Note: Experimental Design for 8RCR1A has been revised. See Third Addendum to the Eighth Work Plan
Note: Schedule for 8RCR1A has been revised. See First Addendum to the Eighth Work Plan
Note: Schedule for 8RCR1B has been revised. See First Addendum to the Eighth Work Plan
Note: Experimental Design for 8RCR1C has been revised. See Third Addendum to the Eighth Work Plan
Note: Schedule for 8RCR1C has been revised. See First Addendum to the Eighth Work Plan
Note: Schedule for 8RCR1D has been revised. See First Addendum to the Eighth Work Plan
Note: 8RCR1D has been cancelled. See Third Addendum to the Eighth Work Plan

Collaborating Institutions
Objectives
Significance
Anticipated Benefits
Identification of Beneficiaries
Collaborative Arrangements
Investigations

Methods for Strain Variations in Sex Ratio Inheritance
Methods for Androgenesis Techniques Applicable to Tilapia
Methods for Contribution from the Male and Female Genome to Sex Inheritance
Methods for Development of YY Lines of Male and Female O. niloticus

Collaborating Institutions

Auburn University

Ronald Phelps

University of Oklahoma

William Shelton

Objectives

1) Provide a greater understanding of sex determination in tilapia.

2) Determine if there are strain differences in sex determination in Oreochromis niloticus.

3) Develop androgenesis techniques applicable to tilapia.

4) Develop YY lines of male and female Oreochromis niloticus.

5) Evaluate in the field the practicality of producing all-male populations of tilapia using YY broodstock (Years 4 & 5).

6) Compare the growth and survival of male tilapia produced by hormone sex reversal to those from a YY breeding program (Years 4 & 5).

Significance

Limited knowledge of reproductive physiology and breeding of culture species has been identified as one of the key constraints to aquaculture in the PD/A CRSP Continuation Plan 1996-2001. Specifically, effective and practical control of reproduction is the major constraint in tilapia culture. Uncontrolled reproduction can result in less than 25% of the adults being greater than 250 g after a 6 month culture period with the majority of the population being progeny smaller than 10 g each. Inter- and intraspecific breeding programs can result in populations with highly skewed sex ratios but often give inconsistent results. Interspecific crosses have not proven to be practical due to difficulties in maintaining the parent species integrity.

Intraspecific breeding programs have been developed to exploit the sex inheritance mechanism in the tilapia Oreochromis niloticus. Females are said to be homogametic (XX) and males heterogametic (XY) but the sex ratio in the progeny from a single pair often do not conform to the expected 50:50. This lack of conformity to a simple XX:XY sex inheritance complicates the intraspecific breeding approach of developing YY males that would give all-male progeny. The sex ratio of individual spawns have been studied in only a limited number of strains of O. niloticus and the variance in sex ratio seen may be related to strain differences. The identification of tilapia populations with a minimum variation in progeny sex ratios from individual spawns would be a significant contribution to the development of a YY male breeding program.

Sex inheritance in tilapia may also be autosomally influenced, and to the extent that cytoplasmic inheritance in the egg may be involved, these sources cannot be altered through androgenesis. However, considering only the role of sex chromosomes in sex determination, androgenesis transfers the male genome into the egg and in such cases should produce either female progeny (XX) or male progeny (YY). The androgenetic approach to developing YY males thus simplifies the identification of YY males as all males produced should be of the YY genotype. In the hormonal approach to obtaining YY males, YY males are produced in the second generation and can only be identified by producing a third generation of progeny. The production of YY males through androgenesis not only would be more direct, it would be able to produce XX females that should be free of autosomal influence. Such a line of fish should breed true to a XX:XY sex inheritance scheme thus making a YY male program practical.

Consequently, these studies will provide additional information on the genetic basis of sex determination in Oreochromis niloticus. Results of these studies have the potential to lead to the development of a chemical-free alternative to steroid-induced monosexed YY-male (a protocol currently tested in the Philippines) with a global application throughout the tilapia culture areas. Components of this effort are linked with studies to be conducted in Kenya on progeny sex ratio testing of tilapia strains.

Anticipated Benefits

Tilapia culture is one of the fastest growing forms of finfish aquaculture in the world. It has broadened its base from a subsistence oriented technology to be a component of world commerce as a high quality filet product exported to Europe and the USA. CRSP host countries of Honduras and Thailand have rapidly growing tilapia industries as well as do other countries in those regions. These industries must have monosex tilapia available to remain competitive. The proposed research would result in all male tilapia production techniques that would avoid hormone treated fish being offered to the consumer.

The proposed work would clearly establish whether there is an autosomal influence on sex inheritance in O. niloticus and by determining strain differences in sex inherence would identify strains most appropriate for use in breeding programs. This study would demonstrate the practicality of a breeding program to control sex.

Identification of Beneficiaries

Consumers- will benefit from having tilapia products available that have not been hormone treated.

Producers- will have an alternative technique for producing male tilapia less subject to government regulation.

Researchers- will have a clearer understanding of sex inheritance in tilapia which may also give insights as to the inheritance of other traits.

Collaborative Arrangements

In the US, a collaborative effort would be made between Auburn University and the University of Oklahoma to conduct the proposed research. Letters of collaboration are enclosed.

Each of the proposed CRSP country programs are currently involved in some aspect of monosex seed production research and are equipped to conduct the proposed research. The proposal would permit each site to continue this line of research while contributing to a more global project beginning in Year 4.

Note: Experimental Design for 8RCR1A has been revised. See Third Addendum to the Eighth Work Plan
Note: Schedule for 8RCR1A has been revised. See First Addendum to the Eighth Work Plan

Objective: Determine the variability in sex ratios from pair mating of tilapia

Significance: The few populations of O. niloticus that have been studied give mean population sex ratios of 50:50 that would be expected from a XX:XY inheritance pattern but with considerable variation from 50:50 when individual pairs are considered. The source of this variation is unknown and maybe a characteristic of the species or only the strain which was evaluated. The proposed study would address that question.

Null Hypothesis: All strains of tilapia have similar variations in sex ratios.

Experimental Design:

Site: Auburn University.

Study Period: Years 1 and 2.

Experimental Procedure: The variation in sex ratio of non-hormone-treated Oreochromis niloticus stocks from a minimum of five unique origins will be described. A minimum of 50 pair spawns will be made in outdoor hapas for each strain. Fry will be collected and reared as individual sets to a minimum of 5 cm in length. The sex ratio of each set of progeny will be determined by examining the gonads of a minimum of 100 fish per set of progeny. Broodstock of those stocks having the least variation from a 50:50 sex ratio will be held for use in a "YY" breeding program.

Sex ratio data from each spawn would be analyzed by Chi square to determine whether it differs from the expected 50:50. The frequency distribution of all spawns within a strain would also be determined and compared across strains.

Deliverables: Strains of O. niloticus with the minimum variation in sex ratios between pairs will be identified for use in breeding studies.

Schedule/Time line: This study will begin in August 1996 and be completed by March 1999.

Final Report Submittal: A final report for Study RCR 1A will be completed by April 1999.

Note: Schedule for 8RCR1B has been revised. See First Addendum to the Eighth Work Plan

Objective: Establish appropriate techniques for the androgenic production of YY male tilapia.

Significance: The success of androgenesis is dependent on being able to sterilize the genetic message of the egg or sperm without doing additional damage to the cell. This can be done with ultraviolet light or other forms of radiation. The strength and duration of exposure must be well defined for success. This information has yet to be established for tilapia and would be the focus of this phase of the work plan.

Null Hypothesis: No concentration or duration of ultraviolet treatment of tilapia eggs combined with pressure shocking of fertilized eggs will produce YY male tilapia.

Experimental Design:

Site: University of Oklahoma.

Study Period: This work would begin in Year 1 and refinements in technique would continue until project completion.

Experimental Procedures: Androgenetic tilapia will be produced by treating unfertilized eggs with various intensities and durations of UV light to kill the chromosome material. Treated eggs would be fertilized with untreated sperm and pressure shocked before the first cleavage to obtain diploid zygotes. The resultant embryos would have either a female XX or male YY genotype. An untreated set of eggs would also fertilized, a portion incubated without further pressure shock and another portion pressure shocked. Eggs would be hatched and the fry cultured to a sexable size. This work would begin in Year 1 at the University of Oklahoma and refinements in technique would continue until project completion.

The results of these studies would be evaluated as to the percent hatch obtained, the percentage of deformities and the sex ratios obtained. Adult fish would be held for use in RCR 1C.

Deliverables: A procedure for producing androgenetic male tilapia would be established.

Schedule/Time line: This study will begin in August 1996 and would continue past Year II of this work plan.

Final Report Submittal: A progress report for RCR 1B will be completed by April 1999.

Note: Experimental Design for 8RCR1C has been revised. See Third Addendum to the Eighth Work Plan
Note: Schedule for 8RCR1C has been revised. See First Addendum to the Eighth Work Plan

Objective: Determine whether there is an autosomal influence on the sex ratio in O. niloticus.

Significance: There are a number of theories to explain the deviation in sex ratios in tilapia. The theory of autosomal influence on sex inheritance can be evaluated through androgenesis. Using the procedures developed in RCR 1B, androgenic zygotes will develop either as XX females or YY males without the influence of cytoplasmic chromatin material. Such XX females mated to untreated males should produce progeny with only a 50:50 sex ratio if the XX:XY inheritance holds true. This theory would be tested in the following study.

Null Hypothesis: There is no autosomal influence on sex inheritance

Experimental Design:

Sites: Auburn University and at the University of Oklahoma

Study Period: Years II to Year IV.

Experimental Procedures: Androgenetic females would be created in RCR 1B and when mature would be pair spawned with normal males and androgenic YY males. The same set of males would also be mated with normal females. Each set of progeny would be cultured to a sexable size. The sex ratio of the progeny from these combinations would be determined through gonadal examination.

Sex ratio data would be evaluated using Chi square to determine if the observed sex ratios conformed with the expected ratios.

Schedule/Time line: This study will begin in August 1996 and would continue past Year II of this work plan.

Final Report Submittal: A progress report for Study 1C will be completed by April 1997.

Note: Schedule for 8RCR1D has been revised. See First Addendum to the Eighth Work Plan
Note: 8RCR1D has been cancelled. See Third Addendum to the Eighth Work Plan

Objective: Develop a pure YY line of male and female Oreochromis niloticus.

Significance: For a YY breeding program to be practical it is necessary to produce large quantities of broodstock. Androgenetic techniques are a promising way to develop YY males and may become refined enough to make it a practical approach for providing broodstock. Another approach is to develop true YY breeding populations. Androgenetically produced YY fry can be feminized through the administration of an estrogen resulting in a phenotypic female of a YY genotype. Such females when crossed with YY males will give all YY progeny.

Experimental Design:

Sites: Auburn University and at the University of Oklahoma

Study Period: Years II to IV.

Experimental Procedures: The strain or strains giving sex ratios that most closely conform to a XX/XY sex determination system will be used to develop YY broodfish. YY females will be developed through hormone feminization of males and confirmed through progeny testing. YY males produced in Studies II and III will be mated with YY females to produce additional YY males. A portion of the offspring will be given estrogen to produce fish that are phenotypic female/genotypic YY male. Such females will be mated with YY males to produce all YY offspring to perpetuate the line without having to use androgenesis.

Deliverables: YY females

Schedule/Time line: This study will begin in June 1998 and would continue past Year II of this work plan.

Final Report Submittal: A progress report for RCR 1D will be completed by April 1999.