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Development of a new rapid measurement technique for fish embryo membrane permeability studies using impedance spectroscopy.

Zhang T, Wang RY, Bao QY, Rawson DM - Theriogenology (2006)

Bottom Line: The optimum frequency was identified at 10(3.14) or 1,380 Hz which provided good sensitivity and reproducibility.Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants.The results agreed well with those obtained from conventional volumetric based studies.

View Article: PubMed Central - PubMed

Affiliation: Luton Institute of Research in the Applied Natural Sciences, University of Luton, The Spires, 2 Adelaide Street, Luton, Bedfordshire LU1 5DU, UK. tiantian.zhang@luton.ac.uk

ABSTRACT
Information on fish embryo membrane permeability is vital in their cryopreservation. Whilst conventional volumetric measurement based assessment methods have been widely used in fish embryo membrane permeability studies, they are lengthy and reduce the capacity for multi-embryo measurement during an experimental run. A new rapid 'real-time' measurement technique is required to determine membrane permeability during cryoprotectant treatment. In this study, zebrafish (Danio rerio) embryo membrane permeability to cryoprotectants was investigated using impedance spectroscopy. An embryo holding cell, capable of holding up to 10 zebrafish embryos was built incorporating the original system electrods for measuring the impedance spectra. The holding cell was tested with deionised water and a series of KCl solutions with known conductance values to confirm the performance of the modified system. Untreated intact embryos were then tested to optimise the loading capacity and sensitivity of the system. To study the impedance changes of zebrafish embryos during cryoprotectant exposure, three, six or nine embryos at 50% epiboly stage were loaded into the holding cell in egg water, which was then removed and replaced by 0.5, 1.0, 2.0 or 3M methanol or dimethyl sulfoxide (DMSO). The impedance changes of the loaded embryos in different cryoprotectant solutions were monitored over 30 min at 22 degrees C, immediately following embryo exposure to cryoprotectants, at the frequency range of 10-10(6)Hz. The impedance changes of the embryos in egg water were used as controls. Results from this study showed that the optimum embryo loading level was six embryos per cell for each experimental run. The optimum frequency was identified at 10(3.14) or 1,380 Hz which provided good sensitivity and reproducibility. Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants. The results agreed well with those obtained from conventional volumetric based studies.

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Effect of embryo loading levels on impedance change. Embryos (three, six or nine) were exposed to METH at the frequency of 103.14 Hz over 30 min. (a) 0.5 M, (b) 1.0 M and (c) 2.0 M. The impedance values were normalised with respect to those obtained in egg water.
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fig4: Effect of embryo loading levels on impedance change. Embryos (three, six or nine) were exposed to METH at the frequency of 103.14 Hz over 30 min. (a) 0.5 M, (b) 1.0 M and (c) 2.0 M. The impedance values were normalised with respect to those obtained in egg water.

Mentions: The change of impedance values not only depended on frequency used but also embryo loading level. Fig. 4 shows normalised impedance values of different embryo loading levels (three, six and nine) in METH (0.5–3.0 M) at the optimum frequency of 103.14 Hz over 30 min. All three embryo loading levels in METH resulted in impedance value change. However, six-embryo loading level was the only one that resulted in steady impedance changes (decreased impedance values over exposure time) at all concentrations. Therefore, six-embryo loading level was confirmed as the optimum loading level and was used in subsequent experiments.


Development of a new rapid measurement technique for fish embryo membrane permeability studies using impedance spectroscopy.

Zhang T, Wang RY, Bao QY, Rawson DM - Theriogenology (2006)

Effect of embryo loading levels on impedance change. Embryos (three, six or nine) were exposed to METH at the frequency of 103.14 Hz over 30 min. (a) 0.5 M, (b) 1.0 M and (c) 2.0 M. The impedance values were normalised with respect to those obtained in egg water.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851733&req=5

fig4: Effect of embryo loading levels on impedance change. Embryos (three, six or nine) were exposed to METH at the frequency of 103.14 Hz over 30 min. (a) 0.5 M, (b) 1.0 M and (c) 2.0 M. The impedance values were normalised with respect to those obtained in egg water.
Mentions: The change of impedance values not only depended on frequency used but also embryo loading level. Fig. 4 shows normalised impedance values of different embryo loading levels (three, six and nine) in METH (0.5–3.0 M) at the optimum frequency of 103.14 Hz over 30 min. All three embryo loading levels in METH resulted in impedance value change. However, six-embryo loading level was the only one that resulted in steady impedance changes (decreased impedance values over exposure time) at all concentrations. Therefore, six-embryo loading level was confirmed as the optimum loading level and was used in subsequent experiments.

Bottom Line: The optimum frequency was identified at 10(3.14) or 1,380 Hz which provided good sensitivity and reproducibility.Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants.The results agreed well with those obtained from conventional volumetric based studies.

View Article: PubMed Central - PubMed

Affiliation: Luton Institute of Research in the Applied Natural Sciences, University of Luton, The Spires, 2 Adelaide Street, Luton, Bedfordshire LU1 5DU, UK. tiantian.zhang@luton.ac.uk

ABSTRACT
Information on fish embryo membrane permeability is vital in their cryopreservation. Whilst conventional volumetric measurement based assessment methods have been widely used in fish embryo membrane permeability studies, they are lengthy and reduce the capacity for multi-embryo measurement during an experimental run. A new rapid 'real-time' measurement technique is required to determine membrane permeability during cryoprotectant treatment. In this study, zebrafish (Danio rerio) embryo membrane permeability to cryoprotectants was investigated using impedance spectroscopy. An embryo holding cell, capable of holding up to 10 zebrafish embryos was built incorporating the original system electrods for measuring the impedance spectra. The holding cell was tested with deionised water and a series of KCl solutions with known conductance values to confirm the performance of the modified system. Untreated intact embryos were then tested to optimise the loading capacity and sensitivity of the system. To study the impedance changes of zebrafish embryos during cryoprotectant exposure, three, six or nine embryos at 50% epiboly stage were loaded into the holding cell in egg water, which was then removed and replaced by 0.5, 1.0, 2.0 or 3M methanol or dimethyl sulfoxide (DMSO). The impedance changes of the loaded embryos in different cryoprotectant solutions were monitored over 30 min at 22 degrees C, immediately following embryo exposure to cryoprotectants, at the frequency range of 10-10(6)Hz. The impedance changes of the embryos in egg water were used as controls. Results from this study showed that the optimum embryo loading level was six embryos per cell for each experimental run. The optimum frequency was identified at 10(3.14) or 1,380 Hz which provided good sensitivity and reproducibility. Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants. The results agreed well with those obtained from conventional volumetric based studies.

Show MeSH
Related in: MedlinePlus