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The microbiome plays a key role in homeostasis and health and it has been also linked to fertility and semen quality in several animal species including swine. Despite the more than likely importance of sperm bacteria on the boar's reproductive ability and the dissemination of pathogens and antimicrobial resistance genes, the high throughput characterization of the swine sperm microbiome remains scarce. We carried RNA-seq on 40 ejaculates each from a different Pietrain boar and found that a proportion of the sequencing reads did not map to the Sus scrofa genome. The current study aimed at using these reads not belonging to pig to carry a pilot study to profile the boar sperm bacterial population and its relation with 7 semen quality traits. We found that the boar sperm contains a broad population of bacteria. The most abundant phyla were Proteobacteria (39.1%), Firmicutes (27.5%), Actinobacteria (14.9%) and Bacteroidetes (5.7%). The predominant species contaminated sperm after ejaculation from soil, faeces and water sources (Bacillus megaterium, Brachybacterium faecium, Bacillus coagulans). Some potential pathogens were also found but at relatively low levels (Escherichia coli, Clostridioides difficile, Clostridium perfringens, Clostridium botulinum and Mycobacterium tuberculosis). We also identified 3 potential antibiotic resistant genes from E. coli against chloramphenicol, Neisseria meningitidis against spectinomycin and Staphylococcus aureus against linezolid. None of these genes were highly abundant. Finally, we classified the ejaculates into categories according to their bacterial features and semen quality parameters and identified two categories that significantly differed for 5 semen quality traits and 13 bacterial features including the genera Acinetobacter, Stenotrophomonas and Rhodobacter. Our results show that boar semen contains a bacterial community, including potential pathogens and putative antibiotic resistance genes, and that these bacteria may affect its reproductive performance., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The mammalian spermatozoon has a unique chromatin structure in which the majority of histones are replaced by protamines during spermatogenesis and a small fraction of nucleosomes are retained at specific locations of the genome. The sperm's chromatin structure remains unresolved in most animal species, including the pig. However, mapping the genomic locations of retained nucleosomes in sperm could help understanding the molecular basis of both sperm development and function as well as embryo development. This information could then be useful to identify molecular markers for sperm quality and fertility traits. Here, micrococcal nuclease digestion coupled with high throughput sequencing was performed on pig sperm to map the genomic location of mono- and sub-nucleosomal chromatin fractions in relation to a set of diverse functional elements of the genome, some of which were related to semen quality and early embryogenesis. In particular, the investigated elements were promoters, the different sections of the gene body, coding and non-coding RNAs present in the pig sperm, potential transcription factor binding sites, genomic regions associated to semen quality traits and repeat elements. The analysis yielded 25,293 and 4,239 peaks in the mono- and sub-nucleosomal fractions, covering 0.3% and 0.02% of the porcine genome, respectively. A cross-species comparison revealed positional conservation of the nucleosome retention in sperm between the pig data and a human dataset that found nucleosome enrichment in genomic regions of importance in development. Both gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and the identification of putative transcription factor binding motifs within the mono- and the sub- nucleosomal peaks showed enrichment for processes related to sperm function and embryo development. There was significant motif enrichment for Znf263, which in humans was suggested to be a key regulator of genes with paternal preferential expression during early embryogenesis. Moreover, enriched positional intersection was found in the genome between the mono-nucleosomal peaks and both the RNAs present in pig sperm and the RNAs related to sperm quality. There was no co-location between GWAS hits for semen quality in swine and the nucleosomal sites. Finally, the data evidenced depletion of mono-nucleosomes in long interspersed nuclear elements and enrichment of sub-nucleosomes in short interspersed repeat elements. These results suggest that retained nucleosomes in sperm could both mark regulatory elements or genes expressed during spermatogenesis linked to semen quality and fertility and act as transcriptional guides during early embryogenesis. The results of this study support the undertaking of ambitious research using a larger number of samples to robustly assess the positional relationship between histone retention in sperm and the reproductive ability of boars. [ABSTRACT FROM AUTHOR]

Background: Genetic pressure in animal breeding is sparking the interest of breeders for selecting elite boars with higher sperm quality to optimize ejaculate doses and fertility rates. However, the molecular basis of sperm quality is not yet fully understood. Our aim was to identify candidate genes, pathways and DNA variants associated to sperm quality in swine by analysing 25 sperm-related phenotypes and integrating genome-wide association studies (GWAS) and RNA-seq under a systems biology framework. Results: By GWAS, we identified 12 quantitative trait loci (QTL) associated to the percentage of head and neck abnormalities, abnormal acrosomes and motile spermatozoa. Candidate genes included CHD2, KATNAL2, SLC14A2 and ABCA1. By RNA-seq, we identified a wide repertoire of mRNAs (e.g. PRM1, OAZ3, DNAJB8, TPPP2 and TNP1) and miRNAs (e.g. ssc-miR-30d, ssc-miR-34c, ssc-miR-30c-5p, ssc-miR-191, members of the let-7 family and ssc-miR-425-5p) with functions related to sperm biology. We detected 6128 significant correlations (P-value ≤ 0.05) between sperm traits and mRNA abundances. By expression (e)GWAS, we identified three trans-expression QTL involving the genes IQCJ, ACTR2 and HARS. Using the GWAS and RNA-seq data, we built a gene interaction network. We considered that the genes and interactions that were present in both the GWAS and RNA-seq networks had a higher probability of being actually involved in sperm quality and used them to build a robust gene interaction network. In addition, in the final network we included genes with RNA abundances correlated with more than four semen traits and miRNAs interacting with the genes on the network. The final network was enriched for genes involved in gamete generation and development, meiotic cell cycle, DNA repair or embryo implantation. Finally, we designed a panel of 73 SNPs based on the GWAS, eGWAS and final network data, that explains between 5% (for sperm cell concentration) and 36% (for percentage of neck abnormalities) of the phenotypic variance of the sperm traits. Conclusions: By applying a systems biology approach, we identified genes that potentially affect sperm quality and constructed a SNP panel that explains a substantial part of the phenotypic variance for semen quality in our study and that should be tested in other swine populations to evaluate its relevance for the pig breeding sector. [ABSTRACT FROM AUTHOR]

Understanding the molecular basis of cell function and ultimate phenotypes is crucial for the development of biological markers. With this aim, several RNA-seq studies have been devoted to the characterization of the transcriptome of ejaculated spermatozoa in relation to sperm quality and fertility. Semen quality follows a seasonal pattern and decays in the summer months in several animal species. The aim of this study was to deeply profile the transcriptome of the boar sperm and to evaluate its seasonal changes. We sequenced the total and the short fractions of the sperm RNA from 10 Pietrain boars, 5 collected in summer and 5 five sampled in winter, and identified a complex and rich transcriptome with 4,436 coding genes of moderate to high abundance. Transcript fragmentation was high but less obvious in genes related to spermatogenesis, chromatin compaction and fertility. Short non-coding RNAs mostly included piwi-interacting RNAs, transfer RNAs and microRNAs. We also compared the transcriptome of the summer and the winter ejaculates and identified 34 coding genes and 7 microRNAs with a significantly distinct distribution. These genes were mostly related to oxidative stress, DNA damage and autophagy. This is the deepest characterization of the boar sperm transcriptome and the first study linking the transcriptome and the seasonal variability of semen quality in animals. The annotation described here can be used as a reference for the identification of markers of sperm quality in pigs. [ABSTRACT FROM AUTHOR]