Circulating non-esterified fatty acids as biomarkers for fat content and composition in pigs

Circulating non-esterified fatty acids (NEFA) can reflect the composition of dietary fat or adipose tissues depending on the fasting conditions. Therefore, circulating NEFA may be valuable as biomarkers for meat quality traits, such as intramuscular fat content and fatty acid composition in finishing pigs. Genetic variants that regulate lipid metabolism can also modulate the circulating NEFA. We conducted an experiment with 150 heavy Duroc pigs to evaluate fluctuations in the circulating NEFA composition due to age, fasting duration and two genetic polymorphisms, one in the leptin receptor (LEPR; rs709596309) and one in the stearoyl-CoA desaturase (SCD; rs80912566) gene. Circulating NEFA were more saturated and less monounsaturated than the subcutaneous and intramuscular adipose tissues. Absolute circulating NEFA content was more influenced by fasting duration than age. The SCD polymorphism did not impact NEFA content or composition. The LEPR polymorphism affected the content but not the fatty acid composition. Circulating oleic acid NEFA content after a short fasting was positively correlated with intramuscular fat content and, after a long fasting, with intramuscular oleic acid content. We conclude that circulating NEFA reflect environmental and genetic metabolic changes but are of limited value as biomarkers for intramuscular fat content and fatty acid composition.This Research was founded by the Spanish Ministry of Economy and Competitiveness and the European Union Regional Development Funds (grants AGL2012-33529, AGL2015-65846-R, and RTI2018-101346-B-I00)

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

[EN] 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.This research was funded by the Spanish Ministry of Economy and Competitiveness (MINECO; grants AGL2012-33529 and AGL2015-65846-R).Ros-Freixedes, R.; Gol, S.; Pena, R.; Tor, M.; Ibañez Escriche, N.; Dekkers, J.; Estany, J. (2016). 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. PLoS ONE. 11(3). https://doi.org/10.1371/journal.pone.0152496S113Cameron, N. ., Enser, M., Nute, G. ., Whittington, F. ., Penman, J. ., Fisken, A. ., … Wood, J. . (2000). Genotype with nutrition interaction on fatty acid composition of intramuscular fat and the relationship with flavour of pig meat. Meat Science, 55(2), 187-195. doi:10.1016/s0309-1740(99)00142-4Christophersen, O. A., & Haug, A. (2011). Animal products, diseases and drugs: a plea for better integration between agricultural sciences, human nutrition and human pharmacology. Lipids in Health and Disease, 10(1), 16. doi:10.1186/1476-511x-10-16Ntawubizi, M., Colman, E., Janssens, S., Raes, K., Buys, N., & De Smet, S. (2010). Genetic parameters for intramuscular fatty acid composition and metabolism in pigs1. Journal of Animal Science, 88(4), 1286-1294. doi:10.2527/jas.2009-2355Ros-Freixedes, R., Reixach, J., Tor, M., & Estany, J. (2012). Expected genetic response for oleic acid content in pork1. Journal of Animal Science, 90(12), 4230-4238. doi:10.2527/jas.2011-5063Clop, A., Ovilo, C., Perez-Enciso, M., Cercos, A., Tomas, A., Fernandez, A., … Noguera, J. L. (2003). Detection of QTL affecting fatty acid composition in the pig. Mammalian Genome, 14(9), 650-656. doi:10.1007/s00335-002-2210-7Kim, Y., Kong, M., Nam, Y. J., & Lee, C. (2006). A Quantitative Trait Locus for Oleic Fatty Acid Content on Sus scrofa Chromosome 7. Journal of Heredity, 97(5), 535-537. doi:10.1093/jhered/esl026Sanchez, M.-P., Iannuccelli, N., Basso, B., Bidanel, J.-P., Billon, Y., Gandemer, G., … Le Roy, P. (2007). Identification of QTL with effects on intramuscular fat content and fatty acid composition in a Duroc × Large White cross. BMC Genetics, 8(1), 55. doi:10.1186/1471-2156-8-55Guo, T., Ren, J., Yang, K., Ma, J., Zhang, Z., & Huang, L. (2009). Quantitative trait loci for fatty acid composition in longissimus dorsi and abdominal fat: results from a White Duroc × Erhualian intercross F2population. Animal Genetics, 40(2), 185-191. doi:10.1111/j.1365-2052.2008.01819.xC.M. Dekkers, J. (2012). Application of Genomics Tools to Animal Breeding. Current Genomics, 13(3), 207-212. doi:10.2174/138920212800543057Uemoto, Y., Nakano, H., Kikuchi, T., Sato, S., Ishida, M., Shibata, T., … Suzuki, K. (2011). Fine mapping of porcine SSC14 QTL and SCD gene effects on fatty acid composition and melting point of fat in a Duroc purebred population. Animal Genetics, 43(2), 225-228. doi:10.1111/j.1365-2052.2011.02236.xUemoto, Y., Soma, Y., Sato, S., Ishida, M., Shibata, T., Kadowaki, H., … Suzuki, K. (2011). Genome-wide mapping for fatty acid composition and melting point of fat in a purebred Duroc pig population. Animal Genetics, 43(1), 27-34. doi:10.1111/j.1365-2052.2011.02218.xEstany, J., Ros-Freixedes, R., Tor, M., & Pena, R. N. (2014). A Functional Variant in the Stearoyl-CoA Desaturase Gene Promoter Enhances Fatty Acid Desaturation in Pork. PLoS ONE, 9(1), e86177. doi:10.1371/journal.pone.0086177Ramayo-Caldas, Y., Mercadé, A., Castelló, A., Yang, B., Rodríguez, C., Alves, E., … Folch, J. M. (2012). Genome-wide association study for intramuscular fatty acid composition in an Iberian × Landrace cross1. Journal of Animal Science, 90(9), 2883-2893. doi:10.2527/jas.2011-4900Muñoz, M., Rodríguez, M. C., Alves, E., Folch, J. M., Ibañez-Escriche, N., Silió, L., & Fernández, A. I. (2013). Genome-wide analysis of porcine backfat and intramuscular fat fatty acid composition using high-density genotyping and expression data. BMC Genomics, 14(1), 845. doi:10.1186/1471-2164-14-845Yang, B., Zhang, W., Zhang, Z., Fan, Y., Xie, X., Ai, H., … Ren, J. (2013). Genome-Wide Association Analyses for Fatty Acid Composition in Porcine Muscle and Abdominal Fat Tissues. PLoS ONE, 8(6), e65554. doi:10.1371/journal.pone.0065554Zhang, W., Zhang, J., Cui, L., Ma, J., Chen, C., Ai, H., … Yang, B. (2016). Genetic architecture of fatty acid composition in the longissimus dorsi muscle revealed by genome-wide association studies on diverse pig populations. Genetics Selection Evolution, 48(1). doi:10.1186/s12711-016-0184-2Kim, E.-S., Ros-Freixedes, R., Pena, R. N., Baas, T. J., Estany, J., & Rothschild, M. F. (2015). Identification of signatures of selection for intramuscular fat and backfat thickness in two Duroc populations1. Journal of Animal Science, 93(7), 3292-3302. doi:10.2527/jas.2015-8879Bosch, L., Tor, M., Reixach, J., & Estany, J. (2009). Estimating intramuscular fat content and fatty acid composition in live and post-mortem samples in pigs. Meat Science, 82(4), 432-437. doi:10.1016/j.meatsci.2009.02.013AOAC. 1997. Supplement to AOAC Official Method 996.06: Fat (total, saturated, and monounsaturated) in foods hydrolytic extraction gas chromatographic method. Page 18 in Official Methods of Analysis (16th ed). Association of Official Analytical Chemists, Arlington, VA.ÓVILO, C., FERNÁNDEZ, A., NOGUERA, J. L., BARRAGÁN, C., LETÓN, R., RODRÍGUEZ, C., … TORO, M. (2005). Fine mapping of porcine chromosome 6 QTL and LEPR effects on body composition in multiple generations of an Iberian by Landrace intercross. Genetical Research, 85(1), 57-67. doi:10.1017/s0016672305007330Amills, M., Villalba, D., Tor, M., Mercad, A., Gallardo, D., Cabrera, B., … Estany, J. (2008). Plasma leptin levels in pigs with different leptin and leptin receptor genotypes. Journal of Animal Breeding and Genetics, 125(4), 228-233. doi:10.1111/j.1439-0388.2007.00715.xPurcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M. A. R., Bender, D., … Sham, P. C. (2007). PLINK: A Tool Set for Whole-Genome Association and Population-Based Linkage Analyses. The American Journal of Human Genetics, 81(3), 559-575. doi:10.1086/519795Bouwman, A. C., Janss, L. L., & Heuven, H. C. (2011). A Bayesian approach to detect QTL affecting a simulated binary and quantitative trait. BMC Proceedings, 5(S3). doi:10.1186/1753-6561-5-s3-s4Legarra, A., Croiseau, P., Sanchez, M., Teyssèdre, S., Sallé, G., Allais, S., … Elsen, J.-M. (2015). A comparison of methods for whole-genome QTL mapping using dense markers in four livestock species. Genetics Selection Evolution, 47(1), 6. doi:10.1186/s12711-015-0087-7Kass, R. E., & Raftery, A. E. (1995). Bayes Factors. Journal of the American Statistical Association, 90(430), 773-795. doi:10.1080/01621459.1995.10476572Barrett, J. C., Fry, B., Maller, J., & Daly, M. J. (2004). Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics, 21(2), 263-265. doi:10.1093/bioinformatics/bth457Wolc, A., Arango, J., Settar, P., Fulton, J. E., O’Sullivan, N. P., Preisinger, R., … Dekkers, J. C. M. (2012). Genome-wide association analysis and genetic architecture of egg weight and egg uniformity in layer chickens. Animal Genetics, 43, 87-96. doi:10.1111/j.1365-2052.2012.02381.xChen, E. Y., Tan, C. M., Kou, Y., Duan, Q., Wang, Z., Meirelles, G., … Ma’ayan, A. (2013). Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics, 14(1), 128. doi:10.1186/1471-2105-14-128Rabbit programme. 2012. Available from: http://www.dcam.upv.es/dcia/ablasco/Programas/THE%20PROGRAM%20Rabbit.pdfHu, Z.-L., Park, C. A., & Reecy, J. M. (2015). Developmental progress and current status of the Animal QTLdb. Nucleic Acids Research, 44(D1), D827-D833. doi:10.1093/nar/gkv1233Óvilo, C., Fernández, A., Fernández, A. I., Folch, J. M., Varona, L., Benítez, R., … Silió, L. (2010). Hypothalamic expression of porcine leptin receptor (LEPR), neuropeptide Y (NPY), and cocaine- and amphetamine-regulated transcript (CART) genes is influenced by LEPR genotype. Mammalian Genome, 21(11-12), 583-591. doi:10.1007/s00335-010-9307-1Muñoz, G., Alcázar, E., Fernández, A., Barragán, C., Carrasco, A., de Pedro, E., … Rodríguez, M. C. (2011). Effects of porcine MC4R and LEPR polymorphisms, gender and Duroc sire line on economic traits in Duroc×Iberian crossbred pigs. Meat Science, 88(1), 169-173. doi:10.1016/j.meatsci.2010.12.018Galve, A., Burgos, C., Silió, L., Varona, L., Rodríguez, C., Ovilo, C., & López-Buesa, P. (2012). The effects of leptin receptor (LEPR) and melanocortin-4 receptor (MC4R) polymorphisms on fat content, fat distribution and fat composition in a Duroc×Landrace/Large White cross. Livestock Science, 145(1-3), 145-152. doi:10.1016/j.livsci.2012.01.010UEMOTO, Y., KIKUCHI, T., NAKANO, H., SATO, S., SHIBATA, T., KADOWAKI, H., … SUZUKI, K. (2011). Effects of porcine leptin receptor gene polymorphisms on backfat thickness, fat area ratios by image analysis, and serum leptin concentrations in a Duroc purebred population. Animal Science Journal, 83(5), 375-385. doi:10.1111/j.1740-0929.2011.00963.xHirose, K., Ito, T., Fukawa, K., Arakawa, A., Mikawa, S., Hayashi, Y., & Tanaka, K. (2013). Evaluation of effects of multiple candidate genes (LEP,LEPR,MC4R,PIK3C3, andVRTN) on production traits in Duroc pigs. Animal Science Journal, 85(3), 198-206. doi:10.1111/asj.12134López-Buesa, P., Burgos, C., Galve, A., & Varona, L. (2013). Joint analysis of additive, dominant and first-order epistatic effects of four genes (IGF2,MC4R,PRKAG3andLEPR) with known effects on fat content and fat distribution in pigs. Animal Genetics, 45(1), 133-137. doi:10.1111/age.12091Mackowski, M., Szymoniak, K., Szydlowski, M., Kamyczek, M., Eckert, R., Rozycki, M., & Switonski, M. (2005). Missense mutations in exon 4 of the porcine LEPR gene encoding extracellular domain and their association with fatness traits. Animal Genetics, 36(2), 135-137. doi:10.1111/j.1365-2052.2005.01247.xLi, X., Kim, S.-W., Choi, J.-S., Lee, Y.-M., Lee, C.-K., Choi, B.-H., … Kim, K.-S. (2010). Investigation of porcine FABP3 and LEPR gene polymorphisms and mRNA expression for variation in intramuscular fat content. Molecular Biology Reports, 37(8), 3931-3939. doi:10.1007/s11033-010-0050-1Tyra, M., & Ropka-Molik, K. (2011). Effect of the FABP3 and LEPR gene polymorphisms and expression levels on intramuscular fat (IMF) content and fat cover degree in pigs. Livestock Science, 142(1-3), 114-120. doi:10.1016/j.livsci.2011.07.003Muraoka, O., Xu, B., Tsurumaki, T., Akira, S., Yamaguchi, T., & Higuchi, H. (2003). Leptin-induced transactivation of NPY gene promoter mediated by JAK1, JAK2 and STAT3 in the neural cell lines. Neurochemistry International, 42(7), 591-601. doi:10.1016/s0197-0186(02)00160-2Wood, J. D., Enser, M., Fisher, A. V., Nute, G. R., Sheard, P. R., Richardson, R. I., … Whittington, F. M. (2008). Fat deposition, fatty acid composition and meat quality: A review. Meat Science, 78(4), 343-358. doi:10.1016/j.meatsci.2007.07.019Clément, K., Vaisse, C., Lahlou, N., Cabrol, S., Pelloux, V., Cassuto, D., … Guy-Grand, B. (1998). A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature, 392(6674), 398-401. doi:10.1038/32911Dubern, B., & Clement, K. (2012). Leptin and leptin receptor-related monogenic obesity. Biochimie, 94(10), 2111-2115. doi:10.1016/j.biochi.2012.05.010Lim, K.-S., Kim, J.-M., Lee, E.-A., Choe, J.-H., & Hong, K.-C. (2014). A Candidate Single Nucleotide Polymorphism in the 3′ Untranslated Region of Stearoyl-CoA Desaturase Gene for Fatness Quality and the Gene Expression in Berkshire Pigs. Asian-Australasian Journal of Animal Sciences, 28(2), 151-157. doi:10.5713/ajas.14.0529Saatchi, M., Garrick, D. J., Tait, R. G., Mayes, M. S., Drewnoski, M., Schoonmaker, J., … Reecy, J. M. (2013). Genome-wide association and prediction of direct genomic breeding values for composition of fatty acids in Angus beef cattlea. BMC Genomics, 14(1). doi:10.1186/1471-2164-14-730Chen, L., Ekine-Dzivenu, C., Vinsky, M., Basarab, J., Aalhus, J., Dugan, M. E. R., … Li, C. (2015). Genome-wide association and genomic prediction of breeding values for fatty acid composition in subcutaneous adipose and longissimus lumborum muscle of beef cattle. BMC Genetics, 16(1). doi:10.1186/s12863-015-0290-

Genetic biomarkers for fat content and fatty acid composition in pork

Aquesta tesi doctoral forma part d'una línia de recerca sobre la millora genètica de la qualitat de la carn en porcí. Un dels recents objectius inclòs en algunes línies paternes seleccionades per mercats de qualitat és aconseguir un nivell òptim de greix intramuscular i de composició en àcids grassos sense penalitzar el rendiment magre. Aquesta tesi està formada per quatre estudis realitzats en una línia pura de porcs Duroc destinats a la producció de càrnics d'alta qualitat. Els estudis es van dissenyar amb l'objectiu d'entendre millor la variabilitat genètica subjactent al contingut de greix i a la composició en àcids grassos i d’identificar marcadors potencials a la selecció. El primer estudi va examinar els paràmetres genètics de la ruta de l'àcid linoleic (C18:2) a l'àcid araquidònic (C20:4). En particular, es va demostrar que s’espera que la selecció pel valor absolut de C18:2 proporcioni una resposta a la selecció similar a la selecció pel greix intramuscular a greix dorsal restringit. Aquests resultats van conduir a investigar el gen de la desaturasa-2 dels àcids grassos (FADS2), com a gen candidat per a la ruta metabòlica de C18:2. Així, el segon estudi va avaluar els efectes d'una variant (el polimorfisme rs321384923 es va utilitzar com a marcador) al promotor del gen FADS2. Els resultats van demostrar que aquest polimorfisme afecta el perfil d'àcids grassos n-6 millorant l'eficiència de desaturació de C18:2 a C20:4. Addicionalment, es va avaluar l'associació dels gens de les perilipines (PLIN) i del gen de la proteína d’unió al guanilat 1 (GBP1) amb caràcters de creixement i de qualitat de la carn. Així, el tercer estudi va examinar els efectes de dos polimorfismes als gens PLIN1 i PLIN2, relacionats amb l'emmagatzematge i la mobilització de lípids. Els resultats indiquen que el polimorfisme rs333231747 a PLIN2 està associat al creixement primerenc i al pes magre. L'últim estudi va demostrar que GBP1 té dos senyals de poliadenilació actius i que el seu ús depèn del genotip rs80800372. Els porcs portadors de l'al·lel G, associat a una menor viremia després de la infecció pel virus reproductiu i respiratori porcí, van tenir transcripcions més llargues i una menor expressió gènica. En condicions no epidèmiques, l'al·lel G va augmentar el contingut de greix intramuscular, però va disminuir el pes magre. El contingut de C18:2 i els marcadors genètics investigats es poden utilitzar per dissenyar estratègies de selecció adequades per millorar la qualitat de la carn i el creixement magre.Esta tesis doctoral es parte de una línea de investigación sobre la mejora genética de la calidad de la carne en porcino. Uno de los recientes objetivos incluido en algunas líneas paternas seleccionadas para mercados de calidad es lograr un nivel óptimo de grasa intramuscular y de composición en ácidos grasos sin penalizar el crecimiento magro. Esta tesis comprende cuatro estudios realizados en una línea pura de cerdos Duroc destinados a la producción de cárnicos de alta calidad. Los experimentos se diseñaron con el objetivo de comprender mejor la variabilidad genética subyacente en el contenido de grasa y la composición en ácidos grasos e identificar marcadores potenciales a la selección. El primer estudio examinó los parámetros genéticos de la ruta del ácido linoleico (C18:2) al ácido araquidónico (C20:4). En particular, se demostró que se espera que la selección del valor absoluto de C18:2 proporcione una respuesta similar a la selección por grasa intramuscular a grasa dorsal restringida. Estos hallazgos llevaron a investigar el gen de la desaturasa-2 de los ácidos grasos (FADS2), como gen candidato para la ruta del C18:2. Por lo tanto, el segundo estudio evaluó los efectos de una variante (el polimorfismo rs321384923 se usó como marcador) en el promotor del gen FADS2. Los resultados mostraron que este polimorfismo afecta el perfil de ácidos grasos n-6 al aumentar la eficiencia de desaturación de C18:2 a C20:4. Además, se evaluó la asociación de los genes de las perilipinas (PLIN) y el gen de la proteína de unión a guanilato 1 (GBP1) con caracteres de crecimiento y de calidad de la carne. De este modo, el tercer estudio examinó los efectos de dos polimorfismos en PLIN1 y PLIN2, relacionados con la deposición y la movilización de lípidos. Los resultados indicaron que el polimorfismo rs333231747 en PLIN2 se asocia con el crecimiento temprano y con el peso magro. El último estudio mostró que GBP1 tiene dos señales de poliadenilación activas y que su uso depende del genotipo rs80800372. Los cerdos portadores del alelo G, asociado con una menor viremia después de la infección por el virus reproductivo y respiratorio porcino, tenían transcritos más largos y una menor expresión génica. En condiciones no epidémicas, el alelo G aumentó el contenido de grasa intramuscular pero disminuyó el peso magro. El contenido de C18:2 y los marcadores genéticos investigados se pueden usar para diseñar estrategias de selección adecuadas para mejorar la calidad de la carne y el crecimiento magro.This PhD dissertation is part of a research line on the genetic improvement of pork quality. One of the latest goals to be included in some sire lines selected for premium markets is to achieve an optimum level of intramuscular fat and fatty acid composition without penalizing lean growth performance. This thesis is comprised of four studies conducted on a purebred Duroc line used for producing high-quality meat products. The experiments were designed with the aim to better understand the genetic variability underlying fat content and fatty acid compostion and to identify potential markers for breeding. The first study examined the genetic parameters of the linoleic acid (C18:2) to arachidonic acid (C20:4) pathway. In particular, it was showed that selection for the absolute value of C18:2 is expected to deliver a similar response outcome as selection for intramuscular fat at restrained backfat thickness. These findings led to investigate the fatty acid desaturase-2 (FADS2) gene, as a candidate gene for C18:2 metabolism route. Thus, the second study evaluated the effects of a variant (rs321384923 was used as a tag single nucleotide polymorphism) in the promoter of the FADS2 gene. Results showed that this polymorphism affects the n-6 fatty acid profile by enhancing the desaturation efficiency of C18:2 to C20:4. Additionally, the association of perilipin (PLIN) genes and guanylate-binding protein-1 (GBP1) gene with growth and meat quality traits was assessed. Thus, the third study examined the effects of two polymorphisms in PLIN1 and PLIN2, which have been related to lipid storage and mobilization. Results indicated that the rs333231747 polymorphism on PLIN2 is associated to early growth and lean weight. The last study showed that GBP1 has two active polyadenylation signals and that their usage depends on the rs80800372 genotype. The pigs carrying the G allele, which has been associated with lower viraemia after porcine reproductive and respiratory virus infection, had longer transcripts and lower gene expression. In non-epidemic conditions, the G allele increased intramuscular fat content but decreased lean weight. Linoleic acid content and the investigated genetic markers can be used to design appropriate selection strategies to enhance meat quality and lean growth

Genetic biomarkers for fat content and fatty acid composition in pork

Aquesta tesi doctoral forma part d'una línia de recerca sobre la millora genètica de la qualitat de la carn en porcí. Un dels recents objectius inclòs en algunes línies paternes seleccionades per mercats de qualitat és aconseguir un nivell òptim de greix intramuscular i de composició en àcids grassos sense penalitzar el rendiment magre. Aquesta tesi està formada per quatre estudis realitzats en una línia pura de porcs Duroc destinats a la producció de càrnics d'alta qualitat. Els estudis es van dissenyar amb l'objectiu d'entendre millor la variabilitat genètica subjactent al contingut de greix i a la composició en àcids grassos i d’identificar marcadors potencials a la selecció. El primer estudi va examinar els paràmetres genètics de la ruta de l'àcid linoleic (C18:2) a l'àcid araquidònic (C20:4). En particular, es va demostrar que s’espera que la selecció pel valor absolut de C18:2 proporcioni una resposta a la selecció similar a la selecció pel greix intramuscular a greix dorsal restringit. Aquests resultats van conduir a investigar el gen de la desaturasa-2 dels àcids grassos (FADS2), com a gen candidat per a la ruta metabòlica de C18:2. Així, el segon estudi va avaluar els efectes d'una variant (el polimorfisme rs321384923 es va utilitzar com a marcador) al promotor del gen FADS2. Els resultats van demostrar que aquest polimorfisme afecta el perfil d'àcids grassos n-6 millorant l'eficiència de desaturació de C18:2 a C20:4. Addicionalment, es va avaluar l'associació dels gens de les perilipines (PLIN) i del gen de la proteína d’unió al guanilat 1 (GBP1) amb caràcters de creixement i de qualitat de la carn. Així, el tercer estudi va examinar els efectes de dos polimorfismes als gens PLIN1 i PLIN2, relacionats amb l'emmagatzematge i la mobilització de lípids. Els resultats indiquen que el polimorfisme rs333231747 a PLIN2 està associat al creixement primerenc i al pes magre. L'últim estudi va demostrar que GBP1 té dos senyals de poliadenilació actius i que el seu ús depèn del genotip rs80800372. Els porcs portadors de l'al·lel G, associat a una menor viremia després de la infecció pel virus reproductiu i respiratori porcí, van tenir transcripcions més llargues i una menor expressió gènica. En condicions no epidèmiques, l'al·lel G va augmentar el contingut de greix intramuscular, però va disminuir el pes magre. El contingut de C18:2 i els marcadors genètics investigats es poden utilitzar per dissenyar estratègies de selecció adequades per millorar la qualitat de la carn i el creixement magre.Esta tesis doctoral es parte de una línea de investigación sobre la mejora genética de la calidad de la carne en porcino. Uno de los recientes objetivos incluido en algunas líneas paternas seleccionadas para mercados de calidad es lograr un nivel óptimo de grasa intramuscular y de composición en ácidos grasos sin penalizar el crecimiento magro. Esta tesis comprende cuatro estudios realizados en una línea pura de cerdos Duroc destinados a la producción de cárnicos de alta calidad. Los experimentos se diseñaron con el objetivo de comprender mejor la variabilidad genética subyacente en el contenido de grasa y la composición en ácidos grasos e identificar marcadores potenciales a la selección. El primer estudio examinó los parámetros genéticos de la ruta del ácido linoleico (C18:2) al ácido araquidónico (C20:4). En particular, se demostró que se espera que la selección del valor absoluto de C18:2 proporcione una respuesta similar a la selección por grasa intramuscular a grasa dorsal restringida. Estos hallazgos llevaron a investigar el gen de la desaturasa-2 de los ácidos grasos (FADS2), como gen candidato para la ruta del C18:2. Por lo tanto, el segundo estudio evaluó los efectos de una variante (el polimorfismo rs321384923 se usó como marcador) en el promotor del gen FADS2. Los resultados mostraron que este polimorfismo afecta el perfil de ácidos grasos n-6 al aumentar la eficiencia de desaturación de C18:2 a C20:4. Además, se evaluó la asociación de los genes de las perilipinas (PLIN) y el gen de la proteína de unión a guanilato 1 (GBP1) con caracteres de crecimiento y de calidad de la carne. De este modo, el tercer estudio examinó los efectos de dos polimorfismos en PLIN1 y PLIN2, relacionados con la deposición y la movilización de lípidos. Los resultados indicaron que el polimorfismo rs333231747 en PLIN2 se asocia con el crecimiento temprano y con el peso magro. El último estudio mostró que GBP1 tiene dos señales de poliadenilación activas y que su uso depende del genotipo rs80800372. Los cerdos portadores del alelo G, asociado con una menor viremia después de la infección por el virus reproductivo y respiratorio porcino, tenían transcritos más largos y una menor expresión génica. En condiciones no epidémicas, el alelo G aumentó el contenido de grasa intramuscular pero disminuyó el peso magro. El contenido de C18:2 y los marcadores genéticos investigados se pueden usar para diseñar estrategias de selección adecuadas para mejorar la calidad de la carne y el crecimiento magro.This PhD dissertation is part of a research line on the genetic improvement of pork quality. One of the latest goals to be included in some sire lines selected for premium markets is to achieve an optimum level of intramuscular fat and fatty acid composition without penalizing lean growth performance. This thesis is comprised of four studies conducted on a purebred Duroc line used for producing high-quality meat products. The experiments were designed with the aim to better understand the genetic variability underlying fat content and fatty acid compostion and to identify potential markers for breeding. The first study examined the genetic parameters of the linoleic acid (C18:2) to arachidonic acid (C20:4) pathway. In particular, it was showed that selection for the absolute value of C18:2 is expected to deliver a similar response outcome as selection for intramuscular fat at restrained backfat thickness. These findings led to investigate the fatty acid desaturase-2 (FADS2) gene, as a candidate gene for C18:2 metabolism route. Thus, the second study evaluated the effects of a variant (rs321384923 was used as a tag single nucleotide polymorphism) in the promoter of the FADS2 gene. Results showed that this polymorphism affects the n-6 fatty acid profile by enhancing the desaturation efficiency of C18:2 to C20:4. Additionally, the association of perilipin (PLIN) genes and guanylate-binding protein-1 (GBP1) gene with growth and meat quality traits was assessed. Thus, the third study examined the effects of two polymorphisms in PLIN1 and PLIN2, which have been related to lipid storage and mobilization. Results indicated that the rs333231747 polymorphism on PLIN2 is associated to early growth and lean weight. The last study showed that GBP1 has two active polyadenylation signals and that their usage depends on the rs80800372 genotype. The pigs carrying the G allele, which has been associated with lower viraemia after porcine reproductive and respiratory virus infection, had longer transcripts and lower gene expression. In non-epidemic conditions, the G allele increased intramuscular fat content but decreased lean weight. Linoleic acid content and the investigated genetic markers can be used to design appropriate selection strategies to enhance meat quality and lean growth

A polymorphism in the fatty acid desaturase-2 gene is associated with the arachidonic acid metabolism in pigs

Arachidonic acid (C20:4) is related to a wide range of biological effects including lipid homeostasis. The fatty acid desaturase-2 (FADS2) gene encodes for the delta-6-desaturase, which is involved in the biosynthesis of C20:4 from linoleic acid (C18:2). The purpose of this study was to characterise mutations in the promoter of the porcine FADS2, evaluating in particular the effect of one haplotype tagging polymorphism (rs321384923A > G) on the biosynthesis pathway of C20:4. A total of 1,192 Duroc barrows with records on fatty acid composition in muscle and subcutaneous fat were genotyped. Pigs carrying the A allele showed, irrespective of fat content, both enhanced FADS2 expression and higher C20:4 in muscle and exhibited increased ratios of C20:4 to C18:2 and of C20:4 to eicosadienoic acid (C20:2) in both muscle and adipose tissue. Despite the inverse relationship observed between C20:4 and fat content, the rs321384923 polymorphism had no impact on lean weight. It is concluded that the haplotype encompassing the rs321384923 polymorphism at the porcine FADS2 affects the n-6 fatty acid profile by specifically modifying the desaturation efficiency of C18:2 to C20:4 rather than by concomitant variations in C18:2 following changes in fat content.This article is published as Gol, Sofia, Ramona N. Pena, Max F. Rothschild, Marc Tor, and Joan Estany. "A polymorphism in the fatty acid desaturase-2 gene is associated with the arachidonic acid metabolism in pigs." Scientific reports 8 (2018): 14336. doi: 10.1038/s41598-018-32710-w.</p

A polymorphism in the fatty acid desaturase-2 gene is associated with the arachidonic acid metabolism in pigs

Abstract Arachidonic acid (C20:4) is related to a wide range of biological effects including lipid homeostasis. The fatty acid desaturase-2 (FADS2) gene encodes for the delta-6-desaturase, which is involved in the biosynthesis of C20:4 from linoleic acid (C18:2). The purpose of this study was to characterise mutations in the promoter of the porcine FADS2, evaluating in particular the effect of one haplotype tagging polymorphism (rs321384923A > G) on the biosynthesis pathway of C20:4. A total of 1,192 Duroc barrows with records on fatty acid composition in muscle and subcutaneous fat were genotyped. Pigs carrying the A allele showed, irrespective of fat content, both enhanced FADS2 expression and higher C20:4 in muscle and exhibited increased ratios of C20:4 to C18:2 and of C20:4 to eicosadienoic acid (C20:2) in both muscle and adipose tissue. Despite the inverse relationship observed between C20:4 and fat content, the rs321384923 polymorphism had no impact on lean weight. It is concluded that the haplotype encompassing the rs321384923 polymorphism at the porcine FADS2 affects the n-6 fatty acid profile by specifically modifying the desaturation efficiency of C18:2 to C20:4 rather than by concomitant variations in C18:2 following changes in fat content