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Chromatin condensation but not DNA integrity of pig sperm is greater in the sperm-rich fraction

Journal of Animal Science and Biotechnology 2024年第1期15卷 171-181页

作者： Estel Viñolas-Vergés Jordi Ribas-Maynou isabel barranco Camila Peres Rubio Sergi Bonet Jordi Roca Marc Yeste Unit of Cell Biology Department of BiologyFaculty of SciencesUniversity of GironaGironaSpain Biotechnology of Animal and Human Reproduction(TechnoSperm) Institute of Food and Agricultural TechnologyUniversity of GironaGironaSpain Department of Medicine and Animal Surgery Faculty of Veterinary ScienceUniversity of MurciaMurciaSpain Catalan Institution for Research and Advanced Studies(ICREA) S08010 BarcelonaSpain

Background Protamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo development.In some mammals,like pigs,ejaculates are emitted in three separate fractions:pre-sperm,sperm-rich(SRF)and post sperm-rich(PSRF).These fractions are known to vary in volume,sperm concentration and quality,as well as in the origin and composition of seminal plasma(SP),with differences being also observed within the SRF one.Yet,whether disparities in the DNA integrity and chromatin condensation and pro-tamination of their sperm exist has not been interrogated.Results This study determined chromatin protamination(Chromomycin A3 test,CMA_(3)),condensation(Dibromobi-mane test,DBB),and DNA integrity(Comet assay)in the pig sperm contained in the first 10 m L of the SRF(SRF-P1),the remaining portion of the sperm-rich fraction(SRF-P2),and the post sperm-rich fraction(PSRF).While chromatin protamination was found to be similar between the different ejaculate fractions(P>0.05),chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF(P=0.018 and P=0.004,respectively).Regarding DNA integrity,no differences between fractions were observed(P>0.05).As the SRF-P1 has the highest sperm concentra-tion and ejaculate fractions are known to differ in antioxidant composition,the oxidative stress index(OSi)in SP,calcu-lated as total oxidant activity divided by total antioxidant capacity,was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF(0.42±0.06 vs.0.23±0.09 and 0.08±0.00,respectively;P<0.01);this index,in addition,was observed to be correlated to the sperm concentration of each fraction(Rs=0.973;P<0.001).Conclusion While sperm DNA integrity was not found to differ between ejaculate fractions,SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the PSRF.This could be related to the OSi of each fraction.

关键词： Chromatin Condensation DNA integrity Ejaculate fractions Pig Protamination Sperm

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Oxytocin in pig seminal plasma is positively related with in vivo fertility of inseminated sows

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Journal of Animal Science and Biotechnology 2022年第2期13卷 430-440页

作者： Lorena Padilla Marina López-Arjona Silvia Martinez-Subiela Heriberto Rodriguez-Martinez Jordi Roca isabel barranco Department of Medicine and Animal Surgery Faculty of Veterinary MedicineUniversity of MurciaE-30100 MurciaSpain Department of Biomedical&Clinical Sciences(BKV) BKH/Obstetrics&GynaecologyFaculty of Medicine and Health SciencesLinköping UniversitySE-58185 LinköpingSweden Department of Veterinary Medical Sciences University of BolognaIT-40064 Ozzano dell’EmiliaBolognaItaly

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 SPOXT 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℃.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.

关键词： Artificial insemination Ejaculate Fertility Oxytocin Pig Semen quality Seminal plasma

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Metabolomic fingerprinting of pig seminal plasma identifies in vivo fertility biomarkers

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Journal of Animal Science and Biotechnology 2022年第2期13卷 473-487页

作者： Yentel Mateo-Otero Pol Fernández-López Ariadna Delgado-Bermúdez Pau Nolis Jordi Roca Jordi Miró isabel barranco Marc Yeste Biotechnology of Animal and Human Reproduction(TechnoSperm) Institute of Food and Agricultural TechnologyUniversity of GironaES-17003 GironaSpain Department of Biology Unit of Cell BiologyFaculty of SciencesUniversity of GironaES-17003 GironaSpain Centre d’Estudis Avançats de Blanes(CEAB) Spanish Research Council(CSIC)ES-17300 GironaSpain Magnetic Nuclear Resonance Facility Autonomous University of BarcelonaBellaterraES-08193 Cerdanyola del VallèsSpain Department of Animal Medicine and Surgery Faculty of Veterinary MedicineUniversity of MurciaES-30100 MurciaSpain Equine Reproduction Service Department of Animal Medicine and SurgeryFaculty of Veterinary MedicineAutonomous University of BarcelonaBellaterraES-08193 Cerdanyola del VallèsSpain Department of Veterinary Medical Sciences University of BolognaIT-40064 Ozzano dell’EmiliaBolognaItaly

Background:Metabolomic approaches,which include the study of low molecular weight molecules,are an emerging-omics technology useful for identification of biomarkers.In this field,nuclear magnetic resonance(NMR)spectroscopy has already been used to uncover(in)fertility biomarkers in the seminal plasma(SP)of several mammalian species.However,NMR studies profiling the porcine SP metabolome to uncover in vivo fertility biomarkers are yet to be carried out.Thus,this study aimed to evaluate the putative relationship between SPmetabolites and in vivo fertility outcomes.To this end,24 entire ejaculates(three ejaculates per boar)were collected from artificial insemination(AI)-boars throughout a year(one ejaculate every 4 months).Immediately after collection,ejaculates were centrifuged to obtain SP-samples,which were stored for subsequent metabolomic analysis by NMR spectroscopy.Fertility outcomes from 1525 inseminations were recorded over a year,including farrowing rate,litter size,stillbirths per litter and the duration of pregnancy.Results:A total of 24 metabolites were identified and quantified in all SP-samples.Receiver operating characteristic(ROC)curve analysis showed that lactate levels in SP had discriminative capacity for farrowing rate(area under the curve[AUC]=0.764)while carnitine(AUC=0.847),hypotaurine(AUC=0.819),sn-glycero-3-phosphocholine(AUC=0.833),glutamate(AUC=0.799)and glucose(AUC=0.750)showed it for litter size.Similarly,citrate(AUC=0.743),creatine(AUC=0.812),phenylalanine(AUC=0.750),tyrosine(AUC=0.753)and malonate(AUC=0.868)levels had discriminative capacity for stillbirths per litter;and malonate(AUC=0.767)and fumarate(AUC=0.868)levels for gestation length.Conclusions:The assessment of selected SP-metabolites in ejaculates through NMR spectroscopy could be considered as a promising non-invasive tool to predict in vivo fertility outcomes in pigs.Moreover,supplementing AI-doses with specific metabolites should also be envisaged as a way to improve their fertility potential.

关键词： Artificial insemination in vivo fertility Metabolomics NMR Pregnancy outcomes Seminal plasma

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GSTM3, but not IZUMO1, is a cryotolerance marker of boar sperm

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Journal of Animal Science and Biotechnology 2019年第4期10卷 931-941页

作者： Marc Llavanera Ariadna Delgado-Bermúdez Beatriz Fernandez-Fuertes Sandra Recuero Yentel Mateo Sergi Bonet isabel barranco Marc Yeste Biotechnology of Animal and Human Reproduction (Techno Sperm) Unit of Cell Biology Department of Biology Institute of Food and Agricultural Technology University of Girona

Background: Cryopreservation is currently the most efficient method for long-term preservation of mammalian gametes and is extensively used in swine artificial insemination(AI) centres. However, it is well-known that cryopreservation procedures induce changes in the water phase in both intra and extracellular compartments,which alter the content and localisation of several proteins and ends up curtailing the structural integrity of functional sperm(i.e., cryoinjuries). Alterations and deficiencies of sperm-oocyte binding proteins during gamete recognition are one of the causes of reproductive failure both in vitro and in vivo. In this sense, characterisation of cryopreservation effects upon oocyte-binding proteins of sperm, such as IZUMO1 and GSTM3, is essential when assessing the impact of this technique in swine reproduction.Results: Cryopreservation was found to induce changes in the localisation of IZUMO1 and GSTM3 in boar sperm.However, the relative content of both proteins was not altered after thawing. Furthermore, whereas IZUMO1 content was found not to be related to the cryotolerance of boar sperm, GSTM3 content was observed to be higher in poor(PFE) than in good(GFE) freezability ejaculates in both pre-frozen(1.00 INT·mm^2± 0.14 INT·mm^2 vs.0.72 INT·mm^2± 0.15 INT·mm^2;P < 0.05) and post-thawed(0.96 INT·mm^2± 0.20 INT·mm^2 vs. 70 INT·mm^2± 0.19 INT·mm^2;P < 0.05) samples. Moreover, GSTM3 levels were found to be higher in those spermatozoa that exhibited low mitochondrial activity, high reactive oxygen species(ROS) production, and high membrane lipid disorder postthaw(P < 0.05).Conclusions: The difference in GSTM3 content between GFE and PFE, together with this protein having been found to be related to poor sperm quality post-thaw, suggests that it could be used as a cryotolerance marker of boar spermatozoa. Furthermore, both IZUMO1 and GSTM3 relocate during cryopreservation, which could contribute to the reduced fertilising capacity of frozen-thawed boar sperm.

关键词： Boar Cryopreservation GSTM3 IZUMO1 ROS Sperm

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Aquaglyceroporins but not orthodox aquaporins are involved in the cryotolerance of pig spermatozoa

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Journal of Animal Science and Biotechnology 2020年第1期11卷 20-31页

作者： Ariadna Delgado-Bermúdez Marc Llavanera Leira Fernández-Bastit Sandra Recuero Yentel Mateo-Otero Sergi Bonet isabel barranco Beatriz Fernández-Fuertes Marc Yeste Biotechnology of Animal and Human Reproduction(TechnoSperm) Unit of Cell BiologyDepartment of BiologyFaculty of SciencesInstitute of Food and Agricultural TechnologyUniversity of GironaC/Maria Aurèlia Campany69Campus MontiliviE-17003 GironaSpain

Background:Aquaporins(AQPs)are a family of transmembrane water channels that includes orthodox AQPs,aquaglyceroporins(GLPs)and super AQPs.AQP3,AQP7,AQP9 and AQP11 have been identified in boar sperm,and they are crucial for sperm maturation and osmoregulation.Water exchange is an important event in cryopreservation,which is the most efficient method for long-term storage of sperm.However,the freezethaw process leads to sperm damage and a loss of fertilizing potential.Assuming that the quality of frozenthawed sperm partially depends on the regulation of osmolality variations during this process,AQPs might play a crucial role in boar semen freezability.In this context,the aim of this study was to unravel the functional relevance of the different groups of AQPs for boar sperm cryotolerance through three different inhibitors.Results:Inhibition of different groups of AQPs was found to have different effects on boar sperm cryotolerance.Whereas the use of 1,3-propanediol(PDO),an inhibitor of orthodox AQPs and GLPs,decreased total motility(P<0.05),it increased post-thaw sperm viability,lowered membrane lipid disorder and increased mitochondrial membrane potential(MMP)(P<0.05).When acetazolamide(AC)was used as an inhibitor of orthodox AQPs,the effects on post-thaw sperm quality were restricted to a mild increase in MMP in the presence of the intermediate concentration at 30 min post-thaw and an increase in superoxide levels(P<0.05).Finally,the addition of phloretin(PHL),a GLP inhibitor,had detrimental effects on post-thaw total and progressive sperm motilities,viability and lipid membrane disorder(P<0.05).Conclusions:The effects of the different inhibitors suggest that GLPs rather than orthodox AQPs are relevant for boar sperm freezability.Moreover,the positive effect of PDO on sperm quality suggests a cryoprotective role for this molecule.

关键词： Acetazolamide Aquaporins Boar Phloretin Propanediol Sperm

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