Introduction:Interleukin-6 (IL-6) has recently emerged as a promising supplement for in vitro maturation (IVM), enhancing pig blastocyst development and reducing apoptosis following in vitro fertilization (IVF). This study assessed whether IL-6 supplementation during IVM is associated with differences in gene expression in day-7 in vitro-produced blastocysts. Methods:Oocytes from prepubertal gilts were matured in IVM medium with (IL-6 IVM group) or without (control group) 100 ng/mL IL-6 and subsequently subjected to IVF. The resulting presumptive zygotes were cultured in vitro for 7 days. Six pools of seven viable blastocysts per group were flash-frozen in liquid nitrogen and stored at -80 °C until microarray analysis. The embryos were analyzed using the GeneChip® Porcine Genome Array (Affymetrix). Differentially expressed genes (DEGs) were identified by ANOVA using a significance threshold of p < 0.01 and a fold change > |1.5|. Results:Compared to the control, the IL-6 IVM group showed 91 up-regulated and 47 down-regulated DEGs. KEGG analysis of up-regulated genes revealed enrichment in 13 pathways including genes involved in key processes related to lipid metabolism, embryo development, cell proliferation, and immune system. Only two pathways were enriched among down-regulated genes, comprising genes not previously associated with essential roles in embryonic development. Discussion:Blastocysts derived from oocytes matured in vitro with 100 ng/mL of IL-6 exhibited differences in activity in several immune-related and lipid metabolism pathways through the overexpression of specific genes associated with cell proliferation, embryo development, and apoptosis reduction. These findings provide insights consistent with a role of IL-6 during porcine IVM and its association with oocyte developmental competence.

The vertebrate zygote results from the merging of two highly specialized gamete cells, namely the oocyte and the spermatozoon, and has the outstanding potential of creating all cells in the future developing embryo. For this to occur, however, the genome of the gametes is mostly striped of "epigenetic marks," or proteins and methyl groups attached to the DNA. Epigenetic marks in the genome constitute the so-called epigenome and have the potential for long term regulation of gene expression. Environmental insults during the highly susceptible and delicate period of germ cell development could, by altering the epigenome of spermatozoa, affect the phenotype of future generations (transgenerational epigenetic inheritance). A brief review of how epigenetics can act transgenerationally through the male germline is hereby presented.

This study aimed to evaluate daily variations in sperm quality, testosterone levels, cryoresistance, and the relocation of AQP3 in plasma membrane domains in wild and domestic goats. Samples were collected from five Murciano-Granadina bucks and seven Iberian ibex at 12:00 h (noon) and 00:00 h (midnight), during the rutting season. Sperm motility, viability, acrosome integrity, DNA fragmentation, and immunolocalization of AQP3 were assessed in fresh and frozen-thawed semen samples. Serum testosterone concentrations were also determined at both times. In domestic goats, kinematic parameters were greater (p < 0.05) in semen collected at midnight, whereas the cryoresistance ratio was greater (p < 0.05) for these variables in semen recovered at noon. In ibexes, fresh semen collected at midnight was greater (p = 0.05) for VCL, VSL, VAP, and ALH values. After freezing-thawing, a significant increase in AQP3 labelling at the acrosomal region was observed in samples collected at midnight for both species, as well as in samples collected at noon for bucks (p < 0.05). An increase in the proportion of spermatozoa with AQP3 labelling in the mid-piece was detected in bucks in frozen-thawed samples collected at noon (p < 0.05). Testosterone concentrations were higher (p < 0.05) at midnight in bucks but no differences were found in ibexes. The findings reveal daily variations in sperm quality in both domestic and wild goats, which do not exactly coincide with the daily changes in cryoresistance, thus calling for attention when processing semen.

The WNT signaling pathway is a central regulator of cell polarity, adhesion, cytoskeletal dynamics, and lineage specification during early embryonic development. Although its roles have been extensively studied in murine and human models, the temporal regulation and pathway architecture of WNT signaling during early porcine development remain poorly defined. Here, we performed a comprehensive transcriptomic analysis to characterize WNT pathway dynamics across key stages of pig in vitro development, including immature oocytes (IMO), mature oocytes (MO), zygotes (ZY), cleaved embryos (2-4 cells; CL), and blastocysts (BL). Global analyses revealed major transcriptomic transitions (FDR <0.05; |Fold Change| ≥2) during oocyte maturation and blastocyst formation, whereas zygotes and cleaved embryos exhibited highly similar expression profiles. Module-based and gene-level analyses showed that oocyte maturation is associated with increased expression of extracellular WNT antagonists and components of the β-catenin destruction complex, together with selective regulation of Frizzled receptors, consistent with tight control of canonical WNT signaling at the MII stage. Following fertilization, this inhibitory configuration was partially relieved, alongside transient upregulation of specific WNT ligands, transcriptional mediators, and adhesion-related components during zygotic genome activation and early cleavage. At the blastocyst stage, WNT signaling became increasingly associated with planar cell polarity and epithelial organization modules. Together, the data reveal a highly dynamic and stage-specific restructuring of WNT signaling during early porcine development. Our findings indicate that precise temporal modulation-rather than uniform activation-of WNT pathway components accompanies the porcine oocyte-to-embryo transition, providing a molecular framework to better understand early developmental regulation and offering insights relevant to reproductive biotechnology and developmental biology.

Efficient superovulation protocols are essential to improve efficiency and facilitate scheduling in porcine embryo transfer (ET) programs. Although hCG is widely used to synchronize ovulation following eCG-based superovulation, constraints in its availability have highlighted the need for hCG-free strategies. GnRH agonists could expand ovulation induction options within eCG-based superovulation protocols. In this study, multiparous weaned donor sows received eCG alone (1000 IU, 24 h post-weaning), eCG plus buserelin, a GnRH agonist (10 µg at estrus onset), or no hormonal treatment (control), and embryos were recovered on Day 6 after estrus to evaluate ovulatory response, embryo production efficiency, developmental synchrony, and blastocyst transcriptomic profiles. Both hormonal protocols independently increased ovulatory response by approximately 48% (~37 vs 25 corpora lutea; P < 0.001) and doubled transferable embryo production efficiency compared with controls (P < 0.001), without affecting pregnancy and fertilization rates, embryonic viability, total cell number, or apoptosis. Untreated sows showed the greatest developmental dispersion and a higher proportion of hatched blastocysts, whereas eCG-treated sows showed intermediate variability. The addition of buserelin further improved ovulation-embryo synchrony, with most embryos at the blastocyst stage and all viable embryos classified as transferable. Transcriptomic profiling identified a limited number of differentially expressed genes between treated and control embryos. In conclusion, both hCG-free treatments increased embryo production in weaned sows without adversely affecting the Day-6 blastocyst quality parameters assessed, whereas the addition of buserelin further improved embryo-stage synchrony at collection. Overall, both protocols represent practical hCG-free strategies for in vivo porcine embryo production under commercial conditions.

[This corrects the article DOI: 10.3389/fvets.2025.1716588.].

Despite the apparent progress in reproductive technologies in wild ruminant species, healthy live births have been limited. Acquiring a sound knowledge of the molecular basis of most functional aspects of spermatozoa will improve the effectiveness of reproductive techniques and optimise conservation programs for threatened species. CatSper channels, opioid receptors and CD44 are involved in sperm capacitation of humans and domestic animals, but their presence in wild ruminants is yet undisclosed. The aim of this study was to determine the presence and localisation of CatSper 1-4, μ, δ and κ-opioid receptors and CD44 in three wild ruminant species spermatozoa (aoudad [n = 5], Iberian ibex [n = 5], mouflon [n = 5]), which show different resistance to freezing-thawing processes. Western blotting (WB) and immunocytochemistry (ICC) performed with commercially available antibodies revealed that aoudad, Iberian ibex and mouflon are equipped with the aforementioned channels and receptors, sharing localisation with other domestic animals' spermatozoa but presenting species-particularities. WB revealed homogeneous results in CatSper 1, Catsper 2, Catsper 3 and CatSper 4 among the spermatozoa of the three species, unlike μ, δ and κ-opioid and CD44 receptors that showed substantial inter-species differences in the number of bands. ICC showed inter-species differences in the location of CatSper 1-4, μ, δ and κ-opioid and CD44 receptors. Data confirmed their presence and putative role on sperm function in wild ruminant species. Inter-species differences in the location of CatSper 1-4, μ, δ and κ-opioid and CD44 receptors might underlie the variable response to reproductive technologies in these species.

Embryo transfer (ET) has transformed swine biotechnology, enabling genetic advancements and disease control. However, its success remains inconsistent, partly due to immune-mediated challenges at the maternal-fetal interface. This study explores the immunological environment of hemi-allogeneic pregnancies (via artificial insemination, AI) versus allogeneic pregnancies (via ET) in pigs during the critical implantation phase. Sows were categorized into groups based on pregnancy type and fetal counts, reflecting varying outcomes. Endometrial immune cell populations, including T lymphocytes, regulatory T cells (Tregs), natural killer (NK) cells, and macrophages, were analyzed using immunohistochemistry. Notably, allogeneic pregnancies with poor outcomes displayed elevated NK cell and macrophage infiltration alongside reduced Treg presence, contributing to a pro-inflammatory environment. In contrast, allogeneic pregnancies with favorable outcomes exhibited immune profiles resembling hemi-allogeneic pregnancies, suggesting enhanced maternal-fetal tolerance. These findings underscore the immunological variability driving embryo survival rates in ET pregnancies and highlight key cellular targets for improving reproductive efficiency. Further research on immunomodulatory strategies is vital to optimize ET success in swine and other species.

Embryo transfer (ET) is a valuable reproductive technology in pigs, albeit its efficiency remains significantly lower than that of natural mating or artificial insemination (AI), owing to high embryonic death rates. Critical for embryo survival and pregnancy success is the placenta, which supports conceptus development through nutrient exchange, hormone production, and immune modulation. Alterations in placental development and function may therefore underlie the reduced efficiency of ET. To investigate the molecular mechanisms underlying this dysfunction, crossbred (Landrace × Large White) recipient sows were hormonally synchronized and either inseminated on the onset of estrus (Day 0), (Control group; n = 8) or surgically transferred 23 morulae on Day 5 post-estrus (Allogeneic group; n = 8). Placental samples were collected from four sows per group on Day 18 (attachment phase) and Day 24 (early placentation) of pregnancy. Libraries were prepared using the TruSeq Stranded mRNA kit (Illumina). Sequencing was performed on a NextSeq 550 platform (2 ×75 bp paired-end), and reads were aligned to the Sus scrofa 11.1 genome. Differential gene expression analysis (FC>2/<-2, P < 0.05) revealed significant downregulation of key angiogenic and immune-regulatory genes in allogeneic placentas. On Day 18, VEGFA, NOS3, FGF9, HIF1A, STAT1, STAT3, SMAD1, SMAD4, SMAD5, and ETS1 were markedly reduced, indicating impaired vascular remodeling and immune modulation. By Day 24, SMAD2 and additional immune-related genes remained dysregulated. Conversely, ANGPTL2 was upregulated on Day 24, possibly due to a compensatory angiogenic response. The data suggest that full allogeneicity disrupts placental transcriptomic programs crucial for angiogenesis and immune tolerance, contributing to the high embryonic mortality observed after ET in swine.

Claw lesions (CLs) are highly prevalent in sow herds, affecting animal welfare and productivity. This study aimed to evaluate the incidence of CLs in pregnant sows and their impact on litter performance. Additionally, we assessed hair cortisol, cortisone, and oxytocin levels to determine whether CLs cause any stress in sows. The study involved 693 hyper-prolific sows, initially assessed for CLs at farrowing. After weaning, they were housed in individual AI crates, where their hair was shaved. At 28 days post-insemination, pregnant sows were moved to group pens. One week before expected farrowing, sows (n = 507) were transferred to individual farrowing crates where newly grown hair samples were collected, and CLs reassessed. Claw lesion severity was scored from 0 to 3 (SS0: no lesions, SS1: mild, SS2: moderate, SS3: severe). Sows were then classified into 3 categories (CAT): CAT1 (SS0 + SS1), CAT (SS1 + SS2, not SS3), and CAT3 (SS1 + SS2 + SS3). The total number of CL per sow and the final sow score were calculated as the sum of CLs observed on the claws and the sum of all CL severity scores, respectively. Only 4,1

Interleukin 6 (IL-6) is a cytokine physiologically expressed within the reproductive tract, where it plays a crucial role in cellular communication and processes such as ovulation and implantation. These processes and the presence of embryos alter the immune response of the female reproductive tract in vivo, a physiological event of utmost relevance. Mimicking such physiological environment may optimize porcine in vitro embryo production (IVP). This study aimed to assess the impact of IL-6 supplementation during in vitro maturation (IVM) and/or in vitro culture (IVC) on the developmental competence of pig oocytes fertilized in vitro. In the first experiment, cumulus-oocytes complexes (COCs) were matured in media containing graded IL-6 concentrations (0-200 ng/mL). IL-6 did not affect maturation, fertilization, cleavage, or blastocyst formation rates. However, 100 ng/mL IL-6 significantly (P < 0.05) improved chromosome alignment, meiotic spindle formation, blastocyst efficiency, and total cell number compared to controls. In the second experiment, COCs were matured and fertilized without IL-6 supplementation. The resulting presumptive zygotes were cultured in IVC media with graded concentrations of IL-6 (0-200 ng/mL). IL-6 supplementation during IVC did not enhance developmental competence. Conversely, the highest IL-6 concentration significantly (P < 0.05) reduced cleavage rate. In the third experiment, combined supplementation of 100 ng/mL IL-6 during IVM and 10 ng/mL during IVC showed no additional improvements compared to IL-6 supplementation during IVM alone. In conclusion, the results of this study indicate that IL-6, particularly when added during oocyte maturation, positively impacts porcine in vitro embryo production.

BACKGROUND:Predicting male fertility in farm animals remains a challenge. Seminal plasma (SP) contains a high amount of heterogeneous seminal extracellular vesicles (sEVs), believed involved in reproductive processes and maybe key to understanding male fertility. AIMS:To identify the sEV proteins that are differentially expressed between more and less fertile boars and that could be candidates for fertility biomarkers in boars used in artificial insemination (AI) programs. MATERIALS AND METHODS:Small (S) and large (L) sEV subsets from SP samples of AI boars with differences in fertility: high (H) or low (L) farrowing rate (FR) and large (L) or small (S) litter size (LS). The S- and L-sEV subsets were isolated by size exclusion chromatography and characterized according to the Minimal Information for Studies of Extracellular Vesicles (MISEV2023) guidelines. Proteomic analyses (three biological replicates per fertility group and sEV subset) were performed using a Bruker timsTOF fleX™ instrument with data-independent acquisition parallel accumulation-serial fragmentation (diaPASEF) technology. RESULTS:A total of 470 and 726 proteins were quantified in S-sEVs and 1801 and 1834 proteins in L-sEVs from FR and LS boars, respectively. Differentially expressed sEV proteins (log2fold change ≥±1, p ≤ 0.05 and effect size d of Cohen >2.0) were found between the fertility groups: seven in S-sEVs and 52 in L-sEVs between H-FR and L-FR boars, and 47 in S-sEVs and 52 in L-sEVs between L-LS and S-LS boars. Many of these differentially expressed sEV proteins are involved in reproductive processes, particularly in sperm function and sperm-zona pellucida binding, but also in embryo development and implantation. CONCLUSIONS:The sEV proteome differs between more and less fertile boars, with many of the differentially expressed proteins known as involved in reproductive processes. This would suggest that sEVs may be involved in male fertility and that some of the differentially expressed sEV proteins could be potential fertility markers for AI boars.

It was back in 1677 when most researchers agreed that spermatology started, supported by van Leeuwenhoek's report about sperm discovery when demonstrating his novel microscope. Since then, multiple approaches have been applied to elucidate key aspects of the morphology of the male gamete and whether they would prognose fertility. Curiosity advanced in parallel with the many analytical tools, including the currently highlighted omics, which opened a yet unexplored scenario where researchers are worldwide trying to describe in detail not only the structure but also the mechanism behind sperm function.

In vitro embryo production (IVP) is of great importance to the porcine industry, as well as for basic research and biomedical applications. Despite the large efforts made in laboratories worldwide to address suboptimal culture conditions, porcine IVP remains inefficient. Nobiletin (Nob, 5,6,7,8,3´,4´ hexamethoxyflavone) supplementation to in vitro culture (IVC) medium, enhances in vitro embryo development in various species. However, its impact on the quality and developmental capacity of in vitro-produced pig embryos is yet to be established. This study evaluated the effects of different concentrations (2.5 and 5 μM) of Nob during the early culture of in vitro-produced pig embryos on embryo developmental competence, mitochondrial activity, lipid content, intracellular Reactive Oxygen Species (ROS) and Glutathione (GSH) content, Total Cell Number (TCN) per blastocyst, and expression of genes related to embryo development, quality and oxidative stress. Embryos cultured in medium without Nob supplementation and in medium supplemented with 0.01% dimethyl sulfoxide (DMSO-vehicle for Nob) constituted the Control and DMSO groups, respectively. Embryo development rates were evaluated on Days 2, 6 and 7 of IVC. Additionally, a representative group of embryos was selected to assess mitochondrial activity, lipid, ROS and GSH content (on Days 2 and 6 of IVC), TCN assessment and gene expression analyses (on Day 6 of IVC). No significant differences were observed in any of the parameters evaluated on Day 2 of IVC. In contrast, embryos cultured under the presence of Nob 2.5 showed higher developmental rates on Days 6 and 7 of IVC. In addition, Day 6 embryos showed increased mitochondrial activity, with decreased levels of ROS and GSH in the Nob 2.5 group compared to the other groups. Both Nob 2.5 and Nob 5 embryos showed higher TCN compared to the Control and DMSO groups. Furthermore, Nob 2.5 and Nob 5 upregulated the expression of Superoxide dismutase type 1 (SOD1) and Glucose-6-phosphate dehydrogenase (G6PDH) genes, which could help to counteract oxidative stress during IVC. In conclusion, the addition of Nob during the first 48 h of IVC increased porcine embryo development rates and enhanced their quality, including the upregulation of relevant genes that potentially improved the overall efficiency of the IVP system.

microRNAs play pivotal roles during mammalian reproduction, including the cross-talk between gametes, embryos and the maternal genital tract. Mating induces changes in the expression of mRNA transcripts in the female, but whether miRNAs are involved remains to be elucidated. In the current study, we mapped 181 miRNAs in the porcine peri-ovulatory female reproductive tract: Cervix (Cvx), distal and proximal uterus (Dist-Ut, ProxUt), Utero-tubal-junction (UTJ), isthmus (Isth), ampulla (Amp), and infundibulum (Inf) when exposed to semen (natural mating (NM) or artificial insemination (AI-P1)) or to infusions of sperm-free seminal plasma (SP): the first 10 mL of the sperm rich fraction (SP-P1) or the entire ejaculate (SP-E). Among the most interesting findings, NM decreased mir-671, implicated in uterine development and pregnancy loss prior to embryo implantation, in Cvx, Dist-UT, Prox-UT, Isth, and Inf, while it increased in Amp. NM and SP-E induced the downregulation of miRlet7A-1 (Dist-UT, Prox-UT), a regulator of immunity during pregnancy. miR-34C-1, a regulator of endometrial receptivity gene expression, was increased in Dist-UT, UTJ and Amp (NM), in Prox-UT (AI-P1), and in Amp (SPP1). miR-296, a modulator of the inflammatory response and apoptosis, was upregulated in the UTJ (all treatments). NM elicited the highest miRNA activity in the sperm reservoir (UTJ), suggesting that key-regulators such as miR-34c or miR-296 may modulate the metabolic processes linked to the adequate preparation for gamete encounter in the oviduct. Our results suggest that SP should be maintained in AI to warrant miRNA regulation within the female genital tract for reproductive success.

This work was aimed to identify androgen receptors (AR) in the spermatozoa of wild and domestic ruminants and to assess the effect of testosterone on sperm localization of aquaporin-3 (AQP3) and cryopreservation process. Sperm samples from wild species were incubated with testosterone (T group), 1,3-propanediol (PDO group), phloretin (PHL group), PDO+T group, PHL+T group. Western blot identified the presence of AR as a single band of about 48 KDa. Immunolabelling of AR was located in the equatorial segment of the sperm head. In mouflons, the cryoresistance ratio for acrosome integrity was lower (p < 0.05) in the PHL+T than in Control and T groups. In ibexes, the cryoresistance ratio for acrosome integrity was lower (p < 0.05) in the PHL+T, PHL, and T group than in the Control group; the cryoresistance ratios for sperm kinematic variables were lower (p < 0.05) in PDO+T than in Control. No changes were found among treatments in the proportion of spermatozoa showing AQP3 in the different membrane domains after incubation and thawing in both mouflon and ibex. In conclusion, testosterone negatively affected sperm cryoresistance expressed as acrosome integrity, enhancing the effects of the AQP blocker PHL. Our findings provide a sound knowledge of the molecular mechanisms that explain the seasonal variation in sperm freezability from ruminants.

Despite decades of research and handling of semen for use in artificial insemination (AI) and other assisted reproductive technologies, 5-10% of selected boar sires are still considered sub-fertile, escaping current assessment methods for sperm quality and resilience to preservation. As end-product, the ejaculate (emitted spermatozoa sequentially exposed to the composite seminal plasma, the SP) ought to define the homeostasis of the testes, the epididymis, and the accessory sexual glands. Yet, linking findings in the ejaculate to sperm production biology and fertility is suboptimal. The present essay critically reviews how the ejaculate of a fertile boar can help us to diagnose both reproductive health and resilience to semen handling, focusing on methods -available and under development- to identify suitable biomarkers for cryotolerance and fertility. Bulk SP, semen proteins and microRNAs (miRNAs) have, albeit linked to sperm function and fertility after AI, failed to enhance reproductive outcomes at commercial level, perhaps for just being components of a complex functional pathway. Hence, focus is now on the interaction sperm-SP, comparing in vivo with ex vivo, and regarding nano-sized lipid bilayer seminal extracellular vesicles (sEVs) as priority. sEVs transport fragile molecules (lipids, proteins, nucleic acids) which, shielded from degradation, mediate cell-to-cell communication with spermatozoa and the female internal genital tract. Such interaction modulates essential reproductive processes, from sperm homeostasis to immunological female tolerance. sEVs can be harvested, characterized, stored, and manipulated, e.g. can be used for andrological diagnosis, selection of breeders, and alternatively be used as additives to improve cryosurvival and fertility.

Objective: To detect whether intravaginal exposure to prepared seminal plasma led to an absolute increase in live birth rate (LBR) after in vitro fertilization (IVF) by 10% compared with placebo. It has been suggested that intravaginal deposition of seminal plasma after ovum pick -up (OPU) for IVF treatment, increases pregnancy and LBRs. Design: Double -blind, placebo-controlled prospective study. An outcome assessment was made before the type of intervention was unblinded. The outcome data were analyzed according to an intention-to-treat protocol. Setting: University Hospital. Patients: Couples scheduled for an IVF treatment cycle: in total, 792 couples (393 in the seminal plasma group and 399 in the control group) were recruited over a 5-year period of inclusion in a single -center setting. Intervention: On the day of OPU, the couples were randomized into groups receiving either vaginal deposition of prepared seminal plasma from the partner or saline. Both participants and the physician were blind to the grouping. Main Outcome Measures: The primary outcome was a live birth (LB). The secondary outcomes were a positive pregnancy test, de fi ned as human chorionic gonadotropin identi fi ed in urine 3 weeks after OPU , and clinical pregnancy, de fi ned as an intrauterine viable pregnancy assessed using transvaginal sonography after 5 - 7 weeks. Results: In the index group, 35.4% had a positive pregnancy test (relative risk [RR],0.93; 95% con fi dence interval {CI} 0.78 - 1.10), 28.8% had a clinical pregnancy (RR 1.00, 95% CI 0.97 - 1.03), and 26.5% had a LB (RR 0.86; 95% CI 0.70 - 1.07), adjusted for day of transfer, female age, and number of fertilized oocytes. Corresponding rates in the control group were 37.3%, 33.6%, and 29.8%. No statistically signi fi cant differences regarding outcomes between the two intervention groups were found. Conclusion: Prepared seminal plasma applied in the vagina directly after OPU did not increase the rates of LB or clinical pregnancies. The importance of immunological factors to allow the implantation of an embryo is not questioned, but no improvement in the LBRs in IVF treatment by introducing the male partner ' s prepared seminal plasma after OPU could be found. Clinical Trial Registration Number: Clinicaltrials.gov, ID NCT02716753. Registration date 17 March, 2016, fi rst enrollment November, 2016, completed March, 2023. (Fertil Steril (R) 2024;122:131 - 9. (c) 2024 by American Society for Reproductive Medicine.) El resumen est & aacute; disponible en Espa & ntilde;ol al final del art & iacute;culo.

This study examined how the vitrification of pig blastocysts using either the superfine open pulled straw (SOPS) or Cryotop method affects the expression profile of embryonic microRNA (miRNA) transcriptomes, as well as its relation to changes in the expression of target genes (TGs). Surgically collected pig blastocysts were vitrified using either the SOPS method (n = 60; 4 - 6 embryos/device) or the Cryotop system (n = 60; 20 embryos/device). Embryos were cultured in vitro for 24 h after warming. Fresh blastocysts (n = 60) cultured for 24 h served as controls. After in vitro culture, five pools of eight viable blastocysts from each group were prepared for miRNA expression analysis based on a microarray approach. Then, biological interpretation of miRNAs profiles and integrative analysis of miRNA and mRNA transcriptome data were performed. Survival after 24 h of in vitro culture was similar ( >96 %) for both the vitrification systems and the control group (100 %). Compared with the controls, the SOPS -vitrified blastocysts had 94 (one upregulated and 93 downregulated) differentially expressed (DE) miRNAs, and the Cryotop-vitrified blastocysts had 174 DE miRNAs (one upregulated and 173 downregulated). One DE miRNA (miR-503) in the SOPS group and three DE miRNAs (miR-7139 - 3p, miR-214 and miR-885 - 3p) in the Cryotop group were annotated for Sus scrofa . The integrative analysis showed that 27 and 61 DE TGs were regulated by the DE miRNAs in blastocysts vitrified with the SOPS and Cryotop systems, respectively. The TGs enriched one pathway (the TGF- beta signaling pathway) for the SOPS system and four pathways (HIF-1, Notch, ascorbate and aldarate metabolism and glycosphingolipid biosynthesis-ganglio series) for the Cryotop system. In summary, vitrification via the SOPS and Cryotop systems dysregulates miRNAs, with slight differences between methods. The altered miRNAs identified in this study were related mainly to cell proliferation, apoptosis, and the response to cell stress. Further studies are needed to clarify the consequences of dysregulation of miRNAs involved in the TGF- beta (SOPS -vitrified blastocyst) and Notch (Cryotop-vitrified blastocyst) signaling pathways, particularly if they can affect embryonic development.