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Additional file 1: Table S1. Eigenvalues of each parameter in the three PCs for bull sperm head morphometry found in sperm migrated or not migrated by thermotaxis.

Additional file 5: Fig. S3. Boxplots showing the distribution of the morphometric variables of the four subpopulations along with mean comparison P-values obtained by the t-test.

Additional file 2: Table S2. Mean values (± S.D.) of each morphometric parameter corresponding to different SPs from bull sperm head morphometry found in sperm migrated or not migrated by thermotaxis.

Additional file 3: Fig. S1. Density plots showing the distribution of the 8 morphometric variables in both migrated and not-migrated spermatozoa after thermotaxis.

Additional file 4: Fig. S2. Distribution of sperm subpopulations according to their PC values.

16 Pág., Spermatogenic cells express more alternatively spliced RNAs than most whole tissues; however, the regulation of these events remains unclear. Here, we have characterized the function of a testis-specific IQ motif-containing H gene (Iqch) using a mutant mouse model. We found that Iqch is essential for the specific expression of RNA isoforms during spermatogenesis. Using immunohistochemistry of the testis, we noted that Iqch was expressed mainly in the nucleus of spermatocyte and spermatid, where IQCH appeared juxtaposed with SRRM2 and ERSP1 in the nuclear speckles, suggesting that interactions among these proteins regulate alternative splicing (AS). Using RNA-seq, we found that mutant Iqch produces alterations in gene expression, including the clear downregulation of testis-specific lncRNAs and protein-coding genes at the spermatid stage, and AS modifications - principally increased intron retention - resulting in complete male infertility. Interestingly, we identified previously unreported spliced transcripts in the wild-type testis, while mutant Iqch modified the expression and use of hundreds of RNA isoforms, favouring the expression of the canonical form. This suggests that Iqch is part of a splicing control mechanism, which is essential in germ cell biology., This study was funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (PID2021-122507OB-I00 and PID2020-117491GB-I00) and the European Union NextGenerationEU/PRTR. P.N.-L. was supported by a pre-doctoral fellowship from the Ministerio de Ciencia e Innovación (PRE2019-088813) and M.L. was supported by a Juan de la Cierva postdoctoral contract (FJC2019-040385-I) from the Ministerio de Ciencia e Innovación. Open access funding provided by Consejo Superior de Investigaciones Cientıficas. Deposited in PMC for immediate release.

11 Pág. Departamento de Reproducción Animal​, [Background], Sperm migration by thermotaxis is a guidance mechanism that operates along the oviduct and it has proved to be a valid method for selecting spermatozoa with low DNA fragmentation (SDF) in mice, humans, and stallions. This study aimed to analyse if bull spermatozoa could be selected by thermotaxis and to assess their quality in terms of SDF as well as determine the presence of a specific sperm subpopulation based on sperm morphometry and assess their fertilizing capacity by ICSI., [Methods], We used frozen-thawed sperm from 6 bulls and sperm selection by thermotaxis was performed with TALP medium supplemented with 25 mmol/L of HEPES and 5 mmol/L of caffeine. In these conditions, sperm selection was achieved, obtaining a net thermotaxis of 3.6%. Subsequently, we analysed the SDF of the migrated and not-migrated spermatozoa using the neutral COMET assay, and we evaluated the size of the sperm head using Hemacolor® staining with Motic Images Plus 3 software. Additionally, migrated and not-migrated spermatozoa by thermotaxis were used to fertilize bovine in vitro matured (IVM) oocytes by ICSI, a very inefficient procedure in cattle that is only successful when the oocyte is artificially activated., [Results], The results showed lower SDF (χ², P < 0.001, 13.3% reduction, n = 8) and lower head size parameters (length and width, P < 0.01; and perimeter and area, P < 0.001; n = 4) in those spermatozoa migrated in comparison to those not-migrated. The distribution of sperm subpopulations structure varied between groups, highlighting cluster 2, characterized by spermatozoa with small head size, and high ellipticity and elongated heads, as the most abundant in the thermotaxis migrated group. When performed ICSI (without oocyte artificial activation) with the thermotactic sperm, the blastocyst rate was 32.2% ± 9.3% in the group microinjected with the thermotactic spermatozoa vs. 8.3% ± 7.8% in the group of not-migrated sperm (χ², P < 0.05)., [Conclusion], Our results showed that bull sperm selection by thermotaxis has a much higher DNA integrity, small and elongated head size parameters, and different sperm subpopulation structure than the not-selected spermatozoa. Additionally, we evidenced that thermotactic spermatozoa improve ICSI success rates., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This research was funded by the Spanish Ministry of Science and Innovation-MCIN (RTI2018-093548-B-I00 and PID2021-122507OB-I00 to A. Gutierrez-Adan and PID2019-111641RB-I00 to D. Rizos, funded by MCIN/ AEI/10.13039/501100011033/ and European Union “NextGenerationEU”/PRTR). M.L. was supported by a Juan de la Cierva postdoctoral contract (FJC2019- 040385‐I) from the MCIN. S.R.D. was supported by a “Doctorados Industriales 2018” fellowship of Comunidad de Madrid (IND2018/BIO‐9610). R.M. and P.N.L. were supported by FPI scholarships from the MCIN (PRE2020-094452 and PRE2019-088813 respectively).

11 Pàg. Departamento de Reproducción animal., This report reviews current knowledge of sperm metabolomics analysis using proton nuclear magnetic resonance spectroscopy (1 H-NMR) with particular emphasis on human and farm animals. First, we present the benefits of NMR over other techniques to identify sperm metabolites and then describe the specific methodology required for NMR sperm analysis, stressing the importance of analyzing metabolites extracted from both the hydrophilic and lipophilic phases. This is followed by a description of advances produced to date in the use of NMR to diagnose infertility in humans and to identify metabolic differences among the sperm of mammalian herbivore, carnivore, and omnivore species. This last application of NMR mainly seeks to explore the possible use of lipids to fuel sperm physiology, contrary to previous theories that glycolysis and oxidative phosphorylation (OXPHOS) are the only sources of sperm energy. This review describes the use of NMR to identify sperm and seminal plasma metabolites as possible indicators of semen quality, and to examine the metabolites needed to maintain sperm motility, induce their capacitation, and consequently, to predict animal fertility., This research was funded by grant RTI2018–093548-BI00 from the Spanish Ministry of Science and Innovation (MICINN). M.L. is supported by a Juan de la Cierva postdoctoral contract (FJC2019-040385-I) from the MICINN. S.R.D. is supported by a “Doctorados Industriales 2018” fellowship of Comunidad de Madrid (IND2018/BIO-9610).

12 Pág. Departamento de Reproducción animal, Heart development requires a precise temporal regulation of gene expression in cardiomyoblasts. Therefore, the transcriptional changes in differentiating cells can lead to congenital heart diseases. Although the genetic mutations underlie most of these alterations, exposure to environmental contaminants, such as bisphenol A (BPA), has been recently considered as a risk factor as well. In this study we investigated the genotoxic and epigenotoxic effects of BPA throughout cardiomyocyte differentiation. H9c2 cells (rat myoblasts) were exposed to 10 and 30 μM BPA before and during the last two days of cardiac-driven differentiation. Then, we have analysed the phenotypic and molecular modifications (at transcriptional, genetic and epigenetic level). The results showed that treated myoblasts developed a skeletal muscle cell-like phenotype. The transcriptional changes induced by BPA in genes codifying proteins involved in heart differentiation and function depend on the window of exposure to BPA. The exposure before differentiation repressed the expression of heart transcription factors (Hand2 and Gata4), whereas exposure during differentiation reduced the expression of cardiac-specific genes (Tnnt2, Myom2, Sln, and Atp2a1). Additionally, significant effects were observed regarding DNA damage and histone acetylation levels after the two periods of BPA exposure: in cells exposed to the toxicant the percentage of DNA repair foci (formed by the co-localization of γH2AX and 53BP1) increased in a dose-dependent manner, whereas the treatment with the toxicant triggered a decrease in the epigenetic marks H3K9ac and H3K27ac. Our in vitro results reveal that BPA seriously interferes with the process of cardiomyocyte differentiation, which could be related to the reported in vivo effects of this toxicant on cardiogenesis., This work was funded by the Spanish Ministry of Economy and Competitiveness (Project AGL2014-53167-C3-3-R). We would like to thank Dr. Beatriz Martín-Fernández for helping us with the cell line establishment.

20 Pág. Departamento de Reproducción animal, Environmental pollution is becoming one of the major concerns of society. Among the emerging contaminants, endocrine-disrupting chemicals (EDCs), a large group of toxicants, have been the subject of many scientific studies. Besides the capacity of these compounds to interfere with the endocrine system, they have also been reported to exert both genotoxic and epigenotoxic effects. Given that spermatogenesis is a coordinated process that requires the involvement of several steroid hormones and that entails deep changes in the chromatin, such as DNA compaction and epigenetic remodelling, it could be affected by male exposure to EDCs. A great deal of evidence highlights that these compounds have detrimental effects on male reproductive health, including alterations to sperm motility, sexual function, and gonad development. This review focuses on the consequences of paternal exposure to such chemicals for future generations, which still remain poorly known. Historically, spermatozoa have long been considered as mere vectors delivering the paternal haploid genome to the oocyte. Only recently have they been understood to harbour genetic and epigenetic information that plays a remarkable role during offspring early development and long-term health. This review examines the different modes of action by which the spermatozoa represent a key target for EDCs, and analyses the consequences of environmentally induced changes in sperm genetic and epigenetic information for subsequent generations., This work was funded by the Spanish Ministry of Economy and Competitiveness (Project AGL2014-53167-C3-3-R; PhD Grant BES-2015-071885).