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2.
  • Cruz, Raquel, et al. (author)
  • Novel genes and sex differences in COVID-19 severity
  • 2022
  • In: Human Molecular Genetics. - : Oxford University Press. - 0964-6906 .- 1460-2083. ; 31:22, s. 3789-3806
  • Journal article (peer-reviewed)abstract
    • Here, we describe the results of a genome-wide study conducted in 11 939 coronavirus disease 2019 (COVID-19) positive cases with an extensive clinical information that were recruited from 34 hospitals across Spain (SCOURGE consortium). In sex-disaggregated genome-wide association studies for COVID-19 hospitalization, genome-wide significance (P < 5 × 10−8) was crossed for variants in 3p21.31 and 21q22.11 loci only among males (P = 1.3 × 10−22 and P = 8.1 × 10−12, respectively), and for variants in 9q21.32 near TLE1 only among females (P = 4.4 × 10−8). In a second phase, results were combined with an independent Spanish cohort (1598 COVID-19 cases and 1068 population controls), revealing in the overall analysis two novel risk loci in 9p13.3 and 19q13.12, with fine-mapping prioritized variants functionally associated with AQP3 (P = 2.7 × 10−8) and ARHGAP33 (P = 1.3 × 10−8), respectively. The meta-analysis of both phases with four European studies stratified by sex from the Host Genetics Initiative (HGI) confirmed the association of the 3p21.31 and 21q22.11 loci predominantly in males and replicated a recently reported variant in 11p13 (ELF5, P = 4.1 × 10−8). Six of the COVID-19 HGI discovered loci were replicated and an HGI-based genetic risk score predicted the severity strata in SCOURGE. We also found more SNP-heritability and larger heritability differences by age (<60 or ≥60 years) among males than among females. Parallel genome-wide screening of inbreeding depression in SCOURGE also showed an effect of homozygosity in COVID-19 hospitalization and severity and this effect was stronger among older males. In summary, new candidate genes for COVID-19 severity and evidence supporting genetic disparities among sexes are provided.
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3.
  • Abelev, Betty, et al. (author)
  • Underlying Event measurements in pp collisions at root s=0.9 and 7 TeV with the ALICE experiment at the LHC
  • 2012
  • In: Journal of High Energy Physics. - 1029-8479. ; :7
  • Journal article (peer-reviewed)abstract
    • We present measurements of Underlying Event observables in pp collisions at root s = 0 : 9 and 7 TeV. The analysis is performed as a function of the highest charged-particle transverse momentum p(T),L-T in the event. Different regions are defined with respect to the azimuthal direction of the leading (highest transverse momentum) track: Toward, Transverse and Away. The Toward and Away regions collect the fragmentation products of the hardest partonic interaction. The Transverse region is expected to be most sensitive to the Underlying Event activity. The study is performed with charged particles above three different p(T) thresholds: 0.15, 0.5 and 1.0 GeV/c. In the Transverse region we observe an increase in the multiplicity of a factor 2-3 between the lower and higher collision energies, depending on the track p(T) threshold considered. Data are compared to PYTHIA 6.4, PYTHIA 8.1 and PHOJET. On average, all models considered underestimate the multiplicity and summed p(T) in the Transverse region by about 10-30%.
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4.
  • Cambra, Josep M., et al. (author)
  • Allogeneic Embryos Disregulate Leukemia Inhibitory Factor (LIF) and Its Receptor in the Porcine Endometrium During Implantation
  • 2020
  • In: Frontiers in Veterinary Science. - : Frontiers Media S.A.. - 2297-1769. ; 7
  • Journal article (peer-reviewed)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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5.
  • Cuello, Cristina, et al. (author)
  • Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model
  • 2021
  • In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:3
  • Journal article (peer-reviewed)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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6.
  • Cuello, Cristina, et al. (author)
  • Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae
  • 2021
  • In: Frontiers in Veterinary Science. - : Frontiers Media SA. - 2297-1769. ; 8
  • Journal article (peer-reviewed)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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7.
  • Gonzalez-Plaza, Alejandro, et al. (author)
  • Cryotop vitrification of large batches of pig embryos simultaneously provides excellent postwarming survival rates and minimal interference with gene expression
  • 2023
  • In: Theriogenology. - : ELSEVIER SCIENCE INC. - 0093-691X .- 1879-3231. ; 206
  • Journal article (peer-reviewed)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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8.
  • Gonzalez-Ramiro, Henar, et al. (author)
  • 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
  • In: Journal of Animal Science. - : Oxford University Press. - 0021-8812 .- 1525-3163. ; 100:11
  • Journal article (peer-reviewed)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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9.
  • Gonzalez-Ramiro, Henar, et al. (author)
  • The Use of a Brief Synchronization Treatment after Weaning, Combined with Superovulation, Has Moderate Effects on the Gene Expression of Surviving Pig Blastocysts
  • 2023
  • In: Animals. - : MDPI. - 2076-2615. ; 13:9
  • Journal article (peer-reviewed)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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10.
  • Kattge, Jens, et al. (author)
  • TRY plant trait database - enhanced coverage and open access
  • 2020
  • In: Global Change Biology. - : Wiley-Blackwell. - 1354-1013 .- 1365-2486. ; 26:1, s. 119-188
  • Journal article (peer-reviewed)abstract
    • Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives.
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11.
  • Martinez, Cristina A., et al. (author)
  • Prevention of hatching of porcine morulae and blastocysts by liquid storage at 20 degrees C
  • 2019
  • In: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
  • Journal article (peer-reviewed)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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12.
  • Martinez, Cristina A., et al. (author)
  • Seminal Plasma Induces Overexpression of Genes Associated with Embryo Development and Implantation in Day-6 Porcine Blastocysts
  • 2020
  • In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 21:10
  • Journal article (peer-reviewed)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.
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13.
  • Martinez, Cristina A., et al. (author)
  • The overlaying oil type influences in vitro embryo production: differences in composition and compound transfer into incubation medium between oils
  • 2017
  • In: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
  • Journal article (peer-reviewed)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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14.
  • Martinez, Emilio A., et al. (author)
  • Achievements and future perspectives of embryo transfer technology in pigs
  • 2019
  • In: Reproduction in domestic animals. - : WILEY. - 0936-6768 .- 1439-0531. ; 54
  • Journal article (peer-reviewed)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.
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15.
  • Martinez Serrano, Cristina, et al. (author)
  • 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
  • In: Antioxidants. - : MDPI. - 2076-3921. ; 11:6
  • Journal article (peer-reviewed)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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16.
  • Martinez Serrano, Cristina, et al. (author)
  • 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
  • In: Biology. - : MDPI. - 2079-7737. ; 10:2
  • Journal article (peer-reviewed)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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17.
  • Martinez-Serrano, Cristina, et al. (author)
  • Seminal Plasma Modifies the Transcriptional Pattern of the Endometrium and Advances Embryo Development in Pigs
  • 2019
  • In: Frontiers in Veterinary Science. - : Frontiers Media S.A.. - 2297-1769. ; 6
  • Journal article (peer-reviewed)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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18.
  • Maside, Carolina, et al. (author)
  • Supplementation with exogenous coenzyme Q10 to media for in vitro maturation and embryo culture fails to promote the developmental competence of porcine embryos
  • 2019
  • In: Reproduction in domestic animals. - : WILEY. - 0936-6768 .- 1439-0531. ; 54, s. 72-77
  • Journal article (peer-reviewed)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.
  •  
19.
  • Muscarella, Robert, et al. (author)
  • The global abundance of tree palms
  • 2020
  • In: Global Ecology and Biogeography. - : Wiley. - 1466-822X .- 1466-8238. ; 29:9, s. 1495-1514
  • Journal article (peer-reviewed)abstract
    • AimPalms are an iconic, diverse and often abundant component of tropical ecosystems that provide many ecosystem services. Being monocots, tree palms are evolutionarily, morphologically and physiologically distinct from other trees, and these differences have important consequences for ecosystem services (e.g., carbon sequestration and storage) and in terms of responses to climate change. We quantified global patterns of tree palm relative abundance to help improve understanding of tropical forests and reduce uncertainty about these ecosystems under climate change.LocationTropical and subtropical moist forests.Time periodCurrent.Major taxa studiedPalms (Arecaceae).MethodsWe assembled a pantropical dataset of 2,548 forest plots (covering 1,191 ha) and quantified tree palm (i.e., ≥10 cm diameter at breast height) abundance relative to co‐occurring non‐palm trees. We compared the relative abundance of tree palms across biogeographical realms and tested for associations with palaeoclimate stability, current climate, edaphic conditions and metrics of forest structure.ResultsOn average, the relative abundance of tree palms was more than five times larger between Neotropical locations and other biogeographical realms. Tree palms were absent in most locations outside the Neotropics but present in >80% of Neotropical locations. The relative abundance of tree palms was more strongly associated with local conditions (e.g., higher mean annual precipitation, lower soil fertility, shallower water table and lower plot mean wood density) than metrics of long‐term climate stability. Life‐form diversity also influenced the patterns; palm assemblages outside the Neotropics comprise many non‐tree (e.g., climbing) palms. Finally, we show that tree palms can influence estimates of above‐ground biomass, but the magnitude and direction of the effect require additional work.ConclusionsTree palms are not only quintessentially tropical, but they are also overwhelmingly Neotropical. Future work to understand the contributions of tree palms to biomass estimates and carbon cycling will be particularly crucial in Neotropical forests.
  •  
20.
  • Nishi, Stephanie K., et al. (author)
  • Mediterranean, DASH, and MIND Dietary Patterns and Cognitive Function : The 2-Year Longitudinal Changes in an Older Spanish Cohort
  • 2021
  • In: Frontiers in Aging Neuroscience. - : Frontiers Media SA. - 1663-4365. ; 13
  • Journal article (peer-reviewed)abstract
    • Background and Aims: Plant-forward dietary patterns have been associated with cardiometabolic health benefits, which, in turn, have been related to cognitive performance with inconsistent findings. The objective of this study was to examine the relationship between baseline adherence to three a priori dietary patterns (Mediterranean, DASH, and MIND diets) with 2-year changes in cognitive performance in older adults with overweight or obesity and high cardiovascular disease risk. Methods: A prospective cohort analysis was conducted within the PREDIMED-Plus trial, involving 6,647 men and women aged 55–75 years with overweight or obesity and metabolic syndrome. Using a validated, semiquantitative 143-item food frequency questionnaire completed at baseline, the dietary pattern adherence scores were calculated. An extensive neuropsychological test battery was administered at baseline and 2-year follow-up. Multivariable-adjusted linear regression models were used to assess associations between 2-year changes in cognitive function z-scores across tertiles of baseline adherence to the a priori dietary patterns. Results: Adherence to the Mediterranean diet at baseline was associated with 2-year changes in the general cognitive screening Mini-Mental State Examination (MMSE, β: 0.070; 95% CI: 0.014, 0.175, P-trend = 0.011), and two executive function-related assessments: the Trail Making Tests Part A (TMT-A, β: −0.054; 95% CI: −0.110, − 0.002, P-trend = 0.047) and Part B (TMT-B, β: −0.079; 95% CI: −0.134, −0.024, P-trend = 0.004). Adherence to the MIND diet was associated with the backward recall Digit Span Test assessment of working memory (DST-B, β: 0.058; 95% CI: 0.002, 0.114, P-trend = 0.045). However, higher adherence to the DASH dietary pattern was not associated with better cognitive function over a period of 2 years. Conclusion: In older Spanish individuals with overweight or obesity and at high cardiovascular disease risk, higher baseline adherence to the Mediterranean dietary pattern may be associated with better cognitive performance than lower adherence over a period of 2 years.
  •  
21.
  • Nohalez, Alicia, et al. (author)
  • Eventual re-vitrification or storage in liquid nitrogen vapor does not jeopardize the practical handling and transport of vitrified pig embryos
  • 2018
  • In: Theriogenology. - : ELSEVIER SCIENCE INC. - 0093-691X .- 1879-3231. ; 113, s. 229-236
  • Journal article (peer-reviewed)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.
  •  
22.
  • Parrilla, Inmaculada, et al. (author)
  • Blastocyst-Bearing Sows Display a Dominant Anti-Inflammatory Cytokine Profile Compared to Cyclic Sows at Day 6 of the Cycle
  • 2020
  • In: Animals. - : MDPI. - 2076-2615. ; 10:11
  • Journal article (peer-reviewed)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.
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23.
  • Parrilla, Inmaculada, et al. (author)
  • Boar seminal plasma: current insights on its potential role for assisted reproductive technologies in swine
  • 2020
  • In: Animal Reproduction. - : BRAZILIAN COLL ANIMAL REPRODUCTION. - 1806-9614 .- 1984-3143. ; 17:3
  • Journal article (peer-reviewed)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.
  •  
24.
  • Perez-Fatino, Cristina, et al. (author)
  • A New In-Depth Analytical Approach of the Porcine Seminal Plasma Proteome Reveals Potential Fertility Biomarkers
  • 2018
  • In: Journal of Proteome Research. - : AMER CHEMICAL SOC. - 1535-3893 .- 1535-3907. ; 17:3, s. 1065-1076
  • Journal article (peer-reviewed)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.
  •  
25.
  • Adrian-Martinez, S., et al. (author)
  • A first search for coincident gravitational waves and high energy neutrinos using LIGO, Virgo and ANTARES data from 2007
  • 2013
  • In: Journal of Cosmology and Astroparticle Physics. - : IOP Publishing. - 1475-7516. ; :6
  • Journal article (peer-reviewed)abstract
    • We present the results of the first search for gravitational wave bursts associated with high energy neutrinos. Together, these messengers could reveal new, hidden sources that are not observed by conventional photon astronomy, particularly at high energy. Our search uses neutrinos detected by the underwater neutrino telescope ANTARES in its 5 line configuration during the period January - September 2007, which coincided with the fifth and first science runs of LIGO and Virgo, respectively. The LIGO-Virgo data were analysed for candidate gravitational-wave signals coincident in time and direction with the neutrino events. No significant coincident events were observed. We place limits on the density of joint high energy neutrino - gravitational wave emission events in the local universe, and compare them with densities of merger and core-collapse events.
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26.
  • Alkmin, Diego V., et al. (author)
  • Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate
  • 2014
  • In: Cryobiology. - : Elsevier. - 0011-2240 .- 1090-2392. ; 69:2, s. 203-210
  • Journal article (peer-reviewed)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.
  •  
27.
  • Alvarez-Rodriguez, Manuel, et al. (author)
  • Mating modifies the expression of crucial oxidative-reductive transcripts in the pig oviductal sperm reservoir: is the female ensuring sperm survival?
  • 2023
  • In: Frontiers in Endocrinology. - : FRONTIERS MEDIA SA. - 1664-2392. ; 14
  • Journal article (peer-reviewed)abstract
    • BackgroundMating induces large changes in the female genital tract, warranting female homeostasis and immune preparation for pregnancy, including the preservation of crucial oxidative status among its pathways. Being highly susceptible to oxidative stress, sperm survival and preserved function depend on the seminal plasma, a protection that is removed during sperm handling but also after mating when spermatozoa enter the oviduct. Therefore, it is pertinent to consider that the female sperm reservoir takes up this protection, providing a suitable environment for sperm viability. These aspects have not been explored despite the increasing strategies in modulating the female status through diet control and nutritional supplementation. AimsTo test the hypothesis that mating modifies the expression of crucial oxidative-reductive transcripts across the entire pig female genital tract (cervix to infundibulum) and, particularly in the sperm reservoir at the utero-tubal junction, before ovulation, a period dominated by estrogen stimulation of ovarian as well as of seminal origin. MethodsThe differential expression of estrogen (ER) and progesterone (PR) receptors and of 59 oxidative-reductive transcripts were studied using a species-specific microarray platform, in specific segments of the peri-ovulatory sow reproductive tract in response to mating. ResultsMating induced changes along the entire tract, with a conspicuous downregulation of both ER and PR and an upregulation of superoxide dismutase 1 (SOD1), glutaredoxin (GLRX3), and peroxiredoxin 1 and 3 (PRDX1, PRDX3), among other NADH Dehydrogenase Ubiquinone Flavoproteins, in the distal uterus segment. These changes perhaps helped prevent oxidative stress in the area adjacent to the sperm reservoir at the utero-tubal junction. Concomitantly, there were a downregulation of catalase (CAT) and NADH dehydrogenase (ubiquinone) oxidoreductases 1 beta subcomplex, subunit 1 (NDUFB1) in the utero-tubal junction alongside an overall downregulation of CAT, SOD1, and PRDX3 in the ampullar and infundibulum segments. ConclusionsNatural mating is an inducer of changes in the expression of female genes commanding antioxidant enzymes relevant for sperm survival during sperm transport, under predominant estrogen influence through the bloodstream and semen. The findings could contribute to the design of new therapeutics for the female to improve oxidative-reductive balance.
  •  
28.
  • Barranco, Isabel, et al. (author)
  • Active paraoxonase 1 is synthesised throughout the internal boar genital organs.
  • 2017
  • In: Reproduction (Cambridge, England). - : Bioscientifica. - 1741-7899. ; 154:3, s. 237-243
  • Journal article (peer-reviewed)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.
  •  
29.
  • Barranco, Isabel, et al. (author)
  • Extracellular vesicles isolated from porcine seminal plasma exhibit different tetraspanin expression profiles
  • 2019
  • In: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
  • Journal article (peer-reviewed)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.
  •  
30.
  • Barranco, Isabel, et al. (author)
  • 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
  • In: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 11:9
  • Journal article (peer-reviewed)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.
  •  
31.
  • Barranco, Isabel, et al. (author)
  • High total antioxidant capacity of the porcine seminal plasma (SP-TAC) relates to sperm survival and fertility
  • 2015
  • In: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 5:18538
  • Journal article (peer-reviewed)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.
  •  
32.
  • Barranco, Isabel, et al. (author)
  • Levels of activity of superoxide dismutase in seminal plasma do not predict fertility of pig AI-semen doses
  • 2019
  • In: Theriogenology. - : ELSEVIER SCIENCE INC. - 0093-691X .- 1879-3231. ; 140, s. 18-24
  • Journal article (peer-reviewed)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.
  •  
33.
  • Barranco, Isabel, et al. (author)
  • Measurement of Activity and Concentration of Paraoxonase 1 (PON-1) in Seminal Plasma and Identification of PON-2 in the Sperm of Boar Ejaculates
  • 2015
  • In: Molecular Reproduction and Development. - : Wiley-Blackwell. - 1040-452X .- 1098-2795. ; 82:1, s. 58-65
  • Journal article (peer-reviewed)abstract
    • This study revealed and characterised the presence of the antioxidant enzymes paraoxonase (PON) type 1 (PON-1, extracellular) and type 2 (PON-2, intracellular) in boar semen. To evaluate PON-1, an entire ejaculate from each of ten boars was collected and the seminal plasma was harvested after double centrifugation (1,500g for 10min). Seminal plasma was analysed for concentration as well as enzymatic activity of PON-1 and total cholesterol levels. Seminal-plasma PON-1 concentration ranged from 0.961 to 1.670ng/ml while its enzymatic activity ranged from 0.056 to 0.400 IU/ml, which represent individual variance. Seminal-plasma PON-1 concentration and enzymatic activity were negatively correlated (r=-0.763; Pless than0.01). The activity of seminal-plasma PON-1 negatively correlated with ejaculate volume (r=-0.726, Pless than0.05), but positively correlated with sperm concentration (r=0.654, Pless than0.05). Total seminal-plasma cholesterol concentration positively correlated with PON-1 activity (r=0.773; Pless than0.01), but negatively correlated with PON-1 concentration (r=-0.709; Pless than0.05). The presence of intracellular PON-2 was determined via immunocytochemistry in spermatozoa derived from artificial insemination. PON-2 localised to the post-acrosomal area of the sperm head and principal piece of the tail in membrane-intact spermatozoa. In summary, PON is present in boar semen, with PON-1 at low levels in seminal plasma and PON-2 within the spermatozoa. Further studies are needed to characterise the relationship between antioxidant PONs with sperm and other seminal-plasma parameters. Mol. Reprod. Dev. 82: 58-65, 2015. (c) 2014 Wiley Periodicals, Inc.
  •  
34.
  • Barranco, Isabel, et al. (author)
  • Seminal Plasma Cytokines Are Predictive of the Outcome of Boar Sperm Preservation
  • 2019
  • In: Frontiers in Veterinary Science. - : Frontiers Media S.A.. - 2297-1769. ; 6
  • Journal article (peer-reviewed)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.
  •  
35.
  • Barranco, Isabel, et al. (author)
  • The Seminal Plasma of the Boar is Rich in Cytokines, with Significant Individual and Intra-Ejaculate Variation
  • 2015
  • In: American Journal of Reproductive Immunology. - : WILEY-BLACKWELL. - 1046-7408 .- 1600-0897. ; 74:6, s. 523-532
  • Journal article (peer-reviewed)abstract
    • Problem The boar, as human, sequentially ejaculates sperm-rich and sperm-poor fractions. Seminal plasma (SP) spermadhesins (PSP-I/PSP-II) induce a primary endometrial inflammatory response in female sows, similar to that elicited by semen deposition in other species, including human. However, the SP is also known to mitigate such response, making it transient to allow for embryo entry to a cleansed endometrium. Although cytokine involvement has been claimed, the exploration of cytokines in different SP fractions is scarce. This study determines Th1, Th2, Th17 and Th3 cytokine profiles in specific ejaculate SP fractions from boars of proven fertility. Methods SP samples from the sperm-rich fraction (SRF) and the sperm-poor post-SRF fraction (post-SRF) of manually collected ejaculates from eight boars (four ejaculates per boar) were analysed by commercial multiplex bead assay kits (Milliplex MAP, Millipore, USA) for interferon-gamma, interferon gamma-induced protein 10, macrophage-derived chemokine, growth-regulated oncogene, granulocyte-macrophage colony-stimulating factor, monocyte chemo-attractant protein-1, interleukins (IL)-6, IL-8, IL-10, IL-15, IL-17 and transforming growth factor (TGF)-beta 1-beta 3. Results Cytokine concentrations differed between the ejaculate fractions among boars, being highest in the post-SRF. Conclusion Boar SP is rich in Th1, Th2, Th17 and Th3 cytokines, with lowest concentrations in the sperm-peak-containing fraction, indicating its main immune influence might reside in the larger, protein-rich sperm-poor post-SRF.
  •  
36.
  • Caballero, Ignacio, et al. (author)
  • Immunolocalization and possible functional role of PSP-I/PSP-II heterodimer in highly extended boar spermatozoa
  • 2006
  • In: Journal of Andrology. - : American Society of Andrology. - 0196-3635 .- 1939-4640. ; 27:6, s. 766-773
  • Journal article (peer-reviewed)abstract
    • PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma which is able to preserve, in vitro, the viability, motility, and mitochondrial activity of highly extended boar spermatozoa for at least 5 hours. However, little is known about the binding pattern of the heterodimer to the sperm plasma membrane and its eventual relation with the maintenance of the sperm functionality. The present study investigated the effect of exposing highly extended boar spermatozoa (11 million/mL) to 1.5 mg/mL of PSP-I/PSP-II for 0.5, 5, and 10 hours at 38 degrees C on sperm characteristics and the changes in PSP-I/PSP-II localization as a result of both the addition of PSP-I/PSP-II to the extender and the incubation time. Exposure of the spermatozoa to PSP-I/PSP-II preserved sperm viability, motility, and mitochondrial activity when compared to nonexposed spermatozoa. This protective effect lasted for 10 hours (P less than.05). After immunolabeling of highly extended semen with rabbit monospecific polyclonal antibody against PSP-I/PSP-11, the percentage of immunopositive spermatozoa declines over time from 71% (0.5 hours) to 49% (10 hours). However, more than 80% of spermatozoa remained labeled during the 10-hour incubation period if PSP-I/PSP-11 was added. Scanning electron microscopy revealed 4 different binding patterns. The heterodimer was mainly localized to the acrosomal area, being redistributed to the postacrosomal area or lost during in vitro incubation. In conclusion, the protective effect of the heterodimer appears to be related to its adhesion to the acrosomal area, and the loss of this protective effect coincides with a stepwise redistribution of PSP-I/PSP-II during incubation.
  •  
37.
  • Cambra, Josep M., et al. (author)
  • Cytokine profile in peripheral blood mononuclear cells differs between embryo donor and potential recipient sows
  • 2024
  • In: Frontiers in Veterinary Science. - : FRONTIERS MEDIA SA. - 2297-1769. ; 11
  • Journal article (peer-reviewed)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.
  •  
38.
  • Cambra, Josep M., et al. (author)
  • Transcriptional Profiling of Porcine Blastocysts Produced In Vitro in a Chemically Defined Culture Medium
  • 2021
  • In: Animals. - : MDPI. - 2076-2615. ; 11:5
  • Journal article (peer-reviewed)abstract
    • Simple Summary The development of chemically defined media has become a particularly important task for in vitro embryo production systems, which require maintained reproducible results when new additives are tested for culture, beyond observational studies. Specifically, we need studies measuring the impact of these media on the embryonic transcriptome, particularly those negatively affecting embryo quality. Consequently, this study evaluated by using a microarray approach the transcriptome of porcine embryos produced in vitro, cultured in a defined vs. an undefined medium and contrasted with in vivo-derived embryos. No significantly altered genes were found between in vitro-produced embryos, despite the theoretical limitations that usually accompany defined media. However, when they were compared with in vivo-derived embryos, many altered genes were observed, reflecting how current culture conditions deeply modify the embryonic transcriptome. A better understanding of these alterations may offer new ways to improve in vitro embryo production systems. Likewise, developing a chemically defined medium capable of producing embryos of a similar quality to traditional media may contribute to this task. The development of chemically defined media is a growing trend in in vitro embryo production (IVP). Recently, traditional undefined culture medium with bovine serum albumin (BSA) has been successfully replaced by a chemically defined medium using substances with embryotrophic properties such as platelet factor 4 (PF4). Although the use of this medium sustains IVP, the impact of defined media on the embryonic transcriptome has not been fully elucidated. This study analyzed the transcriptome of porcine IVP blastocysts, cultured in defined (PF4 group) and undefined media (BSA group) by microarrays. In vivo-derived blastocysts (IVV group) were used as a standard of maximum embryo quality. The results showed no differentially expressed genes (DEG) between the PF4 and BSA groups. However, a total of 2780 and 2577 DEGs were detected when comparing the PF4 or the BSA group with the IVV group, respectively. Most of these genes were common in both in vitro groups (2132) and present in some enriched pathways, such as cell cycle, lysosome and/or metabolic pathways. These results show that IVP conditions strongly affect embryo transcriptome and that the defined culture medium with PF4 is a guaranteed replacement for traditional culture with BSA.
  •  
39.
  • Gil, Maria A., et al. (author)
  • In-depth proteome characterization of endometrium and extraembryonic membranes during implantation in pig
  • 2024
  • In: JOURNAL OF ANIMAL SCIENCE AND BIOTECHNOLOGY. - : BMC. - 1674-9782. ; 15:1
  • Journal article (peer-reviewed)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.
  •  
40.
  • Gonzalez-Plaza, Alejandro, et al. (author)
  • Equilibration time with cryoprotectants, but not melatonin supplementation during in vitro maturation, affects viability and metaphase plate morphology of vitrified porcine mature oocytes
  • 2022
  • In: Reproduction in domestic animals. - : Wiley. - 0936-6768 .- 1439-0531. ; 57:S5, s. 58-63
  • Journal article (peer-reviewed)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.
  •  
41.
  • Gonzalez-Plaza, Alejandro, et al. (author)
  • The Open Cryotop System Is Effective for the Simultaneous Vitrification of a Large Number of Porcine Embryos at Different Developmental Stages
  • 2022
  • In: Frontiers in Veterinary Science. - : Frontiers Media SA. - 2297-1769. ; 9
  • Journal article (peer-reviewed)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.
  •  
42.
  • Gonzalez-Ramiro, Henar, et al. (author)
  • A Short-Term Altrenogest Treatment Post-weaning Followed by Superovulation Reduces Pregnancy Rates and Embryo Production Efficiency in Multiparous Sows
  • 2021
  • In: Frontiers in Veterinary Science. - : Frontiers Media S.A.. - 2297-1769. ; 8
  • Journal article (peer-reviewed)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.
  •  
43.
  • Hernandez, Marta, et al. (author)
  • Cryo-scanning electron microscopy (Cryo-SEM) of semen frozen in medium-straws from good and sub-standard freezer AI-boars
  • 2007
  • In: Cryobiology. - : Elsevier. - 0011-2240 .- 1090-2392. ; 54:1, s. 63-70
  • Journal article (peer-reviewed)abstract
    • A major limiting factor for commercial cryopreservation of boar semen for artificial insemination (AI) is the large individual variation to cooling, where the degree of cell dehydration during ice (re)shaping seems to play a major role. This study investigated, in the frozen state, the degree of dehydration and ice crystal distribution in boar semen doses whose spermatozoa displayed different viability after thawing. Cross-sectioned medium-straws (0.5 mL, n = 10) from a total of 10 stud boars classified as "good" (n = 5) or sub-standard (e.g., "bad" freezers, n = 5) by conventional analyses (computer assisted motility and sperm viability) were examined by Cryo-scanning electron microscopy (Cryo-SEM) to determine whether differences between groups could be already distinguishable prior to thawing. The degree of hydration was monitored in relation to the areas of ice crystal formed extracellularly (lakes), the areas of frozen, concentrated extender (veins) where spermatozoa were located and the degree of compartmentalization (number of lakes) present. Irrespectively of the region studied, the gradient of main dehydration (as lakes) observed along the cross-section area of the straws was very irregular. Most spermatozoa were enclosed in the freezing extender matrix and no obvious signs of external membrane damage were observed. None of the Cryo-SEM variables significantly correlated with post-thaw sperm parameters (p greater than 0.05). However, we identified significant differences (p less than 0.0001) among boars for all ultrastructure variables studied, including the size of the veins, where differences in solute concentration is expected. We concluded that despite the large variability in ice crystal formation during the conventional freezing process among boars, this is unrelated to inter-boar post-thaw sperm differences. (c) 2007 Elsevier Inc. All rights reserved.
  •  
44.
  • Hernandez, Marta, et al. (author)
  • Differences in SCSA outcome among boars with different sperm freezability
  • 2006
  • In: International Journal of Andrology. - : Wiley-Blackwell. - 0105-6263 .- 1365-2605. ; 29:6, s. 583-591
  • Journal article (peer-reviewed)abstract
    • Spermatozoa from some boars sustain the process of cryopreservation poorly and yield poor fertility after artificial insemination. Poor freezability has not been disclosed using conventional semen analyses. A defective chromatin can, if present in a substantial number of spermatozoa, affect the fertilizing ability of spermatozoa. Here we tested the hypothesis that nuclear DNA instability could explain differences in freezability among boars, and complement or supersede conventional tests for sperm quality such as sperm motility or membrane assessments. Frozen-thawed (FT) spermatozoa from a total of 44 stud boars were assessed by the sperm chromatin structure assay (SCSA), in relation to computer-assisted sperm analysis-derived sperm motility variables and sperm viability (triple fluorescent microscopic staining), including three experiments. The first trial, including 24 boars, evaluated the relationship between the sperm motility and viability with levels of DNA integrity. The SCSA showed that most spermatozoa had intact DNA [levels of DNA fragmentation index (%DFI) ranging from 0.63% to 11.85%] significantly correlated (albeit weakly) with current sperm quality variables. The second trial, on 15 boars, assessed the influence of two different thawing rates (20 s at 37 degrees C vs. 8 s at 70 degrees C) and the post-thaw incubation times (0, 30, 150 and 300 min) at 37 degrees C on FT-boar sperm quality. The highest sperm survival (p less than 0.05) and the lowest DNA damage (p less than 0.01) were achieved when thawing was carried out at 70 degrees C for 8 s, without any change during the first 150 min of incubation. Finally, the third experiment studied if differences in sperm freezability showed by stud boar semen, as good or bad freezers by conventional analyses, could be attributed to differences in chromatin structure. All SCSA parameters were low, but significantly (p less than 0.05-0.001) higher for bad freezers, showing they had less homogeneous sperm chromatin than the good freezers. The results indicate that SCSA outcome complements conventional assessment of FT-boar spermatozoa, disclosing differences in their ability to sustain freezing and thawing. However, the low overall DNA damage observed in FT spermatozoa seems to have poor biological significance.
  •  
45.
  • Joglar, Vanessa, et al. (author)
  • Cobalamin and microbial plankton dynamics along a coastal to offshore transect in the Eastern North Atlantic Ocean
  • 2021
  • In: Environmental Microbiology. - : John Wiley & Sons. - 1462-2912 .- 1462-2920. ; 23:3, s. 1559-1583
  • Journal article (peer-reviewed)abstract
    • Cobalamin (B12) is an essential cofactor that is exclusively synthesized by some prokaryotes while many prokaryotes and eukaryotes require an external supply of B12. The spatial and temporal availability of B12 is poorly understood in marine ecosystems. Field measurements of B12 along with a large set of ancillary biotic and abiotic factors were obtained during three oceanographic cruises in the NW Iberian Peninsula, covering different spatial and temporal scales. B12 concentrations were remarkably low (<1.5 pM) in all samples, being significantly higher at the subsurface Eastern North Atlantic Central Water than at shallower depths, suggesting that B12 supply in this water mass is greater than demand. Multiple regression models excluded B12 concentration as predictive variable for phytoplankton biomass or production, regardless of the presence of B12-requiring algae. Prokaryote production was the best predictor for primary production, and eukaryote community composition was better correlated with prokaryote community composition than with nutritional resources, suggesting that biotic interactions play a significant role in regulating microbial communities. Interestingly, co-occurrence network analyses based on 16S and 18S rRNA sequences allowed the identification of significant associations between potential B12 producers and consumers (e.g. Thaumarchaeota and Dynophyceae, or Amylibacter and Ostreococcus respectively), which can now be investigated using model systems in the laboratory.
  •  
46.
  • Li, Junwei, et al. (author)
  • Is boar sperm freezability more intrinsically linked to spermatozoa than to the surrounding seminal plasma?
  • 2018
  • In: Animal Reproduction Science. - : ELSEVIER SCIENCE BV. - 0378-4320 .- 1873-2232. ; 195, s. 30-37
  • Journal article (peer-reviewed)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.
  •  
47.
  • Li, Junwei, et al. (author)
  • Post-thaw boar sperm motility is affected by prolonged storage of sperm in liquid nitrogen. A retrospective study
  • 2018
  • In: Cryobiology. - : Elsevier. - 0011-2240 .- 1090-2392. ; 80, s. 119-125
  • Journal article (peer-reviewed)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.
  •  
48.
  • Li, Junwei, et al. (author)
  • Seminal plasma antioxidants are directly involved in boar sperm cryotolerance
  • 2018
  • In: Theriogenology. - : ELSEVIER SCIENCE INC. - 0093-691X .- 1879-3231. ; 107, s. 27-35
  • Journal article (peer-reviewed)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.
  •  
49.
  • Martinez, Francisco, et al. (author)
  • Short lead(II) soaps: from weakly fluorescent crystals to strongly phosphorescent and structurally varied vitreous phases. A thermal, structural and spectroscopic study
  • 2014
  • In: Journal of Materials Chemistry C. - : Royal Society of Chemistry (RSC). - 2050-7526. ; 2:44, s. 9489-9496
  • Journal article (peer-reviewed)abstract
    • Short lead(II) alkanoates, from propionate to heptanoate, show a very intricate and reversible thermal behaviour, presenting crystalline phases and three different glass states (regular or amorphous, liquid crystal and rotator glasses) with different degrees of ordering depending on the alkyl chain length. A thorough thermal study was carried out in order to study the different phases and to analyze the thermodynamic parameters. The crystal structures of the compounds were solved by X-ray diffraction, showing similar arrangements of the 2D molecular stacking. PDF analyses of the local order in the glass structures showed shorter first neighbour lead-lead interatomic distances than in the crystalline structures. This allows establishment of a direct relationship between the structure and optical properties. Luminescence properties are, in fact, impressively enhanced in the glass states, passing from weak fluorescence at 77 K in the crystal phase to strong phosphorescence in the frozen glasses, which persists at room temperature. The high variability and the structure-property relationship described here pave the way for the design of materials with varied luminescence properties based on fine-tuning of their local structure.
  •  
50.
  • Parrilla, Inmaculada, et al. (author)
  • Cryopreservation of highly extended pig spermatozoa remodels its proteome and counteracts polyspermic fertilization in vitro
  • 2023
  • In: Andrology. - : WILEY. - 2047-2919 .- 2047-2927.
  • Journal article (peer-reviewed)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.
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