In recent years the mitochondria of the stallion spermatozoa is becoming focus of major interest; together to their role as a source of ATP, other functions in fertilization, regulation of sperm lifespan through the activation of an apoptosis like mechanism, their role as source of reactive oxygen species, and their special sensibility to osmotic shocks make these organelles as key regulators of many changes induced by reproductive biotechnologies and sperm malfunction in the stallion. In spite all these evidences their role in regulation of sperm motility remains under debate, and has been proposed that the equine spermatozoa is highly dependent of oxidative phosphorylation (1).  for motility while other species mostly relay on glycolysis to provide ATP for sperm movement. In spite of this is under general consensus that functional mitochondria are needed for proper stallion sperm function. In order to disclose whether oxidative phosphorylation was the main source of ATP for stallion sperm motility oxidative phosphorylation was suppressed by carbonyl cyanide m-chlorophenylhydrazone (CCCP).Semen was obtained from 7 Pure Spanish horses (PRE) (three ejaculates each) individually housed at the Veterinary Teaching Hospital of the University of Extremadura, Cáceres, Spain.  The ejaculate was extended 1:1 in INRA-96 centrifuged (600g x 10 min) and re-suspended in BWW media supplemented with 1% PVA to 50x10⁶ spermatozoa/mL. All the experiments followed a split sample design with every ejaculate divided in controls and treatment groups . Sperm motility and kinematics were assessed using a CASA system (ISASÒ Proiser Valencia Spain). Flow cytometric analyses were conducted using a MACSQuant Analyser 10 (Miltenyi Biotech) flow cytometer equipped with three lasers emitting at 405 nm, 488 nm, and 635 nm and 10 photomultiplier tubes (PMTs) and  Simultaneous flow cytometric assessment of subtle membrane changes, viability and oxidative stress was performed as previously described in our laboratory (2). Evaluation of mitochondrial membrane potential was performed using the lipophilic cationic compound 5,5’,6,6’–tetrachloro-1,1’,3,3’ tetraethylbenzymidazolyl carbocianyne iodine (JC-1), and Intracellular ATP content in sperm lysates was measured using the ATP determination Kit (A22066) (Molecular Probes, Leiden Holland) following the instructions of the manufacturer, and previous protocols from our laboratory (3). Concentrations of CCCP greater than 500 nM significantly decreased mitochondrial membrane potential in a dose dependent manner (p<0.05).  At a concentration of 100nM CCCP no changes in mitochondrial membrane potential occurred. ATP content was reduced in presence of the uncoupler. 

Concentrations of CCCP higher than 500 nM reduced total motility after one and three hours of incubation, with the higher effect at 500 nM (p<0.01), this latter concentration also reduced progressive motility (p<0.05). However at 100nM, CCCP induced also a decrease in the percentage of progressive motile sperm after three hours of incubation (p<0.05). Uncoupling mitochondria also had a profound impact on sperm kinematics, with significant (p<0.05) reductions in all sperm velocities at CCCP concentrations higher than 500 nM both after 1 and 3 hours of incubation.  The presence of the uncoupler had no effect in the intactness or permeability of the membrane during the time of incubation, suggesting that only a decrease in ATP is not enough to compromise the sperm membrane. The uncoupler increased reactive oxygen species after 1 hour of incubation at 100 nM and decreased ROS at 10mM. After 3 hours of incubation no effect was apparent. Disruption of the mitochondrial function leads to reduced motility, that can be attributable either to reduced ATP production or increased reactive oxygen species production. Both parameters were assessed in the present study. The CCCP reduced ATP production but no effect was seen in reactive oxygen species production; thus the effect is mainly attributed to decreased ATP production.