| 1. |
- Barranco, Isabel, et al.
(författare)
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Delays in processing and storage of pig seminal plasma alters levels of contained antioxidants
- 2021
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Ingår i: Research in Veterinary Science. - : ELSEVIER SCI LTD. - 0034-5288 .- 1532-2661. ; 135, s. 416-423
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Tidskriftsartikel (refereegranskat)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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| 2. |
- Barranco, Isabel, et al.
(författare)
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Seminal Plasma Modulates miRNA Expression by Sow Genital Tract Lining Explants
- 2020
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Ingår i: Biomolecules. - : MDPI. - 2218-273X. ; 10:6
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Tidskriftsartikel (refereegranskat)abstract
- The seminal plasma (SP) modulates the female reproductive immune environment after mating, and microRNAs (miRNAs) could participate in the process. Considering that the boar ejaculate is built by fractions differing in SP-composition, this study evaluated whether exposure of mucosal explants of the sow internal genital tract (uterus, utero-tubal junction and isthmus) to different SP-fractions changed the profile of explant-secreted miRNAs. Mucosal explants retrieved from oestrus sows (n = 3) were in vitro exposed to: Medium 199 (M199, Control) or M199 supplemented (1:40 v/v) with SP from the sperm-rich fraction (SRF), the post-SRF or the entire recomposed ejaculate, for 16 h. After, the explants were cultured in M199 for 24 h to finally collect the media for miRNA analyses using GeneChip miRNA 4.0 Array (Affymetrix). Fifteen differentially expressed (False Discovery Rate (FDR) < 0.05 and Fold-change ≥ 2) miRNAs (11 down- versus 4 up-regulated) were identified (the most in the media of uterine explants incubated with SP from post-SRF). Bioinformatics analysis identified that predicted target genes of dysregulated miRNAs, mainly miR-34b, miR-205, miR-4776-3p and miR-574-5p, were involved in functions and pathways related to immune response. In conclusion, SP is able to elicit changes in the miRNAs profile secreted by female genital tract, ultimately depending SP-composition.
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| 3. |
- Martinez, Emilio A., et al.
(författare)
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Achievements and future perspectives of embryo transfer technology in pigs
- 2019
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Ingår i: Reproduction in domestic animals. - : WILEY. - 0936-6768 .- 1439-0531. ; 54
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Tidskriftsartikel (refereegranskat)abstract
- Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows non-surgical ET and short- and long-term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20 years. This review includes several aspects of non-surgical ET technology and different issues affecting ET programmes and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short-term use in pig production.
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| 4. |
- Martinez Serrano, Cristina, et al.
(författare)
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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
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Ingår i: Biology. - : MDPI. - 2079-7737. ; 10:2
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary Although endometrial immune regulation in pigs during the early preimplantation period is poorly documented, particularly under conditions of embryo transfer (ET), it is recognized that seminal plasma (SP) induces molecular changes in the reproductive tract, influencing numerous reproductive functions. A principal constituent of SP is the cytokine transforming growth factor beta 1 (TGF-beta 1), which has an important role in embryo development, pregnancy establishment, and progression. The present study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, porcine TGF-beta 1 in an extender, or an extender alone (control)) by mimicking an ET scenario in so-called "donor" (inseminated) and "recipient" (uninseminated) sows. We investigated the effects of these treatments on day 6 embryo development ("donors") and endometrial explants cytokine production ("donors" and "recipients"). SP infusion positively influenced embryo development compared with TGF-beta 1 or extender infusions. Infusion treatments differentially affected endometrial cytokine production, with the effects being stronger in "donors" than in "recipients." Increased knowledge of the effects of SP or some of its active components on the female immune system may help to develop strategies for increasing the reproductive efficiency for the benefit of pig ET. Seminal plasma (SP) in the female genital tract induces changes that affect multiple reproductive processes. One of the active components in SP is the transforming growth factor beta 1 (TGF-beta 1), which has major roles in embryo development and pregnancy. Embryo transfer (ET) technology is welcomed by the pig industry provided that embryo quality at embryo collection as well as the fertility and prolificacy of the recipients after the ET is increased. This study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, TGF-beta 1 cytokine in the extender, or the extender alone (control)) by mimicking an ET scenario in so-called "donor" (inseminated) and "recipient" (uninseminated) sows. On day 6 (day 0-onset of estrus), all "donors" were laparotomized to determine their pregnancy status (presence and developmental stage of the embryos). In addition, endometrial explants were collected from pregnant "donors" and cyclic "recipients," incubated for 24 h, and analyzed for cytokine production. SP infusions (unlike TGF-beta 1 infusions) positively influenced the developmental stage of day 6 embryos. Infusion treatments differentially influenced the endometrial cytokine production, mainly in donors. We concluded that SP infusions prior to AI not only impacted the porcine preimplantation embryo development but also influenced the endometrial cytokine production six days after treatment, both in donors and recipients.
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| 5. |
- Martinez-Serrano, Cristina, et al.
(författare)
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Seminal Plasma Modifies the Transcriptional Pattern of the Endometrium and Advances Embryo Development in Pigs
- 2019
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Ingår i: Frontiers in Veterinary Science. - : Frontiers Media S.A.. - 2297-1769. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Background: Boar seminal plasma is rich in cytokines, which could influence the capability of spermatozoa to tolerate preservation.Objectives: To evaluate the involvement of boar seminal plasma cytokines in the changes experienced by boar spermatozoa during their storage, either in liquid or frozen state.Materials and Methods: In two separated experiments, semen samples from healthy and fertile boars were split in two aliquots, one centrifuged twice (1,500 ×g for 10 min) to harvest seminal plasma, whereas the other was either commercially extended (3 × 107 sperm/mL) and liquid-stored at 17°C during 144 h (n = 28, Experiment 1) or frozen-thawed using a standard 0.5 mL protocol (n = 27, Experiment 2). Sixteen cytokines were quantified using Luminex xMAP®. Sperm attributes (CASA-evaluated total and progressive motility; flow cytometry-evaluated sperm viability, production of intracellular H2O2 and O2•-" role="presentation" style="box-sizing: border-box; display: inline; line-height: normal; word-spacing: normal; overflow-wrap: normal; white-space: nowrap; float: none; direction: ltr; max-width: none; max-height: none; min-width: 0px; min-height: 0px; border: 0px; padding: 0px; margin: 0px; position: relative; outline: 0px !important;">O∙−2O2•- and levels of lipid peroxidation in viable spermatozoa) were evaluated either at 0, 72, or 144 h of liquid storage (Experiment 1) or before freezing and at 30- and 150-min post-thawing (Experiment 2).Results: Multiple linear regression models, with Bayesian approach for variable selection, revealed that the anti-inflammatory TGF-β2, TGF-β3, IL-1Ra, and IL-4 and the pro-inflammatory IL-8 and IL-18, predicted changes in sperm motility for liquid-stored semen while the anti-inflammatory IFN-γ was included in the models predicting changes in all sperm attributes for cryopreserved semen.Conclusion: Specific boar seminal plasma cytokines would contribute to modulate the structural and metabolic changes shown by spermatozoa during preservation, either in liquid or frozen state.
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| 6. |
- Maside, Carolina, et al.
(författare)
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Supplementation with exogenous coenzyme Q10 to media for in vitro maturation and embryo culture fails to promote the developmental competence of porcine embryos
- 2019
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Ingår i: Reproduction in domestic animals. - : WILEY. - 0936-6768 .- 1439-0531. ; 54, s. 72-77
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Tidskriftsartikel (refereegranskat)abstract
- The coenzyme Q10 (CoQ10) is a potent antioxidant with critical protection role against cell oxidative stress, caused by the mitochondrial dysfunction. This study evaluated the effects of CoQ10 supplementation to in vitro maturation (IVM) or embryo culture media on the maturation, fertilization and subsequent embryonic development of pig oocytes and embryos. Maturation (Experiment 1) or embryo culture (Experiment 2) media were supplemented with 0 (control), 10, 25, 50 and 100 mu M CoQ10. The addition of 10-50 mu M CoQ10 to the IVM medium did not affect the percentage of MII oocytes nor the fertilization or the parameters of subsequent embryonic development. Exogenous CoQ10 in the culture medium neither did affect the development to the 2-4-cell stage nor rates of blastocyst formation. Moreover, the highest concentration of CoQ10 (100 mu M) in the maturation medium negatively affected blastocyst rates. In conclusion, exogenous CoQ10 supplementation of maturation or embryo culture media failed to improve the outcomes of our in vitro embryo production system and its use as an exogenous antioxidant should not be encouraged.
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| 7. |
- Padilla, Lorena, et al.
(författare)
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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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Ingår i: Frontiers in Veterinary Science. - : FRONTIERS MEDIA SA. - 2297-1769. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Introduction : Pig seminal plasma (SP) is rich in active forms of all three isoforms (1-3) of transforming growth factor beta (TGF-beta), a chemokine modulatory of the immune environment in the female genital tract once semen is delivered during mating or artificial insemination (AI). The present study aimed to examine how TGF-beta s are secreted by the epithelium of the male reproductive tract and how they are transported in semen, emphasizing the interplay with seminal extracellular vesicles (sEVs). Methods : Source of TGF-beta s was examined by immunohistochemistry in testis, epididymis, and accessory sex glands, by immunocytochemistry in ejaculated spermatozoa, and by Luminex xMAP((R)) technology in SP and sEVs retrieved from healthy, fertile male pigs used as breeders in AI programs. Results : All three TGF-beta isoforms were expressed in all reproductive tissues explored and would be released into ductal lumen either in soluble form or associated with sEVs. Ejaculated spermatozoa expressed all three TGF-beta isoforms, both inside and outside, probably the outer one associated with membrane-bound sEVs. The results confirmed that pig SP contains all three TGF-beta isoforms and demonstrated that a substantial portion of them is associated with sEVs. Discussion : Seminal EVs would be involved in the cellular secretion of the active forms of seminal TGF-beta isoforms and in their safe transport from the male to the female reproductive tract.
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| 8. |
- Padilla, Lorena, et al.
(författare)
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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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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pro-inflammatory cytokine identified in boar seminal plasma (SP) but until now unexplored in terms of place of production and its association to spermatozoa. This study aimed to explore these aspects by evaluating the presence of GM-CSF in porcine reproductive organs (testes, epididymis and accessory sex glands), SP and mature spermatozoa (from cauda epididymis and ejaculated) using Western blot (WB), immunohistochemistry and immunocytochemistry. Positive labelling was obtained in tissues, SP and spermatozoa. In reproductive organs, WB revealed three forms of GM-CSF with different glycosylation degrees (15, 31 and 40 kDa). In SP and epididymal fluid, the GM-CSF appeared only in its active form while in spermatozoa the GM-CSF form present varied among sperm sources. Non-viable spermatozoa showed more GM-CSF than viable spermatozoa (14.87 +/- 1.98 RU vs. 7.25 +/- 0.52 RU) of fluorescence intensity. In conclusion, GM-CSF is widely present in the reproductive tract of male pigs, attached to the spermatozoa already in the epididymis as well as verted to SP. Consequently, the GM-CSF ought to regulate male genital tract and sperm function as well as mediating initial inflammatory responses and further mediating later immune actions by the female to semen deposition.
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| 9. |
- Padilla, Lorena, et al.
(författare)
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Measurable Cytokine Concentrations in Pig Seminal Plasma Are Modified by Semen Handling and Storage
- 2020
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Ingår i: Biology. - : MDPI. - 2079-7737. ; 9:9
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Tidskriftsartikel (refereegranskat)abstract
- Sample handling and storing are critical steps for the reliable measurement of circulating biomolecules in biological fluids. This study evaluates how cytokine measurements in pig seminal plasma (SP) vary depending on semen handling and SP storage. Thirteen cytokines (GM-CSF, IFN gamma, IL-1 alpha, IL-1 beta, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and TNF alpha) were measured using Luminex xMAP(R) technology in individual seminal plasma (SP) samples (n = 62) from healthy breeding boars. Three separate experiments explored the delay (2 h and 24 h) in SP collection after ejaculation (Experiment 1) and SP storage, either short-term (5 degrees C, -20 degrees C and -80 degrees C for 72 h, Experiment 2) or long-term (at -20 degrees C and -80 degrees C for two months, Experiment 3), before analysis. Levels in fresh SP-samples were used as baseline control values. Delays in SP harvesting of up to 24 h did not substantially impact SP cytokine measurements. Some cytokines showed instability in stored SP samples, mainly in long-term storage. Ideally, cytokines in pig SP should be measured in fresh samples harvested within 24 h after ejaculation. If storage of SP is imperative, storage conditions should be adjusted for each cytokine.
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| 10. |
- Padilla, Lorena, et al.
(författare)
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Period of Boar Ejaculate Collection Contributes to the Yearly Intra-Male Variability of Seminal Plasma Cytokines
- 2020
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Ingår i: Biology. - : MDPI. - 2079-7737. ; 9:5
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Tidskriftsartikel (refereegranskat)abstract
- The concentrations of cytokines in seminal plasma (SP) fluctuate over time in healthy males, weakening their practical usefulness as diagnostic tools. This study evaluated the relevance of intra-male variability in SP cytokines and to what extent the period of the year when ejaculate is collected contributes to such variability. Thirteen cytokines (GM-CSF, IFN gamma, IL-1 alpha, IL-1 beta, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and TNF alpha) were measured using the Luminex xMAP(R) technology for 180 SP samples of ejaculate collected over a year from nine healthy and fertile boars. The SP samples were grouped into two annual periods according to decreasing or increasing daylight and ambient temperature. Intra-male variability was higher than inter-male variability for all cytokines. All SP cytokines showed concentration differences between the two periods of the year, showing the highest concentration during the increasing daylength/temperature period, irrespective of the male. Similarly, some cytokines showed differences between daylength/temperature periods when focusing on their total amount in the ejaculate. No strong relationship (explaining more than 50% of the total variance) was found between annual fluctuations in SP-cytokine levels and semen parameters. In conclusion, the period of the year during which ejaculates were collected helps explain the intra-male variability of SP-cytokine levels in breeding boars.
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