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

F. Szász, S. Sirivaidyapong, F. P. Cheng, W. F. Voorhout, A. Marks, B. Colenbrander, L. Solti, B. M. Gadella

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

The sensitivity of dog sperm cells for extracellular Ca2+/Ca2+- 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 Ca2+), without (control sample) and with 2.5 μM Ca2+-ionophore (induced sample) and incubated for 60 min at 38°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 I week in liquid nitrogen after a computer-driven three-step freeze protocol and subsequently thawed for 50 sec in a 37°C 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 Ca2+- ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca2+-ionophore treatment correlated very negatively (r = -0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca2+- 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 Ca2+-ionophore treatment followed by simultaneous determination PNA- FITC and EthD-1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors. (C) 2000 Wiley-Liss, Inc.

Original languageEnglish
Pages (from-to)289-298
Number of pages10
JournalMolecular Reproduction and Development
Volume55
Issue number3
DOIs
Publication statusPublished - 2000

Fingerprint

Calcium Ionophores
Semen
Spermatozoa
Ionophores
Dogs
Membranes
Acrosome
Acrosome Reaction
Tromethamine
Cryopreservation
Peanut Agglutinin
Fluorescein
Atmosphere
Baths
Fluorescence Microscopy
Glycerol
Flow Cytometry
Coloring Agents
Nitrogen
Staining and Labeling

Keywords

  • Cryopreservation
  • Dog sperm
  • Ionophore test
  • Membrane damage
  • Motility

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology
  • Cell Biology

Cite this

Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen. / Szász, F.; Sirivaidyapong, S.; Cheng, F. P.; Voorhout, W. F.; Marks, A.; Colenbrander, B.; Solti, L.; Gadella, B. M.

In: Molecular Reproduction and Development, Vol. 55, No. 3, 2000, p. 289-298.

Research output: Contribution to journalArticle

Szász, F. ; Sirivaidyapong, S. ; Cheng, F. P. ; Voorhout, W. F. ; Marks, A. ; Colenbrander, B. ; Solti, L. ; Gadella, B. M. / Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen. In: Molecular Reproduction and Development. 2000 ; Vol. 55, No. 3. pp. 289-298.
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