Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential.
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%.
Veterinary Teaching Hospital, Laboratory of Equine Reproduction, Faculty of Veterinary Medicine, University of Extremadura, Cáceres, Spain.
B
B
Macías García
C
C
Ortega Ferrusola
I M
IM
Aparicio
A
A
Miró-Morán
A
A
Morillo Rodriguez
J M
JM
Gallardo Bolaños
L
L
González Fernández
C M
CM
Balao da Silva
H
H
Rodríguez Martínez
J A
JA
Tapia
F J
FJ
Peña
D000199:Actins / Q000378:metabolism; D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects*; D015925:Cryopreservation / Q000662:veterinary; D003451:Cryoprotective Agents / Q000633:toxicity*; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D005990:Glycerol / Q000633:toxicity*; D006736:Horses; D015227:Lipid Peroxidation / Q000187:drug effects*; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D009997:Osmotic Pressure; D013094:Spermatozoa / Q000187:drug effects*
D000199:Actins; D003451:Cryoprotective Agents; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D005990:Glycerol