World Aquaculture Magazine - June 2020

WWW.WA S.ORG • WORLD AQUACULTURE • JUNE 2020 53 (FCI). Factors that affect FCI are not fully understood but differ among populations, year to year within the same population, and are affected by temperature and age of the ova post-ovulation (Weber and Hostuttler 2012). As the FCI appears to be consistent throughout 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 tetraploids have low survival, high rates of deformity, and poor-quality eggs compared with diploid fish, and sperm that is often too large to pass through the egg micropyle to successfully fertilize the egg. Second generation tetraploids are much improved in all these areas except sperm size (Weber and Hostuttler 2012). In the laboratory, all offspring of tetraploid by tetraploid crosses are tetraploid, and tetraploid by diploid crosses are triploids and sterile. The intercross triploids (3NC) produced by crossing tetraploid and diploid parents is superior to the more common pressure shock induced triploids (3NP) in several performance traits, including growth and disease resistance (Weber et al. 2013, 2014). Family values for growth are more consistent with their diploid 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 diploid phenotype. Nevertheless, genetic gains for each trait are generally more improved following each generation of selection than they are between 3NC and 3NP fish. Furthermore, including tetraploidy into a selective breeding program likely delays availability of genetic gains to production seedstock. Although approaches to making 3NC seedstock have been developed, and advantages of cultivars have been identified, the practice has not been widely adopted. Here we present recent examples of efforts to establish tetraploid lines for 3NC production. Two all-female tetraploid broodstocks derived from our genetically improved lines are maintained at our research center. Broodstock were initiated by inducing tetraploidy in a pool of embryos from among the neomales and females with the highest breeding values for the traits of interest. The plan is to maintain about 80 tetraploid females per generation so that we always have tetraploid females that are at least second generation and therefore have high-quality eggs, and then cross these females with about 50 de novo tetraploid males derived from a pool of the most improved diploid broodstock families to continually improve the quality of the germplasm. This approach will allow maintenance of a small group of tetraploid broodstock with improved genetic traits, although behind that of the diploid select lines from which they are derived, that can then be amplified if mass production of tetraploids is required. This requires tetraploid induction of a single pool of embryos each year, that are then masculinized and confirmed to be tetraploid using flow cytometry. In all, this requires a minimum of effort and resources to maintain tetraploid broodstocks. Paint Bank Fish Culture Station (PBFCS; Virginia Department of Game and Inland Fisheries) has also established tetraploid broodstock that is mixed sex and for which active genetic improvement is not practiced. Furthermore, triploidy is a preference but not a requirement for waters stocked with triploids by the hatchery. These attributes and requirements contribute to make 3NC production a viable alternative for PBFCS. The PBFCS recently produced its third generation of tetraploids from its own stocks. The first generation of tetraploids, and therefore the entire tetraploid broodstock program, was derived from a single tetraploid-induction event of a pool of embryos. Primary impediments for production of tetraploids have been sperm size (Fig. 5) effects on fertility and the cost and time required for ploidy confirmation. At PBFCS, we have been using flow cytometry to confirm a limited number of males per generation as tetraploid and selecting those with high fertility to make the next generation. Some male rainbow trout have narrower sperm than others, and therefore have higher fertilization rates and that this is a heritable trait (Blanc et al. 1993). Fertility is determined by taking sperm from each male and fertilizing eggs from a pool of eggs and determining fertility within 24 h of fertilization. The fertility test is to screen for males with narrow sperm. It may be better to evaluate tetraploid male fertility based on embryos reaching later stages of development because occasionally there are more non- viable eggs sorted as viable eggs by mechanical egg pickers when tetraploid males are used. If all males are confirmed as tetraploid, then the fish in the next generation will be either tetraploid if the female is tetraploid, or triploid and sterile if the female is diploid, indicating no further need to test for ploidy in the next generation. Nonetheless, we suggest evaluating sperm size before using a tetraploid male because it is easy to differentiate tetraploid from diploid milt under 400× magnification. Production data for 3NCs are not yet available from this broodstock. Physiological Constraints on Temperature and Hypoxia Tolerances in Triploid Salmonids Tillmann Benfey, Krista Latimer and Rebecca Porter Although numerous approaches to producing sterile fish are under investigation or in pilot-scale evaluation, the only method currently used for commercial production is triploidy induction. However, despite the simplicity and reliability of the protocols used for producing triploids, their benefits as sterile animals have rarely ( C O N T I N U E D O N P A G E 5 4 ) FIGURE 5. Hemocytometer comparing the head size of tetraploid and diploid sperm.