Browsing by Subject "Ejaculate"

Now showing 1 - 8 of 8
  • Thumbnail Image
    Publication
    Restricted
    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.
  • Thumbnail Image
    Publication
    Open Access
    Characterization of the porcine seminal plasma proteome comparing ejaculate portions
    (2016-06-16) Pérez Patiño, Cristina; Barranco, Isabel; Parrilla, Inmaculada; Valero, M. Luz; Martínez, Emilio A.; Rodríguez Martínez, Heriberto; Roca, Jordi; Medicina y Cirugía Animal
    Full identification of boar seminal plasma (SP) proteins remains challenging. This study aims to provide an extensive proteomic analysis of boar SP and to generate an accessible database of boar SP-proteome. A SP-pool (33entire ejaculates/11 boars; 3ejaculates/boar) was analyzed to characterize the boar SP-proteome. Twenty ejaculates (5 boars, 4ejaculates/boar) collected in portions (P1: first 10mL of sperm rich ejaculate fraction (SRF), P2: rest of SRF and P3: post-SRF) were analyzed to evaluate differentially expressed SP-proteins among portions. SP-samples were analyzed using a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS followed by functional bioinformatics. The identified proteins were quantified from normalized LFQ intensity data. A total of 536 SP-proteins were identified, 409 of them in Sus scrofa taxonomy (374 validated with ≥99% confidence). Barely 20 of the identified SP-proteins were specifically implicated in reproductive processes, albeit other SP-proteins could be indirectly involved in functionality and fertility of boar spermatozoa. Thirty-four proteins (16 identified in S. scrofa taxonomy) were differentially expressed among ejaculate portions, 16 being over-expressed and 18 under-expressed in P1-P2 regarding to P3. This major proteome mapping of the boar SP provides a complex inventory of proteins with potential roles as sperm function- and fertility- biomarkers. Biological significance: This proteomic study provides the major characterization of the boar SP-proteome with >250 proteins first reported. The boar SP-proteome is described so that a spectral library can be built for relative 'label free' protein quantification with SWATH approach. This proteomic profiling allows the creation of a publicly accessible database of the boar SP-proteome, as a first step for further understanding the role of SP-proteins in reproductive outcomes as well as for the identification of biomarkers for sperm quality and fertility.
  • Thumbnail Image
    Publication
    Open Access
    Cryopreservation differentially alters the proteome of epididymal and ejaculated pig spermatozoa
    (2019-04-11) Perez-Patiño, Cristina; Barranco, Cascales; Li, Junwei; Padilla, Lorena; Martínez, Emilio A; Rodriguez-Martinez, Heriberto; Roca, Jordi; Parrilla, Inmaculada; Medicina y Cirugía Animal
    Cryopreservation induces differential remodeling of the proteome in mammalian spermatozoa. How these proteome changes relate to the loss of sperm function during cryopreservation remains unsolved. The present study aimed to clarify this issue evaluating differential changes in the proteome of fresh and frozen-thawed pig spermatozoa retrieved from the cauda epididymis and the ejaculate of the same boars, with clear differences in cryotolerance. Spermatozoa were collected from 10 healthy, sexually mature, and fertile boars, and cryopreserved using a standard 0.5 mL-straw protocol. Total and progressive motility, viability, and mitochondria membrane potential were higher and membrane fluidity and reactive oxygen species generation lower in frozen-thawed (FT) epididymal than ejaculated spermatozoa. Quantitative proteomics of fresh and FT spermatozoa were analyzed using a LC-ESI-MS/MS-based Sequential Window Acquisition of All Theoretical Spectra approach. Cryopreservation quantitatively altered more proteins in ejaculated than cauda epididymal spermatozoa. Differential protein-protein networks highlighted a set of proteins quantitatively altered in ejaculated spermatozoa, directly involved in mitochondrial functionality which would explain why ejaculated spermatozoa deteriorate during cryopreservation.
  • Thumbnail Image
    Publication
    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.
  • Thumbnail Image
    Publication
    Open Access
    Oxytocin in pig seminal plasma is positively related with in vivo fertility of inseminated sows
    (BMC, 2021-09-13) Padilla, Lorena; López Arjona, Marina; Martínez Subiela, Silvia; Rodríguez Martínez, Heriberto; Roca, Jordi; Barranco, Isabel; Medicina y Cirugía Animal
    Background: identification of relevant in vivo biomarkers for fertility remains a challenge for the livestock industry. Concentrations of the small peptide hormone oxytocin (OXT), involved in male reproductive function and present in the seminal plasma (SP) of several species could be a robust one. This study characterized concentrations of SP-OXT in ejaculates from boars used in artificial insemination (AI) programs aiming to evaluate its relationship with sperm quality variables and in vivo fertility of their liquid-stored AI-semen. Seminal OXT concentrations (ng/mL) were measured in 169 ejaculates from 61 boars of the Duroc, Pietrain, Landrace and Large White breeds using a direct competitive immunoassay test based on AlphaLISA® technology. Ejaculate (ejaculate volume, sperm concentration, total sperm count) and sperm parameters (motility, viability, intracellular generation of reactive oxygen species, plasma membrane fluidity) were assessed at 0 h and 72 h in AI-semen samples stored at 17 °C. In vivo fertility included only 18 Large White and Landrace boars whose AI-semen was used to inseminated > 100 sows and evaluated both farrowing rate and litter size of 3,167 sows. Results: The results showed that SP-OXT differed between boars and between ejaculates within boar (P < 0.05) but not between breeds (Duroc, Pietrain, Landrace and Large White). Ejaculates with higher SP-OXT concentration/mL (hierarchically grouped; P < 0.001) had larger volume and came from younger boars (P < 0.05). Ejaculates of boars showing positive farrowing rate deviation exhibited higher (P < 0.05) SP-OXT concentration/mL than those with negative farrowing rate deviation. Conclusion: The SP concentrations of OXT are boar, ejaculate and age dependent, and positively related with ejaculate volume and farrowing rates of liquid-stored semen AI-doses.
  • Thumbnail Image
    Publication
    Open Access
    Reproductive physiology of the boar: what defines the potential fertility of an ejaculate?
    (Elsevier, 2024-04-18) Rodríguez Martínez, Heriberto; Martínez Serrano, Cristina A.; Álvarez Rodríguez, Manuel; Martínez, Emilio A.; Roca, Jordi; Medicina y Cirugía Animal
    Despite decades of research and handling of semen for use in artificial insemination (AI) and other assisted reproductive technologies, 5-10% of selected boar sires are still considered sub-fertile, escaping current assessment methods for sperm quality and resilience to preservation. As end-product, the ejaculate (emitted spermatozoa sequentially exposed to the composite seminal plasma, the SP) ought to define the homeostasis of the testes, the epididymis, and the accessory sexual glands. Yet, linking findings in the ejaculate to sperm production biology and fertility is suboptimal. The present essay critically reviews how the ejaculate of a fertile boar can help us to diagnose both reproductive health and resilience to semen handling, focusing on methods -available and under development- to identify suitable biomarkers for cryotolerance and fertility. Bulk SP, semen proteins and microRNAs (miRNAs) have, albeit linked to sperm function and fertility after AI, failed to enhance reproductive outcomes at commercial level, perhaps for just being components of a complex functional pathway. Hence, focus is now on the interaction sperm-SP, comparing in vivo with ex vivo, and regarding nano-sized lipid bilayer seminal extracellular vesicles (sEVs) as priority. sEVs transport fragile molecules (lipids, proteins, nucleic acids) which, shielded from degradation, mediate cell-to-cell communication with spermatozoa and the female internal genital tract. Such interaction modulates essential reproductive processes, from sperm homeostasis to immunological female tolerance. sEVs can be harvested, characterized, stored, and manipulated, e.g. can be used for andrological diagnosis, selection of breeders, and alternatively be used as additives to improve cryosurvival and fertility.
  • Thumbnail Image
    Publication
    Open Access
    Testicular ultrasound patterns, sperm quality, seminal microbiota, caprine arthritis encephalitis virus and Coxiella burnetii infection in bucks
    (Elsevier, 2026-04-25) Bailón-Larrañaga, N.; Gomis, J.; Contreras de Vera, Antonio; Toledo-Perona, R.; Toquet, M.; Reyes, L.E.; Quereda, J.J.; Gonzalez-Torres, P.; Gñpmez-Marín, A.; Sanidad Animal
    This study assesses the frequency and severity of ultrasound-detected testicular alterations across age groups (young and adult) in 219 bucks. Sperm quality and testicular echotexture were evaluated, and their associations with reproductive pathogens (Coxiella (C.) burnetii, caprine arthritis encephalitis virus (CAEV), Chlamydia (C.) abortus and Mycoplasma (M.) agalactiae) were analyzed. Additionally, qPCR was also performed on ejaculates from seropositive bucks. Seminal microbiota was characterized in three groups: 1 (seronegative without lithiasis), 2 (seropositive with lithiasis), and 3 (qPCR-CAEV-positive). A 68.5% of bucks showed testicular microlithiasis (50.2% bilateral and 18.2% unilateral). Moreover, frequency of bilateral lesions differed according to age, being higher in adults, who also showed larger microlithiasis area (P < 0.05). Age was negatively correlated with sperm concentration (P < 0.05). Seropositivity was 35.8% for C. burnetii and 58.8% for CAEV. Adults showed higher CAEV seropositivity (P < 0.001), and 4.2% of ejaculates tested qPCR positive for this pathogen. Bacterial richness was greater (P < 0.001) in group 2 than in group 1. Firmicutes dominated groups 1 and 2, whereas Fusobacteriota prevailed in group 3. The most frequent genus in all three experimental groups was Oceanivirga, with higher abundance in group 1 than in group 2 (P < 0.05). These findings suggest that ejaculate microbiota may be influenced by ultrasound abnormalities and exposure to CAEV and C. burnetii, supporting the use of testicular ultrasound to detect bucks prone to reproductive problems and associated health risks, while also highlighting the need to include males in CAE and Q fever control programs.
  • Thumbnail Image
    Publication
    Open Access
    The proteome of pig spermatozoa Is remodeled during ejaculation
    (Elsevier, 2019-01) Pérez-Patiño, Cristina; Parrilla, Inmaculada; Li, Junwei; Barranco, Isabel; Martínez, Emilio A.; Rodríguez-Martínez, Heriberto; Roca, Jordi; Medicina y Cirugía Animal
    Proteins are essential for sperm function, including their fertilizing capacity. Pig spermatozoa, emitted in well-defined ejaculate fractions, vary in their functionality, which could be related to different sperm protein composition. This study aimed (i) to update the porcine sperm proteome and (ii) to identify proteins differentially expressed in mature spermatozoa from cauda epididymis and those delivered in separate ejaculate fractions. Ejaculates from nine mature and fertile boars were manually collected in three separate portions: the first 10 ml of the sperm-rich ejaculate fraction (SRF), the rest of the SRF and the post-SRF. The contents of cauda epididymides of the boars were collected post-mortem by retrograde duct perfusion, generating four different semen sources for each boar. Following centrifugation, the resulting pellets of each semen source were initially pooled and later split to generate two technical replicates per source. The resulting eight sperm samples (two per semen source) were subjected to iTRAQ-based 2D-LC-MS/MS for protein identification and quantification. A total of 1,723 proteins were identified (974 of Sus scrofa taxonomy) and 1,602 of them were also quantified (960 of Sus scrofa taxonomy). After an ANOVA test, 32 Sus scrofa proteins showed quantitative differences (p < 0.01) among semen sources, which was particularly relevant for sperm functionality in the post-SRF. The present study showed that the proteome of boar spermatozoa is remodeled during ejaculation involving proteins clearly implicated in sperm function. The findings provide valuable groundwork for further studies focused on identifying protein biomarkers of sperm fertility.