Karyotype analysis is a major work in the process of triploid abalone production for the purpose of productivity and quality improvement. However, the metaphase spreads for karyotype analysis have been prepared just from the larvae at trochophore stage, which has restricted the spectrum of sample correction inhibiting more efficient analysis. Here, we investigated the feasibility of preparing metaphase spreads from the larvae at veliger stage that is the next developmental stage of trochophore. For this, diploid and triploid larvae at trochophore and veliger stages from Pacific abalone (Haliotis discus hannai ) were subjected to metaphase spread preparation and its efficiencies were measured and compared each other. As the results, although the efficiencies of metaphase spread preparation were significantly lower in the larvae at veliger stage compared to the ones at trochophore stage regardless of ploidy status, we found that the preparation of metaphase spreads, which showed the clear chromosomal images containing the normal number of chromosomes, was possible from the veliger stage larvae. On the other hands, all larvae used in this study regardless of developmental stage and ploidy did not show colchicine sensitivity. Moreover, no significant difference was observed in cell cycle distribution of the cells comprising larvae between two developmental stages regardless of ploidy status. These suggested that the details of protocol to prepare metaphase spreads from abalone larvae should be optimized depending on its developmental stages. Taken together, we demonstrated the feasibility of preparing metaphase spreads from H. discus hannai veliger stage larvae for karyotype analysis.

This study was conducted to investigate cryopreservation of pearl oyster, Pinctada fucata martensii larvae. Four cooling rates (-0.25, -0.5, -0.75 and -1.0/min.) were used to examine a proper cooling rate during cryopreservation of trochophores before seeding temperature (-12). Seven developmental stages (early and late trochophores, early and late D-shaped larvae and early, middle and late umbo stage larvae) and different sugars (fructose, glucose and sucrose) were used to investigate optimal larval stage and effective sugar in cryopreservation of larvae. The survival rates of frozen-thawed trochophores increased at cooling rate of -1.0/min. As larval developing, survival rate of frozen-thawed larvae increased, except umbo stage larvae, and especially late D-shaped larvae highly survived as 91%. Addition of sugar revealed positive effect on cryopreservation in this experiment and 0.2 M glucose and sucrose mixed with 2.0 M dimethyl sulfoxide significantly enhanced survival rate of larvae (P<0.05). The results of our study indicate that desirable cooling rate, developmental stages of larvae and effective sugar far cryopreservation of pearl oyster, P. fucata martensii larvae are -1/min, late D-shaped larvae and 0.2 M glucose and sucrose, respectively.