DigitalUM :: Browsing by Subject "Seminal plasma"

Browsing by Subject "Seminal plasma"

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Open Access
1H Nuclear Magnetic Resonance of pig seminal plasma reveals intra-ejaculate variation in metabolites
(MDPI, 2020-06-15) Mateo Otero, Yentel; Fernández-López, Pol; Gil-Caballero, Sergi; Fernandez-Fuertes, Beatriz; Bonet, Sergi; Barranco Cascales, Isabel; Yeste, Marc; Medicina y Cirugía Animal; Facultad de Veterinaria
In pigs, ejaculate is expelled in fractions, mainly the sperm-rich fraction (SRF) and the post-SRF (PSRF), which differ in both sperm content and origin. In addition, intra-ejaculate variability between fractions in terms of sperm reproductive characteristics has been previously reported, the highest sperm quality being observed in the first 10 mL of the SRF (SRF-P1). As seminal plasma (SP) composition has been purported to influence sperm physiology, the aim of this study was to profile pig SP metabolite composition and to find putative differences between the ejaculate portions (SRF-P1, the rest of SRF [SRF-P2], PSRF) and entire ejaculate (EE). To this end, ejaculates (n = 8, one per boar) were collected in fractions and SP was analyzed using 1H Nuclear Magnetic Resonance spectroscopy. We identified 19 metabolites present in all ejaculate portions and the EE, and reported correlations between the metabolites. Additionally, and for the first time in mammals, we found intra-ejaculate variability in the SP metabolites, observing different relative abundances in choline, glycerophosphocholine and glycine. Regarding their influence in sperm physiology, we hypothesize that these metabolites may explain the specific reproductive characteristics of each ejaculate portion. Finally, the reported SP metabolites could serve as a first steppingstone in the study of quality, functionality, and fertility biomarkers.
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A Size-Exclusion Chromatography-Based Procedure for Isolating Extracellular Vesicle Subsets from Porcine Seminal Plasma
(Humana Press, 2024-04-10) Martínez Díaz, Pablo; Ana Parra; Christian M Sanchez-López; Antonio Marcilla; Bucci, Diego; Roca Aleu, Jorge; Barranco Cascales, Isabel; Medicina y Cirugía Animal; Facultad de Veterinaria
Extracellular vesicles (EVs), membrane nanoparticles (30-to-1000 nm diameter) secreted and released by most of the body functional cells, have emerged as powerful cell-to-cell messengers transferring their bioactive cargo (proteins, lipids, and nucleic acids) from donor to recipient cells. The promising potential utility of EVs as both noninvasive biomarkers and therapeutic carriers for several pathologies, including some types of cancers, has attracted increasing scientific interest. EVs can be found in all body biofluids, including seminal plasma, a complex fluid consisting mainly of a mixture of secretions of the epididymis and accessory sex glands. Seminal EVs are involved in modulating both sperm physiological processes and immune environment of the internal female genital tract, thus playing an essential indirect role in fertilization and embryo development. Seminal plasma, alike other biofluids, contains a heterogenous population of EV-subsets. However, the lack of consensus on the most accurate procedure for isolating EV-subsets has led to a poor definition of their composition/function. Currently, size exclusion chromatography (SEC), a size-selective separation method, is one of the most promising EV-isolation procedures, allowing the isolation of EVs from biological fluids in a purer, easier, cheaper, and more scalable way compared to other alternative isolation procedures. This chapter reports a SEC-based protocol, combined with differential centrifugation and ultrafiltration, to isolate two subsets of seminal EVs differing in size (large and small EVs) in the ejaculate of pigs, a livestock species of great productive interest and an outstanding animal model for human reproduction.
Open Access
Artificial insemination of all ejaculated sperm fractions accelerates embryo development and increases the uterine vascularity in the pig
(Elsevier, 2024-04-15) Toledo Guardiola, Santa María; Luongo, Chiara; García Vázquez, Francisco A.; Soriano Úbeda, C.; Matás, C.; Párraga Ros, Ester; Seva Alcaraz, Juan; Anatomía y Anatomía Patológica Comparadas
The semen of boar is characterized by ejaculation in well-differentiated fractions with specific concentration, composition, and volume. The ‘sperm-rich fraction' (SRF), the most concentrated seminal fraction, is habitually collected in insemination centers to make artificial insemination (AI) doses. The absence of the other fractions in AI doses could alter the uterine reaction to AI and not trigger essential responses that could maximize fertility. Thus, there is an urge to ascertain the impact of different ejaculate fractions on the uterus after AI to optimize the semen doses. This work analyzed specific parameters related to fertility in pregnant artificially inseminated sows (n = 15) with ac-cumulative fractions of the semen of boars (n = 6): F1, composed of the sperm-rich fraction (SRF); F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF. Non-inseminated sows (n = 5) were included as control (C). The different types of seminal dose did not affect the number of ovulated follicles (CL; corpora lutea, p > 0.05) but did affect the embryo development (p < 0.05). The proportion of embryos in morula stages was significantly higher in AI-F1 sows (84.4%, p < 0.05). Morulas and blastocysts were balanced in AI-F2 or AI-F3 (p > 0.05). Independently of the type of seminal dose (F1, F2, or F3), we observed by immunohistochemistry that AI significantly increased uterine vascularization, although with some anatomical differences. The cranial region of the uterine horns was significantly more vascularized in AI-F1 or AI-F2 sows (26.7 ± 2.3 and 28.6 ± 2.0%, respectively), and AI-F3 showed significantly less vascularization at that point (17.8 ± 1.6%, p < 0.05). To summarize, the synergistic effect of all ejaculate fractions accelerates embryo development, at least during the preimplantation period, and increases the uterine reaction to AI in certain parts of the uterus.
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Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate
(Elsevier, 2014-07-15) Alkmin, Diego V.; Pérez Patino, Cristina; Barranco Cascales, Isabel; Parrilla Riera, Inmaculada; Vázquez, Juan M.; Váquez, Juan M.; Martínez Navarro, Emilio; Rodríguez Martínez, Heriberto; Roca Aleu, Jorge; Medicina y Cirugía Animal
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 24h at 15-17°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 24h 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 < 0.01) than BE samples. Control samples showed higher (p < 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 < 0.05) than EP3. These results showed that boar sperm from BE are more cryosensitive than those from the SRF, particularly when held 24h before freezing, which would be attributable to the cryonegative effects exerted by the SP from post SRF.
Open Access
Cryogenic electron microscopy reveals morphologically distinct subtypes of extracellular vesicles among porcine ejaculate fractions
(Nature Research, 2024-07-13) Parra, Ana; Barranco, Isabel; Martínez Díaz, Pablo; González, Esperanza; Albóniga, Oihane E.; Cabrera, Diana; Falcón Pérez, Juan M.; Roca, Jordi; Medicina y Cirugía Animal
Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin.
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Delays 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 Animal
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 °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.
Open Access
Epididymal and ejaculated sperm functionality is regulated differently by periovulatory oviductal fluid in pigs
(Wiley, 2020-09-13) Soriano-Úbeda, Cristina; Avilés-López, Karen; García Vázquez, Francisco Alberto; Romero Aguirregomezcorta, Jon; Matas Parra, Carmen; Anatomía y Anatomía Patológica Comparada; Facultad de Veterinaria
Background: The current results of in vitro reproduction techniques in pigs, such as in vitro fertilization (IVF) and embryo development, show high performance with both epididymal and ejaculated spermatozoa. However, the results using ejaculated spermatozoa are even better. Ejaculated spermatozoa are exposed to the secretions of the accessory seminal glands: the seminal plasma (SP). It has been reported that exposure of spermatozoa to reproductive fluids, such as SP or periovulatory oviductal fluid (pOF), modulates sperm functionality both in vivo and in vitro. But whether or not this modulating effect of pOF depends on the origin of the spermatozoa being epididymal or ejaculated, is still unknown. Objectives: To determine and compare the effect of pOF on epididymal and ejaculated sperm functionality. Material and methods: The effects of incubating spermatozoa from the epididymis and ejaculate with pOF in capacitating conditions were investigated by analyzing sperm motility, phosphorylation of protein kinase A substrates and proteins in tyros ine (pPKAs and pTyr, respectively), the interaction of the spermatozoa with the oocyte in IVF and intracytoplasmic sperm injection (ICSI), and, finally, the spermatozoa chromatin condensation status.Results: the pOF modified events related to capacitation in epididymal spermatozoa by decreasing motility, pPKAs and pTyr. In the interaction with the oocyte after sperm capacitation, pOF regulated the epididymal and ejaculated spermatozoa differently. While pOF decreased the number of spermatozoa bound to the zona pellucida (Spz/ZP) and increased oocyte activation after ICSI with epididymal spermatozoa, with the ejaculated spermatozoa, it decreased the mean number penetrating each oocyte (Spz/O). Additionally, pOF significantly increased the nuclear decondensation of the epididymal spermatozoa after the fertilization of the oocyte.Conclusion: The modulation of sperm functionality by pOF is conditioned by the origin of the spermatozoa.
Open Access
Exploring 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 Animal
Glutathione 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.
Open Access
Extensive dataset of boar seminal plasma proteome displaying putative reproductive functions of identified proteins
(Elsevier, 2016-07-29) Pérez-Patiño, Cristina; Barranco, Isabel; Parrilla, Inmaculada; Martínez, Emilio A.; Rodríguez-Martínez, Heriberto; Roca, Jordi; Medicina y Cirugía Animal
A complete proteomic profile of seminal plasma (SP) remains challenging, particularly in porcine. The data reports on the analysis of boar SP-proteins by using a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS from 33 pooled SP-samples (11 boars, 3 ejaculates/boar). A complete dataset of the 536 SP-proteins identified and validated with confidence ≥95% (Unused Score >1.3) and a false discovery rate (FDR) ≤1%, is provided. In addition, the relative abundance of 432 of them is also shown. Gene ontology annotation of the complete SP-proteome complemented by an extensive description of the putative reproductive role of SP-proteins, providing a valuable source for a better understanding of SP role in the reproductive success. This data article refers to the article entitled "Characterization of the porcine seminal plasma proteome comparing ejaculate portions" (Perez-Patiño et al., 2016) [1].
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Extracellular vesicles in seminal fluid and effects on male reproduction. An overview in farm animals and pets
(2022-11) Roca Aleu, Jorge; Rodríguez Martínez, Heriberto; Padilla, Lorena; Lucas, Xiomara; Barranco Cascales, Isabel; Medicina y Cirugía Animal
Extracellular vesicles (EVs) are lipid bilayer nanovesicles released by most functional cells to body fluids, containing bioactive molecules, mainly proteins, lipids, and nucleic acids having actions at target cells. The EVs have essential functions in cell-to-cell communication by regulating different biological processes in target cells. Fluids from the male reproductive tract, including seminal plasma, contain many extracellular vesicles (sEVs), which have been evaluated to a lesser extent than those of other body fluids, particularly in farm animals and pets. Results from the few studies that have been conducted indicated epithelial cells of the testis, epididymis, ampulla of ductus deferens and many accessory sex glands release sEVs mainly via apocrine mechanisms. The sEVs are morphologically heterogeneous and bind to functional cells of the male reproductive tract, spermatozoa, and cells of the functional tissues of the female reproductive tract after mating or insemination. The sEVs encapsulate proteins and miRNAs that modulate sperm functions and male fertility. The sEVs, therefore, could be important as reproductive biomarkers in breeding sires. Many of the current findings regarding sEV functions, however, need experimental confirmation. Further studies are particularly needed to characterize both membranes and contents of sEVs, as well as the interaction between sEVs and target cells (spermatozoa and functional cells of the internal female reproductive tract). A priority for conducting these studies is development of methods that can be standardized and that are scalable, cost-effective and time-saving for isolation of different subtypes of EVs present in the entire population of sEVs.
Open Access
Extracellular vesicles isolated from porcine seminal plasma exhibit different tetraspanin expression profiles
(2019-08-09) Barranco, Isabel; Padilla, Lorena; Parrilla, Inmaculada; Alvarez-Barrientos, Alberto; Pérez-Patiño, Cristina; Peña, Fernando; Martínez, Emilio A; Rodriguez-Martínez, Heriberto; Roca, Jordi; Roca, Jordi; Medicina y Cirugía Animal
Seminal extracellular vesicles (EVs) include exosomes (ø 40-120 nm) and microvesicles (MVs, ø 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 < 0.001 and P < 0.05, respectively) than in exosomes, while the opposite was true for CD81; higher (P < 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.
Open Access
Extracellular vesicles would be involved in the release and delivery of seminal TGF-β isoforms in pigs
(Frontiers Media, 2023-02-10) Padilla, Lorena; Barranco, Isabel; Parra, Ana; Parrilla, Inmaculada; Rodríguez Martínez, Heriberto; Lucas, Xiomara; Roca, Jordi; Martínez Hernández, Jesús; Pastor García, Luis Miguel; Medicina y Cirugía Animal
Introduction: pig seminal plasma (SP) is rich in active forms of all three isoforms (1-3) of transforming growth factor β (TGF-β), 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-βs 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-βs was examined by immunohistochemistry in testis, epididymis, and accessory sex glands, by immunocytochemistry in ejaculated spermatozoa, and by Luminex xMAP® technology in SP and sEVs retrieved from healthy, fertile male pigs used as breeders in AI programs. Results: All three TGF-β 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-β isoforms, both inside and outside, probably the outer one associated with membrane-bound sEVs. The results confirmed that pig SP contains all three TGF-β 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-β isoforms and in their safe transport from the male to the female reproductive tract.
Open Access
High total antioxidant capacity of the porcine seminal plasma (SP-TAC) relates to sperm survival and fertility
(2015-12-21) Barranco, Isabel; Tvarijonaviciute, Asta; Perez-Patiño, Cristina; Parrilla, Inmaculada; Cerón, Jose J; Martínez, Emilio A; Rodriguez-Martinez, Heriberto; Roca, Jordi; Medicina y Cirugía Animal
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 (n° = 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 °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 (n° = 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.
Open Access
Immunophenotype 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 Animal
Background: 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.
Open Access
Impact of seminal plasma antioxidants on DNA fragmentation and lipid peroxidation of frozen-thawed horse sperm
(MDPI, 2024-03-06) Catalán, Jaime; Yánez-Ortiz, Iván; Torres-Garrido, Marc; Ribas-Maynou, Jordi; Llavanera, Marc; Barranco Cascales, Isabel; Yeste, Marc; Miró, Jordi; Medicina y Cirugía Animal; Facultad de Veterinaria
Cryopreservation is a stressful process for sperm, as it is associated with an increased production of reactive oxygen species (ROS). Elevated ROS levels, which create an imbalance with antioxidant capacity, may result in membrane lipid peroxidation (LPO), protein damage and DNA fragmentation. This study aimed to determine whether the membrane LPO and DNA fragmentation of frozen-thawed horse sperm relies upon antioxidant activity, including enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and paraoxonase type 1 (PON1)); non-enzymatic antioxidant capacity (Trolox-equivalent antioxidant capacity (TEAC), plasma ferric reducing antioxidant capacity (FRAP) and cupric reducing antioxidant capacity (CUPRAC)); and the oxidative stress index (OSI) of their seminal plasma (SP). Based on total motility and plasma membrane integrity (SYBR14+/PI-) after thawing, ejaculates were hierarchically (p < 0.001) clustered into two groups of good- (GFEs) and poor-(PFEs) freezability ejaculates. LPO and DNA fragmentation (global DNA breaks) were higher (p < 0.05) in the PFE group than in the GFE group, with LPO and DNA fragmentation (global DNA breaks) after thawing showing a positive relationship (p < 0.05) with SP OSI levels and ROS production. In addition, sperm motility and membrane integrity after thawing were negatively (p < 0.05) correlated with the activity levels of SP antioxidants (PON1 and TEAC). The present results indicate that LPO and DNA fragmentation in frozen-thawed horse sperm vary between ejaculates. These differences could result from variations in the activity of antioxidants (PON1 and TEAC) and the balance between the oxidant and antioxidant components present in the SP.
Open Access
Impact of Seminal Plasma Antioxidants on Donkey Sperm Cryotolerance
(MDPI, 2022-02-18) Catalán, Jaime; Yáñez Ortiz, Iván; Tvarijonaviciute, Asta; González Arostegui, Luis Guillermo; Peres Rubio, Camila; Yeste, Marc; Miró, Jordi; Barranco Cascales, Isabel; Medicina y Cirugía Animal
This study investigated whether the activities of the antioxidant components of donkey seminal plasma (SP)-both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant capacity (TEAC))-and oxidative stress index (OSI) are related to sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected and split into two aliquots. The first one was used for measuring the activities levels of enzymatic and non-enzymatic antioxidants and OSI in SP, whereas the other aliquot was cryopreserved. Before cryopreservation, sperm quality parameters (concentration, motility, and viability) were evaluated. After thawing, sperm motility, plasma membrane integrity, lipid disorder, mitochondrial membrane potential, reactive oxygen species (ROS), and calcium intracellular levels were also determined. Based on the percentages of total motility (TM) and of sperm with an intact plasma membrane (SYBR14+/PI-) after thawing, samples were classified as good-freezability (GFE) or poor-freezability (PFE) ejaculates through cluster analyses. The SP activity levels of enzymatic (SOD and PON1) and non-enzymatic antioxidants (CUPRAC, FRAP, and TEAC) were higher (p < 0.05) in GFE than in PFE, whereas SP-OSI was higher (p < 0.05) in PFE than in GFE. In addition, the activity levels of SOD, PON1, GPX, CUPRAC, FRAP, and TEAC were positively (p < 0.05) related to post-thaw sperm motility and plasma membrane integrity and negatively to intracellular ROS levels. The SP-OSI was negatively correlated (p < 0.05) to post-thaw sperm quality parameters and positively to intracellular ROS levels. It can thus be concluded that donkey SP antioxidants are related to sperm cryotolerance and that measurements of antioxidants PON1, SOD, CUPRAC, FRAP, and TEAC, as well as SP-OSI, could be used as markers of sperm cryotolerance. Further research addressing the relationship of these antioxidants and SP-OSI with sperm cryotolerance and their potential use as freezing markers is warranted.
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Is boar sperm freezability more intrinsically linked to spermatozoa than to the surrounding seminal plasma?
(Elsevier, 2018-08) Li, Junwei; Roca Aleu, Jorge; Pérez Patiño, Cristina; Barranco Cascales, Isabel; Martínez García, Emilio; Rodríguez Martínez, Heriberto; Parrilla Riera, Inmaculada; Medicina y Cirugía Animal
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 °C overnight before being frozen in 0.5 mL straws using a standard protocol. Sperm motility (total and progressive) deteriorated (P < 0.05) when P1- or P2-sperm when incubated overnight in SP from post-SRF, while sperm viability differed between P1 and P2 (P < 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 < 0.05) and viability (P < 0.01), as well as plasma membrane fluidity (P < 0.05) or lipid peroxidation values (P < 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.
Open Access
Isolation and characterization of extracellular vesicle subsets in donkey seminal plasma
(Elsevier, 2025-05-22) Catalán, Jaime; Martínez Díaz, Pablo; Parra, Ana; Bonet, Sergi; Yeste, Marc; Roca, Jordi; Barranco Cascales, Isabel; Miró, Jordi; Medicina y Cirugía Animal; Facultad de Veterinaria
Seminal plasma (SP), a fluid composed of secretions from the male genital tract, is rich in seminal extracellular vesicles (sEVs), nano-sized particles surrounded by a lipid bilayer membrane and loaded with functionally active molecules. Seminal EVs are secreted by functional cells of the male genital tract and play a key role in modulating reproductive processes, including sperm function and immune response in the female genital tract. The aim of this study was to isolate and characterize sEVs from donkey SP for the first time. Nine SP samples were collected from nine healthy and reproductive active donkeys. The SP samples were randomly pooled to create three pools (three SP samples per pool). The SP pools were subjected to differential centrifugation and size-exclusion chromatography to separately isolate two subsets of sEVs: small (S-) and large (L-). Orthogonal characterization of sEV samples was performed according to MISEV 2023 guidelines, including morphology (by cryogenic electron microscopy), concentration (by total protein concentration and total and CFSE positive particles by flow cytometry [FC]), particle size distribution (by dynamic light scattering), purity (by albumin assessment by FC), and specific EV protein markers (tetraspanins CD9, CD63, and CD81, and HSP70 by FC). The results showed that donkey SP is highly enriched in sEVs. Size differences were found between both sEV subsets, with S-sEVs being smaller (∼160 nm) and L-sEVs larger (∼290 nm). Both sEV subsets were positive for the four EV protein markers. However, the percentage of CD81-positive events was higher in S-sEV samples than in L-sEV samples (P < 0.05). This study is the first to isolate and characterize sEVs in donkey SP, demonstrating their heterogeneity and suggesting differences in biogenesis and function between S-sEVs and L-sEVs.
Open Access
Measurable cytokine concentrations in pig seminal plasma are modified by semen handling and storage
(MDPI, 2020-09-07) Padilla, Lorena; Barranco, Isabel; Parrilla, Inmaculada; Lucas, Xiomara; Rodríguez-Martínez, Heriberto; Roca, Jordi; Medicina y Cirugía Animal
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, IFNγ, IL-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and TNFα) were measured using Luminex xMAP® 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 °C, -20 °C and -80 °C for 72 h, Experiment 2) or long-term (at -20 °C and -80 °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.
Open Access
Measurement of oxidative stress index in seminal plasma can predict in vivo fertility of liquid-stored porcine artificial insemination semen doses
(MDPI, 2021-07-27) Barranco, Isabel; Rubio, Camila P.; Tvarijonaviciute, Asta; Rodríguez Martínez, Heriberto; Roca, Jordi; Medicina y Cirugía Animal
The study evaluated the relation between the oxidative stress index (OSI) in porcine seminal plasma (n = 76) with sperm resilience and in vivo fertility (farrowing rate and litter size of 3137 inseminated sows) of liquid-stored artificial insemination (AI) semen doses. The OSI was assessed as the ratio of advanced oxidation protein products to Trolox-equivalent antioxidant capacity, both measured using an automated analyzer. Sperm motility (computer-assisted sperm analyzer) and viability (flow cytometry) were evaluated in semen AI-doses at 0 and 72 h of storage at 17 °C. Sperm resilience was defined as the difference between storage intervals. Semen AI-doses were hierarchically clustered as having high, medium and low seminal OSI (p < 0.001) with those of low displaying higher resilience (p < 0.01). Boars were hierarchically clustered into two groups (p < 0.001) as having either positive or negative farrowing rate and litter size deviation; the negative one showing higher seminal OSI (p < 0.05). In sum, seminal OSI was negatively related to sperm motility and the in vivo fertility of liquid-stored boar semen AI-doses, with the receiver operating characteristic curve presenting seminal OSI as a good predictive biomarker of in vivo fertility of AI-boars (area under the curve: 0.815, p < 0.05).