Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen
✍ Scribed by F. Szász; S. Sirivaidyapong; F.P. Cheng; W.F. Voorhout; A. Marks; B. Colenbrander; L. Solti; and B.M. Gadella
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 279 KB
- Volume
- 55
- Category
- Article
- ISSN
- 1040-452X
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✦ Synopsis
The sensitivity of dog sperm cells for extracellular Ca 2ϩ /Ca 2ϩ -ionophore challenge was compared to the detrimental effects of an optimized freeze/thawing protocol. Three sperm-rich fractions of ejaculates from 9 dogs were obtained, and one aliquot of each ejaculate was washed in a modified Tyrode's medium (HBT containing 0.1 mM Ca 2ϩ ), without (control sample) and with 2.5 M Ca 2ϩ -ionophore (induced sample) and incubated for 60 min at 38ЊC 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 week in liquid nitrogen after a computer-driven three-step freeze protocol and subsequently thawed for 50 sec in a 37ЊC water bath and reconstituted into HBT. The acrosome integrity was determined using fluorescein-conjugated peanut agglutinin (PNA-FITC) as an acrosomal marker, while the vitality of the sperm cells was simultaneously assessed with the membrane impermeable 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. Remarkably, the percentage sperm cells that underwent acrosome reactions induced by Ca 2ϩ -ionophore correlated very positively (r ϭ 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca 2ϩ -ionophore treatment correlated very negatively (r ϭ Ϫ0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca 2ϩ -ionophore induced acrosome reaction and motility parameters for frozen/thawed dog sperm cells were not found, suggesting that the generation of acrosome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded that Ca 2ϩ -ionophore treatment followed by simulta-neous determination PNA-FITC and EthD-1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors.