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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., Funding Agencies|Ministerio de Economia y Competitividad-FEDER, Madrid, Spain [AGL2013-43211-R]; Junta de Extremadura-FEDER [GR 15029, IB16030]; Swedish Research Councils VR [521-2011-6353]; Formas [221-2011-512]; Ministerio de Educacion, Cultura y Deporte, Madrid Spain [FPU13/03991]; Ministerio de Economia y Competitividad Juan de la Cierva [IJCI-2014-21671]

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., Funding Agencies|Ministerio de Economia y Competitividad-FEDER, Madrid, Spain [AGL2013-43211-R]; Junta de Extremadura-FEDER [GR 15029, IB16030]; Swedish Research Councils VR [521-2011-6353]; Formas [221-2011-512]; Ministerio de Educacion, Cultura y Deporte, Madrid Spain [FPU13/03991]; Ministerio de Economia y Competitividad Juan de la Cierva [IJCI-2014-21671]

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., Funding Agencies|Ministerio de Economia y Competitividad-FEDER, Madrid, Spain [AGL2013-43211-R]; Junta de Extremadura-FEDER [GR 15029, IB16030]; Swedish Research Councils VR [521-2011-6353]; Formas [221-2011-512]; Ministerio de Educacion, Cultura y Deporte, Madrid Spain [FPU13/03991]; Ministerio de Economia y Competitividad Juan de la Cierva [IJCI-2014-21671]

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., Funding Agencies|Ministerio de Economia y Competitividad-FEDER, Madrid, Spain [AGL2013-43211-R]; Junta de Extremadura-FEDER [GR 15029, IB16030]; Swedish Research Councils VR [521-2011-6353]; Formas [221-2011-512]; Ministerio de Educacion, Cultura y Deporte, Madrid Spain [FPU13/03991]; Ministerio de Economia y Competitividad Juan de la Cierva [IJCI-2014-21671]

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., Funding Agencies|Ministerio de Economia y Competitividad-FEDER, Madrid, Spain [AGL2013-43211-R]; Junta de Extremadura-FEDER [GR 15029, IB16030]; Swedish Research Councils VR [521-2011-6353]; Formas [221-2011-512]; Ministerio de Educacion, Cultura y Deporte, Madrid Spain [FPU13/03991]; Ministerio de Economia y Competitividad Juan de la Cierva [IJCI-2014-21671]

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., Funding Agencies|Ministerio de Economia y Competitividad-FEDER in Madrid, Spain [AGL2013-43211-R]; Gobierno de Extremadura-FEDER [GR 10010, PCE1002]

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., Funding Agencies|Ministerio de Economia y Competitividad-FEDER in Madrid, Spain [AGL2013-43211-R]; Gobierno de Extremadura-FEDER [GR 10010, PCE1002]

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., Funding Agencies|Ministerio de Economia y Competitividad-FEDER in Madrid, Spain [AGL2013-43211-R]; Gobierno de Extremadura-FEDER [GR 10010, PCE1002]

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., Funding Agencies|Ministerio de Economia y Competitividad-FEDER in Madrid, Spain [AGL2013-43211-R]; Gobierno de Extremadura-FEDER [GR 10010, PCE1002]

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., Funding Agencies|Ministerio de Economia y Competitividad-FEDER in Madrid, Spain [AGL2013-43211-R]; Gobierno de Extremadura-FEDER [GR 10010, PCE1002]

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.

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.

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.

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.

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.

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., Funding Agencies|Ministerio de Ciencia e Innovacion-FEDER in Madrid, Spain|AGL 2010-20758BFU2011-30261|Junta de Extremadura, Merida, Spain-FEDER|10010PCE1002

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.

The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an apparent molecular mass of approximately 83 kDa. On the other hand, the Ab developed against the NOS3 showed a band of approximately 96 kDa in fresh and FT sperm lysates. NO production was positively correlated with sperm motility and velocity after thaw, suggesting an NO role for the functionality of cryopreserved stallion spermatozoa; but the production of NO is compromised in egg yolk-containing extenders.

Lipid peroxidation (LPO) of stallion spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezability. Innate levels of LPO were very low in fresh spermatozoa but increased after thawing, a change that was largely stallion-dependent. The level of LPO in fresh spermatozoa was not correlated with that of the thawed spermatozoa. Negative correlations existed between LPO and intact membranes post-thaw (r= -0.789, Pless than0.001), and also between LPO and spermatozoa with high mitochondrial membrane potential (Delta psi m) post-thaw (r= -0.689, Pless than0.001). LPO was also highly and significantly correlated with caspase activity. The correlation between caspase activity in ethidium positive cells and LPO was r=0.772, Pless than0.001. This LPO is unlikely to represent, per se, a sign of cryopreservation-induced injury, but it is apparently capable of triggering apoptotic-like changes that could result in the sub-lethal cryodamage often seen among surviving spermatozoa. Reproduction (2009) 138 55-63, DOI does not work: 10.1530/RFP-08-0484

Although sperm head shape and relative dimensions are considered reliable indicators of sperm quality, their quantification is most often operator-driven, e.g., subjective. Artificial insemination semen doses from 35 mature stud boars of known fertility and belonging to three breeds and two hybrid breeds (Duroc, Large White, Landrace, respectively, Yorker and Risco) were used in this study. Sperm samples were extended to 100 x 10(6) Cells per mL and 10 mu L of the sperm suspension used to made smears which, stained, were examined using phase contrast microscopy interfaced with an automated sperm morphology analyzer (ASMA, 2 ISAS (R)). Each sperm head was measured for four primary parameters [area (A) mu m(2), perimeter (P) mu m, length (L) mu m, width (M mu m], and four derived parameters of head shape [(L/W, (4 pi A/P-2), ((L - W/(L + M), (pi LW/4A)]. Definition of head size was statistically performed. The threshold for each class was established on the basis of the area values, considering the 25th percentile as small and the 75th percentile as large spermatozoa. In a second step, sperm head shape was determined as normal, elliptic, abnormal (rugose) contour, long or irregular and percentiles set as above to define spermatozoa with normal values for each shape parameter. Significant differences were found among breeds in the size of morphologically normal spermatozoa, which were significantly larger and more elliptic (P less than 0.001) in the Duroc breed. Sperm chromatin integrity was studied using the SCSA-assay, with significant differences observed in the degree of fragmentation intensity (DFI) although this value was consistently low in all animals studied. The hereby-validated ASMA was able to determine significant differences in sperm shape and dimensions among breeds, which were not accompanied by deviations in chromatin structure neither within nor between fertile AI-boars. (c) 2007 Elsevier Inc. All rights reserved.