| 1. |
- Balao da Silva, C M., et al.
(author)
-
Sex sorting increases the permeability of the membrane of stallion spermatozoa
- 2013
-
In: Animal Reproduction Science. - : Elsevier Masson. - 0378-4320 .- 1873-2232. ; 138:3-4, s. 241-251
-
Journal article (peer-reviewed)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.
|
|
| 2. |
- Munoz, P. Martin, et al.
(author)
-
Redox cycling induces spermptosis and necrosis in stallion spermatozoa while the hydroxyl radical (OH center dot) only induces spermptosis
- 2018
-
In: Reproduction in domestic animals. - : WILEY. - 0936-6768 .- 1439-0531. ; 53:1, s. 54-67
-
Journal article (peer-reviewed)abstract
- Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2-methyl-1,4-naphthoquinone (menadione), or hydroxyl radical formation after FeSO4 exposure. Either exposure induced significant increases (p amp;lt; 0.05) in two markers of lipid peroxidation: 8-iso-PGF(2) and 4-hydroxynonenal (4-HNE). While both treatments induced changes indicative of spermptosis (caspase-3 activation and decreased mitochondrial membrane potential) (p amp;lt; 0.01), menadione induced sperm necrosis and a dramatic reduction in motility and thiol content in stallion spermatozoa. Thus, we provided evidence that oxidative stress underlies spermptosis, and thiol content is a key factor for stallion sperm function.
|
|
| 3. |
- Ortega Ferrusola, C., et al.
(author)
-
Stallion spermatozoa surviving freezing and thawing experience membrane depolarization and increased intracellular Na
- 2017
-
In: Andrology. - : WILEY. - 2047-2919 .- 2047-2927. ; 5:6, s. 1174-1182
-
Journal article (peer-reviewed)abstract
- In order to gain insight of the modifications that freezing and thawing cause to the surviving population of spermatozoa, changes in the potential of the plasma membrane (Em) and intracellular Na+ content of stallion spermatozoa were investigated using flow cytometry. Moreover, caspase 3 activity was also investigated and the functionality of the Na+-K+ ATPase pump was investigated before and after freezing and thawing. Cryopreservation caused a significant (pamp;lt;0.001) increase in the subpopulation of spermatozoa with depolarized sperm membranes, concomitantly with an increase (pamp;lt;0.05) in intracellular Na+. These changes occurred in relation to activation of caspase 3 (pamp;lt;0.001). Cryopreservation reduced the activity of the Na-K+ pump and inhibition of the Na+-K+ ATPase pump with ouabain-induced caspase 3 activation. It is concluded that inactivation of Na+-K+ ATPase occurs during cryopreservation, an inhibition that could play a role explaining the accelerated senescence of the surviving population of spermatozoa.
|
|
| 4. |
- Balao da Silva, C. M., et al.
(author)
-
Effect of Overnight Staining on the Quality of Flow Cytometric Sorted Stallion Sperm: Comparison with Tradtitional Protocols
- 2014
-
In: Reproduction in domestic animals. - : Wiley. - 0936-6768 .- 1439-0531. ; 49:6, s. 1021-1027
-
Journal article (peer-reviewed)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.
|
|
| 5. |
- Macias Garcia, B, et al.
(author)
-
Toxicity of glycerol for the stallion spermatozoa: Effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential
- 2012
-
In: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 77:7, s. 1280-1289
-
Journal article (peer-reviewed)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%.
|
|
| 6. |
- Balao da Silva, C. M., et al.
(author)
-
Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA
- 2016
-
In: Reproduction in domestic animals. - : WILEY-BLACKWELL. - 0936-6768 .- 1439-0531. ; 51:1, s. 18-25
-
Journal article (peer-reviewed)abstract
- To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa - including sex sorting - as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry-based assays. After sorting, oxidative stress increased from 26% to 33% in pre-and post-incubation controls, to 46% after sex sorting (p < 0.05). Proportions of DNA fragmentation index post-sorting were approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure.
|
|
