Chromatin condensation but not DNA integrity of pig sperm is greater in the sperm-rich fraction

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.

Sperm function, mitochondrial activity and in vivo fertility are associated to their mitochondrial DNA content in pigs

Background Despite their low abundance in sperm, mitochondria have diverse functions in this cell type, includ-ing energy production, signalling and calcium regulation. In humans, sperm mitochondrial DNA content(mtDNAc) has been reported to be negatively linked to sperm function and fertility. Yet, the association between mtDNAc and sperm function in livestock remains unexplored. For this reason, this study aimed to shed some light on the link between mtDNAc and sperm function and fertilising potential in pigs. A qPCR method for mtDNAc quantification was optimised for pig sperm, and the association of this parameter with sperm motility, kinematics, mitochondrial activity, and fertility was subsequently interrogated.Results First, the q PCR method was found to be sensitive and efficient for mtDNAc quantification in pig sperm. By using this technique, mtDNAc was observed to be associated to sperm motility, mitochondrial activity and in vivo, but not in vitro, fertility outcomes. Specifically, sperm with low mtDNAc were seen to exhibit greater motility but decreased mitochondrial activity and intracellular reactive oxygen species. Interestingly, samples with lower mtD-NAc showed higher conception and farrowing rates, but similar in vitro fertilisation rates and embryo development, when compared to those with greater mtDNAc.Conclusions These findings enrich our comprehension of the association of mtDNAc with sperm biology, and lay the foundation for future research into employing this parameter as a molecular predictor for sperm function and fer-tility in livestock.

GSTM3, but not IZUMO1, is a cryotolerance marker of boar sperm

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.

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.

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.