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Sökning: WFRF:(Parrilla Inmaculada)

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1.
  • Martinez, Cristina A., et al. (författare)
  • The overlaying oil type influences in vitro embryo production: differences in composition and compound transfer into incubation medium between oils
  • 2017
  • Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
  • Tidskriftsartikel (refereegranskat)abstract
    • The oil overlay micro-drop system is widely used for cultures of mammalian gametes and embryos. We evaluated hereby the effects of two unaltered commercial oils-Sigma mineral oil (S-MO) and Nidoil paraffin oil (N-PO)-on in vitro embryo production (IVP) outcomes using a pig model. The results showed that while either oil apparently did not affect oocyte maturation and fertilization rates, S-MO negatively affected embryo cleavage rates, blastocyst formation rates, and, consequently, total blastocyst efficiency of the system. No differences in the oxidation state were found between the oils or culture media incubated under S-MO or N-PO. Although both oils slightly differed in elemental composition, there were no differences in the concentrations of elements between fresh media and media incubated under oils. By contrast, we demonstrated clear oil-type differences in both the composition of volatile organic compounds (VOC) and the transfer of some of these VOCs (straight-chain alkanes and pentanal and 1,3-diethyl benzene) to the culture medium, which could have influenced embryonic development.
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2.
  • Alkmin, Diego V., et al. (författare)
  • Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate
  • 2014
  • Ingår i: Cryobiology. - : Elsevier. - 0011-2240 .- 1090-2392. ; 69:2, s. 203-210
  • Tidskriftsartikel (refereegranskat)abstract
    • Boar bulk ejaculates are now being collected instead of usual sperm-rich fractions (SRF) for artificial insemination purpose. The present study evaluated the influence of holding boar sperm samples before freezing surrounded in their own seminal plasma (SP), from either fractions/portions or the entire ejaculate, on post-thawing sperm quality and functionality. Ejaculates collected as bulk (BE) or as separate (first 10 mL of SRF [P1] and rest of SRF [P2]) from 10 boars were held 24 h at 15-17 degrees C and then frozen. Some bulk ejaculate samples were frozen immediately after collections as Control. In addition, epididymal sperm samples from the same 10 boars were collected post-mortem and extended in SP from P1 (EP1), P2 (EP2) and post SRF (EP3), and also held 24 h before freezing for a better understanding of the influence of SP on boar sperm cryopreservation. The sperm quality (motility, evaluated by CASA, and viability, evaluated by flow cytometry) and functionality (flow cytometry assessment of plasma membrane fluidity, mitochondrial membrane potential and intracellular generation of reactive oxygen species [ROS] in viable sperm) were evaluated at 30, 150 and 300 min post-thaw. Post-thawing sperm quality and functionality of P1 and P2 were similar but higher (p less than0.01) than BE samples. Control samples showed higher (p less than 0.01) post-thaw sperm quality and functionality than BE samples. Post-thawing sperm quality and functionality of EP1 and EP2 were similar but higher (p less than 0.05) than EP3. These results showed that boar sperm from BE are more cryosensitive than those from the SRF, particularly when held 24 h before freezing, which would be attributable to the cryonegative effects exerted by the SP from post SRF.
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3.
  • Barranco, Isabel, et al. (författare)
  • Active paraoxonase 1 is synthesised throughout the internal boar genital organs.
  • 2017
  • Ingår i: Reproduction (Cambridge, England). - : Bioscientifica. - 1741-7899. ; 154:3, s. 237-243
  • Tidskriftsartikel (refereegranskat)abstract
    • The paraoxonase type 1 (PON1) is an enzyme with antioxidant properties recently identified in the seminal plasma (SP) of several species, including the porcine. The aims of the present study were to (1) describe the immunohistochemical localisation of PON1 in the genital organs of fertile boars and (2) evaluate the relationship among PON1 activity and high-density lipoprotein cholesterol (HDL-C) concentration in fluids of the boar genital organs. Immunohistochemical analysis demonstrated that PON1 was present in testis (specifically in Leydig cells, blood vessels, spermatogonia and elongated spermatids), epididymis (specifically in the cytoplasm of the principal epithelial cells, luminal secretion and in the surrounding smooth muscle) and the lining epithelia of the accessory sexual glands (cytoplasmic location in the prostate and membranous in the seminal vesicle and bulbourethral glands). The Western blotting analysis confirmed the presence of PON1 in all boar genital organs, showing in all of them a band of 51 kDa and an extra band of 45 kDa only in seminal vesicles. PON1 showed higher activity levels in epididymal fluid than those in SP of the entire ejaculate or of specific ejaculate portions. A highly positive relationship between PON1 activity and HDL-C concentration was found in all genital fluids. In sum, all boar genital organs contributing to sperm-accompanying fluid/s were able to express PON1, whose activity in these genital fluids is highly dependent on the variable HDL-C concentration present.
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4.
  • Barranco, Isabel, et al. (författare)
  • Extracellular vesicles isolated from porcine seminal plasma exhibit different tetraspanin expression profiles
  • 2019
  • Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
  • Tidskriftsartikel (refereegranskat)abstract
    • Seminal extracellular vesicles (EVs) include exosomes (phi 40-120 nm) and microvesicles (MVs, phi 120-1000 nm), which would be involved in multiple functional reproductive roles. The study aimed to establish which EV subtypes are present in pig semen, using a high-resolution flow cytometer to explore differences in their tetraspanin expression profile. The EVs were isolated from 12 pig ejaculates using serial ultracentrifugation and characterized by dynamic light scattering and electron microscopy for size and morphology as well as for tetraspanin expression using flow cytometry with Carboxyfluorescein succinimidyl ester (CFSE) and antibodies against CD9, CD63 and CD81. Pig semen contained a heterogeneous EV-population regarding size and morphology. Flow cytometric analysis demonstrated that the proportion of EVs expressing CD63 and CD9 was higher in MVs (P amp;lt; 0.001 and P amp;lt; 0.05, respectively) than in exosomes, while the opposite was true for CD81; higher (P amp;lt; 0.001) in exosomes than in MVs. In conclusion, (1) the new generation of flow cytometers are able to accurately identify EVs and to gate them in two size-different populations named exosomes and MVs. (2) Tetraspanins CD9, CD63 and CD81 are present in both seminal EVs, albeit with exosomes and MVs differing in expression profiles, suggesting dissimilar cargo and binding affinity.
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5.
  • Barranco, Isabel, et al. (författare)
  • Glutathione Peroxidase 5 Is Expressed by the Entire Pig Male Genital Tract and Once in the Seminal Plasma Contributes to Sperm Survival and In Vivo Fertility
  • 2016
  • Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 11:9
  • Tidskriftsartikel (refereegranskat)abstract
    • Glutathione peroxidase-5 (GPX5) is an H2O2-scavenging enzyme identified in boar seminal plasma (SP). This study attempted to clarify its origin and role on sperm survival and fertility after artificial insemination (AI). GPX5 was expressed (Western blot and immunocytochemistry using a rabbit primary polyclonal antibody) in testes, epididymis and accessory sex glands (6 boars). SP-GPX5 concentration differed among boars (11 boars, P amp;lt; 0.001), among ejaculates within boar (44 ejaculates, P amp;lt; 0.001) and among portions within ejaculate (15 ejaculates). The first 10 mL of the spermrich fraction (SRF, sperm-peak portion) had a significantly lower concentration (8.87 +/- 0.78 ng/mL) than the rest of the SRF and the post-SRF (11.66 +/- 0.79 and 12.37 +/- 0.79 ng/mL, respectively, P amp;lt; 0.005). Spermmotility of liquid-stored semen AI-doses (n = 44, at 15-17 degrees C during 72h) declined faster in AI-doses with low concentrations of SP-GPX5 compared to those with high-levels. Boars (n = 11) with high SP-GPX5 showed higher farrowing rates and litter sizes than those with low SP-GPX5 (a total of 5,275 inseminated sows). In sum, GPX5 is widely expressed in the boar genital tract and its variable presence in SP shows a positive relationship with sperm quality and fertility outcomes of liquid-stored semen AI-doses.
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6.
  • Barranco, Isabel, et al. (författare)
  • High total antioxidant capacity of the porcine seminal plasma (SP-TAC) relates to sperm survival and fertility
  • 2015
  • Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 5:18538
  • Tidskriftsartikel (refereegranskat)abstract
    • The study attempted to clarify the role of total antioxidant capacity of seminal plasma (SP-TAC) on boar sperm survival and fertility after artificial insemination (AI). SP-TAC differed (P < 0.001) among boars (no = 15) and, to a lesser degree, among ejaculates within male (4 ejaculates/boar). SP-TAC also differed (P < 0.001) among ejaculate fractions (43 ejaculates and 3 fractions per ejaculate), of which the sperm-peak portion of the sperm rich ejaculate fraction (SRF) had the highest SP-TAC. SP-TAC was not correlated with sperm quality (motility and viability) or functionality (intracellular ROS generation and lipid peroxidation) of liquid AI-semen samples stored at 17 degrees C for 72 h (90 AI-samples), but the decline in sperm quality was larger (P < 0.05) in ejaculates with low, compared with high SP-TAC (hierarchically grouped). The SP-TAC differences among ejaculate portions agree with sperm cryosurvival rates (14 ejaculates from 7 boars), showing sperm from sperm-peak portion better (P < 0.01) post-thaw quality and functionality than those from the entire ejaculate (mainly post-SRF). Boars (no = 18) with high SP-TAC (hierarchically grouped) had higher (P < 0.05) fertility outcomes (5,546 AI-sows) than those with low SP-TAC. Measurement of SP-TAC ought to be a discriminative tool to prognosis fertility in breeding boars.
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7.
  • Barranco, Isabel, et al. (författare)
  • Levels of activity of superoxide dismutase in seminal plasma do not predict fertility of pig AI-semen doses
  • 2019
  • Ingår i: Theriogenology. - : ELSEVIER SCIENCE INC. - 0093-691X .- 1879-3231. ; 140, s. 18-24
  • Tidskriftsartikel (refereegranskat)abstract
    • Superoxide dismutase (SOD) is a major antioxidant enzyme in boar seminal plasma (SP). This study evaluated how SP-SOD affected sperm attributes when semen of boars of various breeds, included in commercial artificial insemination (Al)-programs, was extended and liquid-stored at 17 degrees C for AI; as well as their in vivo fertility (farrowing rate and litter size of 10,952 AI-sows). SP-SOD-activity was assessed in 311 ejaculates (100 boars) while sperm motility (by CASA), viability and intracellular H2O2 generation in viable spermatozoa (by flow cytometry) were measured at 0 and 72 h of liquid storage. SP-SOD activity was not affected by breed but differed (P amp;lt; 0.001) between boars (n = 50), ranging from 1.16 +/- 0.11 to 7.02 +/- 0.75 IU/mL. Semen Al-doses (n =44) hierarchically grouped (P amp;lt; 0.001) with low SP-SOD activity showed lower (P amp;lt; 0.05) sperm motility and intracellular H2O2 at 72 h of liquid storage. Fertility did not differ between AI-boars (n = 39) hierarchically grouped (P amp;lt; 0.001) with high or low SP-SOD activity. In conclusion, SP-SOD activity is boar dependent and positively related with sperm functionality of liquid stored semen AI-doses. However, this positive effect is not reflected on in vivo fertility post-AI. (C) 2019 Elsevier Inc. All rights reserved.
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8.
  • Barranco, Isabel, et al. (författare)
  • Seminal Plasma Cytokines Are Predictive of the Outcome of Boar Sperm Preservation
  • 2019
  • Ingår i: Frontiers in Veterinary Science. - : Frontiers Media S.A.. - 2297-1769. ; 6
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Boar seminal plasma is rich in cytokines, which could influence the capability of spermatozoa to tolerate preservation.Objectives: To evaluate the involvement of boar seminal plasma cytokines in the changes experienced by boar spermatozoa during their storage, either in liquid or frozen state.Materials and Methods: In two separated experiments, semen samples from healthy and fertile boars were split in two aliquots, one centrifuged twice (1,500 ×g for 10 min) to harvest seminal plasma, whereas the other was either commercially extended (3 × 107 sperm/mL) and liquid-stored at 17°C during 144 h (n = 28, Experiment 1) or frozen-thawed using a standard 0.5 mL protocol (n = 27, Experiment 2). Sixteen cytokines were quantified using Luminex xMAP®. Sperm attributes (CASA-evaluated total and progressive motility; flow cytometry-evaluated sperm viability, production of intracellular H2O2 and O2•-" role="presentation" style="box-sizing: border-box; display: inline; line-height: normal; word-spacing: normal; overflow-wrap: normal; white-space: nowrap; float: none; direction: ltr; max-width: none; max-height: none; min-width: 0px; min-height: 0px; border: 0px; padding: 0px; margin: 0px; position: relative; outline: 0px !important;">O∙−2O2•- and levels of lipid peroxidation in viable spermatozoa) were evaluated either at 0, 72, or 144 h of liquid storage (Experiment 1) or before freezing and at 30- and 150-min post-thawing (Experiment 2).Results: Multiple linear regression models, with Bayesian approach for variable selection, revealed that the anti-inflammatory TGF-β2, TGF-β3, IL-1Ra, and IL-4 and the pro-inflammatory IL-8 and IL-18, predicted changes in sperm motility for liquid-stored semen while the anti-inflammatory IFN-γ was included in the models predicting changes in all sperm attributes for cryopreserved semen.Conclusion: Specific boar seminal plasma cytokines would contribute to modulate the structural and metabolic changes shown by spermatozoa during preservation, either in liquid or frozen state.
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9.
  • Barranco, Isabel, et al. (författare)
  • Seminal Plasma Modulates miRNA Expression by Sow Genital Tract Lining Explants
  • 2020
  • Ingår i: Biomolecules. - : MDPI. - 2218-273X. ; 10:6
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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10.
  • Cambra, Josep M., et al. (författare)
  • Allogeneic Embryos Disregulate Leukemia Inhibitory Factor (LIF) and Its Receptor in the Porcine Endometrium During Implantation
  • 2020
  • Ingår i: Frontiers in Veterinary Science. - : Frontiers Media S.A.. - 2297-1769. ; 7
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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11.
  • Cambra, Josep M., et al. (författare)
  • Cytokine profile in peripheral blood mononuclear cells differs between embryo donor and potential recipient sows
  • 2024
  • Ingår i: Frontiers in Veterinary Science. - : FRONTIERS MEDIA SA. - 2297-1769. ; 11
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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12.
  • Cuello, Cristina, et al. (författare)
  • Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model
  • 2021
  • Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:3
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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13.
  • Cuello, Cristina, et al. (författare)
  • Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae
  • 2021
  • Ingår i: Frontiers in Veterinary Science. - : Frontiers Media SA. - 2297-1769. ; 8
  • Tidskriftsartikel (refereegranskat)abstract
    • 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 developmental potential, despite the relatively high survival rates after warming observed in vitro. In conclusion, vitrification altered the gene expression pattern of porcine morulae produced in vivo, generating alterations in the transcriptome that may interfere with subsequent embryo development and pregnancy after embryo transfer.
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14.
  • Cuello, Cristina, et al. (författare)
  • Vitrification of pig embryos dysregulates the microRNA transcriptome profile
  • 2024
  • Ingår i: Theriogenology. - : ELSEVIER SCIENCE INC. - 0093-691X .- 1879-3231. ; 226, s. 243-252
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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15.
  • Gil, Maria A., et al. (författare)
  • In-depth proteome characterization of endometrium and extraembryonic membranes during implantation in pig
  • 2024
  • Ingår i: JOURNAL OF ANIMAL SCIENCE AND BIOTECHNOLOGY. - : BMC. - 1674-9782. ; 15:1
  • Tidskriftsartikel (refereegranskat)abstract
    • BackgroundProteome characterization of the porcine endometrium and extraembryonic membranes is important to understand mother-embryo cross-communication. In this study, the proteome of the endometrium and chorioallantoic membrane was characterized in pregnant sows (PS) during early gestation (d 18 and 24 of gestation) and in the endometrium of non-pregnant sows (NPS) during the same days using LC-MS/MS analysis. The UniProtKB database and ClueGO were used to obtain functional Gene Ontology annotations and biological and functional networks, respectively.ResultsOur analysis yielded 3,254 and 3,457 proteins identified in the endometrium of PS and NPS, respectively; of these, 1,753 being common while 1,501 and 1,704 were exclusive to PS and NPS, respectively. In addition, we identified 3,968 proteins in the extraembryonic membranes of PS. Further analyses of function revealed some proteins had relevance for the immune system process and biological adhesion in endometrium while the embryonic chorion displayed abundance of proteins related to cell adhesion and cytoskeletal organization, suggesting they dominated the moment of endometrial remodeling, implantation and adhesion of the lining epithelia. Data are available via ProteomeXchange with identifier PXD042565.ConclusionThis is the first in-depth proteomic characterization of the endometrium and extraembryonic membranes during weeks 3 to 4 of gestation; data that contribute to the molecular understanding of the dynamic environment during this critical period, associated with the majority of pregnancy losses.
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16.
  • Gonzalez-Plaza, Alejandro, et al. (författare)
  • Cryotop vitrification of large batches of pig embryos simultaneously provides excellent postwarming survival rates and minimal interference with gene expression
  • 2023
  • Ingår i: Theriogenology. - : ELSEVIER SCIENCE INC. - 0093-691X .- 1879-3231. ; 206
  • Tidskriftsartikel (refereegranskat)abstract
    • 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 SOPS group. In summary, vitrification with the OC system altered fewer genes related to apoptosis and activated genes related to cell proliferation. We conclude that vitrification with either the OC or SOPS system has a moderate to low effect on the transcriptome of in vivo-derived porcine blastocysts. Further investigation is needed to elucidate how the differences in the transcriptome of embryos vitrified with these systems affect their subsequent developmental ability after ET.
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17.
  • Gonzalez-Plaza, Alejandro, et al. (författare)
  • Equilibration time with cryoprotectants, but not melatonin supplementation during in vitro maturation, affects viability and metaphase plate morphology of vitrified porcine mature oocytes
  • 2022
  • Ingår i: Reproduction in domestic animals. - : Wiley. - 0936-6768 .- 1439-0531. ; 57:S5, s. 58-63
  • Tidskriftsartikel (refereegranskat)abstract
    • 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 parameters.
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18.
  • Gonzalez-Plaza, Alejandro, et al. (författare)
  • The Open Cryotop System Is Effective for the Simultaneous Vitrification of a Large Number of Porcine Embryos at Different Developmental Stages
  • 2022
  • Ingår i: Frontiers in Veterinary Science. - : Frontiers Media SA. - 2297-1769. ; 9
  • Tidskriftsartikel (refereegranskat)abstract
    • 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 developmental stages and provides comparable viability and quality results to fresh embryos subjected to 24 h of in vitro culture.
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19.
  • Gonzalez-Ramiro, Henar, et al. (författare)
  • A Short-Term Altrenogest Treatment Post-weaning Followed by Superovulation Reduces Pregnancy Rates and Embryo Production Efficiency in Multiparous Sows
  • 2021
  • Ingår i: Frontiers in Veterinary Science. - : Frontiers Media S.A.. - 2297-1769. ; 8
  • Tidskriftsartikel (refereegranskat)abstract
    • 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 in sows from the SS groups were immature oocytes. In conclusion, although superovulation of weaned sows was highly efficient, short-term administration of Altrenogest in combination with superovulation had negative effects on most of the reproductive parameters assessed, particularly affecting the overall efficiency of pregnancy and embryo production.
  •  
20.
  • Gonzalez-Ramiro, Henar, et al. (författare)
  • Combined synchronization and superovulation treatments negatively impact embryo viability possibly by the downregulation of WNT/beta-catenin and Notch signaling genes in the porcine endometrium
  • 2022
  • Ingår i: Journal of Animal Science. - : Oxford University Press. - 0021-8812 .- 1525-3163. ; 100:11
  • Tidskriftsartikel (refereegranskat)abstract
    • 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 of beta-catenin (Ctnnb1) gene expression in the SS-7 endometrium, suggesting that this treatment influences embryo-uterine dialogue by triggering a cascade of events leading to embryo maldevelopment. These data explain the proliferative defects in SS-7 embryos and suggest a novel mechanism of a porcine embryo-maternal crosstalk. Lay Summary Methods for porcine superovulation (increasing the number of ovulated oocytes per cycle) and estrus synchronization (grouping estrus sows on the same day) are available for assisted reproductive technologies, using hormonal treatments. The main goal of the present study was to understand how hormones used for these purposes influence gene expression patterns in the female reproductive tract (ovaries and endometrium). We observed that hormonal treatments (synchronization combined with superovulation) have the potential to alter ovarian and endometrial gene expression patterns, triggering improper follicle development and oocyte growth, and leading to abnormal embryonic development before implantation. Genes involved in two key metabolic pathways for embryo development (Wnt/beta-catenin and Notch signaling pathways) were dysregulated in reproductive tissues.
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21.
  • Gonzalez-Ramiro, Henar, et al. (författare)
  • The Use of a Brief Synchronization Treatment after Weaning, Combined with Superovulation, Has Moderate Effects on the Gene Expression of Surviving Pig Blastocysts
  • 2023
  • Ingår i: Animals. - : MDPI. - 2076-2615. ; 13:9
  • Tidskriftsartikel (refereegranskat)abstract
    • 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, such as the glutathione metabolism pathway, with down-regulated genes related to cellular detoxification of reactive oxygen species, namely GSTK1 and GSTO1, could depress the embryos response to oxidative stress, thereby impairing subsequent embryo development. The gene expression changes observed in the present study in SS7 embryos, along with previous reports indicating SS7 can negatively affect fertilization, embryo production, and reproductive tract gene expression, make its use in embryo transfer programs unrecommendable.
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22.
  • Li, Junwei, et al. (författare)
  • Is boar sperm freezability more intrinsically linked to spermatozoa than to the surrounding seminal plasma?
  • 2018
  • Ingår i: Animal Reproduction Science. - : ELSEVIER SCIENCE BV. - 0378-4320 .- 1873-2232. ; 195, s. 30-37
  • Tidskriftsartikel (refereegranskat)abstract
    • This study aimed to elucidate the effect of seminal plasma (SP) from post-SRF on boar sperm freezability and, in addition, to determine the relevance of sperm itself to sustain cryopreservation, regardless of the SP surrounding them. Twelve ejaculates from three boars were manually collected in fractions/portions, P1: the first 10 mL of the SRF, P2: the rest of the SRF and the post-SRF. Immediately, samples were centrifuged to separate spermatozoa from the surrounding SP. Spermatozoa from P1 and P2 were then incubated with its own SP or that from post-SRF, diluted in BTS (1:1, v/v) at 17 degrees C overnight before being frozen in 0.5 mL straws using a standard protocol. Sperm motility (total and progressive) deteriorated (P amp;lt; 0.05) when P1- or P2-sperm when incubated overnight in SP from post-SRF, while sperm viability differed between P1 and P2 (P amp;lt; 0.05) regardless of the SP they were incubated in. Post-thaw sperm quality and functionality differed between P1 and P2, regardless of the SP used for overnight pre-freezing incubation. Post-thaw motility (P amp;lt; 0.05) and viability (P amp;lt; 0.01), as well as plasma membrane fluidity (P amp;lt; 0.05) or lipid peroxidation values (P amp;lt; 0.01) were best in P1 sperm compared to those of P2. The protein profile of sperm from P1 and P2, analyzed by 2D-PAGE, showed qualitative differences, which suggest that sperm rather than SP would explain differences in sperm freezability between ejaculate fractions/portions. Use of P1 fraction spermatozoa seems thus optimal for cryopreservation.
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23.
  • Li, Junwei, et al. (författare)
  • Post-thaw boar sperm motility is affected by prolonged storage of sperm in liquid nitrogen. A retrospective study
  • 2018
  • Ingår i: Cryobiology. - : Elsevier. - 0011-2240 .- 1090-2392. ; 80, s. 119-125
  • Tidskriftsartikel (refereegranskat)abstract
    • Owing to the quick genetic turnover of the pig industry, most AT-boar sires live 2-3 yr, a period during which for 1-2 yr their semen is extended and used in liquid form for AI. Despite showing low cryosurvival, affecting fertility after AI, boar semen is frozen for easiness of transport overseas and reposition of valuable genetics. For the latter, semen is stored in liquid nitrogen (LN2, cryostorage) for many years, a controversial practice. Here we studied how length of cryostorage could affect sperm quality. Straws (0.5 mL) frozen using the same cryopreservation protocol at one specific location from AI-sires of proven fertility were stored in LN2 for up to 8 yr. Post thaw sperm quality was evaluated after 2, 4 or 8 yr of cryostorage, always compared to early thawing (15 d after freezing). Sperm motility and kinematics were evaluated post-thaw using CASA and sperm viability was cytometrically evaluated using specific fluorophores. Sperm viability was not affected by length of cryostorage, but total and progressive sperm motility were lower (p amp;lt; 0.01) in sperm samples cryostored for 4 or 8 yr compared to those thawed 15 d after freezing. Cryostorage time affected sperm kinetics, but with greater intensity in the samples cryostored for 4 yr (p amp;lt; 0.001) than in those for 2 yr (p amp;lt; 0.01). The fact that the major phenotypic characteristic of boar spermatozoa, motility, is constrained by time of cryostorage should be considered when building cryobanks of pig semen. Attention should be placed on the finding that amp;gt; 2 yr of cryostorage time can be particularly detrimental for the post-thaw motility of some sires, which might require increasing sperm numbers for AI.
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24.
  • Li, Junwei, et al. (författare)
  • Seminal plasma antioxidants are directly involved in boar sperm cryotolerance
  • 2018
  • Ingår i: Theriogenology. - : ELSEVIER SCIENCE INC. - 0093-691X .- 1879-3231. ; 107, s. 27-35
  • Tidskriftsartikel (refereegranskat)abstract
    • Boar ejaculates are ejected in fractions with a specific composition in terms of sperm numbers and seminal plasma (SP), which is reflected in the varying sperm cryotolerance observed among different fractions. As boar sperm are particularly sensitive to oxidative stress, this study evaluated the role of SP antioxidants in the observed differences in sperm cryotolerance among ejaculate fractions. Ten ejaculates from five boars were manually collected in fractions: the first 10 mL of the sperm-rich fraction (SRF), the rest of the SRF and the post-SRF. Semen samples comprising the entire ejaculate (EE) were created by proportionally mixing the three fractions described above. Each of the 40 resulting semen samples was split into two aliquots: one was used for sperm cryopreservation following a standard protocol utilizing 0.5-mL straws, and the other was used to collect SP for antioxidant assessment. Frozen-thawed (FT) sperm from the SRF (the first 10 mL of the SRF and the rest of the SRF) and those from post-SRF were of the highest and worst quality, respectively, which was measured in terms of total and objective progressive motility and viability (P amp;lt; 0.01). Viable FT sperm from the post-SRF generated more reactive oxygen species and experienced more lipid peroxidation than those from the SRF (both the first 10 mL and the rest of the SRF) (P amp;lt; 0.01). The percentage of FT sperm exhibiting fragmented nuclear DNA did not differ among ejaculate fractions and the EE. Catalase, glutathione peroxidase and glutathione peroxidase 5 (GPx-5) were lowest in SP from the first 10 mL of the SRF (P amp;lt; 0.001), whereas superoxide dismutase (SOD) and paraoxonase 1 (PON-1) were highest in SP of the SRF (both the first 10 mL and the rest of the SRF) (P amp;lt; 0.01). Trolox-equivalent antioxidant capacity (TEAC) and the ferric-reducing ability of plasma (FRAP) were highest in SP from the first 10 mL of the SRF and lowest in the post-SRF (P amp;lt; 0.001), whereas cupric-reducing antioxidant capacity was lowest (P amp;lt; 0.05) in SP from the first 10 mL of the SRF. Regression analyses indicated that certain SP antioxidants had good predictive value for post-thaw recovery rates of total motility (R-2 = 54.8%, P amp;lt; 0.001; including SOD, TEAC and FRAP) and viability (R-2 = 56.1%, P amp;lt; 0.001; including SOD, PON-1, GPx-5 and TEAC). These results demonstrated that certain SP antioxidants are positively involved in boar sperm cryotolerance, minimizing the oxidative stress imposed by cryogenic handling. (C) 2017 Elsevier Inc. All rights reserved.
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25.
  • Martinez, Cristina A., et al. (författare)
  • Prevention of hatching of porcine morulae and blastocysts by liquid storage at 20 degrees C
  • 2019
  • Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
  • Tidskriftsartikel (refereegranskat)abstract
    • Vitrification is the ideal method for long-lasting storage of porcine embryos. However, both strict airline regulations for transport of liquid nitrogen dewars and the technical problems experienced when vitrified embryos are transferred using non-surgical procedures have led to the introduction of alternative storage methods, such as preserving embryos in liquid state. This study evaluated whether a pH-stable medium containing high concentrations of either foetal calf serum (FCS; 50%) or BSA (4%) combined with storage at temperatures of 17 degrees C or 20 degrees C maintained in vivo-derived morulae and blastocysts alive and unhatched (a sanitary requirement for embryo transportation) during 72 h of storage. Neither FCS nor BSA supplements were able to counteract the negative effect of low temperatures (17 degrees C) on embryonic survival after storage. At 20 degrees C, the protective effect of FCS or BSA depended on embryo stage. While FCS successfully arrested embryo development of only blastocysts, BSA arrested the development of both morulae and blastocysts. Over 80% of BSA arrested embryos restarted development by conventional culture and progressed to further embryonic stages, including hatching. In conclusion, porcine morulae and blastocysts can survive and remain unhatched during at least 72 h when stored at 20 degrees C in a BSA-containing medium.
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26.
  • Martinez, Cristina A., et al. (författare)
  • Seminal Plasma Induces Overexpression of Genes Associated with Embryo Development and Implantation in Day-6 Porcine Blastocysts
  • 2020
  • Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 21:10
  • Tidskriftsartikel (refereegranskat)abstract
    • The infusion of boar seminal plasma (SP) before artificial insemination (AI) positively alters the expression of endometrial genes and pathways involved in embryo development. This study aimed to determine which transcriptome changes occur in preimplantation embryos in response to SP infusions during estrus. Postweaning estrus sows received 40-mL intrauterine infusions of either SP (N = 6) or BTS extender (control group; N = 6) 30 min before each of two post-cervical AIs. On Day 6, embryos were surgically collected and analyzed for differential gene expression. Microarray analysis of embryos revealed 210 annotated genes, differentially expressed (p-value < 0.05 and fold change 2) in SP-blastocysts, compared to controls. Most of these genes were associated with biological, cellular, metabolic and developmental processes. The pathways enriched among the upregulated genes related to signal transduction, cellular processes and the endocrine system. Among altered genes involved in these pathways, the SP-group showed a conspicuous overexpression of ApoA-I, CDK1, MAPK1, SMAD2, PRKAA1 and RICTOR, with reported key roles in embryo development, implantation, or progression of pregnancy. In conclusion, the results demonstrate that SP infusions prior to AI upregulates the expression of embryo development related genes in Day 6 pig embryos.
  •  
27.
  • Martinez, Emilio A., et al. (författare)
  • Achievements and future perspectives of embryo transfer technology in pigs
  • 2019
  • Ingår i: Reproduction in domestic animals. - : WILEY. - 0936-6768 .- 1439-0531. ; 54
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
28.
  • Martinez Serrano, Cristina, et al. (författare)
  • Exogenous Melatonin in the Culture Medium Does Not Affect the Development of In Vivo-Derived Pig Embryos but Substantially Improves the Quality of In Vitro-Produced Embryos
  • 2022
  • Ingår i: Antioxidants. - : MDPI. - 2076-3921. ; 11:6
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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29.
  • Martinez Serrano, Cristina, et al. (författare)
  • Intrauterine Infusion of TGF-beta 1 Prior to Insemination, Alike Seminal Plasma, Influences Endometrial Cytokine Responses but Does Not Impact the Timing of the Progression of Pre-Implantation Pig Embryo Development
  • 2021
  • Ingår i: Biology. - : MDPI. - 2079-7737. ; 10:2
  • Tidskriftsartikel (refereegranskat)abstract
    • 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 mimicking an ET scenario in so-called "donor" (inseminated) and "recipient" (uninseminated) sows. On day 6 (day 0-onset of estrus), all "donors" were laparotomized to determine their pregnancy status (presence and developmental stage of the embryos). In addition, endometrial explants were collected from pregnant "donors" and cyclic "recipients," incubated for 24 h, and analyzed for cytokine production. SP infusions (unlike TGF-beta 1 infusions) positively influenced the developmental stage of day 6 embryos. Infusion treatments differentially influenced the endometrial cytokine production, mainly in donors. We concluded that SP infusions prior to AI not only impacted the porcine preimplantation embryo development but also influenced the endometrial cytokine production six days after treatment, both in donors and recipients.
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30.
  • Martinez-Serrano, Cristina, et al. (författare)
  • Seminal Plasma Modifies the Transcriptional Pattern of the Endometrium and Advances Embryo Development in Pigs
  • 2019
  • Ingår i: Frontiers in Veterinary Science. - : Frontiers Media S.A.. - 2297-1769. ; 6
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Boar seminal plasma is rich in cytokines, which could influence the capability of spermatozoa to tolerate preservation.Objectives: To evaluate the involvement of boar seminal plasma cytokines in the changes experienced by boar spermatozoa during their storage, either in liquid or frozen state.Materials and Methods: In two separated experiments, semen samples from healthy and fertile boars were split in two aliquots, one centrifuged twice (1,500 ×g for 10 min) to harvest seminal plasma, whereas the other was either commercially extended (3 × 107 sperm/mL) and liquid-stored at 17°C during 144 h (n = 28, Experiment 1) or frozen-thawed using a standard 0.5 mL protocol (n = 27, Experiment 2). Sixteen cytokines were quantified using Luminex xMAP®. Sperm attributes (CASA-evaluated total and progressive motility; flow cytometry-evaluated sperm viability, production of intracellular H2O2 and O2•-" role="presentation" style="box-sizing: border-box; display: inline; line-height: normal; word-spacing: normal; overflow-wrap: normal; white-space: nowrap; float: none; direction: ltr; max-width: none; max-height: none; min-width: 0px; min-height: 0px; border: 0px; padding: 0px; margin: 0px; position: relative; outline: 0px !important;">O∙−2O2•- and levels of lipid peroxidation in viable spermatozoa) were evaluated either at 0, 72, or 144 h of liquid storage (Experiment 1) or before freezing and at 30- and 150-min post-thawing (Experiment 2).Results: Multiple linear regression models, with Bayesian approach for variable selection, revealed that the anti-inflammatory TGF-β2, TGF-β3, IL-1Ra, and IL-4 and the pro-inflammatory IL-8 and IL-18, predicted changes in sperm motility for liquid-stored semen while the anti-inflammatory IFN-γ was included in the models predicting changes in all sperm attributes for cryopreserved semen.Conclusion: Specific boar seminal plasma cytokines would contribute to modulate the structural and metabolic changes shown by spermatozoa during preservation, either in liquid or frozen state.
  •  
31.
  • Maside, Carolina, et al. (författare)
  • Supplementation with exogenous coenzyme Q10 to media for in vitro maturation and embryo culture fails to promote the developmental competence of porcine embryos
  • 2019
  • Ingår i: Reproduction in domestic animals. - : WILEY. - 0936-6768 .- 1439-0531. ; 54, s. 72-77
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
32.
  • Nohalez, Alicia, et al. (författare)
  • Eventual re-vitrification or storage in liquid nitrogen vapor does not jeopardize the practical handling and transport of vitrified pig embryos
  • 2018
  • Ingår i: Theriogenology. - : ELSEVIER SCIENCE INC. - 0093-691X .- 1879-3231. ; 113, s. 229-236
  • Tidskriftsartikel (refereegranskat)abstract
    • This study aimed (1) to evaluate the in vitro post-warming survival of porcine embryos after re vitrification and (2) to assess the efficacy of transport of embryos in dry shipper (DS) in maintaining the viability and quality of vitrified embryos for a 3-day period. Embryos at the compacted or cavitating morula (CCM) and unhatched blastocyst (UBL) stages were surgically obtained from weaned, crossbred sows. In the first experiment, more than 85% of the embryos survived an initial vitrification and warming and achieved comparable survival rates to those of their fresh counterparts. In contrast, those embryos subjected to a second vitrification and warming had clearly lower survival rates (60% and 64% for re vitrified embryos from the CCM and UBL groups, respectively) compared to the survival rates of the initial vitrification and fresh control groups (P amp;lt; 0.01). Hatching rates were similar in re-vitrified blastocysts derived from vitrified CCMs and fresh control groups (50.8% and 55.3%, respectively). However, differences (P amp;lt; 0.01) in hatching rates were recorded in re-vitrified blastocysts derived from vitrified UBLs and fresh control blastocysts (14.7% and 90.0%, respectively). In the second experiment, vitrified embryos were stored in a liquid nitrogen tank for one month. Then, the straws containing the embryos were transferred to a DS (DS group) or to another liquid nitrogen tank (control group) for an additional three days. Embryos from the DS and control groups had similar survival and hatching rates, regardless of the embryonic stage considered. The DS storage of CCMs and UBLs did not affect their development after culturing, including total cell numbers, compared to the control, although their apoptotic index was slightly higher (P amp;lt; 0.05), regardless of the developmental stage. In conclusion, although re-vitrification negatively affects embryo survival, this study demonstrated that amp;gt;60% of vitrified embryos could be successfully re-vitrified and re-warmed. The present study also showed the effectiveness of the DS for the storage of vitrified porcine CCMs and UBLs for at least three 3 days. (C) 2018 Elsevier Inc. All rights reserved.
  •  
33.
  • Padilla, Lorena, et al. (författare)
  • Extracellular vesicles would be involved in the release and delivery of seminal TGF-beta isoforms in pigs
  • 2023
  • Ingår i: Frontiers in Veterinary Science. - : FRONTIERS MEDIA SA. - 2297-1769. ; 10
  • Tidskriftsartikel (refereegranskat)abstract
    • Introduction : Pig seminal plasma (SP) is rich in active forms of all three isoforms (1-3) of transforming growth factor beta (TGF-beta), a chemokine modulatory of the immune environment in the female genital tract once semen is delivered during mating or artificial insemination (AI). The present study aimed to examine how TGF-beta s are secreted by the epithelium of the male reproductive tract and how they are transported in semen, emphasizing the interplay with seminal extracellular vesicles (sEVs). Methods : Source of TGF-beta s was examined by immunohistochemistry in testis, epididymis, and accessory sex glands, by immunocytochemistry in ejaculated spermatozoa, and by Luminex xMAP((R)) technology in SP and sEVs retrieved from healthy, fertile male pigs used as breeders in AI programs. Results : All three TGF-beta isoforms were expressed in all reproductive tissues explored and would be released into ductal lumen either in soluble form or associated with sEVs. Ejaculated spermatozoa expressed all three TGF-beta isoforms, both inside and outside, probably the outer one associated with membrane-bound sEVs. The results confirmed that pig SP contains all three TGF-beta isoforms and demonstrated that a substantial portion of them is associated with sEVs. Discussion : Seminal EVs would be involved in the cellular secretion of the active forms of seminal TGF-beta isoforms and in their safe transport from the male to the female reproductive tract.
  •  
34.
  • Padilla, Lorena, et al. (författare)
  • Granulocyte-macrophage colony stimulating factor (GM-CSF) is fully expressed in the genital tract, seminal plasma and spermatozoa of male pigs
  • 2020
  • Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 10:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pro-inflammatory cytokine identified in boar seminal plasma (SP) but until now unexplored in terms of place of production and its association to spermatozoa. This study aimed to explore these aspects by evaluating the presence of GM-CSF in porcine reproductive organs (testes, epididymis and accessory sex glands), SP and mature spermatozoa (from cauda epididymis and ejaculated) using Western blot (WB), immunohistochemistry and immunocytochemistry. Positive labelling was obtained in tissues, SP and spermatozoa. In reproductive organs, WB revealed three forms of GM-CSF with different glycosylation degrees (15, 31 and 40 kDa). In SP and epididymal fluid, the GM-CSF appeared only in its active form while in spermatozoa the GM-CSF form present varied among sperm sources. Non-viable spermatozoa showed more GM-CSF than viable spermatozoa (14.87 +/- 1.98 RU vs. 7.25 +/- 0.52 RU) of fluorescence intensity. In conclusion, GM-CSF is widely present in the reproductive tract of male pigs, attached to the spermatozoa already in the epididymis as well as verted to SP. Consequently, the GM-CSF ought to regulate male genital tract and sperm function as well as mediating initial inflammatory responses and further mediating later immune actions by the female to semen deposition.
  •  
35.
  • Padilla, Lorena, et al. (författare)
  • Measurable Cytokine Concentrations in Pig Seminal Plasma Are Modified by Semen Handling and Storage
  • 2020
  • Ingår i: Biology. - : MDPI. - 2079-7737. ; 9:9
  • Tidskriftsartikel (refereegranskat)abstract
    • Sample handling and storing are critical steps for the reliable measurement of circulating biomolecules in biological fluids. This study evaluates how cytokine measurements in pig seminal plasma (SP) vary depending on semen handling and SP storage. Thirteen cytokines (GM-CSF, IFN gamma, IL-1 alpha, IL-1 beta, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and TNF alpha) were measured using Luminex xMAP(R) technology in individual seminal plasma (SP) samples (n = 62) from healthy breeding boars. Three separate experiments explored the delay (2 h and 24 h) in SP collection after ejaculation (Experiment 1) and SP storage, either short-term (5 degrees C, -20 degrees C and -80 degrees C for 72 h, Experiment 2) or long-term (at -20 degrees C and -80 degrees C for two months, Experiment 3), before analysis. Levels in fresh SP-samples were used as baseline control values. Delays in SP harvesting of up to 24 h did not substantially impact SP cytokine measurements. Some cytokines showed instability in stored SP samples, mainly in long-term storage. Ideally, cytokines in pig SP should be measured in fresh samples harvested within 24 h after ejaculation. If storage of SP is imperative, storage conditions should be adjusted for each cytokine.
  •  
36.
  • Padilla, Lorena, et al. (författare)
  • Period of Boar Ejaculate Collection Contributes to the Yearly Intra-Male Variability of Seminal Plasma Cytokines
  • 2020
  • Ingår i: Biology. - : MDPI. - 2079-7737. ; 9:5
  • Tidskriftsartikel (refereegranskat)abstract
    • The concentrations of cytokines in seminal plasma (SP) fluctuate over time in healthy males, weakening their practical usefulness as diagnostic tools. This study evaluated the relevance of intra-male variability in SP cytokines and to what extent the period of the year when ejaculate is collected contributes to such variability. Thirteen cytokines (GM-CSF, IFN gamma, IL-1 alpha, IL-1 beta, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and TNF alpha) were measured using the Luminex xMAP(R) technology for 180 SP samples of ejaculate collected over a year from nine healthy and fertile boars. The SP samples were grouped into two annual periods according to decreasing or increasing daylight and ambient temperature. Intra-male variability was higher than inter-male variability for all cytokines. All SP cytokines showed concentration differences between the two periods of the year, showing the highest concentration during the increasing daylength/temperature period, irrespective of the male. Similarly, some cytokines showed differences between daylength/temperature periods when focusing on their total amount in the ejaculate. No strong relationship (explaining more than 50% of the total variance) was found between annual fluctuations in SP-cytokine levels and semen parameters. In conclusion, the period of the year during which ejaculates were collected helps explain the intra-male variability of SP-cytokine levels in breeding boars.
  •  
37.
  • Parrilla, Inmaculada, et al. (författare)
  • Blastocyst-Bearing Sows Display a Dominant Anti-Inflammatory Cytokine Profile Compared to Cyclic Sows at Day 6 of the Cycle
  • 2020
  • Ingår i: Animals. - : MDPI. - 2076-2615. ; 10:11
  • Tidskriftsartikel (refereegranskat)abstract
    • 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 environment displayed by the blastocyst-bearing endometria. These findings suggest that sperm, seminal plasma (SP) and/or early embryos modify the uterine environment by inducing an immune-tolerant cytokine profile already visible at Day 6. Whether the SP or some of its active components may help to develop strategies to maximize the reproductive performance of recipients after ET needs further investigation.
  •  
38.
  • Parrilla, Inmaculada, et al. (författare)
  • Boar seminal plasma: current insights on its potential role for assisted reproductive technologies in swine
  • 2020
  • Ingår i: Animal Reproduction. - : BRAZILIAN COLL ANIMAL REPRODUCTION. - 1806-9614 .- 1984-3143. ; 17:3
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
39.
  • Parrilla, Inmaculada, et al. (författare)
  • Cryopreservation of highly extended pig spermatozoa remodels its proteome and counteracts polyspermic fertilization in vitro
  • 2023
  • Ingår i: Andrology. - : WILEY. - 2047-2919 .- 2047-2927.
  • Tidskriftsartikel (refereegranskat)abstract
    • 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, further research is necessary to comprehend how the disturbance of specific proteins affects sperm fertilization ability.
  •  
40.
  • Parrilla, Inmaculada, et al. (författare)
  • Immunological uterine response to pig embryos before and during implantation
  • 2022
  • Ingår i: Reproduction in domestic animals. - : Wiley. - 0936-6768 .- 1439-0531. ; 58:S5, s. 4-13
  • Forskningsöversikt (refereegranskat)abstract
    • 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.
  •  
41.
  • Parrilla, Inmaculada, et al. (författare)
  • Neither frozen-thawed seminal plasma nor commercial transforming growth factor-beta 1 infused intra-utero before insemination improved fertility and prolificacy in sows
  • 2022
  • Ingår i: Reproduction in domestic animals. - : Wiley. - 0936-6768 .- 1439-0531. ; 57:S5, s. 86-89
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
42.
  • Parrilla, Marc, et al. (författare)
  • Wearable Potentiometric Ion Patch for On-Body Electrolyte Monitoring in Sweat : Toward a Validation Strategy to Ensure Physiological Relevance
  • 2019
  • Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 91:13, s. 8644-8651
  • Tidskriftsartikel (refereegranskat)abstract
    • Herein, the reproducibility and a double validation of on-body measurements provided by new wearable potentiometric ion sensors (WPISs) is presented. Sweat collected during sport practice was first analyzed using the developed device, the pH-meter, and ion chromatography (IC) prior to onbody measurements (off-site validation). Subsequently, the accuracy of onbody measurements accomplished by the WPISs was evaluated by comparison with pH-meter readings and IC after collecting sweat (every 10-12.5 min) during sport practice. The developed device contains sensors for pH, Cl-, K+, and Na+ that are embedded in a flexible sampling cell for sweat analysis. The electrode array was fabricated employing MWCNTs (as an ion-to-electron transducer) and stretchable materials that have been exhaustively characterized in terms of analytical performance, presenting Nernstian slopes within the expected physiological range of each ion analyte (Cl-, 10-100 mM; 10-10 mM; and Na+, 10- 100 mM and pH, 4.5-7.5), drift suitable for midterm exercise practice (0.3 +/- 0.2 mV h(-1)), fast response time, adequate selectivity for sweat measurements, and excellent reversibility. Besides that, the designed sampling cell avoids any sweat contamination and evaporation issues while supplying a passive sweat flow encompassing specifically the individual's perspiration. The interpretation of ion concentration profiles may permit the identification of personal dynamic patterns in sweat composition while practicing sport.
  •  
43.
  • Perez-Fatino, Cristina, et al. (författare)
  • A New In-Depth Analytical Approach of the Porcine Seminal Plasma Proteome Reveals Potential Fertility Biomarkers
  • 2018
  • Ingår i: Journal of Proteome Research. - : AMER CHEMICAL SOC. - 1535-3893 .- 1535-3907. ; 17:3, s. 1065-1076
  • Tidskriftsartikel (refereegranskat)abstract
    • A complete characterization of the proteome of seminal plasma (SP) is an essential step to understand how SP influences sperm function and fertility after artificial insemination (AI). The purpose of this study was to identify which among characterized proteins in boar SP were differently expressed among AI boars with significantly different fertility outcomes. A total of 872 SP proteins, 390 of them belonging specifically to Sus Scrofa taxonomy, were identified (Experiment 1) by using a novel proteomic approach that combined size exclusion chromatography and solid-phase extraction as prefractionation steps prior to Nano LC-ESI-MS/MS analysis. The SP proteomes of 26 boars showing significant differences in farrowing rate (n = 13) and litter size (n = 13) after the AI of 10 526 sows were further analyzed (Experiment 2). A total of 679 SP proteins were then quantified by the SWATH approach, where the penalized linear regression LASSO revealed differentially expressed SP proteins for farrowing rate (FURIN, AKR1B1, UBA1, PIN1, SPAM1, BLMH, SMPDL3A, KRT17, KRT10, TTC23, and AGT) and litter size (PN-1, THBS1, DSC1, and CAT). This study extended our knowledge of the SP proteome and revealed some SP proteins as potential biomarkers of fertility in AI boars.
  •  
44.
  • Perez-Patino, Cristina, et al. (författare)
  • Characterization of the porcine seminal plasma proteome comparing ejaculate portions
  • 2016
  • Ingår i: Journal of Proteomics. - : ELSEVIER SCIENCE BV. - 1874-3919 .- 1876-7737. ; 142, s. 15-23
  • Tidskriftsartikel (refereegranskat)abstract
    • Full identification of boar seminal plasma (SP) proteins remains challenging. This study aims to provide an extensive proteomic analysis of boar SP and to generate an accessible database of boar SP-proteome. A SP-pool (33 entire ejaculates/11 boars; 3 ejaculates/boar) was analyzed to characterize the boar SP-proteome. Twenty ejaculates (5 boars, 4 ejaculates/boar) collected in portions (P1: first 10 mL of sperm rich ejaculate fraction (SRF), P2: rest of SRF and P3: post-SRF) were analyzed to evaluate differentially expressed SP-proteins among portions. SP-samples were analyzed using a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS followed by functional bioinformatics. The identified proteins were quantified from normalized LFQ intensity data. A total of 536 SP-proteins were identified, 409 of them in Sus scrofa taxonomy (374 validated with amp;gt;-99% confidence). Barely 20 of the identified SP-proteins were specifically implicated in reproductive processes, albeit other SP proteins could be indirectly involved in functionality and fertility of boar spermatozoa. Thirty-four proteins (16 identified in S. scrofa taxonomy) were differentially expressed among ejaculate portions, 16 being over expressed and 18 under-expressed in Pl-P2 regarding to P3. This major proteome mapping of the boar SP provides a complex inventory of proteins with potential roles as sperm function- and fertility- biomarkers. Biological significance: This proteomic study provides the major characterization of the boar SP-proteome with amp;gt;250 proteins first reported. The boar SP-proteome is described so that a spectral library can be built for relative label free protein quantification with SWATH approach. This proteomic profiling allows the creation of a publicly accessible database of the boar SP-proteome, as a first step for further understanding the role of SP-proteins in reproductive outcomes as well as for the identification of biomarkers for sperm quality and fertility. (C) 2016 Elsevier B.V. All rights reserved.
  •  
45.
  • Perez-Patino, Cristina, et al. (författare)
  • Cryopreservation Differentially Alters the Proteome of Epididymal and Ejaculated Pig Spermatozoa
  • 2019
  • Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 20:7
  • Tidskriftsartikel (refereegranskat)abstract
    • Cryopreservation induces differential remodeling of the proteome in mammalian spermatozoa. How these proteome changes relate to the loss of sperm function during cryopreservation remains unsolved. The present study aimed to clarify this issue evaluating differential changes in the proteome of fresh and frozen-thawed pig spermatozoa retrieved from the cauda epididymis and the ejaculate of the same boars, with clear differences in cryotolerance. Spermatozoa were collected from 10 healthy, sexually mature, and fertile boars, and cryopreserved using a standard 0.5 mL-straw protocol. Total and progressive motility, viability, and mitochondria membrane potential were higher and membrane fluidity and reactive oxygen species generation lower in frozen-thawed (FT) epididymal than ejaculated spermatozoa. Quantitative proteomics of fresh and FT spermatozoa were analyzed using a LC-ESI-MS/MS-based Sequential Window Acquisition of All Theoretical Spectra approach. Cryopreservation quantitatively altered more proteins in ejaculated than cauda epididymal spermatozoa. Differential protein-protein networks highlighted a set of proteins quantitatively altered in ejaculated spermatozoa, directly involved in mitochondrial functionality which would explain why ejaculated spermatozoa deteriorate during cryopreservation.
  •  
46.
  • Perez-Patino, Cristina, et al. (författare)
  • The proteome of frozen-thawed pig spermatozoa is dependent on the ejaculate fraction source
  • 2019
  • Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
  • Tidskriftsartikel (refereegranskat)abstract
    • The preservation of sperm functional parameters and fertility post-cryopreservation largely varies in the porcine, a species with a fractionated ejaculate. Although intrinsic individual differences have primarily been linked to this variation, differences in protein abundance among frozen-thawed (FT)-spermatozoa are far more relevant. This study, performed in two experiments, looked for proteomic quantitative differences between FT-sperm samples differing in post-thaw viability, motility, apoptosis, membrane lipid peroxidation and nuclear DNA fragmentation. The spermatozoa were either derived from the sperm-rich ejaculate fraction (SRF) or the entire ejaculate (Experiment 1) or from the first 10 mL of the SRF, the remaining SRF and the post-SRF (Experiment 2). Quantitative sperm proteomic differences were analysed using a LC-ESI-MS/MS-based SWATH approach. In Experiment 1, FT-spermatozoa from the SRF showed better preservation parameters than those from the entire ejaculate, with 26 Sus scrofa proteins with functional sperm relevance showing relative quantitative differences (FC amp;gt;= 1.5) between sperm sources. In Experiment 2, FT-spermatozoa from the first 10 mL of the SRF and the remaining SRF were qualitatively better than those from the post-SRF, and 187 proteins showed relative quantitative differences among the three ejaculate sources. The results indicate that quantitative proteome differences are linked to sperm cryosurvival.
  •  
47.
  • Perez-Patino, Cristina, et al. (författare)
  • The Proteome of Pig Spermatozoa Is Remodeled During Ejaculation
  • 2019
  • Ingår i: Molecular & Cellular Proteomics. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 1535-9476 .- 1535-9484. ; 18:1, s. 41-50
  • Tidskriftsartikel (refereegranskat)abstract
    • Proteins are essential for sperm function, including their fertilizing capacity. Pig spermatozoa, emitted in well-defined ejaculate fractions, vary in their functionality, which could be related to different sperm protein composition. This study aimed (i) to update the porcine sperm proteome and (ii) to identify proteins differentially expressed in mature spermatozoa from cauda epididymis and those delivered in separate ejaculate fractions. Ejaculates from nine mature and fertile boars were manually collected in three separate portions: the first 10 ml of the sperm-rich ejaculate fraction (SRF), the rest of the SRF and the post-SRF. The contents of cauda epididymides of the boars were collected post-mortem by retrograde duct perfusion, generating four different semen sources for each boar. Following centrifugation, the resulting pellets of each semen source were initially pooled and later split to generate two technical replicates per source. The resulting eight sperm samples (two per semen source) were subjected to iTRAQ-based 2D-LC-MS/MS for protein identification and quantification. A total of 1,723 proteins were identified (974 of Sus scrofa taxonomy) and 1,602 of them were also quantified (960 of Sus scrofa taxonomy). After an ANOVA test, 32 Sus scrofa proteins showed quantitative differences (p amp;lt; 0.01) among semen sources, which was particularly relevant for sperm functionality in the post-SR F. The present study showed that the proteome of boar spermatozoa is remodeled during ejaculation involving proteins clearly implicated in sperm function. The findings provide valuable groundwork for further studies focused on identifying protein biomarkers of sperm fertility.
  •  
48.
  • Roca, Jordi, et al. (författare)
  • Proteomics in fresh and preserved pig semen: Recent achievements and future challenges
  • 2020
  • Ingår i: Theriogenology. - : ELSEVIER SCIENCE INC. - 0093-691X .- 1879-3231. ; 150, s. 41-47
  • Tidskriftsartikel (refereegranskat)abstract
    • Proteins in semen, either in spermatozoa (SPZ) or seminal plasma (SP), are directly involved in molecular processes and biological pathways regulating sperm function, including fertilizing ability. Therefore, semen proteins are candidates of choice for biomarkers discovery for fertility and for sperm (dys)function. Mass spectrometry (MS)-based proteomics has opened up a new era for characterizing and quantifying the protein profile of SP and SPZ, as well as for unveiling the complex protein interactions involved in the activation of sperm functionality. This article overviews existing literature on MS-based proteomics regarding porcine semen, with a specific focus on the potential practical application of the results achieved so far. The weaknesses of current studies and the perspectives for future research in MS-based pig semen proteomics are also addressed. (C) 2020 Elsevier Inc. All rights reserved.
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