Optimization of tetraploid induction in Pacific oysters, Crassostrea gigas, using first polar body as a natural indicator

Tetraploid Crassostrea gigas were first successfully produced in 1993 by in hibiting the first polar body of eggs from triploids that had been fertiliz ed with sperm from diploids (Guo and Alien method). However, attempts to re peatedly produce high yields of tetraploids were inconsistent. Because of t hese uncertainties, we examined some of the fundamental aspects of tetraplo id production in an attempt to optimize tetraploid induction using the Guo and Alien method. Varying the duration of the treatment to inhibit polar bo dy 1 (PB 1) of triploid eggs had clear effects on ploidy of progeny. Short treatments (15-35 min after fertilization - about half the period of meiosi s 1 in triploid eggs) yielded tetraploid and heptaploid cells. Long treatme nts (7-43 min - about three quarters of the period of meiosis I in triploid eggs) yielded only heptaploid cells among the embryos. Tetraploid inductio n was most consistent when treatments were accomplished on eggs from indivi dual triploid females rather than pooled from a number of females, and when treatments were metered according to biological landmarks. That is, eggs f rom individual triploids were fertilized and 0.5 mg/l cytochalasin B (CB) a dded after 10 min. A subsample of the fertilized eggs was kept aside untrea ted. When 50% of the untreated eggs showed PB 1 extrusion (as judged by mic roscopic examination of dividing, untreated eggs), the CB treatment was dis continued. In eight treatments based on these "biological criteria," propor tions of tetraploids ranged from 13% to 92% after 8 days for an average of 55%, and seven of eight replicates went through metamorphosis and settlemen t. At settlement, the percentage of tetraploids ranged from 7% to 96%, aver aging 45%. Average survival in all the replicates at 8 days was 4.4%, which is acceptable considering tetraploid progeny are destined for use;Is brood stock. (C) 2000 Elsevier Science B.V. All rights reserved.