Browsing by Subject "Pig"
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- PublicationOpen AccessCo-localization of the zinc transporter ZnT8 (slc30A8) with ghrelin and motilin in the gastrointestinal tract of pigs(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Schweiger, Markus; Steffl, Martin; Amselgruber, Werner M.Zinc is an important co-factor for insulin storage in pancreatic β-cells of different species and the uptake of this ion into insulin containing secretory vesicles is managed by the zinc transporter, ZnT8, a member of the slc30A gene family. Recent studies indicate that this protein is a major autoimmune target in human type 1A diabetes and has also been implicated by genome-wide association studies in type 2 diabetes. Since individuals suffering from type 1 diabetes often develop gastrointestinal motility disorders, we investigated the expression of ZnT8 in the porcine gastrointestinal tract. For this purpose, we studied the cell-type specific expression of ZnT8 in the gut and its co-expression with endocrine hormones that are closely linked to intestinal motility regulation. Nested RT-PCR and immunostaining of sequential serial sections, as well as double-immunostaining using antibodies directed against ZnT8, ghrelin, motilin, neurotensin, serotonin and glucagon-like peptide 1, indicated that ZnT8 is colocalized with ghrelin and motilin. Our findings provide important information about the cell-type specific expression of ZnT8 in the porcine gastrointestinal system. The selective and exclusive expression of ZnT8 in two endocrine cell-types that are engaged in motility functions may be of particular interest for further investigations into type I diabetes-associated gastrointestinal dysfunctions.
PublicationOpen AccessData for compounds included in the article: "A sensitive immunoassay for the quantitation of Pig-MAP in pig saliva samples"(2024-10-05) Piñeiro, M; Matas-Quintanilla, M; Miralles, A; Gutiérrez, AM; Medicina y cirugía animal- PublicationEmbargoDelays in processing and storage of pig seminal plasma alters levels of contained antioxidants(2021-03) Barranco, Isabel; Tvarijonaviciute, Asta; Padilla, Lorena; Rodríguez-Martínez, Heriberto; Roca, Jordi; Xiomara, Lucas; Medicina y Cirugía AnimalSeminal plasma (SP) antioxidants are considered biomarkers of sperm function and fertility for AI-boars. The current protocol for their measurement implies the SP was harvested immediately after ejaculation and prompt stored at -80 °C until analysis. Such protocol may be impractical for AI-centers. This study evaluated how SP levels of antioxidants were influenced by delays in (1) SP-harvesting (0 [control], 2 or 24 h at 17 °C after ejaculate collection), in (2) SP-freezing (0 [control] or 24 h at 17 °C after SP-harvesting) or (3) the temperature of storage (-80 °C [control] or - 20 °C). The SP-antioxidants evaluated were: glutathione peroxidase [GPx], superoxide dismutase [SOD], paraoxonase-1 [PON-1], trolox equivalent antioxidant capacity [TEAC] and oxidative stress index [OSI]. A total of 120 aliquots from 10 entire ejaculates were handled in three trials. They were centrifuged (1500 g, 10 min) for harvesting SP and antioxidants were measured with an Automatic Chemistry Analyzer. A 24 h-delay in harvesting the SP led to an increase (p˂0.001) in TEAC and SOD SP-levels, and a decrease (p˂0.05) of OSI and PON-1. Similarly, a 24 h-delay to freeze the SP increased (p˂0.01) TEAC values and decreased (p˂0.01) PON-1 and GPx activity levels. Finally, storing the SP at -20 °C decreased (p˂0.001) SP-levels of TEAC, PON-1 and GPx, and increased (p˂0.01) OSI values. Strong positive relationships (p˂0.001) were found between antioxidant SP-levels in processed samples and their respective controls. In sum, handling and SP storage influence antioxidant measurements in AI-boars. Reliable levels of SP-antioxidants can only be warranted if a strict protocol for harvesting and SP storage is followed.
- PublicationOpen AccessExploring seminal plasma GSTM3 as a quality and in vivo fertility biomarker in pigs-relationship with sperm morphology(MDPI, 2020-08-12) Llavanera, Marc; Delgado-Bermúdez, Ariadna; Mateo-Otero, Yentel; Padilla, Lorena; Romeu, Xavier; Roca, Jordi; Barranco, Isabel; Yeste, Marc; Medicina y Cirugía AnimalGlutathione S-transferases Mu 3 (GSTM3) is an essential antioxidant enzyme whose presence in sperm has recently been related to sperm cryotolerance, quality and fertility. However, its role in seminal plasma (SP) as a predictor of the same sperm parameters has never been investigated. Herein, cell biology and proteomic approaches were performed to explore the presence, origin and role of SP-GSTM3 as a sperm quality and in vivo fertility biomarker. GSTM3 in SP was quantified using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit specific for Sus scrofa, whereas the presence of GSTM3 in testis, epididymis and accessory sex glands was assessed through immunoblotting analysis. Sperm quality and functionality parameters were evaluated in semen samples at 0 and 72 h of liquid-storage, whereas fertility parameters were recorded over a 12-months as farrowing rate and litter size. The presence and concentration of GSTM3 in SP was established for the first time in mammalian species, predominantly synthesized in the epididymis. The present study also evidenced a relationship between SP-GSTM3 and sperm morphology and suggested it is involved in epididymal maturation rather than in ejaculated sperm physiology. Finally, the data reported herein ruled out the role of this antioxidant enzyme as a quality and in vivo fertility biomarker of pig sperm.
- PublicationOpen AccessExpression of transforming growth factor-B3 -GF-B3- in the porcine ovary during the oestrus cycle(Murcia : F. Hernández, 2008) Steffl, M.; Schweiger, M.; Amselgruber, W.Transforming growth factor-ß (TGF-ß) proteins are growth factors that have been shown to be involved in regulation of ovarian follicular development. Ovarian expression, activity and functional significance of TGF-ß1 and TGF-ß2 isoforms were extensively studied in most species. However, little is known about the biological role of TGF-ß3 previously shown to be expressed independently of the other two isoforms. Therefore, expression of TGF-ß3 mRNA and protein was evaluated by RT-PCR and immunohistochemistry, respectively, in porcine ovaries collected during different phases of the oestrus cycle. Results of RT-PCR analysis showed that TGF-ß3 mRNA is expressed throughout the oestrus cycle. The level of TGF-ß3 mRNA expression was found to be higher at metoestrus and dioestrus. Weak TGF-ß3 immunoreactivity was present in follicular epithelial cells and oocytes of preantral follicles in all stages examined. TGF-ß3 protein expression was exclusively present in theca interna cell layer of antral follicles, and was particularly prominent in large antral follicles. Immediately after ovulation, almost all theca cells outside of the granulosa cell layer were intensively stained with anti-TGF-ß3. Immunostaining of TGF-ß3 in theca lutein cells rapidly decreased during corpus luteum development. It is suggested that TGF-ß3 may play an important role in modulating theca cell function of pre- and postovulatory follicles of the pig.
- PublicationOpen AccessImmunophenotype profile by flow cytometry reveals different subtypes of extracellular vesicles in porcine seminal plasma(2024-01-23) Barranco, Isabel; Alvarez-Barrientos, Alberto; Parra, Ana; Martinez-Diaz, Pablo; Lucas, Xiomara; Roca, Jordi; Medicina y Cirugía AnimalBackground: Porcine seminal plasma (SP) is endowed with a heterogeneous population of extracellular vesicles (sEVs). This study evaluated the immunophenotypic profile by high-sensitivity flow cytometry of eight sEV subpopulations isolated according to their size (small [S-sEVs] and large [L-sEVs]) from four different SP sources, namely three ejaculate fractions (the first 10 mL of the sperm rich fraction [SRF-P1], the remaining SRF [SRF-P2], and the post-SRF [PSRF]) and entire ejaculate (EE). Methods: Seminal EVs were isolated using a size exclusion chromatography-based protocol from six SP pools (five ejaculates/pool) of each SP source and characterized using complementary approaches including total protein (BCA™assay), particle size distribution (dynamic light scattering), morphology (transmission electron microscopy), and purity (albumin by Western blot). Expression of CD9, CD63, CD81, CD44 and HSP90β was analyzed in all sEV subpopulations by high-sensitivity flow cytometry according to MIFlowCyt-EV guidelines, including an accurate calibration, controls, and discrimination by CFSE-labelling. Results: Each sEV subpopulation exhibited a specific immunophenotypic profile. The percentage of sEVs positive for CD9, CD63, CD81 and HSP90β differed between S- and L-sEVs (P < 0.0001). Specifically, the percentage of sEVs positive for CD9 and CD63 was higher and that for CD81 was lower in S- than L-sEVs in the four SP sources. However, the percentage of HSP90β-positive sEVs was lower in S-sEVs than L-sEVs in the SRF-P1 and EE samples. The percentage of sEVs positive for CD9, CD63, and CD44 also differed among the four SP sources (P < 0.0001), being highest in PSRF samples. Notably, virtually all sEV subpopulations expressed CD44 (range: 88.04-98.50%). Conclusions: This study demonstrated the utility of high-sensitivity flow cytometry for sEV immunophenotyping, allowing the identification of distinct sEV subpopulations that may have different cellular origin, cargo, functions, and target cells.
- PublicationOpen AccessMetabolite Profiling of Pig Seminal Plasma Identifies Potential Biomarkers for Sperm Resilience to Liquid Preservation(Frontiers Media, 2021-05-28) Mateo-Otero, Yentel; Fernández-López, Pol; Ribas-Maynou, Jordi; Roca, Jordi; Miró, Jordi; Yeste, Marc; Barranco, Isabel; Medicina y Cirugía AnimalMetabolomic approaches allow the study of downstream gene expression events since metabolites are considered as the products of cell signaling pathways. For this reason, many studies in humans have already been conducted to determine the influence of the metabolites present in seminal plasma (SP) on sperm physiology, and to identify putative biomarkers. However, in livestock species, these relationships are yet to be uncovered. Thus, the present study aimed to explore: (i) if concentrations of metabolites in pig SP are related to sperm quality and functionality, and (ii) if they could predict the sperm resilience to liquid storage at 17°C. To this end, 28 ejaculates were individually collected and split into three aliquots: one was used for SP analysis through nuclear magnetic resonance (NMR) spectroscopy; another served for the evaluation of sperm concentration and morphology; and the last one was utilized to determine sperm functionality parameters using computer-assisted sperm analysis (CASA) and flow cytometry after 0 h and 72 h of liquid-storage at 17°C. NMR analysis allowed the identification and quantification of 23 metabolites present in pig SP which, except for fumarate, were not observed to follow a breed-dependent behavior. Moreover, specific relationships between metabolites and sperm variables were identified: (i) glutamate, methanol, trimethylamine N-oxide, carnitine, and isoleucine were seen to be related to some sperm quality and functionality parameters evaluated immediately after semen collection; (ii) leucine, hypotaurine, carnitine and isoleucine were found to be associated to the sperm ability to withstand liquid storage; and (iii) Bayesian multiple regression models allowed the identification of metabolite patterns for specific sperm parameters at both 0 h and 72 h. The identification of these relationships opens up the possibility of further investigating these metabolites as potential sperm functional biomarkers.
- PublicationOpen AccessParacervical ganglion in the female pig during prenatal development: morphology and immunohistochemical characteristics(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Franke-Radowiecka, AmeliaThe present study investigated the development of the paracervical ganglion in 5-, 7- and 10-week-old porcine foetuses using double labelling immunofluorescence method. In 5-week-old foetuses single PGP-positive perikarya were visible only along the mesonephric ducts. They contained DβH or VAChT, and nerve fibres usually were PGP/VAChT-positive. The perikarya were mainly oval. In 7-week-old foetuses, a compact group of PGP-positive neurons (3144±213) was visible on both sides and externally to the uterovaginal canal mesenchyme of paramesonephric ducts. Nerve cell bodies contained only DβH (36.40±1.63%) or VAChT (17.31±1.13%). In the 10-week-old foetuses, the compact group of PGP-positive neurons divided into several large and many small clusters of nerve cells and also became more expanded along the whole uterovaginal canal mesenchyme reaching the initial part of the uterine canal of the paramesonephric duct. The number of neurons located in these neuronal structures increased to 4121±259. Immunohistochemistry revealed that PGP-positive nerve cell bodies contained DβH (40.26±0,73%) and VAChT (30.73±1.34%) and were also immunoreactive for NPY (33.24±1,27%), SOM (23.6±0,44%) or VIP (22.9±1,13%). Other substances studied (GAL, NOS, CGRP, SP) were not determined at this stage of the development. In this study, for the first time, the morphology of PCG formation in the porcine foetus has been described in three stages of development. Dynamic changes in the number of neurons and their sizes were also noted, as well as the changes in immunochistochemical coding of maturing neurons.
- PublicationOpen AccessPlacental vascularization in in vitro-derived pigs: a preliminary study(Colégio Brasileiro de Reprodução Animal - CBRA, 2022-09) Úrsula Álvarez Martín; Pilar Coy; Juan Seva; Raquel Romar Andrés; Ester Párraga Ros; FisiologiaThe placenta plays a critical role in maintaining and protecting the developing fetus. Placental vascularization abnormalities, including a decrease in arterial number, lumen size, and branching, have been extensively described in humans born from in vitro-produced (IVP) embryos (Riesche and Bartolomei, Seminars Reprod Med, 36:240-247, 2018) but studies on IVP pigs are very limited (Ao et al., Placenta 57:94-101, 2017). The objective of this study was to compare the placental vascularization in pigs born from in vitro- and in vivo-produced embryos (the latter born by artificial insemination; AI group). IVP embryos were produced after in-vitro fertilization (IVF) of in vitro matured oocytes and further culture (IVC) up to blastocyts stage in media supplemented with or without 1% porcine oviductal fluid and 1% uterine fluid (more details in Paris-Oller et al., J AnimSci and Biotech 12:32-44, 2021). Blastocysts produced with (RF-IVP group) and without (C-IVP group) reproductive fluids were surgically transferred at day 7 post-IVF. Both AI and IVP embryos were produced with spermatozoa from the same boar. After birth, placenta samples were collected at 3-5 cm from the insertion of umbilical cord, and fetal parameters were recorded. The placenta of 9 animals (3 per group) from different litters was selected following these criteria among animals: similar uterus position, birth weight, and crown-rump length; and a close male/female ratio among groups. Samples were fixed (10% formaldehyde solution) and paraffin embedded. Two complete placental sections (5 μm thickness) were stained (hematoxylin–eosin), photographed at 5x (ZEN 3.2, ZEN lite, Zeiss) and images processed (ImageJ) for a detailed study to record vessel number, area occupied by each vessel (µm2), and total vascular area (%). Based on their size and histological characteristics, vessels were categorized by an expert operator as capillary (1-500 µm2), arteriole/venule (501-1000 µm2), small artery/vein (1001-3000 µm2), medium-sized artery/vein (3001-30000 µm2), and large artery/vein (>30000 µm2). Data (mean±SEM) were analyzed by one-way ANOVA (Systat v13.1), and differences (P<0.05) were compared by Tukey’s test. The total placental area observed, and total number of vessels analyzed was higher in AI (86.1±7.5 mm2, 726 vessels) than C-IVP (45.9±6.8 mm2, 544 vessels), and RF-IVP (52.8±5.1 mm2, 637 vessels) (P<0.05). However, no differences were found in the total vascular area being 14.9±3.3% (AI), 19.9±2.7% (C-IVP), and 17.8±2.2 (RF-IVP) with similar pattern distribution in all groups: over 85% microvessels, 10-15% medium-size vessels and 5% macrovessels. However, the vascular area occupied by medium-sized vessels (arteries and veins) was significantly higher in the AI group (7.2±0.5%) than in IVP groups (2.1±0.3% and 1.8±0.2%) regardless of the addition of reproductive fluids (P<0.05). No differences in vascular areas of micro and macrovessels were observed. Preliminary results show that impaired placental vascularization in ART-derived pigs might occur due to a reduction of medium size vessels.
- PublicationOpen AccessRelative transcript abundance in porcine cumulus cells collected from different sized follicles(2020) Moros-Nicolás, C.; Izquierdo-Rico, M.J.; Li, Y.; González-Brusi, L.; Romar, R.; Funahashi, H.; Biología Celular e Histología
- PublicationOpen AccessSeminal plasma cytokines are predictive of the outcome of boar sperm preservation(2019-12-04) Barranco, Isabel; Padilla, Lorena; Perez-Patiño, Cristina; Vazquez, Juan M; Martinez, Emilio A; Rodriguez-Martinez, Heriberto; Roca, Jordi; Parrilla, Inmaculada; Medicina y Cirugía AnimalBackground: 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 O∙−2 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.
- PublicationOpen AccessSeminal Plasma Modifies the Transcriptional Pattern of the Endometrium and Advances Embryo Development in Pigs(2019-12-18) Martínez, Cristina A.; Cambra, Josep M.; Parrilla, Inmaculada; Roca, Jordi; Ferreira-Dias, Graça; Pallarés, Francisco J.; Lucas, Xiomara; Vázquez, Juan M.; Martínez, Emilio A.; Gil, María A.; Rodríguez-Martínez, Heriberto; Cuello, Cristina; Álvarez-Rodríguez, Manuel; Medicina y Cirugía AnimalBackground: Seminal plasma (SP) promotes sperm survival and fertilizing capacity, and potentially affects embryo development, presumably via specific signaling pathways to the internal female genital tract. Objectives: This study evaluated how heterologous SP, infused immediately before postcervical artificial insemination (AI) affected embryo development and the transcriptional pattern of the pig endometria containing embryos. Materials and Methods: Postweaning estrus sows (n = 34) received 40-mL intrauterine infusions of either heterologous pooled SP or Beltsville Thawing Solution (BTS; control) 30 min before AI of semen extended to 10% of homologous SP. Embryos (all sows) and endometrium samples (3 sows/group) were removed during laparotomy 6 days after the infusion of SP or BTS to morphologically evaluate the embryos to determine their developmental stage and to analyze the endometrial transcriptome using microarrays (PORGENE 1.0 ST GeneChip array, Affymetrix) followed by qPCR for further validation. Results: Embryo viability was equal between the groups (~93%), but embryo development was significantly (P < 0.05) more advanced in the SP-treated group compared to control. A total of 1,604 endometrium transcripts were differentially expressed in the SP group compared to the control group. An enrichment analysis showed an overrepresentation of genes and pathways associated with the immune response, cytokine signaling, cell cycle, cell adhesion, and hormone response, among others. Conclusions: SP infusions prior to AI positively impacted the preimplantation embryo development and altered the expression of the endometrial genes and pathways potentially involved in embryo development.
- PublicationOpen AccessThe transcriptome of pig spermatozoa, and its role in fertility(MDPI, 2020-02-25) Álvarez Rodríguez, Manuel; Martínez, Cristina; Wright, Dominic; Barranco, Isabel; Roca, Jordi; Rodríguez Martínez, Heriberto; Medicina y Cirugía AnimalIn the study presented here we identified transcriptomic markers for fertility in the cargo of pig ejaculated spermatozoa using porcine-specific micro-arrays (GeneChip® miRNA 4.0 and GeneChip® Porcine Gene 1.0 ST). We report (i) the relative abundance of the ssc-miR-1285, miR-16, miR-4332, miR-92a, miR-671-5p, miR-4334-5p, miR-425-5p, miR-191, miR-92b-5p and miR-15b miRNAs, and (ii) the presence of 347 up-regulated and 174 down-regulated RNA transcripts in high-fertility breeding boars, based on differences of farrowing rate (FS) and litter size (LS), relative to low-fertility boars in the (Artificial Insemination) AI program. An overrepresentation analysis of the protein class (PANTHER) identified significant fold-increases for C-C chemokine binding (GO:0019957): CCR7, which activates B- and T-lymphocytes, 8-fold increase), XCR1 and CXCR4 (with ubiquitin as a natural ligand, 1.24-fold increase), cytokine receptor activity (GO:0005126): IL23R receptor of the IL23 protein, associated to JAK2 and STAT3, 3.4-fold increase), the TGF-receptor (PC00035) genes ACVR1C and ACVR2B (12-fold increase). Moreover, two micro-RNAs (miR-221 and mir-621) were down- and up-regulated, respectively, in high-fertility males. In conclusion, boars with different fertility performance possess a wide variety of differentially expressed RNA present in spermatozoa that would be attractive targets as non-invasive molecular markers for predicting fertility.