Effects of extender, cryoprotectants and thawing protocol on motility of frozen-thawed stallion sperm that were refrozen for intracytoplasmic sperm injection doses

Gonzalez-Castro, Raul A.; Trentin, J. M.; Carnevale, E.M.; Graham, James K.

doi:10.1016/j.theriogenology.2019.06.030

Cited by 12 publications

(14 citation statements)

References 51 publications

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“…61 In a previous study, motility was less affected when frozen spermatozoa were thawed at 5 • C and then refrozen in diluents containing glycerol and methylformamide. 32 In this study, refrozen spermatozoa had better survival after thawing at low temperature in the first cycle. Subsequently, refrozen sperm samples that exhibit higher proportions of motile spermatozoa can be further sorted by swim-up, density gradient centrifugation, or microfluidics, 62 facilitating the selection of individual spermatozoon for ICSI, which potentially has higher PLCZ1 content.…”

Section: Discussionmentioning

confidence: 54%

“…In clinical practice, individual sperm selection for ICSI in horses is performed under X200 or X400 magnification and based on gross morphology and motility of individual spermatozoon 61 . In a previous study, motility was less affected when frozen spermatozoa were thawed at 5°C and then refrozen in diluents containing glycerol and methylformamide 32 . In this study, refrozen spermatozoa had better survival after thawing at low temperature in the first cycle.…”

Section: Discussionmentioning

confidence: 60%

“…Spermatozoa were packaged into straws, sealed, cooled at 5°C for 30 min and refrozen above liquid nitrogen as described above. For the second protocol, all procedures (thawing, diluting, and packing) occurred in a 5°C cold room 32 . Eight straws of frozen spermatozoa from each stallion were thawed individually immersed in a water bath at 37°C for 12 s and quickly removed into a cold‐water bath at 5°C (LT, refrozen low temperature).…”

Section: Methodsmentioning

confidence: 99%

“…For the second protocol, all procedures (thawing, diluting, and packing) occurred in a 5 • C cold room. 32 Eight straws of frozen spermatozoa from each stallion were thawed individually immersed in a water bath at 37 • C for 12 s and quickly removed into a cold-water bath at 5 • C (LT, refrozen low temperature). Thawed spermatozoa were pooled and diluted 10-fold in E-Z Freezin CryoMax MFR5.…”

Section: Semen Collection and Cryopreservation Proceduresmentioning

confidence: 99%

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Cryopreservation of equine spermatozoa reduces plasma membrane integrity and phospholipase C zeta 1 content as associated with oocyte activation

Gonzalez‐Castro,

Whitcomb,

Pinsinski

et al. 2023

Andrology

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BackgroundPhospholipase C zeta (PLCZ1) is considered the major sperm‐borne oocyte activation factor. Cryopreserved stallion spermatozoa are commonly used for intracytoplasmic sperm injection (ICSI). However, plasma membrane damage and protein modifications caused by cryopreservation could impair sperm structure and function, leading to a reduction of PLCZ1 and oocyte activation after ICSI.ObjectivesWe compared membrane integrity and PLCZ1 abundance in populations for fresh, frozen, and refrozen stallion spermatozoa, either thawed and refrozen at room or low temperature; and examined the effect of relative PLCZ1 content on cleavage after ICSI.Materials and methodsWestern blotting, ELISA, and immunofluorescence were conducted in stallion spermatozoa, freezing extenders, and detergent‐extracted sperm fractions to detect and quantify PLCZ1. Retrospectively, PLCZ1 content and cleavage rate were analyzed. Fresh, frozen, and refrozen at room and low temperatures spermatozoa were evaluated for acrosomal and plasma membrane integrity and PLCZ1 content using flow cytometry.ResultsWestern blotting, ELISA, and immunofluorescence revealed significant reduction of PLCZ1 in spermatozoa after cryopreservation and confirmed PLCZ1 detection in extenders. After detergent extraction, a PLCZ1‐nonextractable fraction remained in the postacrosomal region of spermatozoa. Plasma membrane integrity was significantly reduced after freezing. Acrosomal and plasma membrane integrity were similar between frozen and refrozen samples at low temperature, but both were significantly higher than samples refrozen at room temperature. Acrosomal and plasma membrane integrity significantly correlated to PLCZ1 content. Percentages of PLCZ1‐labeled spermatozoa and PLCZ1 content were reduced after freezing but not after refreezing. Relative content and localization of PLCZ1 were associated with cleavage rates after ICSI.Discussion and conclusionSperm PLCZ1 content associates with cleavage rates after ICSI. Cryopreservation is detrimental to sperm plasma membrane integrity and PLCZ1 retention. However, refreezing did not result in additional PLCZ1 loss. Refreezing stallion spermatozoa at a low temperature resulted in better survival but did not improve PLCZ1 retention.

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Section: Discussionmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 60%

Section: Methodsmentioning

confidence: 99%

Section: Semen Collection and Cryopreservation Proceduresmentioning

confidence: 99%

See 2 more Smart Citations

Cryopreservation of equine spermatozoa reduces plasma membrane integrity and phospholipase C zeta 1 content as associated with oocyte activation

Gonzalez‐Castro,

Whitcomb,

Pinsinski

et al. 2023

Andrology

Self Cite

View full text Add to dashboard Cite

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“…It can be used to maintain the frozen sperm quality of bulls [115], boars [29], goats [116], and dogs [117]. In stallions, it is preferable to use DMSO or methyl formamide alone or combined with glycerol in skim milk extenders as cryoprotectants and alternatives to extenders containing glycerol only [118,119]. A study that compared three cryoprotectants, glycerol, ethylene glycol, and DMSO, revealed that the post-thaw motility and fertilization capacity of glycerol were higher than those of ethylene glycol and DMSO in dogs [120].…”

Section: Glycerol As An Anti-shockmentioning

confidence: 99%

Semen extenders: An evaluative overview of preservative mechanisms of semen and semen extenders

Bustani

Baiee

2021

Vet World

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Reproduction is fundamental for all living things as it ensures the continued existence of a species and an improved economy in animal husbandry. Reproduction has developed since history, and diverse processes, such as artificial insemination and in vitro fertilization, have been developed. Semen extenders were discovered and developed to protect sperm from harmful factors, such as freeze and osmotic shock, oxidative stress, and cell injury by ice crystals. Semen extenders preserve sperm by stabilizing its properties, including sperm morphology, motility, and viability and membrane, acrosomal, and DNA integrity. Therefore, semen extenders must provide a favorable pH, adenosine triphosphate, anti-cooling and anti-freeze shock, and antioxidant activity to improve semen quality for fertilization. Hence, this review provides precise data on different semen extenders, preservative mechanisms, and essential additives for semen extenders in different animals.

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Thawing of cryopreserved sperm from domestic animals: Impact of temperature, time, and addition of molecules to thawing/insemination medium

Pezo,

Contreras,

Zambrano

et al. 2024

Animal Reproduction Science

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Effects of extender, cryoprotectants and thawing protocol on motility of frozen-thawed stallion sperm that were refrozen for intracytoplasmic sperm injection doses

Cited by 12 publications

References 51 publications

Cryopreservation of equine spermatozoa reduces plasma membrane integrity and phospholipase C zeta 1 content as associated with oocyte activation

Cryopreservation of equine spermatozoa reduces plasma membrane integrity and phospholipase C zeta 1 content as associated with oocyte activation

Semen extenders: An evaluative overview of preservative mechanisms of semen and semen extenders

Thawing of cryopreserved sperm from domestic animals: Impact of temperature, time, and addition of molecules to thawing/insemination medium

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