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Introduction: The future health of the offspring can be influenced by longstanding maternal anxiety and depression disorders during pregnancy. The present study aimed to explore the effect of psychiatric disorders during pregnancy on placental epigenetics. Methods: We measured DNA methylation patterns in term-placentas of women either suffering longstanding anxiety and depression symptoms (Index group, with overt symptoms), or a healthy population (Control, none/ only mild symptoms). Whole genome DNA methylation profiling was performed using the TruSeq (R) Methyl Capture EPIC Library Prep Kit (Illumina, San Diego, CA, USA) for library preparation and NGS technology for genomic DNA sequencing.Results: The results of high-throughput DNA methylation analysis revealed that the Index group had differential DNA methylation at epigenome-wide significance (p < 0.05) in 226 genes in the placenta. Targeted enrichment analyses identified hypermethylation of genes associated with psychiatric disorders (BRINP1, PUM1), and ion homeostasis (COMMD1), among others. The ECM (extracellular matrix)-receptor interaction pathway was significantly dysregulated in the Index group compared to the Control. In addition, DNA methylation/mRNA integration analyses revealed that four genes with key roles in neurodevelopment and other important processes (EPB41L4B, BMPR2, KLHL18, and UBAP2) were dysregulated at both, DNA methylation and transcriptome levels in the Index group compared to Control.Discussion: The presented results increase our understanding of how maternal psychiatric disorders may affect the newborn through placental differential epigenome, suggesting DNA methylation status as a biomarker when aiming to design new preventive techniques and interventions., Funding Agencies|Medical Research Council of Southeast Sweden [392061, 472721, 891663]; European Union [2017-00946]; Swedish Research Council FORMAS, Stockholm, Sweden [2019-00288]; [661011]

Mammalian follicles are constituted of a complex structure composed of several layers of granulosa cells surrounding the oocyte and of theca cells that reside beneath its basement membrane. During folli-culogenesis, granulosa cells separate into two anatomically and functionally distinct sub-types; the mural cells lining the follicle wall and the oocyte-surrounding cumulus cells, i.e. those in intimate metabolic contact with the oocyte. The cumulus cells connecting with the oocyte have trans-zonal cytoplasmic projections which, penetrating the zona pellucida, form the cumulus-oocyte complex. The connections through gap junctions allow the transfer of small molecules between oocyte and cumulus cells, such as ions, metabolites, and amino acids necessary for oocyte growth, as well as small regulatory molecules that control oocyte development. The bi-directional communication between the oocyte and cumulus cells is crucial for the development and functions of both cell types. Our current knowledge of the relationship between the oocyte and its surrounding cumulus cells continues to change as we gain a greater understanding of factors regulating oocyte development and folliculogenesis. This review will mainly focus on the reciprocal interaction between oocytes and cumulus cells during the latter stages of follicle development i.e. through antral development to peri-ovulatory events including oocyte maturation, expansion, and degradation of the cumulus matrix.(c) 2023 Published by Elsevier Inc., Funding Agencies|JSPS Postdoctoral Fellowship for Research in Japan [JSPS/Ref: PE21723]

During pregnancy, the immune system is modified to allow developmental tolerance of the semi-allogeneic fetus and placenta to term. Pregnant women suffering from stress, anxiety, and depression show dysfunctions of their immune system that may be responsible for fetal and/or newborn disorders, provided that placental gene regulation is compromised. The present study explored the effects of maternal chronic self-perceived stress, anxiety, and depression during pregnancy on the expression of immune-related genes and pathways in term placenta. Pregnancies were clinically monitored with the Beck Anxiety Inventory (BAI) and Edinburgh Postnatal Depression Scale (EPDS). A cutoff threshold for BAI/EPDS of 10 divided patients into two groups: Index group (>10, n = 11) and a Control group (<10, n = 11), whose placentae were sampled at delivery. The placental samples were subjected to RNA-Sequencing, demonstrating that stress, anxiety, and depression during pregnancy induced a major downregulation of placental transcripts related to immune processes such as T-cell regulation, interleukin and cytokine signaling, or innate immune responses. Expression differences of main immune-related genes, such as CD46, CD15, CD8 alpha & beta ILR7 alpha, and CCR4 among others, were found in the Index group (P < 0.05). Moreover, the key immune-like pathway involved in humoral and cellular immunity named "Primary immunodeficiency" was significantly downregulated in the Index group compared with Controls. Our results show that mechanisms ruling immune system functions are compromised at the maternal-fetal interface following self-perceived depressive symptoms and anxiety during pregnancy. These findings may help unveil mechanisms ruling the impact of maternal psychiatric symptoms and lead to new prevention/intervention strategies in complicated pregnancies. Summary Sentence Mechanisms ruling immune system functions are compromised at the maternal-fetal interface followin

Artificial insemination (AI) for pigs relies on liquid storage of extended semen at 17 degrees C, which preserves sperm quality and ensures its fertilizing capacity. Routine quality controls include the evaluation of sperm motility, viability and capacitation status. The physiological functions of all these features depend on transmembrane aquaporins (AQPs), proteins playing key roles in osmoadaptation. In this study, we made a relative quantification, using RT-qPCR, of the mRNA of several sperm AQPs in AI-liquid semen doses before and after a 48-hr incubation period, aiming to determine possible quantitative compromising expression changes during the process that could serve as a diagnostic tool. Our results showed a decrease in classical sperm motility variables (total and progressive motility and velocity) and sperm viability after 48-hr storage, whereas capacitation status increased overtime. mRNA expression increased in the orthodox AQP4 and AQP6 after 48-hr incubation, relative to control (0 hr) and 24-hr time-points. Moreover, mRNA expression of aquaglyceroporins AQP3, AQP7 and AQP10 was higher after 48-hr incubation, confirmed by AQP7-protein validation using Western blot. Our results indicate that expression levels of AQPs-mRNA can change in ejaculated pig spermatozoa under conditions of ex-vivo incubation that could modify sperm homeostasis, suggesting it could eventually become a relevant molecular biomarker to assess the efficiency of liquid storage of pig semen., Funding Agencies|Ministerio de Ciencia, Innovacion y Universidades; Svenska Forskningsradet Formas

Simple Summary The present study searched for the presence and abundance of porcine spermatozoa small RNA sequences (microRNAs) that have the potential to alter gene expression patterns. Four different sperm sources were compared: spermatozoa from three different sections of the ejaculate and from the caudal epididymis, also classed as spermatozoa from higher (HF) or lower (LF) fertility boars. Sperm miRNAs were compared using high-output small RNA sequencing. We identified five sperm miRNAs not previously reported in pigs. Differences in abundance of four miRNAs known to affect the expression of genes with key roles in fertility were related to boar fertility. These miRNAs could be used as fertility markers in artificial insemination programs. MicroRNAs (miRNAs) are short non-coding RNAs (20-25 nucleotides in length) capable of regulating gene expression by binding -fully or partially- to the 3-UTR of target messenger RNA (mRNA). To date, several studies have investigated the role of sperm miRNAs in spermatogenesis and their remaining presence toward fertilization and early embryo development. However, little is known about the miRNA cargo in the different sperm sources and their possible implications in boar fertility. Here, we characterized the differential abundance of miRNAs in spermatozoa from the terminal segment of the epididymis and three different fractions of the pig ejaculate (sperm-peak, sperm-rich, and post-sperm rich) comparing breeding boars with higher (HF) and lower (LF) fertility after artificial insemination (AI) using high-output small RNA sequencing. We identified five sperm miRNAs that, to our knowledge, have not been previously reported in pigs (mir-10386, mir-10390, mir-6516, mir-9788-1, and mir-9788-2). Additionally, four miRNAs (mir-1285, mir-92a, mir-34c, mir-30), were differentially expressed among spermatozoa sourced from ejaculate fractions and the cauda epididymis, and also different abundance was found between HF and LF groups in mir

Uterine homeostasis is maintained after mating by eliminating pathogens, foreign cells, and proteins by a transient inflammation of the uterus. Such inflammation does not occur in the oviductal sperm reservoir (utero-tubal junction, UTJ), colonized by a population of potentially fertile spermatozoa before the inflammatory changes are triggered. Semen entry (spermatozoa and/or seminal plasma) modifies the expression of regulatory genes, including cell proliferation and differentiation-related transcripts. Considering pigs display a fractionated ejaculation, this study aims to determine whether different ejaculate fractions differentially modulate cell proliferation and differentiation-related transcripts in the sow reproductive tract during the peri-ovulatory stage. Using species-specific microarray analyses, the differential expression of 144 cell proliferation and differentiation-related transcripts was studied in specific segments: cervix (Cvx), distal and proximal uterus (DistUt, ProxUt), UTJ, isthmus (Isth), ampulla (Amp), and infundibulum (Inf) of the peri-ovulatory sow reproductive tract in response to semen and/or seminal plasma cervical deposition. Most mRNA expression changes were induced by mating. In addition, while mating upregulates the fibroblast growth factor 1 (FGF1, p-value DistUt = 0.0007; ProxUt = 0.0253) transcript in the endometrium, both its receptor, the fibroblast growth factor receptor 1 (FGFR1, p-value DistUt = 2.14 e(-06); ProxUt = 0.0027; UTJ = 0.0458) transcript, and a potentiator of its biological effect, the fibroblast growth factor binding protein 1 (FGFBP1), were downregulated in the endometrium (p-value DistUt = 0.0068; ProxUt = 0.0011) and the UTJ (p-value UTJ = 0.0191). The FGFBP1 was downregulated in the whole oviduct after seminal depositions (p-value Isth = 0.0007; Amp = 0.0007; Inf = 6.87 e(-05)) and, interestingly, FGFR1 was downregulated in the endometrium in the absence of semen (p-value DistUt = 0.0097; ProxUt = 0.0456). I, Funding Agencies|Research Council FORMAS, Stockholm [2017-00946, 2019-00288]; FEDER funds (EU)European Commission; Government of CataloniaAGAURAgencia de Gestio DAjuts Universitaris de Recerca Agaur (AGAUR)Generalitat de Catalunya; European Social FundEuropean Social Fund (ESF) [2018 FI_B 00236]; Government of Spain-Ministry of Education, Culture and Sports [FPU15/06029]; [PID2019-108320RJ-I00]; [IJCI-2015-24380]; [MCIN/AEI/10.13039/501100011033]

Simple Summary The detection of sub-fertile boars has been a difficult task, and despite their prevalence being low, its impact is very significant because it implies economic drawbacks for artificial insemination (AI) centers and farms. Unfortunately, some crucial reproductive processes fall beyond the routine analysis performed in the porcine model, such as sperm capacitation, which is a necessary event for fertilization. A synergistic action of bicarbonate (HCO3-) with calcium (Ca2+) is needed to achieve capacitation. The transport of Ca2+ is mediated by CatSper channels and Canonical Transient Potential Channels (TRPC). We quantified mRNA transcripts of different subunits of CatSper (beta, gamma and delta) and TRPC (1, 3, 4, 6 and 7) before and after in vitro capacitation by HCO3- ions. Our results showed that in vitro capacitation using HCO3- increases the relative abundance of mRNA transcripts of almost all subunits of Ca2+ channels, except CatSper-delta and TRPC1, which were significantly reduced. More studies are needed to elucidate the specific roles of the TRPC channels at a physiological and functional level. Sperm capacitation is a stepwise complex biochemical process towards fertilization. It includes a crucial early calcium (Ca2+) transport mediated by CatSper channels and Canonical Transient Potential Channels (TRPC). We studied the relative abundance of mRNA transcripts changes of the CatSper beta, gamma and delta subunits and TRPC-channels 1, 3, 4, 6 and 7 in pig spermatozoa, after triggering in vitro capacitation by bicarbonate ions at levels present in vivo at the fertilization site. For this purpose, we analyzedfive5 ejaculate pools (from three fertile adult boars) before (control-fresh samples) and after in vitro exposure to capacitation conditions (37 mM NaHCO3, 2.25 mM CaCl2, 2 mM caffeine, 0.5% bovine serum albumin and 310 mM lactose) at 38 degrees C, 5% CO2 for 30 min. In vitro capacitation using bicarbonate elicits an increase in the relati, Funding Agencies|Research Council FORMAS, Stockholm [2017-00946, 2019-00288]; MCIN/AEI (Spain) [PID2019-108320RJ-I00, IJCI-2015-24380]; FEDER funds (EU)European Commission [PID2019-108320RJ-I00, IJCI-2015-24380]; MECD ("Ministerio de Educacion, Cultura y Deporte"), Madrid, Spain [16/05745]; Spanish Ministry of Science, Innovation and Universities (MICINN), Madrid, Spain [RTI2018-095183-B-I00]

The Superfine Open Pulled Straw (SOPS) system is the most commonly used method for vitrification of pig embryos. However, this system only allows the vitrification of four to seven embryos per straw. In this study, we investigated the effectiveness of the open (OC) and closed (CC) Cryotop (R) systems to simultaneously vitrify a larger number of porcine embryos. Morulae, early blastocysts and full blastocysts were vitrified with the open Cryotop (R) (n = 250; 20 embryos per device) system, the closed Cryotop (R) (n = 158; 20 embryos per device) system and the traditional superfine open pulled straw (SOPS; n = 241; 4-7 embryos per straw) method. Fresh embryos from each developmental stage constituted the control group (n = 132). Data expressed as percentages were compared with the Fishers exact test. The Kruskal-Wallis test was used to analyze the effect of the different vitrification systems on the embryo quality parameters and two-by-two comparisons were accomplished with the Mann-Whitney U test. Differences were considered statistically significant when p < 0.05. Vitrified and control embryos were incubated for 24 h and examined for viability and quality. At the warming step, the embryo recovery rate for the CC system was 51%, while all embryos were recovered when using OC and SOPS. There were no differences between the vitrification and control groups in the postwarming viability of full blastocysts. In contrast, morulae and early blastocysts that were vitrified-warmed with the SOPS system had lower viability (p < 0.01) compared to those from the OC, CC and control groups. The embryonic viability was similar between the OC and control groups, regardless of the developmental stage considered. Moreover, the embryos from the OC group had comparable total cell number and cells from the inner cell mass and apoptotic index than the controls. In conclusion, the OC system is suitable for the simultaneous vitrification of 20 porcine embryos at different devel

Cloned and transgenic pigs are relevant human disease models and serve as potential donors for regenerative medicine and xenotransplantation. These technologies demand oocytes and embryos of good quality. However, the current protocols for in vitro production (IVP) of pig embryos give reduced blastocyst efficiency and embryo quality compared to in vivo controls. This is likely due to culture conditions jeopardizing embryonic homeostasis including the effect of reactive oxygen species (ROS) influence. In this study, the antioxidant melatonin (1 nM) in the maturation medium, fertilization medium, or both media was ineffective in enhancing fertilization or embryonic development parameters of in vitro fertilized oocytes. Supplementation of melatonin in the fertilization medium also had no effect on sperm function. In contrast, the addition of melatonin to the embryo culture medium accelerated the timing of embryonic development and increased the percentages of cleaved embryos and presumed zygotes that developed to the blastocyst stage. Furthermore, it increased the number of inner mass cells and the inner mass cell/total cell number ratio per blastocyst while increasing intracellular glutathione and reducing ROS and DNA damage levels in embryos. Contrarily, the addition of melatonin to the embryo culture medium had no evident effect on in vivo-derived embryos, including the developmental capacity and the quality of in vivo-derived 4-cell embryos or the percentage of genome-edited in vivo-derived zygotes achieving the blastocyst stage. In conclusion, exogenous melatonin in the embryo culture medium enhances the development and quality of in vitro-derived embryos but not in in vivo-derived embryos. Exogenous melatonin is thus recommended during embryo culture of oocytes matured and fertilized in vitro for improving porcine IVP efficiency., Funding Agencies|MCIN/AEI; "ERDF A way of making Europe", Madrid, Spain [RTI2018-093525-B-I00]; European Union [891663]; Fundacion Seneca, Murcia, Spain [19892/GERM/15]; Swedish Research Council FORMAS, Stockholm, Sweden [2019-00288]

The aims of this study were to investigate the effects of different equilibration times with cryoprotectants on viability and metaphase plate morphology of vitrified-warmed porcine mature oocytes (Experiment 1) and to evaluate the effects of supplementation with 10(-9) M melatonin during in vitro maturation on these parameters (Experiment 2). In Experiment 1, 2,392 mature oocytes were vitrified using different equilibration times of oocytes with cryoprotectants (3, 10, 15, 20, 30, 40, 60 and 80 min). Fresh oocytes matured in vitro for 44 hr (n = 509) were used as controls. In Experiment 2, a total of 573 COCs were used. COCs were matured with 10(-9) M melatonin supplementation or without melatonin (control). Some oocytes from each group were vitrified with a 60-min equilibration time with cryoprotectants according to the results of Experiment 1. The remaining oocytes from each maturation group were used as fresh control groups. In both experiments, oocytes were stained with 2 ,7 -dichlorodihydrofuorescein diacetate and Hoechst 33342 to assess viability and metaphase plate morphology, respectively. Vitrification and warming affected (p < .01) oocyte viability compared with controls, which were all viable after 44 hr of IVM. In Experiment 1, the longer the equilibration time with cryoprotectants, the higher the viability. Oocytes equilibrated for 60 and 80 min had the highest (p < .05) viability and similar metaphase plate characteristics to the fresh control oocytes. In Experiment 2, supplementation with melatonin during in vitro maturation had no effect on oocyte viability or metaphase plate morphology of vitrified-warmed oocytes. In conclusion, under our experimental conditions, vitrified porcine mature oocytes equilibrated with cryoprotectants for 60 or 80 min exhibited the highest viability and similar metaphase plate characteristics to fresh controls. Furthermore, supplementation with 10(-9) M melatonin during in vitro maturation had no effect on these par, Funding Agencies|Madrid, Spain [MCIN/AEI/10.13039/501100011033]; ERDF A way of making Europe, Madrid, Spain; Fundacion Seneca, Murcia, Spain [19892/GERM/15]

Successful establishment of pregnancy includes the achievement of a state of immune tolerance toward the embryos (and placenta), where the well-coordinated maternal immune system is capable of recognizing conceptus antigens while maintaining maternal defense against pathogens. In physiological pregnancies, following natural mating or artificial insemination (AI), the maternal immune system is exposed to the presence of hemi-allogeneic embryos, that is, embryos containing maternal self-antigens and foreign antigens from the paternal side. In this scenario, the hemi-allogeneic embryo is recognized by the mother, but the immune system is locally modified to facilitate embryo implantation and pregnancy progression. Pig allogeneic pregnancies (with embryos containing both paternal and maternal material foreign to the recipient female), occur during embryo transfer (ET), with conspicuously high rates of embryonic death. Mortality mainly occurs during the peri-attachment phase, suggesting that immune responses to allogeneic embryos are more complex and less efficient, hindering the conceptuses to survive to term. Reaching a similar maternal tolerance as in conventional breeding would render ET successful. The present review critically summarizes mechanisms of maternal immune recognition of pregnancy and factors associated with impaired maternal immune response to the presence of allogeneic embryos in the porcine species., Funding Agencies|Svenska Forskningsradet Formas; H2020 Marie Sklodowska-Curie Actions

The epididymis is responsible for peripheral immune tolerance of maturing spermatozoa even though these have xeno-antigens foreign to the male and female immune system. The epididymis also produces factors required for fertilization and serves as a sperm repository until the time of ejaculation. These reproduction-relevant epididymal functions occur in the mesonephros-derived duct-system that is composed of absorptive and secretory epithelial cells with the capacity for merocrine and apocrine secretion of proteins, antioxidative- and electrolyte/pH-regulating enzymes and small, non-coding RNAs (sncRNAs), many stored in epididymosomes for sperm adhesion and long-lasting modifications of sperm functions. This paper provides a review summary of current and new knowledge of how the boar epididymis affects the quality of spermatozoa in the ejaculate of breeding boars. There is a particular focus on sperm maturation, survival, function and the role of signaling to the female immune system in fertility modulation. Furthermore, aspects related to the ductus epithelial contributions regarding electrolyte control, protein production, release of epididymosomes that contain sncRNAs are emphasized as are novel associations with fertility of the male, sperm quiescence during storage in the cauda epididymis, and on changes occurring in sperm subsequent to ejaculation., Funding Agencies|Research Council FORMAS, Stockholm [2017-00946, 2019-00288]; MICINN [PID2020-113493RB-I00]; Madrid, Spain; European Union, Brussels, Belgium [891663]

The combination of estrus synchronization and superovulation treatments introduces molecular modifications whose effects are yet to be disclosed. Here, reproductive parameters and gene expression changes in ovaries and endometrium were explored on day 6 after artificial insemination (AI), when synthetic progestin altrenogest (ALT) was combined with gonadotropins. Sows were administered ALT for 7 d beginning on the day of weaning and superovulated with equine chorionic gonadotropin (eCG) 24 h later and human chorionic gonadotropins (hCG) at the onset of estrus (SS-7 group; n = 6). The controls were either superovulated sows with eCG 24 h postweaning and hCG at the onset of estrus (SC group; n = 6) or sows with postweaning spontaneous estrus (NC group; n = 6). Ovary examination and embryo and tissue collection were performed in all sows via laparotomy on day 6 post-AI. RNA-Seq was conducted to analyze differentially expressed genes (DEGs) between groups. Statistical analysis of the reproductive parameters was conducted with ANOVA and Tukey post hoc tests. DEGs were analyzed with an ANOVA (fold changes >= 2 or <= 2, P value <0.05). Hormonal treatments almost doubled (P < 0.03) the number of corpora lutea (39.8 +/- 10.2 and 38.3 +/- 11.1 in SS-7 and SC sows, respectively) compared with that in the NC group (23.1 +/- 3.8). In contrast, embryo viability significantly decreased (P < 0.003) in response to SS-7 treatment (75.1% +/- 15.2%) compared to SC and NC groups (93.8 +/- 7.6% and 91.8 +/- 6.9%, respectively). RNA-Seq analyses revealed 675 and 1,583 DEGs in the SS-7 group compared to both SC and NC groups in endometrial and ovarian samples, respectively. Interestingly, many genes with key roles in the Wnt/beta-catenin and Notch signaling pathways were differentially expressed in SS-7 sows relative to SC and NC groups (e.g., Ctnnb1, Myc, Gli3, Scyl2, Ccny, Daam1, Ppm1n, Rbpj, and Usp8). A key finding in this study was the downregulation, Funding Agencies|Ministerio de Ciencia e Inovacion/Agencia Estatal de Investigacion/European Regional Development Fund [RTI2018-093525-B-I00]; Seneca Foundation [19892]; Swedish Research Council FORMAS Stockholm, Sweden [2019-00288]; European Unions Horizon 2020 research and innovation program under the MSCA [891663]

Pig pregnancy succeeds thanks to a well-coordinated system ruling both maternal immune activation and embryonic antigen tolerance. In physiological pregnancies, the maternal immune system should tolerate the presence of hemi-allogeneic conceptuses from the pre-implantation phase to term, while maintaining maternal defence against pathogens. Allogeneic pregnancies, as after embryo transfer (ET), depict high embryo mortality during the attachment phase, calling for studies of the dynamic modifications in immune processes occurring at the maternal-foetal interface, for instance, of interferon (IFN)-stimulated genes (ISGs). These ISGs are generally activated by IFN secreted by the conceptus during the process of maternal recognition of pregnancy (MRP) and responsible for recruiting immune cells to the site of embryo attachment, thus facilitating cell-antigen presentation and angiogenesis. We performed RNA-Seq analysis in peri-implantation (days 18 and 24) endometrial samples retrieved from artificially inseminated sows (hemi-allogeneic embryos (HAL) group) or sows subjected to ET (allogeneic embryos (AL) group) to monitor alterations of gene expression that could be jeopardising early pregnancy. Our results showed that endometrial gene expression patterns related to immune responses differed between hemi- or allogeneic embryo presence, with allogeneic embryos apparently inducing conspicuous modifications of immune-related genes and pathways. A decreased expression (P < 0.05; FC < -2) of several interferon ISGs, such as CXCL8, CXCL10, IRF1, IRF9, STAT1, and B2M, among others was detected in the endometrium of sows carrying allogeneic embryos on day 24 of pregnancy. This severe downregulation of ISGs in allogeneic pregnancies could represent a failure of ET-embryos to signal IFN to the endometrium to warrant the development of adequate immunotolerance mechanisms to facilitate embryo development, thus contributing to elevated embryo death. (C) 2022 The Author, Funding Agencies|Swedish Research Council FORMAS, Stockholm, Sweden [2019-00288]; European Unions Horizon 2020 research and innovation programme under the MSCA [891663]

Oxidative stress unbalance is a major factor causing impairment of sperm function and, ultimately, sperm death. In this study, we identified transcriptomic and proteomic markers for oxidative-related protectors from the generation of reactive oxygen species (ROS) in spermatozoa from breeding boars with documented high- or lowfertility. Particular attention was paid to glutathione peroxidases, and to transcripts related to DNA stabilization and compaction, as protamine and transition proteins. mRNA cargo analysis was performed using porcinespecific micro-arrays (GeneChip (R) miRNA 4.0 and GeneChip (R) Porcine Gene 1.0 ST) and qPCR validation. Differences between fertility-classed boars were ample among biomarkers; some upregulated only at protein level (catalase (CAT), superoxide dismutase 1 (SOD1) and glutathione proteins), or only at the mRNA level (ATOX1, Antioxidant Protein 1). In addition, protamines 2 and 3, essential for sperm DNA condensation and also transition proteins 1 and 2 (TNP1 and TNP2), required during histone-to-protamine replacement, were overexpressed in spermatozoa from high-fertile boars. This up-regulation seems concerted to reduce DNA accessibility to ROS attack, protecting the DNA. The upregulated intracellular phospholipid hydroperoxide glutathione peroxidase (GPx4), in high-fertile boars at mRNA level, can be considered a most relevant biomarker for fertility disclosure during sperm evaluation., Funding Agencies|Research Council FORMAS, Stockholm [2017-00946, 2019-00288]; FEDER funds (EU)European Commission; European Unions Horizon 2020 research and innovation program under the MSCA [891663]; [AGL2015-69738-R]; [PID2019-108320RJ-100]; [IJCI-2015-24380]; [MCIN/AEI/10.13039/501100011033]

Despite the reported promising farrowing rates after non-surgical and surgical transfers of vitrified porcine morulae and blastocysts produced in vivo (range: 70-75%), the pregnancy loss is 5-15 fold higher with vitrified than with fresh embryos. The present study aimed to investigate whether vitrification affects the transcriptome of porcine morulae, using microarrays and RT-qPCR validation. Morulae were obtained surgically from weaned sows (n = 13) on day 6 (day 0 = estrus onset). A total of 60 morulae were vitrified (treatment group). After 1 week of storage, the vitrified morulae were warmed. Vitrified-warmed and non-vitrified fresh morulae (control; n = 40) were cultured for 24 h to assess embryo survival by stereomicroscopy after. A total of 30 vitrified/warmed embryos that were deemed viable and 30 fresh control embryos (three pools of 10 for each experimental group) were selected for microarray analysis. Gene expression was assessed with a GeneChip (R) Porcine Genome Array (Affymetrix). An ANOVA analysis p-unadjusted <0.05 and a fold change cut-off of +/- 1.5 were set to identify differentially expressed genes (DEGs). Data analysis and biological interpretation were performed using the Partek Genomic Suite 7.0 software. The survival rate of morulae after vitrification and warming (92.0 +/- 8.3%) was similar to that of the control (100%). A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated), compared to the control group. Nine pathways were significantly modified. Go-enrichment analysis revealed that DEGs were mainly related to the Biological Process functional group. Up-regulated DEGs were involved in glycosaminoglycan degradation, metabolic pathways and tryptophan metabolism KEGG pathways. The pathways related to the down-regulated DEGs were glycolysis/gluconeogenesis, protein export and fatty acid elongation. The disruption of metabolic pathways in morulae could be related to impaired embryo quality and dev

Simple Summary Although endometrial immune regulation in pigs during the early preimplantation period is poorly documented, particularly under conditions of embryo transfer (ET), it is recognized that seminal plasma (SP) induces molecular changes in the reproductive tract, influencing numerous reproductive functions. A principal constituent of SP is the cytokine transforming growth factor beta 1 (TGF-beta 1), which has an important role in embryo development, pregnancy establishment, and progression. The present study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, porcine TGF-beta 1 in an extender, or an extender alone (control)) by mimicking an ET scenario in so-called "donor" (inseminated) and "recipient" (uninseminated) sows. We investigated the effects of these treatments on day 6 embryo development ("donors") and endometrial explants cytokine production ("donors" and "recipients"). SP infusion positively influenced embryo development compared with TGF-beta 1 or extender infusions. Infusion treatments differentially affected endometrial cytokine production, with the effects being stronger in "donors" than in "recipients." Increased knowledge of the effects of SP or some of its active components on the female immune system may help to develop strategies for increasing the reproductive efficiency for the benefit of pig ET. Seminal plasma (SP) in the female genital tract induces changes that affect multiple reproductive processes. One of the active components in SP is the transforming growth factor beta 1 (TGF-beta 1), which has major roles in embryo development and pregnancy. Embryo transfer (ET) technology is welcomed by the pig industry provided that embryo quality at embryo collection as well as the fertility and prolificacy of the recipients after the ET is increased. This study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, TGF-beta 1 cytokine in the extender, or the extender alone (control)) by mimi, Funding Agencies|Spanish Ministry of Economy and Competitiveness-European Regional Development Fund, Madrid, Spain [MINECO-FEDER: GL2015-69735-R]; Seneca Foundation, Murcia, SpainFundacion Seneca [19892/GERM/15]; Spanish Ministry of Science and Innovation/Spanish State Research Agency/European Regional Development Fund MCI/AEI/FEDER,UE), Madrid, Spain [RTI2018-093525-B-I00]; European UnionEuropean Commission [891663]; Research Council for the Environment, Areal Industries and Community Development (FORMAS), Stockholm, Sweden [2017-00946, 2019-00288]; Ministry of Economy and Competitiveness (Madrid, Spain) [BES-2016-077869]

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of +/- 1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFG beta, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes., Funding Agencies|Fundacion Seneca, Murcia, SpainFundacion Seneca [19892/GERM/15]; MICIU/FEDER, Madrid, Spain [RTI2018-093525-B-I00]; European UnionEuropean Commission [891663]; Swedish Research Council FORMAS, Stockholm, Sweden [2017-00946, 2019-00288]

Simple Summary The process of mammalian fertilization is dependent on many mechanisms mediated by regulatory genes and proteins expressed in the gametes and/or the female genital tract. This study aimed to determine the expression and location of the cytokine complex CXCL12:CXCR4 in the porcine gametes: oocytes and spermatozoa. This complex is known to play a pivotal role for sperm attraction towards the oocyte prior to internal fertilization in several mammalian species. Gene and protein expressions were analyzed in female and male porcine gametes. The results showed that the CXCL12 gene expression was higher in mature cumulus cells, and CXCR4 was higher in capacitated spermatozoa, both being requisites for gametes to accomplish fertilization. Moreover, for the first time, the CXCL12 protein was located in the cytoplasm of cumulus cells from mature COCs, and the CXCR4 protein was expressed in the midpiece and principal piece of uncapacitated spermatozoa and also in the sperm head of capacitated spermatozoa. These findings increase our current knowledge on porcine physiology of fertilization and reproduction, leading to possible improvements in the performance of reproductive technologies. Successful internal fertilization in mammals depends on several mechanisms, including those triggering the so-called "sperm attraction" towards the oocyte, which include some oocyte-derived sperm chemoattractants and interactive protein complexes, such as the cytokine C-X-C motif chemokine 12/C-X-C chemokine receptor type 4 (CXCL12-CXCR4) receptor complex. The presence and precise localization of these crucial proteins was determined hereby, for the first time, in porcine cumulus-oocyte complexes (COCs) and spermatozoa. CXCL12 was overexpressed in the cumulus cells of in vitro matured, compared to immature COCs (p < 0.05), with its receptor (CXCR4) being up-regulated in capacitated spermatozoa (p < 0.03) compared to uncapacitated spermatozoa. The CXCR4 appeared speci, Funding Agencies|European Unions Horizon 2020 research and innovation program under the MSCA [891663]; Swedish Research Council FORMAS, Stockholm, Sweden [2017-00946, 2019-00288]

Despite its advantages for pig breeding, embryo transfer (ET) has a major handicap: high embryo mortality during the pre- and implantation period, probably caused by divergent phenomena of tolerance between the immunologically unrelated (i.e., allogeneic) embryos and the recipient sow. Thus, to reach a similar maternal tolerance as in conventional breeding by artificial insemination (AI) would be the key to ET-success. For this reason, we studied the expression of the leukemia inhibitory factor (LIF) cytokine and its receptor in the pig endometrium during the implantation period (days 18 and 24) in sows subjected to ET (AL group) vs. post-cervical-AI controls (Hemi-AL group). Quantification of expression was performed at both mRNA (rt-qPCR) and protein (WB) levels. The expression of endometrial LIF on day 24 was considerably lower in ET than in AI pregnancies. Correlations between endometrial mRNA levels of LIF and LIF-R showed that, contrary to early AI-pregnancies, ET-pregnancies lack an inverse relation between cytokine and receptor levels. In conclusion, ET-pregnancies lack sufficient endometrial levels of LIF to develop adequate immunotolerance mechanisms to prevent the rejection of allogeneic ET-embryos., Funding Agencies|Fundacion Seneca, Murcia, SpainFundacion Seneca [19892/GERM/15]; MICIU/FEDER, Madrid, Spain [RTI2018-093525-B-I00]; European Unions Horizon 2020 research and innovation program [H2020-MSCA-IF-2019-891663]; Swedish Research Council FORMAS, Stockholm, Sweden [2017-00946, 2019-00288]

Simple Summary Glucocorticoids are steroid hormones modulating different functions in mammals, including reproduction, that act through the glucocorticoid receptor, encoded by the gene called NR3C1. Here, we describe how the expression levels of the glucocorticoid receptor change along the different compartments of the female rabbit internal reproductive tract 20 h after insemination with sperm-free seminal plasma or natural mating (whole semen) (Experiment 1) and how these levels change at 10, 24, 36, 68, and 72 h post-mating, during specific stages over time, i.e., ovulation, fertilization and the interval of early embryo development to the morula stage occurs (Experiment 2). NR3C1-upregulation was found in the infundibulum at 20 h after all treatments, especially after sperm-free seminal plasma infusion compared to mating (Experiment 1). In Experiment 2, the receptor gene expression levels increased in a spatio-temporal sequence, corresponding to the assumed location of the rabbit embryos (particularly morulae) in the oviductal various segments and timepoints (particularly 72 h), compared to down-expression at uterine regions. We conclude that NR3C1 may play a relevant role in the rabbit female reproductive tract. Rabbits are interesting as research animal models for reproduction, due to their condition of species of induced ovulation, with the release of endogenous gonadotropin-releasing hormone (GnRH) due to coitus. Glucocorticoid (GC) signaling, crucial for physiological homeostasis, is mediated through a yet unclear mechanism, by the GC receptor (NR3C1/GR). After mating, the female reproductive tract undergoes dynamic modifications, triggered by gene transcription, a pre-amble for fertilization and pregnancy. This study tested the hypothesis that when ovulation is induced, the expression of NR3C1 is influenced by sperm-free seminal plasma (SP), similarly to after mating (whole semen), along the different segments of the internal reproductive tract of female r, Funding Agencies|Research Council FORMAS, Stockholm [2017-00946, 2019-00288]; Swedish Research Council (Vetenskapradet, VR) [2015-05919]; Juan de la Cierva Incorporacion Postdoctoral Research Program (MICINN) [IJDC-2015-24380]; Generalitat de Catalunya, Agency for Management of University and Research Grants; European Social FoundEuropean Social Fund (ESF) [2018 FI_B 00236]; Government of Spain Ministry of Education, Culture and Sports [FPU15/06029]; Seneca Foundation Murcia (Spain)Fundacion Seneca [20780/PD/18]

Seminal plasma (SP) supports not only sperm function but also the ability of spermatozoa to withstand biotechnological procedures as artificial insemination, freezing or sex sorting. Moreover, evidence has been provided that SP contains identifiable molecules which can act as fertility biomarkers, and even improve the output of assisted reproductive technologies by acting as modulators of endometrial and embryonic changes of gene expression, thus affecting embryo development and fertility beyond the sperm horizon. In this overview, we discuss current knowledge of the composition of SP, mainly proteins and cytokines, and their influence on semen basic procedures, such as liquid storage or cryopreservation. The role of SP as modulator of endometrial and embryonic molecular changes that lead to successful pregnancy will also be discussed.

Simple Summary A proper uterine environment is basic for obtaining optimal embryo transfer outputs in domestic species, including the pig. However, scarce information is available about the uterine immune response of recipient (uninseminated) sows when receiving embryos during embryo transfer. Endometrial cytokine profile is among the main factors regulating uterine receptivity to embryos. In this study, using Luminex MAP(R) technology, we found important differences in the endometrial production in most of the 16 cytokines analyzed between recipient sows and embryo-bearing (inseminated) sows six days after estrus, with a predominant cytokine anti-inflammatory environment in the embryo-bearing endometria. These observations suggest that insemination components and/or early embryos induce an endometrium immune-tolerant cytokine profile at Day 6 of the cycle. The findings could contribute importantly to design strategies to maximize the reproductive performance of recipients after embryo transfer in swine. In the context of porcine embryo transfer (ET) technology, understanding the tightly regulated local uterine immune environment is crucial to achieve an adequate interaction between the transferred embryos and the receiving endometrium. However, information is limited on the uterine immune status of cyclic-recipient sows when receiving embryos during ET. The present study postulated that the anti- and proinflammatory cytokine profile 6 days after the onset of estrus differs between endometria from uninseminated cyclic sows and blastocyst-bearing sows. On Day 6 of the cycle, endometrial explants were collected from sows inseminated or not inseminated during the postweaning estrus and cultured for 22 h. The culture medium was then analyzed for the contents of a total of 16 cytokines using Luminex MAP(R) technology. The results showed important differences in the endometrial production of most cytokines between the sow categories, with a predominant anti-inflammatory e, Funding Agencies|MINECO-FEDER, Madrid, Spain [AGL2015-69735-R]; Fundacion Seneca, Murcia, SpainFundacion Seneca [19892/GERM/15]; MICIU/FEDER, Madrid, Spain [RTI2018-093525-B-I00]; European Unions Horizon 2020 research and innovation program under the MSCA [891663]; Swedish Research Council FORMAS, Stockholm, Sweden [2017-00946, 2019-00288]; European Union (H2020-MSCA-IF-2019)European Union (EU) [891663]; Ministry of Economy and Competitiveness (Madrid, Spain) [BES-2016-077869]

This study evaluated the effects of different concentrations (1, 10, 25, 50, and 100 mu M) of the antioxidant N-(2-mercaptopropionyl)-glycine (NMPG), during the culture of in vitro-fertilized porcine oocytes. While the highest concentrations of NMPG (50 and 100 mu M) were toxic to the developing embryos during the first two days of culture, 25 mu M NMPG achieved cleavage rates that were similar to those achieved by the control but did not sustain blastocyst production by Day 7 of culture. Compared to the control culture medium, the culture medium supplemented with 10 mu M NMPG increased (P<0.05) the rates of blastocyst formation, decreased (P<0.05) the intracellular levels of reactive oxygen substances, and downregulated (P<0.05) the expression of the oxidative stress related gene GPX1. In conclusion, these results demonstrated that supplementation of porcine embryo culture medium with 10 M NMPG can attenuate oxidative stress and increase the yield of in vitro production of blastocysts., Funding Agencies|Ministry of Science, Innovation and Universities/the European Regional Development Fund, Madrid, Spain [RTI2018-093525-B-I00]; European UnionEuropean Union (EU) [891663]; Seneca Foundation, Murcia, SpainFundacion Seneca [19892/GERM/15, 20780/PD/18]; Research Council FORMAS, Stockholm, Sweden [2017-00946, 2019-00288]

Anxiety, chronical stress, and depression during pregnancy are considered to affect the offspring, presumably through placental dysregulation. We have studied the term placentae of pregnancies clinically monitored with the Becks Anxiety Inventory (BAI) and Edinburgh Postnatal Depression Scale (EPDS). A cutoff threshold for BAI/EPDS of 10 classed patients into an Index group (>10,n= 23) and a Control group (<10,n= 23). Cortisol concentrations in hair (HCC) were periodically monitored throughout pregnancy and delivery. Expression differences of main glucocorticoid pathway genes, i.e., corticotropin-releasing hormone (CRH), 11 beta-hydroxysteroid dehydrogenase (HSD11B2), glucocorticoid receptor (NR3C1), as well as other key stress biomarkers (Arginine Vasopressin, AVP and O-GlcNAc transferase, OGT) were explored in medial placentae using real-time qPCR and Western blotting. Moreover, gene expression changes were considered for their association with HCC, offspring, gender, and birthweight. A significant dysregulation of gene expression for CRH, AVP, and HSD11B2 genes was seen in the Index group, compared to controls, while OGT and NR3C1 expression remained similar between groups. Placental gene expression of the stress-modulating enzyme 11 beta-hydroxysteroid dehydrogenase (HSD11B2) was related to both hair cortisol levels (Rho = 0.54;p< 0.01) and the sex of the newborn in pregnancies perceived as stressful (Index,p< 0.05). Gene expression of CRH correlated with both AVP (Rho = 0.79;p< 0.001) and HSD11B2 (Rho = 0.45;p< 0.03), and also between AVP with both HSD11B2 (Rho = 0.6;p< 0.005) and NR3C1 (Rho = 0.56;p< 0.03) in the Control group but not in the Index group; suggesting a possible loss of interaction in the mechanisms of action of these genes under stress circumstances during pregnancy., Funding Agencies|Medical Research Council of Southeast Sweden (FORSS) [661011, 392061, 472721]; European Unions Horizon 2020 research and innovation program under the MSCA [891663]

The cytokine platelet factor 4 (PF4) enhances differentiation and cell viability of different stem cells lines in vitro. This study investigated whether PF4 addition to customary pig embryo semi-defined culture media can improve their developmental outcome (Experiment 1) and ultimately replace the need for bovine serum albumin (BSA, Experiment 2). Experiment 1 added PF4 (100-1000 ng/mL, 0 = control) to NCSU-23 with 0.4 mg/mL BSA culturing 3430 presumptive zygotes. Experiment 2 added PF4 (100 -1000 ng/mL, 0 = Control-PVA) to a BSA-free medium (NCSU-23 with 0.3 mg/mL PVA) culturing 3820 presumptive zygotes. Zygote culture in NCSU-23 with 0.4 mg/mL BSA was used as overall control. All groups of Experiment 1 displayed similar rates of day 2-cleavage (range: 65.0 +/- 10.9 to 70.0 +/- 5.8%); of day 7-blastocyst rates (range: 46.6 +/- 10.0 to 56.4 +/- 8.2%) and of total day 7-blastocyst efficiency (range: 32.3 +/- 8.3 to 37.2 +/- 7.3%). Addition of PF4 did not affect total cell numbers of day 7 blastocysts (range: 44.1 +/- 23.2 to 50.5 +/- 26.4). In Experiment 2, PF4 accelerated embryo development, increasing (P < 0.01) blastocyst yield compared to 0-PF4, and blastocyst formation by day 5 adding PF4 100-500 ng/mL (range: 29.9 +/- 7.8 to 31.8 +/- 5.5%; P < 0.05) compared with BSA-control (17.2 +/- 8.2%) and PF4 1000 ng/mL (15.5 +/- 7.9%); showing similar blastocyst rates (range: 42.0 +/- 11.5 to 49.3 +/- 10.0%), total efficiency (28.0 +/- 8.2 to 32.3 +/- 7.1%) total cell numbers (range: 42.6 +/- 19.3 to 45.7 +/- 23.9) as BSA-controls. In conclusion, although PF4 did not show additive improvement under usual semi-defined, BSA-supplemented embryo media, it successfully replaced BSA sustaining porcine blastocyst production in chemically defined conditions. (C) 2019 Elsevier Inc. All rights reserved., Funding Agencies|Ministry of Science, Innovation and Universities-ERDF, Madrid, Spain [RTI2018-093525-B-I00]; Seneca Foundation, Murcia, SpainFundacion Seneca [19892/GERM/15]; Research Council FORMAS, Stockholm, Sweden [2017e00946]

The seminal plasma (SP) modulates the female reproductive immune environment after mating, and microRNAs (miRNAs) could participate in the process. Considering that the boar ejaculate is built by fractions differing in SP-composition, this study evaluated whether exposure of mucosal explants of the sow internal genital tract (uterus, utero-tubal junction and isthmus) to different SP-fractions changed the profile of explant-secreted miRNAs. Mucosal explants retrieved from oestrus sows (n = 3) were in vitro exposed to: Medium 199 (M199, Control) or M199 supplemented (1:40 v/v) with SP from the sperm-rich fraction (SRF), the post-SRF or the entire recomposed ejaculate, for 16 h. After, the explants were cultured in M199 for 24 h to finally collect the media for miRNA analyses using GeneChip miRNA 4.0 Array (Affymetrix). Fifteen differentially expressed (False Discovery Rate (FDR) < 0.05 and Fold-change ≥ 2) miRNAs (11 down- versus 4 up-regulated) were identified (the most in the media of uterine explants incubated with SP from post-SRF). Bioinformatics analysis identified that predicted target genes of dysregulated miRNAs, mainly miR-34b, miR-205, miR-4776-3p and miR-574-5p, were involved in functions and pathways related to immune response. In conclusion, SP is able to elicit changes in the miRNAs profile secreted by female genital tract, ultimately depending SP-composition., Funding agencies: FEDER funds (EU)European Union (EU) [AGL2015-69738-R]; Seneca Foundation Murcia, SpainFundacion Seneca [19892/GERM-15]; Swedish Research Council FORMAS, Stockholm, Sweden [2017-00946, 2019-00288]; MEFP (Spain) [CAS19/00116]; MICIU (Spain) [FJCI-2017-31689

In the study presented here we identified transcriptomic markers for fertility in the cargo of pig ejaculated spermatozoa using porcine-specific micro-arrays (GeneChip((R)) miRNA 4.0 and GeneChip((R)) Porcine Gene 1.0 ST). We report (i) the relative abundance of the ssc-miR-1285, miR-16, miR-4332, miR-92a, miR-671-5p, miR-4334-5p, miR-425-5p, miR-191, miR-92b-5p and miR-15b miRNAs, and (ii) the presence of 347 up-regulated and 174 down-regulated RNA transcripts in high-fertility breeding boars, based on differences of farrowing rate (FS) and litter size (LS), relative to low-fertility boars in the (Artificial Insemination) AI program. An overrepresentation analysis of the protein class (PANTHER) identified significant fold-increases for C-C chemokine binding (GO:0019957): CCR7, which activates B- and T-lymphocytes, 8-fold increase), XCR1 and CXCR4 (with ubiquitin as a natural ligand, 1.24-fold increase), cytokine receptor activity (GO:0005126): IL23R receptor of the IL23 protein, associated to JAK2 and STAT3, 3.4-fold increase), the TGF-receptor (PC00035) genes ACVR1C and ACVR2B (12-fold increase). Moreover, two micro-RNAs (miR-221 and mir-621) were down- and up-regulated, respectively, in high-fertility males. In conclusion, boars with different fertility performance possess a wide variety of differentially expressed RNA present in spermatozoa that would be attractive targets as non-invasive molecular markers for predicting fertility., Funding Agencies|Research Council FORMAS, Stockholm [2017-00946, 2019-00288]; Swedish Research Council (Vetenskapsradet, VR)Swedish Research Council [2015-05919]; MINECO; FEDER Madrid (Spain) [AGL2015-69738-R]; Seneca Foundation Murcia (Spain)Fundacion Seneca [20780/PD/18]

Simple Summary In mammals, the expression of regulatory genes is modified by the interaction between semen and the female reproductive tract. This study intends to unveil how mating or insemination with sperm-free seminal plasma, as well as the presence of preimplantation embryos, affects inflammation and angiogenesis in different segments of the reproductive tract of female rabbits. Gene expression of anti-inflammatory cytokines and angiogenesis mediators was analyzed in segmented tracts (cervix to infundibulum) in response to mating and sperm-free seminal plasma infusion. Moreover, the gene expression at different times post-mating was also analyzed. Results showed that gene expression changes were mainly localized in the uterus in the natural mating group, describing a clear temporal variation, while limited to the oviduct in the sperm-free seminal plasma group. These changes suggest an early response in the uterus and late modulation in the oviduct, distinctly demonstrating that semen and seminal plasma, through their interaction with the female reproductive tract, can differentially modulate the expression of anti-inflammatory and angiogenesis mediators. The maternal environment modulates immune responses to facilitate embryo development and ensure pregnancy. Unraveling this modulation could improve the livestock breeding systems. Here it is hypothesized that the exposure of the female rabbit reproductive tract to semen, as well as to early embryos, modulates inflammation and angiogenesis among different tissue segments. qPCR analysis of the gene expression changes of the anti-inflammatory interleukin-10 (IL10) and transforming growth factor beta family (TGF beta 1-3) and the angiogenesis mediator vascular endothelial growth factor (VEGF-A) were examined in response to mating or insemination with sperm-free seminal plasma (SP). Reproductive tract segment (cervix to infundibulum) samples were obtained in Experiment 1, 20 h after gonadotropin-releasing hormone (G, Funding Agencies|Research Council FORMAS, Stockholm [2017-00946, 2019-00288]; Swedish Research Council (Vetenskapradet, VR) [2015-05919]; Juan de la Cierva Incorporacion Postdoctoral Research Program (MICINN) [IJDC-2015-24380]; Generalitat de Catalunya, Agency for Management of University and Research Grants; Eropean Social Found [FI_B 00236]

The avian seminal fluid (SF) is a protein-rich fluid, derived from the testis, the rudimentary epididymis and, finally, from the cloacal gland. The SF interacts with spermatozoa and the inner cell lining of the female genital tract, to modulate sperm functions and female immune responsiveness. Its complex proteome might either be free or linked to extracellular vesicles (EVs) as it is the case in mammals, where EVs depict the tetraspanin CD9; and where those EVs derived from the epididymis (epididymosomes) also present the receptor CD44. In the present study, sperm-free SF from Red Jungle Fowl, White Leghorn and an advanced intercross (AIL, 12th generation) were studied using flow cytometry of the membrane marker tetraspanin CD9, Western blotting of the membrane receptor CD44 and electron microscopy in non-enriched (whole SF) or enriched fractions obtained by precipitation using a commercial kit (Total Exosome Precipitation Solution). Neither CD9- nor CD44 could be detected, and the ultrastructure confirmed the relative absence of EVs, raising the possibility that avian SF interacts differently with the female genitalia as compared to the seminal plasma of mammals., Funding Agencies|VetenskapsradetSwedish Research Council [2015-05919]; Forskningsradet i Sydostra Sverige [473121, 745971]; Lions Forskningsfond [DNR LIU-2016-00641]; Swedish Research Council FORMASSwedish Research CouncilSwedish Research Council Formas [2017-00946]

Funding Agencies|Ministry of Economy and Competitiveness (Madrid, Spain)/the European Regional Development Fund [AGL2015-69735-R]; Seneca Foundation, Murcia, SpainFundacion Seneca [19892/GERM/15]; Research Council FORMAS, Stockholm, Sweden [2017-00946]

The coenzyme Q10 (CoQ10) is a potent antioxidant with critical protection role against cell oxidative stress, caused by the mitochondrial dysfunction. This study evaluated the effects of CoQ10 supplementation to in vitro maturation (IVM) or embryo culture media on the maturation, fertilization and subsequent embryonic development of pig oocytes and embryos. Maturation (Experiment 1) or embryo culture (Experiment 2) media were supplemented with 0 (control), 10, 25, 50 and 100 mu M CoQ10. The addition of 10-50 mu M CoQ10 to the IVM medium did not affect the percentage of MII oocytes nor the fertilization or the parameters of subsequent embryonic development. Exogenous CoQ10 in the culture medium neither did affect the development to the 2-4-cell stage nor rates of blastocyst formation. Moreover, the highest concentration of CoQ10 (100 mu M) in the maturation medium negatively affected blastocyst rates. In conclusion, exogenous CoQ10 supplementation of maturation or embryo culture media failed to improve the outcomes of our in vitro embryo production system and its use as an exogenous antioxidant should not be encouraged., Funding Agencies|Seneca Foundation, Murcia, SpainFundacion Seneca [20027/SF/16, 19892/GERM/15]; Junta de Comunidades de Castilla-La Mancha, SpainJunta de Castilla y Leon [SBPLY/17/180501/000500]; Ministry of Economy and Competitiveness [BES-2013-064087, BES-2016-077869]; MINECO-FEDER [AGL2015-69735-R]; Research Council FORMAS, Stockholm [2017-00946]

Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows non-surgical ET and short- and long-term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20 years. This review includes several aspects of non-surgical ET technology and different issues affecting ET programmes and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short-term use in pig production., Funding Agencies|MICINN-FEDER (Madrid, Spain)European Union (EU)Ministry of Science and Innovation, Spain (MICINN) [AGL2004-07546, AGL2009-12091]; MINECO-FEDER (Madrid, Spain) [AGL2012-38621, AGL2015-69735-R]; CDTI (Madrid, Spain) [IDI-20140140, IDI-20140142]; Fundacion Seneca (Murcia, Spain)Fundacion Seneca [GERM 04543/07, 19892/GERM/15]; Research Council FORMAS, Stockholm, Sweden [2017-00946]; Fundacion Seneca, (Murcia, Spain)Fundacion Seneca [20780/PD/18]; Junta de Comunidades de Castilla-La Mancha (Castilla-La Mancha, Spain, PRT programme) [SBPLY/17/180501/000500]; MINECO (Madrid, Spain) [BES-2016-077869]