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Backfat is an important trait in pork production, and it has been included in the breeding objectives of genetic companies for decades. Although adipose tissue is a good energy storage, excessive fat results in reduced efficiency and economical losses. A large QTL for backfat thickness on chromosome 5 is still segregating in different commercial pig breeds. We fine mapped this QTL region using a genome-wide association analysis (GWAS) with 133,358 genotyped animals from five commercial populations (Landrace, Pietrain, Large White, Synthetic, and Duroc) imputed to the porcine 660K SNP chip. The lead SNP was located at 5:66103958 (G/A) within the third intron of the CCND2 gene, with the G allele associated with more backfat, while the A allele is associated with less backfat. We further phased the QTL region to discover a core haplotype of five SNPs associated with low backfat across three breeds. Linkage disequilibrium analysis using whole-genome sequence data revealed three candidate causal variants within intronic regions and downstream of the CCND2 gene, including the lead SNP. We evaluated the association of the lead SNP with the expression of the genes in the QTL region (including CCND2) in a large cohort of 100 crossbred samples, sequenced in four different tissues (lung, spleen, liver, muscle). Results show that the A allele increases the expression of CCND2 in an additive way in three out of four tissues. Our findings indicate that the causal variant for this QTL region is a regulatory variant within the third intron of the CCND2 gene affecting the expression of CCND2.

Resumen del póster presentado a la 38th International Conference on Animal Genetics (ISAG), celebrada virtualmente del 26 al 30 de julio de 2021., In animals, the chromatin structure of the mature spermatozoon is ultra-compacted due to the replacement of histones by protamines during spermatogenesis. However, a small fraction of nucleosomes remains bound to DNA at specific sites of the genome and it has been linked to sperm biology and embryogenesis. The genomic characterization of nucleosome occupancy in the sperm chromatin could help identifying molecular markers for sperm quality and fertility traits. Nonetheless, these maps are not yet available for most livestock species, including swine. In this study, we performed micrococcal nuclease digestion followed by high-throughput sequencing on pig ejaculated spermatozoa and mapped the mono-nucleosomal and sub-nucleosomal chromatin fractions. We found 25,293 mono-nucleosomal and 4,239 sub-nucleosomal peaks covering 0.3% and 0.02% of the porcine genome, respectively. We detected positional conservation of the nucleosome-associated DNAs in sperm between human and pig. We also carried gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and also searched for putative transcription factor binding motifs within the mono-nucleosomal peaks and found an enrichment for sperm function and embryo development-related processes. Remarkably, we detected enrichment for the canonical binding site of Znf263. In humans, this transcription factor has been suggested as a key regulator of the genes with paternal preferential expression during early embryo development. In addition, we also observed co-occupancy of the RNAs present in pig sperm and these RNAs related to sperm quality, with the mono-nucleosomal peaks. We also found a co-location trend between GWAS hits for semen quality in swine and the mono-nucleosomal sites. The results obtained in this study clearly indicate that there is a relationship between nucleosome positioning in sperm with sperm phenotypes and embryo development.

Resumen del póster presentado a la 38th International Conference on Animal Genetics (ISAG), celebrada virtualmente del 26 al 30 de julio de 2021., The study of the sperm transcriptome is often preceded by the removal of non-sperm cells carrying a larger load of RNA. This purification is usually done by density gradient centrifugation to obtain viable spermatozoa from fresh ejaculates or artificial insemination doses, which limits the processing rate and the remoteness of the samples that can be analyzed in one experiment. To evaluate the impact of purification by density gradient centrifugation with BoviPure on the pig sperm, 4 boar ejaculates were purified with this commercial reagent. The purified and paired nonpurified samples were sequenced by RNA-seq and their transcriptomes were compared. 7,519 protein-coding genes were detected. Correlation, cluster and principal component analysis showed high resemblance between the purified and the nonpurified ejaculates. However, 368 and 4 genes displayed decreased and increased RNA levels after purification, respectively. There was an enrichment of translation, transcription and metabolic processes and specifically, of ribosomal protein genes. Moreover, the catalogs of differentially abundant genes and unaltered genes were contrasted against a list of genes with tissue-specific expression in pigs (ArrayExpress expression atlas from the European Bioinformatics Institute; E-MTAB-5895) and humans (GTEx portal). The list of differentially abundant genes was clearly enriched (1.3%) for genes from epididymal origin when compared with the list of unaltered genes (0.2%). On the contrary, most of the testis specific genes present in the boar sperm, showed unaltered RNA levels after purification. The list of differentially abundant genes also included several genes playing important roles during spermatogenesis. We detected no differences between both gene lists in the proportions of prostate, white blood, lymph node, tonsil, duodenum, skeletal muscle, liver and mammary gland specific genes. In summary, the BoviPure-based purification impacts on the RNA levels of a small number of genes, which is most likely caused by the removal of non-sperm cells or seminal exosomes with a distinct load of RNAs.

Póster presentado a la 54th Annual Conference Physiology and Pathology of Reproduction, celebrada virtualmente del 10 al 12 de febrero de 2021.

Additional file 12: Table S10. Parameter estimates for the significant RNA models. For each of the phenotypes, the model outputs the estimated values for the 10 genes obtained from the GRM regression analysis. The lower the value of Pr >|t|, the higher the involvement of the gene abundance on the total phenotypic variance.

Additional file 2: Figure S2. Correlation across boar sperm quality traits. Heatmap plot of the correlations among the 25 sperm traits measured on 300 boars. CON = concentration; VIAB_5 = viability 5 min; VIAB_90 = viability 90 min; ORT = osmotic resistance test; HABN = head abnormalities; NABN = neck abnormalities; TABN = tail abnormalities; PDROP = proximal droplets; DDROP = distal droplets; MT_5 = motility 5 min; VAP_5 = average path velocity 5 min; VCL_5 = curvilinear velocity 5 min; VSL_5 = straight line velocity 5 min; MT_90 = motility 90 min; VAP_90 = average path velocity 90 min; VCL_90 = curvilinear velocity 90 min; VSL_90 = straight line velocity 90 min; ACRO_5 = abnormal acrosomes 5 min; ACRO_90 = abnormal acrosomes 90 min; R_MT = ratio motility; R_VAP = ratio average path velocity; R_VCL = ratio curvilinear velocity; R_VSL = ratio straight line velocity; R_VIAB = ratio viability; R_ACRO = ratio acrosomes. Figure S3. SNP based dendrogram for the 25 semen parameters. Dendrogram of the standardized SNP effects across the 25 sperm traits.

Resumen del póster presentado a la 37th International Conference on Animal Genetics (ISAG), celebrada en Lleida (España) del 7 al 12 de julio de 2019., Male fertility in swine has been associated with sperm motility parameters. Sperm movement is regularly assessed in Artificial Insemination studs and provides an objective and reproducible measurement of semen quality. As sperm transcripts are highly fragmented and present at low abundances we seek to investigate circular RNAs (circRNAs), a novel class of non-coding RNAs with a closed loop structure, more stable than linear transcripts and potentially acting as miRNA sponges. We have performed total and small RNA-seq from 40 boar sperm samples. The objective of this study was to characterize the porcine circRNA repertoire, assess their potential role as miRNA sponges and the correlation between their abundance and sperm motility. We identified 1,393 potential circRNAs across all the 40 samples. The majority of the circRNAs were derived from exonic regions (63.6%), formed from less than 4 exons and had an average lenght below 500 bp. circRNA abundances ranged between 0.18 and 137 CPMs. Compared with other pig tissues, the sperm circRNA repertoire resembled most to testis, even thought 75% of the sperm circRNAs were sperm-specific. Gene ontology analysis revealed that sperm circRNAs involved genes related to epigenetic regulation, spermatogenesis and cilium assembly functions. We identified 69 circRNA-miRNA interactions. For example, miR-28–5p, a miRNA that is dysregulated in human infertile patients, presented 2 different target sites in a circRNA from ZMYND10, a gene required for motile ciliary function. We identified 148 circRNAs that correlated with different sperm motility parameters. We selected 10 circRNAs and validated the closed loop structure by RT-qPCR and Sanger Sequencing. The RNA abundance of 6 cirRNAs was validated by RT-qPCR and compared in 38 additional animals presenting extreme motility parameters. Three circRNAs presented significant differences in abundance between the groups. These included circRNA from LIN7A (P-value: 0.008), LRBA (P-value: 0.04) and PAPOLA (P-value: 0.026) genes, the functions of these genes are related to generating and maintaining cell membrane channels, vesicle trafficking and RNA and ATP binding, respectively.

Resumen del trabajo presentado a la 37 International Society for Animal Genetics Conference (ISAG), celebrada en Lleida (España) del 7 al 12 de julio de 2019.

Resumen del póster presentado a la 37th International Conference on Animal Genetics (ISAG), celebrada en Lleida (España) del 7 al 12 de julio de 2019., For the last decades, boars have been selected for their genetic merit on carcass and meat quality traits. However, breeders and researchers are now paying attention to additional phenotypes including sperm quality. We carried a GWAS and a semen RNA-seq experiment in pigs with the aim to identify SNP markers for 25 sperm quality traits. The GWAS included 288 boars and genotypes from the Affymetrix Axiom Porcine 660K Genotyping Array. Sperm from 40 of these pigs were subjected to total and to small RNA-seq. The GWAS resulted in 345 SNPs significantly associated mostly to sperm head abnormalities and motility. The RNA-seq evidenced 4,120 protein coding genes and 95 miRNAs. 3,053 genes showed significant correlations with at least one semen quality trait. Independently for each phenotype, we then searched for eQTLs using only the GWAS SNP hits and the genes that correlated with the given semen trait. This yielded 119 eQTLs. Several of these hits involve genes with known function related to semen quality. For example, we found a hit on ACTR2 and the percentage of head abnormalities. ACTR2 has been involved in the morphogenic modeling of the sperm head. We also detected a hit between NDUFS8 and sperm motility. NDUFS8 is a known mitochondrial NADH subunit involved in the respiratory chain which is important for sperm capacitation and motility. Using the RNA-seq data, we also searched for the subset of 10 RNAs that explained the largest percentage of the variability for the semen quality phenotypes. To achieve this objective, we retrieved the genes that (i) were co-associated with traits at the SNP level by building an Associated Weight Matrix and finding correlations with PCIT, and (ii) also displayed co-abundant RNA levels (by PCIT). The resulting list of genes, together with the information of their correlated phenotypes, was used for network analysis. We identified a subset of 10 genes, which included EFHC1, ATP9A and THADA, that explained between 16 to 67% of the different sperm quality parameters.

Understanding the cell molecular changes occurring as a results of climatic circumstances is crucial in the current days in which climate change and global warming are one of the most serious challenges that living organisms have to face. Sperm are one of the mammals' cells most sensitive to heat, therefore evaluating the impact of seasonal changes in terms of its transcriptional activity can contribute to elucidate how these cells cope with heat stress events. We sequenced the total sperm RNA from 64 ejaculates, 28 collected in summer and 36 collected in autumn, from 40 Manchega rams. A highly rich transcriptome (11,896 different transcripts) with 90 protein coding genes that exceed an average number of 5000 counts were found. Comparing transcriptome in the summer and autumn ejaculates, 236 significant differential abundance genes were assessed, most of them (228) downregulated. The main functions that these genes are related to sexual reproduction and negative regulation of protein metabolic processes and kinase activity. Sperm response to heat stress supposes a drastic decrease of the transcriptional activity, and the upregulation of only a few genes related with the basic functions to maintain the organisms' homeostasis and surviving. Rams' spermatozoids carry remnant mRNAs which are retrospectively indicators of events occurring along the spermatogenesis process, including abiotic factors such as environmental temperature., This work has been supported by the RTA2013-00041 INIA project