Fish Reproduction:Fish Reproduction:Special Genetic IssuesSpecial Genetic Issues

Dr. Craig KasperDr. Craig Kasper

FAS 2263CFAS 2263C

Genetics: What is it?Genetics: What is it?

• GeneticsGenetics: The science of heredity and: The science of heredity and variation. variation.

• HeredityHeredity: transmission from generation to : transmission from generation to generation (usually through the process of generation (usually through the process of reproduction) factors which cause the reproduction) factors which cause the offspring to resemble their parents (or not.)offspring to resemble their parents (or not.)

Genetics TermsGenetics Terms

• Selective breedingSelective breeding-the choosing of -the choosing of individuals of a single strain and spp.individuals of a single strain and spp.

• HybridizationHybridization-the crossing of different spp.-the crossing of different spp.

• CrossbreedingCrossbreeding-the mating of unrelated -the mating of unrelated strains of the same spp. to avoid strains of the same spp. to avoid inbreedinginbreeding

Selective BreedingSelective Breeding

• ““Artifical selection” as opposed to natural selection, Artifical selection” as opposed to natural selection, results in reducing genetic variability in a population. results in reducing genetic variability in a population. Could be considered inbreeding if not closely monitored.Could be considered inbreeding if not closely monitored.

• If selective breeding involves excessive inbreeding If selective breeding involves excessive inbreeding physical abnormalities, metabolic deficiencies, and physical abnormalities, metabolic deficiencies, and developmental abnormalities may occur.developmental abnormalities may occur.

• Inbreeding depression-loss of fitness due to inbreedingInbreeding depression-loss of fitness due to inbreeding– more likely to observe occurence of recessive traitsmore likely to observe occurence of recessive traits– Decrease in heterozygosityDecrease in heterozygosity

Selective BreedingSelective Breeding

• Although potential for deleterious results, Although potential for deleterious results, selective breeding rationalized by:selective breeding rationalized by:– size size – colorcolor– shapeshape– better FCRbetter FCR– reproductive capacityreproductive capacity– disease resistancedisease resistance

HybridizationHybridization

• Already looked at hybrid striped bass. Already looked at hybrid striped bass. What other examples are out there??What other examples are out there??

Splake-brook trout/lake trout, Tiger Musky-northern pike/muskySplake-brook trout/lake trout, Tiger Musky-northern pike/musky

Sunfish hybrids (many combinations)Sunfish hybrids (many combinations)

• Hopefully achieve “hybrid vigor,” at least in Hopefully achieve “hybrid vigor,” at least in some trait.some trait.

• Most true hybrids are sterile, but not all.Most true hybrids are sterile, but not all.

GynogenesisGynogenesis

• Development of an ovum without fusion of Development of an ovum without fusion of gametesgametes– usually haploid and dieusually haploid and die– sometimes diploid larva develop sometimes diploid larva develop

• requires retention of 2requires retention of 2ndnd polar body during meiosis polar body during meiosis• Amazon mollies, cruzian carp do thisAmazon mollies, cruzian carp do this

• Induced by:Induced by:– radition, chemical, electrical currents, sperm radition, chemical, electrical currents, sperm

from related speciesfrom related species

Regulation of PloidyRegulation of Ploidy

Monosex, polyploidy, and Monosex, polyploidy, and beyond…beyond…

1.Visual selection

2. Hybridization

3. Sex-reversal

4. Genetic manipulation

Male Populations

Mono-sex male tilapia populations can be produced by:

How DO you sex a tilapia??How DO you sex a tilapia??• Begin with 25 to 30 g fish (any smaller and you’ll go blind Begin with 25 to 30 g fish (any smaller and you’ll go blind

looking for genital openings.)looking for genital openings.)

• Males and females can be separated by visual inspection of Males and females can be separated by visual inspection of the the genital papillagenital papilla (next slide). (next slide).

• The male genital papilla is The male genital papilla is pointedpointed and contains only the and contains only the small urogenital pore which is small urogenital pore which is difficult to observedifficult to observe. .

• The female papilla is The female papilla is roundedrounded and contains a small opening and contains a small opening to the urinary duct and a larger oviduct. to the urinary duct and a larger oviduct.

• Selection is usually based on the presence or absence of the Selection is usually based on the presence or absence of the oviduct. oviduct.

How DO you sex a tilapia??How DO you sex a tilapia??

• Males are stocked for further growth while females Males are stocked for further growth while females can be used as brood stock or discarded. can be used as brood stock or discarded.

• Visual selection of male tilapia is costly because of Visual selection of male tilapia is costly because of the extra labor needed to separate males from the extra labor needed to separate males from females.females.

Male papilla

Female papilla with oviduct

anus

anus

Visual Selection of the Genital Papilla

Using Two SpeciesUsing Two Species

• Two species of tilapia can be crossed to yield Two species of tilapia can be crossed to yield all-male offspring. all-male offspring.

• Male Hornorum tilapia can be hybridized with the Male Hornorum tilapia can be hybridized with the female Nile tilapia and offspring are all-male. female Nile tilapia and offspring are all-male.

• However, the hybrid is However, the hybrid is fertilefertile and can backcross and can backcross with female Nile and Hornorum tilapia. with female Nile and Hornorum tilapia.

Using Two SpeciesUsing Two Species

• Only genetically pure Hornorum and Nile tilapia Only genetically pure Hornorum and Nile tilapia will result in 100% male offspring. will result in 100% male offspring.

• Maintaining pure lines of Nile and Hornorum Maintaining pure lines of Nile and Hornorum tilapia over a long period of time is difficult. tilapia over a long period of time is difficult.

• Additionally, hybrid fry yield per female is low Additionally, hybrid fry yield per female is low compared with fry yield from pure species compared with fry yield from pure species spawns.spawns.

Male Hornorum tilapia

Female Nile tilapia All-male hybrid tilapia

ZZ

XX

XZ

Hybridization

Sex determination by hormonesSex determination by hormones

• A popular way to produce all-male populations is with A popular way to produce all-male populations is with hormone sex reversal of fry. hormone sex reversal of fry.

• Tilapia fry (18 day) are fed a diet containing a male Tilapia fry (18 day) are fed a diet containing a male steroid (methyl testosterone) for 20 to 28 days. steroid (methyl testosterone) for 20 to 28 days.

• Females develop as “functional males” following Females develop as “functional males” following hormone treatment. hormone treatment.

• All-male populations are hard to produce!! All-male populations are hard to produce!!

• ~95 to 98% males are commonly produced~95 to 98% males are commonly produced..

Sex Reversal of Tilapia Fry

1) XXF + XYM XX + XY normal

Genetic Manipulation

More genetic manipulationMore genetic manipulation

• What if female Nile tilapia are homozygous (XX) What if female Nile tilapia are homozygous (XX) and male Nile tilapia are heterozygous (XY) for and male Nile tilapia are heterozygous (XY) for sex.sex.

• A pairing of genetically normal male and female A pairing of genetically normal male and female Nile tilapia will result in 50% male and 50% Nile tilapia will result in 50% male and 50% female offspring.female offspring.

• Now if we feed normal fry estrogen for 28 days, Now if we feed normal fry estrogen for 28 days, we get phenotypic all-female fry. we get phenotypic all-female fry.

1) XXF + XYM estrogen

XXF + XY”F”

Genetic Manipulation

• However, 50% of these “feminized fry” are However, 50% of these “feminized fry” are genetic females (XX) and 50% are genetic genetic females (XX) and 50% are genetic males (XY). males (XY).

• These females are individually paired with These females are individually paired with normal males. normal males.

• A genetic female (XX) paired with a genetic male A genetic female (XX) paired with a genetic male (XY) will have offspring that are 50% male and (XY) will have offspring that are 50% male and 50% female. 50% female.

• Females from this crossing are discarded. Females from this crossing are discarded.

1) XXF + XYM estrogen

XXF + XY”F”

2) XY”F” x XYM

XXF + 2 XYM + YYM

Genetic Manipulation

• When a genetically male female (XY) is crossed When a genetically male female (XY) is crossed with a genetic male (XY) the offspring will be 25% with a genetic male (XY) the offspring will be 25% female (XX) and 75% males (50% XY and 25 % female (XX) and 75% males (50% XY and 25 % YY).YY).

• Again, individual pairings with a genetic female Again, individual pairings with a genetic female (XX) are required to determine which male is YY.(XX) are required to determine which male is YY.

• The YY males or “supermales” will sire 100% The YY males or “supermales” will sire 100% male offspring (XY) when paired with genetic (XX) male offspring (XY) when paired with genetic (XX) females.females.

1) XXF + XYM estrogen

XXF + XY”F”

2) XY”F” x XYM

XXF + 2 XYM + YYM

3) XXF x YYM

100% XYM ??

Genetic Manipulation

Transgenics: How do you get a transgenic fish?Transgenics: How do you get a transgenic fish?

• TransgenicTransgenic or or genetically engineeredgenetically engineered animals are animals are developed by inserting single or multiple genes from one developed by inserting single or multiple genes from one species into the DNA of another species.species into the DNA of another species.

• Three basic steps:Three basic steps:

– IsolateIsolate and identify the genes that posses the desired trait. and identify the genes that posses the desired trait. Genes may come from similar fish species, plants, bacteria, Genes may come from similar fish species, plants, bacteria, animals, and even humans. animals, and even humans.

– Insert Insert isolated material into the intended target fish’s eggs. isolated material into the intended target fish’s eggs.

– GrowGrow your fish!! your fish!!

Differences in terms?Differences in terms?

• The terms genetically engineered, The terms genetically engineered, genetically modified, and transgenic are genetically modified, and transgenic are used synonymously. used synonymously.

• The term transgenic arises from the The term transgenic arises from the procedure of transgenesis, one method in procedure of transgenesis, one method in which scientists have successfully inserted which scientists have successfully inserted genes from one species to another.genes from one species to another.

What is a triploid fish?What is a triploid fish?

• TriploidTriploid fish contain three sex chromosomes. fish contain three sex chromosomes.

• Heat and or pressure applied to females’ eggs to Heat and or pressure applied to females’ eggs to generate the extra chromosome.generate the extra chromosome.

• Proponents of G-EN suggest environmental risks Proponents of G-EN suggest environmental risks associated with their release into open water would be associated with their release into open water would be rendered harmless. rendered harmless.

• No guarantee of sterility!!No guarantee of sterility!!

• Competition is a reality for sterile fish too!Competition is a reality for sterile fish too!

PolyploidyPolyploidy

• Polyploidy has been well-studied in fish and Polyploidy has been well-studied in fish and shellfish. shellfish.

• Channel catfish triploids become larger than Channel catfish triploids become larger than diploidsdiploids

• Triploids converted feed more efficiently than Triploids converted feed more efficiently than diploids (Wolters et al., 1982), had six percent diploids (Wolters et al., 1982), had six percent greater carcass yield at three years of age.greater carcass yield at three years of age.

Intersexed fishIntersexed fish

• Hermaphrodites-contain ovotestes, can go Hermaphrodites-contain ovotestes, can go either way...either way...– MT treatment female after sex determination MT treatment female after sex determination

occurs (ovary has already started to develop)occurs (ovary has already started to develop)– intersexed fish must be killed to determine if intersexed fish must be killed to determine if

the process work! Not very good for the process work! Not very good for aquaculture?aquaculture?

Questions??