Background Sperm migration by thermotaxis is a guidance mechanism that operates along the oviduct and it has proved to be a valid method for selecting spermatozoa with low DNA fragmentation(SDF)in mice,humans,and stal...Background Sperm migration by thermotaxis is a guidance mechanism that operates along the oviduct and it has proved to be a valid method for selecting spermatozoa with low DNA fragmentation(SDF)in mice,humans,and stallions.This study aimed to analyse if bull spermatozoa could be selected by thermotaxis and to assess their quality in terms of SDF as well as determine the presence of a specific sperm subpopulation based on sperm morphometry and assess their fertilizing capacity by ICSI.Methods We used frozen-thawed sperm from 6 bulls and sperm selection by thermotaxis was performed with TALP medium supplemented with 25 mmol/L of HEPES and 5 mmol/L of caffeine.In these conditions,sperm selection was achieved,obtaining a net thermotaxis of 3.6%.Subsequently,we analysed the SDF of the migrated and not-migrated spermatozoa using the neutral COMET assay,and we evaluated the size of the sperm head using Hemacolor■ staining with Motic Images Plus 3 software.Additionally,migrated and not-migrated spermatozoa by thermotaxis were used to fertilize bovine in vitro matured(IVM)oocytes by ICSI,a very inefficient procedure in cattle that is only successful when the oocyte is artificially activated.Results The results showed lower SDF(χ^(2),P<0.001,13.3%reduction,n=8)and lower head size parameters(length and width,P<0.01;and perimeter and area,P<0.001;n=4)in those spermatozoa migrated in comparison to those not-migrated.The distribution of sperm subpopulations structure varied between groups,highlighting cluster 2,characterized by spermatozoa with small head size,and high ellipticity and elongated heads,as the most abundant in the thermotaxis migrated group.When performed ICSI(without oocyte artificial activation)with the thermotactic sperm,the blastocyst rate was 32.2%±9.3%in the group microinjected with the thermotactic spermatozoa vs.8.3%±7.8%in the group of not-migrated sperm(χ^(2),P<0.05).Conclusion Our results showed that bull sperm selection by thermotaxis has a much higher DNA integrity,small and elongated head size parameters,and different sperm subpopulation structure than the not-selected spermatozoa.Additionally,we evidenced that thermotactic spermatozoa improve ICSI success rates.展开更多
基金funded by the Spanish Ministry of Science and Innovation-MCIN (RTI2018-093548-B-100 and PID202M225070B-100 to A.Gutierrez-Adan and PID2019-1l1641 RB-100 to D.Rizos,funded by MCIN/AEI/10.13039/501100011033/and European Union"NextGeneration EU"/PRTR)supported by a Juan de la Cierva postdoctoral contract (FJC2019-040385-1)from the MCIN+1 种基金supported by a"Doctorados Industriales2018"fellowship of Comunidad de Madrid (IND2018/BIO-9610)supported by FPI scholarships from the MCIN (PRE2020-094452 and PRE2019-088813 respectively)。
文摘Background Sperm migration by thermotaxis is a guidance mechanism that operates along the oviduct and it has proved to be a valid method for selecting spermatozoa with low DNA fragmentation(SDF)in mice,humans,and stallions.This study aimed to analyse if bull spermatozoa could be selected by thermotaxis and to assess their quality in terms of SDF as well as determine the presence of a specific sperm subpopulation based on sperm morphometry and assess their fertilizing capacity by ICSI.Methods We used frozen-thawed sperm from 6 bulls and sperm selection by thermotaxis was performed with TALP medium supplemented with 25 mmol/L of HEPES and 5 mmol/L of caffeine.In these conditions,sperm selection was achieved,obtaining a net thermotaxis of 3.6%.Subsequently,we analysed the SDF of the migrated and not-migrated spermatozoa using the neutral COMET assay,and we evaluated the size of the sperm head using Hemacolor■ staining with Motic Images Plus 3 software.Additionally,migrated and not-migrated spermatozoa by thermotaxis were used to fertilize bovine in vitro matured(IVM)oocytes by ICSI,a very inefficient procedure in cattle that is only successful when the oocyte is artificially activated.Results The results showed lower SDF(χ^(2),P<0.001,13.3%reduction,n=8)and lower head size parameters(length and width,P<0.01;and perimeter and area,P<0.001;n=4)in those spermatozoa migrated in comparison to those not-migrated.The distribution of sperm subpopulations structure varied between groups,highlighting cluster 2,characterized by spermatozoa with small head size,and high ellipticity and elongated heads,as the most abundant in the thermotaxis migrated group.When performed ICSI(without oocyte artificial activation)with the thermotactic sperm,the blastocyst rate was 32.2%±9.3%in the group microinjected with the thermotactic spermatozoa vs.8.3%±7.8%in the group of not-migrated sperm(χ^(2),P<0.05).Conclusion Our results showed that bull sperm selection by thermotaxis has a much higher DNA integrity,small and elongated head size parameters,and different sperm subpopulation structure than the not-selected spermatozoa.Additionally,we evidenced that thermotactic spermatozoa improve ICSI success rates.
