Barranco Cascales, Isabel

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Barranco Cascales, Isabel

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Barranco Cascales, Isabel

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Universidad de Murcia. Departamento de Medicina y Cirugía Animal

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Season of ejaculate collection influences the freezability of boar spermatozoa
(Elsevier, 2013-12-03) Barranco Cascales, Isabel; Ortega, María D.; Martínez Alborcia, María J.; Vázquez, Juan M.; Martínez García, Emilio; Roca Aleu, Jorge; Medicina y Cirugía Animal
The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008-2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24h at 17°C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P<0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season's influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.
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The seminal plasma of the boar is rich in cytokines, with significant individual and intra-ejaculate variation
(Wiley, 2015-12) Barranco Cascales, Isabel; Rubér, Marie; Pérez Patiño, Cristina; Atikuzzaman, Mohammad; Martínez García, Emilio; Roca Aleu, Jorge; Rodríguez Martínez, Heriberto; Medicina y Cirugía Animal
Problem: The boar, as human, sequentially ejaculates sperm-rich and sperm-poor fractions. Seminal plasma (SP) spermadhesins (PSP-I/PSP-II) induce a primary endometrial inflammatory response in female sows, similar to that elicited by semen deposition in other species, including human. However, the SP is also known to mitigate such response, making it transient to allow for embryo entry to a cleansed endometrium. Although cytokine involvement has been claimed, the exploration of cytokines in different SP fractions is scarce. This study determines Th1, Th2, Th17 and Th3 cytokine profiles in specific ejaculate SP fractions from boars of proven fertility. Methods: SP samples from the sperm-rich fraction (SRF) and the sperm-poor post-SRF fraction (post-SRF) of manually collected ejaculates from eight boars (four ejaculates per boar) were analysed by commercial multiplex bead assay kits (Milliplex MAP, Millipore, USA) for interferon-γ, interferon gamma-induced protein 10, macrophage-derived chemokine, growth-regulated oncogene, granulocyte-macrophage colony-stimulating factor, monocyte chemo-attractant protein-1, interleukins (IL)-6, IL-8, IL-10, IL-15, IL-17 and transforming growth factor (TGF)-β1-β3. Results: Cytokine concentrations differed between the ejaculate fractions among boars, being highest in the post-SRF. Conclusion: Boar SP is rich in Th1, Th2, Th17 and Th3 cytokines, with lowest concentrations in the sperm-peak-containing fraction, indicating its main immune influence might reside in the larger, protein-rich sperm-poor post-SRF.
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The activity of paraoxonase type 1 (PON-1) in boar seminal plasma and its relationship with sperm quality, functionality, and in vivo fertility
(Wiley, 2015-01-19) Tvarijonaviciute, Asta; Pérez-Patiño, Cristina; Alkmin, Diego V.; Cerón, José J.; Martínez, Emilio A.; Rodríguez-Martínez, Heriberto; Roca, Jordi; Barranco Cascales, Isabel; Medicina y Cirugía Animal
Paraoxonase 1 (PON-1) is a hydrolytic enzyme present in body fluids, capable of protecting cells against oxidative stress. The hypothesis was hereby to test that PON-1, present in seminal plasma (SP), acts protecting boar spermatozoa when showing a reasonable high activity in the ejaculate. SP-PON-1 activity differed (p < 0.001) among boars (from 0.10 to 0.29 IU/mL). Intra-boar variability was also observed (p < 0.05), but only in two of the 15 boars. SP-PON-1 activity differed among ejaculate portions, showing the spermatozoa-peak portion of spermatozoa-rich ejaculate fraction the highest levels (0.35 ± 0.03 IU/mL, ranging from 0.12 to 0.69) and the post-sperm ejaculate fraction the lowest levels (0.12 ± 0.01 IU/mL, ranging from 0.03 to 0.21). SP-PON-1 activity was positively correlated with the percentage of spermatozoa with rapid and progressive movement (p < 0.01) and negatively correlated with the generation of intracellular reactive oxygen species (p < 0.01) in semen samples after 72 h of liquid storage. SP-PON-1 activity was highest (p < 0.01) in boars with highest farrowing rates. In conclusion, SP-PON-1 activity differed among boars and ejaculate fractions/portions. SP-PON-1 activity was positively correlated with sperm quality and functionality of liquid-stored semen samples and it evidenced a positive association with in vivo fertility.
Open Access
Updating research on extracellular vesicles of the male reproductive tract in farm animals: a systematic review
(MDPI, 2024-10-31) Martínez Díaz, Pablo; Parra, Ana; Montesdeoca, Marina; Barranco Cascales, Isabel; Roca Aleu, Jorge; Medicina y Cirugía Animal
This systematic review examined research studies on extracellular vesicles (EVs) of the male reproductive tract in livestock species to summarize the research topics and methodologies used, key findings, and future directions. PubMed and Scopus were searched for time ranges up to 1 September 2024, and 1383 articles were identified. The application of screening and eligibility criteria resulted in the selection of 79 articles focusing on male reproductive EVs in livestock. Porcine and bovine male reproductive EVs were the most studied. A variety of EV isolation techniques were used, with ultracentrifugation being the most common. Characterization of male reproductive EVs in livestock was a weak point, with only 24.05% of the articles characterizing EVs according to MISEV guidelines. Inadequate characterization of EVs compromises the reliability of results. The results of 19 articles that provided a good characterization of EVs showed that male reproductive EVs from livestock species are phenotypically and compositionally heterogeneous. These papers also showed that these EVs would be involved in the regulation of sperm functionality. Research on male reproductive EVs in livestock species remains scarce, and further research is needed, which should include appropriate characterization of EVs and aim to find efficient methods to isolate them and assess their involvement in the functionality of spermatozoa and the cells of the female genital tract.
Open Access
Seminal plasma anti-müllerian hormone: a potential AI-boar fertility biomarker?
(MDPI, 2020-04-10) Barranco Cascales, Isabel; Fernández Fuertes, Beatriz; Padilla, Lorena; Delgado-Bermúdez, Ariadna; Tvarijonaviciute, Asta; Yeste, Marc; Medicina y Cirugía Animal; Facultad de Veterinaria
The anti-Müllerian hormone (AMH), a Sertoli cell-secreted glycoprotein that is present in seminal plasma (SP), is considered as a marker of spermatogenesis in humans. This study aimed to evaluate the presence of this hormone in boar SP, together with its putative relationship with sperm quality, function, and in vivo fertility parameters in liquid-stored semen samples. The concentration of SP-AMH was assessed in 126 ejaculates from artificial insemination (AI)-boars (n = 92) while using a commercial Enzyme-Linked ImmunoSorbent Assay (ELISA) kit with monoclonal antibodies specific for Sus scrofa AMH (CEA228Po, Cloud-clone). Sperm quality (concentration, motility, viability, and acrosome damage) and functionality (membrane lipid disorder and intracellular H2O2 generation) were assessed in semen samples at 0 and 72 h of liquid-storage. In addition, fertility parameters from 3113 sows inseminated with the AI-boars were recorded in terms of farrowing rate, litter size, number of stillbirths per litter, and the duration of pregnancy over a 12-month period. The results revealed that the SP-AMH concentration varied widely among boar ejaculates, with no differences among breeds. Moreover, the SP-AMH concentration proved to be a good predictive biomarker for sperm concentration (p ˂ 0.05), but poor for other sperm quality, functionality, and in vivo fertility parameters of liquid-stored semen samples from AI-boars.
Open Access
Deactivation of the JNK Pathway by GSTP1 is essential to maintain sperm functionality
(Frontiers Media, 2021-02-25) Llavanera, Marc; Mateo-Otero, Yentel; Delgado-Bermúdez, Ariadna; Recuero, Sandra; Olives, Samuel; Barranco Cascales, Isabel; Yeste, Marc; Medicina y Cirugía Animal; Facultad de Veterinaria
Fifty percent of male subfertility diagnosis is idiopathic and is usually associated with genetic abnormalities or protein dysfunction, which are not detectable through the conventional spermiogram. Glutathione S-transferases (GSTs) are antioxidant enzymes essential for preserving sperm function and maintaining fertilizing ability. However, while the role of GSTP1 in cell signaling regulation via the inhibition of c-Jun N-terminal kinases (JNK) has been enlightened in somatic cells, it has never been investigated in mammalian spermatozoa. In this regard, a comprehensive approach through immunoblotting, immunofluorescence, computer-assisted sperm assessment (CASA), and flow cytometry analysis was used to characterize the molecular role of the GSTP1-JNK heterocomplex in sperm physiology, using the pig as a model. Immunological assessments confirmed the presence and localization of GSTP1 in sperm cells. The pharmacological dissociation of the GSTP1-JNK heterocomplex resulted in the activation of JNK, which led to a significant decrease in sperm viability, motility, mitochondrial activity, and plasma membrane stability, as well as to an increase of intracellular superoxides. No effects in intracellular calcium levels and acrosome membrane integrity were observed. In conclusion, the present work has demonstrated, for the first time, the essential role of GSTP1 in deactivating JNK, which is crucial to maintain sperm function and has also set the grounds to understand the relevance of the GSTP1-JNK heterocomplex for the regulation of mammalian sperm physiology.
Open Access
Redox profiling of preovulatory follicular fluid in the donkey is species-specific, and contributes to modulate sperm function
(Nature Research, 2025-02-23) Catalán, Jaime; Padilla, Lorena; Maside, Carolina; Yánez-Ortiz, Iván; Tvarijonaviciute, Asta; Barranco Cascales, Isabel; Bonet, Sergi; Miró, Jordi; Yeste, Marc; Martínez Hernández, Jesús; Medicina y Cirugía Animal; Facultad de Veterinaria
The follicular fluid (FF) is crucial for providing oocytes with an ideal environment that promotes their development and maturation. Not only does this fluid supply nutrients and hormones, but also other components that protect both follicular cells and the oocyte itself from potential harmful factors, such as those inducing oxidative stress (OS). The FF has also been suggested to have beneficial effects on sperm when they reach the oviduct. The objectives of this study were to describe the presence and activity levels of redox biomarkers in the preovulatory follicular fluid (PFF), and to analyze the impact of PFF on the motility, viability and reactive oxygen species (ROS) levels of donkey sperm. For this purpose, ten PFF samples obtained from Catalan jennies and nine ejaculates collected from Catalan donkeys were used. Redox biomarkers -including enzymatic and non-enzymatic antioxidants, and oxidative biomarkers- were analyzed in jennies' PFF. After collection, each semen sample was split into two aliquots of equal volume. The two aliquots were centrifuged to remove the seminal plasma; one pellet was resuspended in Tris Buffered Medium (TBM), and the other was resuspended in TBM supplemented with 20% PFF. Motility parameters (CASA) and other semen quality biomarkers (flow cytometry) were assessed after 0, 60 and 120 min of incubation at 38 °C. Exposure of donkey sperm to PFF reduced intracellular ROS levels and helped maintain sperm motility and viability. These findings suggest that, in the oviduct, the PFF components protect donkey sperm from oxidative stress. Furthermore, knowing better the composition of donkey PFF in terms of antioxidant biomarkers may be used to improve the formulation of media for oocyte maturation and fertilization in this and other species.
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.
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Measurement of activity and concentration of paraoxonase 1 (PON-1) in seminal plasma and identification of PON-2 in the sperm of boar ejaculates
(Wiley, 2015-01) Barranco Cascales, Isabel; Roca Aleu, Jorge; Tvarijonaviciute, Asta; Rubér, Marie; Vicente Carrillo, Alejandro; Atikuzzaman, Mohammad; Cerón Madrigal, José Joaquín; Martínez Navarro, Emilio; Rodríguez Martínez, Heriberto; Martínez García, Emilio; Medicina y Cirugía Animal
This study revealed and characterised the presence of the antioxidant enzymes paraoxonase (PON) type 1 (PON-1, extracellular) and type 2 (PON-2, intracellular) in boar semen. To evaluate PON-1, an entire ejaculate from each of ten boars was collected and the seminal plasma was harvested after double centrifugation (1,500g for 10 min). Seminal plasma was analysed for concentration as well as enzymatic activity of PON-1 and total cholesterol levels. Seminal-plasma PON-1 concentration ranged from 0.961 to 1.670 ng/ml while its enzymatic activity ranged from 0.056 to 0.400 IU/ml, which represent individual variance. Seminal-plasma PON-1 concentration and enzymatic activity were negatively correlated (r = -0.763; P < 0.01). The activity of seminal-plasma PON-1 negatively correlated with ejaculate volume (r = -0.726, P < 0.05), but positively correlated with sperm concentration (r = 0.654, P < 0.05). Total seminal-plasma cholesterol concentration positively correlated with PON-1 activity (r = 0.773; P < 0.01), but negatively correlated with PON-1 concentration (r = -0.709; P < 0.05). The presence of intracellular PON-2 was determined via immunocytochemistry in spermatozoa derived from artificial insemination. PON-2 localised to the post-acrosomal area of the sperm head and principal piece of the tail in membrane-intact spermatozoa. In summary, PON is present in boar semen, with PON-1 at low levels in seminal plasma and PON-2 within the spermatozoa. Further studies are needed to characterise the relationship between antioxidant PONs with sperm and other seminal-plasma parameters.
Open Access
Glutathione S-Transferases play a crucial role in mitochondrial function, plasma membrane stability and oxidative regulation of mammalian sperm
(MDPI, 2020-01-24) Llavanera, Marc; Delgado-Bermúdez, Ariadna; Olives, Samuel; Mateo Otero, Yentel; Recuero, Sandra; Bonet, Sergi; Fernández-Fuertes, Beatriz; Yeste, Marc; Barranco Cascales, Isabel; Medicina y Cirugía Animal; Facultad de Veterinaria
Glutathione S-transferases (GSTs) are essential sperm antioxidant enzymes involved in cell protection against oxidative stress and toxic chemicals, preserving sperm function and fertilising ability. Artificial insemination (AI) in pigs is commonly carried out through the use of liquid-stored semen at 17 °C, which not only reduces sperm metabolic activity but also sperm quality and AI-farrowing rates within the 72 h of storage. While one may reasonably suggest that such enzymes are implicated in the physiology and maintenance of mammalian sperm function during liquid-storage, no previous studies conducted on any species have addressed this hypothesis. Therefore, the objective of the present work was to characterise the presence and function of sperm GSTs in mammalian sperm, using the pig as a model. In this regard, inhibition of such enzymes by ethacrynic acid (EA) during semen storage at 17 °C was performed to evaluate the effects of GSTs in liquid-preserved boar sperm by flow cytometry, immunofluorescence, and immunoblotting analysis. The results of this study have shown, for the first time in mammalian species, that the inhibition of GSTs reduces sperm quality and functionality parameters during their storage at 17 °C. These findings highlight the key role of such enzymes, especially preserving mitochondrial function and maintaining plasma membrane stability. In addition, this study has identified and localised GSTM3 in the tail and equatorial subdomain of the head of boar sperm. Finally, this study has set grounds for future investigations testing supplementation of semen extenders with GSTs, as this may improve fertility outcomes of swine AIs.