Extracellular vesicles from oviductal and uterine fluids supplementation in sequential in vitro culture improves bovine embryo quality

Background:In vitro production of bovine embryos is a well-established technology,but the in vitro culture(IVC)system still warrants improvements,especially regarding embryo quality.This study aimed to evaluate the effect of extracellular vesicles(EVs)isolated from oviductal(OF)and uterine fluid(UF)in sequential IVC on the development and quality of bovine embryos.Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum(dFCS)in the presence(BSA-EV and dFCS-EV)or absence of EVs from OF(D1 to D4)and UF(D5 to D8),mimicking in vivo conditions.EVs from oviducts(early luteal phase)and uterine horns(mid-luteal phase)from slaughtered heifers were isolated by size exclusion chromatography.Blastocyst rate was recorded on days 7-8 and their quality was assessed based on lipid contents,mitochondrial activity and total cell numbers,as well as survival rate after vitrification.Relative mRNA abundance for lipid metabolism-related transcripts and levels of phosphorylated hormonesensitive lipase(pHSL)proteins were also determined.Additionally,the expression levels of 383 miRNA in OF-and UF-EVs were assessed by qRT-PCR.Results:Blastocyst yield was lower(P<0.05)in BSA treatments compared with dFCS treatments.Survival rates after vitrification/warming were improved in dFCS-EVs(P<0.05).EVs increased(P<0.05)blastocysts total cell number in dFCS-EV and BSA-EV compared with respective controls(dFCS and BSA),while lipid content was decreased in dFCSEV(P<0.05)and mitochondrial activity did not change(P>0.05).Lipid metabolism transcripts were affected by EVs and showed interaction with type of protein source in medium(PPARGC1B,LDLR,CD36,FASN and PNPLA2,P<0.05).Levels of pHSL were lower in dFCS(P<0.05).Twenty miRNA were differentially expressed between OF-and UF-EVs and only bta-miR-148b was increased in OF-EVs(P<0.05).Conclusions:Mimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming,total cell number,lipid content,and relative changes in expression of lipid metabolism transcripts and lipase activation.Finally,EVs miRNA contents may contribute to the observed effects.

Bull spermatozoa selected by thermotaxis exhibit high DNA integrity,specific head morphometry,and improve ICSI outcome

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.