Quality assessment of cryopreserved Portuguese oyster (Crassostrea angulata) sperm through ultrastructural and flow cytometry analysis

Abstract

We investigated the effects of cryopreservation on the quality of Portuguese oyster (Crassostrea angulata) sperm, which were examined before and after freezing; sperm motility, fertilizing capacity, and ultrastructural morphology were analyzed. The motility percentage and fertilizing capacity of the cryopreserved sperm (mean +/- standard error) were 16% +/- 1% and 17% +/- 8%, respectively. In the pre-freezing sperm, these were 58% +/- 2% and 76% +/- 4%, respectively. The sperm sustained substantial morphological and ultrastructural damage during cryopreservation. The morphological changes varied considerably in nature and extent, ranging from no apparent damage to virtual disintegration. Sperm were stained with fluorescent dyes to assess viability, plasma membrane integrity, mitochondrial activity, acrosomal membrane integrity, oxidation level, and DNA fragmentation and examined through flow cytometry. The methods used for the flow cytometry assays were slightly modified from those used for evaluating the semen quality of livestock. Relative to the pre-freezing sperm, the frozen-thawed sperm exhibited lower acrosomal membrane integrity (acrosomal damage, 59.86 +/- 5.29; P < 0.05) and substantially higher oxidation levels (free radicals, 60.06 +/- 0.82; P < 0.003). Oxidation level was found to be the most sensitive indicator of cryodamage. Along with ultrastructural analysis, we used flow cytometry to measure the qualitative and quantitative characteristics of Portuguese oyster sperm before and after cryopreservation rapidly, objectively, and accurately. This is the first study to assess the quality of Portuguese oyster sperm through these methods.