Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen

The sensitivity of dog sperm cells for extracellular Ca2+/Ca2+-ionophore ch allenge was compared to the detrimental effects of an optimized freeze/thaw ing protocol. Three sperm-rich fractions of ejaculates from 9 dogs were obt ained, and one aliquot of each ejaculate was washed in a modified Tyrode's medium (HBT containing 0.1 mM Ca2+), without (control sample) and with 2.5 mu M Ca2+-ionophore (induced sample) and incubated for 60 min at 38 degrees C in humidified atmosphere. Another aliquot from the same semen fractions was diluted, washed in a Tris buffer, and packed into 0.5-ml straws with a Tris buffer containing 7.5 vol % glycerol. The samples were stored for 1 we ek in liquid nitrogen after a computer-driven three-step freeze protocol an d subsequently thawed for 50 sec in a 37 degrees C water bath and reconstit uted into HBT. The acrosome integrity was determined using fluorescein-conj ugated peanut agglutinin (PNA-FITC) as an acrosomal marker, while the vital ity of the sperm cells was simultaneously assessed with the membrane imperm eable DNA supravital stain ethidium homodimer 1 (EthD-1) using fluorescence microscopy and flow cytometry. The motility of frozen/thawed sperm samples was evaluated by microscopic as well as computerized motility analyses. Re markably, the percentage sperm cells that underwent acrosome reactions indu ced by Ca2+-ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, t he degree of cellular damage induced by Ca2+-ionophore treatment correlated very negatively (r = -0.99) with the relative amount of sperm cells that r emained motile after cryopreservation. Such clear correlations between Ca2-ionophore induced acrosome reaction and motility parameters for frozen/tha wed dog sperm cells were not found, suggesting that the generation of acros ome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded t hat Ca2+-ionophore treatment followed by simultaneous determination PNA-FIT C and EthD-1 staining can be used to predict the cryopreservability of ejac ulates from individual dogs used as donors. Mel. Reprod. Dev. 55:289-298, 2 000. (C) 2000 Wiley-Liss, Inc.