Tetraploid (4N) induction rates of ~80% are routinely achieved in rainbow trout (Oncorhynchus mykiss ) when eggs are exposed to 9000 PSI for 8 minutes starting at 62-65% of the first cleavage interval (FCI). Factors that affect FCI are not fully understood but has been found to differ among populations, year to year within the same population, and is affected by temperature and age of the ova post-ovulation . Since the FCI appears to be consistent through-out the spawning season for a genetically similar population of fish held under similar conditions, FCI only needs to be determined for a few females at the start of the season. First generation 4Ns have low survival, high rates of deformity, and poor-quality eggs compared with diploid (2N) fish , and sperm that is often too large to pass through the egg micropyle to successfully fertilize the egg.
Second generation 4N s are much improved in all these areas except sperm size. It has been our experience that in the laboratory, all offspring of 4N x 4N crosses are 4N, and 4N x 2N crosses are triploids (3N) and sterile. We have found the intercross triploids (3NC) produced by crossing a 4N and 2N parent, to be superior to the more common pressure-shock-induced triploids (3NP) in several performance traits including growth and disease resistance. We have also found family values for growth to be more consistent with their 2N values when the families are 3NC as opposed to 3NP, supporting a greater potential to improve 3NC growth performance compared to 3NP performance when genetic selection is based on the 2N phenotype. Nevertheless, genetic gains for each trait are generally more improved following each generation of selection, then they are between 3NC and 3NP fish, and including tetraploidy into a selective breeding program likely delays availability of genetic gains to production seedstock.
Two all-female 4N broodstocks derived from our genetically improved lines are maintained at NCCCWA. The lines are enriched each generation with de novo 4N males derived from the most improved 2N broodstock . Paint Bank F ish C ulture S tation (PBFCS; Virginia D epartment of G ame and I nland F isheries) has recently produced its 3rd generation of 4N s from its own stocks. Primary impediments are sperm size and the cost and time required for ploidy confirmation. At PBFCS, we have been using flow cytometry to confirm a limited number of males as 4N and selecting those with high fertility. Fertility is determined by taking sperm from each male and fertilizing eggs from a pool of eggs, and determin ing fertility within 24 hours of fertilization. The fertility test is to in theory screen for males with narrow sperm. We are concerned it may be better to evaluate 4N male fertility based on reaching later stages of development as we sometimes find there are more bad eggs sorted as good eggs by mechanical egg pickers when 4N males are used. If all males are confirmed as 4N, then the fish in the next generation will be either 4N if the female is 4N, or 3N and sterile if the female is 2N meaning no further need to test for ploidy. Nonetheless , w e suggest inspecting the sperm for size before using a 4N male since it is easy to differentiate 4N from 2N milt under 400X magnification.