Quality assessment of cryopreserved black-lip pearl oyster Pinctada margaritifera spermatozoa

Abstract

High quality of sperm is essential to a high fertilization rate, especially post- cryopreservation. Assessment of sperm integrity, motility and energy reserves before cryopreservation is necessary for selection of milt with optimal fertilizing potential. We describe the effect of cryopreservation on the quality of black-lip pearl oyster, Pinctada margaritifera var. cumingii sperm. Evaluated quality indices of fresh and frozen/thawed P. margaritifera spermatozoa, included morphology, ultrastructure and motility characteristics relative to the energy content (ATP) and its capacity to be sustained by mitochondrial respiration. Morphology and ultrastructure were quantitatively evaluated using images obtained by optical microscopy assisted by the Image J software and TEM, respectively. Sperm motility was assessed using Image J software combined with a computer assisted sperm analysis plugin adapted for assessing P. margaritifera spermatozoa. Other sperm quality parameters evaluated included O2 consumption, ATP content, and creatine kinase activity. Frozen/thawed spermatozoa exhibited damage to the head but retained a compact spherical shape. Sperm motility indicators showed a significant decrease in quality resulting from the freeze/thaw process. The percent of motile cells was 54% compared to 84% in fresh sperm, O2 consumption was 4.8 compared to 44 nanomol min−1, ATP content was 0.72 nmol/109 spermatozoa in the activating medium compared to 4.54 nmol/109 spermatozoa, and creatine kinase activity was 9.06 × 10−5 IU mg−1 protein compared to 12.5 × 10−5 IU mg−1 protein.The cryopreservation protocol allowed obtaining an acceptable motility rate after thawing, confirming the predictive value of sperm motility measurements before cryopreservation in terms of their ability to withstand freezing process.