{"id":16011,"date":"2026-06-17T12:35:44","date_gmt":"2026-06-17T12:35:44","guid":{"rendered":"https:\/\/www.proefschriftmaken.nl\/portfolio\/pedro-sa\/"},"modified":"2026-06-17T12:35:53","modified_gmt":"2026-06-17T12:35:53","slug":"pedro-sa","status":"publish","type":"us_portfolio","link":"https:\/\/www.proefschriftmaken.nl\/en\/portfolio\/pedro-sa\/","title":{"rendered":"Pedro Sa"},"content":{"rendered":"","protected":true},"excerpt":{"rendered":"","protected":true},"author":7,"featured_media":16012,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":""},"us_portfolio_category":[45],"class_list":["post-16011","us_portfolio","type-us_portfolio","status-publish","post-password-required","hentry","us_portfolio_category-new-template"],"acf":{"naam_van_het_proefschift":"Environmental, genetic and epigenetic regulation of male reproduction in pigs","samenvatting":"Zowel commerci\u00eble varkens als veel bedreigde zoogdierpopulaties zijn afhankelijk van de bijdrage van een beperkt aantal mannetjes aan de volgende generatie. In de varkensfokkerij versterkt kunstmatige inseminatie (KI) dit patroon, waardoor een kleine groep eliteberen duizenden nakomelingen kan verwekken. Soortgelijke dynamieken treden op bij zoogdiersoorten met kleine effectieve populatiegroottes, waaronder bedreigde of kleine populaties, waar de reproductieve capaciteit van mannetjes de levensvatbaarheid van de populatie op lange termijn sterk kan be\u00efnvloeden. Begrip van de biologische regulatie van mannelijke reproductieve prestaties is daarom niet alleen essentieel voor de verbetering van de veestapel, maar ook zeer relevant voor het beheer en behoud van zoogdierpopulaties.\n\nHoewel sperma-kenmerken routinematig worden geregistreerd in commerci\u00eble KI-centra met behulp van geautomatiseerde systemen, blijven de biologische mechanismen die variatie in mannelijke reproductieve prestaties reguleren onvolledig begrepen. Sperma-eigenschappen worden be\u00efnvloed door additieve en niet-additieve genetische effecten, evenals permanente omgevingsfactoren met onderliggende moleculaire regulatiemechanismen die op transcriptomisch en epigenetisch niveau werken. Het ontleden en ontwarren van dit netwerk is essentieel voor zowel de overdracht van genetische vooruitgang in commerci\u00eble populaties als voor het karakteriseren van geconserveerde mechanismen bij zoogdieren die de genotype-naar-fenotype architectuur van mannelijke reproductie reguleren.\n\nHet algemene doel van dit proefschrift was het kwantificeren van de omgevings-, genetische, transcriptomische en epigenetische bronnen van variatie die ten grondslag liggen aan mannelijke reproductieve kenmerken bij varkens. Door gebruik te maken van grootschalige longitudinale sperma-gegevens gecombineerd met genomische, transcriptomische en epigenomische informatie, biedt dit proefschrift een multi-omics perspectief op mannelijke reproductie in een commerci\u00eble varkenspopulatie.\n\nIn Hoofdstuk 2 hebben we de kennis voorafgaand aan dit proefschrift over de genomische, transcriptomische en epigenomische regulatie van spermacellen bij varkens beoordeeld. We toonden aan dat sperma-eigenschappen een lage tot matige erfelijkheid hebben, wat consistent is met sterke zuiverende selectie op vruchtbaarheidsgerelateerde eigenschappen. Op moleculair niveau dragen spermacellen een complexe lading van gefragmenteerde eiwitcoderende RNA's, samen met lange en kleine niet-coderende RNA's, en specifieke DNA-methyleringspatronen, die allemaal kunnen bijdragen aan de vorming en functie van sperma en de vroege embryonale ontwikkeling van nakomelingen.\n\nIn Hoofdstuk 3 richtten we onze analyse op een commerci\u00eble varkenspopulatie. We schatten genetische parameters en ouderlijke en vroege-levenseffecten op 449.966 ejaculaten van 5.692 KI-beren. De erfelijkheidsgraad varieerde van 0,11 tot 0,27. Een centrale bevinding was de detectie van significante maternale omgevingseffecten, die tussen 2,3% en 4,6% van de fenotypische variatie verklaarden.\n\nIn Hoofdstuk 4 onderzochten we of sperma-eigenschappen gemeten op verschillende leeftijden genetisch verschillende eigenschappen vertegenwoordigen. De resultaten toonden aan dat deze eigenschappen gedurende het leven van een beer genetisch vrijwel identiek blijven, hoewel de additieve genetische variantie toeneemt naarmate beren ouder worden.\n\nIn Hoofdstuk 5 voerden we een genoombrede associatiestudie (GWAS) uit en identificeerden we tien loci die sperma-eigenschappen be\u00efnvloeden, waaronder vier loci met recessieve schadelijke allelen. We identificeerden missense-varianten in biologisch relevante genen zoals MEIOB, CFAP74 en UBE2B.\n\nIn de Hoofdstukken 6 en 7 breidden we de analyse uit naar het sperma-transcriptoom. We identificeerden duizenden RNA's en eQTL's die hun overvloed reguleren. Hoofdstuk 8 richtte zich op DNA-methylering, waarbij substanti\u00eble veranderingen werden waargenomen tijdens de seksuele rijping en meQTL-hotspots werden ge\u00efdentificeerd, waaronder een variant in het DNMT3B-gen.\n\nConcluderend toont dit proefschrift aan dat mannelijke reproductieve prestaties bij varkens worden gereguleerd door een ge\u00efntegreerde architectuur van omgeving, genetica en epigenetica.","summary":"Both commercial pigs and many endangered mammalian populations rely on the contribution of a limited number of males to the next generation. In pig breeding, artificial insemination (AI) amplifies this pattern, allowing a small group of elite boars to sire thousands of offspring. Similar dynamics occur in mammalian species with small effective population sizes, including endangered or small populations, where reproductive capacity of males can strongly influence long-term population viability. Understanding the biological regulation of male reproductive performance is therefore not only essential for livestock improvement but also highly relevant for the management and conservation of mammalian populations.\n\nAlthough semen traits are routinely recorded in commercial AI centers using automated systems, the biological mechanisms regulating variation in male reproductive performance remain incompletely understood. Semen characteristics are influenced by additive and non-additive genetic effects, as well as permanent environmental factors with underlying molecular regulatory mechanisms operating at transcriptomic and epigenetic levels. Dissecting and disentangling this network is essential both for the transmission of genetic progress in commercial populations, and for characterizing conserved mechanisms in mammals that regulate the genotype-to-phenotype architecture of male reproduction.\n\nThe overall objective of this thesis was to quantify the environmental, genetic, transcriptomic, and epigenetic sources of variation underlying male reproductive traits in pigs. Using large-scale longitudinal semen data combined with genomic, transcriptomic and epigenomic information, this thesis provides a multi-omics perspective on male reproduction in a commercial pig population.\n\nIn Chapter 2, we reviewed the knowledge prior to this thesis on the genomic, transcriptomic, and epigenomic regulation of sperm cells in pigs. We showed that semen traits have low to moderate heritabilities, consistent with strong purifying selection acting on fertility-related traits. At the molecular level, sperm cells carry a complex cargo of fragmented protein-coding RNAs, along with long and small non-coding RNAs, and specific DNA methylation patterns, all of which may contribute to sperm formation, function, and early offspring embryonic development. Although several genomic and transcriptomic studies have been conducted in pigs, most have focused on cataloguing molecular profiles on a restricted set of boars rather than establishing functional links or extensively exploring environmental modulation. As a result, causal regulatory mechanisms connecting DNA variants, RNA abundances, and DNA methylation to sperm and semen phenotypes and environmental effects remain largely unresolved. This lack of evidence motivated the multi-level approach developed in the subsequent chapters of this thesis.\n\nIn Chapter 3, we focused our analysis on a commercial pig population which served as the subject of this thesis work. We estimated genetic parameters and parental and early-life effects on 449,966 ejaculates recorded from 5,692 AI boars. Sixteen semen traits measured on fresh semen and six motility traits measured after storage were analysed. Heritability estimates ranged from 0.11 to 0.27 across traits, confirming low to moderate additive genetic control. Repeatability ranged from 0.39 to 0.65 after transformation of skewed traits. Motility traits measured after storage showed heritabilities between 0.20 and 0.24, similar to fresh semen motility, but repeatabilities were substantially lower (0.35\u2013 0.40 compared to those of 0.50\u20130.58 for fresh motility), indicating reduced permanent environmental effects after storage. More interestingly, genetic correlations between fresh and stored motility traits were moderate (0.52 \u2013 0.76) rather than close to unity, demonstrating that sperm motility after storage represents distinct traits. A central finding of Chapter 3 was the detection of significant maternal environmental effects. Maternal environment explained between 2.3% and 4.6% of the phenotypic variance for sperm motility traits, with the largest effect observed for total motility of fresh semen (4.6%). Maternal effects also explained 3.1% of the variance for total morphological abnormalities and 1.5% for ejaculate volume. When maternal environment was included in the model, heritability estimates for motility traits decreased. These results demonstrate that ignoring maternal environmental effects leads to overestimation of additive genetic variance. In contrast, paternal environmental effects were negligible, and mitochondrial DNA and Y chromosome effects did not significantly contribute to phenotypic variation. The main message of Chapter 3 is therefore that semen traits have low heritabilities and are substantially influenced by early-life maternal environment.\n\nIn Chapter 4, we investigated whether semen traits measured at different ages represent genetically distinct traits. Using a trivariate approach, boars were divided into three age classes: 7\u201313 months, 14\u201323 months, and 24\u201360 months. Additive genetic correlations across age classes were consistently high. For example, genetic correlations for ejaculate volume ranged from 0.89 to 0.97 across age comparisons, and for motility traits correlations ranged from 0.81 to 0.98. Most morphology traits exhibited correlations above 0.90 across ages. These high genetic correlations indicated that semen traits are genetically the same trait throughout the life of a commercial boar. However, additive genetic variance increased with age for semen quantity traits, implying that genetic differences among boars become more evident as boars mature. Thus, the main message of Chapter 4 is that age does not change the underlying genetic architecture in AI boars but modulates the magnitude of genetic effects.\n\nIn Chapter 5, we performed a genome-wide association study (GWAS) using 286,025 ejaculates from 2,936 boars to identify loci affecting 15 semen traits. Ten quantitative trait loci (QTL) were detected, including four loci exhibiting recessive deleterious alleles. Several lead SNPs affected multiple traits simultaneously. A prominent locus on SSC6 was associated with distal cytoplasmic droplets and showed effects on tail abnormalities and sperm motility in follow-up analyses. Using whole-genome sequence data, we identified six missense variants in high linkage disequilibrium (LD) with lead SNPs in biologically relevant genes, including the MEIOB (meiotic recombination), CFAP74 (sperm flagellar structure), and UBE2B (spermatogenesis) genes. Notably, allele frequencies of some variants with predicted deleterious effects increased between 2013 and 2019, likely due to indirect selection pressures and pleiotropy with commercially relevant traits. The main message of Chapter 5 is that male fertility in pigs is influenced by specific loci with additive and dominance\/recessive effects.\n\nIn Chapters 6 and 7, we extended the analysis to the sperm transcriptome. Approximately 200 semen samples collected throughout a period of two and half years were used for RNA isolation and sequencing. The analysis of total RNA-Seq data in Chapter 6 revealed thousands of protein-coding and long non-coding RNAs in sperm. The abundance of these RNAs showed low to moderate heritabilities, and environmental factors including age and season significantly influenced abundance levels of only a small set of RNAs. This was the first study that described heritability and genetic contributions to the abundance of RNAs in pig sperm. Expression quantitative trait locus (eQTL) analyses identified predominantly cis-regulatory variants affecting protein-coding and long non-coding RNAs. In Chapter 7, we used short RNA-Seq of the same samples to further characterize small non-coding RNA population, demonstrating that their abundance is also genetically regulated and environmentally sensitive. Analyses of eQTL affecting small non-coding RNAs resulted in a regulatory hotspot on chromosome SSC2 (136.6 \u2013 136.9 Mb) which was co-localized with a QTL for semen quality traits (Chapter 5) located in the UBE2B gene. This observation supports a possible mechanistic link between genomic variation in the UBE2B gene affecting regulatory RNA abundance and sperm characteristics. The main messages of Chapter 6 and 7 is that the transcriptome of sperm cells is affected by both genetics and environmental effects and that some of these genetic effects may underly phenotypic differences in semen traits.\n\nIn Chapter 8, we investigated DNA methylation in sperm using reduced representation bisulfite sequencing. Approximately 460 semen samples also collected throughout a period of two and half years were used for DNA isolation and sequencing. Our analyses revealed that sperm cells have a highly methylated genome. We observed substantial genome-wide changes in DNA methylation levels as boars age, particularly during sexual maturation. DNA methylation showed low to moderate heritability, and widespread cis-methylation QTL (meQTL) were detected. Three trans-methylation QTL hotspots were detected. In one such hotspot, we highlighted a missense variant in the DNMT3B gene, a de novo DNA methyltransferase, associated with DNA methylation levels at over 7,500 CpG sites genome-wide. This provides evidence that genetic variation directly influences epigenetic programming in sperm.\n\nLastly, Chapter 9 contextualized the findings of this thesis within a broader biological and methodological framework. The results collectively demonstrated that spermatogenesis and semen characteristics are influenced by a complex interplay between genetics and environmental conditions from early life to semen collection mediated by transcriptomic and epigenetic programming. In this chapter, I identified three main areas where knowledge remains incomplete. First, I highlighted the need to better understand how genetic and environmental factors interact during critical developmental windows during embryonic development and spermatogenesis and how these interactions shape adult reproductive performance. I also emphasize the need to shift the focus of the field from single-ejaculate averages toward studying inter-ejaculate variation. Second, I addressed the unresolved functional role of the sperm molecular cargo, mainly related to the importance of sperm RNAs and DNA methylation patterns to fertility outcomes and addressed potential epigenetic inheritance. Lastly, in a third section, I reflected on methodological challenges in quantitative multi-omics research, stressing that rigorous statistical modelling and careful integration of high-dimensional molecular data with quantitative genetics are essential for drawing reliable biological conclusions.\n\nThis thesis demonstrates that male reproductive performance in pigs is regulated by an integrated environmental, genetic and epigenetic architecture. By integrating quantitative genetics with multi-omics approaches, this work advances understanding of genetic and environmental interactions in germ cells and provides a mechanistic framework for studying male reproduction in pigs and other mammals.","auteur":"Pedro Sa","auteur_slug":"pedro-sa","publicatiedatum":"10 juli 2026","taal":"EN","url_flipbook":"https:\/\/ebook.proefschriftmaken.nl\/ebook\/pedrosa?iframe=true","url_download_pdf":"https:\/\/ebook.proefschriftmaken.nl\/download\/2d61c86b-b55c-48fa-8483-b52d09369915\/optimized","url_epub":"","ordernummer":"18936","isbn":"978-94-6534-445-4","doi_nummer":"","naam_universiteit":"Wageningen University","afbeeldingen":16013,"naam_student:":"","binnenwerk":"","universiteit":"Wageningen University","cover":"","afwerking":"","cover_afwerking":"","design":""},"_links":{"self":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio\/16011","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio"}],"about":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/types\/us_portfolio"}],"author":[{"embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/users\/7"}],"replies":[{"embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/comments?post=16011"}],"version-history":[{"count":1,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio\/16011\/revisions"}],"predecessor-version":[{"id":16014,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio\/16011\/revisions\/16014"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/media\/16012"}],"wp:attachment":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/media?parent=16011"}],"wp:term":[{"taxonomy":"us_portfolio_category","embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio_category?post=16011"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}