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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

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 degrees C to 35 degrees C (40 degrees C/min), from 35 degrees C to 65 degrees C (17 degrees C/min), and then from 65 degrees C to 85 degrees C (3 degrees C/min); or Protocol 2 (three-step accelerating cooling): from +5 degrees C to 5 degrees C (4 degrees C/min), from 5 degrees C to 110 degrees C (25 degrees C/min), and then from 110 degrees C to 140 degrees C (35 degrees C/min); or Protocol 3 (two-step accelerating cooling), from +5 degrees C to 10 degrees C (5 degrees C/min), and then from 10 degrees C to 130 degrees C (60 degrees 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 degrees 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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Submitted on : Tuesday, May 26, 2020 - 7:06:57 AM
Last modification on : Thursday, August 5, 2021 - 3:51:58 AM

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Diego A. Galarza, Antonio López-Sebastián, Henri Woelders, Elisabeth Blesbois, Julián Santiago-Moreno. Two-step accelerating freezing protocol yields a better motility, membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols. Cryobiology, Elsevier, 2019, 91, pp.84 - 89. ⟨10.1016/j.cryobiol.2019.10.007⟩. ⟨hal-02622979⟩

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