Collaborating Institution
University of Oklahoma

William Shelton

Objective
To increase the overall sustainability of aquacultural systems through production optimization by developing short- and long-term solutions to reproduction technology problems. Specifically, use androgenetic procedures to develop direct induction of YY-male Nile tilapia, Oreochromis niloticus, for the production of all-male offspring.

Significance
One of the major constraints to aquaculture development is the reliability and sustainability of seed organisms for culture. Within the context of tilapia farming, this limitation involves effective and practical control of unmanaged recruitment. Various approaches to monosexing fishes have been applied to culture, but the use of chromosome manipulation is one of the more recently implemented practices (Shelton, 1989; Dunham, 1990). Gynogenesis, which is the production of progeny with only a maternal genome has been investigated widely, but the development of techniques to induce androgenesis, i.e., progeny with only a paternal genome, is much less studied (Thorgaard and Allen, 1986). Androgenesis can provide a mechanism for the development of unique broodstock to use in producing all-male progeny without the use of sex-reversing steroids. Within the contemporary atmosphere of increasing governmental regulation on use of chemicals on food fish, continued dependency on steroid-induced monosexing places the culture of tilapias in a precarious position. The recently developed protocol to develop YY-male tilapia relies on estrogen treatment, and even though fish to be cultured are one generation removed from the treatment the protocol still depends on progeny testing to identify target broodstock (Scott et al., 1989; Mair et al., 1997). In contrast, androgenesis offers the potential of direct induction for YY-male tilapia, without chemical treatment, and without the need for progeny testing to identify the unique male broodstock. Although the system is based on incompletely characterized genetics of sex determination (Wohlfarth and Wedekind, 1991; Mair, 1993), the perceived complications might be minimized by using select strains. 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), therefore, deviations in the expected all-male progeny from YY-male breeding should be anticipated. Thus, this study will involve not only an investigation of a protocol to produce androgenotes (progeny with only paternal genome), but also examination of the basic mechanism of sex determination. Androgenesis should result in offspring of equal sex ratio; females will be XX, but males will 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 and YY-male androgenotes (a full sibling cross) will be compared with outcrosses of YY-males with females from other stocks. This will provide a basis to collaborate with the PD/A CRSP investigators at Auburn University on their study of strain variations in sex ratio inheritance (Eighth Work Plan). Ouputs from our study will be the number of androgenotes (males and females); each will be progeny tested as a component of examining the genetic basis of sex determination. The YY-males will be the foundation for developing a unique broodstock that will produce all-male progeny, and will provide insight into the expected variation in the sex determining system.

Anticipated Benefits
Tilapia culture is one of the fastest growing forms of finfish aquaculture in the world. Production efficiency and product marketability in most economies depend on the capacity to control unwanted reproduction. Techniques for managing recruitment have evolved from traditional means to the current practice of steroid-induced sex reversal, which has been the industry standard for monosexing during the last couple of decades. However, chemical treatment of food fish has become increasingly constrained, and since this monosexing tool could be withdrawn, an alternative should be available. The monosex breeding program using YY-male broodstock might provide such a program. Through androgenesis, YY-males can be produced directly without the use of steroids, and without the need for progeny-test identification.

Research Design
Site: University of Oklahoma in collaboration with Auburn University. Techniques for androgenesis in tilapia will continue to be researched at the University of Oklahoma. The experiment will be an extension of Study II and the initiation of Study III. The latter was discussed as a component of the total program chronology, but not one that could be completed within the two years of the Eighth Work Plan of the PD/A CRSP Continuation Plan 1996-2001.

Plan: Techniques developed during year one of the Eighth Work Plan (Shelton, in review) will be incorporated in the protocol for the experiment to be done under the Ninth Work Plan. The developments include managed aquarium spawning (Rothbard and Pruginin, 1975; Yeheskel and Avtalion, 1986; Myers and Hershberger, 1991), incubation of artificially propagated zygotes (Rana, 1986) and application of Developmental Rate (mitotic interval - t₀) (Dettlaff and Dettlaff, 1961) to diploidization (shock) protocol (Shelton and Rothbard, 1993; Shelton et al., 1997). Induction of androgenesis for the Nile tilapia must focus on two components within the chromosome manipulation regimen: (1) Gamete (egg) treatment with Ultra-Violet (UV) to deactivate the maternal DNA, and (2) Diploidization of activated haploid paternal zygotes through endomitotic shock treatment.

Optimization of UV treatment. Freshly ovulated eggs will be collected from Nile tilapia. Separate groups of eggs will be exposed to UV irradiation using a UV-crosslinker. Dosage will range from 0 (control) to 1,000 Joules/m² (J/m²) in increments of 100. Maternal DNA in tilapia eggs is reported to be dimerized between 200 and 600 J/m2 (Myers et al., 1995). Ova will be activated with conspecific spermatozoa and incubated to hatching. Hatch rate will decrease with increasing UV dose and zero hatch will indicate complete female genome deactivation. Haploids may develop to hatch, but they will be recognizable by various malformations. Trials with several females will be required to determine the range of UV dose which will dependably destroy the genome. Progress is expected on this component during the second year of study II of the Eighth Work Plan; the developed UV treatment will be incorporated into androgenetic protocol.

Diploidization through endomitotic shock. Optimization of shock protocol will require testing the combination of several parameters. Thermal shocks will be tested because of the ease of application and its effectiveness in diploidizing by either polar body (early) or endomitotic (late) shock. The use of pressure shock may be comparatively examined, but only after thorough evaluation of thermal trials. Both cold shock and heat shock have been effective (Don, 1989; Hussain et al., 1993). We will compare the two types of thermal shock based on chromosome manipulation information from the literature; magnitude and duration have been optimized, but the times of application for heat and cold shock in these earlier studies are not in agreement (Don, 1989; Mair, 1993; Myers et al., 1995). This is the rationale for referencing shock administration in tau units and for the comparison of shock types. Heat shock will be applied at 42.5°C and cold shock will be applied at 11°C; the duration of heat will be 3.5 min while the cold application will be for 60 min. Paired tests will compare induction in UV-treated eggs from individual females. Time of initiation should coincide with metaphase of the first mitosis, which is temperature dependent (Saat, 1993). Pre-shock incubation temperature will be controlled, but diploidization rate will be related to tau-units as well as in absolute time. Shock time (ts- minutes) expressed in terms of Developmental Rate (t₀) or t_s /t₀ will provide a dimensionless treatment index which will permit adjustment of absolute shock time with reference to different incubation temperatures.

Broodstock for diploidization studies will have a genetic marker as a treatment control. The eggs from a color mutant (red) of the Egyptian Nile tilapia strain will be used in the androgenetic studies (McAndrew et al., 1988). This color is controlled by a homozygous dominant allele. Normal color males will be used. Progeny from a control cross will be of the red phenotype, while diploid androgenotes will be normal color like the male parent. Any progeny with the red phenotype will be discarded. Offspring should include females and males in a 1:1 sex ratio, but the male genotype is expected to be YY.

Progeny from androgenote males are expected to be only male (XY), but this must be verified to demonstrate the viability of the YY-genotype, and to test variability in sex determination. One component of the progeny test will be full-sibling crosses, i.e. androgenote males with androgenote females (Table 1); outcrosses with females from other families or strains will be compared to the sib-matings. This phase will address the collaboration with the Auburn studies and provide a basis for comparing genetic variation in sex determination. Stocks tested at Auburn which have been identified as having progeny that most closely conform to the expected sex ratio of 1:1 will be logical broodstock for the outcross tests.

Table 1. Expected progeny sex ratios from crosses of androgenotes (full-siblings) and outcross males and females

.	XY(normal male)	YY(androgenate)
XX(normal female)	1:1(control)*	0:1(outcross)
XX(androgenote female)	1:1(outcross)	0:1(sib-cross)

* female: male sex ratio

Regional Integration
The output of androgenotes and progeny tests will integrate studies at Auburn on strain testing. The testing at Auburn also integrates with the East Africa Work Plan.

Schedule
Experiments were started under the Eighth Work Plan. Optimization of androgenesis will continue through the end of the program (2001). Study II of the Eighth Work Plan began June 1997; results will be used in the present proposed study; a progress report for androgenesis will be provided in July 1999 and for progeny testing in July 2000. A final report will be provided by 30 April 2001.

References
Dettlaff, T.A. and A.A. Dettlaff, 1961. On relative dimensionless characteristics of the development duration in embryology. Arch. Biol., 72:1-16.
Don, J., 1989. Study of ploidy and artificial induction of gynogenesis in tilapias. Ph.D. dissertation, Bar-Ilan University, Tel-Aviv, Israel (in Hebrew).
Dunham, R.A., 1990. Production and use of monosex or sterile fish in aquaculture. Rev. Aquat. Sci., 2:1-17.
Hussain, M.G., D.J. Penman, B.J. McAndrew, and R. Johnstone, 1993. Suppression of first cleavage in the Nile tilapia, Oreochromis niloticus L.—A comparison of the relative effectiveness of pressure and heat shocks. Aquaculture, 111:263-270.
Mair, G.C., 1993. Chromosome-set manipulation in tilapia—Techniques, problems and prospects. Aquaculture, 111:227-244.
Mair, G.C., J.S. Abucay, D.O.F. Skibinski, T.A. Abella, and J. A. Beardmore, 1997. Genetic manipulation of sex ratio for the large-scale production of all-male tilapia, Oreochromis niloticus. Can. J. Fish. Aquat. Sci., 54:396- 404.
McAndrew, B.J., F.R. Roubal, R.J. Roberts, A.M. Bullock, and M. McEwen, 1988. The genetics and histology of red, blond and associated colour variants in Oreochromis niloticus. Genetica, 76:127-137.
Myers, J.M. and W.K. Hershberger, 1991. Artificial spawning of tilapia eggs. J. World Aquacult. Soc., 22:77-82.
Myers, J.M., and seven coauthors, 1995. Induction of diploid androgenetic and mitotic gynogenetic Nile tilapia (Oreochromis niloticus L.). Theor. Appl. Genet., 90:205-210.
Rana, K.J., 1986. An evaluation of two types of containers for the artificial incubation of Oreochromis eggs. Aquacult. Fish. Mgt., 17:139-145.
Rothbard, S. and Y. Pruginin, 1975. Induced spawning and artificial incubation of tilapia. Aquaculture, 5:315-321.
Saat, T., 1993. The morphology and chronology of oocyte final maturation and fertilization in fish. In: B.T. Walther and H.J. Fyhn (Editors), Physiological and Biochemical Aspects of Fish Development. University of Bergen, Norway, pp 71-85.
Scott, A.G., D.J. Penman, J.A. Beardmore, and D.O.F. Skibinski, 1989. The ‘YY’ supermale in Oreochromis niloticus (L) and its potential in aquaculture. Aquaculture 78:237-251.
Shelton, W.L., In Review. Artificial propagation of Nile tilapia for chromosome manipulation. PD/A CRSP Annual Report, 1996-97, Work Plan 8, Study II - Develop androgenesis techniques applicable to tilapia.
Shelton, W.L., 1989. Management of finfish reproduction for aquaculture. Rev. Aquat. Sci., 1:497-535.
Shelton, W.L. and S. Rothbard, 1993. Determination of the developmental duration (t₀) for ploidy manipulation in carps. Israeli J. Aquacult. - Bamidgeh, 45:73-81.
Shelton, W.L., F.H. Meriwether, K.J. Semmons, and W.E. Calhoun, 1983. Progeny sex ratios from intraspecific pair spawning of Tilapia aurea and Tilapia nilotica. In: L. Fishelson and Z. Yaron (Editors), International Symposium on Tilapia in Aquaculture. Tel Aviv University, Israel,
pp. 270-280.
Shelton, W.L., S.D. Mims, J.A. Clark, A.E. Hiott, and C. Wang, 1997. A temperature-dependent index of mitotic interval (t₀) for chromosome manipulation in paddlefish and shovelnose sturgeon. Prog. Fish-Cult., 59:152- 168.
Thorgaard, G.H. and S.K. Allen, Jr., 1986. Chromosome manipulation and markers in fishery management. In: N. Ryman and F. Utter (Editors), Population Genetics and Fishery Management Washington Sea Grant Program, University of Washington Press, Seattle, pp. 319-331.
Wohlfarth, G.W. and H. Wedekind, 1991. The heredity of sex determination in tilapias. Aquaculture, 92:143-156.
Yeheskel, O. and R.R. Avtalion, 1986. Artificial fertilization of tilapia eggs, a preliminary study. In: Reproduction in Fish—Basic and Applied Aspects in Endocrinology and Genetics. Les Colloques de l’INRA no. 44, Paris, pp 169-175.