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Updated: Jan 4, 2026
A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization
Published on: July 6, 2009
Long-term sperm cryopreservation does not affect post-thaw survival rates
Juliana R Pariz1,2,3,4, Rosa Alice C Monteiro1, Jorge Hallak1,2,3,4
1Androscience, Science and Innovation Center in Andrology and High-Complex Clinical and Andrology Laboratory. São Paulo, Brazil.
Objective:
To compare cryosurvival rates of human spermatozoa in a prolonged period of cryopreservation.
Methods:
This retrospective study involved 33 cryopreserved semen samples from patients with cancer, between 2002 and 2011. The semen sample was obtained by masturbation and initial semen analysis was performed. The cryoprotectant solution was added and samples were frozen in liquid nitrogen in a slow step-wise process. For thawing, the samples were incubated at 25.0°C for 15 min, followed by incubation at 36.7°C for 15 min. The cryosurvival rate (CS) was calculate by CS= [(% total motile sperm post-thaw) x100/(% total motile sperm/tube)]. Each study sample was divided into three aliquots (Study Group; n=23): (I) official patient sample, which was kept cryopreserved for subsequent Assisted Reproduction procedure, cryopreserved between 2002 and 2011; (II) sample destined to post-thaw tests, performed after the sample had been kept cryopreserved for 24 hours; and (III) study sample. Only in 2014, after 3-12 years of cryopreservation, the study samples were thawed and evaluated. To validate the study design, a Validation Group was created including 10 samples obtained between 2014 and 2016, using the same methodology in the study samples. The data was analyzed using the T-test, with a significant p-value of 5%.
Results:
The mean age was 29.93±9.57 years in the Study Group and 21.80±6.49 years in the Validation Group. No significant difference between the Validation and Study Groups was found in the initial semen analysis (p>0.05). After 24 hours of cryopreservation, the cryosurvival rate was 26.11±46.36% in the Study Group and 23.71±57.06% in the Validation Group. Aliquots of the same sample preserved from 3-12 years demonstrated 23.71±57.06% of cryosurvival rate. Thus, no significant difference was found vis-à-vis the cryosurvival rates (p=0.56).
Conclusion:
We concluded that the method introduced in the late 1990s, which enables the removal of debris, potentially toxic elements and generators of reactive oxygen species from the seminal sample before cryopreservation, exhibited efficiency in maintaining the same cryosurvival rate after an extended period.
