| 1. |
- Balao da Silva, C. M., et al.
(författare)
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Effect of Overnight Staining on the Quality of Flow Cytometric Sorted Stallion Sperm: Comparison with Tradtitional Protocols
- 2014
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Ingår i: Reproduction in domestic animals. - : Wiley. - 0936-6768 .- 1439-0531. ; 49:6, s. 1021-1027
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Tidskriftsartikel (refereegranskat)abstract
- Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre-sorting storage at 5 degrees C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.
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| 2. |
- Balao da Silva, C M., et al.
(författare)
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Sex sorting increases the permeability of the membrane of stallion spermatozoa
- 2013
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Ingår i: Animal Reproduction Science. - : Elsevier Masson. - 0378-4320 .- 1873-2232. ; 138:3-4, s. 241-251
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Tidskriftsartikel (refereegranskat)abstract
- At present, the only repeatable means of selecting the sex of offspring is the Beltsville semen sorting technology using flow cytometry (FC). This technology has reached commercial status in the bovine industry and substantial advances have occurred recently in swine and ovine species. In the equine species, however, the technology is not as well developed. To better understand the changes induced in stallion spermatozoa during the sorting procedure, pooled sperm samples were sorted: sperm motility and kinematics were assessed using computer assisted sperm analysis, sperm membrane integrity was assessed using the YoPro-1 assay, while plasmalemmal stability and lipid architecture were assessed using Merocyanine 540/SYTOX green and Annexin-V, respectively. Lipid peroxidation was also investigated with the probe Bodipy(581/591)-C11. All assays were performed shortly after collection, after incubation and after sex sorting using FC. In order to characterize potential molecular mechanisms implicated in sperm damage, an apoptosis protein antibody dot plot array analysis was performed before and after sorting. While the percentage of total motile sperm remained unchanged, sex sorting reduced the percentages of progressive motile spermatozoa and of rapid spermatozoa as well as curvilinear velocity (VCL). Sperm membranes responded to sorting with an increase in the percentage of YoPro-1 positive cells, suggesting the sorted spermatozoa had a reduced energy status that was confirmed by measuring intracellular ATP content.
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| 3. |
- Diaz-Gallo, L. M., et al.
(författare)
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Analysis of the influence of PTPN22 gene polymorphisms in systemic sclerosis
- 2011
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 1468-2060 .- 0003-4967. ; 70:3, s. 454-462
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Tidskriftsartikel (refereegranskat)abstract
- Objective Two functional single nucleotide polymorphisms (SNP) in the PTPN22 gene (rs24746601 and rs33996649) have been associated with autoimmunity. The aim of this study was to investigate the role of the R263Q SNP for the first time and to re-evaluate the role of the R620W SNP in the genetic predisposition to systemic sclerosis (SSc) susceptibility and clinical phenotypes. Methods 3422 SSc patients (2020 with limited cutaneous SSc and 1208 with diffuse cutaneous SSc) and 3638 healthy controls of Caucasian ancestry from an initial case--control set of Spain and seven additional independent replication cohorts were included in our study. Both rs33996649 and rs2476601 PTPN22 polymorphisms were genotyped by TaqMan allelic discrimination assay. A meta-analysis was performed to test the overall effect of these PTPN22 polymorphisms in SSc. Results The meta-analysis revealed evidence of association of the rs2476601 T allele with SSc susceptibility (p(FDRcorrected) = 0.03 pooled, OR 1.15, 95% CI 1.03 to 1.28). In addition, the rs2476601 T allele was significantly associated with anticentromere-positive status (p(FDRcorrected) = 0.02 pooled, OR 1.22, 95% CI 1.05 to 1.42). Although the rs33996649 A allele was significantly associated with SSc in the Spanish population (p(FDRcorrected) = 0.04, OR 0.58, 95% CI 0.36 to 0.92), this association was not confirmed in the meta-analysis (p = 0.36 pooled, OR 0.89, 95% CI 0.72 to 1.1). Conclusion The study suggests that the PTPN22 R620W polymorphism influences SSc genetic susceptibility but the novel R263Q genetic variant does not. These data strengthen evidence that the R620W mutation is a common risk factor in autoimmune diseases.
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| 4. |
- Macias Garcia, B, et al.
(författare)
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Fatty acids and plasmalogens of the phospholipids of the sperm membranes and their relation with the post-thaw quality of stallion spermatozoa
- 2011
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Ingår i: THERIOGENOLOGY. - : Elsevier Science B.V., Amsterdam.. - 0093-691X. ; 75:5, s. 811-818
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Tidskriftsartikel (refereegranskat)abstract
- Fatty acids and plasmalogens were extracted from the phospholipids of the plasma membrane of stallion spermatozoa, to determine their relation with sperm quality after freezing and thawing. Sperm quality was rated using a quality index that combined the results of the analysis of sperm motility and velocity (CASA analysis), membrane status and mitochondrial membrane potential (flow cytometry) post thaw. Receiving operating system (ROC) curves were used to evaluate the value of specific lipid components of the sperm membrane herein studied as forecast of potential freezeability. From all parameters studied the ratio of percentage of C16 plasmalogens related to total phospholipids was the one with the better diagnostic value. For potentially bad freezers, the significant area under the ROC-curve was 0.74, with 75% sensitivity and 79.9% specificity for a cut off value of 26.9. Also the percentage of plasmalogens respect to total phospholipids gave good diagnostic value for bad freezers. On the other hand, the percentage of C18 fatty aldehydes related to total phospholipids of the sperm membrane properly forecasted freezeability with an area under the ROC curve of 0.70 with 70% sensitivity and 62.5% specificity for a cut off value of 0.32.
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| 5. |
- Macias Garcia, B., et al.
(författare)
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Membrane Lipids of the Stallion Spermatozoon in Relation to Sperm Quality and Susceptibility to Lipid Peroxidation
- 2011
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Ingår i: Reproduction in domestic animals. - : Blackwell Publishing. - 0936-6768 .- 1439-0531. ; 46:1, s. 141-148
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Tidskriftsartikel (refereegranskat)abstract
- Contents: Lipids were extracted from ejaculated spermatozoa from seven individual stallions to distinguish neutral lipids (NL) and polar lipids (PL) and determine their variation among stallions and their relationship with sperm quality and sperm susceptibility to lipid peroxidation. The isolated fatty acids were correlated with sperm quality (membrane integrity, mitochondrial membrane potential (ΔΨm) and expression of active caspases) and the sensitivity of the sperm plasma membrane to LPO. The miristic (C14: 0), palmitic (C16: 0), stearic (C18: 0) and oleic (C18: 1n9) acids were predominant among the NLs. Within the phospholipid fraction, the docosapentanoic acid (C22: 5n6) was dominant, albeit varying among stallions. Surprisingly, the percentage of polyunsaturated fatty acids was positively correlated with sperm quality and a low propensity for LPO, probably because these particular fatty acids provide a higher fluidity of the plasma membrane. The stallion showing the poorest sperm membrane integrity plus a high level of LPO in his ejaculate had a lower percentage (p less than 0.05) of this fatty acid in his sperm plasma membranes. © 2010 Blackwell Verlag GmbH.
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| 6. |
- Macias Garcia, B, et al.
(författare)
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Toxicity of glycerol for the stallion spermatozoa: Effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential
- 2012
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Ingår i: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 77:7, s. 1280-1289
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Tidskriftsartikel (refereegranskat)abstract
- Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sperm parameters evaluated included cell volume, membrane integrity, lipid peroxidation, caspase 3, 7, and 8 activation, mitochondrial membrane potential, and integrity of the cytoskeleton. Glycerol exerted toxicity at concentrations 3.5% and the maximal toxicity was observed at 5%. The actin cytoskeleton was especially sensitive to glycerol presence, inducing rapid F actin depolymerization at concentrations over 1.5%. The sperm membrane and the mitochondria were other structures affected. The toxicity of glycerol is apparently related to osmotic and nonosmotic effects. In view of our results the concentration of glycerol in the freezing media for stallion spermatozoa should not surpass 2.5%.
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| 7. |
- Morillo Rodriguez, A, et al.
(författare)
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Freezing stallion semen with the new Caceres extender improves post thaw sperm quality and diminishes stallion-to-stallion variability
- 2011
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Ingår i: Animal Reproduction Science. - : Elsevier Masson. - 0378-4320 .- 1873-2232. ; 127:1-2, s. 78-83
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Tidskriftsartikel (refereegranskat)abstract
- Ejaculates from 7 stallions were split and simultaneously frozen in three different extenders, INRA 96 egg yolk glycerol, Ghent and the newly developed extender Caceres. After thawing, samples were evaluated for motility (CASA system) sperm membrane integrity and early membrane changes (YoPro-1/Eth staining), acrosome integrity (FICT-PNA), and mitochondrial membrane potential (JC-1) (flow cytometry). Samples frozen in Caceres extender consistently showed the best results in post-thaw motility (increases ranging from 11 to 17%, p andlt; 0.05) and velocity (p andlt; 0.05), membrane integrity (increases ranging from 11 to 14%, p andlt; 0.05) and mitochondrial membrane potential (p andlt; 0.05). It is concluded that this new extender should be included in a freezeability test to determine the best extender for each individual.
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| 8. |
- Rueda, B., et al.
(författare)
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BANK1 functional variants are associated with susceptibility to diffuse systemic sclerosis in Caucasians
- 2010
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 1468-2060 .- 0003-4967. ; 69:4, s. 700-705
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Tidskriftsartikel (refereegranskat)abstract
- Objective To investigate the possible association of the BANK1 gene with genetic susceptibility to systemic sclerosis (SSc) and its subphenotypes. Methods A large multicentre case-control association study including 2380 patients with SSc and 3270 healthy controls from six independent case-control sets of Caucasian ancestry ( American, Spanish, Dutch, German, Swedish and Italian) was conducted. Three putative functional BANK1 polymorphisms (rs17266594 T/C, rs10516487 G/A, rs3733197 G/A) were selected as genetic markers and genotyped by Taqman 5' allelic discrimination assay. Results A significant association of the rs10516487 G and rs17266594 T alleles with SSc susceptibility was observed (pooled OR=1.12, 95% CI 1.03 to 1.22; p=0.01 and pooled OR=1.14, 95% CI 1.05 to 1.25; p=0.003, respectively), whereas the rs3733197 genetic variant showed no statistically significant deviation. Stratification for cutaneous SSc phenotype showed that the BANK1 rs10516487 G, rs17266594 T and rs3733197 G alleles were strongly associated with susceptibility to diffuse SSc (dcSSc) (pooled OR=1.20, 95% CI 1.05 to 1.37, p=0.005; pooled OR=1.23, 95% CI 1.08 to 1.41, p=0.001; pooled OR=1.15, 95% CI 1.02 to 1.31, p=0.02, respectively). Similarly, stratification for specific SSc autoantibodies showed that the association of BANK1 rs10516487, rs17266594 and rs3733197 polymorphisms was restricted to the subgroup of patients carrying anti-topoisomerase I antibodies (pooled OR=1.20, 95% CI 1.02 to 1.41, p=0.03; pooled OR=1.24, 95% CI 1.05 to 1.46, p=0.01; pooled OR=1.26, 95% CI 1.07 to 1.47, p=0.004, respectively). Conclusion The results suggest that the BANK1 gene confers susceptibility to SSc in general, and specifically to the dcSSc and anti-topoisomerase I antibody subsets.
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