Determination of double-and single-stranded DNA breaks in bovine sperm is predictive of their fertilizing capacity

Background:The analysis of chromatin integrity has become an important determinant of sperm quality.In frozenthawed bovine sperm,neither the sequence of post-thaw injury events nor the dynamics of different types of sperm DNA breaks are well understood.The aim of the present work was to describe such sperm degradation aftermath focusing on DNA damage dynamics,and to assess if this parameter can predict pregnancy rates in cattle.Results:A total of 75 cryopreserved ejaculates from 25 Holstein bulls were evaluated at two post-thawing periods(0-2 h and 2-4 h),analyzing global and double-stranded DNA damage through alkaline and neutral Comet assays,chromatin deprotamination and decondensation,sperm motility,viability,acrosomal status,and intracellular levels of total ROS,superoxides and calcium.Insemination of 59,605 females was conducted using sperm from the same bulls,thus obtaining the non-return to estrus rates after 90 d(NRR).Results showed an increased rate of double-stranded breaks in the first period(0-2 h:1.29±1.01%/h vs.2-4 h:0.13±1.37%/h;P<0.01),whereas the rate of sperm with moderate+high single-stranded breaks was higher in the second period(0-2 h:3.52±7.77%/h vs.2-4h:21.06±11.69%/h;P<0.0001).Regarding sperm physiology,viability decrease rate was different between the two periods(0-2 h:-4.49±1.79%/h vs.2-4 h:-2.50±3.39%/h;P=0.032),but the progressive motility decrease rate was constant throughout post-thawing incubation(0-2 h:-4.70±3.42%/h vs.2-4 h:-1.89±2.97%/h;P>0.05).Finally,whereas no correlations between bull fertility and any dynamic parameter were found,there were correlations between the NRR and the basal percentage of highly-damaged sperm assessed with the alkaline Comet(Rs=-0.563,P=0.003),between NRR and basal progressive motility(Rs=0.511,P=0.009),and between NRR and sperm with high ROS at 4 h post-thaw(Rs=0.564,P=0.003).Conclusion:The statistically significant correlations found between intracellular ROS,sperm viability,sperm motility,DNA damage and chromatin deprotamination suggested a sequence of events all driven by oxidative stress,where viability and motility would be affected first and sperm chromatin would be altered at a later stage,thus suggesting that bovine sperm should be used for fertilization within 2 h post-thaw.Fertility correlations supported that the assessment of global DNA damage through the Comet assay may help predict bull fertility.

Expression of miR‑138 in cryopreserved bovine sperm is related to their fertility potential

Background MicroRNAs(miRNAs)are small,single-stranded,non-coding RNA molecules of 22–24 nucleotides that regulate gene expression.In the last decade,miRNAs have been described in sperm of several mammals,including cattle.It is known that miRNAs can act as key gene regulators of early embryogenesis in mice and humans;however,little is known about the content,expression,and function of sperm-borne miRNAs in early bovine embryo.In this study,total sperm RNA was isolated from 29 cryopreserved sperm samples(each coming from a separate bull)using a RNeasy kit and treatment with DNase I.RNA concentration and purity were determined through an Epoch spectrophotometer and an Agilent Bioanalyzer.The expression of 10 candidate miRNAs in bovine sperm(bta-miR-10a,bta-miR-10b,bta-miR-138,bta-miR-146b,bta-miR-19b,bta-miR-26a,bta-miR-34a,bta-miR-449a,bta-miR-495 and btamiR-7),previously identified in testis and/or epididymis,was evaluated with RT-qPCR.The cel-miR-39-3p was used as a spike-in exogenous control.Nonparametric Mann–Whitney tests were run to evaluate which miRNAs were differentially expressed between bulls with high fertility[HF;non-return rates(NRR)ranging from 39.5 to 43.5]and those with subfertility(SF;NRR ranging from 33.3 to 39.3).Several sperm functionality parameters(e.g.,viability,membrane stability or oxygen consumption,among others)were measured by multiplexing flow cytometry and oxygen sensing technologies.Results RNA concentration and purity(260/280 nm ratio)(mean±SD)from the 29 samples were 99.3±84.6 ng/μL and 1.97±0.72,respectively.Bioanalyzer results confirmed the lack of RNA from somatic cells.In terms of the presence or absence of miRNAs,and after applying the Livak method,8 out of 10 miRNAs(bta-miR-10b,-138,-146b,-19b,-26a,-449a,-495,-7)were consistently detected in bovine sperm,whereas the other two(bta-miR-10a,and-34a)were absent.Interestingly,the relative expression of one miRNA(bta-miR-138)in sperm was significantly lower in the SF than in the HF group(P=0.038).In addition to being associated to fertility potential,the presence of this miRNA was found to be negatively correlated with sperm oxygen consumption.The expression of three other miRNAs(bta-miR-19b,bta-miR-26a and bta-miR-7)was also correlated with sperm function variables.Conclusions In conclusion,although functional validation studies are required to confirm these results,this study suggests that sperm bta-miR-138 is involved in fertilization events and beyond,and supports its use as a fertility biomarker in cattle.