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Introduction Pregnancy success relies on the establishment of a delicate immune balance that requires the early activation of a series of local and systemic immune mechanisms. The changes in the immunological profile that are normally occurring in the pregnant uterus does not take place in cyclic (non-pregnant) uterus, a fact that has been widely explored in pigs at the tissue local level. Such differences would be especially important in the context of embryo transfer (ET), where a growing body of literature indicates that immunological differences at the uterine level between donors and recipients may significantly impact embryonic mortality. However, whether components of peripheral immunity also play a role in this context remains unknown. Accordingly, our hypothesis is that the immune status of donor sows differs from potential recipients, not only at the tissue local level but also at the systemic level. These differences could contribute to the high embryonic mortality rates occurring in ET programs.Methods In this study differences in systemic immunity, based on cytokine gene expression profile in peripheral blood mononuclear cells (PBMCs), between embryo-bearing donor (DO group; N = 10) and potential recipient sows (RE group; N = 10) at Day 6 after the onset of the estrus were explored. Gene expression analysis was conducted for 6 proinflammatory (IL-1 alpha, IL-1 beta, IL-2, GM-CSF, IFN-gamma, and TNF-alpha) and 6 anti-inflammatory (IL-4, IL-6, IL-10, IL-13, TGF-beta 1, and LIF) cytokines.Results and discussion All cytokines were overexpressed in the DO group except for IL-4, suggesting that stimuli derived from the insemination and/or the resultant embryos modify the systemic immune profile in DO sows compared to RE (lacking these stimuli). Our results also suggest that certain cytokines (e.g., IL-1 alpha and IL-1 beta) might have a predictive value for the pregnancy status., Funding Agencies|MCIN/AEI; ERDF, Madrid, Spain [2019-00288]; Swedish Research Council FORMAS, Stockholm, Sweden; [PID2022-137645OB-I00]

The combination of estrus synchronization and superovulation (SS) treatments causes alterations in ovarian and endometrial gene expression patterns, resulting in abnormal follicle and oocyte growth, fertilization, and embryo development. However, the impact of combined SS treatments on the transcriptome of the surviving embryos remains unidentified. In this study, we examined gene expression changes in day 6 blastocysts that survived a brief regimen of synchronization treatment combined with superovulation. The sows were included in one of three groups: SS7 group (n = 6), sows were administered Altrenogest (ALT) 7 days from the day of weaning and superovulated with eCG 24 h after the end of ALT treatment and hCG at the onset of estrus; SO group (n = 6), ALT nontreated sows were superovulated with eCG 24 h postweaning and hCG at the onset of estrus; control group (n = 6), weaned sows displaying natural estrus. Six days after insemination, the sows underwent a surgical intervention for embryo collection. Transcriptome analysis was performed on blastocyst-stage embryos with good morphology. Differentially expressed genes (DEGs) between groups were detected using one-way ANOVA with an un-adjusted p-value < 0.05 and a fold change 1.5. The effect of SO treatment on the number of altered pathways and DEGs within each pathway was minimal. Only four pathways were disrupted comprising only a total of four altered transcripts, which were not related to reproductive functions or embryonic development. On the other hand, the surviving blastocysts subjected to SS7 treatments exhibited moderate gene expression changes in terms of DEGs and fold changes, with seven pathways disrupted containing a total of 10 transcripts affected. In this case, the up-regulation of certain pathways, such as the metabolic pathway, with two up-regulated genes associated with reproductive functions, namely RDH10 and SPTLC2, may suggest suboptimal embryo quality, while the down-regulation of others

The most commonly used technique to vitrify pig embryos is the super open pulled straw (SOPS), where a maximum of 6 embryos can be vitrified simultaneously per device without compromising the minimum volume necessary for optimal preservation. Since optimal embryo transfer (ET) demands a transfer of 20-40 embryos per recipient, the customary use of SOPS complicates embryo warming and ET in field conditions. Such complications could be avoided when using the Cryotop® (OC) system, which has been proven to be an effective option for vitrifying at least 20 porcine embryos simultaneously. This study aimed to investigate the changes in the transcriptome of blastocysts caused by vitrification using both systems. In vivo-derived blastocysts were OC- (n = 60; 20 embryos/device) and SOPS- (n = 60; 4-6 embryos/device) vitrified and cultured for 24 h after warming. Nonvitrified blastocysts (n = 60) cultured for 24 h postcollection acted as controls. At the end of culture, 48 viable embryos from each group (6 pools of 8 embryos) were selected for microarray (GeneChip® Porcine Genome Array, P/N 900624, Affymetrix) analysis of differentially expressed genes (DEGs). The survival rate of embryos vitrified with the OC and SOPS systems (>97%) was similar to that of the control embryos (100%). Microarray analysis of each vitrification system compared to the control group showed 245 DEGs (89 downregulated and 156 upregulated) for the OC system and 210 (44 downregulated and 166 upregulated) for the SOPS system. Two pathways were enriched for the DEGs specifically altered in each vitrification system compared to the control (glycolysis/gluconeogenesis and carbon metabolism pathways for the OC system and amino sugar and nucleotide sugar metabolism and lysosome pathways in the SOPS group). The OC group showed 31 downregulated and 24 upregulated genes and two enriched pathways (mineral absorption and amino sugar and nucleotide sugar metabolism pathways) when compared to the SOPS group. In sum

The combination of estrus synchronization and superovulation (SS) treatments causes alterations in ovarian and endometrial gene expression patterns, resulting in abnormal follicle and oocyte growth, fertilization, and embryo development. However, the impact of combined SS treatments on the transcriptome of the surviving embryos remains unidentified. In this study, we examined gene expression changes in day 6 blastocysts that survived a brief regimen of synchronization treatment combined with superovulation. The sows were included in one of three groups: SS7 group (n = 6), sows were administered Altrenogest (ALT) 7 days from the day of weaning and superovulated with eCG 24 h after the end of ALT treatment and hCG at the onset of estrus; SO group (n = 6), ALT nontreated sows were superovulated with eCG 24 h postweaning and hCG at the onset of estrus; control group (n = 6), weaned sows displaying natural estrus. Six days after insemination, the sows underwent a surgical intervention for embryo collection. Transcriptome analysis was performed on blastocyst-stage embryos with good morphology. Differentially expressed genes (DEGs) between groups were detected using one-way ANOVA with an un-adjusted p-value < 0.05 and a fold change 1.5. The effect of SO treatment on the number of altered pathways and DEGs within each pathway was minimal. Only four pathways were disrupted comprising only a total of four altered transcripts, which were not related to reproductive functions or embryonic development. On the other hand, the surviving blastocysts subjected to SS7 treatments exhibited moderate gene expression changes in terms of DEGs and fold changes, with seven pathways disrupted containing a total of 10 transcripts affected. In this case, the up-regulation of certain pathways, such as the metabolic pathway, with two up-regulated genes associated with reproductive functions, namely RDH10 and SPTLC2, may suggest suboptimal embryo quality, while the down-r, Funding Agencies|MCIN/AEI/ERDF a way of making Europe, Madrid, Spain [RTI2018-093525-B-I00]; Fundacion Seneca, Murcia, Spain [19892/GERM/15]; Swedish Research Council FORMAS, Stockholm, Sweden [2019-00288]

The most commonly used technique to vitrify pig embryos is the super open pulled straw (SOPS), where a maximum of 6 embryos can be vitrified simultaneously per device without compromising the mini-mum volume necessary for optimal preservation. Since optimal embryo transfer (ET) demands a transfer of 20-40 embryos per recipient, the customary use of SOPS complicates embryo warming and ET in field conditions. Such complications could be avoided when using the Cryotop (R) (OC) system, which has been proven to be an effective option for vitrifying at least 20 porcine embryos simultaneously. This study aimed to investigate the changes in the transcriptome of blastocysts caused by vitrification using both systems. In vivo-derived blastocysts were OC-(n 1/4 60; 20 embryos/device) and SOPS-(n 1/4 60; 4-6 embryos/device) vitrified and cultured for 24 h after warming. Nonvitrified blastocysts (n 1/4 60) cultured for 24 h postcollection acted as controls. At the end of culture, 48 viable embryos from each group (6 pools of 8 embryos) were selected for microarray (GeneChip (R) Porcine Genome Array, P/N 900624, Affymetrix) analysis of differentially expressed genes (DEGs). The survival rate of embryos vitrified with the OC and SOPS systems (>97%) was similar to that of the control embryos (10 0%). Microarray analysis of each vitrification system compared to the control group showed 245 DEGs (89 downregulated and 156 upregulated) for the OC system and 210 (44 downregulated and 166 upregulated) for the SOPS system. Two pathways were enriched for the DEGs specifically altered in each vitrification system compared to the control (glycolysis/gluconeogenesis and carbon metabolism pathways for the OC system and amino sugar and nucleotide sugar metabolism and lysosome pathways in the SOPS group). The OC group showed 31 downregulated and 24 upregulated genes and two enriched pathways (mineral absorption and amino sugar and nucleotide sugar metabolism pathways) when compared to the, Funding Agencies|Spanish Ministry of Science and Innovation; ERDF "A way of making Europe", Madrid, Spain; Fundacion Seneca, Murcia, Spain; Research Council FORMAS, Stockholm, Sweden [19892/GERM/15]; [2019-00288]; [PRE2019-090508]; [RTI2018-093525-B-I00]

Background: Currently, high polyspermy remains a significant obstacle to achieving optimal efficiency in in vitro fertilization (IVF) and in vitro embryo production (IVP) systems in pigs. Developing strategies that would prevent polyspermy is essential in overcoming this challenge and maximizing the potential of this reproductive biotechnology. Previous results have demonstrated that using boar spermatozoa subjected to a high-extension and reconcentration procedure and then cryopreserved resulted in significant improvements in IVF/IVP systems with high rates of monospermy and penetration.Objective: The aim of the present study was to unveil the molecular mechanisms that may underlie the changes in fertilization patterns exhibited by highly extended and cryopreserved boar spermatozoa.Materials and methods: To achieve this goal, we used quantitative proteomic analysis (LC-ESI-MS/MS SWATH) to identify differentially abundant proteins (DAPs) between highly extended (HE) and conventionally (control; CT) cryopreserved boar spermatozoa. Prior to the analysis, we evaluated the in vitro post-thawing fertilizing ability of the sperm samples. The results demonstrated a remarkable improvement in monospermy and IVF efficiency when using HE spermatozoa in IVF compared with CT spermatozoa.Results: At the proteomic level, the combination of high-extension and cryopreservation had a significant impact on the frozen-thawed sperm proteome. A total of 45 proteins (24 downregulated and 21 upregulated) were identified as DAPs (FC > 1 or <= 1; p < 0.05) when compared with CT spermatozoa. Some of these proteins were primarily linked to metabolic processes and the structural composition of sperm cells. The dysregulation of these proteins may have a direct or indirect effect on essential sperm functions and significantly affect spermatozoa-oocyte interaction and, therefore, the sperm fertilization profile under in vitro conditions. While these findings are promising, fur, Funding Agencies|Swedish Research Council FORMAS; ERDF [MCIN/AEI/10.13039/501100011033]; Fundacion Seneca [RTI2018-093525-B-I00]; Murcia, Spain [19892/GERM/15]; Stockholm, Sweden; [2019-00288]

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]

The establishment of a successful pregnancy can only occur through a concerted functioning of the entire female reproductive system, allowing for fertilization, subsequent embryo development and implantation of the conceptus. In this context, the uterine immunological responses responsible for rejection or tolerance of the conceptus are of critical importance. The aim of the present review is to summarize our current knowledge about those cellular and molecular immunological events occurring at the uterine level during pre-implantation and implantation stages of pregnancy in the pig. Advancing our understanding of the immune mechanisms involved in the success or failure of pregnancy will provide cues to develop novel strategies augmenting endometrial receptivity, finally increasing the efficiency of assisted reproductive technologies in pigs., Funding Agencies|Ministerio de Ciencia e Innovacion; Council FORMAS [2019-00288]; Fundacion Seneca [19892/GERM/15]

Seminal plasma (SP) affects reproduction, inducing cell and molecular changes in the female genital tract. A main active component in SP is the modulatory transforming growth factor-beta (TGF-beta), particularly its TGF-beta 1 isoform, which affects the synthesis of other cytokines as granulocyte-macrophage colony-stimulating factor, relevant for embryo development and pregnancy. This study evaluated the effect of pooled frozen-thawed SP and commercial TGF-beta 1 infused during oestrus in sows post-cervically inseminated with liquid extended semen, containing similar to 4 ml of residual SP, on their fertility and prolificacy. For this, 250 sows in their post-weaning oestrus were used. Sows were randomly assigned to one of the following groups to be post-cervically treated 30 min before insemination: (i) SP group: infused with 40 ml of SP (N = 57); ii) Group TFG(beta 1): infused with 40 ml of BTS extender containing 3 ng/ml of porcine TGF-beta 1 (N = 64); iii) BTS group: infused with 40 ml of BTS extender (N = 60); and iv) Control Group: sows catheterized but not infused prior to Al (N = 69). Farrowing rates (range: 86.7% to 91.3%) and numbers of live-born piglets (range: range: 12.8 +/- 2.9 to 13.4 +/- 3.1) were not affected by any treatment compared with Controls, indicating that neither pre-infusions of SP nor TGF-beta 1 30 min before Al influenced subsequent fertility and prolificacy., Funding Agencies|Research Council FORMAS; Fundacion Seneca; Ministerio de Economia y Competitividad

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]

Oviductal extracellular vesicles (oEVs) have been identified as important components of the oviductal fluid (OF) and have been pointed to as key modulators of gamete/embryo-maternal interactions. Here, we determined the functional impact of oEVs on embryo development and the embryonic transcriptome in porcine. Experiment 1 examined the effect of oEVs and OF on embryo development. In vitro-produced embryos were cultured with oEVs or OF for 2 or 7 days using an in vitro sequential system or without supplementation (control). Experiment 2 analyzed transcriptomic alterations of EV-treated embryos versus control and the oEVs RNA cargo by RNA-sequencing. Two days of EV treatment enhanced embryo development over time when compared to other treatments. Different RNA expression profiles between embryos treated with EVs for two or seven days and untreated controls were obtained, with 54 and 59 differentially expressed (DE) genes and six and seven DE miRNAs, respectively. In oEV RNA cargo, 12,998 RNAs and 163 miRNAs were identified. Integrative analyses pointed to specific oEV components that might act as modulators of the embryonic transcriptome, such as S100A11, ANXA2 or miR-21-5p. Overall, the findings suggested that oEVs could be a potential strategy to improve porcine IVP outcomes, particularly by using two days of EV treatment.

Although embryo transfer (ET) is a biotechnology ready for the swine industry, there are factors to be solved, the availability of embryo donors as one. Multiparous sows as donors ought to be considered since weaning is a natural and efficient method for estrus synchronization. In addition, superovulation treatments at weaning are effective in increasing the efficiency of donor embryo production. However, ET programs typically require more donors than those available from a single weaning, imposing grouping several weanings to establish a batch for ET. Since short-term administration of Altrenogest is effective in delaying estrus after weaning without effects on ovulation and embryo development, we investigated how Altrenogest combined with superovulation would affect reproductive parameters and embryo quality and quantity of weaned multiparous donor sows. The sows were administered Altrenogest from the day of weaning for 14 (SS-14 group; N = 26), 7 (SS-7 group; N = 31) and 4 (SS-4 group; N = 32) days. The sows were superovulated with eCG 24 h after the last administration of Altrenogest and with hCG at the onset of estrus. Sows not treated with Altrenogest that were superovulated with eCG 24 h post-weaning and hCG at the onset of estrus (SC group; N = 37) and sows with natural estrus after weaning (C group; N = 34) were used as control groups. The percentage of sows showing estrus within 10 days was not affected by the treatment, but the interval from Altrenogest withdrawal to estrus was longer (P < 0.05) in the SS groups than the interval from weaning to estrus in the controls. SS treatments increased (P < 0.05) the percentage of sows with ovarian cysts and the development of polycystic ovaries. The pregnancy and the fertilization rates, and the overall embryo production efficiency were also negatively affected by the SS treatments (P < 0.05). Interestingly, almost 70% of the structures classified as unfertilized oocytes or degenerated embryos, Funding agencies: MCIN/AEI/10.13039/501100011033 and by ERDF a way of making Europe (RTI2018-093525-B-I00), Madrid, Spain; Fundacion Seneca (19892/GERM/15), Murcia, Spain; and the Swedish ResearchCouncil FORMAS (Projects 2017-00946 and 2019-00288), Stockholm, Sweden.

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 The development of chemically defined media has become a particularly important task for in vitro embryo production systems, which require maintained reproducible results when new additives are tested for culture, beyond observational studies. Specifically, we need studies measuring the impact of these media on the embryonic transcriptome, particularly those negatively affecting embryo quality. Consequently, this study evaluated by using a microarray approach the transcriptome of porcine embryos produced in vitro, cultured in a defined vs. an undefined medium and contrasted with in vivo-derived embryos. No significantly altered genes were found between in vitro-produced embryos, despite the theoretical limitations that usually accompany defined media. However, when they were compared with in vivo-derived embryos, many altered genes were observed, reflecting how current culture conditions deeply modify the embryonic transcriptome. A better understanding of these alterations may offer new ways to improve in vitro embryo production systems. Likewise, developing a chemically defined medium capable of producing embryos of a similar quality to traditional media may contribute to this task. The development of chemically defined media is a growing trend in in vitro embryo production (IVP). Recently, traditional undefined culture medium with bovine serum albumin (BSA) has been successfully replaced by a chemically defined medium using substances with embryotrophic properties such as platelet factor 4 (PF4). Although the use of this medium sustains IVP, the impact of defined media on the embryonic transcriptome has not been fully elucidated. This study analyzed the transcriptome of porcine IVP blastocysts, cultured in defined (PF4 group) and undefined media (BSA group) by microarrays. In vivo-derived blastocysts (IVV group) were used as a standard of maximum embryo quality. The results showed no differentially expressed genes (DEG) between the PF4 and BSA group, Funding Agencies|Fundacion Seneca, Murcia, SpainFundacion Seneca [19892/GERM/15]; MCI/AEI/FEDER, UE, Madrid, Spain [RTI2018-093525-B-I00]; Swedish Research Council FORMAS, Stockholm, Sweden [2017-00946, 2019-00288]

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]