Joan Estany

Acetyl-CoA carboxylase and stearoyl-CoA desaturase protein expression in subcutaneous adipose tissue is reduced in pigs selected for decreased backfat thickness at constant intramuscular fat content.

The objectives of this study were 1) to determine whether selection toward less subcutaneous fat thickness at constant intramuscular fat content in pigs is related to tissue-specific changes in the expression of lipogenic enzymes acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase (SCD), and Delta(6)-desaturase (Delta6d); and 2) to investigate tissue specific distribution of the porcine ACC, SCD, and Delta6d. The study was conducted on 20 purebred Duroc barrows. Ten animals were from a group selected for decreased subcutaneous fat thickness at constant intramuscular fat content (experimental group). The other 10 animals were from the unselected (control) group. Distribution of ACC, SCD, and Delta6d was investigated in semimembranosus muscle (SM), subcutaneous adipose tissue (SA), liver (L), kidney (K), heart (H), diaphragm (D), rectus capitis muscle (RCM), and abdominal fat (AF). The enzyme expression was studied in 10 animals in the case of SM and SA and in 4 animals in the case of other tissues. The following expression pattern was established for ACC: SM <or= H = K <or= D < RCM < L < AF = SA, whereas the expression patterns for SCD and Delta6d proteins were SM < H < RCM < D < L < K < AF = SA and RCM = SM = D < L <or= H < SA < K < AF, respectively. Expression of ACC and SCD proteins was less in subcutaneous adipose tissue of the experimental animals when compared with the control group (P < 0.001). However, no difference (P > 0.1) in ACC and SCD protein expression between the control and experimental groups was observed in SM. Expression of Delta6d protein did not differ between the control and experimental groups for SA (P = 0.47) or SM (P = 0.31). There was a positive relationship between muscle SCD protein expression and intramuscular fat content (r = 0.48, P < 0.05). Intramuscular fat content did not correlate with ACC or Delta6d protein expression (P = 0.23 and P = 0.80, respectively). We conclude that SCD might be an effective potential biomarker for intramuscular fat deposition.

Age-related changes in intramuscular and subcutaneous fat content and fatty acid composition in growing pigs using longitudinal data

The evolution of intramuscular fat (IMF) and subcutaneous backfat (SB) content and fatty acid composition with age was investigated. A repeated sampling scheme (n=517) for longissimus dorsi (LM) and SB specimens was performed from 160 to 220 days on 216 purebred Duroc barrows. IMF content increased linearly with age at a rate of 0.05%/day, with major compositional changes occurring in C18:1 and C18.2, which increased and decreased by around 4%, respectively. Backfat thickness increase rate declined with age, showing similar compositional changes to IMF but halved in magnitude. Fat content itself also influenced fat composition. At a fixed age, both saturated (SFA) (C16:0) and monounsaturated (MUFA) (C18:1) increased with IMF but only SFA (C16:0 and C18:0) with SB. Aging mostly affected MUFA while fatness SFA. A delay in age at slaughter will enhance IMF and C18:1 in relation to overall fatness but at expense of decreasing body and loin growth rate.

A Functional Variant in the Stearoyl-CoA Desaturase Gene Promoter Enhances Fatty Acid Desaturation in Pork

There is growing public concern about reducing saturated fat intake. Stearoyl-CoA desaturase (SCD) is the lipogenic enzyme responsible for the biosynthesis of oleic acid (18∶1) by desaturating stearic acid (18∶0). Here we describe a total of 18 mutations in the promoter and 3′ non-coding region of the pig SCD gene and provide evidence that allele T at AY487830:g.2228T>C in the promoter region enhances fat desaturation (the ratio 18∶1/18∶0 in muscle increases from 3.78 to 4.43 in opposite homozygotes) without affecting fat content (18∶0+18∶1, intramuscular fat content, and backfat thickness). No mutations that could affect the functionality of the protein were found in the coding region. First, we proved in a purebred Duroc line that the C-T-A haplotype of the 3 single nucleotide polymorphisms (SNPs) (g.2108C>T; g.2228T>C; g.2281A>G) of the promoter region was additively associated to enhanced 18∶1/18∶0 both in muscle and subcutaneous fat, but not in liver. We show that this association was consistent over a 10-year period of overlapping generations and, in line with these results, that the C-T-A haplotype displayed greater SCD mRNA expression in muscle. The effect of this haplotype was validated both internally, by comparing opposite homozygote siblings, and externally, by using experimental Duroc-based crossbreds. Second, the g.2281A>G and the g.2108C>T SNPs were excluded as causative mutations using new and previously published data, restricting the causality to g.2228T>C SNP, the last source of genetic variation within the haplotype. This mutation is positioned in the core sequence of several putative transcription factor binding sites, so that there are several plausible mechanisms by which allele T enhances 18∶1/18∶0 and, consequently, the proportion of monounsaturated to saturated fat.

Estimating intramuscular fat content and fatty acid composition in live and post-mortem samples in pigs.

Repeated muscle sampling (n=732) on 216 pigs was performed to evaluate the effect of live sampling and sample size on intramuscular fat (IMF) content and fatty acid (FA) composition. The sampling scheme consisted of 1-3 biopsies of longissimus dorsi (LM), a small and a big post-mortem sample of LM, and a big post-mortem sample of gluteus medius (GM). IMF was determined by quantitative gas chromatography after direct transesterification. Data on LM were jointly analyzed using a mixed model on age with heterogeneous residual variances across sampling methods. Biopsies overestimated IMF and polyunsaturated FA content and underestimated monounsaturated FA content with decreasing sample size. Potential for bias and not sampling variance is the major limitation of using live samples for assessing changes in IMF with age. Small post-mortem samples of LM were as informative as big samples of GM for inferring IMF content but not IMF composition.

Genome-Wide Association Study Singles Out SCD and LEPR as the Two Main Loci Influencing Intramuscular Fat Content and Fatty Acid Composition in Duroc Pigs.

Intramuscular fat (IMF) content and fatty acid composition affect the organoleptic quality and nutritional value of pork. A genome-wide association study was performed on 138 Duroc pigs genotyped with a 60k SNP chip to detect biologically relevant genomic variants influencing fat content and composition. Despite the limited sample size, the genome-wide association study was powerful enough to detect the association between fatty acid composition and a known haplotypic variant in SCD (SSC14) and to reveal an association of IMF and fatty acid composition in the LEPR region (SSC6). The association of LEPR was later validated with an independent set of 853 pigs using a candidate quantitative trait nucleotide. The SCD gene is responsible for the biosynthesis of oleic acid (C18:1) from stearic acid. This locus affected the stearic to oleic desaturation index (C18:1/C18:0), C18:1, and saturated (SFA) and monounsaturated (MUFA) fatty acids content. These effects were consistently detected in gluteus medius, longissimus dorsi, and subcutaneous fat. The association of LEPR with fatty acid composition was detected only in muscle and was, at least in part, a consequence of its effect on IMF content, with increased IMF resulting in more SFA, less polyunsaturated fatty acids (PUFA), and greater SFA/PUFA ratio. Marker substitution effects estimated with a subset of 65 animals were used to predict the genomic estimated breeding values of 70 animals born 7 years later. Although predictions with the whole SNP chip information were in relatively high correlation with observed SFA, MUFA, and C18:1/C18:0 (0.48–0.60), IMF content and composition were in general better predicted by using only SNPs at the SCD and LEPR loci, in which case the correlation between predicted and observed values was in the range of 0.36 to 0.54 for all traits. Results indicate that markers in the SCD and LEPR genes can be useful to select for optimum fatty acid profiles of pork.

Investigating reference genes for quantitative real-time PCR analysis across four chicken tissues

Accurate normalization of data is required to correct for different efficiencies and errors during the processing of samples in reverse transcription PCR analysis. The chicken is one of the main livestock species and its genome was one of the first reported and used in large scale transcriptomic analysis. Despite this, the chicken has not been investigated regarding the identification of reference genes suitable for the quantitative PCR analysis of growth and fattening genes. In this study, five candidate reference genes (B2M, RPL32, SDHA, TBP and YWHAZ) were evaluated to determine the most stable internal reference for quantitative PCR normalization in the two main commercial muscles (pectoralis major (breast) and biceps femoris (thigh)), liver and abdominal fat. Four statistical methods (geNorm, NormFinder, CV and BestKeeper) were used in the evaluation of the most suitable combination of reference genes. Additionally, a comprehensive ranking was established with the RefFinder tool. This analysis identified YWHAZ and TBP as the recommended combination for the analysis of biceps femoris and liver, YWHAZ and RPL32 for pectoralis major and RPL32 and B2M for abdominal fat and across-tissue studies. The final ranking for each tool changed slightly but overall the results, and most particularly the ability to discard the least robust candidates, were consistent between tools. The selection and number of reference genes were validated using SCD, a target gene related to fat metabolism. Overall, the results can be directly used to quantitate target gene expression in different tissues or in validation studies from larger transcriptomic experiments.

Genetic Marker Discovery in Complex Traits: A Field Example on Fat Content and Composition in Pigs

Among the large number of attributes that define pork quality, fat content and composition have attracted the attention of breeders in the recent years due to their interaction with human health and technological and sensorial properties of meat. In livestock species, fat accumulates in different depots following a temporal pattern that is also recognized in humans. Intramuscular fat deposition rate and fatty acid composition change with life. Despite indication that it might be possible to select for intramuscular fat without affecting other fat depots, to date only one depot-specific genetic marker (PCK1 c.2456C>A) has been reported. In contrast, identification of polymorphisms related to fat composition has been more successful. For instance, our group has described a variant in the stearoyl-coA desaturase (SCD) gene that improves the desaturation index of fat without affecting overall fatness or growth. Identification of mutations in candidate genes can be a tedious and costly process. Genome-wide association studies can help in narrowing down the number of candidate genes by highlighting those which contribute most to the genetic variation of the trait. Results from our group and others indicate that fat content and composition are highly polygenic and that very few genes explain more than 5% of the variance of the trait. Moreover, as the complexity of the genome emerges, the role of non-coding genes and regulatory elements cannot be disregarded. Prediction of breeding values from genomic data is discussed in comparison with conventional best linear predictors of breeding values. An example based on real data is given, and the implications in phenotype prediction are discussed in detail. The benefits and limitations of using large SNP sets versus a few very informative markers as predictors of genetic merit of breeding candidates are evaluated using field data as an example.

A polymorphism in the stearoyl-CoA desaturase gene promoter increases monounsaturated fatty acid content in dry-cured ham

Data on 125 dry-cured hams from purebred Duroc pigs were used to examine whether the favorable effect of the T allele in the promoter region of the stearoyl-CoA desaturase gene (AY487830:g.2228T>C) on monounsaturated fatty acid (MUFA) content in green ham is maintained after the curing process. It is shown that pigs carrying the T allele produced dry-cured hams with increased C16:1, C18:1n-9, C18:1n-7, and MUFA and decreased C18:0 and saturated fatty acid (SFA) content. The TT pigs had 1.39% more MUFA and 1.62% less SFA than the CC animals, while gilts had 0.74% more MUFA and 0.34% less SFA than barrows. The correlation between MUFA in green and dry-cured hams (n=53) was high (r=0.88), with TT pigs being more effective in retaining increased MUFA in green hams until the end of the curing period. It is concluded that increasing the presence of the T allele could have more impact than gender to produce hams with a high level of MUFA.

A WUR SNP is associated with European Porcine Reproductive and Respiratory Virus Syndrome resistance and growth performance in pigs

Porcine reproductive and respiratory syndrome (PRRS) causes decreased reproductive performance and respiratory problems in pigs. The goals of the current study were 1) to examine whether individual variation applies to infection with PRRSV European strains and 2) to investigate the association of a single nucleotide polymorphism (SNP) WUR10000125 (WUR) at the interferon-inducible guanylate-binding protein 1 gene (GBP1) with average daily gain (ADG) in PRRSV infected and uninfected pigs. The experimental procedure consisted of two trials in which pigs from negative PRRSV farms were infected with a wild-type (n=80) or vaccinated with an attenuated European PRRS virus strain (n=40) and then monitored after infection or vaccination. Viral load and ADG were determined for each pig. In a third trial, the ADG for PRRSV-free pigs was monitored. All pigs were genotyped for WUR at the GBP1 gene (AA and AG genotype were defined). Results indicated that there was individual variation in the viral load from pigs challenged with a wild-type or low virulent European PRRSV strain. Secondly, our data showed that WUR SNP was associated to ADG in vaccinated pigs. Thus, ADG in AG pigs was significantly higher than in AA ones after vaccinating with an attenuated PRRSV strain. However, the reverse happened in a PRRSV-free environment where the AA pigs were those that grew faster. Based on these results, there is a scope for selecting pigs according to their responses to PRRS virus infection with European strains and that WUR SNP may play a role in causing PRRSV resistance.

Identification of signatures of selection for intramuscular fat and backfat thickness in two Duroc populations

Intramuscular fat (IMF) content is an important trait affecting the quality of pork. Two Duroc populations, one under positive selection for IMF and the other selected for decreased backfat but under stabilizing selection for IMF, were used to identify signatures of selection associated with IMF using 60,000 single-nucleotide polymorphism data. The effects of selection were analyzed between 2 lines or groups representing selected and control animals within each population using a discriminant analysis of principal components and Wright’s fixation index (FST). Moreover, extended haplotype homozygosity-based approaches were used to examine the changes in haplotype frequency due to recent selection. Each statistical method identified 10–20 selection signatures. A few haplotype-based signatures of selection agreed with results from a genome-wide association study (GWAS), while FST measures showed a better agreement with GWAS results. Agreement of marker-trait associations and signatures of selection was limited, and further examination will be necessary to understand the effect of selection on IMF and why some regions identified by GWAS did not appear to respond to the selection practiced. The genes in 21 consensus selection signatures were examined. Several genes with an effect on overall fatness were identified, but further research is needed to assess whether or not some of them could have a specific effect on IMF.