M.F.W. te Pas

The adipocyte fatty acid-binding protein locus: characterization and association with intramuscular fat content in pigs

Abstract. The porcine A-FABP gene (FABP4) was isolated and sequenced to study the role of A-FABP in the differentiation of intramuscular fat (IMF) accretion in pigs. The coding sequence of the porcine A-FABP gene is highly conserved across human, mouse, and rat. Moreover, all the functionally important amino acids are conserved. This high similarity extends into the first 270 bp of the 5′ upstream region. Within this region, a 56-bp nucleotide sequence was completely identical with the corresponding sequence in the mouse A-FABP gene, which contains the transcription factor binding sites for C/EBP and AP-1, and is implicated in the differentiation-dependent regulation of A-FABP. The A-FABP gene was assigned to porcine Chromosome (Chr) 4 by a porcine sequence-specific PCR on a cell hybrid panel, fully consistent with comparative mapping data with human and mouse. In the first intron of the porcine A-FABP gene, a microsatellite sequence was detected that was polymorphic for all six pig breeds tested. This genetic variation within the A-FABP gene was associated with differences in IMF content and possibly growth in a Duroc population, whereas no effect on backfat thickness and drip loss of the meat were detected. A considerable and significant contrast of approximately 1% IMF was observed between certain genotype classes. We conclude that the A-FABP locus is involved in the regulation of intramuscular fat accretion in Duroc pigs.

Characterization, chromosomal localization, and genetic variation of the porcine heart fatty acid-binding protein gene

The purpose of this study was to detect genetic variation in the porcine H-FABP gene, a candidate gene for meat quality traits in pigs. Lambda phages containing the porcine H-FABP gene were isolated by plaque hybridization with human H-FABP cDNA. The coding and flanking intronic sequences of the porcine H-FABP gene were determined as well as 1.6 kb of the 5 ′ upstream region. The various potential regulatory sequences in this region are in accordance with the function and expression of the protein in muscle and mammary tissue. Furthermore, comparison with the homolog region of the mouse identified a highly conserved 13-bp element (CTTCCT [A/C] TTTCGG) that may be involved in regulation of expression. The porcine H-FABP gene was localized on Chromosome (Chr) 6 by porcine sequence-specific PCR on DNA from a pig/rodent cell hybrid panel. In addition, part of the H-FABP gene was screened for genetic variation by PCR-RFLP analysis. Three PCR-RFLPs were detected, one in the upstream region (HinfI) and two in the second intron (HaeIII and MspI). In most pig breeds the corresponding alleles have a variable distribution, possibly a consequence of selective breeding. This genetic variation will enable us to investigate the role of the H-FABP locus in porcine production and meat quality traits.

Comparison of prenatal muscle tissue expression profiles of two pig breeds differing in muscle characteristics.

The objective of this study was to compare purebred Duroc and Pietrain prenatal muscle tissue transcriptome expression levels at different stages of prenatal development to gain insight into the differences in muscle tissue development in these pig breeds. Commercial western pig breeds have been selected for muscle growth for the past 2 decades. Pig breeds differ for their muscle phenotypes (i.e., myofiber numbers and myofiber types). Duroc and Pietrain pig breeds are extremes; Duroc pigs have redder muscle fiber types with more intramuscular fat, and Pietrain pigs have faster-growing and whiter muscle fiber types. Pietrain pigs are more muscular than Duroc pigs, whereas Duroc pigs are fatter than Pietrain pigs. The genomic background underlying these breed-specific differences is poorly known. Myogenesis is a complex exclusive prenatal process involving proliferation and differentiation (i.e., fusion) of precursor cells called myoblasts. We investigated the difference in the prenatal muscle-specific transcriptome profiles of Duroc and Pietrain pigs using microarray technology. The microarray contained more than 500 genes affecting myogenesis, energy metabolism, muscle structural genes, and other genes from a porcine muscle cDNA library. The results indicated that the expression of the myogenesis-related genes was greater in early Duroc embryos than in early Pietrain embryos (14 to 49 d of gestation), whereas the opposite was found in late embryos (63 to 91 d of gestation). These findings suggest that the myogenesis process is more intense in early Duroc embryos than in Pietrain embryos but that myogenesis is more intense in late Pietrain fetuses than in Duroc fetuses. Transcriptomes of muscle structural genes followed that pattern. The energy metabolism genes were expressed at a higher level in prenatal Pietrain pigs than in prenatal Duroc pigs, except for d 35, when the opposite situation was found. Fatty acid metabolism genes were expressed at a higher level in early (14 to 49 d of gestation) Duroc embryos than in Pietrain embryos. Better understanding of the genomic regulation of tissue formation leads to improved knowledge of the genome under selection and may lead to directed breed-specific changes in the future.

Genetic variation at the porcine MYF-5 gene locus. Lack of association with meat production traits

Abstract. The number of muscle fibers at birth appears to determine the maximal lean meat growth capacity in pigs and in cattle. Development of muscle fibers is regulated by the MyoD gene family consisting of MyoD1, myf-5, myf-6, and myogenin. Myf-5 is expressed in proliferating myoblasts. Here we report the genomic sequence of the porcine myf-5 gene with three microsatellites and two RFLPs located close to the coding sequences. Two of the microsatellites are located in the promoter region. The allelic distribution differs between breeds and selection lines. In two GY selection lines, 1216 pigs of two-generation families were genotyped for the HinfI RFLP, which was segregating in the GY breed. The other polymorphic loci are physically linked to this RFLP locus, and therefore the results can be extrapolated to these loci. Statistical analysis revealed no association with birth weight, growth rate, weight at slaughter age, carcass meat weight, and backfat thickness. Thus, in this study myf-5 did not explain genetic variation in meat (muscle) development in pigs.

Leptin gene polymorphisms and their phenotypic associations

In an era of rapidly increasing prevalence of human obesity and associated health problems, leptin gene polymorphisms have drawn much attention in biomedical research. Leptin gene polymorphisms have furthermore drawn much attention from animal scientists for their possible roles in economically important production and reproduction traits. Of the polymorphisms reported for exonic, intronic, and promoter regions of the leptin gene, 16 have been included in association studies in humans, 19 in cattle, and 6 (all exonic or intronic) in pigs. In humans, associations have been found with overweight or (early-onset) obesity, non-insulin-dependent diabetes mellitus, prostate cancer, and non-Hodgkins lymphoma. In cattle, associations have been found with feed intake, milk yield traits, carcass traits, and reproduction-related traits, and in pigs with feed intake, average daily gain, carcass traits (backfat/leanness), and reproduction performance traits. Many of the polymorphisms were only included in a limited number of association studies, or the phenotypes studied varied largely for a given polymorphism between studies. Therefore, many of the associations found for these polymorphisms need to be confirmed in future studies before firm conclusions can be drawn.

Advances in research on the prenatal development of skeletal muscle in animals in relation to the quality of muscle-based food. I. Regulation of myogenesis and environmental impact

Skeletal muscle development in vertebrates - also termed myogenesis - is a highly integrated process. Evidence to date indicates that the processes are very similar across mammals, poultry and fish, although the timings of the various steps differ considerably. Myogenesis is regulated by the myogenic regulatory factors and consists of two to three distinct phases when different fibre populations appear. The critical times when myogenesis is prone to hormonal or environmental influences depend largely on the developmental stage. One of the main mechanisms for both genetic and environmental effects on muscle fibre development is via the direct action of the growth hormone-insulin-like growth factor (GH-IGF) axis. In mammals and poultry, postnatal growth and function of muscles relate mainly to the hypertrophy of the fibres formed during myogenesis and to their fibre-type composition in terms of metabolic and contractile properties, whereas in fish hyperplasia still plays a major role. Candidate genes that are important in skeletal muscle development, for instance, encode for IGFs and IGF-binding proteins, myosin heavy chain isoforms, troponin T, myosin light chain and others have been identified. In mammals, nutritional supply in utero affects myogenesis and the GH-IGF axis may have an indirect action through the partitioning of nutrients towards the gravid uterus. Impaired myogenesis resulting in low skeletal myofibre numbers is considered one of the main reasons for negative long-term consequences of intrauterine growth retardation. Severe undernutrition in utero due to natural variation in litter or twin-bearing species or insufficient maternal nutrient supply may impair myogenesis and adversely affect carcass quality later in terms of reduced lean and increased fat deposition in the progeny. On the other hand, increases in maternal feed intake above standard requirement seem to have no beneficial effects on the growth of the progeny with myogenesis not or only slightly affected. Initial studies on low and high maternal protein feeding are published. Although there are only a few studies, first results also reveal an influence of nutrition on skeletal muscle development in fish and poultry. Finally, environmental temperature has been identified as a critical factor for growth and development of skeletal muscle in both fish and poultry.

Postmortem proteome degradation profiles of longissimus muscle in Yorkshire and Duroc pigs and their relationship with pork quality traits.

Conversion of muscle to meat is regulated by complex interactions of biochemical processes that take place during postmortem storage of the carcass. Enzymatic proteolysis, among other postmortem biochemical phenomena; e.g. glycolysis; changes tough intact muscle tissue into more tender meat. Knowledge on proteome-wide proteolysis of muscle tissue in relation to meat quality is limited and potential breed-specific differences have received little attention. Therefore, we investigated meat quality traits and proteolysis profiles of the longissimus proteome of five Yorkshire and five Duroc pigs at slaughter and after 1, 2, 3, 7, and 10days of ageing. Drip loss increased with ageing while cooking loss was unchanged in both breeds. Shear force varied between animals and decreased with ageing. Analysis of the proteomes showed four types of temporal expression profiles. Association analysis suggested several potential protein biomarkers for drip loss and shear force in both breeds, but none for cooking loss.

Longissimus muscle transcriptome profiles related to carcass and meat quality traits in fresh meat Pietrain carcasses

High quality pork is consumed as fresh meat, whereas other carcasses are used in the processing industry. Meat quality is determined measuring technical muscle variables. The objective of this research was to investigate the molecular regulatory mechanisms underlying meat quality differences of pork originating from genetically different Piétrain boars. Piétrain boars were approved for high meat quality using a DNA marker panel. Other Piétrain boars were indicated as average. Both groups produced litters in similar Piétrain sows. The LM were sampled from 9 carcasses produced by approved boars and 8 carcasses of average boars. Total RNA was isolated, and an equal portion of each sample was pooled to make a reference sample representing the mean of all samples. Each sample was hybridized on microarrays against the reference in duplicate using a dye swaps design. After normalization and subtraction of 2 times the background, only genes expressed in at least 5 carcasses were analyzed. For all analyses the mean of the M-values relative to the reference (i.e., fold change), were used. Sixteen genes showed significant linear or quadratic associations between gene expression and meat color (Minolta a* value, Minolta L* value, reflection, pH 24 h) after Bonferroni correction. All these genes had expression levels similar to the reference in all carcasses. Studying association between gene expression levels and meat quality using only genes with expression statistically differing from the reference in at least 5 carcasses revealed 29 more genes associating with the technological meat quality variables, again with meat color as a main trait. These associations were not significant after Bonferroni correction and explained less of the phenotypic variation in the traits. Bioinformatics analyses with The Database for Annotation, Visualization and Integrated Discovery (DAVID) using the list of genes with more than 2-fold changed expression level revealed that these genes were mainly found in muscle-specific processes, protein complexes, and oxygen transport, and located to muscle-specific cellular localizations. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed pathways related to protein metabolism, cellular proliferation, signaling, and adipose development differing between the 2 groups of carcasses. Approved meat carcasses showed less variation in gene expression. The results highlight biological molecular mechanisms underlying the differences between the high meat quality approved and average boars.

Selection for growth rate or against back fat thickness in pigs is associated with changes in growth hormone axis plasma protein concentration and mRNA level

Selection for increased growth rate or decreased back fat thickness results in concomitant changes in endocrine and metabolic status. Growth hormone (GH) changes in blood plasma concentration related to selection for growth rate and fat deposition were reported in pigs. The molecular mechanisms regulating selection-induced changes in GH plasma concentration remain largely unknown. We investigated selection-associated changes in GH axis parameters in 2 pig lines selected for increased growth rate (F-line), or decreased back fat thickness (L-line), respectively. First, we investigated selection-associated changes in GH pulse parameters. In both selection lines we found each generation a declining GH peak maximum concentration and area under the GH curve. GH pulse width was not associated with generation number. In both lines generation number was associated with a declined pulse interval, indicating that the number of pulses per day increased on average with 1 pulse per 24 h per generation. Second, plasma concentration of GH axis related Insulin-like growth factor-I (IGF-I) and insulin were investigated. Plasma IGF-I concentration was not associated with generation number in the F-line. Mean plasma insulin concentration declined each generation in both lines. Third, we investigated changes in GH and Pit-1 mRNA levels. In both selection lines GH and Pit-1 mRNA levels increased approximately 50% each generation. The high SD of the GH mRNA levels in both lines may suggest that the GH mRNA levels are pulsatile in vivo. We postulate a molecular mechanism that may explain how selection is associated with increased GH mRNA levels and GH pulse numbers, while lowering GH release per pulse.

Estimation of the extent of linkage disequilibrium in seven regions of the porcine genome

ABSTRACT Linkage disequilibrium (LD) refers to the correlation among neighboring alleles, reflecting non-random patterns of association between alleles at (nearby) loci. A better understanding of LD in the porcine genome is of direct relevance for identification of genes and mutations with a certain effect on the traits of interest. Here, 215 SNPs in seven genomic regions were genotyped in individuals of three breeds. Pairwise linkage disequilibrium was calculated for all marker pairs. To estimate the extent of LD, all pairwise LD values were plotted against the distance between the markers. Based on SNP markers in four genomic regions analyzed in three panels from populations of Large White, Dutch Landrace, and Meishan origin, useful LD is estimated to extend for approximately 40 to 60 kb in the porcine genome.