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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Open Access
Seminal plasma antioxidants are related to sperm cryotolerance in the horse
(MDPI, 2022-06-28) Catalán, Jaime; Yánez Ortiz, Iván; Tvarijonaviciute, Asta; González Aróstegui, Luis Guillermo; Peres Rubio, Camila; Barranco Cascales, Isabel; Yeste, Marc; Miró, Jordi; Medicina y Cirugía Animal
The objective of this study was to determine the relationship of enzymatic (superoxide dismutase, SOD; glutathione peroxidase, GPX; catalase, CAT; and paraoxonase type 1, PON1) and non-enzymatic antioxidants (measured in terms of: Trolox equivalent antioxidant capacity, TEAC; cupric-reducing antioxidant capacity, CUPRAC; and ferric-reducing ability of plasma, FRAP), as well as the oxidative stress index (OSI) in seminal plasma (SP) with the resilience of horse sperm to freeze-thawing. Twenty-one ejaculates (one per individual) were collected and split into two aliquots: the first was used to harvest the SP and assess the activity levels of antioxidants and the OSI, and the second one was cryopreserved. The following post-thaw sperm quality parameters were evaluated: sperm motility, plasma membrane and acrosome integrity, mitochondrial membrane potential, intracellular levels of reactive oxygen species (ROS), and plasma membrane lipid disorder. Based on post-thaw total motility (TM) and plasma membrane integrity (SYBR14+/PI−), ejaculates were hierarchically (p < 0.001) clustered into two groups of good (GFE) and poor (PFE) freezability. The SP activity levels of PON1, SOD, and TEAC were higher (p < 0.05) in GFE than in PFE, showing a positive relationship (p < 0.05) with some sperm motility parameters and with plasma membrane (PON1 and TEAC) and acrosome (SOD and TEAC) integrity. In contrast, OSI was higher (p < 0.05) in the SP of PFE than in that of GFE, and was negatively correlated (p < 0.05) to some sperm motility parameters and to plasma membrane and acrosome integrity, and positively (p < 0.05) to the percentage of viable sperm with high plasma membrane lipid disorder. In conclusion, enzymatic (PON1 and SOD) and non-enzymatic (TEAC) antioxidants of SP are related to horse sperm cryotolerance. In addition, our results suggest that PON1 could be one of the main antioxidant enzymes involved in the control of ROS in this species. Further investigation is needed to confirm the potential use of these SP-antioxidants and OSI to predict sperm cryotolerance in horses.
Embargo
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; 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.
Embargo
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.
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
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.
Embargo
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.
Embargo
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
Proteomic profiling of porcine seminal extracellular vesicles reveals potential in vivo fertility biomarkers
(Wiley, 2025-07-04) Barranco Cascales, Isabel; Martínez Díaz, Pablo; Parra, Ana; Martínez-Alborcia, María José; Lucas Arjona, Xiomara; Rodríguez-Martínez, Heriberto; Roca, Jordi; Medicina y Cirugía Animal; Facultad de Veterinaria
Background: Predicting male fertility in farm animals remains a challenge. Seminal plasma (SP) contains a high amount of heterogeneous seminal extracellular vesicles (sEVs), believed involved in reproductive processes and maybe key to understanding male fertility. Aims: To identify the sEV proteins that are differentially expressed between more and less fertile boars and that could be candidates for fertility biomarkers in boars used in artificial insemination (AI) programs. Materials and methods: Small (S) and large (L) sEV subsets from SP samples of AI boars with differences in fertility: high (H) or low (L) farrowing rate (FR) and large (L) or small (S) litter size (LS). The S- and L-sEV subsets were isolated by size exclusion chromatography and characterized according to the Minimal Information for Studies of Extracellular Vesicles (MISEV2023) guidelines. Proteomic analyses (three biological replicates per fertility group and sEV subset) were performed using a Bruker timsTOF fleX™ instrument with data-independent acquisition parallel accumulation-serial fragmentation (diaPASEF) technology. Results: A total of 470 and 726 proteins were quantified in S-sEVs and 1801 and 1834 proteins in L-sEVs from FR and LS boars, respectively. Differentially expressed sEV proteins (log2fold change ≥±1, p ≤ 0.05 and effect size d of Cohen >2.0) were found between the fertility groups: seven in S-sEVs and 52 in L-sEVs between H-FR and L-FR boars, and 47 in S-sEVs and 52 in L-sEVs between L-LS and S-LS boars. Many of these differentially expressed sEV proteins are involved in reproductive processes, particularly in sperm function and sperm-zona pellucida binding, but also in embryo development and implantation. Conclusions: The sEV proteome differs between more and less fertile boars, with many of the differentially expressed proteins known as involved in reproductive processes. This would suggest that sEVs may be involved in male fertility and that some of the differentially expressed sEV proteins could be potential fertility markers for AI boars.
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
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.