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 ...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.展开更多
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 an...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.展开更多
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 separa...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.展开更多
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)spectro...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.展开更多
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 crucia...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.展开更多
基金support from Ministry of Science,Innovation and Universities,Spain(Grants:RYC-2014-15581,AGL2017–88329-R and FJCI-2017-31689)European Commission(H2020-MSCA-IF-79212)
文摘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.
基金supported by MICINN and FEDER [AGL2016-79096-R and PID2020-113493RB-I00] Madrid (Spain)Seneca Foundation [19892/GERM/15]Murcia (Spain)+2 种基金European Union’s Horizon 2020 research and innovation program (H2020-MSCA-IF-2019-891382)financially supported by MINECO (BES-2016-076404)MECD (FPU16/02170),Spain,respectively。
文摘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.
基金This research was supported by the European Union’s Horizon 2020 research and innovation scheme under the Marie Skłodowska-Curie grant agreement No.801342(Tecniospring INDUSTRYGrant:TECSPR-19-1-0003)+4 种基金the Ministry of Science and Innovation,Spain(Grants:PID2020-113320RB-I00,PID2020-113493RB-I00,RYC2021-034546-I and RYC2021-034764-I)the Catalan Agency for Management of University and Research Grants,Regional Government of Catalonia,Spain(Grants:2017-SGR-1229 and 2021-SGR-00900)the Seneca Foundation,Regional Government of Murcia,Spain(Grant:21935/PI/22)La Marato de TV3 Foundation(Grant:214/857-202039)and the Catalan Institution for Research and Advanced Studies(ICREA).
文摘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.
基金The present study was funded by the Ministry of Science and Innovation,Spain(Grants:RYC-2014-15581,AGL2017–88329-R and FJCI-2017-31689)the Seneca Foundation Murcia,Spain(19892/GERM-15)the Regional Government of Catalonia(Grants:2017-SGR-1229 and 2020-FI-B-00412).
文摘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.
基金the European Commission(H2020-MSCA-IF-79212)the Ministry of Science,Innovation and Universities,Spain(Grants:RYC-2014-15581,AGL2016–81890-REDT,AGL2017–88329-R and FJCI-2017-31689)the Regional Government of Catalonia,Spain(2017-SGR-1229).
文摘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.
