| 1. |
- Balao da Silva, C, et al.
(författare)
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Effect of Hoechst 33342 on stallion spermatozoa incubated in KMT or Tyrodes modified INRA96
- 2012
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Ingår i: Animal Reproduction Science. - : Elsevier Masson. - 0378-4320 .- 1873-2232. ; 131:3-4, s. 165-171
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Tidskriftsartikel (refereegranskat)abstract
- The only known means of effectively separating populations of X and Y bearing sperms is the Beltsville sexing technology. The technology implies that each individual sperm is interrogated for DNA content, measuring the intensity of the fluorescence after staining the spermatozoa with Hoechst 33342. Because there are no data regarding the effect of the staining on stallion sperm, ejaculates were incubated up to 90 min in presence of 0, 4.5, 9, 22.5, 31.5, 45, 54, 67.5, 76.5 and 90 mu M of Hoechst 33342, in two media, KMT or INRA-Tyrodes. After 40 and 90 min of incubation, motility (CASA) and membrane integrity (flow cytometry after YoPro-1/Eth staining) were evaluated. In KMT extender sperm motility significantly decreased after 45 min of incubation when sperm were incubated in the presence of concentrations of Hoechst of 45 mu M or greater (P andlt; 0.05). When incubated in modified INRA96, stallion spermatozoa tolerated greater concentrations of Hoechst, because sperm motility only decreased when incubated in presence of 90 mu M (P andlt; 0.05) and membrane integrity was not affected. After 90 min of incubation the same effect was observed, but in this case at concentrations over 45 mu M the percentage of total motile sperm was also reduced although only in samples incubated in KMT. To produce this effect in samples incubated in Tyrodes modified INRA 96, Hoechst had to be present at concentrations over 67.5 mu M. Apparently, the detrimental effect of Hoechst to stallion spermatozoa varies depending on the media, and INRA modified extender may be an alternative to KMT.
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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- 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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| 6. |
- Ortega Ferrusola, C., et al.
(författare)
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Inhibition of the mitochondrial permeability transition pore reduces "apoptosis like" changes during cryopreservation of stallion spermatozoa
- 2010
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Ingår i: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 74:3, s. 458-465
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Tidskriftsartikel (refereegranskat)abstract
- In order to evaluate to what extent the changes that occur during cryopreservation involve the mitochondrial permeability transition pore (PT-pore), a specific inhibitor was used during the cryopreservation process of stallion spermatozoa. Four ejaculates from each of 7 stallions were frozen using a standard protocol. Before freezing, each ejaculate was split into three subsamples. The first was supplemented with 2.5 mu M bongkrekic acid (BA) and the second with 5 mu M BA. The third subsample served as control. The BA significantly reduced the percentage of spermatozoa depicting active caspases after thawing, reduced the percentage of spermatozoa with increased membrane permeability, and increased the mitochondrial membrane potential of thawed sperm. Sperm motility was reduced as a result of the treatment. It is concluded that the mitochondrial pathway of apoptosis seems to be an important factor involved in the sublethal damage that equine spermatozoa experience after freezing and thawing, and that sperm motility in the equine species is largely dependent on mitochondrial ATP produced by oxidative phosphorylation. (C) 2010 Elsevier Inc. All rights reserved.
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| 7. |
- 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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| 8. |
- 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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