In recognition of the open-access use of Ensembl resources, SNPchiMp v.3 was officially credited as an Ensembl E!mpowered tool. Availability at http://bioinformatics.tecnoparco.org/SNPchimp.

Differential mRNA expression of genes in the porcine adrenal gland associated with psychosocial stress

To gain insight into the adrenal stress response, we analysed differential mRNA expression of genes associated with psychosocial stress in the pig (Sus scrofa domestica). Various levels of psychosocial stress were induced by mixing groups of unfamiliar pigs with different aggressiveness. We selected two experimental groups for comparison, each comprising eight animals, which differed significantly in aggressive behaviour and plasma cortisol levels. To identify differentially expressed genes, we compared the adrenal transcriptome of these two groups of pigs, using the Affymetrix GeneChip porcine Genome Array. Bioinformatic analysis revealed that psychosocial stress induced upregulation of transcripts enriched for functions associated with cholesterol accumulation and downregulation of transcripts enriched for functions associated with cell growth and death. These responses are similar to those induced by ACTH stimulation. Nevertheless, the majority of the differentially expressed genes were so far not described as ACTH responsive. Some, such as GAL and GALP, may have responded to sympathoadrenal stimulation. Several of the differentially expressed transcripts, such as AGT, are associated with processes modulating steroidogenic response of adrenocortical cells to ACTH. One of the most significant findings was upregulation of LOC100039095, comprising a precursor of the microRNA miR-202, pointing to a previously unrecognised layer of regulation of adrenal steroidogenesis by microRNA. Our study, performed under entirely physiological conditions, complements previous studies focusing either on a single adrenal tissue and/or on a single stimulus, and contributes to understanding of the fine-tuning of adrenal stress response.

USE OF TYPE I DNA MARKERS FOR INITIAL GENETIC CHARACTERIZATION OF TWO PORTUGUESE SWINE BREEDS

Para ser efectivos, las estrategias para la conservacion y mejora de las razas porcinas locales deben incluir idealmente tanto su caracterizacion genetica como fenotipica. En este estudio, la informacion fenotipica fue recogida de individuos pertenecientes a las razas portuguesas Alentejana y Bisaro. Las muestras de ADN de estos animales fueron entonces analizadas para la variacion de 6 loci Tipo I para profundizar en el origen y la integridad de estas razas. La variacion detectada en estos loci fue muy baja en el Alentejano y de baja a intermedia en el Bisaro. La asociacion de cada marcador con una serie de caracteres productivos fueron analizadas en el Bisaro. Los resultados sugieren que algunos de estos genes pueden estar asociados con variaciones significativas de esos caracteres. La informacion derivada de la caracterizacion de las razas porcinas locales a nivel molecular deben apoyar los esfuerzos en conservacion y deben tambien ser utilizadas en el futuro en programas de seleccion asistida por marcadores.

THE USE OF MC1R AND KIT GENOTYPES FOR BREED CHARACTERISATION

Describimos la caracterizacion de dos loci determinantes del color de la capa en cerdos y demostramos como esta informacion puede ser utilizada para la identificacion racial. Estos avances pueden ser utiles en esquemas para asegurar la calidad y la trazabilidad cuya demanda se esta incrementando en los consumidores.