45 Ultra-rapid freezing yields a higher cryoresistance than conventional-slow freezing of epididymal guinea pig (Cavia porcellus) spermatozoa

D. A. Galarza; C. A. Hernández; A. J. Salinas; J. M. Duma

doi:10.1071/RDv37n1Ab45

RESEARCH ARTICLE

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45 Ultra-rapid freezing yields a higher cryoresistance than conventional-slow freezing of epididymal guinea pig (Cavia porcellus) spermatozoa

D. A. Galarza ^A ^B , C. A. Hernández ^A , A. J. Salinas ^A and J. M. Duma ^A

+ Author Affiliations

- Author Affiliations

^A Laboratorio de Biotecnología de La Reproducción Animal, Facultad de Ciencias Agropecuarias, Universidad de Cuenca, Cuenca, Azuay, Ecuador

^B Centro Latinoamericano de Formación de Especies Mayores y Menores, Cuenca, Azuay, Ecuador

Reproduction, Fertility and Development 37, RDv37n1Ab45 https://doi.org/10.1071/RDv37n1Ab45

The spermatozoa of the guinea pig (Cavia porcellus) are characterized by large head dimensions, which can influence cryosurvival depending on the freezing method used. This study evaluated the cryogenic response of guinea pig epididymal spermatozoa cryopreserved by conventional slow (CS) or ultra-rapid (UR) freezing methods. For this purpose, 40 epididymides (left and right) were retrieved from 20 orchiectomized healthy adult guinea pigs. Epididymal sperm samples were recovered by retrograde flushing using TCG-EY medium (Tris, citric acid, glucose, and 10% egg yolk). Sperm samples from the right epididymis were cryopreserved using the CS freezing method with TCG-EY medium supplemented with 3% glycerol and 3% dimethylformamide, with samples in 0.25-mL straws exposed to static liquid nitrogen (LN₂) vapors. In contrast, sperm samples from the left epididymis were cryopreserved using the UR freezing methods with TCG-EY medium supplemented with 100 mM sucrose and 1% BSA, with 20-µL drops directly submerged in LN₂. The kinematic and morphometric parameters of prefreezing and post-thawing sperm were examined using CASA system (SCA®, Microptic). Post-thaw integrity of sperm membranes (plasma/acrosome) and chromatin structure were assessed using fluorescence tests: PI/PNA-FITC and acridine orange staining, respectively. Factorial ANOVA and Bonferroni test (P < 0.05) were employed to assess the effects between the freezing method (CS and UR) and sperm type (prefreezing and post-thawing) on sperm quality variables. The results showed a significant reduction (P < 0.01) in sperm motility after thawing with both freezing methods: motility, CS = 72.9 ± 1.81% versus 6.3 ± 0.23%, and UR = 76.0 ± 1.73% versus 16.9 ± 0.40%. Notably, post-thaw values of total and progressive motilities, curvilinear and rectilinear velocities with rapid progression, and the amplitude of lateral head displacement were greater after UR freezing compared with CS freezing (P < 0.05). No significant differences were observed between prefreezing and post-thawing samples of sperm head dimensions in both CS and UR freezing methods (P > 0.05). Following UR freezing, the percentage of simultaneous integrity of plasma and acrosome membranes, and chromatin structure were higher than those observed with CS freezing: 19.2 ± 0.28% versus 8.2 ± 0.16%, and 97.1 ± 0.20% vs. 95.2 ± 0.27%, respectively. In conclusion, UR freezing resulted in greater sperm cryoresistance based on improved kinematics and integrity of membranes and chromatin compared with CS freezing. These findings represent a significant advancement for sperm cryopreservation in this species.