Nalaganje, sestava in metabolizem lipidov pri različnih metabolnih kategorijah prašičev

Fat deposition contributes importantly to various aspects of pig production such as meat and fat quality. It is influenced by various factors, including genetics (i.e. different breeds) and non-genetic factors (e.g. sex). This study aimed to identify mechanisms underlying these differences. The general objective of the present research was to determine how genetic variants and gene expression networks influence lipid-related traits in different metabolic types of pigs (i.e. among local breeds, between local breeds versus modern pig crossbreed and between entire males versus immunocastrated versus surgically castrated pigs). In the first part of the research, 20 European local pig breeds were phenotypically distributed according to stature, growth performance and fatness traits. Principal component analysis of phenotypic data distinguished between larger and leaner breeds with better growth potential from breeds that were smaller, fatter, and had low growth efficiency. The phenotypic data and genetic variants were used for a genome scan for selection signatures associated with phenotype. Several candidate genes were identified that may have adapted to specific phenotypic traits, such as the ANXA4 and ANTXR1 genes for stature, the TLL1 and KCTD16 genes for growth performance, and the DNMT3A and POMC genes for fatness. Several discovered genes in the genotype-phenotype selection scan were further tested for differential gene expression in adipose tissue between the local Krškopolje and the modern crossbreed. However, no significant differences in expression were found between the studied groups. Additionally, the expression of genes involved in lipid metabolism was further investigated in the same groups to discover possible underlying mechanisms for fatty phenotype in Krškopolje pigs. Genes involved in adipogenesis (i.e. PPARγ) and lipogenesis (i.e. FASN, ACACA) were upregulated in Krškopolje compared to modern crossbreed. These results were further supported by a higher quantity of adipose tissue depots together with a higher desaturation capacity in Krškopolje pigs than in modern crossbreed. However, lipogenic enzyme activities of subcutaneous adipose tissue were lower in Krškopolje than in modern pigs. In the second part of the research, male sex categories were compared at different levels. RNA-sequencing analysis revealed 83 differentially expressed genes between entire males and immunocastrated pigs, 15 between immunocastrated and surgically castrated pigs and 48 between entire males and surgically castrated pigs. Upregulated genes in entire males compared to immunocastrated and surgically castrated pigs were related to extracellular matrix dynamics and adipogenesis, whereas downregulated genes were involved in carbohydrate and lipid metabolism. A candidate gene expression approach using quantitative PCR demonstrated that genes involved in lipogenesis (i.e. FASN, ME1, ACACA) were downregulated in entire males compared to immunocastrated pigs. The obtained transcriptomic results were further associated with the phenotype. It was shown that entire males were the leanest compared to castrated pigs, exhibiting the smallest adipocytes and lobulus surface area in the backfat. In addition, activities of lipogenic enzymes in backfat were similar to surgically castrated pigs, while the immunocastrated pigs had increased activity compared to entire males and surgically castrated pigs. The higher activity of lipogenic enzymes in the backfat of immunocastrated pigs was not consistent with the fatty acid composition, as immunocastrates were still similar to entire males (lower saturated and higher polyunsaturated fatty acid content compared to surgically castrated pigs). To conclude, the present study discovered genetic variants and differentially expressed genes responsible for distinct lipid metabolism in different metabolic types of pigs.Nalaganje maščobnega tkiva vpliva na proizvodnjo in kakovost prašičjega mesa in maščobe, nanj pa vplivajo genetski (npr. različne pasme) ali negenetski faktorji (npr. kategorije spola). V tej raziskavi smo poskušali odkriti genetske variante in razlike v izražanju genov, ki bi lahko bili odgovorni za razlike v nalaganju maščob pri različnih metabolnih tipih prašičev, kot so lokalne pasme ali kategorije spola (nekastrirani, imunokastrirani in kirurško kastrirani prašiči). V prvem delu raziskave smo evropske lokalne pasme prašičev fenotipsko razporedili glede na postavo, rastnost in zamaščenost. Z metodo glavnih komponent smo ločili večje in bolj mesnate lokalne pasme z boljšim rastnim potencialom od lokalnih pasem, ki so manjše, bolj zamaščene in ki izražajo manjši rastni potencial. Pridobljene fenotipske podatke smo skupaj z genetskimi variantami uporabili za iskanje selekcijskih podpisov povezanih s fenotipom. Našli smo več kandidatnih genov, ki bi lahko bili pod vplivom adaptacije na specifične fenotipske lastnosti, kot so ANXA4 in ANTXR1 pri fenotipski skupini »postava«, TLL1 in KCTD16 pri skupini »rastnost« in DNMT3A ter POMC pri skupini »zamaščenost«. Pri nekaterih od teh genov smo preverili razlike v izražanju v podkožnem maščobnem tkivu pri lokalni pasmi krškopoljski prašič v primerjavi z modernim križancem. Pri primerjavi obeh skupin nismo našli razlik. Dodatno smo pri pasmi krškopoljski prašič v primerjavi z modernim križancem ugotavljali razlike v izražanju genov, ki so vključeni v lipidni metabolizem. V podkožnem maščobnem tkivu krškopoljskega prašiča v primerjavi z modernim križancem je bilo povečano izražanje genov vključenih v adipogenezo (PPARγ) in lipogenezo (FASN, ACACA). Omenjene razlike so sovpadale z večjo debelino podkožnega maščobnega tkiva in z večjo vsebnost enkrat nenasičenih maščobnih kislin pri krškopoljskem prašiču. V nasprotju s tem pa smo pri krškopoljskem prašiču ugotovili zmanjšano aktivnost lipogenetskih encimov v primerjavi z modernim križancem. V drugem delu raziskave smo kategorije spola primerjali na različnih nivojih. S pomočjo metode sekvenciranja RNK smo pri primerjavi nekastriranih in imunokastriranih prašičev odkrili 83 diferencialno izraženih genov, medtem ko je primerjava transkriptoma pri imunokastriranih in kirurško kastriranih prašičih ter nekastriranih in kirurško kastriranih prašičih pokazala 15 oziroma 48 diferencialno izraženih genov. Geni, ki so imeli povečano izražanje pri nekastriranih v primerjavi z imuno- in kirurško kastriranimi prašiči, so bili povezani z dinamiko ekstracelularnega matriksa in adipogenezo, medtem ko so bili geni z znižanim izražanjem vključeni v metabolizem ogljikovih hidratov in lipidov. Metoda kandidatnih genov z uporabo kvantitativne PCR je dodatno pokazala znižano izražanje genov vključenih v lipogenezo pri nekastriranih samcih v primerjavi z imunokastriranimi prašiči (FASN, ME1, ACACA). Poleg izražanja genov, pa smo pri kategorijah spola ugotavljali tudi fenotipske razlike. Ugotovili smo, da so nekastrirani samci bolj mesnati ter imajo manjše adipocite in površine lobulov v podkožnem maščobnem tkivu. Poleg tega je bila aktivnost lipogenetskih encimov pri nekastriranih samcih podobna kot pri kirurško kastriranih prašičih. V nasprotju s tema dvema skupinama so imeli imunokastrirani prašiči povečano lipogenetsko encimsko aktivnost, ki pa ni sovpadala z maščobno-kislinsko sestavo, saj so bili imunokastrati še vedno podobni nekastriranim prašičem (podkožno maščobno tkivo je imelo nižjo vsebnost nasičenih in višjo vsebnost večkrat nenasičenih maščobnih kislin v primerjavi s kirurškimi kastrati). S to raziskavo smo odkrili genetske variante in gene, ki bi lahko bili odgovorni za različno nalaganje lipidov pri metabolno različnih tipih prašičev ter to povezali z biokemijskimi in histomorfološkimi značilnostmi obravnavanih skupin