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ISSN 2052-1472 (online)

Reproduction Abstracts (2014) 1 P229 | DOI: 10.1530/repabs.1.P229

Trehalose in glycerol-free freezing extender preserves mitochondria membrane potential of post-thaw boar spermatozoa

Rukmali Athurupana & H Funahashi


Okayama University, Okayama, Japan.


Introduction: Movement of spermatozoa and the penetration of zona-pellucida depend on the energy produced in mitochondria. Major steps of cryopreservation can exert stress on sperm mitochondria membrane. Better post-thaw motility, viability and acrosome integrity of spermatozoa was observed when extended in trehalose in a previous study. Objective was to evaluate the effect of trehalose on mitochondria integrity of post-thawed boar spermatozoa.

Materials and method: Spermatozoa were diluted in egg yolk extender containing 0.25% Equex STM and glycerol (100 mM) or trehalose (0, 50, 100, 150, 200, and 250 mM) and cryopreserved with straw freezing procedure. Frozen sperms were thawed at 39 °C for 30 s and analyzed for motility with CASA and mitochondrial membrane potential (MMP) with JC-1/PI staining. Data were analyzed with ANOVA and Pearson’s product moment correlation (mean±S.E.M).

Results and discussion: Motility in glycerol (100 mM) and trehalose (0, 50, 100, 150, 200, and 250 mM) was 21.2±2.3b, 9.5±3.8c, 24.5±2.1ab, 32.9±2.9a, 25.4±0.6ab, 23.5±1.1ab, and 20.8±1.3ab while the spermatozoa with high MMP was 38.4±2.1b, 18.6±4.1c, 41.0±6.9ab, 54.7±3.6a, 46.9±4.4ab, 53.2±1.4ab, and 45.9±1.8ab respectively. Motility and MMP was significantly high in 100 mM trehalose compared to 100 mM glycerol (P<0.0001, n=6). Significant negative correlation was observed between motility and trehalose concentration after 100 mM (r=−0.95, P=0.049) although the MMP was not significantly correlated (r=−0.59, P=0.41). Even though the high concentrations of trehalose slightly reduced the motility, probably due to the viscosity of the medium, it retained the MMP. Differences in motility were observed with previous study, possibly due to the seasonal and intra-ejaculate variations. In conclusion, along with previous data, glycerol-free trehalose extender preserved post-thaw survival including MMP in boar spermatozoa.

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