Rapid thawing and stabilizing procedure improve postthaw survival and in vitro penetrability of boar spermatozoa cryopreserved with a glycerol-free trehalose-based extender

Rukmali Athurupana, Sumire Ioki, Hiroaki Funahashi

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8 Citations (Scopus)

Abstract

Thawing process is important in semen cryopreservation as it brings back the sperm cell to physiologic temperature reactivating the metabolism. Aims of the present study were to evaluate survival rate and in vitro penetrability of boar frozen spermatozoa after rapid and transient thawing at a high temperature followed by a warming procedure at 39 °C. Ejaculated semen samples were diluted in an egg yolk-based glycerol-free extender containing 100-mM trehalose and then cryopreserved in 0.5-mL straws according to a common protocol. In experiment 1, when temperature inside the straws was monitored after thawing at 40 °C, 60 °C, 70 °C, and 80 °C, the calculated average warming rate in the straws from -196 °C to 15 °C was much faster when thawed at 70 °C and 80 °C than at 40 °C (P <0.01). The warming temperature rate inside the straw was 7 to 12 folds faster during the first 2 seconds than the second 2 seconds after immersing in high temperatures. In experiment 2, when frozen straws were thawed at 80 °C for 9 seconds, the viability, motility, and acrosomal integrity were significantly improved (P <0.05), as compared with controls (at 39 °C). In experiment 3, frozen straws were thawed at 39 °C, 60 °C, 70 °C, and 80 °C for 60, 10, 8, and 6 seconds, respectively, and then maintained at 39 °C for 0, 50, 52, and 54 seconds. Higher viability, motility, mitochondria membrane potential, and acrosome integrity were observed (P <0.05) when frozen straws were thawed at 70 °C for 8 seconds and then maintained at 39 °C for 52 seconds as compared with the control (39 °C for 60 seconds). In experiment 4, in vitro penetrability of frozen spermatozoa thawed at 70 °C for 8 seconds and maintained at 39 °C for 52 seconds was higher than that of controls. In conclusion, the rapid transient thawing at 70 °C for 8 seconds followed by stabilizing procedure at 39 °C for 52 seconds maintained the viability, motility, mitochondria membrane potential, acrosome integrity, and in vitro penetrability of spermatozoa frozen in a glycerol-free trehalose extender and recommended as an optimum thawing conditions.

Original languageEnglish
Pages (from-to)940-947
Number of pages8
JournalTheriogenology
Volume84
Issue number6
DOIs
Publication statusPublished - Oct 1 2015

Fingerprint

Trehalose
trehalose
boars
thawing
Glycerol
Spermatozoa
glycerol
straw
spermatozoa
Temperature
Acrosome
Semen
Membrane Potentials
Mitochondria
viability
acrosome
temperature
membrane potential
semen
Egg Yolk

Keywords

  • Cryopreservation
  • Pig
  • Sperm
  • Thawing
  • Trehalose

ASJC Scopus subject areas

  • Animal Science and Zoology
  • Equine
  • Food Animals
  • Small Animals

Cite this

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title = "Rapid thawing and stabilizing procedure improve postthaw survival and in vitro penetrability of boar spermatozoa cryopreserved with a glycerol-free trehalose-based extender",
abstract = "Thawing process is important in semen cryopreservation as it brings back the sperm cell to physiologic temperature reactivating the metabolism. Aims of the present study were to evaluate survival rate and in vitro penetrability of boar frozen spermatozoa after rapid and transient thawing at a high temperature followed by a warming procedure at 39 °C. Ejaculated semen samples were diluted in an egg yolk-based glycerol-free extender containing 100-mM trehalose and then cryopreserved in 0.5-mL straws according to a common protocol. In experiment 1, when temperature inside the straws was monitored after thawing at 40 °C, 60 °C, 70 °C, and 80 °C, the calculated average warming rate in the straws from -196 °C to 15 °C was much faster when thawed at 70 °C and 80 °C than at 40 °C (P <0.01). The warming temperature rate inside the straw was 7 to 12 folds faster during the first 2 seconds than the second 2 seconds after immersing in high temperatures. In experiment 2, when frozen straws were thawed at 80 °C for 9 seconds, the viability, motility, and acrosomal integrity were significantly improved (P <0.05), as compared with controls (at 39 °C). In experiment 3, frozen straws were thawed at 39 °C, 60 °C, 70 °C, and 80 °C for 60, 10, 8, and 6 seconds, respectively, and then maintained at 39 °C for 0, 50, 52, and 54 seconds. Higher viability, motility, mitochondria membrane potential, and acrosome integrity were observed (P <0.05) when frozen straws were thawed at 70 °C for 8 seconds and then maintained at 39 °C for 52 seconds as compared with the control (39 °C for 60 seconds). In experiment 4, in vitro penetrability of frozen spermatozoa thawed at 70 °C for 8 seconds and maintained at 39 °C for 52 seconds was higher than that of controls. In conclusion, the rapid transient thawing at 70 °C for 8 seconds followed by stabilizing procedure at 39 °C for 52 seconds maintained the viability, motility, mitochondria membrane potential, acrosome integrity, and in vitro penetrability of spermatozoa frozen in a glycerol-free trehalose extender and recommended as an optimum thawing conditions.",
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author = "Rukmali Athurupana and Sumire Ioki and Hiroaki Funahashi",
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T1 - Rapid thawing and stabilizing procedure improve postthaw survival and in vitro penetrability of boar spermatozoa cryopreserved with a glycerol-free trehalose-based extender

AU - Athurupana, Rukmali

AU - Ioki, Sumire

AU - Funahashi, Hiroaki

PY - 2015/10/1

Y1 - 2015/10/1

N2 - Thawing process is important in semen cryopreservation as it brings back the sperm cell to physiologic temperature reactivating the metabolism. Aims of the present study were to evaluate survival rate and in vitro penetrability of boar frozen spermatozoa after rapid and transient thawing at a high temperature followed by a warming procedure at 39 °C. Ejaculated semen samples were diluted in an egg yolk-based glycerol-free extender containing 100-mM trehalose and then cryopreserved in 0.5-mL straws according to a common protocol. In experiment 1, when temperature inside the straws was monitored after thawing at 40 °C, 60 °C, 70 °C, and 80 °C, the calculated average warming rate in the straws from -196 °C to 15 °C was much faster when thawed at 70 °C and 80 °C than at 40 °C (P <0.01). The warming temperature rate inside the straw was 7 to 12 folds faster during the first 2 seconds than the second 2 seconds after immersing in high temperatures. In experiment 2, when frozen straws were thawed at 80 °C for 9 seconds, the viability, motility, and acrosomal integrity were significantly improved (P <0.05), as compared with controls (at 39 °C). In experiment 3, frozen straws were thawed at 39 °C, 60 °C, 70 °C, and 80 °C for 60, 10, 8, and 6 seconds, respectively, and then maintained at 39 °C for 0, 50, 52, and 54 seconds. Higher viability, motility, mitochondria membrane potential, and acrosome integrity were observed (P <0.05) when frozen straws were thawed at 70 °C for 8 seconds and then maintained at 39 °C for 52 seconds as compared with the control (39 °C for 60 seconds). In experiment 4, in vitro penetrability of frozen spermatozoa thawed at 70 °C for 8 seconds and maintained at 39 °C for 52 seconds was higher than that of controls. In conclusion, the rapid transient thawing at 70 °C for 8 seconds followed by stabilizing procedure at 39 °C for 52 seconds maintained the viability, motility, mitochondria membrane potential, acrosome integrity, and in vitro penetrability of spermatozoa frozen in a glycerol-free trehalose extender and recommended as an optimum thawing conditions.

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KW - Cryopreservation

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KW - Trehalose

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