Search

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., Funding Agencies|Secretaria de Estado de Investigacion, Desarrollo e Innovacion [AGL2013-43211-R, IJCI-2014-21671]; Ministerio de Educacion Cultura y Deporte [FPU13/03991]; Ministerio de Economia y Competitividad-FEDER [AGL2013-43211-R]; Junta de Extremadura-FEDER [IB16030, GR 15029]; Swedish Research Councils [521-2011-6353]; Formas [221-2011-512]

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]

Techniques such as mass spectrometry have led to unprecedented knowledge of the proteins that are present in the spermatozoa of humans and other mammals. However, in spite of their high-throughput and fractioning techniques, most of the techniques in use only offer average values for the entire sperm population. Yet, ejaculate is very heterogeneous, and average values may mask relevant biological information. The application of flow cytometry may overcome this disadvantage, allowing proteomic analysis at the single-cell level. Moreover, recent advances in cytometry, allowing multiple analyses within a single cell combined with powerful statistical tools, as an expanding subfield in spermatology, are described. The increased use of advanced flow cytometers in andrology laboratories will allow the rapid development of multiparametric, multicolour flow cytometry in andrology that will expand the clinical applications and research possibilities of flow cytometry-based proteomic approaches, especially in the subfields of clinical andrology and sperm biotechnology., Funding Agencies|Secretaria de Estado de Investigacion, Desarrollo e Innovacion [AGL2013-43211-R, IJCI-2014-21671]; Ministerio de Economia y Competitividad-FEDER, Madrid, Spain [AGL2013-43211-R]; Junta de Extremadura-FEDER [GR 10010, IB 16030]; Swedish Research Councils VR [521-2011-6353]; Formas [221-2011-512]; Ministerio de Educacion, Cultura y Deporte [IJCI-2014-21671]

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.