Monosex Tilapia Production through Androgenesis

Control of reproduction is vital to aquaculture and includes artificial propagation as well as management of unwanted recruitment. Developments in manipulation of the reproductive system provide options to enhance production. Nile tilapia, Oreochromis niloticus, spawning was managed by photoperiod and temperature manipulation. A controlled light cycle of 20L:4D and water temperature of 26 ± 2°C directed spawning to a predictable time frame. A developmental rate (t0) relationship was described and applied to chromosome manipulation. Blond Nile tilapia are homozygous recessive for a color mutation that was used as a phenotypic marker in the development of protocol for androgenetic induction, while the color pigmentation for red Nile tilapia is dominant over the wild type color pattern. Androgenotes were produced by neutralizing the female genome of normal color Nile tilapia or that of red tilapia (600 J m-2 UV dose), eggs were activated with sperm from blond males or Ghana males, respectively, and then the eggs were diploidized with cold shock (11 ± 0.5°C for 60 min) applied at various times after incubation at 28 ± 0.2°C. Shock applied at 69 min post-activation produced greater numbers of androgenotes than shocks applied at 59 or 79 min post-activation; the shock application time of 69 min was used for induction with red tilapia stocks. Production of viable diploid androgenotes for crosses involving either red or blond and Ghana stocks was very low, and no progeny survived to maturity. Thus, neither verification of sex determination in androgenotes nor testing of monosex breeding was accomplished. NINETEENTH ANNUAL TECHNICAL REPORT 46 tions in sex ratio inheritance. Androgenotes from the present study were to be progeny-tested (males and females) along with other strains as a component of examining the genetic basis of sex determination. Assuming that sex determination is effected by control from a single pair of chromosomes, a monosex breeding program using YY-male broodstock might provide such a solution. Through androgenesis, YY-males can be induced directly, independent of steroids, and after verification, used without the need for subsequent progeny-test identification. Sex determination is characterized by a homogametic/heterogametic genotype; however, autosomal modifier genes may alter the theoretical 1:1 progeny sex ratio (Shelton et al., 1983; Wohlfarth and Wedekind, 1991; Mair, 1993). Therefore, deviations in the expected all-male progeny from YY-male breeding should be anticipated. Thus, this study proposed to investigate a protocol to produce androgenotes (progeny with only a paternal genome) and also to examine the basic mechanism of sex determination. Androgenesis should result in offspring of equal sex ratio; females would be XX and males would be YY. Progeny testing will be required during experimental development to confirm that the males are fertile and that only male offspring (XY) will result when spawned with normal females (XX). Sex ratio of progeny from crosses of androgenote females with normal males should be 1:1, and presumptive YY-male androgenote crossed with normal females would be expected to produce only male progeny; these results would verify the assumptions of monochromosomal sex determination. The YY-males would then be a basis for developing a unique broodstock that would produce all-male progeny and add insight into the stability and fidelity of the sex-determining system in tilapia. Tilapias do not respond well to hormonal induction for spawning control, but interruption of aquarium spawning so as to collect gametes might be sufficient to proceed with chromosome manipulation studies. Chromosome manipulation involves one or two basic treatments after obtaining fresh gametes (Thorgaard and Allen, 1986). For androgenesis the first treatment is the deactivation of the female genome. Ultraviolet (UV) irradiation is preferred for simplicity and safety but also because it dimerizes the DNA rather than fragmenting it. Egg activation with untreated spermatozoa then requires diploidization of the haploid zygote by some form of shock to interrupt the first mitotic division. Shock is most often physical, e.g., thermal (cold or hot) or pressure. Thermal treatment is usually preferred because of the ease of application and equipment simplicity. In order to prevent chromosome segregation, the shock must be timed to coincide with a cytological event, such as disruption of the spindle fibers during metaphase to prevent karyokinesis, or interference with the cell duplication during cytokinesis. Thus, shock type and intensity, duration, and time of application must be optimally combined into a protocol for maximum yield of diploid progeny. Further, because the rate of development is inversely temperature dependent, either the preshock incubation temperature must be standardized or the shock time must be calibrated to account for the temperature effect. Absolute shock time (minutes post-activation) can be transformed with reference to an index of development rate or mitotic interval (τ0), also in minutes (Dettlaff and Dettlaff, 1961). Shock time (τs) can be related to tau (τs/τ0) to report shock protocol in a dimensionless index, which is temperature compensated (Dettlaff, 1986). A tau curve for the Nile tilapia and gametic treatment with UV were described during an earlier segment of this study (Shelton, 1999), and one phase of the diploidization protocol was reported (Shelton, 2000). Both gametic treatments (UV and shock) are near lethal, increasing direct mortality, and further, the genomic diploidization will increase homozygosity, thereby reducing fitness. Thus, survival of viable androgenotes to maturity is expected to be quite low. This report describes the initial development of a protocol for the production of androgenetic progeny of Nile tilapia, Oreochromis niloticus. METHODS AND MATERIALS