Conservation of fish genetic diversity is important to those who rely on fish as their income, to those using fish as a food source, but also to all who understand that to maintain biodiversity is snyonymous to maintaing "a healthy planet." In order to ensure that genetic diversity is maintained for the future, we propose:
to develop relatively simple techniques for sperm cryopreservation under conditions typical of commercial production;
to develop genetically engineered strains of fish to enhance production;
to establish a Fish Genetics Resource Bank of cryopreserved sperm as a storage site for preservation of genetic diversity for many species;
to develop androgenetic progeny productioon in srugeon and sperm cryopreservation.

This research will support an increase in the genetic diversity and productivity of commercial fish production. At the same time, it will provide the facilities and techniques necessary to maintain or assist in recovery of wild fish populations, some of which may be endangered.

Current techniques of sperm cryopreservation will be tested and improved using several species of fish. Functional parameters of sperm motility will be monitored using computer-assisted sperm motion analysis, and the integrity of sperm morphology will be monitored using electron microscopy. The quality and genetic diversity of brood stock will be improved using techniques of sex determination.

On November 12, 1997, gynogenetic rainbow trout were produced by fertilizing eggs with UV-irradiated rainbow trout sperm (UV dose = 400 microwatts/cm² for 2.5 min) and exposing the activated eggs to heat shock at 29.5°C for 10 minutes starting 10 min after fertilization. Golden variety of rainbow trout (London strain, London State Fish Hatchery, London, Ohio) was used in the gynogenesis and sex reversal experiments. The treatments included i) diploid control (normal eggs +normal sperm, no heat shock), ii) haploid control (normal eggs +UV-irradiated sperm, no heat shock), and iii) gynogens (normal eggs +UV-irradiated sperm, with heat shock). Individual females differed in their response to treatments, which probably resulted from differential egg quality. In female 1, 14.5% of eggs survived to the eyed-stage in the haploid control group, whereas 47.6% of eggs survived to the eyed-stage in the gynogen group. In female 2, 90.5% of eggs survived to the eyed stage in the haplo id control group, whereas 7.4% of eggs survived to the eyed-stage in the gynogen group. All surviving embryos from the haploid groups of females 1 and 2 and the gynogen group of female 2 failed to hatch and showed typical characteristics of haploid syndrome (deformed, curved body, underdeveloped eyes). These results suggest that our parameters of UV-irradiation were effective in completely inactivating paternal DNA, despite some variation in haploid yields observed between females. Only 30.2% of the surviving embryos from the gynogen group of female 1 hatched and the remaining 17.4% manifested similar haploid syndromes. The occurrence of haploids in the gynogen group of female 1 suggests that not all eggs were diploidized during the heat shock treatment. The failure of surviving embryos from female 2 to hatch suggests that the heat shock treatment was not effective in diploidizing the maternal gene complement of eggs from female 2. Normal eggs fertilized with normal sperm showed 71% hatching rate in female 1 and 46% hatching rate in female 2. All hatched alevins from both females developed normally.

One week after 50% of the gynogenetic eggs hatched (December 14, 1997), alevins were immersed in 400 microgram/L of 17a-methyltestosterone (MT) for 2 hours at 11.9°C to induce sex reversal. Control fish were immersed in 0.04% ethyl alcohol, a common solvent used for steroid hormones.

Sex identification of 9-month old fish (September 30, 1998) using the "gonad-squash" technique showed that the untreated gynogens (immersed in 0.04% alcohol only) were 100% females (n=15) and the MT-treated gynogens (n=20) were 30% males. On February 16, 2000, ten, 28-month old rainbow trout (1020.9 ± 162.5 g body weight) from the MT-treated gynogen group were examined morphologically to determine sex. Six fish were females with an average gonadosomatic index (GSI) of 12.5 ± 4.2%, three fish had no eggs or sperm, and 1 fish was spermiating. Surgery was carried out on the three fish and examination revealed immature gonads. Sperm from the MT-treated gynogen male and three control males were used to fertilize separate egg batches from six female rainbow trout (Utah strain, Utah Department of Natural Resources). Sperm from the MT-treated gynogen male was cryopreserved following techniques described in Ciereszko and Dabrowski (1996). Progenies were reared for nine months and on November 16, 2000, approximately ten fish from each cross were sacrificed to determine sex by histology. The cross between MT-treated gynogen male and Utah female 2 produced 100% females while crosses with Utah females 1, 3, 4, 5, and 6 yielded only 30-80% females. The crosses between control males and Utah females yielded an average of 56% females.

We initially expected that all crosses would have produced 100% female progenies since males were hypothesized to have XX sex chromosomes because of their gynogenetic origin (all-maternal contribution). However, our results suggest four possibilities: i) paternal genes must have been introduced into the gynogens because UV-irradiated sperm came from the same species (homologous sperm donor), ii) environmental conditions must have an influence on sex determination, iii) maternal variability must have influence sex ratios of progenies, and iv) the golden variety of rainbow trout lacks genetic purity. Further studies are needed to evaluate these hypotheses.