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

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Marc Llavanera ◽  
Jordi Ribas-Maynou ◽  
Ariadna Delgado-Bermúdez ◽  
Sandra Recuero ◽  
Rodrigo Muiño ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sperm Quality ◽  
Chromatin Condensation ◽  
Pearson Correlation ◽  
Correlation Coefficients ◽  
Quality Parameters ◽  
Sperm Chromatin ◽  
Fertility Rates ◽  
Sperm Chromatin Condensation

Abstract 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.

scholarly journals Free-radical production after post-thaw incubation of ram spermatozoa is related to decreased in vivo fertility

2015 ◽  
Vol 27 (8) ◽  
pp. 1187 ◽  
Author(s):  
Enrique Del Olmo ◽  
Alfonso Bisbal ◽  
Olga García-Álvarez ◽  
Alejandro Maroto-Morales ◽  
Manuel Ramón ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Pregnancy Rate ◽  
Male Fertility ◽  
Sperm Quality ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Sperm Chromatin ◽  
High Fertility ◽  
Ros Production ◽  
Sperm Viability

The aim of the present study was to evaluate the effect of sperm reactive oxygen species (ROS) production and DNA changes on male fertility. For that purpose, six rams with significantly different pregnancy rates were used; these were classified as having high fertility, i.e. 59.4% average pregnancy rate, or low fertility, i.e. 23.1% average pregnancy rate. Sperm quality was assessed after a two-step process of sample thawing followed by an incubation of 2 h, either in the freezing extender (37°C) or after dilution in synthetic oviductal fluid (SOF; 38°C, 5%CO2). Sperm viability (YO-PRO-1), ROS production (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein acetyl ester (CM-H2DCFDA)) and undamaged chromatin (sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, chromomycin A3) were evaluated by flow cytometry. Although no significant differences in sperm viability were observed, our results showed increased ROS production during incubation in the freezing extender as well as in SOF medium. Comparison between fertility groups showed significant differences in ROS production after 2 h of incubation for the two treatments. Regarding DNA integrity, our results showed no significant differences either between treatments and incubation times or fertility groups. Linear regression analysis showed that ROS production determined by CM-H2DCFDA was a good indicator parameter for in vivo male fertility of SOF-incubated samples, yielding a fair correlation between both parameters (r = –0.92). These results indicate that detection of ROS production by CM-H2DCFDA and flow cytometry after 2 h of incubation in SOF could be a useful procedure for predicting fertility of ram spermatozoa.

scholarly journals The Drosophila chromosomal protein Mst77F is processed to generate an essential component of mature sperm chromatin

Open Biology ◽  
2016 ◽  
Vol 6 (11) ◽  
pp. 160207 ◽  
Author(s):  
Shuhei Kimura ◽  
Benjamin Loppin
Keyword(s):  
Chromatin Condensation ◽  
Rapid Evolution ◽  
Mature Sperm ◽  
Sperm Chromatin ◽  
Chromosomal Protein ◽  
Sperm Dna ◽  
Cross Links ◽  
Sperm Chromatin Condensation

In most animals, the bulk of sperm DNA is packaged with sperm nuclear basic proteins (SNBPs), a diverse group of highly basic chromosomal proteins notably comprising mammalian protamines. The replacement of histones with SNBPs during spermiogenesis allows sperm DNA to reach an extreme level of compaction, but little is known about how SNBPs actually function in vivo . Mst77F is a Drosophila SNBP with unique DNA condensation properties in vitro , but its role during spermiogenesis remains unclear. Here, we show that Mst77F is required for the compaction of sperm DNA and the production of mature sperm, through its cooperation with protamine-like proteins Mst35Ba/b. We demonstrate that Mst77F is incorporated in spermatid chromatin as a precursor protein, which is subsequently processed through the proteolysis of its N-terminus. The cleavage of Mst77F is very similar to the processing of protamine P2 during human spermiogenesis and notably leaves the cysteine residues in the mature protein intact, suggesting that they participate in the formation of disulfide cross-links. Despite the rapid evolution of SNBPs, sperm chromatin condensation thus involves remarkably convergent mechanisms in distantly related animals.

scholarly journals Molecular morphology and function of bull spermatozoa linked to histones and associated with fertility

Reproduction ◽  
2013 ◽  
Vol 146 (3) ◽  
pp. 263-272 ◽  
Author(s):  
Rodrigo V de Oliveira ◽  
Sule Dogan ◽  
Lauren E Belser ◽  
Abdullah Kaya ◽  
Einko Topper ◽  
...  
Keyword(s):  
Chromatin Condensation ◽  
Mammalian Species ◽  
Sperm Concentration ◽  
Low Fertility ◽  
Sperm Chromatin ◽  
High Fertility ◽  
Expression Levels ◽  
Molecular Morphology ◽  
Sperm Chromatin Condensation

Sub-par fertility in bulls is influenced by alterations in sperm chromatin, and it might not be solved with increased sperm concentration in artificial insemination. Appropriate histone retention during sperm chromatin condensation plays critical roles in male fertility. The objective of this study was to determine failures of sperm chromatin condensation associated with abnormal persistence or accessibility of histones by aniline blue (ANBL) test, expression levels, and cellular localizations of one variant and two core histones (H3.3, H2B, and H4 respectively) in the spermatozoa of low-fertility (LF) vs high-fertility (HF) bulls. The expression levels and cellular localizations of histones in spermatozoa were studied using immunoblotting, immunocytochemistry, and staining methods. The bioinformatics focused on the sequence identity and evolutionary distance of these proteins among three mammalian species: bovine, mouse, and human. We demonstrated that ANBL staining was different within the LF (1.73 (0.55, 0.19)) and HF (0.67 (0.17, 0.06)) groups (P<0.0001), which was also negatively correlated within vivobull fertility (r=−0.90,P<0.0001). Although these histones were consistently detectable and specifically localized in bull sperm cells, they were not different between the two groups. Except H2B variants, H3.3 and H4 showed 100% identity and were evolutionarily conserved in bulls, mice and humans. The H2B variants were more conserved between bulls and humans, than in mice. In conclusion, we showed that H2B, H3.3, and H4 were detectable in bull spermatozoa and that sperm chromatin condensation status, changed by histone retention, is related to bull fertility.

scholarly journals The age and mouse sperm quality: a flow cytometry investigation

2021 ◽  
Author(s):  
Federica Zacchini ◽  
Michal Bochenek ◽  
Simona Bisogno ◽  
Alan Chan ◽  
Grazyna E Ptak
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
Sperm Quality ◽  
Chromatin Condensation ◽  
Mammalian Species ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Male Gametes ◽  
First Child ◽  
Aging Men

Postponement of fatherhood is growing worldwide due to socio-economic factors. The choice to conceive the first child above the age of 35 years is often associated with reduced fertility and poor pregnancy outcome. As widely known, several factors (e.g., lifestyle, environment, health problems) can affect spermatogenesis leading to poor reproductive outcome. Currently, the debate on the influence of aging on male gametes and safety/risk of conception at advanced age is still ongoing. Controversial results have been published so far on the changes in semen features of aging men and other mammalian species (mainly rodents). In this study, we aimed to assess how aging affects sperm quality in an inbreed mouse model, without underlying infertility, using a flow cytometry approach. Our data showed that aging is associated with increased sperm chromatin condensation, but not changes in the DNA integrity, metabolic activity or viability. These data suggest a mild effect of aging on sperm quality in a mouse model without underlying infertility.

308 GUANACO SPERM CHROMATIN EVALUATION USING TOLUIDINE BLUE

2010 ◽  
Vol 22 (1) ◽  
pp. 310 ◽  
Author(s):  
M. I. Carretero ◽  
S. Giuliano ◽  
A. Agüero ◽  
M. Pinto ◽  
M. Miragaya ◽  
...  
Keyword(s):  
Toluidine Blue ◽  
Chromatin Condensation ◽  
Positive Control ◽  
Sperm Chromatin ◽  
Chromatin Decondensation ◽  
South American Camelids ◽  
Splitting Factor ◽  
Dark Violet ◽  
High Degree ◽  
Sperm Chromatin Condensation

Guanacos, a wild species of South American camelids, have a high-quality fiber with great economic potential. To evaluate reproductive aptitude in guanacos, our laboratory has developed a reliable semen collection technique using electroejaculation and has applied various methods for evaluating semen characteristics. Studies for evaluating the state of sperm chromatin have also been initiated. Toluidine blue (TB) is a cationic stain that unites with the phosphate groups in the DNA, thus permitting differentiation between sperm heads according to the degree of chromatin decondensation. The objectives of this study were to determine the TB staining patterns of guanaco sperm chromatin, establish a positive control for the stain, and evaluate the effect of collagenase on sperm chromatin condensation. Semen was collected from 4 guanacos, between 6 and 9 years old, using electroejaculation. In Experiment 1, to establish a positive control for the stain, equal quantities of 1% dithiothreitol (DTT) and raw semen were incubated at room temperature for 30 s, 1.5 min, and 3 min. After incubation, smears were made and then dried, to avoid continuing the reaction, and finally were stained with 0.02% TB. A split-plot design was used with time as the splitting factor and considering the males as a block. In Experiment 2, raw semen was divided into 2 aliquots, one diluted 4 : 1 in 0.1% collagenase in HEPES-TALP-BSA medium and the other left without enzyme. Both aliquots were incubated 4 min at 37°C and, after centrifugation to remove the enzyme, smears were made and stained with TB. Spermatozoa were classified according to the degree of chromatin decondensation. Analysis of variance was performed using the males as a blocking factor and the treatment as a fixed factor. According to the degree of chromatin decondensation, three patterns of staining with TB were observed: light blue (negative, without alteration of chromatin condensation), light violet (intermediate, some degree of decondensation), and dark violet (positive, high degree of decondensation). A significant increase (P < 0.05) of sperm with highly decondensed chromatin was observed in semen incubated for 3 min with DTT when compared to 30 s of incubation. Therefore, 3 min of incubation with DTT was chosen as the positive control for Experiment 2. No significant differences in any of the 3 patterns of TB staining were observed between semen incubated with or without 0.1% collagenase. In conclusion, it is possible to use TB to evaluate the degree of chromatin decondensation in guanaco spermatozoa and to use DTT as a positive control for the stain. Treatment of guanaco semen with 0.1% collagenase did not affect sperm chromatin condensation; therefore, this enzyme can be used to decrease semen viscosity and aid handling in the laboratory.

scholarly journals Prediction of crude protein digestibility of animal by-product meals for dogs by the protein solubility in pepsin method

2014 ◽  
Vol 3 ◽  
Author(s):  
Iris M. Kawauchi ◽  
Nilva K. Sakomura ◽  
Cristiana F. F. Pontieri ◽  
Aline Rebelato ◽  
Thaila C. Putarov ◽  
...  
Keyword(s):  
Crude Protein ◽  
Pearson Correlation ◽  
Protein Solubility ◽  
Correlation Coefficients ◽  
Basal Diet ◽  
Protein Digestibility ◽  
Large Variability ◽  
The Relationship

AbstractAnimal by-product meals have large variability in crude protein (CP) content and digestibility. In vivo digestibility procedures are precise but laborious, and in vitro methods could be an alternative to evaluate and classify these ingredients. The present study reports prediction equations to estimate the CP digestibility of meat and bone meal (MBM) and poultry by-product meal (PM) using the protein solubility in pepsin method (PSP). Total tract CP digestibility of eight MBM and eight PM samples was determined in dogs by the substitution method. A basal diet was formulated for dog maintenance, and sixteen diets were produced by mixing 70 % of the basal diet and 30 % of each tested meal. Six dogs per diet were used to determine ingredient digestibility. In addition, PSP of the MBM and PM samples was determined using three pepsin concentrations: 0·02, 0·002 and 0·0002 %. The CP content of MBM and PM ranged from 39 to 46 % and 57 to 69 %, respectively, and their mean CP digestibility by dogs was 76 (2·4) and 85 (2·6) %, respectively. The pepsin concentration with higher Pearson correlation coefficients with the in vivo results were 0·0002 % for MBM (r 0·380; P = 0·008) and 0·02 % for PM (r 0·482; P = 0·005). The relationship between the in vivo and in vitro results was better explained by the following equations: CP digestibility of MBM = 61·7 + 0·2644 × PSP at 0·0002 % (P = 0·008; R2 0·126); and CP digestibility of PM = 54·1 + 0·3833 × PSP at 0·02 % (P = 0·005; R2 0·216). Although significant, the coefficients of determination were low, indicating that the models were weak and need to be used with caution.

scholarly journals The Cannabinoid Receptor CB1 Stabilizes Sperm Chromatin Condensation Status During Epididymal Transit by Promoting Disulphide Bond Formation

2020 ◽  
Vol 21 (9) ◽  
pp. 3117 ◽  
Author(s):  
Teresa Chioccarelli ◽  
Francesco Manfrevola ◽  
Veronica Porreca ◽  
Silvia Fasano ◽  
Lucia Altucci ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Cannabinoid Receptor ◽  
Chromatin Condensation ◽  
Sperm Chromatin ◽  
Histone H4 ◽  
Nuclear Condensation ◽  
Heterozygous Condition ◽  
Knock Out ◽  
The Impact ◽  
Sperm Chromatin Condensation

The cannabinoid receptor CB1 regulates differentiation of spermatids. We recently characterized spermatozoa from caput epididymis of CB1-knock-out mice and identified a considerable number of sperm cells with chromatin abnormality such as elevated histone content and poorly condensed chromatin. In this paper, we extended our findings and studied the role of CB1 in the epididymal phase of chromatin condensation of spermatozoa by analysis of spermatozoa from caput and cauda epididymis of wild-type and CB1-knock-out mouse in both a homozygous or heterozygous condition. Furthermore, we studied the impact of CB1-gene deletion on histone displacement mechanism by taking into account the hyperacetylation of histone H4 and players of displacement such as Chromodomain Y Like protein (CDYL) and Bromodomain testis-specific protein (BRDT). Our results show that CB1, via local and/or endocrine cell-to-cell signaling, modulates chromatin remodeling mechanisms that orchestrate a nuclear condensation extent of mature spermatozoa. We show that CB1-gene deletion affects the epididymal phase of chromatin condensation by interfering with inter-/intra-protamine disulphide bridges formation, and deranges the efficiency of histone removal by reducing the hyper-acetylation of histone H4. This effect is independent by gene expression of Cdyl and Brdt mRNA. Our results reveal a novel and important role for CB1 in sperm chromatin condensation mechanisms.

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