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

Galarza, Daniel; Woelders, H.; Blesbois, E.; Santiago-Moreno, J.

doi:10.1016/j.cryobiol.2019.10.007

Cited by 23 publications

(24 citation statements)

References 39 publications

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“…15 °C/ min) transition through the critical temperature range of -10 °C to -25 °C, which is the most dangerous for ram sperm (Salamon and Maxwell 2000). Our results are in accordance with Galarza et al (2019), who demonstrated that the lowest ram sperm damage was associated with the fastest (60 °C/min) transition through the critical temperature range of -10 °C to -25 °C. Based on the above mentioned, sperm freezing with the use of Curve 2 should be recommended to cryopreserve Wallachian ram sperm.…”

Section: Optimizing the Conventional Freezing Methods For Wallachian Ram Sperm Cryopreservationsupporting

confidence: 91%

Optimizing the conventional method of sperm freezing in liquid nitrogen vapour for Wallachian sheep conservation program

Savvulidi¹,

Ptáček²,

Málková³

et al. 2021

Czech J. Anim. Sci.

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The aim of the present study was to optimize the conventional method of sperm freezing in liquid nitrogen (LN2) vapour for successful cryopreservation of Wallachian ram sperm, the genetic resources of the Czech Republic. Sperm in straws were frozen using the conventional freezing method via a static exposure of sperm doses to LN2 vapour, or by four different modified freezing methods. Under modified freezing, straws were frozen by a discontinuous, time-dependent decremental change in the distance between the straws and the surface of LN2. The viability of sperm was evaluated by flow cytometry after sperm equilibration, and immediately after thawing. Besides the observed inter-sire and daily variation, the obtained results suggest the methodological weakness of the conventional freezing method via the static exposure of sperm doses to LN2 vapour. With the use of the optimized freezing procedure, all parameters of thawed sperm were significantly (P < 0.05) improved in comparison with the conventional method: percentage of thawed sperm viability increased up to 48.3%, percentage of sperm with plasma membrane damage after thawing decreased to 6.58%, percentage of sperm with acrosome damage decreased to 24.4%, and percentage of sperm with deteriorated mitochondrial activity decreased to 6.28%. In conclusion, our results suggest that an optimized freezing procedure should be routinely used instead of the conventional method to cryopreserve Wallachian ram sperm.

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Section: Optimizing the Conventional Freezing Methods For Wallachian Ram Sperm Cryopreservationsupporting

confidence: 91%

Optimizing the conventional method of sperm freezing in liquid nitrogen vapour for Wallachian sheep conservation program

Savvulidi¹,

Ptáček²,

Málková³

et al. 2021

Czech J. Anim. Sci.

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“…Using ram sperm, the same two-step accelerating protocol used in the present work was compared with a three-step decelerating and a three-step accelerating rate protocol, and returned better post-thaw survival and functionality results than either of the latter two (Galarza et al, 2019). The authors of that study indicate the accelerating two-step cooling rate to cause less damage to mitochondrial membranes, allowing for better motility than the other protocols (Galarza et al, 2019). However, in the present work, no…”

Section: Discussionmentioning

confidence: 99%

“…In decelerating methods, the initial high cooling rate achieved with nitrogen vapour—i.e. 40°C/min from 5 to −35°C—involves exposure to the harmful effects of unfrozen, extracellular, hypertonic solutions over the critical −5 to −15°C range when ice growth occurs (Galarza et al., 2019). In addition, such exposure could lead to ‘cold shock’ which, along with any ice crystals formed, could cause injury to sperm cell membranes (Hammadeh et al., 2001; Mazur, 1984).…”

Section: Discussionmentioning

confidence: 99%

“…The frozen-thawed sperm viability and acrosome integrity values obtained with the two-step accelerating protocol were equal to or greater than those published previously for ferrets (van der Horst et al, 2009;Leigh, 2010). With this method, the cooling rate of 60°C/min around the point of ice nucleation might have caused less cryodamage as exposure to the negative effects of unfrozen extracellular hypertonic solutions would have been reduced (Galarza et al, 2019). Moreover, the sperm may have become less shrunken and dehydrated.…”

Section: Discussionmentioning

confidence: 99%

“…The aim of the present work was to improve ferret semen cryopreservation by examining the effect of two freezing protocols, one with a decelerating and one with an accelerating freezing rate, on the quality of frozen‐thawed sperm. It is hypothesized that the use of acceleration cooling rates, using slow initial speeds to avoid cold shock and severe cellular dehydration, followed by faster cooling rates can produce less damage to sperm cells and better post‐thaw quality, as has been demonstrated in the freezing of sperm from other species (Esteso et al., 2018; Galarza et al., 2019). Transrectal massage was combined with electrical stimuli in a preliminary attempt to reduce the need for the latter.…”

Section: Introductionmentioning

confidence: 99%

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Cryopreservation of ferret (Mustela putorius furo) sperm collected by rectal massage and electroejaculation: Comparison of a decelerating and an accelerating freezing rate protocol

Toledano-Dı́az

Castaño

Velázquez

et al. 2020

Veterinary Medicine & Sci

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The domestic ferret (Mustela putorius furo) provides a good model for developing new reproductive technologies for use with threatened related species. Such technologies could also be used in the reproductive management of this pet species. The present work reports an improved freezing protocol for ferret sperm. Semen was collected by electroejaculation plus rectal massage (in an attempt to reduce the electrical stimulation necessary) from five adult male ferrets, and then subjected to one of two freezing protocols: (a) from 5 to −35°C at 40°C/min, then from −35 to −65°C at 17°C/min, and finally from −65 to −85°C at 3°C/min—a decelerating freezing rate; and (b) from 5 to − 10°C at 5°C/min, and then from −10 to −130°C at 60°C/min—an accelerating freezing rate. After thawing, the viability and acrosomal integrity of the sperm frozen via the two‐step accelerating method were better than those frozen via the three‐step decelerating method (43.3 ± 3.5% and 71.2 ± 3.4% compared with 29.7 ± 3.7% and 58.8 ± 3.4% respectively; p < .05). No differences were seen between the methods with respect to sperm motility variables; most sperm (>90%) remained static with both freezing methods. In conclusion, although the method with accelerating freezing rate was associated with better post‐thaw sperm viability and acrosome integrity values, neither of the two freezing methods tested provided adequate motility results after thawing. Combining rectal massage with electrical stimuli seemed to reduce the number of the latter required for successful sperm collection.

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Effect of resveratrol supplementation in conventional slow and ultra-rapid freezing media on the quality and fertility of bull sperm

Galarza,

Jaramillo,

Amón

et al. 2024

Animal Reproduction Science

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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

Cited by 23 publications

References 39 publications

Optimizing the conventional method of sperm freezing in liquid nitrogen vapour for Wallachian sheep conservation program

Optimizing the conventional method of sperm freezing in liquid nitrogen vapour for Wallachian sheep conservation program

Cryopreservation of ferret (Mustela putorius furo) sperm collected by rectal massage and electroejaculation: Comparison of a decelerating and an accelerating freezing rate protocol

Effect of resveratrol supplementation in conventional slow and ultra-rapid freezing media on the quality and fertility of bull sperm

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