Two-step accelerating freezing protocol yields a better motility, membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols

Diego A. Galarza*, Antonio López-Sebastián, Henri Woelders, Elizabeth Blesbois, Julián Santiago-Moreno

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

1 Citation (Scopus)

Abstract

The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to −10 °C (5 °C/min), and then from −10 °C to −130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.

Original languageEnglish
Pages (from-to)84-89
JournalCryobiology
Volume91
Early online date16 Oct 2019
DOIs
Publication statusPublished - Dec 2019

Fingerprint

rams
Freezing
Spermatozoa
freezing
cooling
Semen
spermatozoa
Cooling
Membranes
DNA
Ice
ice nucleation
semen
Acrosome
Sperm Motility
Mitochondrial Membranes
Nucleation
Nitrogen
Cell Membrane
acrosome

Keywords

  • Cooling
  • DNA integrity
  • Ram
  • Semen cryopreservation
  • Semen quality

Cite this

Galarza, Diego A. ; López-Sebastián, Antonio ; Woelders, Henri ; Blesbois, Elizabeth ; Santiago-Moreno, Julián. / Two-step accelerating freezing protocol yields a better motility, membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols. In: Cryobiology. 2019 ; Vol. 91. pp. 84-89.
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title = "Two-step accelerating freezing protocol yields a better motility, membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols",
abstract = "The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to −10 °C (5 °C/min), and then from −10 °C to −130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.",
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Two-step accelerating freezing protocol yields a better motility, membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols. / Galarza, Diego A.; López-Sebastián, Antonio; Woelders, Henri; Blesbois, Elizabeth; Santiago-Moreno, Julián.

In: Cryobiology, Vol. 91, 12.2019, p. 84-89.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Two-step accelerating freezing protocol yields a better motility, membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols

AU - Galarza, Diego A.

AU - López-Sebastián, Antonio

AU - Woelders, Henri

AU - Blesbois, Elizabeth

AU - Santiago-Moreno, Julián

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N2 - The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to −10 °C (5 °C/min), and then from −10 °C to −130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.

AB - The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to −10 °C (5 °C/min), and then from −10 °C to −130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.

KW - Cooling

KW - DNA integrity

KW - Ram

KW - Semen cryopreservation

KW - Semen quality

U2 - 10.1016/j.cryobiol.2019.10.007

DO - 10.1016/j.cryobiol.2019.10.007

M3 - Article

VL - 91

SP - 84

EP - 89

JO - Cryobiology

JF - Cryobiology

SN - 0011-2240

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