The kinematics of the appearance of apoptotic markers was studied by flow cytometry and immunoblot assays in equine spermatozoa subjected to freezing and thawing. Caspase activity, low mitochondrial membrane potential, and increases in sperm membrane permeability were observed in all of the phases of the cryopreservation procedure. Freezing and thawing caused an increase in membrane permeability and changes in the pattern of caspase activity; decreases in mitochondrial membrane potential were observed after centrifugation and cooling to 4uC and after freezing and thawing. It is proposed that sperm mitochondria may be directly involved in the subtle damage that is present in most spermatozoa surviving freezing and thawing.

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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 (rZK0.789, P!0.001), and also between LPO and spermatozoa with high mitochondrial membrane potential (Djm) post-thaw (rZK0.689, P!0.001). LPO was also highly and significantly correlated with caspase activity. The correlation between caspase activity in ethidium positive cells and LPO was rZ0.772, P!0.001. 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.

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Strong evidences suggest that many different sperm subpopulations co-exist within the mammalian ejaculate. These subpopulations have been identified in a number of species; however, to the best of our knowledge, no data exist regarding the existence of sperm subpopulations within the canine ejaculate. Ejaculates were obtained by masturbation from four mongrels and processed using a standard freezing protocol. Motility data were analysed before and after cryopreservation using a computer assisted sperm analysis (CASA) system ISAS. On raw data, a principal component analysis (PCA) was performed to reduce the number of motility descriptors to a few informative variables, and then a K-means cluster procedure was performed and then a regression analysis to validate the clusters obtained in the second analysis. ANOVAs and chi-squared analyses were used to compare clusters and males. PCA revealed that two principal components represented more that the 88% of the variance with eigenvalues of 3.25 and 3.02, respectively. The clustering and discriminant analysis using curvilinear velocity and linear velocity as variables revealed the existence of 11 sperm subpopulations--four of them characterized by high velocities, two by medium values and five by low velocities. After freezing-thawing, nine subpopulations were found--four of high velocities, two of medium and three of low velocities. It is concluded that freezing-thawing not only impairs sperm motility but also produces changes in the sperm subpopulation structure in the canine ejaculate, that the evaluation of the sperm structure subpopulations is a better indicator of semen quality and freezeability than the use of mean values, and that two sperm motility quality indexes can be used to resume of the variables obtained from the CASA analysis.

To investigate the hypothesis that oxidative phosphorylation is a major source of ATP to fuel stallion sperm motility, oxidative phosphorylation was suppressed using the mitochondrial uncouplers CCCP and 2,4,-dinitrophenol (DNP) and by inhibiting mitochondrial respiration at complex IV using sodium cyanide or at the level of ATP synthase using oligomycin-A. As mitochondrial dysfunction may also lead to oxidative stress, production of reactive oxygen species was monitored simultaneously. All inhibitors reduced ATP content, but oligomycin-A did so most profoundly. Oligomycin-A and CCCP also significantly reduced mitochondrial membrane potential. Sperm motility almost completely ceased after the inhibition of mitochondrial respiration and both percentage of motile sperm and sperm velocity were reduced in the presence of mitochondrial uncouplers. Inhibition of ATP synthesis resulted in the loss of sperm membrane integrity and increased the production of reactive oxygen species by degenerating sperm. Inhibition of glycolysis by deoxyglucose led to reduced sperm velocities and reduced ATP content, but not to loss of membrane integrity. These results suggest that, in contrast to many other mammalian species, stallion spermatozoa rely primarily on oxidative phosphorylation to generate the energy required for instance to maintain a functional Na/K gradient, which is dependent on an Na-K antiporter ATPase, which relates directly to the noted membrane integrity loss. Under aerobic conditions, however, glycolysis also provides the energy required for sperm motility.

While, for a long time, the role of mitochondria in sperm physiology and pathology has been largely ignored, recent research points out the mitochondria as a major organelle with key roles in sperm function both under physiological and biotechnological conditions. This paper briefly reviews these novel findings regarding the role of mitochondria in sperm, paying special attention to the most practical, readily applicable, aspects of the topic such as their role as a major source of the sublethal damage that sperm experiments after cryopreservation.