Sperm chromatin condensation as an in vivo fertility biomarker in bulls:a flow cytometry approach

Background:Genetic selection in cattle has been directed to increase milk production.This,coupled to the fact that the vast majority of bovine artificial inseminations(AI)are performed using cryopreserved sperm,have led to a reduction of fertility rates over the years.Thus,seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive efficiency.In humans,sperm chromatin condensation evaluated through chromomycin A3(CMA3)has recently been purported to be a powerful biomarker for sperm functional status and male infertility.The objectives of the present study were:a)to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability,and b)to test whether this parameter could be used as a predictor of in vivo fertility in bulls.The study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein males.Reproductive outcomes of each sire were determined by non-return rates,which were used to classify bulls into two groups(highly fertile and subfertile).Results:Chromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow cytometry.Sperm quality parameters(morphology,viability,total and progressive motility)were also assessed.Pearson correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo fertility.Sperm morphology,viability and total motility presented an area under the ROC curve(AUC)of 0.54,0.64 and 0.68,respectively(P>0.05),and thus were not able to discriminate between fertile and subfertile individuals.Alternatively,while the percentage of progressively motile sperm showed a significant predictive value,with an AUC of 0.73(P=0.05),CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in bulls.Specifically,the percentage of viable sperm with poor chromatin condensation showed better accuracy and precision to predict in vivo fertility,with an AUC of 0.78(P=0.02).Conclusions:Chromatin condensation evaluated through CMA3/Yo-Pro-1 and flow cytometry is defined here as a more powerful tool than conventional sperm parameters to predict bull in vivo fertility,with a potential ability to maximising the efficiency of dairy breeding industry.

Metabolomic fingerprinting of pig seminal plasma identifies in vivo fertility biomarkers

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.

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

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.