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A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs

Daniel de Souza Ramos Angrimani, Luana de Cassia Bicudo, Núria Llamas Luceno, Bart Leemans (UGent) , Marcilio Nichi, Camila Infantosi Vannucchi and Ann Van Soom (UGent)
(2022) CRYOBIOLOGY. 105. p.56-62
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Abstract
In order to accurately analyze the possible side effects of sperm cryopreservation, an in-depth screening of post-thaw sperm status is necessary. Thus, this study aimed to identify thorough effects of sperm cryopreservation, by evaluating the integrity of all specific structures of the canine spermatozoa. Thirteen (n = 13) mature dogs of different breeds were selected. Six dogs (n = 6) were subjected to sperm cryopreservation, whereas seven dogs (n = 7) were used as semen donors to validate a simultaneous assessment of sperm plasmatic, acrosomal, and mitochondrial membranes (triple stain) by fluorescent probes. Fresh and post-thaw semen samples were evaluated through a computer-assisted analysis of sperm motility, sperm morpho-functional evaluation, triple stain and sperm DNA integrity. Post-thaw semen samples had lower total and progressive motility, as well as higher percentage of minor and major defects. Moreover, post-thaw samples had higher percentage of sperm with plasma membrane and mitochondrial damage but intact acrosome, and also sperm with simultaneous damaged plasma, acrosomal and mitochondrial membranes. Furthermore, post-thaw sperm had higher protamination deficiency and DNA fragmentation. In conclusion, cryopreservation has a broad impact in sperm morphology and function, altering motility patterns, plasma, acrosome and mitochondrial membranes integrity, as well as sperm DNA.
Keywords
ARTIFICIAL-INSEMINATION, MITOCHONDRIAL-MEMBRANES, SEMEN, CRYOPRESERVATION, FLOW-CYTOMETRY, DNA INTEGRITY, DEGREES-C, SPERMATOZOA, PLASMA, FRESH, APOPTOSIS, DNA fragmentation, DNA protamination, Triple stain, CASA, Canine

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Citation

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MLA
Ramos Angrimani, Daniel de Souza, et al. “A Triple Stain Method in Conjunction with an In-Depth Screening of Cryopreservation Effects on Post-Thaw Sperm in Dogs.” CRYOBIOLOGY, vol. 105, 2022, pp. 56–62, doi:10.1016/j.cryobiol.2021.12.001.
APA
Ramos Angrimani, D. de S., Bicudo, L. de C., Llamas Luceno, N., Leemans, B., Nichi, M., Vannucchi, C. I., & Van Soom, A. (2022). A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs. CRYOBIOLOGY, 105, 56–62. https://doi.org/10.1016/j.cryobiol.2021.12.001
Chicago author-date
Ramos Angrimani, Daniel de Souza, Luana de Cassia Bicudo, Núria Llamas Luceno, Bart Leemans, Marcilio Nichi, Camila Infantosi Vannucchi, and Ann Van Soom. 2022. “A Triple Stain Method in Conjunction with an In-Depth Screening of Cryopreservation Effects on Post-Thaw Sperm in Dogs.” CRYOBIOLOGY 105: 56–62. https://doi.org/10.1016/j.cryobiol.2021.12.001.
Chicago author-date (all authors)
Ramos Angrimani, Daniel de Souza, Luana de Cassia Bicudo, Núria Llamas Luceno, Bart Leemans, Marcilio Nichi, Camila Infantosi Vannucchi, and Ann Van Soom. 2022. “A Triple Stain Method in Conjunction with an In-Depth Screening of Cryopreservation Effects on Post-Thaw Sperm in Dogs.” CRYOBIOLOGY 105: 56–62. doi:10.1016/j.cryobiol.2021.12.001.
Vancouver
1.
Ramos Angrimani D de S, Bicudo L de C, Llamas Luceno N, Leemans B, Nichi M, Vannucchi CI, et al. A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs. CRYOBIOLOGY. 2022;105:56–62.
IEEE
[1]
D. de S. Ramos Angrimani et al., “A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs,” CRYOBIOLOGY, vol. 105, pp. 56–62, 2022.
@article{8761675,
  abstract     = {{In order to accurately analyze the possible side effects of sperm cryopreservation, an in-depth screening of post-thaw sperm status is necessary. Thus, this study aimed to identify thorough effects of sperm cryopreservation, by evaluating the integrity of all specific structures of the canine spermatozoa. Thirteen (n = 13) mature dogs of different breeds were selected. Six dogs (n = 6) were subjected to sperm cryopreservation, whereas seven dogs (n = 7) were used as semen donors to validate a simultaneous assessment of sperm plasmatic, acrosomal, and mitochondrial membranes (triple stain) by fluorescent probes. Fresh and post-thaw semen samples were evaluated through a computer-assisted analysis of sperm motility, sperm morpho-functional evaluation, triple stain and sperm DNA integrity. Post-thaw semen samples had lower total and progressive motility, as well as higher percentage of minor and major defects. Moreover, post-thaw samples had higher percentage of sperm with plasma membrane and mitochondrial damage but intact acrosome, and also sperm with simultaneous damaged plasma, acrosomal and mitochondrial membranes. Furthermore, post-thaw sperm had higher protamination deficiency and DNA fragmentation. In conclusion, cryopreservation has a broad impact in sperm morphology and function, altering motility patterns, plasma, acrosome and mitochondrial membranes integrity, as well as sperm DNA.}},
  author       = {{Ramos Angrimani, Daniel de Souza and Bicudo, Luana de Cassia and Llamas Luceno, Núria and Leemans, Bart and Nichi, Marcilio and Vannucchi, Camila Infantosi and Van Soom, Ann}},
  issn         = {{0011-2240}},
  journal      = {{CRYOBIOLOGY}},
  keywords     = {{ARTIFICIAL-INSEMINATION,MITOCHONDRIAL-MEMBRANES,SEMEN,CRYOPRESERVATION,FLOW-CYTOMETRY,DNA INTEGRITY,DEGREES-C,SPERMATOZOA,PLASMA,FRESH,APOPTOSIS,DNA fragmentation,DNA protamination,Triple stain,CASA,Canine}},
  language     = {{eng}},
  pages        = {{56--62}},
  title        = {{A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs}},
  url          = {{http://doi.org/10.1016/j.cryobiol.2021.12.001}},
  volume       = {{105}},
  year         = {{2022}},
}
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