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| 2. |
- Barranco, Isabel, et al.
(author)
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Delays in processing and storage of pig seminal plasma alters levels of contained antioxidants
- 2021
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In: Research in Veterinary Science. - : ELSEVIER SCI LTD. - 0034-5288 .- 1532-2661. ; 135, s. 416-423
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Journal article (peer-reviewed)abstract
- Seminal 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 degrees 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 degrees C after ejaculate collection), in (2) SP-freezing (0 [control] or 24 hat 17 degrees C after SP-harvesting) or (3) the temperature of storage (-80 degrees C [control] or - 20 degrees 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 degrees 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.
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| 3. |
- Barranco, Isabel, et al.
(author)
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Extracellular vesicles isolated from porcine seminal plasma exhibit different tetraspanin expression profiles
- 2019
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In: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
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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.
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| 4. |
- Barranco, Isabel, et al.
(author)
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Levels of activity of superoxide dismutase in seminal plasma do not predict fertility of pig AI-semen doses
- 2019
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In: Theriogenology. - : ELSEVIER SCIENCE INC. - 0093-691X .- 1879-3231. ; 140, s. 18-24
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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.
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| 5. |
- Barranco, Isabel, et al.
(author)
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Seminal Plasma Cytokines Are Predictive of the Outcome of Boar Sperm Preservation
- 2019
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In: Frontiers in Veterinary Science. - : Frontiers Media S.A.. - 2297-1769. ; 6
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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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| 6. |
- Barranco, Isabel, et al.
(author)
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Seminal Plasma Modulates miRNA Expression by Sow Genital Tract Lining Explants
- 2020
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In: Biomolecules. - : MDPI. - 2218-273X. ; 10:6
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Journal article (peer-reviewed)abstract
- The seminal plasma (SP) modulates the female reproductive immune environment after mating, and microRNAs (miRNAs) could participate in the process. Considering that the boar ejaculate is built by fractions differing in SP-composition, this study evaluated whether exposure of mucosal explants of the sow internal genital tract (uterus, utero-tubal junction and isthmus) to different SP-fractions changed the profile of explant-secreted miRNAs. Mucosal explants retrieved from oestrus sows (n = 3) were in vitro exposed to: Medium 199 (M199, Control) or M199 supplemented (1:40 v/v) with SP from the sperm-rich fraction (SRF), the post-SRF or the entire recomposed ejaculate, for 16 h. After, the explants were cultured in M199 for 24 h to finally collect the media for miRNA analyses using GeneChip miRNA 4.0 Array (Affymetrix). Fifteen differentially expressed (False Discovery Rate (FDR) < 0.05 and Fold-change ≥ 2) miRNAs (11 down- versus 4 up-regulated) were identified (the most in the media of uterine explants incubated with SP from post-SRF). Bioinformatics analysis identified that predicted target genes of dysregulated miRNAs, mainly miR-34b, miR-205, miR-4776-3p and miR-574-5p, were involved in functions and pathways related to immune response. In conclusion, SP is able to elicit changes in the miRNAs profile secreted by female genital tract, ultimately depending SP-composition.
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| 7. |
- Lopes, Tania P., et al.
(author)
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Ovarian Follicle Growth during Lactation Determines the Reproductive Performance of Weaned Sows
- 2020
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In: Animals. - : MDPI. - 2076-2615. ; 10:6
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Journal article (peer-reviewed)abstract
- Simple Summary In this field study, the ovaries of weaned (n= 191, experiment 1) and lactating (n= 40, experiment 2) sows were transrectally scanned to measure the diameter of the follicles. Both the weaned and lactating sows showed great variability in the diameter of the ovarian follicles, indicating that the variability at weaning already existed during early lactation and was carried over to weaning. Sows with small follicles at weaning showed low reproductive performance and were more frequent among those with fewer farrowings and those weaned in summer-autumn. Factors causing variability in ovarian follicle size among weaned sows are not well known. This field study aimed to disclose influencing factors and evaluate if the differences at weaning were established during lactation. Ovaries were scanned using transrectal ultrasound. The first experiment was conducted over a year with 191 randomly chosen sows that were hierarchically grouped (p <0.001) according to ovarian follicle diameter reached at weaning: Small (0.20-0.30 cm;n= 37), medium (0.31-0.39 cm;n= 75), and large (0.40-1.00 cm;n= 69). Sows with small follicles showed a higher incidence of post-weaning anestrus (p< 0.01), longer wean-to-estrus/ovulation intervals (p <0.01) and farrowing smaller litters (p< 0.05). Ovaries with small follicles were more common among sows weaned in summer-autumn than in winter-spring (p <0.01) and among sows of lower parity (1-3) (p <0.05). In the second experiment, with 40 sows randomly chosen at farrowing, the ovaries were scanned at 7, 14, and 21 d post-partum. Sows showed great variability in ovarian follicular size during lactation with a consistent relationship between the three measurement times (r = 0.84,p <0.01). Follicle size was smaller in sows nursing in summer-autumn than in winter-spring (p <0.05). In conclusion, early lactation dictates the great variability in ovarian follicular diameter at weaning shown by sows. Sows with smaller follicles at weaning had longer intervals for estrus and ovulation and smaller litters at farrowing and they were in greater numbers among sows weaned during the summer and fall and among those with fewer previous farrowing.
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| 8. |
- Lopes, Tania P., et al.
(author)
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Weaned Sows with Small Ovarian Follicles Respond Poorly to the GnRH Agonist Buserelin
- 2020
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In: Animals. - : MDPI. - 2076-2615. ; 10:11
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Journal article (peer-reviewed)abstract
- Simple Summary This study evaluated the influence of mean ovarian follicle size and the season of weaning on the effectiveness of administering the GnRH agonist buserelin to synchronize ovulation in weaned sows. The results from 352 sows demonstrated that sows with small follicles (<0.5 cm in diameter) at treatment are poor responders, a condition more frequent among sows weaned in summer-autumn than in those weaned in winter-spring. The GnRH agonist buserelin (GnRH), used to synchronize ovulation in weaned sows, attains only 70-80% effectivity, owing to several reasons of ovarian origin. This study evaluated in particular whether mean ovarian follicle size at treatment and the season of weaning are among those influencing GnRH responsiveness. The experiment was carried out in a temperate-region farm with 352 sows of 1-6 parities weaned either in winter-spring (WS, 174 sows) or in summer-autumn (SA, 178 sows). The sows were randomized into two groups: GnRH (10 mu g of buserelin acetate at 86 h after weaning, 172 sows) and control (180 sows). The ovaries were transrectally scanned from weaning to ovulation and the sows clustered according to their mean follicular size at treatment time: small (<0.5 cm in diameter), medium (0.5 to 0.64 cm) and large (0.65 to 1.09 cm). In total, 88.33% of the GnRH-treated sows ovulated, with 82% of them within the expected time window (120-132 h after weaning). In contrast, 95.45% of the unresponsive sows had small follicles at the time of treatment and were mostly weaned in SA (20.45%) than in WS (4.76%). In conclusion, the conspicuous presence of sows having small ovarian follicles at treatment time compromises the efficiency of the GnRH agonist buserelin to synchronize ovulation in weaned sows, which occurs more frequently in summer-autumn weaning.
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| 9. |
- Padilla, Lorena, et al.
(author)
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Extracellular vesicles would be involved in the release and delivery of seminal TGF-beta isoforms in pigs
- 2023
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In: Frontiers in Veterinary Science. - : FRONTIERS MEDIA SA. - 2297-1769. ; 10
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Journal article (peer-reviewed)abstract
- Introduction : Pig seminal plasma (SP) is rich in active forms of all three isoforms (1-3) of transforming growth factor beta (TGF-beta), a chemokine modulatory of the immune environment in the female genital tract once semen is delivered during mating or artificial insemination (AI). The present study aimed to examine how TGF-beta s are secreted by the epithelium of the male reproductive tract and how they are transported in semen, emphasizing the interplay with seminal extracellular vesicles (sEVs). Methods : Source of TGF-beta s was examined by immunohistochemistry in testis, epididymis, and accessory sex glands, by immunocytochemistry in ejaculated spermatozoa, and by Luminex xMAP((R)) technology in SP and sEVs retrieved from healthy, fertile male pigs used as breeders in AI programs. Results : All three TGF-beta isoforms were expressed in all reproductive tissues explored and would be released into ductal lumen either in soluble form or associated with sEVs. Ejaculated spermatozoa expressed all three TGF-beta isoforms, both inside and outside, probably the outer one associated with membrane-bound sEVs. The results confirmed that pig SP contains all three TGF-beta isoforms and demonstrated that a substantial portion of them is associated with sEVs. Discussion : Seminal EVs would be involved in the cellular secretion of the active forms of seminal TGF-beta isoforms and in their safe transport from the male to the female reproductive tract.
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| 10. |
- Padilla, Lorena, et al.
(author)
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Granulocyte-macrophage colony stimulating factor (GM-CSF) is fully expressed in the genital tract, seminal plasma and spermatozoa of male pigs
- 2020
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In: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 10:1
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Journal article (peer-reviewed)abstract
- Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pro-inflammatory cytokine identified in boar seminal plasma (SP) but until now unexplored in terms of place of production and its association to spermatozoa. This study aimed to explore these aspects by evaluating the presence of GM-CSF in porcine reproductive organs (testes, epididymis and accessory sex glands), SP and mature spermatozoa (from cauda epididymis and ejaculated) using Western blot (WB), immunohistochemistry and immunocytochemistry. Positive labelling was obtained in tissues, SP and spermatozoa. In reproductive organs, WB revealed three forms of GM-CSF with different glycosylation degrees (15, 31 and 40 kDa). In SP and epididymal fluid, the GM-CSF appeared only in its active form while in spermatozoa the GM-CSF form present varied among sperm sources. Non-viable spermatozoa showed more GM-CSF than viable spermatozoa (14.87 +/- 1.98 RU vs. 7.25 +/- 0.52 RU) of fluorescence intensity. In conclusion, GM-CSF is widely present in the reproductive tract of male pigs, attached to the spermatozoa already in the epididymis as well as verted to SP. Consequently, the GM-CSF ought to regulate male genital tract and sperm function as well as mediating initial inflammatory responses and further mediating later immune actions by the female to semen deposition.
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