Optimisation of handling, activation and assessment procedures for Bufo marinus spermatozoa

C. Fitzsimmons, E. A. McLaughlin, M. J. Mahony, J. Clulow

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

In the present study, we investigated handling, activation and assessment procedures for cane toad (Bufo marinus) spermatozoa. Optimisation of these techniques will facilitate the maintenance of sperm viability during cryopreservation and during in vitro fertilisation (IVF) techniques in reproduction technologies for endangered species. Spermatozoa were taken from testicular macerates and assessed using plasma membrane integrity assays (live/dead stains) and quantitative scores of motility parameters. In the assessment of sperm viability using live/dead stains, there were small but significant differences in the percentage of sperm from cryopreserved samples staining positive with propidium iodide, Hoechst H33258 and Trypan blue; these differences were not large and all stains performed acceptably. Spermatozoa were activated by dilution of testicular macerates in water at one of two dilution ratios (1:6 or 1:20) with or without 0.1-5.0 mm theophylline. Sperm plasma membrane integrity (unstained spermatozoa) was unaffected by either dilution ratio (osmolarity) or theophylline concentration. However, sperm motility was significantly affected by osmolarity and theophylline concentration. The stimulation of sperm motility increased with higher theophylline concentrations and these strongly interacted with lower osmolarities through a higher dilution ratio of sperm macerates with water. Spermatozoa were exposed to increasing centrifugation forces to determine tolerance to physical stresses encountered during washing procedures. Forces between 50 and 800g were associated with a significant reduction in motility (mean 56 ± 3% decreasing to 27 ± 3%), but did not affect staining. In conclusion, centrifugation should be minimised in anuran sperm washing procedures; osmotic shock associated with higher dilution ratios reduces the capacity of anuran sperm to achieve high percentages of motile sperm, leading to a likely trade-off between dilution required for activation and sperm motility to optimise IVF fertilisation rates; and optimal conditions for sperm motility after activation occur at lower dilutions of suspensions with 5.0 mm theophylline. The present study has improved protocols for the handling of anuran sperm during pre- and post-cryopreservation procedures.

Original languageEnglish
Pages (from-to)594-601
Number of pages8
JournalReproduction, Fertility and Development
Volume19
Issue number4
DOIs
Publication statusPublished - 10 May 2007
Externally publishedYes

Fingerprint

Bufo marinus
Spermatozoa
spermatozoa
theophylline
Theophylline
Sperm Motility
sperm motility
osmolarity
Cryopreservation
cryopreservation
Osmolar Concentration
dyes
in vitro fertilization
Fertilization in Vitro
Centrifugation
centrifugation
washing
Coloring Agents
plasma membrane
Cell Membrane

Cite this

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title = "Optimisation of handling, activation and assessment procedures for Bufo marinus spermatozoa",
abstract = "In the present study, we investigated handling, activation and assessment procedures for cane toad (Bufo marinus) spermatozoa. Optimisation of these techniques will facilitate the maintenance of sperm viability during cryopreservation and during in vitro fertilisation (IVF) techniques in reproduction technologies for endangered species. Spermatozoa were taken from testicular macerates and assessed using plasma membrane integrity assays (live/dead stains) and quantitative scores of motility parameters. In the assessment of sperm viability using live/dead stains, there were small but significant differences in the percentage of sperm from cryopreserved samples staining positive with propidium iodide, Hoechst H33258 and Trypan blue; these differences were not large and all stains performed acceptably. Spermatozoa were activated by dilution of testicular macerates in water at one of two dilution ratios (1:6 or 1:20) with or without 0.1-5.0 mm theophylline. Sperm plasma membrane integrity (unstained spermatozoa) was unaffected by either dilution ratio (osmolarity) or theophylline concentration. However, sperm motility was significantly affected by osmolarity and theophylline concentration. The stimulation of sperm motility increased with higher theophylline concentrations and these strongly interacted with lower osmolarities through a higher dilution ratio of sperm macerates with water. Spermatozoa were exposed to increasing centrifugation forces to determine tolerance to physical stresses encountered during washing procedures. Forces between 50 and 800g were associated with a significant reduction in motility (mean 56 ± 3{\%} decreasing to 27 ± 3{\%}), but did not affect staining. In conclusion, centrifugation should be minimised in anuran sperm washing procedures; osmotic shock associated with higher dilution ratios reduces the capacity of anuran sperm to achieve high percentages of motile sperm, leading to a likely trade-off between dilution required for activation and sperm motility to optimise IVF fertilisation rates; and optimal conditions for sperm motility after activation occur at lower dilutions of suspensions with 5.0 mm theophylline. The present study has improved protocols for the handling of anuran sperm during pre- and post-cryopreservation procedures.",
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Optimisation of handling, activation and assessment procedures for Bufo marinus spermatozoa. / Fitzsimmons, C.; McLaughlin, E. A.; Mahony, M. J.; Clulow, J.

In: Reproduction, Fertility and Development, Vol. 19, No. 4, 10.05.2007, p. 594-601.

Research output: Contribution to journalArticle

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KW - Theophylline

KW - Vital stains

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